Rates of change in liver copper concentration in cattle given a copper-deficient diet, with or without pre-treatment with tetrathiomolybdate, for evaluation of two parenteral copper supplements.

AIM: To minimise the impact of initial variation in liver copper (Cu) on assessments of Cu supplements for cattle in depletion/repletion experiments. METHODS: Efficacy of two Cu injections was assessed with 18 calves, weighing 200-250 kg, given a Cu-deficient barley diet, containing 4.1 mg Cu/kg dry matter (DM) and added molybdenum (3 mg/kg) and sulphur (3 g/kg). Initial liver biopsy Cu ranged from 3.15-14.17 mmol/kg DM and nine calves with the highest values were given three subcutaneous injections of 235 mg tetrathiomolybdate (TTM) after 42-46 days depletion to lower liver Cu. Untreated (L) and TTM-treated (H) calves were ranked separately for liver Cu after 50 days depletion and allocated to one of three treatments: 100 mg Cu given subcutaneously as CuCaEDTA in either a paraffin (CuP) or aqueous base (CuA) after 56 days depletion (Day 0) or no injection (O). Thereafter, plasma and liver biopsy Cu were measured every 2-4 weeks for 16 weeks. Responses in liver Cu to Cu injections were compared with and without loge transformation and by linear regression. RESULTS: Prior to Cu injection, the fractional decline in liver Cu concentration (FDLCu) after 50 days depletion was 0.64 (SE 0.066) and 0.80 (SE 0.090) in H and L calves, respectively (p=0.09) and mean liver Cu did not differ on Day -6 (6.65 (SE 0.516) and 4.91 (SE 0.681) mmol/kg DM, respectively). Mean plasma Cu was higher in H than L calves on Day 0 (16.6 (SE 0.52) and 13.3 (SE 0.49) µmol/L, respectively (p<0.001)). Rates of decline in loge liver Cu between Days 0-84 in treatments L and H were: 0.0138 and 0.0071 for Groups O; 0.0033 and 0.0016 for Groups CuP; 0.0073 and 0.0049 for Groups CuA (pooled SE 0.0014) mmol/kg DM/day, respectively. Between Days 84-114, FDLCu was uniformly high across experiments and groups (0.59 (SE 0.042)). Cu injections did not affect plasma Cu, which remained 3.1 (SE 0.41) umol/L higher in Experiment H than in L (p=0.017). CONCLUSIONS: The use of rates of change in liver copper concentrations improved the assessment of efficacy for two parental copper supplements and that of pre-treatment with tetrathiomolybdate, which, contrary to expectation, slowed Cu turnover by mechanisms that remain unclear.

Responsiveness of prehaemolytic copper poisoning in sheep from a specific pathogen-free environment to a relatively high dose of tetrathiomolybdate.

Efficacy of ammonium tetrathiomolybdate (3.4 mg/kg LW, TTM(3.4)) was monitored in nine, specific pathogen-free sheep with mild-to-severe, prehaemolytic copper poisoning (pre-HCP). Five sheep were given three subcutaneous injections over seven days and four began a shorter, five-day course four days later. Plasma bile acid (BA) and glutamate dehydrogenase (GDH) had fallen significantly after six days but BA briefly rose again between days 10 to 18. Aspartate aminotransferase (AST), δ-glutamyl transferase (GGT) were later to decline but presented little evidence of hepatotoxicity by day 45. Erythrocyte superoxide dismutase (ESOD) was rapidly inhibited, activities falling by 65 per cent within three days and taking 25 days to recover. Trichloroacetic (TCA)-insoluble copper increased by 6 to 8 µmol/l after three days but had largely disappeared by day 18. Six lambs had become hypercupraemic by day 18 due to a rise in TCA-soluble Cu. ESOD activity fell again by 188 ± 29.5 U/gHb between days 25 and 45 but had largely recovered by day 62. TTM(3.4) gave better control of hepatotoxicity than TTM(1.7) had done in more severely affects cohorts but at the expense of greater inhibition of ESOD. Treatment of pre-HCuP should rely more on reducing copper absorption until more is known of the side effects of TTM on cuproenzymes.

Residual effects of Mycobacterium avium infection on susceptibility of sheep to copper toxicity and efficacy of treatment with tetrathiomolybdate.

Posthaemolytic copper poisoning (post-HCP) in one of six, one-year-old, uninfected sheep (group O) on a Mycobacterium avium experiment prompted an evaluation of copper status and hepatotoxicity in 17 surviving cohorts. Group O had higher mean plasma Cu and δ-glutamyl transferase (GGT) activity and more variable bile acid (BA) concentrations and glutamate dehydrogenase activities (GDH) than two groups infected with M avium soon after birth. Ammonium tetrathiomolybdate (TTM; 3 x 1.7 mg/kg LW) was given subcutaneously over seven days and the pelleted, complete diet replaced by hay, low in copper. Plasma BA immediately declined and was followed by GDH, but erythrocyte superoxide dismutase activity (ESOD) became severely inhibited and took 18 days to recover. Plasma BA and GDH rose sharply after 18 days in uninfected sheep and they became hypercupraemic. TTM treatment was repeated from day 42 and had removed all group differences by day 110 but only after further inhibition of ESOD. M avium infections probably lessened the severity of pre-HCP by reducing copper retention but may predispose grazing livestock to hypocupraemia. The capacity of TTM to reduce liver Cu has probably been overestimated and side effects on cuproenzyme activity underestimated.

Relationships between the concentrations of trichloroacetic acid-soluble copper and caeruloplasmin in the serum of cattle from areas with different soil concentrations of molybdenum.

The concentrations of trichloroacetic acid (tca)-soluble copper and caeruloplasmin were determined in 345 serum samples taken from cattle in March 1998 by eight Scottish Agricultural College veterinary disease surveillance centres serving areas with soils ranging from being 'high' in molybdenum (Thurso) to 'low' (Perth and St Boswells). The mean concentrations varied significantly between the centres, with Thurso having the lowest values for both variables. There were strong linear regressions (r>0.8) between caeruloplasmin and tca-soluble copper for each centre but no significant differences in slope or intercept between the areas with the highest and lowest soil molybdenum, and the pooled regression accounted for 88 per cent of the variation. The distribution of the ratios of caeruloplasmin to tca-soluble copper, unlike those of the individual variables, was not normal, and 70 per cent of the values fell within 10 per cent of the mean ratio of 20.3 mg/micromol and close to the 22 mg/micromol copper expected in pure caeruloplasmin. Low ratios were generally associated with low tca-soluble copper. Ratios above 24 were found in 8 per cent of the samples and were probably attributable to acute-phase reactions and the non-specificity of the assay for caeruloplasmin.

Predicting the risk of cobalt deprivation in grazing livestock from soil composition data.

The objective was to derive predictive equations for acetic acid-extractable cobalt (A-ECo) in soils so that extensive national databases for total (T) Co in soils and stream sediments could be converted to 'plant available' concentrations for the purpose of predicting risk of Co deficiency in grazing livestock. Data on the chemical and physical properties of 103 soils from 15 different parent materials and 54 soil series in England and Wales were used. Ranges for the mean values for parent materials were: TCo, 5.0-20.4 and A-ECo, 0.20-1.30 mg kg-1; percentage (P) A-ECo, 3.4-13.5; soil manganese (Mn) 268-1174 mg kgDM-1; pH, 3.7-8.0. There were significant effects of parent material on all parameters with Chalks, Old and New Red Sandstones particularly low in A-ECo. Multiple linear regression yielded the following equation for predicting A-ECo, which accounted for 56% of the variance with 12 outliers, including the lowest pH values, omitted: [figure: see text] REML was used on the complete, unbalanced, log-transformed data set to fit a Generalised Mixed Model with parent material as random effect and soil Mn and pH as fixed effects; the effect of parent material was no longer significant. It was concluded that A-ECo can be satisfactorily predicted for most soils in England and Wales from TCo, TMn and soil pH.

The effects of breed, neonatal age and pregnancy on the plasma copper status of goats in Oman.

There is a history of copper deficiency in grazing Omani livestock and the copper status of three economically important goat breeds, Jabal Akhdar (JA), Batina (B) and Dhofari (D) were therefore compared in October/November (cool season) and June (dry season) in a penned flock given a plentiful dietary supply of copper. In the cool season, 62 lactating does (5 JA, 12 B and 33 D), their 0-5-day-old kids (17 JA, 19 B and 22 D) and 25 dry does (17 JA, 5 B and 3 D) were blood sampled. In the dry season, the does sampled were either barren (15 JA, 16 B and 13 D) or pregnant (9 JA, 13 B and 33 D). The samples were analysed for total copper (TCu) and trichloroacetic acid (TCA)-soluble copper (TCA-sol Cu). There were no effects of breed on TCu or TCA-sol Cu in the cool season, the overall means being 0.75 (SE 0.049) and 0.59 (SE 0.052) mg/L. The mean TCu was low in kids at birth (0.59 mg/L) but had increased to 0.86 mg/L by 4 days of age (p < 0.001); breed differences were found (p < 0.002), the pooled values for JA, B and D being 0.77, 0.59 and 0.68 (SE 0.033-0.044) mg/L, respectively. By the dry season, the mean TCu had risen in barren does to 0.96 (0.045) mg/L but not in pregnant does (0.76 (0.047) mg/L: p < 0.002) and breed differences had emerged, the mean for D being 20% lower than those for JA and B (p < 0.05). The highest TCu values were found in the 7-month-old kids in June ( 1.17 (0.039) mg/L) but the breeds did not then differ. Some effects on TCA solubility were found but were considered unreliable. Breed effects may have been diminished by the generous supply of copper and early stage of lactation studied.

Effects of dietary molybdenum on nematode and host during Haemonchus contortus infection in lambs.

The addition of molybdenum (0.05 mmol kg-1 dry-matter) to the diet of lambs given a trickle infection of Haemonchus contortus larvae (500 third stage larvae d-1 over six weeks) reduced mean faecal egg counts (epg) from 3952 to 2312 +/- 402 by 32 days (P less than 0.02) and greatly reduced the mean number of worms recovered from the abomasum 14 days after infection ceased (907 compared with 4167: P less than 0.01). Infection reduced haemoglobin concentrations less in lambs given molybdenum although the difference was small relative to the reduction in worm burden. Lambs not given molybdenum had low intraepithelial mast cell counts in the abomasal mucosa and less abomasal hypertrophy than expected from abomasal parasitism. Molybdenum did not consistently reduce the copper status of the host or the parasite. Previous exposure to molybdenum greatly reduced protein but not proteinase activity in, or secreted by, adult worms cultured for eight hours. It is suggested that molybdenum either increased the inflammatory response which preceded worm rejection or that it indirectly enhanced that reaction by reducing the effectiveness of copper-dependent, anti-inflammatory enzymes in the gastrointestinal mucosa.

Effects of dietary molybdenum on nematode and host during Trichostrongylus vitrinus infection in lambs.

The addition of molybdenum (0.05 mmol kg-1 dry matter) to the diet of lambs exposed for four weeks to a trickle (2500 third stage larvae per day) infection with Trichostrongylus vitrinus reduced the number and length of adult worms retrieved from the small intestine 11 days later: both effects were particularly marked in female worms from female lambs (P less than 0.01). Worms from lambs given molybdenum contained less proteinase enzyme activity and secreted less proteinases in culture irrespective of the sex of the host. Pathogenicity was not attenuated by molybdenum. Damage to the intestinal mucosa was severe in both dietary groups but infected females given molybdenum developed lower plasma albumin concentrations and lighter dressed carcases than those not given molybdenum. Neither the effects on the parasite nor those on the host could be attributed simply to molybdenum-induced copper depletion, using conventional measures of copper status. Molybdenum may be toxic to T vitrinus but may also facilitate or enhance the inflammatory process limiting larval establishment or increasing parasite rejection.

Recent developments in trace element metabolism and function: trace elements, disease resistance and immune responsiveness in ruminants.

Evidence for the influence of trace elements on disease resistance in ruminants is reviewed with emphasis on susceptibility to infection in vivo during the more common deficiencies (copper, selenium and cobalt). Copper deficiency associated with increases in pasture molybdenum increased the susceptibility of lambs to microbial infections. Under experimental conditions, dietary molybdenum decreased the establishment of abomasal and intestinal nematodes but not their pathogenicity to lambs. Molybdenum may enhance inflammatory responses leading to parasite rejection by the host. Decreased incidence of metritis in selenium-treated dairy cows provides a rare example of an association between selenium deficiency and decreased disease resistance. Improved antibody responses following selenium administration have also been found in sheep. Cobalt deficiency has reduced lamb survival and increased susceptibility to parasitic infection transiently in cattle and lastingly in sheep. In copper-, selenium- or cobalt-deficient sheep and cattle, there are many reports of impaired leucocyte and lymphocyte responses to in vitro challenges, but their relevance to disease resistance in vivo is unproven. Disease resistance may have priority for limited micronutrient supplies, leaving other processes vulnerable.

An assessment of the efficacy and safety of selenium and cobalt included in an anthelmintic for sheep.

A trial was devised to assess whether the administration of selenium and cobalt together with the anthelmintic mebendazole (Ovitelmin S&C) was safe and could improve the supplies of selenium and cobalt for adult sheep fed a whole grain diet, low in both elements, which produced a steady decrease in blood glutathione peroxidase (GSHPx) and plasma vitamin B12 concentrations. Ovitelmin S&C, when given orally in a single dose as a suspension containing 0.34 mg selenium/ml, and 0.44 mg cobalt/ml (to provide 0.11 mg selenium and 0.15 mg cobalt/kg liveweight) significantly increased the GSHPx activity in blood. After a second dose given 28 days later the rate of change increased from 2.5 to 3.5 u/g haemoglobin/day. The responses in GSHPx were similar for a preparation which contained twice the concentration of selenium. Ovitelmin S&C increased the concentration of vitamin B12 in the plasma by about 1000 pg/ml for four to seven days after each dose and the increases were similar to those observed in sheep treated with an Ovitelmin preparation containing 45 times more cobalt (providing 6.7 mg cobalt/kg liveweight). After 63 days, liver vitamin B12 concentrations were 43 per cent higher in the cobalt treated than in the untreated groups (P less than 0.01) with no differences among the groups given cobalt. Neither adverse reactions nor signs of toxicity followed the administration of Ovitelmin S&C or Ovitelmin containing the higher concentrations of selenium and cobalt.

Heinz body anaemia in lambs with deficiencies of copper or selenium.

1. The progression of Heinz body anaemia was studied in groups of lambs of low- and high-copper status, produced through breeding or Cu supplementation, when they were transferred from improved pasture to rape (Brassica napus L.) in autumn. Some lambs had previously received selenium by injection. The Cu and Se supplements markedly increased superoxide dismutase (EC 1.15.1.1; SOD) and glutathione peroxidase (EC 1.11.1.9; GSHPx) activities respectively in the erythrocytes, and both supplements had elicited growth responses at pasture. 2. At the time of transfer to rape, lambs not treated with Cu had lower whole-blood haemoglobin (Hb) concentrations and a higher percentage of erythrocytes containing Heinz bodies (6.6 v. 3.7%, P less than 0.01) than Cu-treated lambs: the corresponding effects of Se treatment were similar in direction but lower in magnitude (P less than 0.05). 3. After grazing rape for 2 weeks the mean Hb concentration had fallen by 30 g/l while Heinz body count had increased from 5 to 25%. However, counts were negatively correlated with the initial values and were unaffected by the Cu and Se treatments which maintained high plasma Cu concentrations and SOD and GSHPx activities. 4. The results provide the first evidence that Cu deficiency can induce Heinz body formation and the anaemia in grazing Cu-deficient lambs may be partly haemolytic in origin. The concomitant Se deficiency added marginally to the problem but neither the separate nor combined deficiencies increased the susceptibility of lambs to brassica anaemia.

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.

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

Five groups of three or four crossbred steers, mean initial liveweight 220 kg, were given a diet of barley and hay ad libitum. Each animal received a single oral dose of 0, 5, 10, 20 or 40 g cupric oxide particles. A dose of 5 g cupric oxide particles increased liver copper stores for about 240 days and higher doses increased liver stores for longer but 40 g was no more effective than 20 g (85 mg kg-1 liveweight). Variation among individuals was marked but the highest liver copper concentration recorded (7.59 mmol kg-1 dry matter) produced no biochemical evidence of copper toxicity. Earlier, cupric oxide particles were separated into three fractions, clumps, short rods and long rods and 5 mg kg-1 liveweight of each fraction given to steers of 173 kg mean liveweight. The form of the particles did not affect either their retention in the alimentary tract or the accumulation of copper in the liver.