Research in Veterinary Science 87 (2009) 473–478

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Research in Veterinary Science

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Clinical observations of cattle and buffalos with experimentallyinduced chronic copper poisoning

Antonio H.H. Minervino a, Raimundo A. Barrêto Júnior a, Rodrigo N.F. Ferreira a, Frederico A.M.L. Rodrigues a,Selwyn A. Headley b, Clara S. Mori a, Enrico L. Ortolani a,*

a Department of Clinical Science, College of Veterinary Medicine, University of Sao Paulo, USP, São Paulo, SP, Brazilb Department of Basic Veterinary Sciences, School of Veterinary Medicine, St. Matthew’s University, Grand Cayman, Cayman Islands, UK

a r t i c l e i n f o

Article history:Accepted 5 May 2009

Keywords:Copper toxicosisCattleBuffaloZinc

0034-5288/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.rvsc.2009.05.002

* Corresponding author. Tel.: + 55 11 30911342; faE-mail address: ortolani@usp.br (E.L. Ortolani).

a b s t r a c t

The susceptibility of cattle and buffalos to chronic copper poisoning (CCP) was compared by using cattle(n = 10) and buffalo (n = 10) steers distributed into two copper supplemented (n = 6) and two control(n = 4) groups. Supplemented animals received 2 mg copper (Cu)/kg body weight daily for one week, withan additional 2 mg weekly until the end of the experiment (day 105). Three liver biopsies (day 0, 45, and105) were obtained for mineral analyses; clinical examinations and blood samples were obtained every15 days. Three supplemented cattle and two buffalos with typical manifestations of CCP died. There wereno differences in the frequency of mortality between cattle and buffalos; hepatic copper concentrationwas higher in cattle than buffalos. These findings suggest that buffalos and cattle might be equally sus-ceptible to CCP. However, buffalos accumulate less liver copper than cattle and have a lower threshold ofhepatic Cu accumulation, which leads to clinical manifestation of CCP.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction capacity is exceeded, which results in hepatocellular necrosis and

Chronic copper poisoning (CCP) is one of the main diseasesassociated with the death of sheep in Brazil, resulting in great eco-nomic losses to the local sheep industry (Ortolani, 2003). Althoughthe frequency of CCP is relatively reduced in other species of rumi-nants, Cu toxicosis in cattle has recently evolved as an emergingdisease, with descriptions of acute and chronic cases in severalcountries, including Brazil (Perrin et al., 1990; Bradley, 1993; De-voy, 2002; Ortolani et al., 2004). Bidewell et al. (2000) have relatedthat there is an increase in the number of cases of this disease incattle, and have described 14 outbreaks during a period of sixmonths. However, in buffalos there is only one description of thistoxicosis, during which young animals that received a reconsti-tuted copper-enriched powder milk based-diet for a few months,demonstrated signs indicative of CCP, and died shortly (Zicarelliet al., 1981).

It was estimated that the prevalence of Cu toxicosis is beyondthe number of published cases, since diagnostic confirmation isnot always established and CCP might be confused with other dis-eases that have hematological crises as part of their clinical mani-festations (Bidewell and Livesey, 2002).

In CCP, copper is gradually deposited in the liver without pro-ducing any significant clinical sign, until the hepatic copper storage

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the liberation of copper from the liver into the blood stream, pro-ducing intense hemolysis, jaundice, and renal insufficiency (Under-wood and Suttle, 1999). These characteristics relative to thepathogenesis of CCP makes an early diagnosis very difficult, andeven during the hemolytic crisis CCP can be confused with otherhemolytic diseases. In several clinical reports, the authors’ sus-pected different diseases before a diagnosis of CCP was established,these diseases included nitrate/nitrite poisoning, bacillary haemo-globinuria, plant poisoning, babesiosis, leptospirosis, post-parturi-ent haemoglobinuria and even Johne’s disease, hypercalcemia andindigestion (Perrin et al., 1990; Bradley, 1993; Auza et al., 1999;Bidewell and Livesey, 2002).

The range of clinical manifestations associated with CCP islarge. Generally, cattle demonstrate anorexia, reduced bodyweight, dehydration, and alterations in feces, followed by icterus,hemoglobinuria, and death (Gummow, 1996; Auza et al., 1999;Bidewell and Livesey, 2002). In one study with ruminants (Zicarelliet al., 1981), only buffalos demonstrated signs of CCP, while cattlethat received the same ration were not affected; these authorshave suggested that buffalos might have been more susceptibleto CCP than cattle. Additionally, it was related that buffalos absorbcopper more efficiently than cattle (Cardoso et al., 1997); however,during this study, reduced or normal concentration of copper wereadministered in the diet, and there was no indication that the buf-falos were exposed to elevated doses of copper.

This study is important and opportune since we compared thesusceptibility of buffalos and cattle to Cu toxicosis, and evaluated

474 A.H.H. Minervino et al. / Research in Veterinary Science 87 (2009) 473–478

the clinical manifestations and biochemical alterations associatedwith this disease in these animals.

2. Materials and methods

This study was realized in accordance with adequate ethicalstandards and animal care utilization, and was approved by theBioethics Committee of the College of Veterinary Medicine, Univer-sity of Sao Paulo (Protocol no. 746/2005), São Paulo, Brazil.

Two months prior to the experiment, all animals were dewor-med, and vaccinated against clostridial diseases. They were thensubmitted to surgery for the implantation of the ruminal latextube.

Twenty male animals were utilized during this study: 10mixed-breed cattle and 10 Murah breed of buffalos. During theexperiment, all animals received a total daily ration calculated at2.7% of BW DM, which composed of 75% of coast-cross (Cynodondactylon) hay (with 4.2 ppm of Cu) and 25% of commercial concen-trate (Novil� – Socil Evialis Nutrição Animal, São Paulo, Brazil) with14% crude protein (with 10 ppm of Cu). All animals had free accessto water and received daily 35 g of commercial mineral supple-ment (Fosbovi� 20 – Tortuga Cia. Zootécnica Agrária, São Paulo,Brazil) that contained 1530 ppm of Cu. This diet corresponded toan average daily ingestion of 80 mg of Cu per animal.

All animals were eight-months-old and weighed approximately170 kg, and were divided into two groups. The experimental group(n = 12; six bovines and six buffalos) received daily increasingdoses of Cu via ruminal fistula. The initial dose was 2 mg/kg bodyweight (BW) of Cu (copper sulphate pentahydrate – CuSO4 � 5H2O,in an aqueous solution) administered during the first seven days;this was increased by 2 mg every seven days until the 105th day,when the final dose was 28 mg/BW. The control group (n = 8; 4 bo-vine and four buffalos) received only normal ration and water byruminal fistula daily. If copper toxicosis, manifested by hemoglo-binuria, was observed in any animal, the administration of Cuwas suspended immediately and the animal treated with ammo-nium tetrathiomolybdate (3.4 mg/kg live weight) as recommended(Humphries et al., 1986; Ortolani, 2003).

Three serial hepatic biopsies were performed in all animals todetermine the concentration of Cu: (1) day 0, at the beginning ofthe experiment, before the administration of Cu; (2) 45 days afterfirst administration of Cu; and (3) at the end of the experiment(after 105 days of Cu administration). If any animal died, liverand kidney samples were obtained during routine necropsy; a partof these samples was fixed in neutral buffered formalin solutionand routinely processed for histopathological evaluation. Approxi-mately 2 g of tissue obtained from each organ were digested in5 ml of concentrated nitric acid (Merck, PA) and 2 ml of 30% w/vhydrogen peroxide in a open digestion system. The digested sam-ples were transferred into polypropylene tubes and diluted with10 ml with Clodric acid (Merck, PA). Metal concentrations in thedigesters were determined by spectrophotometric atomic absorp-tion (Varian� SpectrAA – Palo Alto – CA, USA) on a dry-matterbasis.

Eight blood samples were obtained from the jugular vein byusing vacuum collecting tubes: one at the beginning of the exper-iment, and thereafter at intervals of 15 days until the end of thestudy (day 105). The serum concentrations of Cu, AST, GGT, urea,and creatinine were determined by biochemical automatic ana-lyzer (Liasys�, AMS�; Rome, Italy), using commercial kits (Randox�

– Antrim, United Kingdom; BioSystems� – Barcelona, Spain; SIG-MA� – St. Louis, USA, respectively).

Blood samples for hematological evaluation was collected invacutainers� with EDTA at the same previously described intervals.To determine the whole globular volume, capillary microcentrifu-

gation was used. The live weight of all animals was recorded atthe beginning of the experiment, and thereafter each 15 daysthroughout the experimental period.

The frequency of mortality associated with copper supplemen-tation between cattle and buffalos were analyzed by the Fishertest. The pattern of all data was analyzed for its distribution bythe Kolmogorov–Smirnov test. Data with normal distribution wereinitially submitted to the variance analysis (F test), and in case ofsignificant differences, the treatments were compared by the Tur-key test. Data with non-parametric distribution were analyzed bythe Kruskal–Wallis test, and expressed as median values. Regres-sion analyses and their respective coefficients of determinationwere utilized to verify the relationship between pairs of variables.The significance obtained by linear regression was evaluated by theF test. Most statistical analyses were done by using the softwareMinitab (Minitab, 2000).

3. Results

3.1. Clinical manifestations

Cattle and buffalos from the control groups did not demonstrateany clinical alteration during the experiment. However, three cat-tle and two buffalos from the Cu treatment groups demonstrated,during the last 15 days of the experiment, manifestations indica-tive of CCP, and eventually died. No statistical difference(P = 0.87) was observed when the frequency of mortality betweencattle and buffalos was compared.

Two distinct clinical manifestations were observed in the exper-imental group: one indicative of CCP and another with atypicalmanifestations of CCP. Two animals (one from each species) dem-onstrated the classical signs of CCP: hemoglobinuria, apathy, disor-derly gait, reduced ruminal movements, severe dehydration, ictericmucous membranes, mild diarrhea, and oliguria. These manifesta-tions were more severe in the buffalo relative to the bovine; theskin of the buffalo was also icteric.

Atypical clinical manifestations of CCP included: dehydration,ruminal hypotony, oliguria, with severe and progressive apathy.These alterations were observed in three animals (cattle #2 and3, and buffalo #9). The classical signs of icterus and hemoglobinu-ria, associated with CCP, were not observed. Additionally, theseanimals demonstrated progressive hyporexia (during 18 days), fol-lowed by anorexia (during 4–5 days); this resulted in the loss of15%, 19% and 22% of the live body weight of the buffalo and thetwo cattle, respectively. Weight loss was related to progressivehyporexia (10 days), followed by anorexia (two days), and collec-tively represented a loss of 12% and 9.4% of live weight of cattleand buffalos, respectively. However, all animals from the experi-mental group were diagnosed with CCP based on elevated hepaticcopper concentrations.

3.2. Gross and histopathological manifestations of toxicosis

Animals with clinical signs associated with the classical mani-festation of Cu toxicosis demonstrated icteric mucosal and serosalmembranes; yellow and severely enlarged liver; severely darkenedkidneys; discrete splenomegaly; severe edematous fluid within thetrachea; severe congestion and small ulcers at the abomasalmucosa.

The histologic hepatic alterations of both species with classicalsigns of CCP were similar, being more severe in buffalos comparedto cattle, and included: moderate to severe diffuse portal fibrosis,centrilobular hepatocellular necrosis, canalicular biliary stasis,mild multifocal hepatitis, and microvesicular lipidosis. Addition-ally, the buffalo that died earlier with classical manifestations of

Table 1Hematological and biochemical variables and levels of hepatic copper in cattle and buffalos with copper poisoning.

Variables Specie of animal Normal range*

Ox 2 Ox 3 Ox 5 Buffalo 7 Buffalo 9

Clinical manifestation Atypical Atypical Classic Classic Atypical –Hepatic copper (ppm) 4901 4389 3987 2053 2051 <400Kidney copper (ppm) 35 72 1105 707 29 <25Serum copper (lMol/L) 24.9 19.1 92.5 48.3 20.5 <15Serum GGT (U/L) 35 39 27 84 23 6–17Serum AST (U/L) 348 116 162 4001 190 78–132Urea (mMol/L) 16 24 12 10 22 7–11Creatinine (lMol/L) 133 148 158 169 288 88–177Packed cell volume (%) 23 17 22 28 40 –

* Normal biochemical values for cattle (kaneko et al., 1997). Copper normal levels in dry-matter basis according to Underwood and Suttle (1999).

Table 3Mean values of hepatic copper and zinc (ppm) and serum copper (lMol/L) observedin the last biopsy samples (105 d) from poisoneda, resistanta, and control animals.

Groups Liver copper(ppm)

Liver zinc(ppm)

Serum copper(lMol/L)

Cattle poisoned 4425 549 45.5Cattle resistant 1159 129 14.6Cattle control 109 90 12.2Buffalos poisoned 2052 126 34.4Buffalos resistant 1455 143 11.8Buffalo control 176 77 9.4

a Animals poisoned and resistant are from copper-treated groups. Poisoned ani-mals show signs of copper toxicosis and resistant did not.

A.H.H. Minervino et al. / Research in Veterinary Science 87 (2009) 473–478 475

CCP demonstrated areas of discrete hepatocellular apoptosis andmoderate swelling of hepatocytes, while the hepatic lesion of thecow was restricted to discrete proliferation of epithelial cells of bileducts. Renal lesions were characterized by moderate tubularnecrosis and the discrete accumulation of protein within renal tu-bules and some glomeruli.

The animals with atypical clinical manifestations of toxicosiswere severely cachectic with marked accumulation of dried ingestawithin the rumen, abomasum, and omasum. The liver of these ani-mals was slightly hypertrophic; the kidneys were unaffected, butthe urine was concentrated and light red in color. The histologichepatic lesions in these animals were characterized by canalicularbiliary stasis and swelling of hepatocytes; discrete proliferation ofepithelial cells of bile ducts occurred only in the buffalos. Signifi-cant renal lesions included discrete lymphoplasmacytic interstitialnephritis, acute tubular nephrosis with foci of protein cast in somerenal tubules.

3.3. Liver copper

The median values of the concentration of hepatic Cu observedthroughout the experiment are given in Table 2. Ruminants fromthe experimental group demonstrated elevated hepatic Cu concen-trations when compared to those from the control group. Therewas progressive increase in the hepatic concentrations of Cu inthe experimental group; this was observed after the second liversample. No statistical differences were observed between animalsfrom the control group during the experiment.

When the groups of animals that received Cu were evaluated, itwas observed that some had clinical evidence of CCP while otherswere apparently unaffected (resistant, R). A descriptive analysis ofthe hepatic Cu concentration between the experimental groups(intoxicated and resistant animals) and the control group is givenat Table 3. A marked numerical difference between animals (cattleand buffalos) considered as resistant and those that demonstratedsigns of CCP occurred. However, statistical analyses were not madedue to the small number of animals contained within the sub-

Table 2Median values of hepatic copper and zinc (ppm) of cattle and buffalos.

Animals Median values of hepatic copper and zinc (ppm)

Copper

Time of samples (days) 1st (0) 2nd (45)

Cu-treated cattle 168cB 524bA

Cu-treated buffalo 262cB 649bA

Cattle control 92B 102B

Buffalo control 197B 170B

P 0.270 0.002

Different small letters within the same line and different large letters within the same c

groups. Nevertheless, resistant animals in both species apparentlyretain less hepatic Cu than intoxicated animals.

3.4. Liver zinc

Cattle demonstrated comparatively more elevated hepatic accu-mulations of zinc relative to buffalos when samples from the firsttwo biopsies were evaluated (Table 3). However, during the finalstage of the experiment, animals with clinical evidence of CCP fromboth species demonstrated more elevated hepatic zinc values thananimals from the control group. When the accumulation of zincwas elevated at the sub-group level, cattle with classical signs ofCCP demonstrated elevated values of hepatic zinc relative to Cu(Table 3).

3.5. Relationship between Cu and zinc in the liver

A significantly elevated coefficient of determination (R2 = 0.88)was obtained when the hepatic accumulations of Cu and zinc forcattle and buffalo were observed, indicating the predominant influ-ence of hepatic Cu on zinc (Fig. 1). This influence was predominantin cattle relative to buffalo, thus when the coefficient of determina-

Zinc

3rd (105) 1st (0) 2nd (45) 3rd (105)

2620aA 120 A 122A 219A

1698aA 87bB 89bB 133aA

92B 136A 117A 101B

175B 73B 76B 77C

0.003 0.002 0.002 0.002

olumn for each one of minerals indicates significant difference (P < 0.001).

Fig. 1. Relationship between the concentration of hepatic copper and zinc in cattleand buffalo from poisoned and control groups during the experiment.

476 A.H.H. Minervino et al. / Research in Veterinary Science 87 (2009) 473–478

tion for the species was compared, cattle demonstrated more ele-vated values (R2 = 0.90) than buffaloes (R2 = 0.74).

3.6. Serum copper

Cattle from the experimental group demonstrated significantlymore elevated values of serum Cu relative to the control group dur-ing the last 30 days of the experiment (Table 4). The death of twobuffalos (after day 75) during the experimental period affected theinterpretation of serum Cu values for these animals, since the med-ian values of serum Cu for buffalos decreased. Additionally, cattleshowed a marked difference in the serum Cu values between the

Table 4Median values of serum copper (Cu) of ruminants during the experiment.

Periodicity of samples (days) Median values of serum copper (lMol/L)

Cu-treated cattle Cu-treated bu

0 11.0B 10.815 8.4B 11.430 9.9B 11.145 9.5B 10.560 16.6A 18.175 14.6A 17.2*

90 14.2aA 13.4ab

105 17.6aA 12.8ab

P 0.001 0.105

Different small letters within the same line and different uppercase letters in the same* Death of intoxicated buffalos.

Table 5Median values of serum GGT activity of ruminants during the experiment.

Periodicity of samples (days) Median values of serum GGT (U/L)

Cu-treated cattle Cu-treated bu

0 10C 13B

15 10C 16B

30 13C 15B

45 13C 16B

60 16BC 18B

75 20B 21B

90 27aAB 28aAB

105 37aA 26aA

P 0.001 0.006

Different small letters within the same line and different uppercase letters in the same

first three samples (up to day 45), and the last sample (day 105);having a two-fold increase in serum values.

3.7. Serum activity of GGT and AST

The results for the GGT and AST activities are presented inTables 5 and 6, respectively. Cattle and buffalo from Cu-treatedgroups demonstrated elevated GGT activity with effect from 75and 90 days post-toxicosis. Differences in the GGT activity wereobserved between the Cu-treated animals (buffalos and cattle)from the control groups on days 90 and 105 of the experiment.

Increases in the activity of AST were observed in cattle with ef-fect from 75 days of the experiment; buffalos did not demonstratesignificant differences within the Cu-treated group during theexperiment, probably because of the large standard deviation ob-served. However, during most of the experimental period buffalosdemonstrated higher AST activity compared to cattle. In cattle andbuffalos affected by the classical and atypical forms of CCP, the GGTand AST activities increasing significantly, five to twenty times pre-vious values.

3.8. Serum urea and creatinine, and packed cell volume

Significant differences between the mean values of serum ureaand creatinine of the animals from the different groups were notobserved throughout the experimental period (data not shown).However, cattle and buffalos with CCP demonstrated elevated val-ues of serum urea (Table 1).

Significant differences in the value of the packed cell volume(PCV) between and within the different groups of animals (controlsand treated) were not observed throughout the experiment. Cattlewith CCP demonstrated a reduction in PCV value (up to 10%). Thiswas not seen in buffalos with CCP probably due to the elevated de-gree of dehydration observed in these animals.

P

ffalo Cattle control Buffalo control

8.9 11.9 0.6089.4 14.3 0.7006.0 10.2 0.05810.3 11.9 0.07410.5 13.0 0.2088.4 10.8 0.0907.8c 10.1b 0.00910.9b 11.4b 0.041

0.140 0.106 -

column indicates significant differences (P < 0.05).

P

ffalo Cattle control Buffalo control

10 8 0.1189 13 0.13212 12 0.19013 1 0.59815 12 0.22511 11 0.07312 b 12 b 0.05011 b 9 b 0.003

0.382 0.399 –

column indicates significant differences (P < 0.05).

Table 6Mean and standard deviation values of serum AST activity of ruminants during the experiment.

Periodicity of samples (days) Mean and standard deviation values of serum AST (U/L) P

Cu-treated cattle Cu-treated buffalo Cattle control Buffalo control

0 40 ± 8.8cB 118 ± 28.4b 61 ± 9.2c 187 ± 33.9aB 0.00115 45 ± 3.7cB 136 ± 40.5b 76 ± 12.1c 210 ± 25.6aA 0.00130 51 ± 6.7cB 149 ± 19.4a 86 ± 20.7b 153 ± 15.6aB 0.00145 52 ± 7.0cB 140 ± 12.4a 60 ± 8.4c 125 ± 19.9bC 0.00160 57 ± 17.5bB 136 ± 33.2a 56 ± 9.9b 118 ± 13.2aC 0.00175 89 ± 64.7AB 125 ± 24.7 76 ± 20.6 118 ± 19C 0.21590 76 ± 31.1bB 144 ± 40.2a 78 ± 16.2b 146 ± 10.1aB 0.001105 162 ± 95.0A 815 ± 1563 80 ± 10.2 149 ± 28.4B 0.490

P 0.001 0.362 0.130 0.001 –

Different small letters within the same line and different uppercase letters in the same column indicates significant differences (P < 0.05).

A.H.H. Minervino et al. / Research in Veterinary Science 87 (2009) 473–478 477

4. Discussion and conclusions

Atypical manifestations attributed to CCP were observed in fewcattle and buffalos during this experiment; these atypical signs (ca-chexia and dried ingesta within the gastrointestinal tract) wouldhave made a clinical diagnosis of CCP difficult, if not impossible. Inthis case, a diagnosis of CCP was supported by laboratory evalua-tions particularly due to the elevated hepatic and serum Cu concen-trations, which were similar to those animals with the classicalmanifestation of CCP. Atypical manifestations were also describedin an outbreak of CCP in milking cows (Bradley, 1993); one of the af-fected animal demonstrated anorexia, dried ruminal ingesta, ictericmucus membranes, darkened kidneys but without hemoglobinuriaand with elevated concentrations of hepatic and renal copper.

Other uncommon clinical manifestations of CCP previously de-scribed include ruminal atony and bloat, and nervous symptomssuch as restlessness, incoordination, and lethargy (Perrin et al.,1990; Bradley, 1993). Therefore, in ruminants, CCP must be con-firmed by laboratory evaluations and not based only on clinic-pathological manifestations. Further, in the clinical reports of CCPwithout classical symptoms, the affected animals demonstrated le-sions consistent with that of hemoglobinuric nephrosis (Bradley,1993); which were not observed in our study. Perhaps the clinicalsigns and lesions in the atypical CCP animals could have been asso-ciated with the experimental design, so additional cases wouldhave to be examined to confirm the manifestation of these atypicalassociated lesions.

The pathological manifestations observed during this study areindicative of Cu toxicosis (Stalker and Hayes, 2007); similar resultshave been described in naturally occurring cases in young animals(Sullivan et al., 1991; Hamar et al., 1997; Steffen et al., 1997; De-voy, 2002). Further, the renal and hepatic Cu values observed dur-ing this study were indicative of CCP and comparatively similar tothose described in other reports (Hamar et al., 1997; Steffen et al.,1997). Additionally, of these tissue accumulations, elevated serumvalues of Cu were also observed in these animals reflecting anacute manifestation of CCP, due to the liberation of Cu into theblood stream during hemolytic crisis (Stalker and Hayes, 2007).

The results from this study suggest that buffalos and cattlemight be equally susceptible to CCP, being different from the the-ory of Zicarelli et al. (1981), since significant differences in the fre-quency of mortality were not observed when these two specieswere compared. A larger number of animals would have been idealto characterize these differences, but this was not possible due tobioethical standards and restrictions. Additionally, we believe thatif buffalos are more susceptible than cattle, all buffalos should havedemonstrated clinical signs of CCP, or at least, elevated liver Cu be-fore similarly affected cattle.

Nevertheless, this study revealed features that are directly re-lated to the metabolism of Cu in buffalos. Affected buffalos demon-

strated comparatively less accumulated hepatic Cu relative tocattle (Table 2). However, buffalos were apparently less susceptibleto high dietary copper concentrations but developed CCP at lowerliver concentrations compared to cattle. The liver acts as a reservoirfor excessive accumulations of Cu, but when this threshold concen-tration has been surpassed, hepatocellular necrosis occurs and Cuis leaked into the circulatory system resulting in the classic mani-festations of CCP, including hemolysis and secondary renal failure(Underwood and Suttle, 1999). During this experiment, the thresh-old limit in cattle was twice that of buffalos, but both species re-ceived equal amounts of Cu.

These findings suggest that buffalos absorb/accumulate less Cuthan cattle. This could possibly occur due to the elevated activity ofmetallothionein, occurring either within the intestine duringwhich metallothionein binds with dietary Cu and inhibits itsabsorption (Cousins, 1985), or in the liver, where hepatic metallo-thionein promotes an increase in the biliary excretion of Cu (López-Alonso et al., 2005). However, additional studies must be done toconfirm this mechanism.

It was suggested that buffalos might absorb Cu more efficientlythan cattle (Cardoso et al., 1997). Therefore, it can be theorized thatbuffalos either retain comparatively less accumulated hepatic Cuor excrete comparatively larger quantities of Cu relative to cattle,which should make these animals more resistant to toxicosis thancattle. Therefore, buffalos may possibly have a more efficientmechanism to regulate Cu concentration than cattle during whichthese animals increase the absorption in deficient diets, and pro-mote its reduction when the concentration of Cu is elevated.

The values of the serum creatinine observed during this studyindicate that most animals did not demonstrate significant renaldysfunction. Similar results have been described in dairy cattle(Bradley, 1993). Sheep with Cu toxicosis demonstrated severe re-nal insufficiency (Soares, 2004). Although the kidney might serveas an indicator of CCP in some species, and renal Cu concentrationswere elevated in cattle and buffalos with CCP during this study, theliver is the main organ associated with the storage and regulationof Cu (Stalker and Hayes, 2007).

Acknowledgements

E.L. Ortolani and R.A. Barrêto Júnior thank CNPq (Brazil) fortheir fellowships. A.H.H. Minervino received a FAPESP fellowship.This research was supported by FAPESP (Process no. 2005/03204-0). The authors are grateful to Dr. Pierre Castro Soares for their lab-oratorial assistance.

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