MICROBIAL QUALITY OF FRESH AND FROZEN EQUINE SEMEN OF INDIAN HORSES

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Veterinary Practitioner Vol. 13 No. 2 December 2012

DEVELOPMENT OF BUFFALO EMBRYONIC STEM CELL CLONES INHANGING DROP USING DIFFERENT CULTURE MEDIUM

Gopal Puri1, B. C. Das2 and Sadhan Bag3

Indian Veterinary Research Institute, Izatnagar-243 122, Uttar Pradesh, India

ABSTRACT

The present experiment was carried out to study the development of buffalo embryonic stem cell colonies in hanging drop with usingdifferent culture media. The 8-16 cell stage IVF derived buffalo embryos were made zona free. The clumped blastomere werecultured in hanging drops with different culture condition viz. control (C-I), M-I: C-I+ stem cell factor, SCF, M-II: C-I+ SCF+ bFGF and M-III:C-I+SCF+bFGF+IGF1 at 37oC, 5% CO2 and 90% relative humidity in CO2 incubator. Once the clumped blastomere cells multiplied andmade stem cell clones in hanging drops, they were passaged mechanically and further cultured for propagation of cell numbers. A totalof 25 number of zona free embryos were cultured in each medium of culture condition. The efficiency of embryonic blastomere to stemcell clones formation and propagation were significantly higher when ES cell medium was supplemented with only stem cell factorSCF (M-I) or supplemented with SCF, bFGF and IGF1 (M-III) than control and supplementation of SCF+bFGF (M-II). Media I and III werefound to be significantly (P<0.01) better followed by Media II. There was no significant difference between Media II and control. It wasobserved that the ES cell colony formation was better in hanging drop. However, when medium was supplemented with SCF, bFGFand IGF1 alone or in combination, the colony formation was almost similar.

Key words: Buffalo, ES cells, hanging drop

IntroductionStem cell research has emerged as the most vibrant

biological research activity. This is evidently a reflection of thefact that these cells have the capacity of self-renewal and thepotential to differentiate into one or more cell types dependingon the in vivo signals (Smith et al., 1992). Traditionally, stemcells are cultured on the embryonic fibroblast cells inactivatedby mitomycin-c. The effects of some factors such as calf serum,feeder cell, culture medium and additives on the efficacy ofisolation and culture of bovine embryonic stem cells werestudied using bovine and murine feeder (Li Long et al., 2002).However, many efforts have recently been made to culture stemcells on feeder free condition. Embryonic stem cells can bewell maintained without feeder layer on gel or extra cellularmatrix substrate in conditioned media or in LIF supplementedmedia (Wiles, 1993). Matrigel™ has been showed as asubstrate for feeder-free propagation of undifferentiated mEScells (Greenlee et al., 2007).

Embryonic stem cell (ES) has been recognized as one ofthe most promising therapeutic tools for the next decade. Apartfrom the tremendous potential of stem cell research in humanaspect, aspect, it has great scope in animal research andproduction as well. Generating pluripotent ES cells in farmanimals may be a key step in facilitating genome manipulationand the generation of cloned, transgenic animal and may putin the forefront of animal biotechnology. Stem cells could alsomake animal testing obsolete in toxicological testing and drugdiscovery and could be used as alternatives to animal models.In valuable livestock species, genetic engineering technologies,such as SCNT with ES cell technology, may result in synergisticgains in the ability to create and study precise geneticalterations. Very limited work has been done on isolation, cultureand characterization of embryonic stem cells from early stage

1#Part of Ph.D Thesis, Present Address: Associate Professor, Department of Physiology and Biochemistry, College of Veterinary Sciences and Animal Husbandry, Navsari Agricultural University, Navsari-396450, Gujarat.2,3Senior Scientist, Physiology and Climatology Division, IVRI, Izanagar, Bareilly, (U.P.)

buffalo embryos. Here we have attempted to derive embryonicstem cells using a new technique called hanging drop method.

Materials and MethodsBuffalo ovaries of unknown reproductive status were

collected from local slaughter house and carried to thelaboratory in normal saline solution (NSS, 0.85% NaCl) fortifiedwith gentamicin (50 µg/ml) in a thermo flask at 30-35°C within2 hours of slaughter. Oocytes were aspirated from all the visiblenon atretic surface follicles (2-8 mm diameter) by a sterile 18G needle attached to a 5 ml syringe containing oocyte collectionmedium and searching of oocytes were carried out understereo zoom microscope (Bausch and Lomb). The cumulusoocyte complexes (COCs) were evaluated and graded by themethods followed by Kobayashi et al. (1994). The processedCOCs were cultured in 50 µl droplets (20-25 oocytes/droplet)of maturation media in 35 mm sterile Petri dish. The dropletswere covered with warm nontoxic mineral oil and cultured inCO2 incubator with an atmosphere of 5% CO2, 21% O2, 37°Cand 95% relative humidity (RH) for 24 hours. After maturationin maturation media, the oocytes were first treated with 1 mg/ml hyaluronidase in OCM followed by pipetting to remove theexpanded cumulus cells. After partial removal of cumulus cells,the oocytes were washed several times with sperm TALP andfinally with fertilization TALP media and transferred to 50 µldrop of fertilization TALP media. To this, 20 µl of capacitatedspermatozoa were added and the droplets were covered withwarm mineral oil and finally incubated in CO2 incubator at 37°C,5% CO2, 21% O2 and 95% relative humidity. After 18-20 hoursof sperm-oocyte co-incubation, the presumptive zygotes weretaken out of fertilization drops and washed 8-10 times inmodified synthetic oviductal fluid (mSOF) (Walker et al., 1996)and cultured in 100 µl drop of the same media and embryo

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Table 1: Culture of embryonic stem cell in hanging drops in different culture medium

Control Media: DMEM+FBS; Media 1: DMEM+FBS+SCF; Media 2: DMEM+FBS+SCF+bFGF; Media 3:DMEM+FBS+SCF+bFGF+IGF-1,P0: Primary clone; P1: First Passage; P2: Second Passage; P3: Third Passage

Fig. 1: (A-C) Development of ES clones in Media-1 & 3, A. ES clone, D2 (10x), M-1; B. ES clone, D6 (10x), M-1; C.ES clone, D2 (10x) M-3

developments were assessed by observing the cleavage rateafter 48-72 hours of in vitro culture (IVC). The embryos of 16-32 cells were treated with protease-K for the dissolution ofzonapellucida. After complete dissolution of zonapellucida, theclumped blastomere cells were cultured in hanging drops EScell medium supplemented with fetal bovine serum anddifferent growth factors containing culture condition viz. Control(C-I), media-I, media-II and media-III. The medium used were:Control Media: DMEM+FBS; Media-1:DMEM+FBS+SCF; Media-2:DMEM+FBS+SCF+bFGF; Media-3: DMEM+FBS+SCF+bFGF+IGF-1. The clumped blastomeres were culturedat 37°C, 5% CO2 and 90% relative humidity in CO2 incubator.

For making hanging drop, ES cell medium of 25-30 µlwas put on the lid of tissue culture dish. After adding theclumped blastomere, the lid was put on the other parts of thedish in such a manner, that the drops now hang due to surfacetension. For observation of ES clone, the lid was set down sideup and saw the cells under microscope. After that again the lidwas put as above and kept in CO2 incubator.

Results and DiscussionIn the present study, different percentage (Table 1) of

primary ES cell clone was observed (Fig. 1A-C). Out of 80 zonafree embryos, cultured by hanging drop method, 64 (80%) EScolonies were formed. The efficacies of colony developmentwere 70%, 90%, 75% and 85% in control media, media-1,media-2 and media-3, respectively (Table 1). During the study,it has been observed that Media-1 and Media-3 were found tobe significantly (P<0.01) better than other media. However,there was no significant difference between Media-2 andControl media. The quality of ES colonies judged by theappearance of colonies was comparatively better by this methodthan those developed in synthetic matrices. Hanging dropculture provided slightly higher number of embryonic stem cellcolonies when compared with feeder and feeder free cultureconditions. The feeder cell layer is one of the most importantfactors affecting ES cell culture. It serves as an attachmentmatrix for cells and can secrete some kinds of cytokines, suchas LIF, that may stimulate ES cell growth and inhibit their

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differentiation. But many efforts have recently been made toeliminate animal derived components and to culture stem cellson feeder free condition. Embryonic stem cells can also bemaintained without feeder layer on gel or extra cellular matrixsubstrate like in conditioned media or in LIF supplementedmedia (Wiles, 1993). There are also reports of propagatingES cells on extracellular matrix for survival and growth, insteadof feeder layer. Matrigel™ shows promise as a substrate forfeeder-free propagation of undifferentiated mES cells forembryo toxicity endpoints (Greenlee et al., 2007). Rosler et al.(2004) examined the long-term stability of three hES cell linesin feeder free culture. Growth factors support the growth andproliferation of embryonic stem cells over long term culturesand multiple passages, without compromising their stemness. For growth and proliferation of undifferentiated embryonicstem cells for a long term culture, several growth factors viz.basic fibroblast growth factor (bFGF), insulin like growth factor-1 (IGF-1), epidermal growth factor (EGF) and stem cell factor

(SCF) are needed (Shim and Anderson, 1998).The quality ofES colonies developed in hanging drop was judged by theappearance of clone. Thus, in the present study, hanging dropcan be used as a method for better ES cell clone development.

AcknowledgementsThe authors are thankful to DBT, Government of India, for

providing financial support and Director, IVRI for providingfacilities for conducting this research work.

ReferencesGreenlee, A. R. et al. (2007) Toxicology in Vitro. 21(8):1695.Kobayashi, K. et al. (1994) J. Reprod. Fertility. 12:439-446.Li Long, A. N. et al. (2002) Chinese J. Vet. Sci. 22: 187-190.Rosler, E. S. et al. (2004) Developmental Dynamics. 229: 259-274.Shim, H. and Anderson, G. B. (1998) Theriogenology. 49: 521-528.Smith, A. G. et al. (1992) Dev. Biol. 151: 339-351.Walker, S.K. et al. (1996) Theriogenology. 45:111-120.Wiles, M.V. (1993) Methods. Ezymol. 225: 900-911.

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IntroductionTrypanosomiasis caused by Trypanosoma evansi is one

of the most important and widespread haemoprotozoandisease of equines and other domestic animals severelylimiting their working ability and productivity in almost all thetropical and subtropical regions of the world. It is an arthropodborn blood protozoan disease commonly known as Surra(Hoare, 1964) is caused by Trypanosoma evansi andtransmitted by Tabanus spp. (Radostits et al., 2000). This wasthe first trypanosome identified in mammals by Sir GriffithEvans in Dera Ismail Khan (Now in Pakistan). It is widelyprevalent in livestock of Africa, Asia and South America (Hunterand Leuckins, 1990) and is endemic in china, the Indian subcontinent, Northern America, The Philippines, Bulgaria, partsof the former U.S.S.R. and parts of Indonesia (O.I.E., 2008). InIndia, T. evansi infection is more prevalent, particularly in theareas where the environment for the breeding of insect vectorslikes tabanids flies is suitable for transmission and is endemicin north India, particularly in Punjab, Haryana, Rajasthan andUttar Pradesh. However, incidences are not uncommon in otherstates of the country and the disease is more common nearthe end of August and remains prevalent until mid winter, whichcoincides with the increase in number of blood sucking flies(Bhatia, 2000). An increasing prevalence of infection associatedwith increasing age is commonly observed (Davison et al.,2000), which may be due to persistent infection or due to

CHEMOTHERAPY AND EVALUATION OF DRUG EFFICACY IN EQUINESINFECTED WITH T. EVANSI WITH ANTRYCIDE PROSALT AND

ISOMETAMEDIUM CHLORIDE

Randhir Singh1, S.K. Gupta2 and Shailendra UpadhyayDivision of Veterinary Clinical Medicine and Jurisprudence

Faculty of Veterinary Sciences and Animal HusbandryS.K.U.A.S.T.-J, R.S.Pura-181102, Jammu, India

ABSTRACT

Trypanosomiasis caused by Trypanosoma evansi is one of the most important and widespread haemoprotozoan diseaseof equines and other domestic animals severely limiting their working ability and productivity in almost all the tropical andsubtropical regions of the world. Two drugs were selected for the present study i.e., Lytrip (Antrycide Prosalt)-Lyka andTrypashish (Isometamedium chloride) - Ashish Life Science Pvt. Ltd. A total of twelve animals (equines) showing clinicalsigns of disease as well as found positive for presence of T. evansi either by PW BF, MHCT or Giemsa’s staining methodwere taken up for treatment and drug evaluation. These animals were first processed for different hemato-biochemicalparameters (n=12) and divided in two groups of six animals each and were treated with Lytrip and Trypashish respec-tively. Lytrip was given @ 5 ml/100 kg body weight s/c single dose and Trypashish was given @ 0.5 mg/kg body weightdeep i/m single dose (2.5 ml of 2% solution per 100 kg body weight). Haemato-biochemical examination one week after thetherapy revealed significant restoration of TEC in both the treatment groups. Lymphocyte count was found to be increasedin group I and decreased in group II. There was also marked improvement in glucose level in both the groups followingtreatment. All other haemato-biochemical parameters were unaffected.

Key words: Equines, haemato-biochemical, trypanosomiasis, antrycide prosalt, isometamedium chloride.

reduced infection in young animals as a result of them beingheld in protected housing conditions (Njiru et al., 2004). Inmost of the domestic and wild animals T. evansi is pathogenicwith clinical signs depending on strain pathogenicity, hostspecies, general stresses on the host and localepidemiological conditions (Hoare 1972; Dieleman 1986).Disease may manifest as an acute or chronic condition, andin the later cases may persist for several months. The clinicalsigns of Surra are not sufficiently pathognomic and diagnosismust be confirmed by laboratory methods. The disease insusceptible animals is manifested by pyrexia, progressiveanaemia, loss of condition, recurrent episodes of fever,parasitaemia, oedema, particularly of the lower parts of thebody, urticarial plaques and petechial haemorrhages of theserous membranes are often observed (Radostits et al., 2000).There are indications that the disease causesimmunodeficiency (Onah et al., 1998). Long standing untreatedcases of trypanosomiasis in equines may cause neurologicalsigns like incordination of gait, paddling moment, head tilt,wide stance and falling down. Several chemotherapeutic drugshave been tried against Surra, but their use is limited becauseof toxicity, inability to bring about complete sterilization and nonavailability. The successful control of Surra requires acombination of immunological methods, chemotherapy andvector control (Chaudhri, 2007). The choice of drug, dosageand route of administration vary with the species affected,

1Part of M.V.Sc. Thesis of Senior Author2Reprint request and corresponding Author: Dr. S.K. Gupta, Dean, F.V.Sc., SKUAST-J, R.S. Pura-181102., Mob: 09419124146; Email: [email protected]

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Table 1: Mean ± SE Haemato-biochemical profile of diseased equine before and afterreceiving different treatment.

Parameter (units) Diseased (n= 12) Group- I (n= 06)

Group-II (n= 06)

Hb (gm/dl) 7.53 ± 0.37 7.61 ± 0.69 7.68 ± 0.25

PCV (%) 23.21 ± 0.83 24.50 ± 1.23 24.84 ± 1.17

TEC (106/cmm) 4.50 ± 0.42a 6.42 ± 0.47b 6.56 ± 0.61b

TLC (103/cmm) 3.67 ± 0.40 3.83 ± 0.54 4.5 ± 0.67

Neutrophils (%) 51.33 ± 0.80 52.23 ± 1.19 50.83 ± 0.60

Lymphocytes (%) 54.67 ± 0.82a 57.90 ± 0.93a 51.33 ± 0.77b

Monocytes (%) 5.67 ± 0.58 5.66 ± 0.76 4.16 ± 0.65

Eosinophils (%) 0.42 ± 0.15 0.33 ± 0.21 0.33 ± 0.21

Basophils (%) 0.25 ± 0.13 0.16 ± 0.16 0.16 ± 0.16

BUN (mg/dl) 22.94 ± 0.154 22.43 ± 0.17 22.65 ± 0.21

Total protein (g/dl) 4.53 ± 0.13 4.7 ± 0.23 4.6 ± 0.15

Albumin (g/dl) 2.00 ± 0.07 2.15 ± 0.10 2.06 ± 0.10

Sodium (mmol/l) 130.33 ± 0.84 131 ± 1.41 131 ± 0.81

Potassium (mmol/l) 3.72 ± 0.09 3.6 ± 0.14 3.73 ± 0.16

Iron (µmol/l) 9.04 ± 0.30 9.86 ± 0.52 8.78 ± 0.15

Creatinine (mg/dl) 1.52 ± 0.07 1.5 ± 0.09 1.41 ± 0.10

Glucose (mg/dl) 78.50 ± 1.78a 89.66 ± 2.45b 87.16 ± 1.40b

Values bearing different superscript in a row differs significantly (P<0.05)

Graph 1: Haematological profile of diseased equines before and after receiving different treatments

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local preferences and presence or absence of drug resistance(Burn et al., 1998). Currently available drugs in India includediminazine aceturate (Berenil), quinapyramine sulphate andquinapyramine chloride (Triquin, Antrycide Prosalt) for treatmentof Surra in domestic animals (Juyal et al., 2007). Treatment ismost effective in the early stages of infection, with late stagetreatment and animals with nervous signs often relapsing.Under dose of trypanocidal drugs may lead to drug resistanceand incomplete sterilization.

Materials and MethodsTwo drugs were selected for the present study i.e., Lytrip

(Antrycide Prosalt)-Lyka and Trypashish (Isometamediumchloride) - Ashish Life Science Pvt. Ltd. A total of twelve animals(equines) showing clinical signs of disease as well as foundpositive for presence of T. evansi either by PWBF, MHCT orGiemsa’s staining method were taken up for treatment anddrug evaluation. These animals were first processed fordifferent haemato-biochemical parameters (n=12) and dividedin two groups of six animals each and were treated with Lytripand Trypashish respectively. Lytrip was given @ 5 ml/100 kgbody weight s/c single dose and Trypashish was given @ 0.5mg/kg body weight deep i/m single dose (2.5 ml of 2% solutionper 100 kg body weight). Supportive therapy with i/v fluidadministration and multi vitamin injection (Belamyl) were alsogiven. Single dose of anti histaminic drug (Anhistamin @ 5 ml/animal i/m) was also given to avoid any allergic reaction beforeadministration of Lytrip and Trypashish. Blood samples werecollected one week after treatment and examined for presenceof parasite by PWBF, MHCT and Giemsa’s staining methodand processed for various haemato-biochemical parameters.

Haemato-biochemical studiesWhole blood was processed for routine haematological

parameters like Hb, PCV, TLC, TEC, DLC as per the standardtechniques described by Jain et al. (1986). Serum wasseparated from blood by centrifugation at 3000 rpm for 15 minand was used for estimation of total protein (Biuret Method),albumin (BCG Dye method) blood urea nitrogen (GLDH-Ureasemethod) using commercially available kits of Transasiabiomedicals, Ltd. H.P., India, creatinine by Jaff’s Method (ErbaMann Heim Pvt. Ltd.) sodium and potassium (Thiocyanatemethod) using commercially available kits of Crest Biosystemsand iron by Bichromatic method using commercial kit (ReconDiagnostics Pvt. Ltd. Gujarat., India). Estimation of glucosewas done on the spot of sample collection by using glucometer(Accu-Chek Active).

The data generated from the present study was subjectedto the test of significance (one way ANOVA) as per the methoddescribed by Snedecor and Cochran (1994).

Results and DiscussionThe therapeutic efficacy of Antrycide Prosalt and

Isometamedium chloride was evaluated on the basis ofpresence/absence of Trypanosoma evansi from the blood ofclinically infected animals (examined via PWBF, MHCT andGiemsa’s staining) one week post treatment. The efficacy wasalso evaluated by taking into account the improvement inhaemato-biochemical alterations being made by these twodrugs one week post treatment (Table 1). In both the group ofanimals receiving Antrycide prosalt and isometamediumchloride, respectively, Trypanosoma evansi was absent (PWBF,MHCT, Giemsa’s staining) one week post treatment.

Haemato-biochemical examination one week after thetherapy revealed significant restoration of TEC in both thetreatment groups. Lymphocyte count was found to be increasedin group I and decreased in group II. There was also markedimprovement in glucose level in both the groups following

Graph 2: Biochemical profile of diseased equines before and after receiving different treatments

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treatment. All other haemato-biochemical parameters wereunaffected. Under present study two different Trypanocidaldrugs were used. Both the drugs were able to clear the parasitefrom blood of infected equines one week post treatment withno visible outward reactions. Haemato-biochemical studiesone week post treatment showed a significant rise in TEC andblood glucose in both groups were as lymphocytes weresignificantly decreased in group II while all other parametersshowed no significant change. The rise in TEC may beattributed to the trypanocidal effect of both the drugs leading toelimination of T. evansi from blood stream thus significantlydecreasing the mechanical destruction to red blood cells dueto lashing movement and partly also due to reducedphagocytosis of RBC’s and reduced levels of toxins afterparasite elimination. Also there was a significant increase inblood glucose levels after treatment which is due to the factthat trypanosomes are voracious consumers of glucose andtheir elimination from blood stream after treatment lead toincrease in blood glucose level. Both the drugs acted equallyagainst Trypanosoma evansi and showed no significantdifference in their efficacy in restoring haemato-biochemicalparameters to normal level. Thus both the drugs were equallyeffective in treatment of acute trypanosomiasis in equines.

Similar recordings were made by Soodan et al. (2007)who successfully treated five horses suffering fromTrypanosoma evansi infection with Antrycide Prosalt. Likewise,Waheed et al. (2003) treated 1151 infected equines in Pakistanwith isometamedium chloride out of which 1129 (98.09%)responded to treatment and recovered while 22 (1.91%) died.Adedapo et al. (2005) accessed the prophylactic dose ofisometamedium chloride (Samorin) on haematological andserum biochemical parameters of rabbits experimentallyinfected with Trypanosoma spp. And found that all the haemato-biochemical parameters reverted to normal levels within 21days of drug administration.

ReferencesAdedapo, A. A. et al. (2005) Folia Veterinaria. 49(3): 138-142.Bhatia, B.B. (2000) Text book of Veterinary Protozoology. pp. 368.

ICAR.Burn, R. et al. (1998) Vet. Parasitol. 79: 95-107.Chaudhri, S.S. (2007) Diagnosis and control of surra in buffaloes. In:

Proceedings of the National Seminar on “Recent diagnostictrends and control strategies for Haemo-protozoaninfections in livestock”, Feb’ 9-11, 2007. S.D. AgriculturalUniversity, Sardar Krushinagar, Gujarat, India. pp. 12-17.

Davison, H.C. et al. (2000) Epidemiological Infections. 124: 163-172.Dieleman, E.F. (1986) The Vet. Quarterly. 8: 250-256.Hoare, C.A. (1964) J. of Protozoology. 11: 200.Hoare, C.A. (1972) The trypanosomes of mammals, A zoological

monograph. Blackwell Scientific Publications, Oxford, U.K.pp. 749.

Hunter and Luckins, A. G. (1990) Trypanosomiasis. Handbook onAnimal Diseases in the Tropics. 4th ed. Eds. Sewell, M. M.H., D. W. Brocklesby, pp. 204-226.

Jain, N. C. (1986) Schalm’s Veterinary Hematology. 4th ed. Lea andFebiger, Washington square, Philadelphia, USA. pp. 30-34.

Juyal, P.D. et al. (2007) Control strategies against Trypanosomosis(Surra) due to Trypanosoma evansi in India. In: Proceedingsof the National Seminar on “Recent diagnostic trends andcontrol strategies for Haemo-protozoan infections inlivestock”, Feb. 9-11, 2007. S.D. Agricultural University,Sardar Krushinagar, Gujarat, India. pp. 4-7.

Njiru, Z.K. et al. (2004) Veterinary Parasitology. 124: 187-199.O.I.E. (2008) Manual of diagnostic tests and vaccines for terrestrial

animals 2008. Ch 2.1.17, Trypanosoma evansi infections(including surra), pp. 352-360.

Onah, D.N. et al. (1998) Vet. Immunology and Immunopathol. 63: 209-222.

Radostits, O.M. et al. (2000) Veterinary Medicine: A Textbook of theDiseases of Cattle, Sheep, Pigs, Goats and Horses. 9th ed.W.B. Saunders Company Ltd. pp. 1329-1338.

Snedecor, G.W. and Cochran, W.G. (1994) Statistical Methods. 6th ed.Oxford and IBH Publishing Company, Calcutta.

Soodan, J. S. et al. (2007) Centaur. 23(3): 43-45.Waheed, M. A. et al. (2003) Pakistan Vet. J. 23(3): 153-154.

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IntroductionCereal straws are increasingly being used as basal feeds

for cattle and buffaloes in the arid region of Rajasthan due toshortage of green. Efficiency of utilization of these poor qualityroughages can be maximized by the supply of deficient nutrientslike nitrogen, micronutrients and use of feed additives in the rationof animals. In this context, the role of medicinal plants, as feedadditives, in ration of animals is of vital significance because oftheir eco-friendly and non hazardous nature. Herbal feed additivescould either influence feeding pattern or influence the growth offavourable microorganism in the rumen or stimulate the secretionof various digestive enzymes, which in turn may improve theefficiency of utilization of nutrients.

Materials and MethodsEight male Rathi calves (age 8-12 months) were divided into

control and treatment group having four in each group as perRandomized Block Design (RBD). Control group was fed withconcentrate and wheat straw while treatment group was fed with

EFFECT OF ASHWAGANDHA (WITHANIA SOMNIFERA)SUPPLEMENTATION ON NUTRIENT UTILIZATION IN RATHI CALVES#

M. S. Meel*, T. Sharma and R. K. DhuriaDepartment of Animal Nutrition, College of Veterinary and Animal Science

Rajasthan University of Veterinary and Animal Sciences, Bikaner-334001, Rajasthan, India

ABSTRACT

Eight male Rathi calves (about 8-12 months of age) were randomly divided into control and treatment group. Calves were fed with wheatstraw and concentrate in the ratio of 60:40 to meet their maintenance requirement. In treatment group Ashwagandha was supplemented @3% in ration. There was no significant effect on dry matter intake (DMI), digestibility of NDF and hemicellulose, balance of nitrogen, calcium andphosphorus, digestible crude protein intake (DCPI) and total digestible nutrient intake (TDNI), whereas, the digestibility of DM, OM, CP, EE, CF,FE, ADF and average daily gain (ADG) was significantly higher in treatment group of calves. Thus it can be concluded that supplementationof Ashwagandha @ 3% of dry matter intake improves the digestibility of nutrients and average daily gain in Rathi calves.

Key words: Rathi, calves, digestibility, balance

1#Part of Ph.D. thesis. Corresponding author :[email protected]

concentrate, wheat straw and Ashwagandha @ 3% of dry matterof ration. All calves were kept under uniform managementconditions by individually housing them in well ventilated cementfloored sheds. The calves were treated for ecto and endoparasiteswith Butox spray and Panacur bolus, respectively, before the startof study. Clean, wholesome drinking water was provided twicedaily on ad libitum basis.

All calves were fed with concentrate mixture and wheat strawin 40:60 ratio for their maintenance requirement. Concentratemixture was composed of deoiled rice bran, maize, GNC, ricebran, Guar korma, Bajra, molasses, Mineral mixture and commonsalt. The samples of feed offered, residue and faeces wereanalyzed for proximate principles and minerals (calcium andphosphorous) as per the protocol described in AOAC (1995). Fibrecomposition was determined as per the methods of Robertsonand Van Soest (1981). The data obtained in the experiment wereanalyzed using statistical procedures as suggested by Snedecorand Cochran (1994).

Table 1: Effect of supplementation of Ashwagandha on nutrient utilization in Rathi calvesAttributes Control Treatment Significance ADG (g/d) 253.33a 390.00b **

DMI (g/d) 3551.44 3768.16 NS DCPI (g/d) 303.08 335.68 NS TDNI (g/d) 1926.68 2213.55 NS DM digestibility (%) 59.60a 63.01b ** OM digestibility (%) 59.99a 64.66b ** CP digestibility (%) 60.88a 63.57b ** EE digestibility (%) 64.24a 69.94b ** CF digestibility (%) 50.17a 53.03b * NDF digestibility (%) 53.05 55.64 NS ADF digestibility (%) 42.79a 45.14b * Nitrogen balance(g/d) 18.66 21.78 NS Calcium balance (g/d) 18.40 19.93 NS Phosphorous balance (g/d) 8.01 8.76 NS

Note: Means superscripted with any one different letters within a row differ significantly from each other** = Significant at 1% level; * = Significant at 5% level; NS = Non Significant

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Results and DiscussionThere was no significant effect on dry matter intake (DMI),

digestibility of NDF and hemicellulose, balance of nitrogen,calcium and phosphorus, digestible crude protein intake(DCPI) and total digestible nutrient intake (TDNI), whereas,the digestibility of DM, OM, CP, EE, CF, FE, ADF and averagedaily gain (ADG) was significantly higher in treatment group ofcalves (Table 1).

ReferencesAOAC, (1995) Official method of analysis, 16th ed., Association of

Official Analytical Chemists, Washington, D.C., USA.Robertson, J.B. and Van Soest, P.J. (1981) The Detergent System of

Analysis and its Application to Human Foods. CornellUniversity, DC, USA.

Snedecor, G.W. and Cochran, W.C. (1994) S t a t i s t i c a lMethods. 8th ed. Oxford and IBH Publishing Co. New Delhi,India.

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IntroductionThe impact of fungal toxins on animals and poultry extends

far beyond the obvious effect of causing death. The economicimpact of lowered productivity, lower weight gain, decreasedfeed efficiency, increased disease incidence because ofimmune system suppression, is many times greater than thatof immediate morbidity and mortality. Besides injurious effectsin animals, these fungal toxins pose food borne hazards tohumans as the toxic metabolites are finally excreted throughmeat and eggs leading to human health hazards. In a recentglobal disease survey, mycotoxins have been regarded asone of the top ranking health concerns in Asia (Sluis andHunton, 2000). A number of fumonisins have since beenisolated and characterized, but FB1 remains the most toxiccompound (Gelderblom et al., 1992). FB1 either in naturallycontaminated maize or maize-based feeds or in purified form,has been reported to cause equine leukoencephalomalacia(Marasas et al., 1988), porcine pulmonary oedema andhydrothorax syndrome (Harrison et al., 1990). FB1 also causesliver toxicity and liver cancer in rats, and atherosclerosis inmonkeys (Norred, 1993).

Ochratoxins (OTA), produced mainly by Aspergillusochraceus (now called A. alutaceus) and Penicillumverrucosum, causes significant loses to the poultry industrydue to its effects on performance and health. It causes areduction in growth rate and feed consumption, poorer feedconversion and increased mortality (Pecham et al., 1971). OTAinduces degenerative changes and an increase in weight ofthe kidneys and liver, as well as decrease in weight of lymphoidorgans (Stoev et al., 2002).

The majority of reports on the toxic effects of FB1 in avian

STUDY OF TOXIC INTERACTION BETWEEN FUMONISIN-B1 ANDOCHRATOXIN-A FOR CERTAIN SERUM BIOCHEMICAL

PARAMETERS IN JAPANESE QUAIL#

Manzoor Ahmad Khan1*, Asif Iqbal2, Abid Ali Bhat3 and Rajesh Kumar Asrani4

Department of Veterinary PathologyCollege of Veterinary and Animal Sciences

CSK HP Agricultural University, Palampur-176 062, Himachal Pradesh, India

ABSTRACT

A total of 300 one-day-old quail chicks (Coturnix coturnix japonica) were divided into 4 groups (3 replicates per treat-ment), viz. control (CX), fumonisin B1 (FX), ochratoxin A (OX) and combination (FO) containing 75 birds in each group. Birdsin the control group (CX) were fed quail mash alone, where as birds in group FX were fed 200 ppm of fumonisin B1 (FB1)from Fusarium verticillioides culture material; group OX was fed 2 ppm of ochratoxin A from Aspergillus ochraceousNRRL-3174; and group FO was fed a combination of 200 ppm of FB1 and 2 ppm of OTA. Diets were fed from day 1 to 28to study serum biochemistry. The mean treatment effect across the time period revealed a signif icant ly higher meancreatinine values in group FX as compared with the other groups (CX and FO). The mean treatment effect at the conclusionof the experiment across the age revealed a significantly higher (P<0.05) mean uric acid values in the groups OX and FOas compared to that of control group (CX).

Key words: Serum biochemistry, Japanese quail, Fusarium verticilloides , Ochratoxin A

1#Part of M.V.Sc. Thesis and corresponding author, Email: [email protected] of Veterinary Epidemiology and Preventive Medicine, SKUAST- Jammu, India3Department of Veterinary Medicine, COVAS, CSK HPKV, 176 062, Himachal Pradesh, Palampur, India4Associate Professor, Department of Veterinary Pathology, COVAS, CSK HPKV, Palampur, India

species pertain to chickens and turkeys, in which it has beenfound that when fed at high levels to be associated with reducedbody weight and feed intake, diarrhoea, poor performance,and alterations in haematological and biochemical parameterswith increased activity of the enzymes, alanine transaminaseand aspartate transaminase (Bermudez et al., 1996; Espadaet al., 1997; Henry et al., 2000). Unlike aflatoxins andochratoxins, the susceptibility of quail to FB1 is not well known.In earlier studies, we reported the effects of feeding Fusariumverticilloides culture material (FCM) supplying 150 ppm of FB1to Japanese quail (Deshmukh et al., 2005b,c). The presentstudy was thus undertaken to determine the effects of feedingFCM containing 200 ppm of FB1 and Aspergillus ochraceousculture material (ACM) containing 2 ppm of OTA in relation toserum biochemistry in Japanese quail.

Materials and MethodsThe present studies were conducted on three hundred

one-day-old Japanese quail chicks procured from the CentralPoultry Development Organization, Chandigarh. The birds werekept under strict hygienic conditions throughout the period ofthe experiment. The animal care and experimental protocolwere approved by the University and by the Committee for thePurpose of Control and Supervision of Experiments on Animals(CPCSEA). The quail chicks were maintained on chick mash(Quail mash procured from the Department of Animal Nutrition,COVAS, CSK HPKV, Palampur) from day one until the end ofthe experiment. Feed was autoclaved for 15 minutes at 15pounds pressure before feeding or mixing with Fusariumculture material (s). Boiled (for 15 minutes) and subsequentlycooled water was given to the birds throughout the experiment.

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Table 1: Dietary treatments starting from day one until the end of the experiment.

Group Treatment Total level of culture material (s) used

Level of mycotoxin (s) supplied (ppm)

CX Chick mash alone 0 % 0 FX FB1 alone 3.25 % 200 OX OTA alone 2.5% 2 FO FB1 and OTA 3.25% and 2.5% 200 FB1 + 2 OTA

Table 2: Effects of fumonisin B1 and ochratoxin A on creatinine levels (mg/dl) in the serum of Japanese quail1

Group Days post-feeding Mean treatment

effect 7 14 21 28

CX 0.57±0.10b 0.63±0.11a 0.60±0.08ab 0.60±0.08b 0.60±0.04z

FX 1.97±0.27a 0.49±0.06a 0.69±0.04a 0.71±0.06ab 0.97±0.12x OX 1.54±0.24a 0.77±0.11a 0.40±0.06b 0.83±0.03a 0.88±0.09xy FO 0.66±0.19b 0.77±0.10a 0.56±0.06ab 0.84±0.02a 0.71±0.05yz

Mean age Effect

1.18±0.14A 0.66±0.05B 0.56±0.03B 0.74±0.03B

Age x treatment effect 8.35HS a-bValues within columns (between groups CX, FX, OX and FO) with different superscripts are significantly different

by ANOVA (P<0.05).x-zValues within a column with different superscripts showing mean treatment effect are signif icantly d ifferent byANOVA (P<0.05)HSF-value indicating interaction between different treatments and age of quail chicks (HS = highly signif icant) byANOVA (P<0.01).A-BValues within a row with different superscripts showing mean age effect are signif icantly different by ANOVA (P<0.05).1Data are means ± SE of three replicate pens of 3 quail each. CX = birds fed quail mash alone; FX = birds fed fumonisinB1; OX = birds fed ochratoxin A; and FO = birds fed fumonisin B1 and ochratoxin A.

Table 3: Effects of fumonisin B1 and ochratoxin A on uric acid levels (mg/dl) in the serum of Japanese quail1

Group Days post-feeding Mean treatment

effect 7 14 21 28

CX 11.28±0.79b 8.27±0.82a 7.23±1.35b 6.12±1.33a 8.23±0.62y

FX 16.28±1.34a 10.23±1.38a 7.81±0.51ab 5.64±0.98a 9.99±0.85xy

OX 16.51±0.71a 10.64±1.94a 9.68±1.59ab 7.18±1.03a 11.00±0.88x

FO 11.82±0.45b 10.31±1.10a 12.23±2.25a 8.65±2.39a 10.75±0.86x

Mean age effect

13.97±0.59A 9.86±0.66B 9.24±0.81B 6.90±0.76C

Age x treatment effect 1.70 NS a-bValues within columns (between groups CX, FX, OX and FO) with different superscripts are significant ly different byANOVA (P<0.05).x-yValues within a column with different superscripts showing mean treatment effect are signif icantly d ifferent byANOVA (P<0.05)NSF-value indicating interaction between different treatments and age of quail chicks (NS = non signif icant) by ANOVA(P<0.05).A-CValues within a row with different superscripts showing mean age effect are signif icantly different by ANOVA(P<0.05).1Data are means ± SE of three replicate pens of 3 quail each. CX = birds fed quail mash alone; FX = birds fed fumonisinB1; OX = birds fed ochratoxin A; and FO = birds fed fumonisin B1 and ochratoxin

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Feed and water were given ad libitum, and no medication wasgiven during the period of the experiment. The feed sampleswere found to contain 12 ppb of aflatoxin- B1 and were freefrom ochratoxin-A, citrinin, zearalenone, aflatoxin-B2, aflatoxin-G1, aflatoxin-G2 and T-2 toxin. The mycotoxins for the presentstudies i.e. fumonisin-B1 and ochratoxin-A was supplied byFusarium verticillioides M-1325 culture material (FCM) andAspergillus ochraceous NRRL-3174 (Courtesy: Dr. G. E.Rottinghaus, University of Missouri, Columbia, USA). Fusariumculture material containing 6200 mg FB1 per kg and ochratoxinculture material containing 80 mg OTA per kg was incorporatedat the rate of 3.25 per cent and 2.5 per cent in the chick mash tosupply 200 ppm FB1 and 2 ppm OTA, respectively. The fumonisinculture material and ochratoxin culture material were notincorporated in the diet of control group (CX).

Three hundred, one-day-old Japanese quail chicks wererandomly divided into four groups i.e. FX (fumonisin B1), OX(ochratoxin-A), FO (fumonisin B1+ochratoxin A), and CX (control)with 75 birds apiece in each of the four groups. The presentstudy was conducted using three pen replicates of 25 quailper pen in each of the four groups for a period of 28 days.Various dietary treatments starting from day one until the endof the experiment are presented in Table 1.

After weighing, 2 to 3 ml of blood was collected via cardiacpuncture from randomly selected nine birds (three quail perreplicate) from each treatment group at 7, 14, 21 and 28 dayspost-feeding for determination of uric acid and creatinine. Allthe biochemical determinations were done using diagnostickits (Bayer Diagnostics India Ltd., Baroda, India) in a fullyautomated Blood Chemistry Analyzer (RA-50 Auto ChemistrySystem) according to the manufacturer’s recommendations.The birds were euthanized by cervical dislocation aftercollection of blood samples at each of the aforementionedintervals.

Data were subjected to statistical analysis for drawinginferences using a standard procedure (Snedecor andCochran, 1967). Treatment means were compared usingDuncan’s Multiple Range Test (2-way ANOVA) to determinethe effect of treatment, age and their interactions (Duncan,1955). All levels of significance were based on the 95 per centlevel of probability.

Results and DiscussionBiochemical parameters revealed a significant variation

between quail fed chick mash alone and those given culturematerial amended diets. Mean values of creatinine for each ofthe experimental groups are presented in (Table 2). They weresignificantly higher in groups, FX and OX than those in groupsCX and FO at 7 DPF. The mean creatinine values for groupsOX and FO were more or less comparable at 14 DPF andonwards. At 28 DPF, however, the serum creatinine levels ingroups OX and FO were found to be significantly higher(P<0.05) than that in group CX. The mean treatment effectacross the time period revealed a significantly higher meancreatinine values in group FX as compared with the other

groups (CX and FO). The overall treatment effect in relation tothe progressing age of birds was found to be highly significant(P<0.01). Significantly increased serum creatinine values asobserved in our study were also reported in turkey poults afterfeeding 300 mg/kg fumonisin B1 and 3 mg/kg ochratoxin A for20 days (Kubena et al., 1997). While studying the interactionbetween fumonisin B1 and moniliformin, Sharma et al. (2008)noted a significantly higher mean serum creatinine values inthe combination (fumonisin B1 + moniliformin) group at 28DPF.

The mean uric acid concentration was significantly higher(P<0.05) in group OX and FX at 7 DPF when compared withgroups CX and FO. The mean values in groups OX and FOwere higher at subsequent intervals but the difference wasfound to be statistically significant (P<0.05) only in thecombination group (FO) from that of group CX at 21 DPF (Table3). The mean treatment effect at the conclusion of theexperiment across the age revealed a significantly higher(P<0.05) mean uric acid values in the groups OX and FO ascompared to that of control group (CX). The mean age effectrevealed that uric acid values were maximum at 7 DPF andminimal at 28 DPF. The overall treatment effect in relation tothe progressing age of birds was found to be non-significant.

Increased serum uric acid values as observed in our studycoincided with the observations made by Kubena et al. (1997),who noticed a significant increase in serum uric acid values inturkey poults after feeding 300 mg/kg fumonisin B1 and 3 mg/kg ochratoxin A. In a study on interaction between fumonisin B1and aflatoxin, Kubena et al. (1994) reported significantly higherserum uric acid values in the combination group in turkey poults.

AcknowledgementsAuthors are grateful to Dean CSK HPKV and Dr. S. K.

Sharma, Associate Professor, Department of Veterinary Surgery,CSK Himachal Pradesh Agricultural University, Palampur fortheir assistance in the completion of this study.

ReferencesBermudez, A.J. et al. (1996) Avian. Dis. 40: 231-235.Deshmukh, S. et al. (2005b,c) Avian. Dis. 49: 274-280.Duncan, D.B. (1955) Multiple range and multiple F tests. Biometrics

11:1-42.Espada, Y. et al. (1997) Avian. Dis. 41: 73-79.Gelderblom, W.C.A. et al. (1992) Mycopathologia. 117: 11-16.Harrison, L.R. et al. (1990) J. Vet. Diag. Invest. 2: 217-221.Henry, M.H. et al. (2000) Poul. Sci. 79: 1378-1384.Kubena, L.F. et al. (1994) Poul. Sci. 73: 408-415.Kubena, L.F. et al. (1997) Poul.Sci. 76: 256-264.Marasas, W.F.O. et al. (1988) J. Vet. Res. 55: 197-203.Norred, W.P. (1993) J. Tox. and Env. Heal. 38: 309-328.Peckham, J.C. et al. (1971) App. Micro. 21: 492-494.Sharma, D. et al. (2008) Poul. Sci. 87: 1039-1051.Sluis, W. Vander and Hunton, P. (2000) Wor. Poult. 16: 26-30.Snedecor, G. W. and Cochran, W. C. (1967) Statistical Methods. 6th

ed., Iowa State University Press, Ames. Pp. 593.Stoev, S.D. et al. (2002) Vet. Res. Comm. 26: 189-204.

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IntroductionWithin any cell in any animal there needs to be a balance

between oxidants and antioxidants. If the cell becomes moreoxidative, then oxidative stress results, which can produce celldamage and often leads to cell death. During metabolism,several powerful oxidants are produced, such as the freeradicals, superoxide (O2

-), peroxyl radical (ROO-), and hydroxylradical (OH ) and other non-radical oxidants such as hydrogenperoxide (H2O2) and singlet oxygen (O). The damaging effectsof free radicals are lipoperoxidation, DNA damage and proteindestruction which are believed to be the causes of cancer,inflammatory diseases, atherosclerosis and aging (Mayes,1993). Free radical and peroxide oxidants have been termedreactive oxygen molecules or metabolites (ROM) (Powell,1991). When cells are unable to control ROM production orremoval, one consequence is tissue membrane destruction.Membrane destruction results in enzymes leaking from cellsand entering the circulation. Oxygen-derived free radicals suchas superoxide anions are detoxified to hydrogen peroxide bysuperoxide dismutase (Deby and Goutier, 1990). The hydrogenperoxide is then reduced to water by enzyme catalase orglutathione peroxidase.

In an earlier study, aqueous extract of Chlorophytumborivilianum significantly ameliorated the elevated levels ofplasma glucose, triglycerides, and cholesterol and serumcorticosterone as effectively as the standard drug (diazepam)in rats. Hence, in the present study, the adaptogenic andantioxidant effect of Chlorophytum borivilianum vis a vis vitaminC, a natural antioxidant, on lipid peroxidation and antioxidantenzymes have been investigated in the rats subjected to chroniccold restraint stress.

EFFECT OF CHLOROPHYTUM BORIVILIANUM (SANTA PAU AND FERNANDES) VIS A VISVITAMIN C ON LIPID PEROXIDATION AND ANTIOXIDANT ENZYMES IN STRESS

INDUCED RATS#

Narjeet Singh, Rajesh Nigam1, Pawanjit Singh and Vijay PandeyDepartment of Biochemistry

College of Veterinary Science and Animal HusbandryDeen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go Anusandhan Sansthan

Mathura-281001, Uttar Pradesh, India

ABSTRACT

The aqueous extract of Chlorophytum borivilianum (CB) when administered orally to the rats under chronic cold restraintstress brought about a signif icant reversal in the augmented lipid peroxidation levels in blood plasma, liver and kidneyhomogenates akin to that observed for vitamin C supplementation in the study. Further, the pretreatment with CB extractsignificantly improved upon the shrunken levels of superoxide dismutase in blood plasma, liver and kidney homogenates asobserved for vitamin C. Catalase levels in blood haemolysate and liver homogenate were signif icantly elevated to nearcontrol values when the animals were pretreated with CB extract or vitamin C. The catalase activity in the kidney tissuedecreased to the values statistically similar to control level when pretreated with CB extract. The CB extract produced50% inhibition at the dose rate of 1750 µg/ml for DPPH* free radical and 300 µg/ml for thiobarbituric acid reactive sub-stances showing considerable antioxidant activity. The study concluded that CB extract produced similar adaptogenic andanti-oxidative effects as observed in case of vitamin C in the rats with chronic cold restraint stress.

Key words: Stress, Chlorophytum borivilianum, antioxidant enzymes, lipid peroxidation, rats.

#Part of M.V.Sc. Thesis of first author.1Corresponding author. Phone: 0565-2471815 and E-mail: [email protected]

Materials and MethodsThe crude root powder of Safed Musli, identified botanically

as Chlorophytum borivilianum (CB) belonging to the familyLiliaceae, was procured from pharmacology laboratory of thecollege. The powdered sample was used for preparation ofhot aqueous extract (HAE).

Preparation of hot aqueous extract (HAE) of Chlorophytumborivilianum: Forty five grams of coarsely ground powder ofChlorophytum borivilianum was placed in a porous cellulosethimble in soxhlet apparatus. The thimble was placed in theextraction chamber above the flask containing 300 ml of tripleglass distilled water. The flask was heated and the solventwas allowed to evaporate. Temperature was adjustedaccording to boiling temperature of the solvent in the flask.Extraction process was allowed to continue for approximately48 hrs involving 20-22 cycles till the solvent siphon tube ofextractor became completely colourless. Thereafter, the extractwas collected in a rotatory vacuum evaporator andconcentrated under reduced pressure to a semisolid massand per cent yield was calculated. The extract was transferredinto air tight containers and stored in refrigerator till further use.

Animals: Study was conducted on healthy, male Wistar albinorats weighing between 100-120 grams obtained fromLaboratory Animal Resources (LAR) section of Indian VeterinaryResearch Institute (IVRI), Izatnagar (U.P.). The rats were keptin smooth impervious poly-propylene cages under standardhousing conditions with 12:12 hours light and dark cycle.Animals were provided with nutritionally adequate standardlaboratory diet which was procured from Ashirwad Industries

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Limited, district- Ropar, Punjab, India. All the procedures wereperformed in accordance with the recommendations andapproval of the Institutional Animal Ethics Committee (IAEC)constituted as per the directions of CPCSEA.

Procedure: The rats were divided into following four groups of 6each: Control [non-stress] group I, Stress group II, CB extract (250mg/kg body weight) treated group III with stress and vitamin C(200 mg/kg body weight) treated group IV with stress. Theexperimental model used was Chronic Cold Restraint Stressmodel (Kenjale et al., 2007). Briefly, the treatments (CB extract orvitamin C) were administered orally 45 minutes prior to the stressregimen up to seven consecutive days except that the rats waskept fasted overnight on the 6th day before drug feeding and stressexposure. The stress was produced by restraining inside anadjustable cylindrical plastic tube (6.2 cm diameter, 20 cm long).Therats were confined individually and exposed continuously to coldstress at 4oC for 50 minutes, once only for 7 consecutive days. Onday seven the rats were sacrificed immediately after stress bydecapitation, and the blood, liver tissue and kidney tissue werecollected. Blood plasma, haemolysate and tissue homogenatesamples were preserved using standard methods for biochemicalanalysis.

Biochemical estimations: Estimation of different biochemicalparameters in blood haemolysate, plasma, liver tissue and kidneytissue homogenates were carried out as described below.Catalase activity was assayed by the UV spectrophotometericmethod (Bergmeryer, 1983), Superoxide dismutase (SOD) by thespectrophotometric method (Madhesh and Balasubramaniam,1998). The extent of lipid peroxidation was evaluated in terms ofMDA (Malondialdehyde) production, determined by thiobarbituricacid (TBA) method (Rehman, 1984).

In vitro anti-oxidant activity: To measure antioxidant activity ofthe herb, the 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radicalscavenging assay and Thiobarbituric acid reactive substances(TBARS) assay were carried out by spectrophotometericmethods (Kenjale et al., 2007).

Statistical analysis: All the data obtained were statisticallyanalyzed by Duncan’s multiple range test (SPSS 16). P values<0.05 were considered significant.

Results and DiscussionEffect of Chronic cold restraint stress on lipid peroxidation(LPO) and antioxidant enzymes: Stress resulted in asignificant increase in LPO levels of blood plasma, liver andkidney tissue. The antioxidant enzyme, SOD recorded asignificant fall in their levels of blood plasma, liver and kidneytissue when rats were subjected to chronic cold restraint stress.Though the catalase activity showed similar trend in the bloodplasma and liver tissue but kidney tissue recorded an increasein the levels after stress (Table 1).

Effect on lipid peroxidation (LPO) levels: LPO levels weremeasured in terms of malondialdehyde (MDA) produced.Pretreatment with Chlorophytum borivilianum (CB) aqueousextract or vitamin C brought about the significant reversal in theaugmented LPO levels in blood plasma, liver and kidney

homogenates. As shown in the Table 1, the values observedfor both the treatment groups III and IV were found to besignificantly lower when compared to the stress group II.

Effect on super oxide dismutase (SOD) levels: Pretreatmentwith Chlorophytum borivilianum (CB) aqueous extract or vitaminC significantly improved upon the shrunken levels of SOD inblood plasma, liver and kidney homogenates since the valuesobserved for both the treatment groups III and IV were found tobe significantly higher when compared to the stress group II(Table 1). Moreover, the SOD levels in the liver and kidney tissuein the treatment groups were statistically similar to the controlvalues.

Effect on catalase levels: Catalase levels in blood haemolysateand liver homogenate were found to be significantly elevated tonear control values when the animals were pretreated with CBextract or vitamin C. The enzyme activity in the kidney tissue recordeda decrease to the values statistically similar to control level. Thelevels for treatment groups (III and IV) in blood haemolysate andliver homogenate differed significantly from that of control (group I)values while kidney homogenate levels did not differ significantlyamong groups I, III and IV (Table 1).

Anti-oxidant activity: As shown in figure 1 and 2, theChlorophytum borivilianum produced 50% inhibition at thedose rate of 1750 µg/ml for DPPH* free radical and 300µg/mlfor Thiobarbituric Acid Reactive Substances (TBARS).

During stress, the energy requirement of the organism isincreased which results into enhanced generation of free radicals.These free radicals bring about oxidation of vital macromoleculessuch as nucleic acids and protein. Free radicals also damagebiomembrane as reflected by increase in LPO that affects the cellintegrity and its function. Therefore, the stress generating conditionsresult into weaker body defence system due to reducedantioxidants. Adaptogens like macro and micronutrient foodsupplements, minerals and herbs can help the biological systemto adapt in such stressful conditions.

In the present study the roots of Chlorophytum borivilianumand vitamin C have been evaluated for their adaptogenic activity inthe rats subjected to chronic cold restraint stress. In general thechronic cold restraint stress releases corticosteroids which inducehyperinsulinaemia causing increased synthesis of cholesterol. Itclearly indicates that stress has profound effect on metabolicfunction of the body (Mulay, 2004).

In the present study, the SOD levels were found to decreasesignificantly after stress in all the tissue and blood which is inagreement with earlier studies (Yildirim et al., 2010; Gupta et al.,2010). It was suggested that the free radicals produced in thestress condition inhibited the activity of anti-oxidative enzymes suchas SOD, Catalase and GPX. Similar studies (Pigeolet et al., 1990)reported the inactivation of these enzymes by hydroxyl radical andhydrogen peroxide. These researchers further observed that thedepression in SOD activity in all the tissue of stressed rats mayresult in cellular injury by superoxide radical which could havepropagated the chain reaction producing one hydrogen peroxideand one GSSG for each turn of cycle.

The pretreatment with Chlorophytum borivilianum or vitaminC were found to augment the SOD activity in the blood, liver and

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kidney tissue to the control levels. In a related study, thehypercholestremic rats treated with doses of Chlorophytumborivilianum exhibited an increase in the SOD and ascorbicacid levels (Visavadiya and Narasimhacharya, 2007).

The catalase activity determined in the rats after chroniccold restraint stress in the present study were found to decreasesignificantly in the blood and liver tissue as compared to thecontrol. However, in the kidney tissue, the catalase activity wassignificantly increased after chronic cold restraint stress. Thefindings were similar to those recorded in an earlierinvestigations (Kaushik and Kaur, 2003; Selman et al., 2000).They suggested relative contribution of catalase and GPX indecomposition of endogenously produced H2O2 that followtissue specificity with catalase being more important in therenal tissue. In the present study, the lowered levels of catalaseobserved after giving stress to the rats were found to increaseto the levels comparable to the values recorded for control inthe blood and liver tissue. Similarly in the kidney tissue,increased concentration of catalase was lowered aftersupplementation with Chlorophytum borivilianum or vitaminC to the level of control. This clearly indicated the anti-oxidativeeffect of Chlorophytum borivilianum and vitamin C on theCatalase activity. It was argued that vitamin C is more effectiveantioxidant than protein thiols, bilirubin, urates and tocopherolin plasma in in-vitro systems based on earlier studies(Jayachandran et al., 1996; Frei et al., 1989). The increase inanti-oxidant activity with vitamin C may be due its effective radicalscavenging action.

The LPO was found to increase significantly whencompared to the control in the stressed rats in all the cases.Similar observations were reported by an earlier study. Theprobable reason for increase in LPO in the stressed rats maybe related to biomembrane injury and cellular damage causedby free radicals .The oxidative damage to the membrane, mayalso disrupt the ionic channels membrane transport proteinand inactivate membrane associated enzymes. The lipidbilayer itself becomes more permeable due to oxidativedamage to membrane, may therefore trigger various diseases(Kaur et al., 2006), including cancer, cardio vascular diseases,immune function decline, brain dysfunction and cataract aswell as in the degenerative process associated with ageing(Ames et al.,1993).

The Lipid peroxidation was found to be reduced

significantly after the supplementation with Chlorophytumborivilianum or vitamin C in liver and kidney tissue includingblood in the present study. Thus, the crucial property ofantioxidant compounds like vitamin C by virtue of which theyinhibit LPO is clearly exhibited by Chlorophytum borivilianum,the wonder herb. The study confirmed the findings of someearlier studies on different herbs (Kaur et al., 2006) and vitaminC supplementation (Jayachandran et al., 1996).

Further, the crude aqueous extract of Chlorophytumborivilianum produced significant percent inhibition in the levelsof DPPH* free radical and TBARS as observed in this study.Similar observations were recorded in some earlier reports(Kenjale et al., 2007; Kaur et al., 2010).

ReferencesAmes, B.N. et al. (1993) Oxidants, antioxidants and degenerative

diseases of ageing. Proc. N & l. Acad. Sci., USA. 90: 481.Bergmeryer, H. U. (1983) U.V method of catalase assay. In: Methods

of enzymatic analysis. Vol. III 3rd ed. Weinheim, DecrfieldBeah, Florida, Basal, pp. 273.

Deby, C. and Goutier, G. (1990) Biochem. Pharmacol. 39: 399.Frei, B. et al. (1989) Ascorbate is an outstanding antioxidant in human

blood plasma. Proc. N & l Acad. Sci., USA. 86: 6377.Gupta, V. et al. (2010) Food and Chemical Toxicol. 48: 1019.Jayachandran, M. et al. (1996) J. Nutri. Biochem. 7: 270.Kaur, G. et al. (2006) J. Ethnopharmacol. 108: 340.Kaur, R. et al. (2010) J. Chinese Clinic. Med. 5(1): 7.Kaushik, S. and Kaur, J. (2003) Clinica. Chimica. Acta. 333: 69.Kenjale, R.D. et al. (2007) Ind. J. Experimental Biology. 45: 974.Madesh, M. and Balasubramanian, K. A. (1998) Ind. J Biochem. Biophys.

35: 184.Mayes, P.A. (1993) Lipids of physiological significance. In: Harpers’

Biochemistry. 23rd ed., (Edt.) Murray, R.K., Granner, D.K.,Mayes, P.A. and Rodwell, V.W., Appleton and Lange Norwalk,CT. pp. 142.

Mulay, M. (2004) Evaluation of herbals for adaptogenic activity. M.Tech.Thesis, University of Mumbai, Mumbai, India.

Pigeolet, E. et al. (1990) Mech. Ageing Dev. 51: 283.Powell, D.W. (1991) Immune physiology of intestinal electrolyte

transport. In: Handbook of Physiology 6. The gastrointestinalsystem IV. Intestinal absorption and secretion. AmericanPhysiology Society, Bethesda, M.D. pp. 591.

Rehman, S. U. (1984) Toxicol. Lett. 21: 333.Selman, C. et al. (2000) Free Radic. Biol. Med. 28: 1279.Visavadiya, N.P. and Narasimhacharya, A.V.R.L. (2007) Clin. Exp.

Pharmacol. Physiol. 34(3): 244.Yildirim, N. C. et al. (2010) Turkish J. Bioch. 35(2): 140.

Table 1: Effect of Chlorophytum borivil ianum and vitamin C supplementation on lipid peroxidation (LPO),superoxide dismutase (SOD) and catalase levels in blood, liver and kidney of control, stress induced and treatmentgroups of Wistar rats. [Values are mean ± SE from 6 animals in each group].

Parameters Group I (Control)

Group II (Stress)

Group III (Stress + AP)

Group IV (Stress + VC)

Blood LPO (nM MDA / ml) 4.74a±0.34 8.89b±0.30 6.54c±0.23 6.9c±0.18 SOD (U/mg Hb) 30.51a±1.19 20.29b±1.33 26.80c±1.05 25.63c±1.08 CAT (mM H2O2 utilized/min/mg Hb) 133.56a±2.91 114.27b±1.96 125c±1.54 121.36c±2.04

Liver LPO (nM MDA / ml) 2.26a±0.09 3.98b±0.21 2.85c±0.07 2.94c±0.08 SOD (U/mg protein) 47.70a±2.79 37.78b±0.92 44.57a±1.55 43.66a±0.96 CAT (mM H2O2 utilized/min/mg protein) 296.73a±4.80 266.45b±4.14 290.87ac± 3.85 279.75c±3.94

Kidney LPO (nM MDA / ml) 1.81a±0.11 3.00b±0.11 2.32c±0.11 2.56c±0.14 SOD (U/mg protein) 11.78a±1.24 8.17b±0.40 10.35a±0.82 10.53a±0.54 CAT (mM H2O2 utilized/min/mg protein) 216.05a±5.23 241.26b±5.05 221.17a±3.03 223.97a±3.87

#Superscripts differ in a row, denotes signif icant (P<0.05) difference, otherwise non-signif icant

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IntroductionIndian Gazelle or Chinkara, an antelope is mainly found

in the Indian subcontinent. Chinkara is included in theschedule I of the Wildlife Protection Act (1972), India and is inthe “least concern” category as per the IUCN red data listassessed by Mallon (2008). Thar desert of Rajasthan is havinga largest stronghold of its population and hence it is the stateanimal of Rajasthan. According to Brohi (2006) the Chinkarais found in the grasslands and desert areas of India, parts ofIran and Pakistan.

The aim of this study is to investigate the skeleton offorelimb of Chinkara, thereby making a contribution in fillingthe gap of knowledge in this field. Till date no detailed study ofthe skeleton of forelimb of Chinkara has been carried out. Asper knowledge, in many vetero-legal cases one fails to identifythe bones of this animal and confuse them with those of someother small ruminants. This investigation will help the fieldveterinarians as well as zoo veterinarians. Radiographs of thebones will help understanding contour, density, nature of thebone and will provide better approach for surgical interventionin osteological affections.

Materials and MethodsIn this study four metacarpii of adult Indian gazelle

(Chinkara) were used. The permission for the specimen’scollection was sought from Ministry of Environment and Forest,Government of India.

The skeleton were collected from the Bikaner zoo [Videapproval letter No. F.No. 1-4/2007 WL-I (pt), Dated 4th January2010 with dispatch No. 479, dated 17-1-10, from InspectorGeneral of Forest (WL), Ministry of Environment and Forest,Govt. of India].

The skeletons were dug out from the graveyard located inthe premises of the office of Deputy Conservator of Forest(WL), Bikaner. Afterwards, the specimens were put into hotwater for maceration in a large aluminum bowl. The boneswere washed with bleaching powder to remove the offensivesmell and were sundried afterwards for two to three days.

GROSS ANATOMICAL AND RADIOGRAPHIC STUDY OF THEMETACARPUS IN CHINKARA (GAZELLA GAZELLE BENNETTII)#

D. K. Jangir1, R. Mathur, A. Dangi and S. JoshiDepartment of Veterinary Anatomy

College of Veterinary and Animal ScienceRajasthan University of Veterinary and Animal Sciences, Bikaner-334001, Rajasthan

ABSTRACT

In the present study four metacarpii of adult Chinkara were used. The metacarpus was comprised of three metacarpalbones. The large metacarpal bone consisted of fused metacarpal III and IV, after confirmation by radiographs. Two smallmetacarpals, metacarpal II and me;tacarpal V were also present, which was the most peculiar f inding differentiatingChinkara from other ruminants.

Key words: Metacarpal, radiograph study, Chinkara(Gazella gazelle bennettii)

The gross study was carried out in the Department ofVeterinary Anatomy and Histology, CVAS, Bikaner, Rajasthan,under supervision of the zoo personals.The following studieswere conducted on the collected specimens.

1. Gross study2. Biometrical study3. Following measurements were taken with the help

of Vernier caliper and ordinary scale.Large metacarpal (Fig. 1)Lg: Greatest lengthBp: Maximum breadth of proximal extremityBlc: Maximum breadth of lateral condyleBlc: Maximum breadth of lateral condyleBs: Maximum breadth of shaft

Small metacarpals (Fig. 2)Lgl: Greatest length of metacarpal VLgm: Greatest length of metacarpal IIIn statistical analysis (Table 1 & 2), arithmetic mean (AM),

standard deviation (SD) and standard error (SE) werecalculated using statistical method described by Snedecorand Cochran (1989).

4. Radiographic anatomyThe radiographs were taken in the Department of

Veterinary Surgery and Radiology, College of Veterinary andAnimal Science, Bikaner, Rajasthan and analyzed on the basisof contour to confirm the gross study and presence of medullarycavity of bones.The terminology for describing the bones wasadopted from NominaAnatomicaVeterinaria 5 th ed.(Anonymous, 2005).

Results and DiscussionIn the present study the metacarpus of Chinkara

comprised of three metacarpal bones. The large metacarpalbone consisted of fused III and IV metacarpals similar to thefindings of Chauveau (1905), Sisson (1911), McFadyean (1953)and Raghavan (1964) in ox, Chauveau (1905) in sheep andgoat, Getty (1975) in sheep, Budras and Robert (2003) in

1# Part of M.V.Sc. thesis and corresponding author. Email:[email protected]

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bovine, Garrett (1988) in goat, Konig and Liebich (2006) andAkers and Denbow (2008) in ruminants. The septum of thefusion became partially absorbed. The radiograph confirmedthe fusion of the two bones. Two small metacarpals, metacarpalII and V were also present in Chinkara in accord with the reportsof Getty (1975), Konig and Liebich (2006), Akers and Denbow(2008) and Frandson et al. (2009) in horse where two splintbones namely metacarpal II and IV, were present. However, 5metacarpals are present in cat and rabbit (Chauveau, 1905),in dog (Chauveau, 1905; Miller et al., 1964 and Konig andLiebich, 2006), in the African elephant (Smuts andBezuidenhout, 1993). Four metacarpals are present in pig(Konig and Liebich, 2006; Akers and Denbow, 2008 andFrandson et al., 2009). In black Bengal goat (Siddiqui et al.,2008) and sheep (Getty, 1975), splint bones were missing.

The shaft presented two surfaces and two borders. Thecranial surface was smooth and semi-cylindrical in outline,similar to the findings of Raghavan (1964) in ox, Getty (1975) inhorse. This surface presented a shallow dorsal longitudinalgroove, which is deep in ox, sheep and goat (Chauveau, 1905).It lodged a proximal and a distal foramen, which is similar to

the reports of Chauveau (1905) in ox, sheep and goat, Sisson(1911) and Raghavan (1964) in ox, Budras and Robert (2003)in bovine. The palmar surface (Fig. 4) was flat in outline. Itpresented a deep longitudinal groove which possessedsimilar foramina. Those foramina communicated with thesimilar foramina on the caudal surface through transversecanals. These findings are identical to the reports of Chauveau(1905) in ox, sheep and goat, Sisson (1911) and Raghavan(1964) in ox, Budras and Robert (2003) in bovine.

In the present study proximal extremity (Fig. 6) presentedtwo slightly concave facets, separated by a median ridge infront and a shallow groove behind in Chinkara, similar to theobservations of Sisson (1911) and Raghavan (1964) in ox.The medial facet was larger than the lateral one. These facetsarticulated with fused second and third and fourth carpals,respectively in Chinkara, unlike horse (Getty, 1975) anddromedary (Smuts and Bezuidenhout, 1987), where thissurface articulated with second, third and fourth carpals. Justbelow this articular surface on the medial and lateral sides,was a small facet for articulation with the small metacarpalbones in Chinkara similar to horse (Getty, 1975), while a single

Table 1: Different measurements of large metacarpal

Specimen no.

Description Greatest Length

(Lg)

Maximum breadth of proximal extrimity

(Bp)

Maximum breadth of

distal extrimity

(Bd)

Maximum breadth of shaft (Bs)

Maximum breadth of

medial condyle (Bmc)

Maximum breadth of

lateral condyle

(Blc) 1. Adult

female-1 Left 16.6 1.81 1.75 1.05 0.81 0.79 Right 16.6 1.82 1.74 1.07 0.82 0.81

2. Adult female-2

Left 16.7 1.84 1.79 1.09 0.84 0.81 Right 16.7 1.83 1.76 1.11 0.84 0.84

3. Adult male -1

Left 17.3 1.87 1.86 1.11 0.83 0.81 Right 17.3 1.86 1.85 1.12 0.83 0.82

4. Adult male -2

Left 16.6 1.86 1.87 1.16 0.84 0.81 Right 16.6 1.85 1.86 1.17 0.83 0.82

Average 16.80 1.84 1.81 1.11 0.83 0.81 SD 0.31 0.02 0.06 0.04 0.01 0.01 SE 0.11 0.01 0.02 0.01 0.00 0.00

Table 2: Different measurements of small metacarpals

Specimen No. Description

Greatest length of Metacarpal II (Lgm)

Greatest length of metacarpal V (Lgl)

1. Adult female-1 Left 11.6 9.6 Right 11.5 9.6

2. Adult female-2 Left 10.1 9.4 Right 9.9 9.7

3. Adult male -1 Left 9.2 8.9 Right 9.3 9.1

4. Adult male -2 Left 9.4 8.9 Right 9.3 9.1

Average 10.04 9.29 SD 0.99 0.33 SE 0.35 0.12

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Fig. 1: Measurements oflarge metacarpal

Fig. 2: Measurements ofsmall metacarpal

Fig. 3: Dorsal view of left metacarpus (a. metacarpal II, b. proximalforamen, c. metacarpal V, d. large metacarpal, e. distal foramen, f andg medial and lateral condyles)

Fig. 4: Palmar view of left metacarpus (a. proximal foramen, b.metacarpal V, c. Metacarpal II, d. large metacarpal, e. distal foramen, fand g lateral and medial condyles)

Fig. 5: Small metacarpals

Fig. 6: Proximal view of left large metacarpal (a and b. medial andlateral facets, c. shallow groove)

Fig. 7: Distal view of left large metacarpal (a. abaxial articular area, b.axial articular area, c. dorso-palmar ridge, d. dorso-palmar groove)

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facet placed caudo-laterally was reported in ox, sheep andgoat (Chauveau, 1905). There was an eminence, themetacarpal tuberosity at the dorso-medial aspect, which wassmall in accordance with the findings of Raghavan (1964) inox and in horse (Getty, 1975).

In the present study, the distal extremity (Fig. 7) was dividedby a dorso-palmar cleft into two condyles in Chinkara simulatingthe reports of Chauveau (1905) in ox, sheep and goat,McFadyean (1953) and Raghavan (1964) in ox, Garrett (1988)in goat and Budras and Robert (2003) in bovine. In contrast itwas undivided in horse (Getty, 1975). However, in dromedarythe divided condyles are not straight but angulated (Smutsand Bezuidenhout, 1987). A dorso-palmar ridge divided eachcondyle into two articular areas. The abaxial articular area washigher than the axial one. These observations obey the reportsof McFadyean (1953) and Raghavan (1964) in ox.

In the present study small metacarpals (Fig. 5) were cordlike bones in Chinkara, which reached the distal third of thelarge metacarpal. The lateral one (metacarpal V) was slightlylarger than the medial one (metacarpal II).

The shaft presented two variably curved surfaces; on thecontrary there are three surfaces in horse (Getty, 1975). Thedorsal surface (Fig. 3) was rough and articular, while the palmarsurface (Fig. 4) was smooth and non-articular.

The proximal extremity (Fig. 6) was comparatively morecurved in metacarpal V than in metacarpal II. It possessed acurved articular facet for articulation with the correspondingfacet on the large metacarpal in accordance with the findingsof Getty (1975) in horse. It was comparatively more curved inmetacarpal II than the metacarpal V.

The distal extremity (Fig. 7) was pointed like needle, whileit was nodular in horse (Getty, 1975).

ReferencesAkers, R.M. and Denbow, M. (2008) Anatomy and Physiology of

Domestic Animals. Blackwell Publishing, pp: 152-156.

Anonymous (2005) Nominaanatomicaveterinaria. 5 th ed. W orldAssociation of Veterinary Anatomists, Hannover, Columbia,Gent, Sapporo.

Baishya, G. et al. (2001) Indian Vet. J. 78(9): 824-825.Brohi, M. A. (2006) Balochistan Rec. Zool. Surv. Pakistan. 17: 11- 14.Budras, K. D. and Robert, E. (2003) Bovine Anatomy: An Illustrated

Text. 1st ed. Schlutersche, pp: 2-3.Chauveau, A. (1905) The Comparative Anatomy of the Domesticated

Animals. 2nd English ed. (Translated and edited by GeorgeFleming). New York D. Appleton and Company, pp. 98-119.

Frandson, R.D. et al. (2009) Anatomy and Physiology of Farm Animals.Willey Blackwell, pp. 71-74.

Garrett, P.D. (1988) Guide to Ruminant Anatomy Based on theDissection of the Goat. pp. 55.

Getty, R. (1975) Sisson and Grossman’s The Anatomy of the DomesticAnimals. W.B. Saunders Comp. Philadelphia.

Konig, H.E. and Liebich, H.G. (2006) Veterinary Anatomy of DomesticAnimals. 3rd ed., Schattauer, Stuttgart Germany. pp. 49-104, 145-164.

Mallon, D.P. (2008) Gazella bennettii. In: IUCN 2009. IUCN Red List ofThreatened Species.Version 2009.2.

McFadyean (1953) Osteology and Arthrology of Domestic Animals,4th ed. Edts. Hughes, H.V. and Dransfield, J.W. LondonBailliere, Tindall and Cox. pp. 130-172.

Miller, M.E. et al. (1964) Anatomy of the Dog. WB Saunders Company,Philadelphia, USA. pp. 64-78.

Raghavan, D. (1964) Anatomy of ox. Indian Council of AgriculturalResearch, New Delhi. pp. 97-117.

Siddiqui, M.S. et al. (2008) Bangladesh J. of Vet. Med. 6(1): 59-66.Sisson, S. (1911) A Text Book of Veterinary Anatomy. W. B. Saunders

Company, Philadelphia and London, pp. 127-131.Smuts, M.M. and Bezuidenhout, A.J. (1993) Onderstepoort J. Vet.

Res. 60(1):1-14.Smuts, M. W. and Bezuidenhout, A. J. (1987) Anatomy of the Dromedary.

Clarendon Press, Oxford, UK., pp. 24-34.Snedecor, G.W. and Cochran, W.G. (1989) Statistical Method. 8th ed.

Lowa State University Press, Ames, Lowa- 50010. pp. 26-37.

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157


IntroductionThe total global horse population is estimated as 58

million (FAOSTAT, 2007). Equines have a prominent positionin the agricultural systems of many developing countries asthey act as a mean of transport for men and material andprovide livelihood to a number of rural and semi-urbanpopulation of India. India holds about 0.751 million horsesand ponies (Census, 2003). Total population of horses inRajasthan is 24.74 thousand (Census, 2007). They do sufferfrom a number of diseases. Parasitic infestation is a majorcause of illness. Documentation of parasitic infestations ofequines in our country is lacking. Monitoring equines numbersand habitat conditions as well as ascertaining parasiticdisease surveillance become extremely important inmaintaining adequate numbers of healthy equines. Scantyreports are available on the occurrence of parasitic infestationsin equines from plains of India (Chaudhri et al., 1985; Senguptaand Yadav, 1997, 1998 and 2001). In Rajasthan there hadbeen a few studies on endoparasites in horses. Also noattention has been paid to determine the potential losses inequines population. In the livestock rich state of Rajasthanlarge number of animal fairs are held every year at differentplaces. The animal fairs held at Nagaur, Merta city, Tilwara,Hanumangarh and Pushkar are important fairs of Rajasthanin which horses are brought in good numbers for businessand sale. In these fairs horses are brought not only fromRajasthan but also from adjoining states.

Materials and MethodsA total of 719 faecal samples of horses from five animal

fairs (153 from Pushkar, 153 from Nagaur, 107 from Merta City,156 from Hanumangarh and 150 from Tilwara) were collectedrandomly. To know the prevalence of parasitic infestations of

PREVALENCE OF GASTROINTESTINAL PARASITOSES INHORSES OF ANIMAL FAIRS IN RAJSTHAN#

P. K. Pilania, G. S. Manohar and A. K. BhanDepartment of Veterinary Parasitology

College of Veterinary and Animal ScienceRajasthan University of Veterinary and Animal Sciences, Bikaner-334001, Rajasthan, India

ABSTRACT

A total of 719 faecal samples were collected from the horses of f ive animal fairs. The faecal samples were examined bydirect smear, flotation and sedimendation methods for detection of any of parasitic infestation based on the presence/absence of parasitic eggs/oocysts. The overall prevalence of gastrointestinal parasites in horses from all the five fairswas 33.24%. Habronema (14.04) showed maximum prevalence followed by strongyle (10.01), Parascaris equorum(7.09), Strongyloides westeri (4.86), Draschia (0.27), Eimeria leuckarti (0.27), Oxyuris equi (0.13), Anoplocephala(0.13) and Schistosoma (0.13%). Gastrointestinal parasites showed maximum prevalence in horses of Tilwara (46.66)fair. Habronema infestation was most prevalent in animals of Tilwara (26.66), Hanumangarh (25.00) and Merta City(17.75) fairs. Strongyle infestation predominated in animals of Nagaur (12.41) and Tilwara (10.66) f airs. Habronemainfestation predominated followed by strongyle, Parascaris equorum and Strogyloides westeri infestations in all the threeage groups of horses i.e. below 3 years, 3-5 years and above 5 years.

Key words: Gastrointestinal parasites, horses

#Part of M.V.Sc. thesis of first author. Corresponding author: Email: [email protected]

horses, the collected faecal samples were processed andexamined by direct smear, floatation and sedimentationmethods for detection of any of parasitic infestation based onthe presence/absence of parasitic eggs/oocysts.

Results and DiscussionResults of the studies are presented in Table 1 and

depicted in Plate 1. It was found that 33.24% of horses of fivedifferent fairs were suffering from subclinical gastrointestinalparasitism. The prevalence of gastrointestinal parasites inhorses has been recorded earlier by many workers in differentparts of world. It has been reported that 93.14% horses inBrazil (Mundim et al., 2000), 77.75% in Jammu and Kashmir,India (Khajuria et al., 2004), 75% in Faisalabad, Pakistan(Mahfooz et al., 2008) and 76.47% in Patiala, Punjab, India(Kaur and Kaur, 2008) were infested with gastrointestinalparasites.

Maximum prevalence (46.66%) of gastrointestinalparasites was recorded in the horses of Tilwara fair. Being thelargest fair of Rajasthan, horses are brought at Tilwara fromdifferent geographical areas of Rajasthan as well as fromadjoining states which might be responsible for the higherprevalence in comparison to Hanumangarh (44.23%), Nagaur(26.79%), Merta City (35.00%) and Pushkar (13.72) fairs wherehorses were mainly brought from adjoining districts of fair.

Prevalence of gastrointestinal parasites in the horses hasbeen reported from India and abroad earlier by many workers(Chaudhri and Singh, 2000; Sengupta and Yadav, 2001, 2003).However, such study on horses of animal fairs could not bedetected in the literature. In a similar study in animals (camels)of animal fairs Kumar et al. (1993) observed that 90.16%animals of Gogameri fair were suffering from subclinicalgastrointestinal parasitism.

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Season and climatic conditions like humidity, temperature,availability of vectors for parasites having indirect life cycle beforeand during the time of fair may also play an important role inthe prevalence of parasites (Gundach et al., 2004). The effectof dry and wet climate on the prevalence of parasites has beenreported by Chaudhri et al. (1985) who recorded prevalence ofgastrointestinal parasitic infestations in the horses of wet anddry districts of Haryana. In wet areas, might be due to the highhumidity, probably the free living stages of parasites survive forsufficient length of time and continuous cycle of infestation ismaintained between the host and pasture. In dry areas,summer and autumn seasons are suitable for the developmentof infestations in equines.

The rate of infestation varied from place to place in a districtand season to season with in the same region (Sengupta andYadav, 1998).

During the present research horses were also sufferingfrom mixed infestation of different gastrointestinal parasites.The mixed infestations of gastrointestinal parasites in horseshave been reported earlier also (Khajuria et al., 2004 and Altaset al., 2007).

In the present study, slightly higher prevalence (35.91%)of gastrointestinal parasites was found in the horses of 3-5years of age group and below 3 years (32.99%) than above 5years (30.49%) of age groups which might be due to highersusceptibility of these animals than older animals to parasiticinfestations. Fischer and Stoye (1983) and Chaudhri et al.(1985) recorded influence of age on the prevalence ofgastrointestinal nematodes. They observed high infestation

in young foals than adults. Fries (1982) also observed thatinfestations were higher among horses aged up to 4 yearsthan in older groups. Fischer and Stoye (1983) recorded higherinfestation rates in foals as compared to that of adults. Silobad(1987) recorded higher prevalence of gastrointestinal parasitesin foals of below 1 year than the horses 1-2 years and adults.

In the present study the prevalence of gastrointestinalparasites was found to be higher (38.54%) in males incomparison to females (31.48%). Kornas et al. (2006) alsoreported 12% tapeworm infestation in geldings and stallionsand 5.2% in mares while conducting a study in horses fromstud farms and individual breeding systems in Poland.However, no significant difference in the prevalence of parasiteswith regard to sex has also been reported (Singh et al., 2002and Eslami et al., 2005).

In the present investigation, among helminths, subclinicalinfestation of Habronema was maximum (14.04%) followedby strongyle (10.01%), Parascaris equorum (7.09%),Strongyloides westeri (4.86%), Draschia (0.26%), Schistosoma(0.13%), Anoplocephala (0.13%) and Oxyuris equi (0.13%).The prevalence of similar helminths was also recorded earlierby many workers (Banerjee et al., 2002; Pandit et al., 2008 andKhan et al., 2010). The higher prevalence of Habronemaobserved in present study is in conformity with the findings ofIslam (1986) who also observed a higher prevalence ofHabronema while conducting a study in Zambia. Pandey et al.(1981) also reported a very high prevalence of Habronemainfestation while conducting a study on stomach worm ofhorses in Morocco. Next to Habronema, strongyle infestation

Table 1: Prevalence of gastrointestinal parasites in horses of animal fairs in Rajasthan

Criteria Faeal

samples examined (numbers)

Faecal samples positive for (numbers)

Gastro-intestinal parasites

Helminths (species) Protozoa Strongyle

type Strongyloides

westeri Parascaris equorum Habronema Draschia Schistosoma Anoplo-

cephala Oxyuris

equi Eimeria leuckarti

1. Fair

Pushkar 153 21(13.72) 15(9.80) 9(5.88) 2(1.30) - - - - 1(0.65) -

Nagaur 153 41(26.79) 19(12.41) 10(6.53) 11(7.18) 3(1.96) - - - - 2(1.30)

Hanumangarh 156 69(44.23) 12(7.69) 5(3.20) 16(10.25) 39(25.00) 1(0.64) - - - -

Tilwara 150 70(46.66) 16(10.66) 8(5.33) 13(8.66) 40(26.66) 1(0.66) 1(0.66) 1(0.66) - -

Merta City 107 38(35.00) 10(9.34) 3(2.80) 9(8.41) 19(17.75) - - - - -

2. Age

Below 3 Years 397 131(32.99) 41(10.32) 23(5.79) 34(8.56) 44(11.08) - - - - 01(0.25)

3-5 Years 181 65(35.91) 17(9.39) 7(3.86) 11(6.07) 38(20.99) 2(1.10) 1(0.55) 1(0.55) - 1(0.55)

Above 5 Years 141 43(30.49) 14(9.92) 5(3.54) 6(4.25) 19(13.47) - - - 1(0.70) -

3. Sex

Male 179 69(38.54) 26(14.52) 10(5.58) 15(8.37) 31(17.31) - - - - -

Female 540 170(31.48) 46(8.51) 25(4.62) 36(6.66) 70(12.96) 2(0.37) 1(0.18) 1(0.18) 1(0.18) 2(0.37)

OVERALL 719 239(33.24) 72(10.01) 35(4.86) 51(7.09) 101(14.04) 2(0.27) 1(0.13) 1(0.13) 1(0.13) 2(0.27)

Note: Figures In the parentheses indicate per cent.

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was observed to be very common in horses in this study. Thestrongyle infestation has been reported to be predominant inhorses in similar studies conducted by many workers (Epe etal., 2004 and Uslu and Guclu, 2007). Moreover, Habronemaeggs are not very easy to detect in faecal examination owing totheir thin and transparent nature of egg shell.

Among protozoa, a low (0.27%) overall prevalence ofEimeria leuckarti only was recorded in present study. Variationsin protozoal infections have been earlier reported by variousworkers from different places. Rehbein et al. (2002) alsorecorded 0.1% and 0.3% incidence of E. leuckarti infestationin Germany and Austria, respectively. Bakirci et al. (2004)recorded 5.88% prevalence of E. leuckarti infestation in Turkey.

Pandit et al. (2008) also recorded 0.34% incidence of Eimeriaspp. infestation in Kashmir valley of Jammu and Kashmir state.

ReferencesAltas, M.G. et al. (2007) Indian Vet. J. 84: 1093-1094.Bakirci, S. et al. (2004) Acta. Parasitol. Turcica. 28: 35-37.Banerjee, P.S. et al. (2002) Centaur. 19: 7-9.Census (2003) A technical note on the 17th All India Livestock Census.

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Census (2007) 18th Livestock census-2007. Board of Revenue forRajasthan, Ajmer.

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Gundach, J.L. et al. (2004) Medycyna Weterynaryjna. 60: 1089-1092.Islam, A.W.M.S. (1986) Livestock Adviser. 11: 44-46.Kaur, H. and Kaur, D. (2008) J. Vet. Parasitol. 22: 25-28.Khajuria, J.K. et al. (2004) Centaur. 21: 58-61.Khan, A. et al. (2010) J. Equine Vet. Sci. 30: 155-158.Kornas, S. et al. (2006) Medycyna Weterynaryjna, 62: 821-823.Kumar, D. et al. (1993) Prevalence of subclinical gastrointestinal

parasitism in the dromedary camel. Proc. V. National Cong.Vet. Parasitol., Udgir, 21-23 April, 1993.

Mahfooz, A. et al. (2008) Pak. Vet. J. 28: 76-78.Mundim, M.J.S. et al. (2000) Veterinaria Noticias. 6: 133-137.Pandey, V.S. et al. (1981) J. Helminthol. 55: 155-160.Pandit, B.A. et al. (2008) Vet Scan. 3: 8-10.Rehbein, S. et al. (2002) Pferdeheilkunde. 18: 439-449.Sengupta, P.P. and Yadav, M.P. (1997) Indian J. Anim. Sci. 67: 460-

462.Sengupta, P.P. and Yadav, M.P. (1998) Indian J. Anim. Sci. 68: 1218-

1220.Sengupta, P.P. and Yadav, M.P. (2001) J. Vet. Parasitol. 15: 163-164.Sengupta, P.P. and Yadav, M.P. (2003) Indian J. Anim. Sci., 73: 394-

396.Silobad, S. (1987) Veterinarski Glasnik. 41: 331-338.Singh, B. et al. (2002) Indian J. Anim. Sci. 72: 861-862.Uslu, U. and Guclu, F. (2007) Bull. Vet. Inst. Puawy. 51: 237-240.

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IntroductionThe future and economics of any livestock farm or dairy industry

depends upon a successful programme of raising calves.Diseases of calves result in economic loss of up to 20% and calfmortality reduces dairy net profit by 38% (Khan and Khan, 1991).Diarrhoea has been recognized as an important condition ofanimals for last several decades because of high morbidity andmortality and has been ranked as one of the main six causes of alldeaths from infectious diseases (Kapikian, 1996; Murray andLopez, 1997; Dhama et al., 2009). Calf diarrhoea/scour is a clinicalsyndrome associated with several factors; in addition to variedenvironmental, nutritional, physiological and management factorsmany infectious agents (bacteria, virus, and protozoa) had beenincriminated either alone or in association as cause of diarrhoeain neonates (Rai, 1979; Snodgrass et al., 1986; Holland, 1990;Khan, and Khan, 1991; Swain and Dhama, 1999; Smith, 2002;Deng et al., 2003; Scott et al., 2004). Amongst them, Rotavirus,Coronavirus, Cryptosporidium, and Escherichia coli collectivelyare responsible for 75-95% of infection in neonatal calvesworldwide; and especially rotavirus and coronavirus are accountingabout 27-36% and 20-26%, respectively (Holland, 1990; Dar et al.,1998; Mallik et al., 2005; Gumusova et al., 2007; Uhde et al., 2008;Dhama et al., 2009).

Calf scours is easy to diagnose based on clinical signs butan aetiological diagnosis can be reached only in laboratory. InIndia, no comprehensive information is available on the prevalenceof infectious causes of neonatal diarrhoea at national level, thoughreports are available on individual pathogens responsible for calfdiarrhoea. The aim of the present study was to determine theprevalence of rotavirus, coronavirus, E. coli and Cryptosporidium,the main infectious agents responsible for calf diarrhoea.

PREVALENCE OF ROTAVIRUS, CORONAVIRUS AND ESCHERICHIA COLI:THE MAIN AGENTS RESPONSIBLE FOR CALF DIARRHOEA

T. Suresh, R.B. Rai*, K. Dhama, P. Bhatt1, P.M. Sawant2 and A.K. SharmaDivision of Veterinary Pathology

Indian Veterinary Research Institute, Izatnagar-243 122, Uttar Pradesh, India

ABSTRACT

The present study was carried out to investigate the prevalence of important enteric pathogens of neonatal calf diarrhoea (NCD) inorganized dairy farms of Bareilly (Uttar Pradesh) and Namakkal (Tamil Nadu), India. A total of 112 diarrhoeic samples, collected fromclinical cases of diarrhoea (103) and from dead calves (9), were screened by enzyme linked sorbent assay (ELISA) for the presenceof rotavirus, coronavirus, Escherichia coli and cryptosporidium. Enteric pathogens were found in 46.42% of the faecal samples. Theindividual overall prevalence rate of 24.10%, 6.25%, and 16.07% was detected for rotavirus, coronavirus and E. coli, respectively,but none of the samples was found positive for cryptosporidium. The prevalence rate at Namakkal and Bareilly was recorded forrotavirus, coronavirus and E. coli to be 21.35%, 5.82% and 14.56%, respectively in clinical cases and 55.55%, 11.11%, and 33.33%in dead cases. Mixed infections of rotavirus, coronavirus and E. coli were also observed in few cases. Age-wise and breed-wiseanalysis of the data is also presented. Of the infectious agents, rotavirus and E. coli were observed to be mainly involved in thecausation of NCD in winter months which leads to high mortality and morbidity in young calves. Under the paucity of comprehensiveprevalence data on infectious agents of calf diarrhoea, the present prevalence study along with further extensive investigations willhelp devise suitable strategies for the prevention and control of neonatal calf diarrhoea.

Key words: Prevalence, rotavirus, coronavirus, E. coli, cryptosporidium, ELISA, India

Materials and Methods

Collection of SamplesDuring the period from September (2008) to March (2009),

faecal samples were collected from 103 calves (clinical cases)with the history of diarrhoea from organized dairy farms in andaround Namakkal (Tamil Nadu) and Military dairy farm, Bareilly(Uttar Pradesh). Affected calves showed signs of diarrhoea,anorexia, weakness and dehydration. All the samples werecollected from calves below three months of age and of differentbreeds namely non-descript, Frieswal, crossbreds of HolsteinFriesian and Jersey (Table 2 and 3). During the same periodintestinal contents were collected from 9 spontaneous dead calveswith the history of diarrhea and gross lesions of enteritis from thepost mortem room, Division of Pathology, Indian VeterinaryResearch Institute (I.V.R.I), Izatnagar.

Screening of samples by ELISAAll the 112 clinical samples were screened for the detection

of rotavirus, coronavirus, E. coli and cryptosporidium, the mainagents responsible for calf diarrhoea, employing ELISA kit (Bio-XDiagnostics, Belgium) based on the principle of sandwitch ELISAdetecting antigens of respective pathogens, and followingmanufacturer’s protocol and instructions for processing and testingof the clinical samples.

Results and DiscussionThe prevalence of the four main agents responsible for calf

diarrhoea viz., rotavirus, coronavirus, E. coli and cryptosporidium(overall, age-wise and breed-wise prevalence) in Bareilly (UttarPradesh) and Nammakkal (Tamil Nadu) region is shown in Table1, 2 and 3 and Fig. 1, 2 and 3. A total of 103 diarrhoeic faecal

*Corresponding author: [email protected] Clinics, College of Veterinary and Animal Sciences, G.B. Pant University of Agriculture and Technology, Pantnagar- 263 145, Uttarakhand, INDIA2Ph.D. Scholar, Immunology Section, Indian Veterinary Research Institute, Izatnagar- 243 122, Bareilly, Uttar Pradesh, INDIA

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samples from clinical cases and 9 intestinal contents from deadcalves were screened. Enteric pathogens (rotavirus, coronavirusand E. coli) were found in 46.42% of the faecal samples. Of the112 samples, 27 (24.10%) samples were found positive forrotavirus, 7 (6.25%) samples were positive for coronavirus, and18 (16.07%) samples were positive for E. coli. None of the sampleswas found positive for cryptosporidium. The prevalence rate atNamakkal and Bareilly was recorded for rotavirus, coronavirusand E. coli to be 21.35%, 5.82% and 14.56%, respectively in clinicalcases. Mixed infections of rotavirus and coronavirus were seen in2 (1.94%) cases and mixed infections of rotavirus and E. coli wereseen in 4 (3.9%) cases (Table 1). Out of 9 calf carcasses died ofdiarrhea, 5 (55.55%) cases were positive for rotavirus, 3 (33.33%)cases were positive for E. coli, 1 (11.11%) case positive forcoronavirus and 2 (22.22%) cases were mixed infection of rotavirusand E. coli.

The present prevalence study for four important entericpathogens of neonatal calf diarrhoea (NCD) in organized dairyfarms of Bareilly (Uttar Pradesh) and Namakkal, India revealed anoverall prevalence rate of 24.10%, 6.25%, and 16.07% for rotavirus,coronavirus and E. coli, respectively, with no presence ofcryptosporidium. The prevalence rate at Namakkal and Bareillywas recorded for rotavirus, coronavirus and E. coli to be 21.35%,5.82% and 14.56%, respectively in clinical cases and 55.55%,11.11%, and 33.33% in dead cases. Mixed infections of rotavirus,coronavirus and E. coli were also observed in few cases (1.94 to33.33%). The incidence of diarrhoea in calves is highest duringthe winter months and calves are under greatest risk of diarrhoeaduring first few months of life (Scott et al., 2004; Uhde et al., 2008).For these reasons, the samples were collected from diarrhoeiccalves below three months during September (2008) to March(2009). The samples were collected from calves with acutediarrhoea as well as from dead calves with history of diarrhoea.

The prevalence rate of rotavirus (24.10%) observed in ourstudy was in agreement with earlier reports (Kaushik et al., 1983;Singh et al., 1985; Vanamayya, 1990; Singh and Pandey, 1986;Chauhan and Singh, 1996; Gulati et al., 1999; Jindal et al., 2000;Kumar, 2006; Dhama et al., 2009; Nataraju et al., 2009; Mayameeiet al., 2009) in which the prevalence rate of 11-43% had beenrecorded. This supports the fact that rotavirus plays an importantrole in causing the diarrhoea and/or mortality. The prevalence ofrotavirus infection was more during first two weeks of age and is inagreement with finding of Mebus et al. (1971). De Leeuw et al.(1980) recorded two types of syndromes: a short lasting earlydiarrhoea within the first three days of life and late diarrhoeaoccurring between 4-14 day of age, which was more common. Incow the colostral rotavirus antibodies decrease dramatically intransition from colostrum to milk and this coincides with rotavirusinfection in calves at 4-14 days of life after that the occurrence getsreduced gradually as age increases (Mebus et al., 1973a). Inconcurrence with these findings we observed decrease inprevalence of rotavirus after two weeks of age.

The relatively low prevalence of coronavirus (6.25%) agreeswith the results of other studies of Reynolds et al. (1986) (14%);Snodgrass et al. (1986) (3.64%); Vanamayya (1990) (20%);Mayameei (2009) (3.1%) in which the prevalence rate of 3-20%had been recorded. This showed lower prevalence of coronavirusdiarrhoea in Bareilly and Namakkal dairy farms. The combinedprevalence of these two viral infections was 28.15%. The overall

prevalence rate of both rota and corona viruses observed in ourstudy was in agreement with earlier report of Vanamayya (1990)which was about 36%. Coronavirus infections were observedduring 2-8 weeks of age in our study which was in agreement withthe earlier reports of Mebus et al. (1973b) and Vanamayya (1990).

Mixed infection of rotavirus and coronavirus in our study wasobserved in 2 cases (1.78%) which is in agreement with the earlierreports where multiple viral infections were reported to be commonin neonatal diarrhoea of calves (Singh and Pandey, 1986;Vanamayya, 1990). In an epidemiological study of selected calfpathogens such as rotavirus, coronavirus and E. coli infections,Waltner Toews et al. (1986) observed that farms positive for one orthe other viruses had increased odds of experiencing calf mortalityrelative to the virus free farms. A recent prevalence study regardingdetection of four enteropathogens in the faeces of young diarrhoeicdairy calves revealed the prevalence of Cryptosporidium parvum,rotavirus, bovine coronavirus (BCV), and enterotoxigenicEscherichia coli (E. coli K99) to be 55%, 58.7%, 7.8% and 5.5%,respectively in calves aged one to 21 days on 71 dairy farms inwestern Switzerland during the winter of 2005 to 2006 (Uhde et al.,2008). The prevalence of different infectious agents varies withcalf’s age and multiple infections are common. Diarrhoea is oneof the most common disease manifestations reported in calvesup to 3 months of age (Svensson et al., 2003). Neonatal calf mortalityin the first month of age was accounted to be 84 per cent of the totalmortality (Chauhan and Singh, 1996) and it was particularly highin the third week of age (Umoh, 1982).

The prevalence rate of E. coli (16.07%) observed in first twoweeks of age is in agreement with earlier reports from Reynoldset al. (1985) (3%); Snodgrass et al. (1986) (3.64%); Debnath et al.(1987) (20%); and Samad et al. (2004) (37%) in which theprevalence rate of 3-37% have been recorded. Prior or simultaneousrotavirus infection may enable E. coli colonisation of the intestinein calves (Snodgrass et al., 1982). The mixed rotavirus and E. coliinfection (5.35%) in present infection may be result of prior orsimultaneous infection of these two agents.

In tropical countries, prevalence of cryptosporidiosis is highestin rainy season (Nagamani et al., 2007; Paul et al., 2008).Prevalence of Cryptosporidium parvum in India and its associationwith diarrhoea in neonatal dairy calves has been reported (Singhet al., 2006). In our study samples were taken only once in winter.Single sampling may give false negative results because rotavirus,BCV and cryptosporidia are excreted in cyclical pattern (Singh etal., 2006). Furthermore, intermittent shedding of pathogens hasbeen reported in both healthy and diseased animals. The abovementioned reason may be behind no detection of cryptosporidiain the present study.

The difference of sex susceptibility to rotavirus, coronavirusand E. coli infections could not be evaluated as most of the samplestaken were incidentally from female calves. However, Hasso andPandey (1986) noticed that female calves were readily susceptibleto rotavirus infections than male calves. In our study, it was observedthat rotavirus infection was more in crossbred Holstein Friesian(53.19%) followed by crossbred Jersey (53.84%), Frieswal(21.21%), Non-descript (40%). The overall prevalence rate ofcrossbred cows observed in our study was in agreement withearlier report of Vanamayya (1990) in which the prevalence rate of26-66% had been recorded. In our study, high prevalence of entericinfection especially rotavirus infection was more in winter months

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163


Fig. 1: Overall prevalence of rotavirus, coronavirus and E. coli

164


Fig. 2: Breed-wise prevalence of rotavirus, coronavirus and E. coli in clinical cases

Fig. 3: Age-wise prevalence of rotavirus, coronavirus and E. coli in clinical cases

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which was in agreement with the report of Singh et al. (1985),Chauhan and Singh (1996) and Uhde et al. (2008).

In conclusion, prevalence of rotavirus, coronavirus and E.coli, important infectious pathogens responsible for calf diarrhoeais reported from dairy farms of northern and southern parts ofIndia. Of these agents, rotavirus and E. coli are mainly involved inthe causation of calf diarrhea in neonatal calves, which leads tohigh mortality and morbidity in young calves. In India, it is difficult tocontrol calf mortality due to unorganized rearing of dairy animals.Prevalence studies will help devise suitable approach for the controlof calf diarrhoea and appropriate advice on colostrum feeding, calfnutrition, hygiene and therapeutic regimens can be given. Further,prevalence studies will contribute in future for designing ofvaccination programme against infectious agents of neonatal calfdiarrhoea which is not currently practiced in India. In calf diarrhoea,apart from clinical signs laboratory detection of aetiological agentis necessary to reach the conclusive diagnosis. Seeing the paucityof comprehensive information regarding prevalence of infectiouscauses of neonatal calf diarrhoea in India, the present prevalencereport regarding identification of causative agents adds to theepidemiological data of the important infectious pathogensresponsible for calf diarrhoea.

ReferencesChauhan, R. S. and Singh, N. P. (1996) International J. Ani. Sci. 11: 221-

223.Dar, A. M. et al. (1998) J. Vet. Diagn. Invest. 10(2): 152-157.De Leeuw, P. W. et al. (1980) Res. Vet. Sci. 29: 135-141.Debnath, N. C. et al. (1987) Indian J. Anim Sci. 57: 1035-1038.Deng, Y. et al. (2003) J. Clin. Microbiol. 41: 2300-2305.Dhama, K. et al. (2009) Vet. Res. Communi. 33(1): 1-23.Gulati, R. B. et al. (1999) J. Clin. Microbiol. 37(6): 2074-2076.Gumusova, S.O. et al. (2007) Medycnya Weterinaria. 63: 62-64.Hasso, S. A. and Pandey, R. (1986) Canadian J. Vet. Res. 50: 287-288.Holland, R.E. (1990) Clinical Microbiol. Rev. 3:345-375.Jindal, S. R. et al. (2000) Rev. Sci. Tech. 19(3): 871-6.Kapikian, A. Z. (1996) Arch. Virol. (suppl) 12: 7-19.Kaushik, A. K. et al. (1983) Zbl. Vet. Med. 30:156-158.Khan, A. and Khan, M. Z. (1991) J. Islamic Academy of Sci. 4(2):159-165.

Kumar, M. (2006) Polymerase chain reaction for rapid detection of importantzoonotic diarrhoeal pathogens. M. V. Sc Thesis, IndianVeterinary Research Institute, Izatnagar.

Malik, S.V.S. et al. (2005) Data sheet on Rotaviruses (Global status ofRotavirus infections in man and animals). In: Animal Health andReproduction Compentium, CAB International, Wallingford, UK.

Mayameei, A. et al. (2009) Comparative Clin. Pathol. 19(6): 553-557.Mebus, C. A. et al. (1971) Can. Vet. J. 12:69-72.Mebus, C. A. et al. (1973a) J. Amer. Vet. Med. Assoc. 163: 880-883.Mebus, C. A. et al. (1973b) Vet. Path. 10: 45-64.Murray, C. J. and Lopez, A. D. (1997) Lancet. 349: 1436-1442.Nagamani, K. et al. (2007) Indian J. Med. Microbiol. 25(2):133-6.Nataraju, S.M. et al. (2009) European Rev. Med. Pharmacol. Sci. 13: 7-11.Paul, S. et al. (2008) Vet. Parasitol. 153(1-2): 143-146.Rai, R. B. (1979) Clinico-etiopathological studies on calf scours. M .V. Sc.

Thesis. G.B. Pant Univ. of Agri. and Technology, Pantnagar(Uttarakhand), India.

Reynolds, D. J. et al. (1985) Res. Vet. Sci. 38:264-269.Reynolds, D. J. et al. (1986) Vet. Rec. 119: 2: 34-39.Samad, M. A. et al. (2004) Bangl. J. Vet. Med. 2(1):49-54.Scott, P.R. (2004) Calf Diarrhoea In: Bovine Medicine. Eds. Andrews,

A.H., Blowey, R.W., Boyd H. and Eddy, R.G. Blackwell Publishing204.

Singh, A. and Pandey, R. (1986) Indian. J. Virol. 2:16-21.Singh, A. et al. (1985) Indian J. Virology. 1:127-132.Singh, B. B. et al. (2006) Vet. Parasitol. 141(3/4): 330-333.Smith, B. P. (2002) Neonatal ruminant diarrhea: Large animal internal

medicine. 3rd ed. Mosby Inc. Missouri, USA -63146.Snodgrass, D.R. et al. (1982) Vet. Microbiol. 7(1): 51-60.Snodgrass, D. R. et al. (1986) Vet.Rec. 119(2): 31-34.Svensson, C. et al. (2003) Morbidity in Swedish dairy calves from birth to

90 days of age and individual calf-level risk factors for infectiousdiseases. Preventive Veterinary Medicine. Elsevier ScienceB.V., Amsterdam, Netherlands: 58(3/4): 179-197.

Swain, P. and Dhama, K. (1999) Indian Farming. 48: 25-27.Uhde, F.L. et al. (2008) Vet. Record, 163: 362-366.Umoh, J.U. (1982) British Vet. J. 138: 507-514.Vanamayya, P. R. (1990) Pathological studies on pneumoenteritis in

calves with special reference to viral infections. Ph.D. Thesis,Indian Veterinary Research Institute, Izatnagar.

Waltner, T.D. et al. (1986) Can. J. Vet. Res. 50: 307-313.

Fig. 3b: Namakkal region

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IntroductionBuffaloes play a prominent role in rural livestock production

in Asia and India in particular. Silent oestrus is perhaps themost important factor leading to poor reproductive efficiency inbuffaloes (Kanai and Shimizu, 1983; Prakash et al., 2002;Suthar and Dhami, 2010). As the signs of oestrus in buffaloesare less obvious than in cattle, the oestrus detection accuracyis one of the major problems. Various oestrus synchronizationprotocols induce oestrus with varying intensity and behaviouralsymptoms with many of them being pronounced in one andsilent in other. Awasthi and Kharche (1989) and Sirmour (1999)devised a score card pattern to decide oestrus intensity whichcan be utilized as a tool to detect optimum breeding period forbetter conception rate.

Materials and MethodsThe present investigation was carried out on 18

postpartum anoestrus buffaloes of College Dairy Farm andthe organized dairy farms in and around College of VeterinaryScience and Animal Husbandry, Mhow. The buffaloes weredivided into group A, B and C with 6 in each group. Group Abuffaloes were subjected to Ovsynch hormone protocol,consisting of an injection of GnRH (Receptal, 5.0 ml, i/m) on0th day followed by an injection of PGF2 (Lutalyse, 5.0 ml, i/m)on 7th day and another injection of GnRH (Receptal, 5.0 ml, i/m) on 9th day. Group B buffaloes were subjected to heat synchprotocol, consisting of an injection of GnRH (5.0 ml, i/m) on 0th

day followed by an injection of PGF2 (Lutalyse, 5.0 ml, i/m) on7th and an injection of estradiol valerate (Progynon depot. 0.1ml, i/m) on 8th day. Group C were subjected to CIDR protocol,in which an intra vaginal devise CIDR was inserted along withan injection of estradiol valerate (Progynon depot., 0.1 ml, i/m)on 0th day followed by an injection of PGF2 (Lutalyse, 5.0 ml,

COMPARATIVE STUDIES ON OESTRUS BEHAVIOUR AND INTENSITY OFINDUCED OESTRUS USING DIFFERENT HORMONE PROTOCOLS IN

POSTPARTUM ANOESTRUS BUFFALOES#

Rameez Ali1, S.P. Shukla2 and S.P. Nema3

Department of Animal Reproduction, Gynaecology and ObstetricsCollege of Veterinary Science and Animal Husbandry, MPPCVVV, MHOW-453446, M.P., India

ABSTRACT

The present investigation was carried out to study the clinical manifestations and intensity of induced oestrus in 18 postpartumanoestrus buffaloes treated with three different hormone protocols. Six buffaloes each in treatment group A, B and C weresubjected to Ovsynch, Heatsynch and CIDR hormone protocol, respectively. Observations on reproductive organs status atinduced oestrus revealed that tumefication of vulva were intense in 71.42 per cent, moderate in 21.42 per cent and mild in 7.14per cent buffaloes. Cervico-vaginal mucus discharge was copious in 64.28 per cent, moderate in 21.42 per cent and absent in14.28 per cent buffaloes. Vulvar mucous membrane was intense pink in 28.57 per cent, pink in 57.14 per cent and pale in 14.28per cent buffaloes. External os open and cervix was fully relaxed in 100 buffaloes. Uterine tonicity was intense in 64.28 percent, moderate in 21.42 per cent and weak in 14.28 per cent buffaloes. Mature follicle was found in 83.72 per cent anddeveloping in 14.28 per cent buffaloes. The overall oestrus intensity ranged from 29-92 with mean weighted score of 74.79±4.20.It ranged from 29-87, 66-92 and 63-88 with mean weighted score of 61.67±17.14, 78.83 ± 3.85 and 77.80±4.21 in treatmentgroups A, B and C, respectively. Statistical analysis revealed significant variation (P<0.05) between the treatment groups

Key words: Oestrus, PGF2, GnRH, CIDR

#1Part of M.V.Sc. thesis and Corresponding author:E-mail: [email protected]&3Professor, Department. of ARGO, College of Veterinary Science and A.H., MHOW, Indore, Madhya Pradesh, India

i/m) on 7th and a 2nd injection of estradiol valerate (Progynondepot., 0.1 ml, i/m) on 8th day.

The status of reproductive organs was ascertained by theprocedure described by Zemjanis (1962). These buffaloeswere further gynaeco-clinically examined on day of treatmentand day of oestrus. Vulva: The intensity of tumefication of vulvar lips wascategorized as intense, moderate or mild.Vulvar mucous membranes: The colour of vaginal mucousmembrane was observed and recorded as intense pink, pinkor pale.Cervix: The cervical relaxation was judged as external os openand fully or partially relaxed.Uterus: The tonicity of uterus was judged and recorded asintense, moderate or mild.Gonads: The ovarian status was characterized by presence ofmature graffian follicle or developing follicle, corpus luteum ornone of these.

Oestrus intensity was numerically scored, based on aproper weightage given for each of the parameter comprisingoestrual changes in the external genitalia, internal genitaliaand behavioural expression. For this purpose, the scorecarddevice of Singh and Kharche (1985), Awasthi and Kharche(1989) and Sirmour (1999) was modified and is detailed inTable 1.

On the basis of weighed score sought by individual buffalothe oestrus intensity was classified into intense, moderateand weak as given in Table 2.

Results and DiscussionObservations on reproductive organs status at induced

oestrus revealed that tumefication of vulva were intense in71.42 per cent buffaloes; it was moderate in 21.42 per cent

167


and mild in 7.14 per cent buffaloes. The present findings fortumefication of vulvar lips are comparable with the finding of Mohanet al. (2010) who found intense tumefication of vulvar lips in inducedoestrus buffaloes. Cervico-vaginal mucus discharge was copiousin 64.28 per cent buffaloes, it was moderate in 21.42 per centbuffaloes and absent in 14.28 per cent buffaloes. Vulvar mucousmembrane was intense pink in 28.57 per cent buffaloes; it waspink in 57.14 per cent buffaloes and pale in 14.28 per cent buffaloes.The pink and congested mucosa is due to increased vascularity(hyperaemia) under the influence of oestrogen (Roberts, 1986)which is a supportive sign of oestrus detection as reported bySingh et al. (1984). External os open and cervix was fully relaxed in100 buffaloes and none of the buffaloes had partially relaxed cervixon oestrus. Uterine tonicity was intense in 64.28 per cent; it wasmoderate in 21.42 per cent buffaloes and weak in 14.28 per centbuffaloes. Sharma et al. (2003) suggested that cervical relaxationshould be considered as better parameter for oestrus confirmationrather than the uterine tone which was not as marked as in cowson the basis of gynaecological investigation. Mature follicle wasfound in 83.72 per cent; it was developing in 14.28 per cent buffaloes.The present findings for the ovarian status are comparable withthe reports of Rao and Kodagali (1982), Sharma et al. (2003) andMohan et al. (2010).

The overall oestrus intensity ranged from 29-92 with meanweighted score of 74.79±4.20. It ranged from 29-87, 66-92 and63-88 with mean weighted score of 61.67±17.14, 78.83±3.85 and

77.80±4.21 in treatment groups A, B and C, respectively. Statisticalanalysis revealed significant variation (P<0.05) between thetreatment groups. Oestrus intensity score of buffaloes rangedfrom 76-92, 66-71 and 29-63 in intense, moderate and weak oestrusintensities, respectively, with mean weighted score of 83.22±1.76,68.67±1.45 and 46.00±17.00, respectively. Statistical analysisrevealed significant variation between oestrus intensity score atinduced oestrus. Variation between groups affirms the view ofRao and Rao (1981) that it is possible to differentiate oestrusintensity using score card device.

ReferencesAwasthi, M. K. and Kharche, K.G. (1989) Livestock Advisor. 14(1): 20-24.Kanai, Y. and Shimizu, H. (1983) Theriogenology. 19(4):593-602.Mohan, K. et al. (2010) Trop. Ani. Hlth. Prod. 42(1): 21-26.Prakash, B. S. et al. (2002) J. Immunol. Methods. 270:281-290.Rao, N.M. and Kodagali, S.B. (1982) Indian J. Ani. Reprod. 2: 12-14.Rao, S.V. and Rao, A.R. (1981) Indian Vet. J. 58: 881-884.Roberts, S.J. (1986) Veterinary Obstetrics and Genital Disease

(Theriogenology). 2nd ed. Edward Brothers, Michigan, U.S.A.Sharma, R.K. et al. (2003) Intas Polivet. 4(2): 152-155.Singh, G. et al. (1984) Theriogenology. 21(6):849-858Singh, M.M. and K.G. Kharche (1989) Livestock Advisor. 10(4): 9-13.Sirmour, S.K. (1999) Therapeutic and biochemical studies in anoestrus

crossbred heifers. M.V.Sc. Thesis. JNKVV. Jabalpur (M.P.) India.Suthar, V. S. and A. J. Dhami (2010) Veterinary World. 3(2): 94-96.Zemjanis, R. (1962) Diagnostic and Therapeutic Techniques in Animal

Reproduction. Williams and Wilkiams. Baltimore. pp. 238.

Table 1: Score card device on the status of reproductive organsS. No. Status of reproductive

organs Weight Source of pattern

1 Tumefication of vulva lips

10 Intense 10 Moderate 06 Mild 04

2 Cervico-vaginal mucus discharge

10 Copious 10 Moderate/scanty 06 Absent 04

3 Vulvar mucous membrane

10 Intense pink 10 Pink 06 Pale 04

4 Cervical relaxation 10 External os open & cervix fully relaxed 10 External os open & cervix partially relaxed

04

5 Uterine tonicity 10 Intense 10 Moderate 06 Weak 04

6 Ovarian status 10 Nature of follicle mature 10 Developing 04

7 Teaser mounting 10 Stands to mount 10 Allow mount but escapes 06 Teaser refuse to mount 04

8 Bending of tail 10 Present 10 Absent 04

9 Micturition 10 Frequent 10 Normal 04

10 Bellowing 10 Present 10 Absent 04

Table 2:Oestrus intensity classification system

S. No. Score range Types of oestrus

1 More than 75 Intense

2 65 to 74 Moderate

3 Less than 65 Weak

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IntroductionEthnoveterinary medicine is defined as a holistic

comprehension of the indigenous systems of animal health,their interpretation through western medicine and thedevelopment of effective and appropriate technologies(McCorkle, 1986). Modulation of immune responses to alleviatethe diseases has been of interest for many years and theconcept of ‘Rasayana’ in Ayurveda is based on related principles(Patwardhan et al., 1990). Apart from being specificallystimulatory or suppressive, certain agents have been shownto possess activity to normalize or modulate pathophysiologicalprocesses and are hence called immunomodulatory agents.A large number of plant products are being investigated forimmune response modifying activity (Upadhyay, 1997).

Tinospora cordifolia (Guduchi) commonly known asGiloya, is a large, glabrous, deciduous climbing shrubbelonging to the family Menispermaceae. Guduchi is widelyused in veterinary folk medicine/ayurvedic system of medicinefor its general tonic, antiperiodic, anti-spasmodic, anti-inflammatory, antiarthritic, anti-allergic and anti-diabeticproperties. The plant is used in ayurvedic, “Rasayanas” toimprove the immune system and the body resistance againstinfections. The root of this plant is known for its antistress, anti-leprotic and anti-malarial activities (Zhao et al., 1991 andNayampalli et al., 1982). In present study, attempts were madeto evaluate the effect of Tinospora cordifolia on humoral andcell mediated immunity in acephate intoxicated mice sinceacephate is a well known immuno toxicant pesticide.

Materials and MethodsThe present study was conducted on 6-8 weak old healthy

BALB/c mice. The mice were procured from Cadila

EVALUATION OF EFFECT OF TINOSPORA CORDIFOLIA EXTRACTON HUMORAL AND CELL MEDIATED IMMUNITY IN ACEPHATE

INTOXICATED MICE#

L. N. Sankhala1, S. M. Tripathi, S. K. Bhavsar and A. M. ThakarDepartment of Pharmacology and Toxicology

College of Veterinary and Animal ScienceAnand Agriculture University, Anand-388001, Gujarat, India

ABSTRACT

Present study was planned to investigate the effect of Tinospora cordifolia extract on humoral and cell mediated immunityin acephate intoxicated mice. A total number of 64 balb/c mice were exposed to different doses of acephate eitherindividually or in combination with Tinospora cordifolia stem extract (100 mg/kg) once daily for 28 days. There wassignif icant decrease in antibody titre against SRBC in high dose acephate treated group (T3) as compared to pesticidevehicle control group (C1). Mice given T. cordifolia along with acephate at different doses have higher antibody titreagainst SRBC as compared to only acephate treated mice. Significant increase in skin thickness was observed in plantcontrol group (C2) in comparison to pesticide vehicle control group after 24 hrs of challenge whereas nonsignif icantincrease in skin thickness was observed in animals that received T. cordifolia extract along with acephate in comparisonto pesticide vehicle control group as well as only acephate treated groups, respectively, after 24 and 48 hrs of challenge.

Key words: Acephate, cell mediated immunity, humoral immunity

#1 Part of M.V.Sc. Thesis and corresponding author: Instructor, Department of Pharmacology and Toxicology, College of Veterinary and Animal Science, Bikaner-334001. [email protected]

Pharmaceuticals, Dholka, and kept in cages at laboratoryanimal house, Veterinary College, Anand Agricultural University,Anand. The mice were provided with ad libitum standardpelleted feed and water. A prior approval was obtained byinstitutional animal ethics committee for the study protocol.After one week of acclimatization mice were used forexperiment.

Experimental designAll the mice were randomly divided into eight groups (C1,

C2, T1, T2, T3, T4, T5 and T6) each containing eight mice.Apparent LD50 of Acephate 351 mg/kg was taken intoconsideration for calculation of different dose groups (Barnettand David, 1984). Mice were treated with pesticide and plantextract as described below.Group C1 - Pesticide vehicle control (administered normal

saline)Group C2 - Plant control (100 mg/kg aqueous extract of

Tinospora cordifolia stems)Group T1 - 1/40th of LD50 (8.78 mg/kg) of Acephate Group T2 - 1/30th of LD50 (11.70 mg/kg) of AcephateGroup T3 -1/20th of LD50 (17.55 mg/kg) of AcephateGroup T4 - Acephate (dose LD50/40) + aqueous extract of T.

cordifolia stem (100 mg/kg).Group T5 -Acephate (dose LD50/30) + aqueous extract of T.

cordifolia stem (100 mg/kg).Group T6 -Acephate (dose LD50/40) + aqueous extract of T.

cordifolia stem (100 mg/kg).Acephate in normal saline and aqueous extract of T.

cordifolia were administered directly in esophagus byusing mice oral feeding needle with 1 ml BD syringe for28 days.

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Preparation of aqueous extraction of Tinospora cordifoliaPlant material (stem) was procured from Department of

Medicinal and Aromatic Plants, Anand Agricultural University,Anand. The stems were shade-dried, powdered and stored inairtight containers. The dried coarse powdered crude plant(100 gm) was transformed to a round bottle flask and two litreof distilled water was added to round bottle flask and soakedfor twelve hours. This was then boiled for 5-6 hours in rotaryevaporator under reduced pressure at 40°C temperature.Extract was decanted in a beaker and then concentrated to 1/6th of total volume by keeping it in hot water bath cum shaker.The extract was preserved by adding few drops of chloroformand kept in the refrigerator.

Assessment of humoral immune response Immunization

Sheep red blood cells (SRBCs) were collected in Alsevier’ssolution, washed in large volumes of sterile 0.9% normalsaline thrice and adjusted to a concentration of 5 X 109 cellsper ml, were used for immunization. Animals were immunizedby injecting 0.2 ml SRBC suspension interaperitoneally 7 th

days prior to sacrifice. Blood was collected from retro orbitalplexus under ether anaesthesia on 29th day and serum wasseparated from blood to determine the antibody titer byhaemagglutination test.

Antibody titerAntibody titre was carried out according to protocol of Puri

et al. (1994). In briefly, two fold dilution of test serum was madein 0.15 M phosphate buffer saline (PBS) and aliquoted in “U”bottomed microtiter plates.1% SRBC suspended in PBS wasdispended in each well and mixed thoroughly. The plates wereincubated for 4 hrs at 370C and then observed visually forhaemagglutination. The highest dilution of the test serum givinghaemagglutination was taken as antibody titre.

Assessment of cell mediated immune responseCell mediated immune response was assessed by the

method as described by Lagrange et al. (1974). All the animalsunder various groups were immunized by injecting 20 ìl of5x109 SRBC per ml subcutaneously into the right footpad on19th day of treatment. Thickness of left footpad was measuredusing vernier calipers on 6th day of treatment. The mice werethen challenged by injecting 20 μl of 5x109 SRBC per mlintradermally on the left hind foot pad (time 0). Foot padthickness was measured after 24 and 48 h of challenge. Thedifference between the thickness of left foot just before andafter challenge in mm was taken as a measure of delayedtype hypersensitivity (DTH)

Statistical analysisOne-way-analysis of variance (ANOVA) was used to

Table 1: Mean ± S.E. values showing effect of T. cordifolia extract on antibody titre against SRBC in acephate intoxicated mice

Groups Antibody titre (Mean±SE) (n=4) C1 320 ± 64.00b C2 768 ± 147.80c T1 208 ± 102.45ab T2 80 ± 16.00ab T3 64 ± 22.62a T4 256 ± 90.51bc T5 160 ± 32.00ab T6 112 ± 16.00ab

Means having different superscripts at a particular period of treatment differs significantly (P<0.05)

Table 2: Mean ± S.E. values showing effect of T. cordifolia extract on cell mediated immunity in acephate intoxicated mice

Skin thickne ss (mm) (n=8) (Mean±SE)

Group s

0 hrs 24 hrs 48 hrs C1 0.2850 ± 0.0105 0.3388 ± 0.0100ab 0.3225 ± 0.0036 C2 0.2563 ± 0.0092 0.3550 ± 0.0042c 0.3250 ± 0.0086 T1 0.2700 ± 0.0108 0.3400 ± 0.0037abc 0.3063 ± 0.0129 T2 0.2688 ± 0.0106 0.3325 ± 0.0045ab 0.3075 ± 0.0055 T3 0.2588 ± 0.0114 0.3250 ± 0.0026a 0.3112 ± 0.0047 T4 0.2588 ± 0.0124 0.3400 ± 0.0026abc 0.3150 ± 0.0119 T5 0.2688 ± 0.0099 0.3425 ± 0.0052bc 0.3338 ± 0.0053 T6 0.2563 ± 0.0119 0.3388 ± 0.0029ab 0.3250 ± 0.0012

Means having different superscripts at a particular period of treatment differs significantly (P<0.05)

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compare the effects of treatment of acephate and T. cordifoliaextract on different body weight, haematological, biochemical,and immunological variables in control and treated mice byusing software SPSS (version 12.1 ).

Results and DiscussionEffect of Tinospora cordifolia extract on humoral and cell

mediated immunity in acephate intoxicated mice is presentedin Table 1 and Table 2.

The results indicated dose related reduction in antibodytitres against SRBC at 28 days exposure of acephate. Similarly,Suppression of humoral immune response was reportedfollowing administration of malathion and chlordimeform inmice (Cushman and Street, 1983; Shopp et al., 1985). Inpresent study, results also indicated that there was significantincrease in antibody titre against SRBC in plant control groupand there is less decrease in antibody titre in mice that weregiven plant extract with different doses of acephate. It suggeststhat T. cordifolia extract lessened the immunotoxic effect ofacephate. Similarly, administration of methanol extract ofSphaeranthus indicus and its fractions produced increase inhumoral antibody response in mice. Increase in antibody titrewas also observed following administration of AshtamangalGhrita (polyherbal formulation) (300 mg/kg/day) in rats (Fulzeleet al., 2002).

In the present study, delayed type hypersensitivity (DTH)response was checked by increased footpad thicknessfollowing challenge with SRBC. There was non significantincrease in skin thickness in acephate treated mice ascompared to control. This result suggests that acephate hasnot altered cell mediated immune response at dose ratesadministered in the present study. Similarly, quinalphos,triphenyl phosphate (TPP) and aldicarb in mice (Jha et al.,1990; Hinton et al., 1987) did not cause suppression of cellmedeated immunity (CMI).

Significant increase in skin thickness was observed inplant control group (C2) in comparison to pesticide vehiclecontrol group after 24 hrs of challenge whereas non significanteffect was observed on skin thickness after 48 hrs of challange.Similarly nonsignificant increase in skin thickness wasobserved in animals that received T. cordifolia extract alongwith acephate in comparison to pesticide vehicle control groupas well as only acephate treated groups respectively, after 24and 48 hrs of challenge. Similar observations were reportedwhen Cyclosporine (25 mg/kg) with alcoholic extract T. cordifoliawas given to mice (Agrawal et al., 2002). Dose dependentincrease in thickness of footpad of mice was also observedafter administration of ethanolic extract of Chlorophytumborivilianum in mice (Thakur et al., 2006) and AshtamangalGhrita (polyherbal formulation) in rats (Fulzele et al., 2002).

ReferencesAgrawal, A. et al. (2002) Indian J. Pharmacol. 34: 339-349.Barnett, A.R. and David, J.H. (1984) Archives of Environ. Conta.

Toxicol. 13: 483-491.Cushman, J.R. and Street, J.C. (1983) Toxicol. Appl. Pharmacol. 70:

29.Fulzele, S.V. et al. (2002) Indian J. Pharmacol. 34: 194-197.Hinton, D.M. et al. (1987) Toxicol. Hlth. 3: 71-89.Jha, G.J. et al. (1990) Acta Vet. Hung. 38(1): 55-60.Lagrange, P.H. et al. (1974) J. Exp. Med. 139: 1529-1539.Mccorkle, C.M. (1986) J. Ethnobiol. 6: 129-149.Nayampalli, S. et al. (1982) Indian J. Pharm. 14: 64-66.Patwardhan, B. et al. (1990) Indian Drugs. 28(2): 56-63.Puri, A. et al. (1994) J. Ethnopharmacol. 42: 31-37.Shopp, G.M. et al. (1985) J. Toxicol. Environ. Hlth. 15: 293-304.Thakur, M. et al. (2006) F. eCAM. 1: 1 -5.Upadhyay, S.N. (1997) Plant products as immune response

modulators. Proceedings of the International AyurvedaConference 97. Sanjay Gandhi Post Graduate Institute ofMedical Sciences, Lucknow. pp.10.

Zhao, T.F. et al. (1991) Planta Med. 57: 505.

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IntroductionAnaemia is defined as reduction in normal number of

the erythrocytes and/or haemoglobin concentration per mlof blood. It is characterized physiologically by insufficientcirculating haemoglobin and clinically by reduced exercisetolerance and pale mucous membrane (Armour et al., 1991and Fraser et al., 1991) and increased destruction or lossof red blood cells (Radostits et al., 2007). Further, most ofthe goats in urban areas are kept as stall fed animal whichmay also lead to nutritional problems and deficiencydiseases. The copper level in blood of animals play vitalrole in haematopoiesis. Copper acts as a catalyst in theassimilation of iron, which is needed in the production ofhaemoglobin in liver and act as integral part of cytochromesystem. In view of the heavy haemosiderin deposits intissues of copper deficient animals, it is probable that copperis necessary for the reutilization of iron liberated from thetissues of copper deficient animals (Radostits et al., 2007).In the present study, an attempt was made explore the roleand status of copper in amelioration of nutritional anaemiain goats.

Materials and MethodsEight clinically weak and anaemic goats negative for

parasitic load were brought to the TVCC of College ofVeterinary and Animal Science, Bikaner. Diagnosis ofanaemia was arrived on the basis of the history, generalexamination, clinical manifestations and laboratoryexamination. The goats having haemoglobin values (g/dl)less than mean -2 standard deviation of healthy goat (8.07g%) were considered as anaemic goat (Welchman et al.,1988). The clinical parameters viz., body temperature, pulseand respiration rate were noted in all animals before andafter treatment. Blood samples collected before and aftercompletion of treatment were analyzed for haemoglobin

EFFICACY OF CURAN-9 IN THE TREATMENT OF NUTRITIONALANAEMIA IN GOATS#

Deepika Goklaney1, A.P. Singh, R.K. Dhuria2 and Anil AhujaDepartment of Clinical Veterinary Medicine, Ethics and Jurisprudence

College of Veterinary and Animal ScienceRajasthan University of Veterinary and Animal Sciences, Bikaner-334 001, Rajasthan, India

ABSTRACT

An experiment, conducted on eight weak and anaemic goats revealed pale conjunctivae, increased heart rate, rough haircoat, dullness and depression. The haematological profile showed signif icantly decreased haemoglobin (7.01 ± 0.24 g/dl,PCV (22.25 ± 0.83%), TEC (8.53 ± 0.14 millions/cumm), MCH (8.22 ± 0.24 pg) and MCV (26.06 ± 0.79 fl) . The serum levelsof total protein, albumin, A: G ratio and blood glucose levels were signif icantly reduced. Biochemical analysis revealeddecreased serum copper, cobalt and iron levels. Use of Curan-9, injection of copper glycinate with mineral mixture yieldedvery good results in goats for amelioration of nutritional anaemia.

Key words: Anaemia, goats, nutritional, treatment, amelioration

(Hb), packed cell volume (PCV), total erythrocyte count (TEC),total leucocyte count (TLC), differential leucocyte count (DLC),mean corpuscular volume (MCV), mean corpuscularhaemoglobin (MCH) and mean corpuscular haemoglobinconcentration (MCHC) as per the standard methoddescribed by Jain (1986). Biochemical analysis of serumsamples was also carried out before and after treatment toestimate serum total protein, albumin, globulin,albumin:globulin (A: G) ratio and blood glucose by themethods of Tietz, (1990), using standard kit. Serum copper,cobalt and iron levels were estimated by Atomic absorptionspectrophotometer as per the method of Pinta (1979). Thetreatment of anaemic goats was done with injections ofCuran-9 (Care Vet Pharma, Ludhiana). Curan-9 is an organicpreparation containing 75 mg/ml as copper glycinatesuspension/emulsion administered 1.5 ml subcutaneously;second injection was administered after 7 days of firstinjection, along with Bestmin gold mineral mixture (Vetcare,Tetragon Chemie Pvt Ltd, Bangalore) orally @10 g daily for1 month. (each 1.2 kg of Bestmin gold contains calcium255 g, phosphorus 127.5 g, magnesium 6.0 g, manganese1.5 g, iron 1.5 g, iodine 325 mg, copper 4.2 g, zinc 9.6 g,cobalt 150 mg, sulphur 7.2 g, potassium 100 mg, sodium 6mg, selenium 10 mg, vitamin A 700000 IU, vitamin D3 70000IU, vitamin E 250 mg and nicotinamide 1000 mg). After 30days of therapeutic trial haemato-biochemical studies wereconducted to note the efficacy of treatment. Data obtainedwere analyzed as per the methods of Snedecor and Cochran(1994).

Results and DiscussionThe mean haemato-clinical observation before and after

treatment are presented in Table 1. The average bodytemperature revealed normal with extremely elevated pulseand respiration rate in anaemic goats. The higher cardiac

#1Part of M.V.Sc thesis and Corresponding address: Veterinary Officer on deputation, College of Veterinary and Animal Science, Bikaner. Email: [email protected] Professor, Department of Animal Nutrition, College of Veterinary and Animal Science, RAJUVAS, Bikaner-334001, Rajasthan

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rate might be due to the effects of compensatory mechanismto decrease the circulation time of erythrocytes (Katoch andMandial, 2003). Significant increase in respiratory rate wasa constant feature and observed in almost all anaemicgroups of goats. This possibly be due to reduction in oxygencarrying capacity of red blood cells as well as consequencesof certain physiological adjustment designed to increasethe deficiency of the reduced circulating red cell mass (Jain,1986). The clinical signs of anaemia as dullness,depression, rough hair coat, pale mucous membrane, lossof weight, weakness, tachycardia and dyspnoeacorroborated with the findings of Bhikane et al. (2006) ingoats. Comparison of the haemato-biochemical profile ofhealthy and anaemic goats indicated significantly (P<0.01)lower values of haemoglobin, PCV, TEC, MCH and MCV inanaemic goats. The decreased level of haematologicalstatus in anaemic goats may be due to nutritional deficiency.Total leucocyte and differential leucocyte count arecomparable in healthy and anaemic goats and agrees withthe observations of Wadhwa et al. (2001). Non-significantchanges in neutrophils, lymphocytes monocytes,eosinophils and basophils values indicated that thedevelopment of caprine anaemia might be due to nutritionaldeficiency either in feed, fodder or soil. Similar finding werealso recorded by Sarkar and Mishra (1991) in nutritionallyanaemic goats.

The values of total serum protein, albumin, albumin-

globulin (A:G) ratio and blood glucose in goats sufferedfrom anaemia was significantly (P<0.01) lowered ascompared to healthy control goats (Table 2). However, anon-significant change was appreciable in values ofglobulin. These findings corroborates with those of Sarkaret al. (1992), who attributed it to low intake of protein andmicro-mineral deficiencies. The goats of anaemic groupshowed a significantly decreased (P<0.01) serum copper,cobalt and iron level in comparison to healthy goats. TheCu and Co defic iencies are the most severe minerallimitations to grazing livestock in tropical countries (McDowell et al., 1984). Treatment of anaemic goats wasundertaken with injections of Curan-9 administered 1.5 mlsubcutaneously; second injection was administered after 7days of first injection along with Bestmin gold mineralmixture orally at the rate of 10 g daily for 1 month. There wasrecovery in the clinical manifestations and the symptomsalso subsided after the administration of the treatment. Theline of treatment adopted against the development ofnutritional anaemia in goats for 4 weeks was highly effectiveas evident from increase in Hb, PCV, TEC, MCH and MCVvalues. The significant (P<0.01) improvement inhaematological parameters reflects the quantitativeregeneration of the erythropoisis which in turn could be dueto copper, cobalt and iron supplementation from therapy(Sarkar et al.,1995). There were no significant variations inthe neutrophils, lymphocytes, monocytes, eosinophils and

Table 1: Clinico-haematological observations before and after treatment in anaemic goats

Anaemic goats Parameters Healthy goats

Before treatment After treatment

Temperature (oF) NS 103.83 ± 0.29 104.25 ± 0.19 103.57 ± 0.36

Pulse (rate/minute)* 81.75 ± 1.03a 89.87 ± 2.10b 87.12 ± 1.17b

Respiration (rate/minute)* 25.12 ± 0.51a 29.75 ± 0.75b 27.71 ± 0.94ab

Haemoglobin (g/dl)** 10.37 ± 0.35b 7.01 ± 0.24a 9.76 ± 0.26b

Packed cell volume (%)** 32.62 ± 0.88b 22.25 ± 0.83a 31.00 ± 1.06b

TEC (million/cumm)** 10.08 ± 0.30b 8.53 ± 0.14a 9.46 ± 0.12b

TLC (thousand/cumm)* 9.07 ± 0.22a 10.86 ± 0.58b 9.58 ± 0.22a

MCV (fl)** 32.60 ± 1.54b 26.06 ± 0.79a 32.76 ± 1.14b

MCH (pg)** 10.37 ± 0.50b 8.22 ± 0.24a 10.32 ± 0.32b

MCHC (g/dl)NS 31.79 ± 0.60 31.55 ± 0.41 31.57 ± 0.52

Neutrophil (%)NS 38.87 ± 1.76 41.87 ± 1.52 40.37 ± 0.98

Lymphocyte (%)NS 53.37 ± 1.22 49.62 ± 1.64 51.75 ± 0.94

Monocyte (%)NS 3.25 ± 0.36 3.87 ± 0.66 3.37 ± 0.49

Eosinophil (%)NS 4.00 ± 0.46 4.37 ± 0.37 4.00 ± 0.37

Basophil (%)NS 0.50 ± 0.19 0.45 ± 0.16 0.50 ± 0.18

Value bearing different superscript in a row differs significantly NS- Non significant; *Significant at (P<0.05); **Significant at (P<0.01)

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basophils at pre-treatment and post-treatment stage. The totalserum protein, serum albumin, A:G ratio and blood glucoselevel of anaemic goats increased significantly (P<0.01) aftertreatment. Whereas, globulin level in anaemic goats increasednon-significantly.

The serum iron, cobalt and copper concentration inanaemic goats increased after treatment. Treatment could leadto improvement in the levels of biochemical parameters butcould not bring the parameters at par with control in a treatmentperiod of 30 days. Dey et al. (2004) recorded improvement invalues of plasma copper in anaemic goats after treatmentfrom 0.57±0.01 to 0.62±0.05 µg/dl as compared to control(0.68±0.09 µg/dl). Therapy of anaemia by administratinginjection Curan-9 was also tried by Kumar (2007) who opinedthat injection of copper glycinate was considered efficaciousin copper deficiency anaemia due to its quick absorption, lackof toxicity and prevention of copper deficiency states. Similarobservations were also recorded by Mondal et al. (2007) inBlack Bengal goats with supplementation of two sources ofcopper i.e. copper sulpate and copper proteinate.

These finding of study in text indicated that injectablecopper preparation given twice helped not only in increasingthe values of serum copper but iron and cobalt also.Supplementation of mineral mixture (Bestmin gold) alsohelped to increase the serum copper and iron levels. Use ofCuran-9, injection of copper glycinate with mineral mixtureyielded very good results in goats for amelioration of nutritionalanaemia.

ReferencesArmour, J. et al. (1991) The Merck Veterinary Manual. 7th ed. pp. 17-

33.Bhikane, A.U. et al. (2006) Indian Vet. J. 83:320-322.Dey, A.K. et al. (2004) Indian J. Anim. Hlth. 43(2): 163-165.Fraser, C.M. et al. (1991) The Merck Veterinary Manual, 7th ed., Merck

and Co., USA. pp. 15-19, 27.Jain, N.C. (1986) Schalm’s Veterinary Haematology. 4th ed. Lea and

Febiger, Philaldelphia. pp. 15-81, 356-404.Katoch, A. and Mandial, R.K. (2003) Indian J. Vet. Med. 23(2):75-78.Kumar, H. (2007) Clinico-therapeutic studies on anaemia in camels

(Camelus dromedarius) cows with reference to copper,cobalt and Iron. M.V.Sc. thesis submitted to RajasthanAgricultural University, Bikaner.

McDowell, L.R. et al. (1984) Proceedings of symposium on herbivorenutrition in sub tropics and tropics- problems andprospects. Chapter 3 pp. 67, Pretoria, South Africa.

Mondal, M.K. et al. (2007) Ani. Nutri. and Feed Technol. 7:37-46.Pinta, M. (1979) Modern Methods for Trace Element Analysis. Ann.

Arbor Science Publishers, Inc. Michingen (USA).Radostits, O.M. et al. (2007) Veterinary Medicine. 10th ed., W.B.

Saunders Co. Ltd. London.Sarkar, S. et al. (1992) Indian J. Ani. Sci. 62(2): 100-102.Sarkar, S. and Mishra S.K. (1991) Indian Vet. J. 68(8): 769-774.Sarkar, S. et al. (1995) Indian J. Vet. Pathol. 19(2): 108-111.Snedecor, G.W. and Cochran W.H. (1994) Statistical Methods. Oxford

IBH Publishing Co., Calcutta.Tietz, N.W. (1990) Clinical Laboratory Tests. 2nd ed. W.B. Saunders

Co. Philadelphia. Pp. 26, 246 and 470.Wadhwa, D.R. et al. (2001) Indian J. Vet. Med. 21:21-24.Welchman, D. et al. (1988) Vet. Rec. 123(20): 505-510.

Table 2: Biochemical observations before and after treatment in anaemic goats

Anaemic goats Parameters Healthy goats

Before treatment After treatment

Total serum protein (g/dl)** 7.30 ± 0.08b 5.57 ± 0.23a 6.81 ± 0.18b

Albumin (g/dl)** 4.14 ± 0.19b 2.32 ± 0.03a 3.28 ± 0.13b

Globulin (g/dl)NS 3.15 ± 0.22 3.24 ± 0.21 3.53 ± 0.25

A:G ratio** 1.39 ± 0.17c 0.74 ± 0.05a 0.99 ± 0.12b

Blood glucose (mg/dl)** 64.96 ± 1.21c 53.75 ± 1.01a 57.57 ± 1.61b

Serum copper (µg/dl)** 94.82 ± 4.68c 46.97 ± 2.44a 80.43 ± 1.90b

Serum cobalt (µg/dl)** 28.02 ± 0.85c 15.06 ± 1.31a 24.17 ± 1.06b

Serum iron (µg/dl)** 169.35 ± 5.79c 109.02 ± 3.61a 143.28 ± 3.82b

Value bearing different superscript in a row differs signif icantly: NS- Non signif icant; **Significant at (P<0.01)

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IntroductionMastitis is a global problem of dairy farmers and is a

serious hindrance in the development of dairy industry (Dhillonet al., 2000). In dairy animals it results in heavy economiclosses mainly due to subclinical mastitis (Sena and Sahni,2001) or we can say economically mastitis is the mostdisastrous disease of dairy animals (Dhillon et al., 1995). Thesubclinical mastitis causes three times more loss than theclinical mastitis (Shukla and Supekar, 1982). Bovine mastitisis the single most common cause for antibiotic use in lactatingdairy cattle (Moore and Heider, 1984; Kaneene and Miller, 1992).Therapy treating this disease is also the most common sourceof illegal antibacterial residues in marketed milk (Erskine,1996). Antibacterial therapy of bacterial induced disease incattle has been incriminated as a catalyst for resistance inbacteria isolated form treated animals, other animals withinthe herd and food derived from cattle for human consumption(Berghash et al., 1983; Griggs et al., 1994). Additionally,antibacterial use has been suggested as a selective force indetermining the bacterial ecology of bovine mastitis (Myllys etal., 1994).

In India, the disease causes financial drain to the tune ofRs.160.71 million annually (Singh and Singh 1994) and upsetsmargin of safety in dairy industry. It is a disease that leads toreduced milk yield and an increased number of clinicaltreatments and early cow culling (Shook 1989, Gill et al., 1990,Beaudeau et al., 1993, Lescourret and Coulon, 1994).

Mastitis without antibiotic is preferred because of resistanteffect of antibiotic leading to a threat to safe milk supply, high

THERAPEUTIC EFFICACY OF VETADE IN RECURRENT CASES OFMASTITIS IN DAIRY COWS#

S. Tiwari1 and M. P. Gupta2

Department of Epidemiology and Preventive Veterinary MedicineGuru Angad Dev University of Veterinary and Animal Sciences, Ludhiana, Punjab

ABSTRACT

Eighteen animals selected from private dairy farm of Ludhiana region were divided into three groups of six animals each. First groupserved as healthy control, 2nd as infected control and 3rdas treated group. Treatment was given with inj. Vetade @ 10 ml/500 kg b.wt.s.i.d. for 4 days on 7th day of observation. Vitamin E level was assayed on 0th, 7th, 14thand 21st day of observation. pH of milk rangedfrom 6.65±0.199 to 6.80±0.289,7.583±0.422 to 8.29± 0.480 and 6.83± 0.047 to 8.23±0.467 in 1st, 2nd and 3rd group, respectively. SCCof milk ranged from 0.166± 0.235 lac cells/ml to 0.166±0.235 lac cells/ml, 25.41±16.66 to 31.33±15.33 lac cells/ml and 2.41±2.89 to26.91±18.40 lac cells/ml in 1st, 2nd and 3rd group, respectively. SLS test showed 1+, 4+ and 1+ in 1st, 2nd and 3rd group, respectively. CSTshowed no bacterial growth, three isolates of E. coli and two isolates of Kliebsiella, two isolates of E. coli and one isolate ofKliebsiellain 1st, 2nd and 3rd group, respectively. The mean value of vitamin E were 4.341±0.216 to 4.365±0.239 mmol/l, 3.12±0.0824 to3.278±0.0527 mmol/l and 3.303±0.1576 to 6.73±0.1474 mmol/l in 1st, 2nd and 3rd group, respectively. In 3rd group, former indicates pre-treatment value and latter indicates post treatment value which is significantly different. After infection, vitamin E level decreases by28% and after treatment its level increases by 49%. Five animals out of six were completely cured showing recovery rate of 83.33%.This shows that vitamin ADE3 can act as adjunct therapy in treatment of mastitis.

Key words: Mastitis, pH, SCC, CST, vetade, vitamin E

cost of antibiotic therapy per clinical case and development ofresistance and cross resistance to various antibiotics amongpathogens of both animals and human origin (Mellenberger,1996). Also in view of WTO regulation, milk with antibiotic is notconsidered fit for processing in milk plant.

Deficiency of many vitamins and micronutrients particularlyvitamin A, vitamin D, vitamin E, selenium, copper in diet leadsto increased incidence of mastitis with infection of longerduration and more severe clinical sings. For lactating dairyanimals, nutrient supplementation for trace minerals andvitamins go beyond correcting for deficiencies but are aimedrather at minimizing stress and optimizing production efficiency(Mcdowell, 2002).

Free radicals can be extremely damaging to biologicalsystems (Padh, 1991). Also, phagocytic granulocytes undergorespiratory burst to produce oxygen radicals to destroyintracellular pathogens. However, these oxidative products canin turn, damage healthy cells if they are not eliminated.Antioxidants serve to stabilize these highly reactive free radicalsthereby maintaining the structural and functional integrity ofcells (Chew, 1995). Therefore, antioxidants are very importantto immune defence and health of animals.

Tissue defence mechanisms against free radical damagegenerally include vitamin A, vitamin C, vitamin E and sometrace mineral like selenium. These are also critical in protectingthe internal cellular constituents from oxidative damage. Bothin vitro and in vivo studies show that these nutrients generallyenhance different aspects of cellular and non-cellular immunity.So, the antioxidant function could at least in part enhance

#1Part of M.V.Sc. Thesis. Present address: Assistant Professor, Division of VC & TH, SKUAST-J, R.S. Pura, Jammu. Email: [email protected] Phone no: +9194191572752Professor and Head

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immunity by maintaining the functional and structural integrityof important immune cells. A compromised immune systemwill result in reduced animal production efficiency throughincreased susceptibility to diseases thereby leading to increaseanimal morbidity and mortality. Antioxidant supplementationcould decrease the duration, incidence and severity of clinicalmastitis (Erskine et al., 1989; Smith et al., 1984) and wasassociated in lower prevalence of intra mammary infectionscaused by contagious pathogens (Erskine et al.,1987).Keeping the above facts in view, this study was planned toevaluate therapeutic efficacy of anti-oxidants like vitamin A,vitamin E, vitamin D3 etc. in recurrent cases of mastitis inbuffaloes.

Materials and MethodsEighteen buffaloes selected from private dairy farm of

Ludhiana region were divided into three groups of six animalseach. First group served as healthy control, 2ndas infected controland 3rd astreated group. Treatment was given on 7th day ofobservation with inj. Vetade** @ 10 ml/500 kg b.wt. s.i.d. for 4days in 3rd group. Pre-treatment and post-treatment milk andblood sampling had been done on 0th, 7th, 14th and 21st day of

observation. Milk sample was processed for estimation of pH,SLS, SCC and CST whereas blood sample was processedfor estimation of vitamin E (estimated within 48 hrs.).

pHpH of milk sample was estimated by using standardized

pH meter.

Sodium lauryl sulphate testThe test reagent was prepared by dissolving three grams

of sodium lauryl sulphate in 100 ml of distilled water andsuspension was heated to 50oC to make it a clear solution.The pH of solution was adjusted to 8.0 with the help of pHmeter. Bromocresol purple in the final concentration of 1:10,000was added to the solution. For this purpose 0.5 per centaqueous solution of bromocresol purple was used and 2 mlof this solution was added to 100 ml of prepared reagent. Thetest was conducted in a plastic paddle with four cups markedas LF, LH, RF and RH. The milk and test reagent was added inequal quantity (3 ml each) in each cup of paddle. The contentswere well mixed by giving a circular motion to the paddle alongthe horizontal plane. The results were recorded within 10seconds and interpreted as:

No mastitis (-) Liquid with no precipitate Doubtful (+) Traces of precipitate with little gel formation Positive (+) Precipitate thickens and moves at the centre on swirling.

When movement was stopped, mixture leveled again covering bottom of cup.

Strong positive (+++) A distinct gel which tended to stick to bottom of paddle and a central peak was seen during swirling (A change in colour to purple in alkaline milk and yellow in acidic milk was observed).

Somatic cell countSomatic cell count of milk sample was done according

to the standard microscopic counting method of Indianstandard institute (1960). After thoroughly mixing 0.01 mlof milk sample was spread over an area of 1 sq. cm. on aclean grease free glass slide using a 4 mm platinumloop. The smear was allowed to dry at room temperatureand stained with Levowitz-Weber modification (1967) ofNewman-Lampert stain. Staining of dried milk films wasdone by placing the slides for 2 minutes in Levowitz-Weberstain in a covered coplin jar. The excess stain drained offby standing the slides on absorbent paper and air dried.The slides were then rinsed in three changes of tap waterat 100-110oF, drained and dried rapidly in air.

**Vit.ADE3 injection. each ml contains vit. A - 250000 IU, Vit.D3 -25000 IU and vit. E- 100 IU. Marketed by SarabaiChemicals, Animal Health Ltd. Administration Building, Gorwa Road, Vadodhara-390023, India.

CalculationAt least 20-50 microscopic fields were examined to

find out the average number of leukocytes per field. Thisaverage number o f leukocytes was multip lied w ithmicroscopic factor (MF) to get total leukocyte per ml.

The diameter of the microscopic field was measuredas 0.14 with the help of a stage micrometer (ruled in 0.01mm). MF was calculated using the following formula:

100 X 100 MF =

r2

By substituting the value for radius (r) of microscopic field,the MF was calculated to be 5, 00,000. The interpretationof result was done under:

Norm al (-) : Be low 1, 00,000 ce lls per m l

D oubtfu l (+) : 1, 00,000 to 5, 00,000 c ells per m l Pos it ive (++) : 5, 00,000 to 50 , 00,000 cel ls per m l S trong ly pos it ive (+++) : 50 , 00 ,000 and above c ells per m l

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Culture sensitivity test (Cruickshank et al., 1970)The organisms isolated from subclinical as well as clinical

cases of mastitis were tested for sensitivity to variouschemotherapeutic agents. Standard discs of thoseantimicrobial agents were preferred for which intramammarypreparations or injectables are easily available in the market.The following antimicrobial discs were used for sensitivitytests:

Standard disc method described by Ward and Bates(1983) was followed for sensitivity test. The growth inhibitionzones were measured with a scale after 24 hours of incubationat 37oC and results were interpreted as sensitive or resistantin comparison with the standard chart supplied along withdiscs.

Vitamin E was estimated by method of Kayden et al.(1973).

S. No. Antimicrobial agent disc Concentration/disc 1 Ampicillin 10 mcg 2 Amikacin 10 mcg 3 Pefloxacin 5 mcg 4 Gentamicin 10 mcg 5 Streptomycin 10 mcg 6 Neomycin 30 mcg 7 Lincomycin 10 mcg 8 Polymyxin B 50 units 9 Erythromycin 15 mcg

Statistical analysisStatistical analysis was done by using completely

randomized design (CRD) and randomized block design (RBD)as per method of Snedecor and Cochran (1968).

Results and DiscussionObservation of animals of first groupHistory and symptoms

The animals taken in this group had never suffered frommastitis. The animals were of age group 3-5 years. Mammaryglands of these animals revealed normal glandularparenchyma. The animals were mostly recently calved. Colour,consistency, opacity and thickness of milk were normal. Therewas absence of sany flakes or blood in milk.

pHThe maen valus of pH ranged from 6.65±0.199 to

6.80±0.289 (Table 2 and Fig.1 ). There was non-significantvariation in pH of milk during whole observation period. Theobserved pH value is in accordance to normal pH of milk whichis 6.5 (Dhillon et al., 1989).

Sodium lauryl sulphate test (SLS)The animals gave negative test with SLS reagent. There

was no gel formation when milk from the quarters was mixedwith SLS (Table 1). The milk sample gave 1 + which iscategorized normal (Mulkalwar et al., 1999).

Somatic cell count (SCC)The mean value of SCC ranged from 0.166 ± 0.235 lac

cells/ml to 0.166 ± 0.235 lac cells/ml of milk (Table 2 and Fig.2). There was no significant variation in SCC of milk fromquarter to quarter of these animals. This finding is similar to

findings of Munoz et al. (2002) and Maunsell et al. (1998). Fouranimals out of six were having nil SCC. Absence of SCCcorresponds to absence of bacterial growth i.e. there is associationbetween bacteriological growth and SCC.

Culture Sensitivity Test (CST)There was no bacterial growth when milk samples were

streaked on trypticase soya agar media and blood agar media.Bacteriologically negative glands were bacteriologically negativeat four sampling times (Table 1). This finding is similar to finding ofMaunsell et al. (1998).

Vitamin EThe mean value of vitamin E in plasma ranged from

4.341±0.216 to 4.365±0.239 mmol/l (Table 2 and Fig 3). Therewas no significant variation in vitamin E level during wholeobservation period. These findings are lower than findings of Jukolaet al. (1996) but in agreement with finding of Ndiweni et al. (1991)who states that mean plasma vitamin E level above 4 mg/ml i.e.8.46 mmol/l is regarded as adequate for cattle.

Plasma concentration of greater than 3.5-4 mg/ml of -tocopherol are considered adequate as evidenced by therelationship between intra cellular kill of bacteria by neutrophilsand plasma vitamin E concentration (Hogan et al., 1993a).Observation of animals of second group (vitamin E deficient)History and symptoms

The animals of this group were in age group 3 to 5 years, in 4th

to 5th lactation and recently calved. The mammary glands ofthreeanimals out of six were inflamed, swollen, oedematous, hotand painful to touch. Two animals out of six had erythematous andenlarged mammary glands .The incidence was highest during firstthree months of lactation. There was presence of flakes in milk ofaffected quarter of 5 animals out of six. There was presence ofblood clots in milk of one animal out of six.

pH Mean pH value of milk varied from 7.5830.422 to

8.290.480 (Table 4 and Fig. 1). There was no significantvariation in pH during whole observation period. Whilecomparing pH of milk of these animals with healthy animalsthere was significant difference (CD 5% - 0.235804) (Fig. 1).This finding is similar to findings of Dhillon et al. (2000). Theincreased alkalinity of mastitic milk is due to decreased lactoseproduction and increased transudation of alkaline salts fromudder tissue (Schalm et al., 1971).

Sodium lauryl sulphate test (SLS)There was severe SLS reaction of milk samples from

affected quarters of these animals (Table 3). There was thick

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Table 1: pH, SLS, SCC, CST and vitamin E values in cows of group first.

SLS Days of observation pH

LF LH RF RH SCC (lac cells/ ml) CST Vit. E (mol/L)

Cow No. 1 0 day 6.87 - - ++ ++ 0.5 No growth 4.20 7 day 6.85 - - ++ + 0.5 No growth 4.20

14 day 6.88 - - + + 0.5 No growth 4.15 21 day 6.87 - - + + 0.5 No growth 4.25

Cow No. 2 0 day 6.90 - - + - Nil No growth 4.56 7 day 6.89 - - + - Nil No growth 4.55

14 day 7.19 - - + - Nil No growth 4.35 21 day 7.08 - - + - Nil No growth 4.40

Cow No. 3 0 day 7.10 - - + - Nil No growth 4.10 7 day 6.7 - - + - Nil No growth 4.10

14 day 7.0 - - + - Nil No growth 4.15 21 day 6.8 - - + - Nil No growth 4.20

COW NO. 4 0 day 6.1 - - - - Nil No growth 4.55 7 day 6.4 - - - - Nil No growth 4.54

14 day 6.3 - - - - Nil No growth 4.55 21 day 6.37 - - - - Nil No growth 4.55

Cow No. 5 0 day 6.61 - - - - Nil No growth 4.66 7 day 6.70 - - - - Nil No growth 4.70

14 day 6.97 - - - - Nil No growth 4.70 21 day 7.18 - - - - Nil No growth 4.65

Cow No. 6

0 day 6.5 - - - - 0.5 No growth 4.00

7 day 6.4 - - - - 0.5 No growth 4.10

14 day 6.4 - - - - 0.5 No growth 4.15

21 day 6.5 - - - - 0.5 No growth 4.10

gel formation in milk of affected quarters of animals. This findingis similar to finding of Sena and Sahani (2001) who found thatwhen pH of milk ranges from 7.5-8 gives SLS reaction asstickiness with slight gel formation.

Somatic cell count (SCC)The mean value of SCC varied from 25.41±16.66 to

31.33±15.33 lac cells/ml of milk (Table 4 and Fig. 2). Therewas no significant variation in SCC value over wholeobservation period. While comparing SCC value of milk sampleof infected control animals to those of healthy animals, therewas significant difference (CD 5% - 8.12491) (Fig.2) betweenpH of milk of these two groups. This value is significantly higherthan what is observed by Green et al. (2004) who described itsrange from 0.6-2.0 cells/ml of milk in healthy animals.

Culture sensitivity test (CST)There were three isolates of E. coli, two isolates of

Kliebsiella and in milk of one animal, there was no growth ofbacteria. The sensitivity pattern for these isolates for antibiotic

that these isolates are sensitive to pefloxacin, tetracycline andstreptomycin. There was intermediate sensitivity for neomycin,gentamicin and polymyxin B but these were resistant toampicillin and amikacin (Table 3). This finding is similar tofinding of Erskine et al. (2002) who isolated E. coli andKliebsiella from mastitis milk and found that they are sensitiveto gentamicin and tetracycline.

Vitamin EThe mean value of vitamin E of animals of this group

ranged from 3.12±0.0824 to 3.278±0.0527 mmol/l (Table 4and Fig. 3). There was significant difference (CD at 5%0.1019801) between values of vitamin E level during wholeobservation period. There was decrease in vitamin E levelfrom 7 day to 14 day of observation period. This finding is lowerthan the value obtained by Jukola et al. (1996) who obtained itas 12.48 mmol/l. While comparing vitamin E level of animalsof this group to vitamin E level of animals of healthy group,there was significant difference (CD 5% - 0.115355) i.e. theseanimals were vitamin E deficient animals.

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Table 2: Mean values of pH, SLS, SCC, CST and vitamin E value of cows of first group

Days of observati on pH S LS S CC (Lac cells /

m l) CS T V it. E (ml/L)

0 day 6.68 0.3 25 + 0.166 0.235m ax No g ro wth 4.345 0.254 7 day 6.65 0.1 99m in + 0.166 0.235 No g ro wth 4.365 0.239m ax 14 day 6.79 0.3 25 - 0.166 0.235 No g ro wth 4.341 0.216 m in

21 day 6.80 0.2 89m ax - 0.166 0.235m in No g ro wth 4.358 0.194

Table 3: pH, SLS, SCC, CST and vitamin E levels in cows of group second.

SLS Days of observation pH LF LH RF RH

SCC (lac cells/ml) CS T Vit. E

(m ol/L) Cow No. 7 E. coli

0 day 7.46 - ++ + - 16.5 N– I, P–I 3.35 7 day 7.80 - ++ + - 15 G-I, A-R 3.30 14 day 8.10 - +++ ++ - 20 T-S, St-S 3.28 21 day 8.30 - +++ +++ - 22 Am-R,Pe-S 3.25

Cow No. 8 E. coli 0 day 7.46 - - + ++++ 21 N–S, P–R 3.26 7 day 7.9 - + ++ +++ 25 G-I, A-R 3.10 14 day 8.0 - + ++ +++ 25 T-S, St-S 3.00 21 day 8.5 - +++ +++ ++++ 30 Am-R,Pe-S 3.10

Cow No. 9 0 day 8.00 ++ +++ - ++ 60 No growth 3.21 7 day 8.20 ++ +++ + + 60 No growth 3.11 14 day 8.4 + +++ ++ ++ 65 No growth 3.10 21 day 8.6 +++ +++ +++ +++ 63 No growth 3.01

Cow No.10 Kliebsiella

0 day 6.78 ++ + ++ - 29 N– I, P–I 3.22 7 day 6.86 ++ ++ + - 30 G-S, A-R 3.20 14 day 6.99 +++ ++ ++ + 30 T-S, St-S 3.19 21 day 7.24 +++ +++ +++ +++ 35 Am-R, Pe-S 3.11

Cow No. 11 Kliebsiella 0 day 8.00 ++ ++ ++ - 18 N–R, P–I 3.30 7 day 8.2 ++ ++ ++ - 18 G-S, A-R 3.30 14 day 8.5 +++ ++ +++ + 20 T-S, St-S 3.15

21 day 8.5 +++ +++ ++++ +++ 20 Am-R, Pe-S 3.05

Cow No. 12 E. coli 0 day 7.80 + + ++ +++ 8 N–R, P–I 3.33 7 day 8.30 ++ ++ +++ +++ 10 G-S, A-R 3.25 14 day 8.60 ++ ++ +++ ++++ 10 T-R, St-R 3.20 21 day 8.60 +++ +++ +++ ++++ 18 Am-R, Pe-S 3.20

Plasma concentrations of -tocopherol are low during

peripartum period (Goff and Stabel, 1990; Weiss et al., 1990a;Weiss et al., 1994; Hogan et al.,1997).

The periparturient period is associated with decreasedactivity of PMN and with increased susceptibility to mastitis.Vitamin E level increased after parturition could improve thecows immune responses and thus leads to lower incidenceof mastitis (Ndiweni et al., 1991).

Vitamin E is an integral component of all lipid membranesand serves to protect lipid membranes from attack by reactiveoxygen species. Polyunsaturated fatty acids of membranesare particularly vulnerable to attack by reactive oxygen speciesAND ROS can initiate a chain reaction of lipid destruction that

destroys the membrane of the cell. Vitamin E can quenchperoxidation reactions in membranes.

Arachidonic acid metabolism is altered in animalsdeficient in vitamin E, selenium or both (Atroshi et al., 1989,Aziz and Klesius, 1986). Vitamin E may function to controlperoxidation of arachidonic acid or its unstable metaboilites.The arachidonic acid metabolites are important for PMNfunction and the amplification of inflammatory responsefollowing pathogen invasion of tissues including the mammarygland (Aziz et al., 1984). The speed with which PMN can bemobilized following pathogen invasion and the efficiency ofintracellular kill are events of critical importance to protection ofthe mammary gland from infection (Smith et al., 1997).

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Table 4: Mean values of pH, SLS, SCC, CST, vitamin E level in cows of group second.

Days of observation pH SLS SCCsp

(lac cells/ml) CS T Vit. E (m ol/L)

0 day 7.583 0.422 ++ 25.41 16.66 3.278 0.0527x 7 day 7.876 0.487 +++ 26.33 16.39 3.21 0.0816x

14 day 8.098 0.538 +++ 28.33 17.48 3.153 0.0874x 21 day 8.29 0. 480 +++ 31.33 15.33

Gram –ve bacilli N–I, P–I G-I, A-R

T-S, St-S Am-R, Pe-S 3.12 0.0824x

x shows signif icant difference at 5%

Table 5: pH, SLS, SCC, CST and vitamin E level in cows of group third.SLS Days of

observation pH LF LH RF RH SCC (lac cells /ml) CS T Vit. E

(m g/lit) Cow No. 13 E. coli

0 day 8.9 +++ ++++ - ++ 23.5 N-I, P-I 3.20 7/0day# 9.1 +++ ++++ - +++ 25.0 G-I, A-R 3.15 14/7day 8.0 - + - ++ 5.0 T-S, St-S 6.74

21/14day 6.9 - + - + 2.0 Am-R,Pe-S 4.22 Cow No. 14 E. coli

0 day 7.9 ++ ++++ +++ +++ 50 N-S, P-S 3.55 7/0day# 8.2 +++ ++++ + ++++ 52.5 G-I, P-R 3.40 14/7day 7.1 ++ ++ + ++ 2.5 T-S, A-R 6.79

21/14day 6.8 - - - + Nil Am-S, St-S 4.40 Cow No. 15

0 day 7.5 - - - ++++ 6.4 No g rowth 3.64 7/0day# 8.1 - - - ++++ 7.5 No g rowth 3.55 14/7day 7.5 - - - ++ Nil No g rowth 6.80

21/14day 6.8 - - - + Nil No g rowth 4.24 Cow no. 16

0 day 7.8 ++++ - - + 6.4 No g rowth 3.69 7/0day# 8.2 ++++ - - ++ 6.5 No g rowth 3.41 14/7day 7.5 ++ - - + 2.0 No g rowth 6.89

21/14day 6.8 + - - - Nil No g rowth 4.14 Cow No. 17 Gram –ve bacilli

0 day 7.7 ++++ +++ +++ ++++ 47 N-R, P-I 3.26 7/0day# 8.3 ++++ ++++ +++ ++++ 50 G-I, A-R 3.15 14/7day 7.1 + + - + 11 T-R, St-I 6.42

21/14day 6.8 + - - + 5 Am-S, Pe-S 4.15 Cow No. 18 Kliebsiella

0 day 6.9 ++++ - + - 17.5 N-R, P-I 3.33 7/0day# 7.5 ++++ - ++ - 20 G-R, A-R 3.16 14/7day 6.9 ++ - - - 10 T-R, St-R 6.74

21/14day 6.9 + - - - 7.5 Am-R,Pe-S 4.44 #Treatment started with vetade (10 ml i/m) daily for 5 days

Vitamin E and selenium play essential roles in these eventsand dietary deficiencies of either leads to impaired PMNfunction and increased incidence of intramammary infectionin dairy cows (Hogan et al., 1993a).

Observation of animals of third group (treatment group-treated with injection Vetade)

History and symptomsThe animals in this group were in third to fifth lactation and

recently calved. Out of six animals, flakes were present in milkof three animals, pus was present in milk of two animals andwatery fluid like consistency was present in milk of one animal.

In two animals, swollen quarters were found and in other twoanimals, painful and oedematous mammary glands weredetected. Colour of milk had taken reddish tinge in two animalsout of six.

pHThe mean value of pH of milk varied from 6.83±0.047 to

8.23±0.467 (Table 6 and Fig. 1) where former indicates pH ofmilk during post treatment period and later indicates pH ofmilk during pre-treatment period. There was significantdifference (CD 5%-0.644410) (Table 6) between pH values of0 to 7 day but not 14 day post treatment and 21 day posttreatment.

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Table 6: Mean values of pH, SLS, SCC, CST and vitamin E level in cows of group third.

Days of observation PH SLS SCC (lac cells/ml) CST Vit. E (mol/L)

0 day 7.78 0.595 ++++ 25.13 17.60x 3.445 0.1899x

7/0day# 8.23 0.467 x ++++ 26.91 18.40x 3.303 0.1576x

14/7day 7.35 0.369x + 5.08 4.10x 6.73 0.1474 x 21/14day 6.83 0.047 - 2.41 2.89x

Gram –ve bacilli N-I, P-IG-I, A-RT-S,

St-S,Am-R, Pe-S

4.265 0.1157x

x shows significant difference at 5%

Fig. 1: Comparison of pH of different groups

Fig. 2: Comparison of SCC of different groups

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Fig. 3: Comparison of vitamin E of different groups

While comparing pH of milk of animals of infected controlgroup which were vitamin E deficient to pH of milk of animalsof this group, there was significant difference (CD 5%-0.376488) (Fig. 1). So, it indicates that treatment with injectionVetade was effective which acts as antioxidant.

While comparing pH of milk of animals of this group topH of milk of animals of healthy group, there was significantdifference (CD 5%-0.256317) (Fig. 1).

Sodium lauryl sulphate (SLS) testAll animals gave severe SLS reaction i.e. ++++ reaction.

There was thick gel formation in milk samples from all affectedquarter. There was progressive decrease in SLS reaction astreatment started and continued. All animals got cured andSLS reaction came within normal range i.e. +ve after 14 daysof treatment (Table 5).

Somatic cell count (SCC)The mean value of SCC ranges from 2.41 ± 2.89 to 26.91

± 18.40 lac cells/ml of milk (Table 6 and Fig 2) where formerindicates SCC value during post treatment period and latterindicates SCC value during pre-treatment period. There wassignificant difference (CD 5%-13.3692) (Table 6) in SCC valuesof animals of this group during whole observation period.

While comparing SCC value of animals of infected controlgroup which were vitamin E deficient to SCC values of animalsof this group, it was found that there was significant difference(CD 5%-9.32496) (Fig. 2). It showed that treatment with injectionVetade is effective which act as antioxidant.

While comparing SCC values of animals of this group toSCC values animals of healthy group, there was significantdifference (CD 5% - 4.57857) (Fig. 2).

Cultural sensitivity test (CST)Two isolates of E. coli and one isolate of Kliebsiella, were

identified. There was no growth of bacteria in two animals outof six.

The isolates were sensitive to pefloxacin, tetracycline,streptomycin, intermediately sensitive to neomycin, polymyxinB and gentamicin and resistant to ampicillin and amikacin

(Table 5).

Vitamin EThe mean value of vitamin E ranged from 3.303±0.1576

to 6.73±0.1474 mmol/l (Table 6 and Fig. 3) where formerindicates vitamin E levels during pre- treatment period andlatter indicates vitamin E level during post treatment period.

There was significant difference (CD 5% - 0.202185)between vitamin E level of animals of this group from 7 to 14days and 14 to 21 days i.e. treatment was effective (Table 6and Fig. 3). There was significant difference (CD 5% - 1.0806)between vitamin E level of animals of infected control groupand vitamin E levels of animals of this group.

There was also significant difference (CD 5%-0.202185)between vitamin E levels of animals of this group during wholeobservation period. There was non-significant differencebetween vitamin E levels of animals of this group to vitamin Elevels of animals of healthy group (Fig. 3).

Mammary gland infections and clinical mastitis were notaffected by treatment, but milk production was increased bytreatment but milk production was increased by feeding highamount of vitamin A (Weiss, 1998).

Vitamin D also is involved with immune function (Reinhardtand Hustmyer, 1987). Increased lumphocyte proliferation wasobserved when Jersey cows were infused subcutaneouslywith 50 mg of 1, 25-dihydroxy vitamin D/day for 7 days (Hustmyeret al., 1994).

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424-43.Shook, G. E. (1989) J. Dairy Sci. 72: 1349-62.Shukla, P. C. and Supekar, P. G. (1982) Livestock Adv. 7: 43-47.Singh, P. J. and Singh, P. B. (1994) Indian J. Dairy Sci. 47: 265-72.Smith, K. L et al. (1984) J. Dairy Sci. 67: 1293-1300.Smith, K. L. et al. (1997) J. Anim. Sci. 75: 1659-65.Snedecor, G. W. and Cochran, W. G. (1968) Statistical methods. 6th

ed. Allied Pacific, Bombay.Ward, G. E. and Bates, F. (1983) Mod. Vet. Prac. 64: 795-98.Weiss, W. P. (1998) J. Dairy Sci. 81: 2493-2501.Weiss, W. P. et al. (1990a) J. Dairy Sci. 73: 281.Weiss, W. P. et al. (1994) J. Dairy Sci. 77: 1422-29.

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IntroductionHaemorrhagic septicaemia (HS) is an acute, fatal disease of

cattle and buffaloes, caused by Pasteurella multocida (P. multocida)type B:2, B:2,5 and B:5 in Asian countries and in African countries,type E:2. The disease is common to all wet tropical countries inAsia and still remains as one of the most serious animal diseasescausing huge economic losses to livestock farmers (De Alwis,1999). In India, disease is prevalent in almost all states of countryand is responsible for approximately 50 to 60% bovine mortalityand other species of animals and has been quantified as numberone killer among cattle and buffaloes (Singh et al., 1996).

The diagnosis of HS depends on conventional phenotypicmethods including bacteriological techniques, serotyping,pathogenicity test and biochemical tests. However, thesetechniques are laborious, time consuming and are not free fromcertain limitations. The limitation of currently employed techniqueshas led to significant problems in understanding the diseaseoutbreaks, virulence characteristic of organism and in determiningits incidence and economic importance.

During the last two decades the development of molecularmethods such as Polymerase Chain Reaction (PCR) assays,which are rapid, specific and highly sensitive, are efficiently beingemployed for early detection and differentiation of various isolatesof different micro-organisms from single/different outbreaks(Owen, 1989). Townsend et al. (1998) have developed PCR basedassays for species- and type-specific identification of P. multocidaand of P. multocida type B, the causal agent of HS. Rapididentification of P. multocida and presumptive confirmation of theHS-causing serotype have the potential to reform HS diagnosis.These nucleic acid-based assays are useful in the detection of

MOLECULAR CHARACTERIZATION AND ANTIBIOGRAM OFPASTEURELLA MULTOCIDA ISOLATED FROM CATTLE AND BUFFALO

IN UTTAR PRADESH#

Avinash Kumar, S.D. Qureshi1, B.C. Pal, Sharad Kumar Yadav, Udit Jain,Subuhi Khan2 and Puneet Varshney1

Department of Epidemiology and Preventive MedicinePandit Deen Dayal Upadhyay, Pashuchikitsa Vigyan Vishvavidhyalaya Evom Go

Anusandhan Sansthan, Mathura-281 001, Uttar Pradesh, India

ABSTRACT

The applicability of molecular methods for rapid detection and typing of Pasteurella multocida (P. multocida) isolated from haemorrhagicsepticaemia (HS) affected Indian cattle and buffaloes and their antibiogram was studied. A 25 samples were revealed the presence of P.multocida by PM-PCR, which yielded 25 isolates on isolation and were further subjected to phenotypic and genotypic characterization. All theisolates were further confirmed as HS causing serogroup B by HSB-PCR. The PCR based techniques were found to be specific and sensitivefor rapid detection and typing of isolates. The results indicated that molecular methods of detection and typing are superior to conventionalmethods for rapid epidemiological investigations of HS. HSB-PCR based diagnosis can be useful for early detection of the HS causingserogroup B isolates for rapid and efficient treatment of the disease. Most of the isolates were sensitive to chloramphenicol (100%) followedby enrofloxacin (84%), ciprofloxacin (80%), gentamicin (80%) but none of the isolates were susceptible for trimethoprim. On the basis ofthese antibiogram results it was deduced that most sensitive antibiotics can be used to contain HS in cattle and buffaloes.

Key words: Cattle, buffalo, haemorrhagic septicaemia, P. multocida, PCR.

#Part of M.V.Sc. thesis of the f irst author1*Department of Microbiology and Immunology, Pandit Deen Dayal Upadhyay, Pashuchikitsa Vigyan Vishvavidhyalaya Evom Go Anusandhan Sansthan, Mathura-281001. Corresponding author: Cell: 91-946-9075089, E. mail: [email protected] of Biotechnology, GICTS College of Life Sciences, Gwalior-474 001, M.P.

organisms directly from clinical samples or from small amountsof cultured bacterial cells, thus dramatically improving the sensitivityand decreasing the time required for bacterial identification.

Due to acute nature of HS caused by P. multocida the use ofantimicrobial agents for treatment has widely reported. However,the development of resistance among various strains hasreported, which is being results of indiscriminate use of antibioticsfor a long period (Shivachandra et al., 2004; Arora et al., 2005;Kumar et al., 2009). The present investigation was undertaken toidentify various isolates of P. multocida of cattle and buffalo originfrom different parts of Uttar Pradesh, India using PCR and to studythe antibiotic sensitivity pattern among these isolates to find outthe most effective antibiotic for field use.

Materials and MethodsSampling and isolation of bacteria

A total of 25 P. multocida isolates were isolated from theclinical cases (showing symptoms of HS) or dead (supposed tobe died of HS) cattle and buffalos of different places like organizeddairy farms, veterinary hospitals and villages of Uttar Pradesh,India during 2005 to 2006. The samples were collected directly asblood from jugular vein at high rise of temperature in a sterile testtube with EDTA aseptically or from nasal mucosa by sterile swabs(live animals) and heart blood, lung, liver and spleen piecesaseptically from dead animals after post-mortem and carried tolaboratory on ice. The pieces were immediately inoculated intoblood agar (HiMedia) and incubated aerobically at 37°C for 24hours. The well-separated colonies were picked up onto bloodagar slants as pure culture and subjected to standardmorphological, biochemical tests as described by Cowan andSteel (1993) to ascertain their identity as P. multocida. The P. mult

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Table 1: In vitro antimicrobial susceptibility pattern of P. multocida isolatesCattle Buffalo Total S.

No. Antimicrobial

Agent S I R S I R S I R 1 Ampicillin (A) 3(12) 6 (24) - 7 (28) 9(36) - 10 (40) 15 (60) - 2 Amoxicillin (Am) 6 (24) 3(12) - 9(36) 7(28) - 15 (60) 10 (40) - 3 Chloramphenicol (C) 9(36) - - 16(64) - - 25(100) - - 4 Ciprofloxacin (Cf) 6(24) 4(16) - 14(56) 2(8) - 20(80) 5(20) - 5 Co-trimoxazole (Co) - 9(36) - 7(28) 9(36) - 7(28) 18(72) - 6 Ery thromycin (E) 5(20) 4(16) - 3(12) 13(52) - 8(32) 17(68) - 7 Enrofloxacin (Ex) 7(28) 2(8) - 14(56) 2(8) - 21(84) 4(16) - 8 Gentamicin (G) 5(20) 4(16) - 15(60) 1(4) - 20(80) 5(20) - 9 Nitrofurantoin (Nf) 4(16) 5(20) - 8(32) 8(32) - 12(48) 13(52) - 10 Oxy tetracyclin (O) 6(24) 3(12) - 11(52) 5(20) - 17(68) 8(32) - 11 Streptomycin (S) 6(24) 3(12) - 7(28) 9(36) - 13(52) 12(48) - 12 Trimethoprim (Tr) - - 9(36) - - 16(64) - - 25(100)

S= sensitive, I= intermediately sensitive, R- resistant. Figure in parenthesis indicate percentage.

M 1 2

620 460

bp 600 500 400

200

Fig. 1: PM-PCR and HSB-PCRLane M: Marker,100 bp DNA ladder;Lane 1: Buffalo (B:2); 2: Cattle (B:2)

multocida P52 (B:2) obtained from Division of Bacteriology andMycology, Indian Veterinary Research Institute, Izatnagar,-243122,U.P., was used as standard reference strain in this study.

Extraction of DNAFor the rapid detection, blood, nasal swab or pure colony

were inoculated to 2 ml of BHI broth. After 18 h of incubation at37°C, broth was processed for extraction of DNA by genomic DNApurification kit ((MBI Fermentas) according to manufacturer’sguidelines.

PCR assaysTwo PCR assays were conducted by using two primers sets

designed from the sequences of the KMTIT7 (5’- ATC CGC TATTTA CCC AGT GG-3’), KMTT1SP6 (5’- GCT GTA AAC GAA CTCGCC AC-3’) and KTT72 (5’-AGG CTC GTT TGG ATT ATG AAG-3’),KTSP61 (5’-ATC CGC TAA CAC ACT CTC-3’) as described byTownsend et al. (1998), for the species specific detection of P.multocida (PM-PCR) and HS causing serogroup-B isolates (HSB-PCR), respectively. Briefly, 25 µl reaction mixture containing 2 µl oftemplate, 1x PCR buffer, 2 mM of MgCl2, 200 µM of each of the fourdNTPs, 20 pmol of each primer and 1 U Taq DNA polymerasewas prepared. The reaction mixture was subjected for amplificationin Thermal cycler (Eppendorf, Germany) according to cyclicconditions:initial denaturation at 95°C for 4 min followed by 30cycles of denaturation at 95°C for 45 sec; annealing at 55°C for 45sec, extension at 72°C for 45 sec and a final extension of 72oC for6 min. Amplified products were separated by agarose gelelectrophoresis (1.5% agarose in 0.5 X TBE) at 5 V/cm for 2 h andstained with ethidium bromide (0.5 µg/ml). DNA fragments wereobserved by UV transilluminator

In vitro antimicrobial susceptibility assayIn vitro antimicrobial sensitivity test of P. multocida isolates

were determined by the disc diffusion technique as advocated byBauer et al. (1966). The 12 different antimicrobial discs(HiMedia)used were ampicillin (10 mcg), amoxicillin (30 mcg),chloramphenicol (30 mcg), ciprofloxacin (5mcg), enrofloxacin (10mcg), nitrofurantoin (300 mcg), erythromycin (10 mcg), co-trimoxazole (25 mcg), gentamicin (10 mcg), streptomycin (10 mcg),trimethoprim (5mcg), and oxytetracycline (30 mcg).

An overnight culture of each isolate in BHI broth was platedwith the help of spreader on Muller-Hinton agar medium enriched

with 5% sheep blood. The culture was allowed to adsorb for 5 minand then the antibiotic discs were placed onto the plate at anappropriate distance from each other. The agar plates wereincubated aerobically at 37°C for 24 h. The interpretation of resultsonthe basis of size of inhibition zones of various antibiotics assensitive, intermediate and resistant was done as per themanufacturer’s instructions.

Results and DiscussionHaemorrhagic septicaemia is one of the important bacterial

diseases of domestic animals causing various type of syndromes,both acute and severe form of disease. Even though the diseaseis known to us from more than 200 years, nothing is being up tomark to reduce the incidence of the disease and to eradicate itcompletely. The region behind this being lack of quick methodoreffective tool in early detection of disease. In recent pastdevelopment of molecular diagnostic method would seem to beeffective tool in early detection of the disease, which in turn to go along way to eradicate the disease.

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In present study a total of 25 P. multocida isolates recoveredfrom samples obtained following standard bacteriologicaltechniques. On blood agar plates the isolated bacterial coloniesof all the isolates were small glistening mucoid dew drop like andappeared Gram negative, cocobacilli in Grams’s stained smear.They did not grow on McConkey’s agar and were found non-haemolytic on blood agar. Biochemical tests revealed that all theisolates were oxidase, catalase and indole positive and foundnegative for citrate and urease.

Regarding molecular detection, an amplicon of ~460 bp wasrecovered from all the isolates subjected to species specific PM-PCR assay (Fig.1). An amplified product of ~460 bp in PM-PCRhas been reported to be confirmatory for the species identificationof P. multocida (Townsend et al., 1998). Many other workers havesuccessfully been used this PCR assay for preliminary screeningof various Pasteurella isolates. Authentication of the PM-PCR wasdone by Dutta et al. (2001) for identification of 18 Indian P. multocidaisolates and nine non Pasteurellae organisms and reported a~460 bp amplicon for all Pasteurella isolates, while no suchamplification for other bacterial species. Further, other workers(Kumar et al., 2009; Biswas et al., 2004; Dey et al., 2007) reportedthe usefulness of PM-PCR in molecular detection of P. multocidaisolates. Hence, our study further accentuated the advantage ofPM-PCR over the laborious or time consuming conventionaltechniques for the diagnosis of HS.

Subsequently, all isolates yielded an amplicon of ~620 bp(Fig.1) in HSB-PCR assay revealed that isolated P. multocida werebelonging to the HS causing serogroup-B, frequently involved inHS outbreaks. Similar observation was attained by Townsend etal. (1998), as only DNA from P. multocida serotype B was amplified.This amplified product was unique to pathogenic serotype B:2strains of P. multocida, mainly responsible for HS in cattle andbuffalo, thus potentially becoming a diagnostic marker for HScausative agents in Asia (Brickell et al., 1998; Dziva et al., 2008).

The results of antimicrobial sensitivity testing indicated thatall the isolates were most sensitive to chloramphenicol as 100%of the isolates were susceptible to this drug, followed by enrofloxacin(84%), ciprofloxacin (80%), gentamicin (80%), oxytetracycline (68%)while rest of the antibiotics were moderately effective (Table 1).None of the isolates were susceptible for trimethoprim. Besides,chloramphenicol; enrofloxacin, ciprofloxacin, gentamicin andoxytetracycline were most effective against the isolates, most ofthe isolates showed only intermediate sensitivity to rest of drugs(Table 1). Amoxicillin was moderately effective as 60% of the isolateswere found sensitive to it. Streptomycin another antibiotic waseffective only against 52% of isolates. Nitrofurantoin, ampicillin,erythromycin and co-trimoxazole were effective against leastnumber of isolates i.e. 48%, 40%, 32% and 28%, respectively.Dutta et al. (2009) reported similar type of observation on pigisolates. Higher efficacy of chloramphenicol and enrofloxacin in astudy conducted on P. multocida isolates has also been reported(Kumar et al., 2009; Sharma et al., 2005). In similar reportschloramphenicol and enrofloxacin were found quite effectiveagainst pasteurellosis by several other workers (Salmah, 2004;Arora et al., 2005).

In present study, ciprofloxacin and gentamicin were foundmoderately effective, however, earlier reports suggest the higherefficacy of this drug for treatment of HS (Kumar et al., 2009; Guptaet al., 1996; Verma et al., 2004). It indicates the gradual developmentof resistance among field strains of Pasteurella against these

drugs as it is widely employed in therapeutics. Catry et al. (2005)has also observed the acquired resistance to gentamicin in P.multocida isolates obtained from the veal calf herds. In our studymoderate to low efficacy of amoxicillin, ampicillin and erythromycinis in accordance with the previous report of Kumar et al. (2009).Further, in this study, the resistance pattern of drugs was almostsimilar in both species of animal studied (Table 1). Theseobservations reflected that the development of resistance againsta particular antibiotic is proportional to its indiscriminate use for along period of time irrespective of animal species infected. Moreover,it could be due to the selective use of antimicrobial agents or dueto transposon mediated drug resistance (Kehrenberg et al., 2001).Increased antimicrobial resistance in P. multocida has also beenassociated with certain genes responsible for synthesis of altereddrug target (Kehrenberg and Schwarz, 2005) or mutation in 16SrRNA (Kehrenberg and Schwarz, 2007).

The present study indicated that PM-PCR is efficient tools forrapid diagnosis of Pasteurella organisms especially inepidemiological studies. HSB-PCR based diagnosis can be usefulfor early detection of the HS causing serogroup B isolates for rapidand sensible treatment of the disease. Results of antimicrobialsensitivity testing provide efficient and rational use of antibiotics forthe treatment of HS.

AcknowledgementsThe authors are thankful to the Vice Chancellor, Pt. DDU

Pashuchikitsa Vigyan Vishvavidhyalaya Evom Go AnusandhanSansthan, Mathura, UP, India. Thanks are also due to the Head,Division of Bacteriology and Mycology, IVRI, Izatnagar, UP, India.

ReferencesArora, A.K. et al. (2005) Indian J. Ani. Sci. 75: 749-752.Bauer, A.W. et al. (1966) Amer. J. Clin. Pathol. 45: 493-496.Biswas, A. et al. (2004) Vet. Res. Comm. 28: 287-298.Brickell, S.K. et al. (1998) Vet. Microbiol. 59: 295-307.Catry, B. et al. (2005) Microbial Drug Resis. 11: 387-94.Cowan, S.T. and Steel, K.J. (1993) In: Cowan and Steel’s Manual for the

Identification of Medical Bacteria. 3rd ed., (eds. G.I. Barrow andP.K.A. Feltham). University Press, Cambridge, UK.

De Alwis, M.C.L. (1999) Haemorrhagic Septicaemia. Aus. Center for Int.Agr. Res. Canberra, Australia.

Dey, S. et al. (2007) Indian J. Ani. Sci. 77: 317-319.Dutta, T.K. et al. (2001) Indian J. Ani. Health. 40: 101-107.Dutta, T.K. et al. (2009) Indian J. Ani. Sc. 79: 1130-31.Dziva, F. et al. (2008) Vet. Microbiol. 128: 1-22.Gupta, V. et al. (1996) Indian J. Comp. Microbiol. Immunol. Inf. Dis. 17:

171-73.Kehrenberg, C. and Schwarz, S. (2005) Antimicrobial Agents Chemo.

49: 414-17.Kehrenberg, C. et al. (2001) Vet. Res. 32: 323-39.Kehrenberg. C. and Schwarz, S. (2007) Antimicrobial Agents Chemo.

51: 2244-46.Kumar, P. et al. (2009) Trop. Ani. Hlth. Prod. 41: 573-78.Owen, R. J. (1989) J. Med. Microbiol. 30: 89-99.Salmah, I. (2004) J. Ani. Vet. Adv. 3: 595-97.Sharma, P.C. et al. (2005) Haryana Vet. 44: 73-74.Shivachandra, S.B. et al. (2004) Trop. Ani. Hlth. Prod. 36:743-50.Singh, V.P. et al. (1996) Significance of HS in Asia: India. International

Workshop on diagnosis and control of HS. Bali, Indonesia,May 28-30.

Townsend, K.M. et al. (1998) J. Clin. Microbiol. 36: 1096-1100.Verma, S.C. et al. (2004) Indian J. Ani. Sci. 38: 14-19.

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IntroductionJohne’s disease (paratuberculosis) is a chronic intestinal

disease of cattle and ruminants caused by Mycobacterium aviumsub sp. paratuberculosis. The organism is acid fast slow growingfastidious bacterium requiring exogenous mycobactin for its growth.This organism has also been implicated to be associated withcases of Crohn’s disease, a multifactorial chronic inflammatorybowel disease (IBD) in humans. A recent survey estimated that21.6% of dairy herds in the United States are infected with M. a.paratuberculosis (Ott et al., 1999). Stabel et al. (1996) estimatedthat it causes about $1 billion loss per annum to the. A estimationof the annual losses due to paratuberculosis to dairy farmers areto the extent of US $ 100 per cow in moderately infected herds,rising to over US $ 200 in heavily infected herds (Kennedy andBenedictus, 2001). Genetic variation of MHC II DRB region is alsoresponsible for susceptibility and resistance of animal for thisdisease (Singh et al., 2012). Effective control or eradication ofJohne’s disease has been hindered by lack of specific diagnostictests to identify infected animals prior to onset of overt clinicalsigns of disease. At present definitive diagnosis requires isolationof M. a. paratuberculosis from faeces or tissues. The infectedanimals occasionally respond to mammalian tuberculin. At presentthere is no reliable therapy or vaccination available. Thechemotherapy also has its limitations due to non-availability ofsuitable and potent therapeutic agent(s). Like other mycobacteria,the paratuberculous bacilli are also susceptible to manyantimycobacterial agents in vitro but these agents do not proveeffective in curing the disease (Angus and Gilmour, 1977). Theexisting animal paratuberculous vaccines are not effectivelyprotective (Harris and Barletta, 2001). Field studies demonstratedthat only partial protection was induced after vaccination loweringthe number of clinically diseased cows amongst bacteriologicallyor histologically positive cows (Chiodini and Kruiniugen, 1984;Van Schaik et al., 1996; Okwumbua et al., 2010). The vaccinesinduce production of antibodies and induction of delayed typehypersensitivity (DTH), presenting a problem in differential

EUKARYOTIC EXPRESSION AND STUDIES ON MICE IMMUNIZATIONOF A DNA CONSTRUCT ENCODING 35-KDA PROTEIN FROM

MYCOBACTERIUM AVIUM PARATUBERCULOSIS

D. Kumar1 and P.P. GoswamiDivision of Veterinary Biotechnology

Indian Veterinary Research Institute, Izatnagar-243 122, Bareilly, Uttar Pradesh, India

ABSTRACT

Till date, no vaccine is available for Johne’s disease (JD), caused by Mycobacterium avium sub sp. paratuberculosis (MAP). In the presentstudy a 35-kDa encoding gene of Mycobacterium avium paratuberculosis, a potent T and B cell antigen, was amplified by polymerase chainreaction (PCR). The fragment obtained was cloned in vector pTZ57R T/a cloning vector. Sub cloning of the 35-kDa gene fragment in eukaryoticvector pVAX 1, generated a recombinant plasmid pVAX-35. Transfection of pVAX-35 DNA in BHK 21 cell line expressed the protein, whichgave a band of 35-kDa on SDS-PAGE. The protein could bind to polyclonal sera against E. coli expressed recombinant 35-kDa protein onwestern blot. Mice were inoculated with plamid pVAX-35 elicited the humoral immune response as detected by ELISA. Further in vivo cellmediated immunity test and challenge studies are in progress to test the potential of this construct as a DNA vaccine.

Key words: Mycobacterium avium paratuberculosis , 35-kDa protein, eukaryotic expression

diagnosis between vaccinated and naturally infected animals.Vaccine does not preclude subsequent infection. Further,mycobacterial FAP (A potential immunoadjuvant for DC-basedtumour vaccination) improves the DC-based immune responsevia the TLR4 signaling pathway (Noh et al., 2012).

Eukaryotic expression and especially the DNA vaccination ofT cell antigens may open new horizons for effective vaccinationagainst paratuberculosis initiating potent cell-mediated immuneresponses by inducing CTL and Th-1 type cytokines. Byestablishing CD4+ T cell response characterized by gammainterferon (IFN-g) secretion and CD8+Cytotoxic T lymphocyte (CTL)response, DNA vaccination may be suited to achieve such a shiftbecause of an immunostimulatory effect of plasmid DNA itself. Inour laboratory a gene from M. a. paratuberculosis, T cell specific35-kDa encoding gene has been characterized and expressed inE. coli (Basagaudanavar et al., 2004). The studies carried out sofar strongly suggest that this antigen closely resembles those ofM. leprae and M. avium in its ability to induce T-cell response(Tricass et al., 1996, 1998). In the present study we described thetransfection of the pVAX-35 DNA in BHK 21 cell line, and preliminarycharacterization of the immune response elicited in mice.

Materials and MethodsBacterial strains and plasmid

The M. a. paratuberculosis strain 316 F was obtained fromcentral Diegenees Kuding Tieh institute Leystad, Netherlands,and is maintained at Biological Products Division, IVRI, IzatnagarIndia. It was grown and maintained in Middlebrook 7H10 agar(Difco Laboratories, Detroit, Mich., USA) enriched with 0.1%glycerol (v/v), 10%OADC (Difco, USA) and additionalsupplement of 2 mg/l mycobactin J (Allied monitor, Fayette.Missouri, USA).

For manipulation of plasmid host strain DH5a of E. coli byM/s Promega, USA, Expression vector pVAX 1 is from InvitrogenUSA. The vector contains a CMV promoter and a kanamycinresistant marker. pTZ57R vector was supplied in InsT/A clone ™

1Gene Expression of Virus Laboratory. Corresponding author E-mail address: [email protected]

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PCR product cloning kit by MBI Fermatas.

Preparation of bacterial DNABacteria were harvested by centrifugation, washed with PBS

and resuspended in TE buffer. The genomic DNA from M. a.paratuberculosis was isolated by the method of Portillo et al. (1991)with certain modifications. The bacterial colonies were scrappedform 2 months old Middlebrook 7H10 agar slants or 100 mg netweight was taken from liquid culture in 1.5 ml microfuge tube,washed thrice with 1X TE and resuspended in 500 ml of 1X TE.Lysozyme was added to the final concentration of 5 mg/ml andincubated at 37°C for 2 hrs. SDS and proteinase K were added toa final concentration of 1% and 250 µg/ml, respectively andincubated at 65ºC for 30 min. Further 80 µl of 5M NaCl was addedand vortexed. Then 64 µl of CTAB/NaCl solution was added andvortexed. The suspension was again incubated at 65ºC for 30min. DNA was extracted once with phenol, once with phenol:chloroform (1:1) and finally with chloroform: iso amyl alcohol (24:1).The DNA was washed with 80% ethanol, dried and redissolved in200 µl of 1 X TE. Finally RNAse (100 µg/ml) treatment for 1 hour at37ºC followed by phenol: chloroform extraction and ethanolprecipitation were carried out. The DNA pallet was washed with70% ethanol.

Oligonucleotide primersThe gene encoding a 35-kDa protein of M a. paratuberculosis

was amplified by a polymerase chain reaction. The 35-kDa genespecific primers have taken which were based on the nucleotidesequence information of 35-kDa protein of M. a. paratuberculosis(EMBL Accession No. AJ250887) (forward) 5’-CTC TCT AGA TCACTT GTA CTC ATC GAA CTG -3’ and (reverse) 5’-CGC GGA TCCGAT GAC GTC GGC TCA G-3’ 25 (Genei, India) was used forspecific amplification of the 35-kDa encoding gene. The primershad linkers at 5’ end for Bam HI and XbaI restriction endonucleasesites. For nested PCR the primer sequences were 5’-CCAGGCGAC GCC GGA-3’ (forward) and 5’-TTG TCG GAC GGG ATC-3’(reverse).

Standardization of PCR protocolAmplification reaction was performed in 50 ml reaction volume

each containing 2 ml (100 mg) of template DNA, 5 ml of Taq DNApolymerase buffer [10 mM Tris HCl (pH 9.0)], 50 nM KCl, 1.5 mMMgCl2 and 0.01 (w/v) gelatin, 10 ml of dNTP mixture containing200 mm of each dNTP, 1 ml (0.5 mm) each of forward and reverse35-kDa gene specific primer and 2.0 U of Taq DNA polymerase.The final volume was made with sterile distilled water. The reactionwas carried out in PTC-100 (MJ Research Inc, Waltham, USA), for30 cycles. PCR conditions were 94°C for 1 min, 52°C for 1 minand 70°C for 1 min. Size of amplified product was determined bysubmarine gel electrophoresis (Genei, Bangalore India)(Sambrook et al., 1989) on 1% agarose gel. The purification of theamplified gene was conducted by using QIAEX II gel extraction kit(Qiagen). About 50 ng of eluted 35-kDa gene PCR product and 25ng of pTZ57R vector (Fermantas) were ligated in a 10 ml ligationreaction with 1x ligase buffer (MBI Fermantas), 3 unit of T4 ligaseenzyme from the kit and 1 mM ATP, into two different tubes. Thereaction was carried out at 22°C for 18-20 h and subsequentlyused for transformation. The E. coli DH5a cells harbouring therecombinant plasmid were screened by plating on LB agar(Sambrook et al., 1989) containing Ampicilline (50 mg/ml). Plasmidisolation from Ampicilline resistant transformants was done by

miniprep plasmid isolation method and further confirmation ofclones done by restriction analysis of plasmid BamHI and XbaIand colony PCR (Sambrook et al., 1989). Marker pUC 18/Sau 3A I-pUC 18/TaqI digest was used as a standard molecular weightmarker for agarose gel electrophoresis (Genei). The 35-kDa genefrom pTZ-35 plasmid construct was sub cloned in BamHI andXbaI digested eukaryotic expression vector pVAX 1. For purposeof cloning about 2 mg of the PCR product was double digestedwith BamHI and XbaI, purified using QIAEX II gel extraction kit(Qiagen) and ligated (16 h at 22°C with 2 U of T4 DNA ligase, MBIFerments) to pVAX 1 predigested with BamHI and XbaI. Theresulting plasmid pVAX-35 was used to transform E. coli DH5acells. The E. coli DH5a cells harbouring the recombinant plasmidwere screened by plating on LB agar (Sambrook et al., 1989)containing Kanamycin (20 mg/ml). Plasmid isolation fromKanamycin resistant transformants was done by miniprep plasmidisolation method and further confirmation of clones done byrestriction analysis of plasmid BamHI and XbaI (Sambrook et al.,1989). 100 bp ladders, was used as a standard molecular weightmarker (Fermantas) for agarose gel electrophoresis.

One dimensional gel electrophoresisProteins were electrophoretiacally separated by sodium

dodicyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)under denaturing conditions. The 12.5% separating and 4%stacking polyacrylamide gel was used. The sample was boiled inan equal volume of 2X Laemmli buffer. After loading of the gelelectrophoresis was carried out with Tris-Glycine electrode buffer(tris base, 25 mM/L; glycine 250 mM/L; SDS 0.1%; sterilize byautoclaving) at 150 volt for 2 h. A molecular weight range from 14-97 kDa was used as molecular weight marker. The gel was stainedwith Comassie brilliant blue R250.

Western blot analysisAfter electrophoresis, proteins were transferred on to

nitrocellulose membrane (0.45nm) at 0.8 mA/Cm2 using semidryblotting, blocking was done in 5% milk powder in PBS for 2 hr atroom temperature. Washing of nitrocellulose membrane wascarried out 3 times for 5 minuets each with PBS Tween-20. Themembrane was incubated for 2 hrs at 37ºC with rabbit anti pPRO-35 (1:2000 in PBS). The membranes were then incubated with1:500 dilution of HRP labelled goat anti rabbit IgG (Genei) for 1 hrVisualization was achieved by adding 0.02% diaminobenzidineand 0.03% hydrogen peroxide in PBS, pH 7.4 for a min.

Transfection of eukaryotic cellsThe column purified recombinant plasmid pVAX-35 was

transfected to 60-70% confluent BHK 21 cells using SuperfectRtransfection reagents kit, following manufacturer’s instructions.Briefly BHK 21 cell monolayer was sub-cultured and the cellswere seeded in 25cm2 tissue culture flask. When 50-60%monolayer was achieved, the cells were used for transfection.5µg of DNA in 20 µl TE (pH 7.5) was diluted with cell growth mediumcontaining the serum proteins, or antibiotic to a total volume of 150µl. Then 30 µl of SuperfectR transfection reagent was added toDNA solution. Afterwards growth medium was aspirated from thedish and cells were washed with 2 ml DMEM (without serum andwithout antibiotic). Then 0.8 ml DMEM (without serum andwithout antibiotic) was added to the reaction tube containingthe transfection complexes and mixed properly. The complexeswere layered over onto the cells and after 6 h incubation at

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37ºC, DMEM with 10% FCS was added to the cells and wasincubated overnight. Next day the medium was replaced with freshgrowth medium and incubated further for 48 hours at 37ºC.

Inoculation of animals with pVAX-35Swiss albino male mice supplied by Laboratory animal

section were maintained on ration comprising wheat dalia 62%,maize 30%, wheat bran 7%, salt 1% and mineral mixture 25 ppmwith 5 ml milk per mice. The animals were divided in to sevengroups (3 mice in each group) five immunized groups and twocontrol groups. The QIAGEN midi kit purified recombinant plasmidpVAX-35 was used to immunize the mice. Mice were inoculatedbetween 8 and 12 weeks of age with 50 µg of plasmid byintramuscular injection into tibialis anterior muscle of each hindleg. Control mice were immunized with PBS.

Mice of Group I, II and control I were immunized at day 0, 14,28 mice of group III, IV, V, and control-II were immunized at day 0,21, 28 and blood was collected at day 28 and 35 from retro orbitalroute.

Antibodies determinationThe optimum concentration of antigen and conjugate were

determined using a block titration as previously described by Enzvalland Pearlman (1971). Mice were bled, serum was collected andthe presence of antibodies specific to 35-kDa protein was detected.The serum was diluted in 0.05 m coating buffer (pH 9.6) and 100µl was added to each well in ELISA plate (Nune). ELISA plate(Greines Geonay) well coated with 100 ml of 1:2 dilution and 1:4dilution of antigen in carbonate bi-carbonate buffer and the platewas incubated at 4ºC overnight. The plate was washed thrice withPBS-Tween 20 (PBS-T) and blocked with 2% BSA for 2 h at 37ºC.Then 1:50 and 1:100 dilution of serum in 50 µl volume were addedin duplicate and incubated at 37ºC for one hour. The plate waswashed thrice with PBS-T with 2 minutes at each wash. Conjugateanti-mouse IgG HRPO (Genei) with the dilution of 1: 1000 in 50 µlvolume was added in each well and plate was incubated for 1hour at 37ºC. The plate was washed 3 times with PBS-T and colorwas developed with 10 ml of 1 mg/ml OPD with 10 ml of 30% H2O2in substrate buffer. After 10-15 min adding 1N H2SO4 stoppedenzyme activity and the plates were read at 490 nm.

Results and DiscussionAlthough good management practices to control JD will lead

to a reduce incidence, eradication is dependent on availability ofeffective vaccination procedure (Sweeny et al., 1996; Harris andBarletta, 2001). Protective antigens for paratuberculosis are illdefined at present because vaccination methods and specificadjuvants for the generation of strong cellular immunity are notreadily available. One vaccination strategy is immunization withDNA plasmid in eukaryotic vector encoding microbial gene. Thisapproach has successful application in respect to viral, bacterialand protozoan infections in animals (Lai et al., 1995; Tascon et al.,1996).

A gene coding for a 35-kDa protein of M. a. paratuberculosishomologous to T-cell specific 35-kDa protein of M. leprae and M.avium (Tricass et al., 1998) was identified and sequenced(Banasure et al., 2001). The 35-kDa gene was expressed in E.coli expression system, which stimulates strong T cell response(Basagaudanavar et al., 2004). The 35-kDa protein is animmunodominant antigen in humans, which, respond to M. leprae(Tricass et al, 1996) and is also recognized during murine infectionwith M. avium (Tricass et al., 1998). The aim of present study was

to construct a eukaryotic expression system encoding 35-kDaantigen of M. paratuberculosis. Attempt was also made to studythe immunoreactivity of the expressed protein and also to evaluatethe antibody and cell mediated immune response induced byimmunizing mice with eukaryotic plasmid construct. The specificprimers were designed from the available sequence of 35-kDagene of M. a. paratuberculosis and gene was successfully amplifiedthrough polymerase chain reaction. Various eukaryotic systemswere used for expressing mycobacterial genes (Faircloth et al.,1999; Martin et al., 2000; Fonseca et al., 2001). A PCR product of943 bp was obtained. Nested PCR with internal primers generateda product of 486 bp length as analyzed on 1% agarose gel (Fig. 1).Restriction digestion of recombinant plasmid pTZ57R-35 withBamHI and XbaI released an identical sized fragment on 1%agarose gel, which was also confirmed by nested PCR (Fig. 2).Restriction digestion of recombinant plasmid pVAX-35 with BamHIand XbaI released a band corresponding to 943 bp on a 1%agarose gel (Fig. 3).

Expression of MPT64, Ag85B and ESAT6 of M. tuberculosishas been achieved in the eukaryotic expression vector pJW4303,which possesses cytomegalovirus early immediate promoter(Kamath et al., 1999). Expression of the 35-kDa gene of M. avium(Martin et al., 2000) was reported in pJW4303 vector. The expressionof 38-kDa protein of M. tuberculosis has similarly been reported inpCDNA3 and VR1012 vectors under the control of cytomegaloviruspromoter. The 35-kDa gene was cloned in eukaryotic expressionvector pVAX 1. The resultant plasmid was designated as pVAX-35.The positive clone was confirmed by the release of identical sizefragment on restriction endonuclease analysis. Eukaryoticexpression driven by the cytomegalovirus (CMV) immediate earlypromoter in pVAX 1 was also used for the expression of 35-kDagene. Eukaryotic vector pVAX 1 was contemplated in the presentstudy because; the eukaryotic DNA sequence is limited only tothose regions marked for expression in order to minimize thepossibility of chromosomal integration.

A wide variety of cell lines have been used for transfection ofmycobacterial antigens. CHO-7 cells were used for transfectionwith DNA for expressing 35-kDa gene of M. avium (Martin et al.,2000) Murine 3T3 fibroblast cell line was used for expressing 65-kDa, 85b antigen of M. avium and BCG 85 antigen of M. bovis(Faircloth et al., 1999). Further ESAT-6, Mpt-64, KatG, HBHA of M.tuberculosis antigens were expressed in rhabdomyosarcoma cells(Li et al.,1999). This cell line however required higher glucosecontent. Transient transfection of 38-kDa antigen of M. tuberculosiswas reported using Ex-38 (H-2b), a thyoma cell line (Fonseca etal., 2001). The purified recombinant plasmid DNA was used totransfect BHK 21 cells. Transfected BHK 21 cell expressedrecombinant 35-kDa protein that co migrated along with E. coliexpressed purified 35-kDa on SDS-PAGE. To ensure that the pVAX-35 construct was functional we analyzed expression in vitro bytransient transfection of BHK 21 cells. A predominant band of size35-kDa was observed in 48 hr post transfected BHK 21 cell lysatecomparable to E. coli expressed recombinant 35-kDa protein. Nosuch band was observed in mock-transfected BHK 21 cells. Theeukariotically expressed 35-kDa protein also reacted with polyclonalantisera raised against E. coli expressed 35-kDa protein onwestern blot. Available polyclonal serum raised against E. coliexpressed 35-kDa protein could bind to pVAX-35 transfected BHK21 cell lysate as well as purified E. coli expressed 35-kDa protein,as detected in western blot analysis. No such band was observed

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Fig. 1: An ethidium bromide-stained agarose gel of PCR and NestedPCR of 35-kDa gene of Mycobacterium avium paratuberculosis.Lane M, Marker pUC 18/Sau 3A I-pUC 18/TaqI digest; lane 1, PCRamplified 35-kDa gene product; lane 2 represents Nested PCRproduct.

Fig. 2: An ethidium bromide stained agarose gel showing Cloning of 35-kDagene in pTZ57R T/A cloning vector; Lane M, 100 bp DNA ladder, lane 1Colony PCR of pTZ-35 cell lysate; lane 2, PCR amplified 35-kDa geneproduct; lane 3, represents Bam HI and XbaI released 35-kDa gene insertand linear pTZ57R vector of 2.8 kb.

Fig. 3: Agarose gel showing sub cloning of 35-kDa gene in pVAX1eukaryotic expression vector. Lane M, 100 bp DNA ladder; lane 1, PCRamplified 35-kDa gene product; Lane 2, Bam HI and XbaI released 35-kDagene insert and linear pVAX1 vector around 3.0 kb size.

Fig. 4: SDS-PAGE and Western Blot analysis of 48 hr post incubatationculture of BHK 21cell transfected wtih pVAX-35 plasmid construct. LaneA1, SDS-PAGE of Whole cell extract of BHK 21cells; lane A2, SDS-PAGEof Whole cell extract of transfencted BHK 21cells with pVAX-35 plasmidconstruct; lane A3, SDS-PAGE of E. coli purified recombinant 35-kDaprotein; Lane B1, Western blot of purified recombinant 35-kDa protein; laneB2, Western blot of BHK 21 cell transfected with pVAX-35 plasmidconstruct; lane B3, Western blot of mock transfected BHK 21 cell.

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in mock transfected BHK 21 cell (Fig. 4).DNA vaccination with promising results has been reported

using the hsp-65 (Tascon et al., 1996), Ag85A (Huygen et al., 1996),Ag85B (Kamath et al., 1999) and 38-kDa antigens. DNA vaccinationmay open new horizons for effective vaccination againstparatuberculosis because strong cell-mediated immuneresponses, including CTL and Th-1 type cytokine can be induced.Several proteins of Mycobacterium tuberculosis have beenevaluated as DNA vaccines in experimental models. Theseinclude the 65 kDa heat shock protein (hsp65), the 36 kDa proline-rich antigen (Tascon et al., 1996), Ag 85A (Huygen et al., 1996) Ag85B and Ag 85C (Lozes et al., 1997), the 38 kDa protein, hsp 70, 36and 6 kDa proteins (Lowrie et al., 1997), MPT64 (23-kDa proteins),Ag 85B (30-kDa protein) and ESAT-6 (6-kDa) protein (Kamath etal., 1999), and the phosphate binding proteins PstS-1, PstS-2 andPst-3 (Tanghe et al., 1999; Fonseca et al., 2001). The pVAX-35plasmid construct produced marked immune response in micemodel. The native 35-kDa protein formed multimers expressionconformational determinants, which stimulated strong IgGantibody responses (Tricass et al., 1998). Such an apparentadvantage does not seem possible with E. coli expressed protein.The advantage of DNA immunization of pVAX-35-kDa constructover E. coli expressed 35-kDa protein (Basagaudanavar et al.,2004) as a recombinant subunit vaccine is that in DNA immunizationprocedure the protein is expressed in side the host body system,which fabricate the antigen with its own post transcriptionalmachinery. Thus the protein produced will be in the rightconformation and is able to induce CTL (Cytotoxic T lymphocyteresponse), which is essential to confer protection for an intracellularpathogen like M. a. paratuberculosis.

Martin et al. (2000) also used the intra muscular route toimmunize mice with eukaryotic construct of 35-kDa protein of M.avium. Challenge studies with pathogenic mycobacteria strainare further required to establish the protection ability of pVAX-35construct. Polyclonal serum obtained 28 and 35 days postimmunization in mice with plasmid pVAX35 could react with E. coliexpressed 35-kDa protein but no reaction occurred with eithercoating buffer or dot, which were developed with sera of controlgroup. Expression of 35-kDa protein in mice was confirmed bydetecting specific antibodies by indirect ELISA which showed morethan three times OD 490 than the control serum and blank (Figure5A and 5B).

The ELISA was used to determine plasmid DNA inducedantibodies in mice against various mycobacterial antigens. Amongthem the DNA expressing 22-kDa cell surface antigen of M. bovis(Lef’evre et al., 2000) MPT64, Ag85B and KatG antigens (Kamathet al., 1999) and Ag85A of M. tuberculosis (D’Souza et al., 2002)

and also 35-kDa antigen of M. avium (Martin et al., 2000). Variabilityin the antibody titers in above study however observed amongstthe different breed of mice used. Mice immunized with pVAX-35DNA eliciting increased titre of antibodies as detected by plateELISA and dot ELISA. The eukaryotic expression and ability to elicithumoral and cell mediated immune response, of pVAX-35 hasprovided an opportunity for its further characterization. Further invivo cell mediated immunity test and challenge studies are neededto establish the potential of pVAX-35 as a candidate DNA vaccine.

AcknowledgementsThe author is grateful to the Director, IVRI, Izatnagar, for

providing necessary facilities and to Dr P.K. Gupta, Senior Scientist,for providing cell culture facilities.

ReferencesAngus, K. W. and Gilmour, N.J.L. (1977) J. Compa. Pathol. 81: 227-234.Banasure, K.D. et al. (2001) FEMS Microbiol. Lett. 196: 195-199.Basagaudanavar, S.H. et al. (2004) Vet. Res. Commun. 28: 209-214.Bjerrum, O.J. and Schafter-Nielson, C. (1986) Buffer systems and transfer

parameters for semidry electro blotting with a horizontalapparatus. In: Dunn (Ed.) Electrophoresis VCH, Weinheim,Germany, 86: 315-327.

Chiodini, R. J. and Kruiningen, H.J. (1984) Cornell Vet. 74: 218-262.D’ Souza, S. et al. (2002) Infect. Immun. 70: 3681-3368.Enzwal, E. and Pearlman, P. (1971) Immunochem. 8: 871-874.Faircloth, M. et al. (1999) Infect. Immun. 67: 4243-4250.Fonseca, D. P. A. J. et al. (2001) Infect. Immun. 69: 4839-4845.Harris, N. B. and Barletta, R. G. (2001) Clin. Micro. Rev. 14(3): 489-512.Huygen, K. et al. (1996) Nat. Med. 2: 893-898.Kamath, A.T. et al. (1999) 67: 1702-1707.Kennedy, D. J. and Benedictus, G. (2001) Rev. Sci.Tech.O. I. E. 20: 151-

179.Lai, W.C. et al. (1995) Cell Biol. 14: 643-648.Lef’evre, P. et al. (2002) Infect. Immun. 68: 1040-1047.Li Z., et al. (1999) Infect. Immun. 67: 4780-4786.Lowrie, D. B. et al. (1997) Vaccine. 15: 834-838.Lozes, E. et al. (1997) Vaccine. 15: 830-833.Martin, E. et al. (2000) Infect. Immun. 68: 3090-3096.Okwumabua, O. et al. (2010) J. Vet. Diagn. Invest. 22(1): 67-69.Noh, K.T. et al. (2012) Exp. Mol. Med. (In press).Ott, S. L. et al. (1999) Prevent. Vet. Med. 40: 179-192.Portillo, P.D. et al. (1991) Plasmid. 39: 100-113.Sambrook, J. et al. (1989) Molecular cloning: A laboratory manual. 2nd ed.

Cold Spring Harbor Laboratory Press, Cold spring Harbor, NY.Singh, P.K. et al. (2012) Int. J. Immunogenet. (In press).Stabel, J. R. (1996) J. Vet. Diagnos. Invest. 8: 345-350.Sweeney, R. W. et al. (1996) Vet. Clinics of North Am. Food Ani. Pract.

12: 305-312.Tascon, R.E. et al. (1996) Nat. Med. 2: 888-892.Triccas, J.A. et al. (1996) Infect. Immun. 64: 5171-5177.Triccas, J.A. et al. (1998) Infect. Immun. 66: 2684-2690.Van Schaik, et al. (1996) Vet. Rec. 139: 624-627.

Fig. 5A, B: Change in 35-kDa specific polyclonal antibodies in serum of mice. An ELISA bar diagram. Serum was diluted to 1: 50 and1: 100and time interval is plotted against optical density.

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IntroductionPseudomonas aeruginosa is a highly versatile ubiquitous

organism which is present almost everywhere in theenvironment. It is an increasingly prevalent opportunisticpathogen found in nosocomial and community acquiredinfections which can infect almost any external site or organ. P.aeruginosa is comparatively more resistant to most ofantibiotics and disinfectants. Only a few antibiotics i.e.aminoglycosides, fluoroquinolones, -lactams, polymyxin-B,collistin etc. are effective against P. aeruginosa, which areknown as antipseudomonal drugs (Bellido and Hancock,1993). W ide variations have been reported in antibioticresistance pattern among P. aeruginosa isolates obtained fromdifferent environments. Environmental isolates were found tobe highly susceptible to the most of the antibiotics tested byRuiz et al. (2004). On contrary, Gad et al. (2007) reportedenvironmental P.aeruginosa isolates to be more resistant tothe antibiotics tested. P. aeruginosa isolates from wound swaband pus were found relatively more resistant than those fromother specimens by Rashid et al. (2007). Further, the isolateswith mucoid phenotype were found to be more susceptible tothe antibiotics than the non-mucoid phenotype by many workers(Shawar et al., 1999 and Srifuengfung et al., 2004) but Eftekharet al. (2003) reported no significant difference in antibioticsensitivity between mucoid and non-mucoid strains in vitro.

The objective of the current study was to determineantibiotic resistance patterns among P. aeruginosa isolatesfrom different environments and observe whether any differenceoccurs among the three groups of the isolates regardingantibiotic resistance.

Materials and Methods A total 296 sample were collected from veterinary college

clinical complex, various animal farms, human medical centres

ANTIBIOTIC RESISTANCE AMONG PSEUDOMONAS AERUGINOSAISOLATES OBTAINED FROM DIFFERENT ENVIRONMENTS#

D.G. Gupta1, S. Maherchandani2, A.Vijay3 and S.K. Kashyap4

College of Veterinary and Animal ScienceRajasthan University of Veterinary and Animal Sciences, Bikaner-334001, Rajasthan, India

ABSTRACT

Antibiogram of Pseudomonas aeruginosa isolates from different sources i.e. clinical samples, samples from apparently healthyanimals and from soil and water were observed in the present study. Out of total 296 samples collected from clinical, non-clinical andenvironmental sources, 43 isolates could be typed as P. aeruginosa. The confirmed 43 P. aeruginosa isolates were subjected toresistotyping with 13 antibiotics of different groups. Highest resistance was observed to chloramphenicol (resistance-53.49%) andcephotaxime (resistance-51.16%) followed by colistin (resistance-20.93%), ticarcillin and ceftazidime (resistance-18.60%) whileless resistance was observed to amikacin (resistance-9.30%), imepenem (resistance-6.98%), gentamicin, tobramicin and ofloxacin(resistance-4.65%), ciprofloxacin and carbanicillin (resistance-2.32%). All the P. aeruginosa isolates of three groups were abso-lutely sensitive to polymyxin (resistance-0%). Resistance pattern of environmental isolates was similar to that of other two groupsfor most of the antibiotics except for colistin and cephotaxime to which environmental isolates were comparatively more sensitive.

Key words: Antibiogram, ribotyping, resistotyping, antibiotic abuse, indiscriminate

and environment. These were divided into three groups forconvenience of the study. The first group comprised of 129clinical samples collected from various clinical sources(mastitis milk samples, pus samples, post-operative surgicalwounds in animals and burn samples from human burnpatients). In the second group, total 99 non-clinical samples(faecal and nasal swabs) were collected from apparentlyhealthy animals. In group three, a total of 68 environmental(soil and water) samples were collected from environment.Isolation and identification was done using standard methods(Cowan and Steel, 1974). Then the isolates were subjectedfor 16S rRNA based genotypic confirmation by using speciesspecific primers taken from previous studies (Spilker et al.,2004).

The confirmed P. aeruginosa isolates, were subjected toresistotyping using 13 antibiotics of different groups by discdiffusion method of Bauer et al. (1966).

Results and DiscussionA total 43 P. aeruginosa isolates i.e. 13 isolates from

clinical origin, 18 from non-clinical origin and 12 isolates fromenvironmental origin were subjected to resistotyping with 13different antibiotics (Table 1) and response of isolates wasinterpreted as sensitive, intermediate and resistant. Theirpatterns of resistance have been shown in Table 1.

The highest resistance was observed for chloramphenicol(resistance 53.49%). In other similar studies comapartivelylower resistance was reported among P. aeruginosa isolatesto chloramphenicol (98.3% Kohanteb et al., 2007; 88% Gadet al., 2007 and 84.31% Brown and Izundu, 2004). Theisolates of clinical origin showed more resistance tocephotaxime (resistance 51.16%) than the isolates from non-clinical and environmental sources (Table 1). Our finding arein agreement with Ndip et al. (2005) who reported 96.1 per

#1Part of M.V.Sc. Thesis of First Author and Present address: Assistant Professor, Dept. of VMC, MGVC, NH-11, Agra Road, Bharatpur, Rajasthan.2Associate Professor, Dept. of Microbiology and Biotechnology, CVAS, RAJUVAS, Bikaner.3Research Associate, CSWRI, Avikanagar, Tonk, Rajasthan.4Associate Professor and Head, Dept. of Microbiology and Biotechnology, CVAS, RAJUVAS, Bikaner.

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Table 1: Sensitivity patterns of different type of isolates to various antibiotics tested

Sensitive Interm ediat e Resistan ce S . No.

Name of Antibiotic Clinical Non

ClinicalEnv iron mental

T otal (%)

Clinical Non Clinical

Env ironmental

T otal (%)

Clinical Non Clinical

Environmental

T otal (%)

1. Amikacin (Ak) 10 13 8 31 (72.09% ) 3 3 2 8

(18.60% ) 0 2 2 4 (9.30% )

2. Chloramphenicol (C) 3 3 0 6

(13.95% ) 5 3 6 14 (32.56% ) 5 12 6 23

(53.49% ) 3. Ceftazidime

(CA) 7 4 4 15 (34.88% ) 4 11 5 20

(46.51% ) 2 3 3 8 (18.60% )

4. Carbanicillin (CB) 12 18 11 41

(95.35% ) 0 0 1 1 (2.32% ) 1 0 0 1

(2.32% ) 5. Cephotaxime

(CE) 0 2 0 2 (4.65% ) 2 10 7 19

(44.18% ) 11 6 5 22 (51.16% )

6. Ciprofloxacin (CF) 11 14 11 36

(83.72% ) 2 3 1 6 (13.95% ) 0 1 0 1

(2.32% ) 7. Colistin (CL) 8 16 10 34

(79.07% ) 0 0 0 0 (0% ) 5 2 2 9 (20.93% )

8. Gent amicin (G) 12 13 10 35

(81.39% ) 0 4 2 6 (13.95% ) 1 1 0 2

(4.65% ) 9. Imepenem (I) 12 17 11 40

(93.02% ) 0 0 0 0 (0% ) 1 1 1 3

(6.98% ) 10. O floxacin (OF) 11 17 11 39

(90.70% ) 1 1 0 2 (4.65% ) 1 0 1 2

(4.65% ) 11. Polym yxin-B

(PB) 13 18 12 43 (100% ) 0 0 0 0

(0% ) 0 0 0 0 (0% )

12. T obramycin (T B) 12 16 11 39

(90.70% ) 0 1 1 2 (4.65% ) 1 1 0 2

(4.65% ) 13. T icarcillin (T I) 12 13 10 35

(81.39% ) 0 0 0 0 (0% ) 1 5 2 8

(18.60% )

cent resistance among P. aeruginosa isolates againstcephotaxime with isolates from clinical origin showing higherresistance than environmental and non-clinical origin.

For colistin, overall 20.93 per cent P. aeruginosa isolateswere found to be resistant with clinical isolates showingcomparatively higher resistance (Table 1). In earlier studies,Eftekhar et al. (2003) reported absolute sensitivity (resistance-0%) to colistin. 18.6 per cent of the P. aeruginosa isolatesshowed resistance to ticarcillin and ceftazidime. Brown andIzundu, (2004) also reported almost similar resistance (19%)to ceftazidime among P.aeruginosa isolates.

Resistance rate to other antibiotics was 9.3%-amikacin,6.98%-imepenem and tobramycin, 4.65%-gentamicin andofloxacin and 2.12%.-carbanicillin and ciprofloxacin (Table 1).All the isolates of three groups were sensitive to polymyxin(resistance-0%). Earlier Eldere (2003) reported 23.5%, 19.5%,8% and 0.5% resistance to gentamicin, tobramycin, amikacinand ciprofloxacin respectively. Brown and Izundu (2004)reported 21.57%, 25.5%, 0% 19.61% and 9.8% resistance togentamicin, tobramycin, amikacin, ciprofloxacin andimepenem, respectively. Kohanteb et al. (2007) reported68.3%, 65%, 63.3%, 53.3%, 38.3% and 93.3% resistance togentamicin, tobramycin, amikacin, ciprofloxacin, imepenemand carbanicillin respectively. Not much difference could beobserved among the three groups of the isolates regardingthe resistance patterns except for colistin and cephotaxime towhich comparatively less resistance was observed amongenvironmental isolates. This increasing trend of resistance inisolates from environment is indicative of antibiotic abuse whichneeds to be sorted out by preventing the indiscriminate use ofthe antibiotics. Resistance pattern of environmental isolates

was similar to that of other two groups for most of the antibioticsexcept for colistin and cephotaxime to which environmentalisolates were comparatively more sensitive.

AcknowledgementsThe authors are thankful to department of veterinary

microbiology and biotechnology for providing the facilities toconduct this work.

ReferencesBauer, A.W. et al. (1966) Am. J. Clin. Pathol. 45(4): 493-496.Bellido, F. and Hancock, R.E.W. (1993) Susceptibility and resistance

of Pseudomonas aeruginosa to antimicrobial agents. In:Campa, M., Bendinelli, M. and Friedman, H. (Eds.),Pseudomonas aeruginosa as an opportunistic infection.Plenum Press, New York. pp. 321-348.

Brown, P.D. and Izundu, A. (2004) Rev. Panam. Salud. Publica. 16(2):125-130.

Cowan, S.T. and Steel, K. J. (1974) In: Cowan and Steel’s manual forthe identification of medical bacteria. 2nd ed. CambridgeUniversity Press, Cambridge, New York. pp: 90-93.

Eldere, J. V. (2003) J. Antimicrob. Chemother. 51: 347-352.Eftekhar, F. et al. (2003) Iran. J. Biotechnol. 1(2): 95-100.Gad, G.F. et al. (2007) J. Antimicrob. Chemother. 60: 1010-1017.Kohanteb, J. et al. (2007) Pakistan. J. Biol. Sci. 10(11): 1817-1822.Ndip, R.N. et al. (2005) Trop. Med. Int. Health. 10(1): 74-81.Rashid, A. et al. (2007) Bangladesh J. Med. Microbiol. 1(2): 48-51.Ruiz, L. et al. (2004) Arch. Med. Res. 35: 251-257.Shawar, R. et al. (1999) Antimicrob. Agents. Chemother. 43(12):

2877-2880.Spilker, T. et al. (2004) J. Clin. Microbiol. 42(5): 2074-2079.Srifuengfung, S. et al. (2004) Southeast Asian J. Trop. Med. Public

Health. 35(4): 893-896.

195


IntroductionPresent demand for feed and fodder is much higher than

their supply for the Indian livestock. The annual requirement issupposed to be over 25 million tones of concentrates, 308million tones of green fodder and 353 million tones of dryfodder. Looking into the requirement the supply is only littleover 16 million tones of concentrate, 261 million tones of greenfodder and 308 million tones of dry roughages. As theconventional feed resources are quite limited and there is awide gap between the demand and supply of animal feeds inour country. Since the concentrates are in direct competitionwith human and industrial utilization and even if available arecostlier. Energy can be supplied through concentrates likemaize and barley. The major factor that limits animal productionfrom these feed resources is nutritional imbalance (Misra etal., 1999). They are low in nitrogen and high in fibre and lignin,characteristics that restrict intake and digestibility in animals.Improvement in the nutritional value of straws and their voluntaryintake following ammoniation makes it a method of choice fornutritional enrichment of wheat straw (Pachauri et al., 2010).Urea ammonization has been found to be most promisingand practical chemical method for improving the nutritive valueof crop residues (Yadav and Yadav 1989; Devendra 1997;Chenost and Kayouli 1997; Singh and Prasad 2002; Hanafi etal., 2012) for ruminants. It increases the palatability, digestibilityand adds a significant amount of nitrogen into the straw Vermaet al. (1995). Several studies (Misra et al., 1999; Verma et al.,1995) have reported significant increase in live weight gain ofanimals fed urea treated straw. The objective of the presentstudy was, therefore, to assess the effect of feeding urea-ammoniated barley straw (UABS) and urea-ammoniated wheatstraw (UAWS) and crushed barley grain on growth rate incrossbred calves reared under stall-fed.

STUDY OF COMPARATIVE EFFECT OF UREA AMMONIATEDSTRAW FEEDING ON GROWTH OF CROSSBRED CALVES#

Rijusmita Sarma Deka1, I.A. Siddique2 and Devendra S. Manohar3

Department of Animal NutritionCollege of Veterinary Science and Animal Husbandry

Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go Anusandhan Sansthan,Mathura-281001, Uttar Pradesh, India

ABSTRACT

A study was conducted to assess the effect of ad lib feeding urea ammoniated straw (UAS) on growth rate, feed intake and nutrientutilization in Holstein Friesian X Hariana crossbred bull calves reared under stall-fed. The study was completed in two phases. In thefirst phase the calves were fed urea ammoniated barley straw (UAS) and in the second phase the same calves were fed crushedbarley grain over and above the urea ammoniated straw (UAS). In both the phases the animals were maintained under stall-fedcondition for a period of 60 days under each phase of experiment. Same calves were continued during second phase and they werefed urea ammoniated straw ad lib supplemented with barley @ 1 kg per 100 kg body weight. The data collected under the said trialdemonstrated that addition of barley in 4% urea ammoniated cereal straw based ration of growing calves have a positive effect ongrowth rate as well as digestibility coefficient of all the organic nutrients except crude fibre.

Key words: Ammoniated straw, crossbred calves, growth performance and nutrient utilization

Materials and MethodsThe study was conducted at College of Veterinary Science

and Animal husbandry, Mathura, situated at 178 m above MSLon 27°24' N latitude and 77°5' E longitude in the South westSemi arid zone having an annual rainfall of 202 mm. The regionis having the deepest soil in India and constitutes the wheatand the rice bowls of India. Wheat and rice being the maincultivated crops, their straws form the basal diet of ruminantsin the area and are fed throughout the year. During the studyperiod, the mean minimum ambient temperature ranged from16°C to 26°C and maximum temperature ranged from 38°C to43°C, with an average ambient temperature of 40.4°CThe totalrainfall received during the experiment was about 44.6 mm.

Ammoniation of wheat straw was done with four per centurea (w/w), 50 per cent moisture, 1% iodized common saltand 1% chalk. Weighed amount of wheat straw was treatedwith urea solution (40 kg in 500 litre water per 1000 kg straw).This treated straw was packed with black polythene sheet andwas sealed to develop anaerobic condition for twenty eightdays. The pit was opened after four weeks from one side totake out the straw. The average ambient temperature at thetime of treatment of straw was 40.4°C. Urea-ammoniated strawwas aerated for 24 hrs prior to feeding to facilitate the escapeof free ammonia. The treated straw was having 33.2%moisture.

The experiment was conducted on four male crossbredcalves (Holstein Friesian x Haryana) of about 12-14 months ofage with an average body weight of 90.25 kg. The study wasconducted in two phases. In the first phase the calves were fedammoniated straw and in the second phase calves were fedcrushed barley grain @ 1 kg barley per 100 kg body weightand ammoniated straw. All the calves were provided cleandrinking water twice a day. The calves were weighed at

1#Part of M.V.Sc. thesis of first Author. Present Address .Ph.D. Scholar, DCN Division, NDRI, Karnal, Haryana.2Retd. Professor and Head, DUVASU, Mathura, Uttar Pradesh3Assistant Professor, Apollo College of Veterinary Medicine, Jamdoli, Agra Road, Jaipur, Rajasthan.

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Table 1: Comparative plane of nutrition and rate of growth

Pla ne of nutrition Am m onia te d stra w fe e ding

Am m onia te d stra w supple m e nte d w ith ba rle y

Dry matter Intak e (DM I) kg 2.521 ±0.3 28 2.636 ±0.2 74

Total P rotein (TP) kg 0.157 ±0.0 20 0.182 ±0.0 19

Digestible Crud e Protein (D CP ) kg 0.035 ±0.0 04 0.077 ± 0.00 4

Total Dig estible N utrie nts (TD N) kg 1.098 ±0.1 37 1.528 ±0.1 23

Body weight gain/day (g ) 120.5 ±1 8.37 160.6 6± 19.3 4

fortnightly intervals for two consecutive days during the feedingtrial and at the beginning and end of the digestibility trial beforeoffering the feed and water.

The feed and faeces samples were analyzed for proximateprinciples (AOAC, 1990). Mineral extracts of feed, faeces andurine were prepared (AOAC, 1990) and analyzed for Ca and P(Talapatra et al., 1940). Statistical methods for calculating thedata were applied as described by Snedecor and Cochron(1989).

Results and DiscussionA feeding trial on growth was conducted done on cross

bred calves fed ammoniated straw and ammoniated strawsupplemented with barley grains. Performance of calves undertwo different schedules was noted. During the first phase ofexperiment calves were fed urea ammoniated straw as a solefeed while during second phase same calves were fed ureaammoniated straw supplemented with 1 kg barley per hundredkg body wt.

In the first phase of feeding ammoniated straw was fed,the initial body wt of experimental calves just at starting of theexperiment was recorded. The body weight ranged from 63 to110 with an average of 84.25±10.0945 kg. The average bodywt during the first 1st fortnight was 88.00 ± 8.869 kg. In 2nd, 3rd

and 4th fortnight the average body wt gain was 92.62 ± 9.049kg, 85.75 ± 9.944 and 91.5± 9.032 kg. The total gain in 60 dayswas recorded as 7.25± 1.108 kg wt gain per day ranged from83 to 166 with an average 120.5 ± 18.377 g. It may be seenfrom above table that average daily wt. gain was only choicefor source of nitrogen in ruminants remain synthetic urea whichis cheap. Such a low growth rate on this type of feeding hasalso been reported by (Ghebrehiwet et al.,1988). They showeda growth rate of 93 g/day for urea treated straw and they alsoreported a negative growth of 123 g/day of feeding untreatedstraw. Nurazzamal et al. (1981) also reported daily gain of 109g/animal on animals fed treated straw. Pachauri et al. (2010)reported better response in growth rate and digestibility ofnutrients on feeding urea ammoniated straw. Similar resultswere reported by Verma et al. (1995).

In the second phase of feeding the initial body weight ofthe calves ranged from 73 to 115 with an average of90.33±10.969.The total gain in body weight in 60 days rangedfrom 7 to11 kg with an average of 9.66±1.154 kg. The weightgain per day varied from 116 to 183 with an average of160.66±19.341. It may be seen from Table 1 that average dailyweight gain was 160 gms, it was observed that there wassome improvement in body weight gain by extra feeding ofbarley grain in comparison to sole feeding of urea treated strawduring the first phase of experiment . This type of body wt gain

was reported by (Ghebrehiwet et al., 1988). They supplementedurea treated straw with 500 g rice bran to each calf and foundgrowth rate of 185 g/day. The DM intake (DMI), total protein(TP), digestible crude protein (DCP) and total digestiblenutrients (TDN) were 2.521±0.328 kg, 0.157±0.02 kg,0.035±0.004 kg and 1.098±0.134 kg, respectively. The planeof nutrition during second phase of feeding were 2.636±0.274kg, 0.182±0.019 kg, 0.077± 0.004 kg and 1.528±0.123 kg perDMI, TP, DCP and TDN, respectively. The utilization of all thesenutrients was high in the 2nd phase of feeding where animalswere offered barley grain along with ammoniated straw. Thegrowth rate was however, increased to 160.66±19.34g/day.

In the first phase of feeding the body wt gain ofexperimental calves during 60 days of feeding ureaammoniated straw feeding was an average of 120±18.7g/day.The growth rate of animal was very low due to sole feedingof ammoniated straw. In the second phase of feeding ureaammoniated straw along with crushed barley grain feedinggrowth was at an average of 160±19.34 g/day when animalswere offered barley grains along with ammoniated wheat straw,it was observed that some amount of cereal grains like barleyis beneficial when added with ammoniated crop residue toget some growth in growing calves.

ReferencesAOAC. (1990) Official Methods of Analysis. 15th ed. Association of

Official analytical Chemist, Arlington, Virginia, USA.Chenost, M. and C. Kayouli. (1997) Roughage utilization in warm

climates. FAO animal production and health paper 135.Food and Agriculture Organization of the United Nations,Rome. pp. 226.

Devendra, C. (1997) Crop residues for feeding animals in Asia:Technology development and adoption in crop/livestocksystem. In: Crop residues in sustainable mixed crop/livestock farming systems. (C Renard editor). CABInternational, Oxen, UK. pp. 241-267.

Hanafi, E.M. et al. (2012) World Applied Sci. J. 16(3): 354-361.Ghebrehiwet, T. et al. (1988) Biological Wastes. 25:269-280.Mishra, A. K. et al. (1999) Indian J. Ani. Nutr. 16: 149-159.Nuzzaramal, Khan. et al. (1981) Effect of treating paddy straw with

ammonia (generated from urea) on performance of localand cross breed lactating cows. In maximum livestockproduction from Minimum land (eds. M.G.Jackson,F.Dolberg, C.H.Davis, M.Hague and M.Saadullah).Joyedebpur, Bangladesh, pp. 168-180.

Pachauri, S.K. et al. (2010) Indian J. Ani. Nutr. 27(1): 73-76.Snedecor, G. W. and Cochran, W.G. (1989) Statistical Methods. 8th ed.

Oxford and IBH Publication Co., Calcutta, India.Sundstol, F. et al. (1978) World Ani. Rev. 26:13-21.Talapatra, S. K. et al. (1940) Indian J. Vet. Sci. and Ani. Husb. 10: 243.Verma, D.N. et al. (1995) Indian J. of Ani. Nutr. 12(1): 25-30.Yadav, B.P. and Yadav, I.S. (1989) Indian J. Ani. Nutr. 6: 215-222.

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IntroductionTrypanosomosis, is one of the most widely distributed

pathogenic mechanically transmitted vector bornehaemoprotozoan, (Trypanosoma evansi) disease of domesticand wild animals in India. T. evansi is quiet prevalent in cattle,buffaloes and horses. Being a potential killer of livestockcauses huge economic losses to the farmers in terms ofmorbidity, mortality, abortion, infertility, reduced milk yield,indiscriminate use of trypanocides and also by interferencewith vaccination programmes of domestic animals. Commonlyused drugs against trypanosomosis in domestic animals inIndia include diminazene aceturate (berenil) quinapyraminesulphate and chloride (Triquin, Antrycide Prosalt) andquinapyramine sulphate (Triquin-S and Antrycide) and recentone isometamidium, homidium for treatment and prophylacticuse. Isometamidium chloride (ISMM) (a phenanthridinearomatic amidine) has been widely used in tropical countriesfor the control of animal tryponosomiosis. Attempts have beenmade to establish the efficacy of the drug againsttrypanosomosis in goat (Braid and Eghianruwa, 1980; Kinaboand Mc Kellar,1990), camel (Ali and Hassan, 1984), dog(Kaggwa et al., 1988), pig (Waiswa, 2005) and cattle (Dowleret al., 1989). But lack of clinical efficacy trials in Indian Buffalohas limited its use as trypanocides in the country. Consideringthese facts, the present study was carried out with the objectivedetermine the plasma-protein binding activity of intravenousIsometamidium in experimentally induced trypanosomosisinfected Indian buffalo calves. In this study, the haematologicalprofile as well as biochemical profile was also evaluated in

EFFICACY OF ISOMETAMIDIUM CHLORIDE AGAINSTTRYPANOSOMA EVANSI INFECTION IN BUFFALO

Shweta Anand1, T.K. Mondal2 and Suprita Sinha3

Department of Pharmacology and ToxicologyWest Bengal University of Animal and Fishery Science

P.O.: Krishi Viswavidyalaya Nadia-741252, Kolkata, West Bengal

ABSTRACT

The eff icacies of different doses of isometamidium chloride against Trypanosoma evansi infection in buffalo calves werestudied by analysis of plasma protein binding activity, haematological and biochemical changes in blood. The percentageof protein binding activity of ISMM ranged from 89 to 93% against the concentration ranged between 24.10±0.57 and4.43±0.03 mg ml-1. The association constant of ISMM was greater than the dissociation constant. A significant decrease(P<0.01) in haemoglobin and total erythrocyte count were found, while significant increase (P<0.05) in neutrophil, lympho-cyte and monocyte count were recorded after trypanosomiosis. The respective values started to decrease after admin-istration of ISMM. A signif icant decrease (P<0.01) in serum creatinine, blood urea nitrogen and blood glucose level wereobserved, while mild alteration in serum ALT and no signif icant alteration in serum sodium and potassium level aftertrypanosomiosis were seen. The experimentally infected buffalo calves responded in better way @ 0.5 mg/kg i/v ISMM ascompared to 1 mg and 0.25 mg/kg body weight.

Key words: ISMM, intravenous, Trypanosoma, buffalo, heamo-biochemical, plasma protein binding.

order to demonstrate the benefit derived from intravenousIsometamidium chloride (ISMM)

Materials and MethodsThirty, clinically healthy calves, including of both the sexes,

(6 months age, weighing between 45-50 kg) after 21 days ofdeworming and 7 days of acclimatization in experimentalenvironment, were used in the study. The calves were groupedinto 5 different clusters each containing 6 animals viz: I (Control)and, II, III, IV and V as experimental groups in which infection(Trypanosomiosis) was induced. Strain of Trypanosoma evansibrought from IVRI, Izatnagar, Bareilly, U.P was seriallypassaged in mice in the department of Veterinary Parasitology,West Bengal University of Animal and Fishery Science. About 1ml of heart blood was taken from mice and was mixed withequal volume of Alsevers solution. The mixed solution (2 ml)was administered in the calves subcutaneously for inductionof trypanosomiosis. Experimentally infected buffalo calves wereshowing symptoms like dullness, staring gaze, corneal opacity,emaciation, anorexia, pyrexia (Temp 104-105°F), posteriorparalysis etc. on 28 days after post infection of Trypanosomaevansi.. The induction was confirmed by mice inoculation testand development of sign and symptoms in calves.Isometamidium chloride/hydrochloride, a trypanocidal drug(technical grade) with purity of the compound >90% wasprocured from M/s Alembic Ltd., Veterinary division, Mumbai(India). Group II served as experimental control (withouttreatment), while ISMM was administered as a singleintravenous dose @ 0.25, 0.5 and 1 mg/kg (as 1% solution in

1Corresponding Author: Ph.D. Scholar, VPT. House No. 1, Baker Street, Infront of IVRI Gate No.2, Izatnagar, Bareilly-243122, E mail: [email protected] M: 99173806162Prof and Head, Department of Veterinary Pharmacology and Toxicology, Faculty of Veterinary and Animal Sciences, West Bengal University ofAnimal and Fishery Science, 37 Kshudiram Bose Sarani, P.O.: Belgachia, Kolkata-700037, E- mail:[email protected] M: 94331361163Ph.D.Scholar, VPT, C.V.Sc.& A.H., A.A.U., Anand (Gujrat), E-mail:[email protected] Mb: 9033414305

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normal saline) to animals of Groups III, IV and V, respectively.Blood samples were collected from jugular vein of calves intest tubes containing EDTA (99.5%) at 0.08, 0.16, 0.33, 0.66,1, 2, 4, 6, 8, 12, 24 and 36 hours. Plasma separated bycentrifugation at 3000 rpm for 20 min, was utilized for theanalysis of plasma protein binding of ISMM. Equilibrium dialysistechnique as described by Sisodia et al. (1965) and Banerjeeet al. (1969) was used in this experiment to determine plasma-protein binding activity of ISMM The protein content of plasmaof each sample was estimated by the Biuret method (Wooton,1974).The binding capacity, dissociation and associationconstants were calculated by the method of least squareregression technique described by Pilloud, 1973.Haematological and haemato-biochemical study of bloodcollected on day 0 (induction of trypanosomiosis), 28(confirmation of infection) and 30 (48 hrs. post administrationof ISMM) was made. Haemoglobin level was determined byindirect acid haematin method as described by Coffin (1953)and expressed as gm/dl. Total erythrocyte count and differentialleukocyte count were done following standard method ofWintrobe as described by Schalm et al. (1995). In biochemicalparameters, Blood urea nitrogen (BUN)

Coulambe and Favrean,1965), creatinine (CRT) ( Varleyet al.,1980), glucose (Trinder,1969), protein estimated by Biuretmethod (Wooton, 1974), alanine amino transferase (ALT) andaspartate amino transferase (AST) as per the methoddescribed by Yatazidis (1960), sodium (Weinbach, 1935) andpotassium concentration (Looney and Dyer, 1942) wereestimated. Analysis of variance (ANOVA) using SPSS (10) wasused for analysis of data where applicable.

Results and DiscussionPlasma protein binding of ISMM

Results obtained in relation to plasma protein binding ofISMM in calves are presented in Table 1 and the binding capacity,association constant and dissociation constant of ISMM withplasma protein in calves have been summarized in Table 2. Itcould be seen that the equilibrium association constant (K’a:0.50x106±0.05x106 L mol-1) was greater compared todissociation constant (Kb: 2.04x10-6±0.18x10-8 mol L-1. Thepercentage of protein binding activity was above 89% whichhas been reflected by higher binding capacity (Bi: 1.51x10-

8±0.29x10 -8 mol/g) and greater equilibrium associationconstant of ISMM with the plasma protein.

Haematological profileHaemoglobin and total erythrocyte count

Table 3 and Fig. 1 describes the effect of ISMM onhaemoglobin level and total erythrocyte count in calves aftersingle dose intravenous administration at different doses. Itwas observed that haemoglobin level and total erythrocyte countwas significantly (P<0.01) decreased in calves of groups II, III,IV, and V on day 28 and 30 than that of the respective value atday ‘0’ (control). Soulsby (1982) reported that Trypanosomaspp. causes bone marrow depletion leading to decrease levelof haemoglobin and total erythrocyte count. Silva et al. (1995)proposed that trypanosome leads to increased red blood celldestruction and extravascular and intravascular haemolysisby immune reaction (trypanosome Ag-Ab complex and anti

erythrocyte Ab). Raina et al. (1986) also observed a significantdecrease in PCV and haemoglobin in buffalo calves inducedwith trypanosomiosis. Ahmed and Malik (2004) reported thatNubian goats inoculated with 7.5x105 T. evansi showedmicrocytic and normochromic type of anemia with reduction inhaematocrit and haemoglobin count. The decreasedhaemoglobin level and total erythrocyte counts in calves ofgroups II, III, IV and V on 28 day corroborate the above findings.

Differential leucocyte count(Neutrophil, eosinophil, lymphocyte, monocyte and basophil)

Mean values with S.E. of total neutrophil count, totaleosinophil count, total lymphocyte count, total lymphocyte countand basophile count in calves of groups I, II, III, IV and V followingsingle dose i/v administration of ISMM have been presented inTable 3 and Fig.1. It is evident from Table 5 that a significantincrease (P<0.05) in neutrophil count in calves of group II, III, IVand V on day 28 compared to day 0 were recorded. But theneutrophil count started to decrease after treatment with ISMMfrom day 30th in calves of groups III, IV and V. It has been statedthat Trypanosoma spp. causes non-specific phagocyticactivation, and also cellular damage in interstitial tissue leadingto mild exudative inflammatory changes which results increasedneutrophil count (Dargantes et al., 2005). This result correlatesthe present finding establishing that Trypanosoma evansi mighthave led to inflammatory responses which have subsided withadministration of administration of ISMM.

The values of total eosinophil count among calves ofdifferent groups on day 0, 28 and 30 were not altered. It istranspired from Table 3 that lymphocyte count was significantlyincreased (P<0.05) in calves of groups II, III, IV and V on day 28and 30 compared to respective day 0. Soulsby (1982) showedthat Trypanosoma spp. causes enlargement of lymph nodeleading to increase in total lymphocyte count. Dargantes et al.(2005) studied cytopathology of different organs to beimmunological in nature characterized by mononuclearinfiltration of interstitial tissues with minor cellular damage intrypanosomiosis. B and T-cell responses were observed inlymphatic system resulting lymphocytosis. Womack et al.(2006) observed an increase total lymphocyte count in goatsinfected with Trypanosoma evansi. The present result onincreased count of lymphocyte in trypanosomiosis in calves ofgroups II, III, IV and V at 28 day is in agreement with the abovecited findings. It was also observed that monocyte count wasincreased significantly (P<0.01) in calves of group II, III, IV andV. on day 28 and 30 compared to respective 0 day. Womack etal. (2006) observed an increase total lymphocyte and monocytecounts in goats infected with Trypanosoma evansi. Darganteset al (2005) also studied cytopathology of different organs tobe immunological in nature characterized by mononuclearinfiltration of interstitial tissues with minor cellular damage intrypanosomiosis leading to monocytosis. Basophil was notdetected in any of the groups at day 0, 28 and 30.

Biochemical profile(Serum alanine transaminase, serum aspartatetransminase, serum creatinine, blood urea nitrogen, bloodglucose, serum sodium and serum potassium level)

Table 4 and Fig 2 show the mean values with SE of serumalanine transaminase activity (SALT), serum AST, serum

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creatinine, blood urea nitrogen, blood glucose, serum sodiumand serum potassium level in calves of groups I, II, III, IVand V on days 0, 28 and 30 after intravenous administration ofISMM.

A significant increase in serum alanine transaminase(P<0.05) activity in calves of groups II, III, IV and V on day 28 and30 than that the values of respective ‘0’ day was found (Fig. 2).Cytopathology of liver characterized by mononuclear infiltrationof interstitial tissues in trypanosomiosis with minor cellulardamage might be the cause of increased serum ALT activity(Dargantes et al., 2005). Therefore, the increased activity ofSALT in the present experiment in groups II, III, IV and V may bedue to trypanosomiosis and not due to ISMM.

There was significant increase (P<0.05) in serum ASTactivity in group II on day 28 and 30. Multiplication oftrypanosome in cells of reticuloendothelial system and striatedmuscle results in cellular damage (Soulsby, 1982). Further,trypanosome causes direct traumatic effect on striated muscleleading to increase in serum AST activity. Hilali et al. (2004)also reported significant alteration in the levels of serum ASTand LDH enzymes in buffalo calves infected with T. evansi. Butit is difficult to explain the possible reason of unalteration ofAST activity in calves of groups III, IV and V in the presentexperiment.

The serum creatinine level and blood urea nitrogen levelwas found to decrease significantly (P<0.01) in calves of GroupsII, III, IV and V at day 28 and 30 compared to respective day 0which might be due to infection of Trypanosoma evansi. Hilaliet al. (2004) also observed a significant decrease in serumcreatinine and blood urea nitrogen level in buffalo calvesinfected with T. evansi. Normally, measurement of creatinineand BUN are being done to evaluate the functional status of

kidney. Any xenobiotic damages the nephron, both creatinineand BUN level in blood are increased and considered as indexof nephropathy. Both creatinine and BUN are the by-product ofamino acid metabolism more specifically protein metabolism.These by-products are generally formed by degradation ofprecursor amino acid. If this process is hampered or interfered,obviously the formation of by-product will be lesser. In thisexperiment, histopathological studies of kidney and liver tissueshas not been carried out, only transferase activity has beenmeasured. In trypanosomiosis, the ALT activity in blood hassignificantly increased compared to the non-inductive period.Therefore, it is expected that some interference in the aminotransferase activity has occurred. Since, the above parametersspecially the status of liver and kidney has not been evaluated,it might happen that trypanosoma itself may have interferedprotein or amino acid metabolism. Review of literature doesnot indicate that this type of study has been carried out.Therefore, this explanation might be attributed as a cause offall of BUN and creatinine level in the blood of trypanosomainduced calf. However, this requires further evaluation andconfirmation.

The blood glucose level also was decreased significantly(P<0.01) in calves of groups II, III, IV and V at day 28 and 30compared to respective 0 day. Trypanosome feeds on bloodsugar, causing hypoglycaemia (Soulsby, 1982). The decreasedglucose level in present experiment may be caused byTrypanosoma evansi not by ISMM.

No significant alteration in serum sodium and serumpotassium level was found among the calves of differentgroups.

From the above findings it may be concluded that ISMM @0.5 mg/kg b.wt. intravenously is quiet effective for the treatmentof Trypanosomosis in Indian Buffalo.

Table 1: Plasma protein binding activity of Isometamidium in calves after i/v administration (Mean value of 3 replicates with S.E.)

Concentration of ISMM after dialysis (μg/ml)

Time (hr)

Plasma concentration of ISMM before dialysis (μg/ml ) Plasma Buffer

Bound protein %

0.08 24.10 0.57 20.98 0.48 2.64 0.48 89.03 0.31 0.16 14.27 0.54 12.54 0.38 1.32 0.08 91.22 0.27 0.50 6.76 0.72 6.02 0.63 0.56 0.09 92.44 0.63 0.66 4.43 0.03 3.87 0.08 0.30 0.006 93.55 0.35

Table 2: Plasma protein binding constant (bi), Dissociation rate constant (Kb) and Association rate constant (Ka) of Isometamidium

Time (hr) i (mol g-1) K (mol L -1) Ka (L mol -1) 0.16 1.49 x 10-8 2.09 x 10-6 0.48 x 10-6 0.50 1.01 x 10-8 2.32 x 10-6 0.43 x 106

0.66 2.02 x10-8 1.7 x10-6 0.59 x 106 Mean ±S.E. 1.51 x 10-8 0.29 x10-8 2.04 x 10-6 0.18 x 10-8 0.50 x 106 0.05 x

106

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Table 3: Effect of Isometamidium on hematological parameters in calves after single dose i/v administration at 3 dose levels(Mean of six replicates with S.E)

A, 0 day (induction of trypanosomiosis); B, after confirmation of infection (28 day); C, 48 hr post-administration of isometamidium (30 day) *Gr. I : Control; Gr II : Experimental control / untreated control; Gr. III : at the dose rate of 0.25 mg/kg ; Gr. IV : at the dose rate of 0.5 mg/kg ; Gr.V : at the dose rate of 1.0mg/kg *Mean value with dissimilar superscript in a column vary significantly (p<0.01) and denoted as superscript (x, y)

Table 4: Effect of Isometamidium on heamobiochemical parameters in calves after single dose i/v administration at 3 dose levels(Mean of six replicates with S.E)

A, 0 day (induction of trypanosomiosis); B, after confirmation of infection (28 day); C, 48 hr post-administration of isometamidium (30 day) *Gr. I : Control;Gr II : Experimental control / untreated control; Gr. III : at the dose rate of 0.25 mg/kg ; Gr. IV : at the dose rate of 0.5 mg/kg; Gr.V : at the dose rate of 1.0 mg/kg *Mean value with dissimilar superscript in a column vary significantly (p<0.01) and denoted as superscript (x, y)

Serum ALT activity (µg pyruvate ml-1 hr-1 ) Time in day Gr. I Gr. II Gr. III Gr. IV Gr.V A 26.75 ± 2.43 23.50 x ±2.49 21.00x ±1.42 20.42x ±1.71 25.33x±2.61

B 25.42 ±2.18 28.33 y ±2.47 26.58y ±1.08 25.50y ±2.41 25.58x ±1.06

C 25.42 ± 2.08 31.42 y ±1.58 28.25y ±1.20 29.00z ±1.76 27.08 y ±1.51 Serum AST activity (µg pyruvate ml-1 hr-1 )

A 63.42±0.04 66.42 x ±3.05 60.92±4.04 63.08±5.06 62.20±6.08 B 63.83±2.56 70.25 y ±3.11 60.50±4.82 63.00±3.76 60.20±5.55 C 66.25±2.29 74.17 y ±2.89 64.00±4.37 63.92±3.71 59.80±5.79

Serum creatinine (µmol L-1) A 125.50±5.30 112.30x ±4.70 129.90x±9.70 131.70x±7.90 124.60x ±7.10

B 128.20±5.30 77.80y ±4.20 83.90 y ±5.30 81.30y± 7.90 83.10y ±10.60 C 131.70±4.40 75.10y ±3.90 84.90y±6.20 97.20y ± 7.90 91.90y±19.40

BUN (mmol L-1) A 7.40± 0.60 5.60 y ± 0.50 6.60x±0.50 6.70x±0.70 6.80 x ±0.80

B 7.80± 0.60 3.10y ± 0.50 3.90 y±0.60 4.10y±0.60 3.70 y ±0.50 C 8.10 ±0.50 3.00y ± 0.40 4.40y ±0.50 4.80y ±0.50 4.30 y±0.50

Blood glucose (mmol L-1) A 4.98 ±0.22 4.78 y ±0.16 5.27y ± 0.27 4.93 y ±0.23 4.95 xy ± 0.28

B 5.19±0.18 3.57x ±0.16 4.06 x ±0.28 3.64 x ±0.31 3.70y ± 0.36 C 5.26 ±0.18 3.43x ±0.18 3.89 x ±0.31 3.89xy ±0.31 3.90x ± 0.31

Sodium (mEq L-1) A 140.00±2.94 138.70±3.47 143.30±2.98 139.30±3.15 136.70±3.71 B 139.70±3.02 136.70±3.51 140.30±3.18 140.00±3.06 135.00±3.55 C 139.30±2.53 136.70±3.14 138.70±3.31 139.70±2.41 136.30±4.41

Potassium ( mEq L-1) A 3.96 ± 0.35 4.06 ± 0.38 4.10 ± 0.30 4.13 ± 0.28 3.72 ± 0.28 B 3.98 ± 0.34 4.03 ± 0.29 4.06 ± 0.26 3.88 ± 0.23 3.53 ± 0.24 C 4.05 ± 0.33 4.01 ± 0.32 3.93 ± 0.28 4.00 ± 0.20 3.60 ± 0.22

Haemoglobin level (gm/dl) Time in day Gr. I Gr. II Gr. III Gr. IV Gr.V

A 10.70±0.17 11.00 x ± 0.27 10.70x ±0.22 8.10x± 0.47 10.16x ± 0.49 B 10.66 ±0.20 8.33 y ± 0.43 8.40y ± 0.54 6.73y ± 0.51 8.46y ± 0.25 C 10.81±0.18 8.08y ± 0.57 8.67y ± 0.49 6.20 y ±0.51 7.70 y ± 0.30

Total Erythrocyte Count (x1012 cells/l) A 4.22 ± 0.12 4.44 x ± 0.27 4.3 2x ± 0.29 4.03 x ± 0.38 4.10 x ± 0.35

B 4.26 ±0.20 2.79 y ± 0.27 3.11 y ± 0.31 2.30 y ± 0.31 2.50y ± 0.32 C 4.55 ±0.22 2.85 y ± 0.49 3.17 y ± 0.31 2.39 y ± 0.33 2.70y ± 0.26

Neutrophil count (x109 cells/L)

A 1.86 ± 0.05 1.80 x ± 0.03 1.92x ± 0.05 1.93x ± 0.03 1.92x ± 0.03

B 1.89 ± 0.05 2.04y ± 0.06 2.14y ± 0.07 2.17y ± 0.05 2.07y ± 0.05 C 1.89 ± 0.02 2.09y ± 0.05 1.93x ± 0.07 1.89x ± 0.04 1.93x ± 0.03

Eosinophil count (x109cells /L)

A 0.49 ±0. 02 0.54 ± 0.03 0.48 ±0.03 0.51 ± 0.04 0.44 ± 0.01 B 0.54 ± 0.01 0.45 ± 0.03 0.48 ± 0.02 0.48 ± 0.02 0.51 ± 0.02 C 0.52 ± 0.02 0.44 ± 0.01 0.52 ± 0.03 0.48 ± 0.02 0.53 ±0.02

Lymphocyte count (x109 cells/L) A 3.99 ± 0.06 3.99x ±0.04 3.92x ± 0.07 3.84x ±0.06 3.88x ± 0.05 B 3.98 ± 0.03 4.18y ± 0.04 4.14 y ± 0.05 4.16y ±0.05 4.17y ± 0.06 C 3.95 ± 0.04 4.22y ±0.05 3.99 x ± 0.07 3.97x ±0.06 4.07xy±0.04

Monocyte count (x109cells /L) A 0.46 ± 0.04 0.52x ±0.03 0.50x ± 0.04 0.53x ± 0.03 0.52x ± 0.03

B 0.50 ± 0.02 0.58y ±0.03 0.62y± 0.03 0.68 y± 0.04 0.72y ± 0.02

C 0.50 ± 0.03 0.60y ±0.03 0.53x ± 0.03 0.52x ± 0.03 0.62z ± 0.02

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ReferencesAhmed, E.A. and Malik K.H. (2004) The effect of experimental

infection of with T evansi. 73rd General Session, WorldOrganisation for Animal health, International Committee,Paris.

Ali, H. and Hassan, A.P. (1984) Indian Vet. J. 71: 191-192.Banerjee, N.C. et al. (1969) J. Exp. Biol. 7:102-103.Braide, V.B. and Eghianruwa, K.I. (1980) Res. Vet. Sci. 29: 111-

113.Coffin, D.L. (1953) Manual of Veterinary Clinical Pathology. 3rd

ed. Comstock Publishing company, Inc. Ithaca, NewYork.

Coulambe, G. C and Favrean, L. A. (1965) Clin. Chem. 11: 624.Dargantes A.P. et al. (2005) Pathology. 133(4):267-276.Dowler, M.E. et al. (1989) Tropical Ani. Hlth. Prod. 21(1): 4-10.Hilali, M. et al. (2004) Haematological and biochemical changes

in buffalo calves in Egypt infected with T. evansi. 73rd

General Session, World Organisation for Animal health,International Committee, Paris.

Kaggwa, E. et al. (1988) Vet. Parasitol. 27(3-4):199-208.Kinabo, L.D.B. and McKellar, Q. A. (1990) Br. Vet. J. 146: 405-

412.

Looney and Dyer (1942) J. Lab Clin. Med. 28: 355.Pilloud, M. M. (1973) Residue Vet. Sci. 15:224-230.Raina,S. A. et al. (1986) Vet. Parasitol. 102 : 291-297.Schalm, O.W. et al. (1995) Veterinary Haematology. 3rd ed. Lea

Febiger, Philidelphia.Silva, R.A. et al. (1995) Memory of Oswald Cruz,Rio de Janeiro.

Vol. 90:299.Sisodia, C.S. et al. (1965) Indian Vet. J. 42:7-16.Soulsby, E.L.J. (1982) Helminths, Arthropods and Protozoa of

Domestic Animals. 7 th ed. Balliere Tindall, London.Trinder, P. Ann (1969) Clinical Biochem. 6(24).Varley H. et al. (1980) Practical Clinical Chemistry. Vol. I General

I top- ‘scomnoner test 5s1 ed. London, William medicalbooks Ltd

Waiswa, C. (2005) Bulg. J. Vet. Med. 8(1): 59-68.Weinbach (1935) J. Biol. Chem. 110: 95.Womack Sarah et al. (2006) J. Vet. Parmacol. Therap. 16: 291-

300.Wooton, I.D.P. (1974) Estimation of Protein by Biuret method. In

microanalysis in Medical Biochemistry. 5th ed. ChurchillLivingstone Edinburgh and London. pp. 156-158

Yatazidis, H. (1960) Nature. 18: 79-80.

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IntroductionIodine is essential element for animal species and

humans, mainly because it is an integral component of thyroidhormones (Preedy et al., 2009). Thyroid hormones viz., thyroxine(T4), and Triiodothyronine (T3) have a versatile role in regulationof tissue differentiation, BMR, growth, development,reproduction and lactation as well as in lipid, carbohydrate,nitrogen (protein) and energy metabolism (McDonald, 2005).The present study is most beneficial to us because of thehigher sensitivity of this element to animals and somedifferences in their dietary requirements and energymetabolism. This would be helpful in setting the lower limits oftoxicity standards for iodine and would allow the better use ofanimal by-products in human nutrition (NRC, 2007). Thepresent investigation was, therefore, undertaken to record thechanges in complete serum protein profile of Marwari goatssubjected to potassium iodide supplementation.

Materials and MethodsThe present study was conducted on six Marwari male

goats of 8 months to 1 year of age selected from Animal nutritionfarm, College of Veterinary and Animal Science, Bikaner. Theseanimals were provided with standard ration and water ad libduring the course of study. All experimental animals were keptisolated from the rest of the animals. They were housed inclean and well ventilated sheds. The experimental plan wasdivided into following phases:

Control (Phase 1)Initially blood samples were collected from the normal

animals, referred as control group. The samples of theseanimals were estimated for complete serum protein profile.These parameters were treated as control parameters.

Experimental phaseEach control animal was further subjected to potassium

iodide supplementation to observe its effect on protein profileduring the course of study. To carry out the objective of presentstudy, potassium iodide was used as a drug of choice in

IODINE OVERDOSAGE EFFECTS ON COMPLETE PROTEINPROFILE IN MARWARI GOATS#

S. Gupta1, M. Sareen and A. MoolchandaniDepartment of Veterinary Biochemistry, College of Veterinary and Animal Science


ABSTRACT

The present investigation was planned to determine the effect of iodine over dosage on complete serum protein profile inMarwari goats. The blood samples were collected from Marwari male goats of 8 months to 1 year of age belonging to AnimalNutrition farm, College of Veterinary and Animal Science, Bikaner and the serum samples were analysed in response toincreasing doses of potassium iodide. The results of present investigation revealed a significant (P<0.01) effect of potassiumiodide overdosage on total serum protein and albumin whereas globulin and A:G ratio remains unaltered.

Key words: Iodine, Marwari goats, protein profile

increasing doses. This stage was further sub-divided into threephases:

T1 - KI @ 13 mg/kg b.wt (Phase 2)T2 - KI @ 20 mg/kg b.wt (Phase 3)T3 - KI @ 26 mg/kg b.wt (Phase 4)

The serum of blood samples were separated bycentrifugation at 2500 rpm for 10 minutes and stored at -200Cuntil analysed. Serum samples were analyzed for total protein,albumin, globulin and A:G ratio by using commercially availablediagnostic kits on spectrophotometer-169 of Systronics. Thedata were analyzed using one-way ANOVA (Snedecor andCochran, 1994).

Results and DiscussionIn experimental group of present study, the total serum

protein tend to be higher in high treatment of KI than low of KI orcontrol treatments which might be due to increase in proteinsynthesis, digestion of protein at T2 and T3 phase and increasein thyroid hormones like T3 and T4. This increase may be theresult of the elevation of anabolic hormone secretion areresponsible for utilization of amino acids and otherphysiological functions related to metabolic rate (Freeman,1983). The present study substantiate the findings of Shetaewiet al. (1991) in lambs, El-Masry and Habeeb (1989) in lactatingFriesian cows and Vsyakikh et al. (1992) in cows.

The level of albumin was observed to be higher in all KIsupplemented groups, but the level was significantly highestin T2 phase as compared with other groups. The globulinconcentration and A:G ratio increased but they showed a non-significant effect. In our present study, the increase in serumprotein and globulin that occurred in relation to KIsupplementation is believed due to release of globulinfrom blood cells and is a normal response to increasedadrenal hormones (Turner et al., 1955). Also, thyroglobulinappears to be bound to peripheral blood cells and maybe a further source of globulin. Earlier studies by variousauthors stated that thyroid hyperfunction in response toiodine supplementation led to acceleration of the main

1#Part of M.V.Sc. Thesis submitted to RAJUVAS, Bikaner-334001, Corresponding author. Email:[email protected]

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metabolism, however, oxidation and phosphorylationprocesses were d isbalanced, less energy wasaccumulated in macroenergetic compounds, and moreheat was emitted. Furthermore, protein metabolism andg luconeogenesis in the liver become ac tivated(Spakauskas et al., 2008; Leonard and Visser, 1986;Nikolic et al., 2001). Our data is in agreement with theresults that thyroid hormones activate protein synthesis(Kepaliene et al., 2006).

The apparent adverse effects of prolonged excessiveiodide intake suggest that dietary iodine should be limitedto nutritional requirements and that prolonged use ofprophylactic or therapeutic amounts should be avoided.

AcknowledgementsThe authors are highly thankful to the Dean, College

of Veterinary and Animal Sciences, Rajasthan Universityof Veterinary and Animal Sciences, Bikaner, for providingnecessary facilities to execute the research.

ReferencesEl-Masry, K.A. and Habeeb, A.A. (1989) Thyroid functions in

lactating Fresian cows and water buffaloes underwinter and summer Egyptian conditions. Proc. of 3rd

Egypt-Brit ish Conf . on Animal Fish and Poult ryProduction. Vol 2, Alex, Egypt, pp.613.

Table 1: Mean ± S.E. concentration of serum total protein, albumin, globulin and A:G ratio according to the effect of KI supplementationin Marwari goats.

Total protein* (g/L) Albumin**(g/L) Globulin NS(g/L) A:G Ratio Phases

No.of Obs. Mean ± SE %Inc. Mean ± SE %Inc. Mean ± SE %Inc. Mean ± SE %Inc.

12 56.05 ± 1.49a 38.85 ± 1.1a 17.2 ± 1.93 3.69 ± 0.19 Control (46.9 – 63.4) - (33.5 -44.6) - (2.3 - 23.6) - (1.49-19.39) - T1 12 75.36± 6.53b 34.45 49.23 ± 3.2b 26.71 26.13 ± 5.0 51.91 5.24 ± 2.22 42.00

(KI @ 13 mg/kg b.wt)

(54.1- 130.9) (38.5 - 79.9) (2.4 - 57.7) (0.87- 24.5)

T2 12 66.46 ± 5.3b 18.57 46.05 ± 1.24b 18.53 20.41 ± 5.45 18.66 6.59 ± 2.18 78.59 (KI @ 20

mg/kg b.wt) (48.2 -107.6) (41.1 - 55.5) (2.3 - 60.6) (0.77-21.04)

T3 12 75.93 ± 6.21b 35.46 48.63 ± 1.41b 25.17 27.31 ± 6.27 58.77 4.21 ± 1.34 14.09 (KI@26

mg/kg b.wt) (50.3 - 109.3) (41.3 - 60.9) (2.8 - 58.6) (0.79-16.96)

Note: Mean comparison have been made within dif ferent phases. Mean superscripted with different let ters differ signif icantly(P<0.05) from each other. Data shown in parenthesis are representing respective range.

Freeman, B.M. (1983) Physiology and biochemistry of domesticbowel. Vol 4, Academic Press, New York and London,pp.181.

Kepaliene, I. et al. (2006) Veterinarijia ir Zootechnika.T. 36(58):39-43.

Leonard, J .L. and Visser, T.J . (1986) Thyroid HormoneMetabolism. pp. 189-229.

McDonald, L.E. (2005) Vet. Endocrinol. Reprod. 3rd ed. pp. 42-59.

Nikolic, J.A. et al. (2001) Acta Vet. (Belgr.) 51: 73-88.NRC (2007) Nutrient Requirement of small ruminants: sheep,

goats, cervids, and new world camelids . By NationalResearch Council (U.S.) ; Committee on Nutr ientRequirement of small Ruminants; National AcademyPress, Washington, pp.130-131.

Preedy, V.R. et al. (2009) Comprehensive handbook of Iodine:Nutritional, Biochemical, Pathological and TherapeuticAspects. pp. 421-428.

Shetaewi, M.M. et al. (1991) Assiut Vet. Med. J. 26(51): 98-107.Snedecor, G.W. and Cohran, W.G. (1994) Statistical Methods .

8th ed. The Iowa State Univ. Press, Inc. Ames. Iowa,USA.

Spakauskas, V. et al. (2008) Veterinarija ir Zootechnika. 43(65):90-95.

Turner, C. Donnell (1955) General Endocrinology. 2nd ed. W.B.Saunders Co. pp.176.

Vsyakikh, A.S. et al. (1992) Vestnik Sel’Skokhozyaistvennoi-Nauki-Moskva- Russia. 5: 104. (Soviet Agric., Sci.) 6:50.

204


IntroductionListeria monocytogenes, long known as an animal

pathogen, has now been recognized as an importantfoodborne pathogen associated with human disease. It hasbeen reported that only in the United States, food-borne illnesscauses over 76 million cases per year including 3,25,000hospitalizations and 5,000 deaths at a projected cost of $233million (Mead et al., 1999). Ready-to-eat (RTE) cooked meatsare frequently contaminated with L. monocytogenes duringpostprocessing steps (Beresford et al., 2001).

The widespread distribution of L. monocytogenes innature and its association with livestock makes the occasionalpresence of these bacteria on raw meats almost unavoidable.Contamination of ready to eat meat products with L .monocytogenes poses a public health hazard because of theorganism’s ability to grow at refrigeration temperatures andits pathogenecity within certain segments of population.Moreover, the most common route of infection of humans isconsumption of foods contaminated by L. monocytogenes (Jay,1996). It is logical that one of the hygienic requirements for rawmaterials and foods of animal origin is the absence ofpathogenic microorganisms, including L. monocytogenes(Regulation 91/1999 Sb., CZ, 1999). This microorganism,nevertheless, has been found in foods of animal origin(Jedlickova et al., 1991; Karpiskova, 1998). The objective ofthis study was to determine the contamination of retail poultry,sheep and goat meat in Punjab by L. monocytogenes.

Materials and MethodsTotal 50 samples each of sheep, goat and poultry raw

meat were collected from local market of Ludhiana city. Eachsamples weighing about 50-60 gm was collected in sterilizedpolythene pouch and processed within 2 to 3 hours aftercollection.

EnrichmentIn primary enrichment, 25 gms of meat sample was rinsed

in normal saline solution and homogenized in 225 ml of listeria

PREVALENCE OF LISTERIA MONOCYTOGENES IN DIFFERENTTYPES OF MEAT

Mohd. Ashraf Malik1, J.K. Sharma and Neelesh Sharma2

Department of Veterinary Public Health and EpidemiologyCollege of Veterinary Science, Ludhiana, INDIA

ABSTRACT

Contamination of ready to eat meat products with L. monocytogenes poses a public health hazard because of theorganism’s ability to grow at refrigeration temperatures and its pathogenecity within certain segments of population. Total50 samples each of sheep, goat and poultry raw meat were collected from local market of Ludhiana city and found overallincidence of L. monocytogenes in raw meat was 4.6%. Individually, the prevalence L. monocytogenes in poultry meat was8%, followed by in sheep and goat meat was 2% in each.

Key words: Prevalence, Listeria monocytogenes, meat

enrichment broth. This broth was incubated at 30°C for 24hours. For secondary enrichment, 0.1 ml of the primaryenrichment broth was transferred to a 10 ml tube of secondaryenrichment broth. This was incubated at 30°C for 24 hours.After incubation, secondary enrichment broth was plated on toModified McBride Listeria Agar (MMA) and incubated at 30°Cfor 24 to 36 hours and finally identification was done as per themethod of Buchanan and Gibbons (1974) and Conner et al.(1989). The incidence of Listeria monocytogenes wascalculated in percentage values.

Results and DiscussionOverall incidence

The incidence of L. monocytogenes reported in fresh meatranges from zero per cent (Ternstorm and Molin, 1987) to 92per cent (Lowry and Tiong, 1988). These figures representenvironmental contamination together with animal bornelisteriae. In the present study, overall incidence of L .monocytogenes in raw meat was 4.6%.

Incidence in sheep meatFifty samples of sheep meat (mutton) were analysed for

the isolation of L. monocytogenes and the organism wasisolated from two samples, thus indicating an incidence of4%. These observations are in agreement with those ofBrahmbhatt and Anjaria (1993), who also observed anincidence of 3.7% of L. monocytogenes in sheep meat. In asimilar study, Nitcheva et al. (1990) have reported the incidenceof Listeria to be 7.1% and Barbuddhe et al. (2000) reported7.4% in sheep. However, a higher incidence of (60%) of L.monocy togenes in boneless lamb meat has beenreported by Farber and Peterkin (1991).

Incidence in goat meatSimilarly, fifty samples of goat meat (chevon) were

screened and L. monocytogenes could be isolated fromone sample showing an incidence of 2%. Barbuddhe etal. (2000) reported 6.66% incidence of L. monocytogenes

1Present address: Division of Veterinary Hygiene and Public Health, F.V.Sc. & A.H., SKUAST-J, R.S. Pura, Jammu. Email: [email protected] address: Division of Veterinary Clinical Medicine and Jurisprudence, F.V.Sc. & A.H., SKUAST-J, R.S. Pura, Jammu- 181 102, INDIA, Email: [email protected]

205


in goat from Bareilly, India. Our results are in variation to thoseof Brahmbhatt and Anjaria (1993) who could not isolate L.monocytogenes out of 54 samples of goat meat analysed.The probable reason could be that the samples collected bythese workers might have been free from environmental andanimal-borne listeriae.

Incidence in poultry meatIncidence of L. monocytogenes was 8 per cent in poultry

meat (chicken) as 4 samples, out of 50 screened, was positivefor L. monocytogenes. Our results are in agreement with thoseof Nitcheva et al. (1990), and Katyal (1996), who have reportedthe incidence to be 7.8 and 8.0%, respectively. However, ahigher incidence of 60% of L. monocytogenes in fresh andfrozen chicken has been reported by Pini and Gilbert (1988).Similarly, Jemmi and Stephan (2006) had also been reported60% incidence of Listeria in chicken breast meat and Ceylanet al. (2008) found 32.76% incidence of L. monocytogenes inchicken from Turkey.

The ubiquitous nature of L. monocytogenes and its abilityto grow at refrigerated temperature makes L. monocytogenesa significant threat to the safety of ready-to-eat (RTE) meatproducts. Listeria monocytogenes and other Listeria specieshave been isolated from many different types of raw andprocessed food, but the main sources and routes ofcontamination are still not fully understood. There is a need formore knowledge, and data are needed for risk assessmentand for improved preventive measures. In order to prevent andcontrol contamination of the environment and food productswith this pathogen, it is important to detect the most importantsources of contamination and to understand the mechanismsof growth, including relationships with other bacteria. It hasbeen demonstrated that normal pasteurization processes areeffective in the destruction of this pathogen so conventionalcooking would also be expected to eliminate this organism(Norrung, 2000).

ReferencesBarbuddhe, S.B. et al. (2000) Small Rum. Res. 38(2): 151.Beresford, M.R. et al. (2001) J. Appl. Microbiol. 90:1000-1005.Brahmbhatt, M.N. and Anjaria, J.M. (1993) Indian J. Anim. Sci. 63: 667.Buchanan, R.S. and Gibbons, N.E. (1974) Bergey’s manual of

determinative bacteriology. 8th edn., Williams and WilkinsCo., Baltimore, Maryland.

Ceylan, Z.G. et al. (2008) J. Food Quality. 31(1): 121.Conner, D.E. (1989) J. Food Sci. 54: 1553.Farber, J.M. and Peterkin, P.I. (1991) Microbiol. Rev. 55: 476.Jay J.M. (1996) Foodborne listeriosis, 478-499. In: Jay J.M. (ed.):

Modern Food Microbiology. Chapman & Hall, New York. Pp.661.

Jedlickova, Z. et al. (1991) Listeria monocytogenes (in Czech). Actahygienica, epidemiologica et microbiologica, Prague, 21:9-19.

Jemmi, T. and Stephan, R. (2006) Rev. Sci. Tech. Off. Int. Epiz. 25(2):571.

Karpiskova, R. (1998) Study of the occurrence of listeriae in foodstuffsin 1998 (in Czech). The Bulletin of Centre for the Hygiene ofFood Chains in Brno. 7: 8-9.

Katyal, P. (1996). Incidence of Listeria monocytogenes in raw meatand vegetables. Thesis, Punjab Agric. Univ., Ludhiana.

Lowry, P.D. and Tiong, I. (1988) In: Proc. 34th International Congr. MeatSci. Technol. Part-B: 528.

Mead, P.S. et al. (1999) Emerg. Infect. Dis. 5: 607-625.Nitcheva, L. et al. (1990) Acta. Microbiol. Hung. 37: 223.Norrung, B. (2000) International J. Food Microbiol. 62: 217.Pinni, P.N. and Gilbert, R.J. (1988) International J. Food Microbiol. 6:

317.Regulation by the Ministry of Health No. 91/1999 Coll. of Laws to

amend the regulation by the Ministry of Health No. 294/1997Coll. of Laws on microbiological requirements for foodstuffs,methods of their control and evaluation (in Czech).

Ternstrom, A. and Molin, G. (1987) J. Food Prot. 50: 141.

FEED BACK The editorial team would greatly solicit feed back from the readers on the information

provided in the form of various articles in each issue. The feed back may be in theform of their own observation on the clinical aspects including treatment of aparticular disease published in the journal. This will help in strengthening ofknowledge of other readers. This will be a permanent column in each issue.

206


IntroductionYak is the unique multipurpose bovid of high altitude (Cai

and Gerald, 2003). The natural habitat of yaks is between 3000to 6000 m above (Mean Sea Level) MSL, which remain snowcovered in almost all the times of year. In India are mostlyreared under semi migratory free range system in high reachesof Jammu and Kashmir, Arunachal Pradesh, Sikkim andHimachal Pradesh (Rahman et al., 2010). In winter, yaks arekept in comparatively lower altitude (3000 m above MSL) closeto the villages and during summer they are taken to the highaltitude alpine pasture (4500 m and above MSL) (Pal andBarari, 1994). The normal haematology and biochemicalparameters in yaks reared under organized sedentary farmcondition have been studied by several workers (Barari et al.,1995, Mondal et al., 1998 and Chatterjee et al., 2004); however,reports on haematology and different biochemical parametersin yaks in the summer/winter grazing ground and during thephase of migration and under traditional system are scanty.Hence, the present investigation was designed to find out thephysiological response of the yaks to high altitude when theyare in the summer as well as winter grazing ground undertraditional management system.

Materials and MethodsThe present study was conducted on thirty apparently

healthy adult yaks of either sex reared under traditionalmigratory free range system of rearing. The selected animalswere grouped into two categories: The first group consisted ofthose animals grazing in winter grazing ground which is around2800-3000 meter above MSL (Mandala Phudung area of WestKameng district, Arunachal Pradesh) and in second group,

BIOCHEMICAL AND MICRO-MINERAL STATUS OF YAK (POEPHAGUSGRUNNIENS L.) REARED UNDER HIGH ALTITUDE TRADITIONAL

MIGRATORY SYSTEMR. Pourouchottamane1, A. Chatterjee1, B.C. Saravanan1, M.A. Kataktalware2,

A. Mishra3 and P.K. Pankaj1*

National Research Centre on Yak (ICAR)Dirang 790101, Arunachal Pradesh, India

ABSTRACT

The present s tudy was conducted on a total of thirty adult and apparently healthy yaks of either sex reared undertraditional migratory free range system of rearing. Blood samples were analyzed for normal haematology, serum biochemi-cal parameters and micro-mineral status of the yaks when they are in summer grazing ground and winter grazing ground.Haemoglobin content in yaks in summer grazing grounds was significantly (P<0.01) higher than that of animals in thewinter pasture. RBC count, WBC count, ESR and PCV were similar in yaks in both summer and winter pasture. Similarly, nosignificant difference was found in any of the biochemical parameters. The mean cobalt and zinc values were significantlyhigher in the animals grazing in the summer grazing ground than those in winter grazing ground. The mean copper valueobtained in the present study was lower than the critical value indicating possible copper deficiency.

Key words: Yak, migratory system, high altitude, physiological response, trace element, haematological, biochemical

1*Corresponding Author: Senior Scientist (LPM) Central Research Institute for Dryland Agriculture (CRIDA), ICAR, Santoshnagar, Hyderabad,Andhra Pradesh-500 059, India2Scientist3Assistant Professor

those animals grazing in the summer grazing ground (Selaarea of Tawang District, Arunachal Pradesh) which is 4600meter above MSL.

Managemental PracticesThe yak starts descending from the high altitude alpine

pasture in November with the onset of snowing and shall beherded at a lower altitude up to April. From May they again startascending to the greater Himalayas in search of green alpinepasture. The winter pastures are often poor and the yak eatlichen, dry leaves and tree loppings where available. Naturally,the animals lose condition. Summer pastures are much higherquality and provide entirely for the growth and production of theyak.

No separate housing was provided for yaks. Only simpleenclosures with out any roof are provided to calves where theyare tied at night times. In the winter grazing grounds, animalsare left in the jungle for grazing. Supplementary feeding wasnot followed in the farmer’s herd except for maize or wheatflour fed to the lactating animals; only grazing and feeding oftree lopping was followed with salt.

The traditional way of maintaining the animals is to allowthem to put on weight as much as possible in summer andutilize the fat as an energy reserve for survival in winter months,when there is scarcity of feed. The availability of the herbage,its growth stage and nutritive value largely influences theproduction performance of yaks, as the supply of supplementaryfeeding is limited.

From each animal, blood samples were collected induplicate from jugular vein to study the normal haemogram,serum biochemical parameters and micro mineral status of

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the yaks. EDTA was used as anticoagulant for haematologicalexamination of parameters like RBC, WBC, PCV, Haemoglobinand erythrocyte sedimentation rate as per standard procedure(Jain, 1986). The serum samples were analyzed within 24hours of collection for different biochemical parameters likeglucose, total serum protein (Vatzidis, 1977). Biochemicalenzymes SGPT and SGOT were analyzed by UVspectrophotometer using Ranbaxy kits (Ranbaxy diagnosticsLtd, India) and alkaline phosphatase by the method of Kindand King (1954). The serum trace elements copper, cobaltand zinc were estimated by atomic absorptionspectrophotometer (Perkin Elmer, 1996) using the standardconditions (Perkin Elmer, 1996). Standards were prepared bydiluting the stock standard solutions (MERCK, Germany) usingthe same diluents as in case of samples.

Statistical analysisThe data generated were analyzed as per Snedecor and

Cochran (1989) to find any variation in the physiologicalresponse of the yaks when they are in winter and summergrazing ground.

Results and DiscussionThe haemato-biochemical parameters and trace element

status of yaks in winter and summer grazing grounds hasbeen presented in the Table 1.

Haematological parametersHaemoglobin content in yaks in summer grazing grounds

was significantly (P<0.01) higher than that of animals in thewinter pasture. RBC count and PCV were at higher side (non-significant) on summer pasture as compared to winter pasturein yaks. The increase in Hb values and higher values of RBC

count and PCV with the increase of altitude indicates theadaptive feature of the animals living in the high altitude toadapt in the environment having low oxygen content. Winter etal. (1989) also observed that yaks reared at an altitude of 4000m above MSL had an Hb value of 13.5 while Hb value was12.01 in the yaks reared at an altitude of 2750 m MSL(Pourouchottamane et al., 2004). Zhang et al. (1994) also notedthat Hb content in Tibetan yaks increased with the increase inaltitude.

WBC count and ESR values were similar in both summerand winter grazing grounds (Table 1). Zhang Rongchang (1989)also reported WBC count in the range of 9.1 to 12.5 x 103 in theTianzhu white Yak. In general, WBC and ESR values denotethe well being of the animals and any increase in the valueindicates the presence of infections.

Biochemical parametersThe serum protein levels were similar in both winter and

summer grazing grounds (7.16±0.15 and 7.23±0.14 g/dl,respectively). Chatterjee et al. (2004) also reported similarlevels of serum protein in yaks reared under organized farm.The glucose levels were slightly higher (63.61±2.86 vs.61.23±2.46 mg/dl) in high altitude summer grazing groundsthan the low altitude winter grazing grounds.

The activity of the metabolic enzymes SGOT and SGPTwere slightly higher in high altitude summer grazing groundcompared to those grazing in winter grounds (Table 1). Eventhough the difference was statistically not significant, the trendindicates that metabolic activity was more in yaks reared atcold high altitude. The mean enzyme levels obtained in thepresent study were in higher range than those yaks rearedunder sedentary farm conditions (Chatterjee et al., 2004). Thealkaline phosphatase levels were similar in both the groups.

Table 1: Haematological, biochemical and trace element profile in Yaks

Parameters Winter grazing ground

Summer grazing ground

Overall mean

Haematological parameters RBC(106/ μl) 4.74 ± 0.08 4.99 ± 0.122 4.89 ± 0.08 Haemoglobin (gm/dl) 11.04 ± 0.43** 13.72 ± 0.488 10.22 ± 0.75 WBC(103/ μl) 9.28 ± 0.14 9.93 ± 0.18 9.73 ± 0.14 PCV (%) 40.97 ± 1.31 41.03 ± 1.076 41.0 ± 0.82 ESR (mm/ 24 hr) 10.00 ± 3.57 10.78 ± 1.38 10.53 ± 1.45 Biochemical parameters Serum protein (g/dl) 7.16 ± 0.15 7.23 ± 0.14 7.20 ± 0.108 Glucose (mg/dl) 61.23 ± 2.46 63.61 ± 2.86 62.57 ± 1.91 Serum glutamate oxaloacetate transaminase (SGOT) IU/L

38.37 ± 2.24 40.18 ± 3.75 39.39 ± 2.29

Serum glutamate pyruvate transaminase (SGPT) IU/L

25.55 ± 2.78 31.84 ± 5.37 29.11 ± 3.27

Alkaline phosphatase IU/L 186.3 ± 16.63 185.5 ± 9.19 185.8 ± 8.67 Trace elements Copper (mg/L) 0.193 ± 0.008 0.296 ± 0.058 0.255 ± 0.037 Cobalt (mg/L) 0.457 ± 0.028** 0.785 ± 0.065 0.654 ± 0.058 Zinc (mg/L) 0.789 ± 0.114* 1.247 ± 0.108 1.064 ± 0.097 * Significant at 5% level (P<0.05), ** Significant at 1% level (P< 0.01)

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Micro-mineral statusThe overall mean values (mg/L) for copper, cobalt and

zinc were 0.255±0.037, 0.654±0.058 and 1.064±0.097,respectively. The concentration of copper was lowest and evennon-detectable in some animals. The mean values obtainedfor cobalt and zinc were higher than the critical level (McDowell,1985), whereas, the mean value for copper was much lowerthan the critical level (0.654±0.058 mg/L) reported by McDowell(1985), indicating possible copper deficiency. Clauss andDierenfeld (1999) in a review reported the susceptibility of yakto copper deficiency.

The mean cobalt and zinc values are significantly higherin the animals grazing in the summer grazing ground thanthose in winter grazing ground. The difference in the serummineral concentration might be due to variation in the soil typeand pH, plant species, stage of maturity of plant, etc.

Several factors like species, age, sex, climate, productionlevel, etc affects the haemato-biochemical profile of an animal.The result obtained in the present study indicates variation inthe physiological response of the yaks to the high altitudesummer grazing ground and winter grazing ground and canbe considered as a preliminary baseline reference for theseparameters in yaks reared under traditional migratory system.

ReferencesBarari, S. K. et al. (1995) J. Applied Ani. Res. 7: 99-103.Cai, L. and Gerald, W. (2003) The Yak. 2nd ed. Pub. FAO regional office

for Asia and the Pacific.

Chatterjee, A. et al. (2004) Indian J. Ani. Sci. 74(6): 633-34.Clauss, M. and Dierenfeld E.S. (1999) Vet. Rec. 145: 436-37.Jain, N. C. (1986) Schalm’s Veterinary Haematology. 4th ed. Lea and

Febiger, Philadelphia. pp. 1040-68.Kind, P. R. N. and King, E. J. (1954) J. Cli. Pathol. 7: 322-24.McDowell, L.R. (1985) Nutrition of Grazing Ruminants in Warm

Climates. Academic Press, New York.Mondal, D. B. et al. (1998) Indian J. Ani. Sci. 68(5): 478-79.Pal, R. N. and Barari, S. R. (1994) Yak Production and Health Advances

in Veterinary Research and their impact on Animal Healthand Production. Bareilly, India. pp. 21-27.

Perkin, Elmer. (1996) Analytical Method: Atomic AbsorptionSpectroscopy. The Perkin Elmer Corporation, Norwalk, USA.

Pourouchottamane, R. et al. (2004) Influence of altitude on the haemato-biochemical changes in yak (Poephagus grunniens L).Proceedings of the fourth International congress on Yakheld on September, 19-26, 2004 at Chengdu, SichuanPublishing House of Science and Technology, Sichuan,P.R.China pp.309-12.

Rahman, H. et al. (2010) Indian J. Ani. Sci. 80(3): 195-98.Snedecor, G.W. and Cochran, W.G. (1989) Statistical Methods. 6th ed.

The Iowa State University Press, Ames, Iowa, USA.Vatzidis, H. (1977) Clinical Che. 23: 908-11.Winter, H. et al. (1989) Australian Vet. J. 66: 299-301.Zhang, Rongchang et al. (1994) Anatomical physiology of yak adapting

to the low oxygen on the high plateau. Proceedings of the1st International Congress on Yak, 4-9 September, 1994.Lanzhou, P. R. China pp. 236-40.

Zhang, Rongchang. (1989) Yak of China. Lanzhou, Gansu Scienceand Technology Publishing house. pp. 75-194.

X-PERTS ANSWER

All the practicing veterinarians are requested to send their problems/querrieson any clinical aspects faced by them. The solution to the problem will be obtainedfrom national level experts of the concerned field. The solution suggested by theexpert will be sent by post and the problem as well as the solution will be published inthe next issue of this Journal for the benefit of other readers. This will be a permanentfeature of this Journal in forth coming issues.

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IntroductionRole of sheep is paramount especially for small farmers.

The knowledge of blood cells is important in diagnosis of manydiseases in an mals. In spite of great advancement in the fieldof research and diagnostics related to livestock like sheep,goat etc. still the blood examination is an important weapon inthe arena of disease diagnosis. The studies on the bloodcells are not only important from anatomical point of view butalso with respect to physiology, clinical pathology and clinicalveterinary medicine. So the present study is undertaken tostudy the ultrastructure of on blood cells of sheep with specialemphasis on granulocytes and platelets.

Materials and MethodsThe study was conducted on ten apparently healthy sheep

maintained at livestock production and management farm ofCollege of Veterinary and Animal Sciences, G.B.P.U.A.T.,Pantnagar. For transmission electron microscopy (TEM), fivemillilitre of blood was taken from the jugular vein in a sterilizedand siliconized tube with EDTA as anticoagulant and centrifugedat 2500 rpm for 30 minutes. The excess of plasma was drainedoff leaving a small amount over the buffy coat. Then 2-3 ml ofmodified Karnovsky’s fluid was poured along the sides of testtube drop by drop without disturbing the buffy coat for fixationand formation of buffy coat plug. The buffy coat plug along witha layer of red blood cells was taken out of the tube with the helpof hooked needle or wire and placed in Petri dish containingphosphate buffer. The plug was cut into thin and small slicesof approximately 1 mm thickness. The samples/slices weresubmitted in PBS at pH 7.4 to the electron microscopy facility atAIIMS, New Delhi for further processing. The samples weredehydrated in graded acetone solutions and embedded inbeam capsule (CY 212 Araldite). Ultrathin sections of 60-80nm thickness were cut using an ultra cut microtome and thesections were stained in alcoholic uranyl acetate (5 min) andlead citrate (2 min). These sections were then placed on grids

ULTRASTRUCTURAL STUDIES ON THE BLOOD CELLS OF SHEEP

Aditya Kumar1, Ishwar Singh2 and Meena Mrigesh3

Department of Veterinary Anatomy, College of Veterinary and Animal SciencesG.B. Pant University of Agriculture and Technology,Pantnagar-263145, Uttarakhand, India

ABSTRACT

The transmission electron microscopy (TEM) and scanning electron microscopy (SEM) was conducted on blood cells of sheep. Ultrastructurally leukocytes varied greatly in shape and size. Under transmission electron microscopy, cytoplasmic granules of theneutrophils varied greatly in shape, size and density. Eosinophils were round in outline and had five types of cytoplasmic granulesdistributed throughout the cytoplasm. The basophils ultrastructurally were roughly spherical in outline with variable size cytoplasmicgranules. The erythrocytes under SEM showed biconcavity. Under scanning electron microscopy (SEM) five types of leukocyteswere observed.

Key words: Erythrocytes, granulocytes, sheep, TEM, SEM

and viewed under transmission electron microscope(Morgagni 268) operated at 60-80 kv (David et al., 1973) atAIIMS, New Delhi.

For scanning electron microscopy (SEM), five millilitre ofblood was taken from the jugular vein in a sterilized andsiliconized tube with heparin as anticoagulant. The blood wascentrifuged at 2500 rpm for 30 minutes. The buffy coat rich inleucocytes along with some erythrocytes was taken out with apasteur pipette in phosphate buffer and washed three timeswith the buffer. Then primary fixation was done with modifiedKarnovsky’s fluid. The supernatant was drained off andsamples were rewashed three times with the phosphate buffer.These samples of fixed cells were sent to SAIF AIIMS in 0.1M phosphate buffer (pH 7.4).The cells were resuspended indistilled water and repeated washing for 5 times wasperformed in distilled water. The film of the blood cells wasmade on a clean cover slip and air dried. Then blood cells filmwere coated with gold sputter coating and observed underLEO 435 variable pressure Scanning electron microscope.

Results and DiscussionTransmission electron microscopy

Under transmission electron microscopy the erythrocyteswere elongated to round in appearance depending upon planeof section (Fig. 1). The elongated erythrocytes were havingcentrally depressed area with rounded ends. The cellsappeared homogenously granular without any cytoplasmicorganelles. Singh and Singh (2003) reported that theerythrocytes in buffalo calf were biconcave discs with deepconcavity and smooth cell surface.

The neutrophils were roughly round in shape withcytoplasmic processes of varying size and length (Fig 1). Thenuclear lobes varied from 2-7 and differed greatly in shapeand size. The cytoplasm was full of membrane boundcytoplasmic granules, showing pleomorphism. The cytoplasmhad electron lucent granules, the electron dense granules and

1Ph.D. student, Division of Veterinary Anatomy, LLRUVAS, Hisar, Haryana, India. Email- [email protected] author, Professor and Head, Department of Veterinary Anatomy, College of Veterinary and Animal Sciences, GBPUA&T, Pantnagar-263145,Uttarakhand, India.email– [email protected] Professor, Department of Veterinary Anatomy, College of Veterinary and Animal Sciences, GBPUA&T, Pantnagar-263145, Uttarakhand, India.

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Fig. 1: A transmission electron microphotograph showing elongatedhomogenously granular erythrocyte (a), neutrophil with electron densegranule (b) and electron lucent granule (c). Uranylacetate and lead citratex 1990c

Fig. 2: A transmission electron microphotograph of neutrophil showinggranule with thin electron dense bacilli rod like lines (a). Uranylacetate andlead citrate x 6630c

Fig. 3: A transmission electron microphotograph of eosinophil showinghomogeneously electron lucent granule (a), granule with one highly electrondense rod (b), granule with two highly electron dense rod (c), and electrondense granule (d). Uranylacetate and lead citrate x 3500

Fig. 4: A transmission electron microphotograph of eosinophil showinggranule with one highly electron dense rod (a), granule with two highlyelectron dense elongated rod (b), and granule with three highly electrondense elongated rod (c). Uranylacetate and lead citrate x 5600

Fig. 5 : A transmission electron microphotograph of basophil showinghighly electron dense granule (a) and less electron dense granule (b).Uranylacetate and lead citrate x 1990c

Fig. 6: A transmission electron microphotograph of thrombocyte showinglarge, round and electron lucent granules (a) and elongated crystalloidgranules with finger like impressions (b). Uranylacetate and lead citrate x13700c

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these granules were having thin electron dense bacilli rod likelines within them (Fig. 2). Similiarly electron dense and electronlucent granules in camel neutrophils were observed by Singhet al. (1997). Sonoda and Kobayashi (1970) reported that thecamel neutrophils had three types of granules i.e. compacthomogeneous ones with high electron density, moderatelydense ones with granular like structure and low dense oralmost vacant ones. Gennaro et al. (1978) observedhomogeneously electron dense spherical, elongated, rod anddumbbell shaped granules in bovine neutrophils. Singh (2000)and Menaka and Singh (2004) observed four different typeddouble membrane bound, different size and density granulesin the neutrophils of the buffalo calves and goat respectively.

Eosinophils were round in outline, had large and smallcytoplasmic processes and lobed nuclei (Fig. 3 & 4). Thenuclear heterochromatin was mostly peripheral with centrallyplaced euchromatin and inside that patches ofheterochromatin were there (Fig. 3). The cytoplasmic granuleswere distributed throughout the cytoplasm and varied greatlyin shape and size, and interior details (Fig. 3 & 4). The one typeof granules were homogeneously electron lucent. The secondtype of granules were comparatively large in size and having asingle highly electron dense rod. The third type of granuleswere having two highly electron dense elongated rods out ofwhich one was longer and other was smaller. The fourth typeof granules were having three electron dense elongated rods,which were arranged in a triangular fashion. The fifth type ofgranules had homogenously electron dense material, whichwere also very less in number. Yamada (1970) in sheep reportedthat the eosinophils had an electron dense middle plate in thespecific granules. In addition to these specific granules, thehomogeneous granules without any structure, the granuleswith myelin like structure, the granules with homogeneousdense round substance and the granules with mixture of someof these structures were also seen. Jain (1993) reported thatcat and sheep eosinophil granules were distinctive in that theyappeared crystalloid whereas equine eosinophil granuleswere homogeneous. Singh et al. (1997) observed that theeosinophils in camel were of two types depending upon typesof granules. The first type of the cell had pleomorphic electrondense granules with homogeneously placed material. Thesecond type of cell had elongated or oval granules with anelectron dense at central and electron lucent at peripheralportion. Singh (2000) reported that in the buffalo calves theeosinophilic cytoplasmic granules were mainly rounded butexhibited a wide variation in their structure and size. Some ofthe granules were homogeneous while some had 2 to 3 zonesof different electron densities. In some of such type of granules,the inner zone had a peripheral cap like highly electron densearea while some of the granules had clearly defined fingerprint like crystalloid material. Menaka and Singh (2004)reported that goat eosinophils had mainly round oval andelongated membrane bound granules of different sizes.

The basophils ultrastructurally were roughly spherical inoutline with few cytoplasmic processes. The cytoplasmicgranules showed pleomorphism. The one type of cytoplasmicgranules was highly electron dense while the other type wasless dense with patches of electron dense area. The secondtype granules were larger in size. Beside that vesicles were

also present inside cytoplasm (Fig. 5). Yamada (1970) reportedthree types of granules in sheep basophils. These were thehomogenously electron dense ones, with coarse granularstructure and moderately or slightly dense ones with finegranular structure. Brown (1987) described fairlyhomogeneous granules in basophils of domestic mammals.Singh et al. (1997) in camel observed that the basophilcytoplasm contained round membrane bound granules, whichwere homogeneously electron dense and uniformly circular.Some of these granules had a slight empty space of variablesize at their periphery. Singh (2000) reported that in the buffalocalves the basophilic cytoplasmic granules had two differentelectron density areas. The peripheral area of such granuleswas less electron dense seen as rim of centrally placedelectron dense area.

Thrombocytes were elongated, oval or circular in outlinedepending upon the plane of section (Fig. 6). The granulesshowed great variation in their size and structure. The one typeof granules was electron dense in nature. The second type ofgranules was electron lucent. The third type of granules wascrystalloid in appearance. The fourth type of granules was largein size and having centrally or eccentrically placed electrondense material. Few vesicles of different sizes enclosingvariable amount of lysed material were also seen. Menakaand Singh (2002) reported that goat platelets appeared aselongated, elliptical or oval in outline depending upon the planeof section. First type of granules had electron dense margin.The second type was filled with granular material. The thirdtype of granules were having an eccentrically placed highlyelectron dense material of variable size.

Scanning electron microscopyUnder scanning electron microscopy erythrocytes were

biconcave with smooth outline. Few erythrocytes showed smallpinpoint surface projections (Fig. 7).

Leukocytes were five different types under SEM. One typeof leukocyte was largest in the size and showing numerousruffle/plate like broad based processes on the surface (Fig. 7).Second type of leukocyte also showed numerous ruffle/platelike broad based processes on the surface, but was smallerin size and had a few long cytoplasmic processes(Fig. 8). Thethird type of leukocyte was showing blunt, rounded mulberrytype outgrowth. The fourth type of leukocyte was also showingblunt rounded mulberry type out growth but smaller in size.The fifth type of leukocytes were round in shape showing shortvilli like projections and smallest in size (Fig. 8). Singh andSingh (2003) examined two types of leukocytes under scanningelectron microscopy in buffalo calves. The smaller ones wereround and had small villi-like projections of the surface. Thelarger one was also rounded and had broad based plate likenumerous folds throughout the surface of the cell.

Under scanning electron microscopy, thrombocytes werethe smallest cells which were cylindrical to roughly round inoutline and showing numerous fine projections on the surface(Fig. 7). Singh and Singh (2003) observed that in buffalo calvesthe platelets appeared irregular in outline and Most of theplatelets had long three to four cylindrical cytoplasmicprocesses. Eurell and Frappier (2006) stated that restingplatelets had a smooth surface with random indentation.

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AcknowledgementsAuthors are thankful to Director Research, Pantnagar and

AIIMS, New Delhi for providing necessary facilities for carryingout this research work.

ReferencesBrown, E. M. (1987) Blood and bone marrow. In: Textbook of

Veterinary Histology . 3 rd ed., Lea and Febiger,Philadelphia, pp. 71-99.

David, G. F. X. et al. (1973) J. Anat. 115: 79.Eurell, J.A. and Frappier, B.L. (2006) Blood and bone marrow.

Dellmann’s, Textbook of Veterinary Histology. 6th

ed., Blackwell Pub., U.S.A.

Gennaro, R. et al. (1978) Proc. Soc. Exp. Biol. Med. 157: 342-347.Jain, N. C. (1993) Essentials of Veterinary Hematology. Lea and

Febiger, Philadelphia. pp. 19-40.Menaka, R. and Singh, I. (2002) Ind. J. Vet. Anatomy. 14: 30-34.Menaka, R. and Singh, I. (2004) Ind. J. Ani. Sci. 74: 1135-1136.Singh, G. et al. (1997) J. Camel Prac. Res. 4: 1-12.Singh, I. (2000) Light and ultrastructural studies on the blood

cells of normal and dexmethasone treated buffalocalves. Ph.D. thesis, CCS HAU, Hisar.

Singh, I. and Singh, G. (2003) Ind. J. Vet. Anatomy. 15: 62-64.Sonoda, M. and Kobayashi, K. (1970) Japan J. Vet. Res. 18:

37-41.Yamada, Y. (1970) Jap. J. Vet. Res. 18: 9.

Fig. 7: A scanning electron microphotograph of blood cells showinglargest sized leukocyte with numerous ruffle/plate like broad basedprocesses on the surface (a) biconcave erythrocyte with smoothsurface (b) and thrombocyte with numerous varying sized cytoplasmicprocesses on the surface (c). x 5000

Fig. 8: A scanning electron microphotograph of blood cells showingsmall sized leukocyte with numerous ruffle/plate like broad basedprocess on the surface with few long cytoplasmic processes (a) andsmallest sized leukocyte with short villi like projections (b). x 5000

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IntroductionOn the Indian subcontinent, the Dromedarian camel has

been incessantly playing an unique role, being importantcomponent of the desert ecosystem. This large ruminant iswell adapted both anatomically as well as physiologically, tothrive well upon throny bushes and fodder trees leaves in aridand semi arid Thar desert, where epizooteologicaldeterminants are not favourable for faster propagation,transmission and survival of metazoan parasites, especiallyhelminths (Raisinghani, 1992, Partani et al., 1995 and Chhabraand Gupta, 2006). Adverse impact of helminths on body weightgain, nutritent utilization, growth and work performance etc.and at time terminating into death of the host, have beendocumented elsewhere (Sharma and Satija, 1974; Irfan, 1984and Sharma, 2000). This prompted the authors to undertakeprevalence of gastrointestinal metazoan parasites in camel.

Materials and MethodsTwo hundred and fifty camels irrespective of sex, age and

breed exhibiting nonspecific and indiscriminately clinical signswere coprologically screened for helminthioses. The animalswere presented before the clinicians at Clinic, Department ofClinical Veterinary Medicine, Ethics and Jurisprudence, Collegeof Veterinary and Animal Science, Bikaner, India. The animalswere brought up and maintained traditionally in the nearbyvillages Udasar, Pemasar, Udramsar, Ridmalsar, Sagar andbesides those in and around Bikaner city.

Individual coprosamples, weighing 5-10 gram, from eachsuspected animals were collected in labeled polythene bagsduring morning hours brought to laboratory and processed formicroscopic examinations using standard techniques (Hansenand Perry1990; Bowman et al., 2003).

PREVALENCE OF GASTROINTESTINAL HELMINTHIOSES IN CAMEL(CAMELUS DROMEDARIUS) IN AND AROUND BIKANER#

S.R. Gupta1, A.P. Singh, S.K. Dixit2 and D.S. Meena3

Department of Clinical Veterinary Medicine, Ethics and JurisprudenceCollege of Veterinary and Animal Science


ABSTRACT

Prevalence of gastrointestinal metazoan parasites was investigated in 250 camels, aged 5 to 12 years, of either sex. Outof 250, 97 animals were positive for naturally acquired nematode parasites, with mean EPG 953.75 ranged 300-2000 pergram of faeces. The overall prevalence of gastrointestinal helminths was 38.8%. In the 97 positive animals, monospeciesinfestation was more prevalent (86.60%) than infestation with more than one spp. (13.40%). None of the sample waspositives for trematodes and/or cestodes infections. The difference were signif icant (P<0.01). Trichostrongyle spp.infestation predominated. Where as only eight animals (8.25%) had concurrent infestation with Trichostrongyle spp. withStrongyloides spp. and five (5.15%) were positive for mixed infestation with Strongyloides spp. plus Trichuris spp. Hotand dry climate during major part of the year and browsing of the tree leaves could be attributed to lower prevalence of themetazoan infection in camels than other ruminants in the study area.

Key words: Camel, Trichostrongyle spp., Strongyloides spp. and Trichuris spp.

Results and DiscussionGrossly no adult worms were detected in the faeces of

camels. On the basis of faecal examinations, the overallprevalence of gastrointestinal helminths in camels was 38.8%irrespective of age, sex and breed. Out of 97 positive cases, 84(86.60%) showed single and 13 (13.40%) showed mixedinfestation of parasitic eggs. None of the sample was positivesfor trematodes and cestodes infestation. Similar findings havealso been reported by earlier workers (Pathak et al., 1993b;Tanwar et al., 2005; Rajneesh, 2005; Kamani et al., 2008). Inpresent study low prevalence of gastrointestinal helminthosesis in conformity with Hayat et al. (1998), Rajneesh (2005) andDia (2006) 37.33%, 44.68% and 31.51%, respectively. It couldbe due to the fact that hot and dry climate in and around Bikanerand very low rain fall might have resulted in unfavourablecondition for development of propagative parasites in this studyarea.

Out of 97 positive samples, 84 (86.60%) samples revealedpresence of Trichostrongyle spp. eggs as a single infestation.Where as only eight (8.25%) samples revealed concurrentinfection Trichostrongyle spp with Strongyloides spp. and five(5.15%) samples with Strongyloides spp. plus Trichuris spp.eggs.

Parasitism is one of the most important constraint thataffects growth, development, nutrient utilization and workperformance of the metazoan infested hosts, exhibiting nonspecific clinical signs when brought to the clinics. Thehighest prevalence (86.60%) of Trichostrongyle spp. eggsfollowed by mixed infestation with Strongyloides spp.(8.25%) and Trichris spp. (5.15%). The profile of singleand mixed helminths infestations as reported herein

#1Part of M.V.Sc. Thesis and present posting Assistant Professor, Department of Veterinary Medicine, Apollo College of Veterinary Medicine, Jamdoli, AgraRoad, Jaipur (Raj). Email: [email protected] Scientist, Division of Health, CSWRI, Avikanagar-304501, Distt-Tonk, Rajasthan3Instructor on Deputation, post graduate education and research centre for livestock health and production, Jaipur, Rajasthan. RAJUVAS, Bikaner-334001.

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attributed to stronger ability of the Trichostrongyle wormto with stand and harsh (hot and dry) climate prevailing in theregion and non homogeneous groups of animal of the differentbreeds, age and variable activity levels of endocrine secretionin the circulation of host under study.

Highest prevalence of Trichostrongyle spp. in the presentstudy is in accordance with Chauve et al. (1990); Partani et al.(1996a); and Tekle and Abede (2001), who recordedprevalence of 95.00%, 82.37% and 94.60%, respectively, incamels. The prevalence of Strongyloides spp. (8.25%)recorded in the present study is similar to the earlier reports ofKumar et al. (1993), Tekle and Abede (2001) and Tanwar et al.(2005). Similarly the prevalence recorded for Trichuris spp.(5.15%) in the present study is in agreement to the study ofChauve et al. (1990), whereas, Partani (1998) has recorded22.45% prevalence of Trichuris spp. in camels of Bikanerarea. High prevalence of Strongyle spp. in the area seemssynchronously contributed by the in situ developing arrestedlarvae during post rainy season of short spell (Jacquit et al.,1996).

AcknowledgementsThe authors are grateful to the Dean, College of Veterinary

and Animal Science, Bikaner for providing the requisite facilities.

ReferencesBowman, D.D . et al. (2003) Georgia, Parasitology for

Veterinarian. 8 th ed. Saunders Publication, USA, pp.287-358.

Chauve, M. et al. (1990) Maghreb Veterinaire. 5: 35-36, 38-39.

Chharba, M.B and Gupta, S.K. (2006) J. Camel Pract. Res.13(2):81-87.

Dia, M.L. (2006) Trop. Ani. Hlth Prod. 38(1): 17-21.Hansen, J. and Perry, B. (1990) In: Annual Report of International

Laboratory for Research on Animal Disease, Narobi,Kenya. pp. 62-67.

Hayat, C.S. et al. (1998) J. Camel Pract. Res. 5:251-254.Irfan, M. (1984) Pakistan Vet. J. 4: 25-27.Irfan, M. (1984) Pakistan Vet. J. 4: 25-27.Jacquiet, P. et al. (1996) Veterinary Parasitol. 66:193-204.Kamani, J. et al. (2008) J. Camel Pract. Res. 15(2): 181-182.Kumar, D. et al. (1993) Prevalence of subclinical gastrointestinal

parasitism in the dromedary camels. Proc. V, NationalCongress of Veterinary Parasitology, Udgir, 21-23April., 1993.

Partani, A.K. et al. (1995) J. Camel Pract. Res. (2)1: 33 -36.Partani, A.K. et al. (1996a) J. Vet. Parasitol. 10(1):23-32.Partani, A.K. et al. (1998) J. Camel Pract. Res. 5(2): 255-256.Pathak, K.M.L. et al. (1993b) Indian J. Ani. Sci. 63: 30-31.Raisinghani, P.M. (1992) Helminthic diseases of dromedary camel

in India. Proc.1st International Conference, Dubai. 2-6Feb. 1992. (edt. Allen, W.R., Higgins, A.J., Mayhew,I.G., Snow, D.H. and Wade, J.F.) pp. 105-106.

Rajneesh (2005) Epidemiological studies on some infectiousdisease of camel (Camelus dromedarius). M.V.Sc.Thesis submitted to RAU, Bikaner.

Sharma, S.S. and Satija, K.C. (1974) Indian Vet. J. 51: 231-232.Sharma, N. (2000) Clinical, haemato-biochemical and

therapeutic s tudies in camels suffer ing fromallotr iophagia . M.V.Sc. Thesis submitted to RAU,Bikaner.

Tanwar, R.K. et al. (2005. Vet. Pract. 6(2): 133-134.Tekle, T. and Abebe, G. (2001) J. Camel Pract. Res. 8(1):39-42.

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IntroductionDigestive diseases were affecting the work efficiency and

productive performance and even death of the animal. Thus,ultimately these diseases cause great economic losses tothe owners and indirectly to the national economy also. Variousworkers have observed that disorders of digestive system areresponsible for high mortality in camels (Khanna et al., 1992;Singh and Gahlot, 1999; Al-Rawashdeh et al., 2000; Bengoumiet al., 2000; Nagpal and Purohit. 2001; Mehta et al., 2003;Tanwar et al., 2007 and Sena et al., 2006). The disorders ofdigestive system of camel are the important aspect that evinceschallenges to the veterinary clinician to arrive at a definitediagnosis of a particular problem. Among various disorders ofthe digestive systems inanition is a chronic problem,dehydration, being the major effect in acute diseases and shockis the important physiological disturbance in hyper acutediseases. Some degree of abdominal pain is usual in mostdisorders of the digestive system. Other manifestations includeabnormalities of prehension, mastication, swallowing,diarrhoea, haemorrhage, constipation and scanty faeces(Radostits et al., 2000). Singh et al. (1980) have describedsome common digestive disorders in camels which includespharyngitis, oesophagitis, diarrhoea, abomasitis, gastro-enteritis, impaction of omasum and abomasum, colic, tympanyand nodular disease of intestine, whereas, Rathore (1986)have recorded salivation, pharyngitis, indigestion, constipation,diarrohea, dysentery, spasmodic colic, flatulent colic, volvulus,incarceration and intussusception in camels.

HAEMATOLOGICAL CHANGE IN DIGESTIVE AILMENT OF DIFFERENTMANAGEMENTAL CONDITIONS IN DROMEDARY#

D.S. Meena, A.P. Singh, S. Kachhawaha1, S.K. Dixit2, Anil Ahuja, T. Sharma3 and H. Dadhich4

Department of Clinical Veterinary Medicine, Ethics and JurisprudenceCollege of Veterinary and Animal Science


ABSTRACT

Among haematological parameters the overall mean haemoglobin, PCV, erythrocyte count and total leukocyte count were found to be10.54±0.29 g%, 31.23±0.85%, 7.59±0.16 millions/cumm and 10.62±0.22 thousans/cumm, respectively. A significantly higher Hb, PCVand TEC were recorded in organized and male camels in comparison with unorganized and female camel groups, respectively. Inrespect with various disease conditions significantly lower values were recorded for these parameters in comparison with control.Thetotal leukocytic counts were found to be significantly higher in unorganized camels (9.83±0.10) in comparison with organized camels(9.05±0.11). In respect with age and sex no effect could be recorded on total leukocytic count. In all the disease conditions an increasein TLC was observed in comparison with control except for mange where a disease in TLC was noticed.The differential leukocyticcounts revealed a decrease in female camels compared to male camels for neutrophil counts, an increase in organized, female, adultand lactating camels for monocyte counts compared to unorganized, male, young and non lactating camels, respectively, and asignificantly higher mean value in organized, female and non lactating camels for eosinophils in comparison to organized, male andlactating camels, respectively. As such no effect could be recorded on lymphocyte and basophil counts due to age, sex, lactation andmanage mental conditions. In respect with various disease conditions a decrease in neutrophils on account of mange and as well aseosinophils due to enteritis and mange were recorded on comparison of mean values with values observed for healthy camels.

Key words: Camel, digestive disease and haematology

#Part of Ph.D.Thesis submitted to RAJUVAS, Bikaner, Present address: Instructor on Deputation, Post-graduate Education and Research Centre for Livestock Health and Production, Jaipur, Rajasthan, RAJUVAS, Bikaner-334001.1Ph.D. scholar, Department of Clinical Veterinary Medicine, E & J.; 2Senior Scientist, CSWRI, Avikanagar, Tonk, Rajasthan; 3Director, DPME, RAJUVAS, Bikaner.4Associate Professor, Department of Veterinary Pathology, CVAS, Bikaner

Materials and MethodsA total of 520 camels of both sex and different age groups

(3 year to 16 years) were included in the present study, whichwere either brought to the Medicine and Surgery clinic of Collegeof Veterinary and Animal Science, Bikaner, or belonged toindividual holding of the farmers in and around Bikaner. Thestudy was carried out from January to May. Out of 520 camels,280 clinical cases were selected for study from Medicine andSurgery Clinic of College of Veterinary and Animal Science,Bikaner, Veterinary Polyclinic, Bikaner. These camels werefurther classified according to diseases, having 40 camelseach. Other, 200 camels were selected from field belonging toindividual holding in and around Bikaner. After clinicalexamination of the animals, blood samples were collectedand processed as recommended by Jain (1986). The stasticalanalysis was carried out as per Snedecor and Cochran (1994)

Results and DiscussionHaemoglobin

The overall mean values of haemoglobin (g%) inorganised camels was found to be 12.15±0.20 g%, whereasfor the unorganised/private holdings camels the value was11.32±0.13 g%.

Statistical analyses revealed significant (P<0.05) impactof the type of holding on haemoglobin and it was higher inorganised camels compared to unorganised camels. Inrespect with sex the significantly higher haemoglobin (g%)was recorded in males in comparison with females. Likewise

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lactating females showed lower values for haemoglobincompared with non lactating female camels. However, inrespect with age as such no effect could be recorded as themean values of young and adult camels were comparable.Further while statistically analysing the effect of diseaseconditions on haemoglobin all the diseases showedsignificant decrease in haemoglobin in comparison withhealthy group. The lower mean haemoglobin was recordedfor mange followed by debility, pica, simple indigestion,impaction wound and enteritis.

The overall low means value (10.83±0.17 g%) ofhaemoglobin in female animals in comparison to overallmales (12.07±0.12 g%) could probably be due to the regularexcretion of iron in milk. The higher values of mean haemoglobinin adult female under organised condition in comparison toadult male in organised holdings could possibly be attributedto the better feeding conditions of female in organised sector.

The overall mean value of 12.15±0.20 recorded in animalsfrom organised sector corresponds well with the report of Gupta(2009) for healthy camels. The haemoglobin values recordedin animals under unorganised sector were found to be lower.This could be possibly attributed to the non supplementationof green fodder or mineral mixture in the diet. However, inspiteof low level of haemoglobin in the camel of the present study,no animal showed any clinical symptoms of anaemia.

Packed cell volume (PCV)The overall mean values were found to be 36.23±0.61%

and 33.91±0.41% in organised and unorganised/privateholdings, respectively. The value in organised young male andfemale were 34.8±1.17% and 34.8±1.27%, respectively,whereas these values for organised adult male and adultfemale were 36.2±1.17 and 39.1±0.80%, respectively.

Statistical analysis of data revealed significant (P<0.05)impact of the type of holding on PCV, i.e. organised animalsshowed significant higher values of PCV than unorganisedanimals. The organised group of adult male animals showedsignificant (P<0.05) lower values than organised adult femalecamels. But, under unorganised sector the male animalsshowed significant higher (P<0.05) values than female camels.However, irrespective of holdings male animals showedsignificantly (P<0.05) higher values than female camels. Allthe diseased animals showed significantly lower values ofPCV compared with healthy group of camels.

The overall mean PCV value of organised healthy animalsrecorded to be 36.23±0.61 in this study agrees well with thereports of Rathod (2006) and Gupta (2009) for camels. Rathod(2006) recorded average value of PCV in healthy camel to be35.50±1.45 % which fall in line with the average PCV valuesrecorded in study in text (36.23±0.61%) in animals kept underorganised conditions. In animals kept under unorganisedconditions, comparatively lower values of PCV were obtained.Lower PCV values recorded in the animals could be becauseof low serum copper and total protein level, as reported byRunnels et al. (1976), Seshi Reddy and Mahadevan (1977)and Samanta et al. (1995).

Total erythrocyte count (TEC)The overall mean values in organised camels were found

to be 9.08±0.10 millions/cumm and the values for unorganised/

private holdings were recorded to be 7.31±0.11 millions/cumm.The mean values in organised young male and female were9.06±0.24 and 9.09±0.12 millions/cumm, respectively,whereas these values for organised adult male and adultfemale were 9.39±0.15 and 8.77±0.23 millions/cumm,respectively.

Statistical analysis revealed significant (P<0.05) effect ofthe type of holding on TEC, i.e. organised animals showedsignificantly higher values of TEC than in unorganised animals.The organised group of adult male animals showedsignificantly (P<0.05) higher values than organised andunorganised adult female camels and unorganised adult malecamels than in females.

Overall male animals irrespective of holdings showedsignificantly (P<0.05) higher values than overall femalecamels. All the diseased animals Showed significantly lowervalues than healthy camels with lowest values in simpleindigestion condition. Regarding effect of milk production nonlactating animals showed significantly higher (P<0.05) valuesthan lactating animals.

The mean values of TEC for healthy animals reported byDongre (2000), Raghvendra et al. (2000) Sharma (2000), Mali(2002), Rathod (2006), Gupta (2009) agrees well with theresults of TEC recorded in the present investigation. Whereaslower values recorded in the present studies for variousdisease conditions are also in agreement with Mali (2002),Dixit et al. (2004), Rathod (2006) and Choudhary (2008).

Total leukocyte count (TLC)The overall mean values of TLC in organised camels were

found to be 9.05±0.11 thousand/cumm with 9.83±0.10thousand/cumm for the unorganised/private holdings. Thevalues in young and adult female were 9.50±0.14 thousand/cumm and 9.82±0.10 thousand/cumm, respectively, whereasthese values for organised male and female were 9.61±0.10and 9.78±0.19 thousand/cumm, respectively.

Statistical analysis revealed significant (P<0.05) effect ofthe type of holding on TLC, i.e. unorganised animals showedsignificant higher values of TLC than organised animals. Theorganised group of adult female animals showed significant(P<0.05) higher values than adult male camels, but as suchthere was no change in unorganised holdings. Overall maleanimals irrespective of holdings showed no difference withfemale camels. All the diseased animals showed significantlyhigher values of TLC except for camels suffering from mangeshowed lower mean values and impaction exhibitedcomparable mean values with control group. These twogroups recorded Significant lower (P<0.05) values than healthyanimals. In unorganised group non lactating animals showednon significant difference from the values of lactating animals.

The mean values of TLC for healthy animals reported byDongre (2000), Raghvendra et al. (2000) Sharma (2000), Mali(2002), Rathod (2006), Gupta (2009) agrees well with the resultof TLC recorded in the present investigation.

Differential leukocyte count (DLC)The overall mean values for unorganised and organised

population were found to be 53.27±0.35 and 53.23±0.49%,40.55±0.35 and 39.35±0.42%, 3.28±0.08 and 3.90±0.21%,3.35±0.21 and 2.80±0.07, 0.11±0.02 and 0.18±0.06% for

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neutrophil, lymphocyte, monocyte, eosinophil and basophilcounts, respectively. Statistical analysis of data revealedsignificant effect of type of holdings on monocyte and eosinophilcounts with higher values of monocyte and eosinophil inorganised group compared to unorganised group. Whereas,in case of monocyte, the effect of age appeared to be significantand in respect with sex, the neutrophils were recorded to behigher in males and monocytes as well as eosinophils werenoticed to be higher in females.

Among diseased group of animals neutrophils in mangeand wounded camels were significantly lower than the valuesof healthy animals. The lymphocytes and monocytes ofwounded animals were also significantly (P<0.05) higher thanhealthy group of animals. Similarly, eosinophils weresignificantly (P<0.05) higher in mange suffering camels incomparison to other disease conditions and healthy camels.

Though, statistically significant variations were noticed invarious groups of animals but apparently on observing thedata not much variation could be observed and as such itcould be concluded that there was no effect of organisationalconditions as well as other factors like sex, age and lactationon differential leucocyte count. The values of differentialleucocyte count recorded in various groups were incorroboration with the earlier reports of Talvelkar et al. (1980),Juneja (1996) and Gupta (1999), Dongre (2000), Rathod et al.(2008) and Gupta (2009).

ReferancesAl, Rawashdeh-O.E. et al. (2000) J. Zoo Wildlife Med. 31(3): 335-

358.Bengoumi, M. et al. (2000) Revue-d’ Elevage-et-de-medicine-

veterinaire-des-pays-Trpicaux. 53(2):132-135.Choudhary, B.S. (2008) Clinical studies of digestive disorder in camels

(Camelus dromedarius). M.V.Sc. thesis submitted to RAU,Bikaner.

Dixit, S.K. et al. (2004) Vet. Pract. 5(2): 114-116.Dongre, R.A. (2000) Haemato-biochemical studies in relation to trace

mineral status in camel (Camelus dromedarius). M.V.Sc.Thesis, submitted to RAU, Bikaner.

Gupta, D. (1999) Clinico-biochemical and haematological studieson ketosis and milk fever in cows. M.V.Sc. Thesis, CVAS,RAU, Bikaner.

Gupta, S.R. (2009) Clinico-therapeutic studies on Camel helminthiosisin and around Bikaner. M.V.Sc. Thesis, submitted toRajasthan Agricultural University, Bikaner-334001.

Jain, N. C. (1986) Schalm¢s Veterinary Haematology. 4th ed. Lea andFebiger, Philadelphia.

Juneja, A. (1996) Clinical studies on hypocalcaemia in crossbredcows. M.V.Sc. Thesis, submitted to, RAU, Bikaner.

Khanna, N. D. et al. (1992) Calf mortality in Indian camels. In proceedingsof the First International Camel Conference, Dubai, 2nd - 6th

February 1992.Mali, M. M. (2002) Clinico-therapeutic studies on gastrointestinal

disorders in camels (Camelus dromedarius). M.V.Sc.thesis submitted to RAU, Bikaner.

Mehta, S.C. et al. (2003) Indian J. Ani. Sci. 73(8): 873-874.Nagpal, G.K. and Purohit, G.N. (2001) Indian Vet. J. 78(9): 787-789.Radostits, O.M. et al. (2000) Veterinary Medicine. 9th ed. W.B. Saunders

Co. Ltd. London.Raghvendra, S. et al. (2000) Indian J. Ani. Sci. 70(1): 54-55.Rathod, A. (2006) Therapeutic studies on Sarcopticosis in camels

(Camelus dromedaries). M.V.Sc. thesis submitted to RAU,Bikaner.

Rathod, A. et al. (2008) Vet. Pract. 9(2): 104-109.Rathore, G.S. (1986) Camels and their management. 1st ed. Publication

and Information division, Indian Council of AgriculturalResearch, New Delhi, India. pp. 34- 47, 118-126.

Runnells, R.A. et al. (1976) Principles of Veterinary Pathology. 7th ed.,Scientific Book Agency, Calcutta. pp. 481-484.

Samanta, A.K. et al. (1995) Indian Vet. J. 75(10): 1031-1034.Sena, D.S. et al. (2006) J. Camel Pract. Res. 13(2): 171-172.Seshi Reddy, B. and Mahadevan, V. (1977) Indian Vet. J. 54: 561-

565.Sharma, N. (2000) Clinical, haemato-biochemical and therapeutic

studies in camel suffering from allotriophagia. M.V.Sc.Thesis, submitted to R.A.U., Bikaner.

Singh, A.P. and Gahlot, A.K. (1999) Studies on Sarcoptes scabieiinfestation in dromedary . Paper presented at 17 th

International; Conference of the World Association for theAdvancement of Veterinary Parasitology, held atCopenhagen (Denmark) from 15-19 August 1999.Abst. (C6.10). p. 23.

Singh, R.D. et al. (1980) Disease of camels. 1st ed. Seven Seas,Aghapura, Hyderabad, India.

Snedecor, G. W. and Cochran, W. G. (1994) Statistical Methods. OxfordIBH Publishing Co., Calcutta. pp. 83-106.

Talvelkar, B.A. et al. (1980) Indian Vet. J. 57: 640-643.Tanwar, R.K. et al. (2007) Haematuria associated with calcification of

renal medulla in camels (Camelus dromedarius). Paperpresented in Int. Camel Conf. on Recent Trends in CamelidResearch and Future Strategies for Saving Camels, Org. byCVAS, Bikaner, held between 16-17 Feb., 2007. pp. 12.

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IntroductionRespiratory tract infections are of a common occurrence

in various species of domestic and farm animals (Mohamedand Abdelsalam, 2008). Viruses, bacteria, fungi and parasiteshave been incriminated as the main causative agents ofpneumonia in mammals (Jubb et al., 1993; Cotran et al., 1999).These agents may represents risk to camels, other livestockand even human population (Abou, 2000; Ogunsan et al., 2000;Bardonnet et al., 2002; Teshome et al., 2003). Although camelsare well adapted to their environment and seem to be spreadfrom devastating epidemic infections which threaten otherlivestock species in the same region, there are however anumber of economically important diseases that affect camels(Kane et al., 2003; Dia, 2006). Pulmonary diseases are amongthe emerging problems of camels that are causingconsiderable loss in production and death (Bekele, 2007;Zubair et al., 2004; Kane et al., 2003).

Pasteurellosis is quite prevalent in South West Asiancountries and East Africa among cattle, buffaloes and camels.The disease is usually seen in adult animals but all age groupcan be affected. The morbidity of the disease is low but mortalityrate can reach 50-80% among affected animals (Schewartzand Dioli, 1992). Among acute fatal diseases Haemorrhagicsepticaemia (H.S) has been reported from most of the camelbreeding countries (Higgin, 1986). Respiratory infectionscommonly occurring in camel result from adverse physicaland physiological stress combined with viral and/or bacterial

AN OUTBREAK OF PNEUMONIC PASTEURELLOSIS IN CAMEL(CAMELUS DROMEDARIES): CLINICAL BEHAVIOR, METEOROLOGICAL

VARIABLES, MANAGEMENT AND CONTROL

Subhash Kachhawaha1, D.S.Meena2, M.K. Srivastava1, Dheeraj Singh3,R.K. Tanwar4, A.P. Singh and J.P. Kachhawa5

Department of Clinical Veterinary Medicine and JurisprudenceCollege of Veterinary and Animal Science, Bikaner-334001, Rajasthan

ABSTRACT

The outbreak was correlated with prevailing climatic condition to find out climatic stress factor. The lungs were examined by visual andpalpation for the presence of lesions after the bacteriological and histopathological investigation of collected lungs. A total of 590camels were under study during the outbreak. The overall morbidity, mortality and case fatality rates were 13.2%, 5.42% and 41.02%,respectively in February, 2010. In November-December, 2010, 14 camels died, the overall morbidity, mortality and case fatality rateswere 48.94%, 29.79% and 60.87%, respectively. The clinical signs observed were high temperature (102°F), depression, oedematousswelling of the throat and neck region, dyspnoea and death within 72 hours after the onset of signs. Therapeutic efficacy ofcombination of antibiotic tests were 30% in Group A (amoxycillin + clavlanic acid), 40% in Group B (enrofloxacin + gentamicin) and72.7% in Group C (sulphadimidine + oxytetracycline) during the period of 5 days treatment. Correlation of pneumonia with meteoro-logical parameters indicated that sudden change in temperature and relative humidity occurred before the outbreak. The study showsthat relative humidity in February, 2010 (r=0.547) and in November-December, 2010 (r=0.125) is positively correlated with mortality.

Key words: Camel, pasteurellosis, meteorological, arid region, pneumonia

infections. Pasteurella haemolytica pneumonia occurs asresults of these interactions (Brogden et al., 1998; Martin, 1996).

Pasteurella spp. are important primary and opportunisticpathogens as well as common commensals of the upperrespiratory tract of various domestic animals (Loubinoux, 1999).Animal farm management interventions such as weaning, longdistance houling, draught, climatic factors including suddentemperature changes, poorly ventilated barns andovercrowding are important in worse infection, malnutrition,feed changes and parasitism are some other important factors.

Pneumonia is a major disease of domestic animals.Outbreaks occur in camel as well as other animal’s worldwide(Selman and Wiseman, 1983; Al-doughaym et al., 1999). Theeconomic losses due to pneumonia in camels are representedby loss of weight, losses due to condemnations during meatinspection and mortality rate (Mohmoud et al., 1988; Al-Ani,1990; Mohamed et al., 1990). Survived animals show reducedlung capacity and weight gain efficiency. This infection is stillamong the most economically important disease of camelthroughout the world due to high mortality. Therefore, immediateidentification of disease and management to minimizeeconomic losses is imperative. This paper represents asystematic study on an outbreak of pneumonic pasteurellosisin camels in Pali district of Rajasthan with their clinicalmanifestation, pathological findings, correlation with climaticparameters and their management.

1Ph.D Scholar and corresponding author email:[email protected] on Deputation, Post graduate education & Research centre for Livestock Health and Production, Jaipur, Rajasthan, RAJUVAS, Bikaner-3340013Programme Coordinator, Krishi Vigyan Kendra, Central Arid Zone Research Institure, Pali- Marwar,,Rajasthan, India4Retd. Director Clinic, Rajasthan University of Veterinary and Animal Sciences, Bikaner-334001, Rajasthan5M.V.Sc. Scholar

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Material MethodsRespiratory diseases were noticed on mass scale in

camel with subsequent death in the month of February,November and December, 2010 in Pali district of Rajasthan.The camel flocks were kept under open grazing system ofmanagement. The clinical examinations of affected animalswere conducted as per protocol mentioned in the standardtext of clinical diagnosis nasal swabs from clinical affectedanimals and lung samples from dead animals were collectedunder aseptic conditions and processed microbiologically forthe identification of etiological agents as per the standardtechniques (Cowan and Steel, 1970). All the laboratory studywas done with the help of Incharge, RDDL, Jodhpur(Rajasthan).

A. Tissue samples for histopathological examinationThe lungs were examined by visual examination and

palpation for the presence of lesions; after which samples forbacteriological and histopathology investigations werecollected from lungs. Samples for bacteriology were placed inpolythene bags and kept in flask containing ice and taken tothe laboratory (Barrow and Feltham, 1993; Cheesbrough,2000). Tissue specimens for histopathological examinationswere fixed in 10% neutral, buffered formalin for at least 48hours prior to processing. The tissues were processed byroutine paraffin embedding technique for microscopy (Luna,1968; Humason, 1972)

B. Isolation and identification of bacteriaSwabs from the lungs were streaked on plates containing

blood agar enriched with 5-6% sheep blood and incubated at37°C for 24 hours with further re-incubation for 36-72 hours, ifno growth was observed after 24 h. Single colonies of differentcolony types were picked on plates containing blood and onMacConkey agar. The pure cultures were Gram-stained.Identification of bacterial agents was through cultural,morphological and in some cases biochemical characteristics(Barrow and Feltham, 1993; Cheesbrough, 2000).

The outbreak was correlated with prevailing climaticcondition to find out climatic stress factors on precipitation ofrespiratory disease in these animals. Information’s ofmeteorological parameters (max/min temperature,temperature fluctuation, vapour pressure and relative humidity)were recorded at Regional Research Station of Institute. Formanagement of episode, three different groups of drugs wereadministered. It consisted in group A (Amoxycillin+Clavulanicacid), Group B (Enrofloxacin+ Gentamycin), group C(Sulphadimidine+ Oxytetracycline), administered at theirstandard dose rate along with anti-inflammatory andantihistaminic drugs for a period of 5 days, depending uponresponse in animals.

Results and DiscussionA total 590 camels were under risk of infection during the

outbreak, which revealed morbidity rate 13.2% (78/590),mortality rate 5.42% (32/590) and case fatality rate 41.02%(32/78) indicating highly acute respiratory disease. Thisepisode appeared in the month of February, 2010; whereclimatic experience a change of temperature from lower tohigher side in this arid climatic zone of country. The first case of

outbreak was observed on 7 February and last case wasreported on 28 February, 2010. Daily morbidity and mortalitypattern has been shown (Fig.5, 6 and 7) which indicates themortality started after 6 days of outbreaks and peaked for aperiod of 6-7 days and then declined with immediate medicalintervention. This pattern shows virulence of organism in camel.

In second time outbreak occur in November andDecember. Daily mortality, morbidity and case fatality were29.79% (14/47), 48.94% (23/47) and 60.87% (14/23),respectively, are shown in Fig. 5, 6 and 7 which indicated thatfirst case seen in 22nd November and last in 28th November2010.

In both the outbreak change in humidity, temperature andvapour pressure due to the rain show that the mortality,morbidity and case fatality rates are high.

In the month of November-December, 2010 the relativehumidity (r=0.125) and vapour pressure (r=0.215) are positivelycorrelated with mortality. The morbidity (r=0.005) and mortality(r=0.023) are also positively correlated with temperature butweakly correlated.

In the month of February, 2010 the morbidity (r= -0.061)and mortality (r= -0.399) are negatively correlated withtemperature. The relative humidity (r=0.547) is strongly andpositively correlated with mortality. It shows if relative humidityincreases the mortality of animal increases. There was nopositive effect of vapour pressure because it is negativelycorrelated with morbidity (r= -0.261). The mortality increaseswith the vapour pressure since it is positively correlated(r=0.364).

The main clinical symptoms comprised of polypnoea,respiratory distress, nasal discharge, high fever, listlessness,poor appetite and death in 2-3 days. Similar clinical symptomswere observed by Momin et al. (1987).

Post-mortem findings included congested lungs, pleura,trachea, oesophagus and liver. Similarly typical lesions likewidespread haemorrhages, enlarged lymph nodes, fibrinouspericarditis, haemorrhagic lesions of abomasum and necroticliver. In most of cases, small intestine was haemorrhagic andinflammed. Haemorrhagic entritis in upper alimentary tract iscommonly seen in septicaemia and generalized form. Kidneywas also hyperaemic, necrosed and pulpy in most of cases.Meteorological parameters during outbreaks were correlatedwith occurrence of pneumonia in camel, which have beendepicted in Fig. 5, 6 and 7, which clearly show that animalswere under a period of environmental temperature fluctuation,which suddenly rise and then decline. Similarly vapourpressure and relative humidity fluctuation were also recorded.It appeared that this sudden variation in temperature andrelative humidity might act as precipitating factors to trigger theinfection. It is concluded that in this climatic zone during thesemonths the animals need extra managemental care.

In this clinical episode, authors experienced a suddenvariation in environmental temperature and relative humidityand as a result pneumonia precipitated, similar climatic stressprecipitated respiratory condition were also observed in goat.In a similar study, Mcllroy et al. (1989) found highly significantcorrelation coefficients between the percentage lungcondemnations in sheep and the rain/windchill factor prevailingduring the same months. Heat stress has also been identified

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Fig. 1: Haemorrhage of throat region with enlargement of lymph gland Fig. 2: Intestine haemorrhage

Fig. 3: Gross examination of lung having haemorrhage Fig. 4: Histopathologically lung showed fibrinopurulent bronchopneumoniawith alveoli and bronchioles full of neutrophills associated with congestionand oedema (H & E X 250).

Fig. 5: Comparative study of temperature, morbidity and mortality outbreaksin February, 2010.

Fig. 6: Comparative study of temperature, morbidity and mortality inNovember-December, 2010.

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as manageable risk factors to mitigate pneumonia (Friend,1991).

The cultural examination and subsequent biochemicalcharacterization of the clinical specimens revealed thepresence of Gram negative bipolar organisms, characteristicof Pasteurell multocida.

Fibrinoprulent bronchopneumonia was diagnosed in theaffected lungs. Grossly, consolidation of anteroventral lobesand microscopically, congestion, oedema haemorrhage,necrosis, alveoli and bronchioles full of fibrin and neutrophilsand rare fibrinous thrombi within vessels were observed inaffected area (Fig. 4) lesions similar to what has been observedbefore (Asil et al., 2004).

Treatment of pneumonia often requires antimicrobialtherapy. The decision of antimicrobial therapy depends on thesensitivity of the targeted microorganism and thepharmacokinetics of the drug to achieve the therapeuticconcentration at the site of infection and thus clinical efficacy(Mckellar et al., 2004). Although antimicrobial agents includingpenicillins, tetracyclinees, aminoglycosides and recentlyfluoroquinolones are frequently used for treatment ofpneumonia in camels. There is paucity of information on thesusceptibility of respiratory tract bacterial pathogens in camelsto these drugs (Radostits et al., 2007).

This clinical episode of pneumonia in camel was treatedwith various groups of antibiotics. Numbers of animals ingroups were decided on the basis of response of a particulargroup to save the life of maximum affected camels. Initially,antibacterial drug combination was immediately instituted ingroup A and group B, but unfortunately, only few animalssurvived. This fact is very much associated with drug resistanceto multiple antibiotics, which warrants study in this direction.Therapeutic efficacy of different combinations of antibioticstested were 30% (3/10), 40% (8/20), 72.7% (35/48) in Group A(Amoxycillin + Clavulanic acid), Group B (Enrofloxacin +Gentamicin) Group C (Sulphadimidine + Oxytetracycline)respectively along with melonex 30 ml and Avil 15 mlintramuscular for in 5 days of treatment. This treatment trialindicated the variations in susceptibility of organism to differentclass of antibiotics. Our results showed that oxyteracyclineand sulphadimidine were highly effective in the treatment ofpneumonia in camel. Therefore, it can be predicted thatresistance may exist against the other group of medicine whichis extensively used for treatment of pneumonia in farm animal.

The figure 5 shows that abrupt change in temperatureduring both the outbreaks, it helps spreading the pasturaellosisdue to stress so mortality and morbidity occurs.

The figure 6 shows that increase in relative humidity during

Fig. 7: Comparative study of relative humidity, morbidity and mortality inFebruary, 2010.

Fig. 9: Comparative study of vapour pressure, morbidity and mortalityin February, 2010.

Fig. 8: Comparative study of relative humidity, morbidity and mortalityin different outbreaks in November-December, 2010.

Fig. 10: Comparative study of vapour pressure, morbidity and mortalityin November-December, 2010.

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both the outbreaks helps for the propagation of bacteria alreadypresent in nasopharynx (Rhimler and Rhoades, 1989) due towhich mortality and morbidity occurs.

The figure 7 shows that increase in vapor pressure due tothe relative humidity during both the outbreaks helps to increasethe bacterial growth to spread the diseases due to whichmortality and morbidity occurs.

AcknowledgementsThe authors are thankful to Director, CAZRI, Jodhpur and

Deupty Director, Animal Husbandry, Pali-Marwar for immensesupport and encouragement.

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Asil, T. et al. (2004) Studies on pathology changes of condemnedlungs of one humped camels (Camelus dromedaries).Deutscher Tropentag October 5-7, Berlin.

Ayers, J.L. et al. (1992) Goat hand book. Pennsylvania State University,University Park.

Bardonnet, K. et al. (2002) Royal Soc. Trop. Med. Hyg. 96(4):383-386.Barrow, G. I. and Feltham R.K.A. (1993) Cowan and Steel’s Manual for

the Identification of Medical Bacteria. 3rd ed., CambrigeUniversity Press, pp. 128-238.

Bekele, S.T. (2007) Tropical Ani. Hlth Prod. 40(1):25-28Bhardwaj, B. et al. (2006) Indian J. Vet. Pathol. 30:1Brogden, A.K. et al. (1998) Vet. Res. 29:233-54.Cheesbrough, M. (2000) District Laboratory Practice in Tropical

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Cotran, R.S. et al. (1999) Robbins Pathologic Basis of Disease. 6th

ed. W.B. Saunders, Philadelphia, pp. 697-775.Cowans, S.T. and Steel, K.J. (1970) Manual for identification of medical

bacteria. 2nd ed. Cambridge Univ. Press London. pp. 420-422.

Dia, M.L. (2006) Trop. Ani. Hlth. Prod. 38: 17-21.Dyson, D.A. et al. (1981) T. J. Med. Microbial. 14(1):89-95.

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London.Humason, L.G. (1972) Animal Tissue Technique. 3rd ed. W.H. Preeman

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Mauritaure. Revue Africaine de Sante et de Prod. Animales.1(1): 31-37.

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Martin, W.B. (1996) Comp. Immunol. Microbiol. Infectious Dis. 19:171-179.

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5(2): 207-212.Mohamed, G.E. et al. (1990) The status of camel diseases in the

Sudan and future out load. Proceedings: symposium oncamel breeding, disease and their control, Arab Organizationfor Agriculture Development (AOAD), Algeria. pp. 251-269.

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diseases of Cattle, Horses, Sheep, Pigs and goats. 10th ed.Saunder Elsevier, Philadelphia. USA. pp: 747.

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IntroductionInfectious bursal disease (IBD) is an important

immunosuppressive and acute highly contagious viral infectionof young chickens (three to five weeks of age) with morbidityapproaching 100% and mortality up to 50% depending onvirus strain and flock history. The causative agent of the diseaseis IBD virus (IBDV), which belongs to genus Avibirna virus(Leong et al., 2000) of the family Birnaviridae (Dobos et al.,1979). It has two linear double stranded RNA (dsRNA) enclosedin a non-enveloped icosahedral capsid. There are two serotypesof IBDV, serotype 1 is pathogenic for chickens, whereas serotype2 strain is non-pathogenic and do not cross protect againstserotype 1. The virus is highly stable and has a tendency topersist in the environment despite thorough cleaning anddisinfection. Chicken is the only avian species known to besusceptible to clinical disease. Infections caused by IBDVmay exacerbate infections with other pathogens agents andreduce the hosts’ immune responses to vaccination. IBDinteracts with other respiratory pathogens like IBV (infectiousbronchitis virus), ND (Newcastle disease) and LPAI (lowpathogenic avian influenza) by interfering with effectiveimmunization, enabling exacerbation of respiratory infections(Rosenberger and Gelb, 1978; Toro et al., 2006). IBD is alsoassociated with increased severity of bacterial diseases,including salmonellosis, colibacillosis, staphylococcosis, andclostridial infections. In most respiratory diseases of poultry,E. coli is the final pathogen to express sequentially, followingIBDV and other immunosuppressors, and a primary respiratorydisease (Igbokwe et al., 1996). Diagnosis and confirmation of

INFECTIOUS BURSAL DISEASE (IBD) PLAYING A TRIGGERING ROLEIN E. COLI AND MYCOPLASMA INDUCED RESPIRATORY DISEASE

COMPLEX IN BROILERS

V. Gowthaman, S.D. Singh, K. Dhama, R. Barathidasan, Anjaneya and P. Bhatt1

Avian Diseases Section Division of PathologyIndian Veterinary Research Institute, Izatnagar, Bareilly-243122, U.P

ABSTRACT

Infectious bursal disease (IBD) is an important immunosuppressive and acute highly contagious viral infection of youngchickens. IBDV interacts with other respiratory pathogens like IBV, NDV, E. coli and MG by interfering with effectiveimmunization, enabling exacerbation of respiratory infections. IBD is also associated with increased severity of bacterialdiseases. In most respiratory diseases of poultry, E. coli is the final pathogen to express sequentially, following IBD. A totalof 18 suspected cases brought for disease diagnosis to avian diseases section, IVRI, Izatnagar with the history ofrespiratory diseases were investigated. The general clinical signs consisted of dullness, depression, ruffled feathers,cyanotic combs and wattles, reduced feed intake and water consumption, uneven/stunted growths; watery white diar-rhoea and soiling and pasting of vent. Severe respiratory distress was prominently noticed. Post-mor tem examinationrevealed haemorrhagic tracheitis, caseous plaques in tracheal mucosa, severe diffuse pulmonary oedema, congestionand consolidation, pleurisy, severe thoracic/abdominal airsacculitis, adhesive pericarditis, f ibrinous perihepatitis andascites. Mortality rate ranged between <1 and 60%. Out of 18 samples screened 9 were found to be positive for IBD.E.coli and Mycoplasma was isolated from 12 cases out of 18 cases. It confirms that IBD also playing major role inpredisposing the chicken to E.coli and Mycoplasma-induced respiratory disease complex.

Key words: Infectious bursal disease (IBD) E. coli, Mycoplasma, respiratory disease complex, broilers

1Assistant Professor, C.V.A.Sc., G.B.P.U.A.T, Pantnagar, Uttarakhand, India

an IBDV infection are based on characteristic pathologicalchanges in the bursa of fabricius and histopathologicalinvestigations combined with the demonstration of viralantigens by immunohistochemistry. Viral antigens can bedemonstrated by agar gel precipitation test (AGPT), ELISA(Manoharan et al., 2004). This paper states the role of infectiousbursal disease in E. coli and CRD (chronic respiratory disease)respiratory disease complex in broilers.

Materials and MethodsStudy location and chickens

A total of 18 suspected cases form different poultry farmsbrought for disease diagnosis to avian diseases section, IVRI,Izatnagar with the history of respiratory diseases wereinvestigated. All the farms were located 100-150 km radius ofBareilly region.

Collection of clinical samplesThe history, clinical signs and managemental procedures

were recorded in a standard Proforma. Necropsy was carriedout on freshly dead or euthanized ailing birds with the historyand symptoms of respiratory diseases. The lesions werephotographed and gross findings were recorded. Grosslyaffected bursa, trachea and lungs were collected forhistopathology, bacterial isolation, virus detection and/oragarose gel immunodiffusion test (AGID).

AGID for diagnosis of IBDVThe AGID test was performed as described in Witter (1962).

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Fig.4: Bursa of Fabricius; lymphoid atrophy and vacuolation of folliculararea, and increased interfollicular space

Fig.1: Abdominal air sacs; thickened and opaque air sacs typical ofCRD in poultry.

Fig.2: Liver; Fibrinous or fibrinopurulent pericarditis and perihapatitis;surface of liver covered by thin, opaque, grayish-white gelatinousmaterial typical of colibacillosis.

Fig.3: IBD: (a) Bursa of Fabricius; diffuse haemorrhage on the mucosalfolds, (b) thigh muscles; petichiae to ecchymotic haemorrhages onthe thigh muscles

Briefly, 1% agarose gel was prepared in 8% sodium chloridesolution. Four mL of agarose was layered on a clean glassslide (75x25 mm). After solidification, a set of seven wells (acentral well and six peripheral wells) was cut with the help ofgel cutter. The IBDV reference antiserum (Avian diseasesection, IVRI) was placed in the central well while IBDVsuspected bursal triturates were placed in the surroundingwells. Each well was filled with 25 µL of the sample. Once thesamples were loaded, the plate was left undisturbed in a moistcontainer and incubated at room temperature. The slide wasexamined daily for 2-3 days. A narrow beam of light in a darkbackground was used to examine the presence of precipitationline and the results were recorded.

Bacteriological studiesPieces of internal organs sent to Centre for Animal Disease

Research and Diagnosis (CADRAD), IVRI, Izatnagar forbacterial isolation.

RNA, DNA extraction and polymerase chain reaction (PCR)Total RNA was extracted from tumour tissues and normal

mammary gland with TRI reagent (Product No. T9424, Sigma-Aldrich, USA) as per the manufacturer’s instructions. TheDNase-1-treated RNA was used to synthesize cDNA usingrandom hexamers. DNA from tissue homogenate wasextracted using W izard® Genomic DNA Purification Kit(Promega, USA) as per the manufacturer’s instructions. PCRfor detection of common respiratory viral pathogens wascarried out with published primers (Ottiger et al., 2010).

Results and DiscussionHistory and clinical signs

The broiler birds were reared under deep liter system ofmanagement and had no history of vaccination. The age of thebroiler flocks ranged from 3-5 weeks. The managementalconditions were poor. The general clinical signs consisted of

225


dullness, depression, ruffled feathers, depression, cyanoticcombs and wattles, reduced feed intake and waterconsumption, uneven/stunted growths; hock sitting posture,watery white diarrhoea and soiling and pasting of vent. Severerespiratory distress was prominently noticed. The birds didnot respond to antibiotic treatment. Mortality rate rangedbetween <1 and 60%.

Gross and histopathological findingsPost-mortem examination revealed haemorrhagic

tracheitis, caseous plaques in tracheal mucosa, severe diffusepulmonary oedema, congestion, and consolidation, pleurisy,severe thoracic/abdominal air sacculitis (Fig. 1), adhesivepericarditis and fibrinous perihepatitis (Fig. 2) , ascites,nephritis-nephrosis complex, haemorrhagic/swollen bursawith bloody/creamy exudates in lumen (Fig. 3), and petechialto ecchymotic haemorrhages in fascial sheaths of leg andthigh muscles. Microscopically, haemorrhagic tracheitis,fibrinous necrotizing bronchopneumonia, fibrinohaemorrhagicairsacculitis, fibrinous pericarditis, interstitial nephritis lymphoidatrophy of spleen, and bursa (Fig. 4) were the prominentfindings recorded.

Detection of viral and bacterial pathogensOut of 18 samples screened 9 were found to be positive

for IBDV E. coli and/or Mycoplasma were isolated from 12cases out of 18 cases which include all positive cases of IBDV.Screening for common viral respiratory pathogens did not yieldany positive results.

The entire positive cases (9/9) for IBDV had the infectionof E. coli and/or Mycoplasma. It confirms that IBDV also playinga major role in predisposing the chicken to RDC. This studyconcurs with the reports of Toro et al. (2006) who found IBDVinteractions with other respiratory pathogens like IB, ND andLPAI interfering with effective immunization, enablingexacerbation of respiratory infections. Clinical signs observedin our study include anorexia, depression, ruffled feathers,

diarrhoea, prostration and death. Lesions of bursal atrophy/inflammation, haemorrhages in the thigh and pectoralmuscles were similar to those described previously(Eterradossi and Saif, 2008). In most of the PM cases thelesions like adhesive pericarditis and fibrinous perihepatitiswere suggestive of secondary infection with E. coli and MGand in some cases E. coli was isolated. These findingcorroborate with the studies of Mitra et al. (2004) who suggestedin most respiratory diseases of poultry, E. coli and/orMycoplasma are the final pathogens to express sequentially,following infections with IBDV and other immunosuppressors.The pathogenesis involves the failure of bacterial clearancefrom the circulating blood and colisepticemia is establishedeven by relatively apathogenic strains of E. coli (Rosenbergeret al., 1985). Mycoplasma is known for concurrent infectionswith other infectious agents such as Escherichia coli or virusescausing respiratory infections. Although classified as a primarypathogen, Mycoplasma often causes great damage whenacting together with other pathogens.

ReferencesDobos, P. et al. (1979) J. Virol. 32(2): 593-605.Eterradossi, N. and Saif, Y.M. (2008) Infectious bursal diseqase. In

Diseases of Poultry. 12th ed., Edited by Y. M. Saif. Ames, IA:Blackwell Publishing. pp. 185-208.

Igbokwe, I.O. et al. (1996) Rev Elev Med Vet Pays Trop. 49(2), 110-113.

Leong, J.C. et al. (2000) Birnaviridae. In: Virus Taxonomy.Classification and Nomenclature of Viruses. M.H.V.Regenmortel, C.M. Fauquet, D.H.L. Bishop, et al. (eds),Academic Press, London, 481-490.

Manoharan, S. et al. (2004) Vet. Res. Commun. 28(4): 339-346.Mitra, M. et al. (2004) Trop. Anim. Hlth Prod. 36(7):627-32.Ottiger, H.P. (2010) Biologicals. 38(3):381-8.Rosenberger, J. K. et al. (1985) Avian Dis. 29:1094-1107.Rosenberger, J.K. and Gelb, J., Jr. (1978) Avian Dis. 22(1), 95-105.Toro, H. et al. (2006) Avian Pathol. 35(6): 455-464.Witter, R.L. (1962) Avian Dis. 6: 478-492.

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IntroductionThiacloprid is a member of a new group of insecticides

called neonicotinoids. It is known to act as a nicotinicacetylcholine receptor (nAChR) agonist and binds to andactivates nAChR at its nicotine and acetylcholine binding site,affecting synaptic transmission and leading to disruption ofthe nervous system. The immune system is a frequent targetorgan of toxic insult following chronic or acute exposure toenvironmental chemicals, therapeutic drugs, abused drugsor radiation. The health implications of these immunedysfunctions are increased risk of infectious diseases;development of neoplasia; autoimmune disorders andallergies. The extensive use of insecticides has been criticizedin recent years due to their persistence in the environment andtheir accumulation in the living tissues of organisms.Thiacloprid is a member of a new group of insecticides calledneonicotinoids. It was introduced in response to restrictionson the use of the most commonly used pesticides, chlorpyrifosand diazinon (TDC Environmental, 2003). Since its launch in1999, thiacloprid has been registered in 120 countries for useon termites and households pests (Liu et al., 2005). Becauseof its selectivity for insects (Tomizawa and Casida, 2005),thiacloprid is thought to be safer than other pesticides (Felsotand Ruppert, 2002), and it now has the highest production ofall insecticides worldwide (Ware and Whiteacre, 2004). It isknown to act as a nicotinic acetylcholine receptor (nAChR)agonist (Tomizawa and Casida, 2005) and binds to andactivates nAChR at its nicotine and acetylcholine binding site,affecting synaptic transmission and leading to disruption ofthe nervous system (Anatra-Cordone and Durkin, 2005). Thepotential risks from thiacloprid have gained significant attentiondue to its worldwide use and persistence in crops. Laboratorystudies of it have shown genotoxicity in both rats (Karabay andOguz, 2005) and cultured human human lympho-cytes (Demsiaet al., 2007). Moreover, neurobehavioural deficits were foundin rats exposed to neonicotinoids in the uterus (Abou-Donia etal., 2008). The immune system is a frequent target organ of

IMMUNOLOGICAL EFFECTS OF SUB CHRONIC EXPOSURE TOTHIACLOPRID INSECTICIDE IN GALLUS DOMESTICUS

Saloni Goyal1and H.S.Sandhu2

Department of Veterinary Pharmacology and ToxicologyCollege of Veterinary Science

Guru Angad Dev Veterinary and Animal Science University, Ludhiana-141004, Punjab, India

ABSTRACT

In the present study estimation of total immunoglobulins and enumeration of B and T lymphocytes in blood after repeatedoral administration of thiacloprid at the dose rate of 10 mg/kg/day revealed no significant alterations in Gallus domesticus.The observed findings in the present study indicated that repeated thiacloprid exposure in poultry birds did not adverselyaffect the immune status of Gallus domesticus and thus is immunologically safer insecticide

Key words: Toxicity, Gallus domesticus, thiacloprid, insecticide, immune system

1Research Associate. Email: [email protected] (Reprint requests are solicited at this address).2Dean, College of Veterinary Science, GADVASU, Ludhiana, Email: [email protected]

toxic insult following chronic or acute exposure to environmentalchemicals, therapeutic drugs, abused drugs or radiation. Thehealth implications of these immune dysfunctions areincreased risk of infectious diseases; development ofneoplasia; autoimmune disorders and allergies. (Berlin et al.,1987) Due to its importance in assessing drugs and non drugchemicals, immunotoxicity testing is required by manyregulatory agencies (Holsapple, 2003). Although there is alarge body of literature addressing immune responses duringinsecticide exposure, the immunotoxicity of thiacloprid is poorlyunderstood. Therefore, this study aimed to increase ourunderstanding of the immunotoxic effects of sub chronicThiacloprid exposure.

Materials and MethodsThe present study was conducted on one and half-year

old-layer Gallus domesticus. The birds were procured andhoused in pens at the layer house of the poultry farm, GuruAngad Dev Veterinary and Animal Sciences University,Ludhiana, India. The birds were provided with standard feedand clean water ad libitum and were acclimatized to the layerhouse for ten days prior to the commencement of the study.The thiacloprid (Alanto 240 SC, Thiacloprid 21.7%), used inthis study was commercially obtained from the authorizeddealer of Bayer Cropscience Limited, Sabarkanta, Gujarat.Based on the recommended concentrations of thiacloprid usedfor crop protection, a suitable non-lethal dose of thiaclopridwas selected for the administration by oral route. All animalprocedures were performed in accordance with the guidelinesfor the care and use of experimental animals of the Committeefor Purpose of Supervision of Experiments on Animals(CPCSEA).

Fifty two birds were randomly divided into nine groups. Asinstructed by (CPCSEA) to decrease the number of birds onlyfour groups of 4 birds were kept as healthy control (Group I, IIIII and IV), in which no insectic ide was administered,whereas the birds were stressed giving distilled water by

227


using catheter with 2 ml glass syringe. The birds in group V, VI,VII, VIII, IX and X contained six birds each and were administeredthiacloprid in repeated oral dose of 1 mg/kg/day for 15, 30, 45,60, 75 and 90 days, respectively.

The requisite amount of insecticide was suspended inwater and administered directly into the proventriculus of thebird by using catheter with 2 ml glass syringe and/or tuberculinsyringe. The birds were starved overnight and their bodyweights were recorded before the start of treatment. All thebirds were weighed after 15 days and doses of thiaclopridcorrected according to changes in body weights.

To study the immunological parameters, blood sampleswere collected directly by cardiac puncture on 0, 30, 60 and 90treatment days from Groups I, II, III and IV, respectively and on0, 30, 45, 60, 75 and 90 days in the birds of treatment groupsV,VI ,VII, VIII, IX and X, respectively. EDTA containing blood wasused for enumeration of B and T cells, and blood containingno anticoagulant for estimation of total immunoglobulins. Forcollection of serum, blood samples were collected in non-heparinized test tubes and were allowed to clot at roomtemperature. The serum separated from clot was thencentrifuged at 2500 rpm for 15 min and clear supernatant serumwas collected for estimations

Two widely used procedures for measuringimmunoglobulin levels are single radial immunodiffusion (RID)(Meguire et al., 1976) and zinc sulphate turbidity. The latter is a“salting out” procedure that depends on the biochemicalproperties of immunoglobulin in relation to the characteristicsof zinc sulphate. In this study, this technique was performed aspreviously described (Mcewan et al., 1970; Pfeiffer et al., 1977).Lymphocytes were separated by centrifuging whole blood onpremix commercial preparation of Ficoll (Histopaque, SigmaFicoll- Paque, Pharmacia) as described by Boyum (1968).Separation of T and B lymphocyte was done by Nylon woolcolumn separation method as described by Julius et al. (1973).Statistical analysis was performed using one-way ANOVAfollowed by a Tukey’s HSD post-hoc test for multiplecomparisons with SPSS 16.0 for computer programme. Thevalues of P<0.05 were considered statistically significant.

Results and DiscussionRepeated oral administration of thiacloprid at dose rate of

1 mg/kg/day for 90 consecutive days did not produce anysignificant alterations in the levels of total immunoglobulins(Table 1) in Gallus domesticus. The observed findings on totalimmunoglobulins are in agreement with work of Sharma (2007)after the administration of atrazine but are in contrast to studiesof Chauhan and Tripathi (2002) on malathion and Garg et al.(2004) on fenvalerate, endosulphan where decrease in totalimmunoglobulin levels have been documented. Totalimmunoglobulin levels significantly increased afterImidocloprid administration (Mohanty et al., 2011). Similarobservations have been made by Klucinski et al. (1996), whofound that workers exposed to multiple insecticides hadincreased serum IgM, IgG and IgA concentrations, theseincreases may be attributable to inflammation induced bythese insecticides.

Immunoglobulins are class of glycoproteins that areproduced by immunocompetent cells as a result of antigenicstimulation and react specifically with the given antigen

resulting into different visible reactions (Sinha, 2007).Immunoglobulins thus provide protective covering to wholebody and protect the organisms from attack of different micro-organisms and toxins. The non-significant alteration in totalimmunoglobulins levels in the present study suggested thatthiacloprid has no adverse effect on the immune system ofpoultry birds.

Repeated oral administration of thiacloprid at the doserate of 1 mg/kg/day produced no significant effect on the numberof B and T lymphocytes in birds. Various workers have reportedvariable effects of pesticides on the B and T lymphocytes indifferent species of animals. Suppression of humoral immuneresponse has been reported with lindane in cats and rabbits(Burkatzkaya 1963) and rats (Dewan et al., 1980; Saha andBanerjee, 1993). Depression of cell mediated immunity in miceon exposure to chlordane without adverse effect on humoralimmunity has been found by Menna et al. (1985). Garg et al.(2004) documented decline in B lymphocyte number in broilerchicks after administration of fenvalerate and endosulphanbut not with monocrotophos for 8 weeks and decrease in Tcell count in broiler chicks exposed to fenvalerate,monocrotophos and endosulphan. Dimov and Simenov (1985)however observed stimulatory effect on the immune responseof sheep treated with organophosphate insecticides. Methylparathion in mice elevated humoral immunity whereascypermethrin and carbyl suppressed humoral immunity andpropoxur decreases both B and T lymphocytes count (Sharma,2007).

The lymphocytes are the central cells of the immunesystem. The number and functional capacity of circulatingperipheral blood lymphocytes reflects the overall state ofimmunocompetence of an organism (Talwar and Gupta, 1992).Numerical and maturation changes in the lymphocytepopulation can reflect significant alteration in the immunesystem. Certain xenobiotics are reported to upregulate level ofapoptosis in lymphocytes which may account forimmunosuppression. Xenobiotics including pesticidesinduced oxidative stress may contribute to the immunotoxicity(Burns et al., 1996).

The observed findings in the present study indicated thatrepeated thiacloprid exposure in poultry birds did not adverselyaffect the immune status of Gallus domesticus and thus isimmunologically safer insecticide.

ReferencesAbou-Donia, M. B. (2008) J. Toxicol. Environ. Hlth. 71: 119-130.Anatra, M. and Durkin, P. (2005) Imidacloprid. Human health

assessment and ecological risk assessment. Final report.Syracuse Environmental Research Associates, Inc., NewYork, SERA TR 05- 43-24-03a.

Berlin, A. et al. (1987) Immunotoxicology. Martinus Nijhoff Publishers.Boyum, A. (1968) Scandinavian J. of Clinical and Laboratory

Investigation 21: 1Burkatzkaya, E. N. (1963) Gigiena I Sanitariia 28: 29.Burns, L. A. et al. (1996) The Basic Science of Poisons. In: Klaassen

C D. (ed) Casarett and Doull’s Toxicology 5th ed. McGrawHill, New York. pp. 355-402.

Chauhan, R. S. and Tripathi, B. N. (2002) In: Veterinary ImmunologyPathology Theory and Practice. International BookDistributing Co. Lucknow.

Demsia, G. et al. (2007) Mutat. Res. 634: 32-39.

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Dewan, A. et al. (1980) J. of Environ. Sci. Hlth. 15: 395.Dimov, I. and Simenov, S. P. (1985) Veterinario-Medicinski Nauki. 22:

38-42.Felsot, A.S. and Ruppert, J.R. (2002) J. Agric. Food Chem. 50: 4417-

442Garg, U. K. et al. (2004) International Immunopharmacology. 4:

1709-22.Holsapple, M.P. (2003) Toxicol.185: 193-203.Julius, M. H. et al. (1973) European J.l of Immunology. 3: 645-49.Karabaym, N.U. and Oguz, M.G. (2005) Genet. Mol. Res. 4: 653-662.Klucinski, P. et al. (1996) Int. J. Occup. Med. Environ. Health. 9: 103-

11.Liu, Z. et al. (2005) Proc. Natl. Acad. Sci. U S A. 14: 8420-8425.Mcewan, et al. (1970) Clin. Chim. Acta. 27: 155-163.Meena, J. H. et al. (1985) Toxicological Letter. 24:45-52.Meguir, T.C. et al. (1976) JAVMA. 169: 713-718.Mohanty, M. et al. (2011) African J. Pharmacy Pharmacol. 5(18):

2106-2114.

Saha, S. and Banerjee, D. (1993) Bulletin of EnvironmentalContamination and Toxicology. 51: 795.

Sharma, R. P. (2007) Veterinary Toxicology Basic and ClinicalPrinciples. Academic Press Inc. San Diego. pp. 289-301.

Sinha. B. K. (2007) Principles of Immunology Student’s Compendium.International Book Distributing Co. Lucknow.

Talwar, G. P. and Gupta, S. K. (1992) A Handbook of Practical andClinical Immunology. Vol 1, CBS Publishers and Distributors,New Delhi.

TDC Environmental, (2003) Insecticide market trends and potentialwater quality implications. TDC Environmental, LLC, SanMateo, CA, USA.

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Ware, G.W. and Whitacre, D.M. (2004) An Introduction to Insecticides.4th ed., MeisterPro Information Resources, A division ofMeister Media Worldwide, Willoughby, Ohio, USA.

Table 1: Effect of sub-chronic oral administration of thiacloprid (1 mg/kg/day) on total immunoglobulins (g/100ml) and B and Tlymphocytes (%) in Gallus domesticus. Figures in the bracket indicate number of birds

Tim e (day s) Trea tm ent

GROUPS 0 15 30 45 60 75 90 To tal imm unoglobulins (mean ± S.E)

C ontrol 1.55±0.11a (4)

- 1.72±0.17a

(4) - 1.67±0.20a

(4) - 1.61±0.11a

(4) Th iaclopr id

1.56±0.20a

(6) 1.68±0.15a

(6) 1.59±0.16a

(6) 1.46±0.17a

(5) 1.40±0.15a

(6) .44±0.14a

(5) 1.41±0.15a

(6)

B Ly mphocy tes (m ean ± S.E) C ontrol

1.85 ±0.5a (4)

- 2.50 ±0.6 a

(4) - 2.75±0.9a

(4) - 2.00 ±0.4a

(4) Th iaclopr id

2.50±0.9a

(6) 2.17 ±1.0a

(6) 2.00 ±0.5a

(6) 2.80±0.5 a

(5) 2.67 ±0.7a

(6) 2.40 ±0.4a

(5) 2.17 ±1.0a

(6)

T Ly mphocy tes (M mean± S.E) C ontrol 18.00±1.5a

(4) -

17.50±1.8 a (4)

- 16.80 ±0.8a (4)

- 6.00±1.9a

(4) Th iaclopr id 16.67±1.3a

(6) 16.17±1.4 a

(6) 18.37±0.8a

(6) 6.60±1.7a

(5) 19.17 ±1.1a

(6) 17.40±1. 3a

(5) 15.83 ±1.0a

(6)

Mean with atleast one common superscript are similar within the group (P<0.05)

229


IntroductionDonkeys and mules are mainly used as pack in hilly terrains

and in cart for transportation of materials in plains. These are alsoused in agricultural operations in some parts of India. Indiandonkeys are small in body size and weight. The mules producedthrough covering mares with indigenous donkeys are also smallin size. The breeding for mule production in the mares is mostlynatural except some organized farms where artificial insemination(AI) is done using fresh diluted semen or frozen semen. Due tosmall size of Indian donkeys it is difficult for them to cover themares. To overcome this problem NRCE imported Poitou maleand female donkeys from France during 1990 for the up-gradationof indigenous donkeys and production of superior quality mules.These Poitou donkeys are being maintained at Bikaner situatedin arid zone of Rajasthan, where minimum and maximumtemperature varies widely. These animals have adapted well inarid conditions and produced progenies through AI. The studywas initiated to assess the seminal characteristics of Poitou jacksunder arid conditions of Rajasthan.

Materials and MethodsExperimental animals: The study was carried out on eight

apparently healthy adult jacks (Equus asinus) of Martina Francabreed (Poitu) maintained at Equine Production Campus, Bikanerunder uniform conditions of feed and management. They werehoused individually in open housing system.

Semen collectionThe semen was collected in artificial vagina (AV) using an

oestrus jenny as a dummy in the morning hours before feedingbetween 7-8 AM during summer and 8-9 AM during winter seasons.The erected penis of excited jack was washed with lukewarmwater before mounting over jenny. The semen was collected inpre warmed semen collected bottle attached with AV. Immediatelyafter collection semen was brought to laboratory under coveredposition to protect semen from direct sun light and to avoidtemperature shock for semen evaluation and processing. Semensamples were collected at different intervals from all the jacksthrough out the experimental period.

Macro and microscopic analysis of semenThe colour and consistency of the semen were assessed by

SEMINAL CHARACTERISTICS OF EXOTIC JACKS UNDER ARIDCONDITIONS IN INDIA

R. A. Legha and Yash PalNational Research Centre on Equines, PB No. 80, Bikaner-334 001, Rajasthan, India

ABSTRACT

The present study was conducted on eight adult Poitou jacks to assess the seminal characteristics under arid conditions of Rajasthan.These donkeys were reared under standard feeding and management conditions. The semen was collected from the jacks at regularintervals throughout the year and analyzed for microscopic and macroscopic parameters. Mean gel-free semen volume, pH, initialmotility, progressive motility, sperm concentration, live sperm count were recorded as 45.04±0.60 ml, 7.19±0.01, 84.31±0.27%,75.43±0.30%, 293.39±4.99 x106 ml-1 and 77.47±0.36%, respectively, irrespective of season. Individual jack variation was observed invalues of seminal characteristics. Some seminal parameters were significantly higher during the breeding season.

direct visual observations. The semen volume was directlymeasured in marked (graduated) semen collecting bottle beforeand after removing the gel if any. The pH of the semen wasmeasured by pH paper strip within 5 min after semen collection.The initial and progressive motility of spermatozoa were assessedby putting a drop of fresh semen without any dilution on pre-heatedglass slide and covered with cover slip under a light microscope.Live and dead spermatozoa were counted using eosin-nigrosinstaining dye technique (Dott and Foster, 1972). Spermconcentration of each ejaculate was estimated in gel free semenby standard methods. The data were statistically analyzedaccording to Snedecor and Cochran (1994).

Results and DiscussionAppearance and consistency

The colour of semen was off white to creamy in appearance.In general, creamy appearance was observed very frequentlyirrespective of season as well as jack. Gupta et al. (2003) and Royet al. (2003) also observed that Jack’s semen was ‘off white’ to‘creamy’ in colour, however, the creamy appearance was veryfrequent irrespective of any month or season. The consistency ofthe semen varied among jacks from thin to thick. However, thevariation in consistency was irrespective of the season.

Semen volume: Total semen volume, gel volume and gel-free semen volume was recorded. Total semen volume recordedat different intervals ranged between 10 to 250 ml. The total semenvolume per ejaculation was significantly (P<0.05) higher duringbreeding season (April-October) as compared to non-breedingseason (November-February). Total semen volume wassignificantly (P<0.05) different between jacks (Table 2). Variationsin volume may be due to individual stallion variation, abnormalities,age, workload and length of teasing etc. (Pickett et al., 1988 a, b).

The gel was observed in semen at few occasions in all thejacks’ irresepective of season and its volume ranged between 0-220 ml. Gel volume was significantly (P<0.01) higher duringbreeding season as compared to non-breeding season (Table1). Gel volume showed significant (P<0.05) differences betweenjacks. In general, gel-free semen volume semen variedsignificantly as it was maximum and significantly higher duringsummer than other seasons. However, no much seasonalvariations in semen pH values were observed in present study

Key words :Seminal characteristics, Jack, arid condition

230


Table 1: Jack’s semen characteristicsCharacteristics Pooled Seasons April - October November- February Total volume (ml) 49.58±0.84 (712) 51.24±1.16 (456) 46.39±0.99* (256) Gel volume (ml) 5.06±0.64 (712) 6.30±0.94 (456) 1.70±.48** (256) Gel free volume (ml) 45.04±0.60 (712) 45.14±0.78 (456) 44.92±0.92 (256) Initial motility (%) 84.31±0.27 (712) 84.38±0.34 (456) 83.27±0.49 (256) Progressive motility (%) 75.43±0.30 (712) 76.19±0.35 (456) 74.06±0.53** (256) Consistency Thin to thick Thin to thick Thin to thick Colour Off white to creamy Off white to creamy Off white to creamy pH 7.19±0.01 (712) 7.19±0.01 (456) 7.20±0.02 (256) Sperm concentration (x106 ml-1)

293.39±4.99 (702) 291.49±3.17 (451) 300.06±4.83 (251)

Live sperm (%) 77.47±0.36 (288) 78.10±0.41 (170) 76.55±0.64* (118) Abnormal sperm (%) 2.47±0.04 (278) 2.42±0.05 (167) 2.51±0.07 (111)

Table 2: Jack-wise averages of semen characteristics

Ch 1 2 3 4 5 6 7 8 V 47.26

±1.07ad (133)

51.82 ±4.33aef

(23)

97.33 ±15.29b

(15)

35.0 ±0.35cg

(8)

44.57 ±1.11di (140)

51.61 ±1.73ej

(119)

45.18 ±1.27aci

(135)

54.21 ±2.67f gj

(139) G 0.75

±0.30adf (133)

5.95 ±0.27b

(23)

54.33 ±15.83c

(15)

4.38 ±0.38bdeg

(8)

1.61 ±0.48d

(140)

1.94 ±0.62d (119)

1.85 ±0.54d

(135)

10.72 ±2.03e (139)

Gf 46.50 ±0.99ae

(133)

48.91 ±4.23ade

(23)

43.0 ±5.39aef

(15)

30.63 ±3.58bf g

(8)

42.96 ±1.17cf

(140)

49.91 ±1.46ae

(119)

43.33 ±1.15cd

(135)

43.49 ±1.74cf g

(139) M 78.49

±0.67a

(133)

89.13 ±0.59be

(23)

90.0 ±0.67be

(15)

89.38 ±1.06bde

(8)

84.50 ±0.56c

(140)

86.43 ±0.54d

(119)

87.48 ±0.43de

(135)

83.67 ±0.57c

(139) P 68.65

±0.68a (133)

86.09 ±0.97b

(23)

85.0 ±1.15b

(15)

83.75 ±1.71b

(8)

75.18 ±0.58c

(140)

77.56 ±0.58d

(119)

78.26 ±0.50d (135)

74.32 ±0.61c

(139) C Thin-Thick Thin-Thick Thin-Thick Thin-Thick Thin-Thick Thin-Thick Thin-thick Thin-

Thick Cl Off- white

to creamy Off-white to creamy

Off-white to creamy

Off- white to creamy



Off-white to creamy


pH 7.14 ±0.03acd

(133)

7.09 ±0.03abc

(23)

7.13 ±0.05abcde

(15)

7.15 ±0.03abcde

(8)

7.23 ±0.03bcde

(140)

7.21 ±0.03cde

(119)

7.19 ±.02de

(135)

7.22±0.02e

(139) Cn 272.59

±3.75a (133)

263.0 ±12.47a

(22)

253.26 ±17.98ab

(15)

206.75 ±6.86bf

(8)

309.21 ±3.65c

(140)

338.30 ±4.85d

(116)

355.26 ±5.09e

(131)

217.42 ±4.81f

(137) L 72.30

±0.84a

(54)

- - - 77.66 ±0.66bd

(57)

79.25 ±0.75bcd

(63)

79.50 ±0.64cd

(58)

78.23 ±0.76d

(56) Ab 2.44

±0.09a

(53)

- - - 2.64 ±0.11a

(57)

2.34 ±0.11a

(56)

2.54 ±0.08a

(56)

2.43 ±0.09a

(56) Within parenthesis are number of observations. Superscription with different letters differ significantly (P<0.05) in rows. Ch- Characteristics;V- Total volume (ml); G- Gel volume (ml); Gf- Gel free volume; M- Sperm motility (%); P- Progressive motility (%); C- Consistency; Cl- Colour;Cn- Sperm conc. (x106); L- Live sperm count (%); Ab- Abnormal sperm (%)

Gel-free semen volumes was almost at par during breedingand non-breeding season (Table 1). Gel-free semen volumewas recorded at par between most of the jacks except significant(P<0.05) variations between some jacks (Table 2).

It was also observed that production of gel was more duringbreeding season as compared to non-breeding season. Theanimal variation was observed in regards to total semenproduction and gel production per collection. The average gelfree semen per collection was 45.04±0.60 ml with variation insemen production by individual jack. It was observed that the gelfree semen production was not much affected by season ofbreeding though it was slightly more in breeding season ascompared to non-breeding season. Gupta et al. (2003) reportedthat the gel free semen volume of exotic jacks ranged from 41.25to 52.20 ml/ejaculate and gel free volume was significantly higher

during autumn season (50.31 ml) followed by summer (44.54ml), winter (44.08 ml) and spring (43.26 ml). Similar seasonalvariations have also been reported by Roy et al. (2003). However,in horse stallion, gel-free semen volume was reported maximumin June (Pickett et al., 1976). Such variations in semen volumewere also observed in stallion semen (Pickett and Voss 1972;Squires et al., 1979).

pH of semenIt should also be assessed immediately after collection. Overall

average of the pH value of semen was observed as 7.19±0.01asrecorded at different intervals ranged between 10-110 ml. with slight individual variations in pH value. Gupta et al. (2003)also reported that the semen was neutral or slightly alkalineandranged from 6.98 to 7.40 and during different seasons, pH of

231


due to inclusion of more number of jacks and replications.

Sperm concentrationThe sperm concentration is one of the most important

parameter affecting fertility. It will provide not only an indication as towhether the sample is worth using but it will also determine thenumber of mares that can be covered from a single ejaculate. Thevalues for spermatozoa concentration in semen sample vary widely;ranging from 136 to 550x106 ml-1. All values within this range canbe considered acceptable and appropriate for use in AI (Ricketts,1993). In present study, overall sperm concentration was293.39±4.99 x106 ml-1. Individual animal variation was observedin values of sperm concentration as maximum and minimumsperm concentration was 355.26±5.09 x106 ml-1 and 206.75±6.86x106 ml-1. The values observed were 291.49±3.17 x106 and300.06±4.83 x106 ml-1 for breeding and non-breeding season,respectively. These values are within the range as reported byGupta et al. (2003) and Pal et al. (2009). They reported the range ofsperm concentration from 266.90 x106 to 346.55 x106. Amongdifferent seasons; spermatozoa concentration was significantlyhigher during spring and summer than autumn and winter. Roy etal. (2003) also reported the sperm concentration in exotic donkeyswas significantly (P<0.05) more in summer than in autumn andwinter. In horse stallions, maximum spermatozoa output and libidoof stallions was reported during spring and summer and leastduring fall and winter. These changes in reproductive capacitycoincide with the natural breeding season of the mare (Hafez andHafez, 1993). The present findings also seem to be like that inhorse stallion and indicate spring and the summer as the breedingseason for donkeys. Factors affecting sperm concentrationsinclude season, ejaculation frequency, testicular size, breed, age,environmental factors etc.

Morphology: In this study, the semen was stained withnigrosine-eosin method for live-dead sperm count andexamination of abnormalities of the sperm. The overall average ofthe abnormal sperm count was 2.47±0.04% (range 2.34 to 2.64%).The percentage of abnormal sperm was non-significantly differentbetween the non-breeding and breeding season. Roy et al. (2003)reported that the mean of abnormal sperm percentage wassignificantly different in three seasons. It was highest in summer(5.78) followed by winter (4.74) and autumn (3.03). Fertile stallionwill normally show no more than 10-15% of any singlemorphological defects. Samples with high percentage ofabnormalities can be compensated to a certain extent if the totalvolume and sperm concentration are high; as abnormalspermatozoa have no apparent detrimental effect on viablespermatozoa in the semen sample (Varner and Schumacher,1991). The sperm abnormality was with in the acceptable range inthe present study.

Live:dead ratio: An indication of the percentage of live:deadspermatozoa within a sample can be obtained initially by a motilityassessment. The live:dead ratio gives an accurate indication ofsperm viability. The overall live sperm count observed was77.47±0.36% (range 55.5-93.7%). The live sperm counts weresignificantly (P<0.05) higher in breeding season as compared tonon-breeding season. The individual animal variation was alsoobserved as the highest and lowest being 79.50 and 72.30%. Asample with a live dead ratio in excess of 60% is consideredappropriate for AI (Ricketts, 1993). Gupta et al. (2003) reported live

sperm concentration more than 80%. Further live sperm per centduring September to January was significantly lower than that inMarch to June months. During summer, live sperm counts weremaximum and significantly higher than autumn and winter. Theresults of present study are also matching with the results of Guptaet al. (2003). In horse stallion semen also higher percent of livesperm during spring as compared to winter was observed(Dowsett and Pattie, 1987). However, Roy et al. (2003) reportednon-significant effect of season on live sperm percentage.

Motility: The percentage of motile spermatozoa and inparticular those showing progressive motility is a good indicationof the number of viable spermatozoa. The correlation betweenmotility and morphologically normal spermatozoa is reported tobe 0.63 (Long et al., 1993). In the present study the overall initialsperm motility observed was 84.31% (range 60-95%). The overallprogressive sperm motility observed was 75.43±0.30% (range50-90%). The similar results of sperm mass motility andprogressive motility have been reported by Gupta et al. (2003).They further observed that sperm motility and progressive motilitywere maximum and significantly higher during summer comparedto autumn and winter. Roy et al. (2003) reported that the mean ofmass motility was highest in autumn which was significantly morethan that of summer and winter. The mean motility of spermatozoahas been reported as 81.93±6.27% (Morais et al., 1994). Gastal etal. (1997), however, reported a lower motility score in donkeys(3.8±0.1). The variation in the motility score could also be attributedto the subjective nature of assessment which may differ fromperson to person.

ReferencesDott, H.M. and Foster, G.C. (1972) J. Reprod. and Fert. 29: 443-445.Dowsett, K. F. and Pattie, W. A. (1987) J. Reprod. and Fert. (Suppl.) 35:

645-647.Gastal, M.O. et al. (1997) Theriogenology. 47: 627-638.Gupta, A. K. et al. (2003) Indian J. Ani. Sci. 73: 986-991.Hafez, E. S. E. and Hafez, B. (1993) Horses: Reproduction of Farm

Animals. (eds) Hafez, B and Hafez, E S E. Lippincott Williamsand Wilkins, Philadelphia. pp.192-217.

Long, P.L. et al. (1993) J. Equine Vet. Sci. 10: 298-300.Morais et al. (1994) Brazilian J. Vet. Res. Ani. Sci. 31: 145-151.Pal, Y. et al. (2009) Indian J. Ani. Sci. 79: 1028-1029.Pickett, B. W. and Voss, J. L. (1972) Reproductive management of stallions.

In proceedings of the 18th Annual Convention of Am. Assoc. ofequine practitioners, San Francisco, California. 18: 501-531.

Pickett, B. W. et al. (1976) J. Ani. Sci. 43: 617-625.Pickett, B.W. et al. (1988a) Comparison of seminal characteristics of

stallions that passed or failed seminal evaluations. Inproceedings of 11th International Congress on Animal Productionand Artificial Insemination, June 26-30 Vol. 3 Paper 380. UniversityCollege Dublin, Dublin, Republic of Ireland.

Pickett, B.W. et al. (1988b) Seminal characteristics and total scrotal width(TSW) of normal and abnormal stallions. In proc. of the 33rd

Annual Convention of Am. Assoc. of Equine Pract. pp. 485-518.Ricketts, S.W. (1993) Equine Vet. Edu. 5: 232-237.Roy, A. K. et al. (2003) Indian J. Ani. Sci. 73: 620-22.Snedecor, G.W. and Cochran, W.C. (1994) Statistical Methods. 8th ed.

Oxford and IBH Publishing Co. New Delhi.Squires, E. L. et al. (1979) J. Reproduction and Fert. (Suppl.) 27: 7-12.Varner, D. D. and Schumacher, J. (1991) Equine Medicine and Surgery.

Vol. 2. 4th ed. In: Colahan, P.T., Mayhew, I.G., Merritt, A.M.and Moore, J.N. (eds). Am. Vet. Pub. Incorporated, Goleta,California, pp. 847-948.

232


IntroductionThe skin is the largest organ of the body and the

anatomic and physiological barrier between the animaland environment. The skin infections are caused bybacteria, viruses, parasites and fungi. Dermatitis causedby bacteria is one of the common skin disorder causesproduction losses in the camel. In this study, incidence ofperifolliculitis, folliculitis and furunculosis were recordedby histopathological examinations along with haemato-biochemical parameters and with the identification ofspecific bacteria responsible for their occurrence incamel.

Materials and MethodsFor the present study, 187 camels showing frank skin

lesions were examined to ident ify the var ious skindisorders, which are commonly prevailing in camels.Histopathological aspects of these skin problems werestudied along with the haemato-biochemical parametersand iso lat ion of bacter ial organ isms by standardtechniques. The areas of skin showing frank macroscopiclesions were preserved in 10 per cent formal saline andprocessed mechan ically for paraf f in embedding byacetone and benzene technique (L ill ie, 1965). Thesections of 4-6 micron thickness were cut and stainedwith haematoxylin and eosin method of staining. The bloodsamples were also collected for haemato-biochemicalstudies of these cases by standard techniques.

Results and DiscussionThe present study showed 11.68 per cent cases of

dermatitis. Perifolliculitis grossly showed a wide spread

PATHLOGICAL AND HEMATO-BIOCHEMICAL OBSERVATIONS OFPERIFOLLICULITIS, FOLLICULITIS AND FURUNCULOSIS IN CAMEL

(CAMELUS DROMEDARIUS)

Manisha Mathur, Hemant Dadhich, Sandeep Khare1, A.P. Singh2 and D.S. Meena3

Department of Veterinary PathologyCollege of Veterinary and Animal Science


ABSTRACT

In the present investigation, a total of one hundred eighty seven skin biopsies showing frank macroscopic lesions werecollected from camel for study of histopathological aspects of various types of dermatitis along with haemato-biochemicalparameters and isolation of organisms. Perifolliculitis, folliculitis and frunculosis conditions were recorded in 11.68 percent cases of dermatitis in camel. All haematological parameters showed an increase except lymphocyte count and MCVexhibited decrease, likewise the biochemical parameters i.e. serum glucose, total protein and serum albumin also showedsignificant effect of these conditions. Various bacteria isolated from these conditions were S. aureus, S. epidermidis ,Pseudomonas sp. and Streptococcus sp. in which S. aureus, S. epidermidis were isolated from all the samples.

Key words: Perifolliculitis, folliculitis, furunculosis, camel

1Veterinary Officer, Incharge Veterinary Hospital, Jaitpur, Bikaner.2Associate Professor, Department of Clinical Veterinary Medicine, CVAS, Bikaner3Instructor on Deputation, Post-graduate Education and Research Centre for Livestock Health and Production, Jaipur, Rajasthan, RAJUVAS, Bikaner-334001.

alopecia on the various parts of the body (Fig. 1), whereasin folliculitis and furunculosis erythematous follicularpapules were usually seen on abdominal area.

Microscopically, perifolliculitis revealed exocytosis ofthe cells into follicular epithelium alongwith inflammatoryinfiltration of neutrophils and lymphocytes around the hairfollicle (Fig. 2). In folliculitis, there were hyperplasia ofepidermis and accumulation of inflammatory cells mainlyof neutrophils within follicular wall (Fig. 3) and lumina.Ulceration of follicu lar ep ithelium leading to dermalabscesses, perifollicular neutrophillic abscesses, foreignbody giant cells and vascular reaction. Sometimes therewas grannulomatous inflammation with suppuration andheavy neutrophilic infiltration. In furunculosis, there weredestruction of majority of follicular epithelium which wasdestroyed by the inflammatory reaction (Fig. 4) withrelease of hair shafts and keratin debris in the dermis.Pyogrannu lomatous inf lammatory inf iltration alsoobserved in few cases of furunculosis.

These gross and microscopic findings have alsobeen explained well by Jubb et al. (1993) in large domesticanimals and Scott et al. (1995) in the small animaldermatological disorders.

Haemato-biochemical studiesThe various haematological parameters in the cases

of perifolliculitis, folliculitis and furunculosis revealedsignificant variation in comparison to normal healthyanimals for all haematological parameters except PCV,basophil count and monocytes count. All parametersshowed an increase except lymphocyte count and MCV

233


exhibited decrease, likewise the biochemical parametersi.e. serum glucose, total protein and serum albumin alsoshowed significant effect of perifolliculitis, folliculitis andfurunculosis though both serum albumin and globulinincreased in comparison of normal healthy animals butA:G ratio remained unaffected. Almost similar findingswere reported by Banerjee (1962), Gowda et al. (1982)and Alhadrami (1997).

Bacterial isolationVarious bacteria isolated from these conditions were

S. aureus , S . ep idermid is , Pseudomonas sp . andStreptococcus sp. in which S. aureus, S. epidermidis wereisolated from all the samples where as Pseudomonas

sp. were isolated from 75 per cent of the total cases. S.aureus, S. epidermidis were highly sensitive to ampicillinand oxytetracyclin.

ReferencesAlhadrami, G.A. (1997) J. Camel Pract. Res. 4(1): 13.Banerjee, S. (1962) Am. J. Physio. 203: 1185.Gowda, B.K.K. et al. (1982) Indian J. Vet. Med. 2(1): 29-32.Jubb, K.V.F. et al. (1993) Pathology of Domestic Animals. 4th ed.

Academic Press, Inc. London, U.K.Lill ie, R.D. (1965) Histopathologic Technique and Practical

Histochemistry. Mc-Graw Hill Book Co., New York andLondon, U.K.

Scott, D.W. et al. (1995) Small Animal Dermatology. 5th ed. W.B.Saunders Co. London, U.K.

Fig. 4: Histological section of skin having furunculosis showing destructionof follicular epithelium, inflammatory infiltration predominantly ofmononuclears (H&E 200X).

Fig. 3: Histological section of skin having folliculitis showing accumulationof inflammatory cells in the wall of follicle (H&E 200X).

Fig. 2: Histological section of skin having perifolliculitis showing inflammatoryinfiltration of neutrophils, lymphocytes and haemorrhages around the hairfollicle (H&E 400X).

Fig. 1: Camel perifolliculitis with widespread alopecia

234


IntroductionIncreasing productivity is one of the main objectives in

animal production. Traditional breeding methods have led toincreased gains in some traits but gains are not easilyattainable in traits such as litter size which have low heritability.Exploiting the genetic variations underlying desired phenotypesis the goal of today’s animal producers and consequently,candidate genes of traits of interest have been searched forpossible relationships with such traits. Genetic studies onsheep prolificacy have indicated that litter size and ovulationrate can be determined by the action of single genes, namedfecundity (Fec) genes, with major effects (Davis 2004, 2005).One of them located on the X chromosome and known as theFecX locus is the Bone Morphogenetic Protein 15 (BMP15)gene. Six mutations, labelled FecXR (Rasa Aragonesa),Monteagudo et al. (2009), FecXH (Hanna) and FecXI (Inverdale),Galloway et al. (2000), FecXL (Lacaune), Bodin et al. (2007),FecXG (Galway) and FecXB (Belclare), Hanrahan et al. (2004)have been detected so far within the BMP15 gene. All the sixmutations of sheep BMP15 show the same phenotype:homozygous carrier ewes are sterile and heterozygous carriersshow increased ovulation rate as described by Davis et al.(2006) and Monteagudo et al. (2009). The genetic basis of thisheterozygote advantage is well understood. The BMP15 geneencodes bone morphogenetic protein 15 (also known asgrowth differentiation factor 9B, GDF9B), which is a member ofthe transforming growth factor â (TGFâ) superfamily. BMP15acts through a cascade of other proteins (the SMAD pathway)that are responsible for a huge diversity of cellular behaviours,including oocyte development and maturation. Without BMP15,oocytes continue to grow in the absence of granulosa cellproliferation until they are unable to be supported by the residual

PROLIFICACY GENOTYPES OF BONE MORPHOGENETIC PROTEIN 15(BMP15) GENE IN SHEEP WERE NOT DETECTED IN INDIAN GOATS

Sonika Ahlawat1, Rekha Sharma2 and A. Maitra3

National Bureau of Animal Genetic Resources, Karnal, Haryana 132001, India

ABSTRACT

Different mutations in the BMP15 gene cause increased ovulation rate and infertility in a dosage sensitive manner in sheep. Toexplore the mechanism of caprine prolificacy, the genetic polymorphism of BMP15 gene was studied in 6 Indian breeds of goat.On comparing the sequence of sheep BMP15 gene with indigenous goats, 3 deletions were detected in exon 1 and 5 transitionsand 4 transversions were seen in exon 2. The analysis of the sequence of BMP15 revealed that the six mutations (FecXG,FecXH, FecXI, FecXL, FecXB and FecXR) associated with fecundity in sheep were not identified in indigenous goats by sequencealignment. Two novel Single Nucleotide Polymorphisms (SNPs): G735A and C808G were seen in exon 2 of indigenous goatbreeds. The mutation G735A is synonymous in nature but mutation C808G is non-synonymous leading to change in amino acid(Q270E) in the corresponding protein. BLAST analysis of coding DNA sequences (CDS) of indigenous goat BMP15 generevealed 98% homology with Bos taurus, 99% with Ovis aries and exotic goat. In silico translation of coding DNA sequence ofBMP15 gene in caprines revealed a sequence of 395 amino acids with eight amino acid changes between Indian goat and cattle,4 amino acid changes between sheep and goat and 2 changes between Indian and exotic goat.

Key words: BMP15, FecX, prolificacy, Indian goat, fecundity

granulosa cells, whereupon they degenerate. Curiously,although a lack of BMP15 blocks follicular growth inhomozygotes, inactivation of only one copy of BMP15 increasesovulation rate, conferring a fecundity advantage to theheterozygote as described by Gemmell and Slate (2005).

Reproductive traits are extremely important to the goatindustry because moderate increase in litter size can lead tolarge profit. Thus there is a need to explore the genetic basis ofcaprine prolificacy. Since the tendency of twining and triplicateis inherited and common in both sheep and goat, it will beinteresting to see whether sheep SNPs are associated withfecundity in goat. Thus the polymorphism in the BMP15 genewas investigated in 6 indigenous goat breeds with differentprolificacy.

Materials and MethodsTo explore the genetic variations within BMP15 gene, blood

samples from a panel of Indian goat breeds were used. Thepanel included six breeds with six samples each based onphenotype (prolificacy), geographical distribution and geneticdiversity (Table 1). The blood samples were collected fromjugular vein into EDTA containing vacutainer tubes. GenomicDNA was isolated and purified from the blood cells using thestandard phenol-chloroform-isoamyl alcohol extractionfollowed by ethanol precipitation as described by Sambrookand Russell (2001). Oligonucleotide primers were designedto amplify the coding DNA sequence (Fig. 1) of caprine BMP15gene (EU743938.1) using PRIMERSELECT program ofLASERGENE software (DNASTAR Inc., Madison, WI, USA). Oneprimer was designed for exon 1 and 2 overlapping primerswere designed for exon 2 (Table 2).

Polymerase chain reaction (PCR) was carried out in a

1Scientist and address for correspondence: Core Lab, National Bureau of Animal Genetic Resources, G T Road, Karnal-132001 (Haryana), India,Tel.: +919416161369; Fax: +91 184 2267654 E-mail address: [email protected] Scientist3Senior research fellow

235


Table 1: Indian goat breeds employed in the study

Phenotype Breed Geographical distribution

Twinning percentage % (Acharya, 1982)

Beetal Punjab 52.6 Barbari Uttar Pradesh 49.3

Black-Bengal West Bengal, Bihar, Jharkhand 54 High proli ficacy

Malabari Kerala 42.4 Medium prolificacy Osmanabadi Maharashtra 29

Low proli ficacy Ganjam Orissa 1.6 Table 2: Oligonucleotide primers designed to amplify the coding DNA sequence of caprine BMP15 gene

Region Oligonucleotide sequence Product size (bp)

Annealing temperature (°C)

Exon 1 F-5’-GCGTTATCCTTTGGGCTTTTATC-3 R-5’-TACTTTTCTTCCCCATTTTTCTGC-3’ 443 57.2

F-5’-ACGCTTTGCTCTTGTTCC-3’ R-5’-AATACTGCCTGCTTGACGA-3’

527 54.8 Exon 2

F-5’-TCCCTAAAGGCCTGAAAGAGT-3’ R-5’-GCTGAAGGCAAGGAATAGAATC-3’ 575 55.1

Table 3: Variations in coding DNA sequence (CDS) of BMP15 gene

Region Position Exotic goat (EU743938.1)

Indian goat Sheep (AF236078S1 AF236078S2)

175 T T - 176 T T - 177 C C -

Exon 1 343 C T T

5919 G G T 5920 G G C 5966 C C T 6076 A A G 6187 G A G 6260 C G C 6280 T T G 6353 G A A 6443 T T C

Exon 2

6492 A A G

Table 4: Polymorphic sequence variations in exon 2 of BMP15 gene in sheep

Allele Base change Coding base

Amino acid change

Accession number Reference

FecXG C-T 718 Q239Ter Hanrahan et al. (2004) FecXH C-T 871 Q23stop Galloway et al. (2000) FecXI T-A 896 V31D Galloway et al. (2000) FecXL G-A 962 C53Y Bodin et al. (2007) FecXB G-T 1100 S99I Hanrahan et al. (2004)

FecXR

17 bp deletion (from

nucleotide 525 -541)

AF236078S2

Monteagudo et al. (2009)

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Table 5: SNPs in exon 2 of caprine BMP15 gene

final reaction volume of 25 µl on i-cycler (BIO-RAD, USA). PCRcocktail consisted of 50 to 100 ng of genomic DNA, 200 µM ofeach

dNTPs, 50 pM of each primer, 0.5 units of Taq DNApolymerase and Taq buffer having 1.5 mM MgCl2 for eachreaction. The PCR reaction cycle was accomplished bydenaturation for 1 min at 94°C; 30 cycles of 94°C for 45 sec,annealing at specific temperature for 45 sec, 72°C for 45 secwith a final extension at 72°C for 5 min. The PCR productswere visualized following electrophoresis through a 2%Ethidium bromide stained agarose gel.

The amplified region was sequenced using ABI 3100(Applied Biosystems, USA) Automated DNA Sequencer. Priorto sequencing, PCR products were purified by enzymaticmethod using Exonuclease I and Antarctic Phosphatase (NewEngland Biolab, USA). Raw sequence data were edited usingChromas (Ver. 1.45, http://www.technelysium.com.au=chromas.html). Multiple sequence alignments wereperformed with MegAlign program of LASERGENE software toidentify polymorphisms in the exon 1 and exon 2 of BMP15gene in indigenous goats and phylogenetic analysis wasperformed with CLC Main Workbench version 5.0.2. The

coding sequences of different exonic regions wereconceptually translated to amino acid sequences usingChromaspro software.

Results and DiscussionGenomic DNA of 6 indigenous goat breeds was

successfully amplified by three pairs of primers that cover theentire coding sequence of BMP15 gene. The results showedthat amplification fragment sizes were consistent with the targetones and had good specificity and could be screened for SNPsby direct sequencing. There were a total of 20 nucleotidechanges in coding region of indigenous goat BMP15 gene ascompared to Bos taurus (AY572412) out of which 70% weretransitions whereas 30% were transversions. When theBMP15 sequence of Indian goats was compared with exoticgoat (Jining grey goat, EU743938.1), 1 transition (C343T) inexon 1 and 2 transitions (G6187A, G6353A) and onetransversion (C6260G) in exon 2 were observed. On comparingthe sequence of indigenous goats with sheep, 3 deletions weredetected in exon 1 (AF236078S1) and 5 transitions and 4transversions were seen in exon 2 (AF236078S2) (Table 3).The analysis of the sequence of BMP15 in indigenous goat

Coding base

Base change Genotype frequency

Amino Acid change

735 G-A GG=0.44 AG=0.47 AA=0.08

No change

808 C-G CC=0.63 CG=0.30 GG=0.05

Q270E

Exon 1 Exon 2

1 144 471 5781 6637 6648bp

Fig. 1: Caprine BMP15 gene (EU743938.1)

Fig. 2: Phylogenetic analysis of GDF9 coding DNA sequence of different species following UPGMA algorithm

237


238


239


Fig. 3: Multiple alignment of conceptualized amino acid sequence of BMP15 gene

revealed that the six mutations (FecXG, FecXH, FecXI, FecXL,FecXB and FecXR) associated with fecundity in sheep (Table 4)were not identified in indigenous goats by sequence alignment.All these polymorphic points have the wild type pattern in theinvestigated goat breeds.

The findings of the present study are in line with those ofPolley et al. (2009) who typed the first five mutations by tetra-primer ARMS-PCR in Black Bengal goats and reported thewild type pattern only. Hua et al. (2008) analyzed loci FecXG,FecXH, FecXI and FecXB by forced PCR-RFLP in 6 goat breedsof China. None of these mutations was detected in these goatbreeds and their crossbreds. Forced PCR-RFLP was alsoemployed by Tejangookeh et al. (2009) in Iranian goats to detectFecXG and FecXB mutations. All the goats were monomorphicfor exon 2 of BMP15 gene. The absence of known mutations inBMP15 was reported by He et al. (2010) in some Chinese goatbreeds also. Contrary to all these findings, Lin et al. (2007)reported that FecXB mutation was found in female goats withtriplets in white goat population of Guizhou.

Two novel SNPs (G735A and C808G) were seen in exon 2of indigenous goat breeds in the present study. The mutation

G735A is synonymous in nature but mutation C808G is non-synonymous, leading to change in amino acid (Q270E)) in thecorresponding protein. The position of these SNPs, theirgenotype frequencies and position of amino acid change arepresented in Table 5. It is noteworthy that all the three genotypeswere observed at both these loci. Both the SNPs are differentfrom the previously reported SNPs in BMP15 of exotic goat.Jiao et al. (2007) reported that exon 2 of BMP15 has A963Gand C1050G mutants, which lead to amino acid changesS300G and L329V, respectively. It was reported by Feng et al.(2009) that the A963G mutant of BMP15 gene exon 2 in JiningGrey goats has three genotypes (AA, AG and GG) while only AAgenotype was found in Liaoning cashmere and Inner MongoliaCashmere goats. Boar goat have two genotypes (AG and GG)while Angora and Inner Mongolia Cashmere goats have onlyAA genotype. Feng et al. (2009) also found that genotypedistributions of BMP15 gene were significantly different amongthose goat breeds. Seven substitutions were found in maturepeptide sequence of BMP15 in white goat population of Chinaby Ran et al. (2009). Chu et al. (2007) sequenced BMP15 genein Jining grey goats and found two point mutations (G963A

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and G1050C) in AB genotype in comparison to the AA genotypein which heterozygous AB does had 1.13 kids more than thehomozygous AA does. These results preliminarily showed thatthe BMP15 gene is either a major gene that influences theprolificacy of Jining Grey goats or a molecular genetic markerin close linkage with such a gene. Considering the importantrole played by BMP15 in the folliculogenesis and ovigenesis(McNatty et al., 2005), it may be a candidate gene for goat.Therefore, new SNPs are important and should be exploredfurther for association with fecundity in indigenous goat.

BLAST analysis was carried out to find the percentagehomology in the DNA sequence of Indian goats with otherspecies. BLAST analysis of coding DNA sequences (CDS) ofBMP15 gene revealed homology of 98% of goat sequencewith Bos taurus, 99% with Ovis aries and exotic goat.Phylogenetic analysis of BMP15 gene following UPGMAalgorithm revealed that domestic animals, murines andprimates form distinct clusters (Fig. 2) and small ruminants(sheep and goat) were closer to large ruminants (cattle) ascompared to pig, horse, dog, cat which coincides with the factthat goat, sheep, cattle belong to domestic ruminants and areevolutionarily more related. Chicken, murines and primateswere found to be distinct from domestic animals. In silicotranslation of coding DNA sequence of BMP15 gene in caprinesrevealed a sequence of 395 amino acids. Eight amino acidchanges were observed between Indian goat and cattle while2 such changes were found between Indian goat and exoticgoat. Conceptualised translation of coding DNA sequence ofsheep BMP15 revealed a sequence of 394 amino acids with 4amino acid changes between sheep and goat. This is becauseof 3 consequetive deletions in sheep CDS as compared togoats (Fig 3).

AcknowledgmentsWe acknowledge the financial support provided by

Network Project on Animal Genetic Resources (ICAR) forcarrying out the work.

ReferencesAcharya, R.M. (1982) Sheep and goat breeds of India. FAO Animal

Production and Health Paper 30. FAO of United Nations,Rome, Italy.

Bodin, L. et al. (2007) Endocrinol. 148:393-400.Chu, M. X. et al. (2007) Ani. Biotech. 18:263-274.Davis, G.H. (2004) Ani. Reprod. Sci. 82-83:247-253.Davis, G.H. (2005) Genetics Selection Evolution. 37:S11-S23.Davis, G.H. et al. (2006) Ani. Reprod. Sci. 92: 87-96.Feng, T. et al. (2009) Chinese J. Ani. Vet. Sci. 40(4):468-475.Galloway, S.M. et al. (2000) Nature Genetics. 25:279-283.Gemmell, N.J. and Slate, J. (2006) PLoS ONE 1(1): e125 doi:10.1371/

journal.pone. 0000125Hanrahan, J.P. et al. (2004) Biology of Reprod. 70: 900-909.He, Y. et al. (2010) J. Agric. Sci. 2: 88-92.Hua, G.H. et al. (2008) Ani. Reprod. Sci. 108: 279-286.Jiao, C.L. et al. (2007) Chinese Ani. Vet. Sci. 34(12):52-55.Lin, J.B. et al. (2007) Ani. Husb. Vet. Med. 39(12):21-24.McNatty, K.P. et al. (2005) Genetics Selection Evolution. 37(Suppl. 1):

S25-S38.Monteagudo, L.V. et al. (2009) Ani. Reprod. Sci. 110:139-146.Polley, S. et al. (2009) Small Ruminant Res. 85:122-129.Ran, X.Q. et al. (2009) Zoological Res. 30:593-602.Sambrook, J. and Russell, D.W. (2001) Molecular Cloning: A Laboratory

Manul III. Cold Spring Harbour, Cold Spring Laboratory Press,NY.

Tejangookeh, H.M. et al. (2009) African J. Biotech. 8(13): 2929-2932.

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IntroductionHimachal Pradesh, a north-western hilly state having

livestock population of more than 6.23 millions, of which cattleis of prime importance. The local cows are predominantly non-descript and generally low yielders. Hill cattle are a non-descriptbreed of Himachal Pradesh. Although most of the nativelivestock has now been upgraded with exotic germ-plasm butstill hill cattle, native to state finds its’ importance in mid andhigh hill terrains. In spite of this not much scientific attentionhas been paid to this important breed. The Himalayaneconomy is basically agro-pastoral and is dependent onlivestock. The land holdings being very small, the livestockrearing supplement the income and sustain livelihood of thefarming communities of the hills. Farmers are predominantly(47.5%) rearing cattle, besides sheep and goat. The hill cattlealthough are low in production but are tough, fit for hilly terrains,adapted to cold and harsh climate, disease resistant, thrivingon poor pastures and nutrients. Despite of these qualities;very limited scientific attention has been paid to exploit theirunique physiology, so as to augment production andreproduction. The aim of the present study was to determineblood biochemical profile as well as hormonal indices, in theirnative tract and to assess the variability present to act as indexfor further evaluation of these animals with respect to importantphysiological traits associated with uniqueness of cattle forefficient conservation and propagation of the species.

BLOOD BIOCHEMICAL AND HORMONAL STUDIES IN HILLCATTLE OF HIMACHAL PRADESH

R. Kumar1, M.S. Verma2, Gopal Puri3, V. Sejian4, Varun Sankhyan3 and K.B. Sharma1

Department of Veterinary Physiology and Biochemistry, DGCN, College of Veterinary and Animal Sciences CSK,HPKV, Palampur- 176062, Himachal Pradesh, India

ABSRACT

The study was planned with the objective of exploring the possibility of identifying biochemical and hormonal parameters in Hill cattle,which have prime importance in metabolic pathway and involved in basic adaptation physiology of hill cattle. Twenty adult hill cattlemaintained at livestock farm, Palampur were selected for investigation. Blood samples were collected and plasma was separated foranalysis of biochemical parameters as per standard technique and hormonal assay by RIA method. The average concentration ofAST, ALT, LDH and cholinesterase activity were 65.11±2.92 IU/L, 78.53±3.74 IU/L, 711.11±18.09 IU/L, and 281.22± 17.54 IU/L,respectively; whereas mean level of plasma creatinine, total cholesterol and total plasma protein concentration were found to be1.70±0.05 mg/dl, 127.79±5.31 mg/dl and 7.18±0.12 g/dl, respectively. The average concentration of triiodothyronine hormone was1.736 ± 0.10 nm/l and it varied from 1.090 to 2.787 nm/l; whereas circulating thyroxine level was found to be 243.33 ± 12.11 nm/l, andit varied from 158.5 to 328.6 nm/l and mean level of cortisol hormone was 16.07 ± 4.70 nm/l. There were no significant changes inplasma concentration of ALT, cholesterol, creatinine and total plasma protein in tropical cattle but T3, T4 and cortisol level weresignificantly (P<0.05) high in hill cattle as compared to exotic cattle. The results of our study can be concluded that changes inbiochemical and hormonal level may be of physiological importance to adaptation of the animal in harsh and adverse climatic conditionwhich exists in breeding tract of hill cattle.

Key words: Hill cattle, triiodothyronine, thyroxine, AST, ALT, cortisol

Materials and MethodsTwenty adult hill cattle were randomly selected from

animals from different region of Himachal Pradesh maintainedat livestock farm, Palampur for the present study. Bloodsamples were collected and plasma was stored at -20°C tillanalysis. Plasma from blood samples was separated.Biochemical parameters for aspartate aminotransferase(AST), alanine aminotransferase (ALT), creatinine, cholesterol,total protein, LDH, cholinesterase, were analyzed as perstandard technique immediately using diagnostic kits andtriiodothyronine (T3), thyroxine (T4) and cortisol hormones wereanalyzed by RIA method.

Results and DiscussionThe results on different blood biochemical and hormonal

parameters of the hill cattle have been presented in Table 1.The average concentration of AST, ALT, LDH and cholinesteraseactivity were 65.11±2.92 IU/L, 78.53±3.74 IU/L, 711.11±18.09IU/L, and 281.22± 17.54 IU/L, respectively; whereas mean levelof plasma creatinine, total cholesterol and total plasma proteinwere found to be 1.70±0.05 mg/dl, 127.79±5.31 mg/dl and7.18±0.12 g/dl, respectively. The alanine aminotransferase(ALT) and plasma creatinine concentration were found to behigher in hill cattle as compare to tropical cattle (Nahed, 2010),while cholesterol and plasma protein level were similar. In hillcattle, the average concentration of triiodothyronine hormone

1Corresponding author: Professor, Department of Veterinary Physiology, College of Veterinary and Animal Sciences, CSKHPKV, Palampur (HP)-176062,Email ID:[email protected] officer, Animal Husbandry Department, HP.3Associate Professor, Department of Physiology and Biochemistry, College of Veterinary Sciences and Animal Husbandry, Navsari Agricultural University,Navsari-396450, Gujarat.4Scientist, CSWRI, Avikanagar

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was 1.736 ± 0.101 nm/l and it varied from 1.090 to 2.787 nm/l;whereas circulating thyroxin level was found to be 243.33 ±12.11 nm/l, which varied from 158.5 to 328.6 nm/l and meanlevel of cortisol hormone was 16.007 ± 4.704 nm/l. whereasfindings of Campos et al. (2004) for thyroxine in exotic cows isquite different than our results, this may possibly due to geneticgroup variation of animals. Kreuzer, et al. (1998) found thatplasma protein, albumin, creatinine and liver enzyme activitieswere not affected at high altitude however, our results showssignificantly (P<0.05) higher plasma cortisol level as comparedto exotic cattle breed, possibly due to the metabolic responseat high altitude conditions can be mostly explained by the effortsto cover the additional energy requirements.

The results indicated that increment in the level of differentparameter as compare to tropical cattle exhibit physiologicalresponses in harsh and adverse climatic condition at highaltitude adopted by animals which exist in breeding tract of hillcattle. The physiological responses of the animals toenvironmental stress during the winter and summer and theirenergy balance showed that seasonal heat and cold stresshave profound effects on serum biochemical parameters

Table 1: Biochemical and hormonal parameters in hill cattle of Himachal Pradesh (Mean±SE)

A S T (IU /L)

A LT (IU /L)

LDH (IU /L)

C holines teras e (IU /L)

C holesterol mg/dl

C reatinine mg/dl

To tal P rotein

g/dl

T3 nm/ l

T4 nm/ l

C ortisol nm/ l

65.1 ± 2.92

78.5 ± 3.74

711. 11 ± 8.90

281. 22 ± 17.54

127. 97 ± 5.31

1.70 ± 0.05

7.18 ± 0.12

1.74 ± 0.10

243. 34 ± 2.11

16.0 7 ± 4.70

(Brakat and Abdel-Fattah, 1971). Thyroid hormones are knownto be important modulators of developmental process andgeneral metabolism (Kaneko, 1989), which is in line with ourfindings for T3 and T4. Serum cortisol concentration has alsoshown significant changes during cold and heat stress asreported by Nazifi et al. (2003); which justify higher cortisollevel in hill cattle. The results of our study can be concludedthat some changes in biochemical and hormonal levels maybe of physiological importance to adaptation of the animal inharsh and adverse climatic condition which exists in breedingtract of hill cattle.

ReferencesBarakat, M.Z. and Abdel Fatth (1971) Zentralbl. Veterinary. Med. Seri

A.18:174-178.Campos, R. et al. (2004) Revista-Brasileira-de-Ciencia-

Veterinaria.11(3): 174-177.Kaneko, J.J. (1989) Clinical biochemistry of domestic animals. 4th

ed., New-York. pp. 630-648.Kreuzer, M. et al. (1998) Ani. Sci. 67(2): 237-248.Nahed, S.T.(2010) Global Veterinaria. 4(5):450-455.Nazifi, S. et al. ( 2003) Comp. Clinical Pathol.12:135-139.

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IntroductionMange in pigs resulting from infection with Sarcoptes

scabiei have been recognized in many countries includingIndia. Circumstantial evidence shows that these parasites havea negative effect on growth rate, feed conversion of weanersand fattening pigs resulting in poor economic return (Pennyand Muirhead, 1986). The disease is fatal sometime inneonates. Outbreaks of coughing and scouring have beenrecorded in piglets with mange infection. The parasite affectthe skin of the host causing varying degree of lesions anddisorders like annoyance, irritation, itching, scratching,hyperkeratosis, alopecia, anaemia and loss of production byway of slow body growth and poor quality of meat etc.

Materials and MethodsTwenty five (25) pigs naturally infected and suspected with

Sarcoptes scabiei were selected for the present investigationand were divided into 5 groups viz. Group I (T1), Group II (T2),Group III (T3), Group IV (T4), Group V (T5); consisting 5 pigs ineach group. Samples collected from each group of pigs at 0days and after treatment on 30th day of experiment.

Group-I (T1): The infected animals were treated withivermectin premix at the dose rate of 330 gm/ton of feed for 7days. Group-II (T2): The infected animals were treated withpaste mixture Sulphur-15 parts and Karanj Oil 100 ml. thispaste mixture was applied once daily as local application, aftercleaning the lesion thoroughly with soap and water. Group-III(T3): The infected animals were treated with Teeburb Powder2 gm/100 kg body weight for 30 days. Group-IV (T4): Theinfected animals were treated with Doramectin at the doserate of 200 µg/kg body weight (1 ml/ 50 kg body weight)intramuscular and repeated after 3 weeks. Group-V (T5): Allthe infected animals were kept as untreated control.

HAEMATO-BIOCHEMICAL CHANGES INMANGE INFECTED PIGS TREATED WITH VARIOUS DRUGS#

P. Minj1, S. Haque2, S.N. Mishra3 and N. RoyDepartment of Veterinary Medicine

Ranchi Veterinary College, Bihar Agriculture University, Ranchi- 834 006, Bihar, India

ABSTRACT

The present study was carried out on twenty five (25) pigs naturally infected with Sarcoptes scabiei. The pigs were randomlydivided in five (5) groups, consisting of five pigs in each group, viz. group I (T1), group II (T2), group III (T3), group IV (T4) and groupV (T5). The infected animals under group T1, T2, T3, T4 were treated with Ivermectin, Sulphur and Karanj oil, Teeburb Powder andDoramectin respectively whereas, group T5 were kept as untreated control throughout the period of experiment (i.e.30 days). Studiesrevealed that Hb and PCV (%) level were showing decreasing trend in all infected pigs, but it returned to normal level after treatment.TLC (%) in mange infected pigs was increased. The highest TLC was recorded in control groups. The TLC returned to normal ininfected groups after treatment. Total serum protein and albumin were below the normal range on 0 days and it returned to normal aftertreatment (13 days).

Key words: Haematobiochemical changes, mange, Pigs

#Part of M.V.Sc. Thesis of First Author1T.V.O., Bihar State Government2Head Department of Veterinary Medicine, Ranchi Veterinary College, Ranchi3Assistant Professor (Medicine), Mahatma Gandhi Veterinary College, Bharatpur, Rajasthan (“Reprint requests are solicited at this address”)

Haematology: The haemoglobin content of the bloodsample was estimated by Sahli’s haemoglobinometer as perprocedure described by Coles (1987), the values wereexpressed in gm%. Packed Cell Volume (PCV) was estimatedby capillary haemoglobin technique using adam’s autocutcentrifuge as per method described by Coles (1987). Valueswere expressed in percent (%). Total leucocyte count was doneaccording to the method described by Coles (1987). Thevalues were expressed in 103/cumm. The differentialbiochemical values were estimated before and after proteinand albumin were estimated by modified Biruet and Dumasmethod (Dumas et al., 1971) using diagnostic reagent kitmanufactured by Span diagnostic Ltd. (Surat, India). Date wereanalysed and interpreted as per statistical method describedby Snedecor and Cochran (1989).

Results and DiscussionHaematological upper and lower found. The

haematological parameters in respect of haemoglobin packedcell volume and total leucocyte count (Table 1) were carried outduring the course of Mange infection and their treatment withivermectin premix, paste mixture of Sulphur and Karanj Oil,Teeburb Powder and Doramectin respectively. Analysis ofvariance showed a significant variation and all thehaematological values on 30th day of post treatment whereaseffect was non significant at 0 day on haemoglobin and PCV.There was a significant improvement in haemoglobin andpacked cell volume in all the four treated groups whereas nosuch improvement was noticed in infected control group.Similar trends of results were also reported by Dalapati andBhowmik (1996), Naidu and Rao (1999) in Goats and Prasadet al. (2001) in Pigs. The marked reduction of haemoglobin(gm%) and packed cell volume (%) in Mange infected pigs

244


might be due to its poor condition occurred owing to sucking ofblood and tissue fluid by mites leading to deficiency of nutrientor malnutrition.

Studies on effect of total leucocytes count revealed thatthe difference between various groups of pigs was significantly(P<0.01). The lowest value was recorded in Group IV(12.43±0.31) i.e. the pigs treated with doramectin. Leucocytosiswas recorded in all the pigs infected with sarcoptic mange.The present finding corroborates with the finding of Dalapatiand Bhowmik (1996) in Goat Hirudukar et al. (1997) in Sheep,Naidu and Rao (1999) in Goats. Leucocytosis observed duringthis study may be due to secondary bacterial infection.

Biochemical studiesAnalysis of variance showed a significant variation in all

the biochemical values at 30th day of treatment. There was asignificant improvement in total serum protein and albumin(Table 2) in all the four treated groups whereas suchimprovement was not observed in infected control group.Similar finding was also reported by Dalapati and Bhowmik(1996) in goats, Hirudukar et al. (1997) in sheep and JuneyChandy et al. (2000) in dogs.

The hypoproteinaemia was recorded in this study mightbe due to inadequate production, malnutrition or loss of protein

by the body. The decreased serum albumin concentration inpresent study might be due to wide spread skin lesions,impaired intake of protein, increase catabolism due to mangeinfection, reduce synthesis and impaired intake of protein.

AcknowledgementsAuthors are thankful to the Dean, Ranchi Veterinary

College, B.A.U., Ranchi for providing necessary facilities.

ReferencesColes, E.H. (1987) Veterinary Clinical Pathology. 4th ed., W.B.

Saunders, Philadelphia, P.A.Dalapati M.R. and Bhowmik, M.K. (1996) Indian Vet. J. 73(7):

728-733.Dumas, B.T. et al (1971) Clin. Chem. Acta. 31: 57-76.Hirudukar, U.S. et al. (1997) Indian Vet. J. 74: 834-836.Juney Chandy et al. (2000) Indian Vet. J. 77: 755-757.Naidu, M.M. and Rao, D.V. (1999) Indian Vet. J. 76: 730-732.Penny, R.H.C. and Muirhead M.R. (1986) Diseases of Swine.

6th ed. Lowa, State University Press, Ames, Lowa U.S.A.pp. 90-91.

Prasad, K.D. et al. (2001) J. Vet. Parasitol. 15(1): 63-65.Snedecor, G. W. and Cochran, W.G. (1989) Statistical Methods.

8th ed. Oxford and IBH Publication Co., Calcutta, India.

Table 1: Average value of haemoglobin (g%), packed cell volume (%) and total leucocyte count (th/cumm) indifferent groups of pig

Table 2: Average value of total serum protein and albumin (g/100 ml) in different groups of pig

Mean under the same superscript in a column did not differ significantly

Mean under the same superscript in a column did not differ significantly

Group Haemoglobin (Hb)

Packed Cell Volume (PCV)

Total leucocyte Count (TLC)

0 day 30th day 0 day 30th day 0 day 30th day Group-I 10.360.23 12.760.17a 32.000.70 38.600.92b 15.080.55bc 12.220.53a Group-II 10.800.26 12.980.16a 32.600.92 40.201.28bc 14.270.45ab 12.540.27a Group-III 11.160.27 13.040.14a 33.000.70 40.201.28bc 15.901.25c 13.300.52a Group-IV 11.120.22 13.040.17a 32.600.97 40.600.97b 14.790.67ab 12.430.31a Group-V (Control)

10.440.17 10.080.24b 31.600.50 31.000.44a 13.450.25a 14.970.34b

Group Total Serum Protein Albumin 0 day 30th day 0 day 30th day Group-I 6.210.14 7.130.17b 3.360.27 4.080.13b Group-II 6.120.06 7.300.28b 3.220.08 4.10.08b Group-III 6.690.11 7.300.16b 3.200.15 4.020.08b Group-IV 6.290.18 7.480.23b 3.340.17 4.120.08b Group-V (Control)

6.900.24 5.810.20a 3.080.11 3.260.18a

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IntroductionUrinalysis is an important tool in disease detection, as

well as monitoring and screening pet health. This test isrelatively simple, easy, inexpensive, and can be conducted invery short time i.e. less than 5 minutes. Abnormalities can beindicative of diseases of the urinary system as well as otherorgan systems, including liver function, acid-base status, andcarbohydrate metabolism (Duncan et al., 1994). Completeurinalysis involves gross visual assessment of the urine,microscopic examination, and chemical evaluation. Presentresearch based on the comparative urinalysis of healthy dogsand dogs suffering from renal failure.

Materials and MethodsThe present study was conducted on 21 apparently healthy

non descript dogs of either sex, weighing about 21.78 ± 4.7 kg,height 39.33 ± 2.7 cm and age about 5.72 ± 1.3 year (Group I)and 31 dogs of either sex, breed weighing about 19.64 ± 0.89kg, height 36.53 ± 2.24 cm, age about 5.06 ± 0.27 year andsuffering from renal failure (Group II). Inclusive criterion for therenal failure cases was creatinine level (>2.0 mg/dl). The healthstatus was confirmed by thorough clinical examination, routinehaematology, liver function test and kidney function test.Approximately 10 ml urine sample was collected in a clean,dry container free of any disinfecting or cleaning chemicals, inearly morning by catheterization (Ling, 1995) and subjected toanalysis as per standard procedure (Benjamin, 1986).Catheterization was performed by placing a small hollow tubeinto the urethra to the level of the bladder. Urine was thenwithdrawn from the bladder using a syringe. The analysis wasperformed as soon after collection as possible (within 30minutes of collection) after proper mixing prior to testing. Insome cases test was not performed immediately, in thesescases the sample was covered and refrigerated and allowedto return to room temperature prior to testing.

COMPARATIVE STUDY OF URINALYSIS IN HEALTHY AND RENALFAILURE DOGS

M. K. Srivastava1, R. V. Gaikwad2, S. Kachhawaha3, Ashish Srivastava1 and S. Purohit4

Department of Veterinary Clinical MedicineBombay Veterinary College, MAFSU, Nagpur, Maharashtra, India

ABSTRACT

Present study was undertaken on 21 healthy dogs (Group I) and 31 dogs with renal failure (Group II). Inclusive criterion for the renalfailure cases was creatinine level (>2.0 mg/dl). A non-significant difference was observed in pH and specific gravity of urine of theanimals of the both groups. Moderate to severe proteinuria and bilirubin was observed in dogs of Group II. There was significantpyuria (6.12 ± 3.44/hpf) and haematuria (4.56 ± 2.43/hpf) in dogs of Group II as compare to Group I (0.59 ± 0.50/hpf and 0.56 ± 0.50/hpf, respectively). A significant increase in epithelial cell count was noticed in Group II dogs (5.39 ± 1.92/hpf) in comparison dogs ofGroup I (0.27 ± 0.11 /hpf), beside significant difference in hyaline and epithelial cast count of Group II (2.80 ± 1.60 /lpf) and Group Ihealthy dogs (1.53 ± 0.14).

Key words: Urine analysis, renal failure, dogs

Results and DiscussionThe results of physical, chemical and microscopic

examination of healthy and renal failure dogs were specifiedin Table 1. For healthy dogs all observed analytes were withinnormal range and in accordance with Osborne et al. (1972).Catheterized samples have less contamination from the distalurogenital tract; however, contamination from the urethra maystill occur. Poor catheterization technique may lead to traumaor, less commonly, infection (Chew and DiBartola, 1998). Inorder to obtain accurate results, the urine collection, storageand handling must be sterile and follow standard procedures.1. Physical examination

The colour of urine in renal failure dogs was found to varyfrom yellow- brown to colourless, the yellow- brown colourwas suggestive for presence of bile pigment, might be duepolysystemic disease involving liver while colourless urineindicate a dilute urine, that might be due presentation of dogsin polyuric phase of renal failure, in few cases normal colour ofurine was noticed, same observation was noticed by Osborneand Finco (1995), who stated that significant disease mayoccur when urine is normal in colour, therefore, colour of urineprovides nonspecific information. Presence of WBC, RBC,epithelial cells, mucus and bacteria were suggestive reasonfor hazy to cloudy appearance of urine in renal failure dogs.

Urine pH may be used as a crude index of body acid basebalance, but not always a reliable index of blood pH. The pH ofurine can vary depending on an animal’s diet as well as itsacid-base status. For example, animals that primarily eat highprotein meat-based diets will have acidic urine. On the otherhand, animals that eat more vegetable-based diets will havealkaline urine (Chew and DiBartola, 1998).

A non-significant difference was observed in pH of urineof healthy (6.20 ± 0.48) renal failure (5.93 ± 0.60) dogs. Thisnon-significant reduction in urine pH was might be due topresence of uraemia and previous usages of drugs like

1Assistant Professor Department of Veterinary Clinical Medicine, DUVASU, Mathura, Uttar Pradesh2Associate Professor3SMS, KVK (CAZRI), Pali, Rajasthan4Assistant Professor, Department of Surgery and Radiology, DUVASU, Mathura, Uttar Pradesh

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furosemide that may alter the urine pH (Osborne and Finco,1995).

Urine specific gravity is based on the ratio of weight ofurine to weight of an equivalent volume of pure water. This testis used to measure tubular function. Specific gravity of urinealso showed a non-significant increment in specific gravity ofrenal failure (1.02±0.12) dogs in compassion to healthy(1.01±0.08) dogs. This increased value in urine specific gravitymight be due to acute nephritis, oliguric phase of renal failure,protein loosing and nephrotoxic nephrosis (Osborne et al.,1972). The ability of kidney to regulate specific gravity dependsupon their functional competence and selective re-absorptionat the time urine formation. For clinical significance repeatedanalysis may require to arrive at conclusion merely on specificgravity, therefore could not be used as a mean of prognosis.2. Chemical examinations

Proteins, glucose, ketones, bile salts, bile pigmentsand occult blood were observed in dogs of Group II. Glucosuriawas might due to either an excess amount of glucose reachingthe tubules that cannot be resorbed or, less commonly,decreased tubular resorptive function (Osborne et al., 1972).Glucosuria may be either persistent or transient and multipletests may be needed for differentiation of these conditions.Persistent causes of glucosuria include diabetes mellitus,administration of glucose containing fluids, chronic diseasethat is not related to the kidneys such as hyperadrenocorticism,hyperpituitarism, or acromegaly. Other diseases that may resultin transient hyperglycaemias leading to glucosuria includeshyperthyroidism, acute pancreatitis, stress, postprandial, andadministration of certain drugs.

Dogs normally have small proteins that pass through theglomerular filter, however, a majority of these proteins areresorbed by the renal tubules. Thus, only a very small amount

of protein is normally excreted in the urine, which is not usuallyclinically detectable (Duncan et al., 1994). Moderate to severeproteinuria was observed in dogs of Group II. Positive proteinresults must be evaluated in relationship to the patient’s history,physical examination, method of urine collection, urine specificgravity, and microscopic sediment examination. Proteinuriamay be due to haemorrhage, infection, intravascularhaemolysis, or renal disease. Haemorrhage is confirmed bypresence of red blood cells in the sediment. A urinary infectionor cystitis can be confirmed by observing bacteria and whiteblood cells on sediment examination. Cases of intravascularhaemolysis have haemoglobinuria leading to a positive occultblood test. Proteinuria of renal disease may be due toglomerular and/or tubular lesions. If the proteinuria is due torenal disease, the occult blood test will be negative and thesediment may or may not contain casts. Determination of theurine protein/urine creatinine ratio is helpful in confirming renalproteinuria. Protein results must be analyzed with the urinespecific gravity. Trace proteinuria may represent significantprotein loss with low specific gravity, but not with high specificgravity.

Mild to moderate bilirubin was observed in dogs of GroupII. Suggested reason behind presence of bilirubin in few renalfailure dogs was low threshold for bilirubin in dogs (Duncan etal., 1994) and concurrent polysystemic etiology of renaldysfunction including liver and bile duct (Osborne et al., 1972).3. Microscopic examinations

There was significant pyuria in dogs of Group II 6.12± 3.44/hpf as compare to Group I (0.59 ± 0.50/hpf). This wasindicative for inflammation or infection anywhere in urinary tract(Osborne et al., 1972). Possible reason for significanthaematuria in Group II dogs (4.56±2.43/hpf) as compare GroupI dogs (0.56±0.50/hpf), was presence of renal infarcts, calculi

Table 1: Comparative study of urinalysis in healthy and renal failure dogs

Parameters Healthy dogs (n=21) Mean ± SD

Renal failure dogs (n=31) Mean ± SD

Physical examination Colour Pale yellow to amber Yellow brown to colourless

Appearance Clear Hazy to cloudy Reaction (pH) 6.20 ± 0.48 5.93 ± 0.60 NS Specific gravity 1.01 ± 0.08 1.02 ± 0.12 NS

Chemical examination Proteins Absent Moderate to severe Glucose Absent Absent Ketones Absent Absent Bile salts Absent Mild in few case Bile pigments Absent Moderate in few case

Microscopic examination Pus cells (No./hpf) 0.59 ± 0.50 6.12 ± 3.44* RBCs (No./hpf) 0.56 ± 0.50 4.56 ± 2.43* Epithelial cell (No./hpf) 0.27 ± 0.11 5.39 ± 1.92* Casts (No./lpf) 1.53 ± 0.14 2.80 ± 1.60*

NS Non significant; * significant increase

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and infection of any part of urinary system (Benjamin, 1986). Asignificant increase in epithelial cell count was noticed in GroupII dogs (5.39 ± 1.92/hpf) in comparison to healthy dogs of GroupI (0.27±0.11/hpf). These findings are only indicative of thedisease process in kidney and inconclusive (Osborne et al.,1972). Significant difference was seen in hyaline and epithelialcast count of Group II (2.80±1.60/lpf) and Group I healthy dogs(1.53±0.14). It could be because of active pathologic processat loop of henle, distal tubule and collecting tubule. But absenceof cast does not rule out the disease and number is not areliable index of severity, duration and reversibility orirreversibility of disease (Osborne and Finco, 1995).

ReferencesBenjamin, M.M. (1986) Outline of Veterinary Clinical Pathology. 3rd ed.

The IOWA State University Press. Ames. IOWA. USA.Chew, D.J and DiBartola, S.P. (1998) Interpretation of Canine and

Feline Urinalysis. Ralston Purina Co., St Louis. pp. 1-21.Duncan, J.R. et al. (1994) Veterinary Laboratory Medicine: Clinical

Pathology. 3rd ed. Iowa State University Press, Ames. Pp.162-183.

Ling, G.V. (1995) Techniques of Urine Collection and Handleing. In:Ling G.V. Lower Urinary Tract Diseases of Dogs and Cats.St. Louis, Mosby. pp. 23-28.

Osborne, C.A. and D.R. Finco (1995) Canine and Feline Nephrologyand Urology. Baltimore, Williams and Wilkins, pp.136-305.

Osborne, C.A. et al. (1972) Canine and Feline Urology. W.B. SaundersCompany, Philadelphia. pp. 3-10, 39-84.

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IntroductionAnalysis of dairy herd records to determine the cause of

low reproduction efficiency is an important aspect of dairyingthat affects the life time milk production, the total calf productionand the genetic advancement. The reproductive efficiency istaken in terms of the numbers of inseminations or servicesrequired per conception which is translated in terms ofconception rate. It has been reported that only about one half ofthe total cows are conceived with one service and rest of thecows require more than one service to conceive. Requirementof more than three services for conception by dairy cowsadversely affect the overall performance and such cows areuneconomical. Knowledge of variability in the fertility status ofa breed and the extent to which this influenced by variousenvironmental factors, is therefore, essentially required. Thepresent study was carried out to obtain the information onreproductive status of newly developed crossbred strain,Frieswal.

Materials and MethodsRelevant data for the present investigation were collected

on 3134 gestation records of 1024 Frieswal cows (a new strainof Holstein Friesian X Sahiwal) maintained at Military DairyFarm, Meerut. The data pertained to a period of 21 years (1987to 2007). The entire period of a year was classified in to fourseasons (winter; December-March, summer; April-June, rainy;July-September and autumn; October-November). Breedingof the animals was done by artificial insemination. Conceptionrate was calculated in percentage as a confirmed pregnancyout of total number of services. The frequency distribution ofthe cows was made according to the number of services theyreceived for conception (S/C) during different parities and

INFLUENCE OF SEASON AND LACTATION ORDER ONREPRODUCTIVE EFFICIENCY OF FRIESWAL COWS

Arun Kumar1, Umesh Singh, Sushil Kumar, B.K. Beniwal2 and Satish KumarProject Directorate on Cattle, Grass Farm Road, Cantt, Meerut, Uttar Pradesh, India

ABSTRACT

Fertility records of 3134 lactations of 1024 Frieswal (Holstein x Sahiwal) cows, over a period of 21 years from 1987 to 2007maintained at Military farm, Meerut were analyzed. Frieswal cow of this herd required on an average 2.28±0.09 services to conceiveand with an overall conception rate of 43.9 per cent. Season of calving had highly significant (P<0.01) effect on conception rate andnumber of services per conception, which was more in autumn calvers and lower in summer calvers, revealing the best season forconception is winter followed by autumn. The average numbers of services required for conception were significantly less forheifers (1.74±0.02) than in older cows (2.48±0.08). Further it was observed that differences in services per conception in differentparity from third to sixth were not significant. Incidence of repeat breeding was 22.8 per cent, it was lowest in heifers (5.8%) andhighest in 8th parity (ranging between 10.3 to 28.3%). Season of calving had significant effect on incidence of repeat breeding. It waslowest in autumn calvers (16.4%) and highest in summer calvers (25.2%). The 4.6% repeat breeder cows required more than 5services for conception. The trend in the incidence of problem cows among different parities and seasons was similar to theincidence of repeat breeders. Heritability and repeatability estimates for these traits were found to be very low (less than 0.12)indicating predominance of environmental causes.

Key words: Cow, conception rate, parity order, repeat breeders, season

calving in different seasons. The cows which could not conceivein first three inseminations were considered repeat breeders,while those receiving more than 5 services were termed asproblem cows. Analysis of variance was conducted to test thesignificance of differences in these traits among different levels,considering the trait as a threshold character after makingarcsine transformation of percentile data, using followingmodel:

Yijkl = ì+ Sj +Lk +eijklWhere, ì is the overall mean, Sj is the effect of Jth season of

calving, Lk is the effect of Kth order of lactation and ejkl is therandom error.

Results and DiscussionThe results indicated that a cow in this herd required on

an average 2.28 ± 0.09 services for conception. Almost similarvalue is reported in Hariana cattle by Chaudhari et al. (1984)and crossbred cattle by Arun et al. (1993). The average numberof services required for conception was significantly less(P<0.01) for heifers (1.74±0.02) than older cows (2.48±0.08).The differences in services per conception in different parityfrom third to sixth was not significant, though the older cowsrequired more services. These findings are supported byTomar and Tripathi (1986), Singh et al. (1988) and Arun andSingh (2009). A non significant effect of parity on service perconception is, however, reported by Chaudhari et al. (1984). Itwas observed that variation in service per conception amongdifferent season of calving were significant and autumn calversrequired less services as compared to cows calving in summermonths. Present findings corroborates with findings of Arunand Singh (2009).

The overall average conception rate was 43.9 per cent.

1Corresponding author, Correspondence address: Senior Scientist, Central Sheep and Wool Research Institute, Post: Avikanagar, Tehsil: Malpura,Tonk, Rajasthan, Pin code 304501.2Associate Professor, Department of Animal Breeding and Genetics, RAJUVAS, Bikaner - 334001

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Table 1: Number of services per conception (S/C), overall conception rate (CR), CR with Ist service, repeatbreeders in a herd of Frieswal cows.

Effect Gestation records

Total services (AI’s)

Services/ conception (S/C±S.E.)

Overall conception rate (%)

1st service conception rate

Repeat breeders (%)

Overall (u) 3134 7144 2.28±0.09 43.9 52.3 22.8 Parity order ** ** ** ** 1 1024 1781 1.74±0.02 60.4 74.2 5.8 2 779 1792 1.98±0.04 46.2 52.4 10.3 3 557 1281 2.30±0.05 43.5 46.3 18.5 4 350 816 2.33±0.06 42.8 44.3 22.6 5 215 484 2.24±0.06 44.5 46.5 19.5 6 119 309 2.58±0.08 38.5 42.1 24.3 7 51 137 2.65±0.11 37.2 38.4 26.4 8 20 56 2.80±0.18 35.7 36.6 28.3 9≥ 19 54 2.82±0.23 35.2 35.2 26.3 Season of calving ** ** ** ** Winter 1355 3202 2.36±0.03 42.3 46.4 23.8 Summer 593 1541 2.59±0.11 38.4 43.6 25.2 Rainy 638 1378 2.13±0.09 46.4 57.6 18.4 Autumn 547 1023 1.85±0.21 48.3 52.1 16.8

** P<0.01

This is well within the range as reported in literature for indigenousand crossbred cattle, Mandal and Tayagi (2004) reportedconception rate to be 35.5% for Frieswal cows but they used onlyone year data. Conception rate was highest in heifer stage (60.4%)whereas it ranged from 35.2 to 46.2 per cent among the oldercows. This could be attributed to the fact that older cows get exposedto various reproductive problems and production stress leadingto low fertility. Season of calving had significant effect on conceptionrate. The conception rate was more among the cows calving inautumn followed by those calving in rainy season and it was lowestamong the cows calving during summer month. It might be due tothe reason that the cows calving in autumn (October-November)will come in heat during winter (December-March), when they getgreen fodder and pleasant climate, whereas the cows calvingduring winter and summer will come in heat during hottest andhumid part of summer/rainy months which might have altered theuterine environment and also depressed the energy intake.Significant effect of season on conception rate was reported byArun et al. (1993), Bhagat and Gokhale (1999), Mandal and Tayagi(2004) and Arun and Singh (2009). It was also observed that about52.3 per cent of the total cows conceived with first service and restof them took more than one service for conception. The conceptionrate with first service was highest in heifers (74.2%) and it averaged48.7% for older cows ranging from 35.2 to 52.4%.

The overall incidence of repeat breeding (cow’s required morethan 3 services) was 22.8 per cent. Lower incidences than thepresent study have been reported by Arun (1997) in crossbredcows at military dairy farm (15.7%), Sangeeta et al. (2002) forcrossbred cows (7.1%) and Arun and Singh (2009) for Harianacows (10.8%), whereas higher incidence than the present studyhave been reported by Shukla and Pandit (1989) and Bhattacharyaand Buchoo (2008). A significantly lower incidence in heifers (5.8%)which ranged 10.3 to 28.3 per cent in subsequent parity wasobserved in this study. It was highest among the cows that were intheir 8th parity. The high incidence of repeat breeding among oldercows may be due to the reason that older cows were exposed tothe risk of various reproductive disorders like abortion, retention of

placenta, metritis etc. which leads to decrease in fertility rate. Thesame trend was observed by Arun et al. (1993) for crossbred cowsand Arun and Singh (2009) for Hariana cows.

The incidence of repeat breeding was significantly highamong the summer calvers (25.2%) and low among autumncalvers (16.8%). The high incidence of repeat breeding duringrainy and summer season may be due to uncomfortableenvironmental conditions like high temperature and humidity, whichdepresses the feed consumption resulting in reduction ofconception rate. Significant effect of season on repeat breedinghad also been reported by Arun and Singh (2009) in Harianacows, being low during winter and high during rainy season.However, Shukla and Pandit (1989) and Arun et al. (1993) did notobserve such effect. The frequency of problem cows requiringmore than 5 services for conception among the repeaters was4.6%. The trend in the incidence of problem cows among differentparities and seasons was similar to the incidence of repeatbreeders. Most of the cows (95.4%) settled with first five services.

Heritability and repeatability of these traits were estimatedand the estimates were observed to be low (below 0.12). Lowestimates indicated that there was not much genetic variability toexploit genetic improvement in these traits and the environmentalfactors were more important in causing variation. To minimize theenvironmental effects on reproductive performance farmers shouldreduce the heat stress during summer and rainy months by provingshade, use of fans to provide a good cool air flow etc.

ReferencesArun Kumar (1997) Indian J. Dairy Sci. 50(4):302.Arun Kumar and Singh Umesh (2009) Indian Vet. J. 86:807.Arun Kumar et al. (1993) Indian J. Dairy Sci. 46: 292.Bhagat, R.L. and Gokhale, S. B. (1999) Indian J. Dairy Sci. 74(1): 98.Bhattacharya, H.K. and Buchoo, B.A. (2008) Indian J. Dairy Sci. 61:165.Chaudhuri, G. et al. (1984) Indian Vet. J. 61: 585.Mandal, D.K. and Tayagi, S.K. (2004) Indian J. Ani. Sci. 74(1): 98-99.Sangeeta, C. et al. (2002) Indian J. Dairy Sci. 55:313.Shukla, S. P. and Pandit, R. K. (1989) Indian Vet. J. 66:665.Singh, K. P. et al. (1988) Indian J. Ani. Res. 22: 100.Tomar, S. S. and Tripathi, V. N. (1986) Indian J. Dairy Sci. 39:363.

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IntroductionThe poultry industry is one of the most profitable ventures

of agriculture and provides nutritious meat and eggs for humanconsumption. The shortest incubation period in harvesting thebenefits and its ability to adopt in all the climates provide itsuperiority over the other ventures. In India nearly 30% eggsand 35% meat is produced by the rural poultry productionsystem (Khan, 1996). The situation is more or less similar inother developing countries also. The rapid growth of intensivepoultry production system, mainly comprising broilers andlayers, is dependent on balance feed, quality germplasm,protected housing and other infra-structural facilities to supportthe venture. The availability of quality feed at a reasonable costis a key to successful poultry operation (Basak et al., 2002).FAO programme focuses on increasing the feed baseproduction systems to locally available feed resources indeveloping countries (Sansoucy, 1993). For sustainability ofthe venture, it is necessary that the resources are availablelocally and competition with human food should be minimal. Ifpossible there should be a synergy of activities pertaining tocrop and livestock for the purpose (Preston and Murgucitio,1987).

In developing countries the situation is different and needsapproaches suiting to their socio-economic penury. The majorityof the farmers are resource poor and nearly 80-85% populationis either landless or small land holders. In India, the situationis more or less similar and the increasing human populationpressure, diminishing cultivable land resources owing tourbanization etc., lack of initial capital resources, monopolisticmarketing network and lack of awareness make situation morecomplex (Rai et al., 2011). The situation is worse with the

EVALUATION OF AZOLLA (AZOLLA PINNATA) AS A POULTRY FEED ANDITS ROLE IN POVERTY ALLEVIATION AMONG LANDLESS PEOPLE IN

NORTHERN PLAINS OF INDIA

R.B. Rai1*, K. Dhama2, T. Damodaran3, Hamid Ali4, Sweta Rai5, Balvir Singh6 and P. Bhatt7

Division of PathologyIndian Veterinary Research Institute, Izatnagar, Uttar Pradesh-243 122, India

ABSTRACT

The present study was undertaken to evaluate rural poultry as source of livelihood by reducing the feed cost. The evaluation wasdone in 42 villages in 2 districts of Uttar Pradesh, India. Two strains of poultry viz. Nirbhik (dual purpose) and Shyama (eggs) wereevaluated under semi-range system. The Nirbhik birds raised on Azolla pinnata achieved 1810± 12.5 g body weight by 14th week incomparision with non-azolla fed Nirbhik birds which achieved 1270 ± 12.9 g. The net return per 200 chicks in azolla fed group wasRs. 42330±20.4. The Shyama birds raised on azolla produced 197.6±3.2 eggs in 72 weeks in comparision of 138.4±3.1 eggs of non-azolla fed birds. The rural poultry integrated with in situ azolla cultivation was able to provide sustainable livelihood security as wellas income for diversifying the livelihood base under infra-structure constraints rural areas.

Key words: Azolla (Azolla pinnata), poultry

landless and marginal farmers as they are dependent on theearnings from the meager land available or the wages earnedas agricultural workers in the locality.

To address the situation for providing sustainable livelihoodsecurity and alleviating the poverty level of the families variousinterventions were made. Rural poultry production systemcomprises open range, semi-range and deep litter system ofrearing in backyards. While deep litter system requires externalinputs, the open range and semi-range systems do not requireexternal inputs. Under intensive system nearly 60-65% ofrecurring expenditure goes for the feed itself. Furthermore, forproducing one kg poultry meat under intensive system it isestimated that about 200 litres of water is required, and 4 kggreen house gases are produced. The resource poor farmersare unable to raise the initial capital costs of housing,equipments and chicks besides the risk involved.

The use of Azolla as a feed resource for pigs and poultryhad been tested with favourable results by us and manyworkers (Castillo et al. 1981; Basak et al., 2002; Alalade andIyayi, 2006; Singh and Subudhi, 1978). It is a free- floatingfresh water fern belonging to the family Azollaceae. It growsnaturally in stagnant water. Anabaena azollae, living in thecavity of Azolla leaf, can fix high amount of atmosphericdinitrogen due to the presence of symbolic algae in the leaves(Becking, 1979). Azolla grow luxuriously between 15-250 Ctemperatures and is rich in protein and the total crude proteincontent vary from 22-30% (Basak et al., 2002). The otherconstituents in Azolla are minerals, chlorophyll, carotinoids,amino acids, vitamins etc. Boyd (1968), Subudhi and Singh(1977) and Maurice et al. (1984) reported that inclusion ofaquatic plants at low levels in poultry diets had shown better

1Principal Scientist and P.I. NAIP-3 Project and corresponding author e-mail: [email protected] Scientist,3Senior Scientist, C.S.S.R.I.-R.R.S., Lucknow (U.P.)4Senior Research Fellow,5,6Research Associate, Division of Pathology, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh-243 122, India7Assistant Professor, Veterinary Clinics, College of Veterinary and Animal Sciences, Govind Ballabh Pant University of Agriculture and Technology,Pantnagar, Udham Singh Nagar, Uttarakhand - 263 145, India

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Table1: Status of annual income (Rupees) from sampled populationBarabanki Raebareli

Status Trivediganj Haidergarh Lalganj Sareni & Tera

Average annual income

Land less farmers (0-0.1 ha land) 14000.45 13500.20 16000.08 15000.15 14606 Marginal farmers (0.1-1 ha) 25900.45 24400.82 29100.25 27850.50 26809 Small farmers (1-3 ha) 50400.12 49058.55 52000.00 51400.00 50711 No. of farmers having backyard birds - - - - - No. of farmers Aware (a) Rural poultry production (b) Commercial broiler production tech.

2 -

2 1

1 -

2 -

Table 2: Effect of Azolla feeding on body weight of Nirbhik strain

Group Body weight at 8 week (g)

Body weight at 14 week

Pooled mortality at 14 th week (%)

Group I (n = 18)

782 10.8 1270 12.9 7.01

Group II (n = 18)

803 11.3 1581 11.8 5.2

Group III (n = 18)

839 11.7 1810 12.5 4.98

N = Number of farmers monitored

Table 3: Effect of Azolla feeding on egg production of Shyama strain

Group Total egg production at 40 week

Total egg production at 72 weeks (No. of birds recorded)

Pooled mortality at 14th week (%)

Group I (n = 6)

39.1 4.2 138.4 3.1 (206) 16.9

Group II (n = 6)

42.3 2.4 161.24.6 (215) 15.2

Group III (n = 8)

48.6 2.9 197.6 3.2 (221) 15.1

N = Number of farmers monitored

performance, especially when they supply part of the totalprotein. The cost of cultivation of Azolla is negligible and thusthe expenditure towards feed is saved by resource poorfarmers.

The present study was undertaken to evaluate Azollapinnata as sole feed supplement to poultry under rural poultryproduction system (open range and semi-range) and its rolein poverty alleviation and creating sustainable livelihood securityto landless and marginal land holder families.

Materials and MethodsThe present study was undertaken in villages of Barabanki

and Raebareli districts of U.P., India as an intervention underthe World Bank funded National Agricultural Innovation Projectof the Indian Council of Agricultural Research. The researchproject is being implemented in 2 clusters in each districtcomprising 10-12 villages in each cluster to develop practicalmodels of sustainable livelihood security focusing the ruralfamilies.

Base line survey: Base line survey covering 15% ofhouseholds in each cluster was conducted during April-July,2009 i.e. beginning of the project using random stratifiedsampling method for knowing status of livelihood andresources in rural areas under investigation.

Azolla cultivation: Azolla pinnata was cultivated in smallpits of 4 x 2 x 0.2 m (L x W x D) dimensions and in village ponds.The pits floor was covered with polythene sheets and about 2kg fresh cow dung and 60-70 g single super phosphate wasmixed in a bucket of water and poured in the pit. The pits werefilled with fresh water and about half kg of fresh Azolla wasinoculated in the pit by spreading on the surface. The pits weremade near the shelter of the poultry. The water was replaced atmonthly intervals.

Poultry rearing: Small shelters of approximately 8 x 4 x 4feet were constructed by farmers using mud. Asbestos sheetwas used as roof. One side of the shelter was made of chickenwire mesh and a door of wire mesh in wooden frame was

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made in the other side of shelter. Day old chicks of dual purposestrains of poultry, Nirbhik and Shyama, targeting for growthand egg production, respectively, were provided to the farmersin batches of 50 chicks at interval of 21-35 days to a total of 200chicks in the year. The chicks were fed either chick mesh orfinely grinded wheat for 7-10 days and were allowed to grazeonward.

Evaluation of azolla as sole feed: In the field conditionsand existing situations of non-availability of sufficient grazingspace and concentrate ration, the evaluation of azolla as solefeed resource was evaluated. For the purpose 3 groups offarmers were identified as:

Group I: No supplementary feeding; only grazing inbackyard was practiced.

Group II: Besides grazing in backyard, 0.5-1 kg wastegrain per 50 birds per day was supplemented.

Group III: Azolla pinnata (ad lib.) was provided to thebirds and it was the main feed during grazing.

The birds were monitored for mortality and body weight at8th and 14th week.

Economic evaluation for livelihood security: Farmers inall the three groups were monitored for their cash input cost,cash return, utilization pattern of returns and sustainance whichis vital for livelihood security.

Results and DiscussionBase line survey: The data of 42 villages of Barabanki

and Raebareli districts of U.P., India, surveyed for initialeconomic status of the rural farmers are presented in the Table1. The average family size across the clusters was 7.1. Theaverage income varied to ‘ 14606, 26809 and 50711 per annumfor landless, marginal and small farmers, respectively. Themajor source of livelihood for landless families was workingas agricultural laborers, hawkers or as helpers in nearby villagemarket shops. The major livelihood source for marginal andsmall farmers was agricultural produce i.e. wheat, paddy,pulses, oilseeds and rearing of bovines. None of the farmersout of 42 villages were having any backyard bird but fewoccasionally reared 1-5 birds whenever they got for family use.Only 7 farmers were aware with the rural poultry productiontechnology and one with commercial broiler production.

Azolla cultivation: The northern India has 3 distinctseasons viz., summer, rainy and winter season. Thetemperature during summer reaches 44-45oC and duringwinter months may drop to 2-3oC as lowest peak. The growthof Azolla pinnata was optimum for 9-10 months but decreasewas seen during mid December to January and June months.In peak season when the ideal temperature was available(15-30oC), 7-8 kg fresh fern was harvested per week from

Fig.1: Showing wellness due to poultry feed Azolla (Azolla pinnata) among landless poultry farmers

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each pit. The birds of the Group III were fed the fern and averageconsumption varied from 30-90 g/day/bird. Farmers fed theAzolla daily in the morning and afternoon.

Effect on growth: The growth rate of Nirbhik strain is givenin the Table 2 for all the 3 groups. The body weight gain inAzolla fed group was 1.81 kg at 14th week (tender meat stage)compared to 1.58 kg in Group-II (grain supplementation) and1.27 kg of Group-I (without any supplementation). The mortalityvaried between 4.98 to 7.01%. The causes of mortality werepredation, pneumonia and salmonellosis. In one batch undergroup 1, one farmer suffered chick mortality due to IBD whichclaimed 24 deaths out of 50 chicks supplied.

Evaluation of return and livelihood: The farmers sold thebirds in the local market gradually. Though a total of 604 farmerswere covered under the interventions but data of 18 farmers ineach group was compiled and monitored. The price of livebirds in local market varied between Rs.100-150/kg dependingon the season. Almost 90% farmers sold Nirbhik strain birdswhen they attained body weight between 1.5 -2 kg and realizedthe cash. However, farmers provided with Shyama strainretained nearly 50% of the available total female birds for egglaying but sold the males and about 50% female birds also tofulfill their domestic needs.

The egg laying birds available with farmers in the 3 groupswere monitored for egg production status and effect of Azollafeeding. The results are presented in the Table 3. The Group-III birds produced 197.6 eggs which was comparatively muchlower in Group-II (161.2) and Group-I (138.4 eggs).

The cost of chicks irrespective of strain was Rs.10/- each.The cost of shelter was Rs. 452.5± 6.5 per farmers. The laborcost was not assessed as it was by family itself. The cost ofsingle super phosphate, which was purchased, was Rs. 4/-kg. thus, each pit required cash input cost of Rs. 44/- per annumand produced approximately 300 kg fresh Azolla per annum.

The average income per bird sold was between Rs. 150-210 depending on weight and season. The mortality up to 72weeks was between 15.1-16.9 and the main reasons reportedwere predation, pneumonia, septicaemia, salmonellosis andcolibacillosis.

The income received from 200 birds reared by farmersalso varied. The highest average income was realized fromAzolla fed Nirbihik birds which was Rs. 42,330± 20.4 comparedto non-supplemented group (Rs.26938±10.3) and partiallysupplemented group (Rs.35892 ±12.4)

In the villages under investigation 30.26% population islandless followed by marginal (38.16%) and small farmers(26.98%). The average family size of 7.1 with average annualfamily income of Rs.14000.45 of landless population is acomplex situation and a major challenge to developsustainable on farm models for their livelihood security. Eventhe annual income of small land holders Rs. 26809 of marginaland Rs. 50711 of small farmers are not sufficient to cater theneed of the families such as good education for childrennutritional and health security. The existing models availableneed capital investment which is not available with theseresource poor farmers. Even a small size broiler farm withcapacity of 250-500 chicks needs investment in the range ofRs. 50,000-80,000/-. The estimated feed cost under deep littersystem is 60-65% of total recurring expenditure.

In the present study integration of Azolla in situ cultivation

and feeding to birds under semi-range saved the feed cost bymore than 80%. The higher growth rate, as also reported bymany workers (Castillo et al., 1981; Basak et al., 2002; Alaladeand Iyayi, 2006; Singh and Subudhi, 1978) due to higher crudeprotein content (above 22%), minerals and vitamins availablein the fern containing Azolla were able to meet the majorrequirement of the body, and grazing in the backyardsupplemented with other amino acids and nutrients. The non-Azolla fed group showed lower growth rate and egg productionand, thus, the advantage of Azolla is fully established. Thereports are in conformity with other workers (Basak et al., 2002).The smaller leaf size of Azolla pinnata is suitable for intake bythe chicks as well as grower/adults.

The livelihood security of such resource poor farmers isreal challenge. They lack resources to initiate any venturebesides the technical knowledge. The fear of risk involved withany live and capital intensive venture is beyond their capacity.The rural poultry production commonly in form of open rangesystem can only provide subsidiary income where as smallunit of deep litter system results in poor profits margin due tohigh feed cost and its non-availability in the vicinity. In thisscenario the present approach proved a viable venture. The insitu azolla cultivation at negligible cost was able to sustain thegrowth which was near to the standard parameters of thestrains under farm conditions. In the present study out of 604farmers, 52 upgraded their venture into deep litter broilerproduction besides continuance of the present system in thesubsequent years which yielded about Rs. 6000-10000/ percycle of 250-400 broiler chick units. A few farmers graduallyconstructed/renovated their own houses, purchased lactatingcows/buffalo for milk, also started goatery unit from the earningsin which this integration of poultry with azolla played major roll.Almost all farmers increased their rural poultry intake frominitial 50 chicks to 150-300 per batch within 6 months thoughthe data of separate units are not included in the present report.The most significant result was capacity building and comingout of vicious cycle of poverty. Some of the landless families re-started sending their children to school again which wasdiscontinued due to economic reasons.

The present study is suggestive of a viable on farm modelof rural poultry integrated with in situ Azolla cultivation andfeeding where the initial capital for taking up the desired activitiesfor sustainable livelihood security can be generated.Simultaneously, it can serve itself as source of sustainablelivelihood security, besides assuring nutritional and healthsecurity. Rural poultry production system, particularly semi-range based technology, can be a viable tool for povertyalleviation among landless and small land holder masses.This does not require higher level of technical competence.

AcknowledgementAuthors are thankful to National Agricultural Innovation

Project (NAIP) Project, ICAR, New Delhi for financial supportand Director, IVRI for providing necessary research facilities tocarry out the present research work.

ReferencesAlalade, O.A. and Iyayi, E.A. (2006) International J. of Poultry

Sci. 5:137-141.Basak,B. et al. (2002) International J. of Poultry Sci. 1:29-34.

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Becking, J.H. (1979) Environmental requirement of Azolla forthe use of tropical rice production. In: Nitrogen andRice. International Rice Research Institute, Los Banos,Leguna, Philippines, pp. 345-374.

Boyd, C.E. (1968) Economic Botany. 22:359-365.Castillo, L.S. et al. (1981) Exploratory studies on Azolla and

fermented ricehulls in broiler diets . College, Leguna(Philippines), pp. 6.

Khan, A.G. (1996) Indigenous poultry and strategies for improvingsustainable rural poultry production in India. IndianFarming. 46:19-24.

Maurice, D.V. et al. (1984) Poultry Sci. 63:317-323.Preston, T.R. and Margueitio, E. (1987) Trees and shrub legumes

as protein sources for livestock. In Forage legumes

and other local protein sources as substitute forimportant protein meals. D. Walmsley (ed.), CTA,Wageningen and CARDI, Trinidad, pp. 94-104.

Rai, R.B. et al. (2011) Livestock based specialized integratedfarming system for livelihood security and se lfemployment in Is land ecosystem . Lead paper inInternational conference on tropical islands ecosystem,March, 23-26, Port Blair.

Sansoucy, R. (1993) The FAO program for better utilization oflocal resources in developing countries . In Proc. 7 th

W orld Conference Animal Production. Edmonton,Canada, pp. 77-80.

Singh, P.K. and Subudhi, B.P.R. (1978) Indian Farming. 27:37-39.

Subudhi, B.P.R. and Singh, P.K. (1977) Poultry Sci. 57:378-380.

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IntroductionSheep is an important economic livestock species that

helps to strengthen the backbone of rural economy of north-western arid and semiarid regions of Rajasthan. Marwaribreed of sheep is reared for its medium and coarse qualitycarpet wool and mutton. It is well known for its high diseaseresistance, draught tolerance, faster growth rate and capacityto travel longer distances in search of forage. The economicsof sheep production is greatly affected by the growthperformance as heavier lambs with high growth rate wouldfetch relatively more economic returns in lesser time spancompared to weaker lambs (Narula et al., 2009). With the risingprices of mutton in the market, fast growing and heavier lambsare in great demand. Post- weaning growth performance is asimportant as the pre-weaning performance and is affected bynumerous genetic and non-genetic factors. In our country,generally selection is done at six months of age. Moreover,selection of ram is more profitable when it has grown to its fullmaturity and expresses the desired traits. Also, the impact ofmaternal environment is very negligible after weaning. Thus,the present study was conducted to investigate the effects ofgenetic and non-genetic factors on post-weaning growthperformances in Marwari sheep.

Materials and MethodsPedigree and performance data on 1371 animals used

in the present study was collected from the database of Marwarisheep, maintained at arid region campus of Central Sheepand Wool Research Institute, District Bikaner, Rajasthan, overa period of 12 years (1999-2010), divided into four classes viz.P1 (1999-2001), P2 (2002-2004), P3 (2005-2007) and P4(2008-2010). Performance traits included in the study werebody weight at six (6WT), nine (9WT) and twelve (12WT) months

EFFECT OF GENETIC AND NON-GENETIC FACTORS ONPOST-WEANING GROWTH TRAITS IN MARWARI SHEEP#

H. Singh, U. Pannu, H.K. Narula1, B.K. Beniwal, A. Chopra2 and Vivekanand3

Department of Animal Breeding and GeneticsCollege of Veterinary and Animal Science


ABSTRACT

The present investigation was undertaken to evaluate the effect of genetic and non-genetic factors on post-weaning growth traitsin Marwari flock comprising records of 1371 animals maintained at arid region campus of Central Sheep and Wool Research Institute,Bikaner. The least-squares mean for body weight at 6, 9 and 12 months of ages and average daily gain during weaning to 6 and 6 to12 months of ages were 21.14 ± 0.188 kg, 25.85 ± 0.166 kg, 28.87 ± 0.211 kg, 66.75 ± 1.031 g/day and 38.67 ± 1.170 g/day,respectively. The least-squares analysis of variance revealed highly significant (P< 0.01) effect of sire and sex on all the post-weaning traits. Effect of year of birth and regression of dam’s weight at lambing was a significant (P<0.01) source of variation for allthe post-weaning body weights but found non-significant on average daily gains. Effect of parity was found significant (P<0.01) onlyon nine month’s body weight.

Key words: Marwari sheep, post-weaning growth, genetic and non-genetic factors

of age and post-weaning average daily gains i.e., from three tosix (ADG1) and six to twelve (ADG2) months of ages. Averagedaily gain was calculated by formula (Final weight of the period-Initial weight of the period/final age-initial age).

Since the subclass numbers were unequal anddisproportionate, the data was analyzed by the least-squaresprocedure of fitting constants and using LSMLMW programme(Harvey, 1990). The statistical model included sire as a randomeffect; sex (two classes), period of birth and parity (four classeseach) as fixed effects; and regression of dam’s weight atlambing as a covariate. Duncan’s multiple range test (DMRT)as modified by Kramer (1957) was used for pair wisecomparison among the means.

Results and DiscussionThe overall least-squares mean for body weight at six

(6WT), nine (9WT), twelve (12WT) months of age and averagedaily gain from three to six (ADG1) and six to twelve (ADG2)months of ages were found to be 21.14 ± 0.188 kg, 25.85 ±0.166 kg, 28.87 ± 0.211 kg, 66.75 ± 1.031 g/day and 38.67 ±1.170 g/day, respectively (Table 1). As described earlier, averagedaily gain during 3-6 months (ADG1) was calculated by formula(three months body weight-six months body weight/90), sooverall least-squares mean for weaning (WWT) that comprisedof 1525 records was 15.13 ± 0.138 kg used in this analysis.

The effect of sire was observed to be highly significant(P<0.01) on all post-weaning growth traits under study. Thesignificant effect of sires on these traits studied indicated thatheavier sires could be used effectively for genetic improvementof these traits. Arora et al. (1999) in Avikalin and Malpura sheep,Tomar et al. (2000) in Bharat Merino sheep and Gohil (2010) inMarwari sheep also observed similar significant effect of sireon post-weaning body weights. Gohil (2010) reported

#Part of M.V.Sc. Thesis of first author. Corresponding author E mail: [email protected] region campus, Central Sheep and Wool Research Institute, Bikaner-334001, Rajasthan, India2Central Sheep and Wool Research Institute, Avikanagar-304501, Tonk, Rajasthan, India3M.V.Sc. Scholar

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significant effect of sire also on average daily gains during 3-6and 6-12 months of ages in Marwari sheep.

As expected, sex had a highly significant (P<0.01) influenceon post-weaning live weights and growth rates. Similar resultswere also reported in the literature by Sahani et al. (2002),Nehra and Singh (2006), Dass et al. (2008), Narula et al. (2010)and Gohil (2010) in Marwari lambs. The influence of sex on liveweight difference increased from 13.48% (2.67 kg) at sixmonths to 19.33 % (5.09 kg) at 12 months of age. This mightbe due to different physiological processes operating in thetwo sexes. Also, males grew approximately 24.03% (14.32 g/day) and 21.10% (7.38 g/day) faster than the females from 3-6and 6-12 months of age, respectively. This indicates that sexeffects are more pronounced with age after puberty.

Period of birth had a highly significant (P<0.01) effect onall the post-weaning growth traits under study, except for ADG2.Live weights showed an increasing trend from P1 to P2 andthen decreases subsequently in P3 and P4, for all the bodyweight traits under study. However, maximum growth was

observed during P3 and P4 for average daily gain during 3-6and 6-12 months of age, respectively. Differences in the growthduring different periods are attributed to variation in differentclimatic conditions like rate of annual precipitation and rainfall,temperature and humidity, environmental and managementalconditions which influence the quality and accessibility ofnutrients in pasture. The present result agrees with the findingsof Nehra and Singh (2006), Dass et al. (2008), Narula et al.(2010), and Gohil (2010) in Marwari lambs.

The effect of parity was found non-significant on all thetraits under study, excluding nine month’s body weight whereit was found to be highly significant (P<0.01). After weaning,influence of dam (maternal ability) is negligible on post-weaning growth traits and thus it is not surprising that postweaning weights at 6 and 12 months, along with post-weaningaverage daily gains were not significantly affected by parity.Non-significant effect of parity on post weaning growth traitswas reported by Mishra et al. (2008) in Garole × Malpura sheep.However, Swain et al. (2004) in Bharat Merino and Gohil (2010)

Table 1: Least-squares mean (± S.E) for post-weaning growth traits in Marwari lambs

Traits/ Factors 6WT 9WT 12WT ADG1 ADG2 Overall Mean

(µ) 21.14 ± 0.188

(1371) 25.85 ± 0.166

(850) 28.87 ± 0.211

(612) 66.75 ± 1.031

(1371) 38.67 ± 1.170

(612) Sire ** ** ** ** **

Period ** ** ** ** NS P1 (1999-2001) 17.11 ± 0.398a

(203) 21.33 ± 0.345a

(140) 24.63 ± 0.467a

(71) 48.74 ± 2.199a

(203) 36.66 ± 2.299

(71)

P2 (2002-2004) 23.54 ± 0.414d (194)

27.52± 0.345b (164)

30.54 ± 0.433b (127)

66.07 ± 2.279b (194)

40.82 ± 2.490 (127)

P3 (2005-2007) 22.71 ± 0.338c (259)

27.43± 0.312b (202)

30.20 ± 0.375b (172)

76.28 ± 2.012c (259)

36.36 ± 1.980 (172)

P4 (2008-2010) 21.22 ± 0.242b (715)

27.11 ± 0.247b (344)

30.10 ± 0.305b (242)

75.91 ± 1.317c (715)

40.83 ± 1.712 (242)

Sex ** ** ** ** ** Male

22.48 ± 0.201b

(666) 28.10 ± 0.194b

(384) 31.41 ± 0.248b

(236) 73.91 ± 1.171b

(666) 42.36 ± 1.300b

(236)

Female

19.81 ± 0.201a

(705) 23.60 ± 0.187a

(466) 26.32 ± 0.230a

(376) 59.59 ± 1.173a

(705) 34.98 ± 1.216a

(376) Parity NS ** NS NS NS

1st

21.20 ± 0.215

(493) 25.65 ± 0.208a

(319) 28.69 ± 0.256

(241) 66.87 ± 1.290

(493) 37.95 ± 1.298

(241) 2nd

21.23 ± 0.214

(438) 26.23 ± 0.214b

(272) 29.09 ± 0.262

(194) 68.32 ± 1.295

(438) 38.25 ± 1.318

(194) 3rd

21.26 ± 0.241 (264)

26.24 ± 0.259b (163)

29.08 ± 0.312 (115)

66.33 ± 1.555 (264)

38.24 ± 1.524 (115)

= 4th

20.89 ± 0.270 (176)

25.28 ± 0.315a (96)

28.62 ± 0.388 (62)

65.50 ± 1.819 (176)

40.24 ± 1.786 (62)

Dam’s weight ** ** ** NS NS Regression Coefficient 0.132 ± 0.0229 0.144 ± 0.0299 0.136 ± 0.0329 0.282 ± 0.1736 0.148 ±

0.1376 No. of observations are given in parenthesis. Figure with different superscripts differ significantly. ** - Highly significant (P<0.01); NS -

Non-significant; 6WT= 6-month weight; 9WT= 9-month weight; 12WT= 12-month weight; ADG1= average daily gain from 3-6 months;ADG2= average daily gain from 6-12 months

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in Marwari sheep reported significant effect of parity on ninemonths body weight.

Dam’s weight at lambing was a highly significant (P< 0.01)source of variation in all the post-weaning body weights. Thisrevealed that lambs that were born heavier from heavier damsmaintained their superiority in advanced age i.e. after weaning.However, regression of post-weaning average daily gains ondam’s weight at lambing was found non-significant. Singh etal. (1998), Dass et al. (2008), Narula et al. (2010) and Gohil(2010) also reported similar influence of dam’s weight atlambing on post-weaning body weights in Marwari breed ofsheep.

AcknowledgementsAuthors are thankful to the Director, CSWRI; Head, ARC,

CSWRI; Vice-chancellor, RAJUVAS; and Dean, CVAS; forproviding the facilities for the execution of work. The technicalhelp rendered by Mr. Vimal Mehrotra T-7-8 at ARC, CSWRI,Bikaner, during data collection is deeply acknowledged.

ReferencesArora, A.L. et al. (1999) The Indian J. Small Ruminants. 5(1): 4-8.Dass, G. et al. (2008) Indian J. Ani. Sci. 78(9): 1019-1022.Gohil, G. (2010) Genetic evaluation of growth and reproduction of

Marwari sheep. M.V.Sc. Thesis submitted to RajasthanAgricultural University, Bikaner.

Harvey, H.R. (1990) User’s guide for LSMLMW PC –É Version mixedmodel Least Squares and Maximum Likelihood computerprogramme. LOWA State University, USA.

Kramer C.Y. (1957) Biometrics. 13:13.Mishra A.K. et al. (2008) Indian J. Ani. Sci. 78(7): 746-750.Narula H.K. et al. (2010) Indian J. Ani. Sci. 80(4): 350-353.Narula H.K. et al. (2009) Indian J. Ani. Sci. 79(6): 639-641.Nehra, K.S. and Singh, V.K. (2006) The Indian J. Small Ruminants.

12: 91-94.Sahani M.S. et al. (2002) Indian J. Ani. Sci. 72(8): 704- 705.Singh V.K. et al. (1998) The Indian J. Small Ruminants. 4(1): 1-4.Swain, N. et al. (2004) Indian Vet. J. 81 (6): 679-682.Tomar, A. K. S. et al. (2000) Indian J. Ani. Sci. 70 (6): 647-648.

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IntroductionIndia has about 40 breeds of sheep, out of which 24

are d istinc t . Among these eight b reeds are f romRajasthan alone. These are Magra, Marwari, Pugal,Jaisalmeri, Nail, Malpura, Sonadi and Chokla. Magrabreed (Chakri) is found in Bikaner and Nagour districts ofRajasthan. Bikaneri Chokla is a variant of Magra breedand is abundantly available in Loonkaransar and BikanerTehsils in Bikaner district particularly towards eastern andsouthern parts of Bikaner. Bikaneri Chokla wool deemedto be the best carpet wool in the world . Its wool isextremely white and lusterous. The characteristic ofBikaneri Chokla wool suit to the requirement of idealcarpet in terms of micron, length, strength and resilience.B ikaner i Chok la sheep is medium to large in size,passess white face with brown patches around the eyes,pink skin, small to medium and tubular eyes, both sexespolled and tail medium in length and thin. Magra andBikaner i Chokla breed of sheep have sign if ican tdifferences in wool quality. The threat of extinction ofBikaneri Chokla sheep is because o f unscrupulousbreeding and distortion of the wool quality and alsoquantity. A healthy Bikaneri Chokla sheep produces morethan 2.5 kg of fleece in a year and the need of the hour isto take all measures to save this breed and to improveupon its number by selective breeding programme in aconcerted manner.

Materials and MethodsThe present study was conducted on 580 records of

sheep maintained at Kodamdesar Farm under W oolImprovement Scheme (SWIS), CVAS, RAJUVAS, Bikaner.The data were classified on the basis of sex (male andfemale) and age group (young and adult). The sheepbelow one year of age was grouped as young while aboveone year in adult age group. The att ributes like

STUDY ON WOOL QUALITY ATTRIBUTES IN BIKANERI CHOKLASHEEP IN ARID REGION OF RAJASTHAN

Madhu Prajapati1, R. S. Prajapati2, C. K. Murdia and Urmila PannuDepartment of Animal Breeding and Genetics

College of Veterinary and Animal Scinece, RAJUVAS, Bikaner-334001

ABSTRACT

The study was conducted on wool attributes of Bikaneri Chokla breed of sheep in arid region of Rajasthan. The overallleast- squares means for fibre diameter, medullation, staple length, number of crimps, hairy f ibre, hetero fibre and purefibre were 36±0.353 micron 53.61±1.282 %, 7.77±0.111 cm, 1.02±0.024, 17.41±0.938 %, 36.39±0.929 %, and 46.19±1.274%, respectively. The effect of sex was signif icant on number of crimps only while the effect of age was significant onlyon staple length. The wool quality parameters indicated the wool to be of good carpet quality with average fibre diameterand staple length. However, the medullation percentage was on higher side which needs to be reduced through appropri-ate breeding strategies.

Key words: Wool, Bikaneri Chokla, sheep, arid region

1M.V.Sc. Scholar2Veterinary Officer, Jhabua-457770, Madhya Pradesh

medullation percent, f ib re diameter, staple length ,number of crimps per cm, per cent hetero, hairy and purefibres were analysed in Wool Analysis Laboratory ofAnimal Husbandry Department, Goverment of Rajasthan,Bikaner. The data were subjected to Least-squares andMaximum likelihood computer programme (Harvey, 1990).The statistical model included the fixed effect of agegroups and sexes.

Yijk = µ+ Si + Aj + eijkWhere,

Yijk=kth observation of animal of ith age group and j th

sexµ=overall meanAi=effect due to ith age groupSj=effect due j th sexeijk=random error

Results and DiscussionThe overall least-squares means for fibre diameter,

medullation percentage, and staple length, number ofcrimps per cm, per cent hairy fibre, per cent hetero fibreand per cent pure fibre were 36±0.35 micron, 53.61±1.28%, 7.77±0.11 cm, 1.02±0.02, 17.41±0.94 %, 36.39±0.93%, and 46.19±1.27 %, respectively, as shown in Table 1.

Contrary to the above results Mehta et al. (1998)reported lower fibre diameter, medullation% and staplelength in Magra sheep at three months of age in fieldcondition. Lower fibre diameter, medullation percentage,staple length, number of crimps per cm, hairy fibre%, andhetero fibre% were reported by Narula et al. (2011) inMagra sheep.

In present study sex had non-significant effect on allthe traits except number of crimps per cm. The non-significant effect of sex on fibre diameter, medullation%was reported in Patanwadi and its crosses by Shah andPatel (1989). Similarly sex had no influence on fibre

259


diameter, medullation% and pure fibre% in Marwari breedof sheep as reported by Pannu (1994).

However, the effect of sex was observed to besignificant on fibre diameter and medullation per cent inthe study conducted by Chopra and Chopra (1972) inLohi sheep and Haq and Rahman (1986) in Romney andPerdale g rades o f sheep. The lower medu llat ionpercentage in clips from males was reported by Charyulaand Acharya (1988) in Chokla, Malpura and their crosseswith Rambouillet. The wool from males had significantlyfiner fib res (35.9±0.44 µ) and greater proportion ofmedullated fibre (41.8±1.51%) in comparision to that offemales. Fibre diameter of 37.00±0.38 µ and medullationpercentage of 46.7±1.40% were reported by Taneja et al.(1991) in field data on Magra sheep. Females had higherpercentage of medullated fibre as compared to males asrevealed by Sharma (1998) in Nail breed of sheep. Maleshad higher percentage of medullated fibres as comparedto females in Magra. Ganai et al. (2009) reported fibrelength, staple length and average crimpness % in Karnahsheep on the basis of sex. Fibre length and staple lengthwere higher in females while average crimpness% wasfound to be higher in males. The significant effect of sexon crimp was also reported by Narula et al. (2011) inMagra sheep.

In present study age had no significant effect on allthe traits except staple length. The length of wool fibrewas observed to be more in adult than young one.

ReferencesCharyulu, E. K. and Acharya, R. M. (1988) Indian J. Ani. Sci.

58:1342.Chopra S. C. and Chopra, C. S. (1972) Indian J. Ani. Sci. 42:

363-369.Ganai, T. A. S. et al. (2009) Indian J. Small Ruminants. 15(2):172.Haq, M. A. and Rahman, M. M. (1986) Indian Vet. J. 63:579.Harvey, W. R. (1990) Mixed Model Least-Squares and Maximum

Likelihood Computer Programme PC-2 version. OhioState University, USA.

Mehta, S. C. et al. (1998) Indian J. Ani. Sci. 68:91.Narula, H. K. et al. (2011) Indian Vet. J. 88(5):53.Pannu, U. (1994) Genetic evaluation of performance of Marwari

sheep of different selection lines . M. V. Sc. ThesisSubmitted to RAJUVAS, Bikaner, Rajasthan (India).

Shah, R. R. and Patel, M. M. (1989) Indian J. Ani. Genetics andBreeding. 11:22.

Sharma, M. (1998) Comparative performance of Karakul andMarwari breeds of sheep in arid zone of Rajasthan. M.V.Sc. Thes is Submi tted to RAJUVAS, Bikaner,Rajasthan (India).

Taneja, A. D. et al. (1991) Wool and Woolness of India, Oct-Dec: 27-30.

Table 1: Least-squares means for wool quality traits in Bikaneri Chokla sheep

Particulates Fibre diameter (micron)

Medullation (%)

Staple length (cm)

Number of crimps/cm

Hairy fibre (%)

Hetero fibre (%)

Pure fibre (%)

Overall(580) 36.57±0.35 53.61±1.28 7.77±0.11 1.02±0.02 17.41±0.94 36.39±0.93 46.19±1.27 Sex NS NS NS * NS NS NS Male (382) 36.36±0.38 52.60±1.37 7.72±0.12 1.07b±0.03 16.88±1.003 35.97±0.99 47.14±1.36 Female (198) 36.78±0.47 54.62±1.72 7.82±0.15 0.97a±0.03 17.95±1.26 36.81±1.25 45.24±1.71 Age group NS NS ** NS NS NS NS Young (505) 36.51±0.26 54.86±0.94 7.29a±0.08 1.04±0.02 17.57±0.68 37.30±0.68 45.13±0.93 Adult(75) 36.64±0.65 52.36±2.35 8.25b±0.20 1.00±0.05 17.06±1.72 35.48±1.70 47.26±2.33

NS non-significant; *significant (P<0.05); **highly significant (P<0.01). Figures in parenthesis indicate number of observations.

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IntroductionThe milk production can be enhanced by improving the

production performance of non-descript cattle by planned andorganized breeding with the indigenous breeds of highergenetic merit. Though crossbreeding of native stock with exoticbreeds will give quicker response, yet the crossbred cows areless tolerant to the hot-humid tropical climatic condition of ourcountry and are prone to tropical diseases. In this situation,the indigenous breed like Sahiwal is the most suitable option.Obviously no genetic improvement programme in dairyanimals can succeed unless superior sires can be identifiedand multiplied. The selection of the superior sires withmaximum accuracy is of prime importance, any mistake insire selection will affect all the progenies of the next generationproduced by that bull. Part production records may be helpfulin making an early evaluation of a bull as well as for earlyselection of dairy cow. The preliminary sire evaluation on thebasis of part lactation yield, having higher heritability of partlactation milk yield than first lactation 300 days milk yield givesgenetic correlation approximately same. Hence use of partlactation records in sire evaluation is desirable. Several workers(Dutt et al., 1961; Sikka and Taneja, 1981; Shrivastava andKhan, 1987) have reported about the reliability of using partialrecords.

Materials and MethodsThe data utilized under present analysis were collected

from State Livestock cum Agriculture farm, Chak-ganjaria,Lucknow (U.P.). The performance records of 485 Sahiwal cattlewere collected for genetic analysis. The cows were progeniesof 46 sires born during the years from 1980 to 2003 and calvedduring year 1983 to 2006 (24 years period). Lactation recordsthose were considered abnormal on account of systemicdisorders, abortion and death of the calf during lactation, andrecords below 150 days lactation yield, were omitted from thepresent study. The traits taken for genetic study were production

ESTIMATION OF GENETIC PARAMETERS OF PART LACTATIONRECORD IN SAHIWAL HERD

Sumit Kumar1*, R. Goel1, Sanjiv Kumar2 and Pawanjit Singh3

Department of Animal Genetics and BreedingCollege of Veterinary Science and Animal Husbandry, DUVASU, Mathura- 281001, Uttar Pradesh, INDIA

ABSTRACT

The present investigation was carried out for genetic study of production traits viz. first lactation 300 days milk yield (MY-300), 90 days milk yield (MY-90), 120 days milk yield (MY-120), 150 days milk yield (MY-150), 180 days milk yield (MY-180),first lactation peak yield (FPY), and days to reach peak yield (DTRP). The heritability estimates for 90, 120, 150, 180 dayspart milk yield, peak yield, days to reach peak yield and 300 days milk yield were 0.129 ± 0.042, 0.145 ± 0.045, 0.237 ±0.063, 0.303 ± 0.075, 0.384 ± 0.089, 0.365 ± 0.086 and 0.322 ± 0.078, respectively. The genetic correlations of 90, 120,150, 180 days milk yield and peak yield with 300 days first lactation milk yield were 0.636 ± 0.121, 0.807 ± 0.067, 0.754 ±0.077, 0.611 ± 0.108 and 0.722 ± 0.080, respectively and of days to reach peak yield was high, negative and significant.

Key words: Part production, sire evaluation, heritability.

traits viz. first lactation 300 days milk yield (MY-300), 90 daysmilk yield (MY-90), 120 days milk yield (MY-120), 150 days milkyield (MY-150), 180 days milk yield (MY-180), first lactation peakyield (FPY) and days to reach peak yield (DTRP).

The statistical analysis was carried out by least squareanalysis of variance method (Harvey, 1960) to overcome non-orthogonality of data. The least squares constants for the factorsunder study, when the interactions of these factors were non-existent were fitted according to the following mathematicalmodel:

Yijkn = m + Pi + Sj + Ak + eijknWhere,

Yijkn = nth observation in the jth season of calving in ith periodof calving in kth age group.

m= Population meanPi= Effect of ith period (i=1-8)Sj= Effect of jth season (j=1-3)Ak= Effect of age at first calving group (k=1-8)

eijkn = Random error associated with observations in the(ijk)th cell which is assumed to be normally and independentlydistributed with mean zero and variance s2.

The heritability estimates were calculated by paternal half-sib correlation method described by Hazel and Terril(1945).The standard error of the heritability was computedwith the method given by Dickerson (1959) for half-sibcorrelation.

Genetic correlation was calculated as follows: Cov(sisj)rg ig j = _______________

(Vsi. Vsj)1/2

Where,rg ig j = Genetic correlation coefficients between i th andjthcharacters.Cov(sisj) = Sire component of covariance between ith andjth characters.

1*Department of Animal Genetics and Breeding. Corresponding author; email: [email protected], Mob. +91-94576833062Department of Pathology3Department of Biochemistry.

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Table 1: Estimates of heritability, genetic and phenotypic correlations with ±SE among different traits inSahiwal cattle

T ra its 90 M Y 120 M Y 150 M Y 180 M Y 300 M Y P Y D T R P

90 M Y 0.129 ± 0 .042

0 .829 ± 0 .070

0 .804 ± 0 .073

0.762 ± 0 .084

0 .636 ± 0 .121

0 .612 ± 0 .122

-0.81 3 ± 0 .066

120 M Y 0 .886 ± 0 .020

0 .145 ± 0 .045

0 .823 ± 0 .065

0.795 ± 0 .072

0 .807 ± 0 .067

0 .752 ± 0 .082

-0.64 2 ± 0 .112

150 M Y 0 .728 ± 0 .031

0 .764 ± 0 .029

0 .237 ± 0 .063

0.831 ± 0 .056

0 .754 ± 0 .077

0 .663 ± 0 .098

-0.70 5 ± 0 .089

180 M Y 0 .611 ± 0 .036

0 .582 ± 0 .037

0 .798 ± 0 .027

0.303 ± 0 .075

0 .611 ± 0 .108

0 .709 ± 0 .084

-0.80 5 ± 0 .060

300 M Y 0 .329 ± 0 .043

0 .453 ± 0 .040

0 .340 ± 0 .040

0.375 ± 0 .042

0 .322 ± 0 .078

0 .722 ± 0 .080

-0.72 2 ± 0 .153

P Y 0 .293 ± 0 .043

0 .382 ± 0 .042

0 .284 ± 0 .043

0.321 ± 0 .043

0 .802 ± 0 .027

0 .384 ± 0 .089

-0.83 6 ± 0 .041

D T R P -0 .36 6 ± 0 .042

-0 .44 3 ± 0 .041

-0 .35 4 ± 0 .042

-0 .36 9 ± 0 .042

-0 .80 2 ± 0 .026

-0 .79 5 ± 0 .028

0 .365 ± 0 .086

Diagonal represent the heritability of different traitsAbove the diagonal Genetic correlation between different traitsBelow the diagonal phenotypic correlation between different traits

Vsi = Sire component of variance of ith character.Vsj = Sire component of variance of j th character.

The standard error of the genetic correlation wasestimated by the formula given by Robertson (1959).

The phenotypic correlation coefficients were estimateby the formula given by Searle (1961).

Cov(sisj) + Cov (eiej)rp ip j = _____________________________

[(Vsi + Vei) (Vsj + Vej)]1/2

Where,Cov(sisj) = Sire component of covariance between ith andjth characters.Cov (eiej) =Error component of covariance between ith andjth characters.Vsi = Sire component of variance of ith character.Vsj = Sire component of variance of j th character.Vei =Error component of variance of ith characterVej =Error component of variance of j th character

The standard errors of the phenotypic correlationcoefficient were obtained by the formula given by Panseand Sukhatme (1967).

Results and DiscussionIn present study the heritability estimates for 90, 120,

150, 180 days part yield, peak yield, days to reach peakyield and 300 days milk yield were 0 .129±0.042,0.145±0.045, 0.237±0.063, 0.303±0.075, 0.384 ±0.089,0.365±0.086 and 0.32±0.078, respectively. All theseestimates were medium in magnitude and significant dueto very less standard error. These results agree well with

the observations of singh et al.(1988), Kumar (1990),Yadav et al. (1992) and Gaur and Raheja (1996) for partlactation milk yield and for peak yield agree with the resultof Singh et al. (2001) in Sahiwal cattle. For 300 days milkyield heritability estimate was similar to the results ofGopal and Bhatnagar (1972), Singh (1986) and Pundirand Raheja (1994). These heritability estimates revealedsignificant genetic variability in part lactation milk yields,peak yield and 300 days milk yield. The magnitude ofheritability depends upon the variability in sires for additivegenetic component. So these traits can be improved by applyingselection pressure in these animals.

The genetic correlation of 90 days milk yield with 120, 150,180 days milk yield and peak yield are 0.829±0.070, 0.804±0.073,0.762±0.084 and 0.612±0.122, respectively and phenotypiccorrelations are 0.886±0.020; 0.728±0.031; 0.611±0.036 and0.293±0.043, respectively. The genetic correlation of 120 days milkyield with 150, 180 days milk yield and peak yield are 0.823±0.065,0.795±0.072 and 0.752±0.082, respectively and phenotypiccorrelations are 0.764±0.029; 0.582±0.037 and 0.382±0.042,respectively. The genetic correlation of 90, 120, 150, 180 days milkyield and of peak yield and days to reach peak yield with 300 daysfirst lactation milk yield were 0.636±0.121, 0.807±0.067,0.754±0.077, 0.611±0.108, 0.722±0.080 and 0.722±0.080,respectively. The phenotypic correlation of 90, 120, 150, 180 daysmilk yield and of peak yield and days to reach peak yield with 300days first lactation milk yield were 0.329±0.043, 0.453±0.040,0.340±0.043, 0.375±0.042, 0.802±0.027 and 0.802±0.027,respectively. The genetic and phenotypic correlations were positiveand highly significant. Other workers have also reported positiveand highly significant genetic and phenotypic correlations betweencumulative part yields and first lactation yield (Singh and Gurnani2006). These estimates indicate that first lactation 300 days milk

262


yield would become higher if part lactation yields and peak yieldachieved higher. With such desirable correlation the combinationof part lactation yields and peak yield in selection index wouldproof desirable for simultaneous genetic improvement in 300days milk yield in subsequent generation.

AcknowledgementsThe authors feel extremely thankful to the Dean, Veterinary

College, Mathura for providing necessary financial support andthe other staffs of the Department for their technical and nontechnical support.

ReferencesDickerson, C.E. (1959) Techniques for research in quantitative animal

genetic “in the book’ Techniques and Procedures in AnimalProduction Research,” American Society of Animal Productionpp. 56-105.

Dutt, M. et al. (1961) Indian Vet. J. 41: 273-75.Gaur, G. K. and Raheja, K.L. (1996) Indian J. of Ani. Res. 30(2): 137-138.Gopal, D. and Bhatnagar, D.S. (1972) Effect of age at fist calving and first

lactation yields on lifetime production in Sahiwal cattle. AnnualReport 1969. N.D.R.I., Karnal.

Harvey, W.R. (1960) Least square analysis of data with unequal numbers.U.S. Deptt. Of Agriculture, A.R.S.

Hazel, L.N. and Terrill, C.E. (1945) J. Ani. Sci. 4: 347-58.Kumar, V. (1990) Relative efficiency of different methods of sire evaluation.

M.V.Sc. Thesis submitted to CSA Univ. of Agri. & Tech., Kanpur.Panse, V.G. and Sukhatme, P.V. (1967) Statistical methods for Agricultural

workers. I.C.A.R., New Delhi.Pundir, R. K. and Raheja, K.L. (1994) Indian J. Ani. Sci. 64(11): 1219-1225.Robertson, A. (1959) Biometrics, 15: 469.Searle, S.R. (1961) Biometrics. 17: 474-480.Shrivastava, G.K. and Khan, F.H. (1987) Indian Vet. J. 66: 40-43.Sikka, A.K. and Taneja, V.K. (1981) Indian J. Dairy Sci. 34(3): 347-349.Singh, M. et al. (1988) Indian J. Ani. Sci. 58: 860-861.Singh, M.K. and Gurnani, M. (2006) Indian J. Dairy Sci. 56(6):

384-386.Singh, R. (1986) Selection of optimum combinations of early

traits for maximizing genetic improvement in dairycattle. Ph.D. Thesis. Kurukshtra University, Kurukshtra,India.

Singh, R. et al. (2001) Indian J. Ani. Res. 35(2): 147-149.Yadav, A.S. et al. (1992) J. Dairying Food and Home Sci. 11(2):

81-89.

263


IntroductionEnzymology has made great advances since the

introduction of serum alkaline phosphatase as a diagnosticaid in 1927 by King and Armstrong. Advancement inbioengineering and computer technology linked enzymology,immunology and molecular genetics to produce new clinicaldiagnostic procedures. The normal values of creatine kinaseactivity vary with physical activity, restraint, biopsy, age and sex(Kaneko et al., 1997). Determination of specific elevatedenzymes activity of the serum serves to aid in differentialdiagnosis. ALT (SGPT) and AST (SGOT) are important in thediagnosis of heart and liver damage and ALT, AST, LDH andCK leaks from the injured heart or anaerobic heart muscle(Lehninger et al., 2000). A magnitude of serum CK increase isreported to be a quantifiable marker of exercise (Akatas et al.,1993; Lefebvre et al., 1994; Janssen et al., 1989). Therefore,keeping these in view, the present study has been undertakenwith the objective to study the influence of treadmill exercise onplasma enzymes level with different stages of exercise in bothmale and female of German shepherd dog.

Materials and MethodsThis study was conducted on 12 apparently healthy

German shepherd dogs of either sex, 24-42 months of ageand weighing 38-44 kg. All animals were maintained undersimilar managemental and feeding schedule. Water wasprovided ad libitum. The treadmill used was designed at fitnessworld, U.S.A. for laboratory studies and for Gyms of persons.All treadmill exercises were done in a chamber with atemperature of 24-260C and relative humidity of 60-65%.

The exercise in each case was done four hour aftermorning feeding and one hour rest in the chamber. Theanimals were familiarized to the machine with pilot trials beforeactual experiment. Pre-exercise venous blood samples werecollected to form the base values. Once the dogs started to

EFFECT OF TREADMILL EXERCISE ON PLASMA ENZYMESLEVEL IN GERMAN SHEPHERD DOGS

N.S. Rathore1 , Anil Moolchandani, Meenaxi Sareen and Devi Singh Rajput College of Veterinary and Animal Science, Navania, Vallabhnagar, Udaipur-313601, Rajasthan

Rajasthan University of Veterinary and Animal Sciences, Bikaner-334001, Rajasthan

ABSTRACT

The present paper highlighted the influence of treadmill exercise on plasma enzymes level with diff erent stages ofexercise in both male and female of German shepherd dog. Twelve dogs of either sex were included in present study andsubjected to treadmill exercise at the speed of 9 km/hour. The blood samples were collected at control or per-exercisecondition and at 20 minutes, 40 minutes and 60 minutes during the course of exercise. The mean values of plasmaenzymes (AST, ALT, ALP) showed s ignif icantly (P<0.01) increasing pattern with advancement of exercise, with thehighest concentrations at 60 minutes of exerc ise and lowest concentrations at control or pre-exercise condit ions inpresent investigation. The concentrations of AST increased 38.19%, 72.59% and 109.04%; ALT increased 66.97%,118.79% and 176.97%; ALP increased 8.98%, 19.70% and 31.67% during the course of study at 20 minutes, 40 minutesand 60 minutes of exercise, respectively.

Key words: Treadmill, plasma enzymes, German shepherd dog

walk on treadmill the speed was slowly increased to 9 km/hour. Blood samples were collected by puncturing the cephalicvein with inflicting least stress to the animals under all asepticprecautions, directly into the autoclaved sterile test tubes, byusing 22 guaze, 2.5 cm sterilized needle and 10 ml disposablesyringe. The blood samples were taken at the time of pre-exercise and during exercise (at the interval of 20, 40 and 60minutes during exercise) on treadmill.

For biochemical studies blood samples were taken insterilized tubes for separation of plasma. Blood samples werecentrifuged at 3000 rpm for 15 minutes and plasma sampleswere separated out and preserved at -20 ± 2oC in deep freezertill the biochemical analysis.

Aspartate aminotransferase (AST) and Alanineaminotransferase (ALT), both was estimated by method ofReitman and Frankel (1957) using diagnostic reagent kitmanufactured by Span diagnostic Ltd.

Alkaline phosphatase (ALP) was estimated by Kind andKing’s method (1954) using diagnostic reagent kit suppliedby span diagnostic Ltd.

The statistical analysis of the data was done by leastsignificant difference methods (Snedecor and Cochran, 1967).

Results and DiscussionPlasma AST, ALT and ALP

The mean concentrations of plasma enzymes (AST, ALTand ALP) have been illustrated in Table 1, according to theeffect of exercise and effect of sex including interaction ofexercise and sex.

The mean ± SE concentrations of these plasma enzymes(U/litre) at the different stages of exercise [T1 (control), T2, T3and T4] were measured as : AST : 28.583 ± 0.468, 39.5 ± 0.529,49.333 ± 0.607 and 59.75 ± 0.552; ALT : 27.5 ± 0.399, 45.917 ±0.358, 60.167 ± 0.458 and 76.167 ± 0.386 and ALP : 33.417 ±0.434, 36.417 ± 0.434, 40 ± 0.522 and 44 ± 0.522 U/litre for

1Assistant Registrar, College of Veterinary and Animal Science, Navania, Udaipur- 313601, RAJUVAS, Bikaner-334001

264


Table 1: Mean ± SE concentrations of AST (GOT), ALT (GPT) and alkaline phosphates (ALP) according to effect of treadmill exerciseand effect of sex including interaction between exercise and sex in German Shepherd dogs

AST/GOT (U/litre) ALT/GPT (U/litre) Alkaline phosphatase (U/litre) Main effects Sex 1

(6 males)

Sex 2 (6 females)

Overall mean±SE (12 animals)

% incr.

Sex 1 (6 males)

Sex 2 (6

females)

Overall mean±SE

(12 animals)

% incr.

Sex 1 (6 males)

Sex 2 (6

females)

Overall mean±SE

(12 animals)

% incr.

Control or pre-exercise condition (T1)

28.333 ±0.601

28.333 ±0.760

28.583a ±0.468

– 27.500 ±0.671

27.500 ±0.500

27.500a ±0.399

– 33.667 ±0.667

33.167 ±0.601

33.417a ±0.434

–

At 20 minutes of exercise (T2)

39.833 ±0.749

39.167 ±0.792

39.500b ±0.529

38.19 46.00 ±0.577

45.833 ±0.477

45.917b ±0.358

66.97

36.667 ±0.667

36.167 ±0.601

36.417b ±0.434

8.98


49.667 ±0.803

49.00 ±0.966

49.333c ±0.607

72.59 60.333 ±0.615

60.00 ±0.730

60.167c ±0.458

118.79

40.167 ±0.749

39.833 ±0.792

40.00c ±0.522

19.70


60.167 ±0.543

59.333 ±0.989

59.750d ±0.552

109.04

76.500 ±0.50

75.833 ±0.601

76.167d ±0.386

176.97

44.167 ±0.749

43.833 ±0.792

44.00d ±0.522

31.67

Overall mean ± SE

44.625A ±2.442

43.958A ±2.433

44.292 ±1.706

– 52.583A ±3.776

52.292A ±3.725

52.438 ±2.624

– 38.667A ±0.882

38.250A ±0.895

38.458 ±0.622

–

Note: Mean comparison have been made within main effects. Mean superscripted with different small letters column-wise andcapital letters row-wise differ signif icantly (P<0.05) from each other.

Table 2: Analysis of variance (ANOVA) of AST (GOT), ALT (GPT) and alkaline phosphates (ALP) according to effect of treadmillexercise and effect of sex including interaction between exercise and sex in German Shepherd dogs

AST/GOT (U/litre) ALT/GPT (U/litre) Alkaline phosphates (U/litre)

Source of variation

Degree of freedom

Sum of Square

(SS)

Mean square (MSS)

Sum of Square

(SS)

Mean square (MSS)

Sum of Square

(SS)

Mean square (MSS)

Effect of sex 1 5.333 5.333NS 1.021 1.021NS 2.083 2.083NS Effect of exercise 3 6409.083 2136.361** 15446.56 5148.854** 752.083 250.694** Interaction between exercise and sex

3 0.167 0.056NS 0.729 0.243NS 0.083 0.028NS

Reminder 40 149.333 3.733 83.50 2.088 119.667 2.992 Total 47 6563.916 – 15531.81 – 873.916 –

NS = Non significant (P>0.05) * = Significant at 5% level (P<0.05) ** = Significant at 1% level (P<0.01)

control of pre-exercise condition (T1), at 20 minutes of exercise(T2), at 40 minutes of exercise (T3) and at 60 minutes of exercise(T4), respectively. In case of above parameters (plasma AST,ALT and ALP), highest concentrations were observed at 60minutes of exercise (i.e. end of exercise). While, lowestconcentrations were recorded in control or pre-exercisecondition. In present study, these observations reflect a highlysignificant (P<0.01) effect of treadmill exercise showinggradually increasing pattern with advancement of exercise (T1<T2<T3<T4) on plasma AST, ALT and ALP activities were recorded.AST increased 38.19%, 72.59% and 109.04%; ALT increased66.97%, 118.79% and 176.97% and alkaline phosphates

increased 8.98%, 19.70% and 31.67%, at 20 minutes, 40minutes and 60 minutes, respectively.

The mean ± SE values of plasma AST, ALT and ALPaccording to sex were measured as : AST : 28.833 ± 0.601 and28.333 ± 0.76; ALT : 27.5 ± 0.671 and 27.5 ± 0.5 and ALP :33.667 ± 0.667 and 33.167±0.601 U/litre in male and female(control) animals, respectively. These values also representthe normal values of plasma ALT, ALT and ALP, in both sexes.The overall mean ± SE concentrations of plasma AST, ALT andALP during the course of study [T1 (control), T2, T3 and T4] wererecorded as: AST: 44.625 ± 2.442 and 43.958 ± 2.433; ALT:52.583 ± 3.776 and 52.292 ± 3.725; ALP: 38.667 ± 0.882 and

265


38.250 ± 0.895 U/litre, in males and females, respectively.Male animals showed non significant (P>0.05) but slightlyhigher values than that of female animals. The mean ± SEvalues of plasma AST, ALT and ALP were lying well within thereference range as documented by Hinchcliff et al. (1993) andKaneko et al. (1997), in different breeds of dog.

The statistical analysis (ANOVA) revealed (Table 2), a highlysignificant (P£0.01) effect of treadmill exercise on plasmaenzymes AST, ALT and ALP activities. A non significant (P>0.05)sex related variation was noted in normal plasma AST, ALTand ALP concentrations. Analysis of variance (ANOVA) alsoindicated a non-significant (P>0.05) effect of interactionbetween sex and treatment (exercise) in respect of aboveparameters (i.e. plasma AST, ALT and ALP).

As per the statistical analysis (ANOVA), the plasma AST,ALT and ALP increased gradually and significantly (P £ 0.01) inresponse to treadmill exercise. The increasing pattern of theseplasma enzymes in response to treadmill exercise is well inaccordance with the earlier studies.

The earlier findings in various breeds of dogs documentedby Bolter and Kritz, (1975); Hammel et al. (1977); Lindena et al.(1979, 1984); Ilkiw et al. (1989); Hinchcliff et al. (1993); Burr etal. (1997); Cary et al. (1999) and Chanoit et al. (2002), havesupported the observed findings of present study in G.S. dogs.

Hammel et al. (1977) suggested that in dogs subjected tointensive exercise, increasing trends of AST (GOT) and ALT(GPT) are thought to reflect leakage from cells, predominantlymuscle cells subjected to hypoxia, increased plasmaconcentrations of catecholamine and probably other factors(Bolter and Kritz 1975). The increase in ALT and ALP determinedimmediately after exercise in G.S. dogs probably resulted fromexercise induced changes in blood flow. Further increase inAST activity during the course of exercise indicting muscledamage associated with racing (Ilkiw et al., 1989).

A non significant (P>0.05) sex related effect was observedon plasma AST and ALT activities in the present study. However,male animals showed somewhat higher values of AST incomparison to female animals. Present investigation is in closeagreement with the findings of Otesile and Kasali (1992) inEthiopian high land sheep and Kmiec and Baranowski (1999)in polish long wool lambs. Probably the female sex hormoneshave a depressing effect on the activity of aminotransferases.

Likewise, according to sex, a non significant (P>0.05)effect was also observed in case of plasma ALP in presentinvestigation. The concentration of ALP observed was however,some what higher in male animals than the females. Thefindings of present investigation in relation to sex is in fullaccordance with Al-Sadi (1995), Bickhardt et al. (1999) andYigit et al. (2002) in differed breeds of sheep and Behera et al.(1993) in Black Bengal goats. Lower ALP activity in femalesmay be due to the influence of oestrogen hormone which wasrelated to the earlier epiphyseal fusion of long bones andtherefore, the need for phosphatase activity may be low thanits male counterpart.

ReferencesAkatas, M. et al. (1993) Vet. Res. Communication. 17: 353-369.Al-Sadi, B.K.H. (1995) Iraqi J. Vet. Sci. 6: 217-221.Behera, P.C. et al. (1993) Indian Vet. J. 78: 1042-1045.Bickhardt, K. e t al. (1999) Deutsche-T ierarzt liche-

Wochenschrift. 106 (10): 445-451.Bolter, C.P. and Kritz, J.B. (1975) Proc. Soc. Exptl. Biol. Med.

145: 1359.Burr, J.R. et al. (1997) J. Am. Vet. Med. Assoc. 211(2): 175-179.Cary, L. et al. (1999) Aus. J. Vet. Res. 60(1): 88-92.Chanoit, G.P. et al. (2002) J. Vet. Med. A. Physio. Patho. Clin.

Med. 49 (4): 222-224.Hammel, E.P. et al. (1977) Am. J. Clin. Nutr. 30: 409-418.Hinchcliff, K.W. et al. (1993) J. Am. Vet. Med. Assoc. 202(1):

401-405.Ilkiew, J.E. et al. (1989) Am. J. Vet. Res. 50(4): 583-586.Janssen, G.M.E. et al. (1989) Int. J. Sports. Med. 10: S160.Kaneko, J.J. et al. (1997) Clinical biochemistry of domestic

animals. 5th ed. Academic Press, Sen diego, California92101-4495, pp. 303, 317-318.

Kmiec, M. and Baranowski, P. (1999) Bulletin of the VeterinaryInstitute in Pulawy. 43 : 89-94.

Lefebvre, H.P. et al. (1994) Am. J. Vet. Res., 55: 487.Lehniger, A.L. et al. (2000) Principles of Biochemistry. 3rd ed.,

Replica press Pvt. Ltd., New Delhi, pp. 631.Lindena, J. et al. (1979) Enzyme. 24(2): 120-131.Lindena, J. et al. (1984) Euro. J. Appl. Physio. Occup. Physio.

52(2) : 188-195.Otesile, E.B. and Kasali, O.B. (1992) Bulletin of Animal Health

and Production in Africa. 40(3): 181-184.Snedecor, G. W. and Cochran W. G. (1967) Statistical Methods.

6th ed. Oxford and IBH Publishing Company, Calcutta.Yigit, A. et al. (2002) Ankara-Universitesi-Veteriner-Fakultesi-

Dergisi. 49 : 101-106.

266


IntroductionSperm membrane integrity plays a pivotal role in the

maintenance of biochemical and structural architecture of thespermatozoa. Since the plasma membrane functional activity iscrucial for the viability and fertilising ability of spermatozoa, it is veryimportant to assess the structural and functional activity of thesperm membrane (Zekariya Nur et al., 2005). The membraneintegrity is evaluated by the use of a simple technique known asthe HOST (Jeyendran et al., 1984). When exposed to hypoosmoticsolutions, biochemically-active and intact spermatozoa increasetheir volume in order to establish equilibrium between the fluidcompartment within the spermatozoa and the extracellularenvironment. Swelling causes changes in both cell size and shape(Cabrita et al., 1999). This swelling process ultimately leads to aspherical expansion of the cell membrane covering the tail, andforces the flagellum to coil inside the membrane. Coiling of the tailbegins at the distal end of the tail and proceeds toward the midpiece and head as the osmotic pressure of the suspending mediais lowered (Jeyendran et al., 1984). Since its development, thistest is used in various species of animals for the evaluation ofsperm membrane integrity like in bovines (Rota et al., 2000); equine(Neild et al., 2000); canine (Rodriguez-Gil et al., 1994); Porcine(Perez-Liano et al., 2001); bucks (Foneseca et al., 2005) andrainbow trout (Cabrita et al., 1999). The higher number of positivesperms shows the higher concentration of biochemically activesperm cells with intact functional membrane (Jeyendran et al.,1984).

The per cent of HOST positive sperms to HOS test varies withthe bucks, animals, season, mass activity, progressive motility,sperm count and total sperm with intact acrosome and individualfertility level. These findings were observed both in fresh/neatsemen as reported by (Fonseca et al., 2005) in bucks and incryopreservation semen in other animal species by Prasad et al.(1999). Lodhi et al. (2008) reported that HOS test as a significanttest as a marker for membrane intactness in Nilli-Ravi buffalo and

INFLUENCE OF SEASON AND AGE ON SPERM MEMBRANEINTEGRITY OF PREFREEZE BARBARI BUCK SPERMATOZOA

A. Kumar, T. Kumar, D.K. Swain and S. YadavDepartment of Physiology, College of Veterinary Science and Animal Husbandry

Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya, Mathura-281001, Uttar Pradesh

ABSTRACT

The present investigation was designed to elucidate the influence of different seasons and age of the animal on sperm membraneintegrity in Barbari bucks. The semen was collected round the year and the period was divided into three seasons viz. S1 (July toOctober), S2 (November to February) and S3 (March to June). Eighteen adult bucks were selected and were divided into threedifferent age groups: G1 (7 months to less than 2 years of age), G2 (2 years to less than 4 years of age) and G3 (4 years to 7.5 yearsof age). Twenty four ejaculates were collected from each buck in each season and sperm membrane integrity was assessed by hypoosmotic swelling test (HOST) with 125 mOsm/L hypo osmolar solution. HOST was carried out in the neat semen. The results ofpresent study revealed significant (P<0.01) effect of age and season on membrane integrity of the buck spermatozoa. The HOSTpositive spermatozoa were observed highest in S3 as compared to S1 and S2 seasons where as G2 exhibited highest HOST positivespermatozoa as compared to G1 and G3. From the study it was concluded that the both age and season affect the sperm membraneintegrity and hence the variation in HOST positive spermatozoa was evident in the current study.

Key words: Age, season, sperm membrane integrity, HOST

Sahiwal cow bull semen. These findings were significant in boththe species of the animals and showed a very significant correlationwith fertility levels, sperm motility, sperm viability and morphologicallynormal spermatozoa percentage.

Goat is a seasonal breeder showing a potential regulation ofreproduction with increasing photoperiod. That is why the seasonsplay a critical role in the regulation of semen quality in goats.Reviewing the current literature lacks information regarding theseasonal variation in the sperm membrane integrity in Barbaribreed of goats. Age related alterations in the membrane integrityhave not yet been studied in the animals especially in bucks. Ageenhances the abnormalities in the sperm leading to reduction inthe quality of semen. With this background information, the presentstudy was designed to evaluate the influence of age and seasonon the sperm membrane integrity in the fresh buck semen.

Materials and MethodsThe present investigation was carried out in the Hi-Tech

semen laboratory of Department of Physiology, College ofVeterinary Science and Animal Husbandry, DUVASU, Mathura, UttarPradesh. Eighteen clinically healthy Barbari bucks were selectedfrom the goat farm of the college and divided into three age groupsviz. G1 (7 months to less than 2 years of age), G2 (2 years to lessthan 4 years of age) and G3 (4 years to 7.5 years of age) containingsix animals in each group. The duration of the study was 12 monthswhich was divided into three seasons, S1 (July to October), S2(November to February) and S3 (March to June). Semen wascollected using artificial vagina (length=20cm and diameter=4.5cm) in morning hours and immediately after collection transportedto the laboratory for evaluation of semen characteristics. Twentyfour ejaculates were collected from each buck in each age groupin each season. This was followed by pooling of the semensamples within the same age group.

Evaluation of sperm membrane integrity by HOSTThe hypo-osmotic swelling test was performed according to

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Table 1: Effects of age and season on HOST positiveper cent spermatoza (Mean ± SE) on prefreeze Barbaribuck semen.

SEASONS GROUP S1 S2 S3

G1 77.13bB±0.70 73.72aB±0.55 80.90cB±0.66 G2 81.79aC±0.23 81.83aC±0.49 86.36bC±0.50 G3 67.19aA±0.56 70.07bA±0.70 74.26cA±1.07

Significant level = 1% (P=0.01), a, b, c = Superscript shows thesignif icant difference between season (S1, S2, S3). A, B, C =supersc ript shows the s ignif icant d if ference between agegroups (G1, G2, G3).

Fig.1: Microphotograph showing P=Host positive and N=Hostnegative spermatozoa

P

P P N

N

N P

the method described by Jeyendran et al. (1984) in humanspermatozoa. The protocol was modified according to therequirement of goat semen. Hypo-osmotic solutions of 300 mOsm/L trisodium citrate and fructose based solution was made as perthe chemical composition given by Revell and Mrode (1994). Asproposed by Correa and Zavos (1994), serial dilutions for 4 timeswere carried out in triple distilled water to finally get a hypoosmolarsolution of 125 mOsm/L. The solution was kept in eppendorftubes at -20ºC until use. A total of about 1000 spermatozoa werecounted in different fields on 60X objectives under phase contrastmicroscope. The total proportion of swollen spermatozoa wascalculated by dividing the number of reacted cells by the totalspermatozoa counted in the same area and multiplying the figureby 100. The obtained results were analyzed by using the SPSSsoftware version 14 and the means were compared by computingANOVA and DMRT on the sperm membrane integrity.

Results and DiscussionThe mean per cent of buck spermatozoa of different groups

has been presented in Table 1. The results revealed a significanteffect of (P<0.01) season and age of bucks on sperm membraneintegrity. The highest numbers of HOST positive sperms wereobserved in S3 as compared to S1 and S2 for all the three agegroups. The results of our study are corroborated with ZekariyaNur et al. (2005) in sannen bucks; Kale et al. (2000) in cross bredbucks and Nandre et al. (2007) in buffaloes. These studiesreported significantly high HOST positive spermatozoa in summermonths as compared to winter and rainy months. The rise in the

HOST positive sperms indicated a possible role of increasedphotoperiod and rise in the secretion of testosterone which insteadincreases the semen quality as reported in many earlier studiesby various authors in goats (Zekariya Nur et al. (2005) in Sannenbucks; Bucak et al. (2009) in Angora goats; Batista et al. (2009) inMajorera goat and Barkawi et al. (2006) in Zaraibi goats. Rise inthe testosterone concentration increases the sperm productionas well as maintains the optimal quality of the sperm. This may bea reason for us to get a higher number of HOST positive spermsin S3. Jannet et al. (2003) reported a rise in HOST positivespermatozoa in stallions with rise in photoperiod.

The present study showed significant effect of (P<0.01) agein all the seasons. The highest number of HOST positivespermatozoa was observed in G2 as compared to G1 and G3.The HOST positive sperms were found lower in G1 and lowest inG3. Age related alterations in HOST in buck and other domesticatedanimals were not reported. In human studies it was alreadyreported decrease in HOST positive spermatozoa with ageing.The result of the present study can be correlated with the findingsin human (Kid et al., 2001 and Chen et al., 2003). In the presentstudy HOST positive (%) was found to be decreasing in G3 ascompared to G1 and G2. The results can be correlated to agerelated alterations in membrane structure, composition andintactness. It may be considered that with ageing the membraneintactness decreases and hence the number of HOST positivespermatozoa decreased. Ageing induces rise in the generation offree radicals which are damaging to the sperm membrane bybringing out lipid peroxidation. In our study, this may be a possiblereason for getting a more number of sperms with damaged cellmembrane in aged group (G3).

AcknowledgementsAuthors are thankful to the Vice Chancellor, U.P. Pt. Deen

Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya, Mathura,for providing necessary facilities to carry out this work. The authorsare also thankful to Dean, College of Veterinary Science, for hiscooperation and guidance in carrying out the research work.

ReferencesBarkawi, A. H. et al. (2006) Small Rumin. Res. 66: 209-213.Batista, M. et al. (2009) Theriogenology. 71: 1307-1315.Bucak, M. N. et al. (2009) Small Rumin. Res. 81: 90-95.Cabrita, E. et al. (1999) Anim. Reprod. Sci. 55(3-4): 279-287.Chen, Z. et al. (2003) J. Androl. 24: 226-231.Correa, J.R. and Zavos, P.M. (1994) Theriogenology. 42:351-360.Fonseca, J. F. et al. (2005) Anim. Repod. 2(2): 139-144.Janett, F. et al. (2003) Theriogenology. 60: 453-461.Jeyendran, R. S. et al. (1984) J. Repord. Fertil. 70: 219-228.Kale M.M. et al. (2000) Indian J. Anim. Sci. 70:25-29.Kidd, S.A. et al. (2001) Fertil Steril. 75:237-248.Lodhi, L. A. et al. (2008) Pakistan Vet. J. 28(4): 186-188.Nandre, R. M. (2007) Effect of Preservation of Spermatozoa at Sub-

zero Temperature on DNA Integrity by Comet Assay. M. V.Sc. Thesis submitted to C. V. Sc. & A. H. Anand AgriculturalUniversity, Anand, Gujarat, India.

Neild, D. et al. (2000) Andrologia. 32(6): 351-355.Perez-Llano et al. (2001) Theriogenology. 56(3): 387-398.Prasad, J. K. et al. (1999) Indian J. Anim. Sci. 69: 766-769.Revell, S.G. and Mrode, R.A. (1994) Anim. Reprod. Sci. 36:77-86.Rodriguez-Gil. et al. (1994) Theriogenology. 42(5): 815-829.Rota, A. et al. (2000) Theriogenology. 53(7): 1415-1420.Zekariya Nur. et al. (2005) Bull. Vet. Inst. Pulawy. 49: 183-187.

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IntroductionPuberty in heifers is the culmination of a long and gradual

process that begins before birth and continues until animal issexually mature. The metabolic actions of insulin have beenwidely studied with recent interest aimed towards the directinvolvement of insulin in reproductive functions. Insulinstimulates glucose uptake, general metabolism and mitosisof cultured bovine granulosa cells (Langhout et al., 1991).Pulsatile infusion of bovine insulin increases the ovulationrate in response to FSH in energy-deprived heifers (Harrisonand Randel, 1986). Peripheral concentrations of insulin areassociated positively with nutritional status. In addition toexerting direct effects on the pituitary gland, insulin may alsoinfluence luteinizing hormone releasing hormone (LHRH)release. On the basis of this evidence, we hypothesized thatthe infrequent discharge of LH associated with poor nutritionmay be due to low circulating concentrations of insulin. Theeffect of insulin on reproduction can be well understood byestimating the hormonal profile in the blood. Thus presentstudy was undertaken with an objective to determine the effectof exogenous insulin on estrus induction and hormonal profilein delayed pubertal cross-bred heifers.

Materials and MethodsThe study was conducted on 6 normal pubertal and 6

delayed pubertal cross-bred heifers procured from livestockfarm Adhartal, College of Veterinary Science and AnimalHusbandry, Jabalpur. Body weight and age of each animal

EFFECT OF EXOGENOUS INSULIN ON FERTILITY RESPONSE ANDSERUM HORMONAL PROFILE IN DELAYED PUBERTAL

CROSS-BRED HEIFERS

S.A. Lone1, Ajaz A. Ganie2, M.A. Quadri3, M. Raies Haq4 and I.M. Reshi4Department of Veterinary Biochemistry

College of Veterinary Science and Animal HusbandryMadhya Pradesh Pashu Chikitsa Vigyan Vishwavidyalaya South Civil Lines

Jabalpur-482001 Madhya Pradesh, India

ABSTRACT

Twelve cross-bred heifers were divided into two groups, viz. (untreated control, normal pubertal, n = 6 and insulin treated delayedpubertal heifers n = 6). The animals of the treatment group were administered bovine insulin @ 0.25 IU/kg body weight, dailysubcutaneously for 5 consecutive days. All the treated animals were subjected to oestrus detection twice daily morning and eveningby visual observations and teaser bull. The blood of normal pubertal animals was collected once on day 10th post-oestrus and thatof insulin treated animals on day 0, 5, 12, at oestrus and day 10th post-oestrus for the estimation of oestrogen, progesterone andcortisol. The results indicated that all the insulin treated animals were induced in oestrus with 67% conception rate. The progesteronelevel was higher (4.53±0.23 ng/ml) in normal cycling heifers than in delayed heifers (0.36±0.02 ng/ml), however, upon insulin treatmentthe level reached to 4.44±0.41 ng/ml on 10th day post-oestrus (P<0.05). The oestrogen level (12.01±0.60 pg/ml) and cortisol level(1.30±0.11 µg/dl) were lower in delayed pubertal heifers than in normal cycling heifers, however, upon insulin treatment the levelincreased up to 41.30±1.53 pg/ml (P<0.05) and 3.66±0.12 µg/dl, respectively, on the day of oestrus. Thus, insulin treatment resultedin increased oestrogen, progesterone and cortisol levels and also enhances the fertility in delayed pubertal heifers.

Key words: Bovine insulin, oestrogen, progesterone and cortisol

was recorded. Clinical examination of all experimental animalswas carried out routinely. The animals were divided into 2groups: Group 1 consisted of 6 normal pubertal heifers of thesame age group and requisite body weight acted as controlgroup. Group 2 consisted of 6 delayed pubertal crossbredheifers with the history of not exhibiting signs of oestrus despitehaving achieved normal pubertal age but not the weight servedas experimental group. All the delayed pubertal animals wereexamined per rectal to ascertain non-cyclicity and infantilegenitalia.

Long acting bovine insulin (LONGACT B, Biopharm PvtLtd., Mumbai) @ 0.25 IU/kg b. wt. per day was administratedsubcutaneously for 5 consecutive days to all the animals oftreatment group. All the treated animals were subjected tooestrus detection twice daily morning and evening by visualobservations and teaser bull.

Blood collection and hormonal assayBlood was collected on day 10th post-oestrus from all

animals of Group 1 and on day 0, 5th day, 12th day, at oestrusand thereafter on day 10th post-oestrous from each animal ofGroup 2. The quantitative determination of oestrogen,progesterone and cortisol were made throughradioimmunoassay using the kits supplied by Adaltis Italia(Catalog No. L-14 005K), Biotech Inc. USA. (Catalog No. SH-301) and United Biotech Inc. USA. (Catalog No. SH-101),respectively. For oestradiol analytical sensitivity of the kit was<0.6 pg/ml; the intra- and inter-assay coefficients of variation

1Veterinary Assistant Surgeon, Animal Husbandry, Department, J & K2Ph.D Scholar, corresponding author and reprint request can be made to: DCN Division, NDRI, Karnal-132001, Haryana. E-mail: [email protected], Department of Veterinary Biochemistry, College of Veterinary Science and Animal Husbandry, Jabalpur4PhD Scholars, NDRI, Karnal, Haryana

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were 12.1% and 11.2%, respectively. For progesterone analyticalsensitivity of the kit was 0.05 ng/ml; the intra- and inter-assaycoefficients of variation were 5.8% and 9.0%, respectively.

ObservationsOestrus behaviour of all animals was observed in the

presence of accompanying teaser bull. Further confirmation ofoestrus was done by rectal palpation of genetalia. After treatment,all the animals were examined per-rectal to monitor the ovarianand uterine changes for ascertaining oestrus and development ofcorpus luteum. The time taken for onset of oestrus followingwithdrawal of treatment was recorded.

The data were analyzed statistically for the mean and standarderror and significant variation has been shown by using Duncanmultiple range tests (Steel and Torrie, 1992).

Results and DiscussionFertility response

All insulin treated animals showed oestrus and 4/6 (67%)were conceived, as confirmed by rectal examination on the 45thday of service. Enhanced ovarian functions and fertility responsewere reported in anoestrus buffaloes upon exogenousadministration of insulin by Gupta (2008). Sarath et al. (2007)observed oestrus induction with long acting bovine insulin in 5/7goats (71%) and ovulation occurred in 4/5 (80%) of respondinganimals, as confirmed by detecting CL ultra-sonographically. Butlerand Smith (1989) reported that low insulin concentration mightlimit the responsiveness of ovary to endogenous gonadotropinsecretion thus affecting ovulation. Butler (2000) hypothesized thathypoinsulinaemia during early lactation represents a key indicatorof nutritional status, resulting in delayed ovulation.

Hormonal profileMean concentrations of serum oestrogen, progesterone and

cortisol values are presented in Table 1, which indicates that theoestrogen level was lower (12.01±0.60 pg/ml) in delayed pubertalheifers than in normal cycling heifers (12.22±0.53 pg/ml), however,upon insulin treatment the level was increased and reached41.30±1.53 pg/ml (P<0.05) on day of oestrus (Table 1). Circulatingoestradiol was elevated in treated animals and reached to peakvalue on the day of oestrus, suggesting that hyper-insulinaemiaincreased follicular aromatase activity. Insulin infusion increasedoestradiol secretion by the dominant follicle of the first postpartumfollicular wave in dairy cows and probably not been mediatedthrough changes in pulsatile LH release. Suguna et al. (2008)found that serum concentrations of oestradiol 17β was significantly(P<0.01) higher in insulin treated goats than control. Insulin andIGF-1 given alone or in combination with gonadotropin were found

to have a profound effect on steroidogenesis of cultured bovinegranulosa cells (Gong et al., 1993). The progesterone level washigher (4.53±0.23 ng/ml) in normal cycling heifers than in delayedheifers (0.36±0.02 ng/ml). Ahmed et al. (2010) also reportedundetectable to very low serum progesterone levels in buffaloeswith delayed puberty. However, upon insulin treatment the levelreached to 4.44±0.41 ng/ml on 10th day post-oestrus (P<0.05).Insulin and IGF-1 are effective in stimulating high progesteroneconcentration in late luteal phase, whereas total release ofprogesterone is limited to IGF-1 stimulation alone (Sauerwein etal., 1992). Kumud (1999) reported a gradual decrease inprogesterone that occurred at 42.33±6.33 hr prior to oestrus, whichfurther declined to the lowest level at oestrus in Murrah buffaloes.Shukla (2004) used exogenous insulin in anoestrus cattle andfound that the level of progesterone was comparable to the presentstudy. Serum cortisol in normal cycling heifers were havingsignificantly higher values (3.68±0.26 µg/dl) as compared todelayed pubertal heifers (1.30±0.11 µg/dl), however, upon insulinadministration the value was increased significantly and reached3.66±0.12 µg/dl on the day of oestrus (P<0.05). David et al. (2000)found that Insulin supplementation increased LH pulse frequencyand decreased plasma cortisol. On the other hand Matamoros etal. (2006) reported that systemic LH, FSH, glucagons, cortisol,oestrogen values were not affected by insulin. Thus, insulintreatment resulted in increased oestrogen, progesterone andcortisol levels in delayed pubertal heifers as shown in table 1.

ReferencesAhmad, W.M. et al. (2010) Global Veterinaria. 4: 78-85.Butler, W. R. (2000) Ani. Reprod. Sci. 60:449-457.Butler, W.R. and Smith, R.D. (1989) J. Dairy Sci. 72: 767-783.David, C.B. et al. (2000) Biol. Reprod. 62: 1248-1255.Gong, J.G. et al. (1993) J. Endocrinol. 139 : 67-75.Gupta, V.K. (2008) Studies on ovarian functions and fertility response

using insulin in anestrous buffaloes. M.V.Sc. & A.H. Thesis,JNKVV, Jabalpur, M.P., India.

Harrison, L.M. and Randel, R.D. (1986) J. Ani. Sci. 63:1228-1235.Kumud, N. (1999) Determination of Plasma Oxytocin Profile In Crossbred

Cows And Murrah Buffaloes Using Sensitive Enzyme ImmunoAssay Procedure. PhD. Thesis, N.D.R.I., Karnal.

Langhout, D.J. et al. (1991) J. Ani. Sci. 69:3321-3334.Matamoros, I.A. et al. (2006) J. Ani. Sci. 69:2081-2091.Sarath , T. et al. (2007) Ani. Reprod. Sci. 17:91-98.Sauerwein, H.J. et al. (1992) J. Reprod. Fertil. 96:103-115.Shukla, S.N. (2004) Studies on ovarian function using insulin in anestrous

cattle. M.V.Sc. & A.H. Thesis, I.V.R.I., Izatnagar, Bareilly, U.P.,India.

Steel, R.G.D. and Torrie, J.H. (1992) Principles and Procedures of Statistics:A Biometrical Approach. McGraw Hill, New York.

Suguna, K. et al. (2008) Ani. Reprod. Sci. 111:202-213.

Table 1: Mean ± SE of serum oestrogen, progesterone and cortisol values in cross-bred heifers

Profile

Normal cycling

cross bred heifers

Delayed pubertal cross bred heifers

Hormones 10th day post-oestrus

0th day just before

treatment

5th day of treatment

12th day of treatment

Day of oestrus

10th day post-

oestrus

Oestrogen pg/ml 12.22±0.53b 12.01±0.60b 12.53±0.5b 12.89±0.47b 41.30±1.53a 10.96±1.98b

Progesterone ng/ml 4.53±0.23a 0.36±0.02b 0.40± 0.02b 0.44±0.01b 0.46±0.01b 4.44 ± 0.41a

Cortisol μg/ dl

3.68±0.26a 1.30±0.11c 3.59 ±0.20a 2.05 ± 0.15b 3.66±0.12a 3.40± 0.14a

Note: The means with at least one common superscript does not differ significantly (P<0.05).

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IntroductionRiverine buffalo have established themselves superior to

crossbred cattle in many respects. However, their lowreproductive efficiency remains a major economic problemglobally and its incidence is higher in India (Kumar et al., 2009).The buffalo has been traditionally regarded as a slow breedermanifested mainly as late sexual maturity, long postpartumanoestrus interval, poor expression of oestrus, poor conceptionrates, long calving interval, high stress of lactation andseasonality in breeding pattern (Barile, 2005). Reproductiveefficiency is one of the most primary factors affecting productivity.Low breeding efficiency is one of most serious and frustratingproblems confronting the dairy industry because of economiclosses and problems are difficult to correct due to multipleetiologies. Postpartum anoestrus is the most prevalent formof infertility encountered in buffalo and most frustrating andchallenging problem. Amongst the various factors that causeanoestrus, under-nutrition is a major problem (Francos et al.,1977; Bhaskaran and Patil, 1982). Thyroid hormones playimportant role in regulating metabolic rate and reproductionthrough their action at cellular level and are considered to causea change in sensitivity of gonads to gonadotrophic hormones(Nalbandov, 1976) as they regulate cyclic ovarian functionthrough functional coordination of hypothalamic-pituitary-gonadal axis for secretion of gonadotropins (Brookes et al.,

REPRODUCTIVE HORMONE PROFILES IN POSTPARTUMANOESTRUS AND CYCLIC BUFFALO

Ashok Kumar1,a, S. Mehrotra2,a, S.S. Dangi1,b, G. Singh2,b, M.C. Pathak4,b, Arvind Sharma5,b

K. Nehra3,c, Y.K. Soni1a and L.P. Singh1e

Division of Animal ReproductionIndian Veterinary Research Institute, Izatnagar- 243 122, Bareilly, Uttar Pradesh, India

ABSTRACT

Maintenance of regular calving interval of desirable length depends upon early ovarian rebound and conception after calving.Moreover, in recent years considerable interest has grew to identify specific reproductive endocrinology as a mean to identifyreproductive problems and to adopt appropriate therapeutic measures for augmenting bovine infertility particularly the postpartumanoestrus. Therefore, present study was conducted on cyclic (n=6) and three months postpartal anoestrus Murrah buffalo (n=10)of 4-7 years of age maintained at the Institute Dairy Farm, IVRI, Izatnagar to examine the endocrine profiles. Animals were examinedgynaecologically at ten days interval apart and selected on basis of smooth inactive ovaries as true anoestrus and animals thosecame in spontaneous oestrus, were detected by teaser bull twice daily and selected as cyclic animals. Blood and faecal sampleswere collected at weekly interval in cyclic animals considering day of heat as day 0 in plastic tube from jugular venipuncture and ininterlocking bags from rectum, respectively and stored at -20°C until analysis. Serum and faecal progesterone, triiodothyronine (T3),thyroxine (T4) were measured using RIA. Mean serum and faecal progesterone concentrations ranged from 0.95±0.02 to 5.57±0.38ng/ml and 110.99±5.35 to 142.38±2.54 ng/ of faeces in cyclic and 1.02±0.07ng/ml and 88.13ng/g of faeces in anoestrus buffalo. Meanserum T3 and T4 concentrations in cyclic buffalo on day of oestrus, 1st, 2nd and 3rd week were 1.19±0.1, 1.12±0.23, 1.1±0.21, 1.04±0.04and 89.01±3.14, 77.26±4.83, 85.01±3.95, 87.46±7.5 ng/ml, respectively. Serum T3 and T4 concentration were 0.71±0.12 ng/ml,70.95±1.77 ng/ml, respectively, in anoestrus buffalo. These values are substantially low as compare to reported values for cyclicbuffalo. Low concentration during anoestrus and high concentration during oestrus cycle indicate that T3 and T4 might be metabolicsignals triggering onset of ovarian activity and play a vital role in buffalo reproduction as well.

Key words: Anoestrus buffalo, faecal progesterone, triiodothyronine, thyroxine

1Ph.D. Scholar; 2Sr. Scientist; 3M.V.Sc. Scholar; 4Technical Officer; 5SRFaDivision of Animal Reproduction; bDivision of Physiology and Climatology; dDivision of Bacteriology; eDivision of Poultry Science CARI, IVRI,Izatnagar, Bareilly

1964; Stewart et al., 1994). To some extent these problemscan be detected through metabolic profile tests. Moreover, inrecent years considerable interest has grew to identify specificreproductive endocrinology as a mean to identify reproductiveproblems and to adopt appropriate therapeutic measures foraugmenting bovine infertility particularly the postpartumanoestrus. Therefore, this study was conducted to rule outhormonal profiles in postpartum anoestrus buffalo andcompared with normal cyclic values.

Materials and MethodsThe present investigation was conducted on Murrah

buffalo of 4-7 years of age, maintained at the Institute DairyFarm, LPM section, Indian Veterinary Research Institute,Izatnagar. The selected animals were grouped in as: Group I(anoestrus, n=10) and II (cyclic, n=6). The animals weremaintained under isomanagerial conditions with intensivesystem and housed in a well ventilated brick cemented housewith non-slippery floor and offered standard ration having greenand dry fodder along with concentrate. Animals were examinedper-rectal at ten days interval apart and selected on basis ofsmooth inactive ovaries as true anoestrus and animals camein spontaneous oestrus, were detected by teaser bull twicedaily selected as cyclic animals. Blood samples were collectedat weekly interval in cyclic animals at weekly interval considering

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day of heat as day 0 in plastic tube from jugular venipuncturefollowed by serum separation and stored at -20°C until analysis.

Faecal sampling and steroids extractionFaecal samples (10-20 gm) were collected directly from

rectum simultaneously with blood collection in interlockingpolythene bags and stored at -20°C until analysis. Faecalsteroids extraction was done according to methods describedby Kornmatitsuk et al. (2007) and Isobe et al. (2005) with slightmodifications and extraction was done using methanol basedprocedure used by Arunji (2008). Faeces was dried in hot airoven at 37°C and 0.3 gm of dried faecal samples were takenand homogenized in 5 ml methanol in glass tubes, thenvortexed for 10 minutes. All glass tubes were centrifuged at3000 rpm for 10 minutes. Supernatant was collected and driedin water bath at 37°C and reconstituted in 1 ml absolutemethanol and again vortexed for 1 minute and preserved indeep freezer until assay.

Estimation of progesterone, T3 and T4 using RIAProgesterone in extracted faecal and serum samples,

serum T3 and T4 were measured by RIA using diagnostic I125

kits from BARC, Mumbai. The concentration of faecalprogesterone was multiplied by 3.33 to obtain concentrationsper gram. Analytical sensitivity of kit for estimation ofprogesterone was 0.3 ng/ml; for T3 0.22 ng/ml and for T4 0.5ng/ml, respectively. The intra and inter-assay coefficient ofvariation of progesterone was <8 and <12.0%, for T3 3.3 and8.6%, for T4 6.2 and 8.6%, respectively.

Statistical analysisData for hormonal and NO profiles within groups and

between groups were analyzed by computer package usingSPSS 10 by one way ANOVA, using paired t-test and student’st-test, (Snedecor and Cochran, 1994) and correlation wasobtained using PROC CORR of SAS 9.2 software.

Results and DiscussionProgesterone in cyclic and anoestrus buffalo

Mean serum progesterone concentrations ranged from0.95±0.02 to 5.57±0.38 ng/ml in cyclic buffalo (Table 1). Serumprogesterone concentration reveals a typical pattern of oestruscycle. Estimation of progesterone at weekly interval in cyclicanimals showed a gradual change in progesteroneconcentration in follicular and luteal phase. Serumprogesterone concentration was basal (1.01±0.04 ng/ml) atday of oestrus and maximal (5.57±0.38 ng/ml) at 2 week(day14th) indicating presence of fully matured and functionalcorpus luteum. The concentration rose significantly (P<0.05)on day 7 and further day 14 post-oestrus. The progesterone

concentration during oestrus was higher than finding of Sarvaiyaet al. (1993) who found 0.5ng/ml concentration during oestrusin buffalo; however, peak level at second week was inaccordance with present study. The findings are in agreementwith Booth et al. (1975) and Ahmed et al. (1977) who reporteda significant rise in serum progesterone concentration on day7 after oestrus and peak at 2nd week of cycle in buffalo. Basalserum progesterone (0.97±0.45 ng/ml) on day of oestrus andmaximal (3.59±0.45 ng/ml) during 2 week in buffaloes wasdetected by Arunji (2008).

Mean faecal progesterone concentrations ranged from110.99±5.35 to 142.38±2.54 ng/g in cyclic animals (Table 1).The changes in faecal progesterone concentration werecorresponding to serum values. Concentration of progesteronemetabolites in faeces depicted the functional status of CL asobserved by Hattab et al. (2000) in buffalo. Arunji (2008),monitored the oestrus cycle in buffalo by faecal steroids andreported that pattern of faecal progesterone was virtually similarto that of plasma progesterone with delay time of 2-4 days andfaecal progesterone basal at oestrus (127.7±24.94 ng/g) andthe highest (419±52.62 ng/g) at day 8 of oestrus cycle. Serumand faecal progesterone values were positively correlated incyclic buffaloes (r= 0.797; P<0.0001).

In anoestrus buffalo serum and faecal progesteroneconcentration was recorded as 1.02±0.07 ng/ml and 88.13ng/g of faeces, respectively. Jainudeen (1989) recorded verylow or negligible level of progesterone up to 60 to 90 dayspostpartum in anoestrus swamp buffaloes. In the presentstudy, anoestrus was diagnosed by per-rectal palpation, henceit might be chances that the animal had shown silent heat andunable to palpate embedded or early developing CL thatcaused higher level of the P4 compared to finding of Jainudeen(1989) or higher level may be due to differences in sensitivityand specificity of kit. There is no available report on faecal P4concentration in buffalo and cattle during anoestrus.Serum T3and T4 in cyclic and anoestrus buffaloMean serum T3 and T4 concentrations in cyclic buffalo onday of oestrus, 1 st, 2nd and 3 rd week was 1.19±0.1,1 .12±0.23 , 1 .1±0.21, 1 .04±0.04 and 89 .01±3.14 ,77.26±4.83, 85.01±3.95, 87.46±7.5 ng/ml, respectively,without any significant difference within group. The highervalues was recorded on day of oestrus in both T3 (1.19±0.1ng/ml) and T4 (89.01±3.14 ng/ml) compared to lutealphase. During luteal phase lower T3 and T4 concentrationthan oestrus, in agreement with Soliman and Said (1960)in buffalo and Soliman et al. (1963) in cattle. Khuranaand Madan, (1985) reported the mean T 3 and T 4concentrations between 0.8±0.16 and 1.88±0.10 ng/mland 33.28±5.08 and 50.76±8.23 ng/ml, respectively, in

Table 1: Serum and faecal progesterone concentration (Mean±SEM) in cyclic buffalo

Time period (in weeks) serum progesterone (ng/ml)

faecal progesterone (ng/g)

0 1.01 a ±0.04 115.08 a ±1.53 1 3.79 b ±0.48 138.05 b ±1.00 2 5.57 c ±0.38 142.38 b ±2.54 3 0.95 a ±0.02 110.99 a ±5.35

Means bearing different superscripts differ signif icantly (P<0.05) within columns.

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in buffalo which is in accordance with T3 values obtained underpresent study while considerably lower in case of T4concentrations. Siddiquee and Chaudhary (2004) determinedmuch higher plasma T3 concentration during oestrus (23±1.65ng/ml) in Mehsana buffalo which could be due to breed variationor difference in sensitivity and specificity of kit.

Serum T3 and T4 concentration were 0.71±0.12ng/ml,70.95±1.77ng/ml, respectively, in anoestrus buffaloes. Thesevalues are substantially low as compare to reported values forcyclic buffalo. Low concentration during anoestrus and highconcentration during oestrus cycle indicate that T3 and T4 mightbe metabolic signals triggering onset of ovarian activity andplay a vital role in buffalo reproduction.

AcknowledgementsAuthors are sincerely thankful to Director IVRI, Incharge

LPM, Head of Animal Reproduction Division, ICAR and BRNSProject for facilities provided and financial support.

ReferencesAhmad, A. et al. (1977) Indian J. Exp. Biol. 15: 591-593.Arunji, J.T.K., (2008) Non-invasive monitoring of buffalo estrous cycle.

M. V. Sc. Thesis, IVRI, Izatnagar.Barile V.L. (2005) Livest. Prod. Sci. 92: 83-94.Bhaskaran, R. and R.V. Patil. (1982) J. Ani. Sci. 46: 1522.Booth, W.D. et al. (1975) Vet. Rec. 97: 366-369.Brookes, J. R. et al. (1964) J. Ani. Sci. 23: 54.

Francos, G. et al. (1977) Theriogenology. 7: 105-111.Hattab, S.A. et al. (2000) Theriogenology. 54: 1007-1017.Isobe, N. et al. (2005) Ani. Reprod. Sci. 90: 211-218.Jainudeen, M.R. (1989) Reproduction in the buffalo. In: Arthur’s

Veterinary Reproduction and Obstetrics . 8 th ed. Eds.Noakes, D.E., Parkinson, T.J., England, G.C.W., SaundersHarcourt, India, pp. 789-800.

Khurana, M.L. and Madan, M.L. (1985) Indian J. Dairy Sci. 38: 119-123.

Kornmatitsuk, B. et al. (2007) Reprod. Dom. Ani. 42(3): 238-242.Kumar, R. et al. (2009) Indian Vet. J. 86: 326-327.Nalbandov, A.V. (1976) Reproductive Physiology of Mammals and

Birds. 3rd ed. pp. 377-378. W.H. Freman and Co., San Francis.Sarvaiya, N.P. et al. (1993) Indian J. Ani. Sci. 63: 296-299.Siddiquee, G.M. and Chaudhary, C.G. (2004) Plasma level of tri-

iodothyronine (T3) in various reproductive conditions ofMehsana buffaloes. (Abstract), XXI Annual Convention ofISSAR and National Symposium on: Recent Trends andInnovations in Animal Reproduction: Nov. 23rd -25th, SKAUST,Jammu. pp. 58.

Snedecor, G.W. and Cochran, W.G. (1994) Statistical Methods. 8th ed.Affiliated East West Press, New Delhi.

Soliman, F. A. and Said, A. H. (1960) Hormonal changes in the bloodof cows and buffaloes during the estrous cycle. Proc. 1st

Arab. Vet. Congress, Cairo., pp. 235.Soliman, F. A. et al. (1963) J. Reprod. Fert. 6: 335-340.Stewart, R.E. et al. (1994) Domestic Ani. Endocrinol. 11(1):13-24.

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IntroductionGenetic improvement of Murrah buffaloes for increasing

milk production can be achieved by application of artificialinsemination (A.I.). Successful outcome of A.I. depends onaccurate detection of oestrus and proper adoption of A.I.technique. However, buffaloes rarely show mounting behaviour(Baruselli et al., 2003) and experience poor oestrus expressionand variable duration of oestrus (6-64h) (Ohashi, 1994) makingthe prediction of appropriate time of ovulation difficult. Therefore,the application of A.I. in buffaloes has not been widelyacceptable (De Rensis and Lopez Gatius, 2007). Fixed timeA.I. could be solution to overcome the difficulty of oestrusdetection in buffaloes. Interval to the ovulation is variable (60-156h) depending on the size and the status (growing orregressing) of the dominant follicle at the time of PGF2administration (Brito et al., 2002). Similarly ovulations followinguse of exogenous progesterone-based protocol e.g. controlledInternal drug release (CIDR) occur over a vide time intervaland two to three inseminations are required to reach optimumpregnancy rates. Therefore, the comparative efficacy ofOvsynch in different hormonal combinations was evaluated inMurrah buffaloes in regulation of follicular growth, dominance,ovulation and corpus luteum (CL) formation.

Materials and Methods The present study was conducted during summer season

(May-June) at animal farm of the Central Institute for Researchon Buffaloes Hisar, on twenty four anoestrus Murrah buffaloes(Bubalus bubalis) possessing sound physical health betweenage group of 4 to 7 years weighing more than 500 kg bodyweight. The animals were diagnosed anoestrus on the basisof anamnesis, two per rectal examinations at ten days apartand real time B mode ultrasonography inactive ovaries without

EFFECT OF OVSYNCH PROTOCOL IN DIFFERENT HORMONALCOMBINATIONS ON FOLLICULAR DYNAMICS IN ANOESTRUS

MURRAH BUFFALOES

Sandeep Kumar, R.K. Malik, R.K. Sharma1, Ravi Dutt2, Pardeep Singh,Gyan Singh3 and Meenakshi Virmani

Department of Animal Production PhysiologyLala Lajpat Rai University of Veterinary and Animal Sciences, Hisar-125004, Haryana, India

ABSTRACT

The present study was conducted to study the effect of Ovsynch protocol in different hormonal combinations on follicular dynamicsin twenty four anoestrus Murrah buffaloes (Bubalus bubalis) randomly divided into four equal groups. Group I was kept asuntreated control. Group II received Ovsynch protocol alone where as Ovsynch+CIDR and Ovsynch+hydroxy-progesterone wereadministered in group III and IV, respectively. The mean values of diameter of ovulatory dominant follicles and percentage of animalsthat maintained CL on day 10 post fixed time artificial insemination (FTAI) were 13.70±1.02, 13.03±0.89 and 12.67±0.82 mm and 100,83 and 33% in groups II, III and IV, respectively. From the present investigation it can be concluded that Ovsynch alone is capable ofinducing cyclicity in anoestrus buffaloes.

Key words: Anoestrus, Follicular dynamics, Ovsynch, CIDR, hydroxyprogesterone, buffaloes

any palpable ovarian structures on it, flaccid horns and closedcervix.

A total of 24 true anoestrus buffaloes were dividedrandomly into four equal groups (I, II, III, and IV) having sixbuffaloes in each group. Animals in group I were kept asuntreated control. In group II, a total of six true anoestrusbuffaloes were treated with Ovsynch protocol consisting of 5ml Receptal (GnRH analogue; 10 µg Buserelin acetate)injection on Day 0 (day of start of experiment), 5 ml Lutalyse(PGF2 analogue; 25 mg Dinoprost tromethamine) plus 500I.U Folligon (pregnant mare serum gonadotrophin) on Day 7and 5 ml Receptal on Day 9, followed by FTAI at 24h after the2nd GnRH injection. In group III, buffaloes were treated withOvsynch plus CIDR protocol and the protocol consisted ofGnRH (5 ml Receptal) injection and CIDR insertion on Day 0,5 ml Lutalyse plus injection 500 I.U. Folligon on Day 7, CIDRremoval on day 8 and 5 ml Receptal (2nd GnRH) on Day 9,followed by FTAI at 24h after the 2nd GnRH injection. Similarly,in group IV, buffaloes were treated with 1 ml Duraprogen (250mg hydroxy-progesterone caproate) subcutaneously on Day -6, -3 and 0 followed by GnRH (5 ml Receptal) injection on Day0 (day of start of experiment), 5 ml Lutalyse plus 500 I.U .Folligon on Day 7 and 5 ml Receptal (2nd GnRH) on Day 9,followed by FTAI at 24h after the 2nd GnRH injection.

Ovarian follicular changes were monitored in allexperimental buffaloes with a real time B-mode ultrasoundscanner (Just Vision 200, Model SSA-320A, Toshiba, Japan)equipped with a convex array multi-frequency transducer usingfrequency of 7.0 MHz. Follicular dynamics studies were carriedon day -6, -3, 0, 2, 4, 6, 7, 8, 9, 10 using real time B mode trans-rectal ultrasonography. Follicles were counted and measuredas small (2-4 mm), medium (4-8 mm) and large (>8.0mm)(Malik, 2005). Maximum diameter of ovulatory dominant follicle,

1Division of Buffalo Physiology and Reproduction, CIRB, Hisar2Ph.D Scholar, Department of Veterinary Gynaecology and Obstetrics3Assistant Professor, Teaching Veterinary Clinical Complex, Corresponding author: [email protected], Tel.: +91-9467536697

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dominant follicle (DF) at Ist GnRH, 2nd GnRH and PGF2treatment and of 2nd largest/subdominant follicle wasmeasured. Corpora lutea was confirmed ultrasonographicallyon day 10 post FTAI. The data obtained in the present studywere subjected to appropriate statistical analyses to drawscientific inferences. The students’t-test was used to evaluatethe differences between means where two variables wereinvolved, whereas Duncan’s Multiple Range Test was usedwhere more than two variables were involved (Snedecor andCochran, 1994).

Results and DiscussionThe ultrasonographic imaging of ovarian stroma, follicles

and corpus luteum were distinctly different from each other.The ovarian stroma was echogenic and brighter. Folliclesappeared as black (non-echogenic) and roughly circumscribedstructures whereas corpus luteum appeared grey in colour.Non-significant (P>0.05) increase in small follicle populationwas observed on day 8 in group II as compared to I, III and IV.Similarly, mean number of follicles non-significantly (P>0.05)increased in group III on day 9 compared to I, II and IV. Thediameter of small follicles on day 7 in group II was significantly(P<0.05) higher compared to group I, III and IV. The values ofsmall follicular diameter were significantly (P<0.05) higher onday 8 and 10 in group II, III and IV, compared to group I. On day9 in group II the values of diameter were significantly (P<0.05)higher compared to group I and IV. Similarly, on day 11, ingroup IV the values of diameter were significantly higher(P<0.05) compared to group I, II and III (Fig.1).

The number of medium follicles was found non-significantly (P>0.05) higher in group II on day 10 compared togroup I, III and IV. The mean value of diameter of mediumfollicles was significantly (P<0.05) higher from day 4 to 10 ingroups II, III and IV compared to group I on respective days. Onday 11 the mean value of group II was significantly (P<0.05)higher compared to group I and IV (Fig.2).

No significant difference (P<0.05) was observed intreatment groups upon respective treatments. The diameterwas significantly (P<0.05) higher on day 4 and day 9 in groupII, III and IV compared to group I. On day 6 and day 8, in groupIV the mean values of diameter were significantly (P<0.05)higher compared to group I. On day 7 mean values of diameterin group III and IV were significantly (P<0.05) higher comparedto group I. Mean values of follicular diameter of large follicleson day 10 were significantly (P<0.05) higher of group IIcompared to group I, III and IV. Similarly on the same day themean values of group III and IV were significantly (P<0.05)higher compared to group I. The mean diameter of group II onday 11 was significantly (P<0.05) higher compared to groups I,III and IV. On the same day the mean value of group III wassignificantly (P<0.05) higher compared to groups I and IV (Fig.3).The mean values of total number of follicles varied from7.50±0.50 to 9.17±0.48, 8.50±0.50 to 12.80±1.56, 8.20±1.20to 14.00±4.36 and 7.50±0.64 to 10.00±1.58 in groups I, II, IIIand IV, respectively on different days of examination. The totalfollicles number was non-significantly (P>0.05) higher on day10 in groups II, III and IV compared to control.

The mean number of small, medium, large and totalfollicles did not differ significantly (P> 0.05) between and withinthe group on day -6, -3 and 0. i.e. prior to treatment and within

group in group I, II and III indicating emergence of constantnumber of follicles at any day during anoestrus condition asobserved in cycling animals. In the present study the averagenumber of follicles on both ovaries ranged between 7.50±0.50to 9.33±1.69 in group I, II, III and IV before treatment i.e. inanoestrus condition. Result of the present investigationrevealed the increase in number of small and medium folliclepopulation on day 8, 9 and 10 indicating favourable effects ofexogenous GnRH and PMSG on recruitment and selection offollicles. The increase in diameter of small and medium follicleson day 9 and 10 might have converted them into medium andlarge follicles, respectively, ultimately resulting in maximumdiameter (large follicle) on day 10 i.e. day of oestrus indicatingrole in dominance also. Besides, the increase in total numberof follicles on day 10 again emphasized the effect of PMSG on7th day and GnRH on 9th day. Medium follicular population washighest on day 10 due to conversion of small follicles to mediumand medium into large follicles category towards the end ofthe treatment. PGF2given on day 7 induces luteolysis andpromotes the emergence of a new wave of follicular growth(Bodensteiner et al., 1996). A second dose of GnRH or hCG isrecommended after 48 hrs of PGF2 injection for bettersynchronization of ovulation and to allow FTAI (De Rensis andPeters, 1999).

The mean value of diameter of dominant follicle at Ist GnRH(day 0) were 9.17±1.86, 9.54±0.27 and 8.29±1.66 mm in groupsII, III and IV, respectively. The diameter of dominant follicles atPGF2 (day 7) were 9.49±0.38, 10.23±0.38 and 10.48±0.44mm in group II, III and IV, respectively. Diameter of dominantfollicle at 2nd GnRH (day 9) were 12.83±0.36, 11.90±0.36 and11.45±0.73 mm in groups II, III and IV, respectively. The diameterof dominant follicle at 2nd GnRH treatment was significantly(P<0.05) higher in group II compared to group IV. Results arein agreement with Pursely et al. (1995) where they observedlarger size of ovulatory follicle at 2nd GnRH injection (13.9±0.51mm) which reached peak size of 15.7±0.48 mm beforeovulation. The mean values of diameter of ovulatory dominantfollicles were 13.70±1.02, 13.03±0.89 and 12.67±0.82 mm ingroups II, III and IV, respectively. Similarly diameters ofsubdominant follicles were 9.68±0.52, 9.25±0.23 and8.98±0.22 mm in groups II, III and IV, respectively. There wasno statistical (P<0.05) difference among treatment groups indiameter of ovulatory dominant follicles and second largestfollicles. In the present study 83% in Ovsynch followed by 33%and 50% animals in Ovsynch+CIDR andOvsynch+Hydroxyprogesterone, respectively ovulated to firstGnRH treatment (Table 1). Efficiency of the treatment forsynchronization of ovulations depends on the ovarian responseto the 1st GnRH injection, and this ovarian response has beenrecognized to be an important factor in synchronization of theovulations for FTAI (De Jarnette et al., 2001). Results of thepresent experiment in Ovsynch group are similar to the findingsof Santos et al. (2001) who observed synchronized ovulationsin 80 % treated cows after Ovsynch protocol. The percentageof animals that maintained CL on day 10 post FTAI were 100%(6/6), 83% (5/6) and 33% (2/6) in groups II, III and IV, respectively.Two out of six treated animals in group IV developed follicularcyst after 2nd GnRH treatment in the present investigation whichmay be related to increased release of LH with insensitivity to

275


F ig . 1: E ffe c t of O vsynch in diffe re nt com bina tions on di ame ter of sma l l fol lic le s

2

2.5

3

3.5

4

-6 -3 0 2 4 6 7 8 9 10 11Da y s of obser va ti ons

Dia

met

er o

f fo

llic

les

C ontro l Ovs ync h Ov + C IDR OV + P 4

F i g . 3 : E ffe c t o f O v s yn c h i n d i f fe re n t c o m b i n a ti o n s o n d i a m e te r o f l a r g e fo l l i c l e s

6

7

8

9

1 0

1 1

1 2

1 3

1 4

1 5

-6 -3 0 2 4 6 7 8 9 10 11D a ys o f o b s e r v a ti o n s

Dia

met

er

of

foll

icle

s

C o ntro l O v s y nc h O v + C I D R O V + P 4

Table 1: Percentage of animals Ovulated to 1st GnRH, 2nd GnRH and to both GnRH treatments

E x p e r im e n ta l g r o u p s

A n i m a l s o v u la te d to 1 s t G n R H t re a tm e n t

A n i m a l s o v u la te d to 2 n d G n R H t re a tm e n t

A n i m a l s o v u la te d to b o th 1 st & 2n d G n R H

t r e a tm e n t s C o n t ro l N i l N i l N i l

O vs y n c h 5 / 6 ( 8 3 % ) 6 / 6 ( 1 0 0 % ) 5 / 6 ( 8 3 % )

O v + C ID R 2 / 6 ( 3 3 % ) 5 / 6 ( 8 3 % ) 1 / 6 ( 1 6 % )

O v + P 4 3 / 6 ( 5 0 % ) 4 / 6 ( 6 6 % ) 2 / 6 ( 3 3 % )

estradiol positive feedback (Wiltbank et al., 2002).Besides inducing the synchronization of a new follicular

wave, GnRH also induces an ovulation 24 and 32 h afteradministration, when the CL is regressing (Pursley et al., 1995).This property of GnRH was used to develop FTAI with the so-called GnRH-PGF2-GnRH (Ovsynch) protocol. In the presentinvestigation the maximum diameter of dominant folliclescoincided day of oestrus. It was reported that anoestrusbuffaloes responded better to Ovsynch treatment if a follicle of> 9 mm was present in the ovaries at the start of the treatment(Baruselli et. al., 2003; Rohilla, 2003). The maximum size ofovulatory follicle in the present study (13.03±0.89 mm) iscomparable to 13.0±0.9 mm observed by Duffy et al. (2004) inpostpartum beef cows treated with norgestomet + PMSG. If theDF present at the time of GnRH treatment fails to express LHreceptors, it will not respond to GnRH treatment and will fail toovulate (Kasimanickam et al., 2005). Further, GnRH treatmentis reported to be effective in causing ovulation of DF if it is in thegrowing or plateau phase, but not when it is in the regressingphase (Silcox et al., 1993), which explains why ovulations donot occur in all the treated animals (Macmillan and Thatcher,1991).

It has been hypothesized that progestin treatment

stimulates development and maturation of dominant folliclesin anoestrus cows by enhancing LH secretion and stimulatingdevelopment of LH receptors and secretion of estradiol(Rhodes et al., 2003). The ovulation and conception rates incattle and buffaloes are higher when there is a CL and maturedominant follicle on the day of GnRH injection (Berber et al.,2002), which was not the case in the present investigation inanestrous buffaloes treated with Ovsynch+Hydrox-progesterone.

AcknowledgementsThe authors thank the Director Research, LLRUVAS, Hisar

and Director, Central Institute for Research on Buffaloes, Hisarfor providing necessary facilities and logistics support toconduct the study.

ReferencesBaruselli, P.S. et al. (2003) Braz. J. Vet. Res. Ani. Sci. 40: 431-442.Berber, R.C. et al. (2002) Theriogenology. 57: 1421-1430.Bodensteiner, K.J. et al. (1996) Theriogenology. 45: 1115-1128.Brito, L.F.C. et al. (2002) Ani Reprod. Sci. 73:23-35.De Jarnette, J.M. et al. (2001) Ani. Reprod. Sci. 67: 27-35.De Rensis, F. and Lopez Gatius, F. (2007) Theriogenology. 67:209-216.De Rensis, F. and Peters, A.R. (1999) Reprod. Dom. Ani. 34: 49-59.Duffy, P. et al. (2004) Theriogenology. 61: 725-734.Kasimanickam, R. et al. (2005) Theriogenology. 2550-2559.Macmillian, K.L. and Thatcher, W.W. (1991) Biol. Reprod. 45: 883-889.Malik, R.K. (2005) Ph.D Thesis submitted to CCS Haryana Agricultural

University, Hisar (India).Ohashi, O.M. (1994) Buffalo J.10: 61-64.Pursley, J.R. et al. (1995) Theriogenology. 44: 915-923.Rhodes, F.M. et al. (2003) J. Dairy Sci. 86: 1876-1894.Rohilla, N. (2003) M.V.Sc. Thesis, CCS Haryana Agricultural

University, Hisar (India).Santos, R.M. et al. (2001) Revista Brasilaria Reproducao Animal.

25: 298-300.Silcox, R.W. et al. (1993) J. Ani. Sci. 71: 219 (Abstr.).Snedecor, G.W. and Cochran, W.G. (1994) Statistical Methods.

8th edn. Affiliated East West Press, New Delhi.Wiltbank, M.C. et al. (2002) Theriogenology. 57: 21-52.

276


IntroductionAmongst the viral diseases responsible for gastroenteritis

in dogs, canine parvovirus (CPV) is considered as the mostpathogenic. The disease is well established throughout theworld with high morbidity (100%) and frequent mortality up to10% (Nandi and Kumar, 2010). It is a highly contagious viralinfection. Canine parvovirus infection is an emerging diseasein India, and the incidence is showing an increasing trend(Sanjukta et al., 2008). Adult dogs host the virus withoutdisplaying clinical manifestation (Ettinger and Feldman, 2010).They discharge the virus in faeces and thus facilitate the spreadof infection. This study depicts the prevalence of canineparvovirus gastroenteritis in dogs in Palam valley (H.P) inrelation to age, sex, breed and immunity status.

Materials and MethodsThe present study was conducted on dogs presented in

Clinics of College of Veterinary and Animal Sciences, Palampurwith the history of gastroenteritis. Faecal swabs were collectedand kept in sterile vials containing PBS and tested for canineparvovirus by PCR. A total number of 45 faecal samples werecollected for analysis. After pre-treatment (boiling andtreatment with SDS and proteinase-k) of faecal sample,genomic DNA of CPV-2 was extracted by phenol chloroformmethod (Sambrook and Russel, 2001). The data obtainedwere subjected to statistical analysis by using computersoftware Instat from Graphpad software, 2008.

Results and DiscussionOut of 45 faecal samples screened by PCR assay, 18

samples were found positive for canine parvovirus. Themaximum occurrence of parvoviral gastroenteritis wasrecorded in younger age group. As many as 66.66 per centdogs were below 1 year of age and 88.88 per cent dogs werebelow 3 years of age (Table 1). It is well known that turnoverrate of the cells, lining the intestine is very high in youngpuppies. Increased intestinal epithelial turnover caused bychanges in microflora, diet (weaning) and diminishingmaternal antibody level are the predisposing factors to CPVinfection in pups (Decaro et al., 2004). The canine transferringreceptor is expressed at high density on actively dividing cells,

PREVALENCE OF CANINE PARVOVIRAL GASTROENTERITISIN PALAM VALLEY OF HIMACHAL PRADESH

Abid Ali Bhat, Des Raj Wadhwa and Manzoor Ahmad KhanDepartment of Veterinary Medicine, Dr. G.C. Negi College of Veterinary and Animal Sciences

CSK HPKV, Palampur -176062, Himachal Pradesh, India

ABSTRACT

Canine parvovirus 2 (cpv-2) is the causative agent of acute haemorrhagic gastroenteritis and myocarditis in dogs. The paperdescribes the prevalence of canine parvoviral gastroenteritis with respect to age, sex, breed, vaccination and dewormingstatus of pet dogs in Palam valley, India. The screening of 45 faecal samples collected from canine gastroenteritis cases by PCRassay revealed 40.0 % of the samples were positive for CPV. The maximum occurrence was recorded in younger age group,in males, in German Shepherd and in non-vaccinated dogs.

Key words: Prevalence, canine, parvovirus, gastroenteritis.

and this enhances the pathogenesis of parvovirus infection inyoung pups as the virus needs mitotically active tissues formultiplication (Parthiban et al., 2010). Also, this may be becauseyounger ones are more active than adults and because oftheir inquiring nature, they may unnecessarily try to eat or feelthings around them which may ultimately end up in getting aninfection or ingesting some foreign objects leading to GITdisturbances or in young pups of 2-4 months of age, it couldbe due to weaning stress (Macartney et al., 1984). Also, at thisstage of life, they usually come out from their shelter placesand start roaming their neighbourhood more, thus chances ofingesting garbage and acquiring infection is more (Strombeckand Guilford 1991). Alves et al. (1998) concluded that dogsless than 12 months of age were at increased risk of developingthe haemorrhagic gastroenteritis (90.8%).

The incidence of parvo viral gastroenteritis was more insexually intact males (61.11%) than intact females (38.88%,Table 1), which simulate with the observations reported earlier(Parthiban et al., 2010). Varsheney (2001) concluded thatincidence of roaming and dominance aggression directedtowards other dogs is comparatively high in males. However,no significant sex variation in haemorrhagic gastroenteritiswas noticed by Banja et al. (2002). Breed wise, GermanShepherd was more susceptible (33.33%) followed by Mongrel,Labrador and local/non-descript (16.66% each). Highestsusceptibility to haemorrhagic gastroenteritis (HGE) was alsorecorded in German Shepherd following Spitz, Labrador andDoberman by Banja et al. (2002). This is also in agreementwith the findings of Saxena et al. (2006).

Only 3 (16.66%) dogs were vaccinated against distemper,hepatitis, parvo virus, parainfluenza and leptospirosis while 5(27.77%) were vaccinated against rabies. Deworming wasnot done during the last 6 months in 44.44 per cent dogs. Thisis in concurrence with Kaur et al. (2006) and Saxena et al.(2006) who reported that majority of the cases had poorvaccination status, which might have played the role in thedevelopment of clinical signs. Housten et al. (1996) reportedthat unvaccinated dogs are mostly affected by parvovirusgastroenteritis.

277


ReferencesAlves, P.A.B. et al. (1998) Arquivo Brasileiro de Medic ina

Veterinaria-e-Zootenia. 50: 661-664.Banja, B.K. et al. (2002) Indian Vet. J. 79: 850-851.Decaro, N. et al. (2004) New Mic. 27: 375-379.Ettinger, J.S. and Feldman, E.C. (2010) Text Book of Veterinary

Internal Medicine: Diseases of Dog and Cat. W.B.Saunders Company, Philadelphia. pp 1310-1408.

Houston, D.M. et al. (1996) J. Am. Vet. Med. Assoc. 208: 542-546.Kaur, K. et al. (2006) Indian J. Ani. Sci. 76: 363-365.Macartney, L. et al. (1984) Vet. Rec. 115: 201-210.Nandi, S. and Kumar, M. (2010) Indian J. of Virol. 21: 31-44.Parthiban, S. et al. (2010) Ani. Sci. Rep. 4: 98-102.

Sambrook, J. and Russell, D. (2001) Molecular Cloning: ALaboratory Manual. 3 rd ed. Cold Spring HarbourLaboratory Press,USA.

Sanjukta, R. et al. (2008) Molecular epidemiological study of canineparvovirus in some parts of india. International conferenceon emerging infectious diseases of animals andbiotechnological applications. 28-29 July 2008, TamilnaduUniversity of Veterinary and Animal Sciences (TANUVAS)Compendium and Souvenir, 65.

Saxena, R. (2006) Indian J. Vet. Med. 26: 81-85.Strombeck, D.R. and Guilford, W.G. (1991) Small Animal

Gastroenterology. Wolfe Publishing Limited, London.Varshney, J.P. (2001) Indian J. Vet. Med.21: 1-9.

Table 1: Distribution of parvo viral gastroenteritis in relation to age, sex and breed.

Parameters Number of dogs Per cent Age

1d - 6m 8 44.44 >6m - 1 y 4 22.22 >1y - 3y 4 22.22

Total (1d – 3 y) 16 88.88 >3y 2 11.11

Sex Males 11 61.11

Females 7 38.88 Breed German Shepherd 6 33.33 Mongrel 3 16.66 Labrador 3 16.66 Local/Non-discript 3 16.66 Pomeranian 1 5.55 Lhasa Apso 1 5.55 Cross bred 1 5.55

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InroductionAnimal rearing in India has provided sustenance to

rural economy, particularly to the small and marginalfarmers who constitute nearly 65 % of the rural population(Gautam et al., 2007). These animals suffer with widearray of diseases which accounts economical losses tothe tune of Rs. 100 bill ion per year nat ionally andsustainable control of diseases leads to at least 20- 25per cent more output (Rao, 2006). Out of all diseases,parasitic diseases attain important status as they causeproduction lossess to the livestock industry.

Cryptosporidium spp. is an apicomplexan,intracellular, extracytoplasmic protozoan parasite whichinfect the microvillar epithelium of GI tract in animals,birds, reptiles and man (Sapano et al., 1998). Theinfection causes diarrhoeic complications, retardation ofgrowth rate and some time mortality resulting in economiclosses. No effective drug or vaccine is available againstthe disease. Being a highly zoonotic disease, it poses agreat threat to developing countries like India and holdshigh significance from public health point of view.

A lthough cryptosporidiosis in cat t le has beenreported throughout the globe (Review; Thompson et al.,2005), but there are very few reports regarding theprevalence and risk factors of Cryptosporidium spp.infection in buffalo calves. In India cryptosporidiosis inbovines was first reported by Nooruddin and Sarma(1987). Since than high prevalence of cryptosporidiosis

CROSS-SECTIONAL STUDY AND ANALYSIS OF POTENTIAL RISKFACTORS FOR CRYPTOSPORIDIUM SPP. INFECTION IN

BUFFALO CALVES IN JAMMU

Anish Yadav, Rajesh Katoch, Meenu Katoch1, Rajesh Agrawal2, J.K. Khajuria,Rajesh Godara and Rajesh Kalha3

Division of Veterinary ParasitologyFaculty of Veterinary Sciences and Animal Husbandry

S.K. University of Agricultural Sciences and Technology, R.S.Pura, Jammu- 181102

ABSTRACT

Examination of faecal samples from buffalo calves (up to six months of age) on monthly basis for a year from individual animalunits kept in villages of Jammu district revealed an overall positivity of 11.94% (172/11440) animals using modified Ziehl-Neelsentechnique (mZN). The prevalence was higher (12.96%) in plain irrigated area as compared to kandi area (8.88%). Significantly(P<0.05) higher prevalence was observed in diarrhoeic calves (15.33%) than non diarrhoeic animals (10.05%). According toage highest prevalence (19.58%) was observed in animals of <1 month and it varied significantly (P<0.05) in all the three agegroups. Season wise highest prevalence was observed in monsoon (20.0%) and lowest in summer (5.42%). Diarrhoeic faecalsamples having mucus (35.78%) showed significantly (P<0.05) higher prevalence than those having blood (11.76%). Thechances of occurance of Cryptosporidium spp. in buffalo calves in <1 month age group was 3.78 times higher than >3 monthage group animals. Similarly tnad calves having mucus in diarrohoea was higher.

Key words: Cryptosporidium, buffalo calves, Jammu, modif ied Ziehl-Neelsen technique

in cattle and buffalo calves have been reported throughoutthe country (Kumar et al., 2004; Das et al., 2004; Singh etal., 2006). Considering the importance of livestock sectorin country’s economic growth, high human population atrisk, poor sanitation and drinking water facilit ies andrapidly increasing number of HIV patients in the countrythe present study was, therefore, planned to study theprevalence and r isk fc to rs associated w ithCryptosporidium spp. in buffalo calves of Jammu district.

Materials and MethodsStudy Area

The faecal samples were collected from buffalocalves (up to six months of age) on monthly basis for ayear (March 2008 to February 2009) from individual animalunits kept at the houses of the Jammu district of Jammuand Kashmir. The study area was selected due to the factthat it features a significant development of livestockindustry and has very high human population.

The study area is located between 30 °43’ Northlatitude and 74° 54 ’ East longitudes. The Jammu districtincludes eight blocks, among which six blocks namelyR.S. pura, Bishnah, Satwari, Marh, Khor, and Akhnoor lieson South west of Jammu- Delhi national highway andextending mostly along Indo- Pak international border isplain irrigated belt with canals and ponds as a source ofdrinking water. This area is located at an altitude of 300-325 meter above sea level (masl). Rest of the two blocks

Indian Institute of Integrative Medicine, Canal Road, Jammu2Division of Veterinary Epidemiology and Preventive Medicine, SKUAST-Jammu3Ph. D. Scholar, Division of Veterinary Clinical Medicine, SKUAST-Jammu

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(Dansal and Bhalwal) mainly comprise of kandi belt, whichis located in foot hills of Himalaya at an altitude of 325-500masl. The region has shallow soil, full of boulders withnegligible water.

Collection of faecal samplesFaecal samples of buffalo calves of three age groups

(<1 month, 1-3 month, 3-6 month) were collected from eightvillages (one village from each block) geographically locateddistantly apart of Jammu district. Multistage cluster samplingmethod was used for collection of faecal samples, wherefive faecal samples of buffalo calves per age group per monthper village were collected. At the time of faecal samplecollection, data related to the age, sex, breed, andconsistency of faeces were recorded. To carry out the workthe permission was taken from institutional Animal EthicalCommittee of Sher-e-Kashmir University of AgriculturalSciences and Technology, Jammu.

Sample examinationDirect faecal smear was made from freshly collected

sample on clean grease-free microscopic glass slide andstained by acid fast staining technique using modified Ziehl-Neelsen technique (mZN) (Henricksen and Pohlenz, 1981).Briefly, the air dried smear was fixed with Methanol for 5minutes, air dried, transiently fixed over flame and kept onstaining rack. Concentrated Carbol fuchsin was poured overthe smear and kept for 30-40 minutes. The slide waswashed thoroughly under running tap water for 5 minutes,decolorized using 10% H2SO4 for 15-30 seconds and thenwashed in water. The smear was counterstained with 5%Malachite green for 5 minutes. The slide was again washedin running tap water for 5 minutes. After drying, examinedusing the high-power, dry objective (40 fold magnification)and confirmed the morphology using oil immersion.

The intensity of infection for positive samples (by acidfast staining) was measured based on the number ofoocysts observed under x 40 objective lens (OIE, 2008) i.e.1+; less than 5 oocysts per slide, 2+; 1 to 10 oocysts perfield of view and 3+; 11 or more oocysts per field of view.

Statistical Analysis and permission from ethical commit-tees

The association of Cryptosporidium infection withregard to age, season, faeces consistency etc. wereanalyzed through Chi-square test. A P value <0.05 wasrequired for significance. Odd ratio and 95% Confidenceinterval (C.I.) were calculated using each group as reference.

Results and DiscussionExamination of acid fast smear revealed that

Cryptosporidium spp. oocysts stain dark pinkish, 4-7 µm indiameter having the crescentic forms of the sporozoite (Fig.1). In the present study 11.94% (172/11440) buffalo calveswere found positive for Cryptosporidium spp. oocysts, wherehigher (12.96%) prevalence was recorded in plain irrigatedarea as compared to kandi area (8.88%). Significantly(P<0.05) higher prevalence was observed in diarrhoeiccalves (15.33%) than non diarrhoeic animals (10.05%). Asper age highest prevalence (19.58%) was observed inanimals of <1 month and it varied significantly (P<0.05) in

all the three age groups. Season wise highest prevalencewas observed in monsoon (20.0%) and lowest in summer(5.42%). Diarrhoeic faecal samples having mucus (35.78%)showed significantly (P<0.05) higher prevalence than thosehaving blood (11.76%). The detailed results of prevalenceof Cryptosporidium spp. infection in buffalo calves of Jammudistrict has been summarized in Table 1.

Statistical analysis also revealed that age was stronglyassociated with shedding of C. parvum oocyst. The chancesof occurance of Cryptosporidium spp. in buffalo calves in<1 month age group was 3.78 times higher (OR:3.78, 95%C.I.: 2.39-6.01) than >3 month age group animals. Thechances of occurance of Cryptosporidium spp. in monsoonseason was 4.36 times more than summer (OR:4.36, 95%C.I.: 2.66-7.20), similarly the likelihood of Cryptosporidiuminfection in buffalo calves having diarrhoea with mucus was4.17 times higher than those having bloody diarrhoea(OR:4.17, 95% C.I.: 1.66-11.13).

The number of Cryptosporidium spp. oocysts presentin the faecal smear (stained by mZN technique) of buffalowas assessed, where the higher percentage (55.59%) ofanimals excreated low oocyst number (Fig. 2). Diarrhoeicanimals (15.1%) had higher oocysts intensity (3+) than nondiarrhoeic animals (Fig. 3). Like prevalence results higheroocyst intensity was observed in monsoon season, maleanimals, buffaloe calves of <1 month age and diarrhoeicanimals having mucus in the faeces.

Epidemiology relates to the study of diseases inpopulation and various factors having direct or indirectbearing on the occurrence of diseases. An epidemiologicalstudy of an infectious disease in a population is an initialstep towards the introduction of the proper interventions forcontrolling the disease because the features and thepatterns of isolation of etiologic agents of the disease varyfrom place to place depending on the local meteorology,geography, and socioeconomic elements (Ferreccio et al.,1991).

In Indian context, although animal keeping in form oforganized dairy farms is increasing, but still most of theanimal rearing is practiced as individual animals unitswhere one, two or three animals are kept by the farmers atthe home and they remain in close contact with humanhabitation. Most of the earlier studies from the country pertainto examination of faecal samples of only organized farmsanimals, but the present study included animalsn fromunorganized farming pattern.

Cryptosporidiosis in India is mainly diagnosed bymicroscopical detection of the parasite in the faecalsamples. The absence of internationally accepted referenceantigens for improved serology and standardized DNA basedprotocols are the main hindrances for the confirmatorydiagnosis of the disease in the laboratories (Roy et al.,2006). The rate of prevalence of infection (11.94%) in buffalocalves of Jammu is in almost agreement with other studiestaken in India. In the survey of infection adopting modifiedacid fast staining of faecal smears, Dubey et al. (1992)recorded 17.7 per cent infection whereas, Jeyabal and Ray(2005) recorded 35.5 per cent infection in bovine calvesfrom Izatnagar, UP. In the same manner a prevalence rate of

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11-23 per cent was recorded in the Eastern part of India byChattopadhyay et al. (2000) and Das et al. (2004b). SimilarlyKumar et al. (2004) reported 25% infection from south India.However, difference in rate of infection was also recorded whichcould be due to application of more sensitive and specificPCR technique. Paul et al. (2008) reported 30.20 % infectionfrom neonatal bovine calves across the different regions ofIndia. The actual prevalence of cryptosporidiosis among thebuffalo calves of the target area could also be influenced by thefact that only one sample per calf was examined which couldbe negative during a period when the animal was experiencingintermittent oocyst excretion (Fayer et al., 2007). It has alsobeen observed that overall prevalence of cryptosporidiosis canvary remarkably depending on the age of calves (Santin et al.,2008) and the detection method used because molecularmethods are more sensitive and accurate than microscopicmethods.

Managemental practices which enhance the spread ofcryptosporidiosis include indoor calving, feeding the neonatesin groups, keeping young susceptible animals in close contactwith each other or in contact with faeces of infected animals. Inaddition the infection rate is more in calves housed in penswith concrete floors than in those with earth floors (El-Khoderyand Osman, 2008). The frequency of cleaning (daily or weekly)also changes the level of risk of infection. Moreover, the herdsize also determines the occurrence of Cryptosporidium spp.infections. Mohammed et al. (1999) stated that the higher thedensity of animals, the greater the number of infected calvesand consequently higher prevalence. On contrary, in our studykept by farmers and because of better managemental practices

the prevalence of cryptosporidiosis was less.Among animals of eight blocks of Jammu district highest

prevalence was observed in R.S. Pura and Bishnah blocksand lowest in Dansal and Bhalwal blocks. The difference canbe attributed to demographic location of these blocks as R.S.Pura and Bishnah block are located on plain irrigated belt,wherein round the year agriculture crops are grown and areafor open grazing is very less. Thus, animals are mostly housedin sheds resulting in more close contact. On contrary, Dansaland Bhalwal blocks are located mainly on non irrigated foothills i.e. kandi area where farming is rain dependent andanimals are kept on open grazing which reduce period of directcontact between animals. It has been already established thatcalves kept on pasture through out the year had lower probabilityof Cryptosporidium infection than kept in cow shed (Kvac et al.,2006).

The age related susceptibility of calves to Cryptosporidiuminfection showed highest prevalence in calves of <1 monthage. In non-diarrhoeic calves also higher prevalence wasrecorded up to 1 month of age. This suggests that 0-1 monthage group is more vulnerable for cryptosporidiosis. Presenceof Cryptosporidium infection in clinically asymptomaticanimals indicated that the particular age group of animalsmight be reservoir for the parasite (Mtambo et al., 1997). Thepresent observation also supports the earlier finding of Kumaret al. (2004).

The association of diarrhoea with prevalence ofcryptosporidiosis showed positive correlation as higherprevalence was observed in diarrhoeic bovine calves Thepresent observation of higher intensity of oocysts in diarrhoeicand neonatal calves is in agreement with Singh et al. (2006)and Del Coco et al. (2008), respectively. The above observationmay further be explained by the fact that generally diarrhoeadevelops when intestinal absorption is impaired or secretionis enhanced. Both of these processes are regulated by theintestinal epithelial cells which are infected by Cryptosporidiumspp. which in turn results in impaired glucose- stimulated Na+

and water absorption and/or increased Cl- secretion (Argenzioet al., 1993). In addition to these transport defects,abnormalities in the barrier properties of the intestinalepithelium mediated by intercellular junctional complexes,contribute to Cryptosporidium diarrhoea (Buret et al., 2003).

In India, the male calve are generally neglected as presentday agriculture has become more mechanized, so males arenot used for draught purpose. All these reasons predispose

Fig. 2: Overall intensity of Cryptosporidium oocyst in buffalo calves ofJammu district

Fig. 3: Oocyst intensity of Cryptosporidium oocyst in buffalocalves based on consistency of faeces

Fig. 1: Cryptosporidium oocyst (pink colour) in faeces by ModifiedZiehl-Nelson staining x 1000

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male bovine calves to poor feeding and management, whichin turn lowers their immunity and predispose them to variouspathogens of biological origin.

Association of mucous with Cryptosporidium infection asnoticed in present study has also been reported by Del Cocoet al. (2008). It is because of very superficial location of theparasite (Heine et al., 1984). The co-existence of C. parvum,rotavirus, coronavirus and Salomonella in calves is already onrecords (de la Fuente, 1999) and these factors may also beresponsible for association of blood and Cryptosporidiuminfection as observed in few calves in present study.

A significant association was observed between theseason and prevalence of Cryptosporidium in during thepresent study. The higher infection was recorded in monsoonmonths (July to September) and this finding is in agreementwith other reports from India (Roy et al., 2006 and Paul et al.,2008) and abroad (Chai et al., 2001). The calves were at highrisk of contracting infection by Cryptosporidium spp. duringmonsoon months because of greater contact with source ofinfection. As these animals drink water from canals and pondsand during rains faecal material run off to these water bodiesand increase the contact between infective oocysts and host.

The number of Cryptosporidium spp. oocysts present inthe faecal smear (stained by mZN technique) of buffalo calveswas assessed. It was observed that more number of calvesof <1 month age group, having diarrhoea with presence ofmucus and of cross bred cattle calves of organised farmshowed highest intensity (3+) in acid fast smear. The resultsare in accordance with observation of Singh et al. (2006) andDel Coco et al. (2008).

The study concludes that in Jammu district there is highprevalence and intensity of Cryptosporidium oocysts in young(<1 month) diarrhoeic buffalo calves with mucus. Thus, it isrecommended that there is an utmost need of inclusion ofmZN staining in routine diagnostic procedures for diagnosisof causative agent of diarrhoea in buffalo calves.

AcknowledgementsThe authors are grateful to Sher-e-Kashmir University of

Agricultural Sciences and Technology, Jammu, for theencouragement and facilities provided. We are also grateful tothe farmers for their assistance in sample collection.

ReferencesArgenzio, R. A. et al. (1993) Gastroenterol. 104:440-447.Buret, A.G. et al. (2003) Int. J. Parasitol. 33:1363-1371.Chai, J.Y. et al. (2001) Amer. J. Trop. Med. Hyg. 65: 518-522.Chattopadhyay, et al. (2000) J. Vet. Parasitol. 14:167-168.Das, G. et al. (2004) J. Vet. Pub. Hlth. 2:15-17.de 1a Fuente, R. et al. (1999) Vet. Parasitol. 8: 119-185.Del Coco, V. F. et al. (2008) Vet. Parasitol. 158: 31-35.Dubey, J.P. et al. (1992) J. Vet. Parasitol. 6: 55-56.El- Khodery S. A. and Osman, S. A. (2008) Trop. Ani. Health Prod. 40:

419-426.Fayer, R. et al. (2007) Vet. Parasitol. 145: 260-266.Ferreccio, C. et al. (1991) Amer. J. Epid. 134: 614-627.Gautam, U. S. et al. (2007) Ind. Res. J. Ext. Edu. 7: 1-5.Heine, J. et al. (1984) J. of inf. Dis. 150: 768-775.Henricksen, S.A. and Pohlenz, J.F.L. (1981) Acta Veterinaria

Scandinavica. 22: 594.Jeyabal, L. and Ray, D.D. (2005) J. Vety. Parasitol. 19: 165-166.Kumar, D. et al. (2004) Ind. J. Ani. Sci. 74:261-263.Kváè, M. et al. (2006) Vet. Parasitol. 137: 202-209.Mohammed, H. O. et al. (1999) Vet. Parasitol. 106: 1-10.Mtambo, M.M. et al. (1997) Prev. Vet. Med. 31: 185-190.Nooruddin, M. and Sarma, D.K. (1987) Livestock Advisor. 12: 49.OIE (2008) Cryptosporidiosis. Chapter 2.9.4. In : OIE Terrestrial Manual,

pp. 1192-1215.Paul, S. et al. (2008) Vet. Parasitol. 153: 143-146.Rao, J. R. (2006) Challenges and opportunities in Veterinary

Parasitology. In: Proceedings of 17th National Congress ofVeterinary Parasitology and National Symposium on“Strengths, Challenges and Opportunities in VeterinaryParasitology”. Organised at R.G. College of Veterinary andAnimal Sciences, at Puducherry, pp. 13.

Roy, S.S. et al. (2006) Vet. Parasitol. 141: 330-333.Santin, M. et al. (2008) Vet. Parasitol. 155: 15-23.Singh, B.B. et al. (2006) Vet. Parasitol. 140: 162-165.Spano, F. et al. (1998) Exp. Parasitol. 90: 195-198.Thompson, R.C.A. et al. (2005) Adv. in Parasitol. 59:77-158.Yamashiro,T. et al. (1998) J. Cl. Microbiol. 36: 2195-199.

Table 1: Prevalence of Cryptosporidium spp. in buffalo calves of Jammu district

Total number of animals Parameters Examined Positive % Positive

< 1 month 480 94 19.58 1-3 month 480 49 10.20

Age

3-6 month 480 29 6.04 Male 334 53 15.86

Sex Female 1106 119 10.75 Summer 480 26 5.42 Monsoon 360 72 20.0 Post monsoon 240 20 8.33

Season

winter 360 54 15.0 Murrah 555 62 11.17 Breed Niliravi 885 110 12.43 Diarrhoeic 515 79 15.33 Faeces consistency Non diarrhoeic 925 93 10.05

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IntroductionThe proposed investigation was carried out on Karakul

sheep. Karakul is possibly the oldest breed of domesticatedsheep. Karakuls are medium-sized sheep, but they differradically in conformation from many other breeds In animalhusbandry there are some management stresses which cannot be eliminated from the daily work operations. Stress couldbe physiological, physical, environmental, chemical andemotional. Restraint is one of the most common practices indomestic animal husbandry. Sheep being gregarious animalsare very sensitive to the stress of becoming separated fromthe flock.

Among the domesticated animals sheep usually do notreceive the same research attention as other species, thoughthey are an important livestock resource and live in a largenumber in semi arid region/area.

Although enzymes appear to have only a passive part invarious forms of biotechnology, understanding theirfundamental action is essential to clinical diagnostics andbasic research (Kaneko et al., 1997). In present study clinicalenzymes, viz.: AST (GOT), ALT (GPT), ALP (AlkalinePhosphatase), CK/CPK (Creatine Kinase) and LDH areincluded to study the effect of RIS.

Materials and MethodsThe present study was planned in two phases:

First phase of experimentTen apparently healthy adult female animals procured

from the project scheme in the department of Gynaecologyand Obstetrics, College of Veterinary and Animal Science,Bikaner (Rajasthan), India were included in this study. Theseanimals were housed in a flock undisturbed during theexperimental period for ten days and were treated as controlgroup.

Second phase of experimentEach animal of control group was subjected to restraint

and isolation stress (RIS) for three consecutive days. Thestress treatment comprised of removal of animal from flock

PLASMA ENZYMES VARIATIONS IN RELATION TO RESTRAINT ANDISOLATION (RIS) IN FEMALE KARAKUL SHEEP

M. Sareen and A. MoolchandaniDepartment of Veterinary Biochemistry

College of Veterinary and Animal ScienceRajasthan University of Veterinary and Animal Sciences, Bikaner 334001, Rajasthan, India

ABSTRACT

The study was carried out to observe the effect of restraint and isolation stress (RIS) on plasma enzymes profile in femaleKarakul sheep. In present study ten apparently healthy female adult animals were subjected to restraint and isolation stress. Theblood samples were collected and analysed for plasma AST, ALT, ALP, CK and LDH at control or pre-stress condition and on 1st,2nd and 3rd day of RIS, respectively. A highly significant (P<0.01) effect of RIS was observed on plasma AST, ALT and ALP, whileCK and LDH level did not reveal any significant effect.

Key words: Plasma, Karakul, sheep, enzymes, restraint and isolation stress

and transferring them into separate trial cages away from visualand tactile contact with other animals. Animals were fed withdry roughage with approximately 250 g of concentrate andsome quantity of salt and mineral mixture, as per the standardconditions of feeding and management. Drinking water wasprovided ad libitum.

Collection, processing and preservation of samplesThe blood samples were collected at 9.30 AM each day for

control and 1st, 2nd and 3rd consecutive days of RIS, respectively,under aseptic conditions in sterile heparinized tube (heparinwas added @ 30 IU/ml of blood). For separation of plasma thetest tubes were centrifuged at 3000 rpm for 20 minutes, plasmawas pipetted out in clear dry plasma tubes. After collection,plasma was stored at –20oC.

Estimation of blood/plasma enzymesAll the blood/plasma biochemical enzymes were

estimated by using spectrophotometer-169 of systronics, bymethod as described in diagnostic reagent kit manufacturedby Span diagnostic Ltd.

Statistical analysisThe results were presented as Mean ± SE. The mean

values were determined according to RIS and sex groups.The data were subjected to analysis of variance (ANOVA)(Snedecor and Cochran, 1967). The critical differences amongvarious means were worked out by “least significancedifference (LSD) method.

Results and DiscussionThe mean ± SE concentrations of plasma enzymes (AST,

ALT, ALP, CK and LDH) have been illustrated in Table 1,according to the effect of RIS. The statistical analysis (ANOVA)revealed, a highly significant (P<0.01) effect of RIS on plasmaenzymes AST, ALT and ALP activities and a non-significant (P>0.05) effect of RIS on plasma CK and LDH concentrations.

The mean ± SE values of plasma AST, ALT, ALP, CK andLDH were lying more or less within the reference range asdocumented by Kaneko et al. (1997).

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The findings of our study are well in agreement with thefindings of Apple et al. (1993); Minton et al. (1995) and Marco etal. (1998). They reported significant increase in SGOT (AST)and ALT and on the other hand significant decrease in ALPactivity during the course of RIS, supporting the findings ofpresent study. Similar finding have also been reported in blackBengal goats by Rudramma et al. (2001) in which they reportedsimilar trend for AST and ALT, which is also in close agreementwith our findings. Our findings are also similar to those ofGalyean et al. (1981), that serum alkaline phosphatase levelswere lower in stressed steers than in unstressed controls.Moreover, Schmidt et al. (1970) reported that blood from stresssusceptible pigs had greater concentrations of glutamicoxaloacetic transaminase than did blood from stress-resistantpigs.

Destruction of cardiac or skeletal muscle or hepatic tissuesresulting from RIS would be the most plausible explanation forthe massive increase in serum glutamic oxaloacetictransminase; that is, the enzyme leaked from cells in thesetissues and its concentration increased in serum (Apple et al.,1993). Alkaline phosphatase reflects the activity of theosteoblasts within bone (Harper, 1971).In our study plasmaALP activity decreased gradually due to RIS. The physiologicalsignificance of this finding is unclear as number of factors arecontributing in the activity of ALP, like metabolic status,physiological status, sex, age, nutritional status,managemental conditions of animal.

Normal concentrations of CK and LDH are indicative of nofurther muscular damage as no statistical difference observedin level of CK and LDH. Our finding is in partial agreement withthe report of Marco et al. (1998). In which they reported nostatistical differences in activity of LDH and CK in Mouflon (Ovisammon), however, it is not possible to compare the presentfindings due to the scarcity and non-availability of literature, inthis aspect.

AcknowledgementsThe authors are thankful to the Dean, College of Veterinary

and Animal Science, Bikaner (Rajasthan) for providing facilitiesto undertake the present investigation.

ReferencesApple, J.K. et al. (1993) J. Ani. Sci. 71: 1, 71-77.Galyean, M.L. et al. (1981) J. Ani. Sci. 53: 7.Harper, H.A. (1971) Review of physiological chemistry . Lange

Medical Publications, Los Altos, CA.Kaneko, J. J. et al. (1997) Clinical biochemistry of domestic

animals . 5 th edit ion. Academic Press, San Diego,California, USA. pp. 890-894.

Marco, I. et al.(1998) J. Vet. Med. A. 45: 243-253.Minton, J.E. et al. (1995) J. Ani. Sci. 73: 812-817.Rudramma, B.G. et al. (2001) Indian. J. Ani. Sci. 71(6): 540-542.Schmidt, G.R. et al. (1970) J. Ani. Sci. 31: 1168.Snedecor, G. and Cochran, W.G. (1967) Statistical Methods. 6th

ed. Allied Pacific (P) Ltd., Bombay.

Table 1: Mean ± SE concentrations of plasma enzymes according the effect of RIS in female Karakul sheep

AST/GOT (U/litre)

**

ALT/GPT (U/litre)

**

ALP (U/litre) **

CK (U/litre) NS

LDH (U/litre) NS Main effects

No. of Samples

Values Values Values

Values Values

Control or pre-RIS condition (T1)

10 32.896a ±4.887

20.981a ±1.854

188.833C ±2.372

11.366a ±0.455

239.666a ±0.802

On 1st day of RIS (T2)

10 75.963c ±6.885

46.945b ±3.821

187.00 C ±1.983

12.25a ±0.306

241.333a ±0.557

On 2nd day of RIS (T3)

10 58.876b ±3.987

43.851b ±3.915

184.00b ±2.898

11.633a ±0.388

240.33a ±0.666

On 3rd day of RIS (T4)

10 52.642b ±6.012

45.854b ±4.485

142.333a ±2.951

10.933a ±0.363

239.5a ±0.763

Overall mean ± SE

40 55.875 ±4.184

40.041 ±2.761

175.541 ±4.190

11.545 ±0.204

240.208 ±0.361

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IntroductionGoats can be taken as experimental model for carrying

out experimental work on ruminants, as cost of feeding andrearing them is much less than that of calves. Generally duringdrought particularly in western part of Rajasthan the changesare into environment includes scarcity of water and scarcity orno vegetation. Serum constituents are good indicators of thephysiology of the animal. Serum biochemical constituentsduring different physiological status are essential as they helpnot only in clinical and experimental investigation but also helpsin the diagnosis of various metabolic disease and disorders(Dahate et al., 2004). The liver has great functional reserveand sign of hepatic failure generally develop when 70% ormore of the functional capacity of the liver is lost (Tennant,1999). An early recognition of the ailments affecting hepatictissue is important for assessment of the liver changesoccurred and their treatment Therefore, determination ofenzymes is essential to clinical diagnostics and basic research.

Materials and MethodsThe experiment was designed to evaluate the effect of

stress due to drought on serum enzymes in goat (0 up to 5years of age) .The effects of sex and age were also determinednon various parameters during drought. The animals belongedto farmers’stock of drought stricken area of western part ofRajasthan particularly area in and around Jaislmer district.The animals belonged to similar environmental habitat andmanagemental practices. Field grazing pattern was adoptedfor the animals. Animals browsed Ziziphus nummularia leavesand also received small amounts of loppings of Prosopiscineria trees. Blood was collected from jugular vein undercomplete aseptic condition in to the test tubes without any anticoagulant. All the samples were collected and processed induplicate. After collection of blood test tubes were kept inslanting position for 30 minutes to allow clot formation. Byusing a thin stainless steel wire, clot was separated from wallof the test tubes and test tubes were centrifuged at 3000 rpmfor 20 minutes. Clear serum was pipetted out into anotherclear dry test tube and only non haemolysed serum samples

EFFECT OF DROUGHT ON LIVER FUNCTION TESTS IN GOATS

G.S. Gottam1, N. Kataria and A. K. Kataria2

Department of Veterinary PhysiologyCollege of Veterinary and Animal Science, Bikaner-334001, Rajasthan, India

ABSTRACT

The experiment was designed to assess the effect of drought on serum enzymes in 100 goats of both sex in western part ofRajasthan. All animals belonged to farmers’ stock. The parameters included serum aspartate amino-tranfearase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (AKP) and the overall mean values were 97.830±3.715; 70.130±2.197 and 32.660±0.979IU/L, respectively. The effect of sex and age was also observed. It was found that the sex effect was significant (P<0.05) only onthe mean values of AST in goats. A non significant (P>0.05) age effect was noticed on the mean values of AST, ALT and AKP in goats.

Key words: Age, drought, goat, liver function test, sex

1Senior Assistant Professor, College of Veterinary and Animal Science, Navania, Udaipur-313601, Rajasthan University of Veterinary and AnimalSciences, Bikaner-334001, Rajasthan, India2Associate Professor, Department of Veterinary Microbiology and Biotechnology

were used. After collection, serum was stored at -20ºC in adeep freeze. Aspartate amino-tranfearase (AST), alanine aminotransferase (ALT) was determined by Reitman and Frankel’smethod of kit (Glaxo) and alkaline phosphatase (AKP) by kindand king’s method of kit (Glaxo) (Span). To determine the effectof age and sex the mean values were campared statisticallyby using ‘t’ test from the respective mean value.

Results and DiscussionThe overall values of serum enzymes viz. aspartate amino-

tranfearase (AST), alanine amino transferase (ALT) and alkalinephosphatase (AKP) and their mean values according to sexand age are presented in Table 1.

The overall values of serum enzymes AST, ALT and AKPwere 97.830 ± 3.715, 70.130 ± 1.130 and 32.660 ± 0.979,respectively. The ranges were from 46.08 -184.32, 24.72 -110.95 and 9.44 -52.04, respectively.

The overall mean values of AST and ALT in presentinvestigation were higher than those recorded bySundaravandanan et al. (1989), Kataria et al. (1993) and higherby Lewis (1976) in goats. The overall mean values of AKP islower than those reported by by Lewis (1976) andSundaravandanan et al. (1989) in goats.

The effect of sex was studied on liver function test in goats.The effect was significant (P<0.05) on AST in goats. The valuesof enzyme may differ according to physiological conditions ofanimals with varing nutritional regimens (Negi et al., 1960).Present study revealed non significant (P>0.05) changes inAKP activities due to sex in goats.

The effect of age was studied on liver function test in goats.AKP activitiy in present investigation showed non significant(P>0.05) changes due to age in a goats. However, Adaval et al.(1969) reported significant age effect on AKP activity in goats.Baruah et al. (1983) recorded a decreasing variation in AKPactivity with advancing age in goats. However, in presentinvestigation the mean value of AKP increased in higher agegroup although non significantly (P>0.05). Upadhyay and Rao(1985) observed a significant (P<0.05) change in themean value of serum protein with the age in goats. In

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present study a non significant (P>0.05) effect was observed.These results agreed partially with those of presentinvestigation. ALT activity in goats in present study showed nonsignificant change with advancement of age which was similarto the Kalita and Mahapatra (1998). The recorded age effect onother enzymes viz. AST and ALP.

The overall mean values of different liver functionsanalytes viz. serum AST, ALT and AKP were compared with thestandard normal values reported for goats. It was noticed thatthe mean values of AST for goats were lower than the normalstandard value (167-513 IU/l). Serum ALT values in presentinvestigation were higher while that of AKP was lower than thestandard normal respective values.

The variations in the different indices of liver function testsin sheep indicated the load on the liver due to adverse conditionsduring drought in terms of infection and infestation. Theseresults reveal the functional status of liver (Coles, 1986). Higheractivities of serum AST is generally observed due to liverdamage. Dwivedi and Yousif (1984) reported increased serumAST in infected goats and attributed it to liver damage. Higherserum ALT activities in present investigation indicated towardsliver involvement. Cellular degeneration in liver results in higherserum values of ALT enzymes.

Kaneko et al. (2008) reported that AST and ALT activitiescan be increased in many hepatic diseases of small domesticanimals. AST activities is high in liver, kidney, pancreas anderythrocytes and damage to the cells. Because both enzymesare increased in a variety of hepatic diseases, they are of limitedvalue for differential diagnosis. Although elevations ofaminotranferases are generally considered indicative ofhepatocellular injury, in severe form of liver disease. The highestaminotranferases levels are associated with acute hepaticinjury, but more modest increases in aminotranferases activityare seen in chronic liver disease including chronic

hepatocellular disease, cirrhosis, parasitic hepatopathy, andprimary or metastatic neoplasia.

Alkaline phosphates activities were low in the presentinvestigation in goats. This enzyme hydrolyze phosphoricesters with liberation of in organic phosphorus (Coles,1986).Inthe present study screening of animals revealed a deficiencyof phosphorus leading to low serum phosphorus levels. Thismight be due to low AKP activities. Lower levels indicateddecreased osteoblastic activities in animals (Kaneko et al.,1999). This showed overall depression of growth and metabolicactivities. This supervision resulted from different factors aimingdue to drought conditions.

ReferencesAdaval, S. C. et al. (1969) Indian Vet. J. 46:485-490.Baruah, R. N. et al. (1983) Indian J. Ani. Hlth. 22: 21-23.Coles, E. H. (1986) Haemostasis and coagulation of blood. In:

Veterinary Clinical Pathology . 4 th ed. W.B. SaundersCompany, Philadelphia. pp. 98-113.

Dahate, M.M. et al. (2004) Indian Vet. Medical J. 28 : 217-220.Dwivedi, S. K. and Yousif, Y.A. (1984) Indian Vet. J. 61: 1024-1026.Kalita, D. J. and Mahapatra, M. (1998) Indian J. Ani. Res. 32(1): 38-

40.Kaneko, J.J. et al. (2008) Clinical Biochemistry of Domestic Animals.

6th ed. Acedemic Press, U.S.A.Kataria, A. K. et al. (1993) Indian Vet. J. 70: 761-762.Lewis, J. H. (1976) Am. J. Vet. Res. 37(5): 601-605.Negi, S. S. et al. (1960) Indian Vet. J. 37: 541-547.Reitman, S. and Frankel, S. (1957) Am. J. Clin. Path. 28: 56.Sundaravandanan, V. K. et al. (1989) Indian J. Ani. Sci. 59(9): 1058-

1060.Tennnat, B. C. (1999) Hepatic function. In: Clinical Biochemistry of

Domestic Animals. 5th ed. Eds. Kaneko, J.J.; Harvey, J.W.and Bruss, M.L. pp. 327-352.

Upadhyay, R. C. and Rao, M. V. N. (1985) Indian J. Dairy Sci. 38(3):168-173.

Table 1: Effect of drought on liver function test in goats

S.N. Effects AST(IU/L) ALT(IU/L) AKP(IU/L) 1. Overall (100)

97.830 ± 3.715 (46.08 -184.32)

70.130 ± 2.197 (24.72 -110.95)

32.660 ± 0.979 (9.44 - 52.04)

2. a.

Sex Male(30) 76.320± 6.387 (46.08-184.32)

67.489 ± 4.509 (24.72 -110.95)

31.367 ± 1.704 (9.44 - 42.6)

b. Female (70) 107.048b ± 4.103 (54.72-184.32)

71.262a ± 0.113 (28.74-110.95)

33.206a ± 1.192 (11.78-44.94)

3. Age 0-2Y rs (51)

100.454 ± 5.357 (46.08-184.32)

73.363 ± 2.604 (24.72-106.06)

30.885 ± 1.540 (9.44-44.94)

b. Above 2 Yrs (49)

95.098a ± 5.165 (54.72-184.32)

66.765a ± 3.535 (24.72-110.95)

34.506a ± 1.150 (11.78-52.04)

(i) Figures in the parentheses indicate numbers of animals for the effects and range for the different parameters.(ii) In sex effect mean values of all parameters of female animals have been compared with respective mean values of animals.(iii) In age effect mean values of all parameters of the animals of above 2 years of age group have been compared with respective mean values of animals of below 2 years of age group(iv) Mean superscribed by letter “a” do not differ significantly (P>0.05) and letter “b” differ significantly (P= 0.05) from the respective means (male and 0 to 2 tears age group)

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IntroductionIn veterinary surgical practice, skin closure is achieved by

the use of non absorbable sutures e.g. cotton, silk andpolymerized caprolactum. Their disadvantages are creation ofnidus for the growth of micro organisms, tissue reaction,increase operative time, and increase in anaesthesiaadministration. Infection is avoided with the monofilament,unlike the braided multifilament, such as cotton and silk, whichcan potentially sustain bacterial inocula. The staples areadvantageous in rapid closure of skin, decreased risk ofinfection, improved wound eversion and minimal tissuereactivity (Margaret, 2007).

Materials and MethodsThe present study was undertaken in 12 apparently healthy

mongrel dogs of same sex and approximately same weightwere selected for surgical procedure. The animals procuredwere de-wormed by administering the Praziplus orally at thedose rate of one tab/10 kg body weight. After a gap of one weekdogs were vaccinated against rabies by injecting anti-rabiesvaccine (Inj. Rabivac) 1ml i/m. The animals were randomlydivided into two groups of six animals each. In one group, skinincision was closed with staple sutures and in the other groupthe skin incision was closed with cotton thread. All the animalswere administered general anaesthesia before surgicalprocedure. The animals were premeditated with Inj. atropinesulphate @ 0.04 mg/kg body weight. After 15 min the animalswere administered Inj. Xylazine @ 1.1 mg/kg body weight andInj. Ketamine @ 22 mg/kg body weight. The preferred site waslimb as decided on model experimentation. The skin and fasciawere incised. After procedure, the fascia and subcutaneous

STUDIES ON EFFICACY OF STAPLES SUTURE OVER COTTON SUTUREWITH SPECIAL REFERENCE TO HAEMATO-MICROBIAL ALTERATION

N. Shukla1, S.S. Pandey2, B.P. Shukla3 and B. Bharti4Department of Veterinary Surgery and Radiology

College of Veterinary Science and Animal Husbandry MHOW, Indore -453446, M.P., INDIA

ABSTRACT

The study was conducted on 12 healthy mongrel dogs, divided into two groups comprising of 6 animals in each group. In group A skinincision was closed with staple sutures and in group B the skin incision was closed with cotton thread. The haematogical studieswere done on days 0th, 4th, and 7th. The Hb and PCV value did not have any significant differences between the group or within group.There were statistically significant differences TLC between time interval within the treatment groups A and B (P<0.05). Significantincrease was noticed in TLC from day 0 to 4th day in the treatment group A and B. The TLC increase was more in cotton sutures from0 to 4th day than in staple sutures. All the values of lymphocytes, monocytes and eosinophils did not differ significantly within orbetween the treatment groups. There was a significant increase in the neutrophil count (P<0.05) from day 0 to 4th day postoperativelyin the group A and B. Microbiological study was conducted in 8 animals (4 from each group) at the time of suture removal. Growth wasobserved in both the groups. In group A the growth rate was lower and in only 2 cases, the bacterial growth was observed. In GroupB growth was observed in all the cases under study. This is in accordance with the findings that in staples there is lower woundinfection rates and are more resistant than conventional sutures to infection

Key words: Staple suture material, cotton suture material and haemato-microbial examination.

1,4PG scholar2Professor3Associate Professor

fascia was sutured using 1-0 catgut thread using simplecontinuous manner. Skin suturing was done with 35 Wstainless steel skin staples in group A. Skin staples weredelivered at 600 angle by holding the wound edges togethermanually or with the help of thumb forceps and slightly raisedthe edges with it. In Group B cotton thread in interrupted fashionwas used. In both the groups suturing were done atapproximately 1cm interval.

Haematological changesBlood samples were collected on 0, 4, 7 days for the

following haematological studies. Differential leucocyte count(DLC%), total leucocyte count (TLC in thousand per cumm),haemoglobin concentration (Hb g%) and PCV (%). All thesehaematological parameters were studied as per the proceduredescribed by Jain (1986).

Microbial load (aerobic plate count)In the present study, part of suture material that is

embedded in skin, after removal, were collected aseptically forculture to assess bacterial load. The media used for countingpurpose was plate count agar (PCA). For enumeration purposeten-fold serial dilutions of each sample were prepared in sterilenormal saline (NSS) up to 10-8 dilution. Subsequently, 0.1 mlof each dilution was aseptically transferred with the help of asterile pipette on the surface of sterile media in plate. Theinoculum was then spread on the surface using a sterilespreader. For each dilution triplicate plates of appropriatemedia and different sets of sterile pipettes and spreaders wereused. The inoculated plates were kept at room temperaturefor 30 minutes to allow the inoculum to be adsorbed and thenincubated at 370C for 24 hours. After incubation, the bacterial

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colonies were counted using digital colony counter. Forcalculation of bacterial counts (APC). The plates with 30-300colonies were counted and mean count of the three plateswas further multiplied with the reciprocal of dilution factor. Theresults were expressed in colony forming unit (CFU) pergramme of sample.

Post operative follow upAntiseptic dressing was done with Liq Betadine followed

by application of Oint. Betadine. Inj. Contramol @ 1 mg/kg at12 hrs interval for 3 days was given. Inj.Monocef @ 7 mg/kgbody wt. i/v was given in 12 hrs interval. Dressing of woundwas done on alternate day’s i.e .2, 4, 6, 8, 10 days forassessment of wound. Sutures were removed on 8th day postoperatively using staple remover or suture cutting scissorsexcept in exceptional cases.

The data was analyzed by using simple analysis ofvariance to test the different among the interval under eachgroup, the standard procedure as outlined by Snedecor and

Cochran (1994).

Results and DiscussionThere were no significant differences in the haemoglobin

concentration and PCV between the groups or within group. Allthe values were the normal range. Haematocrit values wereon normal range in all groups this indicate normal hydrationstatus of animals.

There were statistically significant differences TLCbetween time interval within the treatment groups A and B(P<0.05). Significant increase was noticed in TLC from day 0to 4th day in the treatment group A and B. The TLC increase wasmore in cotton sutures from 0 to 4th day than in staple sutures.George and Simpson (1985) also stated that stapling hasdecreased inflammatory response. This increase in totalleucocyte count was in response to inflammatory reaction.

All the values of lymphocytes, monocytes and eosinophilsdid not differ significantly within or between the treatmentgroups. There was a significant increase in the neutrophil count

Table 1: Mean values (±SE) of haematological parameters at different time intervals following two treatments in dogs

Periods (days) Parameters Groups 0 4 7

A 13.43±0.25 13.95±0.21 13.8±0.25 Hb(g/dl) B 13.08±0.21 13.10±0.21 13.10±0.19 A 40.5±0.731 42.76±0.981 40.67±0.74 PCV

(%) B 39.25±0.625 39.34±0.65 39.24±0.82 A 12.08±0.22a 14.18±0.35b 12.±0.32a TLC

(Thousand/cumm) B 12.83±0.22a 15.5±0.27b 13.5±0.24a A 69.17±0.53a 72.67±0.67c 69.42±0.62b Neutrophil

(%) B 69.41±0.41a 74.67±0.82c 70.58±0.57b A 23.83±0.33 22.02±0.66 23.5±0.45 Lymphocytes

(%) B 23.73±0.30 21.43±0.44 23.7±0.45 A 4.75±0.31 4.28±0.48 4.69±0.41 Eosinophils

(%) B 4.83±0.33 4.42±0.31 4.74±0.34 A 1.5±0.14 1.44±0.143 1.48±0.142

D

L

C Monocytes (%) B 1.52±0.186 1.48±0.14 1.5±0.144

Group A: It consists of application of staple sutures for the closure of skin incision after surgical manipulation.Group B: It consists of application of cotton sutures for the closure of skin incision after surgical manipulationMeans within treatment group with different superscripts differ signif icantly (P<0.05) from each other.

Group Case No Type of suture Material Number of colonies cfux103/gm

1 Staple Nil 2 Staple 68 3 Staple Nil

Group A

4 Staple 72 1 Cotton 311 2 Cotton 282 3 Cotton 260

Group B

4 Cotton 251

Table 2: Comparison of bacterial load in Group A and B Dogs

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(P<0.05) from day 0 to 4th day postoperatively in the group Aand B. The value increased from day 0 to 4th day in group A anddecreased in day 7 whereas it increased from day 0 to 4th dayin group B and decreased to in day 7. This is due to increasedinflammatory response.

Bacterial countThe aerobic plate count was determined by spread plate

technique.

Aerobic plate count (APC)In staple suture material samples, the APC was ranging

between 0-72x103cfu/gm. While the APC of cotton suturematerial samples ranged between 251x103 to 311x103cfu/gm.All the counts were higher in cotton suture samples ascompared to staple suture samples. Microbiological study wasconducted in 8 animals (4 from each group) at the time ofsuture removal. Growth was observed in both the groups. Ingroup A the growth rate was lower and in only 2 cases, thebacterial growth was observed. In Group B growth wasobserved in all the cases under study. This is in accordance

with the findings that in staples there is lower wound infectionrates and are more resistant than conventional sutures toinfection (Pickford et al., 1983; Stillman et al., 1984). In noinstances microbiological growth was observed wherestaplers were reused after sterilization with formalin tablets. Incase of cotton suture high microbial growth was observedwhich is in accordance with the findings of Anthony et al. (1989)who opined that cotton potentiates infection because it harboursbacteria.

ReferencesAnthony, S.T. et al. (1989) Techniques of Large Animal Surgery. 11th

ed. pp. 381.Jain, N.C. (1986) Schlams Veterinary Haematology. 4th ed. Lea Febriger,

Philadelphia.George,T.K., and Simpson, D.C. (1985) J. R. Coll. Surg. Edinb. 30:54-

56.Margaret (2007) Materials for wound closure.<http.//

:www.emedicine.medscape.com>Pickford, I.R. et al. (1983) Br. J. Surg.70:226-228.Stillman, R.M. et al. (1984) Arch. Surg.119:821-822.Snedecor, G.W., and W.G. Cochran (1994) Statistical Methods. Iowa

State University Press, Amer, Iowa.pp. 53-57.

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IntroductionIn dogs, general anaesthesia is generally used but in

case of older patient, weak, toxemic and debilitated animals,epidural anaesthesia is employed. Injection of anaestheticwithin spinal canal but outside the duramatar is termed asepidural or extradural anaesthesia. Spinal or epiduralanaesthesia is commonly employed to desensitize hindquarters in animals. The present study was planned to focuson clinico-haemato-biochemical alterations due to the effectof drugs.

Materials and MethodsThe study was conducted on six apparently healthy dogs

of either sex weighing between 10-15 kg. Each experimentalanimal was subjected to two treatments and each treatmentlasted for three days at an interval of 8 days. In treatment Iropivacaine hydrochloride 0.5% @ 0.22 mg/kg body and intreatment II ropivacaine hydrochloride 0.5% @ 0.22 mg/kg andxylazine @ 0.75mg/kg in the lumbosacral space. The rectaltemperature (°F), pulse rate (per min) and respiration rate,(per min) was recorded at 0, 20, 40, 60, 80, 120,180, 240 300,360, 480 min., haematological parameters viz. Hb, PCV, TECand TLC and biochemical parameters i.e. blood glucose,protein, ALT, AkP, bilirubin, BUN and creatinine were studied.Statistical analysis was done as per method described bySnedecor and Cochran (1994).

Results and DiscussionThe onset of analgesia was 1.23± .02 and 0.87± 0.1

minutes in treatment I (Ropivacaine group) and II (Ropivacaine-Xylazine group), respectively. The epidural administration ofRopivacaine and Ropivacaine-Xylazine induced analgesiceffect which lasted for 135.83 ± 3.39 min and 202.50 ± 5.88min in treatment I and II respectively. Results of the clinicalstudies showed that onset of analgesia was quick in group II,as compared to ropivacaine group. The analgesic effect alsolasted maximum in group II. Therefore, clinical studies revealed

COMPARATIVE EVALUATION OF ROPIVACAINE ANDROPIVACAINE-XYLAZINE COMBINATION AS AN EPIDURAL

ANALGESIC IN DOGS

S.S. Khodwe, B. P. Shukla and S. S. PandeyDepartment of Veterinary Surgery and Radiology

College of Veterinary Science and AH, Mhow- 453446 (MP)

ABSTRACT

The study was conducted on six apparently healthy dogs of either sex weighing between 10-15 kg. In treatment I ropivacainehydrochloride 0.5% @ 0.22 mg/kg body and in treatment II ropivacaine hydrochloride 0.5% @ 0.22 mg/kg and xylazine @ 0.75mg/kgin the lumbosacral space. Clinico-haematobiochemical parameters viz. rectal temperature (°F), pulse rate (per min) heart rate (per min)and Respiration rate, (per min) was recorded at 0, 20, 40, 60, 80, 120,180, 240 300, 360, 480 minutes. Hb, PCV, TEC, TLC, bloodglucose, protein, ALT, AkP, bilirubin, BUN and creatinine was studied.in treatment I and II, respectively. Quick onset and longer durationof analgesia duration was observed in treatment II but their was some synergetic effect also observed in treatment II as compared totreatment I. Also in group II their was significantly(P<0.05) changes in haematobiochemical parameters.

Key words: Epidural analgesia, Haemodynamic, Ropivacaine, Xylazine, Biochemical, Dogs.

that ropivacaine group was better ropivacaine and xylazinegroup showed some synergistic effect and effect was betterother group, same finding was observed by Duke et al. (2000).

Treatments Body reflexes I II

Loss of anal reflex

1.23 0.87

Loss of tail reflex

1.55 1.05

Loss of pedal reflex

1.76 1.66

Duration of analgesia

135.83 202.50

Complete recovery

361.33 412.00

Respiration rate

The respiration rate showed the respiration rate did notdiffer significantly (P>0.05) in treatment I,. The values ofrespiration rate fluctuated non significantly between 20 to 480minutes. In treatment II, there was a significant (P<0.05)decrease in respiration rate from the control value and reachedto its minimum value i.e. 20.66 ± 0.33 at 80 minutes afterepidural administration of drugs. Thereafter values startedincreasing and reached to 31.33 ± 0.42 at 480 minutes.

Similar findings were observed by Kelawala et al. (1996),Kumar et al. (1975) who reported significant reduction inrespiratory rate. Yayla and Kilic (2010) observed that nonsignificantly decrease in respiration rate after administrationof ropivacaine and bupivacaine in dogs. Decrease in respirationrate in treatment II was observed which may due to depressionof respiratory centre by these drugs (Lumb and Jones, 1984).There was no decrease in respiration in ropivacaine groupsuggestive of its superiority over other two groups.

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Pulse RateThere were non significant alterations in pulse rate ranged

between 99.50 ± 0.61 to 93.16 ± 2.53 per minute in treatment I. Intreatment II, the values decreased significantly from 0 (101.83 ±0.65) to 120 minute (91.00 ± 1.09) and there after value startedincreasing from 180 minute and reached to maximum at 480minute (101.00 ± 0.36) pulse rate was observed.

Similar finding were also observed and correlated with Yaylaand Kilic (2010).

Kennely and West (1967) suggested that the opioid has directeffect on sinoaterial and artioventricular conduction causingasystole leading to bradycardia. The central neural mediatedmechanism might be primary mechanism of opioid, induced mildto severe bradycardia (Bailey et al. 1994) which might be the causeof reduction in pulse rate in treatment I and II.

Heart rateThere was a non significant alteration in heart rate ranged

between 102.66 ± 0.33 to 100.45 ± 0.34 per minute in group I. Ingroup II, the values decreased significantly from 0 (101.50 ± 0.95)to 120 minute (92.66 ± 0.33) and there after value started increasingfrom 180 minute and reached to maximum at 480 minute (101.50± 0.56). Kelawala et al. (1996) also observed decrease in heartrate after epidural administration of xylazine in diazepampremedicated dogs.

Kennely and West (1967) suggested that the opioid has directeffect on sinoaterial and artioventricular conduction causingasystole leading to bradycardia. The central neural mediatedmechanism might be primary mechanism of opioid, induced mildto severe bradycardia (Bailey et al. 1994) which might be the causeof reduction in pulse rate in treatment I and II.

Rectal TemperaturesIn treatment I, there was non-significant (P>0.05) decrease in

rectal temperature. The highest and lowest values ranged from 0minute (101.86 ± 0.30) to 480 minute. (101.76 ± 0.16). In treatmentII, there was a significant (P<0.05) decrease in rectal temperaturefrom 0 to 120 minutes (101.90 ± 0.16 to 99.15 ± 0.12), there afterrise in temperature was noticed and the value reached to pretreatment value (101.43 ± 0.09) at 480 minute.

Similar finding were stated by Kelawala et al. (1996) afterepidural administration of xylazine. Ishiyama et al. (1997) observeda non significant decrease in rectal temperature afteradministration of ropivacaine in dogs.

In the present study the significant decrease in rectaltemperature in treatment III, might be due to activation of alpha-2agonists and hypothalamus alpha-receptor, inhibiting the heatconserving mechanism. Reduced metabolic rate and muscleactivities might have been resulted in production of less heat inbody and depression of thermoregulation resulted in hypothermia(Ponder and Clark, 1980).

Haemoglobin ConcentrationThe haemoglobin concentration did not vary significantly

(P>0.05) in treatment I, however in treatment II, after epiduraladministration of drugs there was a significant (P<0.05) decreasein Hb concentration up to 6 hours (9.46 ± 0.22 g%), thereaftervalues started increasing, reaching close to control value (10.66 ±0.22 g%) at 72 hours.

The present findings of haemoglobin concentration are in

accordance with finding of Kumar et al. (1997) who reportedsignificant decrease in haemoglobin concentration afteradministration of xylazine in goats. Similar finding was alsoobserved Fani et al. (2008) after administration of epiduraladministration of bupivacaine, xylazine and ketamine in dogs.

The reduction in Hb % after ropivacaine-xylazineadministration might be due to shifting of fluid volume fromextravascular compartment to intravascular compartment in orderto maintain the normal cardiac output of pooling of circulationblood cells in the spleen or other reservoirs secondary tosympathetic tone (Pratap et al., 2001).

Packed Cell VolumeThe Packed cell volume did not vary significantly (P>0.05) in

treatment I after epidural administration of drugs. But in treatmentII, there was a significant (P<0.01) decrease in PCV reachingminimum value (28.08 + 0.25) at 24 hours from the control valueand thereafter value started increasing significantly reaching to33.10 + 0.33 at 72 hours.

Similar finding was also stated by Fani et al. (2008) afteradministration of epidural administration of bupivacaine, xylazineand ketamine in dogs.

Total Erythrocyte CountThere was no significant change in total erythrocyte count

(TEC) in treatment I. In treatment II, there was a significant (P<0.01)decrease in TEC reaching minimum value (5.72 + 0.09) at 6 hoursfrom the control value and thereafter value started increasingsignificantly reaching to 6.49 + 0.07 at 72 hours.

The decreasing trend was observed for TEC, in treatment II,in present investigation it might be due to the sequestration of redblood cells in spleen resulting into decrease in total erythrocytecount in circulation (Lumb and Jones, 1984).

GlucoseThe blood glucose level was increased significantly (P<0.05)

in both treatment groups. Fani et al. (2008) showed a significantlyincrease in blood glucose level after epidural administration ofbupivacaine in dogs.

Increase of glucose concentration, might be due to effects ofstress induced secretion of cortical hormones from adrenal glandunder ACTH. Secreted cortical hormones might have stimulatedgluconeogenesis and also reduced consumption of glucose bycells, and both resulted in increased blood glucose concentration(Guyton and Hall, 2006).

Total ProteinThere was no significant (P>0.05) variation in the values of

total protein in any treatment groups. The observed value variedfrom 6.98 ± 0.29 to 7.15 ±0.32 and 7.53 ± 0.21to 8.11 ± 0.19 g% intreatments I and II respectively.

Similar finding was observed by Raghuvanshi (2008).The transiently increase in total protein values in the present

study might have been due to effect of Xylazine on liver duringbiotransformation of drug which might have caused increase intotal protein levels (Fayed et al., 1989)

Alkaline PhosphataseThere was significant (P<0.05) increase in alkaline

phosphatase level in treatment II. The lowest and highest valueswere 37.53 ±1.00 and 43.02± 1.01 in treatment II. In treatment I,

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Table 1: Haematological parameters recorded in the animals of treatments I and II

Table 2: Biochemical parameters recorded in the animals of treatments I and II

Value with similar superscripts within treatment group does not differ.

Parameters Treatment Intervals (min/hrs)

0 15 3 6 12 24 36 72 Haemoglobin (g%)

I 11.40 ±0.41

11.10 ±0.39

10.80 ±0.43

10.53 ±0.44

10.73 ±0.44

10.93 ±0.44

11.06 ±0.40

11.23 ±0.40

II 10.86a ± 0.22

10.36abc ±0.21

9.83cd ±0.23

9.46d

±0.22 10.0cd

±0.23 10.1bc ±0.23

10.3abc

±0.24 10.66ab ±0.22

Packed cell volume (%)

I 34.53 ±1.31

33.33 ±1.47

32.99 ±1.49

32.87 ±1.40

32.64 ±1.44

33.29 ±1.40

33.43 ±1.44

33.62 ±1.43

II 33.93a ±0.36

32.43b ±0.34

30.6c ±0.51

29.15d ±0.38

28.5de

±0.34 28.0e ±0.25

31.28c ±0.33

33.10ab ±0.33

Total erythrocyte count (million/cumm)

I 5.76 ±0.21

5.55 ±0.24

5.49 ±0.24

5.47 ±0.23

5.44 ±0.24

5.53 ±0.24

5.57 ±0.23

5.60 ±0.23

II 6.66a ±0.06

6.36b ±0.09

6.09c

±0.10 5.72d ±0.09

5.96cd ±0.09

5.92cd ±0.10

6.45ab ±0.06

6.49ab ±0.07

+__+

+

Parameters Treat-ments

Intervals (min/hrs) 0 15 3 6 12 24 36 72

Glucose (mg/dl)

I 78.16 ±1.66

79.69 ±1.72

82.65 ±1.83

82.85 ±2.22

80.67 ±2.05

79.37 ±1.84

78.65 ±1.78

II 80.72cd ±1.99

84.05bcd ±1.81

86.28ab ±1.83

90.52a ±1.70

90.66a ±0.51

85.66abc ±0.87

82.26bcd ±2.55

80.57d ±1.95

Protein (g %)

I 6.98 ±0.29

7.05 ±0.32

7.15 ±0.32

7.01 ±0.32

6.98 ±0.30

6.95 ±0.30

6.96 ±0.30

6.94 ±0.29

II 7.53 ±0.21

7.69 ±0.18

7.94 ±0.18

8.11 ±0.19

7.69 ±0.20

7.52 ±0.25

7.49 ±0.22

7.51 ±0.22

Alkaline Phosphatase (IU/L)

I 36.64 ±1.08

37.62 ±1.11

38.75 ±1.14

39.66 ±1.21

39.30 ±1.16

37.54 ±1.19

36.66 ±1.06

36.62 ±1.09

II 37.53c ±1.00

39.01bc ±1.03

41.66a ±0.99

43.02a ±1.01

40.05abc ±1.18

39.1bcd ±1.14

39.25bcd

±1.09 38.91cd ±1.08

Alanine amino transferase (IU/L)

I 17.83 ±0.39

18.01

±0.41 18.74 ±0.51

19.50 ±0.44

18.95 ±0.33

18.18 ±0.38

18.03 ±0.41

17.81 ±0.39

II 17.05e ±0.34

22.99c ±0.43

25.48b ±0.65

29.52a ±0.82

21.46d ±0.54

18.44e ±0.43

17.27e ±0.22

17.23e ±0.29

Bilirubin (mg/dl)

I 0.49 ±0.01

0.48 ±0.02

0.47 ±0.01

0.47 ±0.03

0.46 ±0.04

0.48 ±0.02

0.48 ±0.01

0.48 ±0.01

II 0.47d ±0.04

0.50c ±0.06

0.54b ±0.01

0.58a ±0.03

0.61a ±0.04

0.54b ±0.02

0.49cd ±0.03

0.48cd ±0.09

Urea nitrogen (mg/dl)

I 14.74 ±0.36

15.35 ±0.33

15.55 ± 0.32

15.52 ± 0.33

15.41 ±0.33

15.16 ±0.32

14.84 ±0.36

14.82 ±0.37

II 14.43d ±0.35

14.93cd ±0.33

15.81bc ±0.37

16.79a

b ±0.36 17.09a ±0.37

17.33a ±0.36

15.41c

d ±0.40 14.78cd ±0.32

Creatinine (mg/dl)

I 0.98 ±0.04

1.03 ±0.04

1.11 ±0.04

1.10 ±0.04

1.06 ±0.05

1.03 ±0.04

0.99 ±0.04

0.97 ±0.04

II 0.95cd ±0.02

0.97cd ±0.02

1.02c ±0.02

1.14b ±0.02

1.25a ±0.02

1.00cd

±0.02 0.95cd ±0.03

0.93d ±0.03

Value with similar superscripts within treatment group does not differ.

292


there was a non significant (P>0.05) increase in alkalinephosphatase level up to 6 hours (39.66 ±1.21) after that valuesstarted decreasing significantly up to 72 hours (36.62 ± 1.09).

Vickers et al. (1984) reported that there is a possibility oftransient liver damage during biotransformation of xylazine, whichmay be the cause of rise in ALP values in the present study.

Alanine Amino Transferase (ALT)In treatment I, there was a non-significant (P>0.05) increase

in ALT values. The highest lowest and highest values recordedwere 19.50 ± 0.44 and 17.81 ± 0.39. However in treatment II, therewas significant (P<0.05) increase in ALT values up to 6 hours.Chang and Glazko (1974) indicated that the local anesthetic drugsare metabolised by the liver in most of the species and excreted inurine, during this there may be transient damage to liver, thusmight be increase in ALT levels.

Total Bilirubin (mg/dl)There was non significant (P>0.05) decrease in serum

bilirubin value upto 6 hours (0.47 ± 0.03) and gradually decreaseand reach to near base value at 72 hours (0.48 ± 0.01) in treatmentI, and treatment II, there was a significant (P<0.05) increase inserum bilirubin. In the present study therefore, there is possibilitythat the metabolism of these in the liver might have caused somedisruption in the liver parenchymal cells leading to non significantincrease in the serum bilirubin level (Chang and Glazko, 1974).

Blood Urea Nitrogen and CreatinineIn treatment I, there was non significant (P>0.05) increase in

blood urea nitrogen values after epidural administration ofropivacaine. The lowest and highest values were 14.74 ±0.36 and15.55 ±0.32. In treatment II, there was significant (P<0.05) increasein BUN values up to 24 hours.

There was significant (P<0.05) increase in serum creatinine

values in treatment II. The lowest and highest values of serumcreatinine 0.95 ± 0.02 and 1.25 ±0.02 in treatment II. However,there was non significant increase (P>0.05) in serum creatininevalues in treatment I up to 3 hours. The increase in creatinine andBUN may be attributed to the temporarily inhibitory effect of drugson the renal blood flow, which in turn might have caused a rise inserum creatinine and BUN values (Wright, 1965). However, thepossibility of renal damage could be ruled out because all thevalues were under normal physiological limits.

ReferencesBailey, P. L. et al. (1994) Intravenous opioid anaesthetic. In: Miller, R. D. (ed)

Anaesthesia 4th ed. Vol. I. 273-376.Chang, T. and Glazko, A.J. (1974) Int. Anaesthesiol. Clin. 12: 157-177.Duke, T. et al. (2000) Vet. Rec. 127 (11): 277-281.Fani, F. et al. (2008) Veterinary World. 1(6): 175-177.Fayed, J.W. et al. (1989) Can. J. Vet. Med. 12 (11): 112 -115Guyton, A.C. and Hall, J. E. (2006) In: Textbook of Medical Physiology. 11th

Edn. Philadelphia. Elsevirer Saunders Inc. 951-954.Kelawala, N.N. et al. (1996) Ind. Vet. J., 73 (5) : 552-557.Kennedy, B. L. and T. C. West (1967) J. Pharmacol. Exp. Ther., 157:149.Kumar, A .and Singh, B. (1995) Ind. Vet. J. 72: 364-369.Kumar, D.D. et al. (1997) Ind. Vet. J. 74: 496-498.Lumb, W.V. and E.W. Jones (1984) Spinal Anaesthesia. In: Veterinary

Anaeshesia. 9th ed. Lea and Febriger, Philadelphia, 393-412.Ponder, S. W. and Clark, W.G. (1980) J. Vet. Pharmacol. 3: 203-207.Pratap, K. et al.(2001) Ind. J. Vet. Surg. 21: 116.Raghuvansi, N. (2008) M.V.Sc. Thesis, JNKVV, Jabalpur (M.P.).Snedecor, G.W. and Cochran, W.G. (1994) Statistical methods. 8th ed. The

Iowa State College Press, Inc. Amer. Iowa, U.S.A.Vickers, M. et al. (1984) Drugs in anaesthetic practice. 6th ed. Publ.

Butterworth, London. 63-95.Wright, S. (1965) Applied Physiology. 9th ed. Publ. Oxford University Press,

London. 1109.Yayla, S., and Kilic, E. (2010) Kafkas Univ. Vet. Fak. Derg. 16(5): 835-840.

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IntroductionVentral hernia is the protrusion of abdominal content(s)

through the defect in the abdominal wall with an intact lining of thesac (Engelsman et al., 2007). The only effective treatment is surgeryto restore integrity of the abdominal wall and prevent incarcerationand strangulation of herniated abdominal contents. Tense sutureapplication to approximate and close the defect led to wounddehiscence and recurrent hernia. The use of mesh allows a tensionfree repair of abdominal hernias. The prosthetic materials suchas polypropylene mesh leads to complications like mesh extrusion,fistula formation, wound infection, seroma and adhesions (Molloyet al., 1991). The synthetic degradable materials do not providegood strength during degradation process (Tyrell et al., 1989).Gulati and Cole (1994) observed less immunogenicity and bettertolerance of acellular grafts in rats and rabbits. Acellular dermalgrafts have been used successfully for the reconstruction ofabdominal wall defects in rats (Chung et al., 2003) and rabbits(Gangwar et al., 2006). Kumar et al. (2004) used glutaraldehydepreserved diaphragm of porcine origin for the repair of largeumbilical hernia in dog with excellent recovery. Carbon fibres andcarbon fibres mesh has been used for the repair of umbilicalhernias in bovines (Gangwar et al., 2008a and b). Decellularizedswine aorta has been used for repair of abdominal wall herniadefects in rat model with excellent results (Bellows et al., 2008). Invivo biocompatibility evaluation of acellular aortic matrix (AAM) ofbuffalo origin in Guinea pig model revealed that AAM isbiocompatible (Devarathnam, 2010), with these results, presentstudy was undertaken to evaluate the efficacy of acellular aorticmatrix of buffalo (Bubalus bubalis) origin for reconstruction ofventral hernia in a buck.

Materials and MethodsPresent study was conducted in the Biomaterial and

Bioengineering Laboratory, Division of Surgery and in the ReferalVeterinary Polyclinic, Indian Veterinary Research Institute, Izatnagar-243122, Uttar Pradesh, India. This communication deals with thesurgical management of ventral hernia in a buck. A Jamunaparibreed buck aged 3 years having body weight of 35 kg was presentedwith the history of swelling in the right ventral abdomen. Clinicalexamination revealed large ventral hernia of abdominal wall. Thesize of hernial ring was 10-12 cm in diameter (Fig. 1). Since thehernia ring was large so it was decided to perform hernioplasty.

Preparation of acellular aortic graftFresh posterior aorta of buffalo origin was collected from

local abattoir and immediately preserved in ice-cold sterilephosphate buffered saline solution (pH 7.4) containing 0.1%amikacin and 0.02% EDTA. The tissue was rinsed with normalsaline before the start of protocol. The maximum time periodbetween retrieval and the initiation of protocol was less than 4 h.The tissue was cut into desired sizes and made acellular using

ACELLULAR AORTIC MATRIX FOR THE REPAIR OF LARGEVENTRAL HERNIA IN A BUCK

A. K. Gangwar, Vineet Kumar, J. Devarathnum, Naveen Kumar,A.K. Sharma, Himani Singh and D.T. Kaarthick

Division of SurgeryIndian Veterinary Research Institute, Izatnagar-243122, Bareilly, Uttar Pradesh, India

1% anionic biological detergent for 24 h followed by 0.25% enzymesolution for 2 h and then again with same 1% anionic biologicaldetergent for next 24 h (Devarathnum, 2010). Finally, the tissuewas thoroughly washed in phosphate buffer saline (PBS) solution.The prepared acellular aortic matrix was stored in PBS solutioncontaining 0.1% amikacin solution at 4°C.

Surgical treatmentThe animal was kept off fed for 24 h before surgery.

Premedication was made with adsorbed tetanus toxoid (TeatanusToxoid®, GSK; 5 Lf unit, intramuscularly), enrofloxacin (Enrocin®,Vetnex RFCL; 5 mg/kg intramuscularly) and, meloxicam (Melonex®,Intas; 0.5 mg/kg intramuscularly). The operation was performedin dorsal recumbency. The surgical site was prepared for asepticsurgery. After aseptic preparation of the site, xylazine (Xylaxin®,Indian immunologicals; 0.1 mg/kg intramuscularly) wasadministered and 2% lignocaine hydrochloride (Xylocaine®,AstraZeneca) was infiltrated at the site. After proper anaesthesiaan elliptical incision was made over the hernial sac and fasciaand muscles were separated from the hernial ring (Fig. 2). Thehernial contents were pushed back in the abdominal cavity. Thehernial ring was repaired by acellular aortic matrix using inlaytechnique. The graft was anchored in position by black braidedsilk No. 2 using horizontal mattress suture pattern (Fig. 3). Finallythe skin was closed by silk sutures (Fig. 4).

Postoperative analgesia was provided by meloxicam (0.5mg/kg i/m, once daily) for 5 days. Enrofloxacin (5 mg/kg i/m, oncedaily) was administered for 7 days. Daily dressing of the sutureline was performed with 5% povidone iodine (Betadine®, Win-Medicare) until healing of surgical wound. The skin sutures wereremoved on 10th postoperative day. The owner was advised tofeed the animal frequently with little quantity. To evaluate the longterm results a telephonic survey was carried out and it revealedthat after 6 months of operation the animal was fine.

Results and DiscussionThe buck recovered completely without clinical signs of wound

dehiscence or infection. In this study, we demonstrated that ventralhernia repaired with AAM result in no herniation up to 3 monthsafter reconstruction. This may reflect the fact that AAM is designedto withstand high pressures such as that seen in the arterialvascular system (Bellows et al., 2008). In this study, decellularizationprotocol preserves distinctive, natural three dimensional structureof collagen and elastin in the aorta. The decellularization approachminimizes the host immune response to the bioprosthetic tissueand generated biomaterial for tissue reconstruction. Theseacellular tissues retained their natural mechanical properties andpromote remodeling of the prosthesis by neovascuarization andrecellularization by the host (Schmidt and Baier, 2000). Collagenis a natural substrate for cellular attachment, growth anddifferentiation in its native state. They are biocompatible and non

294


toxic to tissues. It is universal biological molecule that exhibits onlyvery slight differences from species to species and possessesonly minimal antigenic properties (Davies, 1983). Successful useof glutaraldehyde treated acellular dermal graft in a goat has beenreported by Gangwar et al. (2003). Xenogenic collagen applied toinjured surface of the skin did not cause any foreign body reactionnor any immune rejection reaction or sensitization (Soo et al.,1993). The acellular matrix possesses the appropriate mechanicalproperties and induces appropriate interaction with the host cellsthat resulted in the regeneration of functional tissues (Voytik-Harbinet al., 1998). Decellularized swine aorta has been used for repairof abdominal wall hernia defects in rat model with excellent results(Bellows et al., 2008). Keeping in view the possible advantage ofacellular aortic matrix of buffalo origin, it has been usedsuccessfully for the reconstruction of ventral hernia in a buck.

ConclusionPresent study revealed that acellular aortic matrix proved to

be a good biocompatible biomaterial for the reconstruction ofhernia. It is easy to prepare and can be clinically practiced in otherspecies of animals.

AcknowledgementThe authors acknowledge the financial assistance received

from the Department of Biotechnology (DBT), Ministry of Scienceand Technology, New Delhi, India to carry out this work.

ReferencesBellows, C. F. et al. (2008) Hernia. 12: 351–358.Chung, S. et al. (2003) Plast. Reconstr. Surg. 111: 225-232.Davies, J.W.L. (1983) Burns. 10: 94-103.Devarathnum, J. (2010) M.V.Sc. Thesis submitted to Deemed University,

IVRI, Izatnagar.Engelsman, A.F. et al. (2007) Biomaterials. 28: 2314-2327.Gangwar, A. K. et al. (2003) Vet. Pract. 4(1): 25-26.Gangwar, A. K. et al. (2006) J. South Afr. Vet. Assoc., 77: 79-85.Gangwar, A. K. et al. (2008a) Indian Vet. J. 85: 430-431.Gangwar, A. K. et al. (2008b) Indian Vet J. 85: 658-659.Gulati, A. K. and Cole, G. P. (1994) Acta Neurochir. Wien. 126: 158-164.Molloy, R. G. et al. (1991) Br. J. Surg. 78: 242-244.Kumar, N. et al. (2004) J. Canine Develop. Res. Vol. 4: 43-46.Schmidt, C. E. and Baier, J. M. (2000) Biomaterials. 21: 2215-2231.Soo, C. et al. (1993) J. Dermatol. Surg. Oncol. 19: 431-434.Tyrell, J. et al. (1989) Surg. Gynecol. Obstet. 168: 227-232.Voytik-Harbin, S. L. et al. (1998) Tissue Engineering. 4: 157-174.

Fig. 1: Photograph showing big ventral hernia having hernia ring of 10-12 cm in diameter

Fig. 2: An elliptical incision was made over the hernial sac and fasciaand muscles were separated from the hernial ring

Fig. 3: Anchoring of aortic acellular matrix as inlay graft using horizontalmattress suture pattern

Fig. 4: Closure of skin by silk sutures

295


IntroductionSheep being gregarious animal are very sensitive to the

stress of becoming separated from the flock. Earlier studieshave reported that subjecting sheep to restraint and isolationstress (RIS) caused robust increase in plasma concentrationsof ACTH and cortisol (Minton and Blecha, 1990; Coppinger etal., 1991).

In animal husbandry there are some managementstresses, which cannot be eliminated from the daily workoperations. Stress could be physiological, physical,environmental, chemical and emotional. Restraint is one ofthe most common practices in domestic animal husbandry.Exposure of livestock to stress has been associated withactivation of the hypothalamic-pituitary-adrenal axis (Minton,1994). Among domestic animals the sheep is one of the mostsensitive to emotional factors (Stephens, 1980) and seems tobe a very good model in stress experiment. The stressreactions are characterized by enhanced pituitary-adrenalreactivity, which facilitates return of the body to homeostaticconditions.

Therefore, keeping these in view, the present study wasundertaken with the objectives to estimate the normal cortisolhormonal level in female Karakul sheep and to study the effectof restraint and isolation stress on cortisol hormonal level inKarakul sheep.

Materials and MethodsThe present study was planned in two phases:

First phase of experimentTen apparently healthy adult female animals procured

from the project scheme in the department of Gynaecologyand Obstetrics, College of Veterinary and Animal Science,Bikaner (Rajasthan), India were included in this study. Theseanimals were housed in a flock undisturbed during theexperimental period for ten days and were treated as controlgroup.

Second phase of experimentEach animal of control group was subjected to restraint

and isolation stress for three consecutive days. The stresstreatment comprised of removal of animals from flock andtransferring them into separate trial cages away from visual

INFLUENCE OF RESTRAINT AND ISOLATION STRESS (RIS) ONPLASMA CORTISOL IN FEMALE KARAKUL SHEEP

A. Moolchandani and M. SareenDepartment of Veterinary Biochemistry, College of Veterinary and Animal Science


ABSTRACT

The study was carried out to observe the effect of restraint and isolation stress (RIS) on plasma cortisol hormonal profile in femaleKarakul sheep. In present study ten apparently healthy female adult animals were subjected to restraint and isolation stress. The bloodsamples were collected and analysed at pre-stress condition and on 1st, 2nd and 3rd day of RIS, respectively. A highly significant(P<0.01) effect of RIS on plasma cortisol level with increasing trend was observed.

Key words: Plasma, Karakul, sheep, cortisol, restraint and isolation stress

and tactile contact with other animals. Animals were fed withdry roughage with approximately 250 g of concentrate andsome quantity of salt and mineral mixture, as per the standardconditions of feeding and management. Drinking water wasprovided ad libitum.

Collection, processing and preservation of samplesThe blood samples were collected at 9.30 AM each day for

control and 1st, 2nd and 3rd consecutive days of RIS, respectively,under aseptic conditions in sterile heparinized tube (heparinwas added @ 30 IU/ml of blood). For separation of plasma thetest tubes were centrifuged at 3000 rpm for 20 minutes, plasmawas pipetted out in clear dry plasma tubes. After collection,plasma was stored at -20oC.

Hormonal assay (Cortisol)Plasma cortisol hormonal level was estimated by Radio

Immuno Assay (RIA) technique using coat-A-count kit suppliedby Diagnostic Production Corporation, Los Angeles, USA. TheRIA protocol, specificity of antisera and critical evaluation ofassays have been reported (Rastogi and Agarwal, 1990).

Statistical analysisThe results were presented as Mean ± SE. The mean

values were determined according to RIS and sex groups.The data were subjected to analysis of variance (ANOVA)(Snedecor and Cochran, 1967). The critical differences amongvarious means were worked out by least significance difference(LSD) method.

Results and DiscussionThe mean ± SE concentrations of plasma cortisol in female

karakul during pre-stress and stress are shown in Table 1. Inpresent study, the statistical analysis revealed a highlysignificant effect (P<0.01) of RIS on plasma cortisolconcentration in female karakul sheep. The highest plasmacortisol concentration was observed during 1st day of RIS andlowest in control group. The plasma cortisol concentrationincreased by110% at the 1st day of RIS and thereafter thelevel of cortisol declined during 2nd day and 3rd day of RIS,respectively. However, in spite of declination the concentrationsof cortisol during 2nd and 3rd day of RIS were comparativelyhigher than that of control group. It is concluded from this study

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that the plasma cortisol concentration increased significantlyin response to stress.

In present study, the observed plasma cortisol values werelower than those reported by Bobek et al. (1986) and Kanekoet al. (1997) in sheep. It is also important to be mentioned herethat the cortisol level in blood stream differs with each individualbecause some animals secrete more stress hormones thanothers. Possibly breed difference and different metabolic statusof animals may be considered as factors responsible for suchdifferences. Observed values of mean plasma cortisolconcentration in present study were more or less in agreementwith the findings of Niezgoda et al. (1993). The analysis ofvariance revealed that the mean plasma cortisol level increasedsignificantly (P>0.01) during the course of RIS and this patternwas well in accordance with the earlier reports in reference toRIS in various breeds of sheep (Apple et al., 1993; Bobek et al.,1986 and Niezgoda et al., 1993). Subjecting sheep to restraintand isolation causes robust increase in plasma concentrationof ACTH and cortisol and it has been suggested that stressconditions activates pituitary-adrenal-axis and this is one ofthe important contributing factors for elevated cortisol level(Parrott et al., 1987; Mormede, 1988; Minton and Blecha, 1990;Coppinger et al.1991; Adrichem and Vogt, 1993; Apple et al.,1993)

In our present study the highest cortisol level was observedduring the first day of RIS and thereafter a decline was observedin the cortisol level. These findings are more or less supportedby earlier reports in different sheep breeds (Bobek et al., 1986and Doubek et al., 2003), and also in other species (Cooper etal., 1995 in calves and Antione et al., 1995 in gilts), in which allthey reported overall increase in blood/plasma cortisolconcentration in response to stress. Thus it can be concludedin present study that the presence or absence of socialcompanions has major effects on blood cortisol level. In present

study, it is evident that restraint and isolation stress (RIS) insheep appreciably alters the blood cortisol level. Cortisol isreferred as a stress hormone because its level rises afterexposure to a physiological or physical stress. The increasedplasma cortisol concentration during RIS study, suggests thatthis passive response to stress is due to hypothalamic-pituitary-adrenal axis activation ultimately resulting in significantincrease in plasma cortisol concentration during the course ofRIS study.

AcknowledgementsThe authors are thankful to the Dean, College of Veterinary

and Animal Science, Bikaner for providing facilities to undertakethe present investigation.

ReferencesAdrichem P.W.M. and Van.Vogt, J.E. (1993) Livestock Prod. Sci. 33:

151-159.Antoine, W.M.R. et al. (1995) Am. J. Vet. Res. 56: 1225-1227.Apple, J.K. et al. (1993) J. Ani. Sci. 71: 1, 71-77.Bobek, S.et al. (1986) J. Vet. Med. A, 33: 698-705.Cooper, C. et al. (1995) Can. J. Ani. Sci. 95: 197-201.Coppinger, T.R. et al. (1991) J. Ani. Sci. 69: 2808.Doubek, J. et al. (2003) Czech-J. Ani. Sci. 48: 10, 403-411.Kaneko, J. J. et al. (1997) 5th ed. Academic Press, San Diego, California,

USA. pp. 890-894.Minton, J.E. (1994) J. Ani. Sci. 72: 1891-1898.Minton, J.E. and Blecha, F. (1990) J. Ani. Sci. 68: 3145-3151.Mormede, P. (1988) In: R. Zayan and R. Dantzer (Editors), Social Stress

in Domestic Animals. Dordrecht: Kluwer. pp. 203 - 221.Niezgoda, J. et al. (1993) J. Vet. Med. A 40: 631-638.Parrott, R.F. et al. (1987) J. Endocrinol. 112: 2, 305-310.Rastogi, S.K. and Agarwal, S.P. (1990) Indian J. Ani. Sci. 60: 1047.Snedecor, G. and Cochran, W.G. (1967) Statistical Methods. 6th ed.

Allied pacific (P) Ltd., Bombay.Stephens, D.B. (1980) Adv. Vet. Sci. Comp. Medicine 24: 179-210.

Table 1: Mean ± SE concentrations of plasma cortisol hormone according to the effect of RIS in female Karakul sheep.

Main e ffects

No. o f observations

Plasma cortisol

(nmol/L. ) **

% Increase

Cont rol o r pre-RIS condition (T1)

10 28.46 a ±1.464

-

On 1st day of RIS (T2)

10 58.535 c ±0.426

110.92

On 2nd day o f R IS (T3)

10 32.165 b ±0.784

20.08

On 3rd day o f RIS (T4)

10 31.445 b ±0.765

15.63

O verall mean ± SE 40 37.65 ±2.115

-

Note: Mean comparison have been made within main effects. Mean superscripted by different letters di ffersignificantly (P< 0.05) from each other in columns. **Highly Significant

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Post-partum prolapse of uterus is commonly observeddairy cow, ewe and buffalo. The prolapse of uterus is mostcommonly observed immediately after parturition. Themanagement of such prolapse cases is put on record.

Case history and observationA pluriperous buffalo of five years of age brought to the

veterinary hospital, Jharoda dairy colony with prolapseduterine mass. The cow had delivered a healthy calf five daysbefore in normal parturition. The owner reported that theanimal had showed straining and passed some placentanormally. After one day of parturition the uterine mass cameout. The local para vet has tried to push the organ inside butfailed to do it properly.

On clinical examination of the animal an inflammeduterine mass, some laceration over parenchyma hangingfrom the vagina was observed. The prolapsed mass wasfound oedematous and soiled with dirt. The animal showedsevers tenesmus, arched back. The body temperature wassub normal (98°F) with weak pulse and rapid respiration.The animal was severely depressed and not taking anykind of food, found anorectic.

TreatmentThe animal was restrained in the service crate and the

prolapsed mass was washed with mild antiseptic solution.Potassium permanganate one pinch in 5 litre of clean watersolution made to wash the mass. As the animal showedsevere pain and restlessness epidural anaesthesia 2%Lignocain hydrochloride (Xylocain, Astra Zeneca LP) wasinjected in inter-coccygeal space. A total of 5 ml solutioninjected. The dead tissue, dirt, blood clots, faeces etc wereremoved completely from the organ. Washing was done invery gently and cold ice pack applied over the organ mass.After ice pack application the prolapsed organ shrink andthe size become reduced. The antiseptic lubricant gelapplied over the mass. The organ was lift to the normalvaginal level and mild gentle pressure was applied to pushthe organ inside. The organ was then tried to push inside

CLINICAL MANAGEMENT OF POST-PARTUM PROLAPSEOF UTERUS IN A BUFFALO- A CASE REPORT

P. Goswami and Sanku Borkataki1Veterinary Hospital, Jharoda, Delhi-84,India

and repositioned by arm pressure through vagina. Afterrepositioned a purse ting suture was applied to keep theorgan inside.

The animal was then treated parenterally by injectingPfizer milk 450 ml slow i/v along with infusion of 5 mlDexamethasone sodium vet injection (dexona vet ofSarabhai Zydus). Besides intra muscularly 10% Enrofloxacin(100 mg/ml, Intas @7.5 mg/kg b.wt), Melonex plus(meloxicam and paracetamol, Intas) @ 15 ml and inorganicphosphorous (Tonophosphan, Intervet) @ 15 ml wereinjected. Involon bolus (Natural Remedies) was advised toadminister @ 2 boli twice daily for four days. 200 ml of oralCalup Gel (Vet Care) was also advised to given after 24hours of primary treatment. The animal recovered eventfully,and no prolapse mass was seen even after removing ofsuture after four days of treatment.

DiscussionThe post-parturient prolapse in this case might be due

to hypocalcaemia, as the animal recover successfully afteradministration of calcium therapy. Arthur et al. (2001) alsoreported hypocalcaemia as major cause of uterine prolapsein parturition. The loss of myometrial tone may predisposeto uterine prolapse during the peristaltic contraction of labour.The mean values of serum calcium and inorganicphosphorus were significantly (P<0.01) lower in thebuffaloes with prolapsed uteruses on the day of uterineprolapse (Pandey et al., 2007)

SummaryA case of post-parturient prolapse in a buffalo was

corrected with manual pressure followed by administrationof calcium therapy alongwith other supplemental therapywas described for the successful management of the case.

ReferencesArthur, G.H. et al. (2001) Veterinary Reproduction and Obstetric.8th

ed. Harcourt (India) Private Limited.Pandey, A.K. et al. (2007) Buffalo Bull. 26(1): 20-22.

1Assistant Professor, Division of Parasitology, F.V.Sc. & A.H., R.S.Pura, Jammu, India

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IntroductionFormaldehyde is colourless, flammable gas with pungent

and irritating odour and is widely used as a chemical in theindustries, and for embalming and preservation of tissues inlaboratories (IARC, 1982). Direct exposure to the formaldehydevapours can cause irritation of the eyes and upper respiratorytract to the anatomists, technicians and students, as it is easilyabsorbed by the respiratory mucosa (Casanova et al., 1988;Heck et al., 1985). The occupational exposure of formaldehydein higher concentration causes irritant and allergic reaction onall the vital organs (Siegel et al., 1983). Some of the studieshave indicated the cytotoxic and teratogenic effects offormaldehyde on rat lung (Sheela and Sreedevi, 1991), nasalmucosa (Edling et al., 1988), trachea (Davarian et al., 2005),testis (Golalipour et al. 2007), kidney (Golalipour et al., 2009)and liver (Cikmaz et al., 2010). As the formaldehyde ismetabolized in liver, so present study was designed to observethe hepatotoxicity caused by direct exposure of formaldehydein rabbits with the hypothesis to find the possible relationshipbetween the cytotoxicity and changing pattern of metabolicenzymes. The aim of this study was to elucidate the short andlong term effect of different concentration of formaldehydeexposure on the rabbit liver.

Materials and MethodsThe experiment was conducted on 14 male rabbits of 6-9

months age and 1100-1200 g body weight. The rabbits weredivided into three groups; group I and group II with six animalsin each group, and whereas group III had two animals ascontrol. These animals were kept in ventilated iron cages andwere fed with black grams and green fodder. The rabbits ofgroup I were exposed to formaldehyde solution 40% (37- 41%w/v) for 6 weeks and those of group II were exposed to 10%formaldehyde solution for 12 weeks. The solution was kept in

HEPATO-TOXIC EFFECTS DUE TO DIRECT EXPOSURE TOFORMALDEHYDE IN RABBITS

Varinder Uppal, Neelam Bansal, Devendra Pathak and AnuradhaDepartment of Veterinary Anatomy

Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141004, Punjab, India

ABSTRACT

The experiment was conducted to observe the effect of direct exposure of formaldehyde in different concentrations in the liver.For this purpose, 14 rabbits ageing 3- 6 months and having weight 1100-1200 g were divided into three groups. The animals ofgroup I (n=6) were exposed to 10% formaldehyde solution for 12 weeks, group II (n=6) to 40% formaldehyde solution for 6weeks and group III (n=2) kept as control. After sacrificing the animals, fresh tissue samples were collected from liver for thedemonstration of enzymes and some part of samples were also preserved in 10% NBF and Karnovsky’s fixative to studyhistological and electron microscopic changes. The present study revealed that after an exposure to 40% formaldehydesolution, there was disorganisation of hepatocytes, increased Kupffer cells, pyknosis and karyorhexis of chromatin materialand cytoplasmic vacuolizations. Marked congestion and haemorrhages were observed in the hepatic tissue. Lymphomononuclearcellular infiltration was seen at portal area along with mild oedema. The number of mitochondria increased with decreased insize. The rough endoplasmic reticulum became irregular whereas the smooth endoplasmic reticulum showed dilatation. The lossof various enzymatic activities may be due to the reduced metabolism caused by hepatic degeneration after direct exposure offormaldehyde in rabbits. It is concluded that histopathological and histoenzymological changes were more remarkable in rabbitsexposed to 40% formaldehyde for short duration than those exposed to 10% formaldehyde for longer duration.

Key words: Formaldehyde, liver, rabbits, histopathology, histoenzymology

open containers below the cages and was changed daily. Aftercompletion of experimental periods, the animals weresacrificed and fresh tissue samples were collected from theliver for cryostat sectioning. These sections were incubated indifferent substrates for the demonstration of phosphatases(Barka and Anderson, 1963) and oxidoreductases (Pearse,1972) in the liver. Some part of the liver was also fixed in 10%NBF and processed as per acetone benzene schedule (Luna,1968). Paraffin sections of 5- 6 µm thickness were stainedwith haematoxylin and eosin to observe histomorphologicalchanges. For electronmicroscopic studies, small pieces theliver tissues were also collected in Karnovsky’s fixative andsubjected to dehydration in ascending grades of acetone (30%to absolute) at room temperature. After infiltration, the tissueswere embedded in pure embedding media using beamcapsule and prepared blocks were trimmed by block trimmer(Reichert TM 60) and semi thin sections (0.5-2.0 µm) were cutto scan the tissues under optical microscope. After scanning,the ultra thin sections (70-90 nm) were cut and lifted on coppergrids (100 mesh size) and stained with uranyl acetate (15min)followed by lead citrate (10min) and examined under TEM(Morgagni) for detailed study and required photographs weretaken.

Results and DiscussionI. Histomorphological studies: The rabbits of control group

showed the normal histoarchitecture of the liver. Thehistopathological observations in the rabbits exposed to 40%solution of formaldehyde for 6 weeks included disorganizationof hepatocytes along with nuclear pyknosis and vacuolizationin the cytoplasm. Marked congestion and haemorrhageswere seen in the sinusoids with an increase in the numberof Kupffer cells (Fig. 2). Lymphomononuclear cellular infiltrationwas observed at portal area along with mild oedema (Fig. 3).

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Fig.9: Electronmicrographs showinghaemorrhages in the sinusoids (X 880).

Fig.1: Paraffin section showing mildcongestion and haemorrhages in theliver of animals exposed to 10%formaldehyde solution (H & E X100).

Fig.2: Paraffin section showing markedcongestion, haemorrhages andcytoplasmic granulations of hepatocytesof l iver of animals exposed to 40%formaldehyde solution (H & E X200).

Fig.3: Paraffin section showing marked oedemaand lymphomononuclear inf iltration in the portalarea of l iver of rabbits exposed to 40%formaldehyde solution (H & E X200)

Fig.4: Cryostat section showing SDH activity in liver tissue exposed to40% formaldehyde solution (Nitro BT method X 100).

Fig. 5: Cryostat section MDH activity in liver tissue exposed to 40%formaldehyde solution (Nitro BT method X 100).

Fig.6: Electronmicrographs showinggranulations of hepatocytes, decreased sizeand increased number of mitochondria,irregular rough endoplasmic reticulum, anddilatation of smooth endoplasmic reticulumin the liver of rabbits exposed to 40%formaldehyde solution. (X 880).

Fig.7: Electronmicrographs showingmarked oedema and vacoulations in theliver of rabbits exposed to 40%formaldehyde solution (X 880).

Fig.8: Electronmicrographs showingkaryorhexis and pyknosis in the nuclei ofdegenerating hepatocytes (X 1100).

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Similar observations have been reported on the liver of ratsafter inhalation of formaldehyde (Cikmaz et al., 2010) in rats,and on fatty liver and kidney in adult and developing rats (Kumet al., 2010). The congestion in the sinusoids may be due toinflammation in the hepatocytes leading to hyperaemia andoedema. In present study, focal necrosis and fibrosis wasalso observed in the portal area. Similarly, Dossing et al. (1983)explained that patients exposed to organic solvents forprolonged periods caused marked steatosis, focal necrosis,fibrosis and enlargement of the portal canals. Binucleatedhepatocytes were seen at places after an exposure to 40%solution of formaldehyde may be due to the regenerative attemptof the degeneration cells as reported earlier in the liver ofcorboryl treated rats by Munglang et al. (2009). However,histological changes reported in the liver of rabbit exposed to10% formaldehyde solution showed slight degenerativechanges in the hepatocytes and portal triad area (Fig. 1). Thepresent experiment showed that although the histopathologicalchanges were observed in the liver of both the groups, yet thechanges were more pronounced in the rabbits exposed to40% formaldehyde solution for shorter duration. Theseobservations are in close proximity with the observation ofKamata et al. (1997), who reported that chronic inhalation offormaldehyde causes hepatotoxicity in rats. However,Woutersen et al. (1987) noticed that 6 hrs a day exposure of 10ppm of formaldehyde for 13 weeks did not show anyhepatotoxic effect, while 20 ppm of formaldehyde exposureinduced mild effects.

The electron microscopic studies depicted decreased sizeand increased number of mitochondria, irregular roughendoplasmic reticulum, and dilatation of smooth endoplasmicreticulum (Fig. 6). The degenerating hepatocytes showed lossof cytoplasm with increased granulation and vacuolations (Fig.7). The formation of collagen fibrils and exudate were seen inthe focal necrotic areas. The capillaries were dilated withcongestion and haemorrhages. The karyorhexis and pyknosiswas observed in the nuclei of degenerating hepatocytes,however, some of the nuclei were binucleated (Figs. 8 and 9).The EM findings were inconsistent with the histopathologicalchanges in the liver of rabbits exposed to 40% formaldehydesolution. The present findings are in close proximity to theobservation of Cikmaz et al. (2010) in rat liver.

II. Histoenzymic observations(i) Phosphateses: There was weak AKPase activity in the

hepatocycytes, kupffer cells and sinusoids. The hepatocytesshowed moderate and kupffer cells had strong ACPase activity,where as a weak reaction of AT Pase and G-6-Pase wasobserved in the hepatocytes and Kupffer cells. The loss of AKPase, AT Pase and G-6- Pase in the hepatocytes may be dueto the degeneration of cell membranes leading to leakage ofthese enzymes into blood from the tissues. The increasedreaction of ACPase indicated the lymposomal activity of this

enzyme in degenerating hepatocytes. Similar observationshave been reported in the liver of buffalo calves followingselenium toxicity (Gill, 2000) and lead toxicity (Anuradha et al.,2005).

(ii) Oxidoreductases: There was weak to moderate activityof SDH, LDH and G-6-PD enzymes in the hepatocytes andKupffer cells, whereas sinusoids showed weak reaction (Fig.4). A weak reaction of G-6-PD was observed in the hepaticcomponents may be either due to the reduced level ofglutathione exposure or due to extracellular and intra cellularlipid peroxidation in the liver following formaldehyde as reportedearlier by Kanazawa and Ashida (1991). The activity of NADPHand MDH was moderate to strong in the hepatocytes and weakto moderate in the Kupffer cells and sinusoids (Fig. 5).Thechange in the activity of dehydrogenases is suggestive ofaltered metabolic pathway of the liver following formaldehydeexposure. The present observations are similar to the findingsof Yilmaz et al. (2004), who also found decreased G-6-PDactivity and increased MDH activity in the liver of rats exposedto 20 ppm of formaldehyde for 13 weeks. However, unlike thepresent study, they not detect any difference in LDH activity incontrol and test groups.

ReferencesAnuradha et al. (2005) Indian J. Anim. Sci. 75(7): 782-6.Barka, T. and Anderson, P. J. (1963) Histochemistry: Theory, Practice

and Bibliography. Harper and Row Publishers, Inc., NewYork, pp. 273-316.

Casanova, M. et al. (1988) Food Chem. Toxicol. 26: 715-716.Cikmaz, S. et al. (2010) Toxicol. Indust. Health. 26(2): 113-119.Davarian, A. et al. (2005) Int. J. Morphol. 23: 369-372.Dossing, M. et al. (1983) Eur. J. Clin. Invest. 13: 151-157.Edling, C. et al. (1988) Brit. J. Indust. Med. 45: 761-765.Gill, G.P.S. (2000) Histomorphological, histochemical and

histoenzymological study on organs/systems of buffalo inexperimental selenosis. M.V.Sc. thesis. Punjab AgriculturalUniversity, Ludhiana, India.

Golalipour, M. J. et al. (2009) Int. J. Morphol. 27(4): 1195-1200.Golalipour, M. J. et al. (2007) Folia Morphol. (Warsz). 66(3): 167-71.Heck, Hd’A. et al. (1985) Amer. Ind. Hyg. Assoc. J. 46: 1-3.IARC (International Agency for Research on Cancer). (1982) IARC

Monogr. Eval. Carcinog. Risk Chem. Hum. 29: 345-389.Kamata, E. et al. (1997) J. Toxicol. Sci. 22: 239-254.Kanazawa, K. and Ashida, H. (1991) Arch. Biochem. Biophys. 288:

71-78.Kum, S. et al. (2010) J. Ani. Vet. Adv. 9(2): 396-401.Luna, L.G. 1968. Manual of Histologic Staining Methods of the Armed

Forces Institute of Pathology. 3rd ed., McGraw-Hill BookCo., New York. pp. 33, 94.

Munglang, M. et al. (2009) J. Anat. Soc. India. 58(1): 6-9.Pearse, A.G.E. (1972) Histochemistry: Theoretical and Applied. 3rd

ed. Vol. II. Churchill Livingstone, London.Sheela, R. and Sreedevi, P. (1991) J. Anat. Soc. India. 40: 97-103.Siegel, D. M. et al. (1983) Regul. Toxicol. Pharmacol. 2: 355-371.Woutersen, R. A. et al. (1987) J. Appl. Toxicol. 7(1): 43-49.Yilmaz, H. R. et al. (2004) Eastern J. Med. 9:47-50.

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IntroductionChromium compound are widely used in leather tanning,

oil refinery, fertiliser, stainless steel, dye manufacturingindustries and effluent from these industries contaminate thesurrounding air, water and soil (Barceloux, 1999; Xu et al.,2004). So concentration of chromium in biosphere aroundchromium based industrial plant has been found above thecritical level. Livestock and birds present in these areas maybe continuously exposed to this form of chromium throughfood, air and water.

Chromium can exist in variety of oxidation state but trivalent[Cr (III)] and hexavalent [Cr (VI)] are most stable andpredominant form (Baruthio, 1992). The toxicokinetics of a givenchromium compound depend on the valence state of thechromium atom and the nature of its ligands. Trivalentchromium act as essential trace element of body in animaland human, and has nutraceutical importance whilehexavalent chromium in environment is mostly man madthrough industrial emission and both acute and chronic toxicityare mainly caused by hexavalent chromium compound(Ducros, 1992). The soluble hexavalent chromium Cr (VI) isan environmental contaminant widely recognized ascarcinogen, mutagen, and teratogen toward humans andanimals. The fate of chromium in the environment is dependenton its oxidation state. The intracellular reduction of potassiumchromate i.e. Cr (VI) to Cr (III) results in the generation of reactiveintermediates leading to oxidative tissue damage and cellularinjury (Patlolla et al., 2009). Reactive oxygen species (ROS)generated in excess by these reactions can cause injury tocellular proteins, lipids and DNA, leading to oxidative stress.

Since free radical generation has been implicated in thepathogenesis of chromium toxicity, supplementation ofantioxidants can be considered as the alternative method for

PROTECTIVE EFFECT OF VITAMIN E AND SELENIUM IN POTASSIUMCHROMATE INDUCED SUB-ACUTE TOXICITY OF BROILER CHICK#

Rashmi Rekha Kumari1#, A.K. Chakraborty2, T.K. Ghosh3 and T.K. Mondal4Department of Pharmacology and Toxicology

West Bengal University of Animal and Fishery Sciences, Kolkata-700037

ABSTRACT

The present study was carried out to study the sub-acute toxicity of potassium chromate in broiler chicks and to evaluate theameliorative effect of vitamin E and selenium. There was reduction in superoxide dismutase, reduced glutathione, aspartate transami-nase, alanine transaminase and increase in lipid peroxidation in liver and kidney tissue of chicks in dose dependent manner inpotassium chromate treated groups as compared with control (T1). Also the pathological changes were observed at higher dose inliver and kidney tissue and there was significant increase in chromium concentrations in different tissues of all treatment groups.Simultaneous treatment with vitamin E and selenium mitigated these toxic effects in chicks of group T4.

Key words: Potassium chromate, sub-acute toxicity, broiler chick, vitamin E and selenium

chelation therapy. Selenium (Se) and vitamin E aremicronutrients essential for normal health and maintenancein poultry (Choct and Naylor, 2004). They are involved inpreventing free radical damage to phospholipid membranes,enzymes and other important molecules. Limited literature isavailable on protective effects of vitamin E and Se on potassiumchromate (K2CrO4) induced sub-acute toxicity in broiler chicks.Soudani et al. (2011) reported benficial effects of Se inpreventing K2Cr2O7 induced hepatotoxicity in rats.

Most of the work has been carried out on toxicity study ofhexavalent chromium compound in laboratory animalespecially on its carcinogenic and mutagenic aspect butliterature in relation to toxicity of chromium compounds inanimals and poultry are limited. Therefore, there is a need oftoxicological study of chromium [Cr (VI)] compound in livestockand poultry and measurement of its residues in meat of foodanimals after acute, sub acute and chronic exposure tochromium compound. Considering these, the present studywas undertaken to study the biochemical and histopathologicalchanges in sub-acute toxicity of K2CrO4 in broiler chick atdifferent dose levels. Further, to evaluate the residualconcentration of chromium in different tissues and the role ofvitamin E and Se in amelioration of toxic effect.

Materials and MethodsChemicals and drugs

Potassium chromate (K2CrO4) of E. Merck (India) limitedwas used as test compound. Vitamin E and Se was used asTocosel® supplied by M/s Nandan Remedies 311/8, Nagendraroad, Kolkata. Each 10 g of Tocosel® contained 5 g of tocopherolacetate and 30 mg of sodium selenite.

Design of experimentLD50 value of K2CrO4 in 3 weeks old broiler chicks (Strain-

1#M.V.Sc. Student, Present Address for correspondence: Assistant Professor cum Junior Scientist, Department of Pharmacology, Bihar VeterinaryCollege, Patna-800014, Email: [email protected], Mob: +91-95463917552Retired Professor, Department of Pharmacology and Toxicology, West Bengal University of Animal and Fishery Sciences, Kolkata-7000373Professor, Department of Animal Nutrition, West Bengal University of Animal and Fishery Sciences, Kolkata 7000374Professor, Department of Pharmacology and Toxicology, West Bengal University of Animal and Fishery Sciences, Kolkata- 700037, Email:[email protected], [email protected]

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Vancobb) was calculated before initiating the experiment bygraphical probit analysis method described by Finney (1971).Experiment was conducted using 24, one day old healthyvaccinated broiler chicks. They were randomly divided intofour equal groups (n=6) as T1, T2, T3 and T4. Chicks in group T1was considered as control while T2, T3 and T4 were experimentalgroups. Chicks of each group were maintained up to the ageof 45 day and sacrificed on 46th day. Chicks were fed withbalanced broiler starter feed (Metabolic energy 2890 Kcal/kgand crude protein 22.1%) for first 4 weeks followed by broilerfinisher feed (Metabolic energy 2945 kcal/kg and crude protein19.10% ) for the remaining period of experimentation as perrecommendation of (Bureau of Indian standards) BIS, (1992).Fresh water and feed was supplied ad lib. From 15th to 45th

day of age chicks of group T2 and T3 were fed K2CrO4 @ 55.6mg/kg (LD50/5

th) and 92.65 mg/kg (LD50/3rd), respectively, for

consecutive 30 days. Chicks of group T4 were fed both K2CrO4@ 92.65 mg/kg and vitamin E + selenium for the same duration(15th to 45th day of age). Vitamin E and selenium powder wasorally given @ 600 mg/kg body weight and initiated one weekprior to the feeding of potassium chromate. K2CrO4 dissolvedin water was fed by feeding syringe directly into ventriculuswhile vitamin E and selenium was fed after dissolving in oliveoil at the rate of 600 mg vitamin E and selenium (Tocosel®)/0.5 ml of olive oil directly into proventriculus. Chicks of controlgroup (T1) were provided with basal diet and drinking water.

Estimation of biochemical parameterImmediately after sacrifice a part of liver and kidney tissue

collected in ice-cold normal saline for estimation of variousbiochemical parameters. Tissues were minced, washed inchilled distilled water and potassium hydroxide solution (1.15%w/v) to remove all blood clots and then blotted. 10% homogenatewas prepared by using tissue homogeniser (Remi, RQ127A)maintaining cold condition. Homogenate was centrifuged inrefrigerated centrifuge machine (4°C) at 6000 rpm for 20 minand the supernatant obtained was utilised for estimation ofreduced glutathione (Griffith, 1980), superoxide dismutase(Miesera and Fridovitch, 1972), aspartate transaminase, alanintransaminase (Yatizidis, 1960) and protein (Wooton, 1974).10% homogenate for estimation of lipid peroxidation wasprepared from liver and kidney tissue of each chick by adding0.1 ml of butylated hydroxyl toluein (BHT) in ethanol and 9.9 mlof 1 mM EDTA to 1 g of tissue. Trichloroacetic acid was addedto it, then centrifuged at 6000 rpm for 20 min. Supernatant wasused for lipid peroxidation estimation (Nair and Turner, 1984).

Histopathological studyFor histopathological study a small piece of liver and kidney

were collected from each chick and stored in 10% bufferedformalin solution. Tissues were processed by using standardparaffin embedding and stained with haematoxylin and eosin(H and E) to examine under microscope.

Tissue residue estimationA portion of liver, kidney, heart, muscle, lung and brain

were collected from each chick for chromium residueestimation. Tissue samples were digested by wet oxidationmethod using acid mixture (Stewart and Stolman, 1961).Analysis of chromium (at ë max 357.9 nm) concentration was

done by atomic absorption spectrophotometer (Perkin-Elmer-Analyst 100).

Statistical analysisMean and standard error were calculated and data were

analysed using standard methods of Snedecor and Cochran(1968). Differences at P<0.05 (at least) were considered to besignificant.

Results and DiscussionEffect of potassium chromate in different doses alone and

in combination with vitamin E and selenium on differentbiochemical parameters of liver and kidney have beenpresented in Fig. 1, 2, 3, 4 and 5. K2CrO4 decreased significantly(P<0.05) the superoxide dismutase level in liver and kidneytissue of chicks in group T2 and T3 in dose dependent mannerwhile simultaneous administration of vitamin E and seleniumin chicks of T4 significantly increased the value as comparedwith T3, supporting its protective role.

Again lipid peroxidation level in liver and kidney tissue ofchicks of group T2 and T3 were significantly increased ascompared to control. Lipid peroxidation in liver and kidney tissueof chick of T3 was significantly higher than chicks of T2 whilesimultaneous administration of vitamin E and selenium ingroup T4 brought the value up to normal.

AST level in liver and kidney tissue of chicks of group of T3was significantly decreased (P<0.05) as compared to controlwhile no significant change was observed in chicks of groupT4. Likewise, ALT level in liver and kidney of chicks of group T3were significantly decreased while the same was brought tocontrol value in chicks of group T4. So, AST and ALT level aredecreased only at higher dose.

No histopathological changes were observed in sectionof liver and kidney tissues of chicks of group T2 while mildcongestion, inflammation around portal tract and coagulativenecrosis in liver and congestion and focal haemorrhage inkidney tissue section of chicks of group T3 was observed (Fig.6). Section of liver and kidney tissue of chicks treated withvitamin E and selenium and K2CrO4 showed nohistopathological changes similar to chicks in group T1 (Fig.6). This signifies that K2CrO4 at higher dose producedhistopathological changes.

Clearly from perusal of Table 1, residual chromiumconcentration in different tissue like liver, kidney, heart, brain,lung, muscle indicates increase in dose dependent manneras compared with control. However, increase in chromiumconcentration at both dose levels in muscle tissue is found tobe significant but it is very less as compared to control andpractically appears to be no different from control.

Result of biochemical parameters implies that K2CrO4caused decrease of SOD, reduced glutathione, AST and ALTand increase of lipid peroxidation level in dose dependentmanner while simultaneous administration of vitamin E andselenium prevented the abnormal enzymatic and oxidativechanges. Mammalian cells have evolved numerous protectivemechanisms to minimise the extrinsic and intrinsic toxicchemicals generated from external environment or duringoxidative process of cellular metabolism. SOD and GSH arethe chief component of body’s cellular defence strategiesagainst oxidative damage by reactive oxygen species (ROS).

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Superoxide anion are formed normally in the body duringseveral metabolic pathway such as cytosolic oxidation(xanthine oxidase, aldehyde dehydrogenase, etc.), inelectron transport chain during cellular respiration andothers. Whenever superoxide anion is formed in tissue,it will lead to formation of other free radicals like hydrogenperoxide (H2O2), hydroxyl radical (OH.), hydroperoxy radical(HOO-). All of these radicals are very reactive and toxic tob io logical membrane (Chatterj ea and Sh inde,2000).Super oxide radical reacts with nitric oxide to formperoxynitrite, which decompose to form highly reactiveHO . radical. It can initiate chain reaction leading tooxidation o f polyunsaturated fatty ac id (PUFA) inmembrane. SOD converts superoxide anion into hydrogenperoxide while glutathione peroxidase converts H2O2 intoH2O by oxidising two molecule of reduced glutathione(Chatterjea and Shinde, 2000). Inhibition of SOD andreduced glutathione level in this experiment causedincreased ROS inside the body which initiated increasedlipid peroxidation in chicks of T 2 and T3 resulting inoxidat ive stress. Consequently at higher dose (T 3) ,decreased AST and ALT level significantly in liver andkidney tissue indicates potential damage. Liu and Shi(2001) have demonstrated that Cr (VI) is reduced into Cr(V) in the body and Cr (V) can mediate the generation offree radical. Reactive intermediates and free radicalsgenerated during reduction process may be the rootcause for Cr (VI) toxicity (Bosgelmez et al., 2008). Thereduction of Cr (VI) to Cr (III) results in the formation ofreactive intermediates leading to oxidative tissue damageand cellular injury (Patlolla et al., 2009). Shakin-ketebaumet al. (1991) observed that Cr (VI) inhibited SOD enzymeand degree of inhibition was directly proportional tochromium concentration, reaction time and to substrateconcentration. On the same line, Paurhmad O’Brien(2001) reported that addition of chromium dichromate toisolated rat hepatocyte resulted in rapid glutathioneoxidation , react ive oxygen spec ies fo rmation , lipidperoxidation and lysosomal membrane rupture beforelysis of hepatocyte. Susa et al. (1997) also studiedincubation of primary culture of rat hepatocyte withpotassium d ichromate caused inc reased lipidperoxidation inhibition of antioxidant enzyme like SOD,

Glutathione reductase and level of vitamin E.ROS are highly reactive which can bring about

lipid peroxidation producing lipid peroxide and lipoxide.These radicals constitute a threat to bio membrane whoseoxidation can be prognosed into cell death. Vitamin Eappears to be the 1st line of defence against peroxidationof polyunsaturated fatty acid contained in cellular andsubcellular membrane phospholipid required for cellmembrane integrity. Hence simultaneous administrationof vitamin E and selenium along with K2CrO4 thwartedlipid peroxidation, GSH, AST and ALT to abnormal levelwhich is in agreement with our histopathological findings.In vitro study done by Susa et al. (1996) reportedlydescribed the effect of pre-treatment of á-tocopherolsuccinate (vitamin E) on chromium [Cr(VI)] inducedcytotoxicity. They reported marked decrease of lactatedehydrogenase leakage. Pre-treatment of vitamin E,reduced lipid peroxidation and also normalised the levelo f non-enzymat ic ant i-oxidant such as glutathionesuppressed by potassium chromate. Present study alsocorroborates with the finding of Susa et al. (1997). Asevident from Table 1, there is significant increase inresidual chromium concentration in liver, kidney, heart,brain, lung and muscle tissue of chicks of all experimentalgroups i.e. T2, T3 and T4. No significant difference inchromium concentration was observed in liver and kidneytissue of chicks of group T4 treated with K2CrO4 as well asvitamin E and selenium compared to group T3 treatedwith K2CrO4 alone. However, biochemical result showedthat administration of vitamin E and selenium along withK2CrO4 to chicks of group T4, brought different biochemicalparameter to normal value in liver and kidney comparedto group T 2 and T 3 that was supported by nohistopathological changes in section of liver and kidneytissue of group T4. Sugiyama et al. (1991) also showedthat incubation of cell (Chinese hamster V-79 cell) withsodium chromate (Na2CrO4) resulted in cytotoxicity andpre-treatment with vitamin E reduced such toxicity withoutinterfering uptake of chromate by the cell. On the sameline, Susa et al. (1996) also demonstrated that pre-treatment of primary culture of rat hepatocyte with á-Tocopherol succinate (vitamin E) for 20 hr prior to exposureto K2CrO4 resulted in marked decrease in Cr (VI) induced

Table 1: Mean ± SE chromium residue (ppm) in different organ tissues after daily oral administration of K2CrO4in different treated groups.

Organ Tissue T 1 (n=6) T 2 (n=6) T3 (n=6) T 4 (n=6)

Liver 0.61a0.01 1.40b0.04 1.79c0.03 1.90c0.05

Kidney 0.24a0.007 0.78b0.02 1.27c0.08 1.27c0.05

Bra in 0.230.008 0.57b0.01 0.86c0.02 0.88c0.02

Heart 0.48a0.05 0.57b0.04 0.79c0.02 0.74C0.01

Lung 0.63a0.01 1.08b0.06 1.9 c0.05 1.94c0.04

Muscle 0.11a0.004 0.13b0.005 0.17c0.004 0.20d0.002

Values with different superscripts differ signif icantly

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Fig. 6: Section of Liver showing normal structure (A) in group T1, congestion (C) and focal necrosis (D) in group T3 and section of kidney showingnormal structure (B) in group T1, congestion (E) and focal haemorrhage (F) in group T3 broiler chicks (H & E 100X). Lesions are indicated by arrowmarks.

A B

C D

E F

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Fig.1: Effect of vitamin E and selenium in K2CrO4 induced sub-acute toxicity on superoxide dismutase activity (Units/mg protein ± S.E). A. Liver andB. kidney tissue of broiler chicks

A. Liver B. kidney

Fig. 2: Effect of vitamin E and selenium in K2CrO4 induced sub-acute toxicity on reduced glutathione (n mole of reduced glutathione/mg of wettissue± S.E). A. Liver and B. kidney tissue of broiler chicks

A. Liver B. kidney

Fig. 3: Effect of vitamin E and selenium in K2CrO4 induced sub-acute toxicity on lipid peroxidation (n mole of malonaldehyde/gm of wet tissue). A.Liver and B. kidney tissues of broiler chicks

A. Liver B. kidney

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A. Liver B. kidney

cytotoxicity without affecting cellular uptake and sub-cellulardistribution. Therefore, it may be concluded that vitamin E andselenium had neutralised the damaging effect of K2CrO4 inliver and kidney tissue in spite of high residual metaldeposition.

LD50 of K2CrO4 in broiler chick was calculated as 277.95mg/kg using probit analysis. Although toxic effect on biochemicalparameters i.e. oxidative stress were found in both doses i.e.,55.6 mg/kg b.wt. (LD50/5th) and 92.65 (LD50/3rd); however,histopathological changes were promiscuous at higher dose.

Thus, it may be concluded K2CrO4 (Cr VI) producedoxidative stress and histopathological changes in dosedependent manner and vitamin E and selenium can bepotentially used as a therapy in animals reared in chromiumtoxicity prone areas.

ReferencesBarceloux, D.G. (1999) J. Toxicol. Clin. Toxicol. 37: 173-94.Baruthio, F. (1992) Biol. Trace Elem. Res. 32: 145-53.BIS (1992) Bureau of Indian Standards. Specifications for Poultry

Feeds - Revised: 95:1374-1992. B.I.S., New Delhi, India.Bogelmez II. et al. (2008) Biol. Trace Elem. Res. 125: 46-58.Chatterjea, M.N and Shinde, R. (2000) Textbook of Medical

Biochemistry. 4th ed. pp. 184-187.Choct, M and Naylor, A.J. (2004) Asian-Aust. J. Anim. Sci. 17:1000-

1006.

Ducros (1992) Biol. Trace Elem. Res. 32: 65-77.Finney, D.J. (1971) Probit Analysis. 3rd ed. Cambridge University Press,

Cambridge, UK.Griffith, O.W (1980) Anal Biochem. 106: 207.Liu, K.J. and Shi, X. (2001) Mol. Cell Biochem. 222: 41-47.Miesera, H.P and Fridovich, I (1972) J Biol Chem. 247: 3170-3185.Nair, V and Turner, G.A (1984) Lipids. 19: 804.Patlolla, A.K. et al. (2009) Environ Toxicol. 24: 66-73.Pourahmad, J. and O’Brien, P.J. (2001) Adv Exp Med Biol. 500: 203-

207.Shainkin-Kestenbaum, R. et al. (1991) Trace Elem Electrolytes Health

Dis. 5: 197-201.Snedecor, G.W. and Cochran, W.G. (1989) Statistical Methods, 8th

ed. Iowa State University Press.Soudani, N. et al. (2011) Exp. Toxic. Path. 63: 541-548.Stewart, C.P. and Stolman, A. (1981) Toxicology-Mechanisms and

Analytical Method. Vol. II. Academic Press, New York andLondon. pp. 648-654.

Sugiyama, M. (1991) Environ Health Perspect. 92: 63-70.Susa, N. et al. (1996) Arch Toxicol. 71: 20-24.Susa, N. et al. (1997) Arch Toxicol. 71: 345-350.Wooton, I.D.P (1974) Estimation of protein by biuret method. In:

Microanalysis in medical biochemistry. 5th ed. ChurchillLivingstone, Edinburgh and London, pp. 156-158.

Xu, X.R et al. (2004) Chemosphere. 57:609-613.Yatizidis, H. (1960) Nature. London 18: 79-80.

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Umbilical hernia is one of the major surgical affectionsin animals. It occurs in all domestic animals, but is morecommon in foals, calves and pups (Priester et al., 1970).Umbilical hernias are considered hereditory in cattle andmost commonly occur in the Holstein-Friesian (HF) breed(Zhigachev, 1983; Fubini and Ducharme, 2004). Umbilicalhernia is a developmental anomaly based on chromosomalabnormalities (Baird, 1993). Umbilical hernia result due toimproper closure of the umbilicus at birth due tomaldevelopment or hypoplasia of the abdominal muscles(Singh et al., 1989). An umbilical hernia is the protrusion ofabdominal content(s) through the defect in the abdominalwall with an intact lining of the sac (Fubini and Ducharme,2004; Engelsman et al., 2007). The only effective treatmentis surgery to restore integrity of the abdominal wall andprevent incarceration and strangulation of herniatedabdominal contents. Tense suture application toapproximate and close the defect led to wound dehiscenceand recurrent hernia. The use of mesh allows a tensionfree repair of abdominal hernias. The prosthetic materialssuch as polypropylene mesh leads to complications likemesh extrusion, fistula formation, wound infection, seromaand adhesions (Molloy et al., 1991). The syntheticdegradable materials have not provided sufficient strengthduring the degradation process (Tyrell et al., 1989). Gulatiand Cole (1994) observed less immunogenicity and bettertolerance of acellular grafts in rats and rabbits. Acellulardermal grafts have been used successfully for thereconstruction of abdominal wall defects in rats (Chung etal., 2003) and rabbits (Gangwar et al., 2006). Kumar et al.(2004) used glutaraldehyde preserved diaphragm of porcineorigin for the repair of large umbilical hernia in dog withexcellent recovery. Carbon fibres and carbon fibres meshhas been used for the repair of umbilical hernias in bovines(Gangwar et al., 2008, a and b). Protocols for makingacellular aortic matrices have been developed in ourBiomaterial and Bioengineering Laboratory. In-vitro and in-vivo biocompatibility evaluation of these matrices revealsthat these matrices are biocompatible (Devarathnum, 2010).In present study acellular aortic grafts of Buffalo (Bubalusbubalis ) origin have been used for reconstruction ofumbilical hernia in four HF calves.

Materials and MethodsPresent study was conducted in Biomaterial and

Bioengineering Laboratory, Division of Surgery and in ReferalVeterinary Polyclinic, Indian Veterinary Research Institute,Izatnagar-243122, UP, India. Four HF calves with an averageage of 7 months and average weight of 60 kg were presentedwith the history of swelling at the umbilical region (Fig.1).

ACELLULAR AORTIC GRAFTS FOR THE RECONSTRUCTION OFUMBILICAL HERNIA IN HOLSTEIN-FRIESIAN CALVES

J. Devarathnum, A.K. Sharma, A. K. Gangwar, Vineet Kumar, Naveen Kumar,Himani Singh, D.T. Kaarthick, A. M. Pawde and S. K. Maiti

Division of SurgeryIndian Veterinary Research Institute, Izatnagar-243122, Bareilly, Uttar PradeshI

IntroductoinOn physical examination average hernial ring diameter of 8cm was observed and all these cases were diagnosed ascongenital umbilical hernia.

Preparation of acellular aortic graftFresh posterior aorta of buffalo origin was collected

from local abattoir and immediately preserved in ice-coldsterile phosphate buffered saline solution (pH 7.4)containing 0.1% amikacin and 0.02% EDTA. The tissue wasrinsed with normal saline before the start of protocol. Themaximum time period between retrieval and the initiation ofprotocol was less than 4 h. The tissue was cut into desiredsizes and made acellular using 1% anionic biologicaldetergent for 24 h followed by 0.25% enzyme solution for 2h and then again with same 1% anionic biological detergentfor next 24 h (Devarathnum, 2010). Finally, the tissue wasthoroughly washed in phosphate buffer saline (PBS) solution.The prepared acellular aortic graft was stored in PBSsolution containing 0.1% amikacin solution at 4°C.

Surgical treatmentAll the animals were kept off fed for 24 h before surgery.

The operation was performed in dorsal recumbency. Thesurgical site was shaved and prepared for aseptic surgery.After aseptic preparation of the site, Xylazine was given tocalves @ 0.1 mg/kg body weight intramuscularly and 2%lignocaine hydrochloride was infiltrated at the site in all theanimals. After proper anaesthesia an elliptical incision wasmade over the hernial sac and fascia and muscles wereseparated from the hernial ring. All animals were havingslight to strong adhesions. The hernial contents werepushed back in cases where no adhesions or slightadhesions were present. The adhesions were removed byblunt dissection. The hernial ring was freed and repaired byacellular aortic graft using inlay technique (Fig. 2). The graftwas anchored in position by black braided silk No. 2 usinghorizontal mattress sutures in all the animals (Fig. 3). Finallythe skin was closed by black braided silk No. 2 (Fig. 4).

Post-operative treatment included intramuscularinjection of Enrofloxacin (5 mg/kg) for 7 days and Meloxicam(0.2 mg/kg) for 3 days. Daily dressing of the suture line wasperformed with 5% Povidone iodine. The owner was advisedto give the concentrate diet. The skin sutures were removedon 10-14th postoperative days as per wound healing status.To evaluate the long term results a telephonic survey wascarried out.

Results and DiscussionAll the animals recovered completely and no complication

of wound healing was observed up to 10-14th postoperativedays. Collagen is a natural substrate for cellular attachment,

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growth and differentiation in its native state. It is biocompatibleand non toxic to tissues. It is universal biological molecule thatexhibits only very slight differences from species to speciesand possesses only minimal antigenic properties (Davies,1983). Xenogenic collagen applied to injured surface of theskin did not cause any foreign body reaction nor any immunerejection reaction or sensitization (Soo et al., 1993). Theacellular matrix is produced by controlled process that removesthe epidermis and the cells from the dermis without affectingthe structure of the extracellular matrix and the basementmembrane (Wainwright, 1995). The acellular matrixpossesses the appropriate mechanical properties andinduces appropriate interaction with the host cells that resultedin the regeneration of functional tissues (Voytik-Harbin et al.,1998). Acellular dermal grafts have been used for the repair ofabdominal wall defects in experimental animals with excellentresults (Chung et al., 2003; Gangwar et al., 2006). In-vitro andin-vivo biocompatibility evaluation of acellular aortic graftsreveals that these matrices are biocompatible and can be triedin clinical cases (Devarathnum, 2010). Keeping in view thepossible advantage of acellular aortic matrix of buffalo origin, ithas been used successfully for the reconstruction of umbilicalhernia in HF calves.

ConclusionPresent study concluded that acellular aortic grafts from

buffalo origin is good alternative for the reconstruction of

umblical hernia. It is easy to prepare and can be clinicallypracticed in other species.

AcknowledgementsThe authors acknowledge the financial assistance

received from the Department of Biotechnology (DBT), Ministryof Science and Technology, New Delhi, India to carry out thiswork.

ReferencesBaird, A. N. (1993) J.A.V.M.A. 202:1480-1482.Chung, S. et al. (2003) Plast. Reconstr. Surg. 111: 225-232.Davies, J.W.L. (1983) Burns. 10: 94-103.Devarathnum, J. (2010) M.V.Sc. Thesis submitted to Deemed

University, IVRI, Izatnagar.Engelsman, A.F. et al. (2007) Biomaterials. 28: 2314-2327.Fubini, S. L. and Ducharme, N. G. (2004) Farm Animal Surgery. Saunders,

St. Louis, Missouri, USA, pp. 477-484.Gangwar, A. K. et al. (2006) J. South Afr. Vet. Assoc. 77: 79-85.Gangwar, A. K. et al. (2008a) Indian Vet. J. 85: 430-431.Gangwar, A. K. et al. (2008b) Indian Vet J. 85: 658-659.Gulati, A. K. and Cole, G. P. (1994) Acta Neurochir. Wien. 126: 158-

164.Molloy, R. G. et al. (1991) Br. J. Surg. 78: 242-244.Priester, W.A. et al. (1970) Am. J. Vet. Res. 31:1871-1879.Singh, A. P. et al. (1989) Indian J. Vet. Surg. 10:28-31Soo, C. et al. (1993) J. Dermatol. Surg. Oncol. 19: 431-434.Tyrell, J. et al. (1989) Surg. Gynecol. Obstet. 168: 227-232.Voytik-Harbin, S. L. et al. (1998) Tissue Engineering, 4: 157-174.Wainwright, D. J. (1995) Burns. 21:243-248.Zhigachev, A. I. (1983) Genitica-USSR. 19:312-315.

Fig.3: Anchoring of acellular aortic graft.

Fig.2: Anchoring of acellular aortic graft by inlay technique.Fig.1: Umbilical hernia in HF calf.

Fig.4: Umbilical hernia after repair.

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Red bag delivery is a term used for prematureseparation of the placenta prior to or during a mare’s foaling(Carluccio et al., 2008) leading to appearance of red velvetybag at vulva. Sometimes, the placenta does not rupture andthe foal is delivered while still enclosed in the membrane.The present report puts on record a case of red bag deliveryin a mare associated with dystocia.

Case History and ObservationsA full term mare was presented at Veterinary Clinics,

Guru Angad Dev Veterinary and Animal Sciences University,Ludhiana. The animal was in 3rd parity with incompletegestation period by 20 days. The owner had observed a red“velvety” sack at vulva without any delivery of foal even afterstraining by the mare for 6-8 hours. The size of red bag wasincreasing and decreasing with the straining of the animal.Per-vaginal examination revealed “velvety” sack as a part ofchorioallantois. Deep palpation revealed the presence of afoetus within the bag with its head obstructed at pelvic brim.The case was diagnosed to be red bag delivery associatedwith downward deviation of head.

Treatment and DiscussionThe exposed part of chorioallantois was incised. Within

the chorioallanaotis, the intact amnion was revealed. Theamnion being thicker had to be incised to assess the foal. Adead male foetus was delivered after correcting the slightdownward deviation of head. Placenta was shed within twohours of extraction of the foetus. Thereafter the uterus wasdouched with weak Betadine® solution. Post-obstetricaltreatment involved parenteral administration of antibiotics,intravenous fluids and tetanus toxoid. The mare exhibitednormal foal heat after 10 days of the delivery.

A red bag delivery had been found in mare occasionally(Samuel et al., 1976) and is a common complication ofinduced parturitions (Frazer, 2007). In mares, in cases ofnormal delivery, chorioallantois ruptures within the genitaliaand amnios appears as the first water bag at vulva. Thenormal chorioallantois is relatively thin and breaks easily

A RARE CASE OF RED BAG DELIVERY IN A MARE

P. S. Brar1, A. K. Singh2 and S. Prabhakar3

Department of Veterinary Gynaecology and ObstetricsCollege of Veterinary Science

Guru Angad Dev Veterinary and Animal Sciences UniversityLudhiana-141 004, Punjab, India

during birth. However, some placentas are thickened frominfection or inflammation which cannot be ruptured by thefoetus. Hence chorioallantois appears at the vulva beforeamnios and results in red bag deliveries. Exposure of latepregnant mares to fescue grass can also result in athickened placenta at foaling (Schmidt, 2009).

In the present case, foetus died though the mare couldbe saved. The premature separation of chorioallatoiscauses deprivation of oxygen and nutrients to the foetusleading to death of the foetus. Therefore, such cases shouldbe attended as early as possible to save the life of the foetus.

ReferencesCarluccio, A. et al. (2008) Theriogenology. 69: 918.Frazer, G. S. (2007) Dystocia and Fetotomy. In: Current therapy in

equine Reproduction (Ed) Samper Jc, Pycock, J.F. andMcKinnon, A. O. Saunders Elsevier, USA.

Samuel, C. A. et al. (1976) J. Reprod. Fertil. 48: 257.Schmidt, A. (2009) http://www.horsechannel.com/horse-health/red-

bag-delivery.aspx.

Fig. 1: Red bag hanging out from vulva in mare

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IntroductionObstruction in free passage of cerebrospinal fluid

into the arachnoid space leads to excessive swelling ofcranial cavity during foetal development and the conditionis termed as hydrocephalus (Salunkhe et al., 2001).Considerable variation in the morphology of the conditionis evident, but many reports describe the condition simplyas internal hydrocephalus (Axthelm et al., 1981 and Buck etal., 2009). The incidence of bovine hydrocephalus has beenreported at 1.5 cases/1000 calvings, however, many casesof bovine hydrocephalus go undetected or are incorrectlyreported as abortions or drowning (Priester et al., 1970).Some affected foetuses are stillborn and many are bornprematurely (Leech et al., 1978). Calves with pronouncedcranial enlargement usually die within 48h, but less severelyaffected calves may survive for several weeks or longer(Leech et al., 1978). Occurrence of dystocia due to foetalhydrocephalus is rare in buffaloes (Kumaresan et al., 2003),although this was observed virtually in all breeds of cattle(Greene et al., 1974; Christoferson et al., 1977; Purohit etal., 2006). Management of foetal hydrocephaly diagnosedafter the start of calving process is difficult and per-vaginaldelivery is usually difficult. The present document discussesmanagement and treatment of a hydrocephalic foetus forits successful per-vaginum delivery.

Case history and observationsA Murrah buffalo in third parity with complete gestation

period was presented to University veterinary clinics. Buffalowas straining since 24h and water bags had ruptured beforethe case was presented to clinics. Immediately, variousclinical parameters were ascertained viz. rectal temperaturewas 102°F, mucous membranes were pink, prescapularlymph node size was normal and respiratory rate wasdepressed. Per-vaginal examination revealed soft andrelaxed cervix. The presentation of foetus could not be ruledout as only football-shaped foetal part with some fluid thrillwas palpable in birth passage. During examination, foetalhydrocephalus condition was not recognizable. No foetalextremities were palpable in the birth passage. Absence ofsuckling and eye ball reflex indicated the foetus is not viable.

Treatment and D iscussionFollowing epidural anaesthesia (8 ml, 2% lignocaine

hydrochloride), a stab incision was given on the football-shaped foetal mass and large amount of fluid was drained

PER-VAGINAL DELIVERY OF A MURRAH BUFFALO CALF WITHCONGENITAL HIGH-GRADE HYDROCEPHALUS INTERNUS

R. Upasana, G.R. Bhat, E.E. Varughese, S.P.S. Ghuman1 and M. HonparkheDepartment of Veterinary Gynaecology and Obstetrics

College of Veterinary ScienceGuru Angad Dev Veterinary and Animal Sciences University

Ludhiana-141004, Punjab, India

out. This rendered sufficient space to recognize the foetalhead. After ample lubrication with carboxymethyl cellulosesodium gel (SD Fine Chemicals Limited, Ludhiana), tractionwas applied on the foetal head by holding it with both thehands. Application of continuous pressure facilitated incompressing the head. Thereafter, foetal head was brought,although with some amount of difficulty, in the birth passagealong with both forelimbs and traction was applied tosuccessfully deliver hydrocephalic male calf. The completeplacenta was removed immediately. Next day, the buffalowas discharged with the routine prescription of antibioticsand supportive therapy. Follow up of case revealeduneventful recovery.

Thorough gross examination of the markedly enlargedcalf head and highly deformed cranium revealed thecondition as high-grade hydrocephalus internus (Fig. 1 andFig. 2). The thinning of frontal, parietal and temporal skullbones was noticeable and appeared as loosely organizedbony plates that fell apart when the cavity was opened.Pathological-anatomical assessment revealed thatcerebrum was replaced by the ventricle system that mighthave filled with 1.5 litres of cerebrospinal fluid. The cerebellarhemispheres were ruptured by the dilated fourth ventricle.The vermis and pons were missing and fluid accumulationin the subarachnoidal space going up to the first spinalcord segment was noticeable (Fig.2). The observedmorphologic alterations closely resembled those seen inhuman hvdrocephalus, where simple aqueductal stenosisaccounts for most cases (Leech et al., 1978). Severalhydrocephalus syndromes described in Hereford andShorthorn breed cattle features include cerebellarhypoplasia, microphthalmia, myopathy, and retinal dysplasia(Blackwell et al., 1959).

Etiology of the present case of hydrocephalic foetuswas not related to history of inbreeding. No other affectedcalves from the same parents were known at the farm.Infections and parasitic diseases could be ruled out in thepresent case. Very rare defect alleles might have beeninvolved in the development of this inborn defect asgenetically determined bovine hydrocephalus has beendescribed earlier (Baker et al., 1961 and Blackwell et al.,1959). An autosomal recessive gene is consideredresponsible for many hereditary cases, but intrauterineinfections and nutritional factors have not been fullyevaluated except for the relationship of blue tongue virus to

Reprint requests are solicited at this address (Dr S.P.S.Ghuman, Associate Professor, Department of Veterinary Gynaecology and Obstetrics,Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141004, Punjab, India. Email: [email protected]

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hydranencephaly (Leech et al., 1978). In addition, Neosporacaninum-associated hydrocephalus was observed in anaborted Hereford bovine foetus of 7 months gestational age(Dubey et al., 1998).

ReferencesAxthelm, M.K. et al. (1981) Vet. Med. Small Ani. Clin. 76: 567-570.Baker, M.L. et al. (1961) J. Hered. 32: 135-138.Blackwell, R.L. et al. (1959) J. Hered. 50: 143-148.

Buck, B.C. et al. (2009) Dtsch. Tierarztl. Wochenschr. 116: 220-226.Christoferson, L.A. et al. (1977) Surg. Neurol. 7: 165-170.Dubey, J.P. et al. (1998) J. Comp. Pathol. 118: 169-173.Greene, H.J. et al. (1974) Cornell Vet. 64: 596-616.Kumaresan, A. et al. (2003) Indian J. Ani. Reprod. 24: 82.Leech, R.W. et al. (1978) Am. J. Pathol. 92: 567-570.Priester, W.V.A. et al. (1970) Am. J. Vet. Res. 31: 1871-1879.Purohit, G. N. et al. (2006) Indian J. Ani. Reprod. 27: 98-99.Salunke, S.P. et al. (2001) Indian J. Anim. Reprod. 22: 96.

Fig. 1: High-grade hydrocephalus in buffalo calf. Fig. 2: Cranial cavity showing high-grade hydrocephalus internus, non-recognizable brain tissue and thinning of skull bones.

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IntroductionIngestion of highly fermentable carbohydrate feed in

excessive quantities or decrease amount of well structureroughages and gradually increase in concentrate in ration fora prolonged period in ruminants leads to decrease time ofrumination, reduction in amount of saliva, decline in ruminalpH to such degree that change in microbial flora in rumen andfavour the multiplication of amylolytic bacteria which degradestarch and sugar causing rapid fermentation and productionof large quantity of lactic acid and long chained volatile fattyacids (VFA). This turned normal buffering capacity of rumenresulting ruminal acidosis, ruminal hypotonocity and/or rumenstasis, circulatory impairment, decrease renal blood flow andglomerular filtration rate, dehydration, hypovolumic shock anddeath. (Anderson, 1992; Wendy, 1992).The case fatality ratemay be up to 90 per cent in untreated cases where as intherapeutically managed cases it may exceed to 30 – 40 percent (Radostits et al., 2007).

Although this disease has been extensively studied for itsclinico-biochemical aspects in bovine (Mishra et al., 1972;Mishra and Singh, 1974; Randhawa et al., 1989; Dwivedi,2000), sheep (Hoflund et al., 1948) and experimental goats(Rai and Pandey, 1980; Tanwar and Mathur, 1983; Lai et al.,1989; Basak et al., 1993; Hajikolaei et al., 2006), but reports inclinical cases of ruminal acidosis in goats is meagre. Keepingin view the above facts, the present investigation wasundertaken to study the changes in ruminal activity (ruminalmotility, consistency and pH of ruminal fluid, motility and activityof rumen micro-flora) of goats suffering from different degreesof ruminal acidosis (mild, moderate and severe), which mightbe of immense help in quick and proper diagnosis andtreatment of this disease.

Materials and MethodsThe present study was carried out on twenty goats of Sirohi

breed aged between six months to two years of either sexbrought to the TVCC, Apollo College of Veterinary Medicine,Jaipur.

DIETARY INDUCED METABOLIC ACIDOSIS IN GOATS AND ITSSUCCESSFUL THERAPEUTIC MANAGEMENT

S.R. Gupta, R. Yadav, C.S. Sharma, and A. Gattani1Department of Veterinary Medicine

Apollo College of Veterinary Medicine, Jamdoli, Agra Road, Jaipur, Rajasthan, India

ABSTRACT

The present study is carried out on 10 goats of Sirohi breed which were diagnosed as dietary induced metabolic acidosis. Inpresent study, ruminal pH, ruminal motility, odour, colour, consistency of rumen liquor, ruminal microflora and protozoal motilitydiffered significantly from apparently healthy groups of goats. In haematological parameters, a significant increase observed inhaemoglobin, packed cell volume and total leukocyte counts and biochemistry showed significant increase in blood lactate level,total protein, AST, ALT and serum sodium level while serum potassium level were significantly decreased.

Key words: Goats, metabolic acidosis, ruminal liquor, haemato-biochemical parameters

These goats having history of accidental ingestion of wheatgrain in large quantities or were offered wheat flour prepared“Chapaties” by owner and subsequently the goats developedclinical signs comprising of anorexia, tympany, constipationor pasty diarrhoea, grinding of teeth and regurgitation of ruminalcontents. On detailed clinical examination revealed reducedmotility to atony of rumen, suspended rumination, subnormalto normal rectal temperature (99° to 103.8° F), increase pulserate (70-90/min.) and laboured breathing.

Rumen liquor was collected aseptically directly fromrumen using 16 G needle with syringe for assessment ofphysical characteristics of rumen liquor. pH of rumen liquorwas recorded by portable pH meter. Ruminal protozoa activitywas examine in a drop of rumen liquor under microscope andrumen liquor smear were stain with Gram’s stain whichshowed G-ve micro flora overgrown by G+ve microflora. Basedon these clinico-rumenological indices goats were diagnosedas case of rumen lactic acidosis. In addition to these, 10apparently healthy goats of same breed from an organizedfarm were taken as control group for comparative study.

Blood samples were collected from affected goat forestimation of altered haematological indices viz. haemoglobin(Hb), packed cell volume (PCV), total erythrocyte count (TEC),total leucocyte count (TLC), using standard technique of Jain(1986). Serum was separated for estimation of total protein,ALT and AST using Span diagnostic kits, serum sodium andpotassium concentration were estimated using Flamephotometry (Oser, 1986), and blood lactic acid estimation byIDEXX wet test automated chemistry analyzer. Blood sampleswere also collected from 10 control group of goats once forestimation of same parameters. Data were analyzed usingANOVA “t” test (Sendecor and Cochran, 1994).

TreatmentAll the affected goats were therapeutically managed with

sodium bicarbonate (7.5% w/v) solution @ 2 ml/kg body weight,intravenously, ringer’s lactate solution intravenously @ 25 ml/

1Assistant Professor, Department of Veterinary Biochemistry

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kg body weight, Pheneramine maleate @ 0.5 mg/kg bodyweight, intramuscularly, Oxytetracycline hydrochloride @ 10 mg/kg body weight in divided doses intraruminally andintravenously, and powder Bufzone 50 g once a day orally untilrecovery.

Results and DiscussionIn the present study accidental ingestion of large amount

of wheat grain or Chapaties was responsible for thedevelopment of ruminal acidosis in goats. The reduction ofruminal pH is the cause of most of systemic and clinicalchanges which are present in present study. The clinicalexamination of affected goats revealed increase pulse rate,respiration rate and subnormal to normal rectal temperature,bloat, constipation or yellow pasty diarrhoea, nasal discharge,shrunken eyes, dehydration, sluggish rumen motility andrumen seems to be full and doughy in all diseased goatswhich are accordance with Anderson (1992); Wendy (1992);Basak (1993) and Hajikolaei et al. (2006).

These clinical signs were gradually disappeared aftertreatment. Administration of sodium bicarbonate lessened theeffect of metabolic acidosis, antihistaminic drug Phenareminemaleate has reduced the rumen and blood histamine level,Ringer’s lactate solution have maintain the sodium, potassiumand chloride level of diseased goats. Oxytetracylinehydrochloride prevents the fermentation and powder Bufzonea buffering oral drug containing ideal ruminal buffer, metabolicbooster and yeast for the management of rumen acidosis(Selvaraj, 2007).

The present observations in respect of ruminal motility,

odour, colour, consistency of rumen liquor, protozoal motilityand ruminal microflora were significantly different fromapparently healthy group of goats (Table 1) which are inagreement with the observation of Prasad et al. (1976);Radostits et al.( 2007). Ruminal pH of diseased goats wassignificantly lower than apparently healthy group of goats whichmight be due to excess accumulation of lactic acid in rumen(Smith, 2002; Hajikolaei et al., 2006; Radostits et al., 2007;Ceroni et al., 2012). Acidic rumem pH not only adversely affectsrumen motility but also rumen microbes and promotes theformation of toxic amines (Radostits et al., 2007). Rumen liquorsmear of acidotic goats revealed preponderance of grampositive flora as also reported by Dirkson (1970) in cow. Amonghaematological parameters there was a significant increasein Hb, PCV and TLC values as compared to control group ofgoats (Table 2) which could be attributed to haemocencentrationdue to loss of intra vascular water in rumen (Lal et al., 1992;Meglia et al., 2001; Morgante et al., 2004; Gozho et al., 2007;Morgante et al., 2007 and Ceroni et al., 2012).

A significant increase in ALT and AST might be due tohepatocellular damage as a result of toxic products like alcohol,histamine, thiaminase and other endotoxins produced inrumen epithelium and entering the portal circulation (Radostitset al., 2007). A significant increase in total protein, ALT and ASTwas also reported by Cakala et al. (1974) and Patra and Swarup(1996) in acidotic sheep. Significant increase in serum sodiumlevel and decrease in serum potassium level might be due toretention of sodium and excess excretion of potassium by thekidney which were agreement with Tremblay et al., 1991 andNilolob (1998). The excess accumulation of lactic acid in rumen

Table 1: Physical characteristics of rumen liquor of healthy and affected goats

Parameters Healthy Goats Diseased Goats Ruminal motility (per 5 min.) 7.5±0.72 (7-10) 2.30±0.72 (Nil-4) Ruminal pH 6.9 ±0.24 (6.4-7.2) 4.6 ± 0.21 (4.0-5.4) Colour Greenish Milky gray Odour Aromatic Sweetish sour Consistency Viscous More Watery Protozoal motolity +++ +/0 Microflora Predominant G -ve Predominant G +ve

Table 2: Haemato-biochemical findings in apparently healthy group and affected goats

Parameters Healthy Goats Diseased Goats Hb (gm/dl) 12.25± 0.15 16.43± 0.17 PCV (per cent) 35.79± 0.07 53.78 ± 1.5 TEC (Millions/cumm) 9.86 ± 0.30 10.25± 0.35 TLC (Thousands/cumm) 9.15 ± 0.66 17.65± 1.70 Total Protein (gm/dl) 6.35 ± 0.90 14.34± 2.25 AST 52.85± 7.96 103.89± 4.95 ALT 27.83± 1.42 59.87± 4.97 Serum Sodium (mEq/L) 127.78± 8.2 148.07± 5.4 Serum Patassium (mEq/L) 4.25 ± 3.3 3.17 ± 1.07 Blood lactic acid (mg/dl) 12.35 ± 6.3 25.30 ± 4.5

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subsequently absorbed into blood might be the reason forincrease blood lactic acid level Patra et al., 1996 and Hajikolaeiet al., 2006).

AcknowledgementsAuthors are grateful to the Dean, Apollo College of

Veterinary Medicine, Jaipur for providing the requisite facilities.

ReferencesAnderson, N.V. (1992) Veterinary Gastroenterology. 2nd ed.

Philadelphia, Lea and Febiger, London, pp. 712-722.Basak, D.M. (1993) Ind. Vet. J. 70: 662-663.Basak, D.N. et al. (1993) Ind. J. Ani. Sci. 63: 263-267.Cakala, S. et al. (1974) Pol. Arch.Weter. 17:117-130.Ceroni, V. et al. (2012) J. Ani. and Vet. Advances. 11(7): 927-930.Dirkson, G. (1970) In Proceedings, 3rd International Symposium on the

Physiology of Digestion and Metabolism in ruminants.Phillipson, A.I. (Ed.) Otiel Press. pp. 612.

Dwivedi, S.K. et al. (2000) Veterinarian. 24: 22-25.Gozho, G.N. et al. (2007) J. Dairy Sci. 90: 856-866.Hajikolaei, M.R.H. et al. (2006) Pak. J. Biol. Sci. 9(10): 2003-2005.Hoflund, S. et al. (1948) Vet. Sci. Ani. Hush. 23: 395-409.Jain, N.C. (1986) Schalm’s Veterinary Haematology. 4th ed. Lea and

Febiger, Philadelphia.Lai, S.B. et al. (1989) Ind. J. Vet. Med. 9: 81-85.

Lal, S.B. et al. (1992) Ind. J. Ani. Sci. 63: 952-956.Meglia, G.E. et al. (2001) Acta. Vet. Scand. 42: 139-150.Mishra, S.K. and Singh, U. (1974) Ind. Vet. J. 51: 698-704.Mishra, S.K. et al. (1972) Ind. Vet. J. 49: 463-470.Morgante, M.C et al. (2004) Subclinical rumen acidosis in Italian

dairy herds. Proceeding of 23rd World Buiatrics Congress,July11-16, 2004, Canada.

Morgante, M.C et al. (2007) J. Ani. Physiol. Ani. Nutr. 91:226-234.Nikolob, Y. (1998) Veterinarski Archiv. 68(1):1-9.Oser, B.L. (1986) In: Hawk’s Physiological Chemistry. 14th ed. Tata

Mc Grawhill Publishing Co. Ltd. New Delhi.Patra, R.C. et al. (1996) Small Rum. Res. 19(2): 177-180Prasad, J. et al. (1976) Ind. Vet. J. 53: 297-299.Radostits, et al. (2007) Veterinary Medicine. 10th ed. Sounders

Elsevier Edinburgh, London.Rai, G.S. and Pandey, M.D. (1980) Ind. Vet. J. 57: 120-125.Randhawa, S.S. et al. (1989) Ind. J. Vet. Med. 9: 1-7.Selvaral, S.R. et al. (2007) Intas Polivet. 8: 443-446.Sendocor, G.W. and Cochran, W.G. (1994) Statistical Methods. 8th ed.

Iowa University Press, USA.Smith, B.P. (2002) Large Animal Internal Medicine. 3rd ed., C.V. Missouri.

pp. 747-764, 782-791.Tanwar, R.K. and Mathur, P.D. (1983) Ind. Vet. J. 60: 499-500.Tremblay, Robert, R.M. et al. (1991) Can. Vet. J. 32: 308-310.Wendy, J.U. (1992) Compend. Contin. Edu. 14: 1265-1270.

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IntroductionThe oesophageal obstruction in buffaloes is reported to

be lesser than cattle. Choke occurs in ruminants due to theattempts to swallow of a whole fruit like turnips, apples, clothsor rexin material (Shivaprakash et al., 1998 and Shivaprakash,2003), trichobezoars (Jagadish and David, 1995), phytobezoars(Tyagi and Singh, 1993), stones (Umakanthan, 1995; DilipKumar et al., 1995), pieces of leather (Yadav et al., 2008) andcoconut shell (Madhava Rao et al., 2009). The present studyreports rexin as the cause of choke in buffaloes. The articlealso reports evaluation of prolene (No. 2-0) and silk (No. 2-0)for oesophageal mucosa suturing.

Materials and MethodsTen clinical cases presented to TVCC, Bidar were

randomly divided into two groups of five animals each. Thechoke was diagnosed by clinical examination and wasconfirmed by radiography as per techniques described by

CLINICAL EVALUATION OF PROLENE AND SILK FOR OESOPHAGIALMUCOSAL SUTURING IN BUFFALOES WITH CHOKE

D. Dilip Kumar1, B.V. Shivaprakash2, S.P. Manjunath3, M. N. Kiran Kumar4 andM. Naveen5

Department of Surgery and RadiologyVeterinary College, KVAFSU Bidar-585 401, Karnataka, India

ABSTRACT

A total of ten clinical cases of oesophageal obstruction in buffaloes presented to TVCC, Bidar were treated by performingoesophagotomy and removing the obstruction. Ten cases were divided into two groups containing five animals in each group.In group 1, after performing oesophagotomy mucosa was sutured by using silk (No. 2-0) and in group 2, mucosa was suturedby prolene (No.2-0). In both the groups submucosa, muscularis and adventitia were sutured by catgut (No. 1-0). The suturepattern used for mucosa in both the groups was simple interrupted with knots buried in the lumen (in-out-out-in pattern).Theremaining layers in both the groups were sutured by simple continuous pattern. In the present study none of the animal in bothgroups showed any complications. Both silk (No.2-0) and prolene (No. 2-0) were found to be suitable suture materials for theoesophageal mucosa in buffaloes.

Key words: Oesophagotomy, rexin, choke, prolene, silk

Singh and Nigam (1980) (Fig.1). The choke in all the caseswas in cervical region. The standard left lateraloesophagotomy procedure was followed in all the cases. Theoesophagotomy under local anaesthesia in right lateralrecumbency was done on the dorsal aspect of the oesophagus(Fig.3) with prior ligation of oesophagus in cervical and ruminalend by gauze piece (Fig. 2). Then incision was given on theoesophagus and foreign body rexin was removed (Fig.3). Theoesophagotomy wound in group 1 animals was sutured withprolene (2-0) for mucosa and chromic catgut (No. 1-0) forsubmucosa, muscularis and adventitia. In group 2, the mucosawas sutured by silk (No.2-0) and remaining layers by chromiccatgut (No. 1-0) (Fig. 4). The suture pattern used for mucosa inboth the groups was simple interrupted with knots buried inlumen (in-out-out-in pattern). The remaining layers in both thegroups of animals were sutured by simple continuous pattern.Skin wound was sutured using nylon by vertical mattress pattern.The animals were followed for long term to recordcomplications if any.

Results and DiscussionNone of the animal in both the groups showed any

complication. Both type of sutures showed good results. Thissuccess could be attributed to separate suturing of the mucosawhich is the strongest layer of oesophagus preventing leakageof contents.

Earlier workers had reported inversion pattern of suturessimilar to intestine which have potential threat of fistulations(Singh and Nigam, 1980). The present suture pattern alsoallowed the animal back to normal feeding within three days.

SummaryThe choke in buffaloes is mainly due to rexin. The site of

1Professor2Professor and Head3,4,5M.V.Sc. Students

Fig. 1: Arrow in a radiograph showing foreign body in oesophaghus

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choke was in cervical region. The local infiltration anaesthesiawas suitable for oesophagotomy in buffaloes. From the presentstudy both silk (No.2-0) and prolene (No.2-0) were found to besuitable suture materials for the oesophageal mucosa inruminants. The wound dehiscence and fistulations were notobserved with present pattern of suturing. The present suturepattern showed none of the complication in long term evaluationof oesophagotomy surgeries.

ReferencesDilip Kumar, D. et al. (1995) Indian Vet. J. 72: 385-386.

Jagadish, H. Patel and David, M. Bruce (1995) Can. Vet. J. 36: 774-775.

Madhava Rao, T. et al. (2009) Intas Polivet. 10:1-3.Shivaprakash, B.V. (2003) Intas Polivet. 4(2): 284-288.Shivaprakash, B.V. et al. (1998) Indian Vet. J. 75: 159.Singh, A.P. and Nigam, J. M. (1980) Mod. Vet. Pract. 61: 867.Tyagi, R.P.S. and Jit Singh (1993) The digestive system: Ruminant

Surgery. 1st ed. CBS Publishers, Delhi. pp. 192-193.Umakanthan, T. (1995) Indian Vet. J. 72: 275-276.Yadav, G.U. et al. (2008) Vet. World. 1(5): 149.

Fig.2: Temporary ligation of oesophagus in ruminal end by gauze piece Fig. 3: Removal of rexin after oesophagotomy

Fig. 4: Oesophagus after repair Fig. 5: 7th day after operation

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Torsion of uterus is a major reproductive disorder inlate pregnant dairy buffaloes. If not treated on time, maylead to death of foetus, dam or both. Sharma’s method ofdetorsion has been found to be highly successful inachieving detorsion of uterus in fresh cases (Singh andNanda, 1996). Failure to achieve detorsion of uterus due tofore-stomach impaction in a fresh case of uterine torsion ina buffalo is reported herein.

Case history and observationsA six-year-old buffalo in 4 th parity was presented to

clinical complex teaching hospital with the history of colicypains since 12 hours. As per the history previousparturations of the animal were normal and gestation periodof present parity was complete. The field veterinaniandiagnosed it to be case of uterine torsion and gave 20 rollson either sides (clockwise and anticlockwise) with andwithout external fixation of the uterus with a plank. On generalexamination the animal was alert and standing withintermittent straining. Per-vaginum examination revealedpost-cervical right sided (clockwise) uterine torsion of about90 degree. Cervix was open and foetal muzzle was palpablethrough the site of torsion. Per-rectal examination revealedstretching of broad ligaments and absence of oedema/adhesions. It was diagnosed to be a fresh case of uterinetorsion having fully relaxed sacrosciatic ligaments andengorged udder.

TreatmentThe buffalo was restrained in right lateral recumbancy

and was rolled clockwise with pervaginal fixation of the

FORE-STOMACH IMPACTION-A HINDRANCE TO ACHIEVE DETORSIONOF UTERUS IN BUFFALO - A CASE REPORT

G. Nazir, G. Rasool, E. E. Varugese and P. S. BrarDepartment of Veterinary Gynaecology and Obstetrics

Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141004, Punjab, India

cervix/foetus by hand. However, detorsion could not beachieved. Thereafter two rolls were given by externally fixingthe uterus with plank and per-vaginally with hand. After threerolls, the degree of uterine torsion got reduced to about 45degree. Both forelimbs of the foetus could be brought intovagina and a deviated head could felt on deep palpation.Further rolling and pulling of the foetus did not yield anycorrection in the torsion of uterus. To rule out uterine rupture,tapping was carried out to check for the colour of peritonealfluid which was normal, straw coloured and odourless.Accordingly cesearean section was advised, which theowner refused to got done. Hence the buffalo waseuthenized.

Post-mortem finding and discussionPost-mortem examination revealed severely impacted

omasum with dry contents (Fig.1) with enlarged overfilledrumen. In the months of December and January due toextremely cold weather scarcity of green fodder occurs dueto which wheat straw along with variable amount ofconcentrates is fed to ruminants which leads to omasalimpaction (Toor and Saini, 2008). To compensate thehypoglycaemia developed due to omasal impaction, theanimal consumes more feed which ultimately leads to over-filling of the rumen. The impacted and overfilled fore-somachmight have lead to, pushing uterus caudally, improper fixingof the uterus during rolling and decreased space fordetorsion, once the torsion has occurred. Collectively thesefactors played role in failure to achieve complete detorsionof uterus in the present case. The foetus was lying in theventro-oblique presentation which also might have occurreddue to fore-stomach impaction and contributed to failure inachieving detorsion. Fore-stomach impaction to be causeof uterine torsion could not be ascertained.

Hence, from the present study it is concluded thatomasal impaction can become a hinderance in achievingdetorsion. Giving many rolls before putting the animal tocaesarean section decreases the survivability of the animal(Dhaliwal et al., 1991). Hence from the present and previousstudy it implies that if no improvement is achieved evenafter a good roll in fresh case of uterine torsion, the animalbe assessed for omasal impaction and directly put tocaesarean section for its better survivability post-operation.

ReferencesDhaliwal, G S. et al. (1991) Pakistan Vet. J. 11(3):117-119.Singh, P. and Nanda, A S. (1996) Indian J. Ani. Reprod. 17(1):33-

34.Toor, A S. and Saini, N S. (2008) The Vet. Rec. 162(9): 275-278.Fig.1: Impacted omasum with dry contents

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IntroductionThe Interstitial Nephritis is prominent tubular and interstitial

change without primary involvement of glomerulus. Its incidenceis known to be frequently associated with Leptospirosis (McIntyreand Montgomery, 1952) and infectious viral hepatitis in dogs(Wright, 1976). However, an antibody against tubule basementmembrane i.e. involvement of immune responses (McClusky andKalasen 1973) has also been reported. It has also been reportedto be produced after administration of therapeutic agents.

The study was done on six dogs, two Labrador and fourGerman shepherd breed, regardless of sex, which were beingtreated in the veterinary hospital of Deen Dayal Upadhyaya PashuChikitsa Vigyan Vishwavidyalaya Mathura, and showing symptomsof renal damage. The dogs were admitted after having longtreatment somewhere else with a history of early responses withantibiotics and thereafter showing no improvement. The symptomsobserved were excessive thirst, stomatitis, stiff arched back,vomiting, not eating, reduced urine production and blood in urine.Blood was collected from the cephalic vein for estimation ofhaemoglobin, PCV and differential leucocyte count. Serum wasseparated for estimation of creatinine and blood urea nitrogen.Finally after being treated for about fifteen days, the dogs collapsedand were submitted for post-mortem examination. Gross lesionswere noted and samples from kidneys, liver, intestine and lungswere collected at the time of necropsy and fixed in neutral buffer10% formalin. Histopathology was done by paraffin embeddingtechnique. Sections 5 µm thick were stained with H & E stain.Tissue blocks were also processed for Levaditi’s stain.

Laboratory examination of the blood revealed varying degreeof decrease in Hb and PCV with increased neutrophil count.Serological study showed moderate to high increased values ofserum creatinine (6-15 mg/dl) and BUN (130-250 mg/dl). SGPT(60-80 IU/L) and SGOT (100-120 IU/L) values were also highlyincreased and were due to severe damage of kidneys and liver.

Post-mortem gross examination invariably revealed petechialhaemorhage on serosa, intestine, liver, kidneys, heart, spleen etc.along with hepatomegaly in all the dogs. One of the labradorshowed highly distended urinary bladder with urine and hydrocoelewith spots of abscess. Histopathological studies revealed cloudyswelling of hepatocytes with highly congested sinusoids with focalareas of necrosis in liver. Intestinal blood vessels were congested

INTERSTITIAL NEPHRITIS IN DOGS

Sanjiv Kumar1, Santosh K. Verma2 and Upendra Kumar3

Department of Pathology,College of Veterinary Science and Animal HusbandryPt. Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya, Mathura-281001, Uttar Pradesh, India

ABSTRACT

Interstitial nephritis was observed in six dogs reported at Veterinary hospital of Deen Dayal Upadhyaya Pashu Chikitsa VigyanVishwavidyalaya, Mathura. The dogs showed symptoms of renal affections and were treated for the same. All the dogs were agedabove 4 years. Diffuse infiltration of mononuclear cells with fibrosis was recognized in interstitium of kidneys, some of which havemany extensive tubular degeneration and necrosis. Some of them showed diffuse or focal glomerular lesions characterized bythickening of capillary wall and hyalinization.

Key words: Fibrosis, infiltration, interstitium.

with denudation of epithelial cells at some areas. Lungs alveoliwere severely congested. Kidneys showed infiltration ofmacrophages, lymphocytes and plasma cells either focally ordiffusely in the cortical or medullary interstitium. Interstitial nephritislesions were of non-purulent type. In most cases plasma cellswere predominant. In the diffuse type, there were tubular changesvarying in grades. Some cases with less severe cellular infiltrationhave caused swelling, degeneration and necrosis of epithelialcells without interstitial oedema. In cases having diffuse fibrosisand cellular infiltration in the interstitium tubular changes weremuch more severe and sometime significant vascular lesionswere seen. The tubular epithelium was mostly degenerated orsometimes hypoplastic and regenerative. Dilatation of lumen andthickening, distortion of tubular basement membrane alsooccurred. Glomeruli were also atrophic with hyalinization andperiglomerural fibrosis.

No Leptospirosis were detected on the Levaditis stainednephritic kidneys. No inclusions in the H&E stained sections weresuggestive of absence of canine hepatitis viral infection. Thereseems to be connection between age, as all the cases were ofmore than four years old but no correlation of breeds. The gradesof interstitial mononuclear infiltration and fibrosis varied greatlybetween individual cases. This may probably be due to eitherstages of disease or atrophy or difference in pathology.

Interstitial cellular infiltration and fibrosis may causedisturbance of blood flow to glomerulus and of urinary excretionresulting in the secondary glomerular lesions. Some immunereaction to tubular antigen might concern with glomerularlesions (Anderson, 1968). Recently it has been suggestedthat also tubular and interstitial renal lesions could be mediatedby immune response (Andres and McClusky, 1975).

AcknowledgementsThe authors are thankful to the Head and staff of Pathology,

for providing necessary facilities and support.

ReferencesAnderson, L.J. (1968) J. Path. Bact. 95: 59-65.Andres, G.A. and McClusky, R.T. (1975) Kidney Int. 7: 271-289.McClusky, R.T. and Klasen, J. (1973) New Engl. J. Med. 288: 564-570.McIntyre, W.I.M. and Montgomery, G.I. (1952) J. Path. Bact. 6: 145-160.Wright, N. G. (1976) J. Small Ani. Pract. 17: 25-33.

1Corresponding author: Assistant Professor, Department of Pathology, Bihar Veterinary College Patna-800014, Bihar, Email: [email protected],3M.V.Sc. Scholars

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IntroductionPaecilomyces fungi are ubiquitous. Originally classified

in the genus Penicillium as Penicillium lilacinum, the organismwas classified into the separate genus Paecilomyces in 1974.This organism usually produces disease inimmunocompromised (Tan et al., 1992), transplant patients(Lott et al., 2007; Castro et al., 1990) and those exposed tolong-term corticosteroid treatment and patients onchemotherapy (Booth et al., 2001). This organism has beendocumented to infect nonhuman primate (Fleischman andMcCracken, 1977), reptiles (Maslen et al., 1988) and animals(Rosser, 2003; March et al., 1996; Saberhagen et al., 1997;Foley et al., 2002).

Materials and MethodsCase history: A male calf of Jaisalmeri breed was born to

a completely agalactic dam in the month of Feb., 2011. Duringthis period almost ninety per cent of the camel populationsuffered with upper respiratory tract infection, this infection wasspecies specific and other species of livestock were notaffected. Etiology of this disease could not be ascertained butthe pattern of spread is suggestive of a viral infection. Thisinfection thought to be resulted in agalactia in most of thenewly parturient camels. Since calf could not get colostrum, sothe camel milk feeding through nipple bottle was started onday first, as a usual practice adopted for such calves on thefarm. On the 3rd day calf stopped feeding and suffered withrespiratory insufficiency with deep laboured respirations alongwith abdominal movements and fever of 103°F. Calf becameanaemic with pallor mucous membranes and its haemoglobinwas 8 gm/dl. Differential leucocytic count revealed neutrophilia-58%, lymphocytes-32%, eosinophils-06% and monocytes-04%. The calf was treated with antibiotics, analgesics,corticosteroids and fluid therapy. On the 4th day calf recoveredpartially and on 5th day again abdominal respiration started,calf became recumbent and on 6th day calf died.

Post-mortem examination: Revealed clear lesions in thelungs. Whole of the lung was black in colour with papules andpatchy fibrinous deposit on the surface along with emphysema

MORTALITY OF A NEONATAL CAMEL CALF DUE TO PAECILOMYCOSISINFECTION OF THE LUNGS

F.C. Tuteja, N.V. Patil, S.D. Narnaware, S.S. Dahiya, G. Nagarajan, G. Sivakumar andKashi Nath

National Research Centre on Camel, Jorbeer, Shivbari, P.B.-07, Bikaner-334001, Rajasthan, India

ABSTRACT

A male calf of Jaisalmeri breed was born to a completely agalactic dam. Since calf could not get colostrum, so the camelmilk feeding through nipple bottle was started on day first, as a usual practice adopted for such calves on the farm. On the3rd day calf stopped feeding and suf fered with respiratory insuff ic iency with deep laboured respirations along withabdominal movements and fever of 103°F. The calf was treated with antibiotics, analgesics, corticosteroids and f luidtherapy but the calf died on 6 th day. Post-mortem examination revealed that whole of the lung was black in colour withpapules and patchy fibrinous deposit on the surface along with emphysema. Mycological examination revealed for theisolation and identif ication of Paecilomyces lilacinus .

Key words: Camel, Paecilomycosis, lung infection

(Fig.1, 2) Other lesions observed were anaemic condition ofthe carcass, liver was slightly enlarged and hard along withone hydated cyst of approximately 1.5 centimetre in diameterand intestines were congested. Lung tissue samples werecollected for bacteriological and mycological examination.

Results and DiscussionMycological examination of the lung tissue samples on

Sabourauds dextrose chloramphenicol agar revealed for theisolation and identification of Paecilomyces lilacinus .Bacteriological examination was negative but possibility ofbacterial infection as initiator of the infection or as secondaryinvader cannot be ruled out because calf was under antibiotictreatment since last three days. On Sabourauds dextrosechloramphenicol agar colonies matured in five days. Thecolonies were floccose, pink in colour from front whereasreverse colour was typically orange at 28°C. The subculture ofthe isolates at 37°C revealed yellow brown colour and sweetaromatic odour was observed on 10 th day of this culture.Microscopically septate hyaline hyphae, long (400 to 600 µm),slightly roughened conidiophores giving rise to whorls ofdivergent phialides bearing chains of ellipsoidal conidia (2 x 3µm). Based on these morphological characteristics, the isolatewas identified as P. lilacinus (Fig. 3-7).

The antibody status of this calf was not measured. Thisnew born calf coupled with failure to get colostrum is theexample of a potentially weak immune status in any of theanimal species. The status which can be compared with zeroimmune status. The camel calf comes to life almost deprivedof serum immunoglobulin and depends on colostrum forvirtually all its humoral passive immunity (Ghazi et al., 1994).Failure to achieve adequate transfer of passive immunity hasbeen associated with increased risk of diarrhoea, respiratoryaffections and motility of neonates (McGuire et al., 1975; Reaet al., 1996). Neutrophilia observed in the present case maybe due to deprivation of colostrum. Al-Sultan (2008) reporteddeprivation of camel calf to colostrum results in leukocytosismainly due to neutrophilia.

Paecilomycosis most commonly affects dogs with the

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Pic.4: Colony of P. lilacinusat 280C (5days reverse).

Pic.3: Colony of P. lilacinus at 280C (5days f ront).

Pic.1: Lungs black in colour with caseous nodules on the surface.

Pic.5: Colony of P. lilacinus at 370C (10days f ront)

Pic.6: Colony of P. lilacinus at 370C (10 days reverse).

Pic.2: Lungs with patchy fibrinous deposit on the surface.

disease typically manifesting as a systemically disseminated form(Patnaik et al., 1972; Jang et al., 1971; Littman and Goldschmidt,1987; Patterson et al., 1983; March et al., 1996; Nakagawa et al.,1996; Garcia et al., 2000; Foley et al., 2002; Booth et al., 2001).Clinical manifestations are dependent on the affected organs.The most commonly reported clinical signs are fever, ataxia, paresisor paralysis, weight loss and lameness. However, vestibular signs,bilateral deafness, generalized seizures, chorioretinitis andanaemia have also been reported. In some animals, localized

cutaneous disease is the only clinical sign of occult disseminateddisease (Van den Hoven and McKenzie 1974; Elliott et al., 1984).The German shepherd is the breed most commonly affected withpaecilomycosis or other systemic mycosis (Watt et al., 1995; Day,1998). Adult female dogs are more commonly affected (Booth etal., 2001). Most animals did not have obvious immunosuppressiveconditions (e.g. terminal illness, immunosuppressive drugs) oran alteration of the normal microbial balance by prolonged antibioticuse. German shepherds have a relative deficiency ofimmunoglobulin A (IgA) compared with other purebred and mixed-breed dogs (Griot-Wenk et al., 1999), supporting the possibilitythat local immunity might be defective in German shepherds andmay play a role in the development of fungal infections. IgA is acomponent of mucosal surface immunity which includes the skinand the respiratory, digestive and reproductive tracts. In the presentcase, the potentially weak immune status of the newborn calfcoupled with failure to get colostrum (main source of IgA) possiblyacted as predisposing factors to this infection. Further hugeinvolvement of lung in the five-day-old calf shows that, this organismutilized lung tissue just as growth medium without any resistanceof the in-vivo mechanisms. Lack of supply of colostral IgA in thiscalf, supports the more occurrence of disease in Germanshepherds dogs.

Paecilomycosis has been reported in cats. One cat presentedwith local skin lesions that progressed into disseminated

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paecilomycosis after two months, was treated with ketoconazolefor three months without success (Elliott et al., 1984). Another cathad localized recurrent cutaneous infections of the left metacarpalregion and upper lip. The cat was successfully treated with multipleexcisions of the affected cutaneous tissues, débridement ofsurrounding tissues and postoperative treatment with itraconazole(Rosser, 2003). Pulmonary infection in an immunocompitent catwas treated with itraconazole therapy for six months (Pawloski etal., 2010).

The location of the primary lesion in systemic paecilomycosisis not usually determined in animals. The authors of previouslyreported cases suspected cutaneous or mucosal wounds (Janget al., 1971; Littman and Goldschmidt, 1987; Patterson et al., 1983;Nakagawa et al., 1996; Garcia et al., 2000; Foley et al., 2002; Elliottet al., 1984). But in this camel calf there was no external injury,which could suggest the mucocutaneous route and evenmedication in form of injections were started on 3rd day, did notsuggest so much flaring of the infection in the lungs. Further ifmucocutaneous route could have been there then it could havetaken sufficient time for establishment of the infection anddissemination to the other organs and not only the lungs. So thetypes of lesions are clearly suggestive of entry via inhalation in thepresent case.

Many cases have been reported in which the role of this fungusas a pathogen in healthy patients has been clearly demonstrated.Pastor and Guarro (2006) reviewed 119 cases reported between1964 and 2004 of human infection by P. lilacinus. Most of thecases were of oculomycosis (51.3%) followed by cutaneous andsub-cutaneous infections (35.3%) and a smaller group ofmiscellaneous infections (13.4%).

Foley et al. (2002) reviewed 14 cases of paecilomycosis in atertiary care veterinary hospital and all reports of the disease in theveterinary literature. Paecilomycosis is a rare disease primarily ofdogs, horses, reptiles, and humans. Clinical manifestations inveterinary patients vary but include disseminated disease and

diskospondylitis, particularly in dogs: pneumonia in dogs, horses,and reptiles; keratitis in horses; and miscellaneous local infections.The prognosis for paecilomycosis is poor. Although some treatmentsuccess has been reported and success rates could improve ifadditional information were available regarding fungal speciesoccurring in veterinary patients and drugs to which these fungi aresusceptible.

Although a rare pathogen, P. lilacinus has the capability ofcausing a severe, systemic fatal disease. The precise identificationof the fungus can be made only by isolation in a pure culture. Inmost veterinary reports, paecilomycosis was definitively diagnosedon post mortem examination (Van den Hoven and McKenzie, 1974;Patnaik et al., 1972; Jang et al., 1971; Littman and Goldschmidt,1987; Patterson et al., 1983; Nakagawa et al., 1996; Garcia et al.,2000; Foley et al., 2002; Elliott et al., 1984). As has been diagnosedin the present case.

Treating this rare disease is challenging. Several antifungalmedications have been used, including itraconazole, ketoconazole,and amphotericin B. In vitro susceptibility testing in one studysuggested that amphotericin B and itraconazole are optimalchoices for treating paecilomycosis in veterinary patients (Foley etal., 2002). However, susceptibility-testing results often do notcorrelate with clinical outcome (Castro et al., 1990; Foley et al.,2002; Naidu and Singh, 1992). For this reason, it is difficult tomake specific recommendations regarding treatment.Nevertheless, sensitivity testing of each clinical isolate ofPaecilomyces species is justified because it indicates a definitetrend in susceptibility of the organism to antifungal agents (Boothet al., 2001).

ReferencesAl-Sultan, S. I. (2008) J. Camel Pract. Res.15: 191-193.Booth, M.J. et al. (2001) J. South African Vet. Assoc. 72:99-104.Castro, L.G. et al. (1990) J. Medical Vet. Mycology. 28:15-26.Day, M.J. (1998) Canine disseminated aspergillosis. In: Greene CE, ed.

Infectious diseases of the dog and cat. 2nd ed. Philadelphia, Pa:WB Saunders Co. pp. 409-412.

Elliot, G. S. et al. (1984) J. Am. Vet. Med. Assoc. 1:93-94.Fleischman, R.W. and McCracken, D. (1977) Vet. Pathol. 14: 387-391.Foley, J.E. et al. (2002) J. Vet. Internal Med. 16: 238-243.García, M.E. et al. (2000) J. Vet. Med. A, Physiol., Pathol., Cli. Med.

47:243-249.Ghazi, S.R. et al. (1994) Anat. Histol. Embryol. 23:337-342.Griot-Wenk, M.E. et al. (1999) Res. Vet. Sci. 67:239-243.Jang, S. S. et al. (1971) J. Am. Vet. Med. Assoc. 159:1775-1779.Littman, M.P. and Goldschmidt, M.H. (1987) J. Am. Vet. Med. Assoc. 191:445-

447.Lott, M.E. et al. (2007) J. Drugs in Dermatol. 6: 436-439.March, P.A. et al. (1996) J. Am. Ani. Hosp. Assoc. 32:509-514.Maslen, M. et al. (1988) J. Med. Vet. Mycol. 26:219-225.McGuire, T.C. et al. (1975) J. Am. Vet. Med. Assoc. 166: 71-75.Naidu, J. and Singh, S.M. (1992) Antonie Van Leeuwenhoek. 62:225-230.Nakagawa, Y. et al. (1996) J. Vet. Med. Sci. 58:157-159.Pastor, F.J. and Guarro, J. (2006) Cli. Microbiol. Infection. 12:948-960.Patnaik, A.K. et al. (1972) J. Am. Vet. Med. Assoc. 161:806-813.Patterson, J.M. et al. (1983) J. Am. Ani. Hosp. Assoc.19: 569-574.Pawloski, D. R. et al. (2010) J. Am. Ani. Hosp. Assoc. 46:197-202.Rea D.E. et al. (1996) J. Am. Vet. Med. Assoc. 208: 2049-2051. Rosser, E.J. Jr. (2003) J. Am. Ani. Hosp. Assoc. 39:543-546.Saberhagen, C. et al. (1997) Cli. Infectious Dis. 25:1411-1413.Tan, T.Q. et al. (1992) J. Clinical Microbiol. 30: 2479-2483.Van den Hoven, E. and McKenzie, R.A. (1974) Australian Vet. J. 50:368-

369.Watt, P.R. et al. (1995) J. Am. Vet. Med. Asso. 207:67-70.

Pic .7: Microscopy of P. lilac inus, showing conidiophores,phialides and conidia

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IntroductionPeste des petits ruminants (PPR) is an acute and highly

contagious viral disease of goat and sheep. It was firstdescribed in West Africa (Gargadennec and Lalanne, 1942)and is characterized by necrotizing and erosive stomatitis,enteritis and pneumonia followed by death or recovery. In Asia,the disease was first reported in India (Shaila et al., 1989). Thedisease is highly contagious, with morbidity and mortality ratein goat and sheep as high as 100% and 90%, respectively andhence this disease is also known as “goat plague”. In India,severity of the disease is more pronounced in goats than insheep with a combined susceptible population of about 200million and thus it is one of the major threats to the smallruminant population of the country. PPR is caused by Pestedes Petits ruminant’s virus (PPRV), which is grouped underthe genus Morbilli virus of family Paramyxoviridae of orderMononegavirales. For specific diagnosis of PPR in smallruminants, it is essential to differentiate it clearly fromRinderpest. Conventional serological tests like AGID, CIEPetc. often fail to differentiate between them. Although, crossvirus neutralization can differentiate between these two viruses,it is laborious and difficult especially when sample size islarge. sandwich-ELISA kit have proved to be an important toolfor detecting antigens (Mangal, 2007).In present investigationthe efficacy of six different antiviral herbal drugs was evaluatedon goats found positive for PPR antigen by s- ELISA test.

Materials and MethodsDuring the study, a total of 1068 nasal swab samples

were collected from goats of either sex belonging to all agegroups were selected for the detection of PPR antigen usingPPR sandwich-ELISA kit developed at Rinderpest laboratory,Division of Virology, Indian Veterinary Research Institute,Mukteshwar campus, Uttaranchal. The test was done as perthe protocol supplied along with kit. Under the present

STUDIES ON CLINICO-HAEMATO-BIOCHEMICAL CHANGES INPESTE DES PETITS RUMINANTS IN GOATS#

C.S. Sharma1, H.K. Mehta2, M.M. Prakash3 and P.C. Shukla4

P.G. Department of Zoology, Holkar Science College, D.A.V.V., Indore, M.P.

ABSTRACT

The present epidemiological study was carried out to determine the clinical, haemato-biochemical findings in PPR affected goats. Atotal of 1068 nasal swab samples were collected from goats of either sex belonging to all age groups were selected for the detectionof PPR antigen using PPR sandwich-ELISA kit. The materials (blood, serum and occulonasal samples) were collected from goatsslaughtered for meat purpose in the slaughterhouse, Veterinary hospitals brought for the treatment of various ailments viz diarrhoea,pneumonia, oculo-nasal affections and stomatitis. A total of 70 and 203, 40 and 140 serum samples from non infected and PPR infectedgoats of Veterinary hospital and slaughter group were studied for haemato-biochemical changes respectively. Out of 1068 nasalswab samples tested, 346 (32.39 %) were found to be positive for PPR virus antigen using PPR sandwich-ELISA kit.

Key Words: PPR, incidence, goats, sandwich-ELISA

1#Assistant Professor cum Hospital Superintendent and Corresponding Author2Associate Professor, Department of Veterinary Medicine, Veterinary College, Mhow, Madhya Pradesh, India3Professor and Head, Department of Biotechnology, Holkar Science College, DAVV, Indore, Madhya Pradesh, India.4Professor and University Head, Department of Veterinary Medicine, Veterinary College, Jabalpur, Madhya Pradesh, India

investigation the rectal temperature, pulse rate, respiratory rate,oculo-nasal discharge, mouth lesions, coughing and diarrhoeaclinical parameter were studied. The materials (blood, serumand occulonasal samples) were collected from goatsslaughtered for meat purpose in the slaughterhouse, Veterinaryhospitals brought for the treatment of various ailments vizdiarrhoea, pneumonia, oculo-nasal affections and stomatitis.A total 70 and 203, 40 and 140 serum samples from noninfected and PPR infected goats of Veterinary hospital andslaughter group were studied for haemato-biochemicalchanges, respectively. Blood samples for haematological andserum biochemical changes in PPR infected goats observedat slaughter house and veterinary hospitals were collectedonly once i.e. on the day of observation/visit. The haematologicalparameters included haemoglobin (Hb), packed cell volume(PCV), total erythrocyte count (TEC), total leucocyte count (TLC),mean corpuscular volume (MCV), mean corpuscularhaemoglobin (MCH), differential leucocyte counts (DLC) wereestimated as per the procedure given by Benjamin (1985). Forthese, 2 ml blood was collected in EDTA vials. The serumbiochemical studies were performed on the serum harvestedfrom 5 ml blood in sterilized tubes. The biochemicalparameters studied included total plasma protein (TPP), totalalbumin, globulin, blood glucose (GL), serum alanineaminotransferase (ALT), serum aspartate aminotransferase(AST), serum bilirubin and blood urea nitrogen (BUN) wereestimated using span diagnostic, Surat kits.

Results and DiscussionThe disease in its pattern, symptoms, lesions, severity,

morbidity and mortality simulated involvement of Morbilli virusinfection. The affected animals went off- feed and showed high-rise of temperature. The clinical observations of infected andcontrol goats of above two groups are presented in Table1. The mean rectal temperature of infected and non infected

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Table 1: Body temperature, pulse rate and respiration rate in PPR infected and non infected goats of different goats.

Veterinary hosp. group Slaughter group Physiological parameters Control Infected Control Infected

Body temperature (ºF) 103.2±0.01 105.22±0.33 103.18±0.03 105.32±0.36 Pulse rate (per minute) 84.26±1.88 85.68±1.52 84.24±1.84 85.64±1.55 Respiration rate ( per minute) 23.76±0.65 24.21±0.24 23.75±0.68 24.22±0.26

Table 2: Incidence of PPR at Veterinary hospital in Indore District of M.P.

Location Total Posi tive Percentage positive Govt., Dist. Hospital, Indore 155 51 32.90 Govt., Hospital, Mhow 192 62 32.29 TV CSC, Mhow 110 36 32.72 Govt., Hospital, Depalpur 91 29 31.86 Govt., Hospital, Sanwer 76 25 32.89 Total 624 203 32.53

Table 3: Incidence of PPR at Slaughter House in Indore District of Madhya Pradesh.Location Total Posi tive Percentage positive

Slaughter house, Indore 206 66 32.03 Slaughter house, Mhow 238 77 32.35 Total 444 143 32.20

Table 4: Haematological values of infected, non infected goats in Veterinary and Slaughter groups.Veterinary Hospital group Slaughter house group S.No. Parameters

Non infected

Infected Non infected

Infected

1. Haemoglobin (g/dl) 9.52±0.25 7.22±0.13* 9.54±0.23 7.20±0.11* 2. TEC (million/cmm) 11.36±0.18 8.22±0.44* 11.34±0.16 8.21±0.46* 3. TLC(103/cmm) 9.26±0.14 6.20±0.05* 9.27±0.15 6.21±0.04* 4. PCV (%) 32.92±1.8 18.22±2.6* 32.92±1.7 18.26±2.7* 5. MCV(fl) 30.41±0.94 22.95±0.36* 30.08±0.57 22.99±0.8* 6. MCH (pg/dl) 7.83±0.22 8.38±0.14 7.71±0.29 7.93±0.12 7. MCHC(g/dl) 37.91±0.42 38.44±0.36 37.94±0.44 38.42±0.38 8. DLC (%) a. Lymphocyte% 50.01±1.57 20.22±1.8** 50.04±1.2 26.02±1.62** b. Neut rophil % 38.60±1.50 73.00±1.62** 38.81±1.14 70.06±1.5** c. Eosinophil % 2.88±0.35 1.65±0.05* 2.87±0.16 1.68±0.4* d. Monocyte % 2.02±0.34 1.38± 0.22 2.04±0.43 1.41±0.12 e. Basophil % 0.83±0.14 0.81±0.10 0.79±0.16 0.76±0.10

Table 5: Biochemical values of infected, non infected goats in Veterinary and Slaughter groups.

*Significant (P<0.05) **Significant (P<0.01)

Veterinary Hospital group Slaughter house group S.No. Parameters Non infected Infected Non infected Infected

1 Total protein(g/dl) 6.55±0.31 5.62±0.25 6.50±0.37 5.57±0.10 2 Blood glucose (mg/dl) 43.85±1.38 25.93±1.22** 43.92±1.35 25.88±1.12** 3 ALT(IU/L) 57±4 106±5* 58±3 108±5* 4 Na(m Eq/ liter) 144.07±1.34 143.06±1.30 144.09±1.33 143.09±1.32 5 K(m Eq/ liter) 5.10±0.05 5.03±0.03 5.08±0.07 5.03±0.04

*Significant (P<0.05) **Significant (P<0.01)

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Fig. 3: Gross photograph showing shootingdiarrhea in goats suffering from PPR

Fig. 2: Gross photographs showing a necrotic stomatitis in goats suffering from PPR

Fig. 1: Gross photographs showing profuse occulo-nasal discharge in goats suffering from PPR.

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control goats of hospital and slaughter groups was 105.22°F(±0.33) and 103.2°F (±0.01) and 105.32°F (±0.36) and 103.18°F(±0.03), respectively. A significant increase in the rectaltemperature was noted after the infection. There was suddenrise in body temperature. The temperature usually remainshigh for about 5-8 days before gradually returning to normalproceeding recovery or dropping below normal before death.The ‘t’ test of significance performed to compare the meanvalues of infected and normal healthy goats revealed significantdifference. (Table 1). The rise in body temperature followinginfection in goats have been observed by several workers(Hamdy et al., 1976; Abegunde and Adu, 1977; Ihemelandu etal., 1985; and Brown et al., 1991). The observation for individualgoats in this study is an agreement with the workers mentionedin this context. In the present study, the febrile responses andsignificant rise in body temperature was noticed in all infectedanimals suggesting that PPR virus has initiated the febrilereaction. There was no significant difference in pulse rate andrespiration rate between normal healthy and infected goats ofveterinary hospital and slaughter groups as revealed by ‘t’ test.

There was profuse occulo-nasal discharge, necroticstomatitis revealing ulceration in lips, dorsal and under surfaceof tongue and cheeks and congestion in gums (Fig. 1 and 2).Shooting diarrhoea with foetid odour and blood tinged shreadsdeveloped after 3- 4 day of infection (Fig. 3). The occulo- nasaldischarge turned mucopurulent by a couple of days. Therewas an extreme dehydration, distressing cough, respiratorytrouble and death on 6th - 8th day was the sequele in affectedanimals (624) of hospital group. In some of the cases due tomultiulcerative lesions, skin/mucous membrane of nostrils,lips, gums and tongue were sloughed off and large size,i.e.,1to 5 cm lesions were recorded in these animals. Similar lesionshave also been reported by earlier workers (Aruni et al., 1998;Hussain et al., 1998). The clinical signs like weaknessdebilitation, pale mucous membrane, emaciation were causeddue to the protein gastropathy, increased intestinal movementsdue to irritability leading to decreased absorption ultimatelycaused the sever weakness and associated symptoms.

Out of 1068 nasal swab samples tested, 346 (32.39 %)were found to be positive for PPR virus antigen using PPRsandwich-ELISA kit (Table 2 and 3). Majumdar (1997) havereported presence of virus in 40% nasal swab samples inPPR affected goats in Tripura by immunocapture ELISA.

Haematological alterations occurring during the outbreakhave been depicted in Table 4. There were significant (P<0.05)differences in the haemoglobin (Hb), packed volume (PCV)

and total erythrocyte count (TEC) levels between control andinfected group. The decreased haemoglobin concentrationmight be associated with significantly low TEC and PCVpossibly due to toxaemia. The lower TEC values may be dueto decreased rate of erythropoiesis in such cases. Theleucopoenia in infected animals may be because of the virusinfection. Similar findings have been reported (Raghevendraet al., 2000).The mean corpuscular volume was decreased inthe infected group as compared with the control group. Thedecreased MCV values and normal MCH values indicated amicrocytic normochromic type of anaemia.

The total protein did not show a significant differencebetween the infected and control groups (Table 5). The serumglucose level showed a decrease in the infected group. Theserum alanine aminotransferase (ALT) levels showedsignificant (P<0.05) increase in infected group. The increasedlevel ALT observed in the infected group could be attributed tohepatic damage. Similar observations have been made byKaneko et al. (1997).

Earlier there has not been any report of PPR in this area.Disease might have occurred due to picking up of infection bylocal goats from migratory goats from neighboring state(s) asthere is frequent inter state migration of goats in this area.

ReferencesAbegunde, A.A. and Adu, F.D. (1977) Bull of Ani. Hlth. Prod. Afr. 25:

307-311.Aruni, A.W. et al. (1998) Small Rum. Res. 28(3):233-240.Benjamin, M.M. (1985) Outline of Veterinary Clinical Pathology. 3rd

ed., Kalyani Publishers, New Delhi.Brown, C.C. et al. (1991) Vet. Pathol. 28: 166-170.Gargadennec, L. and Lalanne, A. (1942) Bulletin des Service

Zootechniques, et des Epizooties de I’ Afrique OccidentaleFrancaise. 5: 16-21.

Hamdy, F.M. et al. (1976) Can. J. Comp. Med. 40: 276-284.Hussain, M.M. et al. (1998) Pak. Vet. J. 18(4): 224-226.Ihemelandu, E.C. et al. (1985) Trop. Ani. Hlth. Prod. 17: 83-88.Kaneko, J.J. et al. (1997) Clinical Biochemistry of Domestic Animals.

5th ed., Academic Press, London.Majumder, P. (1997) Indian J. Ani. Hlth. 36(2):169-171.Mangal, Sarita. (2007) Incidence and clinicohaematological studies

on Peste Des Petits Ruminants in goats (Capra hircus).M.V.Sc. & A.H. thesis J.N.K.V.V., Jabalpur.

Mann, E. et al. (1974) Bull. Epizoot. Dis. Afr. 22: 99-103.Raghevendra, L. et al. (2000) Int. J. Ani. Sci. 15(2): 171-173.Shaila, M.S. et al. (1989) Vet. Rec. 125: 602.Snedecor, G.W. and Cochran, W.G. (1994) Statistical Methods. 8thed.

The lowa State College Press, Inc. Amer. lowa USA.

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IntroductionPasteurella multocida, a Gram-negative short rod, is

etiological agent of Haemorhhagic septicaemia (HS), an olddisease of great importance (Pasteur, 1880). Thiscosmopolitan disease causes catastrophic epizootics,especially in Asia and Africa (Bain et al., 1982; De Alwis, 1992).In north India, which is the cradle of high milk yielding buffalobreeds; HS has been estimated to cause huge economiclosses (Dutta et al., 1990; Kumar et al., 2004; Venkataramananet al., 2005). The majority of the HS cases in India are suddenoutbreaks of acute respiratory illness in large ruminants,especially buffaloes, and cattle as well as small ruminantpopulations, mainly in hot and humid rainy and post-monsoonseason. The disease may also present itself in a herd inimmunologically naïve young animals, which may not havebeen vaccinated. The outbreak results in high morbidity andmortality. The disease is presented in 3 forms: respiratory orpulmonary, oedematous, and intestinal forms. Normally anupper respiratory tract commensal, P. multocida sub. spp.multocida is a non-motile, non-sporulating coccobacillus. Asthe main epidemic strain is Pasteurella multocida sub spp.multocida B:2 serotype, it is suspected to be carried by carrierlivestock in a flock.

Epidemiological precipitating factors like managementaland environmental stresses may lead to carrier mediated rapidspread of infectious agent via feed, water and droplets,especially when humidity is high (Ataei et al., 2009). Outbreaksmostly occur in hot and humid weather, indicating failure ofimmunity after vaccination or otherwise. On the basis of

SPORADIC OUTBREAK OF HAEMORRHAGIC SEPTICAEMIA INBUFFALO CALVES IN AN ORGANIZED FARM

R. K. Vaid1#, K. Shanamugasundaram1, Ashok Boora2, T. Riyesh1, B. C. Bera1, B. N. Shukla,Taruna Anand1, N. Virmani3, S. Barua1, N. Rana2, K. P. Singh2, P. Malik1 and R. K. Singh1,2

Veterinary Type Culture CollectionNational Research Centre on Equines

Sirsa Road, Hisar-125001, Haryana, India

ABSTRACT

Water buffalo are of enormous economic significance in the dairy economy of north India, however, they are highly susceptible tobacterial respiratory diseases including haemorrhagic septicaemia (HS) caused by Pasteurella multocida. An outbreak of HS causedby Pasteurella multocida sub spp. multocida occurred in 3 buffalo calves of a discrete population kept in an organized farm. Theperacute episode of disease resulted in early death and samples from animals and post-mortem samples yielded typical bi-polarstaining Gram-negative bacilli. The isolations were made from nasal swabs and intestinal contents. T hree isolates ofPasteurella multocida was isolated in pure culture from 2 calves, and the diagnosis was based on clinical signs, biochemical andphenotypic characterization and PCR confirmation performed on the cultures. The calves were diagnosed with HS due to infectionwith P. multocida capsular type B:2 strain. The cultures were subjected to antimicrobial sensitivity profile, and were found to begenerally susceptible except showing resistance to flouroquinolones. The importance of diagnosis, reporting and isolation, preserva-tion and genetic characterization of isolates with geographical information is discussed.

Key words: haemorhagic septicaemia, buffalo, PCR, Pasteurella multocida

1#Veterinary Type Culture Collection, Sirsa Road, Hisar, 125001, Haryana, India. E-mail: [email protected] Phone: 91-9416786940 Fax: +91-1662-276217 2Central Institute for Research on Buffalo, Sirsa Road, Hisar 125001, Haryana, India3National Research Centre on Equines, Sirsa Road, Hisar, 125001, Haryana, India.

Carter:Heddleston typing scheme, the serotypes of P. multocidaassociated with HS in Asia is B:2 (Rimler and Wilson, 1994).

Materials and MethodsAmong a group of 25 weaned murrah buffalo calves,

cases of dullness, acute respiratory distress, high fever andperacute sudden death was reported in 3 buffalo calves ofabout an year age, in an organized farm, reared under semi-intensive system of management. The group was vaccinatedfor HS, BQ and FMD vaccines. The disease was reported inthe mid-summer season in the month of June. Nasal swabsfrom clinically diseased animals were collected. However,before the bacteriological etiology could be ascertained andwithin few hours of start of antimicrobial treatment, there wasperacute death in 3 calves. Rest of the calves did not report anysickness. PM samples of spleen, lungs, liver and intestinewere collected under aseptic conditions and processed forpathogen isolation. Briefly, swabs were streaked on 5% Sheepblood agar (SBA), and MC Conkey lactose agar (MLA), andincubated at 37°C for 24 hours. The resultant colonies wereexamined for size, zone of haemolysis and were subjected toGrams and Giemsa’s staining and microscopic examination.From the culture plates inoculated with nasal swabs, andintestinal content specimens, three representative colonieswere isolated and purified by repeat streaking. These werecryo-preserved in glycerol and as working stock in SBA slantsat 5°C. The slant cultures were used for biochemicalcharacterization and antibiotic sensitivity testing. Isolateswere characterized on the basis of its cultural, colony,

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morphological and biochemical characteristics at 37ºC (Cowanand Steele, 2004; Quinn et al., 1994). The isolates weresubjected to antimicrobial sensitivity test with the followingantibiotics by disc diffusion assay method (Bauer et al., 1966).The antimicrobial discs, amikacin (30 µg), kanamycin (30 µg),chloramphanicol (30 µg), gentamicin (30 µg), enrofloxacin (10µg), cloxacillin (30 µg), norfloxacin (10 µg), ceftazidime (30 µg),ampicillin (25 µg), amoxycillin (30 µg), erythromycin (15 µg),cephalothin (30 µg), tetracycline (30 µg), nalidixic acid (30 µg),and furazolidone (50 µg) were tested. All tests were conductedat 37ºC.

The three isolates obtained from the outbreak of HS weresubjected to PCR confirmation of genus, species and capsularserotype. The PCR amplification of the SP6-T7 promoter regionof P. multocida using KMT-1 and KTT primer was performed.The identification of the culture isolate obtained was confirmedby using KMT-1 primers; forward T-7 (5’-ATC CGC TAT TTACCC AGT GG-3’) and reverse SP-6 (5’-GCT GTA AAC GAA CTCGCC AC-3’) and for capsular type, forward KTT 72-(5’ AGGCTC GTT TGG ATT ATG AAG -3’) and reverse KTS P61- (5’ ATCCGC TAA CAC ACT CTC -3’) (Townsend et al., 1998).

The PCR reaction was performed in a total volume of 50 ìlconsisting of 3 ìl template DNA, 10× PCR buffer, 200 ìMdeoxynucleoside triphosphates, 0.25 U of Taq DNA polymerase(Fermentas), and 15 pmole of each primer. PCR cyclingcondition consisted of initial denaturation at 95°C for 5 minutes,denaturation at 95°C for 50 sec, annealing at 53°C for 50 secand extension at 72°C for 1 minute. A final step of extensionwas applied at 72°C for 10 minutes. Amplification productswere run in agarose gel electrophoresis (2% agarose in 13TAE) stained with ethidium bromide The PCR product wasvisualized under UV light.

Results and DiscussionThree isolates, 2 from nasal swab and 1 from intestinal

contents were isolated. Two of the isolates were from sameanimal, from nasal swab and intestinal contents. The isolateson 5% SBA were non-haemolytic, shiny, and small to mediumand greyish in colour. Microscopically, the cells were Gramnegative, small rods with characteristic bipolar staining. Thebipolar staining cells showed characteristic morphology inGiemsa’s stained slides.

The cultures were oxidase and catalase positive; non-motile and fermentative on Hugh and Leifson media. TSI slantshowed yellow slant and butt without hydrogen sulphideproduction. Simmon’s citrate and urease were not utilized.Isolates were not able to grow on McConkey’s Agar (MLA) plates;were positive for indole production and nitrate reduction. Allthree isolates showed lysine and ornithine decarboxylasepositive reactions. On Andrade’s peptone water sugar testing,all isolates were positive for glucose, fructose, galactose,mannitol, mannose, sorbitol, sucrose and xylose, whereasnegative result were showed for arabinose, adonitol,cellobiose, dulcitol, inositol, inulin, lactose, maltose, mellibiose,raffinose, rhamnose, salicin and trehalose. Results ofphenotypic bacteriological analysis of the isolates indicatedspecies Pasteurella multocida sub spp. multocida (Quinn etal., 1994; Dousse et al., 2008). The species identification wasconfirmed by molecular biology based techniques. Theamplified PCR products were found to be of 460 bp size andPCR amplification with the primer pair designed (KTSP61-KTT72) specifically produced a product of approximately590 bp from HS causing capsular type B isolates of P. multocida(Fig. 1) which was similar to that reported for species specificPCR (PM-PCR) assay for P. multocida (Townsend et al., 1998).

M 1 2 3 4 5 6 7 8

500 bp

1 kb

Fig. 1: PCR amplification. M= 1kb DNA ladder, L1= negative control for KTT primer, L2-4: amplifiedproduct (590 bp) of KTT primers, L5-7= amplified product (460 bp) of KMT primers & L8: Negativecontrol of KMT primers.

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Phenotypic bacteriological analysis of three isolates fromdifferent samples of PM cases and PCR results revealed thatthese isolates could be assigned to the species Pasteurellamultocida sub sp. multocida, of serotype B:2. Various otherworkers have also reported the amplification of the KMT-1 genefor species specific detection of P. multocida cells (Anupamaet al., 2003; Dutta et al., 2004).

The bacterium, Pasteurella multocida predominantlycauses respiratory diseases and septicaemia, which havebeen correlated in different animal species with one of the fiverecognized capsular serogroups (serogroups A, B, D, E, andF) (Rimler and Rhoades, 1989). In Indian subcontinent, isolatesof capsular types B are well documented to be associatedwith haemorrhagic septicaemia (HS) in cattle and especiallywater buffaloes. Opportunistic pathogen such as P. multocidais commonly present in the nasal cavity, and is kept to safelevels by immune status of animal, however, some animalsare carriers of pathogenic strains, maintaining the organismin the lymphatic tissues associated with the upper respiratorytract, and periodically shedding it in nasal secretions. Whensuch animals gain entry into an immunologically naïve herd,the pathogenic B serotypes organism spread rapidly,transmitted by ingestion-contaminated feed and water,inhalation through aerosols or direct contact. Isolation of B2serotype pasturellae from nasal swabs showed activesecretion of the organism in sick buffalo calves, however,immune status of other vaccinated calves may be the reasonfor restricted spread of disease in the herd. As such an excretionmay be triggered by stress like hot and humid weather, whichin this case was also evident due to hot weather, which mighthave triggered the outbreak. High environmental temperature(37°C) does favour the growth of the bacteria (Hajikolaei et al.,2008). We were successful in isolation of bacteria in pureculture from intestinal contents, as apart from classical forms,like pulmonary and oedematous forms, intestinal forms of HSare also reported on Indian sub-continent. Case report fromOrissa describes signs of pyrexia, muco-purulent nasaldischarge in isolated cases (Nayak et al., 1999). In this casealso, peracute mortality of pyrexic calves and isolation of P.multocida from intestinal contents indicate a predilection forintestinal tract. The serotypes that cause HS are probably shedinto the oropharynx. Dabo et al. (1999b) reported isolation of P.multocida in the digestive tract and upper respiratory tractmucosa of healthy cattle. The pathogenesis of HS is not wellunderstood (Brickell et al., 1998). It would be informative tostudy the gastrointestinal tract pathogenesis of B2 type P.multocida in ruminants.

Traditionally, diagnosis of Pasteurella is based on clinicalfindings, culture and/or serological testing. Although cultureidentification methods are time consuming and costly, theyare definitive, and provide an opportunity for epidemiologicalstudies to be conducted, provided that cultures are preservedby submission in a cultural repository. Molecular detection byPCR, however, offers a highly sensitive, accurate and timelymethod for detecting Pasteurella and confirming currentinfection (Miflin and Blackall, 2001). The clinical picture of acutedistress in buffalo calves, followed by mortality and isolationand biochemical and molecular confirmation of P. multocidaconfirms that the buffalo calves died due to Haemorhhagic

septicaemiaAntimicrobial sensitivity results indicate the isolates were

sensitive to gentamicin, kanamycin, erythromycin, amikacin,cloxacillin, ampicillin, amoxycillin, tetracycline, chloramphanicol,enrofloxacin, cephalothin, ceftazidime, and furazolidone,however, resistance was shown against norfloxacin. In Europe,Pasteurellaceae have been included in the national networkfor the monitoring of antimicrobial resistance within the mainpathogenic bacteria for ruminants (Martel et al., 1995).Antimicrobial susceptibility testing on P. multocida has beenhighly recommended owing to plasmid bearing resistance tosulpha drugs (Quinn et al., 1994). There have been numerousstudies that have reported antimicrobial susceptibilities amongPasteurella species, and, in India, studies has also revealedthe emergence of multidrug-resistant strains of P. multocidaamong poultry and sheep (Shivachandra et al., 2004; Vaid etal., 2008). Various levels of sensitivities of Pasteurella spp. todifferent classes of antimicrobials have been reported (Rajiniet al., 1995; Arora et al., 2005). The presence of Pasteurellaisolates in an environment with pathogens of diferent generaprovides a chance of acquisition of resistance imparting genes(DeFlaun and Levy, 1989). Penicillin is the drug of choice fortreatment of Pasteurella infections, and in case of resistance,third-generation cephalosporins and fluoroquinolones aregood alternatives (Zurlo, 2000), however, in this case, resistanceto flouroquinolones has been detected. Genes for resistanceto flouroquinolones have been reported abroad in Pasteurella(Sellyei et al., 2009).

The restricted nature of HS in the closed herd of calveswith disease occurring only in 3 cases with peracute mortalityis unlike the conventional epidemiological picture, however,as the vaccines currently being used consist of whole cellformalin killed bacterin prepared from strain P52 of P. multocidawith an oil adjuvant, which provide good protection andremarkable thermostability at room temperature (Nangia etal., 1966); the infection in calves may be attributed tomanagement.

Haemorhagic septicaemia is probably the most importantbacterial disease affecting large ruminant population in India,and resulting in enormous economic loss due to dairy animalmorbidity and mortality (Singh et al., 1996). It has beenconsidered to account for about 58.77% of the total bovinemortalities due to five major diseases (Dutta et al, 1990). Fifty-three thousand bovine deaths in India are attributed to thisdisease annually (Dutta et al., 1990). The occurrence of B2serotype related with HS in buffaloes have been confirmed bymany authors (Kumar et al., 1996; Dutta et al., 2011). The per-acute nature of the disease and the brief time period from theonset of clinical symptoms to death, coupled with generallynon-specific clinical and post-mortem findings may invariablyconfound the true incidence of HS in the field. For effectivecontrol of disease an accurate diagnosis of HS is important. Itis also necessary that the bacterial isolates from HS casesneeds to be collected with geographical, meteorological andclinical metadata collection, in order to better understand thenature of disease through epidemiological and geneticinferences drawn by their analysis. The ex-situ availability ofisolates can also help us to draw inferences about antimicrobialresistance trends of P. multocida.

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Accession numbers: The Pasteurella multocida sub spp.multocida isolates described in the study are deposited inVTCC, NRCE Hisar with Accession number(s) VTCCBAA264,VTCCBAA265 & VTCCBAA266.

AcknowledgementsThe facilities provided by Director, NRCE, Hisar, to carry

out the work is thankfully acknowledged.

ReferencesAnupama, M. et al. (2003) Indian J. Ani. Sci. 73: 166-167.Arora, A. K. et al. (2005) Indian J. Ani. Sci. 75: 749-52.Ataei, S. et al. (2009) Res. Vet. Sci. 87: 207-210.Bain, R. V. S. et al. (1982) FAO Animal Production and Health. Paper

No. 33. FAO, Rome, Italy.Bauer, A. W. et al. (1966) Am. J. Clin. Pathol. 45: 496.Brickell, S. K. et al. (1998) Vet. Microbiol. 59:295-307Cowan, S. T. and Steele K. J. (2004) Manual for the Identification of

Medical Bacteria. 3rd ed. Eds. G. I. Barrow and R. K. A.Feltham. Cambridge University Press, London.

Dabo, S. M. et al. (1999b) J. Med. Microbiol. 48:279-86De Alwis, M. C. L. (1992) Br. Vet. J. 148:99-112.DeFlaun, M. F. and Levy, S. B. (1989) In: Levy S B, Miller R V. (Eds.),

McGraw Hill New York, 1-32.Dousse, F. et al. (2008) J. Vet. Diagn. Invest. 20:716-724.Dutta, T. K. et al. (2011) Indian J. Anim. Sci. 81: 242-244.Dutta, J., et al. (1990) Indian Vet. J. 67:893-899.Dutta, T. K. et al. (2004) Ind. J. Comp. Micrbiol. Immunol. Infect. Dis.

72: 752.

Hajikolaei, M.R.H. et al. (2008) J. Vet. Res. 63: 25-29.Kumar, A. A. et al. (2004) Vet. Res. Commun. 28: 657-667.Kumar, A. A. et al. (1996) Ind. J. Comp. Micrbiol. Immunol. Infect.

Dis. 17:120-24.Kumar, A. et al. (2005) Vet. Pract. 6: 119-122.Kumar, A. A. et al. (2004). Vet. Res. Commun. 28: 657-667.Martel, J. L. et al. (1995) Microb. Drug. Resist. 1: 273-283.Harper, M. et al. (2006) FEMS Microbiol Lett. 265:1-10Miflin, J. K. and Blackall, P. J. (2001) Lett. Appl. Microbiol. 33: 216-221.Nangia, S.S. et al. (1966) Indian Vet. J. 43:280-287.Nayak, B. C. et al. (1999) Indian J. Ani. Sci. 69: 664-666.Pasteur, L. (1880) C. R. Acad. Sci. 91: 673-680.Quinn, P. J. et al. (1994) Clinical Veterinary Microbiology. Mosby

International Limited, Wolfe, London. pp. 254-258.Rajini, R. et al. (1995) Indian Vet. J. 72: 115-118.Rimler, R. B. and Rhoades, K.R. (1989) Pasteurella multocida. In C. Adlam

and J. M. Rutter (ed.), Pasteurella and pasteurellosis. AcademicPress Limited, London, United Kingdom. pp. 37-73.

Rimler, R.B. and Wilson, M.A. (1994) Vet. Rec. 134:256.Sellyei, B. et al. (2009) Acta Vet. Hung. 57:357-67.Shivachandra, S.B. et al. (2004) Trop. Ani. Health. Prod. 36:743-50.Singh, V. P. et al. (1996) International Workshop on diagnostics and

control of HS. May 28-30. 1996. Bali, IndonesiaTownsend, K. M. et al. (1998) J. Clin. Microbiol. 36: 1096-1100.Vaid, R. K. et al. (2008) Vet. Pract. 9(1): 32-26.Venkataramanan, R. et al. (2005) Indian J. Ani. Sci. 75: 456-464.Zurlo, J. J. (2000) Pasteurella species. In: Mandell, Douglas and

Bennett’s principles and practice of infectious diseases 5th

ed. Eds. Mandell, G. L., Bennett J.E., Dolin, R. (ChurchillLivingstone, Philadelphia, Pa), pp. 2402-2406.

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IntroductionNitrates (NO3) are generally not much toxic to animals but

their reduced form i.e. nitrite (NO2) is very toxic to animals.Nitrate poisoning in animals occurs only when the nitratecontent of feed in rumen exceeds the capacity of animal toconvert it into non-toxic compounds and their excretion as urea.Some plant species such as sorghum, oats etc. are naturallyknown to be nitrate accumulators; their consumption in highquantity may cause sudden deaths in animals (Burrows et aI.,1989). The present report deals with an outbreak of nitratepoisoning in buffaloes in Haryana state.

Case history and observationsThis outbreak was reported to the Department of Veterinary

Public Health and Epidemiology, College of VeterinarySciences, LLRUVAS, Hisar, with a history of ad lib feeding ofsorghum, oats and berseem to the buffaloes. A total of ninebuffaloes were affected in 5-6 households in a vicinity inSaffidon, Jind district (Haryana), The affected buffaloes showedthe symptoms of laboured breathing, dyspnoea, subnormalbody temperature and excessive salivation. Clinicalexamination of three animals also revealed cyanosis ofmucous membrane of eyes. Blood was collected from jugularvein from these three animals for haematological studies suchas haemoglobin, PCV, DLC and for cultural examination.Haematological parameters (haemoglobin, PCV, DLC) werefound in the normal range. Cultural examination of blood ontryptose soya agar did not reveal bacterial growth, ruling outany septicaemic cause. However, the change of colour ofvenous blood from reddish brown to bright red on exposure toair was observed. Based on history, clinical findings, changeof blood color and laboratory examination; the tentativediagnosis was made as nitrate poisoning.

Treatment and DiscussionAccordingly, the affected animals were given treatment

specific to nitrate poisoning. Methylene blue @ 4mg per kgbody weight was administered as 1% solution as an antidotealongwith 4-5 litres of normal saline solution via theintravenous route. This treatment was repeated after 4 hours.In addition, activated charcoal (1ml/kg body weight; KoalTM)was given as a drench to all the affected buffaloes. The treatedanimals showed dramatic improvement after 24 hours oftreatment. Response to therapy confirms our diagnosis asnitrate poisoning. In present outbreak, none of the affectedanimals died. Cases of nitrate poisoning had also beenreported from Punjab, (Sidhu et al., 2011), Australia (Mckenzieet al., 2004), Canada (Yong et al., 1990) and New Zealand(O’Hara and Fraser, 1975) in diary animals.

AN OUTBREAK OF NITRATE POISONING IN BUFFALOES IN HARYANA

G. Narang, Davinder Singh1, D. Arora , N. Jindal and N.K. MahjanDisease Investigation Laboratory

Department of Veterinary Public Health and EpidemiologyCollege of Veterinary Sciences

LLR University of Veterinary and Animal Sciences, Hisar-125004, Haryana, India

The farmers were advised to strictly follow therecommendations for use of nitrogenous fertilizers in the fields.According to a report, nitrate contents in ground water in partsof, Haryana including Jind district from where these caseswere reported, are higher than the permissible limits(Government of India, 2010). In such situations, the probabilityof crops having more nitrate contents is higher. Feeding ofcrops with higher nitrate contents would increase the chancesof poisoning in ruminants. Because of higher nitrate content incrops like sorghum, oats and toriya, the farmers were alsoadvised to use these crops by mixing with other crops havinga low amount of nitrate so as to reduce the chances of toxicityin dairy animals. Such type of feeding system has been reportedby Sidhu et al. (2011) as a measure to reduce the incidence ofpoisoning in animals. Epidemiological investigations alsorevealed that this region (Saffidon in Jind District) has largenumber of commercial broiler chicken farms along with largenumber of hatcheries which would produce huge quantity ofpoultry manure. According to Radostits et al. (2007), excessivepoultry litter/animal manure is known to build up nitrates in thesoil which gets stored in forage plants thereafter. The excessiveuse of poultry manure in the fields was later confirmed by thefarmers of the village. Hot environmental conditions in monthof April in combination with low moisture content of soil mighthave led to higher concentration of nitrates in forage as alsoreported by Kahn (2005). In very hot summer, the nitrification ofbacteria in soil increases many folds which may lead to nitrateaccumulation in fodder (Sidhu et al., 2011).

Though the cases were reported only from one village,the incidence may be higher in domestic animals which maygo unnoticed due to poor reporting system. Thus there is aneed to give more attention for judicious use of nitrogenousfertilizers particularly in Northern India, which is known as foodbasket of India. Further surveillance and data recording onnitrate contents of feed, forages and soil and their effects onanimals particularly ruminants are needed using integratedapproach.

ReferencesBurrows, G.E. and Tyrl, R.I. (1989) Vet. Clin. North Am. Food Prac. 5: 263.Government of India (2010) Central Ground Water Board, Ministry of Water

Resources, Faridabad, Haryana. pp. 17.Kahn, C.M. (2005) The Merck Veterinary Manual. 9th ed. New Jersey.

Merck & Co., Inc. pp. 2423.Mckenzie, R.A. et al. (2004) Aust. Vet. J. 82(10): 630.O’Hara, P.J. and Fraser, A.J. (1975) New Zealand Vet. J. 23(4): 45.Radostits, O.M. et al. (2007) Veterinary Medicine. 10th ed. W B Saunders

Company Ltd. London, U.K. pp. 1855.Sidhu, P.K. et al. (2011) Toxicol IntI. 74(1): 22.Yong, C. et al. (1990) Can. Vet. J. 31: 118.

1Corresponding author: [email protected]

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Pyometra is a potentially life threatening conditionassociated with cystic endometrial hyperplasia. Cysticendometrial hyperplasia is an abnormal uterine response thatdevelops during dioestrus (luteal phase of cycle) (Fossum,2007). The risk of an intact bitch developing pyometra before10 yrs of age is 23-25% (Egenvall et al., 2001). Pyometra hasa two-step pathogenesis. The primary insult is the developmentof cystic endometrial hyperplasia (CEH), the bacterial infectionbeing the secondary one. CEH is a hormone- dependentchange that develops after repeated oestrus cycles in bitches.This may culminate in to septic shock and renal failure.

A five-and-half-year-old Spitz bitch was presented at thedepartment of Veterinary Surgery and Radiology, RanchiVeterinary College with the anamnesis of vaginal dischargefor past six months. Further, clinical examination revealedmucopurulent and haemorrhagic vulvo-vaginal greenishdischarge with foul odour, which was suggestive of cervicalpatency. The bitch was pyrexic, lethargic, and anorexic, exhibitedslightly distended abdomen, dysuria and dyspnoea, too, wereevident to a milder extent only. An abnormality/ irregularity of theoestrus cycle was pin-pointed by the anamnesis.

Radiography was done to arrive at the diagnosis but thiscould not yield much in undermining the uterine pathology.Based on the clinical symptoms manifested, the case wastentatively diagnosed as cystic endometrial hyperplasia-pyometra complex. The confirmatory diagnosis was arrived atby CBC (Complete Blood Count), serum chemistry profile andurinalysis.

CYSTIC ENDOMETRIAL HYPERPLASIA (CEH) - PYOMETRACOMPLEX IN A SPITZ BITCH

A.K. Sharma1, Hemant Kumar2 and Dayanand Turi3Ranchi Veterinary College, Birsa Agricultural University, Kanke, Ranchi-834006, Jharkhand, India

The CBC revealed marked leucocytosis (32,000/μL) withslight monocytosis (5%) and neutrophilia (80%). Thishaematological finding corroborates with the findings ofFaldyna et al. (2001) and AJadi et al. (2008). Parameters viz.alkaline phosphatase, ALT, total protein and erythrocytesedimentation rate (ESR) were found to be slightly elevated.

Bearing the above-dealt points in mind, OHE(ovariohysterectomy) was advocated. The food for 12-18 hoursand water for 4-6 hours was withheld. Positioning the dog inthe dorsal recumbency, entire ventral abdomen was preparedfor an aseptic surgery. Pre-operatively, Inj. Ceftriaxone @ 10mg/kg b.wt; i/v and Inj. Prednisolone @15-30 mg/kg b.wt; i/vwere administered. After due atropinisation, the patient wasput under general anaesthesia using the combination ofxylazine and ketamine @ 1 mg/kg b.wt i/m and 5 mg/kg b.wt;i/v, respectively.

Ovario-hysterectomy was performed through ventralmidline incision. The laparotomy wound was closed as routinesurgical procedure. Ovario-histerectomy has traditionally beenthe treatment of choice for pyometra, however, it renders theanimal infertile (Tobias and Wheaton, 1995).

Post-operatively, Inj. Ceftriaxone @ 10 mg/kg b.wt; for 7days and Inj. Meloxicam @ 0.5 mg/kg b.wt. for 3 days wasadministered intramuscularly. Daily antiseptic dressing of thesurgical site was undertaken using povidone iodine till healingwas appreciable. Dextrose normal saline @ 200 ml twice inday were administered for two days after surgical intervention.The sutures were taken off on 10th post-operative day with an

Fig. 1: Photograph showing multiple small well defined nodularlesions

Fig.2: Photomicrograph showing hypertrophy of myometriumalong with the cystic lesion (H& E)

1Asstt. Professor and corresponding author. Email: [email protected],3M.V.Sc Scholar, College of Veterinary Science & Animal Husbandry, Ranchi-6

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uneventful recovery of the patient. The reoccurrence was notreported within the follow up period extending for twomonths.

Grossly, the uterine wall showed multiple small well-defined nodular lesions with no observable primary growth(Fig.1). Histopathologically, features of adenomyoma wereobserved with the presence of well-differentiated glandularacini and small cystic lesions embedded in the smooth muscletissue, which is suggestive of cystic endometrial hyperplasia.There was marked myometrial thickening and endometrialhyperplasia (Fig. 2). Pyometra develop as a result of complexetiological factors, which include hormonal influence on uterineenvironment and virulence of the infective bacterial (Frances,2006). Ovariohysterectomy, alone, is reported to cause theresolution of clinical symptoms of pyometra in dogs in 5-15days (Dow, 1957).

The successful surgical management of a clinicallypresented case of CEH-Pyometra in a Spitz bitch byovariohysterectomy in the light of no reported reoccurrencewithin the follow-up period of two months.

ReferencesAjadi, T. A. et al. (2008) Indian J. of Vet. Surg. 29(2):123-125.Dow, C. (1957) Vet. Rec. 69:1409-1415.Egenvall.A. et al. (2001) J. Vet. Intern. Med.15:530Faldyna, M. et al. (2001) J. Small Anim. Prac. 42:5-10.Fossum,T.W. (2007) Surgery of the Abdominal Cavity. In: Small Animal

Surgery. 3rd ed. By Fossum T.W. (eds)., Mosby, St. louis,Missouri. pp. 327.

Frances, O.S. (2006) Theriogenol. 66:610-612.Tobias, K.M.S. and Wheaton, L.G. (1995) Sem. Vet. Med. Surg. (Small

Anim.). 10:30-34.

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IntroductionBrucellosis is the worldwide contagious disease of

zoonotic importance in goats. The disease is characterized byabortion, retention of placenta, decreased milk yield andsubsequent high rate of infertility. During last 3-4 decades,brucellosis in small ruminants has gained much attentionbecause of its role in spread of infection to cattle and humanbeings. Seroprevalence of caprine brucellosis has beenreported from different parts of India and abroad. Thegastrointestinal parasitism causes ill health, high morbidityand sometimes high mortality. Gastrointestinal nematodesare the major cause of impaired productivity in small ruminants(Gordern, 1974; Gupta et al., 1986). Parasitic gastroenteritisdue to gastrointestinal helminthiasis is widely prevalentpathological condition affecting mainly growing kids. It causesdiarrhoea, debility and severe economic losses due to highmorbidity in growing stock and mortality in young stock.

Materials and MethodsThe study was carried out on 300 goats (200 were

between 4-6 month age and were classified as young kids;and 100 were above 6 months age and were classified asadults) brought for slaughter at local abattoir of Bikaner. Bloodsamples were of goats were collected into sterile test tubes atthe time of slaughter from Juglar vein. Serum was separatedand stored at -20ºC till analyses. The procedure suggested byMorgan et al.(1969) was used for RBPT. Coloured antigenprocured from biological products division, IVRI, Izatnagar, U.P.STAT was performed as described by the Alton et al. (1975).Brucella abortus plain antigen procured from biologicalproducts division, IVRI, Izatnagar, U.P. Representative freshfaecal samples were collected from these goats and wereexamined by direct smear method, sedimentation techniqueand floatation method using saturated sodium chloride as perSoulsby (1982).The intensity of Strongyle infection wasassessed by modified Mc-Master technique as suggested byColes et al. (1992).

Results and DiscussionOut of 300 serum samples 18 (6%) were found positive

PREVALENCE OF BRUCELLOSIS AND PARASITICINFESTATION IN GOATS IN BIKANER

A. Maheshwari, Fakhruddin, R.K Tanwar, A. Chahar and A.P. Singh1

Department of Epidemiology and Preventive Veterinary Medicine Veterinary College of Veterinary and Animal Science ,Bikaner 334001

ABSTRACT

Seroprevalence of brucellosis in 300 goats was carried out by performing Rose Bangal plate test and Standard Tube AgglutinationTest. Out of 300 serum samples 18 (6%) were found positive for brucellosis by STAT and 30 (10%) by RBPT. For detection ofgastrointestinal prevalence, faecal examination of 300 goats carried out. A total of 210 goats were found infested with gastrointestinalhelminthosis and Eimeria oocysts infection. The eggs of strongyle spp. and Trichuris spp. were recorded. The overall range of eggs/oocysts per gram of faeces was 800-12000.

Key words: Goats, seroprevalence, brucellosis, STAT, RBPT, gastrointestinal parasites.

1Associate Professor, Department of Clinical Veterinary Medicine, Ethics and Jurisprudence

for brucellosis by STAT and 30 (10%) by RBPT. All the sampleswhich were positive by STAT were also found positive by RBPT.This suggests that RBPT can be used as rapid screening testfor brucellosis in field condition, as also reported by Ghani(1995). A titre value of 40 IU or above was considered positivefor brucellosis whereas 20 IU titre was considered as doubtful(Kapoor et al., 1985). The adult goats showed higherseroprevalence of 12% by RBPT as compared to 4-6 months(9%) age group. Congenital infection may occur in calves bornfrom infected dams but its frequency is low. Latent infectioncan also be acquired from the ingestion of infected colostrumand milk and this may be the reason of presence of positivereactors in young ones (Radostits et al., 2000). Similar to thesefindings Rajeshwari et al. (1996) reported 8.27 and 4.43%seroprevalence of brucellosis in goats by using RBPT andSTAT, respectively. Thakur et al. (2004) also reported 5.15% ofseroprevalence of caprine brucellosis in Bihar using STAT.

The animals of adult age group goats showed higherseroprevalence (8%) by STAT in comparison to 4-6 monthsage group (5%) animals. Chandra et al. (2005) reported higherprevalence in adult (1.65%) than in young goats (0.58%).Susceptibility to brucellosis in adult animals might be attributedto their breeding suitability. Sexual maturity is attained in adultage. Different stress factors may also contribute to increasedsusceptibility of animal towards infection due to release ofnaturally occurring corticosteroids which are stronglyimmunosuppressive in nature (Wadhwa 2007).

On the basis of faecal examination, an overall prevalenceof 70 % (210) mixed infection of helminths and Eimeria spp.was found. The range of helminths eggs per gram of faeceswas 100-1600 and Eimeria oocysts 800-12000. In the presentstudy, mixed infection of Eimeria and helminths infestationwas recorded in 210 goats. Out of these, 30 samples (14.28%)contained Strongyle eggs. No attempt was made to differentiateeggs of different spp. Strongyle infestation in overall was 10%(30/300) which is similar with findings of Agarwal et al. (2004)recorded it as 7.8-47.7% in different districts of Madhya Pradesh.

In 180 faecal samples, Eimeria oocysts were presentalong with eggs of other helminths. The overall prevalence

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of Eimeria oocysts infection in the present study was 60%(180/300). Similar to these findings Maichomo et al. (2001)reported 45% prevalence for Eimeria oocysts. Findings of 60%prevalence of Eimeria spp. in present study is similar withfinding of Gul et al.(2008) who reported 60.54% prevalence ofEimeria species in goats from Hakkari province, Turkey. Kaurand Kaur (2008) also reported high prevalence (85.71%) ofmixed helminths and protozoan infection in sheep and goatsin Patiala and its adjoining area. Trichuris spp. eggs were alsopresent in 15 faecal samples along with Eimeria spp. oocysts.The overall prevalence of Trichuris spp. infestation was 5%(15/300) with range of 100-800 eggs per gram of faeces.Mamatha and D’Souza (2007) reported similar results ofTrichuris spp. infestation with 5.6% prevalence in goats fromdifferent districts of Karnataka. Animals of 4-6 months agegroup showed mixed infection of 80% (160/200) while adultage group goats showed 50% (50/100) over all prevalence.This might be due to that kids maintained with adults are likelyto get infected by oocysts (sporulated) during suckling, lickingand through contaminated food and water. Perhaps dry climaticconditions of Bikaner with high environmental temperature andvery low rainfall might have produced the unfavourableconditions for the development of the parasites and contributeto keeping low profile of parasitic infestation in this area. Therate of prevalence and intensity of various gastrointestinalparasities is severely affected during drought conditions(Chauhan et al., 1981). Similar to present findings Padmaja etal. (2007) also reported 13.91% prevalence of gastrointestinalparasites in goats during pre-monsoon season. They alsoreported 5% overall prevalence of Trichuris spp.

ReferencesAgrawal, M.C. et al. (2004) J. Vet. Parasitol. 18(2):147-149.Alton, G.G. et al. (1975) Laboratory technique in brucellosis. 2nd ed.

Monograph series No. 55, W.H.O, Geneva. pp. 163.Chandra, M. et al. (2005) Indian J. Anim. Sci. 75(2):220-221.Chauhan, P.P.S. et al. (1981) Indian J. Vet. Med. 5:108-117.Coles, G.C. et al. (1992) Vet. Parasitol. 44:35-44.Ghani, M. (1995) Indian Vet. J. 72:976.Gorden, H.M.C.L. (1974) Parasite penalties on production. Proc. Aust.

Soc. Anim. Proc. 10:180.Gul, A. et al. (2008) Indian Vet. J. 85: 564-565.Gupta, R.P. et al. (1986) Indian Vet. J. 63:449-453.Kapoor, P.K. et al. (1985) Indian J. Comp. Microbiol. Immunol. Infect.

Dis. 6(2-3) 96-101.Maichomo, M.W. et al. (2001) Onderstepoort J. Vet. Res. 71(4 ):257-

261.Mamatha, G.S. and D’Souza P.E. (2007) Intas Polivet. 8(1):112-114.Morgan, W.J.B. et al. (1969) Vet. Rec. 85: 636.Padmaja, K. et al. (2007) Intas Polivet. 8(1):115-116.Radostits, O.M. et al. (2000) Veterinary Medicine. A textbook of the

disease of cattle, sheep, pig, goats and horse. 9th ed.,Baillere Tindall, Great Britain. pp. 1004.

Rajeshwari, S. et al. (1996) Indian J. Comp. Microbiol. Immunol.Infect. Dis. 27:1.

Soulsby, E.J.L. (1982) Helminths Arthopods and Protozoa ofdomesticated animals. 7th ed. ELBS Bailliers Tindall andCassell, London. pp. 279.

Thakur, A. et al. (2004) Indian J. Comp. Microbiol. Immunol. Infect.Dis. 25(2): 124-125.

Wadhwa. A. (2007) Survey of Brucellosis, IBR, PPR andgastrointestinal helminthoses in cattle and buffaloes inand around Bikaner. M.V.Sc. thesis submitted to RajasthanAgricultural University, Bikaner-334001 (Raj.).

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IntroductionFoetal ascites is one of the dropsical conditions that

cause dystocia in many species but occurs more frequentlyin cows. It is usually associated with accumulation of fluidin the uterus and mesotheliomas of the foetal abdomen(Roberts, 1971). The present report describes a case ofdystocia due to foetal ascites in a murrah buffalo which wasrelieved through partial foetotomy.

Case history and observationsA 5-year-old Murrah buffalo in her second parity was

presented to the Veterinary Clinics and Teaching Hospital,R. S. Pura, Jammu, with difficulty in parturition for the previous6 h after the rupture of allantochorion. The case wasunsuccessfully handled by a local veterinarian. Head of thefoetus was found hanging from the birth canal and both fore

PERVAGINAL DELIVERY OF ASCITES FOETUS IN BUFFALO-ACASE REPORT

Anil Kumar Pandey*1, Sharad Kumar1, M. Mutha Rao2, Utsav Sharma2,Sudershan Kumar2 and S. K. Gupta3

Division of Veterinary Gynaecology and ObstetricsFaculty of Veterinary Sciences and Animal Husbandry (SKUAST-J)

R. S. Pura, Jammu, India

limbs were amputated from shoulder joint. A detailed pervaginal examination revealed fully dilated birth canal and adead calf with an abnormally distended abdomen. Theanimal was active and there was no straining.

Treatment and DiscussionIn the present case, since delivery by direct traction was

not feasible due to distended abdomen of the foetus, theabdomen of the foetus was punctured using a longconcealed knife. About 25 litres of straw coloured asciticfluid was siphoned off from foetal abdominal cavity using aPVC pipe. Then the foetus was delivered per vaginally bytraction and the placenta was also removed manually. Theanimal was administered four litres of normal saline fluid i/v along with inj. dexamethasone, antibiotic and antipyreticsto prevent temperature shock, pain and infection. Theanimal was released from the clinic after two hours of deliveryof dead ascetic foetus. Gross examination of the foetusrevealed an abnormally distended abdomen with atrophiedlimbs (Fig. 1). Part of large intestine had everted out throughthe foetal rectum. The placenta was leathery in appearancewith several necrotic foci. On the basis of distendedabdomen which was filled with straw coloured fluid the casewas diagnosed as foetal ascites (Honparkhe et al., 2003).Vidya Sagar et al. (2010) had reported a similar case relievedby caesarean operation in buffalo.

ReferencesHonparkhe, M. et al. (2003) Indian J. Ani. Reprod. 24: 83-84.Roberts, S.J. (1971) Veterinary Obstetrics and Genital Diseases.

2nd ed. CBS Publishers, New Delhi.Vidya Sagar, P. et al. (2010) Buffalo Bulletin. 29(2):73-74.

Fig. 1: Ascitic foetus

*Corresponding author: E mail: [email protected] Professor2Associate Professor3Dean, F.V.Sc. & A. H.

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IntroductionThe application of bacteriological surveillance in

management of equine semen meant for AI is not a routinepractice. However, as the semen could act as a vehicle for thewide distribution of pathogens, therefore, it acquires importanceto verify the microbial contamination of semen in order to findout the risk involved in transmission of pathogens to the marein AI. Moreover, increasing international trade in germplasmemphasizes the need for rapid, sensitive, and specificdiagnostic tests for certification of semen free from anincreasing list of pathogenic agents (Metcalf, 2001).

The method of stallion semen cryopreservation is of greatimportance (Reger et al., 2003) as the utilization and importanceof frozen semen is increasing in AI (Loomis, 2001).

However, various non-standard methods have beendescribed for stallion semen production (Graham, 1996;Devireddy et al., 2002; Alvarenga et al., 2005). Normally, thesemen is produced and frozen near equine reproductioncentres where it is stored in small containers: 0.25 or mostly0.5 ml straws filled with about 240×106 progressively motilespermatozoa showing not less than 30% progressive motility(Vidament et al., 1997; Barbacini et al., 1999; Loomis, 2001)and then it is plunged in liquid nitrogen (LN2) for an indefinitetime. Although it is advisable to use aseptic techniques, sterileequipments, and general hygienic measures to collect thesemen; however, the general managemental conditionsprevailing at the time of semen collection in tropical conditions

MICROBIAL QUALITY OF FRESH AND FROZEN EQUINESEMEN OF INDIAN HORSES

R. K. Vaid*1, A. Arangasamy3, T. Talluri4, S. Ravi4, B. C. Bera2, T. Anand2,T. Riyesh2, N. Virmani5, P. Malik6 and R. K. Singh7

National Research Centre on Equines, Sirsa Road, Hisar-125 001, Haryana

ABSTRACT

Artificial insemination (AI) is a practical method for horse breed improvement and equine conservation. The semen can be collectedfrom stallion and stored for varying periods at low temperatures. The semen can then be distributed to the near and far-off locations,throughout the country and can even be exported to other countries. However, even though it is a useful tool for various reproductivemanagement practices and for utilization of genetic potential of desired stallions, the semen could act as a vehicle for the widedistribution of pathogens. We describe a qualitative and quantitative analysis for bacterial contamination of fresh and frozen equinesemen produced on a farm managed on modern practices. Semen samples (11) collected from 11 stallions were divided into 11 freshand 11 frozen semen samples. Fresh semen samples and samples from straws were checked for aerobic pathogenic and non-pathogenic bacteria, and fungi (moulds and yeasts). The total microbial counts (TMCs) were quite variable with an average TMC of2.1×105 CFU/ml in fresh semen and 1.7 x 104 CFU/ml in frozen semen. The significant microbial pathogens of venereal importanceidentified were Pseudomonas spp., Nocardia spp., and Escherichia coli. Other frequently isolated bacteria were Corynebacteriumspp. and those belonging to Gram-negative glucose non-fermentor group. Culturing of Pseudomonas aeruginosa indicates coloniza-tion of exterior genitalia by this opportunistic pathogen. Presence of Nocardia and glucose-non-fermenter group indicates a sourceof environmental contamination. The knowledge of equine frozen semen microbial quality is essential to check out transmission ofvenereal disease and improve the quality of cryopreserved germplasm.

Key words: Stallion, semen, bacteria, Nocardia, Pseudomonas

1*Senior Scientist, Corresponding author, [email protected], 3Senior Scientist; 4Scientist, Equine Production Centre, Jorbeer, NRCE, Bikaner, Rajasthan; 5Senior Scientist;6Principal Scientist; 7Director, Veterinary Type Cultures Collection, National Research Centre on Equines, Sirsa Road, Hisar- 125 001, Haryana, India.

may lead to contamination of semen. In such cases,bacteriological examination also helps to indicate the leveland the source of contamination. When semen is required forAI, the frozen semen dose is thawed and it is utilized directly forAI. The bacteriological counts and controls on fresh, frozen,and on fresh and cooled semen are hardly mentioned(Clément et al., 1993;1995; Pickett et al., 1999; Corona et al.,2006) and the application of bacteriological information are allbut not applied in handling of equine frozen semen (Loomisand Barbacini, 2005). The penis and preputial sheath area ofstallion are inhabited by a variety of commensal, opportunisticor pathogenic bacteria that contaminate sperm at ejaculation(Bristol, 1991; Tischner and Kosiniak, 1992; Varner et al., 1998;Pickett et al., 1999). Such microflora may be cultured from afresh stallion ejaculate as well as from frozen–thawed semen.Opportunistic bacteria such as Pseudomonas aeruginosa,Klebsiella pneumoniae, Streptococcus zooepidemicus ,Taylorella equigenitalis, if carried with AI, may cause infectionand improve inflammation leading to infertility in susceptiblemares (Parlevliet, 1997; Metcalf, 2001; Samper and Tibary,2006).

It is, therefore, important to assess the bacteriologicalquality of equine fresh and frozen semen in order to monitorthe type and species of microbes contaminating thegermplasm (Clément et al., 1993). Furthermore, its importancecan not be overemphasized as the semen is utilized forproduction purpose and contaminated semen may also lead

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to uterine contamination. Furthermore, the frozen semen ofimportant breeds may be shipped and exchanged within aregion or world (Metcalf, 2001; Bielanski et al., 2003; Levy et al.,2004).

Materials and MethodsSemen was collected from 11 stallions (S1-S11) of age 6-

12 years, 4 from stallion of Marwari breed, 4 from Jacks meantfor mule production and 3 (S9-S11) from Zanskari breed ponystallions kept ex-situ or equine conservation studies (Table 1).Fresh semen (S1-S11) was aliquoted into sterile 5 mlcontainers and immediately refrigerated to 4ºC and sent tolaboratory on ice for microbiological examination. The semensamples were also frozen at the equine production campus(S1-S11). Afterwards, it was automatically packaged in 0.5 mlstraws (0.5 cm3 semen straws), immersed in LN2 and storedfrom 1 to 2 years (2010-11). From 11 stallions, 11 fresh semensample ejaculates, and 11 frozen semen samples straws (22samples) were processed for bacteriological examination. Allfrozen samples were thawed in 37ºC water bath for 30seconds.

Bacterial concentration determinationThis calculation was carried out under sterile conditions:

1 ml of frozen–thawed and shaken semen was taken by asterile pipette and diluted 1:10 with cold sterile buffered peptonewater, vortexed vigorously for 1 min for thorough mixing of thesamples, serial decimal dilutions made in cold sterile bufferedpeptone water, and then 100 µl aliquots from each dilutionwere surface plated on Nutrient agar base added with 5%sheep blood in triplicates to calculate the total microbial counts(TMC). The plates were incubated at 37ºC for 48 h aerobically.The number of colonies multiplied by 1000 indicated thenumber of bacteria per ml. or colony forming units per ml (CFU/ml) of semen.

Determination of microbial parametersAnother 100 µl of cold sterile buffered peptone solution

was layered for the following microbial parameters:Staphylococcus spp. in mannitol salt agar (HiMedia),Streptococcus spp. in Columbia blood agar base (HiMedia),members of Enterobactericeae family and other Gram-negative rods in MacConkey (HiMedia), Pseudomonads onPseudomonas agar base (HiMedia), Taylorella equigenitalisin Chocolate agar (HiMedia), and other exigent rods in brainheart agar base (HiMedia). The plates were incubated at 37ºCand bacterial growth was recorded after 48 h. In the case of T.equigenitalis the plates were cultured at 37ºC in 5-10% CO2atmosphere and recorded after 48 h until 10 days. The isolateswere picked up randomly, purified and subjected to biochemicalidentification (Barrow and Feltham, 1993). Each strain isolatedin purity was tested for Gram-reaction, cellular morphology,and cellular arrangement using Gram-stain and modified ZiehlNielson stain. Colonies (Gram-positive and -negative) weretested for haemolysis on 5% sheep blood agar, catalase andoxidase activity (HiMedia), and motility. Bacteria were identifiedby biochemical and enzymatic assays (Barrow and Feltham,1993).

Mycological isolations Isolation of fungi spp. (moulds and yeasts) was carried

out in Sabouraud dextrose agar (Hi media) with antibiotics atabout 30ºC from 2 to 10 days. The identification was based onstructural and morphologic characteristics for moulds andyeasts and lactophenol cotton blue for staining (Beneke andRogers, 1971).

Results and DiscussionSemen samples (S1- S11) from all the eleven stallion’s

showed positive bacterial growth in both fresh semen as wellas frozen-thawed semen. There was a wide variation in theTMC (number of cfu/ml) (Table 1) with a wide variety of speciesof bacteria isolated (Table 2). The quantitative enumeration ofaerobic bacteria showed an overall bacterial count range from1 x102 in S1 to 9 x 105 cfu/ml in S10 frozen semen and 2.8 x103

in S2 to 2.6 x 106 cfu/ml in S10 fresh semen. We found anaverage TMC of 2.1×105 CFU/ml in fresh semen and 1.7 x 104

in frozen semen. Glance of Table 1 shows the variability in theTMC between 11 stallion’s frozen and fresh semen samples(among S1-S11). However, comparison of the TMC of freshand frozen semen revealed decrease in counts in frozensemen.

Table 1 also shows microbial counts on MLA plates withcounts in frozen semen samples ranging from 3 x 102 in S2 to2.1 x 104 in S10. The average count on MLA plates wascalculated as 5 x 103 and 5.2 x 103 in fresh and frozen semen,respectively.

Out of 22 equine semen samples, bacterialcontamination was detected in 100% (22/22) frozen and freshsamplings and a total number of 31 isolates distributed in 10different species were detected. A majority, 21 (68%) were Gram-negative and 10 (32%) were Gram-positive. For a group of 7isolates, it was not possible to identify them with theconventional methods employed.

It was observed that the bacterial counts were lower infrozen samples as compared to fresh semen samples. Of the22 samples examined, none was found to be sterile, and apredominantly mixed flora was isolated.

Most frequently isolated potential pathogen bacteria ofvenereal importance from the equine semen were:Pseudomonas spp. 8(24%) comprising of 4 eachPseudomonas spp. and Pseudomonas aeruginosa strains;Nocardia spp. 3 (9%), and Escherichia coli 2(6%). One isolateof Staphylococcus spp. was also identified. There wasisolation of 3 Corynebacterium spp. also. Of the remainingbacteria, Glucose non-fermentor group predominated; thesewere identified as Flavobacterium spp (1), Acinetobacter (2),Achromobacter spp (1) and Alcaligenes (3). The seven cultureswhich remained unidentified comprised of 4 Gram negativerods and 3 Gram positive rods. Among 2 fungus isolates thedetected moulds were: Aspergillus spp., and among yeasts,Candida albicans was isolated. Significantly, Staphylococcusaureus, Klebsiella pneumoniae, Rhodococcus equi, andTaylorella equigenitalis could not be detected.

The microbial load contamination studies of equinesemen are in themselves few and far between. In this paper,the microbial load contamination of Indian equine’s fresh andfrozen semen has been calculated and microbial isolatesidentified for the first time. Our study is also unique in that weare microbiologically comparing the same set of 11 equinesemen ejaculates obtained from equine of Indian breeds,

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Table 1: Microbial counts of fresh and frozen equine semen

TM C-SBA TM C-M LA Bree d Stallion Fresh Froze n Fresh Froze n

M 1- Jack S1 4.1 x 104 1 x 102 5 x 102 ND Rag hu - M arwari S2 2.9 x 103 4 x 103 1.9 x 102 3 x 102 M 11- Jack S3 3.8 x 103 6.6 x 103 1.6 x 103 7.3 x 103 M 9- Jack S4 4.2 x 103 1.8 x 103 1.4 x 103 6 x 102 M 7- Jack S5 4.9 x 105 8 x 102 1 x 103 ND Paras- M arwari S6 3.4 x 105 7.4 x 103 1.1 x 104 3.2 x 103 Cha man - M a rw ari S7 6.1 x 104 4.8 x 103 3.3 x 102 2.3 x 103 Rajat - M arwa ri S8 2.2 x 104 2.4 x 104 9.2 x 103 9.1x 103 Z-1F -Za nskari S9 3.1 x 105 8x 103 ND 1.1x 103 Z-3F -Za nskari S10 5.5 x 105 5.2x104 2.5x 104 2.1x 104 Z-4F -Za nskari S11 5.5x105 8.5x104 5x102 2x103

Table 2: Bacteria isolated from different samples

Fresh sem en

Isolate Frozen semen

Isolate

S1 Noca rdia sp p.; Unid entified G ram n eg ative rods

S1 -

S2 Unid enti fied Gra m pos it ive rods; Pseud omo nas spp. ; Acinetob acter spp.

S2 Flavob acterium spp.

S3 Pseudom on as aerugin osa; Escherichia coli; Pseudom on as spp. ; Nocardia spp.

S3 Pseudom on as spp.

S4 Alcaligen es spp. S4 Achromob acter sp p.; Pseudom on as spp.

S5 Alcaligen es spp. S5 - S6 Pseudom on as aerugin osa Ac inetob acter spp. S6 Alcaligen es spp. S7 Aspergill us spp S7 Coryneb acterium spp.

Unid enti fied Gra m pos iti ve rods

S8 Pseudom on as aerugin osa S8 - S9 Noca rdia sp p.; Coryn eb acterium sp p. S9 - S10 Pseudom on as aerugin osa; Co ryneb acteri um

spp.; Escherichia coli ; 2 Unide ntified G ram neg ative rods

S10 -

S11 Can did a alb icans ; 1 Unid entifi ed G ram n eg ati ve rods & 1 Gram pos itive rods

S11 Staph ylococcus spp.

Marwari and Zanskari by measuring the total microbial loadand identification of randomly isolated pathogens in freshsemen ejaculates and in same semen samples aliquotedand kept frozen for at least 1 year before being examined forTMC. We are reporting a total microbial load of 2.1×105 CFU/ml in fresh semen, which is higher than total charge of 1.1-2.6×104 CFU/ml, reported in 88 ejaculates (Clèment et al.,1993). However, there is a range of variability between differentsamples from as low as 2.9 x 103 (S2) to high of 5.5 x 105 (S10& 11), so that a generalization on average microbial count infresh semen may not be possible. Burns et al. (1975) havereported a mean population of 5.7 x 105 aerobic bacteria perml of undiluted stallion semen.

We also report an average value of 1.7 x 104 in frozensemen, which is a log cycle less than 1.6×105 CFU/ml TMC inequine frozen semen reported by Corona and Cherchi (2009),

however, the TMC in frozen semen may be a function of initialmicrobial counts in fresh semen, as in our study, we haveobserved a lower microbial counts in 8 (S1, S4, S5, S6, S7, S9,S10 and S11) out of 11 frozen semen sample, as compared tothe TMC in the corresponding fresh samples (Table 1).However, as due to practical contention of researchers toconsider the TMC of frozen semen for single stallion for all theinsemination doses, and as in AI the number of thawed strawsvaries from 6 to 10, therefore it becomes critical to calculatebacteriological control of fresh semen itself before its freezing.The lowering of TMC in frozen semen samples may be due tolow temperature lethality to the bacterial cells contaminatingthe semen samples.

In our study, the most frequently isolated bacteria are thosebelonging to genus Pseudomonas, with 4 isolates identifiedas Pseudomonas aeruginosa and 4 as other unidentified

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species of Pseudomonas. Furthermore, these were isolatedin 6 samples (S2, S3, S4, S6, S8 and S10), indicatingcolonization of penis and prepuce in the source stallions afterthe disruption of normal bacterial flora (Hughes et al., 1967;Johnson et al., 1980). Pseudomonas aeruginosa, which maybe highly contagious, may cause venereal disease in maresbred to carrier stallions (Pickett et al., 1999). Detection of P.aeruginosa has been reported to result in failure of stallions topass fertility tests (Pickett et al., 1987). In another study, thefailure of authors to isolate P. aeruginosa from reproductivetract of healthy stallions also indicates that presence of theseopportunistic pathogens is detrimental to the health and fertilityof equines (Rota et al., 2011). Recently, Tiago et al. (2012) havealso demonstrated that presence of P. aeruginosa carrierstallions leads to a numerically higher incidence of uterinedisease following natural service due to venereal transmission;however, fertility reduction was not reported in mares.

Other significant emerging opportunistic bacterium whichwas isolated from semen was Nocardia which are ubiquitoussoil saprophytes, found in organic material, water and plants.The isolation and identification of Nocardia asteroides fromsemen is significant from the view of recent reports of isolationof Nocardioform Gram positive branching actinomycetes fromcases of placentitis leading to equine abortions (Bolon et al.,1989; Volkmann et al., 2001; Erol et al., 2012). A quick review ofliterature on equine semen microbiology reveals that this isprobably the first ever isolation of Nocardia asteroides complexmember from horse semen. Even though N. asteroides entersthe body via inhalation or traumatic implantation (Biberstein etal., 1985), its presence in semen meant for AI increases therisk for uterine nocardioform infections and abortions. Thedetection of Nocardia spp. in semen probably indicatesenvironment as the source of contamination. Therefore, thepresence of Nocardia spp. in semen should be checked,especially in tropical environments.

Escherichia coli was isolated in one semen sample fromfresh semen, and a single isolate of Staphylococcus spp. wasisolated from frozen semen. Escherichia coli, Pseudomonasspp and Staphylococcus spp., among others are commensalmicroflora of male external genitalia, and may thus be reflectedin freshly ejaculated semen (Rota et al., 2011). Inseminationof mares with semen containing these microbes may causeinfertility in susceptible mare. Escherichia coli have beensometimes associated with endometritis. The Staphylococcusspp. and E. coli are most frequently isolated from fresh semen(Corona et al., 2006; Varner et al., 1998). Low isolation ofcommensal bacteria and high isolation of Pseudomonas spp.and Pseudomonas aeruginosa is significant indicator ofdisturbance in resident microflora of male genitalia of sampledstallions.

Gram-negative glucose non-fermentors constituted alarge group of isolates including Acinetobacter spp.,Alcaligenes spp., Flavobacter spp. and Achromobacter spp.Acinetobacter spp. and, Alcaligenes spp were isolated fromfresh semen whereas all 3 except Acinetobacter spp. wereisolated from frozen semen. These are ubiquitous environmentcontaminant from soil and water, and except as indicator of

environment contamination of semen during collection, theyare of not much importance.

Fungal agents are considered responsible for 2%-9% ofthe endometritis (Pugh et al., 1986) and of up to 10% ofabortions in mares (Giles et al., 1993). Among fungalorganisms, the only detected mould was: Aspergillus spp.and in yeasts: Candida albicans; both of which were isolatedfrom fresh semen. Candida spp. and Aspergillus spp., are notcommon in semen or the genital tract of the stallion, but bothcan be potential pathogens in AI programmes where thehygiene of the collection and processing equipment is not wellmonitored (Samper and Tibary, 2006), although Aspergillusspp. has also been reported from healthy stallion reproductivetract (Rota et al., 2011).

AcknowledgementsAuthors are thankful to Director NRCE, Hisar for providing

facilities for carrying out the work.

ReferencesAlvarenga, M.A. et al. (2005) Ani. Reprod. Sci. 89(1):105-113.Barbacini, S. et al. (1999) Equine Vet. Educ. 11(2):109-112.Barrow, G. I. and Feltham, R. K. A. (1993) Cowan and Steel’s Manual

for the identification of medical bacteria. 3rd ed. CambridgeUniversity Press, Cambridge.

Beneke, E. S. and Rogers, A. L. (1971) Medical mycology manual.Burgess Publishing Company, Minneapolis, Minnesota.

Biberstein, E. L. et al. (1985) J. Am. Vet. Med. Assoc. 186:273-277.Bielanski, A. et al. (2003) Cryobiology. 46:146-152.Bolon, B. et al. (1989) Vet. Pathol. 26:277-278.Bristol, F. (1991) Proc. Ann. Meeting Soc. Theriogenol. 171-173.Burns, S. et al. (1975) J. Reprod. Fertil. (Suppl.) 23:139-142.Clément, F. et al. (1993) Prat. Vet. Equ. 25(1):37-43.Clément, F. et al. (1995) Biol. Reprod. Mono. 1:779-786.Corona, A. and R. Cherchi. (2009) Anim. Repro. Science. 115:103-

109.Corona, A. et al. (2006) Proceedings of the 9th International Symposium

on Equine Reproduction. Ani. Reprod. Sci. 94:85-88(abstract).

Devireddy, R.V. et al. (2002) Biol. Reprod. 66:222-231.Erol, E. et al. (2012) Vet. Microbiol. 158:425-430.Giles, R. C. et al. (1993) J. Am. Vet. Med. Ass. 203:1170 -5.Graham, J.K. (1996) Reprod. Technol. 12:131-147.Hughes, Y.P. et al. (1967) Cornell Vet. 57:53-69.Johnson, T.L. et al. (1980) Proc. Ann. Meeting Am. Assoc. Equine

Practitioners. 111-116.Levy, R. et al. (2004) Andrologia. 36:282-285.Loomis, P.R. (2001) Anim. Reprod. Sci. 68:191-200.Loomis, P.R. and Barbacini, S. (2005) Ippologia. 16(3):31-37.Metcalf, E.S. (2001) Anim. Reprod. Sci. 68:229-237.Parlevliet, J.M. (1997) Theriogenology. 47:1169-1177.Pickett, B. W. et al. (1987) Proc. 33rd Ann. Cony. AAEP 487-518.Pickett, B.W. et al. (1999) J. Equine Vet. Sci. 19:424-469.Pugh, D. G. et al. (1986) J. Equine Vet .Sci. 6:40-3.Reger, H.P. et al. (2003) Equine Vet. Edu. 15(2):101-106.Rota, A. et al. (2011) Theriogenology. 76:464-470.Samper, J. C. and Tibary, A. (2006) Theriogenology. 66:551–559.Tiago, G. et al. (2012) Acta. Vet. Scand. 54:20.Tischner, M. and Kosiniak, K. (1992) Acta Vet. Scand. 88 (Suppl.):83-

90.Varner, D.D. et al. (1998) Theriogenology. 50:559-573.Vidament, M. et al. (1997) Theriogenology. 48: 907-917.Volkmann, D. H. et al. (2001) J. S. Afr. Vet. Ass. 72(4): 235-238.

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IntroductionA camel, popularly known as the “ship of the desert” support

the survival of millions of people around the world in arid andsemi arid regions and is mainstay of economy of desertfarmers. The camel is more reliable milk provider than otherlivestock in arid areas (IDF, 1986); during both dry season anddrought years. Scanty rains, frequent drought and limited feedresources are fully exploited by the remarkable ability of camelto better utilize the scarce resources available in the desertand are well suited to live under extremely hot harsh climaticconditions, where the available water is very less. Camelreduces the vulnerability to milk insecurity during lean period.Camels are used as pack animals, riding and draft purposesunder desert conditions. Moreover, they are known for theirmeat, good quality hair and hide. Apart from all this, they alsoprovide valuable milk through out the year in ample quantitycompared to other animals living under similar conditions.Milk yield of camels in different regions vary widely dependingon quality and quantity of forage, frequency of watering andmilking, age of breeding, climate, parity, calf nursing, presenceof the calf, milking method (hand or machine milking), health,reproductive status, and individual merit. During this year theextreme winter corresponding to calving of camels (calvingduring Oct. 2010 to Feb.2011), lead to respiratory problem inmajority of camels and agalactia in newly parturated animals.Agalactia is lack of milk synthesis and secretion post parturientmay be due to failure of milk let down or synthesis andproduction of milk. It occurs just after parturition or within fewdays after parturition. Environmental factors and hormonedeficiencies often contribute to this problem. Mastitis, agalactiaor hypogalactia was reported in some younger breedingcamels, particularly after first parturition. The fate of calves fromsuch animals is either death or support of foster mother. Bottlefeeding of colostrum from contemporary calved motherssustain life but with certain health problems. In many instanceseither milk production or let down is very limited in camelsparticularly during the first few days post parturition or there iswatery secretion with thick consistency and glycerin likeappearance detracting calf to suckle such mothers. The etiology

INDUCTION OF LACTATION IN AGALACTIC CAMEL USINGDIFFERENT DRUGS

Sajjan Singh, F.C. Tuteja, Kashinath, B.L. Chirania and N.V. PatilNational Research Centre on Camel, P.B.07, Jorbeer, Shivbari, Bikaner-334001, Rajasthan, India

ABSTRACT

Sixty six she camel calved during the year spread between October to February with very good body weight and body conditionscore (BCS) of she camel and birth weight of calves. Out of these sixty six animals calved nineteen animals were agalactic. Theseanimals belong to different breeds. (Kachchi=3, Bikaneri=7, Jaisalmeri=2 and Mewari=7). Initially all these animals were taken into onegroup (Group I) to administer liquid ostovet orally. Subsequently the same were divided into two groups (Group II and Group III) toadminister Agrimin forte Powder plus Galactin to Group II and Agrimin forte plus Galactin plus Nausdin and injection Eminorm to GroupIII. No change of fodder was done during the period of experiment. Just after one month all the agalactic animals initiated lactation andthose who were induced lactation earlier were continuing lactation and producing milk sufficient to meet the demand of their calf.

Key words: Lactation, agalactia, camel, domperidone

of agalactia is unclear in these animals but it could be due tosevere oedema, problem of milk let down, hormonalimbalances, nutritional problem or mastitis (Tibary andAnouassi, 2000). In recent years, veterinarians in the USA havebeen experimenting with the use of Domperidone supplementto increase lactation efficiency. Domperidone is a D2 dopaminereceptor blocker that is used for the prevention of fescuetoxicosis and agalactia in pregnant mares. The same alongwith certain other drugs was successfully tried to induce lactationin she camel.

Materials and MethodsNational research centre located at Bikaner is having a

population of about 365 animals. All these camels are rearedand managed in routine scientific way. This year 66 she camelcalved spread between October to February with very goodbody weight and body condition score (BCS) of she camel andbirth weight of calves. Out of these sixty six animals calvednineteen animals were agalactic. These animals belong todifferent breeds. (Kachchi=3, Bikaneri=7, Jaisalmeri=2 andMewari=7). Initially all these animals were taken into one group(Group I) to administer liquid ostovet orally. Subsequently thesame were divided into two groups ( Group II and Group III) toadminister Agrimin forte powder plus Galactin to Group II andAgrimin forte plus Galactin plus Nausdin and injection Eminormto Group III. Change of fodder was done during the period ofexperiment

Results and DiscussionResults are presented in Table 1, 2, 3 and 4. On analysis

of data it was found that after treatment with ostovet whichlasted for a weak there was no initiation in any of the breeds ofcamel. Just after the end of this treatment a second treatmentwas started taking a clue from equine were domperidone wasused to induce lactation. These animals were divided in twogroups Group II and Group III. Group I was administered Agriminforte plus Galactin and it was observed that during the treatmentsix out of twelve animal started milk synthesis and secretionprior to treatment the secretion was transparent, thick and

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Table 1: Treatment with liquid Ostovet administered orally

S. No.

Tag No Dam

BCS/Wt. Dam

Date of Calving

Weight of calf (kg)

Treatment 24.01.11-31.01.11

After Treatment Lactation

1 K-159 2.5/530 9.11.10 35 Ostovet Not initiated 2 K-141 3/623 13.12.10 28 -do- Not initiated 3 K-143 2/695 died 25.1.11 35 -do- Not initiated 4 B-509 3/644 19.12.10 30 -do- Not initiated 5 B-463 3.5/607 2.1.11 36 -do- Not initiated 6 B-473 3/564 25.12.10 40 -do- Not initiated 7 B-543 3.5/616 15.1.11 32 -do- Not initiated 8 B-541 3/598 18.1.11 40 -do- Not initiated 9 B-523 3.5/572 21.1.11 39 -do- Not initiated 10 B-495 3.5/674 21.1.11 36 -do- Not initiated 11 J-89 2.5/589 3.1.11 34 -do- Not initiated 12 J-113 2.5/617 11.1.11 32 -do- Not initiated 13 M-01 3/764 16.12.10 38 -do- Not initiated 14 M-39 3/520 2.1.11 37 -do- Not initiated 15 M-53 2.5/546 6.1.11 35 -do- Not initiated 16 M-15 3.5/598 14.1.11 36 -do- Not initiated 17 M-93 9.2.11 36 -do- Not initiated

Table: 2 Treatment with Agrimin forte and Galactin

S.No. Tag No mother

BCS Dam

Date of Calving

Weight of calf (Kg)

Treatment 31.1.11-01.02.11


Lactation as on 27.04.11

1. K-159 2.5/530 9.11.10 35 Agrimin +Galactin

Initiated continue

2. K-141 3/623 13.12.10 28 -do- Initiated continue 3. K-143 2/695 D 25.1.11 35 -do- Initiated Calf died

28.1.11 4. B-509 3/644 19.12.10 30 -do- Initiated continue 5. B-463 3.5/607 2.1.11 36 -do- Not initiated 6. B-473 3/564 25.12.10 40 -do- Initiated Initiated

and continue

7. B-543 3.5/616 15.1.11 32 -do- Not initiated Initiated and continue


9. J-89 2.5/589 3.1.11 34 -do- Not initiated Initiated and continue

10. M-01 3/764 16.12.10 38 -do- Initiated continue 11. M-15 3.5/598 14.1.11 36 -do- Not initiated Initiated

and continue

12. M-93 - 9.2.11 36 -do- Not initiated Calf died on 17.2.11

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Table: 3 Treatment with Agrimin forte, Galactin, Nausdin and Eminorm

S.No. Tag No mother

BCS Dam

Date of Calving

Weight of calf (Kg)

Treatment 31.01.11-01.02.11


Lactation as on 27.04.11

1. B-541 3/598 18.1.11 40 -do- Not initiated Initiated and continue


3. J-113 2.5/517 11.1.11 32 -do- Not initiated Initiated and continue

4. M-39 3/520 2.1.11 37 -do- Initiated continue 5. M-53 2.5/546 6.1.11 35 -do- Initiated continue 6. M-13 2.5/527 26.1.11 33 -do- Not initiated Initiated

and continue

7. M-41 3/396 27.1.11 24 -do- Not initiated Initiated and continue

Table: 4 Breed wise response to different drug treatments

S. No Breed No. of animals Group-I Group II Group III 1 Kachchi 3 Nil 2/3 Nil 2 Bikaneri 7 Nil 2/5 0/2 3 Jasalmeri 2 Nil 0/1 0/1 4 Mewari 7 Nil 2/3 2/4 5 Total 19 Nil 6/12(50%) 2/7(28.57%)

glycerin type and calves were reluctant to suckle. In Group IIIwhere we fortified Group II treatment with Nausdin and Eminormresulted in initiation of lactation in two animals out of seven.But just after one month all the agalactic animals initiatedlactation and those who were induced lactation earlier werecontinuing lactation and producing milk sufficient to meet thedemand of their calf. Prior to induction of lactation either thesecalves were bottle fed or were supported by foster mother. Twocalves died due to enteritis. Milk production from these animalsis not recorded but is sufficient to sustain survival of therecalves. Breed wise Bikaneri and Mewari were more agalcticfollowed by Kachchi and Jaisalmeri. Similar reports wererecorded by Kohli et al. (1988) who used Leptaden in cases ofagalactia in camels to respond favorably to Leptaden therapy.The camel calves as in present study were alive and damsexhibited normal motherly instinct towards their calves, thereare reports where nutrition also play role in milk production.Diets enriched with green forages such as alfalfa, barseem orcabbage greatly increase milk yield (Knoess, 1977; Knoess etal., 1986; Richard and Gerard, 1989). Furthermore, camelbreeds or individual animals probably exist with significantlydifferent milk-producing potential that has not been fullyexploited because the selective pressure of humans on thecamel has been minimal compared with other domesticanimals (Richard and Gerard, 1989). Lactation curves in factindicate large differences compared with other lactating

mammals. Some curves indicate low yields during the firsthalf of the lactation period and an increase in the second.Other results report higher yields at the beginning, followed byfalls towards the end. Occasionally, one or two distinct peakscan be observed or, conversely, steady production throughoutthe lactation (Field, 1979; Bachmann and Schulthess, 1987).The high disparity between these various sets of data canprobably be explained by differences in genetic potential,climate, feeding conditions and sampling techniques. Scantyreports about agalactia in camel are available and etiology ofagalactia is unclear in these animals but camel response todifferent drugs was positive in inducing lactation.

ReferencesBachmann, M.R. and Schulthess, W. (1987) Milchwissenschaft. 42:

766-768.Field, C.R. (1979) IFS Workshop on Camels. Khartoum, the SudanInternational Dairy Federation (1986) Int. Dairy Federation 202.

Brussels. 221 pp.Knoess, K.H. (1977) World Animal. Rev. 57: 11-21.Knoess, K.H. et al. (1986) World Animal. Rev. 57: 11-21Kohli, I. S. et al. (1988) Indian Vet. J. 65: 551-552.Richard, D. and Gérard, D. (1989) Rev. Elev. Med. Vét. Pays Trop. 42:

97-103.Tibary, A. and Anouassi, A. (2000) In: Recent Advances in Camelid

Reproduction, Skidmore J.A. and Adams G.P. (Eds.),International Veterinary Information Service (www.ivis.org).

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IntroductionRabbit (Oryctolagus cuniculus) farming is on rise in most

parts of the country. The small animals are reared for meat, furand as pet in urban areas. Rabbitary has advantages that itcan be fed upon a very wide range of feeding stuffs, can bemaintained on very small scale, stocks built up is very rapid,housing difficulty are very easily overcome, relatively unskilledlabours are required and produce meat most economically interm of feed stuffs. The docile animals are also preferred inhouse, educational institutes and in amusement parks.

Mange has become a common and major constraint inrabbit production in India (Ravindran and Subramanium, 2000).With the hot and humid climate of the country, mange amongrabbits is very high (Aulakh et al., 2003). Among various speciesof mites, Sarcoptes scabiei is a deep burrowing mite in theepidermis causing intense itching, pruritis, crust formation,scale production, thickening and wrinkling on skin of affectedarea (Hendrix and Robinson, 2012). The disease producesvariety of other pathological effects resulting in reduced reducedfeed intake, reduction in body weight, fur production and alsopredisposes the animals to various infections of bacterial andviral origin. The mite is very easily transmitted to othersurrounding animals through close contact. Severe infectionespecially in young or debilitated animals causes high mortality(Aiello et al., 1998 and Bornstein et al., 2001). Although themite is host specific but is also cross transmitted to humans(Hengge et al., 2006). Zoonotic aspect of Sarcoptes mites hasbeen documented by various authors (James, 1997 andGlenda et al., 2008). Present communication reports anoutbreak of Sarcoptes scabiei in rabbit farm at Hisar, Haryana,its histopathological manifestations in skin sections and itstherapeutic remedy.

AN OUTBREAK OF SARCOPTIC MANGE IN A RABBITFARM AT HISAR (HARYANA)

Davinder Singh, N. K. Mahajan, Sukhdeep Vohra1, Satyavir Singh1 and P.K. KapoorDisease Investigation Lab., Department of Veterinary Public Health & Epidemiology

LLR University of Veterinary & Animal Sciences, Hisar-125 004, Haryana.

ABSTRACT

In a farm having 120 rabbits (90 adults and 30 young ones), located at Patel Nagar, Hisar. Thirty rabbits were showing alopecia, itchingand crust formation. Twenty two rabbits had died showing these lesions during the month of November-December 2011. The farmwas visited and clinical observations revealed overcrowding and poor hygiene at the farm. Rabbits were malnourished as no properdiet was being fed to the rabbits. Affected areas of the rabbits included nose, around eyes, ear tips and legs. Skin scrappings werecollected in 10% KOH and kept overnight. The microscopic examination revealed high density of adult Sarcoptes scabiei along withtheir eggs. The histopathological of skin sections showed hyperplasia, hyerkeratosis, villus like projections, ballooning degeneration,areas of haemorrhages with infiltration of mononuclear cells and formation of keratin cysts in epidermal area. Various mites in partsof epidermis were also visible. Interestingly, two farm workers closely associated with rabbits were also found affected with lesionslike pruritis, inflammation and papule formation but the mite could not be isolated because of the ongoing treatment by avermectincompound. Consequently, the owner was advised to administer doramectin @ 200 µg/kg body weight subcutaneously to all rabbits.The endectocide was found highly effective and gave complete cure.

Key words: Doramectin, histopathology, rabbit, Sarcoptes scabiei

1Department of Veterinary Parasitology

Case history and investigationFour dead Chinchilla rabbits were brought to Disease

Investigation Laboratory, College of Veterinary Sciences,LLRUVAS, Hisar in the mid of December, 2011 with lesions ofalopecia, crust formation on nostrils, around eyes, ear tipsand legs. Thickening of skin on the affected areas with wrinkleformation on leg was also visible. History revealed mangeinfection in the farm with a stock of 120 rabbits located at Hisar,Haryana (75.98204 Longitude, 29.10259 latitude). There wasmortality of 22 (18.3%) rabbits after showing above mentionedlesions. Thirty closely housed rabbits were also showingsimilar lesions and symptoms.

Collection of samples for diagnosisThe deep skin scrappings were collected in 10% KOH for

examination of mites. Post mortem of the dead animals wascarried out for any infection, heart blood sample were collectedfor bacteriological examination and affected skin area werecollected and preserved in 10% neutral buffer formalin (NBF)for histopathology studies.

Farm visit and examinationOn visit to the farm, it was found that rabbits of all age

groups belonging to either sex were being reared for meatpurpose. All the rabbits were kept together in a small area withovercrowding. There was poor hygiene in the area along withimproper management. The clinical observations revealed hairloss with crust formation and itching at areas of body wherehair growth is comparatively less. There was anorexia, loss ofcondition and decrease in body weight growth. As reported bythe workers in the farm the lesion in the affected rabbits startedon the face near the upper lip, later extending to the full faceand lower jaw, and involved ear pinnae, lips and both eyelids,

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Fig. 1: Infested rabbit showing lesions on tip of ear, around eyes,nose,forelimb and hindlimb.

Fig. 2: The leg of labour showing papule and pustule formation.

Fig. 3: Skin scrappings showing Sarcoptes scabiei var cuniculi mite Fig. 4: Skin section showing various numbers of mites embedded inthe epithelium and in the epidermis (H & E x 62.5)

which were then closed and contained thick whitish exudates.Later the lesions extended to forelimbs and hind limbs (Fig.1). The affected regions showed scales, alopecia and scabformation. The rabbits showed pruritis and were intermittentlyscratching the area with front paws. Later, haemorrhagic crustswere observed on area of lesions. The condition of the affectedrabbits was weak and body coat was ruffled. Furtherinvestigations revealed mortality in weak and young animalsafter showing similar lesions. The disease was persistentsince November, 2011.

Two labours working in the farm and closely associatedwith rabbits also showed intense itching and papule formationon the legs and hands. The papules further formed pustulewith continuous irritation on the affected area (Fig. 2).

Treatment and adviceAll the affected rabbits were treated with doramectin @

200 µg/kg body weight along with anti histaminic drugs.Segregation of the affected rabbits, proper feeding,

improvement in farm management and proper ventilation ofanimal house was advised.

The farm was visited on 7 th, 15 th, and 30 th day posttreatment and the skin scrappings were collected to observeefficacy of treatment. The recovery of affected animals wasalso recorded in terms of reduction in mites and lesions,decrease in irritation, hair regeneration and growth withimprovement in general body condition.

Results and DiscussionSkin scrappings, necropsy and bacteriological

examinationScrappings showed presence of numerous mites along

with their developmental stages. On the basis of round bodywith transverse ridges, triangular scales on dorsal surfaceand presence of terminal anus, these were identified asSarcoptes scabiei var cuniculi (Soulsby, 1982) (Fig. 3). Post-mortem examination of the dead rabbits revealed no majorchanges on the internal organs, except on skin which showed

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alopecia, formation of crust and scales along with thickening.Blood from heart was placed on nutrient broth for bacteriologicalculture but did not show any bacterial growth after 72 hours ofincubation. Thus, ruling out involvement of any possibleinfectious agent.

HistopathologyThe histopathological examination revealed various

numbers of mites embedded in the epidermis. The epidermisshowed changes of hyperplasia, occasional areas ofhaemorrhages and hyperkeratization (Fig 4). Lesionscharacterized by hyperkeratosis were observed. The mites didnot burrow beyond the epidermal region and were present insuperficial layer of epidermis. The epithelium of the epidermisshowed villus like projections, ballooning degeneration andformation of keratin cysts. Sub-epidermal dermatitis wascharacterized by infiltration of mononuclear cells viz.macrophages and lymphocytes.

Post-treatment observationsComplete recovery from itching along with absence of

scabs on body was observed on seventh day post treatment.No mite or its developmental stages were seen during skinscrapping examination. However, the thickening of skin andhair loss was evident. Second observation on 15th day posttreatment revealed that the hair growth has started at the areaof alopecia and by one month post treatment the thickenedskin became normal and there was complete hair growth ataffected areas.

Itching and appearance of lesions on the hands of theworkers involved in handling of these rabbits was also noticedin the present case, which indicates the zoonotic importanceof the mite. However, isolation of the mites from the workerswas not successful because of ongoing treatment byavermectin compound.

Sarcoptes scabiei are highly contagious and burrowsdeep in epidermis of skin (Wall and Shearer, 1997). Occurrenceof infestation in rabbits has been reported by several authors(Kuizheng et al., 1994; Raji et al., 1997; Saha and Mukherjee,1998). The lesions observed in rabbits in present outbreakare similar to those observed by previous authors (Kumar,1999 and Radi, 2004). The disease leading to outbreakoccurred during post rainy season. Similarly, high prevalenceof mites during low atmospheric and high mean relativehumidity was also reported by Ravindaran and Subramanian(2000). External factors such as extreme weather conditionsor reduced food availability may result in increased susceptibilityto the parasite in rabbit populations (Gortazar et al., 1998; Penceand Ueckermann, 2002).

Clinical manifestations such as development of scales,scabs, crusts and alopecia along with a large number of S.scabiei below the crusts as observed in present study wereconsistent with findings of Oraon et al. (2000) and Chandey etal. (2000). Such pathogenic effects of these mites have beenattributed to their burrowing activity and mechanical damagecaused by the parasites during excavation, irritant action oftheir secretions and excretions (Darzi et al., 2007).

In the present outbreak, mortality of 18.3% (22/120) wasobserved. High mortality due to severe mite infestations wasalso reported by other authors (Aiello et al., 1998 and Bornstein

et al., 2001) and may even reach up to 100% in some specificcases as observed by León-Vizcaíno et al. (1999).

The histopathological observations of hyperplasia,haemorrhages, hyerkeratosis and presence of mite inepidermal region observed in present study are in agreementwith Rajeshwari et al. (1995); Singla et al. (1996) and Radi(2004).

The lesions in humans closely associated with miteinfested animals have also been observed by Deshmukh etal. (2010) and Shelley and Currie (2007).

Aulakh et al. (2003) also reported doramectin @ 200 µg/kg introduced subcutaneously once a week for 2 weeks wasan effective treatment of scabies. Single dose of doramectin@ 400 µg/kg along with the supportive treatment for three daysreported to be 100% effective in control of Notoedric mange inrabbits (Vohra et al., 2005) whereas the present observationsindicate that doramectin therapy coupled with propermanagement, disinfection of the rabbit cages and segregationof infected animals is effective in control of mange in rabbits.

ReferencesAiello, S.E. et al. (1998) Rabbits. In Merck Veterinary Manual (S.E.

Aeillo, edt.) Merck Inc, New Jersey, 1386-1396.Aulakh, G.S. et al. (2003) J. Vet. Parasitol. 17:127-129.Bornstein, S. et al. (2001) Parasitic diseases of wild mammals. In W.

M. Samuel, M. J. Pybus, and A. A. Kocan (ed.), Sarcopticscabiei and sarcoptic mange. Iowa State University Press,Ames, Iowa. pp. 107-119.

Chandey, J. et al. (2000) Indian Vet. J. 77: 755-757.Darzi, M.M. et al. (2007) Vet. Arhiv 77: 167-175.Deshmukh, V.V. et al. (2010) Intas Polivet. 11(1):112-114.Glenda, D. et al. (2008) Handbook for Zoonotic Diseases of Companion

Animals. Published by Center for Food Security and PublicHealth, Iowa State University, College of Veterinary Medicine,Ames, Iowa-50011. p. 65.

Gortazar, C. et al. (1998) Z. Jagdwiss. 44: 251–256.Hendrix, C.M. and Ed. Robinson (2012) Diagnostic parasitology for

Veterinary technicians. Elsevier Publication. pp. 236.Hengge, U.R. et al. (2006) Lancet Infect. Dis. 6:769-779.James S. Tan (1997) Arch Intern Med. 157(17):1933-1943.Kumar, N. (1999) Studies on rabbit mange with special reference to

elvaluation of recent chemotherapeut agents against rabbitmange. M. V. Sc Thesis, Punjab Agricultural University,Ludhiana, India.

Kuizheng, C. et al. (1994) Chinese J. Vet. Sci. Technol. 24: 16-17.León-Vizcaíno, L. et al. (1999) J. Wildlife Dis. 35: 647-659.Oraon, B. et al. (2000) Indian J. Ani. Sci. 70: 405-406.Pence, D.B. and Ueckermann, E. (2002) Rev. Sci. Tech. Off. Int. Epizoot.

21: 385-398.Radi, Z.A. (2004) Comparative Medicine. 54: 434-437.Rajeshwari, Y.B. et al. (1995) J. Parasit. and Appl. Anim. Biol. 4:27-

28.Raji, M. A. et al. (1997) Nigerian Vet. J. 18: 97-98.Ravindran, R. and Subramanian, H. (2000) Indian Vet J. 77:991-992. Saha, S.B. and Mukherjee, S. (1998) Indian J. Ani. Hlth. 37: 73.Shelley, F.W. and Currie, B.J. (2007) Clin. Microbiol. Rev. 20(2):268-

279.Singla, L.D. et al. (1996) Parasite. 3: 87-89.Soulsby, E.J.L. (1982) Helminths, Arthropods and Protozoa of

Domesticated Animals. 7th ed. ELBS, Bailliers Tindall andCassel, London.

Vohra, S.S. et al. (2005) J. Vet. Parasitol. 19(1):47-49.Wall, R., Shearer, D. (1997) Veterinary Entomology. 1st ed. Chapman

and Hall, London, UK.

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IntroductionCanine parvovirus infection, defined as a nosological entity

in 1978 (Appel et al., 1979) is a world wide occurring contagiousdisease (Mathys et al., 1983) of puppies, dogs and canids. Onthe basis of its morphological, structural and biologicalcharacteristics, the causative agent isolated from faeces ofdogs suffering form haemorrhagic enteritis has been classifiedas canine parvovirus Type 2 (CPV-2) (Appel et al., 1979). It hasa world wide distribution and is widely prevalent in India.Ramadass and Khader (1982) first confirmed CPV as anetiological agent of the disease in dogs in India. CPV replicatesin several lymphoid tissues and the intestinal epithelium ofdogs, therefore great amount of parvovirus are found in thefaeces of infected dogs. The faecal-oral route of thetransmission is most common in CPV infection and dogs undersix months of age are most susceptible. Canine parvoviruscauses an acute infection characterized by sudden onset ofdiarrhoea, anorexia and fever with resulting high morbidity andmortality in younger dogs (Archna et al., 2009). The clinicalappearances of the diseases vary and may be confused withother gastrointestinal diseases. Infection spread easily amongsusceptible animals, so rapid diagnosis is essential fordisease control and treatment.

The present experiment was conducted for thecomparative study of various immunodiagnostic tests for thediagnosis of CPV along with the biochemical changesproduced in puppies naturally infected with CPV because veryscanty information is available on biochemical changes of thedisease in clinical cases.

MICRO-BIOCHEMICAL STUDIES OF CANINE PARVOVIRUSINFECTION IN PUPPIES

Gurudutt Joshi, Rajesh Singathia*, Anil Gattani1, Rajendra Yadav2 and R. L. LakhotiaDepartment of Veterinary Microbiology

Apollo College of Veterinary Medicine, Agra Road, Jaipur-302031.

ABSTRACT

The present study was conducted on eleven puppies naturally infected with parvoviral gastroenteritis. Five puppies seronegative forcanine parvovirus (CPV) were taken as control. Disease diagnosis was done from puppies faeces using various serological test viz.haemagglutination test using 0.8% swine RBC. Viral antigen was further confirmed by performing direct enzyme linked immunosorbentassay using monoclonal antibody against canine parvovirus. It was found that seven out of eleven samples were positive for thepresence of CPV antigen. Biochemical studies were carried out on all the samples along with control. It was found that the puppieswhich were positive for CPV showed significant hypoglycaemia i.e. 60.14±1.16 mg/dl (P<0.05) along with hypoprotenaemia(5.11±0.061g/dl), hyponatremia (135.4±0.023 mM/L), hypokalaemia (3.89±0.36 mM/L) and hypochloremia (104.81±0.053 mM/L). Bloodurea nitrogen levels were also significantly decreased from 15.62±1.02 mg/dl in seronegative to 9.14±0.091mg/dl in CPV positivepuppies. Further, the levels of serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase were significantlyincreased (P<0.05) i.e. 75.55±0.028 IU/L and 140.24±0.021 IU/L, respectively. This study clearly indicates that ELISA is the mostsensitive test to diagnose canine parvovirus infection. Biochemical studies revealved that there is increased extracellular leakageresulting from increase cell permeability and injured hepatocytes which lead to dysfunction of liver.

Key words: Canine Parvovirus, ELISA, haemagglutination test.

Materials and Methods1. Collection of faecal sample: A total of eleven puppies belowone year of age showing symptom of pyrexia, anorexia,vomition and diarrhoea were presented to the clinicians,Teaching Veterinary Clinical Complex, Apollo College ofVeterinary medicine, Jaipur, Rajasthan. The faecal samples ofall the eleven cases and of five control puppies were collectedwith the help of sterile cotton swab. The samples weresuspended in PBS, pH 7.2, centrifuged at 4000 r.p.m. for 15minutes and stored at -20O till further use.2. Collection of blood for serum separation: Blood sample(1-1.5 ml) was collected from all the cases in sterile test tubes.Blood was allowed to clot at 370 C for 1 hour (hr) in slantingposition and then kept in refrigerator at 40C for further 3-4 hr.Serum was collected by centrifugation at 1000xg at 40C for 10min. After proper labeling sera samples were stored at -200Ctill further use.3. Preparation of 0.8% erythrocytes: The blood of middle whiteYorkshire pig was collected from the marginal ear vein using22G sterile needle under aseptic condition in an equal amountof Alsever’s solution. Further, 0.8 % suspension of swine RBCwas prepared in sterile PBS for conducting different diagnostictests.4. Serological tests: CPV antigen was detected in faecalsamples using various immunodiagnostic tests.Haemagglutination (HA) test test was conducted on faecalsamples of infected dogs as per method of Carmichael et al.(1980). Viral antigen was further confirmed by performing direct

*Corresponding Author: Assistant Professor, Department of Veterinary Microbiology, Apollo College of Veterinary Medicine, Agra Road, Jaipur- 302031.E mail: [email protected] Professor, Department of Veterinary Physiology and Biochemistry2Assistant Professor, Department of Veterinary Medicine

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enzyme linked immunosorbent assay (ELISA) usingmonoclonal antibody (Mab) against CPV (Mab CPV-2A and 2B)and rabbit anti dog IgG Fc fragment horse radish conjugate(VMRD, Pullman, U.S.A).5 Biochemical Tests: Serum samples were processed forbiochemical estimation of sodium and potassium by Flamephotometry (Oser, 1986). Blood glucose, total serum protein,blood urea nitrogen (BUN), chloride, serum glutamicoxaloacetic transaminase (SGOT) and serum glutamic pyruvictransaminase (SGPT) were estimated by commercial kits (SpanDiagnostics Ltd., Surat, India).

Results and DiscussionIn the present investigation a total of 11 faecal samples of

puppies showing symptoms of diarrhoea and vomition andfive contro puppies were tested by HA and direct ELISA and itwere found that 36.36% and 63.63% samples, respectively,were positive for canine parvovirus infection respectively. Outof these tests ELISA was found to be most sensitive whereasHA is suitable for screening the field samples but is notconsidered as a confirmatory test.

In the present investigation, the mean blood glucose levelwas 60.14±1.16 mg/dl in puppies with parvoviralgastroenteritis where as in control puppies it was 110.25±1.83mg/dl. This shows significant decrease in blood glucose levelin infected pups (p < 0.05). According to Sagar et al. (2008) themean blood glucose level in parvoviral infected dogs was40.1±1.26 mg/dl where as in case of healthy dogs it was79.1±2.83 mg/dl which directly co-relates the present results.Similar observation has been reported by Greene (1984).Hypoglcemia might be due to decreased hepatic glucoseproduction because of decreased blood supply to liver withprotosystemic shunt which results in liver atrophy and liver cannot play its normal role in maintaining the blood glucoseconcentration.

The mean values of total serum proteins was 5.11±0.061g/dl in parvoviral infected puppies whereas in control puppies itwas 6.23±0.012g/dl which was also supported by Ram Prabhuet al. (2002). Hypoproteinaemia might be due to the leakage ofserum proteins through damaged capillaries of the villi ofintestine and also due to less absorption through villi (Biswaset al., 2005). But the above findings were not supported byDharmadheeran et al. (2003) who reported that the levels ofserum total proteins did not vary significantly in the experimentalcanine parvoviral infection.

The mean BUN concentration was 15.62±1.02 mg/dl inthe control puppies and 9.14±0.091mg/dl in the CPV infectedpuppies. It shows significant decrease in the BUN level(P<0.05) which may be due to hepatic damage. The meanvalues of sodium, potassium and chloride were143.5±0.19mM/L, 4.85±0.61 mM/L and 109.38±0.13 mM/L in

the control puppies and 135.4±0.023 mM/L, 3.89±0.36 mM/Land 104.81±0.053 mM/L in the infected puppies, respectively.This hyponatraemia, hypokalaemia and hypochloraemia aresequelae of diarrhorea with villous atrophy. Similar observationwere also reported by Haligur et al. (2009).

The mean values of SGOT and SGPT was 58.54±0.031IU/L & 98.74±0.036 IU/L in control puppies and 75.55±0.028IU/L &140.24±0.021 IU/L in the infected puppies, respectively.Elevated SGOT and SGPT levels may be due to their increasedsynthesis and secretion or due to diminished catabolism and/or by increase extracellular leakage resulting from increasedcell permeability or injured hepatocytes (Christ-Crain, 2004).Large quantities of transaminase (SGOT and SGPT) arepresent in myocardium, skeletal muscles, brain, liver, kidneythan other body tissues. Destruction of any of these tissueresulting in liberation of large quatities of these enzymes in theblood (Oser, 1986), where they can be detected. The increasedactivity of SGOT is a marker of soft tissue damage. However,the increased SGPT activity is specific indicator of hepatocytedamage (Kaneko et al., 1999).

The finding of present study indicate that ELISA was foundto be most sensitive test whereas HA is suitable for screeningthe field samples but is not considered as a confirmatory test.Results of various biochemical investigations revealedhypoglycaemia, hypoproteinaemia, hyponatraemia,hypokalaemia, hypochloraemia and elevated SGOT and SGPTlevel in blood serum indicator of diarrhhea, increase cellpermeability and injured hepatocytes which leads to the liverdysfunction.

AcknowledgementsAuthors thanks to DEAN, Apollo College of Veterinary

Medicine, Jaipur for providing necessary facilities during study.

ReferencesAppel, M.J. G. et al. (1979) Vet. Res. 105: 156-159.Archna, et al. (2009) Indian J. Vet. Res. 18: 42-44.Biswas, S. et al. (2005) Indian J. Vet. Med. 25: 16-18.Carmichael, L. E. et al. (1980) Am. J. Vet. Res. 41: 784-791.Christ-Crain, M. et al. 2004) EXCIL J. 3:1-9.Dharmadheeran, J. S. et al. (2003) Indian J. Vet. Med. 23: 9-11.Greene, C.E. (1984) Clinical microbiology and infectious diseases of

dogs and cats. W.B. Saunders, Co., Philadelphia.Haligur, M. et al. (2009) J. of Ani. and Vet. Advances. 8: 720-725.Kaneko, J.J. et al. (1999) Clinical Biochemistry of domestic animals.

5 th ed. Harcourt Brace and company Asia, PTE Ltd,Singapore.

Mathys, A.R. et al. (1983) Am. J. Vet. Res. 44: 150-151.Oser, B.L. (1986) Hawk’s Physiological chemistry.14th ed. Tata McGraw

Hill Publishing Co. Ltd, New Delhi.Ramadass, P. and Khader, T.G.A. (1982) Cherion. 11: 323-328.RamPrabhu, R. et al. (2002) Indain Vet. J. 79: 374-376.Sagar, A. et al. (2008) Intas Polivet. 9:262-265.

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IntroductionAmong respiratory diseases, pneumonia is a very

common disease in animals particularly in sheep. Numerousfactors such as age, season, immune status andmanagemental condition influence its occurrence. Inspite ofrevolution in antimicrobials and other therapeutic agents andappreciable improvement in the field of managemental system,varieties of etiological agents have emerged and increasedthe problems many folds as yet the major disorders associatedwith lamb death are reported to be respiratory (31.4%) followedby others viz. digestive disorders (14.6%), starvation (9.6%),endoparasitism (5.0%), septicaemia and toxaemia (10.1%),accidental (2.1%) and undetermined causes (27.2%)(Sonawane et al., 2012). Several studies have shown thatapproximately 10-35% of lambs die by the 6 months of age(Green and Morgan, 1993; Nash et al., 1996; Haughey, 1991;Gama et al., 1991). Susceptibility of individual animals tomicrobes and increasing resistance, low dose, toxic andresidual problems etc. have drawn the attention of cliniciansand researchers to go through pulmonary medicines.Therefore an attempt was made to include bronchodialatorsin therapeutic regimen and evaluate and compare their efficacyin naturally occurring pneumonic cases in sheep.

Materials and MethodsA group of twelve lambs weighing around 8-15 kg and 3-

7 month of age was observed to be suffering from fever withrespiratory distress. These animals were carefully examinedto record physiological parameters, clinical symptoms andauscultative changes. The nasal swabs/blood samples werecollected and processed for microbial (Merchant and Packer,1983) and haematological examination (Benjamin, 1985).These animals were grouped as G-I and G-II having 6 numberin each and G-I treated with Ceftriaxone sodium (Monocef,Aristo) @ 25 mg/kg intramuscularly 12 hourly for 5 days aloneand the other group G-II was treated with Monocef andbronchodialators-Etophylline and Theophylline (Deriphyllin) @1 ml intramuscularly 8-12 hourly for 3 days and Salbutamol

THERAPEUTIC POTENTIAL OF BRONCHODIALATORS INMANAGEMENT OF PNEUMONIA IN SHEEP

S.K. Dixit, B. N.Tripathi, Jyoti Kumar G.G. Sonawane, and O.P. KoliCentral Sheep and Wool Research Institute

AVIKANAGAR-304501 (Rajasthan)

ABSTRACT

Twelve lambs were clinically examined to record clinical signs viz. rise in temperature, increased shallow respiration with noisy ralesand mostly involving abdominal muscles, watery to mucoid nasal discharge, inappetance, reduced ruminal movements, occasionallachrymation. Blood examination revealed neutrophelia with leucocytosis, marginally reduced Hb but no eosinophelia. Nasal swabs/lung tissue recorded presence of mixed infection (G +ve and G -ve). The group of animals treated with Ceftriaxone sodium (Monocef,Aristo) @ 25 mg/kg intramuscularly 12 hourly for 5 days alone responded well but other group of animals additionally treated withsupport of bronchodialators- Etophylline and Theophylline (Deriphyllin) @ 1-2 ml intramuscularly 8-12 hourly and Salbutamol 1.25 mgper animal per day twice orally for 3-5 days were more comfortable in terms of recovery from respiratory distress.

Key words: Bronchodilators, sheep, pneumonia

1.25 mg per animal per day twice daily orally for 5 days. Thehealth status of these animals was assessed twice dailythrough clinical examination. The data was collected andprocessed as per standard statistical methods (Snedecor andCochran, 1989).

Results and DiscussionAnimals selected for study showed the symptoms such

as dyspnoea, nasal discharge, and high rise in temperature,lachrymation and abdominal respiration. The dyspnoeareflects abnormalities associated respiratory system or maybe a compensatory process of gas exchanges through lungparenchyma (Koneko, 1980). The lungs are part of therespiratory system and are located in the chest, inside the ribcage and above the diaphragm. Lungs are complex organsthat consist of spongy, elastic tissue that is designed for oxygenintake and expiration of carbon dioxide. Oxygen enters the lungswhile inhale a breath. It is distributed throughout the lungs by asystem called the bronchial tree, with branches of decreasingdiameter called bronchi and bronchioles. The bronchial treecarries oxygen to small sacs (alveoli) deep within the lungwhere oxygen, from air that is inhaled, moves from the lunginto the blood stream, and carbon dioxide, a byproduct of ourmetabolism, moves from the blood into the lung to be exhaled.Intake of oxygen and delivery by the blood to tissue is necessaryfor all of the cells in the body to function (Mc Connochie et al.,1987). Removal of carbon dioxide is necessary to maintainthe blood’s pH at an appropriate level as part of the body’ssystem of acid-base balance. Since the breathe air containsmany components from the environment such as dust, pollens,bacteria, viruses, smoke, and volatile chemicals, the lungsmaintain a system of defense against these potentially toxicinvaders (Brigham and Meyrick,1986). The lungs’ defencesystem includes immune cells and secretion of mucus tocontain and remove these unwanted components from thelungs (Cunnigham and Klein, 2007). Lung diseases areconditions in which some function of the lung is adverselyaffected. In some cases, the problem is in the gas exchange

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process at the membrane between the alveoli and blood-impeding efficient uptake of oxygen and removal of carbondioxide. In other cases, the problem is the inability of thebronchial system to effectively deliver air to the alveoli -possiblydue to blockage of the branches of the bronchial tree or due tocompromised ability of the chest muscles to expand andcontract enough to move air through the bronchial tree to thealveoli. Sometimes the problem is the inability of the lung toremove or detoxify foreign substances-possibly because ofan underlying deficiency or because the amount of thesesubstances has overwhelmed the lungs’ defence systems.Infections caused by bacteria, viruses, and more rarely, fungican occur primarily in the lungs, may affect the entire body,including the lungs and pleura, which are the membranessurrounding the lungs (Gunter, 2007).

Inflammation of the bronchi and bronchioles may createairway obstruction. Smoke and other particles in the air, dust,mold, allergens, exercise, cold air, and other factors may triggerto the level of attack (Rahman and Macnee,1996). During theprocess, the lining of the airways swell and the musclessurrounding the bronchi contract, narrowing the airway. Mucussecretion can further inhibit the flow of air; making breathingdifficult and causing the affected animal to wheeze. Mostepisodes do not cause permanent lung damage but frequentlyrequire immediate veterinary attention while in the cases withemphysema; the air sacs in the lungs are slowly destroyed(Brigham and Meyrick, 1986). Damage and scarring to thetissues between the air sacs, inflammation of the air sacs,and stiffening of the lungs may be a result of pulmonary fibrosis.Narrowing of the blood vessels in the lungs, increasing theirblood pressure and causing the heart to work harder to transportblood into the lungs observed in pulmonary hypertension. Thiscondition may co-exist and exacerbate a variety of lung diseasesand can lead to heart failure. Blood clot usually originating inthe veins of the legs or pelvis and traveling to the lungs to blocka blood vessel, causing chest pain, acute shortness of breath,and coughing to the life-threatening level in pulmonaryembolism that is again a very difficult condition to diagnoseand require prompt veterinary attention. Bronchopulmonarydysplasia (BPD), Respiratory Distress Syndrome (RDS), AcuteRespiratory Distress Syndrome (ARDS), Alpha-1 Antitrypsindeficiency are various diseases that are difficult to diagnosein veterinary field due to a variety of limitations and thereforefocus remains collectively on antimicrobials and very least onsupportive medicine, a very discouragable step for veterinaryphysicians. Diagnosing lung diseases to determine theirpossible causes and to evaluate their severity generally dependon careful observation of clinical symptoms and qualityauscultation. Though, pulmonary function tests (PFTs-Spirometry Air flow with a Peak Flow Meter Lung VolumeDiffusing Capacity Measurement), chest x-rays and or CT(computed tomography) scans, MRI (Magnetic resonanceimaging), Ultrasound, nuclear lung scanning, Positronemission tomography (PET) scans have their facilitative meritsbut have limitations in veterinary field in practical terms.

Rectal temperature 104°F and above was recorded inaffected cases and auscultation of the lungs revealed quiteharsh bronchovesicular sounds with or without moist rales. Ina few cases anterior ventral consolidation was recorded in

very narrow lung field. Appreciable recovery was noticedespecially at the time of withdrawal of treatment when only afew symptoms viz. reduced inclination to walk and intakeparticularly to water persisted for longer duration. Thesymptoms such as dullness, depression, inappetance andhigh rise in temperature may be a reflection of the reaction ofthe body to its internal changes occurring due to propagationof a variety of micro organism subsequently release of pyrogensand other related intrinsic factors within the system anddyspnoea, nasal discharge, lachrymation may be the outcomeof a complex interaction of environmental factors producingstress, a variety of micro organisms working synergistically todamage the cells lining the respiratory tract allowingcolonization and invasion of other organisms and acompensated host response(Aiello,1998). Blood examinationrevealed a variable picture of haematogram viz. Hb (7-10 g/dl),PCV (22-32), TLC (6800-13000), TEC (2.8-3.9), DLC (N-58-68, L-25-35, M-3-8, E-1-6, B-0-2). Increased number ofneutrophils with leucocytosis may be a reflection of acutebacterial infection. On bacterial cultural examination, both(Gram positive and Gram negative) kinds of organisms wereobtained. Main identified isolates were Staphylococcus spp.and E. coli, along with numerous unidentified isolates. Thetherapeutic approach used in present treatment has shown agood recovery with Beta-blocker and bronchodialators.Deriphyllin (Theo plus etophylline) inhibits phosphodiesterase,which degrades cyclic nucleotides, hence increased amountof intra cellular CAMP molecules causing smooth musclerelaxation, blockade of adenosine receptors (which enhancerelease of histamine and other inflammatory mediator andbronchospasm). The overall effect of the drug is to producebronchodilation by bronichial muscle relaxation, suppressionof response of airways to stimuli, cardiac stimulation (increasesheart rate and cardiac output) and respiratory stimulation italso induces diuresis. Salbutamol is a short-acting â2-adrenergic receptor agonist used for the relief ofbronchospasm. (Tinkelman et al., 1993; Reed et al., 1998;Ward et al., 1993; Kidney et al., 1995; Kazuhiro et al., 2002).Eosinophil survival induced by IL-5 and GM-CSF is decreasedby low concentrations of theophylline independently from PDEinhibition and changes in cAMP (Ohta et al.,1996; Yasui etal.,1997). The theophylline can markedly reduce histone H4acetylation at the GM-CSF promoter and now histone acetylationis thought to be an important area of regulation to producebronchodilation by bronichial muscle relaxation andsuppression of response of airways to stimuli. Beta adrenergicagonist and xanthene derivative are effective bronchodialators.They relieve bronchospasm and their bronchodialatory actionalso increase mucocilliary clearance possibly by stretchingand thinning the mucus layer thus allowing more effectiveciliary’s activity (Jones et al., 1977). The anti-allergic drugswere weak inhibitors of histamine release, high concentrations(100-1000 µM) producing less than 35% inhibition. Salbutamolproduced 39% inhibition at 10 microM (Church and Hiroi, 1987)which means Salbutamol is a more effective inhibitor ofmediator release from human lung mast cells than anti-allergicdrugs.

Ceftriaxone sodium inhibits bacterial cell wall synthesisthere by effective against both gram positive and gram negative

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bacteria (Laurence et al., 2010) and accordingly has beenevaluated in clinical cases of pneumonia in sheep withsupportive bronchodialators and beta blockers. Theseantibiotics are known to act by inhibiting the synthesis ofbacterial cell walls. It inhibits cross linkage between the linearpeptidoglycan polymer chains that make up a majorcomponent of the cell walls of both Gram positive and Gramnegative bacteria. Use of Beta blockers and bronchodialatorsseem to be good enough in getting the desired results of gasexchange through respiratory tract. Though this is a very smallstudy without screening the functions of vital organs duringand after treatment but primarily indicate of beneficial effects ofdrugs individually and collectively in relieving the patients’sufferings. Further studies are called for on larger scale withmore scientific approach to reach the conclusion.

AcknowledgementsThe authors are thankful to Director Central Sheep and

Wool Research Institute Avikanagar and other supporting stafffor providing necessary facility and support.

ReferencesAiello, Susan, E. (1998) The Merck Veterinary Manual. 8th ed. Merck &

Co. Inc., USA.Benjamin, M. M. (1985) Outline of Veterinary Clinical Pathology. 3rd ed.

Kalyani Publisher, New Delhi.Brigham,K. L. and Meyrick, B. (1986) The Am. Rev. of Respiratory

Dis. 133(5): 913-927.Church, M. K. and Hiroi J. (1987) Br. J. Pharmacol. 90(2): 421.

Cunningham, J. and Klein, B. (2007) Text book of Veterinary Physiology.4th ed. Saunders,

Gama, L.T. et al. (1991) J. Ani. Sci. 69: 2727-2743.Green, L.E. and Morgan, K.L. (1993) Prev. Vet. Med. 17: 251-261.Gunter et al. (2007) Reveiws in minerology and geochemistry. 67:453-

56.Haughey, K.G. (1991) J. S. Afri. Vet. Assoc. 62: 78-91.Jones et al. (1977) Veterinary Pharmacology and therapeutics. Ames

The Lowa Press.Kazuhiro et al. (2002) PNAS June 25, vol. 99(13): 8921-8926.Kidney et al. (1995) Am. J. Respir. Crit. Care Med. 151:1907-1914.Koneko, Jiro. (1980) Clinical Biochemitry of Domestic Animals. 3rd

ed. Academic Press, New York.Laurence, L. Brunton, et al. (2010) Goodman and Gilman’s The

Pharmacological Basis of Therapeutics. 12th ed. McGraw-Hill Professional; New York.

McConnochie et al. (1987) Thorax. 42:342.Merchant, I.A. and Packer, R.A. (1983) Veterinary Bacteriology and

Virology. Delhi: CBS Publishers and Distributors. 255.Newman MW and MacLean SA.

Nash, M.L. et al. (1997) Small Rumin. Res. 26(1-2):53-60.Ohta, et al. (1996) Clin. Exp. Allergy. 26, Suppl. 2:10-15.Rahman, I. and MacNee, W. (1996) Free Redical Biology and Medicine.

21(5): 669-681.Reed et al. (1998) J. Allergy Clin. Immunol. 101:14-23.Snedecor, G.W and Cochran, W.G. (1989) Statistical Methods. 8th ed.

Lowa State University Press London.Sonawane, G.G. et al. (2012) Vet. Pract. 13(1): 121.Tinkelman, et al. (1993) Pediatrics. 92:64-77.Ward, et al. (1993) Am. Rev. Respir. Dis. 147:518-523.Yasui, et al. (1997) J. Clin. Invest. 100:1677-1684.

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IntroductionFever is well known not for a clinician alone but far a

common man to the level that it has acquired the status ofdisease rather than a symptom and for a clinician, a simplethought of clinical health evaluation starts with recording oftemperature. This may be alone or in combination with orsequel of other diseases in all the animals including sheep(Mackenzie, 1980). There are many factors to influence theoccurrence such as presence of kind of etiological agents,transmitters, management system, season, age, region andimmune status of the animals.

In general lambs are at greater risk. Various studiesreported to have approximately 10-35% of lambs’ death within6 months of age in different agro-climatic conditions (Yapi etal., 1990; Green and Morgan, 1993; Nash et al., 1997; Haughey,1991; Gama et al., 1991). The major causes associated withlamb death were respiratory disorders (31.4%), digestivedisorders (14.6%), starvation (9.6%), endoparasitism (5.0%),septicaemia and toxaemia (10.1%), accidental (2.1%) andundetermined causes (27.2%) (Mandal et al., 2007). Feverwith diarrhoea and in some cases with hyperpnoea showscomplexity of disease and attracts attention of farmers/clinicians/researchers to look in to diseases carefully to evolveeffective methodology for rapid diagnosis and effectivetherapeutic management.

Materials and MethodsAround 48 lambs/sheep maintained at different farmers

around 8-20 kms of Central Sheep and Wool ResearchInstitute, Avikanagar, were clinically examined for health statusduring the period from June 2010 to July 2010. It was observedthat few animals were sick with sudden onset of high rise oftemperature followed by digestive disorder. These animalswere put on exhaustive clinical examination with recording oftheir rectal temperature, pulse and respiratory rates, mucousmembrane colour at the conjunctiva, gums and vulva.Examination at the neck for any large, firm swellings and ofrumen for doughness, gases and contractions, assessmentof abdominal contour and auscultation of the heart, lungs wascarried out. The hair or wool around the perineum was

CLINICO-THERAPEUTIC MANAGEMENT OF ENTERICFEVER IN SHEEP

S.K. Dixit, Jyoti Kumar , G.G. Sonawane , B.N. Tripathi and Fateh SinghCentral Sheep and Wool Research Institute

AVIKANAGAR-304501, Rajasthan, India

ABSTRACT

The occurrence of fever with and without diarrhoea in lambs was observed with the clinical signs of dullness, depression and veryoccasional hyperpnoea, rectal temperature varying between 103ºF-105.8ºF, inappetance to anorexia, lethargyness, variable thirst(prefer to lick water at short interval), pasty faeces (watery -very rare) with increased frequency, rough and dull hair coat. Theywere suspected to be suffering from enteric fever (fever with diarrhoea) and treated with injectable Cefotoxime, Acetaminophen andsodium bicarbonate along with fluid replenishment through normal saline and oral substitution of normal gut flora.

Key words: Therapeutics, sheep, enteric fever

inspected for the presence of wet or dry faecal material. Acareful examination of legs and feet was carried out to becertain that a suspected locomotor problem is not, in factsecondary to a neurologic, metabolic or infectious disease. Representative clinical samples (blood) were collected in EDTAon pre treatment for haematological (Hb, PCV, TLC, TEC, andDLC) investigations (Benjamin, 1985). The nasal swabs, bloodand faecal smears were stained for Gram’s and Giemsa’susing standard procedure (Merchant and Packer, 1983) foridentification of organism.

Therapeutic management was carried out usinginjectable Cephalosporin-Cefotoxime @ 5 mg/kg i/m b.d. for4-5 days, sodium bicarbonate 1 ml i/v b.d. on 1st day followedby 5 mg b.d. orally from next day for 2 days and normal saline100 ml b.d. i/v for three infusions only. Paracetamol (Fibrinil) 1-3 ml i/m 6-8 hourly was injected for 1-3 days, only as per needof animals. Gut flora was substituted through lactobacillusacidophilus (sporolac) ½ tablet b.d. for 2-3 days. Recovery inanimals was recorded in terms of improvement in clinicalsymptoms and restoration of derailed physiologicalparameters and accordingly efficacy of tried medication wasassessed.

Disease history: The sheep around the Institute aremaintained by farmers for supportive livelihood in Rajasthan.The maximum temperature during summer (June-July) rangesfrom 28oC to 42oC and occasionally goes up to 48ºC. Averagerainfall is about 260-440 mm and occurs mostly in July-August.The sheep husbandry is not well organised and animals arelet loose to surroundings and other open areas for feeding.Somehow they are maintained with inadequate veterinary andmanagemental practices but the animals were vaccinated forsheep pox, Peste des Petits Ruminants (PPR), andenterotoxaemia as per the schedule in general. On set of rainyseason and sudden downfall in the temperature, lambs andother animals are generally put on direct exposure to the cold.In animals under report, mortality started suddenly in 3-4months old lambs with history of clinical signs such as suddendeath, increased shallow respiration and fever, shivering,unable to walk and loose motion.

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Microbial examinationThe nasal swabs, blood and faecal smears were stained

for Gram’s and Giemsa’s using standard procedure (Merchantand Packer, 1983) for identification of organism. Swabhomogenates (20% w/v) were made in sterile PBS andenriched in bovine heart infusion (BHI) broth at 370C for 48 hrfollowed by streaking on to the sheep blood agar. The bacterialcolonies were further streaked on Mac-Conkeys and eosinmethylene blue (EMB) agar for identification of E. coli(Cruickshank et al., 1975).

Antibiotic sensitivity assayAmong economic antibiotic discs (Himedia) were utilised

to conduct antibiotic sensitivity assay. Ammoxycillin (Am),ampicillin (A), cloxacillin (CX), colistin (Methane sulphonate)(CL), novobiocin (NV), doxycycline hydrochloride (DO),ciprofloxacin (CF), enrofloxacin (EX), levofloxacin, furazolidone(FR), nitrofurantoin (NF), norfloxacin (NX), erythromycin (E),gentamicin (G), chloramphenicol (C), chlortetracycline (CT),penicillin-G (P), streptomycin (S), trimethoprim (TR) andofloxacin (OF) were put on trial to determine as to whichantibiotic the organism will be the most effective for futuretreatment and control of similar symptom appearing disease.The Muller Hinton agar plate was used to record zone ofinhibition around the disks and that was measured with thehelp of the compact zone scale (Himedia), using the referencetable size of zone. The results were recorded as sensitive (S),intermediately sensitive (I) or resistant (R).

Results and DiscussionClinical health examination of affected cases was carried

out during the period of investigation. Nearly all animals wereshowing rectal temperature varying from 103.8-105.4ºF andresting respiratory rates range from 12-20 per minute while inthe excited patient, rates were higher to number varying from30-46 and pulse rates range from 70-90. Rectal temperature,respiratory rate, and heart rate are easily elevated in the anxioussmall ruminant. Normal rectal temperatures range from101.5-104.0°F and may reach to higher side on exertion,excitement, or prolonged exposure to warm or humidenvironment (Aiello, 1998). Other unaffected animalsmaintained under same environment helped in healthevaluation particularly for comparison purpose. Fever due toinfection or other factors may seriously affect usualphysiological functions and in turn may reduce their foodconsumption and weight loss. Evaluation of colour of mucousmembrane at the conjunctiva, gums, or vulvas was notsuggestive of any critical condition in majority of the cases. Afew cases showed pale conjunctiva may be result of mildanaemia and may be correlated with reduced intake (Radostitset al., 2000). Examination at the neck for any large, firmswellings ruled out any abnormality. Auscultation of the lungsrevealed anterio-ventral consolidation in very narrow lung fieldindicative of lower respiratory disease and presence of a bithigh risen rectal temperature 105 to 105.5°F triggers the thoughtof many infections that cause fever by inducing production ofseveral cytokines, of which ingterleukin-6 and tumour necrosisfactor are most likely involved. It is unlikely that the cytokinescross the blood-brain barrier, but act on the organumvasculosum of the laminia terminalis, a circumventricular organ

outside the brain, which results in activation of the preopticarea of the hypothalamus to induce the production ofprostaglandins. A fever is produced by bacterial, viral orchemical pyrogens. It is the result of a “resetting” of thethermoregulatory mechanism to function above the normallevel. In many animals prostaglandins are responsible for thereadjustment (Aiello, 1998). In acute infections, the temperaturemay rise several degrees above normal for a few days. In a fewcases, the depth of respiration was a bit short and shallow butnot associated with obvious abnormal lung sounds. Elicitinga cough by tracheal palpation was not a detectable parameterin any of the case ruling the possibility of severe lunginvolvement. Assessment of abdominal contour did not showany detectable abnormality of immediate clinical significance.The skin and urine examination ruled out presence ofectoparasites and urine disorders. Blood samples analysedfor Hb (9-12g%), PCV (24-36%), TLC (5700-16000/cumm),TEC (2.6-4.1 millions/cumm), DLC (N 55-65%, L 25-35%, M 3-8%, E 1-6% and B 0-2%) showed increased number ofneutrophils, in some cases with leucocytosis may be areflection of acute bacterial infection. Staining revealedpresence of both, Gram’s positive and negative, kind oforganism.

Nearly in all the treated animals’ clinical recovery wasobserved from 12-24 hrs. of treatment as elicited fromimprovement in various symptoms such as dullness,depression, reduction in temperature. Complete recovery tookmore time and days of varying from 5-10 days of treatment.The symptoms such as dullness, depression, inappetanceand high rise in temperature may be a reflection of reactions ofthe body to its internal secretions/changes subsequentlyrelease of pyrogens and other related intrinsic factors withinthe system and in a few cases dyspnoea, respiratory ralesmay be the outcome of a complex interaction of environmentalfactors producing stress, a variety of micro organisms workingsynergistically to damage the cells lining the respiratory tractallowing colonization and invasion of other organisms and acompensated host response.

These relate to similar observations of many workers (Dixitet al., 2010; Stevenson and Robinson, 1970; Lehmkuhl andCutlip,1984 and Robinson, 1983). Cephalosporins in generaland used Cefodoxime in particular is a penicillinase resistantantibacterial agent effective against both spectrum of (gram-positive and gram-negative) bacteria including Chlamydia,Mycoplasma and some methicillin resistant strains (Riviereand Papich, 2009) and therefore was put in use in these clinicalcases with support of antipyretic, blood isotones, Normal Salineand ion replacer which might have acted in one or the otherway individually and/or collectively to produce desiredtherapeutic effect on affected animals. Invasion of the systemiccirculation by pathogenic bacteria, which may causebacteraemia, fever and even septic shock in advance caseswith possible localization in various organs of body (Radostitiset al., 2007) and therefore need to be killed at the earliest.Simultaneously, among pathogens, drug-resistant is anincreasing problem apart from toxicity and residual effects andtherefore, antibiotics alone and with other drugs should beused with caution especially in meat/ milk producing animalsand repeated pharmacological and clinical field trials must be

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carried out covering large number population in differentseasons and geographical area to reduce the otherwiseoccurring problem.

Microbial examinationStaining revealed presence of both kind of organism,

Gram’s positive and negative Staphylococcus sp. Proteus, E.coli along with nonspecific isolates.

Antibiotic sensitivity assayOf the tested antibiotics, ammoxycillin (Am), ampicillin (A),

cloxacillin (CX), colistin (methane sulphonate) (CL), novobiocin(NV), doxycycline hydrochloride (DO), ciprofloxacin (CF),enrofloxacin (EX), norfloxacin (NX), furazolidone (FR),nitrofurantoin (NF), erythromycin (E), gentamicin (G),chloramphenicol (C), chlortetracycline (CT), penicillin-G (P),streptomycin (S), and trimethoprim (TR) for the sensitivity, thesewere sensitive against E. coli while levofloxacin, andcotrimoxazole were intermediately sensitive. Erythromycin,penicillin-G, and ofloxacin and were resistant for these testedorganisms. Changing trend of resistance warns the cliniciansas the latest among fluoroquinilones such as levofloxacin andofloxacin are moderately sensitive or resistant.

Long acting tetracycline has been recommended in thetreatment of acute bacterial infections in sheep (Martin, 1996)and the adverse climate influences the disease resistance ofthe lambs (Wassmuth, 2003; Turkson and Sualisu, 2005).Tetracyclines are being used from centuries with variablesreports but Cephalosporins are comparatively least reportedthat too in sheep with present combinations. Animals treatedwith Cefotoxime, sodium bicarbonate, normal saline, andparacetamol and lactobacillus acidophilus showed goodperformance in terms of recovery in animals. Improvement inclinical symptoms and restoration of derailed physiologicalparameters including brightness/visible activeness matchingwith the claim of other research workers (Laurence et al., 2010)and may be used as a choice of therapeutic regimen insheep af ter their critical health examination and moreexhaustive clinical therapeutic trial.

AcknowledgementsThe authors are thankful to Director Central Sheep and

Wool Research Institute Avikanagar and other supporting stafffor providing necessary facility and support.

ReferencesAiello, Susan, E. (1998) The Merck Veterinary Manual. 8th ed. Merck &

Co. Inc., USA.Benjamin, M. M. (1985) Outline of Veterinary Clinical Pathology. 3rd ed.

Kalyani Publisher, New Delhi.Cruickshank, R. et al. (1975). Medical Microbiology. 12th ed., Churchill

Livingstone, Edinburgh, London and New York.Dixit, S.K. et al. (2010) Vet. Pract. 11(2): 111-113.Gama, L.T. et al. (1991) J. Ani. Sci. 69: 2727-2743.Green, L.E. and Morgan, K.L. (1993) Prev. Vet. Med. 17: 251-261.Haughey, K.G. (1991) J. S. Afri. Vet. Assoc. 62: 78-91.Laurence, L. Brunton, et al. (2010) Goodman and Gilman’s The

Pharmacological Basis of Therapeutics. 12th ed. McGraw-Hill Professional; New York.

Lehmkuhl, H. and Cutlip, R. (1984) Am. J. Vet. Res. 45:260-262.Mackenzie, D. (1980) Goat Husbandry. Oxford and IBH Publishing Co.

New Delhi.Mandal, A. et al. (2007) Small. Rumin. Res. 71: 273-279.Martin, W. B. (1996) Comp. Immun. Microbiol. Infect. Dis. 19(3): 171-

179.Merchant, I.A. and Packer, R.A. (1983) Veterinary Bacteriology and

Virology. Delhi: CBS Publishers and Distributors. 255.Newman MW and MacLean SA.

Nash, M.L. et al. (1997) Small Rumin. Res. 26 (1-2):53-60.Radostitis, O. M. et al. (2007) Veterinary Medicine: A Textbook of the

diseases of cattle, sheep, pigs, goats and horses. 10th ed.W. B. Saunders, Elsevier Science Ltd. Philadelphia, USA.

Radostits et al. (2000) A text book of Veterinary Medicine. 9th ed. W.B.Saunders Book Power.

Riviere, Jim, E. and Papich, Merck, G. (2009) Veterinary Pharmacologyand Therapeutics. 9th ed. North Carolina,Wiley-Blackwell.

Robinson (1983) Vet. Clin. N. Am. L.A. Pract. 5:539-555.Stevenson, R.G. and Robinson, G. (1970) Res. Vet. Sci. 11: 469-474.Turkson, P.K. and Sualisu, M. (2005) Trop. Ani. Health Prod. 37(1):49-

64.Wassmuth, R. (2003). Dtsch. Tierarztl.Wochenschr. 110(5):212-215.Yapi, C.V. et al. (1990). Prev. Vet. Med. 10:145-152.

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IntroductionBovine mastitis is a single most common disease

syndrome in adult dairy cows, accounting for about 38 per centof morbidity (Smith, 1996). Mastitis is also associated withnumber of zoonotic diseases in which milk acts as a vehicle ofinfection. Disease is of great economic importance owing tohigh treatment cost, reduced milk production, culling of highproducing affected animals and extra expenses for themanagement of affected animals. The economic loss per cowin India roughly estimates Rs. 1164 in clinical and Rs. 1016 insubclinical mastitis (Misra et al., 2003). The disease manifestsitself in two forms i.e clinical and subclinical. Most of the dairyfarmers in India are hardly aware of SCM and as such attachno importance to it. Disease is complex in nature withmultifaceted aetiopathogenesis. Identification and knowing theprevalence of these organisms thus becomes essential toevolve proper treatment and control measures. The presentwork was undertaken to study the prevalence of SCM amongcrossbred cattle of Jammu region.

Materials and MethodsA total of 470 milk samples were collected aseptically

from apparently healthy quarters of lactating crossbred cattleof two different organized farms. The relevant date includinganimal number, lactation number and date of calving wererecorded. Milk samples were subjected to sodium laurylsulphate test (SLST) (Pandit and Mehta, 1969) for the diagnosisof SCM. The milk samples found positive by one or manyindirect tests were transferred to the laboratory and weresubjected to bacterial isolation and characterization as per thestandard bacteriological methods (Cruickshank et al., 1982).

Results and DiscussionThe overall animal-wise incidence of SCM among

crossbred cattle was 42.18% and quarter-wise incidence was19.14% (Table 1). Quarter-wise the incidence of SCM was19.14 per cent. Finding of present study corroborates with thefinding of Rahman et al. (2010) and Sharma et al. (2011).However, Nath and Dutta, (2007) reported higher animal wise

PREVALENCE OF SUBCLINICAL MASTITIS IN CROSS BREEDCATTLE FROM JAMMU REGION

A. Sharma, R. Singh, S.A. Beigh and R. K. BhardwajDivision of Veterinary Clinical Medicine and Jurisprudence

Faculty of Veterinary Science and Animal Husbandry (SKUAST-J)R.S. Pura-181102, Jammu, India

ABSTRACT

A total of 470 milk samples were collected from apparently healthy quarters of lactating crossbred cattle of Jammu region to study theprevalence of subclinical mastitis (SCM). Milk samples were subjected to sodium lauryl sulphate test (SLST). Animal wise and quarterwise incidence of SCM was 42.18% and 19.14%, respectively. Right side quarters were having higher incidence (64.44%) comparedwith left quarters. The incidence of SCM was lowest during first lactation which increased with subsequent lactations and washighest in sixth and above lactations. Bacteriological isolation revealed that Staphylococcus aureus (82.22%) was the major pathogenfollowed by coagulase -ve staphylococci (6.66 %), Streptococci spp. (3.33 %), Escherichia coli (3.33 %) and Corynebacterium spp.(4.44 %).

Key words: Subclinical, mastitis, bacteriology and prevalence.

(73.46%) and quarter wise (73.82%) incidence of SCM amongdairy cows. Likewise, 35.25% and 36% quarter-wise andanimal-wise prevalence of SCM among crossbred cattle fromPakistan was reported by Bachaya et al. (2011), respectively.The differences observed among various studies could bedue to the variation in the managemental practices beingfollowed at various farms, prompt treatment of clinical cases,culling of carriers, selective breeding number of cases studied,geographical variations, milking techniques, season of studyand adaption of mastitis control programme.

On individual quarter-wise basis, right sided quartersrevealed had higher incidence (64.44%) of mastitis (Table 1).Contrary to present findings, Khan and Muhammad (2005)reported higher prevalence of mastitis in left sided quarters.The higher incidence of SCM in right-side quarters could beascribed to the fact that cows mostly sit on right-side with theresult right-side quarters are frequently exposed to dung andsoil moreover due to pressure of the body of animal the milkdribbles out through the teats of high yielders and thusincreasing their susceptibility. Dakshinkar et al. (1999)observed higher incidence of mastitis in fore quarterscompared with hind quarters observed in present study.

Lactation wise, the highest prevalence of SCM wasrecorded among the animals in sixth and above lactations(37.03%). In first, second, third, fourth and fifth lactations thepercentage occurrence of SCM was 9.25%, 7.40%, 12.96%,11.11%, and 22.22%, respectively. Radostits et al. (2000)emphasized that generally the incidence peaks at 7 years ofage. Nath and Dutta (2007) reported higher incidence of SCMin the 3rd followed by forth and 2nd lactation. Increasing evidenceof mastitis with advanced ages was recorded by Hawari andAl-Dabbas, (2008). This may be ascribed to gradual loss inthe immune system in the body of the animal with the increasein lactation number, which makes it susceptible to infection.The loosening of sphincter and patency of teat canal in oldercows as cited vide-supra could be responsible for the observedincrements. Moreover, the med ian ligaments whichprovide support to the teat also get relaxed with age

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leading to hanging of udder with age and making it moreprone towards the mastitis. Among the mastitic crossbredcows studied, 42.59 per cent were in first trimester oftheir lactation compared with 25.92 per cent in mid and31.48 per cent in late lactation. The findings were inagreement Datta and Rangnekar (2001) and Peeler et al.(2002). This high incidence could be ascribed to entry oforganisms into udder during the early days of dry periodas keratin plug at teat orifice is formed after 7 days of lastmilk ing and also due to delayed d iapedesis o fpolymorphonuclear neutrophils into the infec tedmammary gland. Moreover, weaning is not practicedso181102 un-weaned calves often cause injury to theudder and create a focus for infection, the calves causeinjury, because of biting, pulling and hitting the udder.Inrelation to milk yield cattle with milk yield above 6 lt. wereobserved to have more prevalence of mastitis (81.48%)compared with the animals having lesser milk yield.Incidence of mastitis is influenced by many factors ofwhich milk yield plays a pivotal role. The stress of overproduction might have potentiated the onset of mastitisin high producers (Radostits et al., 2000).

Out o f 90 bacter ial iso lates f rom sub-c linicallyinfec ted c rossbred catt le majority belonged toStaphylococcus aureus (82.22%) followed by coagulase–ve staphylococci (6.66 %), Streptococci spp. (3.33 %),Escherichia coli (3.33 %), Corynebacterium spp. (4.44%). The present finding corroborates with the finding ofSahoo et al. (2009) who reported Staphylococcus spp.as major pathogen fo llowed by Streptococc i spp . ,Escherichia coli of sub-clinical mastitis. The increasedincidence of staphylococci may be attributed to ubiquitousnature of organism secondly, due to drug resistance. It isbelieved that infections with Staphylococcus aureusrespond poorly to therapy with antimicrobial agents,whether given parenterally or via the intramammary route(Moon et al., 2006). The emergence of antibacterialresistance among pathogens that affect animal health isof growing concern in veterinary medicine. Antimicrobial-resistant pathogens in animals have been incriminatedas a potential health risk for humans from possibletransmission as foodborne pathogens. Particu larly,

Table 1:Distribution of quarter wise prevalence of subclinical mastitis

Total animal Total quarters Quarter wise distribution Examined Posi tive Examined Posi tive LF LH RF RH

128 54 (42.18%)

470 90 (19.14%)

13 (14.44%)

19 (21.11%)

22 (24.44%)

36 (40%)

mastitis is the single greatest cause of antibacterial useon dairy farms (Moon et al., 2006).

Based on study it was concluded that SCM is widelyprevalent in crossbred dairy cows in our field conditions,which ultimately reflects the bad quality of milk availableto the consumers. Thus, regular screening of dairy farmsfor diagnosing SCM should be practiced in the field bythe farmers. As mastitis is managemental problem andmastitis prevention practices like dry cow therapy, pre andpost-milk ing teat dip are not practiced at most of themilking sheds. Most common pathogen of mastitisStaphylococcus aureus is contagious, therefore at thetime of milking non-mastitic animals are exposed to thesepathogens from milkers’ hands. Milking procedure in ourconditions is accompanied with unhygienic conditionsand the teats are exposed to injury with faulty invertedthumb method of milking.

ReferencesBachaya, H. A., et al. (2011) The J. Ani. and Plant Sci. 21(1):

16-19.Cruickshank, R. et al. (1982) Medical Microbiology. 12th ed. Vol.

III. Churchill Livingstone, New York.Dakshinkar, N.P. et al. (1999) Indian Vet. Med. J. 23: 339-340.Datta, S. and Rangnekar, A. (2001) Indian Vet. J. 78: 161-162.Hawari, A.D. and Al-Dabbas, F. (2008) Am. J. Ani. Vet. Sci. 3:

36-39.Khan, A. Z. and Muhammad, G. (2005) Pakistan Vet. J. 25 (1):

9-12.Misra, S. S. et al. (2003) In: IAAVR Proceeding Round Table

Conference on Mastitis, H.P.K.V., Palampur, 14th to 15th

April, 2003. pp. 32-46.Moon, J.S. et al. (2006) J. Dairy Sci. 90: 1716-1724.Nath, A. K and Dutta, J. B. (2007) Indian J. Vet. Med. 27(2): 117-

118.Pandit, A. V. and Mehta, M.L. (1969) India Vet. J. 40: 111-119.Peeler, E.J. et al. (2002) Vet. Rec. 151: 170-176.Radostits, O. M. et al. (2000) Veterinary Medicine. 9th ed., W. B.

Saunders Harcourt Publishers Ltd.Rahman, M. M. et al. (2010) Int. J. Bio. Res. 1(2): 23-28.Sahoo, S. S. et al. (2009) Indian Vet. J. 86: 1298-1299.Sharma, A. et al. (2011) Ani Biotechnol. 22(3):163-9.Smith, B. P. (1996) Large Animal Internal Medicine: Diseases of

Horses, Cattle, Sheep, and Goats . 2nd ed., Mosby-year book, U S A, 1177-1188.

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IntroductionMastitis, defined as inflammation of the parenchyma of

the mammary gland (Radostits et al., 2007) is one of the mostimportant diseases of dairy animals, responsible for heavyeconomic losses. Generally, bovine mastitis is of infectiousnature. Watts (1988) identified 137 different organisms as thecauses of mastitis. However, the majority of mastitis casesare of bacterial origin and five species of bacteria(Staphylococcus aureus, Streptococcus agalactiae,Streptococcus dysgalactiae, Streptococcus uberis andEscherichia coli) account for almost 80% of all diagnoses(Anon, 2001). A bacteriological diagnosis and proper selectionof antibiotic based on antibiotic sensitivity are critical for rationaland effective treatment of mastitis. Therefore, the present studywas carried out to identify the bacterial pathogens of bovineclinical mastitis and antibiogram of each bacterial isolate toselected antibiotics.

Materials and MethodsThe study was carried in cross-bred cows (crosses of

Jersey/Brown Swiss/Haryana/Holstein Friesian) under differentphases of lactation at the cattle and buffalo farm, IndianVeterinary Research Institute, Izatnagar, India. Twelve cases ofclinical mastitis were identified on basis of physicalexamination of udder and milk collected in sterile glass vials.

The udder and teats of cows were examined for signs ofinflammation viz. redness, swelling, heat, pain and depressedmilk yield. The quarters with any physical deformity like atrophyor fibrosis, blind or supranumerary teats etc. were excludedfrom the study. The milk was examined for the presence ofdiscolouration, blood clots, flakes, purulent material etc. Themilk samples were also examined for somatic cell countindirectly by California Mastitis Test as per Schlam andNoorlander (1957) and directly as per Schlam et al. (1971).Bacterial isolation and identification was done by culture of

IDENTIFICATION AND ANTIBIOGRAM OF BACTERIAL ISOLATESFROM DAIRY COWS WITH CLINICAL MASTITIS

R. Jhambh1, U. Dimri, V.K. Gupta and R. Rathore2

Division of MedicineIndian Veterinary Research Institute, Izatnagar-243122, Uttar Pradesh, India

ABSTRACT

The present study was carried out to identify bacterial pathogens of bovine clinical mastitis and their antibiogram to the selectedantibiotics. A total of twelve cases of clinical mastitis in lactating dairy cows were identified on basis of physical examination of udderand milk and examination of somatic cell count in milk. Bacterial isolation and identification was done by culture of milk samplescollected in sterile glass vials and requisite biochemical tests which revealed a predominance of Streptococcus agalactiae, followedby Micrococci and Coliform as cause of mastitis. The antibiogram of each bacterial isolate to standard antibiotics determined by discdiffusion method reflected the highest sensitivity to amoxycillin/sulbactam followed by enrofloxacin and the least sensitivity toamoxycillin. Judicious use of antibiotics based on antibiotic sensitivity in control of mastitis may reduce the chances of treatment failureand ultimately the economic losses.

Key words: Antibiogram, bacterial isolates, clinical mastitis, dairy cows, identification

1Corresponding author and Ph.D. Scholar ([email protected])2Scientist (SS), Centre for Animal Disease Research and Diagnosis, IVRI.

milk samples and requisite biochemical tests by the methodof Quinn et al. (2004) and sensitivity of each bacterial isolate tostandard antibiotics viz. amoxycillin (10 µg/disc), ampicillin/cloxacillin (10/10 µg/disc), erythromycin (15 µg/disc),enrofloxacin (10 µg/disc) and amoxycillin/sulbactam (30/15 µg/disc) was determined by disc diffusion method (Bauer et al.,1966).

Results and DiscussionMean CMT point score and SCC recorded in milk of affected

cows were 3.67±0.13 and 83.12±1.17 x 105 per ml, respectivelywhich corresponds with clinical mastitis as per Schlam et al.(1971). Out of total 12 cases of clinical mastitis, 7 (58.33%)were found positive for Streptococcus agalactiae, 2 (16.66%)positive for Micrococci, 2 (16.66%) positive for Coliform and 1(8.33%) showed no growth on culture media. Antibiogram ofbacterial isolates recovered from mastitic milk samples isdepicted in table 1 which reflects highest sensitivity of isolatesto amoxycillin/sulbactam followed by enrofloxacin, erythromycin,ampicillin/cloxacillin and amoxycillin.

Streptococcus agalactiae is a highly contagious obligateorganism of the bovine mammary gland (McDonald, 1977)which generally causes a low-grade, persistent type of infectionand does not have high self-cure rate (Farnsworth, 1987). Thepresent study revealed predominance of Streptococcusagalactiae in clinical cases of mastitis in cows which correlateswith finding by Gonzalez et al. (1986) of high prevalence withinherd when it is infected with S. agalactiae. Usually the organismis sensitive to wide range of commercially available antibiotics(Radostits et al., 2007) and the present antibiogram reflectedall isolates sensitive to erythromycin, enrofloxacin andamoxycillin/sulbactam, so are the potent options for treatmentof Streptococcus agalactiae mastitis. Micrococci similar tocoagulase negative staphylococci (Quinn et al., 2004) are teatskin opportunistic mastitis pathogens responsible for mild

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clinical mastitis which usually has a spontaneous cure(Radostits et al., 2007). The present study revealedmicrococcus organisms sensitive to enrofloxacin andamoxycill in /su lbac tam but resistant to amoxycill in .Resistance to amoxycillin may be attributed to its frequentuse in mastitis con trol. Co liform wh ich inc ludesEscherichia, Klebsiella, and Enterobacter are consideredenvironmental pathogens o ften isolated from acuteclinical cases of mastitis. Currently available antibioticshave minimal effec t on shortening the duration o fintramammary infections caused by coliform (Hogan andSmith, 2003). However, the present antibiogram reflectedall coliform isolates sensitive to amoxycillin/sulbactamfollowed by enrofloxacin but resistant to erythromycin.

So the overall antibiogram of isolates reflected thehighest sensitivity of isolates to amoxycillin/sulbactamand least sensitivity to amoxycillin. Poor sensitivity toamoxycillin may be due to production of alpha-lactamaseby resistant strains of isolates and sulbactam added toamoxycillin overcomes this alpha-lactamase resistance

(Radostits et al., 2007), therefore, the combinationpresents the potent candidature for mastitis control thatfurther needs to be confirmed through clinical trials.

ReferencesAnon (2001) Veterinary Investigation Surveillance Report .

London, Veterinary Laboratories Agency.Bauer, A.W. et al. (1966) Am. J. Cl. Path. 45: 453-496.Farnsworth, R.J. (1987) Proc. 26 th Annu. Meet Natl. Mastitis

Counc. 26: 151.Gonzalez, R.N. et al. (1986) J. Am. Vet. Med. Assoc. 189: 442.Hogan, J. and Smith, K.L. (2003) Vet. Res. 34: 507.McDonald, J.S. (1977) J. Am. Vet. Med. Assoc. 170: 1157.Quinn, P.J. et al. (2004) Clinical Veterinary Microbiology. Mosby,

Elsevier Limited, Philadelphia, USA.Radostits, O. M. et al. (2007) Veterinary Medicine: Diseases of

cow, buffalo, horse, sheep, goat and pig . 10 th ed.Saunders Elsevier Limited, Philadelphia, USA.

Schalm, O.W. and Noorlander, D.O. (1957) J. Am. Vet. Med.Assoc. 130: 199.

Schlam, O.W. et al. (1971) Bovine mastitis. Lea and Febiger,Philladelphia. pp. 128.

Watts, J.L. (1988) Vet. Microbiol. 16:41.

Table 1: Antibiogram of bacterial isolates recovered from mastitic milk samples

Isolate Sensitivity Antibiotics Amoxycillin Ampicillin/cloxacillin Erythromycin Enrofloxacin Amoxycillin/sulbactam (10 µg/disc) (10 µg/disc) (15 µg/disc) (10 µg/disc) (30/15 µg/disc) S. agalactiae (7)S 3 (42.86%) 5 (71.4%) 7 (100%) 7 (100%) 7 (100%) I 3 (42.86%) 2 (28.57%) 0 0 0 R 1 (14.28%) 0 0 0 0 Micrococci (2) S 0 0 0 2 (100%) 2 (100%) I 0 2 1(50%) 0 0 R 2 (100%) 0 1(50%) 0 0 Coli form (2) S 1 (50%) 1(50%) 0 0 2 (100%) I 0 1(50%) 0 2 (100%) 0 R 1(50%) 0 2 (100%) 0 0 Overall (11) S 4 (36.36%) 6 (54.56%) 7 (63.64%) 9 (81.82%) 11 (100%) I 3 (27.27%) 5 (45.45%) 1 (9.09%) 2 (18.18%) 0 R 4 (36.36%) 0 3 (27.27%) 0 0

S-Sensitive, I-Intermediate, R-Resistant

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IntroductionOestrus ovis is a cosmopolitan fly. It prevails where sheep

and goat herd resides. First instar larvae (L1) are depositedon the nostrils of the host by female flies, and penetrate activelyinto the nasal cavities. Then, L1 migrate to the ethmoid tomoult to second instar larvae (L2). In the sinus, L2 larvae moultto third instar larvae (L3) which are sneezed by the host ontothe ground where they pupate. Occasionally they enter the skullbones and get trapped in the cerebral cavities causing falsegid. Adult flies have a reduced and nonfunctional oral apparatus,and so are not able to feed. Thus larvae, in close contact withmucosa, play an essential role in accumulating nutrients forthe free stages of the life cycle.

The presence of O. ovis larvae leads to stronginflammation associated with mucus hyper secretion andnasal discharge in response to numerous larval depositionsby adult flies and sinusitis in response to the rapid developmentof larvae. In addition to these local effects, some indirect effectssuch as lung abscesses and interstitial pneumonia are notrare (Dorchies et al., 1993). According to breeders, goats arenot heavily infested and oestrosis is only a sheep diseasedespite the common presence of the parasites in goats(Dorchies et al., 2000).

Oestrus ovis larvae are obligatory parasites of nasal andsinus cavities of sheep and goat but occurrence of nasal botsin an aberrant location has also been recorded (Godara et al.,2010). The breeding and rearing of sheep and goats is alsoan important activity in India and an unorganized labour takingcare of animals may have the chances of zoonosis.Jayaparkash and Kathikayan (2008) reported ocular myiasisalong with mucopurulent conjunctivitis in humans indicatingthe zoonotic potential of the parasite. Infestation of nasal botsin sheep has been reported from other regions of India likeBareilly (Parihar, 1986), Bangalore (Jagannath et al., 1989)

INCIDENCE OF OESTRUS OVIS IN SHEEP IN UDAIPURREGION OF RAJASTHAN

Maneesh Sharma1, Abhishek Gupta2*, Chetna Mahajan3 and Sonal Ingle4

Department of Veterinary PathologyCollege of Veterinary and Animal Science


ABSTRACT

The present study was conducted to investigate the incidence of Oestrus ovis in sheep in Udaipur region of Rajasthan. Aconsiderable incidence of 19.53% was observed in the wide spread study area. Oestrus ovis larva is an obligatory parasite ofthe nasal and sinus cavities of sheep and goat. The aberrant migration of the nasal bots was also observed in the present study.Keeping in view the economical losses caused by the fly and its increased concerns about the drug residues on human healthand the environment, it is recommended to conduct more studies on Oestrus ovis to develop better control strategies.

Keywords: Incidence, Oestrus ovis, Rajasthan, sheep, Udaipur.

while O. ovis larvae were reported in both sheep and goatsfrom Hisar (Chhabra and Ruprah, 1978) and from Rajasthan(Pathak, 1992).

Materials and MethodsA total of 128 sheep heads were examined for Oestrus

ovis infection. The study was carried out on sheep slaughteredby local butchers and postmortems conducted in theDepartment of Veterinary Pathology. All larvae present werecollected from the nasal passage, septum and the middlemeatus and the conchae and sinuses.

Results and DiscussionOut of 128 sheep examined, a total of a 25 sheep were

found positive for Oestrus ovis infestation with an incidencerate of 19.53%. The aberrant migration of nasal bots wasobserved in two sheep’s in which one of the bots bored its wayinto the mandibular periosteum (Fig.1) which was also reportedby Godara et al. (2010) in goats. It digested spongy deeperbone tissues using its acidic and enzymic secretions whileother was located in the trachea (Fig. 2). A detailed microscopicexamination of the creamy coloured nasal bots, recovered fromsheep heads and aberrant location, measured 17-25 mm insize and possessed dark coloured transverse bands on dorsalsurface of each segment, anterior end was armed with minutepaired hooks, posteriorly connected with cephalo-thoraxialskeleton and ventral surface of bots had rows of minute spines(Fig.3). Morphological characterstics of ‘D’ shaped, closed,dark black coloured, deep seated stigmal plates with radiallyarranged respiratory holes, confirmed the identification of thebots.

It is known that marked flies present in the sheep penssurvive for up to 37 days and remain at the site or reach othersheep pens up to 6 km away as well (Ternovoi and Mikhailenko,

1Assistant Professor, Department of Veterinary Pathology, CVAS, RAJUVAS2Assistant Professor, Department of Veterinary Parasitology, CVAS, RAJUVAS3Assistant Professor, Department of Veterinary Physiology and Biochemistry, CVAS, RAJUVAS4Assistant Professor, Department of Veterinary Microbiology, CVAS, RAJUVAS

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Fig. 3: Dorsal and ventral view of Oestrus ovis larva

Fig.2: Oestrus ovis larva in tracheaFig.1: Aberrant infestation of Oestrus

1973) external reintroductions of O. ovis can occur very easilyin well-treated sheep flocks. Some farmers do not treat theiranimals and organized farms treat their animals after theappearance of clinical signs. So, inspite of strict treatmentprotocol and use of effective drugs, it becomes very difficult tocontrol the incidence. Recent findings from experiments withtracer lambs showed that low grade infestations occur in thespring (Dorchies et al., 1996). The absence of reactionassociated with spring infestation may be due to the lowlarval dose received which may be under a ‘reaction’threshold. Nasal discharges and sneezing are oftenevident a month or two after the primary attack whenthesecond or third fly generations are active.

Moreover, concerns about the effects of drug residueson consumer health and the environment have focusedattention on the prospect of developing alternative controlstrategies. No baits and no traps are available for adult

O. ovis. Immunisation of sheep with excretory-secretoryproducts o f larvae does not p rotec t against larvalestablishment but provide a transitory inhibition of larvalgrowth (Frugere et al., 2000). At present, the most feasibleand practical alternative approach is to keep the sheepindoors during the warmest hours of the day betweenJuly and September. This strategy seems to be veryeffective in the Atlantic Pyrenees. Sheep breeders do notobserve clinical signs in their flocks when using thismanagement strategy.

Other measures may include providing good farmingpractices and management. Paying special attention tofeeding standards and feeding programs. Dipp ingpractice should be applied to all herd members with allages at least twice, at late spring and August using anefficient insecticide. The present study is indicating aconsiderable incidence of Oestrus ovis in sheep ofUdaipur reg ion o f Rajasthan. Hence it is stronglyrecommended to conduct more studies on Oestrus ovisto develop better con trol strategies against thepathogenesis and zoonosis caused by the parasite.

ReferencesChhabra, M. B. and Ruprah, N. S. (1978) Indian Vet. J. 53: 180-

184.Dorchies, P. et al. (1993) Vet. Rec. 133:325.Dorchies, P. et al. (1996) Bull. Soc. Fr. Parasitol. 14: 20-27.Dorchies, P. et al. (2000) Vet. Parasitol. 88 269-273.Frugere, S. et al. (2000) Vet. Res. 31:527-535.Godara, R. et al. (2010) Trop. Ani. Health Prod. 42: 137-139.Jagannath, M. S. et al. (1989) Indian Vet. J. 66: 677-679.Jayaprakash, K. and Kath ikeyan, A. (2008) Indian J.

Occupational & Environ. Med. 12(1):Parihar, N. S. (1986) Indian J. Ani. Sci. 59: 1268-1272.Pathak, K. M. L. (1992) Indian J. Ani. Sci. 62: 50.Ternovoi, V.I. and Mikhailenko, V.K. (1973) Vet. Parasitol. 7:123-

127.

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IntroductionEthnoveterinary practices are often cheap, safe, long time

tested and based on local resources and strengths. These canprovide useful alternatives to modern animal health care systems(Kumar, 2002; Mc Corkle, 1995). These indigenous practices playa crucial role in the livestock health care of traditional societies.However, due to changing socio-economic and cultural values ofthe traditional communities, the indigenous practices of livestockrearing are gradually declining (Farooquee and Saxena, 1996;Purohit et al., 2002). According to Catley et al. (1998), veterinaryservices are poorly developed in many developing countries andthis situation is more evident in remote and dry land areas inhabitedby pastoral and agro pastoral communities. The large andscattered area, harsh climate, poor infrastructure and relativelysmall human population are some of the characteristics of suchareas. These factors alone have been reported as constraints toanimal health service delivery system through government andprivate sectors. Pastoralist, contacted with traditional healers asfirst preference for common animal health problems due to theireasy accessibility and cost-effectiveness of treatment. Thesetraditional practices have passes from generation to generationthrough oral transaction and skilled learning without anydocumentation. Changing the socio-culture setting of society,introduction of modern technologies, less interest of newgeneration, shrinkage of forest and less availability of some floraare the important factors that responsible for continue extinction ofthese practices from our society.

Materials and MethodsPresent study has conducted in two purposively selected

CONSTRAINTS PERCEIVED BY TRADITIONAL HEALERS ABOUTSUSTENANCE OF THEIR ANIMAL HEALTH PRACTICES IN ARID

ZONE OF RAJASTHAN#

Devi Singh Rajput1 and Hema Tripathi2Department of Veterinary and Animal Husbandry Extension Education

College of Veterinary and Animal Science, Navania, Vallabhnagar, Udaipur-313601Rajasthan University of Veterinary and Animal Sciences, Bikaner, Rajasthan, India

ABSTRACT

Present paper highlights the occupational profile and constraints perceived by traditional healers in arid zone of Rajasthan. Animal husbandryreported as major family occupation followed by crop farming among 33.33 per cent of respondents. About 53 per cent of traditional healershad 10 to 20 years experience of traditional occupation followed by 26.67 per cent of respondents who reported more than 20 years ofexperience. About 60 per cent of respondents had acquired their knowledge from their ancestor and self experience regarding treatment ofanimals. None of the traditional healer had attended any training from any sources regarding treatment of animal diseases. Lack of herbal drugstore in the village was reported major constraints and ranked first by most of the traditional healers with highest RBQ value (95.04), followedby availability of some of plants only in particular season making treatment impossible in off season (79.86), lack of effective therapies forepidemic diseases (77.88), inconvenient and time consuming in obtaining, preparing and administrating some of medicines (46.20), lack oftransport to visit for treatment nearby villages (43.56), inadequate diagnosis due to lack of training resulting ineffective therapy to save theanimal (40.92), extinction of some of the ethno treatment due to non practice and less interest of younger generation (36.96), lack of education(20.46), poor financial base of pastoralists (19.14).

Key words: Traditional healers, pastoralists, constraints, Rajasthan

Bikaner and Jaisalmer districts from arid zone of Rajasthan. Twotehsils were selected purposively on the basis of existence oflarge number of pastoralists and animal health service providersfrom each of the district. Similarly four clusters of villages, eachconsisting three villages were identified from each tehsil. Thus atotal of 12 villages have been selected from 4 identified selectedtehsils of two districts. Since, all the available traditional healersfrom selected villages were contacted for information. A total of 15traditional healers were identified providing traditional animal healthtreatment, covering the areas about 350 kms in selected villageswere taken as respondents for data collection. Interview schedule,discussion with traditional healers and farm families, observationwere the source of data collection.

Rank Based Quotient (RBQ)For calculating the RBQ of different constraints perceived by

the traditional healers, a list of constraints was prepared afterinformal discussion with respondents. Once the constraints wereidentified, each respondent was asked separately to rank the eachproblem without having interaction with the remaining respondents.Thus, each respondent had his own independent opinionregarding the seriousness of the problem faced by him. On thebasis of ranks provided by the respondents, rank based quotient(RBQ) for each problem was calculated by using followingformula:

#Part of Ph.D. thesis1Assistant Professor, Department of Veterinary and Animal Husbandry Extension, College of Veterinary and Animal Science, Navania, Udaipur313601, Rajasthan, India2Principal Scientist and I/C Krishi Vigyan Kendra, IVRI, Izatnagar-243 122, Bareilly, Uttar Pradesh, India

n fi (n+ 1- i) RBQ = —————————-- x 100 i=1 Nn

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Table 1: Occupational profile of traditional healers (N=15)

Occupational characteristics f Percentage Major family occupation Crop farming 5 33.33 Animal husbandry 8 53.33 Ethnoveterinary treatments 2 13.33 Practice experience (in completed years) <10 3 20.00 10-20 8 53.33 >20 4 26.67 Source of knowledge Ancestors + self experience 9 60.00 Ancestors + other contacts 4 26.67 Others + self experience 2 13.33 Person involved in traditional treatment Only self 13 86.67 Self + wife - - Self + children 2 13.33 Self + wife + children - - Procurement of herbal medicine/drugs Homemade 9 60.00 Homemade + herbal drug store 6 40.00 Specialized training attended Yes - - No 15 100

Table 2: Constraints perceived by traditional healers (N= 15)

Perceived Constraints RBQ value Rank Lack of effective therapies for epidemic diseases

77.88 III

Availability of some of plants only in particular season making treatment impossible in off season

79.86 II

Lack of education 20.46 IX Lack of herbal drug store/shop in the village

95.04 I

Extinction of some of ethno treatment due to less interest by new generation

36.96 VIII

Poor financial base of pastoralists

19.14 X

Inconvenient and time consuming in obtaining preparing and administrating some of medicines

46.20 V

Inadequate diagnosis due to lack of training resulting ineffective therapy to save the animal

40.92 VII

Lack of transport to visit for treatment nearby village

43.56 VI

Difficulty in standardizing optimal doses of medicine

70.54 IV

Where,fi=the frequency of respondents for the ith rank of the problemN=the total number of respondentsn=the number of ranks

Results and DiscussionOccupational profile of traditional healers

The findings indicated that majority of the respondentsfell in middle age (30-60) group residing under Joint family

system and majority of them were illiterate or up to primarystandard of education. The possible reason may be that mostof the selected villages possessed up to eighth standard schoolthus to obtaining higher education they need to visit at town/city. Besides it, they were attached with their ancestraloccupation and not give emphasis for higher education in theirfamilies. About forty per cent of traditional healers belonged tolarge farmer’s category and had above 10 acres of land, none

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of them were found landless and majority ( 53 per cent) ofthem were reported animal husbandry as their major familyoccupation followed by crop farming among 33.33 per cent ofrespondents. Sheep and goats were found dominant speciesamong small ruminants, desi cow among large ruminant andcamel as drought animal among their livestock. About 53 percent of traditional healers had 10 to 20 years experience oftraditional occupation followed by 26.67 per cent of respondentswho reported more than 20 years of experience. About 20 percent of the traditional healers were engaged in this professiononly from last 10 years.

Transmission of traditional knowledgeAbout 60 per cent of respondents had acquired their

knowledge from their ancestor and self experience regardingtreatment of animals. About 27 per cent had also received fromancestors and contact with other persons including neighborand other traditional healers. Only about 13 per cent ofrespondents reported their share of knowledge experiencefrom contact with other persons and self experience. Thus,transmission of traditional knowledge from ancestors to newgeneration was found a major channel of knowledgeacquisition among the respondents. De (2003) reported thatmajority of traditional healers (55.55%) acquired knowledgeabout indigenous practice from their fathers and rest of therespondents (44.44%) had got for the first time from their guruin Paschim Midnapore district of West Bengal.

Involvement of family members in occupationMajority (86.67%) of traditional healers reported that they

themselves involved in traditional occupation. Only 13.33 percent reported that their sons also performing traditionaltreatment for diseased animals. None of them statedinvolvement of their wife for treatment of diseased animal butall of them in same view that women in their families help inpreparing the medicine from leaves, root, seeds of differentshrubs and bushes (Table 1).

Procurement of herbal medicine/drugsMajority of respondents (60%) were using homemade

medicine for treatment of diseased animals. However, 40 percent of respondents were also purchasing indigenousmedicine from herbal store along with homemade medicine.

Specialized training attendedThe findings revealed that none of the traditional healer

had attended any training from any sources regarding treatmentof animal diseases. Table further indicates that none of thetraditional healer was maintaining any type of record aboutnumber of cases attended by them, number of casesrecovered, medicine purchased etc. The probable reason maybe that most of the traditional healers were illiterate and theirclients were livestock owners of their own or nearby villageand for that they keep oral records.

Constraints perceived by traditional healersTraditional healers were asked to state their problems,

perceived by them in delivering animal health services to the

pastoralists. After preparing a list of constraints, they wereasked to assign rank to each of the constraints according totheir perceived intensity. Rank Based Quotient (RBQ) wascalculated for each problem on the basis of ranked assignedby traditional healers. The results in Table 2 clearly depictedthat lack of herbal drug store in the village was ranked as firstby most of the traditional healers with highest RBQ value (95.04),followed by availability of some of plants only in particularseason making treatment impossible in off season (79.86),lack of effective therapies for epidemic diseases (77.88),difficulty in standardizing optimal doses of medicine (70.54),inconvenient and time consuming in obtaining, preparing andadministrating some of medicines (46.20), lack of transport tovisit for treatment nearby village (43.56), inadequate diagnosisdue to lack of training resulting ineffective therapy to save theanimal (40.92), extinction of some of the ethno treatment dueto non practice by younger generation (36.96) , lack of education(20.46), poor financial base of pastoralists(19.14).

Lack of herbal drug shop in the village perceived as majorconstraints by traditional healers. They state that they visitedregularly to collect and purchase medicine from nearby city ortown that indirectly increase treatment cost. They also reportedthat some of plant available only in the particular season thusthey were not able to provide effective treatment by using partsof that plant during off seasons. They perceived that collectionof different parts of plants/shrubs from field, herbal store andpreparing them in the form of bolus, powder, paste and solutionwere very time consuming and cumbersome process. Theyalso reported that most of the quantity of drug and dosesprovided by them to animals suffering from different diseaseswere based on their assumption and practical experience, noton appropriate measurement thus, some time it createproblems of overdose in animals. Further, they frankly acceptedthat low level of literacy and lack of special training experience,related to animal health, disease diagnosis and treatmentmake their treatment ineffective and demoralized them to be acompetitive health service provides. Even though, otherproblems like lack of transport facilities in village, poor financialbackground of pastoralists also considerable factors thathindrance for effective provision of animal health services butwith minor importance.

ReferencesAhuja, V. et al. (2002) Livestock services and the poor. Proceedings

and Presentations of the International W orkshop,Bhubaneshwar, India, pp. 21-47.

Catley, A. et al. (1998) Community based animal health services in thegreater horn of Africa. An assessment for USAID-officer offoreign disaster assistance in cooperation with the United Statedepartment for agriculture famine mitigation, OFDA/USAID,Washington, DC .

De, A. (2003) A study of indigenous animal husbandry technical knowledgein Paschim Midnapore district of West Bengal. M.V.Sc. Thesis,IVRI, Izatnagar.

Farooquee, N. A, and Saxena, K. G. (1996) Environ.l Conservation. 23:75-80.

Kumar, D. (2002) Indian. J. Small Ruminants. 82:124-128.Mc Corkle (1995) Agr. and Human Values. 122:52-80.Purohit, A. et al. (2002) Indian J. Traditional Knowledge. 1:72-80.

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IntroductionLivestock sector plays an important role in the livelihood

security and employment generation for rural areas inRajasthan. Dairy farming coverts available feed and foddermaterial in to milk and other products. The population of bovinein the state had positive growth rate of 0.43% per annum mainlydue to high growth in the buffalo and crossbred cattle population(Gupta et. al., 2007). Apart from genetic potential of cattle, itsproduction depends mostly on manage mental practices, whichexhibits variation in different parts of the state. There is a largegap between the technologies generated and adoption by thedairy farmers. The present investigation was carried out toassess the adoption of different management practices by thedairy farmers.

Materials and MethodsThe present investigation was conducted in four tehsils of

Sikar district of Rajasthan. These tehsils were divided into twogroups of organized and unorganized dairy farmers. Eachgroup possessed two tehsils each for the study. For selectionof villages, three villages were selected from each identifiedtehsil. Thus, a total of 12 villages were included for thepresent investigation (six from organized group and six fromunorganized group). To select respondents, 30 dairy cattleowners were randomly selected from each selected village.Therefore, 360 respondents were identified from the selectedvillages. Data were collected through structured scheduledfrom the identified respondents by applying personal interesttechnique. Collected data were analyzed tabulated andinterpreted in the light of the objective of the study.

Results and DiscussionThe literacy percentage among dairy farmers of Sikar

district was high irrespective of organized or unorganized dairyfarmers. In organized farmers, 75 per cent of dairy farmerswere literate while in unorganized dairies, 72.2 per cent farmerswere literate, only 27.32 per cent dairy farmers in organizedfarms and 24.66 per cent in unorganized farms and dairyhusbandry as a primary occupation. Thus, the presentinvestigation revealed that the literacy did not play any significantrole in dairy farmers to take dairy farming as a primaryoccupation. However, Bora (1972) and Hazarika (1994)observed that dairy enterprise in rural Assam could be madesuccessful only when the farmers were properly trained to actas qualified organizers among themselves in their locality.

Herd sizes were significant differences in adoptionbetween different groups with regard to management practices

STUDIES ON ADOPTION OF IMPROVED PRACTICES OFCATTLE REARING

Upendra Kumar1, N.S Dahiya2 and V.K. ChaudharyKrishi Vigyan Kendra

Swami Keshwanand Rajasthan Agricultural University, Bikaner-334 006, Rajasthan, India

(Table 3). As there was higher extent of adoption by big cattleowners in all the practices of management viz. breeding,feeding, housing and health care due to higher knowledge inthese scientific practices where as the medium and smallgroups lagged behind with regard to adoption. The Table 4shows maximum adoption gap was 90.31 per cent for adoptionof health care practices, followed by the breeding (79.55%),management (77.80%) and feeding (72.39%). Minimumtechnological gap of 45.72 per cent about adoption of feedingpractices followed by breeding (54.27), management (61.31)and health care (60.05) in organized dairy group where as, incase of unorganized dairy group the maximum.

The overall analysis of Table shows that minimumtechnological adoption gap i.e. 59.55 per cent was obtained incase of feeding practices followed by the improved breeding(66.91%), management (69.55%) and health care (77.68%).The results revealed that technological information in neededby all the farmers in recommended animal husbandry practices.

Researchers have reported that adoption was highest incase of breeding (83.49%) followed by health care (64.08%),management (62.33%) and breeding practices (49.11%) asreported that by Chug (1995). Similar results have beenobserved by Prasad (1992) who reported that maximum extentof adoption was found in feeding (57.83%) followed bymanagement (52.39%), health care (48.87%) at least inbreeding i.e. 48.75%. Kumar (1987) who reported that the extentof use of clearly management practices was moderate to lessin use among all the farmers. Sihag et al. (1998) and Chug(1995) indicated that the overall adoption behaviour of dairyfarmers was satisfactory but still there existed a widetechnological gap which needed to be built up. The practiceslike navel-cord sealing, weaning the calf, balanced feeding,record keeping and dehorning were adopted by very fewrespondents.

Breeding practicesThere was a significant different in adoption of

breeding practices between the two groups of organized andunorganized dairy owners (Table 5). It was adopted atsignificantly higher level by organized farmers as compared tounorganized farmers. The mean score of breeding practiceswere 4.52 and 2.04, respectively of two groups. Theunorganized dairy farmers adopted less that none of themadopted improved breeding practices of artificial insemination,the reason being, due to extreme temperatures duringsummers. They could not identify the animal in heat, secondlythe availability of green fodder for the crossbred was a problem

1Corresponding author: Email- [email protected]. Prof. and Head, LPM College of Agriculture, Bikaner3Associate Professor, Department of LPM, CVAS, RAJUVAS, Bikaner

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S. No. Characteristics Organized Unorganized Total 1 Literate 135 (75.0) 130 (72.2) 265 (73.6) 2 Illiterate 45 (25.0) 50 (27.8) 95 (26.4) Total 180 (100) 180 (100) 360 (100)

Table 1: Literacy status of the head of the family (in percentage)

Group Agriculture Dairying Service Busine ss Labours Organized 40.05 27.32 23.19 5.01 4.43 Unorganized 55.36 24.66 7.52 2.31 10.15

Table 3 : Herd size effect on management practices

Group No. of Respondents Mean Max. % of obtaining score Small 161 5.85 58.00 Medium 118 7.57 75.70 Big 81 7.72 77.00 SEM± 0.106 CD 1%

0.277

Table 4: Adoption gap of livestock keepers with respect to improved animal husbandry practices

Unorganized Organized Total Improved Practices MPS Adoption

gap % MPS Adoption

gap % MPS Adoption

gap % Breeding 20.45 79.55 45.73 54.27 33.09 66.91 Feeding 27.61 72.39 54.28 45.72 40.94 59.55 Management 22.20 77.80 38.69 61.31 30.44 69.55 Health care 9.69 90.31 34.95 65.05 22.32 77.68

MPS Mean Max. % obtaining score Practice No. of Respondents Organized Un-organized Organized Un-organized Organized Unorganized

SEM±

CD 1%

Breeding 360 45.73 20.45 4.52 2.04 64.50 38.00 0.10 0.28

Feeding 360 54.28 27.61 10.85 5.52 56.00 47.50 0.27 0.75 Housing 360 38.69 22.20 19.34 11.10 77.00 54.00 0.35 0.98 Health care

360 34.95 9.69 6.99 2.19 64.20 44.20 0.18 0.50

MPS = Mean Per cent Score

Table 5: Adoption of cattle management practices

Figures in parenthesis denote percentage

Table 2: Occupational status of dairy farmers (in percentage)

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due to lack of irrigation facilities, the reason for high level ofadoption among organized dairy farmers that they possessedgood knowledge about diagnosis of pregnancy and timelyremoval of placenta, whereas unorganized dairy farmers lackin knowledge related to these aspects.

Feeding practicesTable 5 further shows that the cattle owners of organized

and unorganized groups had a mean score 10.85 and 5.52,respectively with regard to extent of adoption of different feedingpractices. None of the cattle owners belonging to unorganizedgroup provided balanced ration to animals. Dry animals werenot fed concentrate and even milch animals were not givenadequate quality of concentrate due to poor economiccondition. It was further found that all the respondents chaffedthe long Stover’s and provided top feeds of Khejri booner adlibitum as it is found in abundance in the area.

It was further, noted that some of the organized cattleowners were giving mineral mixture and salts to cattle regularlyand also enriched the dry fodder with urea molasses on accountof higher economic status and also due to the reason that theypossessed higher knowledge regarding scientific feedingpractices. Similar findings have also been reported by Chug(1995) and Sihag et al. (1998) where maximum adoption wasin case of scientific feeding practices and least in breedingpractices. Regarding adoption of feeding practices thedifferences among the two groups with respect to feedingpractices being highly significant (P<0.01).

Housing practicesThe significant difference was observed between the

organized and unorganized groups with respect to housingpractices. The mean value given in the (Table 5) indicated thatthere was almost equal and good adoption of scientifichousing practices by the organized dairy cattle owners whereasthe level of adoption was low in respondents of unorganizeddairy cattle owners.

Health care PracticesThere was significant difference in adoption of health care

practices between the two groups as per given in (Table 5).The reason for lesser adoption by unorganized dairy cattleowners may be because the medicine and veterinarytreatments are costly and as the have unorganized, the costalso comes out to be high hence they opt for local treatment

regarding eradication of external parasites of animals. As foras castration is concerned they do not prefer for it, rather theysell the calves and also do not feel the need of dehorning orvaccination the animal till they are seriously ill. The probablereasons may be illiteracy, lower socio-economic status andlack of awareness regarding scientific health care practices.

Kumar (1987) in his study found that they were 68.33 percent adoption in beneficiaries. Whereas, 58.66 per cent extentof adoption in case of breeding practices Contradictory toPrasad (1992) and Chug (1995) reported that there was 48.75per cent and 49.11 per cent extent of adoption in breedingpractices which was seen to be the least. Further results showsthat feeding practices, the unorganized and organized dairyfarmers category have 47.50 per cent and 56.00 per cent levelof adoption which was comparatively higher than the breedingpractices. During the investigation it was found that most of theanimals were fed at ad libitum only during grazing and majorityof the respondents chaffed the long stovers and dry grass tofeed to the cattle. Results showed that unorganized cattle ownernever fed mineral mixture and salts to their cattle, whereasorganized cattle owners fed balanced feed, mineral mixturesalts and urea molasses as per the knowledge regardingfeeding and their economic status. As the organized cattleowners perceived high level of knowledge about proper feedand concentrate mixture during its advanced stage ofpregnancy and time of feed a newly born calf hence extentadoption was also high in case of scientific feeding practices.

ReferencesBora, L.R. (1972) Farmers training are an essential input for livestock

development. IX All India Dairy Industry Conference Souvenir.pp. 53-56.

Chug, M. (1995) Exploratory study of dairy farm owned by ex-serviceman in Karnal District (Haryana). M.Sc. ThesisNDRI Karnal.

Gupta, D. C. et al. (2007) Indian J. Ani. Sci. 77: 494-99.Hazarika, D. N. (1994) Indian J. Ani. Prod. Mgmt. pp. 58-59.Kumar, A. (1987) A study of adoption of dairy husbandry innovation of

beneficiaries and non- beneficiaries of lab to landprogramme. M.Sc. Thesis Kurukshetra, University,Kurukshetra.

Prasad, K. (1992) Training needs of farmers in scientific dairy andcrop farming Practices in rainfed area of Hamirpur (UP).M.Sc. Thesis NDRI, Karnal.

Sihag, et al. (1998) Indian J. Ani. Prod. Mgnt. 2(1):24-29.

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Several initiatives have been taken up by welfareorganization aims at fulfilling the concern for better farm animalwelfare. The assessment of animal welfare is essential uponwhich feedback can be given to livestock owners and animaltraders about the status of welfare at their livestock farm andthe potential future risk to animals. Further, the consumersalso expect that animal related products to be produced withgreater welfare of animals (Harper and Hanson, 2001). Theterm animal welfare has been explained that good welfare isregarded as good physical and mental health of animal.Though, the welfare of animal is multidimensional, dependingupon many aspect of life including the extent to which an animalexperiences positive (those produced by the expression ofmotivated behaviours-Farm Animal Welfare Council, 1992) andnegative (hunger, thrust, pain, fear) affective states. Variousset of standards, guidelines, legal acts under Indian PenalCode and package of animal welfare practices have beenenacted by the concerned agencies and authority (People forAnimals (PFA), People for Ethical Treatment of Animal (PETA),Committee for the Purpose of Control and Supervision ofExperimentation on Animals (CPCSEA), Society for Preventionof Cruelty to Animal (SPCA), Beauty Without Cruelty (BWC),Animal Welfare Board of India (AWBI), National Institute ofAnimal Welfare (NIAW) for appropriate welfare of animals toreduce the unnecessary pain and suffering to the animalswhile at work and in production. The present study wasundertaken to ascertain what is and what ought to be for properanimal welfare by analyzing the existing welfare practicesfollowed by the animal traders and the awareness amongthem about animal welfare standard and their opinion to improvethe animal welfare.

Research methodologyData were collected from animal markets selected from

each of the identified districts (Bareilly, Badaun, Shajhanpurand Pilibhit) of Rohilkhand region in Uttar Pradesh. Further, 10animal traders, involved in trading of large animals, wereselected from each of the identified animal market (total 16animal markets) of each districts to make the total samplesize of 160 respondents. The data were collected by personalinterview method by applying pre-structured interviewschedule.

Results and DiscussionThe findings of the research indicated that majority (43.3%)

of the animal traders were involved in purchasing of milch

FARM ANIMAL WELFARE VS. ANIMAL TRADERS: A CASE OFROHILKHAND REGION IN UTTAR PRADESH, INDIA

B.P. Singh1, Rupasi Tiwari2 and Vijay Kumar3

Joint Directorate of Extension EducationIndian Veterinary Research Institute, Izatnagar- 243 122, Bareilly, Uttar Pradesh, India

animals, followed by draft animals (21.6%), dry, emaciated,weak and old animals for slaughter (19.1%) and only 10% ofanimal traders were involved in calves’ trading. Further, majorityof the animal traders (61.2%) were not providing any veterinarycare to the injured and diseased animals in the markets, whichmay be because of non-availability of veterinary facilities inanimal markets, this findings was similar to Dwivedi (2007)and Kumar (2008). Further, 55.6% of respondents werereported to follow prophylactics measure against contagiousdiseases to their animals.

As far as concern for marketing the animals, it was foundthat oiling the body of animals were practicing by 93.3 per cent,rasping the horn by 57.5 per cent, retaining the milk in udder by65 per cent of animal traders to show the higher yield of animalsto the buyers and further trimming the hooves of animalswere followed by the 10.8 per cent of the animal traders to hidethe age of animals. Oiling the body of animals indicate thatmajority of them were following this, but few were using usedengine oil which may caused skin related problems to theanimals. Pertaining to the feeding, only 60 per cent of animaltraders were feeding their animals during transportation andamong them only 19.7% were feeding twice in a day whereasmajorities (77%) were feeding once in a day duringtransportation. Further, water was provided to the animals duringtransportation by only 16.8 per cent of the respondents.Regarding the effect of feeding and its effect on growth ofanimals several researches unearthed that improper feedingweaken the health of animals, reduced food intake andincreased the risk of metabolic disturbances such as ruminalacidosis (Albright, 1993; Phillips and Rind, 2002).

Regarding frightening the animals, 70 per cent of animaltraders frightened during loading in transport by several waysviz. by piercing a nail in body (16.9%), by beating (48.2%) andby inserting the stick in genital organs (34.8%). Wilkins et al.,(2005) reported that the way that animals are treated dependson many factors including socio-economic conditions, culture,religion and traditions prevailed in the society. It was reportedthat 88.7% per cent were restraining the animal by tying ropein neck of many animals together, 43.7% were tying the legstogether of many animals and 28.1% were tying the legs ofsingle animals .

According to the number of animals carried in differentsize of truck, it was reported that they loaded 15-20 animals ina truck of 17 feet size, which is against the guiding rules. Thereare several reports in various daily newspaper of Rohilkhand

Paper presented in a National Seminar on “Enhancing Efficiency of Extension for Sustainable Agriculture and Livestock Production”,heldat IVRI, Izatnagar1Senior Scientist, email: [email protected] Scientist and I/C ATIC, email: [email protected] (CIRG) and Ph. D. Scholar (IVRI), email: [email protected]

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region about cruelty to animals by overloading in vehicles(Anonymous, 2009a, 2009b). The average space provided percattle (means bull, bullock, buffalo, yaks, calves etc) in railwaywagon or vehicle should not be less than two square metre asprescribed under welfare act. It has been reported that byoverloading and overcrowding the animals become lame, gotinjury and bruises which caused behavioural restriction, painand reduced longevity (Bergston et al., 1998). Further, theanimal traders carried the animals without spreading anti-slippery material in vehicles as it was found to be practiced by16.25 per cent of animal traders. Further, loading facilities inanimal markets like proper ramp was also found either absentor temporary structure. It is reported that handling duringloading/unloading can induce stress (increased cortisol) inanimal and reduce meat quality by declining the pH of meat(Fernandez et al., 1996; Grandin, 2000). Whereas, Villarroel etal. (2001) and Hemswarth and Colaman (1998) reported thatwelfare of livestock depends greatly on the attitude and trainingof stockperson, day to day human-animal interaction and onavailability of appropriate facilities. It was worsen to note thatno animal traders had any certificate (animal fitness, diseasefree etc.) from a qualified veterinarian.

Regarding the awareness among animal traders aboutanimal welfare, only 4.3% of them were aware about the animalwelfare agencies (PETA, PFA , CPCSEA, SPCA, AWBI, NIAW,BWC etc.) those work as watch dog and responsible forimplementation of animal welfare standards and guidelinesfor protection of animal to cruelty. Further, it was found that 5.6% of the respondents were aware about basic animalfreedoms. Only 11.2% of them had knowledge about variouslegislations and Acts enacted to safeguard the animals. Further,23.1% of the animal traders opined that a person causingcruelty to animal should be punished, whereas 41.2% felt thata person failing to provide sufficient feed, water and shelter toanimals should also be punished. Whereas, 26.2 per cent ofthe animal traders were of the view that a person selling theanimal suffering with pain by reason of mutilation, starvation,thirst, overcrowding or other ill treatment should be punished.Appley et al. (1992) reported that public pressure for increasedprotection and welfare of animals comes primarily fromurbanized people. Ouden et al. (1997) reported that animalwelfare has become a major concern for consumer.

SummaryThis study concludes that handling methods, as in case

of straining, and the behaviours of animal traders towardsanimals were not proper, since majority were not providingtimely treatment. Further, majority of them were not educativeabout animal welfare standards. Animal welfare is consideredgood when its nutritional, environmental, health, behavoural

and mental needs are met. It can be advocated that guidelines/legals acts constituted for prevention of cruelty to animals mustbe implemented properly and further, educational programme,exclusively for the animal traders must also be formulated andimplemented to educate the animal traders which in turn willhave impact on animal productivity in terms of work efficiency,milk production, health and meat quality etc. Singh et al. (2009)reported that field veterinarians are also need to be trained infield of animal welfare to reduce the cruelty and suffering toanimal. Hence, it should be a moral duty on the part of tradersto behave with the animals with love and passion which isalso imperative for economic consideration since livestockproduces of good welfare animal can fetch a handsomerevenue to the farmers and animal traders in country.

ReferencesAlbright, J.L. (1993) J. Dairy Sci. 66(10): 2208-20.Anonymous (2009a) Amar Ujala, a daily Newspaper. 12th July, 2009.

pp. 6.Anonymous (2009b) Hindustan. a daily Newspaper. 4th November,

2009. pp. 6.Appleby, M.C. et al. (1992) Poultry production systems: Behaviour,

management and welfare. Wallingford: CAB International,UK.

Bergsten, C. et al. (1998) Claw Traits and foorlesions in swedishdairy cows in relation to trimming interval and housingsystem. A Preliminary report. In: Proc. 10th Int. symposiumon lameness in ruminants. September 7-10, Lucerne,Switzerland, pp: 46-48.

Dwivedi, P. K. (2007) Study on meat animal welfare practices and itsconsciousness in market. M.V.Sc. thesis, IVRI, Izatnagar,U.P.

Farm Animal Welfare Council (1992) FAWC updates the five freedoms.Vet. Rec. (The), 17:357.

Fernandez, X. et al. (1996) J. Anim. Sci. 74: 1576-1583.Grandin, T. (2000) Behavioural principles of handling cattle and other

grazing animals under extensive conditions. In Grandin T.(ed.), Livestock handling and transport. 2nd ed. CABInternational, Wallingford, Oxon, UK: pp. 63-85.

Harper, G.C. and Henson, S.J. (2001) Consumer values and farmanimal welfare-the Comparative Report. The University ofReading. United Kingdom. EU FAIR CT98-3678.

Hemsworth, P.H. and Coleman, G.J. (1998) Human-Livestockinteractions: the stockperson and the productivity andwelfare of intensively farmed animals. CAB International,New York, NY, USA, pp. 158.

Kumar, Vijay (2008) Farm Animal Welfare Practices in MadhubaniDistrict of Bihar. M.V.Sc. Thesis, IVRI, Izatnagar, U.P.

Ouden, M.D. et al. (1997) Livestock Prod. Sci. 48(1):23-37.Phillips, C. J. C. and Rind, I. (2002) J. Dairy Sci. 84: 2424-2429.Singh, B.P. et al. (2009) Indian J. Vet. Med. 29(1): 43-44.Villarroel, M. et al. (2001) Vet Rec. 149(6):173-6.Wilkins, D.B. et al. (2005) Rev. Sci. Tech. 24 (92): 625-38.

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IntroductionThe term acupuncture derives its meaning from the latin

words “acus” meaning “needle” and “pungare” meaning “topractice”. Acupuncture, may be defined as the stimulation of aspecific point on the body, referred to as an “acupoint” on thebody with a specific method, resulting in a therapeutic orhomeostatic effect. Physiological changes in response toacupuncture point stimulation is the basis of clinical treatment.Some of these changes include release of endogenousopoids, immune system stimulation, and blood pressureregulation (Xie and Ortiz, 2006). Thus, there are 3 majorcomponents of acupuncture process: (1) acupuncture point(acupoint), (2) stimulating methods, (3) acupuncture-inducingphysiological effects. It is considered as a useful therapy formany musculoskeletal, neurological, cardiovascular,reproductive and dermatological disorders. Thoughconsidered as a traditional Chinese medicine, acupuncturebasically has its origin from North India dating back to nearly1500 B.C. Advantages of acupuncture analgesia over druginduced analgesia include absence of respiratory depression,cardiovascular depression, decreased intra-operativebleeding and post-operative pain. Neuroendocrine andhormonal theories have been proposed to explain themechanism of acupuncture. The technique has been used totreat diseases in horses, cattle, dogs, cats, and birds andmany clinical studies have documented the benefits providedfrom acupuncture (Li, 1993; Steiss et al., 1989; Still, 1989; Xieet al., 1996).

Origin and historical perspectiveThe origin of acupuncture as a method of treatment can

be traced back to stone age, when warriors observed that lamehorses hit by arrows during battle got cured. Thoughacupuncture is considered as a traditional Chinese medicine,but it has its origin from North India dating back to nearly 1500B.C. Where stone instruments known as Bian, where used totreat elephants. Veterinary acupuncture has been practiced in

ACUPUNCTURE IN VETERINARY MEDICINE- A REVIEW

J. D. Parrah, B.A. Moulvi, F. H. Dedmari, H. Athar and M.O. KalimDivision of Veterinary Surgery and Radiology

FVSC and AH, SKUAST-K, Shuhama, Alusteng, Srinagar-190001, J & K

ABSTRACT

Acupuncture, the stimulation of a specific point on the body, “acupoint” with a specific method, resulting in a therapeutic or homeo-static effect, is used for a variety of clinical conditions in animals, especially in three areas, namely pain management, geriatricmedicine, and sports medicine. Instead of using it as a sole mode of treatment, it is better to follow the holistic approach by using thecombination therapy including acupuncture. The main advantage inherited with this traditional method of treatment is being without anyside effect; however it is contraindicated in many conditions like tumours. This review deals with its different aspects includingphilosophical background, clinical application and with its different forms.

Key words: Acupuncture, sports medicine, traditional Chinese medicine

China for at least 2,000 years (Yu, 1995). The Shang dynastyduring 16th - 11th B.C. advocated the use of bronze needles, byvirtue of their bioelectric conductivity, lead to the discovery ofchannels or meridian systems, and subsequent classificationof acupuncture points for therapeutic purposes. Around 650B.C., General Shun Yan, considered as the father of veterinaryacupuncture, wrote a book, “Bialess Canon”, describing hisgreat skills as veterinary acupuncturist, especially with horses.In 1608, two brothers Yu Ben Yuan and Yu Ben Heng publishedanother book titled “Yuan and Heng’s Therapeutic Treatise ofHorses”, containing all the information known aboutacupuncture up to that time concerning horses, cattle andcamels, enumerating about 107 acupoints in horses. Duringthe past 25 to 30 years there has been tremendous growthand development of acupuncture in animals. Surgery was firstperformed on horses and donkeys using acupuncture in 1969after its use in humans in 1958.

Nowadays veterinary acupuncture is being practicedthroughout the world especially in USA, where “The InternationalVeterinary Acupuncture Society” has also been established.The veterinary professions in Australia, Austria, Britain, France,Germany and Belgium have also given formal recognition tothe validity of veterinary acupuncture.

Philosophical background of acupuncture (Yin - Yangtheory)

Traditional Chinese medicine uses concept of Yin andYang to portray, and attempts to simplify an understanding ofthe dynamic nature of life in all its relationships. Yin = -ive , andYang = +ive energy, just as we say hypo or hyper, or sympatheticand parasympathetic influences of the autonomic nervoussystem. The two together are regulated by the Qi (pronouncedas Chi) force of life. As per the traditional Chinese medicine, adisease will eventually manifest when there exists imbalancebetween the Yin and Yang. This view has been validatedmost recently by the discovery of the relationship betweenbrain chemistry and immune system.

Corresponding author email: [email protected]; [email protected]

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AcupointsIn Chinese medicine, acupoints or acupuncture points

are known as “Shu Xue”, “shu” meaning “passing orcommunication” and “xue”, meaning “hole or outlet”. So shuxue, is a hole in the skin that communicates with one or moreinternal organs by way of a meridian channel (Jing) or itscollaterals (Luo). Meridians are energetic channels locatedthroughout the entire body that form a connection betweenacupoints (Xie and Ortiz, 2006). Most acupoints are found alongmeridians, called Meridian Points. Meridian acupoints areconfirmed by electronic point finders and low skin resistancepoints. There are nearly 176 points in dog (Chan et al., 1996),173 in horse (Klide and Martin, 1989) and 366 in humans.Acupoint communicates with a specific organ and reflects thecondition of that organ. When the acupoints are treated byacupuncture, the effect could readily reach the communicatedorgan through the point and the meridian. The main histologicalfeatures of the acupoints include:• Extensive neural terminals.• A vascular network at or near the site of acupoints.• Higher concentration of the hair follicles.• Loose connective tissue sleeve around the acupoints.

These features of acupoints contribute to retention ofsufficient water and subsequent reduced skin resistance atacupoints. Acupoints are measured by a specially designedohmmeter called as Point Finder.Instruments and basic techniques used in veterinaryacupuncture

1) Needle acupunctureThis is the most commonly employed acupuncture

technique, and involves the insertion of needles into the desiredacupoints. Currently 9 traditional types of needles are in use.The commonly used needles are long, have no lumen and aremade from high quality steel or silver. The needle handle ismade in a spiral form to help insertion (Altman, 2001). Theseneedles are sterilized by autoclaving at 120°C for 30 minutes.Pre-sterilized disposable needles are also available but arecostly. For insertion, the needle handle is held firmly andinserted slowly, while simultaneously exerting pressure toadvance the needle to the desired depth in the tissue. For thewithdrawal, the needle handle is held firmly and removed slowlywhile simultaneously easing the needle out of the tissue. Angleof insertion of the needle may be:• Perpendicular or Straight Insertion: Usually used at heavymuscled area.

• Slant or Angled insertion: Usually used near joints, over thelateral or ventral aspects of thorax or abdomen.

•Parallel or Horizontal insertion : Used in area with littleunderlying muscle tissues.

A needle acupuncture session usually lasts for 10-30minutes. After needle insertion, moderate to strong stimulationis needed, which can be provided by twirling of the needlehandle or by pecking i.e. moving the needle up and down.

2) AcupressureIt is the application of finger pressure to the body surface

at designated acupoints. Eight different forms of transdermalpressure techniques reported include: thrusting, grasping,pressure, rubbing, rolling, pinching, blunt needle pushing

(concentrated acupressure), rubbing between palms andtaping. In veterinary practice, acupressure is mostly used toaugment the needle treatment.

3) Cupping vacuum therapyThis technique involves applying negative pressure to

acupoints. Alcohol is applied to the interior of a glass cup andignited (Altman, 2001). The cup is then firmly applied over thepoint. A vacuum is created as the fire consumes the oxygen inthe cup. This causes the skin and the underlying tissue to bepulled up into the cup.

4) MoxibustionIt involves heating of acupoint by burning a herb (Artemesia

valgaris) on or above the skin. It is usually used to treatdegenerative or chronic disorders. Pieces of moxa rolls (2 cmlong x 1 cm diameter) are placed on the inserted needle andignited. Gentle heat from the smoldering moxa is transmittedthrough the needle to stimulate acupoint indirectly.

5) Electro acupunctureWith the needles already in place, electrical energy with

adjustable frequency and intensity is passed throughacupuncture points and stimulated. The selection of frequencydepends on the therapeutic purpose e.g. low frequency (2-8Hz) usually induces endorphin secretion but high frequency(>100 Hz) induces serotonin production (Angeli et al., 1995).Its indications include neoplasia, analgesia andcontraindications include cardiac arrhythmias, epilepsy, shock,fever and pregnancy (Bihar and Kumar, 1991).

6) SonopunctureUltrasonic stimulation of acupoints is becoming popular,

because of being non-invasive and short duration of treatment(10-30 seconds).

7) Aqua puncture or injection therapyEach acupoint is injected directly with conventional drug

or 0.2-2.0 ml of normal saline (0.9% NaCl) or lidocaine (0.5%in saline). Other suitable solutions for injection include, Vit. B,DMSO, antibiotics and anti inflammatory drugs. The solutionshould be sterile and should be suitable for intramuscularuse (Altman, 2001).

Mechanism of action of acupuncture therapyThe various theories that have been proposed to explain

the mechanism of acupuncture therapy are:

I. Gate control theoryStimuli from the acupuncture points activate interneurons

in the substantia gelatinosa of the spinal cord. Pain stimulifrom other parts of body also project to the same region(Latshaw, 1975). When acupoints are stimulated, the gatesget closed, which inhibits the transmission of pain impulsesvia the ascending spinal tracts to the cerebral cortex, thusinducing analgesia or alleviation of the peripheral pain. Thispain inhibitory effect is due to the release of inhibitoryneurotransmitters such as endorphins, GABA and serotoninin the spinal cord, thalamus and mid brain.

II. Hormonal mechanismVarious hormonal changes occur in the animal receiving

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acupuncture treatment. This has further been proved whenthe CSF from one animal under acupuncture analgesia wastransferred to an animal not receiving acupuncture, and resultedin analgesia in the recipient.

III. Acupuncture interaction with the ANSStimulation of acupoints can cause a reflex arc, resulting

in sympathetic induced segmental superficial and visceralvasodilatation. This explains how acupuncture of somaticstructures can be effective in treatment of internal organdysfunction.

Clinical uses of acupunctureAccording to the Chinese concept the main applications

of acupuncture are:• Control of pain.• Readjustment of organ function.• Sedation.• Musculoskeletal disorders like arthritis.• Antipyretic and immunological actions.• Treatment of paralysis, shock etc.• Treatment of neuro-endocrinal disorders (Sterility etc.).• Acupuncture analgesia for surgery.• Reversal of general anaesthesia.• Neurological disorders like anxiety and epilepsy.• Gastrointestinal disorders.• Dermatological disorders (Sharma, 2000)

Acupuncture can be used for a variety of clinical conditionsin animals, especially chronic diseases. Acupuncture is mostcommonly used in three areas, namely pain management,geriatric medicine, and sports medicine. Acupuncture isreportedly effective for the treatment of various painful conditionsin animals including cervical, thoracolumbar and lumbosacral

hyperpathia, chronic lameness, degenerative joint diseases,and colic. (Li, 1993; Steiss, 1989; Still, 1989). Acupuncturestimulation produces an analgesic effect, generally calledacupuncture analgesia (Bossult et al., 1984). The release ofendorphins may be one of the pathways in which acupuncturerelieves pain (Xie et al., 2001a&b). Geriatric patients sufferfrom a variety of conditions that can be treated with acupuncture,these patients are also too weak to undergo conventionaltherapy and thus require an alternative to treatment that is safeand effective. Acupuncture can effectively treat geriatric patientsand improve their quality of life (Xie, 2004) Animals can sufferfrom exercise-related diseases such as chronic pain, arthritis,injury to tendons and ligaments, joint injuries, muscle injuries,and bone injuries. Acupuncture can treat these diseases byrelieving pain and promote healing to the affected areas byincreasing blood flow (Xie et al., 2005; Bossut et al., 1984)

A list of problems of different species of animals that havebeen corrected using acupuncture is shown in Table.

Advantages of acupunctureAs compared with other modalities of therapy, acupuncture

is much safer because of dual-direction regulation (Yu, 1995;Li, 1993). This means when an acupoint is stimulated, thephysiological response induced matches the body’s need atthe time. Other advantages include:• Very useful for producing analgesia in cardiopulmonarycompromised patients.

• Less cardiopulmonary, physiological changes and quickensthe blood clotting.

• Immunostimulating.• No side effects.• May be useful for therapy in cases where conventionaltreatment methods fail.

Table 1: List of clinical conditions treated by acupuncture in different species (Sharma, 2000)

Ca nine condition s Fe line condition s Equine condition s

M u sculo ske le ta l Lam eness of unknown origin Hip dysplasia Arthrit ic and rhe um atic joints with or witho ut bony exostoses Back pain Cruc iate ligam ent dam age Re spira tory Chronic b ronchit is Cou ghing unrespons ive to treatm ent Kennel coug h Uroge nita l In fe rtility Inco nt inence in bitches Cys titis O va ria n dys fu nc tion Ne rvou s sy stem Epilepsy Dige stive syste m Pers is tent diar rh oea Pers is tent const ipation

M u sculo ske le ta l Lam e ness of unkno wn origin Cruc iate ligam ent dam ag e A rthritic /rhe um atic problem s Back pain Re spira tory Feline as thm a Chronic n asal discharge Chronic cat flu Chronic sneez ing Uroge nita l K idney disease Incontine nce Cyst itis In fe rtility Ne rvou s syste m Epilepsy Dige stive sy ste m Pers istent diar rh oea Pers istent cons tipation

M u sculo ske le ta l Lam e ness of unkno wn origi n Navicula r disease Lam init is Cold back syndrom e Respira tory Chronic n asal discharge Chronic cou gh Hay alle rgy COP D Urogenita l K idney disease Inco ntine nce In fe rt ility Cys tic ovaries Excess ive beh aviou r w hen in season

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Cautions and contraindicationsCertain cautions must be taken when treating weak or

debilitated patients. The use of fewer acupoints is used forweak or geriatric animals. When treating performance animals,wait a couple of hours after training or racing sessions beforestarting any acupuncture treatment. Acupoints around thethoracic cavity (e.g., BL-13 to BL-17) require an insertiondistance for the needles that is shorter for other acupoints. Becautious when using moxibustion in the summer time becauseit warms the body and might lead to too much heat. Be cautiouswhen using points around the eyes as to not puncture theglobe. Although rarely a problem in animals, electro-acupuncture must be done very cautiously in patients withpacemakers (Xie and Ortiz, 2006).

There are no specific diseases that cannot be treatedwith acupuncture. However, there are certain contraindicationsof needle insertion based on the location of the acupoint orhealth status of the patient. Contraindications for certainapplications of acupuncture include the following:1. Never insert a needle directly into a tumor or open wound.2. Never use a needle at acupoint CV-8; only moxibustion is

used at CV-8.3. Never use certain acupoints (i.e., ST-36, SP-6, BL-40, BL-60

and BL-67), as well as points around the lumbar and lowerabdominal regions during pregnancy.

4. Never use electro-acupuncture on seizure patients. (Xie etal., 1996; Janssens et al., 1988; Xie, 1994).

ConclusionAcupuncture can be used to treat a variety of diseases

including pain, geriatric diseases, and exercise-relateddiseases. Different acupoints and different methods ofstimulation can be employed to treat specific diseases.Although few, there are cautions and contraindications to usingacupuncture therapy. Therefore, it is highly recommend that alicensed veterinarian take a course certifying them in veterinary

acupuncture before implementing it as part of their practice.With the increasing amount of clinical trials and research beingperformed using acupuncture therapy, we have a betterunderstanding of acupuncture’s mechanism of action.

ReferencesAltman, S. (2001) Techniques and instruments in veterinary

acupuncture. In: Ancient art to modern medicine, eds. A. M.Shoen. Mosby Publishers, St. Louis. pp. 95-111.

Angeli, A.L. et al. (1995) Vet. Rec. 157: 662-664.Bihar, A. and Kumar, A. (1991) Indian J. of Vet. Surg. 12: 7.Bossut, D. F. B. et al. (1984) Am. J. Vet. Res. 4: 620-625.Chan, W.W. et al. (1996) Vet. Bulletin. 66: 1000-1008.Janssens, L. et al. (1988) Vet. Rec. 122: 355-358.Klide, A. M. and Martin, B. B. (1989) J. of Ani. Vet. Med. Assoc. 195:

1375-1379.Latshaw, W. K. (1975) J. Ani. Vet. Med. Assoc. 201: 1321-1325.Li, K. C. (1993) Electro-acupuncture for treatment of lameness in

horses. Chi. J. Trad. Vet. Sci .1:3-14-15 (in Chinese).Sharma, S. K. (2000) Acupuncture in animals. http://hillagric.ernet.in/

edu/covas/vpharma/ winter%20school/ lectures/23%20Veterinary%20Acupuncture.pdf).

Steiss, J. E. et al. (1989) Can. J. Vet. Res. 53: 239-243.Still, J. (1989) J. Small Ani. Pract. 30: 298-301.Xie, H. et al. (2001b) Influence of acupuncture on experimental

lameness in horses. In: Proceedings Am. Assoc. EquinePract., San Diego. 47: 347-357.

Xie, H. and Ortiz-Umpierre, C. (2006) J. of Am. Ani. Hospital Assoc.42(4):244-248.

Xie, H. (1994) Traditional Chinese Veterinary Medicine. Beijing: BeijingAgricultural University Press.

Xie, H. (2004) Geriatric Medicine: A Holistic Approach. In: Proceedingsof The North American Veterinary Conference: Small AnimalEdition, 17, Orlando, Florida. pp. 65-67.

Xie, H. et al. (1996) J. Equine Vet Sci. 7: 285-290.Xie, H. et al. (2001a) J. Equine Vet. Sci. 21(12): 591- 600.Xie, H. et al. (2005) J. of Ani. Vet. Med. Assoc. 227(2).Yu, C. (1995) Traditional Chinese Veterinary Acupuncture and

Moxibustion. Beijing: Agriculture Press.

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IntroductionThe North-Eastern Region (NER) of India occupies about

seven per cent of total land area and four per cent of totalpopulation of the country where agriculture is the prime sourceof livelihood characterized by subsistence, low input-low output,technologically lagged mixed farming system, and isdominated by smallholders (Kumar et al., 2007). This regionaccounts for 6.20, 28.23, 24.62 and 91.37% of cattle, pig, yakand mithun to the total country’s population. In spite of havingrich genetic bio-resource, the region is deficit in milk, meatand egg production which may be attributed to the fact thatmajority of animals maintained here are of non descript localtype whose productivity is poor. Moreover, the farmers arerearing these animals in low input sustenance system ofrearing with inadequate housing, feeding with locally availablefeed stuffs, lack of health care etc. (Bhowmick and Kalita, 2001).Hence, in order to improve the production in livestock sector,there is an urgent need to introduce new technologies throughfront line demonstration and also training the farmers inadopting the new technologies as well as provide theminstitutional support (Birthal and Singh, 1995).

As systematic procedure for planning and implementationof training programme, KVKs in general starts with identificationof training needs of farmers/rural youth/extension personnel,and hence becomes the most important step in any trainingprogramme by KVKs. Training need refers to the gap between“what is” and “what should be” in terms of the traineesknowledge, skills, attitude and the behaviour in a given situationand time. The present study on training needs assessment of

TRAINING NEEDS ANALYSIS OF LIVESTOCK FARMERS ANDRURAL YOUTHS OF NORTH EASTERN INDIA

R. Pourouchottamane1 , V. Venkatasubramanian2 , A.K. Singha3 ,A. Mishra4 and P.K. Pankaj5 *

Zonal Project Directorate (Zone III)ICAR Research Complex for NEH Region, Barapani, Meghalaya - 783131

ABSTRACT

The present study on training needs assessment of the livestock farmers and rural youths of the North East Region was conducted by theZonal Project Directorate (Zone III) in collaboration with the KVKs of the NE region. The result revealed that the training needs of the farmersand rural youths of different states varies according to the agro climatic regions, farming system prevalent, demography of the state etc. Theresult obtained from the present study helps the KVKs to identify the areas where the training programme has to be conducted on prioritybasis. Moreover, there is an urgent need to increase the number of trainings conducted by the KVKs in the field of livestock sector to bringabout any visible change in the livestock productivity. During the year 2009-10 and 2010-11, 71 KVKs of North Eastern Region conducted 1801and 2979 farmers training courses of which 223 (12.38 %) and 411 (13.79%) courses were in the field of livestock Production andmanagement. The average number of trainings for farmers and rural youth conducted in livestock sector by each KVK came to 3.14 and 2 forthe year 2009-10 and 5.78 and 2.11 for the year 2010-11. Even though considerable efforts have been made by KVKs in training of farmersand rural youths in different aspects of animal husbandry, there still remains a gap which needs to be addressed. The KVKs have to re-orienttheir trainings as well as increase the number of trainings based on these findings to reduce the existing technological and adoption gap amongthe livestock farmers of north east region.

Key words: Training need, north east, livestock, rural youth, adoption, economy

the livestock farmers and rural youths of the North East Regionwas conducted by the Zonal Project Directorate (Zone III) incollaboration with the KVKs of the NE region.

Materials and MethodsSelection of districts

The study covered 41 rural districts of the region (Table 1)which were purposively selected based on following criteria:· The identified districts could represent all the agro-climaticzones (Table 2) of the region.· Major hills and plain farming system of the region were foundin these selected districts.· Krishi Vigyan Kendra are in existence in the districts with fullstaff for at least three years, serving for the farming communityin the respective districts.· The districts were selected considering the productionpotentials in diversified farming and enterprise.

Selection of farmersFrom each selected district, four villages based on

production potential of the different farming system weredrawnup for inclusion in the present study. Onconsultation with theextension functionaries of state agricultural departments, localleader as well as KVK staff, a list of livestock farmers wasprepared for each village. From the individual list of farmersfrom selected village, five farmer respondents were randomlyselected which made twenty farmers from each district. Thusa total of 820 farmer respondents were finally selected for datacollection from forty one districts of the region.

1Senior Scientist (LPM), NRC on Pig, Guwahati2Assistant Director General (AE), Division of Agricultural Extension, ICAR, KAB, New Delhi3Senior Scientist (AE), Zonal Project Directorate, Zone III, Meghalaya4Assistant Professor, Co V. Sc. & A.H., Jabalpur5*Corresponding Author: Senior Scientist (LPM) Central Research Institute for Dryland Agriculture (CRIDA), ICAR, Santoshnagar, Hyderabad,Andhra Pradesh-500 059, India

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Table 1: State-wise distribution of districts under study

State No. of KVKs Name of the districts Assam 12 Karimganj, Lakhimpur, Nalbari, Barpeta, Kamrup, Goalpara, Golaghat,

Sivasagar, Cachar, Sonitpur, Dhemaji, Chirang

Arunachal Pradesh 4 Tirap, West Kameng, East Siang, West Siang

Nagaland 8 Kohima, Zuhenabeto, Mokokchung, Tuensang, Phek, Mon, Dimapur,

Wokha

Mizoram 7 Aizwal, Champai, Serchip, Saiha, Mamit, Lawngthlai, Lunglie

Manipur 5 Imphal East, Churachandpur, Thoubal, Senapati, Bishnupur

Sikkim 3 East Sikkim, West Sikkim, North Sikkim

Meghalaya 1 West Garo

Tripura 1 South Tripura

Total number of districts selected: 41, and total number of farmers interviewed: 820

Table 2: Agro-climatic zone wise coverage of the selected districts in North East Region

Agro-climatic zone Districts North-Bank Plain Zone Sonitpur, North Lakhimpur, Dhemaji Low Brahmaputra Valley Zone Kamrup, Barpeta, Nalbari, Goalpara, Bongaigaon. Barak Valley Zone Cachar, Karimganj Upper Brahmaputra Valley Zone Golaghat, Sibsagar Alpine Zone ( more than 3500 msl)

Parts of West Kameng, West Siang, Northern Parts of North Sikkim and West Sikkim, Parts of East Sikkim

Temperate Sub Alpine ( 1500-3500 msl)

West Kameng, Parts of East Siang, Tuensang and Zonhoboto, Parts of North, West and East Sikkim

Sub Tropical Hills (1000-1500 msl)

Tirap and rest of West Siang, West Garo hills, Mokokchung, Kohima, Wokha and Mon, Parts of East and West Sikkim, Parts of East Siang, Aizawl, Champai, Lunglei, , Serrchip

Sub Tropical Plain (400-1000 msl)

Imphal East, Bishnupur, Senapati, Thoubal

Mild Tropical Hills (200-800 msl)

Parts of Dimapur, Parts of East Sikkim, Mamit, Lawngtlai, Chuarachandpur

Mild Tropical Plain (0-200 msl)

Parts of East Siang and Tirap, Southern parts of Dimapur, South Tripura

Data collection

Data collection from randomly selected respondents wasmade by using pretested ‘structured schedule’ throughpersonal interview method. For the present study a list of 11main thematic areas under animal husbandry and on farmproduction of inputs was prepared.

Data analysisThe farmer’s responses were collected in 3-point

continuum scale such as very important (VI), important (I) andnot important (NI) by assigning scores 3, 2 and 1, respectively.The results were calculated as weighted score for each of the

thrust area identified for the training Weighted scores in therange of 2-3 were ranked and the first five rankings wereidentified as training needs of the farmers of the state.

Results and DiscussionDistrict wise and state wise training needs of livestock

farmers and rural youths engaged in different livestock basedvocations in northeast India are given in Tables 3 to 10. Onperusal of the Table, it might be noted that some of the trainingneeds are not high at state level; however, the same may beimportant at the district level. Hence, while identifying the trainingneeds from the Table, it is important to see whether particular

(No. of VI x 3) + (No. of I x 2) + (No. of NI x 1)Weighted score (WS): ———————————————————————————————

Total No. of VI + I + NI

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area is identified as important at the concerned district eventhough it is not identified as important training need at statelevel.

Arunachal PradeshTraining needs of livestock farmers and rural youths of

Arunachal Pradesh in terms of weighted score and rank aregiven in Table 3. Training on pig rearing (2.56) is most soughtafter by the farmers followed by disease management (2.4)and poultry management (2.35). The farmers are alsointerested in knowing integrated farming system and feedingmanagement of the animals. It is to be noted that the tribalfarmers and rural youths are least interested on dairy farming

Table 3: Training needs of Arunachal PradeshLivestock farm ers of Arunachal Pradesh

(n = 80) Rural yout hs of Arunachal Pradesh in d ifferent

livestock bas ed voc ations (n=80) Them atic Area VI I NI W S Rank Them atic area VI I N I WS R ank

D airy m anagement 19 18 43 1.70 Production of org anic inputs 20 39 21 1.99

Poultry m an agem ent 44 20 16 2.35 3 In tegrated farm ing 33 27 20 2.16 4 Piggery m anagem ent 54 17 9 2.56 1 Verm i-culture 44 31 5 2.49 2

R abbit m anagem ent 2 10 68 1.18 Production of quality anim al p roducts 9 17 54 1.44

D is ease managem ent 48 16 16 2.40 2 Da iry farm ing 16 25 39 1.71

Feed managem ent 34 29 17 2.21 5 Sheep and g oat farm ing 2 25 53 1.36

Production of q uality anim al products 6 21 53 1.41 Qu ail farm ing 1 6 73 1.10

In teg rated farm ing s ys tem 43 18 18 2.32 4 Pig fa rm ing 57 14 9 2.60 1

Verm i-com pos t productio n 60 17 3 2.71 1 Ra bbit farm ing 1 20 59 1.28

Orga nic m anures productio n 62 13 5 2.71 1 Poultry production 42 24 14 2.35 3

Production of l ives to ck feed and fodder

29 25 26 2.04 Para veterinary wo rker 4 13 63 1.26

and as a result, dairying in this region is mostly carried out byNepalese and persons from the north east plains. The farmersare also interested in training programmes on vermi compostand organic manure production. The most important trainingneeds of rural youths engaged in livestock based vocationwere identified as training on different aspects of pig farming,followed by vermi-composting, poultry rearing and integratedfarming.

ManipurTraining needs of livestock farmers and rural youths of Manipur

are given in Table 4. Management of disease outbreaks, pig andpoultry rearing, feeding management and integrated farming

Table 4: Training needs of ManipurLivestock farmers of Manipur

(n=100) Rural youths of Manipur in different livestock based

vocations (n=100) Thematic Area VI I NI WS Rank Thematic area VI I NI WS Rank

Dairy management 32 29 39 1.93 Production of organic inputs 34 33 33 2.01 5

Poultry management 46 27 27 2.19 2 Integrated farming 42 34 24 2.18 4 Piggery management 51 17 32 2.19 2 Vermi-culture 49 32 19 2.3 2

Rabbit management 7 15 78 1.29 Production of quality animal products 22 25 53 1.69

Disease management 54 24 22 2.32 1 Dairy farming 28 30 42 1.86

Feed management 40 23 37 2.03 4 Sheep and goat farming 3 18 79 1.24

Production of quality animal products 23 28 49 1.74 Quail farming 4 6 90 1.14

Integrated farming system 41 20 39 2.02 5 Pig farming 61 7 32 2.29 3

Vermi-compost production 51 28 21 2.30 1 Rabbit farming 7 22 71 1.36

Organic manures production 39 29 32 2.07 2 Poultry production 57 17 26 2.31 1

Production of livestock feed and fodder

19 28 53 1.66 Para veterinary worker 6 22 72 1.34

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Table 5: Training needs of SikkimLive s toc k fa rm e rs of S ik k im

(n=8 0 ) Rura l yout hs of S ik k im in d iffe re nt live s toc k ba s e d voc a tions ( n=8 0 )

The m a tic Are a V I I NI W S Ra nk The m a tic a re a VI I N I W S R a n k

D a iry m a na g em e n t 4 0 1 3 7 2 .5 5 1 P ro d u ction o f o rg a nic in p u ts 1 9 2 2 1 9 2 .0 0 5

Po u l try m a n ag em en t 3 9 1 2 9 2 .5 0 2 In te g ra te d fa rm ing 2 7 1 8 1 5 2 .2 0 3 P ig g e ry m an a gem e n t 2 1 1 1 2 8 1 .8 8 Ve rm i-cu ltu re 3 7 7 1 6 2 .3 5 1

R a b bit m an a gem e n t 3 1 0 4 7 1 .2 7 P ro d u ction o f q u a lity a n im al p ro du c ts 1 7 1 5 2 8 1 .8 2

D is e ase m a n ag em en t 3 7 1 4 9 2 .4 7 3 D a iry fa rm ing 3 0 6 2 4 2 .1 0 4

Fe e d m a n ag em en t 2 8 1 9 1 3 2 .2 5 4 Sh e e p an d g oa t fa rm in g 9 1 4 3 7 1 .5 3

P ro d u ction o f q u a lity a n im al p ro du c ts 1 6 2 2 2 2 1 .9 0 Qu a il fa rm ing 1 6 5 3 1 .1 3

In te g ra te d fa rm ing s ys tem 2 4 7 2 9 1 .9 2 P ig fa rm in g 2 4 4 3 2 1 .8 7

Ve rm i-com pos t p ro d u ctio n

4 1 8 1 1 2 .5 0 1 R a b bit fa rm in g 4 8 4 8 1 .2 7

O rg a n ic m a nu res p ro d u ctio n 3 0 1 3 1 7 2 .2 2 2 Po u l try p ro d u ctio n 3 5 7 1 8 2 .2 8 2

P ro d u ction o f live s to ck fe e d an d fo d d e r

1 6 1 5 2 9 1 .7 8 Pa ra ve te rin a ry w o rke r 1 1 5 4 4 1 .2 8

Table 6: Training needs of Mizoram

Lives toc k fa rm ers of M izoram (n=14 0)

Rura l yout hs of M izoram in diffe rent live s tock based voc ations ( n=140 )

Them atic Area V I I NI W S Ra nk Them a tic a re a VI I N I W S R ank

D a iry m anagem e n t 59 40 41 1 .73 P roduction o f o rganic inpu ts 54 60 2 3 2 .23 5

Pou l try m anagem en t 75 40 25 2 .36 2 In te g ra ted fa rm ing 81 39 1 7 2 .47 2 P igg e ry m an agem en t 86 27 27 2 .42 1 Ve rm i-cu ltu re 93 28 1 6 2 .56 1

R abbit m anagem en t 5 22 113 1 .23 P roduction o f qua lity an im al p roducts 38 56 4 3 1 .96

D is e ase m a nag em en t 74 34 32 2 .3 4 D a iry fa rm ing 49 53 3 4 1 .81

Fee d m a nag em en t 77 32 30 2 .34 3 Sheep and goa t fa rm ing 10 36 9 0 1 .41

P rodu ction o f qua lity an im al p roducts 36 46 58 1 .84 Qu a il fa rm ing 9 22 10 9 1 .29

In teg ra te d fa rm ing s ys tem

62 37 41 2 .15 5 P ig fa rm in g 77 33 2 4 2 .40 3

Verm i-com pos t p rod uction 112 20 8 2 .74 1 R a bbi t fa rm ing 8 25 10 8 1 .29

Organ ic m anu res p rod uction 98 29 13 2 .61 2 Pou ltry p roduction 60 48 2 6 2 .25 4

Produ ction o f lives tock fe ed and fod de r

44 57 39 2 .04 3 Para ve te rina ry w o rke r 25 46 6 6 1 .70

Table 7: Training needs of Nagaland

Livestock farm ers of Nagaland (n=160)

Rural yout hs of Nagaland in different l ivestock based vocations ( n=155)

Them atic Area VI I NI W S Rank Them atic area VI I N I WS Rank

Dairy m anagement 63 44 48 2.10 Production o f organic inputs 79 46 30 2.32 5

Poultry m anagem ent 117 11 27 2.58 2 Integrated fa rm ing 86 41 28 2.37 4

Piggery m anagem ent 116 14 25 2.59 1 Verm i-cu lture 91 46 18 2.47 3

Rabbit m anagem ent 16 58 81 1.58 Production o f Quali ty anim al products 57 58 40 2.11

D is ease managem ent 113 17 25 2.57 3 Dairy fa rm ing 61 39 55 2.04

Feed managem ent 90 37 28 2.4 5 Sheep and goat farm ing 19 58 78 1.62

Production o f qua lity anim al products

55 48 52 2.02 Quail farm ing 9 44 102 1.40

Integrated fa rm ing s ys tem

91 40 24 2.43 4 Pig farm ing 114 29 12 2.66 1

Verm i-com pos t production

111 33 11 2.65 1 Rabbit farm ing 28 64 63 1.77

Organic m anures production 104 37 14 2.58 2 Poultry production 111 22 22 2.57 2

Production o f lives tock feed and fodder

70 46 39 2.2 3 Para ve terina ry worke r

13 45 97 1.46

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Table 8: Training needs of AssamL ive s toc k fa r m e rs of As s a m

(n =2 4 0 ) Ru ra l yo u t h s o f As s a m in d iffe re n t li ve s to c k b a s e d voc a tion s ( n =2 4 0 )

T he m a tic Are a V I I N I W S Ra nk T h e m a tic a re a VI I N I W S R a n k

D a iry m a na g em e n t 1 6 3 5 1 2 6 2 .5 7 1 P ro d u ction o f o rg a nic in p u ts 9 0 8 1 6 9 2 .0 9 5

P o u l try m a n ag em en t 1 5 0 5 4 3 6 2 .4 8 3 In te g ra te d fa rm ing 1 3 6 5 1 5 3 2 .3 5 2 P ig g e ry m an a gem e n t 9 1 5 5 9 4 1 .9 9 Ve rm i-c u l tu re 1 4 0 6 6 3 4 2 .4 4 1

R a b bit m an a gem e n t 2 2 2 9 1 8 9 1 .3 0 P ro d u ction o f q u a li ty a n im al p ro du cts 4 6 7 1 1 2 3 1 .6 8

D is e as e m a n ag em en t 1 6 1 4 5 3 4 2 .5 3 2 D a iry fa rm ing 1 3 7 4 2 6 1 2 .3 2 3

F e e d m a n ag em en t 1 1 5 7 5 5 0 2 .2 7 5 S h e e p an d g oa t fa rm in g 8 1 7 1 8 8 1 .9 7

P ro d u c tion o f q u a li ty a n im al p ro du c ts 4 6 9 1 1 0 3 1 .7 6 Q u a il fa rm ing 1 7 3 7 1 8 6 1 .3 0

In te g ra te d fa rm ing s ys tem 1 3 7 4 7 5 6 2 .3 4 4 P ig fa rm in g 8 4 4 5 1 1 1 1 .8 9

Ve rm i-com pos t p ro d u c tio n 1 4 6 6 3 3 1 2 .4 8 1 R a b bit fa rm in g 2 1 2 7 1 9 2 1 .2 9

O rg a n ic m a nu res p ro d u c tio n 1 1 8 7 5 4 7 2 .3 0 2 P o u l try p ro d u ctio n 1 1 6 5 7 6 7 2 .2 0 4

P ro d u c tion o f l i ve s to ck fe e d an d fo d d e r

8 4 7 7 7 9 2 .0 2 3 P a ra ve te r in a ry w o rk e r 9 5 4 1 7 7 1 .3 0

system were identified as their major training needs by the farmersof Manipur. Even though dairying does not feature as importanttraining need at state level, farmers and rural youths in ImphalEast and Senapati districts identified dairying as their importantarea where training is required. Poultry rearing, vermin culture andpig rearing are the most needed areas under which training isrequired followed by integrated farming system and production oforganic inputs as identified by the rural youths who are engaged inlivestock based vocations. The reports are in the similar line assuggested by Meitei and Devi. (2009)

SikkimTraining needs of livestock farmers and rural youths of Sikkim

are given in Table 5. In Sikkim unlike other north east hill states,dairy management is the most important training needs asperceived by farmers followed by poultry rearing, diseasemanagement and feeding management. Pig rearing andintegrated farming system are given comparatively less importanceby the farmers of this state which might by due to populationdemography of the state, i.e. 20.59 % of the population belongs totribal and majority are either Nepalese or other plain people whoare engaged in dairy farming. It is also important to note the fact thestate is having more number of cattle than pigs (159 thousandsvs. 38 thousands) while other north east hill states comparablenumber of pig and cattle population. Similarly, the per capitaavailability of milk (g/day) is highest among north east states (213g/day as against 74 g/day for NE Region).

Training on vermin-culture, poultry rearing, integrated farming,dairy farming and production of organic inputs were identified astheir needs by the rural youths of Sikkim who are engaged inlivestock based vocations.

Mizoram and NagalandTraining needs of livestock farmers and rural youths of

Mizoram are given in Table 6 while of Nagaland is given in Table 7.In both Mizoram and Nagaland, farmers perceived that training

on the areas of piggery, poultry rearing, disease management,integrated farming system and feeding management are importantfor improving their farm productivity. Further, their training needs

included on farm production of inputs such as vermin composting,organic manure production and production of livestock feed andfodders.

In some districts of Nagaland such as Kohima, Tuensang,Phek, Dimapur and Wokha, farmers perceived dairying includingMithun rearing as one of their training needs. The rural youths ofNagaland and Mizoram perceived piggery, poultry, vermin compost,integrated farming and production of organic inputs were the areaswhere the training is required.

AssamIn Assam, training needs of farmers and rural youths of twelve

districts were collected and results were presented in Table 8.Farmers from almost all districts identified dairying as the

most important area in which training is required. This is in contrastto the north east hill states expect Sikkim where training in pigrearing aspect is considered most important (NEDFI, 2002). Theother areas where training is considered most needed aremanagement of animal diseases, poultry rearing, integratedfarming system and feeding management of livestock. Eventhough pig rearing is not identified as one of thrust area wheretraining is required at state level, the farmers from districts ofLakhimpur, Goalpara, Golaghat, Sivsagar, Sonitpur, Dhemaji andChirang perceived pigerry as one of the important area where thetraining is required. The district wise priority of training needs of thefarmers in detail is given in the table. The training needs of therural youths engaged in livestock based vocations were in theareas of vermin culture production, integrated farming system,dairy farming, poultry rearing and production of organic inputs. Indistricts of Goalpara, Golaghat, Sivsagar and Dhemaji, rural youthsidentified piggery as one of the priority area for conduction of trainingprogrammes. Similarly, farmers are interested in undergoingtraining in area of on farm production of inputs such as vermicompost production and organic manure production. Similarreports have been suggested by CHD-ILRI-DDD, (2006).

TripuraTraining needs of livestock farmers (Table 9) and rural youths

of Tripura are given in Table 10. The training needs of the livestock

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Table 9: Training needs of livestock farmers of Tripura and Meghalaya

The m a tic Ar e a T r ipu ra (n = 4 0 ) M e g ha la ya (n = 3 0 )

V I I N I W S Ra n k V I I N I W S Ra nk

An im a l hus ba n dr y D a iry m a na g em e n t 2 6 1 1 3 2 .5 8 1 1 4 2 1 4 2 5 P o u l try m a n ag em en t 2 0 1 6 4 2 .4 4 1 4 7 9 2 .1 7 2 P ig g e ry m an a gem e n t 1 6 1 2 1 2 2 .1 1 7 2 1 1 2 .2 1 R a b bi t m an a gem e n t 6 1 5 1 9 1 .6 8 1 1 0 1 9 1 .7 3 D is e ase m a n ag em en t 2 3 1 2 5 2 .4 5 3 1 4 6 1 0 2 .1 3 4 Fe e d m a n ag em en t 1 7 1 8 5 2 .3 5 1 1 2 1 7 1 .8 P ro d u c tion o f q u a li ty a n im al p ro d u c ts 1 2 2 0 8 2 .1 1 1 1 1 8 1 .7 7

In te g ra te d fa rm ing s ys tem 2 8 7 5 2 .5 8 1 1 5 5 1 0 2 .1 7 2 O n fa r m pr oduc t ion o f i npu ts Ve rm i -com pos t p ro d u c tio n 2 5 1 1 4 2 .5 3 1 0 1 1 9 1 .7 O rg a n ic m a nu res p rod u c tion 1 9 1 4 7 2 .3 9 1 2 0 1 .6 3 P ro d u c tion o f l ive s to ck fe e d a nd fo d d e r 1 6 1 6 8 2 .2 9 1 2 0 1 .6 3

Table 10: Training needs of rural youths of Tripura and Meghalaya in different livestock based vocations

Thematic Area Tripura (n = 40) Meghalaya (n = 30)

VI I NI WS Rank VI I NI WS Rank Production of organic inputs 20 15 5 2.38 4 11 0 14 1.88 Integrated farming 28 7 5 2.58 1 11 1 13 1.92 Vermi-culture 24 12 4 2.5 2 12 1 12 2 Production of quality animal products

8 17 15 1.83 11 1 13 1.92

Dairy farming 21 14 5 2.4 3 11 1 13 1.92 Sheep and goat farming 16 15 9 2.18 5 10 15 1.8 Quail farming 1 11 28 1.33 10 15 1.8 Pig farming 16 11 13 2.08 10 15 1.8 Rabbit farming 5 11 24 1.53 10 15 1.8 Poultry production 21 14 5 2.4 10 1 14 1.84 Para Veterinary worker 8 10 22 1.65 10 15 1.8

farmers of Tripura are similar to that of Assam, i.e. dairying andintegrated farming system followed by management of animaldiseases, poultry rearing and feeding management of livestock.In addition to training on different aspects of vermi culture, integratedfarming, dairy farming etc. rural youths of Tripura perceived thatthey need training in aspects of sheep and goat production.

MeghalayaTraining needs of livestock farmers (Table 9) and rural youths

of Meghalaya are given in Table 10. Farmers perceived that trainingon the areas of piggery, poultry rearing, integrated farming system,disease management and dairy farming are important forimproving their farm productivity and economic viability.

ConclusionThere is an urgent need to increase the number of trainings

conducted by the KVKs in the field of livestock sector to bring aboutany visible change in the livestock productivity. Even thoughconsiderable efforts have been made by KVKs in training of farmersand rural youths in different aspects of animal husbandry, therestill remains a gap which needs to be addressed. The KVKs haveto re-orient their trainings as well as increase the number oftrainings based on these findings to reduce the existingtechnological and adoption gap among the livestock farmers ofnorth east region.

ReferencesAnnual Report (2009-10) Zonal Project Directorate, Zone III, Barapani,

Meghalaya-793103. pp. 1-44.Annual Report (2010-11) Zonal Project Directorate, Zone III, Barapani,

Meghalaya-793103. 1-52..Birthal, P.S. and Singh, M.K. (1995) Indian J. Agr. Economics. 30(2): 168-

175.Bhowmick, B. C. and Kalita, D.C. (2001) Performance of livestock and

fishery: Constraints for agricultural development andprioritization of strategies. In: Prioritization of Strategies forAgricultural Development in North-Eastern India. Ed: B.C. Barah,Workshop Proceedings 10, National Centre for AgriculturalEconomics and Policy Research, New Delhi.

CHD-ILRI-DDD. (2006) Project Report on Qualitative Assessment of Dairyin Assam (unpublished). Centre for Humanistic Development,Guwahati.

Kumar, A. et al. (2007) Agri.l Economics Res. Rev. 20: 255-272.Meitei, L.S. and Devi, T.P. (2009) Annals of library and information studies.

56: 35-40.NEDFI. (2002) Pre-investment Feasibility Report on Development of Dairy

Industry in the North-Eastern Region. Volume III, State Report-Assam. North-Eastern Development Finance Corporation Ltd.,Guwahati, Assam.

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IntroductionThe work of veterinarians consists of numerous jobs

descriptions like small and large animal practice, the work ofan inspecting veterinary officer, laboratory duties, consultationfrom stable to stable, public and environment health monitoringand various administrative duties, education and research.These tasks involve several factors that have a significantimpact on workers health and the work environment.Veterinarians by virtue of their occupation are prone to healthhazards of animal origin and from materials used in theveterinary practice/ research. These range from an occasionalkick from a reluctant patient to a highly fatal disease like rabies.The growing interest in pet practice has brought new demandsto the training and professional competence of veterinarians.Despites the importance of the occupational hazards, theknowledge of biohazards is scarce, its magnitude isoverlooked. Available reports show the veterinarian’sproneness to one or other type of occupational hazards indeveloped countries. But in India with honourable exceptionsempirical information on veterinarians work related hazards isnot available. The personal characteristics of the veterinariansmay influence the work efficiency and hazards associated withtheir work place. The kind of job and quantum of the exposureof an individual with the hazardous situations at the work place

PROFILE CHARACTERISTICS OF THE VETERINARIANS AND THEWORKING ENVIRONMENT INFLUENCING OCCUPATIONAL

HEALTH HAZARDS

Sariput Landge1 , Hema Tripathi2 and Vaishali Banthiya3

Division of Extension EducationIndian Veterinary Research Institute, Izatnagar-243122, Bareilly, Uttar Pradesh, India

ABSTRACT

Through a expost facto research profile characteristics and the working environment of the veterinarians from the field and researchinstitutes was studied from the state of Maharashtra and Uttar Pradesh. The study revealed that majority of veterinarians were frommiddle age category, from rural background, having specialization in non clinical and paraclinical subjects with medium work experi-ence and working at the district and close to work place. Though the veterinarians had not attended any training on occupationalhealth hazards, mixed practice was predominant for field veterinarians while the research scientist were handling the laboratoryanimals and were working for nearly 48 hours a day. In case of physical infrastructure majority of field veterinarians were satisfiedwith respect to availability of other basic amenities like the stationary (86.89%), electricity (90.77%), fan (94.66%), and technicalliterature (61%), apron (97.08%), gloves (78.64%) and boot (71.35%). While things like type writer (73.2%), computer (42.7%),duplicating machine (58.5%), audio visual equipments (52.4%), mask (63.6%), staff quarter (40.2%) and medical facilities (48.8%)were not available for majority of the field veterinarians. Irrespective of nature of work, majority of the veterinarians in both thecategories were having refrigerator (85.43%), microscope (93.68%), slides (94.17%), stethoscope (72.81%) and thermometer(65.53%) in adequate quantity. The equipments like the sterilizer (82.3%), distillation unit (88.7%) and syringe (58.9%) were reportedadequate by the research scientists while, instruments like hoof trimmer (51.2%), hoof cutter (50.0%), burdizoo castrator (90.2%),drench gun (61.0%), post mortem set (74.4%), muzzle (64.6%), trevis (90.2%) and ropes (69.5%) were reported adequate bymajority of the field veterinarians. However, item like syringe (35.4%), surgical instruments (22.0%) and ropes (25.8%) wereinadequately available at work place reported by field veterinarians.

Key words: Profile characteristics, veterinarians, occupational hazards

may reduce or augment the work productivity. The study focuseson the socio-personal and work environment related aspectsof the veterinarians working in the hazard prone areas.

Materials and MethodsThe present study has been conducted in two purposively

selected states namely; Uttar Pradesh and Maharashtra. Theveterinarians working as Livestock Development Officers (LDO)were selected from Latur division of state of Maharashtra forthe study. The two research institutes from the state of UttarPradesh were selected purposively for the study of occupationalhealth hazards among the veterinary scientists engaged inresearch, academic and transfer of technology in thesepremiere Institutes. Both institutions had total strength of 272at the time of data collection (2008). The scientists engaged insocial science disciplines were excluded from the pool asthey were not engaged in the laboratory oriented research.From the pool of the scientists of both the institutes and LDOs,50 per cent were finalized through proportionate randomsampling. Thus, a total of 82 LDOs and 124 scientists formedthe total sample size of 206 veterinarians. Final data werecollected from the field veterinarians during the monthlymeetings in each of the selected districts of Maharashtra.Likewise, a separately prepared structured interview schedule

1Assistant Professor, Department of Veterinary and Animal Husbandry Extension Education, Nagpur Veterinary College, Nagpur (MS)2Principal Scientist and Program Coordinator (KVK), Division of Veterinary Extension, IVRI, Izatnagar, 243122 (UP)3Assistant Professor, Department of Veterinary and Animal Husbandry Extension Education, Nagpur Veterinary College, Nagpur (MS)

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was used to collect the data from the scientists working in theselected research institutions through personal interview.

Results and DiscussionA. Profile Characteristics1. Age

Majority of veterinarians (44.66) were from middle agecategory followed by nearly 35 per cent in old age group and 20per cent fell under young age category. Respondents groupindicated that majority of field veterinarians (46.35%) belongedto old age (44-54 years) followed by middle (37.80%) and young(15.85%) age groups. In case of respondents, engaged inresearch activities about 49 per cent came from middle agecategory (39–50 years) followed by old (27.42%) and young(23.39%) age groups. Results of the present study are in linewith that of Sen and Chander (2001) who reported that privateveterinary practitioners in West Bengal in India were mainlydominated by male practitioners over 60 years of age. Contraryto it, Ray et al. (2005) reported that majority of veterinary assistantsurgeons (VASs) were middle aged and none fell in youngage (27 years and below) category. In another study conductedby Elbers et al. (1996) revealed that the mean age of theparticipants was 40 years while Boland and Morris (1999)revealed that mean age of veterinarians as 38 years.

2. SexMore than 92 per cent veterinarians in both the categories

were males and the remaining around 7 per cent were femalesamong the selected respondents irrespective of the twoseparate groups of veterinarians. This could be due to the factthat veterinary profession has been traditionally dominated bymales and is still considered as a male dominated profession.Elbers et al. (1996) also reported only six female veterinariansamong 102 Dutch veterinarians. However, Jeyaretnam andJones (2000) reported that the proportion of women in theworkforce has increased from 5 to 35 per cent over the last 20years and in countries with comparable veterinary practicessuch as the UK, USA and Canada, there has been anincreasing proportion of female veterinarians.

3. Educational qualification and area of specializationMajority of the LDO’s (62.2%) had the B.V.Sc. and A. H.

degree only and around 38 per cent were having Master’sdegree. None of the veterinarians working in field as LDO hadPh.D degree. On the other side 94 per cent of the researchscientists were having PhD degrees and only 5.6 per cent hadMaster’s degree. Similar results were reported by Singh (2004);Ray et al. (2005) and Shikhakolanu (2007) who reported thatmajority of the veterinarians were having undergraduatedegree. However, Gautam et al. (2006) revealed that morethan two-third of the faculty members had obtained Ph.D. Datawere also gathered regarding area of specialization during theMaster’s degree which were later classified on the basis of theclinical, para clinical and non clinical aspects of the variousdisciplines. In the overall sample, majority of veterinarians(42.58%) came from non clinical disciplines followed by nearly37.42 and 20 per cent in para clinical and clinical subjects,respectively.4. Present place of posting and engagement in type of work

Majority of (44%) of LDO’s were working at the district

headquarters followed by 34 per cent who were posted in ruralareas and rest 22.00 per cent at block level and most of themwere also involved in the hospitals administration (91.50%).Since both the research institutions selected under study werelocated in district, cent per cent of the research scientists wereplaced at district headquarter and were engaged mainly in theresearch. Teaching and extension activities were also beingperformed by them under the Institute mandate.

5. Job experienceMajority of the field veterinarians (41.46%) had medium

level of work experience followed by high (35.37%) and lowlevel (23.17%) of work experience. However, in case of thescientists, 41 per cent were having medium level of workexperience followed by low (37.09%) and high level (21.78%)of work experience. Shikhakolanu (2007) on the other handreported that majority of Veterinary Assistant Surgeons (73.3%)had low job experience .

6. NativeMajority of the respondents hailed from rural areas

(71.84%). Category wise, about 83 per cent LDO’s and 64 percent of research scientists had rural background. Interestingly,fairly large number of the research scientists (35.50%) camefrom urban areas against 17 per cent LDO’s. The availability ofresources often decides the rate and extent of developmentand this seems to be indeed true for veterinarians as well. Andthis can be evident from the fact that a fairly large number ofscientists came from the urban background.

7. Trainings attended regarding safety precautions for hazardfree working conditions

Neither the LDO’s nor the scientists had attended trainingregarding safety precautions for hazards free workingconditions. Similar results were quoted by Ray et al. (2005).However, the normal precautionary measures to be followedby the veterinarians in the practice were well known to all ofthem. Van Knapen (2000) suggested that continuing educationfor veterinarians with respect to health hazard awarenesstraining for veterinarians is must in the epidemiology and controlof communicable (zoonotic) diseases in man and animals.

8. Type of animals handledCent per cent field veterinarians had handled all production

animals as well as pets as they were involved in a mixedpractice involving all the commonly available species ofdomestic animals. Similarly cent percent research scientistsinvolved in research activities were dealing with the laboratoryanimals along with farm animals for the clinical trails or studies.About 5 per cent of the veterinarians also reported that theyhave been also handling the wild animals either for treatmentor postmortem purpose. Boland and Morris (1989) reportedthat 38 per cent of the veterinarians were in large animal practiceand 31 per cent were engaged in small animal practice andthe remaining 31 per cent were in mixed practice.

9. Average working timeThe official average work time of both the categories of

veterinarians was 48 hours per week because the workingtime of the organized government employee was eight hours

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a day and work for 6 days in a week. Similar results were alsoreported by Reijula et al. (2003) that the average working timefor practicing veterinarians was 44.1 hrs/week. While Jacksonet al. (2004) reported that veterinarians involved in privatepractice were working for 4.7 days per week in New Zealand.

10. Distance of workplace from residence/commutationMore than 90 per cent of the respondents were residing in

less than one kilometer due to having residential campus inclose vicinity of their work place. Groupwise analysis showedthat more than 70 per cent of the field veterinarians weretraveling up to 17 kms. Majority of the research scientists(86.29%) were living with in 1 to 3 kilometers from their workplace. Rahman et al. (2002) studied veterinary field assistants(VFAs) in Assam and found that 39 per cent of them wereworking at short distance (0.1- 03 Km) from their home ismuch similar to the finding of our study in respect to researchscientists.

11. Knowledge of veterinarians for the occupational healthhazards and safety precautions for hazard free workingconditions

The study revealed that only 30 per cent field veterinarians,against 85 per cent of the research scientists had highknowledge about the potentials of the hazards which may occurin veterinary profession and also the safety measures to workunder hazard free conditions. Group wise, it revealed thatmajority of the respondents (63.43%) engaged in the fieldpractice had medium level of knowledge if compared to theresearch scientists working in laboratories who in majority(85.48%) had high level of knowledge (Table 1).

The present study further revealed that nearly 93 per cent

of the veterinarians working as livestock development officer/field veterinarians had medium to high level of knowledge inthe field of occupational health hazards. This could be attributedto one of the serious happenings which occurred in one of theselected district in the study area just before the data collection.This included the sad demise of a fellow veterinarian due toleptospirosis which is a potential occupational hazards and asuicide in another adjacent district by another fellowveterinarian. Thus, the risks of various hazards were directly ofindirectly brought to the notice of the veterinarians working inthis area.

Better knowledge of the research scientist in the field ofoccupational health hazards and the measure to reduce themmay be attributed to have better level of education and followinggood laboratory practices (GLP) work. Nowadays, terms likehazard, hazard identification, hazard characterization, risk, risk-analysis, risk-management and risk-perception are commonlyused in the recent literature of the veterinary practice and thescientists by virtue of their work involving all sorts of hazardsseems to know about these terms (Van Knapen, 2000).

Mean differences in knowledge level among veterinariansTable 2 shows the analysis of data through “t” test to see

the differences with in the two selected group of respondentsregarding their knowledge level on occupational health hazardsand safety precautions for hazard free working conditions

The respective mean score of knowledge among fieldveterinarians (10.68) and research scientists (14.33) weresignificant at one per cent level of significance. This highlightedthat there were significant differences in the level of knowledgewithin the LDO’s. Similarly significant variation was found withinthe group of research scientists.

B. Infrastructure and other basic facilities available at workplace

Majority of the field veterinarians were not having officialjeep for transportation in the rural areas. In case of the personalvehicles, more than fifty per cent of veterinarians reported thatthey had motorcycles. In case of the research scientists, theavailability of the vehicle/jeep is cent per cent on demand.Regarding sitting arrangement, 96 and 91 per cent of the field

veterinarians reported adequate availability of table and chairs,respectively. Majority of them were satisfied with respect toavailability of other basic amenities like the stationary (86.89%),electricity (90.77%), fan (94.66%) and technical literature (61%),apron (97.08%), gloves (78.64%) and boot (71.35%). Whilethings like type writer (73.2%), computer (42.7%), duplicatingmachine (58.5%), audio visual equipments (52.4%), mask(63.6%), staff quarter (40.2%) and medical facilities (48.8%)

Table 2: In-group mean dif ferences in the knowledge level of the veterinarians w ith respect to occupational health hazards Knowledge level Mean Difference "t" value df

Field veterinarians N= 82 10.68** 34.261 81

Research Scientists N=124 14.33** 91.339 123 **P<0.01

Table 1: Know ledge level of the veterinarians about occupational health hazards and safety precautions for hazard free working conditions

Knowledge Field Veterinarians Research Scientists Total Veterinarians level (n =82) f (n=124) f (N = 206) f Low (0-6) 5 (06.09) - 5 (2.43) Medium (7-12) 52 (63.43) 18 (14.52) 70 (33.98) High (13-19) 25 (30.48) 106 (85.48) 131(63.59) Figures in parentheses indicate percentages

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were not available for majority of the field veterinarians asagainst the research scientists who were having theaccessibility of these facilities in adequate amount. Ramkumar(1982) indicated that around 66 per cent of the (VAS) hadmedium number of physical facilities in Punjab. Shikhakolanu(2007) also reported that majority of the VASs (52.2%) in AndhraPradesh had medium physical facility. Rajput (2006) in hisstudy in Rajasthan also reported that 58 pet cent of theveterinary hospitals did not even had regular water supply and66 per cent of these hospitals were not having electricity andelectric operated gadgets.

C. Instruments/equipments available at work placeThe requirement of the various instruments for the

veterinarians in practice and research is quite different.However, an inventory of the needed equipments was given tothe veterinarians to have their responses for availability of theseinstruments at their workplace. Majority of the veterinarians inboth the categories were having refrigerator (85.43%),microscope (93.68%), slides (94.17%), stethoscope (72.81%),and thermometer (65.53%) in adequate quantity. Theequipments like the sterilizer (82.3%), distillation unit (88.7%)and syringe (58.9%) were reported adequate for researchscientist while, instruments like hoof trimmer (51.2%), hoofcutter (50.0%), burdizoo castrator (90.2%), drench gun (61.0%),post mortem set (74.4%), muzzle (64.6%), trevis (90.2%) andropes (69.5%) were reported adequate by majority of the fieldveterinarians as against the research scientists. Item likesyringe (35.4%), surgical instruments (22.0%) and ropes(25.8%) were inadequately available at work place reported bymany respondents working in the field conditions. Rajput (2006)also reported that only 17 per cent hospitals were equipped

with refrigerator, only one hospital with telephone facility andnone of them had vehicle facility in the remote veterinaryhospitals of Rajasthan. Trevis, however, was available in allhospitals. Sandford (1988) found that livestock related servicesin Northern Uganda government also suffer due to lack ofequipments, funds, transport, direction and supervision.

ReferencesBoland, C. J. and Morris, R. S. (1989) New Zealand Vet. J. 37(3): 112-

116.Elbers, A. et al. (1996) Vet. Quarterly. 18:127-131.Gautam, M. et al. (2006) Livestock Res. Rural Develop. 18(7):Jackson, R. et al. (2004) New Zealand Vet. J. 52 (4): 180-188.Jeyaretnam, J. and Jones. (2000) Aust. Vet. J. 78:625-629.Rahman, S. et al. (2002) Indian J. Social Res. 44(2): 165-177.Rajput, D. S. (2006) Animal health delivery system among pastoralists

in arid zone of Rajasthan. Ph.D. Thesis, IVRI, Izatnagar.Ramkumar (1982) Role conflict and its consequences-A study on

Veterinary Assistant surgeons. Ph.D. Thesis, NDRI, Karnal.Ray, M. N. et al. (2005) Indian Vet. J. 82(4): 434-435.Reijula, K. et al. (2003) Am. J. Indust. Med. 44: 46-57.Sandford, R. H. D.(1988) Proposals for Oxfam’s role in Livestock

development in Kotido district. Uganda, Oxfam, UK/Ireland,Kampala.

Sen, A. and Chander, M. (2001) Livestock Res. Rural Develop. (13) 6http://www.cipav.org.co/ lrrd/lrrd13/6/arin136.htm

Shikhakolanu, R. (2007) Livestock service delivery by state departmentof animal husbandry in Andhra Pradesh-A critical analysis.Ph.D. Thesis, IVRI, Izatnagar.

Singh, R. R. (2004) Status of vaccination against foot and mouthdisease: An exploratory study in Bareilly district of UttarPradesh. M.V.Sc. Thesis, IVRI, Izatnagar.

Van Knapen, (2000) The Vet. Quarterly. 22(2): 36-41.

MICROBIAL QUALITY OF FRESH AND FROZEN EQUINE SEMEN OF INDIAN HORSES

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