Effect of chronic exposure to excess dietary copper and dietary selenium supplementation on liver specimens from rats

OBJECTIVE: To determine the effects of chronic exposure to excess dietary copper (Cu) on liver specimens from rats and the effects of dietary selenium (Se) supplementation in experimental Cu toxicosis. ANIMALS: 60 weanling male Fischer 344 rats. PROCEDURE: Rats were randomly assigned to 4 groups of 15 rats each and fed 1 of the following 4 diets: high Cu (500 microg/g)/adequate Se (0.2 microg/g); high Cu (500 microg/g)/supplemented Se (2 microg/g); adequate Cu (18 microg/g)/adequate Se (0.2 microg/g); or, adequate Cu (18 microg/g)/supplemented Se (2 microg/g). Five rats per group were euthanatized after 3, 6, and 12 months, and liver specimens were obtained for histologic examination, histochemistry, metal analysis by atomic absorption spectrophotometry, measurement of glutathione peroxidase activity, and assessment of lipid peroxidation, using quantification of malondialdehyde (MDA) by the thiobarbituric acid reaction. RESULTS: Hepatic Cu concentration was significantly higher in rats fed high Cu diets (range, 9 to 18 microg/g of tissue [wet weight]), compared with rats receiving adequate Cu diets (4.0 to 5.7 microg/g of tissue). Rats fed high-Cu diets for 3, 6, and 12 months had mild multifocal hepatitis often surrounding necrotic foci. However, an increase in hepatic MDA content, indicative of lipid peroxidation, was not detected in these rats. Development of morphologic changes was not prevented by use of dietary Se supplementation. CONCLUSION AND CLINICAL RELEVANCE: Long-term exposure to excess dietary Cu caused mild hepatic lesions in Fischer 344 rats. Dietary Se supplementation did not prevent hepatic damage in rats with Cu toxicosis.     

The failure of selenium supplementation to prevent copper-induced liver damage in Fischer 344 rats.

This study evaluates the ability of selenium (Se) supplementation to prevent experimental copper (Cu)-induced hepatocellular damage. Weanling male Fischer 344 rats were randomly assigned to groups of 15, 3 groups (A,B,C) were fed Cu-loaded diets (containing 2000 microg/g copper, added as CuSO4) and different levels of Se (added as Na2SeO3 x 5H2O) as follows: A) Cu-loaded/Se adequate diet (0.4 microg/g Se, fed basis); B) Cu-loaded/Se-supplemented diet (2 microg/g Se); and C) Cu-loaded/Se-deficient diet (< 0.2 microg/g). Three additional groups (D,E,F) were fed diets containing adequate levels of Cu (14 microg/g Cu, fed basis) and different levels of Se as follows: D) Cu-adequate/Se-adequate diet; E) Cu-adequate/Se-supplemented diet (2 microg/g Se); and F) Cu-adequate/Se-deficient (< 0.2 microg/g) diet. After 4, 8, and 12 weeks on the experimental diets, liver samples were processed for histology, histochemistry, metal analysis, glutathione peroxidase (GSH-Px) measurement, and quantification of malondialdehyde (MDA). Morphologic changes characteristic of Cu-associated hepatitis, without an increase in hepatic MDA levels, were seen in all Cu-loaded rats in each sampling. Similar changes occurred in rats fed Se-adequate, Se-supplemented and Se-deficient diets. This study demonstrates that Fischer 344 rats fed 2000 microg/g Cu develop morphologic changes due to Cu toxicity without evidence of lipid peroxidation. Furthermore, Se supplementation does not result in protection against Cu-induced liver injury.     

Tolerance for excess basic zinc chloride and basic copper chloride in chicks

(1) Four chick experiments were conducted to determine toxicity estimates for basic zinc chloride (BZC) and basic copper chloride (BCC), now being used as sources for these minerals. (2) In experiment 1, New Hampshire x Columbian crossbred chicks were fed 0, 500, 1000, 1500, 3000 and 5000 mg Zn/kg from BZC (Zn5Cl2(OH)8). Broken-line regression analysis showed that the minimal toxic break points for chick weight gain and gain:food were 1720 and 2115 mg Zn/kg, respectively. (3) Crossbred chicks were fed 0, 150, 250, 500, 750 and 1000 mg Cu/kg from BCC (Cu2(OH)3C) in experiment 2. Regression analysis indicated that the minimal toxic break points for chick weight gain and gain:food were 642 and 781 mg Cu/kg, respectively. (4) In experiment 3, commercial broiler chicks were fed 0, 1500, 2000 and 2500 mg supplemental Zn/kg from BZC or 0, 500, 650 and 800 mg supplemental Cu/kg from BCC. Broiler chicks fed those high inclusion rates of Zn did not show reduced weight gain in comparison to chicks fed no supplemental Zn. All high concentrations of supplemental Cu depressed chick weight gain in comparison to control chicks. (5) Experiment 4 involved two separate 4 x 2 factorial designs with supplemental Zn (0, 2500, 3500 and 4500 mg/kg) or Cu (0, 500, 750 and 1000 mg/kg) and two breeds of chicks (crossbred and commercial). Significant interactions for weight gain, food intake, gain:food and liver Cu suggested that the crossbred and commercial chicks responded differently to high concentrations of supplementary dietary Cu.     

Unilateral degeneration of retina and optic nerve in Fischer-344 rats

Unilateral degeneration of the retina and optic nerve was observed among Fischer-344 (F-344) rats fed a semi-purified synthetic feed. Further studies were conducted using standard cereal-based and synthetic diets. Beginning at 4 weeks of age, all experimental rats (169 F-344 rats) were fed various diets and were examined for morphologic and functional changes in the retina and optic nerve. No ocular lesions were observed in any F-344 rats prior to 21 weeks of age, whether fed a synthetic diet or a standard diet; however, approximately 16% (13/86) of the F-344 rats examined between 57 and 64 weeks of age developed unilateral degeneration of the retina and optic nerve. On the other hand, the F-344 rats fed the synthetic diet developed the degenerative lesions by 30 weeks of age, while the F-344 rats fed the standard diet did not develop lesions over this shorter time period. Degenerative changes of the affected retinas and optic nerves were closely related with functional abnormalities evaluated by electroretinogram and visual evoked potentials. In contrast with the F-344 rats, Long-Evans rats that were fed either the synthetic or standard diet up to the age of 68 weeks (77 rats) did not develop the ocular lesions. There was no apparent relationship of the development of the lesions with dietary modification, toxicity or trauma; thus, these observations appear to indicate that spontaneous unilateral degeneration of the retina and optic nerve occurs in F-344 rats and that these ocular lesions may be accelerated by the feeding of certain semi-purified synthetic diets.     

Vitamin D3 requirement of young chicks receiving diets varying in calcium and available phosphorus

1. Three battery experiments were conducted with broiler chicks during the 2nd and 3rd week of life. Graded amounts of cholecalciferol (D3) were added to maize-soyabean meal diets that were designed to be (a) severely deficient in available phosphorus (P), (b) marginally deficient in calcium (Ca) or (c) adequate in both available P and Ca. 2. With diets containing 1.0 g available P and 6.3 g Ca/kg (assay 1), graded doses of D3 between 0 and 37.5 mu/kg produced linear (P 0.05) positive responses in both weight gain and tibia ash. With a D3 concentration of 1250 mug/kg, 250 times the requirement recommended by the NRC, bone ash was increased (P 0.05) over that of birds fed 37.5 mug/kg, and neither weight gain nor food intake were reduced. 3. With a P- adequate diet (4.5 g available P/kg) containing 8.5 g Ca/kg (assay 2), weight gain and bone ash increased linearly (P 0.05) upon supplementing the basal diet with 0, 2.5 and 5.0 mug D3/kg. Higher doses of D3 did not elicit further responses, and chicks fed on a diet containing 1250 mug D3/kg gained as fast and had bone ash values that did not differ from those of chicks receiving 5, 10, 20 or 40 mug D3/kg. 4. When the maize-soyabean meal basal diet was fortified with Ca and P to achieve adequate amounts of Ca (10.1 g/kg) and P (4.5 g available P/kg) in assay 3, dietary additions produced results similar to those obtained in assay 2 where P was adequate and Ca was slightly deficient. Again, chicks receiving a surfeit of D3 (1250 mug/kg) exhibited weight gains and bone ash values that were as great as those of chicks receiving 5, 10, 15 or 30 mug D3/kg. 5. It is apparent that young chicks have a high tolerance for excess D3, and chicks fed on diets that are severely deficient in available P continue to respond to D3 in excess of 37.5 mug/kg.     

Evaluation of the diagnostic value of plasma copper levels in cattle

Copper (Cu) is an essential trace element. Depletion of liver-Cu stores leads to clinical or sub-clinical Cu deficiency. Our objective was to evaluate plasma-Cu levels as a diagnostic test for the assessment of Cu status in cattle. We used liver-Cu levels as the gold standard. The chosen cutoff for plasma Cu was 9 mmol/l; two different cutoffs were chosen for liver-Cu: 20 and 10 microg/g dry matter (DM). When 20 microg/g DM of liver Cu was the cutoff, plasma Cu had sensitivity=69% (58, 79) and specificity=84% (74, 93). The same values if the cutoff in liver was 10 microg/g were 92 (82, 100) and 73% (64, 82), respectively. ROC curves showed that the best cutoff to be used for plasma Cu was 8.26 mmol/l; however, no significant improvement was found in sensitivity or specificity. We concluded that interpretation of plasma-Cu levels significantly varies with the liver-Cu cutoff.     

Interactive effects of dietary calcium, phosphorus and copper on performance and liver stores of pigs

Three experiments involving 304 pigs were conducted to determine the related effects of copper (Cu), calcium (Ca) and phosphorus (P) on the performance and liver Cu stores of growing-finishing pigs. Rate and efficiency of gain were improved by the addition of 250 ppm of Cu to the diets. Improvements in rate of gain averaged 6.6% (652 vs 696 g/d) to 60.5 kg body weight and 1.7% (713 vs 725 g/d) to 94.5 kg body weight. Feed:gain ratio was improved by 1.4% to 60.5 kg and 1.6% to 94.5 kg body weight when Cu was added to the diet. Increasing the dietary Ca and P levels from .65% Ca and .55% P to 1.2% Ca and .86 or 1.0% P resulted in increased (P less than .01) growth rate to 60 and 95 kg (649 vs 699 g/d and 700 vs 737 g/d, respectively), but feed efficiency was not affected (2.86 vs 2.84 and 3.18 vs 3.17 kg feed/kg gain, respectively.) Feeding the higher Ca and P levels resulted in increased liver Cu levels in pigs fed 250 ppm Cu (189 vs 323 ppm), but Ca and P did not affect liver Cu of pigs fed low Cu diets (29 vs 28 ppm). When dietary Ca and P were varied independently, the high Ca level increased liver Cu, but P had little effect on liver Cu. Increasing the dietary P level partially alleviated the effect of Ca on liver Cu.     

Effect of the chemical form of supranutritional selenium on selenium load and selenoprotein activities in virgin, pregnant, and lactating rats

Virgin, pregnant, and lactating rats were used to assess the influence of selenomethionine and selenocystine, fed at four to seven times the daily Se requirement (supranutritional), on Se load and selenoprotein activities. Female Sprague Dawley rats (n = 48; age = 13 wk), reared on a low-Se torula yeast diet, were assigned to one of three reproductive states (n = 16 per reproductive state) to occur simultaneously: virgin, pregnant, and lactating. Once reproductive state was achieved, rats were fed (ad libitum) either l-selenomethionine (n = 24) or L-selenocystine (n = 24) diets providing 2.0 microg Se/g of diet (as-fed basis) for 18 d, and then killed. Lactating rats consuming selenomethionine had the greatest Se concentration in the brain, with pregnant rats being intermediate, and virgin rats having the least (P < 0.02). When selenocystine was fed, the concentration of Se in the brain was greater (P = 0.008) in lactating rats, but not different (P = 0.34) between pregnant and virgin rats. Selenium concentrations in the heart, liver, lung, muscle, spleen, plasma, placenta, uterus, and fetus were greatest (P < 0.001) in rats consuming selenomethionine. Brain, kidney, and liver thioredoxin reductase, and brain, erythrocyte, kidney, and liver glutathione peroxidase activities did not differ (P = 0.13 to P = 0.85) between Se treatments. Lactating rats exhibited the greatest (P < 0.006) Se concentration in the heart, lung, muscle, plasma, and spleen compared with pregnant and virgin rats. Thioredoxin reductase was greatest (P < 0.004) in the brain of pregnant rats, greatest (P < 0.004) in the liver of lactating rats, and greater (P < 0.03) in the kidney of lactating and pregnant vs. virgin rats. Regardless of reproductive state, supranutritional Se (2.0 microg/g of diet) fed as selenocystine resulted in less Se load, and when fed as selenomethionine, was equally available for thioredoxin reductase synthesis as the Se in selenocystine. Independent of dietary Se chemical form, thioredoxin reductase activity was responsive to reproductive state.     

Dietary L-carnitine increases plasma insulin-like growth factor-I concentration in chicks fed a diet with adequate dietary protein level

1. The effect of L-carnitine supplemented into experimental diets with varying dietary protein concentrations (50, 200 and 400 g/kg) on body weight gain and plasma insulin-like growth factor-I (IGF-I) concentration in chicks was examined. 2. Dietary L-carnitine supplementation provided 0, 200, 500 and 1000 mg/kg. Chicks were given the diet ad libitum for 10 d. 3. When L-carnitine was provided as 500 or 1000 mg/kg, body weight gain was significantly improved in birds receiving the 200 and 400 g protein/kg diets. 4. There was an interaction between dietary L-carnitine and protein content on plasma IGF-I concentration. L-carnitine supplementation had little influence on plasma IGF-I concentrations in birds receiving the low protein (50 g/kg) diet. When dietary L-carnitine concentrations were increased from 0 to 1000 mg/kg in the adequate protein (200 g/kg) diet, plasma IGF-I concentrations were also increased. However, when dietary L-carnitine content was more than 500 mg/kg in the 400 g/kg protein group, plasma IGF-I concentration decreased with increasing dietary L-carnitine content. 5. Body weight change correlated significantly with the alteration in plasma IGF-I concentrations in chicks given diets with adequate dietary protein. 6. In conclusion, the improvement in body weight gain caused by dietary L-carnitine supplementation was achieved when chicks were given their dietary protein requirement, which may be partially explained by an increase in plasma IGF-I concentration.     

Effect of excess levels of lysine and leucine in wheat‐based,amino acid‐fortified diets on the mRNA expression of two selected cationic amino acid transporters in pigs

An experiment was conducted to evaluate the effect of excess levels of Leu and Lys on the expression of b^0,+ and CAT‐1 mRNA in jejunum, liver and the muscles Longissimus dorsi (LDM) and Semitendinosus (STM). Twenty pigs with an average initial BW of 16.4 ± 1.7 kg were used in a Randomized Complete Block. Dietary treatments (T) were as follows: T1, basal diet; T2, basal plus 3.5 g l ‐Lys/kg diet; T3, basal plus 1.5 g l ‐Leu/kg diet; T4, basal plus 3.5 g l ‐Lys plus 1.5 g l ‐Leu/kg diet. Diets in T1 and T3 met 100% the requirement of Lys for pigs within the 10 to 20 kg body weight range; diets in T2 and T4 contained 35% excess of Lys. Also, diets in T1 and T2 supplied 104%, whereas diets in T3 and T4 supplied 116% the requirement of Leu. The expression of b^0,+ in jejunum was reduced (p = 0.002) because of the supplementation of l ‐Leu, but l ‐Lys supplementation had no effect (p = 0.738). In contrast, the expression of b^0,+ in STM (p = 0.012) and liver (p = 0.095) was reduced by the high level of Lys, but Leu had no effect (p > 0.100). CAT‐1 expression in STM increased by high Lys (p = 0.023) and Leu (p = 0.007) levels. In liver, the expression of CAT‐1 substantially increased (p = 0.001) because of Lys. In conclusion, excess levels of dietary Lys and Leu affect the expression of cationic amino acid transporters, and this effect varies depending on the studied tissue.     

Dietary L-carnitine supplementation enhances the lipopolysaccharide-induced acute phase protein response in broiler chickens

The objective of the present study was to evaluate the potential effects of dietary L-carnitine supplementation on acute phase protein response upon a lipopolysaccharide (LPS) challenge of male broiler chickens receiving a commercial broiler diet supplemented with 15 or 100 mg L-carnitine/kg or an unsupplemented (control) diet from 14 days of age onwards. At 28 days of age, eight chickens per dietary treatment were weighed and subcutaneously injected with 300 microg LPS from E. coli (100 microg LPS/ml saline) or 3 ml saline (unsupplemented group only). During the next 10 days, blood samples were taken repeatedly and analysed for their hemopexin (HX) and alpha-1 acid glycoprotein (AGP) levels. Extra dietary L-carnitine did not affect broiler performance. At day 1 postinjection, plasma HX and AGP levels were significantly increased in all treatment groups. However, the elevations in circulating HX and AGP levels were more pronounced in the L-carnitine supplemented chickens, especially in the 100mg L-carnitine group. It is concluded that extra L-carnitine in the diet of broiler chickens enhances or advances the acute phase protein response. The exact mode of action needs to be elucidated but seems to be consistent with a glucocorticoid mimicking effect.     

Effects of dietary copper and molybdenum on tansy ragwort (Senecio jacobaea) toxicity in sheep

Dried tansy ragwort (Senecio jacobaea), which contains pyrrolizidine alkaloids (PA), was fed to sheep to determine whether PA consumption influenced liver Cu concentrations. Nine Polypay wether lambs were allotted to 3 groups according to the diets: control diet + 50 micrograms of Cu/g of feed (control + Cu); tansy ragwort (TR) diet + 50 micrograms of Cu/g of feed (TR + Cu); and TR diet + 50 micrograms of Cu/g of feed and 10 micrograms of Mo/g of feed (TR + Cu + Mo). Liver Cu concentrations were sharply increased (13-fold) after 8 weeks of feeding the diets, and then decreased slightly. There were no significant differences in concentrations of liver Cu among treatment groups. All lambs in the group fed TR + Cu + Mo died by the end of 23 weeks, with the usual signs of PA poisoning, including liver necrosis, hepatic megalocytosis, and biliary hyperplasia. The TR intake was about 2.7 to 3.0 kg/kg of their initial body weight. All lambs in the group fed the control + Cu diet survived. The results indicated that sheep fed TR do not preferentially accumulate liver Cu. There was an indication that dietary Mo increased susceptibility to TR intoxication.     

Copper status of ewes fed increasing amounts of copper from copper sulfate or copper proteinate

The Cu status of mature, crossbred ewes fed two sources (CuSO4 vs. Cu proteinate) and three levels (10, 20, or 30 mg/kg) of dietary Cu was determined in a 73-d feeding trial. Ewes (n = 30) were fed a basal diet containing rice meal feed, cottonseed hulls, cottonseed meal, meat and bone meal, cracked corn, and vitamin-mineral supplements at 2.5% of BW to meet NRC requirements for protein, energy, macrominerals, and microminerals, excluding Cu. The basal diet contained 5 mg/kg Cu, 113 mg/kg Fe, .1 mg/kg Mo, and .17% S. Copper sulfate or Cu proteinate was added to the basal diet to supply 10, 20, or 30 mg/kg of dietary copper in a 2x3 factorial arrangement of treatments. Ewes were housed in 3.7- x 9.1-m pens in an open-sided barn. Blood samples were collected on d 28 and 73. Ewes were slaughtered on d 74, and liver and other tissues were collected to determine Cu concentrations. An interaction (P = .08) occurred between source and level for liver Cu. The interaction existed due to an increase in liver Cu concentrations when ewes were fed increasing dietary Cu from CuSO4 but not when fed Cu proteinate diets. There was no source x level interaction (P>.10) for the blood constituents measured. On d 73, plasma ceruloplasmin activity was greater (P<.05) in ewes fed Cu proteinate than in those fed CuSO4 (33.1 vs. 26.8 microM x min(-1) x L(-1)). Increasing the concentration of dietary Cu did not affect (P>.10) plasma ceruloplasmin. Packed cell volume (PCV), red blood cell count (RBC), white blood cell count, whole blood hemoglobin (wHb), plasma hemoglobin, and plasma Cu were similar between sources of Cu. Ewes fed 20 mg/kg Cu had lower (P<.05) PCV, RBC, and wHb than those fed 10 or 30 mg/kg Cu diets. Feeding up to 30 mg/kg Cu from these sources did not cause an observable Cu toxicity during the 73-d period.     

Dietary conjugated linoleic acid improves antioxidant capacity in broiler chicks

1. The influence of dietary conjugated linoleic acid (CLA) on the antioxidant status in the absence or presence of endotoxin exposure was studied with male broiler chicks. 2. In experiment 1, a total of 240 1-d-old broilers were allotted into 4 dietary groups (0, 2.5, 5.0 or 10.0 g pure CLA/kg) to study the influence of CLA on growth performance and antioxidant defence systems. The results showed that growth performance was not altered by 42 d of CLA consumption. Increased total superoxide dismutase (TSOD) activities in liver, serum and muscle were observed in chicks given 10.0 g CLA/kg diet. Dietary CLA at 10.0 g/kg also markedly elevated liver catalase (CAT) activity. Malondialdehyde (MDA), a marker of lipid peroxidation, decreased in liver, serum and muscle in chicks given 5.0 and 10.0 g CLA/kg diet. 3. In experiment 2, a total of 120 1-d-old broilers were fed on a control diet (without CLA) or 10.0 g CLA/kg diet. Half of the birds fed on each diet were injected intraperitoneally with 0.25 mg/kg body weight of Salmonella enteritidis lipopolysaccharide (LPS) at 16, 18 and 20 d of age. Decreased glutathione peroxidase (GSH-Px), TSOD activity and increased ceruloplasmin and MDA concentrations were seen in the challenged chicks. Dietary CLA prevented the loss of body weight gain and feed conversion ratio of chicks followed repeated endotoxin exposure. CLA partially inhibited the increase of serum ceruloplasmin and MDA at 17 and 21 d of age and notably suppressed the decrease of serum TSOD activity at 21 d of age. 4. These results suggested that dietary CLA enhances the activity of antioxidant enzymes including TSOD and CAT. Supplementation of CLA has been shown to ameliorate the antioxidant balance and performance of chicks during oxidative stress.     

Analyses of hemorrhagic diathesis in high-iron diet-fed rats

Iron overload has been well recognized to cause oxidant-mediated cellular/tissue injury; however, little is known about the effects of iron overload on the blood coagulation system. We encountered an unexpected bleeding tendency in rats fed a high-iron diet in a set of studies using iron-modified diets. In this study, we investigated the mechanism of hemorrhagic diathesis induced by dietary iron overload in rats. Six-week-old F344/DuCrlCrlj male rats were fed a standard (containing 0.02% iron) or a high-iron diet (containing 1% iron) for 6 weeks and were then sampled for hematological, blood biochemical, coagulation, and pathological examinations. Serum and liver iron levels increased in rats fed the high-iron diet (Fe group) and serum transferrin was almost saturated with iron. However, serum transaminase levels did not increase. Moreover, plasma prothrombin time and activated partial thromboplastin time were significantly prolonged, regardless of the presence of hemorrhage. The activity of clotting factors II and VII (vitamin K-dependent coagulation factors) decreased significantly, whereas that of factor VIII was unaltered. Blood platelet levels were not influenced by dietary iron overload, suggesting that the bleeding tendency in iron-overloaded rats is caused by secondary hemostasis impairment. In addition, hemorrhage was observed in multiple organs in rats fed diets containing more than 0.8% iron. Our results suggest that iron overload can increase the susceptibility of coagulation abnormalities caused by latent vitamin K insufficiency.     

Effect of copper status, supplementation, and source on pituitary responsiveness to exogenous gonadotropin-releasing hormone in ovariectomized beef cows

The effect of Cu status, supplementation, and source on pituitary responsiveness to exogenous GnRH was evaluated using nine multiparous, nonpregnant, nonsuckling, ovariectomized Angus cows (7.1 +/- 3.3 yr; 622.9 +/- 49.8 kg; BCS = 6.0 +/- 0.5). Cows were considered Cu-deficient based on liver Cu concentrations (< 30 mg of Cu/kg of DM) after receiving a low-Cu, forage-based diet supplemented (DM basis) with 5 mg of Mo/kg and 0.3% S for 216 d. Copper-deficient cows were stratified based on age, BW, BCS, and liver Cu concentration and assigned randomly to repletion-phase treatments. Treatments included 1) control (no supplemental Cu); 2) organic (ORG; 100% organic Cu); and 3) inorganic (ING; 100% inorganic CuSO4). Treatments were formulated to meet all NRC recommendations, except for Cu, which was supplemented to ORG and ING cows at 10 mg of Cu/kg of dietary DM. During the 159-d repletion phase, Cu status was monitored via liver biopsy samples, and all cows received exogenous progesterone. A controlled intravaginal drug-release device (replaced every 14 d) was used to maintain luteal phase progesterone as a means to provide negative feedback on the hypothalamic-pituitary axis. During the repletion phase, liver Cu concentrations did not differ between ORG and ING cows at any time. By d 77 of the repletion phase, all supplemented cows were considered adequate in Cu, and liver Cu concentrations were greater in supplemented than in nonsupplemented control cows on d 77 (P < 0.05) and throughout (P < 0.01) the repletion phase. Beginning on d 99, exogenous GnRH was administered to all cows at low (0, 3, and 9 microg; Exp. 1) and high doses (0, 27, and 81 microg; Exp. 2) at six different times. Cows were catheterized every fifth day, and blood samples were collected every 15 min for 1 h before and 4 h after GnRH administration and analyzed for LH concentration. In Exp. 1, Cu status and supplementation did not affect basal or peak LH concentrations, but total LH released tended (P < 0.07) to be greater in Cu-supplemented vs. control cows when 3 microg of GnRH was administered. In Exp. 2, there was no effect of Cu supplementation or source on basal, peak, or total LH released, regardless of GnRH dose. Pituitary LH concentrations did not differ across treatments. In conclusion, Cu status, supplementation, and source did not affect GnRH-induced LH secretion or pituitary LH stores in ovariectomized, progesterone-supplemented cows in this experiment.     

Use of 2,3,2-tetramine as a hepatic copper chelating agent for treatment of copper hepatotoxicosis in Bedlington terriers

Five Bedlington Terriers with inherited copper (Cu) hepatotoxicosis and with hepatic Cu concentrations ranging from 3,000 to 11,000 micrograms/g of dry weight (normal, less than 350 micrograms/g of dry weight) were treated daily for up to 200 days with 2,3,2-tetramine tetrahydrochloride. During treatment, no change was made in the dietary Cu intake, which ranged from 12 to 16 micrograms/g of dry diet. Concentrations of hepatic and serum Cu, iron, and zinc were determined before and at the conclusion of the treatment period. In one dog, 24-hour urinary Cu concentration was measured before and during treatment. A liver biopsy specimen obtained after treatment had significantly (P less than 0.05) reduced hepatic Cu concentration (3,282 micrograms/g of dry weight; a 54.9% reduction), compared with the pretreatment value (7,281 micrograms/g of dry weight). After treatment, there was an overall general lessening of the extent of hepatic morphologic damage. Cytochemical examination for Cu in rhodanine-stained biopsy specimens revealed decreased numbers of Cu-laden hepatic lysosomes. The mean daily urinary Cu concentration increased as much as 25-fold during 2,3,2-tetramine treatment. Hepatic iron and zinc concentrations and serum Cu concentrations remained within normal ranges after treatment. Clinical or laboratory evidence of 2,3,2-tetramine toxicosis was not detected during treatment. These findings indicated that in affected Bedlington Terriers, 2,3,2-tetramine was a safe and rapid chelating agent of hepatic Cu.     

Effects of dietary protein level and clinoptilolite on the weight gain and liver mineral response of growing lambs to copper supplementation

Growing male Synthetic I (1/2 Finnish Landrace x 1/4 Dorset x 1/4 Rambouillet) lambs were used in two experiments (64 lambs in Exp. 1 and 63 in Exp. 2) to test the hypothesis that dietary CP level (9 or 14% of diet as fed) and(or) clinoptilolite (clino; 0 or 2% of diet) affects growth and tissue mineral concentrations of growing lambs fed supplemental Cu. Lambs were individually fed their respective diets ad libitum and killed after 12 wk (Exp. 1) or 16 wk (Exp. 2) to obtain carcass measurements, organ weights and liver mineral concentrations. In Exp. 1, 20 ppm added Cu (as CuSO4.5H2O) increased mortality and depressed BW gain (P less than .01) and daily feed intake (P less than .05) in the presence or absence of clino and at both levels of CP. Liver Cu concentration was greater (P less than .01) in lambs fed added Cu than in those not fed Cu (408 ppm vs 110 ppm, respectively). Neither CP nor clino affected liver Cu concentration. Clinoptilolite increased daily gain of lambs fed high CP but not low CP (P less than .01). In Exp. 2, clino in the diet had no effect on daily gain or daily feed, but 20 ppm Cu addition depressed daily gain (P less than .01) and gain/feed (P less than .07). Organ weights and levels of trace elements other than Cu in the liver generally were not affected by diet in either experiment. It is concluded that high dietary CP or 2% dietary clino did not protect against toxic signs of Cu when Cu was added to the basal diet (10 ppm Cu) at 10 or 20 ppm.     

The Effect of Dietary Sulphate Copper Supplementation on Growth Performance, Intestinal Digestive Enzymes and Absorptive Transporters in Weanling Pigs

The experiment was conducted to assess the effects of dietary supplementation of Cu on the growth performance,digestive enzymes,tissue minerals and absorptive transporters in small intestinal mucosa of weanling pigs.One hundred crossbred pigs weaned at 28±2 d of age were assigned randomly to one of the following diets with 5 replicates:corn-soybean basal diet with 10,100,175,250 mg/kg of Cu as CuSO4·5H2O.The results showed that 250 mg/kg Cu had a positive effect (P0.05) on average daily gain,daily feed intake and ratio of gain/feed.Compared to 10 mg/kg Cu,higher Cu had significant effect on the apparent digestibility of protein and fat (P0.05).The supplementing of Cu improved amylase and lipase activity in jejunum content and lipase in pancreas (P0.05) and had no effect on intestinal morphology.The liver Cu elevated approximately 4-fold in pigs fed diet with 250 mg/kg Cu compared with pigs fed diet with 10 mg/kg Cu,no increases were observed in pigs receiving the lower level of Cu (100 and 175 mg/kg).Both Fe and Zn contents in kidney and liver were not affected by Cu supplementation.There was no positive effect (P0.05) of Cu supplementation on PepT1 (peptide transporter 1) and SGLT1 (sodium/glucose cotransporter) mRNA abundance in intestinal mucosa.However,higher supplementing level (250 mg/kg) significantly elevated the DMT1 (divalent metal transporter) mRNA abundance in duodenum mucosa.These results suggested that dietary supplementation with 250 mg/kg Cu could improve growth performance,nutrient digestibility and intestinal enzyme activities of weanling pigs.