Effect of Brazilian red propolis administration on hematological, biochemical variables and...

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Tropical Animal Health andProduction ISSN 0049-4747Volume 45Number 7 Trop Anim Health Prod (2013)45:1609-1618DOI 10.1007/s11250-013-0406-3

Effect of Brazilian red propolisadministration on hematological,biochemical variables and parasiticresponse of Santa Inês ewes during andafter flushing periodAmr S. Morsy, Adibe L. Abdalla, YosraA. Soltan, Sobhy M. A. Sallam, Kheir El-Din M. El-Azrak, Helder Louvandini &Severino M. Alencar

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REGULAR ARTICLES

Effect of Brazilian red propolis administration on hematological,biochemical variables and parasitic response of Santa Inêsewes during and after flushing period

Amr S. Morsy & Adibe L. Abdalla & Yosra A. Soltan &

Sobhy M. A. Sallam & Kheir El-Din M. El-Azrak &

Helder Louvandini & Severino M. Alencar

Accepted: 21 March 2013 /Published online: 5 April 2013# Springer Science+Business Media Dordrecht 2013

Abstract Brazilian red propolis was administered orally toSanta Inês ewes, and evaluation was made of general healthand hematological, biochemical, and parasitic responsesduring and after flushing. Thirty mature, nonlactating,nonpregnant Santa Inês ewes (40±2 kg and 2.5 years old)were grazing tropical pasture (Brachiaria decumbens andPennisetum purpureum) and, as flushing after synchroniza-tion, were supplemented with a concentrate–roughage mix-ture at a rate of 4 % body weight (BW). Ewes were dividedaccording to BWand fecal egg count (FEC) into two groups

(n=15 each): control and propolis that received propolisethanolic extract at rates of, respectively, 0 and 3 g/ewe/day.The treatments lasted 21 days until the end of flushingperiod. BW and body condition score (BCS) were recorded,and blood and fecal samples were taken weekly for 8 weeks.Mean values of BWand BCS were not (P>0.05) affected bypropolis administration. Propolis increased (P<0.01) totalleukocytes (WBC), but no significant differences were ob-served for other hematological parameters. Propolis in-creased (P<0.01) total protein and globulin concentrationsand decreased (P<0.01) triglycerides, glutamate oxaloace-tate transaminase, and glutamate pyruvate transaminase.Propolis decreased (P<0.05) FEC. Propolis administrationhad good impact on ewe health and may be a promising feedadditive during critical periods such as flushing.

Keywords Isoflavonoids . Propolis . Parasites . Serumparameters

Introduction

The livestock industry is continually searching for ways tomanipulate the ruminal microbial ecosystem to improveproduction efficiency of domestic ruminants. Feed additiveslike antibiotics or growth-promoting agents have become anintegral part of ruminant production. The ionophores (e.g.,monensin, lasalocid, and sonilomycin) are an especiallyimportant group of feed additives and the positive responsesof livestock to dietary antibiotics supplementation have beenwell documented. However, in recent years, this practice hasfaced reduced social acceptance in many countries, becauseusing antibiotics lead to the risk of transferring residues intomeat and milk and resistant strains of bacteria. Thus, the use of

A. S. Morsy :A. L. Abdalla :Y. A. Soltan :H. LouvandiniCentre for Nuclear Energy in Agriculture, University of Sao Paulo,Piracicaba, Sao Paulo, Brazil

A. L. Abdallae-mail: [email protected]

Y. A. Soltane-mail: [email protected]

H. Louvandinie-mail: [email protected]

A. S. Morsy (*)Agriculture Research Centre, Animal Production Research Institute,Dokki, Giza, Egypte-mail: [email protected]

Y. A. Soltan : S. M. A. Sallam :K. E.-D. M. El-AzrakFaculty of Agriculture, Alexandria University, Alexandria, Egypt

S. M. A. Sallame-mail: [email protected]

K. E.-D. M. El-Azrake-mail: [email protected]

S. M. AlencarCollege of Agriculture “Luiz de Queiroz”, University of Sao Paulo,Piracicaba, Sao Paulo, Brazile-mail: [email protected]

Trop Anim Health Prod (2013) 45:1609–1618DOI 10.1007/s11250-013-0406-3

Author's personal copy

antibiotics in animal nutrition has been prohibited in theEuropean Union since January 2006 (Oeztuerk andSagmanligil 2009). As a consequence, several natural alterna-tive feed additives have been used such as propolis.

Propolis is the product of resinous, gummy, and balsamicsubstances that are collected by bees from buds, flowers,and plant exudates and mixed with their salivary secretions,wax, and pollen. This serves to seal and protect the honey-comb against insect and microorganism attack as well as tomaintain internal temperature and humidity. The chemicalcomposition of propolis is complex and variable because itis intrinsically related to the floristic and ecological compo-sition of the environment visited by the bees. The combina-tion of these factors affects the pharmacological propertiesof propolis. In general, it is composed of 50 % resin andvegetable balsam, 30 % wax, 10 % essential and aromaticoils, 5 % pollen, and 5 % various other substances, includ-ing organic debris (Silva et al. 2007). Propolis contains avariety of chemical compounds such as polyphenols (flavo-noid aglycones, phenolic acids and their esters, phenolicaldehydes, alcohols, and ketones), sesquiterpene quinones,coumarins, steroids, amino acids, and inorganic compounds(Alencar et al. 2007). Propolis has versatile biological andpharmacological activities, such as antibacterial, antioxi-dant, antiviral, antifungal, anti-inflammatory, antitumoral,and immunomodulatory (Alencar et al. 2007; Sforcin 2007).

Earlier studies report the use of propolis in animal produc-tion focusing on meat quality and nutrition. For instance,dietary addition of propolis was shown to enhance the perfor-mance and carcass quality of feedlot-finished bulls (Zawadzkiet al. 2011).Moreover, propolis extract can be potentially usedas food supplement instead of monensin sodium in the diets offeedlot lambs (Ítavo et al. 2011). Propolis can inhibit thegrowth of positive bacteria; thus, it might be a useful additivefor modifying microbial fermentation in the rumen (Ozturk etal. 2010). The ethanol extract of propolis affects fermentationand methanogenesis of continuous microbial culture in rumi-nants; it decreases protozoa population and increases propio-nate levels by 10.3 % (Brodiscou et al. 2000). However, wefound no reports of studies that examined the effect of propolisextraction on the ewe's health including hematological, bio-chemical, and parasitic responses. Such theses parameters ofewes are determined as an index of their health status as well.As a sign of stress specially at critical period as pre-mating(flushing period), the use of hematological, biochemical, andparasitic response methods provides valuable knowledgeabout physiological reactions occurring against changing con-ditions, especially understanding the physiological, biochem-ical, and hematological changes occurring at this perioddealing with propolis treatment.

Considering the above-mentioned observations, to ourknowledge, there have been no investigations about this areaof subject in ewes. Therefore, the present work attempted to

address lack of information by describing the general action ofpropolis ethanolic extract administration on the overall healthof ewes through determining some hematological, biochemi-cal, and parasitic responses of Santa Inês ewes during andafter flushing period.

Material and methods

All chemical analyses of propolis were done at the Departmentof Agri-Food industry, Food and Nutrition, “Luiz de Queiroz”College of Agriculture, University of Sao Paulo (USP),Piracicaba, Brazil. The laboratory analysis and in vivo exper-iment were done at the Centre for Nuclear Energy inAgriculture (CENA, USP) Piracicaba, Brazil. All animals werealways treated in accordance to the Internal Commission forEnvironmental Ethics in Experimentation with Animals ofCENA (USP).

Origin of propolis

Brazilian red propolis (BRP) sample was collected in January2011, from the mangrove region in Marechal Deodoro, a cityin the vicinity of Maceio, capital of Alagoas State, in north-eastern Brazil. In general, the climate in this region is wettropical (9° 42′ 36″ S, 35° 53′ 42″W). Propolis was classifiedas type 13 according to Silva et al. (2007). The collectedsamples were weighed and stored at 4 °C until processing.

Preparation of propolis extraction

Propolis extraction was done according to Alencar et al.(2007) with some modifications as follows: crude propolissample (10 g) was treated with liquid nitrogen and thengrounded to a fine powder and mixed with 100 ml ethanol70 %, and then, the mixture was transferred to ultrasonicequipment (Kerry, ultrasonic limited model, PUL-250,England) for 30 min. The ethanol extract solution was subse-quently filtered through a filter paper (Whatman no. 41). Theextracted solution was kept at the freezer (−5 °C) overnight,and then, it was filtered again. The supernatant was transferredto the rotary evaporator at approximately 42 °C for 30 min inorder to remove all the ethanol, and the concentrated extractrecovered in the volumetric flask was lyophilized for 3 days toget the resultant brownish resin (pure propolis)

Propolis analysis and chemical composition

Gas chromatography–mass spectrometry

The BRP sample was chemically analyzed after methylationof the extracts. Sample of the methylated solutions wasanalyzed by gas chromatography–mass spectrometry (GC-

1610 Trop Anim Health Prod (2013) 45:1609–1618


MS) according to Fernández et al. (2008) with some mod-ifications as a Shimadzu Model GC-2010 Series gas chro-matograph coupled with an Shimadzu series mass-selectivedetector quadrupole mass spectrometer model GCMS-QP2010 plus was used. The GC-MS peaks were identified bycomparison with data from the literature (Piccinelli et al.2005) and the profiles from the NIST 98 library.

Experimental description

Animals and management

Thirty mature, nonlactating and nonpregnant Santa Inês ewes(40±2 kg body weight; 2.5 years old) were used. All animalswere estrus synchronized before start flushing using doubleintramuscular injection of 75 μg D-cloprostenol/animal(Ciosin®, Coopers, Brazil) with interval of 7 days as a sourceof PGF2α.

Animal flushing and propolis treatment

All ewes were grazing tropical grass pasture signal grass(Brachiaria decumbens) and elephant grass (Pennisetumpurpureum) and fed (4 % of body weight; NRC 2007) afixed mixture (50:50 w/w of roughage and concentrate) asflushing diet after synchronizations. The diet was dividedinto two equal proportions to be offered twice a day, at earlymorning (8:00 a.m.) and after they back from the pasture (at4:00 p.m.). Feed ingredients and chemical composition ofthe experimental diet are summarized in Table 1. Water andmineral mixture were available ad libitum to all animalsduring the entire trial. Ewes were divided according to theirbody weight (BW) and fecal egg counts (FEC) into twogroups (n=15each): control and propolis that received0 and 3 g propolis ethanolic extract/ewe/day, respectively.

At the first day of the flushing period, the propolis groupstart to be administrated orally (3 g propolis ethanolicextract/ewe/day) at early morning before access to theirdiet. The treatment lasted 21 days until the end offlushing period as shown in Fig. 1. All animals werefree of diseases, and no behavioral abnormalities weredetected throughout the experimental period.

Data collection

Live body weight and body condition score

Both BW and body condition score (BCS) for each ewewere recorded weekly throughout the experiment in themorning before access to feed and water. Body weight wasdone using a digital standing scale, and body conditionscoring (BCS=1 for emaciated ewes to BCS=5 for obeseewes at 0.5 intervals) has been recorded as described byRussel et al. (1969). The BCS of the ewes was taken bythree trained person, who mastered the technique.

Blood samples and assays

Two blood samples were collected weekly from the jugular veinof each ewe during and after the flushing period as shown inFig. 1. Blood was collected in the morning before access to feedand water and was placed immediately on ice. The first bloodsample was collected by puncturing into sterile glass flasks(5 ml), containing ethylenediamine tetra acetic acid (EDTA) asanticoagulant, whereas the second one was collected withoutusing an anticoagulant. The EDTA blood samples were used fordetermining hematological parameters, while the non-EDTAblood samples were centrifuged at 1,500×g for 10 min to collectserum and were stored at −20 °C till biochemical analysis.

Hematological parameters

The hematological analyses [erythrocyte counts (RBC), totalleukocyte counts (WBC), hemoglobin (Hb) concentration,and packed cells volume (PCV)] were determined using amethodology of Hepler (1966), while other hematologicalparameters were calculated as follows: mean corpuscular vol-ume (MCV)=(PCV×10)/RBC, mean corpuscular hemoglo-bin (MCH)= (Hb×10)/RBC, and mean corpuscularhemoglobin concentration (MCHC)=(Hb×100)/PCV.

Serum biochemical parameters

Blood serum concentrations of glucose (Gl), total protein(TP), albumin (A), triglycerides (TG), total cholesterol(Cho), glutamate oxaloacetate transaminase (GOT), gluta-mate pyruvate transaminase (GPT), urea (UR), creatinine(CR), and total bilirubin (TB) were determined by

Table 1 Feed ingredients andchemical composition (in gramper kilogram dry matter) ofthe experimental diet (fixedmixture of diet 50:50 %)

Composition g/kg

Feed ingredients

Tifton 85 hay 500

Ground corn 337

Soybean meal 163

Chemical composition

Dry matter 899

Organic matter (OM) 923

Crude protein (CP) 152

Neutral detergent fiber(NDF)

548

Acid detergent fiber(ADF)

256

Ether extract (EE) 19

Lignin (ADL) 42

Trop Anim Health Prod (2013) 45:1609–1618 1611


colorimetric kits using a commercial lab test (DiagnósticaS.A.®, Lagoa Santa, MG, Brazil). Globulin (G) concentra-tion was calculated as the difference between TP and A.

Fecal egg counts analysis

The individual fecal samples were collected weekly as sameas the protocol followed for collecting blood samples tomeasure FEC by the modified McMaster technique (Uenoand Gonçalves 1994) for a period of 8 weeks.

Statistical analysis

Data were analyzed by analysis of variance using PROCMIXED procedure of SAS (2002). The model includedtreatment and week, as mean effect and their interaction,as follows:

Yijk ¼ μþ Ti þWj þ TWð Þij þ Akt þ eijk

where Yijk is an observation taken on the kth individual, μ isthe overall mean, Ti is a fixed effect of the ith treatment (i=1to 2), Wj is a fixed effect of the jth week (j=1 to 9), (TW)ij isan interaction between ith treatment and jth week, Akt is therandom effect of the animal (inside treatment), and eijk is arandom error assumed to be independent by and normallydistributed with mean=0 and variance=σ2.

Differences were considered significant if P<0.05. TheFEC was transformed by log10 (x+10) and differences atthe 5 % level were considered significant.

Results

Propolis major constituents

The chromatograms of ethanolic extract and identificationof constituents of BRP were shown in Fig. 2 and Table 2,respectively. The relative percentage of the major identifiedcompound of BRP was isoflavonoid, which presented al-most 42.5 % of total content of propolis, especially whenvestitol, medicarpin, isoliquiritigenin, and formononetinwere 24.62, 13.52, 2.46, and 1.84 %, respectively.

Effect of propolis on BW and BCS

The effect of BRP on BW and BCS are shown in Fig. 3.Mean values of BW and BCS were not significantly(P>0.05) affected by BRP compared with the control group.However, BW and BCS were significantly affected(P<0.05) by weeks throughout pregnancy period. Meanvalues of both parameters increased progressively through-out pregnancy period.

Effect of propolis on hematological parameters

Effects of BRP on hematological parameters and erythro-cyte indexes are presented in Table 3. Propolis resulted in anincrease (P<0.01) of WBC compared with control, whilethere were no significant differences observed for otherhematological parameters included (RBC, Hb, PCV, MCV,

1st PGF2α 2nd PGF2α

Starting mating3-6-2011

End mating3-6-2011 1st Ultrasound 2ndUltrasound

Blood samples collections

Daily propolis oral administration and flushing

7 days

Fig. 1 Schedule of theexperiment

Time (min)

Abu

ndan

ce

Fig. 2 GC-MS chromatogramsof Brazilian red propolis



MCH, and MCHC). Both Hb and PCV were not affected(P>0.05) by the advances of weeks of pregnancy, whileWBC and all erythrocyte indexes (MCV, MCH, and MCHC)tended to increase (P<0.01) by pregnancy progress, and RBCtended to decrease (P<0.01) by advanced pregnancy. There isno effect observed among the treatment weeks interaction.

Biochemical parameters and fecal egg counts

Effect of BRP on blood serum concentrations of Gl, TP, A, G,TG, Cho, GOT, GPT, UR, CR, and TB are presented in Table 4.

Results indicated that BRP increased (P<0.01) TP and G. Onother hand, TG, GOT, and GPT were decreased (P<0.01) bypropolis treatment. Regarding to the effect of weeks, the resultsindicated that all biochemical parameters were affected (P<0.01) by weeks, but in a different manner as glucose, TP, A,TG, Cho, CR, GPT, UR, and GOTwere increased (P<0.01) bythe advances of pregnancy, while TB was not affected signif-icantly, and globulin was fluctuated by the week's progress.

Concerning to the effect of BRP on the FEC, results inFig. 4 showed that propolis resulted in a significant decrease(P<0.05) of FEC compared with control.

Table 2 Identification of con-stituents of Brazilian red propo-lis by gas chromatography–massspectrometry

tR (min)a = retention time inminuteaComponents similarity was mo-re than 50 % of the search resultsby comparison with data fromthe literature and the profilesfrom the NIST 98 library. Theion current generated dependson the characteristics of the com-pound concerned, and it is not atrue quantitation

Brazilian red propolis

Peaks Compounds tR (min)a % Area of eachcomponent e

1 4,4′-bis[(Trimethylsilyl)ethynyl]-2,2′-bithiophene-5,5′-dicarbaldehyde

36.49 0.80

2 Silane, trimethyl[5-methyl-2-(1-methylethyl)phenoxy]a 37.41 4.97

3 Medicarpin 37.55 13.52

4 Benzenepropanoic acid, 3,4-bis[(trimethylsilyl)oxy]-, trimethylsilylestera

38.11 3.93

5 Benzenepropanoic acid, 3,5-bis[(trimethylsilyl)oxy]-, trimethylsilylester

38.24 16.49

6 Vestitol 38.37 24.62

7 4,4′-bis[(Trimethylsilyl)ethynyl]-2,2′-bithiophene-5,5′-dicarbaldehyde

39,07 7.07

8 Hydrocinnamic acid, p-(trimethylsiloxy)-, trimethylsilyl estera 39.43 1.96

9 3,4-Dihydroxy-9-methoxypterocarpan 39.68 1.51

10 3,8-Dihydroxy-9-methoxypterocarpan (3-hydroxy-8,9-dimethoxypterocarpan)

40.13 0.79

11 9h-Fluorene-4,5-diamine, N,N,N′,N′-tetramethyla 40.51 2.88

12 Formononetin 40.86 1.84

13 Silane, 9h-fluoren-9-ylidenebis(trimethyl)a 41.17 0.78

14 2,5,?-tri-OH-phenylacetate 4tms 41.44 1.77

15 Isoliquiritigenin 41.74 2.64

16 2-Propenoic acid, 3-(3,4,5-trimethoxyphenyl)-, methyl estera 42.02 0.70

17 Silane, trimethyl[1-[(trimethylsilyl)ethynyl]-2-naphthalenyl]a 44.91 3.30

18 Propanedioic acid, bis[(trimethylsilyl)oxy]-, bis(trimethylsilyl)estera

45.60 0.78

19 Beta-amyrin trimethylsilyl ether 47.57 2.33

20 Silane, (9,19-cyclo-9.beta.-lanost-24-en-3.beta.-yloxy)trimethyla 48.11 3.09

21 9,19-Cyclolanostan-3-Ol, 24-methylene-, (3.beta.)a 48.78 1.21

22 Lup-20(29)-en-3-yl acetate 49.26 2.54

23 Solanesol 49.88 0.48

0.0

1.0

2.0

3.0

1 2 3 4 5 6 7 8

Bod

y C

ondi

tion

Sco

re

Weeks

BCS

38.0

42.0

46.0

1 2 3 4 5 6 7 8

Lif

e bo

dy w

eigh

t (k

g)

Weeks

BW

ConBRP

Fig. 3 Body weight (BW) andbody condition score of SantaInês ewes after treaded withBrazilian red propolis (BRP)



Discussion

Propolis chemical composition and ewes BW and BCS

The Brazilian propolis has been widely studied to elucidateits chemical composition, and 12 types of Brazilian propolishave been characterized and classified into types 1–12 (Parket al. 2000). The results of GC-MS analysis of the testedBrazilian red propolis confirmed that this type was notincluded among these 12 types that were classified before,and the current propolis was the type 13 (Silva et al. 2007),which first classified by Alencar et al. (2007) since 42.5 %of the total content of propolis were isoflavonoids (Fig. 2and Table 2). The vestitol was the most abundant com-pounds (24.62 %) confirmed by the GC-MS analysis thatwas never before reported in the previously 12 Brazilianpropolis types (Alencar et al. 2007). The data suggest thatthese major compounds may be associated with the

biological properties and main activity of these varieties ofpropolis since isoflavonoids have been reported as an anti-oxidant, antimicrobial, antiprotozoal, and antifungal(Cattani et al. 2012). Many studies reported the stimulationof the immune system by flavonoids. In an investigationconducted by Marquele et al. (2005) on Brazilian propolis,propolis of the indicated origin was determined to exhibitantioxidant effect. As claimed by other researchers, thiseffect was demonstrated to originate from isoflavonoids.

There were no significant effects of BRP on both BWandBCS (Fig. 3). Our results with agreement with Ítavo et al.(2011), who demonstrated that propolis (green and brown)was not affected significantly on lambs BW. Even in thesame study, the authors mentioned that flavonoids and phe-nol levels were higher in green propolis than in brownpropolis extract. As a consequence, the higher concentrationof these compounds in animal feed possibly exerted bacte-ricidal action in lamb rumen, thereby explaining the poor

Table 3 Hematological parameters and erythrocyte indexes of Santa Inês ewes after treaded with of Brazilian red propolis

Hematological parameters Treatment SEM P value

Control Propolis T W T×W

Total leukocyte (m3/103) 11.9b 13.1a 0.45 ** ** NS

Erythrocyte (mm3/106) 13.1 13.0 0.22 NS ** NS

Hemoglobin (g/dl) 11.5 11.3 0.18 NS * NS

Packed cell volume (%) 32.7 32.1 0.44 NS * NS

Erythrocyte indexes

Mean corpuscular volume (μm3) 25.4 25.3 0.39 NS ** NS

Mean corpuscular hemoglobin (pg) 8.9 8.9 0.12 NS ** NS

Mean corpuscular hemoglobin concentration (g/dl) 35.3 35.1 0.29 NS * NS

Data are mean values (n=15). Within a row, means with different letters a and b are significantly different

SEM standard error of the mean, NS not significant, T treatment effect, W week effect, T×W treatment–week interaction (P>0.05)

*P<0.05; **P<0.01

Table 4 Serum biochemical pa-rameters of Santa Inês ewes aftertreaded with Brazilian redpropolis

Data are mean values (n=15).Within a row, means with dif-ferent letters a and b are signifi-cantly different

SEM standard error of the mean,NS not significant, T treatmenteffect, W week effect, T×Wtreatment–week interaction (P>0.05)

*P<0.05; **P<0.01

Biochemical parameters Treatment SEM P value

Control Propolis T W T×W

Glucose (mg/dl) 60.8 59.4 1.85 NS ** *

Total protein (g/dl) 7.1b 7.6a 0.12 ** ** *

Albumin (g/dl) 2.8 2.8 0.05 NS ** *

Globulin (g/dl) 4.4b 4.8a 0.11 ** ** **

Triglycerides (mg/dl) 21.4a 16.6b 0.91 ** ** *

Total cholesterol (mg/dl) 61.3 61.2 1.84 NS ** NS

GOT (U/ml) 32.2a 29.7b 1.41 * ** NS

GPT (U/l) 11.1a 9.9b 0.50 * * NS

Urea (mg/dl) 44.4 45.7 1.27 NS ** NS

Creatinine (mg/dl) 0.57 0.54 0.02 NS ** NS

Total bilirubin (mg/dl) 1.4 1.5 0.12 NS NS NS



feed conversion value, but without affecting on final BW oflambs. The same finding was observed for postweaningHanwoo calves by Sarker and Yang (2010). On the otherhand, Zawadzki et al. (2011) concluded that the addition ofpropolis extract to the diet of feedlot-finished bulls in-creased (P<0.03) BW and improved feed conversions com-pared with sodium monensin.

The gradual increase in BWand BCS with the advance ofpregnancy has been reported by many investigators in sev-eral animal species and was attributed to the growth of thefetus as well as to the accumulation of fetal fluids.Additionally, pregnancy in sheep and probably in most otherspecies is accompanied by an increase in total body waterand blood volume, which contribute to the increase in bodyweight during pregnancy.

Propolis and ewes hematological parameters

The increase (P<0.01) of WBC counts in propolis groupcompared with the control of the current experiment(Table 3) agrees with those of Orsolić and Basic (2005),who reported that water-soluble derivatives of Propol givento mice caused a significant elevation of leukocytes in theperipheral blood. The increase observed in total leukocytecount may indicate an activation of the animal's immunesystem. Previous studies have shown that propolis has anti-inflammatory and immune modulatory activities (Dantas etal. 2006). It has been also reported that propolis treatmentincreased the proliferation of leukocyte precursors frompluripotent stem cell in mice (Orsolić and Basic 2005).Such effect of BRP on WBC count may be related toflavonoids, the major active components of propolis.Many studies reported the stimulation of the immunesystem by flavonoids. Propolis can act as an immunestimulant as ample stimulation of the immune system ofpropolis can increase the total leukocytes value (Talas andGulhan 2009).

Leukocyte counts were increased significantly during thepregnancy weeks. Similarly, Jain (1993) found that leuko-cyte numbers gradually increase during gestation to the dayof parturition. These counts are usually above the normal

values in most domestic animals (Jain 1993). On the otherhand, RBC counts tended to be decreased significantly bypregnancy advances without any effect on both Hb andPCV, and such effect may explain the increasing value ofMCV, MCH, and MCHC by pregnancy progress. Similarresults have been observed in pregnant sheep, mares, andsows, and bitches (Jain 1993). These results during theadvanced stages of gestation could be attributed to thehemodilution effect, resulting from an increase in plasmalevels of RBCs. These findings have also been observed inpregnant does (Azab and Abdel-Maksoud 1999). The he-modilution of the domestic animals may be of physiologicalimportance, as it decreases the blood flow in the capillaryvessels, and it may improve the blood flow through theplacental capillary vessels, especially to increase the diffu-sion of O2 and nutrients to the fetus (Yýlmaz 2000).

Propolis and ewes serum biochemical parameters

In the present study, propolis increased (P<0.01) the totalprotein concentration as shown in Table 4; the same findingwas observed by Eraslan et al. (2007), who demonstratedthat significant increase—compared to control group—wasfound in total protein values of the rat group to which theygave propolis at 200 mg/kg (body weight) with drinkingwater for 21 days. In addition, Çetin et al. (2010) demon-strated that treatment of rats with propetamphos plus prop-olis increased total protein levels compared to the ratstreated with propetamphos, and this suggests that propoliscan modulate protein metabolism.

Globulin concentration was highly significant (P<0.01)in ewes treated with propolis, and such increment could beattributed to stimulation of the immune system by propolisflavonoids (Havsteen 2002) or because the increasing con-centration of TP which occurs without affecting A, whereglobulin was calculated as the difference between TP and A.On the other hand, Sarker and Yang (2010) reported thatpropolis did not affect significantly the G concentration ofpostweaning Hanwoo calves.

Our study indicated that treatment of ewes with propolisdecreased triglyceride, compared to the control group.Decrease in triglyceride levels may be directly related tothe influence of propolis on lipid metabolism. These find-ings are similar to the data reported by Çetin et al. (2010)and Fuliang et al. (2005); hence, propolis caused a decreasein triglyceride levels when administered to rats with diabetesmellitus. In addition, Kolankaya et al. (2002) reported thatpropolis caused a decrease in triglyceride level of rats treat-ed with alcohol.

Among other biochemical parameters, the increase inGOT and GPT activities were found to be related to thedamage in the liver and the change in hepatic functions.Currently, we observed a significant decrease (P<0.01) in

0

100

200300

400

500

600

700

800900

1 2 3 4 5 6 7 8

Eg

g p

er g

ram

Weeks

Con

BRP

Fig. 4 Non-transformed mean eggs per gram of feces for Santa Inêsewes after treaded with Brazilian red propolis (BRP)



both GOT and GPT data presented in Table 4. This decreasesupports the hepatoprotective effect of propolis. Similar re-sults were obtained by Sugimoto et al. (1999) who reportedthat the propolis causes a decrease in GOT activity whenadministered to rats exposed to D-galactosamine. Similarly,in rats, the administration of ethanolic extracts of propolis at aconcentration of 10 mg/kg administered three times per dayfor 3 days was found to significantly reduce concentrations oftransaminase (GOT and GPT) and serum and hepatictriacylglyceride (Lin et al. 1999). Çetin et al. (2010) investi-gated the effectiveness of propolis in alleviating the toxicity ofpropetamphos on hematological and biochemical parametersin rats and concluded that treatment with propetamphos aloneincreased the activities of GPT and GOT in serum. Aftertreatment of rats with propetamphos plus propolis, the activ-ities of GOTand GPTwere normalized to their control values.This decrease supports the hepatoprotective effects of propo-lis. Flavonoids found in propolis may be responsible for thepositive effect of propolis on these enzymes (Sanz et al. 1994).This result is in agreement with the findings that propolisinduced reduction of the activity of plasma GOT and GPT ofrats treated with aluminum chloride and alcohol (Kolankaya etal. 2002). This difference in our study is considered to arisefrom the origin of propolis and its constituents. Furthermore,the duration of exposure, the dose exposed to, and the phys-iological differences of the exposed animal may also lead tothis result.

Regarding to the effect of pregnancy weeks on theseplasma parameters, pregnancy period was found to havesignificant effect on all the experimental parameters inTable 4. Blood parameters increased gradually by the ad-vances of pregnancy, and these results were completely inagreement with that of El-Sherif and Assad (2001) whodemonstrated that in dry ewes, blood parameters remainedconstant, while in pregnant counterparts, these parametersincreased gradually to reach their maximum level beforeparturition. Such changes which occur in blood parameterscould be explained by nutritional requirements of the eweincrease during the pregnancy due to rapid growth of thefetus (Fırat and Özpınar 2002).

Propolis and ewes FEC

Ewes treated with BRP showed significant decrease of FEC(Fig. 4). Our results are in agreement with those of Principalet al. (2002a, b) who observed the efficacy of propolis tocontrol sheep helminthiasis and to decrease significantlyFEC in propolis-treated group compared with control.Moreover, Heinzen et al. (2012) concluded that the anthel-mintic effect of alcoholic extract of propolis was at 30 % ofconcentration, in naturally infected calves where 83 % ofanimals showed an average decrease of 48.48 % of FEC,specially Trichostrongylus sp. and Strongyloides sp.

Principal et al. (2002a, b) tested several levels of propolisextracts to control helminthiasis in West African sheep andfound that a dose of 10 ml of 3 % ethanolic extracts ofpropolis was the most effective in this species. Araújo et al.(2006) and Castagnara et al. (2007) found a reduction ofFEC in Santa sheep breed, by using the 30 % extract ofpropolis. Loureiro (2007) found that the addition of 30 mgof propolis extract to the diet of sheep was effective inreducing the number of FEC, demonstrating its antiparasiticeffect and the possibility of its use in control of worms.Dürrewald et al. (2008) used alcoholic extract of propolis at33 % in 20 cattle (12 months) naturally infected withTrichuris sp., Trichostrongylus sp., and Ascaris sp. whichwas administered in single dose once or twice a day for threeconsecutive days and, 30 days after the start of treatment,found an average reduction of 59.7 % in the FEC countbetween animals receiving treatment and average increaseof 63.6 % in the control animals that did not receive anytreatment.

Propolis possesses variable biological activities. It hadbeen proven to be 100 % effective against some parasitessuch as lethal protozoa and would also decrease inflam-mation associated with parasite infection (Higashi and deCastro 1994). Previous study proved evidence of inhibi-tory activity of propolis on the vitality and hatchability ofimmature Fasciola gigantica eggs (Hegazi et al. 2006).In addition, Fahmy et al. (2010) observed that the ani-mals orally treated with propolis revealed significantdecrease (P<0.05) in parasitological parameters, wherepercentage worm reduction was 68 % for hepatic worms,and intestinal eggs reduction being 68.0 and 70.60 %,respectively.

Moura et al. (1998), on naturally infected New ZealandWhite growing rabbits by Eimeria spp. and treated withhydroalcoholic propolis solution (HPS) (0.0, 4.0, 8.0, 12.0,and 16.0 ml of HPS/l of water), observed that addition ofHPS levels to the drinking water showed a linear decline ofthe oocysts Eimeria spp. in drops of the rabbits feces.Furthermore, Hegazi et al. (2007) suggested an alternativeand effective fasciolicidal drug can be developed usingpropolis extract because propolis has high efficacy againstboth mature and immature F. gigantica flukes. Such reduc-tion of FEC, which occurs in our study, could be attributedto the antiparasitic activity of isoflavonoids, the red propolismain components (Salem et al. 2011). There is anotherexplanation related to the stimulation of the immune systemby propolis, which reflected as increasing the total leuko-cytes value (Talas and Gulhan 2009).

Acknowledgments The authors are grateful to Mr. AlessandroEsteves for providing the propolis samples and to the Third World ofAcademic Science (TWAS) and National Council for Scientific andTechnological Development (CNPq) for their financial foundation.



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