- Email: [email protected]
Effects of high copper supplements on performance, health, plasma copper and enzymes in goats
Small Ruminant Research 41 (2001) 127±139
Effects of high copper supplements on performance, health, plasma copper and enzymes in goats S.G. Solaiman*, M.A. Maloney, M.A. Qureshi, G. Davis, G. D'Andrea1 Department of Agricultural Sciences, George Washington Carver Agricultural Experiment Station, Tuskegee University, 105 Milbank Hall, Tuskegee, AL 36088, USA Accepted 21 May 2001
Abstract Six growing female Nubian goats (average BW 34:8 0:55 kg, 7±8 months of age) were randomly assigned to either a basal diet (BD, 10±15 ppm Cu/DM), or to medium Cu (MC, BD 50 mg Cu), or to high Cu (HC, BD 100 mg Cu) diets for 9 weeks. This level would cause Cu toxicity in sheep, but none occurred in the goats. Therefore, Cu supplementation was then increased to 150 and 300 mg per head per day, for the following 14 weeks; to 300 and 600 mg per head per day, for the next 8 weeks; and to 600 and 1200 mg per head per day, for an additional 4 weeks, in the MC and HC group, respectively. Body weight and vital signs were recorded and blood samples collected at different time intervals. Hematological parameters, plasma Cu, sorbitol dehydrogenase (SDH), glutamic oxaloacetic transaminase (GOT), and g-glutamyl transferase (GGT) were determined. At the termination of the study, tissue Cu concentration in different organs was also determined. During ®rst 23 weeks (<300 mg Cu per day) of the study there were no apparent signs of Cu toxicity. Cu supplementation at 600 mg per head per day in young Nubian does, had no effect on respiration rate (RR), heart rate (HR), and decreased (P < 0:05) rectal temperature (RT) in the HC group only. Leukocyte counts were positively correlated with Cu supplementation (r 0:296, P < 0:02) and negatively correlated (r 0:254, P < 0:05) with RT in the HC group. Plasma SDH increased (P < 0:05) when Cu supplementation was 300 mg per head per day, thus, SDH may serve as an early indicator of Cu toxicosis in goats. Increases (P < 0:05) in GOT were noted when Cu intake was 600 mg per head per day. Contrary to the results observed for SDH and GOT, feeding goats 50 mg Cu per day or more, resulted in an increased plasma GGT as compared to BD goats. Levels of SDH, GOT and GGT of the BD goats were within normal range. Plasma Cu was not indicative of Cu status of animals. Copper improved ADG by 28% at the 100±150 ppm level in diet. No relationship between Cu intake and hair Cu was found in the present study. Highest concentration of Cu was found in liver, followed by duodenum, rumen and brain. Results of this study indicate that goats are more resistant to Cu toxicity than sheep. This is one of the ®rst reports documenting signi®cant differences in Cu requirements and tolerance between goats and sheep. # 2001 Elsevier Science B.V. All rights reserved. Keywords: Copper supplementation; Copper toxicity; Plasma enzymes; Copper in tissues; Goats
1. Introduction *
Corresponding author. Tel.: 1-334-727-8401; fax: 1-334-727-8552. E-mail address: [email protected] (S.G. Solaiman). 1 Present address: State Veterinary Diagnostic Laboratory, Auburn, AL 36830, USA.
Copper is an essential element required by goats and other animals for a number of biochemical functions (Davis and Mertz, 1987). Copper is a normal component of all body tissues and ¯uids, however, ingestion of quantities of Cu slightly higher than
0921-4488/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 1 - 4 4 8 8 ( 0 1 ) 0 0 2 1 3 - 9
128
S.G. Solaiman et al. / Small Ruminant Research 41 (2001) 127±139
required, may cause accumulation within the tissues (Howell and Gawthorne, 1987). The mechanism of Cu uptake and transport through the intestinal wall is not clearly de®ned, however, bile and gastrointestinal (GI) tract are two important routes of Cu excretion (Howell and Gawthorne, 1987; Mertz, 1987). Cu stimulates growth and ADG in swine (Cromwell, 1997) and alters lipid metabolism in steers (Engle and Spears, 2000). From a toxicity standpoint, there is a great variation between species. Clinical toxicity symptoms include hemolytic crises; the animal becomes dull and lethargic, passes soft stool, looses appetite and may have excessive thirst (Ishmael et al., 1971; Howell and Gopinath, 1977). Also there may be marked elevation of liver speci®c enzymes (sorbitol dehydrogenase, SDH; glutamic oxaloacetic transaminase, GOT; g-glutamyl transferase, GGT) in blood (Howell and Gawthorne, 1987; Mertz, 1987; Ishmeal et al., 1972; Schee et al., 1983). Sheep are more sensitive to high Cu supplementation than other agricultural animals (NRC, 1980). Chronic Cu toxicity has also been reported in dairy cows fed 37.5 mg Cu/kg DM during lactation and 22.6 mg Cu/ kg DM during the non lactating period (Bradley, 1993). Searching the literature (Meschy, 2000; Howell and Gawthorne, 1987; Mertz, 1987), there are more reports on Cu de®ciency in goats than on toxicity. This may be an indication that goats can tolerate higher levels of Cu than sheep or cattle. Goats, especially young kids, seem to be less sensitive to Cu intake by having lower hepatic uptake, than lambs, when fed diets containing high levels of Cu (Zervas et al., 1989). The exact amount of Cu required in the goat's diet is currently unknown (Haenlein, 1992) and the current established Cu requirement of goats (8±10 ppm) needs more studies (NRC, 1981; Kessler, 1991; Haenlein, 1992; AFRC, 1997). Signs of Cu toxicity and levels of associated liver speci®c enzymes are not clearly established for goats, therefore, this study was designed to determine some parameters associated with Cu toxicity in goats and to establish some guidelines for feeding Cu to goats. 2. Materials and methods 2.1. Animals and treatments Six growing female Nubian goats (Capra hircus hircus), 7±8 months of age, with average BW of
34:87 0:55 kg were used in this study. Goats were housed at the old dairy barn of Tuskegee University in compliance with Tuskegee University Institutional Animal Care and Use Committee regulations. Each animal was treated for internal parasites (Ivomectin; Meriel, Division of Merck and Co., Rahway, NJ) prior to the start of the experiment. After 2 weeks for adjustment, animals were weighed, randomly assigned to three experimental treatments and housed in three pens (two animals each) with concrete ¯oors covered with old bermudagrass hay (BGH) as bedding. Goats were offered once daily, at 0900, 1 kg, of basal diet (BD; 16.7% CP, 11±15 ppm Cu; Table 1) and free choice bermudagrass hay (7.8% CP, 5±8 ppm Cu), to secure intake of nutrients required for maintenance and 100 g per day growth, in accordance with NRC (1981). Animals had access to water (1.0± 1.5 ppm Cu) at all times and they were not allowed outside the pens except to facilitate the cleaning once a week. Levels of Cu supplement were based on information from sheep and cattle (Howell and Gawthorne, 1987; Mertz, 1987). Initial supplemental levels were 50 mg Cu per head per day (MC 50) and 100 mg Cu per head per day (HC 100), anticipating Cu toxicity within 8±10 weeks, according to the sheep data (Howell and Gawthorne, 1987). Pre-determined amounts of Cu, as Cu sulfate (CuSO45H2O), were placed in gelatin capsules and inserted into the Table 1 Dietary composition of the basal diet used in the experiment Ingredient
%a
Cracked corn Alfalfa meal Whole oats Soybean meal (48% CP) Molasses Masonexb Dicalcium phosphate Limestone Salt Vitamin/mineral mix
43.5 15.0 17.5 16.5 2.5 2.5 0.5 0.5 0.5 1.0
Cu (ppm) Basal diet Bermudagrass hay Water
10.8±15.2 5.8±8.3 1.0±1.5
a b
As fed basis. A commercial pelletizing agent.
S.G. Solaiman et al. / Small Ruminant Research 41 (2001) 127±139
esophagus with a balling gun at the time of feeding. This regiment continued from weeks 0 to 9 (phase I) with no apparent sign of Cu toxicity. Therefore, Cu supplementation was increased to 150 (MC 150) and 300 (HC 300) mg per head per day for the MC 50, and HC 100 groups, respectively, from weeks 10 to 23 (phase II). During this phase of the study, all the animals were healthy, without signs of Cu toxicity, and were checked for estrus, bred by AI, and con®rmed pregnant. During weeks 24±31 (phase III), Cu in the diet of goats in MC 150 and HC 300 group was again increased to 300 (MC 300) and 600 (HC 600) mg per head per day, respectively. Supplemental Cu was increased to 600 (MC 600) and 1200 (HC 1200) mg per head per day for the MC 300 and HC 600 group, respectively, for the last 4 weeks (weeks 32±35, phase IV), because still no toxicity occurred at the 600 mg level. The experiment was terminated when goat no. 174 in the HC 1200 group, exhibited clinical signs of Cu toxicity (Anorexia, dark urine, brown patches on her white hair coat and hemolytic crisis) and died. The dosing regimens and duration are summarized in Table 2. The control group (BD) was fed no supplemental Cu throughout the study. 2.2. General health and clinical observations General condition and reactions of goats to supplemental Cu was observed daily throughout the experiment. Feed intake was monitored and body weights were recorded weekly, for the ®rst 9 weeks and every 2±4 weeks during rest of the experimental period. Clinical evaluation was conducted by a veterinarian prior to dosing, at weekly intervals during the ®rst 9 weeks of the study, and every 2±4 weeks thereafter depending on the general condition of the goats. Vital body signs recorded were respiration rate (RR), heart rate (HR), and rectal temperature (RT).
129
2.3. Blood collection and analysis Blood samples were collected weekly for the ®rst 9 weeks and every 2 weeks thereafter, by jugular venipuncture to measure plasma Cu and enzymes. Blood samples collected into 3 ml vacutainer tubes (Becton Dickinson, Vacutainer System, Franklin Lakes, NJ) containing EDTA, were used to measure packed cell volume (PCV), white blood cells (WBC), neutrophils and lymphocytes. Samples collected in 10 ml vacutainer tubes containing EDTA were used for plasma Cu determination. Blood samples for SDH, GOT and GGT enzyme assays were collected in 10 ml heparinized vacutainer tubes. Blood samples were placed on ice immediately after collection, and transported to the laboratory for further preparation. Packed cell volume was measured following centrifugation in micro-hematocrit capillary tubes with a Hawksley microhematocrit reader (Galbraith et al., 1997). Leukocytes were counted manually with a hemocytometer. Each sample was counted twice and the mean was calculated (Azab and Abdel-Massoud, 1999). Samples for plasma Cu and enzymes were prepared by conventional centrifugation of whole blood at 1000 g. Plasma separated for Cu determination was stored in acid washed (37.5 ml nitric acid/l H2O) containers. Plasma Cu concentrations were determined by diluting plasma 1:4 in deionized water and aspirating into the ¯ame of an atomic absorption spectrophotometer (model 5000, Perkin-Elmer, Norwalk, CT). Liver speci®c enzymes, SDH (Kit # 50-UV, Sigma Chemical Co., St. Louis, MO), GOT (Kit # 505, Sigma Chemical Co.) and GGT (Kit # 545, Sigma Chemical Co.) were assayed in plasma according to procedures outlined in respective commercial kits using a spectrophotometer (Beckman DU-7) set at 340-UV, 500 and 540 nm wavelength, respectively.
Table 2 Dosing regimen for medium Cu (MC) and high Cu supplemented goats Phase
Weeks
Duration (weeks)
MC group (mg Cu per head per day)
HC group (mg Cu per head per day)
I II III IV
0±9 10±23 24±31 32±35
9 14 8 4
50 150 300 600
100 300 600 1200
130
S.G. Solaiman et al. / Small Ruminant Research 41 (2001) 127±139
An ovine SDH (SDH control, Sigma Chemical) in a bovine serum base was used as a quality control measure. Porcine GOT and GGT (Sigma Enzyme Control 2-N, Sigma Chemical) in a buffered bovine albumin base were used as quality controls for GOT and GGT assays, respectively. All samples for SDH were analyzed within 2 h of collection at 22±258C room temperature. The GOT assay was conducted within 4 h of collection and blood samples used in the GGT assay were stored at 48C with the assay being conducted within 24 h of collection. 2.4. Tissue collection and analysis At the termination of the study, remaining animals were euthanized and different body organs and tissues were collected for Cu analysis. Different tissue samples were dried at 1008C for 48 h, weighed, and 1 g sample DM was ashed in a muf¯e furnace at 5008C overnight. Ashed samples were digested in 10 ml of 25% (w/v) HCl, over a hot plate for 2±3 min, ®ltered through no. 41 Whatman ®lter paper and rinsed with additional 15 ml 25% HCl into 50 ml volumetric ¯asks. To reduce the absorptive interference, 10 ml of 20% (w/v) KCl was added to each volumetric ¯ask and was brought up to volume with deionized water. Fat samples were prepared according to procedures described for oils and fats in AOAC (1984). Cu concentration was determined by aspirating the diluted samples into the ¯ame of an atomic absorption spectrophotometer. Different sets of standard solutions were prepared to cover the expected range of Cu concentration in the samples. 2.5. Statistical analysis Data were analyzed by the GLM procedure of SAS (1990). Week and Cu level interactions were not signi®cant. Therefore, hematological parameters, BW and performance data, plasma Cu and enzymes, were averaged over weeks 0±9, 10±23, 24±31 and 32± 35 and analyzed as a complete randomized design with Cu levels as a source of variation. Mean responses were separated with the least squares means procedure and signi®cant differences were observed at P < 0:05, unless otherwise indicated. Regression analysis of the BW responses to various levels of supplemental Cu was conducted to evaluate growth
performance. Correlation coef®cients of plasma Cu and enzymes responses to various levels of supplemental Cu were also determined. Data are presented as simple means or least squares means with S.E. 3. Results and discussion Analysis for vital signs, hematological and toxicological parameters indicated that the effect of week was not signi®cant. Thus, data were subjected to an evaluation of treatment groups identi®ed as basal diet (BD), MC 50, MC 150, MC 300, MC 600, HC 100, HC 300, HC 600 and HC 1200. The BD values represent 31 or in some cases 35 weeks of data collection, MC 50 and HC 100, MC 150 and HC 300, MC 300 and HC 600, and MC 600 and HC 1200, represent data collected during (0±9) 9 weeks, (10±23) 14 weeks, (23± 31) 8 weeks and (32±35) 4 weeks, respectively. 3.1. General animal health and performance During phases I and II of the study, there were no apparent signs of Cu toxicity. Animals, when receiving 600 mg Cu and more (MC 600, HC 600 and HC 1200), exhibited excessive thirst, diarrhea, dehydration, anorexia, loss of BW and were inactive. Beginning week 33 (phase IV), animal no. 174 (HC 1200), exhibited more signs of Cu toxicity by having pale mucus membranes and a change of hair coat color (brown patches) and ®nally death on week 35, at which time this study was terminated. Mean vital signs, PCV, leukocytes and leukocyte differential counts are reported through phase III or for 31 weeks only (Table 3). RR, HR and RT in goats were not affected (P > 0:05) by feeding supplemental Cu for 31 weeks (Table 3). Mean RR ranged from 42.4 to 49.4 breaths/min, which falls within the normal range (20±76.5 breaths/min) reported by others (Brooks et al., 1984; Sleiman and Abi-Saab, 1998; Kasa et al., 1999). Genotype, sex, age, ambient temperature (Sleiman and Abi-Saab, 1998) and level of activity (Kasa et al., 1999) can affect RR in goats. Mean HR ranged from 116.9 to 132.1 beats/min, falling within the normal range (70±135 beats/min) reported by others (Brooks et al., 1984; Sleiman and Abi-Saab, 1998). Genotype and sex can in¯uence HR in goats (Sleiman and Abi-Saab, 1998). Mean RT was lower
Table 3 Effects of medium copper (MC) and high copper (HC) supplement on vital signsa blood cellsb, plasma enzymesc and Cu in growing Nubian female goats (mean S:E:) Item
BDd (0)
MC (Cu mg per head per day) 50
Duration (weeks) RR (breaths/min) (n) HR (beats/min) (n) RT (8C) (n) PCV (%) (n) WBC (103/ml) (n) Lymphocytes (%) (n) Neutrophils (%) (n) SDH (U/l) (n) GOT (U/l) (n) GGT (U/l) (n) Plasma Cu (mg/l) (n) a
0±35 49.4 132.1 39.1 40.3 14.0 58.2 38.8 18.7 46.0 21.2 1.15
2.23 (36) 3.51 (36) 0.08 (36) 1.13 (36) 0.71 (36) 2.45 (26) 2.55 (26) 0.50 (42) 02.82 (40) 0.82 (38) 0.04 (42)
0±9 46.7 126.0 39.4 35.8 14.6 23.7 70.7 16.5 54.0 33.6 1.23
150
3.65 (9) 5.34 (10) 0.11 (9) 1.78 (9) 1.36 (9) 6.0 (3)e 07.2 (3)h 0.77 (12) 03.85 (12) 2.63 (12)f 0.09 (12)
10±23 47.9 128.6 39.3 42.0 14.5 58.1 37.5 18.8 27.6 29.9 1.22
HC (Cu mg per head per day) 300
4.65 (14) 6.95 (14) 0.10 (14) 2.18 (16) 1.23 (16) 1.64 (12) 2.20 (12) 1.79 (16) 03.15 (14) 1.85 (14)i 0.06 (14)
24±31 44.0 130.4 39.3 38.4 14.1 56.4 40.8 26.7 58.0 38.9 1.39
600 2.92 (6) 11.76 (6) 0.13 (6) 0.68 (5) 1.19 (5) 2.91 (5) 3.18 (5) 4.63 (10)i 11.44 (10) 04.26 (8)f 0.09 (12)
32±35 45.0 145.0 39.1 ±j ± ± ± 28.0 86.5 26.3 1.08
100 15.0 (2) 15.0 (2) 0.0 (2)
14.0 25.2 2.25 0.05
(4) (4) (4) (4)
RR: respiration rate; HR: heart rate; RT: rectal temperature. PCV: packed cell volume; WBC: white blood cells. c SDH: sorbitol dehydrogenase; GOT: glutamic oxaloacetic transferase; GGT: g-glutamyl transferase. d BD: basal diet with no supplemental Cu. e Means within a row are different from BD group at P < 0:02. f Means within a row are different from BD group at P < 0:003. g Means within a row are different from BD group at P < 0:0001. h Means within a row are different from BD group at P < 0:05. i Means within a row are different from BD group at P < 0:002. j ±: Not measured. b
0±9 47.4 124.8 39.5 33.3 13.3 20.8 77.5 17.5 54.7 28.8 1.12
300
6.84 4.84 0.14 0.69 1.37 8.25 9.30 1.05 4.71 2.38 0.09
(7) (10) (10) (10)g (10) (4)e (4)e (12) (12) (12)i (12)
10±23 48.9 116.9 39.1 39.0 15.9 59.9 37.2 28.8 34.0 35.4 1.05
600 3.41 (14) 6.22 (14) 0.06 (14) 1.40 (16) 0.99 (16) 2.62 (12) 2.89 (12) 4.95 (15)i 03.51 (13) 2.41 (14)f 0.05 (14)
24±31 42.4 126.7 38.6 39.5 21.2 55.3 41.7 28.6 167.6 73.1 1.40
1200 32±35 5.88 (5) 46.0 14.0 (2) 11.9 (6) 130.0 30.0 (2) 39.5 0.0 (2) 0.18 (6)e 1.55 (4) ± 8.47 (4)h ± 4.84 (3) ± 6.01 (3) ± 4.93 (10)i 29.7 17.4 (4) 54.43 (10)i 258.5 106.1 (4)i 11.1 (8)f 80.5 12.0 (4)f 0.10 (12) 2.28 0.96 (4)i
132
S.G. Solaiman et al. / Small Ruminant Research 41 (2001) 127±139
(P < 0:0001) in HC 600 goats when compared to BD and it ranged from 39.1 to 39.58C for the rest of the animals, which was similar to other reports (38.9± 39.418C) (Brooks et al., 1984; Sleiman and Abi-Saab, 1998; Kasa et al., 1999) for normal goats. Genotype, ambient temperature, and level of activity can also in¯uence RT in goats (Sleiman and Abi-Saab, 1998; Kasa et al., 1999). Packed cell volume ranged from 33.3 to 40.3% (Table 3) which falls within the normal range of 24±48%, reported by Brooks et al. (1984) and Jain (1986), however, it was higher than 27.25% reported by Azab and Abdel-Massoud (1999) for Baladi female goats. Supplemental Cu (up to 600 mg per day) did not affect (P > 0:05) PCV; however, HC 100 goats had lower (P < 0:003) PCV, in phase I (when animals were exposed to high Cu intake initially), compared to BD goats, and it recovered as animals adjusted to high Cu intake. Packed cell volume (%) was unchanged when sheep received 9.1 or 37.3 mg Cu/kg of diet (Buckley and Tate, 1981), however, PCVof 10.5% was observed in Angora kids suffering from Cu toxicosis (Humphries et al., 1987). Ewes fed 20 mg/kg Cu for 73 days had lower PCV, red blood cells and whole blood hemoglobin, than those fed 10 or 30 mg/kg Cu diets in a study reported by Ecckert et al. (1999). In another study (Adams et al., 1977) with Nubian goats, PCV was decreased when goats received oral doses of Cu sulfate at 20, 40, and 80 mg/kg diet for 144 days. Pack cell volume, only in HC goats, was negatively correlated (r 0:31, P < 0:02) with RR and positively correlated (r 0:35, P < 0:01) with HR. The mean leukocyte count for BD goats was 14:01 0:71 103 /ml (Table 3) which falls within the normal range ( 5 14 106 /ml) reported by Brooks et al. (1984) and the 6:9 18:18 106 /ml range, in a literature review by Jain (1986). White blood cell count did not change when ewes were fed 10, 20 or 30 mg/kg Cu in the diet for 73 days (Ecckert et al., 1999). Leukocyte count increased (21:17 106 / ml, P < 0:02) and exceeded the normal upper limit reported for goats, in HC 600 goats when compared to BD goats. Leukocytosis is usually a consequence of an increase in the total number of circulating neutrophils, although in some circumstances other cell types may also be increased. Absolute values for neutrophils and lymphocytes were increased with no changes in leukocyte differential counts in HC 600 goats (Table 3).
Goats in both MC 50 and HC 100, initially, had higher neutrophil:lymphocyte ratio (70.7:23.7, P < 0:003; 77.5:20.8, P < 0:003, respectively) as compared to control (BD) (38.8:58.2), however, the ratio reached that of control goats as the animals' body adjusted to Cu load. Non-infectious diseases resulting in leukocytosis or neutrophilia are those that stimulate a stress reaction. Included in this category are metabolic disturbances, drugs and toxic chemicals. Tissue destruction regardless of its cause will produce an increase in number of circulating neutrophils. Increased absolute number of lymphocytes may be an indication of chronic in¯ammatory effect associated with Cu toxicosis. It was concluded that Cu supplementation at 600 mg per head per day in young Nubian does, had no effect on RR, HR and decreased RT. Packed cell volume (%) temporarily decreased, within the normal range, with initial Cu dose of 100 mg Cu per day. Cu supplementation at 600 mg per day increased (P < 0:02) total leukocytes and stimulated stress reaction in goats with no change in neutrophil:lymphocyte ratio, however this ratio temporarily increased when goats were introduced to Cu in phase I of the study. Leukocyte counts were positively correlated with Cu supplementation (r 0:296, P < 0:02) and negatively correlated (r 0:254, P < 0:05) with rectal temperature in the HC group. 3.2. Plasma enzymes Mean plasma SDH value in goats fed the basal diet (Table 3) was 18:7 0:50 U/l with a CV of 17.3% which falls within the normal range for goats (9.3± 20.7 and 14±23.6 U/l) reported by Boyd (1984) and Brooks et al. (1984), respectively. Feeding supplemental Cu at the rate of 50, 100 or 150 mg per day did not effect (P > 0:05) plasma SDH compared to BD goats, however, plasma SDH was increased (P < 0:05) when goats were fed 300 and 600 mg Cu per day. Elevated SDH values (1.5 times normal) would be an indication of hepatic damage (Bartholomew et al., 1987). The mean plasma SDH for the MC 600 (28:0 13:98 U/l) and HC 1200 (29:7 17:4 U/ l) were 42% higher than the BD goats (18:7 0:5 U/l) but not signi®cant. Goats severely affected by Cu supplementation (MC 600 and HC 1200) tended to exhibit high SDH values followed by abnormally low
S.G. Solaiman et al. / Small Ruminant Research 41 (2001) 127±139
133
Fig. 1. Plasma sorbitol dehydrogenase (SDH) as affected by high Cu (HC) supplementation in goats.
SDH values which is an indication of hemolytic crisis and liver damage in these goats. Elevations in SDH values are transitory and unless samplings are made more frequently during the clinical elocution of hemolytic crisis, they may not be detected (Howell and Gooneratne, 1987). Observed changes (Fig. 1) resulted in lower mean values and a high degree of variation, as is indicated by S.E. of these means. A similar response was also noted in some other studies (Wiesner et al., 1965; Asada and Galambos, 1963), where, SDH values were suppressed or near normal during obstructive jaundice in sheep. Schee et al. (1983) concluded that the activity of the enzyme SDH can be considered as an indicator of high Cu
status of sheep. The increases observed in SDH of goats in the MC and HC groups were not as high as those reported by Shaw (1974) for sheep with liver damage (178 and 395 U/l). However, the amount of plasma SDH has been reported to be a sensitive test for detecting minor hepatic damage and an increase of 1.5 times or more, directly related to hepatic damage (Bartholomew et al., 1987). The observed SDH values for the MC group were positively correlated (r 0:337, P < 0:05) with Cu dosage rate (Table 4), however, similar relationships did not exist for the HC group. Mean plasma GOT in BD goats was 46:0 2:82 U/l, (Table 3) which falls within the normal range
Table 4 Correlation coef®cients for level of Cu, plasma Cu and plasma enzymes in medium (MC) and high Cu (HC) supplemented growing Nubian female goats Cu level
Sorbitol dehydrogenase
Glutamic oxaloacetic transferase
g-Glutamyl transferase
Plasma Cu
MC goats Cu supplement Plasma Cu
0.337* 0.093
0.385** 0.145
0.087 0.311
0.030 ±
HC goats Cu supplement Plasma Cu
0.195 0.281
0.549*** 0.635***
0.700*** 0.515***
0.486*** ±
*
P < 0:05. P < 0:01. *** P < 0:002. **
134
S.G. Solaiman et al. / Small Ruminant Research 41 (2001) 127±139
(43±230 U/l) reported for goats (Brooks et al., 1984; Boyd, 1984). The amount of variation in GOT estimates was high (CV 39%). Others (Boyd, 1984; Brooks et al., 1984) have also found higher analytical and biological ranges (66±230 and 43±132 U/l, respectively) and variation (Boyd, 1984; Jenkins et al., 1982) in GOT estimates compared to other liver speci®c enzymes (CV 1:6±2.7) and CV 1:4± 3.3) those of SDH and GGT, respectively). Differences in individual animal responses to higher Cu dosage also have contributed to high variation in GOT values. Animals in the MC 600 group tended to have elevated GOT (1.8 BD). Goats fed 600 mg and 1200 mg Cu per day, in the HC group, had mean values of GOT ((3.6±5.6) GOT in BD goats) higher (P < 0:05) than BD goats. These changes were comparable to observed changes in cattle, considered to have moderate to severe hepatic damage (Bartholomew et al., 1987). Determination of serum GOT has been proposed as a means of early detection of Cu toxicity as well as monitoring recovery in sheep (Davis and Mertz, 1987; Hidiroglou et al., 1984; Buckley and Tate, 1981), however, elevated levels of GOT were associated with apparent clinical signs of Cu toxicosis in the present study. Plasma GOT was positively correlated (r 0:385, P < 0:01) with the Cu dose for the MC group, and (r 0:549, P < 0:002) HC group (Table 4). Mean plasma g-glutamyl transferase in BD goats (Table 3) was 21:2 0:82 U/l with CV 24%, which falls within the normal range (20±50 U/l, CV 21%) reported by Boyd (1984). Contrary to the results observed for SDH and GOT, feeding goats 50 mg Cu per day or more, resulted in an increased plasma GGT as compared to BD goats. We cannot explain mean plasma GGT observed for the MC 600 group. Different responses between MC 600 and HC 600 group may indicate accumulative effects of Cu on this enzyme. The changes noted in GGT as compared to the BD group were not as great as those reported for cattle with hepatic damage (Bartholomew et al., 1987). Mean plasma GGT was positively correlated (r 0:70, P < 0:002) with Cu dosage in the HC group only (Table 4). Different responses in the MC and HC group may be an indication of accumulative effects of Cu on this enzyme. Data reported in literature for plasma SDH, GOT and GGT for goats (Nubian) are very limited. It can be
concluded that mean plasma SDH, GOT and GGT for control goats were similar to those reported for normal goats and estimates of mean plasma GOT were associated with higher biological and analytical variability compared to the other two enzymes measured in control goats. Goats exhibited elevated plasma SDH when fed 300 mg Cu per day, with yet no signs of apparent Cu toxicity, thus, SDH may serve as an early indicator of Cu toxicosis in goats. Individual animal variation in response to Cu dosage affected variability of mean SDH. Affected animals (especially in the MC 600, HC 600 and 1200 groups) exhibited sharp increases in SDH followed by abnormally low values (clinically silent hemolytic crisis), which contributed to data variation. Plasma GOT was elevated when goats received 600 mg Cu per day and at this time they were showing apparent signs of Cu toxicity. Plasma GGT responding immediately to initial doses of Cu may be more a response to initial stress on liver cells. Accumulative effect of Cu on GGT enzyme was apparent and all enzymes were elevated at high levels of Cu intake. 3.3. Plasma copper Mean plasma Cu measured for 35 weeks for BD goats (Table 3) was 1:15 0:04 which falls within the normal range (0.94±1.34 mg/l) reported for goats by others (Galbraith et al., 1997; Suarez et al., 1958), and reported for all animals (0.5±1.5 mg/l) by Davis and Mertz (1987). Plasma Cu was maintained within the normal range for all goats until the last 4 weeks of study when animals in the HC 1200 group had higher (P < 0:05) plasma Cu than the MC and BD goats, had developed clinical signs of Cu toxicity (de®ned earlier) and had high levels of SDH, GOT and GGT. Therefore, estimation of plasma Cu level is of limited value for diagnosing Cu loading or Cu poisoning in goats. Our ®ndings are similar to those reported by others for sheep (Howell and Gooneratne, 1987; Buckley and Tate, 1981; Ecckert et al., 1999). Plasma Cu was independent of Cu supplementation except at very high levels (>600 mg Cu per day, HC goats only), and it was positively correlated with GOT (r 0:635, P < 0:002) and GGT (r 0:515, P < 0:002) when Cu supplementation increased from 100 to 1200 mg per day (HC goats only, Table 4).
S.G. Solaiman et al. / Small Ruminant Research 41 (2001) 127±139
135
Table 5 Effects of medium Cu and high Cu supplement on BWa and feed ef®ciency in growing Nubian female goats (mean S:E:) Treatment groupb
Item
P
Basal diet Weeks 0±9 Mean BW (kg) Mean ADG (kg) Gain:feed
b
High Cu
Supplemental Cu (mg per head per day) 0 ppm 50 ppm 36.99 0.76 a 34.06 0.35 b 0.089 0.004 0.085 0.004 0.091 0.025 0.110 0.014
Weeks 10±23 Mean BW (kg) Mean ADG (kg) Gain:feed a
Medium Cu
0 ppm 42.31 1.33 ab 0.133 0.004 0.120 0.003
150 ppm 40.48 0.72 b 0.124 0.016 0.114 0.0
100 ppm 33.57 0.67 b 0.114 0.008 0.124 0.008
0.002
300 ppm 43.04 0.61 a 0.144 0.008 0.127 0.019
0.004
Due to BW loss associated with >300 mg per day Cu intake, data were reported for 23 weeks only. LS means within a row with different letters differ.
3.4. Growth performance Mean BW, ADG and gain:feed ratio of goats fed different levels of Cu from 0 to 23 weeks (phases I and II) are presented in Table 5. During phase I (weeks 0± 9), goats receiving 50 and 100 mg Cu per day, had a lower (P < 0:004) mean BW (for 9 weeks) than BD goats. In phase II (weeks 10±23), goats in MC 150 and HC 300 group, had mean BW (for 14 weeks) similar (P > 0:05) to BD goats, however, mean BW of HC 300 was higher (P < 0:05) than those of MC 150. When Cu intake increased to 600 or 1200 mg per head per day in phases III and IV, the animals in the HC group lost weight compared to MC and BD, therefore, data reported are for 23 weeks only. During phase I, Cu supplementation at 100 mg per head per day resulted in 28% improvement in mean ADG compared to BD (0.114 and 0.089 kg per head per day for HC 100 and BD, respectively). Since animals received 1 kg grain mix per head per day (with ad libitum hay intake) throughout the study, improvement in mean
ADG (28%) was used to estimate improved ef®ciency of gain:feed ratio. Even though number of animals were limited, growth rate determined by regression analysis of BW (Table 6), recorded over 23 weeks (phases I and II) for HC goats (b 1:096 0:082), also resulted in an improvement of 66% when compared to BD (b 0:660 0:116). Stimulatory effects of Cu on ADG and gain:feed ratio has also been reported in cattle (Gengelbach et al., 1994; Ward and Spears, 1997), swine (Braude, 1967; Edmonds et al., 1985; Cromwell, 1991, 1997; Cromwell et al., 1998) and poultry (Pesti and Bakalli, 1996). However, Luginbuhl et al. (2000) reported that feed intake, ADG or feed ef®ciency were not affected in growing meat goats fed 0, 10 or 30 mg/kg, supplemental Cu in the diet for 87 days which are lower than supplemental Cu level (100±300 mg per head per day) considered as growth stimulant in the present study. Therefore, results of this experiment warrant further studies on the role of Cu as a growth stimulant in goats.
Table 6 Regression coef®cients of BW as affected by Cu supplement for weeks 0±23, in growing female Nubian goats Cu supplement (mg per head per day) Basal diet only (0) Mediuma Cu (50±150) Highb Cu (100±300) a b
Linear Linear Linear
Goats were fed 50 mg Cu for 9 weeks, then 150 mg Cu for 14 weeks. Goats were fed 100 mg Cu for 9 weeks, then 300 mg Cu for 14 weeks.
b S.E.
P
R2 (%)
0.66 0.116 0.74 0.068 1.10 0.082
0.0001 0.0001 0.0001
56.6 81.5 87.2
136
S.G. Solaiman et al. / Small Ruminant Research 41 (2001) 127±139
3.5. Tissue copper residues Tissue Cu residue (TCR) for different organs and ¯uids are presented in Table 7. The liver is the central organ in Cu metabolism, by removing ionic Cu from blood, disposing a portion into bile, synthesizing ceruloplasmin and storing the remainder. Hepatic Cu of 315 15 for BD goats was within the range of 157±590 ppm or mean of 300 ppm (DM basis), reported by Beck (1956) for goats. TCR in control goats (BD) tended to be lowest in fat, rumen ¯uid, and bile (<1 ppm); followed by reticulum, lungs, ovary, muscle, hair and abomasal content (1±3 ppm); oma-
sum, abomasum, jejunum, ileum, heart, spleen and uterus (3±5 ppm); rumen, colon, and brain (8± 10 ppm); duodenum, and feces (10±20 ppm) and it was highest in liver (230±330 ppm). According to NAS (1977), Cu concentration of the whole human body on average is 2 ppm. Highest concentrations are found in the liver and brain, with lesser amounts in the heart, and spleen, which agreed with our ®ndings for goat. Cu contents of different segments of the alimentary tract (abomasum, duodenum, jejunum, ileum, and colon), spleen, uterus, brain, fat, bile, and muscle reported in our study were within the range reported for human or other mammalian tissues, however, Cu
Table 7 Effects of medium Cu (MC) and high Cu (HC) supplement (0±35 weeks) on tissue Cu concentration (mg/kg DM) in growing Nubian female goats (mean S:E:)a Tissues
Accumulated Cu supplementation (g) BD (0.0) (ppm)
Digestive tract Rumen Reticulum Omasum Abomasum Duodenum Jejunum Ileum Colon Some internal organs Brain Heart Lung Liver Fetus liver Spleen Ovary Uterus Other tissues and fluids Muscle Fat Hair Rumen fluid Abomasal content Feces Bile Blood a
MC (7.35) (ppm)
HC (14.70) (ppm) 47.7* 142.6** 153.2** 4.4 1.0 7.8 1.3 10.6*
8.8 2.4 3.1 4.5 13.7 4.8 4.4 8.5
4.40 0.05 0.40 1.90 a 7.30 1.60 1.25 4.80
12.0 7.0 8.1 9.5 13.9 8.2 5.4 9.8
3.90 0.90 2.70 0.85 a 6.60 1.30 0.0 6.40
419.6 356.6 474.9 20.0 14.3 37.5 5.8 90.1
403.0 346.8 462.3 0.0 b 0.0 31.2 0.0 76.4
8.3 4.2 2.6 315.0 240.0 3.2 2.6 4.6
0.10 0.40 0.65 15.0 a 10.0 0.80 0.15 1.50
7.7 6.0 3.7 890.0 230.0 1.6 5.4 4.9
0.60 0.80 0.50 100 b 0.0 0.35 2.30 2.45
7.5 14.4 16.8 1100.0 590.0 17.3 5.6 8.1
0.85 9.50 12.80 100.0 b 0.0 13.85 4.20 4.30
0.9 3.4 6.3 3.5 2.4 5.4 2.1 1.8
1.8 0.2 2.1 0.3 1.2 20.7 0.5 1.1
0.05 0.02 0.35 0.0 0.05 2.20 a 0.05 a 0.04 a
2.2 0.3 1.8 37.3 59.5 187.2 0.4 1.1
0.15 0.0 0.15 8.10 30.0 59.9 ab 0.13 a 0.05 a
3.2 0.3 2.9 537.1 3805.0 398.1 16.1 2.3
0.7 0.0 1.2 438.0 2500 137.4 b 12.1 b 0.96 b
1.7 1.5 1.4 1790.3*** 3044.0*** 19.2* 29.4* 2.0
LS means within a row with different letters differ (P < 0:05). >10-fold increase. ** >100-fold increase. *** >1000-fold increase. *
HC/BD
S.G. Solaiman et al. / Small Ruminant Research 41 (2001) 127±139
values for heart, lungs, ovary and hair were lower (Forssen, 1972; Tipton and Cook, 1963; Schroeder et al., 1966; Mills and Williams, 1962; Bingley and Dufty, 1972). Data on ruminant tissues, especially goats, is not widely available in the literature. Level of Cu increased (P < 0:05) in feces and bile for the HC group when compared to BD, probably due to increased Cu intake. In all species, a high proportion of ingested Cu appears in feces (Davis and Mertz, 1987), and the billiary system is recognized as the principal excretory pathway of absorbed Cu. However, in sheep, increasing Cu concentration in the diet does not appear to increase the excretion of Cu in bile (Howell and Gooneratne, 1987; Ecckert et al., 1999). Tissue Cu tended to increase in rumen, reticulum and omasum by 48-, 143- and 153-fold, respectively, with the highest increases found in rumen ¯uid and rumen and abomasal contents (>1000-fold) in the HC group (Table 7). Liver and abomasum Cu were higher (P < 0:05) in the HC than BD group. The main storage tissue for Cu is the liver, especially when the animal is loaded with Cu, although appreciable amounts of Cu accumulate in other tissues. No differences (P > 0:10) in TCR were observed between BD and MC groups except for liver Cu. Due to low numbers of experimental animals per treatment, differences were not signi®cant statistically. However, TCR tended to increase when Cu supplementation exceeded 600 mg per head per day. All goats were con®rmed pregnant during weeks of 23±25, however, only four out of six goats, all BD goats, one-half of MC and one-half HC goats remained pregnant. Fetal liver Cu at the termination of the study was 240, 230 and 590 ppm, (DM basis) for the BD, MC and HC groups, respectively. Goats in the MC group consumed a total of 7.35 g Cu for the 35week experiment, accumulated higher (P < 0:05) Cu in the liver (890 ppm, DM basis), with no change in fetal liver Cu. However, when Cu intake was 14.7 g for 35 weeks (HC), fetal Cu increased 2.45-fold (590 ppm, DM basis). The level of Cu in hair has been studied as an indicator of the Cu status in animals (Underwood, 1977). In the present study, random hair samples were collected every 4 weeks throughout the study. Hair Cu content ranged from 1.81 to 2.9 ppm for all animals, which agreed with values (2±4 ppm Cu) reported by Bingley (1974) for sheep. Cunningham and Hogan
137
(1958) reported higher values (8.3±13.3 ppm Cu) in New Zealand sheep and they found no relationship between dietary Cu and hair Cu content. There was no correlation between hair Cu and serum Cu in 800 hair samples quoted by Mertz (1987). No relationship between Cu intake and hair Cu was found in the present study. 4. Conclusions Vital signs and hematological parameters such as PCV, white blood cell counts, and differential white blood cell counts showed little variation with feeding high doses of Cu. Other biochemical changes occurring in blood, as a result of feeding high levels of Cu, appeared to be directly related to a degree of liver injury. One of the earliest biochemical changes observed in the prehemolytic period was an elevation in the activities of liver speci®c enzymes such as SDH. Plasma enzymes were in¯uenced by Cu supplementation to a greater extent than plasma Cu. Of the plasma enzymes measured, only GGT exhibited consistent elevations related to Cu intake. Cu levels as low as 50 mg per head per day resulted in elevated GGT. Plasma SDH was not changed until the amount of Cu supplement exceeded 300 mg per head per day. Supplemental Cu below 600 mg per head per day did not in¯uence GOT. Comparing data of this study with published data for sheep, indicates that goats (Nubian) are more resistant to Cu toxicity than sheep and this ®nding agrees with others (Adams et al., 1977; Soli and Nafstad, 1978) who also found that goats are less susceptible than sheep to repeated administration of Cu. Limited data on growth performance supported the stimulatory effect of 100±300 ppm Cu in the diet of Nubian goats. Extra Cu accumulated in the liver and to a lesser extent in other tissues, and was excreted through the billiary system (bile) and GI tract (feces). Hair Cu was not indicative of Cu status in Nubian goats. This study is a preliminary work to determine Cu requirement and toxicity in goats. Further investigations are needed with larger number of animals to recon®rm these ®ndings. Results of this study warrant further investigation on the role of Cu as a growth promoting factor in goats and on the ®nding that goats are not only more tolerant to high Cu level in the diet but actually require higher Cu in the diet than sheep.
138
S.G. Solaiman et al. / Small Ruminant Research 41 (2001) 127±139
Acknowledgements The authors wish to thank Mr. Joseph Sparks for his invaluable assistance throughout the study. This experiment was supported in part by USDA/CREES grant. Mention of trade names or commercial products is solely for providing speci®c information. References Adams, S.E.I., Was®, I.A., Magzoub, M., 1977. Chronic copper toxicity in Nubian goats. J. Comp. Pathol. 87, 623±627. AFRC, 1997. The nutrition of goats. Technical Committee on Responses to Nutrients, Report 10. Agricultural Food Research Council. Nutr. Abstr. Rev. (Series D) 67, 806±815. AOAC, 1984. Of®cial Methods of Analysis, 14th Edition, Association of Of®cial Analytical Chemists, Arlington, VA, pp. 152±169. Asada, M., Galambos, J.T., 1963. Sorbitol dehydrogenase and hepatocellular injury: an experimental and clinical study. Gastroenterology 44, 578±587. Azab, M.E., Abdel-Massoud, H.A., 1999. Changes in some hematological and biochemical parameters during prepartum and postpartum periods in female Baladi goats. Small Rumin. Res. 34, 77±89. Bartholomew, M.L., Willett, L.B., Liu, T.-T.Y., Morehead, P.D., 1987. Changes in hepatic function test to induce toxicity in bovine liver. J. Anim. Sci. 64, 201±209. Beck, A.B., 1956. The copper content of the liver and blood of some vertebrates. Aust. J. Zool. 4, 1±18. Bingley, J.B., 1974. Effects of high doses of molybdenum and sulphate on the distribution of copper in plasma and blood of sheep. Aust. J. Agric. Res. 25, 467±474. Bingley, J.B., Dufty, J.H., 1972. Distribution of copper in the tissues of the bovine neonate and dam. Res. Vet. Sci. 13, 8±14. Boyd, J.W., 1984. The interpretation of serum biochemistry test results in domestic animals. Vet. Clin. Pathol. 13, 7±14. Bradley, C.H., 1993. Copper poisoning in a dairy herd fed a mineral supplement. Can. Vet. J. 34, 287±292. Braude, R., 1967. Copper as a stimulant in pig feeding (Cuprum propecunia). World Rev. Anim. Prod. 3, 69±81. Brooks, D.L., Tillman, P.C., Niemi, S.M., 1984. Ungulates as laboratory animals. In: Fox, Cohen, Loew (Eds.), Laboratory Animal Medicine. Academic Press, New York, pp. 273±295. Buckley, W.T., Tate, R.M., 1981. Chronic copper toxicity in lambs: a survey of blood constituent responses. Can. J. Anim. Sci. 61, 613±624. Cromwell, G.L., 1991. Antimicrobial agents. In: Miller, E.R., Ullrey, D.E., Lewis, A.J. (Eds.), Swine Nutrition. ButterworthHeinemann, Stoneham, MA, pp. 297±314. Cromwell, G.L., 1997. Copper as a nutrient for animals. In: Richardson, H.W. (Ed.), Handbook of Copper Compounds and Applications. Marcel Dekker, New York, pp. 177±202. Cromwell, G.L., Lindemann, M.D., Monegue, H.J., Hall, D.D., Orr
Jr., D.E., 1998. Tribasic copper chloride and copper sulfate as copper sources for weanling pigs. J. Anim. Sci. 76, 118±123. Cunningham, I.J., Hogan, K.G., 1958. The in¯uence of diet on the copper and molybdenum contents of hair, hoof and wool. N. Z. J. Agri. Res. 1, 841±846. Davis, G.K., Mertz, W., 1987. Copper. In: Mertz, W. (Ed.), Trace Elements in Human and Animal Nutrition, 5th Edition. Academic Press, New York, pp. 301±350. Ecckert, G.E., Green, L.W., Carstens, G.E., Ramsey, W.S., 1999. Copper status of ewes fed increasing amounts of copper from copper sulfate or copper proteinate. J. Anim. Sci. 77, 244±249. Edmonds, M.S., Izquierdo, O.A., Baker, D.H., 1985. Feed additive studies with newly weaned pigs: ef®cacy of supplemental copper, antibiotics and organic acids. J. Anim. Sci. 60, 462± 469. Engle, T.E., Spears, J.W., 2000. Effects of dietary copper concentration and source on performance and copper status of growing and ®nishing steers. J. Anim. Sci. 78, 2446±2451. Forssen, A., 1972. Inorganic elements in the human body. 1. Occurrence of Ba, Br, Ca, Cd, Cs, Cu, K, Mn, Ni, Sn, Sr, Y, and Zn in the human body. Ann. Med. Exp. Biol. 50, 99±162. Galbraith, H., Chigwada, W., Scaife, J.R., Humphries, W.R., 1997. The effect of dietary molybdenum supplementation on tissue copper concentrations, mohair ®ber and carcass characteristics of growing Angora goats. Anim. Feed Sci. Technol. 67, 83±90. Gengelbach, G.P., Ward, J.D., Spears, J.W., 1994. Effect of dietary copper, iron, and molybdenum on growth and copper status of beef cows and calves. J. Anim. Sci. 72, 2722±2727. Haenlein, G.F.W., 1992. Advances in the nutrition of macro- and micro-elements in goats. In: Proceedings of 5th ICG. New Delhi, India, pp. 933±950. Hidiroglou, M., Heaney, D.P., Hartin, K.E., 1984. Copper poisoning in a ¯ock of sheep. Copper excretion patterns after treatment with molybdenum and sulfur or penicillamine. Can. Vet. J. 25, 377±382. Howell, J.McC., Gawthorne, J.M., 1987. Copper in Animals and Men, Vol. II. CRC Press, Boca Raton, FL. Howell, J.McC., Gooneratne, S.R., 1987. The pathology of copper toxicity in animals. In: Howell, J.McC., Gawthorne, J.M. (Eds.), Copper in Animals and Men, Vol. II. CRC Press, Boca Raton, FL, pp. 54±71. Howell, J.McC., Gopinath, C., 1977. Copper toxicity in sheep: the effect of repeated inter-venous injections of copper sulphate. Res. Vet. Sci. 22, 86±94. Humphries, W.R., Morrice, P.C., Mitchell, A.N., 1987. Copper poisoning in Angora goats. Vet. Rec. 121, 231. Ishmael, J., Gopinath, C., Howel, J.McC., 1971. Experimental chronic copper toxicity in sheep. Histological and histochemical changes during the development of the lesions in the liver. Res. Vet. Sci. 12, 358±366. Ishmeal, J., Gopinath, C., Howell, J.McC., 1972. Experimental chronic copper toxicity in sheep. Biochemical and hematological studies during the development of lesions in the liver. Res. In Vet. Sci. 13, 22±29. Jain, N.C., 1986. Schalm's Veterinary Hematology, 4th Edition. Lea & Febiger, Philadelphia, pp. 20±80. Jenkins, S.J., Green, S.A., Clark, P.A., 1982. Clinical chemistry
S.G. Solaiman et al. / Small Ruminant Research 41 (2001) 127±139 reference values of normal domestic animals in various age groups as determination on the ABA-100. Cornell Vet. 72, 403± 415. Kasa, I.W., Thwaites, C.J., Hill, M.K., 1999. Effects of treadmill exercise on sweating in three breeds of goats. Aust. Vet. J. 77, 660±661. Kessler, J., 1991. Mineral nutrition of goat. In: Morand-Fehr, P. (Ed.), Goat Nutrition. EAAP, Pudoc., pp. 104±119. Luginbuhl, J.M., Poore, M.H., Spears, J.W., Brown, T.T., 2000. Effect of dietary copper level on performance and copper status of growing meat goats. Sheep Goat Res. J. 16, 65±71. Mertz, W., 1987. Trace Elements in Human and Animal Nutrition, 5th Edition. Academic Press, Orlando, FL, pp. 301±350. Meschy, F., 2000. Recent progress in the assessment of mineral requirements of goats. Livestock Prod. Sci. 64, 9±14. Mills, C.F., Williams, R.B., 1962. Copper concentration and cytochrome-oxidase and ribonuclease activities in the brains of copper-de®cient lambs. Biochem. J. 85, 629±632. NAS, 1977. Copper: Medical and Biological Effects of Environmental Pollutants, National Academy of Science, Washington, DC, pp. 14±27. NRC, 1980. Mineral Tolerances of Domestic Animals. National Research Council, National Academy of Science, Washington, DC. NRC, 1981. Nutrient Requirements of Domestic Animals. No. 15. Nutrient Requirements of Goats: Angora, Dairy and Meat Goats in Temperate and Tropical Countries. National Research Council, National Academy of Science, Washington, DC, pp. 10±12. Pesti, M.G., Bakalli, R.I., 1996. Studies on the feeding of cupric sulfate pentahydrate and cupric citrate to broiler chickens. Poult. Sci. 75, 1086±1091. SAS, 1990. SAS User's Guide: Statistics, Version 6, 4th Edition. SAS Institute Inc., Cary, NC.
139
van der Schee, W, Schotamn, A.J.H., van der Berg, R., 1983. Levels of enzyme activities in blood serum as indicators of a high copper status in sheep ¯ocks. Zentralbl. Veterinarmed. A 30, 664±673. Schroeder, H.A., Nason, A.P., Tipton, I.H., Balassa, J.T., 1966. Essential trace minerals in man: copper. J. Chron. Dis. 19, 1007±1034. Shaw, F.D., 1974. Sorbitol dehydrogenase in the diagnosis of liver disease of ruminants. Aust. Vet. J. 50, 279±280. Sleiman, F.T., Abi-Saab, S., 1998. In¯uence of environment on respiration, heart rate and body temperature of Filial crosses compared to local Awassi sheep. Small Rumin. Res. 16, 49±53. Soli, N.E., Nafstad, I., 1978. Effect of daily oral administration of copper to goats. Acta Vet. Scand. 19, 561±568. Suarez, R.M., DeBerrocal, H.F., DeOlavarrieta, S.T., Suarez Jr., R.M., Buso, R., Sabater, J., 1958. Studies on blood copper in normal and pathological states. Biol. Assoc. Med. Puerto Rico 50, 1±10. Tipton, I.H., Cook, M.J., 1963. Trace elements in human tissue. Part II. Adult subjects from the United States. Health Phys. 9, 103±145. Underwood, E.J., 1977. Trace Elements in Human and Animal Nutrition, 4th Edition, Academic Press, New York, pp. 56±108. Ward, J.D., Spears, J.W., 1997. Long term effects of consumption of low copper diets with or without supplemental molybdenum on copper status, performance, and carcass characteristics of cattle. J. Anim. Sci. 75, 3057±3065. Wiesner, I.S., Rawnsley, M.S., Brook, F.P., Senior, J.R., 1965. Sorbitol dehydrogenase in the diagnosis of liver disease. Am. J. Digestive Dis. 10, 147±151. Zervas, G., Nikoloay, E., Manzios, A., 1989. Comparative study of chronic copper poisoning in lambs and young goats. In: Proceedings of 6th International Trace Element Symposium. University Leipzig-Jena, Germany, pp. 569±581.
Effects of high copper supplements on performance, health, plasma copper and enzymes in goats S.G. Solaiman*, M.A. Maloney, M.A. Qureshi, G. Davis, G. D'Andrea1 Department of Agricultural Sciences, George Washington Carver Agricultural Experiment Station, Tuskegee University, 105 Milbank Hall, Tuskegee, AL 36088, USA Accepted 21 May 2001
Abstract Six growing female Nubian goats (average BW 34:8 0:55 kg, 7±8 months of age) were randomly assigned to either a basal diet (BD, 10±15 ppm Cu/DM), or to medium Cu (MC, BD 50 mg Cu), or to high Cu (HC, BD 100 mg Cu) diets for 9 weeks. This level would cause Cu toxicity in sheep, but none occurred in the goats. Therefore, Cu supplementation was then increased to 150 and 300 mg per head per day, for the following 14 weeks; to 300 and 600 mg per head per day, for the next 8 weeks; and to 600 and 1200 mg per head per day, for an additional 4 weeks, in the MC and HC group, respectively. Body weight and vital signs were recorded and blood samples collected at different time intervals. Hematological parameters, plasma Cu, sorbitol dehydrogenase (SDH), glutamic oxaloacetic transaminase (GOT), and g-glutamyl transferase (GGT) were determined. At the termination of the study, tissue Cu concentration in different organs was also determined. During ®rst 23 weeks (<300 mg Cu per day) of the study there were no apparent signs of Cu toxicity. Cu supplementation at 600 mg per head per day in young Nubian does, had no effect on respiration rate (RR), heart rate (HR), and decreased (P < 0:05) rectal temperature (RT) in the HC group only. Leukocyte counts were positively correlated with Cu supplementation (r 0:296, P < 0:02) and negatively correlated (r 0:254, P < 0:05) with RT in the HC group. Plasma SDH increased (P < 0:05) when Cu supplementation was 300 mg per head per day, thus, SDH may serve as an early indicator of Cu toxicosis in goats. Increases (P < 0:05) in GOT were noted when Cu intake was 600 mg per head per day. Contrary to the results observed for SDH and GOT, feeding goats 50 mg Cu per day or more, resulted in an increased plasma GGT as compared to BD goats. Levels of SDH, GOT and GGT of the BD goats were within normal range. Plasma Cu was not indicative of Cu status of animals. Copper improved ADG by 28% at the 100±150 ppm level in diet. No relationship between Cu intake and hair Cu was found in the present study. Highest concentration of Cu was found in liver, followed by duodenum, rumen and brain. Results of this study indicate that goats are more resistant to Cu toxicity than sheep. This is one of the ®rst reports documenting signi®cant differences in Cu requirements and tolerance between goats and sheep. # 2001 Elsevier Science B.V. All rights reserved. Keywords: Copper supplementation; Copper toxicity; Plasma enzymes; Copper in tissues; Goats
1. Introduction *
Corresponding author. Tel.: 1-334-727-8401; fax: 1-334-727-8552. E-mail address: [email protected] (S.G. Solaiman). 1 Present address: State Veterinary Diagnostic Laboratory, Auburn, AL 36830, USA.
Copper is an essential element required by goats and other animals for a number of biochemical functions (Davis and Mertz, 1987). Copper is a normal component of all body tissues and ¯uids, however, ingestion of quantities of Cu slightly higher than
0921-4488/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 1 - 4 4 8 8 ( 0 1 ) 0 0 2 1 3 - 9
128
S.G. Solaiman et al. / Small Ruminant Research 41 (2001) 127±139
required, may cause accumulation within the tissues (Howell and Gawthorne, 1987). The mechanism of Cu uptake and transport through the intestinal wall is not clearly de®ned, however, bile and gastrointestinal (GI) tract are two important routes of Cu excretion (Howell and Gawthorne, 1987; Mertz, 1987). Cu stimulates growth and ADG in swine (Cromwell, 1997) and alters lipid metabolism in steers (Engle and Spears, 2000). From a toxicity standpoint, there is a great variation between species. Clinical toxicity symptoms include hemolytic crises; the animal becomes dull and lethargic, passes soft stool, looses appetite and may have excessive thirst (Ishmael et al., 1971; Howell and Gopinath, 1977). Also there may be marked elevation of liver speci®c enzymes (sorbitol dehydrogenase, SDH; glutamic oxaloacetic transaminase, GOT; g-glutamyl transferase, GGT) in blood (Howell and Gawthorne, 1987; Mertz, 1987; Ishmeal et al., 1972; Schee et al., 1983). Sheep are more sensitive to high Cu supplementation than other agricultural animals (NRC, 1980). Chronic Cu toxicity has also been reported in dairy cows fed 37.5 mg Cu/kg DM during lactation and 22.6 mg Cu/ kg DM during the non lactating period (Bradley, 1993). Searching the literature (Meschy, 2000; Howell and Gawthorne, 1987; Mertz, 1987), there are more reports on Cu de®ciency in goats than on toxicity. This may be an indication that goats can tolerate higher levels of Cu than sheep or cattle. Goats, especially young kids, seem to be less sensitive to Cu intake by having lower hepatic uptake, than lambs, when fed diets containing high levels of Cu (Zervas et al., 1989). The exact amount of Cu required in the goat's diet is currently unknown (Haenlein, 1992) and the current established Cu requirement of goats (8±10 ppm) needs more studies (NRC, 1981; Kessler, 1991; Haenlein, 1992; AFRC, 1997). Signs of Cu toxicity and levels of associated liver speci®c enzymes are not clearly established for goats, therefore, this study was designed to determine some parameters associated with Cu toxicity in goats and to establish some guidelines for feeding Cu to goats. 2. Materials and methods 2.1. Animals and treatments Six growing female Nubian goats (Capra hircus hircus), 7±8 months of age, with average BW of
34:87 0:55 kg were used in this study. Goats were housed at the old dairy barn of Tuskegee University in compliance with Tuskegee University Institutional Animal Care and Use Committee regulations. Each animal was treated for internal parasites (Ivomectin; Meriel, Division of Merck and Co., Rahway, NJ) prior to the start of the experiment. After 2 weeks for adjustment, animals were weighed, randomly assigned to three experimental treatments and housed in three pens (two animals each) with concrete ¯oors covered with old bermudagrass hay (BGH) as bedding. Goats were offered once daily, at 0900, 1 kg, of basal diet (BD; 16.7% CP, 11±15 ppm Cu; Table 1) and free choice bermudagrass hay (7.8% CP, 5±8 ppm Cu), to secure intake of nutrients required for maintenance and 100 g per day growth, in accordance with NRC (1981). Animals had access to water (1.0± 1.5 ppm Cu) at all times and they were not allowed outside the pens except to facilitate the cleaning once a week. Levels of Cu supplement were based on information from sheep and cattle (Howell and Gawthorne, 1987; Mertz, 1987). Initial supplemental levels were 50 mg Cu per head per day (MC 50) and 100 mg Cu per head per day (HC 100), anticipating Cu toxicity within 8±10 weeks, according to the sheep data (Howell and Gawthorne, 1987). Pre-determined amounts of Cu, as Cu sulfate (CuSO45H2O), were placed in gelatin capsules and inserted into the Table 1 Dietary composition of the basal diet used in the experiment Ingredient
%a
Cracked corn Alfalfa meal Whole oats Soybean meal (48% CP) Molasses Masonexb Dicalcium phosphate Limestone Salt Vitamin/mineral mix
43.5 15.0 17.5 16.5 2.5 2.5 0.5 0.5 0.5 1.0
Cu (ppm) Basal diet Bermudagrass hay Water
10.8±15.2 5.8±8.3 1.0±1.5
a b
As fed basis. A commercial pelletizing agent.
S.G. Solaiman et al. / Small Ruminant Research 41 (2001) 127±139
esophagus with a balling gun at the time of feeding. This regiment continued from weeks 0 to 9 (phase I) with no apparent sign of Cu toxicity. Therefore, Cu supplementation was increased to 150 (MC 150) and 300 (HC 300) mg per head per day for the MC 50, and HC 100 groups, respectively, from weeks 10 to 23 (phase II). During this phase of the study, all the animals were healthy, without signs of Cu toxicity, and were checked for estrus, bred by AI, and con®rmed pregnant. During weeks 24±31 (phase III), Cu in the diet of goats in MC 150 and HC 300 group was again increased to 300 (MC 300) and 600 (HC 600) mg per head per day, respectively. Supplemental Cu was increased to 600 (MC 600) and 1200 (HC 1200) mg per head per day for the MC 300 and HC 600 group, respectively, for the last 4 weeks (weeks 32±35, phase IV), because still no toxicity occurred at the 600 mg level. The experiment was terminated when goat no. 174 in the HC 1200 group, exhibited clinical signs of Cu toxicity (Anorexia, dark urine, brown patches on her white hair coat and hemolytic crisis) and died. The dosing regimens and duration are summarized in Table 2. The control group (BD) was fed no supplemental Cu throughout the study. 2.2. General health and clinical observations General condition and reactions of goats to supplemental Cu was observed daily throughout the experiment. Feed intake was monitored and body weights were recorded weekly, for the ®rst 9 weeks and every 2±4 weeks during rest of the experimental period. Clinical evaluation was conducted by a veterinarian prior to dosing, at weekly intervals during the ®rst 9 weeks of the study, and every 2±4 weeks thereafter depending on the general condition of the goats. Vital body signs recorded were respiration rate (RR), heart rate (HR), and rectal temperature (RT).
129
2.3. Blood collection and analysis Blood samples were collected weekly for the ®rst 9 weeks and every 2 weeks thereafter, by jugular venipuncture to measure plasma Cu and enzymes. Blood samples collected into 3 ml vacutainer tubes (Becton Dickinson, Vacutainer System, Franklin Lakes, NJ) containing EDTA, were used to measure packed cell volume (PCV), white blood cells (WBC), neutrophils and lymphocytes. Samples collected in 10 ml vacutainer tubes containing EDTA were used for plasma Cu determination. Blood samples for SDH, GOT and GGT enzyme assays were collected in 10 ml heparinized vacutainer tubes. Blood samples were placed on ice immediately after collection, and transported to the laboratory for further preparation. Packed cell volume was measured following centrifugation in micro-hematocrit capillary tubes with a Hawksley microhematocrit reader (Galbraith et al., 1997). Leukocytes were counted manually with a hemocytometer. Each sample was counted twice and the mean was calculated (Azab and Abdel-Massoud, 1999). Samples for plasma Cu and enzymes were prepared by conventional centrifugation of whole blood at 1000 g. Plasma separated for Cu determination was stored in acid washed (37.5 ml nitric acid/l H2O) containers. Plasma Cu concentrations were determined by diluting plasma 1:4 in deionized water and aspirating into the ¯ame of an atomic absorption spectrophotometer (model 5000, Perkin-Elmer, Norwalk, CT). Liver speci®c enzymes, SDH (Kit # 50-UV, Sigma Chemical Co., St. Louis, MO), GOT (Kit # 505, Sigma Chemical Co.) and GGT (Kit # 545, Sigma Chemical Co.) were assayed in plasma according to procedures outlined in respective commercial kits using a spectrophotometer (Beckman DU-7) set at 340-UV, 500 and 540 nm wavelength, respectively.
Table 2 Dosing regimen for medium Cu (MC) and high Cu supplemented goats Phase
Weeks
Duration (weeks)
MC group (mg Cu per head per day)
HC group (mg Cu per head per day)
I II III IV
0±9 10±23 24±31 32±35
9 14 8 4
50 150 300 600
100 300 600 1200
130
S.G. Solaiman et al. / Small Ruminant Research 41 (2001) 127±139
An ovine SDH (SDH control, Sigma Chemical) in a bovine serum base was used as a quality control measure. Porcine GOT and GGT (Sigma Enzyme Control 2-N, Sigma Chemical) in a buffered bovine albumin base were used as quality controls for GOT and GGT assays, respectively. All samples for SDH were analyzed within 2 h of collection at 22±258C room temperature. The GOT assay was conducted within 4 h of collection and blood samples used in the GGT assay were stored at 48C with the assay being conducted within 24 h of collection. 2.4. Tissue collection and analysis At the termination of the study, remaining animals were euthanized and different body organs and tissues were collected for Cu analysis. Different tissue samples were dried at 1008C for 48 h, weighed, and 1 g sample DM was ashed in a muf¯e furnace at 5008C overnight. Ashed samples were digested in 10 ml of 25% (w/v) HCl, over a hot plate for 2±3 min, ®ltered through no. 41 Whatman ®lter paper and rinsed with additional 15 ml 25% HCl into 50 ml volumetric ¯asks. To reduce the absorptive interference, 10 ml of 20% (w/v) KCl was added to each volumetric ¯ask and was brought up to volume with deionized water. Fat samples were prepared according to procedures described for oils and fats in AOAC (1984). Cu concentration was determined by aspirating the diluted samples into the ¯ame of an atomic absorption spectrophotometer. Different sets of standard solutions were prepared to cover the expected range of Cu concentration in the samples. 2.5. Statistical analysis Data were analyzed by the GLM procedure of SAS (1990). Week and Cu level interactions were not signi®cant. Therefore, hematological parameters, BW and performance data, plasma Cu and enzymes, were averaged over weeks 0±9, 10±23, 24±31 and 32± 35 and analyzed as a complete randomized design with Cu levels as a source of variation. Mean responses were separated with the least squares means procedure and signi®cant differences were observed at P < 0:05, unless otherwise indicated. Regression analysis of the BW responses to various levels of supplemental Cu was conducted to evaluate growth
performance. Correlation coef®cients of plasma Cu and enzymes responses to various levels of supplemental Cu were also determined. Data are presented as simple means or least squares means with S.E. 3. Results and discussion Analysis for vital signs, hematological and toxicological parameters indicated that the effect of week was not signi®cant. Thus, data were subjected to an evaluation of treatment groups identi®ed as basal diet (BD), MC 50, MC 150, MC 300, MC 600, HC 100, HC 300, HC 600 and HC 1200. The BD values represent 31 or in some cases 35 weeks of data collection, MC 50 and HC 100, MC 150 and HC 300, MC 300 and HC 600, and MC 600 and HC 1200, represent data collected during (0±9) 9 weeks, (10±23) 14 weeks, (23± 31) 8 weeks and (32±35) 4 weeks, respectively. 3.1. General animal health and performance During phases I and II of the study, there were no apparent signs of Cu toxicity. Animals, when receiving 600 mg Cu and more (MC 600, HC 600 and HC 1200), exhibited excessive thirst, diarrhea, dehydration, anorexia, loss of BW and were inactive. Beginning week 33 (phase IV), animal no. 174 (HC 1200), exhibited more signs of Cu toxicity by having pale mucus membranes and a change of hair coat color (brown patches) and ®nally death on week 35, at which time this study was terminated. Mean vital signs, PCV, leukocytes and leukocyte differential counts are reported through phase III or for 31 weeks only (Table 3). RR, HR and RT in goats were not affected (P > 0:05) by feeding supplemental Cu for 31 weeks (Table 3). Mean RR ranged from 42.4 to 49.4 breaths/min, which falls within the normal range (20±76.5 breaths/min) reported by others (Brooks et al., 1984; Sleiman and Abi-Saab, 1998; Kasa et al., 1999). Genotype, sex, age, ambient temperature (Sleiman and Abi-Saab, 1998) and level of activity (Kasa et al., 1999) can affect RR in goats. Mean HR ranged from 116.9 to 132.1 beats/min, falling within the normal range (70±135 beats/min) reported by others (Brooks et al., 1984; Sleiman and Abi-Saab, 1998). Genotype and sex can in¯uence HR in goats (Sleiman and Abi-Saab, 1998). Mean RT was lower
Table 3 Effects of medium copper (MC) and high copper (HC) supplement on vital signsa blood cellsb, plasma enzymesc and Cu in growing Nubian female goats (mean S:E:) Item
BDd (0)
MC (Cu mg per head per day) 50
Duration (weeks) RR (breaths/min) (n) HR (beats/min) (n) RT (8C) (n) PCV (%) (n) WBC (103/ml) (n) Lymphocytes (%) (n) Neutrophils (%) (n) SDH (U/l) (n) GOT (U/l) (n) GGT (U/l) (n) Plasma Cu (mg/l) (n) a
0±35 49.4 132.1 39.1 40.3 14.0 58.2 38.8 18.7 46.0 21.2 1.15
2.23 (36) 3.51 (36) 0.08 (36) 1.13 (36) 0.71 (36) 2.45 (26) 2.55 (26) 0.50 (42) 02.82 (40) 0.82 (38) 0.04 (42)
0±9 46.7 126.0 39.4 35.8 14.6 23.7 70.7 16.5 54.0 33.6 1.23
150
3.65 (9) 5.34 (10) 0.11 (9) 1.78 (9) 1.36 (9) 6.0 (3)e 07.2 (3)h 0.77 (12) 03.85 (12) 2.63 (12)f 0.09 (12)
10±23 47.9 128.6 39.3 42.0 14.5 58.1 37.5 18.8 27.6 29.9 1.22
HC (Cu mg per head per day) 300
4.65 (14) 6.95 (14) 0.10 (14) 2.18 (16) 1.23 (16) 1.64 (12) 2.20 (12) 1.79 (16) 03.15 (14) 1.85 (14)i 0.06 (14)
24±31 44.0 130.4 39.3 38.4 14.1 56.4 40.8 26.7 58.0 38.9 1.39
600 2.92 (6) 11.76 (6) 0.13 (6) 0.68 (5) 1.19 (5) 2.91 (5) 3.18 (5) 4.63 (10)i 11.44 (10) 04.26 (8)f 0.09 (12)
32±35 45.0 145.0 39.1 ±j ± ± ± 28.0 86.5 26.3 1.08
100 15.0 (2) 15.0 (2) 0.0 (2)
14.0 25.2 2.25 0.05
(4) (4) (4) (4)
RR: respiration rate; HR: heart rate; RT: rectal temperature. PCV: packed cell volume; WBC: white blood cells. c SDH: sorbitol dehydrogenase; GOT: glutamic oxaloacetic transferase; GGT: g-glutamyl transferase. d BD: basal diet with no supplemental Cu. e Means within a row are different from BD group at P < 0:02. f Means within a row are different from BD group at P < 0:003. g Means within a row are different from BD group at P < 0:0001. h Means within a row are different from BD group at P < 0:05. i Means within a row are different from BD group at P < 0:002. j ±: Not measured. b
0±9 47.4 124.8 39.5 33.3 13.3 20.8 77.5 17.5 54.7 28.8 1.12
300
6.84 4.84 0.14 0.69 1.37 8.25 9.30 1.05 4.71 2.38 0.09
(7) (10) (10) (10)g (10) (4)e (4)e (12) (12) (12)i (12)
10±23 48.9 116.9 39.1 39.0 15.9 59.9 37.2 28.8 34.0 35.4 1.05
600 3.41 (14) 6.22 (14) 0.06 (14) 1.40 (16) 0.99 (16) 2.62 (12) 2.89 (12) 4.95 (15)i 03.51 (13) 2.41 (14)f 0.05 (14)
24±31 42.4 126.7 38.6 39.5 21.2 55.3 41.7 28.6 167.6 73.1 1.40
1200 32±35 5.88 (5) 46.0 14.0 (2) 11.9 (6) 130.0 30.0 (2) 39.5 0.0 (2) 0.18 (6)e 1.55 (4) ± 8.47 (4)h ± 4.84 (3) ± 6.01 (3) ± 4.93 (10)i 29.7 17.4 (4) 54.43 (10)i 258.5 106.1 (4)i 11.1 (8)f 80.5 12.0 (4)f 0.10 (12) 2.28 0.96 (4)i
132
S.G. Solaiman et al. / Small Ruminant Research 41 (2001) 127±139
(P < 0:0001) in HC 600 goats when compared to BD and it ranged from 39.1 to 39.58C for the rest of the animals, which was similar to other reports (38.9± 39.418C) (Brooks et al., 1984; Sleiman and Abi-Saab, 1998; Kasa et al., 1999) for normal goats. Genotype, ambient temperature, and level of activity can also in¯uence RT in goats (Sleiman and Abi-Saab, 1998; Kasa et al., 1999). Packed cell volume ranged from 33.3 to 40.3% (Table 3) which falls within the normal range of 24±48%, reported by Brooks et al. (1984) and Jain (1986), however, it was higher than 27.25% reported by Azab and Abdel-Massoud (1999) for Baladi female goats. Supplemental Cu (up to 600 mg per day) did not affect (P > 0:05) PCV; however, HC 100 goats had lower (P < 0:003) PCV, in phase I (when animals were exposed to high Cu intake initially), compared to BD goats, and it recovered as animals adjusted to high Cu intake. Packed cell volume (%) was unchanged when sheep received 9.1 or 37.3 mg Cu/kg of diet (Buckley and Tate, 1981), however, PCVof 10.5% was observed in Angora kids suffering from Cu toxicosis (Humphries et al., 1987). Ewes fed 20 mg/kg Cu for 73 days had lower PCV, red blood cells and whole blood hemoglobin, than those fed 10 or 30 mg/kg Cu diets in a study reported by Ecckert et al. (1999). In another study (Adams et al., 1977) with Nubian goats, PCV was decreased when goats received oral doses of Cu sulfate at 20, 40, and 80 mg/kg diet for 144 days. Pack cell volume, only in HC goats, was negatively correlated (r 0:31, P < 0:02) with RR and positively correlated (r 0:35, P < 0:01) with HR. The mean leukocyte count for BD goats was 14:01 0:71 103 /ml (Table 3) which falls within the normal range ( 5 14 106 /ml) reported by Brooks et al. (1984) and the 6:9 18:18 106 /ml range, in a literature review by Jain (1986). White blood cell count did not change when ewes were fed 10, 20 or 30 mg/kg Cu in the diet for 73 days (Ecckert et al., 1999). Leukocyte count increased (21:17 106 / ml, P < 0:02) and exceeded the normal upper limit reported for goats, in HC 600 goats when compared to BD goats. Leukocytosis is usually a consequence of an increase in the total number of circulating neutrophils, although in some circumstances other cell types may also be increased. Absolute values for neutrophils and lymphocytes were increased with no changes in leukocyte differential counts in HC 600 goats (Table 3).
Goats in both MC 50 and HC 100, initially, had higher neutrophil:lymphocyte ratio (70.7:23.7, P < 0:003; 77.5:20.8, P < 0:003, respectively) as compared to control (BD) (38.8:58.2), however, the ratio reached that of control goats as the animals' body adjusted to Cu load. Non-infectious diseases resulting in leukocytosis or neutrophilia are those that stimulate a stress reaction. Included in this category are metabolic disturbances, drugs and toxic chemicals. Tissue destruction regardless of its cause will produce an increase in number of circulating neutrophils. Increased absolute number of lymphocytes may be an indication of chronic in¯ammatory effect associated with Cu toxicosis. It was concluded that Cu supplementation at 600 mg per head per day in young Nubian does, had no effect on RR, HR and decreased RT. Packed cell volume (%) temporarily decreased, within the normal range, with initial Cu dose of 100 mg Cu per day. Cu supplementation at 600 mg per day increased (P < 0:02) total leukocytes and stimulated stress reaction in goats with no change in neutrophil:lymphocyte ratio, however this ratio temporarily increased when goats were introduced to Cu in phase I of the study. Leukocyte counts were positively correlated with Cu supplementation (r 0:296, P < 0:02) and negatively correlated (r 0:254, P < 0:05) with rectal temperature in the HC group. 3.2. Plasma enzymes Mean plasma SDH value in goats fed the basal diet (Table 3) was 18:7 0:50 U/l with a CV of 17.3% which falls within the normal range for goats (9.3± 20.7 and 14±23.6 U/l) reported by Boyd (1984) and Brooks et al. (1984), respectively. Feeding supplemental Cu at the rate of 50, 100 or 150 mg per day did not effect (P > 0:05) plasma SDH compared to BD goats, however, plasma SDH was increased (P < 0:05) when goats were fed 300 and 600 mg Cu per day. Elevated SDH values (1.5 times normal) would be an indication of hepatic damage (Bartholomew et al., 1987). The mean plasma SDH for the MC 600 (28:0 13:98 U/l) and HC 1200 (29:7 17:4 U/ l) were 42% higher than the BD goats (18:7 0:5 U/l) but not signi®cant. Goats severely affected by Cu supplementation (MC 600 and HC 1200) tended to exhibit high SDH values followed by abnormally low
S.G. Solaiman et al. / Small Ruminant Research 41 (2001) 127±139
133
Fig. 1. Plasma sorbitol dehydrogenase (SDH) as affected by high Cu (HC) supplementation in goats.
SDH values which is an indication of hemolytic crisis and liver damage in these goats. Elevations in SDH values are transitory and unless samplings are made more frequently during the clinical elocution of hemolytic crisis, they may not be detected (Howell and Gooneratne, 1987). Observed changes (Fig. 1) resulted in lower mean values and a high degree of variation, as is indicated by S.E. of these means. A similar response was also noted in some other studies (Wiesner et al., 1965; Asada and Galambos, 1963), where, SDH values were suppressed or near normal during obstructive jaundice in sheep. Schee et al. (1983) concluded that the activity of the enzyme SDH can be considered as an indicator of high Cu
status of sheep. The increases observed in SDH of goats in the MC and HC groups were not as high as those reported by Shaw (1974) for sheep with liver damage (178 and 395 U/l). However, the amount of plasma SDH has been reported to be a sensitive test for detecting minor hepatic damage and an increase of 1.5 times or more, directly related to hepatic damage (Bartholomew et al., 1987). The observed SDH values for the MC group were positively correlated (r 0:337, P < 0:05) with Cu dosage rate (Table 4), however, similar relationships did not exist for the HC group. Mean plasma GOT in BD goats was 46:0 2:82 U/l, (Table 3) which falls within the normal range
Table 4 Correlation coef®cients for level of Cu, plasma Cu and plasma enzymes in medium (MC) and high Cu (HC) supplemented growing Nubian female goats Cu level
Sorbitol dehydrogenase
Glutamic oxaloacetic transferase
g-Glutamyl transferase
Plasma Cu
MC goats Cu supplement Plasma Cu
0.337* 0.093
0.385** 0.145
0.087 0.311
0.030 ±
HC goats Cu supplement Plasma Cu
0.195 0.281
0.549*** 0.635***
0.700*** 0.515***
0.486*** ±
*
P < 0:05. P < 0:01. *** P < 0:002. **
134
S.G. Solaiman et al. / Small Ruminant Research 41 (2001) 127±139
(43±230 U/l) reported for goats (Brooks et al., 1984; Boyd, 1984). The amount of variation in GOT estimates was high (CV 39%). Others (Boyd, 1984; Brooks et al., 1984) have also found higher analytical and biological ranges (66±230 and 43±132 U/l, respectively) and variation (Boyd, 1984; Jenkins et al., 1982) in GOT estimates compared to other liver speci®c enzymes (CV 1:6±2.7) and CV 1:4± 3.3) those of SDH and GGT, respectively). Differences in individual animal responses to higher Cu dosage also have contributed to high variation in GOT values. Animals in the MC 600 group tended to have elevated GOT (1.8 BD). Goats fed 600 mg and 1200 mg Cu per day, in the HC group, had mean values of GOT ((3.6±5.6) GOT in BD goats) higher (P < 0:05) than BD goats. These changes were comparable to observed changes in cattle, considered to have moderate to severe hepatic damage (Bartholomew et al., 1987). Determination of serum GOT has been proposed as a means of early detection of Cu toxicity as well as monitoring recovery in sheep (Davis and Mertz, 1987; Hidiroglou et al., 1984; Buckley and Tate, 1981), however, elevated levels of GOT were associated with apparent clinical signs of Cu toxicosis in the present study. Plasma GOT was positively correlated (r 0:385, P < 0:01) with the Cu dose for the MC group, and (r 0:549, P < 0:002) HC group (Table 4). Mean plasma g-glutamyl transferase in BD goats (Table 3) was 21:2 0:82 U/l with CV 24%, which falls within the normal range (20±50 U/l, CV 21%) reported by Boyd (1984). Contrary to the results observed for SDH and GOT, feeding goats 50 mg Cu per day or more, resulted in an increased plasma GGT as compared to BD goats. We cannot explain mean plasma GGT observed for the MC 600 group. Different responses between MC 600 and HC 600 group may indicate accumulative effects of Cu on this enzyme. The changes noted in GGT as compared to the BD group were not as great as those reported for cattle with hepatic damage (Bartholomew et al., 1987). Mean plasma GGT was positively correlated (r 0:70, P < 0:002) with Cu dosage in the HC group only (Table 4). Different responses in the MC and HC group may be an indication of accumulative effects of Cu on this enzyme. Data reported in literature for plasma SDH, GOT and GGT for goats (Nubian) are very limited. It can be
concluded that mean plasma SDH, GOT and GGT for control goats were similar to those reported for normal goats and estimates of mean plasma GOT were associated with higher biological and analytical variability compared to the other two enzymes measured in control goats. Goats exhibited elevated plasma SDH when fed 300 mg Cu per day, with yet no signs of apparent Cu toxicity, thus, SDH may serve as an early indicator of Cu toxicosis in goats. Individual animal variation in response to Cu dosage affected variability of mean SDH. Affected animals (especially in the MC 600, HC 600 and 1200 groups) exhibited sharp increases in SDH followed by abnormally low values (clinically silent hemolytic crisis), which contributed to data variation. Plasma GOT was elevated when goats received 600 mg Cu per day and at this time they were showing apparent signs of Cu toxicity. Plasma GGT responding immediately to initial doses of Cu may be more a response to initial stress on liver cells. Accumulative effect of Cu on GGT enzyme was apparent and all enzymes were elevated at high levels of Cu intake. 3.3. Plasma copper Mean plasma Cu measured for 35 weeks for BD goats (Table 3) was 1:15 0:04 which falls within the normal range (0.94±1.34 mg/l) reported for goats by others (Galbraith et al., 1997; Suarez et al., 1958), and reported for all animals (0.5±1.5 mg/l) by Davis and Mertz (1987). Plasma Cu was maintained within the normal range for all goats until the last 4 weeks of study when animals in the HC 1200 group had higher (P < 0:05) plasma Cu than the MC and BD goats, had developed clinical signs of Cu toxicity (de®ned earlier) and had high levels of SDH, GOT and GGT. Therefore, estimation of plasma Cu level is of limited value for diagnosing Cu loading or Cu poisoning in goats. Our ®ndings are similar to those reported by others for sheep (Howell and Gooneratne, 1987; Buckley and Tate, 1981; Ecckert et al., 1999). Plasma Cu was independent of Cu supplementation except at very high levels (>600 mg Cu per day, HC goats only), and it was positively correlated with GOT (r 0:635, P < 0:002) and GGT (r 0:515, P < 0:002) when Cu supplementation increased from 100 to 1200 mg per day (HC goats only, Table 4).
S.G. Solaiman et al. / Small Ruminant Research 41 (2001) 127±139
135
Table 5 Effects of medium Cu and high Cu supplement on BWa and feed ef®ciency in growing Nubian female goats (mean S:E:) Treatment groupb
Item
P
Basal diet Weeks 0±9 Mean BW (kg) Mean ADG (kg) Gain:feed
b
High Cu
Supplemental Cu (mg per head per day) 0 ppm 50 ppm 36.99 0.76 a 34.06 0.35 b 0.089 0.004 0.085 0.004 0.091 0.025 0.110 0.014
Weeks 10±23 Mean BW (kg) Mean ADG (kg) Gain:feed a
Medium Cu
0 ppm 42.31 1.33 ab 0.133 0.004 0.120 0.003
150 ppm 40.48 0.72 b 0.124 0.016 0.114 0.0
100 ppm 33.57 0.67 b 0.114 0.008 0.124 0.008
0.002
300 ppm 43.04 0.61 a 0.144 0.008 0.127 0.019
0.004
Due to BW loss associated with >300 mg per day Cu intake, data were reported for 23 weeks only. LS means within a row with different letters differ.
3.4. Growth performance Mean BW, ADG and gain:feed ratio of goats fed different levels of Cu from 0 to 23 weeks (phases I and II) are presented in Table 5. During phase I (weeks 0± 9), goats receiving 50 and 100 mg Cu per day, had a lower (P < 0:004) mean BW (for 9 weeks) than BD goats. In phase II (weeks 10±23), goats in MC 150 and HC 300 group, had mean BW (for 14 weeks) similar (P > 0:05) to BD goats, however, mean BW of HC 300 was higher (P < 0:05) than those of MC 150. When Cu intake increased to 600 or 1200 mg per head per day in phases III and IV, the animals in the HC group lost weight compared to MC and BD, therefore, data reported are for 23 weeks only. During phase I, Cu supplementation at 100 mg per head per day resulted in 28% improvement in mean ADG compared to BD (0.114 and 0.089 kg per head per day for HC 100 and BD, respectively). Since animals received 1 kg grain mix per head per day (with ad libitum hay intake) throughout the study, improvement in mean
ADG (28%) was used to estimate improved ef®ciency of gain:feed ratio. Even though number of animals were limited, growth rate determined by regression analysis of BW (Table 6), recorded over 23 weeks (phases I and II) for HC goats (b 1:096 0:082), also resulted in an improvement of 66% when compared to BD (b 0:660 0:116). Stimulatory effects of Cu on ADG and gain:feed ratio has also been reported in cattle (Gengelbach et al., 1994; Ward and Spears, 1997), swine (Braude, 1967; Edmonds et al., 1985; Cromwell, 1991, 1997; Cromwell et al., 1998) and poultry (Pesti and Bakalli, 1996). However, Luginbuhl et al. (2000) reported that feed intake, ADG or feed ef®ciency were not affected in growing meat goats fed 0, 10 or 30 mg/kg, supplemental Cu in the diet for 87 days which are lower than supplemental Cu level (100±300 mg per head per day) considered as growth stimulant in the present study. Therefore, results of this experiment warrant further studies on the role of Cu as a growth stimulant in goats.
Table 6 Regression coef®cients of BW as affected by Cu supplement for weeks 0±23, in growing female Nubian goats Cu supplement (mg per head per day) Basal diet only (0) Mediuma Cu (50±150) Highb Cu (100±300) a b
Linear Linear Linear
Goats were fed 50 mg Cu for 9 weeks, then 150 mg Cu for 14 weeks. Goats were fed 100 mg Cu for 9 weeks, then 300 mg Cu for 14 weeks.
b S.E.
P
R2 (%)
0.66 0.116 0.74 0.068 1.10 0.082
0.0001 0.0001 0.0001
56.6 81.5 87.2
136
S.G. Solaiman et al. / Small Ruminant Research 41 (2001) 127±139
3.5. Tissue copper residues Tissue Cu residue (TCR) for different organs and ¯uids are presented in Table 7. The liver is the central organ in Cu metabolism, by removing ionic Cu from blood, disposing a portion into bile, synthesizing ceruloplasmin and storing the remainder. Hepatic Cu of 315 15 for BD goats was within the range of 157±590 ppm or mean of 300 ppm (DM basis), reported by Beck (1956) for goats. TCR in control goats (BD) tended to be lowest in fat, rumen ¯uid, and bile (<1 ppm); followed by reticulum, lungs, ovary, muscle, hair and abomasal content (1±3 ppm); oma-
sum, abomasum, jejunum, ileum, heart, spleen and uterus (3±5 ppm); rumen, colon, and brain (8± 10 ppm); duodenum, and feces (10±20 ppm) and it was highest in liver (230±330 ppm). According to NAS (1977), Cu concentration of the whole human body on average is 2 ppm. Highest concentrations are found in the liver and brain, with lesser amounts in the heart, and spleen, which agreed with our ®ndings for goat. Cu contents of different segments of the alimentary tract (abomasum, duodenum, jejunum, ileum, and colon), spleen, uterus, brain, fat, bile, and muscle reported in our study were within the range reported for human or other mammalian tissues, however, Cu
Table 7 Effects of medium Cu (MC) and high Cu (HC) supplement (0±35 weeks) on tissue Cu concentration (mg/kg DM) in growing Nubian female goats (mean S:E:)a Tissues
Accumulated Cu supplementation (g) BD (0.0) (ppm)
Digestive tract Rumen Reticulum Omasum Abomasum Duodenum Jejunum Ileum Colon Some internal organs Brain Heart Lung Liver Fetus liver Spleen Ovary Uterus Other tissues and fluids Muscle Fat Hair Rumen fluid Abomasal content Feces Bile Blood a
MC (7.35) (ppm)
HC (14.70) (ppm) 47.7* 142.6** 153.2** 4.4 1.0 7.8 1.3 10.6*
8.8 2.4 3.1 4.5 13.7 4.8 4.4 8.5
4.40 0.05 0.40 1.90 a 7.30 1.60 1.25 4.80
12.0 7.0 8.1 9.5 13.9 8.2 5.4 9.8
3.90 0.90 2.70 0.85 a 6.60 1.30 0.0 6.40
419.6 356.6 474.9 20.0 14.3 37.5 5.8 90.1
403.0 346.8 462.3 0.0 b 0.0 31.2 0.0 76.4
8.3 4.2 2.6 315.0 240.0 3.2 2.6 4.6
0.10 0.40 0.65 15.0 a 10.0 0.80 0.15 1.50
7.7 6.0 3.7 890.0 230.0 1.6 5.4 4.9
0.60 0.80 0.50 100 b 0.0 0.35 2.30 2.45
7.5 14.4 16.8 1100.0 590.0 17.3 5.6 8.1
0.85 9.50 12.80 100.0 b 0.0 13.85 4.20 4.30
0.9 3.4 6.3 3.5 2.4 5.4 2.1 1.8
1.8 0.2 2.1 0.3 1.2 20.7 0.5 1.1
0.05 0.02 0.35 0.0 0.05 2.20 a 0.05 a 0.04 a
2.2 0.3 1.8 37.3 59.5 187.2 0.4 1.1
0.15 0.0 0.15 8.10 30.0 59.9 ab 0.13 a 0.05 a
3.2 0.3 2.9 537.1 3805.0 398.1 16.1 2.3
0.7 0.0 1.2 438.0 2500 137.4 b 12.1 b 0.96 b
1.7 1.5 1.4 1790.3*** 3044.0*** 19.2* 29.4* 2.0
LS means within a row with different letters differ (P < 0:05). >10-fold increase. ** >100-fold increase. *** >1000-fold increase. *
HC/BD
S.G. Solaiman et al. / Small Ruminant Research 41 (2001) 127±139
values for heart, lungs, ovary and hair were lower (Forssen, 1972; Tipton and Cook, 1963; Schroeder et al., 1966; Mills and Williams, 1962; Bingley and Dufty, 1972). Data on ruminant tissues, especially goats, is not widely available in the literature. Level of Cu increased (P < 0:05) in feces and bile for the HC group when compared to BD, probably due to increased Cu intake. In all species, a high proportion of ingested Cu appears in feces (Davis and Mertz, 1987), and the billiary system is recognized as the principal excretory pathway of absorbed Cu. However, in sheep, increasing Cu concentration in the diet does not appear to increase the excretion of Cu in bile (Howell and Gooneratne, 1987; Ecckert et al., 1999). Tissue Cu tended to increase in rumen, reticulum and omasum by 48-, 143- and 153-fold, respectively, with the highest increases found in rumen ¯uid and rumen and abomasal contents (>1000-fold) in the HC group (Table 7). Liver and abomasum Cu were higher (P < 0:05) in the HC than BD group. The main storage tissue for Cu is the liver, especially when the animal is loaded with Cu, although appreciable amounts of Cu accumulate in other tissues. No differences (P > 0:10) in TCR were observed between BD and MC groups except for liver Cu. Due to low numbers of experimental animals per treatment, differences were not signi®cant statistically. However, TCR tended to increase when Cu supplementation exceeded 600 mg per head per day. All goats were con®rmed pregnant during weeks of 23±25, however, only four out of six goats, all BD goats, one-half of MC and one-half HC goats remained pregnant. Fetal liver Cu at the termination of the study was 240, 230 and 590 ppm, (DM basis) for the BD, MC and HC groups, respectively. Goats in the MC group consumed a total of 7.35 g Cu for the 35week experiment, accumulated higher (P < 0:05) Cu in the liver (890 ppm, DM basis), with no change in fetal liver Cu. However, when Cu intake was 14.7 g for 35 weeks (HC), fetal Cu increased 2.45-fold (590 ppm, DM basis). The level of Cu in hair has been studied as an indicator of the Cu status in animals (Underwood, 1977). In the present study, random hair samples were collected every 4 weeks throughout the study. Hair Cu content ranged from 1.81 to 2.9 ppm for all animals, which agreed with values (2±4 ppm Cu) reported by Bingley (1974) for sheep. Cunningham and Hogan
137
(1958) reported higher values (8.3±13.3 ppm Cu) in New Zealand sheep and they found no relationship between dietary Cu and hair Cu content. There was no correlation between hair Cu and serum Cu in 800 hair samples quoted by Mertz (1987). No relationship between Cu intake and hair Cu was found in the present study. 4. Conclusions Vital signs and hematological parameters such as PCV, white blood cell counts, and differential white blood cell counts showed little variation with feeding high doses of Cu. Other biochemical changes occurring in blood, as a result of feeding high levels of Cu, appeared to be directly related to a degree of liver injury. One of the earliest biochemical changes observed in the prehemolytic period was an elevation in the activities of liver speci®c enzymes such as SDH. Plasma enzymes were in¯uenced by Cu supplementation to a greater extent than plasma Cu. Of the plasma enzymes measured, only GGT exhibited consistent elevations related to Cu intake. Cu levels as low as 50 mg per head per day resulted in elevated GGT. Plasma SDH was not changed until the amount of Cu supplement exceeded 300 mg per head per day. Supplemental Cu below 600 mg per head per day did not in¯uence GOT. Comparing data of this study with published data for sheep, indicates that goats (Nubian) are more resistant to Cu toxicity than sheep and this ®nding agrees with others (Adams et al., 1977; Soli and Nafstad, 1978) who also found that goats are less susceptible than sheep to repeated administration of Cu. Limited data on growth performance supported the stimulatory effect of 100±300 ppm Cu in the diet of Nubian goats. Extra Cu accumulated in the liver and to a lesser extent in other tissues, and was excreted through the billiary system (bile) and GI tract (feces). Hair Cu was not indicative of Cu status in Nubian goats. This study is a preliminary work to determine Cu requirement and toxicity in goats. Further investigations are needed with larger number of animals to recon®rm these ®ndings. Results of this study warrant further investigation on the role of Cu as a growth promoting factor in goats and on the ®nding that goats are not only more tolerant to high Cu level in the diet but actually require higher Cu in the diet than sheep.
138
S.G. Solaiman et al. / Small Ruminant Research 41 (2001) 127±139
Acknowledgements The authors wish to thank Mr. Joseph Sparks for his invaluable assistance throughout the study. This experiment was supported in part by USDA/CREES grant. Mention of trade names or commercial products is solely for providing speci®c information. References Adams, S.E.I., Was®, I.A., Magzoub, M., 1977. Chronic copper toxicity in Nubian goats. J. Comp. Pathol. 87, 623±627. AFRC, 1997. The nutrition of goats. Technical Committee on Responses to Nutrients, Report 10. Agricultural Food Research Council. Nutr. Abstr. Rev. (Series D) 67, 806±815. AOAC, 1984. Of®cial Methods of Analysis, 14th Edition, Association of Of®cial Analytical Chemists, Arlington, VA, pp. 152±169. Asada, M., Galambos, J.T., 1963. Sorbitol dehydrogenase and hepatocellular injury: an experimental and clinical study. Gastroenterology 44, 578±587. Azab, M.E., Abdel-Massoud, H.A., 1999. Changes in some hematological and biochemical parameters during prepartum and postpartum periods in female Baladi goats. Small Rumin. Res. 34, 77±89. Bartholomew, M.L., Willett, L.B., Liu, T.-T.Y., Morehead, P.D., 1987. Changes in hepatic function test to induce toxicity in bovine liver. J. Anim. Sci. 64, 201±209. Beck, A.B., 1956. The copper content of the liver and blood of some vertebrates. Aust. J. Zool. 4, 1±18. Bingley, J.B., 1974. Effects of high doses of molybdenum and sulphate on the distribution of copper in plasma and blood of sheep. Aust. J. Agric. Res. 25, 467±474. Bingley, J.B., Dufty, J.H., 1972. Distribution of copper in the tissues of the bovine neonate and dam. Res. Vet. Sci. 13, 8±14. Boyd, J.W., 1984. The interpretation of serum biochemistry test results in domestic animals. Vet. Clin. Pathol. 13, 7±14. Bradley, C.H., 1993. Copper poisoning in a dairy herd fed a mineral supplement. Can. Vet. J. 34, 287±292. Braude, R., 1967. Copper as a stimulant in pig feeding (Cuprum propecunia). World Rev. Anim. Prod. 3, 69±81. Brooks, D.L., Tillman, P.C., Niemi, S.M., 1984. Ungulates as laboratory animals. In: Fox, Cohen, Loew (Eds.), Laboratory Animal Medicine. Academic Press, New York, pp. 273±295. Buckley, W.T., Tate, R.M., 1981. Chronic copper toxicity in lambs: a survey of blood constituent responses. Can. J. Anim. Sci. 61, 613±624. Cromwell, G.L., 1991. Antimicrobial agents. In: Miller, E.R., Ullrey, D.E., Lewis, A.J. (Eds.), Swine Nutrition. ButterworthHeinemann, Stoneham, MA, pp. 297±314. Cromwell, G.L., 1997. Copper as a nutrient for animals. In: Richardson, H.W. (Ed.), Handbook of Copper Compounds and Applications. Marcel Dekker, New York, pp. 177±202. Cromwell, G.L., Lindemann, M.D., Monegue, H.J., Hall, D.D., Orr
Jr., D.E., 1998. Tribasic copper chloride and copper sulfate as copper sources for weanling pigs. J. Anim. Sci. 76, 118±123. Cunningham, I.J., Hogan, K.G., 1958. The in¯uence of diet on the copper and molybdenum contents of hair, hoof and wool. N. Z. J. Agri. Res. 1, 841±846. Davis, G.K., Mertz, W., 1987. Copper. In: Mertz, W. (Ed.), Trace Elements in Human and Animal Nutrition, 5th Edition. Academic Press, New York, pp. 301±350. Ecckert, G.E., Green, L.W., Carstens, G.E., Ramsey, W.S., 1999. Copper status of ewes fed increasing amounts of copper from copper sulfate or copper proteinate. J. Anim. Sci. 77, 244±249. Edmonds, M.S., Izquierdo, O.A., Baker, D.H., 1985. Feed additive studies with newly weaned pigs: ef®cacy of supplemental copper, antibiotics and organic acids. J. Anim. Sci. 60, 462± 469. Engle, T.E., Spears, J.W., 2000. Effects of dietary copper concentration and source on performance and copper status of growing and ®nishing steers. J. Anim. Sci. 78, 2446±2451. Forssen, A., 1972. Inorganic elements in the human body. 1. Occurrence of Ba, Br, Ca, Cd, Cs, Cu, K, Mn, Ni, Sn, Sr, Y, and Zn in the human body. Ann. Med. Exp. Biol. 50, 99±162. Galbraith, H., Chigwada, W., Scaife, J.R., Humphries, W.R., 1997. The effect of dietary molybdenum supplementation on tissue copper concentrations, mohair ®ber and carcass characteristics of growing Angora goats. Anim. Feed Sci. Technol. 67, 83±90. Gengelbach, G.P., Ward, J.D., Spears, J.W., 1994. Effect of dietary copper, iron, and molybdenum on growth and copper status of beef cows and calves. J. Anim. Sci. 72, 2722±2727. Haenlein, G.F.W., 1992. Advances in the nutrition of macro- and micro-elements in goats. In: Proceedings of 5th ICG. New Delhi, India, pp. 933±950. Hidiroglou, M., Heaney, D.P., Hartin, K.E., 1984. Copper poisoning in a ¯ock of sheep. Copper excretion patterns after treatment with molybdenum and sulfur or penicillamine. Can. Vet. J. 25, 377±382. Howell, J.McC., Gawthorne, J.M., 1987. Copper in Animals and Men, Vol. II. CRC Press, Boca Raton, FL. Howell, J.McC., Gooneratne, S.R., 1987. The pathology of copper toxicity in animals. In: Howell, J.McC., Gawthorne, J.M. (Eds.), Copper in Animals and Men, Vol. II. CRC Press, Boca Raton, FL, pp. 54±71. Howell, J.McC., Gopinath, C., 1977. Copper toxicity in sheep: the effect of repeated inter-venous injections of copper sulphate. Res. Vet. Sci. 22, 86±94. Humphries, W.R., Morrice, P.C., Mitchell, A.N., 1987. Copper poisoning in Angora goats. Vet. Rec. 121, 231. Ishmael, J., Gopinath, C., Howel, J.McC., 1971. Experimental chronic copper toxicity in sheep. Histological and histochemical changes during the development of the lesions in the liver. Res. Vet. Sci. 12, 358±366. Ishmeal, J., Gopinath, C., Howell, J.McC., 1972. Experimental chronic copper toxicity in sheep. Biochemical and hematological studies during the development of lesions in the liver. Res. In Vet. Sci. 13, 22±29. Jain, N.C., 1986. Schalm's Veterinary Hematology, 4th Edition. Lea & Febiger, Philadelphia, pp. 20±80. Jenkins, S.J., Green, S.A., Clark, P.A., 1982. Clinical chemistry
S.G. Solaiman et al. / Small Ruminant Research 41 (2001) 127±139 reference values of normal domestic animals in various age groups as determination on the ABA-100. Cornell Vet. 72, 403± 415. Kasa, I.W., Thwaites, C.J., Hill, M.K., 1999. Effects of treadmill exercise on sweating in three breeds of goats. Aust. Vet. J. 77, 660±661. Kessler, J., 1991. Mineral nutrition of goat. In: Morand-Fehr, P. (Ed.), Goat Nutrition. EAAP, Pudoc., pp. 104±119. Luginbuhl, J.M., Poore, M.H., Spears, J.W., Brown, T.T., 2000. Effect of dietary copper level on performance and copper status of growing meat goats. Sheep Goat Res. J. 16, 65±71. Mertz, W., 1987. Trace Elements in Human and Animal Nutrition, 5th Edition. Academic Press, Orlando, FL, pp. 301±350. Meschy, F., 2000. Recent progress in the assessment of mineral requirements of goats. Livestock Prod. Sci. 64, 9±14. Mills, C.F., Williams, R.B., 1962. Copper concentration and cytochrome-oxidase and ribonuclease activities in the brains of copper-de®cient lambs. Biochem. J. 85, 629±632. NAS, 1977. Copper: Medical and Biological Effects of Environmental Pollutants, National Academy of Science, Washington, DC, pp. 14±27. NRC, 1980. Mineral Tolerances of Domestic Animals. National Research Council, National Academy of Science, Washington, DC. NRC, 1981. Nutrient Requirements of Domestic Animals. No. 15. Nutrient Requirements of Goats: Angora, Dairy and Meat Goats in Temperate and Tropical Countries. National Research Council, National Academy of Science, Washington, DC, pp. 10±12. Pesti, M.G., Bakalli, R.I., 1996. Studies on the feeding of cupric sulfate pentahydrate and cupric citrate to broiler chickens. Poult. Sci. 75, 1086±1091. SAS, 1990. SAS User's Guide: Statistics, Version 6, 4th Edition. SAS Institute Inc., Cary, NC.
139
van der Schee, W, Schotamn, A.J.H., van der Berg, R., 1983. Levels of enzyme activities in blood serum as indicators of a high copper status in sheep ¯ocks. Zentralbl. Veterinarmed. A 30, 664±673. Schroeder, H.A., Nason, A.P., Tipton, I.H., Balassa, J.T., 1966. Essential trace minerals in man: copper. J. Chron. Dis. 19, 1007±1034. Shaw, F.D., 1974. Sorbitol dehydrogenase in the diagnosis of liver disease of ruminants. Aust. Vet. J. 50, 279±280. Sleiman, F.T., Abi-Saab, S., 1998. In¯uence of environment on respiration, heart rate and body temperature of Filial crosses compared to local Awassi sheep. Small Rumin. Res. 16, 49±53. Soli, N.E., Nafstad, I., 1978. Effect of daily oral administration of copper to goats. Acta Vet. Scand. 19, 561±568. Suarez, R.M., DeBerrocal, H.F., DeOlavarrieta, S.T., Suarez Jr., R.M., Buso, R., Sabater, J., 1958. Studies on blood copper in normal and pathological states. Biol. Assoc. Med. Puerto Rico 50, 1±10. Tipton, I.H., Cook, M.J., 1963. Trace elements in human tissue. Part II. Adult subjects from the United States. Health Phys. 9, 103±145. Underwood, E.J., 1977. Trace Elements in Human and Animal Nutrition, 4th Edition, Academic Press, New York, pp. 56±108. Ward, J.D., Spears, J.W., 1997. Long term effects of consumption of low copper diets with or without supplemental molybdenum on copper status, performance, and carcass characteristics of cattle. J. Anim. Sci. 75, 3057±3065. Wiesner, I.S., Rawnsley, M.S., Brook, F.P., Senior, J.R., 1965. Sorbitol dehydrogenase in the diagnosis of liver disease. Am. J. Digestive Dis. 10, 147±151. Zervas, G., Nikoloay, E., Manzios, A., 1989. Comparative study of chronic copper poisoning in lambs and young goats. In: Proceedings of 6th International Trace Element Symposium. University Leipzig-Jena, Germany, pp. 569±581.