Effects of intravenously administered tetra-thiomolybdate on the distribution of copper in the liver and kidney of copper loaded sheep: a histochemical study

The effects of intravenously administered thiomolybdate on the liver and kidney of copper loaded sheep were studied using 16 ewes in three groups. Copper, iron and molybdenum concentrations were determined by spectrophotometry and the distribution of copper in the liver and kidney was studied histochemically. Following thiomolybdate administration, the concentration of copper in the liver was reduced, that of molybdenum increased and the concentration of copper and molybdenum in the kidney increased. The reduction of copper concentration in the liver was associated with reductions in the number and size of granules in hepatocytes which stained positively for copper and in the number of Kupffer cells containing positively staining granules. The decrease in the amount of copper in hepatocytes appeared to be greater than that in Kupffer cells. This effect was greatest in the centrilobular zones and least in the periportal zones. The increased concentration of copper and molybdenum in kidney was associated with an increase in the number and size of granules staining positively for copper in the epithelial cells of the proximal convoluted tubules which suggested an uptake of copper-molybdenum complexes by the lysosomes of these cells.     

锌、铜对实验性钼中毒绵羊肝脏、肾脏中钼含量的影响

小尾寒羊20只,随机分为5组,分别口服钼、铜和锌(mg/kg),Ⅰ组30mg/kg钼、Ⅱ组30mg/kg钼+15mg/kg铜、Ⅲ组30mg/kg钼+15mg/kg锌、Ⅳ组30mg/kg钼+15mg/kg铜+15mg/kg锌,Ⅴ组去离子水。试验90d期满,采取肝脏和肾脏,硫酸-高氯酸消化,原子吸收法测定钼、锌、铜含量,结果显示,Ⅰ组肝脏钼含量与第Ⅱ、Ⅲ、Ⅳ、Ⅴ组差异极显著(P<0.01),Ⅱ、Ⅲ组与Ⅳ、Ⅴ组差异极显著(P<0.01);Ⅳ组肝脏铜含量与Ⅰ、Ⅲ、Ⅴ组差异极显著(P<0.01),与Ⅱ组差异显著(P<0.05),Ⅱ组肝铜含量与Ⅰ、Ⅲ、Ⅴ组差异极显著(P<0.01);组间肝脏锌含量均差异极显著(P<0.01)。Ⅰ、Ⅲ组肾钼含量显著高于Ⅱ、Ⅳ、Ⅴ组(P<0.01);Ⅱ与Ⅳ、Ⅴ组差异极显著(P<0.01);肾铜含量Ⅲ组与Ⅰ组差异极显著(P<0.01);Ⅱ、Ⅲ、Ⅳ组与Ⅰ、Ⅴ组差异极显著(P<0.01);各组肾脏锌含量差异均极显著(P<0.01)。结果表明,30mg的钼可使肝脏钼含量显著增加(P<0.01),锌、铜含量降低。锌对肝脏钼有很好的颉抗作用,铜、锌有效地降低了钼在组织中的蓄积。     

Parenteral administration of copper as cerate to cattle and sheep

Extract

Under the grassland farming regime in New Zealand, copper deficiency in cattle and sheep can be effectively controlled by topdressing all the pastures on the farm each year with 5 pounds of copper sulphate per acre. Such treatment removes the deficiency by increasing the copper content of the herbage to a level that is adequate for grazing animals.     

Breed differences in sheep with respect to the accumulation of copper in the liver

Male lambs of the pure breeds Texel, Flemish milk sheep, and Finnish landrace, and of the crossbreds Texel x Friesian milk sheep, Texel x Finnish landrace, Suffolk x Friesian milk sheep, and Finnish landrace x Ile de France were fed hay and concentrates, the latter differing only in the content of copper. At high (+/- 35 ppm) and at moderately low (+/- 10 ppm) copper levels in the concentrate, breed differences in the accumulation of copper in the liver were studied. Hepatic Cu-levels increased significantly in the case of the high Cu-fed animals, but great differences in the metabolism of excessive amounts of dietary Cu exist between the particular breeds and crossbreds. The Texel breed was most susceptible to an excess of dietary Cu and the Finnish landrace was the least susceptible breed, while the Flemish milk sheep and the crossbreds were intermediate. In blood serum, the enzyme activities of sorbitol dehydrogenase, lactate dehydrogenase, gamma-glutamyl transferase, and glutamic oxaloacetic transaminase were increased to a greater or less extent in the high Cu-fed animals, but the correlation with hepatic Cu-content was poor. For the most susceptible breeds, even a level of 10 ppm Cu in a ration with nearly equal amounts of hay and concentrate is insufficiently low for the prevention of Cu-accumulation in the liver.     

Breed differences in sheep with respect to the accumulation of copper in the liver

Male lambs of the pure breeds Texel, Flemish milk sheep, and Finnish landrace, and of the crossbrech Texel x Friesian milk sheep, Texel x Finnish landrace, Suffolk x Friesian milk sheep, and Finnish landrace x He de France were fed hay and concentrates, the latter differing only in the content of copper. At high (± 35 ppm) and at moderately low (± 10 ppm) copper levels in the concentrate, breed differences in the accumulation of copper in the liver were studied‐Hepatic Cu‐levels increased significantly in the case of the high Cu‐fed animals, but great differences in the metabolism of excessive amounts of dietary Cu exist between the particular breeds and crossbreds. The Texel breed was most susceptible to an excess of dietary Cu and the Finnish landrace was the least susceptible breed, while the Flemish milk sheep and the crossbreds were intermediate. In blood serum, the enzyme activities of sorbitol dehvdrogenase, lactate dehydroge‐nase, γ‐glutamyl transferase, and glutamic oxaloacetic transaminase were increased to a greater or less extent in the high Cu‐fed animals, but the correlation with hepatic Cu‐content was poor. For the most susceptible breeds, even a level of 10 ppm Cu in a ration with nearly equal amounts of hay and concentrate is insufficiently low for the prevention of Cu‐accumulation in the liver.     

Effects of concurrent copper deficiency and gastro-intestinal nematodiasis on circulating copper and protein levels, liver copper and bodyweight in sheep

Sheep were rendered hypocupraemic using parenteral ammonium tetrathiomolybdate (ATM). Fifteen thousand third stage larvae of Trichostrongylus axei and T. colubriformis in the ratio 1:1 were administered three times per week for six weeks, starting four weeks after cessation of ATM treatment. The changes in liver and plasma copper (Cu), caeruloplasmin activity, serum proteins, faecal nematode egg counts and total nematode counts were measured in the sheep for 10 weeks after infection. Decreases in liver Cu, plasma Cu and caeruloplasmin activities were detected soon after infection. There was a significant (P less than 0.05) interaction of the effects of Cu deficiency and nematode infection on these changes. Hypoproteinaemia, attributed to serum albumin loss, was demonstrated seven weeks after infection, but this was not associated with the interaction of Cu deficiency and nematode infection. No changes in serum globulins were detected. Although the results support the contention that gastro-intestinal nematodiasis can significantly exacerbate an existing Cu deficiency in sheep, there was no evidence that hypocuprosis would predispose sheep to higher nematode burdens.     

Histochemical study of the accumulation of copper in the liver of sheep

Six Merino sheep were dosed orally with a 0.2 per cent solution of copper sulphate, six others were undosed controls. Liver biopsies were obtained and stained for copper by the p-dimethylaminobenzylidene rhodanine (DMABR), rubeanic acid (RA) and ferricyanide (FCN) methods for examination by light microscopy. The initial and most marked accumulations of copper were found within the hepatocytes of the centrilobular zones. Increased copper loading resulted in copper deposition extending through the midlobular to the periportal zones. The deposition of copper was unequal between hepatocytes and with increasing copper loading isolated hepatocytes became packed with copper containing granules. Copper appeared within Kupffer cells and macrophages of portal triads. The first Kupffer cells to be positively stained and the greatest number of such cells were adjacent to the central veins. Accumulation of copper was demonstrated with hepatocytes at copper concentrations equivalent to 44.3 micrograms copper g-1 liver wet weight. The FCN method provided a more satisfactory demonstration of intracellular copper than the RA technique and the latter was better than the DMABR method. However, the DMABR technique provided the clearest morphological details.     

Safety and effectiveness of cupric oxide particles for increasing liver copper stores in sheep

Five groups of four, housed Cheviot ewes (mean live-weight 50 kg) were given single doses of 0, 2.5, 5, 10 or 20 g cupric oxide particles in gelatin capsules while receiving a diet of marginal copper content based on pelleted oats. After 65 days liver copper concentrations had increased curvilinearly in relation to dose and all ewes given 10 or 20 g cupric oxide particles showed increases of at least 13.4 mmol kg-1 dry matter (850 ppm). Liver copper concentrations had generally declined after 85 days but biochemical and histological evidence of copper toxicity was recorded in one ewe which had received 20 g cupric oxide particles. Despite marked variations between individual sheep a dose of 0.1 g kg-1 liveweight (5 g) was considered to be safe and did not induce clinical copper toxicity in five sheep of the susceptible North Ronaldsay breed given the same basal diet.     

Effect of faunation on ruminal solubility and liver content of copper in sheep fed low or high copper diets

Naturally fauna-free crossbred rams approximately 5 mo old, 49.3 +/- 1.88 kg live weight, were allotted to four groups. Two groups remained fauna-free, whereas the other two groups were faunated. A diet based on corn silage-soybean meal containing either a low or a high Cu concentration (7 or 15 micrograms/g dry matter) was fed ad libitum to the two groups for 197 d. The rams were killed at the end of the experiment and livers and ruminal contents were analyzed for Cu. Faunated sheep grew faster (P less than .01) and as a group consumed 1.53 kg diet dry matter daily compared with 1.34 for the fauna-free sheep. Faunated sheep had lower final liver Cu concentrations (P less than .01), total liver Cu per unit of Cu intake and ruminal Cu solubilities (P less than .01) when fed both low and high Cu diets. The relative hepatic Cu uptake was higher by rams fed the low Cu than by those fed the high Cu diet. The results are interpreted to show that faunation decreased the availability of Cu in sheep fed a corn silage-soybean meal diet. The magnitude of the effect was independent of the Cu concentration in the diet.     

Influence of sex hormones on the subcellular distribution of copper in sheep liver

At the 14th to 16th week of pregnancy the copper content in the maternal liver was low while that in the fetus was high. After ovariectomy there was a decrease in liver copper and ceruloplasmin which was prevented by treatment with oestrogens but not with progesterone. Five groups of sheep were studied: control (barren ewes), pregnant, ovariectomised and ovariectomised treated with oestrogens or progesterone. In all, with high or low liver copper, the nuclear fraction contained the most copper. This suggests a species specificity of the copper metabolism in sheep.     

Selenium levels in the blood, liver, kidney and muscle of sheep after the administration of iron/selenium pellets or soluble-glass boluses

Slow-release devices composed of elemental selenium and iron or a soluble-glass containing selenium, cobalt and copper were effective in significantly raising blood and tissue selenium levels of sheep at pasture. Three out of six animals given a soluble-glass bolus lost them over the four months of the trial. In contrast, all six animals retained their iron-selenium pellets. Selenium levels in the kidney cortex four months after administration of the devices, when peak levels were most likely to occur, were approximately 12.7 micromol/kg (1mg/kg). This value is 50% of the maximum permitted level in edible tissue in New Zealand. There was no obvious pattern of distribution of selenium throughout the liver. Variations in concentration from one site to another were, in some cases, almost two-fold. Therefore the analysis of liver biopsy samples gives only an approximate assessment of the mean hepatic concentration.     

Breed differences in sheep with respect to the interaction between zinc and the accumulation of copper in the liver

In sheep the effects of Zn‐supplementation on the Cu‐accumulation in the liver were studied.

At a relatively high Cu‐level in the feed, dietary Zn reduced the hepatic Cu‐level in the Texel breed and in the crossbreed Texel x Friesian milksheep, but not in the Friesian milksheep breed.

On account of this result it can be concluded that in sheep the mutual interaction between Cu and Zn may be dependent on genetic factors.     

Lactate administration and fatty liver and kidney syndrome development in biotin-deficient chicks.

Two experiments were carried out to determine whether administration of lactate to biotin-deficient chicks induced fatty liver and kidney syndrome (FLKS). 2. The results suggest that increased serum lactate concentrations are a consequence of the syndrome rather than a contributory factor in its incidence. 3. The increase in liver lipids of birds affected by FLKS was not associated with an increase in the specific activity of the hepatic lipogenic enzyme acetyl CoA carboxylase accept when birds developed FLKS spontaneously in experiment 2. 4. Some biotin-deficient chicks did not show physical symptoms of deficiency although mean liver biotin concentrations were low (0.31 microgram/g liver).     

The effect of lupinosis on liver copper, selenium and zinc concentrations in merino sheep

Merino wether weaners were exposed to toxic lupin stubbles for periods of one, two and six to nine weeks, and the effect on their liver copper, selenium and zinc concentrations studied. After the one week period there was a slight decrease in liver copper and selenium concentrations in sheep severely affected with lupinosis. This was attributed to loss of these elements from necrotic cells, and greatly increased quantities of fat in the liver. In the same period liver zinc concentrations declined and were negatively correlated with the degree of liver injury. After two weeks or more of exposure to toxic lupins a positive correlation existed between both the liver copper and selenium concentrations, and the degree of liver injury. Furthermore, total liver copper and selenium levels were also positively correlated with the degree of liver injury. Liver zinc concentrations were negatively correlated with the degree of liver injury. It is suggested that when the liver is under the influence of the toxins causing lupinosis for more than two weeks, it stores copper and selenium, and loses zinc.     

Level of heavy metals in the liver of sheep in experimental poisoning with copper oxide from industrial emissions

The life span and cumulation of heavy metals in the liver of 12 ewes of the Improved Wallachian breed were investigated in relation to feeding pollutants from a copper works. The total intake of copper, iron, zinc, arsenic, cadmium and lead was 466.8; 1253.93; 111.67; 34.75; 0.091; 2.13 mg per experimental animal/day, respectively. The average life span of ewes in the experimental group was 77 days. The difference between the first and the last animal which died from copper intoxication was 18 days. The concentration of copper in the liver of sheep who died from intoxication by this metal (2138.28 +/- 1090.96 mg. kg-1 in dry matter) was significantly higher (p less than 0.01) if compared with the control ewes (212.9 +/- 58.33 mg. .kg-1 in dry matter). In the course of observations no direct dependence between the concentration of copper in the liver and the life span of experimental animals was confirmed. The content of iron and zinc in the liver of experimental animals was within the reference range; the difference in zinc was statistically significant when compared with the control group (p less than 0.01). The intake of pollutants from the copper works increased significantly the concentration of arsenic and lead in the liver of experimental animals (p less than 0.01) if compared with the control group. The amount of cadmium in the liver of experimental ewes was 0.304 +/- 0.22 mg. .kg-1 and in the control ewes 0.285 +/- 0.10 mg.kg-1 in dry matter; no significant difference was determined at the same time.     

Heliotropium europaeum poisoning of sheep with low liver copper concentrations and the preventive efficacy of cobalt and antimethanogen

In a field experiment in the Mallee district of Victoria, Merlno xBorder Leicester ewes and wethers grazed Heliotropium europaeum (heliotrope) over periods of 3 to 4 months in 4 successive years. By the end of the second year 12% (14 of 120) of the sheep had died; after 4 years the loss attributable to heliotrope was between 18% and 35%. Mortality was not affected by intraruminal treatment with cobalt or antimethanogen. At the end of the experiment the highest concentration of copper in the liver was 1.95 mmol/kg wet weight (approximately 413 micrograms/g dry weight). The relatively low mortality from primary heliotrope poisoning and the low concentration of copper in the liver of sheep grazing the plant are discussed in relation to the contrasting situation that prevails in the Riverina area of New South Wales. The importance of local environmental factors in the management of heliotrope grazing by sheep is emphasised, particularly in relation to the number of seasons in which the plant may be a major component of the diet.     

Injection of sheep with organic compounds of copper

Subcutaneous injections of up to 6 mg copper per kg body-weight as copper methionate (A) produced no deleterious effects in ewes. When copper was given in the form of copper calcium EDTA (B), deaths occurred within 48 hours of the subcutaneous injection of 3 or 4 mg copper per kg body-weight. There was centrilobular necrosis of the liver, necrosis of kidney tubules and excess fluid in the pleural and peritoneal cavities accompanied by the release of liver specific enzymes and a rise in the concentration of urea and creatinine in serum. The injection of 2 mg copper per kg body-weight produced a liver lesion at 48 hours with similar serum changes but recovery took place. Liver and kidney lesions occurred when the copper was in the form of diethylamine copper oxyquinoline sulphonate (C). In this case deaths occurred after the injection of 4, 3 or 2 mg copper per kg body-weight and liver damage followed by recovery was caused by 1 mg copper per kg body-weight. All compounds produced an increase in copper oxidase activity and in the concentration of copper in serum and whole blood. Compound A produced a slow increase in plasma copper concentration to a plateau between 5 and 10 days. Compounds B and C produced a rapid initial increase in the first few hours. The concentration fell rapidly for 12 or 24 hours and then more gradually. It is suggested that rapidity of absorption and transfer of copper to the liver and kidneys may be responsible for the toxic effects of compounds B and C.