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Aim: To determine the impact of sodium molybdate treatment, given weekly, on concentrations of Cu in liver, activity of liver enzymes, and weight gain over 4 weeks, in yearling bulls with elevated concentrations of Cu in liver., Methods: The study was carried on two commercial grazing farms in the Otago region of New Zealand in yearling Friesian bulls (n=150 on Farm A and n=49 on Farm B) with mean concentration of Cu in liver >3,000 µmol/kg fresh weight. On Day 0, all animals were weighed and half were systematically allocated to treatment with sodium molybdate (3 mg/kg liveweight on Farm A and 7 mg/kg liveweight on Farm B); the remainder received no treatment (Control). Sodium molybdate was given as a drench weekly for 4 weeks and all animals were weighed again on Day 28. Ten animals on each farm (five from each treatment group) were systematically selected for blood sampling and liver biopsies on Days 0 and 28. Samples were analysed for concentrations of Cu in plasma, vitamin B 12 in serum, activities of γ-glutamyl transferase, aspartate aminotransferase and glutamate dehydrogenase in serum, and concentrations of Cu and vitamin B 12 in liver. Separate multivariable linear models were used to compare the change in outcome variables between Days 0 and 28 between bulls that had been drenched with sodium molybdate or not., Results: On Farm A, mean concentrations of Cu in liver on Day 28, as a percentage of concentrations on Day 0, for the control group was 55 (95% CI=40-73)% and for the treatment group was 73 (95% CI=43-111)%. On Farm B, the equivalent mean for the control group was 75 (95% CI=42-131)% and for the treatment group was 85 (95% CI=38-134)%. The multivariable linear models indicated that the changes in concentrations of Cu in liver, activities of liver enzymes and weight gain between Days 0 and 28 did not differ between the bulls treated or not with sodium molybdate (p>0.18)., Conclusions and Clinical Relevance: Treatment with sodium molybdate in one bolus at weekly intervals for 4 weeks did not affect concentrations of Cu in liver, activity of liver enzymes or weight gain in animals with high concentrations of Cu liver on two farms.

Aim: To determine the minimum number of samples required to obtain a robust estimate of the Cu and Se status of dairy herds, as assessed by determining liver Cu and serum Se concentrations., Methods: Results were collated from analyses of samples of liver from 18 dairy herds and serum from 19 herds, for concentrations of Cu and Se, respectively. All herds were in either the Manawatu or Rangitikei regions of the North Island of New Zealand. Data were used to determine the required sample size for each herd; firstly to estimate the population mean with 90% confidence with a precision of 27.5 nmol/L for Se in serum, and 100 μmol/kg fresh weight (FW) for Cu in liver; and secondly to ensure that the 90% CI of the sample mean did not include specified thresholds for concentrations of Se or Cu., Results: For Se concentration in serum, the SD of each batch varied from 0.5-147 nmol/L, and for Cu concentration in liver, the SD varied from 173-829 μmol/kg FW. For Se, the minimum sample size required to estimate the population mean to within 27.5 nmol/L with 90% confidence was >10 for 13/19 batches. For Cu, the minimum sample size required to estimate the population mean to within 100 μmol/kg FW was >10 for 17/18 batches. When estimating required sample size based on 90% CI and a threshold value, the minimum sample size to confirm the population mean of Se was >140 nmol/L was four in 17/18 batches where the sample mean was>140 nmol/L. For concentrations of Cu in liver, ≤8 samples would have been sufficient for a threshold of 45 μmol/kg FW in 16/18 batches. For the 95 μmol/kg threshold, the minimum required was 12. For the threshold of 300 μmol/kg FW, 6/17 batches with a mean >300 μmol/kg FW required ≤ 20 samples., Conclusions: From this dataset of 21 herds, the sample size recommendation for ensuring that the population mean of Se concentration was not below the marginal threshold was similar to previous recommendations. For Cu concentrations in liver, the estimated sample size recommendations for ensuring that the population mean was not below the marginal threshold was much larger than currently recommended., Clinical Relevance: In dairy cattle, five to six blood samples per group should be taken to determine Se status, and to effectively monitor Cu status a minimum of 12 liver samples should be taken, preferably in the autumn.

In the goat, diagnosis of copper (Cu) deficiency is often based on measurement of Cu in serum or plasma. Previous research in cattle and sheep has shown that these values are not interchangeable, but data for goats have not been published. Paired serum and heparinised plasma samples taken from 119 goats in eight herds were tested for Cu concentration. Plasma and serum Cu were significantly correlated (r=0.95). On average serum Cu was 3.5 μmol/L lower than plasma Cu, but this difference was related to Cu status (r=0.45). Mean serum Cu concentration was 83% of plasma Cu, with the 95% limits of agreement ranging from 66% to 100%. Similar to cattle and sheep, individual variability in Cu loss during clotting is too great for serum Cu to be used as a measure of Cu status in goats., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Aim: To determine the effect of parenteral supplementation with Cu on the activities of hepatic enzymes in deer with adequate concentration(s) of Cu in liver and serum., Methods: Male red deer aged 12 months were given either 0.8 or 1.6 mg of Cu/kg of a S/C injectable Cu formulation or no Cu (n=10/group) on 03 December 2007 (Day 0). Concentration of Cu in liver was measured in biopsies taken on Days -13 and 17, and that in serum on Days 0, 1, 3 and 17. Liver enzymes, gamma-glutamyl transferase (GGT) and glutamate dehydrogenase (GDH), were measured in serum on Days 3 and 7. Deer were weighed on Days 0 and 17., Results: The mean concentration of Cu in liver was 203 (range 50-460) μmol/kg fresh weight (FW) on Day -13 and that in serum was 14.8 (range 7.8-25.0) μmol/mL on Day 0. Concentrations of Cu in liver were adequate (>100 μmol/kg FW) in 25/30 deer. Injection with 0.8 mg of Cu/kg resulted in elevated mean concentrations of Cu in serum to 20.3 (range 14-26) and 17.4 (range 12-26) μmol/mL on Days 1 and 3 respectively, whereas 1.6 mg/kg resulted in mean concentrations of Cu in serum of 26.7 (range 20-42) and 19.3 (range 16-25) μmol/mL on Days 1 and 3 respectively (p<0.05). The mean concentration of Cu in liver on Day 17 was 1,460 (range 1,000-2,300) after injection with 0.8 mg/kg, 1,666 (range 1,100-2,200) after 1.6 mg/kg, and 280 (range 75-420) μmol/kg FW in controls. Injection with 1.6 mg Cu/kg, but not 0.8 mg/kg, resulted in an increase in the activity of GGT (p=0.05), and in 4/10 deer a marked change in the activity of GDH, between Day 3 and Day 7, indicating that injection of copper induced a moderate degree of subclinical hepatopathy. Mean liveweight gain (g/day) to Day 17 was 105.9 (range -176 to 411) in control animals and 64.7 (range -59 to 176) 64.7 and -61.7 (range -294 to 117) in deer given 0.8 and 1.6 mg/Cu/kg respectively (p=0.003)., Conclusions: S/C administration of 1.6 mg of Cu/kg in deer which had adequate Cu status pre-treatment resulted in elevated activities of GGT and GDH in serum, indicating hepatopathy, and both this dose rate and 0.8 mg/kg resulted in reduced liveweight gain post-treatment compared with untreated controls., Clinical Significance: Cu supplementation should not be administered when the Cu status of deer is adequate or unknown.

Aim: To evaluate the relationship between the concentration of elemental Cu and activity of caeruloplasmin (CP) in the plasma and serum of farmed red deer (Cervus elaphus)., Methods: Paired serum and heparinised plasma samples taken from 142 deer from 10 herds in the North Island of New Zealand were tested for the concentration of elemental Cu and activity of CP. Additionally, liver biopsy samples were taken from 49 of the deer (all one herd). The relationship between individual results for the concentration of elemental Cu and activity of CP was examined using a segmented model, with random slope and intercept and threshold., Results: The activity of CP in plasma and that in serum were strongly correlated (R2=0.994), with a high level of agreement. The concentration of Cu and activity of CP also showed a strong association (R2>0.88 for both plasma and serum), although there were significant differences between herds, with the segmented regression model providing the best fit rather than a linear mixed model. Using the current thresholds for elemental Cu, the relationship between the concentration of Cu and activity of CP suggested that an activity of CP of <4 IU/L indicates Cu deficiency, and that deer with an activity of CP of >or=7 IU/L have adequate Cu status., Conclusions: The relationship between elemental Cu and CP is markedly different from the simple linear relationship previously reported for cattle. Above a threshold of 10 IU/L, the rise in elemental Cu per unit increase in activity of CP was 50% of that below 10 IU/L. This is probably because above this threshold the oxidative activity of CP per enzyme unit was greater., Clinical Relevance: The association between concentration of Cu and activity of CP was robust enough to define provisional thresholds for determining Cu status. However, the proximity of the deficiency threshold to the limit of detection of the CP test, the effect of herd on the association, and the inherent disadvantages of using an enzymatic system, mean that activity of CP should not be used routinely to estimate Cu status in deer. When assessing the Cu status of deer based on blood samples, the concentration of Cu in plasma or serum should be used.

Aim: To determine the relationship between the concentrations of Cu in plasma and serum in red deer, and to compare this relationship with those previously reported in cattle and sheep., Methods: Paired serum and heparinised plasma samples from 114 red deer from 10 herds (n=6-20 per herd) were analysed for concentrations of Cu. Samples were collected either at slaughter (n=84; eight herds) or by jugular venepuncture (n=30; two herds). Thirty-nine of the samples taken at slaughter were from adult hinds from four herds, while other samples were taken from 10-14-month-old males, except for one herd (10 samples) where an equal number of 8-9-month-old males and females were sampled. The effect of age, gender and herd on the relationship between concentrations of Cu in plasma and serum was assessed using univariate ANOVA. The individual results for concentrations of Cu in serum were compared with those in plasma, using limits-of-agreement plotting., Results: The mean concentration of Cu in plasma was not significantly different from that of serum (0.048; 95% CI=-0.14 to 0.24 micromol/L). There was no effect of age, sex or herd on this relationship., Conclusions: In deer, there was no significant difference between concentrations of Cu in plasma and serum regardless of age, sex or herd of origin., Clinical Relevance: In contrast to the situation in cattle and sheep, the concentration of Cu in serum can be used interchangeably with that in plasma for the estimation of concentration of Cu in blood of red deer.

Aim: To assess the relationship between concentrations of copper in serum and plasma in sheep., Methods: Concentrations of Cu were measured in paired serum and heparinised plasma samples collected from 110 sheep in nine flocks. Linear regression was used to evaluate whether flock or gender had a significant effect on the association between concentrations of Cu in serum and plasma. The individual results for concentrations of Cu in serum were then compared with those from plasma, using correlation and limits of agreement plotting., Results: Concentrations of Cu in serum ranged from 7.3 to 22 (mean 14.0) micromol/L, while concentrations in plasma ranged from 9 to 27 (mean 16.3) micromol/L. On average, concentrations of Cu in serum were 2.3 micromol/L lower than in plasma. Over the range of values seen in this study, concentrations of Cu in plasma and serum were significantly correlated (r=0.89), and mean concentrations in serum were 87% of those in plasma. There was no effect of flock or gender on the relationship between concentrations of Cu in serum and plasma. Despite the significant correlation, there was marked variability between individual samples in the proportion of Cu that was lost during clotting, with the 95% limits of agreement for serum Cu ranging from 70 to 104% of the plasma concentration., Conclusions: As in cattle, the individual variability in the loss of Cu during clotting in sheep is too great for concentration of Cu in serum to be used as a substitute for that in plasma., Clinical Relevance: When assessing the blood Cu pool as part of the diagnosis of Cu-responsive disease in sheep, the concentration of Cu in plasma should be measured in preference to that of serum. We suggest that a range of 4.5 to 9 micromol/L in plasma be used to define marginal Cu status in sheep.

In cattle, sequestration of copper (Cu) occurs during the clotting process so that serum Cu concentrations are markedly lower than plasma Cu. It has been suggested that all of the Cu lost during clotting is caeruloplasmin (CP). This study used paired samples from 125 cattle to assess whether this assumption was correct. The regression equations for plasma CP activity against plasma Cu concentration and serum CP against serum Cu had significantly different intercepts suggesting that at zero CP activity the amount of Cu remaining was dependent on sample type. Furthermore, the difference between serum and plasma Cu was unrelated to Cu status, whereas the difference between serum and plasma CP was related to Cu status. The regression equation for the loss of CP activity against change in Cu concentration had an intercept that was different from zero, indicating that a reduction in Cu concentration could occur even if CP activity was unaffected by clotting.

Case History: A 400-cow dairy unit in the Waikato suffered a severe outbreak of facial eczema (FE) despite consistent zinc supplementation and significantly elevated serum zinc concentrations., Clinical Findings: FE prevention had begun in mid-January 2006, via zinc sulphate supplementation in the water. Photosensitisation was reported on 06 April, contemporaneous with a marked increase in the number of Pithomyces chartarum spores in pasture samples. Within 10 days of this first clinical case, 100 affected cows had been dried off, eight of which subsequently died or were culled. Blood samples were collected from ten affected cows; all had serum zinc concentrations >17 micromol/L, and eight had gamma-glutamyl transferase (GGT) activities >200 U/ml, indicating that the photosensitisation was secondary to liver damage, i.e. probably FE. Further investigation identified that this herd had been receiving excess copper supplementation; 4/6 culled cows had liver copper concentrations within or above the marginal range for copper toxicity., Diagnosis: Severe FE despite zinc supplementation that increased serum zinc concentrations above recommended levels., Clinical Relevance: As zinc supplementation significantly reduces apparent copper status, copper supplementation is often used to counteract this. Previous excess copper intake may reduce the efficacy of zinc in preventing FE, thus copper intake should be assessed prior to the start of zinc supplementation.

The ratio of caeruloplasmin activity to copper concentration (CP:Cu) has been suggested as a more accurate determinant of the requirement for additional copper than plasma or liver copper concentrations. Although this test has no peer-reviewed evidence of efficacy, it has been used by a large number of UK veterinarians. However, the available test uses a serum caeruloplasmin (sCP) activity to plasma copper (pCu) concentration ratio which, because of the preferential loss of caeruloplasmin during clotting, is likely to underestimate the true CP:Cu, although it has been suggested that the marginal range accounts for this. This study was undertaken to evaluate the effect of using serum copper (sCu) rather than pCu concentrations in calculating CP:Cu. Using sCu rather than pCu increased CP:Cu by more than was accounted for by the marginal range. Of 48 cattle which were reported as 'low' using sCP:pCu, 22 were 'normal' when sCu was used instead of pCu. All herds with 'marginal' or 'low' mean CP:Cu when the sCP:pCu concentration ratio was used had 'normal' ratios when sCu was used instead of pCu.

Aim: To evaluate the effect of test, either copper (Cu) concentration or caeruloplasmin (CP) activity, and sample type, either serum or plasma, on the diagnosis of blood Cu status in cattle., Methods: Paired serum and heparinised plasma samples taken from 125 cattle in 13 herds were tested for Cu concentration and CP activity. The individual results for serum Cu concentration and serum and plasma CP activities were compared with the plasma Cu concentration results, as were their diagnostic values as determined by reference ranges, i.e. 'marginal', 'adequate', 'excess'., Results: The overall mean serum Cu concentration was 2.92 micromol/L lower than the mean plasma Cu concentration; however, there was significant variability between individual samples, and the 95% limits of agreement ranged from 0.44 micromol/L more to 6.28 micromol/L less. The relationship between CP activity and plasma Cu concentration was less variable; the 95% prediction interval for plasma Cu concentration from CP activity was +/- 2.8 micromol/L, and was unaffected by whether CP activity was measured in plasma or serum. Using the threshold currently recommended for 'marginal' status of <8.0 micromol/L for serum Cu concentration identified a significantly different population of cattle than a threshold of <9.0 micromol/L for plasma samples. Altering the threshold to <7.0 micromol/L for serum Cu concentration produced better agreement. For CP activity, a threshold of 15 IU/L for both serum and plasma identified the same population as a threshold of <9 micromol/L for plasma Cu concentration., Conclusions: Serum Cu concentration is not a suitable substitute for plasma Cu concentration for the detection of 'marginal' blood Cu status in cattle as the individual variability in the apparent loss of Cu during clotting is too great. In this study, CP activity, in both serum and plasma, was found to be a suitable substitute for the detection of 'marginal' blood Cu status., Clinical Relevance: The use of serum Cu concentration rather than plasma Cu concentration in the diagnosis of Cu responsive disease in cattle needs to be re-evaluated as does the way in which individual sample results are used in such tests.

The reference ranges for blood copper (Cu) concentration used by SAC and the UK Veterinary Laboratories Agency have been developed for use with plasma only. Copper sequestration into the clot, primarily in the form of caeruloplasmin, means that serum Cu concentrations are significantly lower than plasma Cu concentrations in the same animals. Serum Cu is commonly used as a surrogate for plasma Cu by applying a proportional conversion factor. However the results of these two studies, which involved a total of 104 pairs of serum and Cu samples from 84 Holstein cattle in one herd, suggest that serum Cu concentrations should not be used in this way. First, the reduction in Cu concentration during clotting was unrelated to initial plasma concentration (varying from 8-59%), which suggests that using a proportional conversion factor is incorrect. Secondly, the correlation between plasma and serum Cu concentration, while significant, was relatively low (r2 proportional 0.6), with the individual variation in Cu loss during clotting ranging from 1.5 to 10.8 micromol/L (mean 4.9 micromol/L). Although this study was undertaken using samples from normocupraemic lactating Holstein cattle in one herd, the data strongly suggest that plasma rather than serum Cu should be used to estimate Cu status unless the laboratory used has developed a reliable serum reference range. We also found that factors other than plasma Cu concentration had a significant effect on loss of Cu during the clotting process. However, no significant effect on the loss of Cu during clotting or by varying the incubation/storage time and temperature of bloods prior to clot removal was noted, nor was there any significant effect of age or Cu intake. Furthermore, when the loss of Cu during clotting was examined in the same heifers at two different timepoints, no correlation between the results was found. Further research is required to establish the factors that affect the loss of Cu during clotting.

The article focuses on a study to assess the relationship between concentrations of copper in serum and plasma of sheep. Linear regression was used to evaluate if the flock or gender had any effect on the association between concentrations of copper in plasma. The individuals were then compared using correlation and limits of agreement plotting. It was found that the concentrations in plasma were more as compared to serum and there was no flock or gender relationship found between concentrations of serum and plasma.