Concurrent changes in serum vitamin B12 and methylmalonic acid during cobalt or vitamin B12 supplementation of lambs while suckling and after weaning on properties in the South Island of New Zealand considered to be cobalt-deficient

AIM: To compare serum analyses of vitamin B12 and methylmalonic acid (MMA) as indices of cobalt/vitamin B12 deficiency in lambs around weaning. METHODS: Lambs on five properties, considered to be cobalt- deficient, were supplemented with either cobalt bullets, or short- or long-acting vitamin B12 preparations. Blood samples, and in some cases liver biopsies, and liveweights were obtained at monthly intervals. Serum samples were assayed for vitamin B12 and MMA and liver for vitamin B12 concentrations. Pasture cobalt concentrations were measured on three of the properties. RESULTS: Pasture cobalt concentrations were generally maintained below 0.07 microg/g dry matter (DM) on the properties sampled. Growth responses to supplementation were observed on only 2/5 properties, despite serum vitamin B12 concentrations being within the currently used 'marginal' reference range (336-499 pmol/L) for at least 3 months on all properties and in the deficient reference range (0-335 pmol/L) for at least 2 months on all farms except one. Serum MMA concentrations in supplemented lambs were <2 micromol/L, except in those animals sampled 1 month after receiving treatment with a short-acting vitamin B12 injection. Serum MMA concentrations in unsupplemented animals on properties on which no growth response to supplementation occurred generally reached peak levels of between 4 and 7 micromol/L at the nadir of serum vitamin B12 concentration. When a growth response was observed, differences in weight gain between supplemented and unsupplemented lambs occurred as mean serum MMA concentrations increased from 9 to 14 micromol/L. On one property where supplementation commenced before weaning, normal growth rates were maintained despite serum vitamin B12 concentrations of 140 pmol/L and serum MMA concentrations in excess of 40 micromol/L serum. CONCLUSIONS: The possibility that current serum vitamin B12 references ranges for diagnosis of cobalt deficiency are set too high and lead to over-diagnosis of responsiveness to cobalt/ vitamin B12 supplementation is discussed. The suggestion is made that serum MMA concentrations in excess of 9-14 micromol/L will provide a more reliable diagnostic test for cobalt deficiency. However, there was sufficient variation between properties in the relationships between cobalt concentrations of pasture and serum vitamin B12 or MMA concentrations to require more rigorous testing of the reliability of using serum MMA concentration for this purpose. The possibility that differences in rumen fermentation and therefore propionate and vitamin B12 production could be involved is discussed. The measurement of serum MMA and vitamin B12 appears to be of little value whilst the lamb is still suckling. CLINICAL SIGNIFICANCE: Serum MMA concentration may offer advantages over serum vitamin B12 concentrations in the diagnosis of a cobalt/vitamin B12 responsiveness in weaned lambs.     

Dose response effects of long-acting injectable vitamin B12 plus selenium (Se) on the vitamin B12 and Se status of ewes and their lambs

AIM: To determine the effect of increasing doses of long-acting injectable vitamin B12 plus selenium (Se) given pre-mating on the vitamin B12 and Se status of ewes and their lambs from birth to weaning. METHODS: Four groups of 24 Poll Dorset ewes each were injected 4 weeks pre-mating with different doses of a long-acting vitamin B12 + Se product, containing 3 mg vitamin B12 and 12 mg Se per ml. The treatment groups received 5 ml (15 mg vitamin B12 + 60 mg Se), 4 ml (12 mg vitamin B12 + 48 mg Se), 3 ml (9 mg vitamin B12 + 36 mg Se), or no vitamin B12 or Se (control). Twelve of the twin-bearing ewes per group were selected for the study. Efficacy of the product was evaluated from changes in the concentrations of vitamin B12 in serum and liver, and of Se in blood, liver and milk in the ewes during gestation and lactation, and in their lambs from birth to weaning. Pasture samples in paddocks grazed by the ewes and lambs were collected at about 2-monthly intervals from 200-m transects. RESULTS: The flock was Se-deficient, as the mean initial concentration of Se in the blood of ewes was 182 (SE 20.3) nmol/L. Compared with untreated controls, all doses significantly (p < 0.01) increased concentrations of Se in the blood of ewes for at least 300 days. Selenium concentrations in milk were likewise increased throughout lactation, as were those in the blood and liver of lambs. The mean concentration of vitamin B12 in the serum of ewes was initially > 1,000 pmol/L, but this decreased within 28 days to < 460 pmol/L. Treatment with the 5-ml and 4-ml doses raised serum vitamin B12 concentrations of ewes for at least 176 days (p < 0.01), while their lambs had significantly greater concentrations of vitamin B12 in serum and liver for less than 37 days after birth. Tissue concentrations and duration of elevation of both vitamin B12 and Se were proportional to the dose administered. The mean concentrations of Se and cobalt (Co) in the pastures were 32 and 74 microg/kg dry matter (DM), respectively. CONCLUSIONS: Injecting ewes from a Se-deficient flock 4 weeks prior to mating with 48 or 60 mg Se and 12 or 15 mg vitamin B12 increased and maintained the Se status of ewes for at least 300 days, and of their lambs from birth to weaning. The vitamin B12 status of ewes was increased for at least 176 days and that of their lambs for less than 37 days. Due to the proportional nature of the response to increasing dosage, the dose rate of the formulation tested can be adjusted according to the severity of Se and Co deficiency in a flock. CLINICAL SIGNIFICANCE: A single subcutaneous injection of vitamin B12 + Se administered pre-mating to Se-deficient flocks is likely to prevent Se deficiency in ewes and their lambs until weaning, as well as increase the vitamin B12 status of ewes and their lambs until 5 weeks after lambing.     

Growth response to increasing doses of microencapsulated vitamin B12 and related changes in tissue vitamin B12 concentrations in cobalt-deficient lambs

AIM: To investigate growth response of cobalt deficient lambs to increasing doses of microencapsulated vitamin B12, and to measure associated changes in serum and liver vitamin B12 concentrations over 243 days. METHODS: From a flock grazing pastures that had low cobalt (Co) levels (about 0.06 mg Co/kg dry matter), 4-6-week-old lambs (n=137) were assigned to four groups and received either no treatment or a subcutaneous injection of 3.0, 4.5 or 6.0 mg of microencapsulated vitamin B12 on Day 1. At approximately monthly intervals, all lambs were weighed and blood samples were collected from a selection (n=10) of monitor animals, up to Day 243. Liver biopsies were also carried out on the monitor lambs (n=8) on Days 1, 124 and 215. RESULTS: The vitamin B12-treated lambs grew significantly faster (p<0.001) than untreated animals. Liveweights after 243 days were 28, 45, 45 and 47 kg for the untreated, 3.0, 4.5 and 6.0 mg vitamin B12-treated lambs, respectively. Of the initial group of untreated lambs, 68% had to be removed before the end of the trial because of substantial weight loss, but none of the treated animals were similarly afflicted. Serum vitamin B12 concentrations increased in all vitamin B12-treated lambs, reaching a peak at Day 25, and those of the 4.5 and 6.0 mg vitamin B12-treated lambs remained significantly higher (except at Day 124) than the untreated lambs to Day 187. However, at Day 124, but not Day 215, the liver vitamin B12 concentrations of treated lambs were two to three times higher than those of controls. CONCLUSIONS: The growth rates of Co deficient lambs were markedly improved by injection of 3.0, 4.5 or 6.0 mg of microencapsulated vitamin B12, and liveweights were maintained for at least 243 days. Serum vitamin B12 concentrations were related to this growth response; concentrations of <220 pmol vitamin B12/l were associated with a 95% probability that lambs were Co deficient and would thus respond to Co/vitamin B12 supplementation. Based on these data, the current New Zealand reference criteria for Co deficiency should be reviewed. CLINICAL SIGNIFICANCE: An injection of 3 mg microencapsulated vitamin B12 given to lambs at tailing will treat Co deficiency and will increase and maintain liveweights in a flock for up to 8 months.     

Changes in serum concentrations of methylmalonic acid and vitamin B12 in cobalt-supplemented ewes and their lambs on two cobalt-deficient properties

AIM: To determine concurrent changes in serum methylmalonic acid (MMA) and vitamin B12 concentrations of ewes and their lambs on cobalt-deficient properties, subsequent to cobalt supplementation. METHODS: Three experiments were carried out on two farms. Groups of ewes (n=25-50) were either supplemented with cobalt bullets during late pregnancy, 23-47 days before the mean lambing date, or left unsupplemented. In two experiments, lambs from within each group were supplemented directly by vitamin B12 injection at 3-weekly intervals from birth, and in the third experiment by injection with micro-encapsulated vitamin B12 at tailing and 3 months later. Pasture samples were obtained for analysis of cobalt content at each sampling time. Blood samples were obtained and liveweight recorded from ewes and lambs at approximately monthly intervals. On one farm (two experiments), liver and milk samples were obtained from ewes and liver samples from lambs. RESULTS: Serum vitamin B12 concentrations in unsupplemented ewes fell below 250 pmol/L during early lactation in all experiments and mean concentrations as low as 100 pmol/L were recorded. MMA concentration was maintained below 2 micromol/L in serum from supplemented ewes but increased to mean concentrations ranging from 7 to 14 micromol/L at the nadir of serum vitamin B12 concentration during peak lactation. A significant liveweight response to supplementation was recorded in ewes on one property, and the vitamin B12 concentration in the ewes' milk and in the livers of their lambs more than doubled. No liveweight-gain response to supplementation was observed in lambs on this property. Mean serum MMA concentrations in lambs ranged from <2 in supplemented, to 19.2 micromol/L in unsupplemented lambs, and the latter had concurrent serum vitamin B12 concentrations of >300 pmol/L. Pasture cobalt concentration was lowest at 0.04-0.09 microg/kg dry matter (DM) on the property on which responses in lambs occurred but considerably higher (>0.09 microg/kg DM) on the property on which responses in ewes occurred. On the second property, serum vitamin B12 concentrations in lambs at tailing were extremely low (100 pmol/L), irrespective of supplementation of dams with cobalt. Mean serum MMA concentration was increased to 20 and 42 micromol/L in lambs from supplemented and non-supplemented ewes, respectively. Weight-gain response to direct supplementation of lambs with vitamin B12 occurred during suckling in the latter, but not the former. Lambs from ewes supplemented with vitamin B12 showed a much bigger increase in serum vitamin B12 concentrations a month after supplementation than did lambs from unsupplemented ewes (+1,400 pmol/L vs + 650 pmol/L). CONCLUSIONS: Serum MMA concentration gave a more precise indication of responsiveness to vitamin B12 or cobalt supplementation than serum vitamin B12 concentrations in ewes and lambs. Neither very low serum vitamin B12 nor elevated MMA concentrations were necessarily indicative of responsiveness to supplementation in suckling lambs, but the latter gave an early indication of impending responsiveness. Supplementation of the ewe with a cobalt bullet appeared to protect the growth performance of the lamb for 90 days and influence the subsequent serum vitamin B12 response in the lamb to vitamin B12 supplementation. CLINICAL SIGNIFICANCE: Supplementing ewes with cobalt bullets in late pregnancy can improve the vitamin B12 status of their lambs, and modify their response to vitamin B12 supplementation.     

Reduction of the prevalence of footrot on New Zealand farms by vaccination

AIM: To compare serum analyses of vitamin B₁₂ and methylmalonic acid (MMA) as indices of cobalt/vitamin B₁₂ deficiency in lambs around weaning.

METHODS: Lambs on five properties, considered to be cobalt- deficient, were supplemented with either cobalt bullets, or short- or long-acting vitamin B₁₂ preparations. Blood samples, and in some cases liver biopsies, and liveweights were obtained at monthly intervals. Serum samples were assayed for vitamin B₁₂ and MMA and liver for vitamin B₁₂ concentrations. Pasture cobalt concentrations were measured on three of the properties.

RESULTS: Pasture cobalt concentrations were generally maintained below 0.07 μg/g dry matter (DM) on the properties sampled. Growth responses to supplementation were observed on only 2/5 properties, despite serum vitamin B12 concentrations being within the currently used ’marginal‘ reference range (336–499 pmol/L) for at least 3 months on all properties and in the deficient reference range (0–335 pmol/L) for at least 2 months on all farms except one. Serum MMA concentrations in supplemented lambs were <2 μmol/L, except in those animals sampled 1 month after receiving treatment with a short-acting vitamin B12 injection. Serum MMA concentrations in unsupplemented animals on properties on which no growth response to supplementation occurred generally reached peak levels of between 4 and 7 μmol/L at the nadir of serum vitamin B12 concentration. When a growth response was observed, differences in weight gain between supplemented and unsupplemented lambs occurred as mean serum MMA concentrations increased from 9 to 14 μmol/L. On one property where supplementation commenced before weaning, normal growth rates were maintained despite serum vitamin B12 concentrations of 140 pmol/L and serum MMA concentrations in excess of 40 μmol/L serum.

CONCLUSIONS: The possibility that current serum vitamin B₁₂ references ranges for diagnosis of cobalt deficiency are set too high and lead to over-diagnosis of responsiveness to cobalt/ vitamin B₁₂ supplementation is discussed. The suggestion is made that serum MMA concentrations in excess of 9–14 μmol/L will provide a more reliable diagnostic test for cobalt deficiency. However, there was sufficient variation between properties in the relationships between cobalt concentrations of pasture and serum vitamin B₁₂ or MMA concentrations to require more rigorous testing of the reliability of using serum MMA concentration for this purpose. The possibility that differences in rumen fermentation and therefore propionate and vitamin B₁₂ production could be involved is discussed. The measurement of serum MMA and vitamin B₁₂ appears to be of little value whilst the lamb is still suckling.

CLINICAL SIGNIFICANCE: Serum MMA concentration may offer advantages over serum vitamin B₁₂ concentrations in the diagnosis of a cobalt/vitamin B₁₂ responsiveness in weaned lambs.     

Foremilk and bovine mastitis

Abstract

Lambs grazing cobalt-deficient pastures and injected with hydroxocobalamine gained significantly more weight and excreted significantly less methylmalonic acid in the urine than untreated controls. Lambs with liver vitamin B₁₂ levels in the range 0.1–0.2?g/g excreted less than 25 ?g of methylmalonic acid per ml of urine, whereas lambs with liver vitamin B₁₂ concentrations of less than 0.1 ?g/g excreted greater amounts. Lambs in both groups had serum vitamin B₁₂ concentrations less than 500 pg/ml.

No consistent diurnal variation in urinary methylmalonic acid concentrations was found for four lambs studied.

There was a decrease in the methylmalonic acid levels of urine after storage for more than 24 hours which could be prevented by acidification of the urine.

A mean urinary methylmalonic acid concentration greater than 30 ?g/ml for 10 animals randomly selected from a flock would indicate a cobalt deficiency in the flock as a whole.     

A critical evaluation of serum methylmalonic acid and vitamin B12 for the assessment of cobalt deficiency of growing lambs in New Zealand

AIM: To derive reference ranges for serum methylmalonic acid (MMA) for the diagnosis of cobalt/vitamin B12-responsiveness in lambs and critique existing serum vitamin B12 reference ranges. METHODS: Individual animal data from earlier supplementation trials, involving 225 ewes, 106 suckling lambs, 301 lambs during the suckling and post-weaning periods and 414 weaned lambs, for which weight gain to supplementation was observed, were used to derive relationships between serum vitamin B12 and MMA, and liveweight gain. RESULTS: Serum MMA concentrations were rarely elevated above the norm of <2 micromol/L when serum vitamin B12 concentrations were >375 pmol/L, and not elevated into the range where a liveweight response to supplementation occurred (>10 micromol/L) unless serum vitamin B12 concentrations were below 200 pmol/L. Suckling lambs were able to maintain high growth rates despite elevated serum MMA concentrations (>20 micromol/L). CONCLUSIONS: The current reference ranges used in New Zealand for serum vitamin B12 are set conservatively high. Serum MMA concentrations appear to allow better differentiation of a responsive condition than vitamin B12 concentrations. Serum MMA concentrations >13 micromol/L indicate responsiveness to supplementation whilst concentrations <7 micromol/L indicate unresponsiveness. In the range 7-13 micromol/L, variation in response was observed and predictability of response is less certain, but supplementation is advisable. CLINICAL RELEVANCE: The current reference ranges for vitamin B12 responsiveness are conservatively high and lead to over-diagnosis of vitamin B12 deficiency in ill-thriftiness of sheep.     

A comparison of vitamin B12 levels in the liver and serum of sheep receiving treatments used to correct cobalt deficiency

The efficacies of four methods, used for the prophylaxis of cobalt deficiency in sheep as measured by the elevation of liver and serum vitamin B12 levels, were compared in marginally deficient sheep over 14 weeks. The methods used were weekly drenches of either cobalt sulphate or cobalt chelate (EDTA) three-weekly injections of hydroxocobalamin, and ruminal cobalt pellets. On the basis of elevated liver and serum vitamin B12 levels, chelated cobalt was shown to be available to rumen microflora for the synthesis of vitamin B12. However, at no stage were liver and serum vitamin B12 levels of sheep receiving the chelate significantly different from those receiving the same amount of cobalt as the sulphate. After five, three-weekly injections of hydroxocabalamin liver vitamin B12 levels were significantly higher (p<0.01) than for the other treatments, with the exception of cobalt sulphate. Cobalt pellets led to an initial rapid and significant rise in serum vitamin B12 when compared with the other treatments. However, at four weeks there was no significant difference between treatment groups for serum vitamin B12. Fourteen weeks after the administration of cobalt pellets, serum and liver.vitamin B12 levels in this group were not significantly different from those of untreated sheep. At this time, three out of 12 sheep had lost their pellets.     

The efficacy of a subcutaneous injection of soluble Vitamin B12 in lambs

AIMS: To assess the efficacy of a soluble Vitamin B12 injection in lambs by measuring changes in the serum and liver Vitamin B12 concentrations. METHODS: Thirty-six lambs were injected subcutaneously with 2 mg of soluble Vitamin B12 while another group of 36 served as untreated controls. Blood and liver biopsy samples for Vitamin B12 determinations were collected just before the injection and at days 1, 2, 5, 8, 16, 24, 30 and 45. RESULTS: The serum Vitamin B12 concentrations of the Vitamin B12 treated lambs increased rapidly compared to the untreated lambs. Concentrations peaked at day 2, decreased rapidly to day 8, and then decreased more slowly until day 24 when there were no longer differences between the groups. Liver Vitamin B12 concentrations of the Vitamin B12 treated lambs were significantly greater over days 8-24. CONCLUSION: A subcutaneous injection of 2 mg of soluble Vitamin B12 was effective in increasing and maintaining the Vitamin B12 status of lambs for about 24 days. CLINICAL SIGNIFICANCE: This Vitamin B12 product is only effective for preventing cobalt deficiency in lambs for about 4 weeks.     

Boluses of controlled release glass for supplementing ruminants with cobalt

Boluses of controlled release glass containing cobalt and weighing approximately either 60 g or 14.5 g were administered to 22 steers and 21 sheep respectively. The steers were housed and slaughtered at intervals between 17 and 145 days after dosing. The boluses released more than 0.85 mg cobalt daily. In both untreated and dosed animals serum and liver vitamin B12 concentrations were at the upper end of the normal range. Two types of glass were administered to sheep. In five wethers one glass released 0.07 mg cobalt per day, and in 16 grazing lambs a second glass released more than 0.15 mg cobalt per day. Fourteen of the boluses were recovered from the lambs up to 276 days after dosing. The concentration of B12 in serum of lambs increased significantly from a mean +/- sd of 1.64 +/- 0.47 to 2.02 +/- 0.04 ng/ml serum and the concentration in liver from 3.84 +/- 0.85 to 4.99 +/- 0.72 micrograms/g dry weight liver.     

Effect of cobalt deficiency in the pregnant ewe on reproductive performance and lamb viability

Reproductive performance and lamb viability in cobalt sufficient and subclinically deficient ewes, and from ewes experiencing repletion from and depletion into cobalt deficiency, were investigated in two experiments. The sheep were fed a cobalt deficient ration and supplementation was by oral dose according to treatment. The treatments had a significant effect (P less than 0.001) on ewe serum vitamin B12 and methylmalonic acid concentrations. There were no significant effects on ewe liveweight, condition score or conception rate. Cobalt deficient ewes produced fewer lambs and had more stillbirths and neonatal mortalities than cobalt sufficient controls. Lambs from deficient ewes were slower to start suckling (P less than 0.05), had reduced concentrations of serum immunoglobulin G and zinc sulphate turbidity levels (P less than 0.05), and had lower serum vitamin B12 and higher methylmalonic acid concentrations (P less than 0.05), than lambs from cobalt sufficient dams. Cobalt supplementation in either the first or second half of pregnancy only did not fully alleviate these adverse effects.     

Absence of a weight gain response to Vitamin B12 supplementation in weaned dairy heifers grazing pastures of marginal cobalt content

AIM: To obtain information on serum and liver vitamin B12 and urinary methylmalonic acid concentrations as diagnostic tests to predict a weight gain response to supplementation with vitamin B12 in young dairy cattle when grazing pasture of low cobalt content. Methodology. Forty dairy cattle (12 Friesian, 14 Friesian x Jersey and 14 Jersey) were allocated to two equal sized groups, treated and untreated, based on liveweight. At monthly intervals for 14 months, all animals were weighed, their serum and urine sampled, their liver biopsied and the pasture sampled from the paddocks they were grazing and going to graze. Serum and liver were assayed for vitamin B12 concentrations. For the first 5 months of the trial, urine was assayed for methylmalonic acid concentrations. Both washed and unwashed pasture samples were assayed for cobalt concentrations. RESULTS: No weight gain response occurred vitamin B12 supplementation in young growing cattle grazing pasture with a cobalt concentration of 0.04-0.06 mg/kg DM. For 5 months of the trial, liver vitamin B12 concentrations from untreated calves were in the range 75-220 nmol/kg and serum vitamin B12 concentrations were as low as 72 pmol/1. There was no associated growth response to supplementation. CONCLUSION: Further trials involving young cattle grazing pastures with cobalt concentrations less than 0.04 mg/kg DM are required to reliably determine liver and serum vitamin B12 concentrations at which growth responses to vitamin B12 or cobalt supplementation are likely under New Zealand pastoral grazing conditions.     

Ovine white-liver disease (OWLD). Changes in blood chemistry

Changes in blood chemistry were examined in vitamin B12 deficient lambs which developed ovine white-liver disease (OWLD), and were compared with values of cobalt/B12 supplemented lambs on the same pastures, as well as clinically healthy, but sometimes B12 deficient, lambs on other pastures (H). In the OWLD group, signs of hepatic damage were seen concurrently with reduction in weight gain, or 1-3 weeks before, and comprised elevation of serum glutamate dehydrogenase (GLDH) and decrease of phospholipid and cholesterol. Drop of plasma glucose and elevation of gamma GT also came in the earlier phase of the disease. All other blood changes developed later, and were partly regarded as reflections of the inappetence or hepatic injury. The changes included a drop in packed cell volume (PCV) and mean corpuscular volume (MCV), elevation of serum iron, and reduction of total serum protein and urea. Generally Co/B12 supplementation prevented hepatic damage and normalized blood values. The clinically healthy H lambs also showed signs of hepatic damage, especially one year when they were B12 deficient, indicating that simple B12 deficiency causes a moderate liver damage as well. For diagnostic purposes, clinical pathology is recommended mainly on a flock basis.     

Sex-related responses to vitamin B12 and trace element supplementation in prime lambs

Bodyweight, plasma vitamin B12 and blood selenium concentrations were monitored in prime lambs given different forms of supplementation at 2 sites in separate years. At the first site treatment groups comprised control, vitamin B12 injection, selenium given orally and a combination of vitamin B12 and selenium. At the second site cobalt and selenium supplied in a glass bullet was compared with an untreated group. Significant sex-related responses were observed to treatment in terms of bodyweight and at site 2 in plasma vitamin B12 concentrations. A marked bodyweight response to glass bullet supplementation was observed in castrated male lambs but not in ewe lambs. These studies show that sex differences should be considered when investigating trace element deficiencies.     

Growth response to increasing doses of microencapsulated vitamin B12 and related changes in tissue vitamin B12 concentrations in cobalt-de cient lambs

AIM: To investigate growth responses of cobalt-deficient lambs to increasing doses of microencapsulated vitamin B₁₂, and to measure associated changes in serum and liver vitamin B₁₂ concentrations over 243 days.

METHODS: From a flock grazing pastures that had low cobalt (Co) levels (about 0.06 mg Co/kg dry matter), 4-6-week-old lambs (n=137) were assigned to four groups and received either no treatment or a subcutaneous injection of 3.0, 4.5 or 6.0 mg of microencapsulated vitamin B₁₂ on Day 1. At approximately monthly intervals, all lambs were weighed and blood samples were collected from a selection (n=10) of monitor animals, up to Day 243. Liver biopsies were also carried out on the monitor lambs (n=8) on Days 1, 124 and 215.

RESULTS: The vitamin B₁₂-treated lambs grew significantly faster (p<0.001) than untreated animals. Liveweights after 243 days were 28, 45, 45 and 47 kg for the untreated, 3.0, 4.5 and 6.0 mg vitamin B₁₂-treated lambs, respectively. Of the initial group of untreated lambs, 68% had to be removed before the end of the trial because of substantial weight loss, but none of the treated animals were similarly afflicted. Serum vitamin B₁₂ concentrations increased in all vitamin B₁₂-treated lambs, reaching a peak at Day 25, and those of the 4.5 and 6.0 mg vitamin B₁₂-treated lambs remained significantly higher (except at Day 124) than the untreated lambs to Day 187. However, at Day 124, but not Day 215, the liver vitamin B₁₂ concentrations of treated lambs were two to three times higher than those of controls.

CONCLUSIONS: The growth rates of Co-deficient lambs were markedly improved by injection of 3.0, 4.5 or 6.0 mg of microencapsulated vitamin B₁₂, and liveweights were maintained for at least 243 days. Serum vitamin B₁₂ concentrations were related to this growth response; concentrations of <220 pmol vitamin B₁₂/l were associated with a 95% probability that lambs were Co-deficient and would thus respond to Co/vitamin B₁₂ supplementation. Based on these data, the current New Zealand reference criteria for Co deficiency should be reviewed.

CLINICAL SIGNIFICANCE: An injection of 3 mg microencapsulated vitamin B₁₂2 given to lambs at tailing will treat Co deficiency and will increase and maintain liveweights in a flock for up to 8 months.     

A comparison of vitamin B12 levels in the liver and serum of sheep receiving treatments used to correct cobalt deficiency

The efficacies of four methods, used for the prophylaxis of cobalt deficiency in sheep as measured by the elevation of liver and serum vitamin B₁₂ levels, were compared in marginally deficient sheep over 14 weeks. The methods used were weekly drenches of either cobalt sulphate or cobalt chelate (EDTA); three-weekly injections of hydroxocobalamin, and ruminal cobalt pellets.

On the basis of elevated liver and serum vitamin B₁₂ levels, chelated cobalt was shown to be available to rumen microflora for the synthesis of vitamin B12. However, at no stage were liver and serum vitamin B₁₂ levels of sheep receiving the chelate significantly different from those receiving the same amount of cobalt as the sulphate.

After five, three-weekly injections of hydroxocabalamin liver vitamin B₁₂ levels were significantly higher (p < 0.01) than for the other treatments, with the exception of cobalt sulphate.

Cobalt pellets led to an initial rapid and significant rise in serum vitamin B₁₂ when compared with the other treatments. However, at four weeks there was no significant difference between treatment groups for serum vitamin B₁₂. Fourteen weeks after the administration of cobalt pellets, serum and liver. vitamin B₁₂ levels in this group were not significantly different from those of untreated sheep. At this time, three out of 12 sheep had lost their pellets.     

Pathological changes in cobalt-supplemented and non-supplemented twin lambs in relation to blood concentrations of methylmalonic acid and homocysteine.

In a controlled field study of three years' duration we evaluated the effect of cobalt supplementation on pathological changes in cobalt/vitamin B12-deficient Texel twin lambs grazing the same cobalt-deficient pasture. Semi-quantitative evaluation of the histopathology of liver and brain was done on 44 sets of twins. Pathological changes were related to blood concentrations of vitamin B12, methylmalonic acid, and homocysteine. Lesions were mainly confined to the liver and brain. Acute hepatic changes were characterized by steatosis, hepatocytic degeneration, and single cell necrosis. Chronic changes consisted of bile duct proliferation, the presence of ceroid containing macrophages, and fibrosis in the portal triads. Many non-supplemented lambs showed polymicrocavitation and Alzheimer type II reaction in the brain. Polioencephalomalacia was observed in three non-supplemented lambs but was regarded as a secondary lesion. Our results indicate that the main lesions found in cobalt/vitamin B12-deficient lambs are acute and chronic hepatitis. These lesions were associated with low concentrations of vitamin B12 and high concentrations of methylmalonic acid and homocysteine in the blood. The liver lesions were also associated with polymicrocavitation of the brain, probably as morphological evidence of hepatoencephalopathy.     

Serum vitamin B12 and methylmalonic acid determinations in the diagnosis of cobalt deficiency in pregnant ewes

Serum vitamin B12 and methylmalonic acid (MMA) concentrations were used to monitor the development of cobalt (Co) deficiency and repletion from the deficient state in housed pregnant hill sheep. Serum MMA concentrations were less variable and provided a more accurate diagnosis of Co deficiency than serum vitamin B12. This was particularly the case for subclinical disease. However, unlike serum MMA, concentrations of the vitamin in serum could be used prognostically. The most precise diagnosis was provided by serum vitamin B12 and MMA data used in conjunction, but where one technique is to be used, serum MMA determinations are preferred.     

Ovine white-liver disease (OWLD). Vitamin B12 and methyl malonic acid (MMA) estimations in blood

At pasture outlet, mean plasma vitamin B12 varied between 210 and 1,200 pmol/l in 1 month old lambs, 19% of them had values below 250 pmol/l. In those put on OWLD pastures, mean values dropped after 2-4 weeks, and mostly stayed below 150 pmol/l throughout grazing. Plasma methylmalonic acid (MMA) rose above 5 mumol/l 2-8 weeks after outlet, and above 15 mumol/l 4 weeks later. Reduced growth occurred 3-8 weeks after plasma B12 dropped below 150 pmol/l, and 4-6 weeks after MMA rose above 5 mumol/l. Clinical OWLD was most often associated with plasma B12 less than 150 pmol/l and MMA greater than 15 mumol/l. Cobalt fertilization of pastures induced satisfactory plasma B12/MMA values for 3 succeeding years. Elevated plasma B12 was found 3 weeks after Co pellet dosing. The use of Co lick resulted in large individual variations in plasma B12/MMA. The control lambs, which were healthy and grew well on pastures which some years contained marginal/deficient cobalt, had plasma B12/MMA values which varied considerably. One year values indicated functional Co deficiency, but none developed OWLD, and growth was satisfactory, but less than other years. In these lambs, high MMA was not always associated with low B12, or depressed growth. OWLD occurred in Co/B12 deficient lambs, but Co/B12 deficient lambs on other pastures did not develop OWLD.     

The effect of increasing the Vitamin B12 status of Romney ewes on foetal liver Vitamin B12, milk Vitamin B12 and liver Vitamin B12 concentrations in suckling lambs

AIM: To determine the effect of increasing the Vitamin B12 status of the ewe on the Vitamin B12 supply to the suckling lamb. METHODS: The Vitamin B12 status of the ewe was increased during gestation and lactation by three injections of a long-acting preparation of Vitamin B12 microencapsulated in an organic acid polymer. The Vitamin B12 status of the ewes and suckling lambs was assessed from changes in serum and liver Vitamin B12 concentrations. RESULTS: Compared to untreated animals, serum and liver Vitamin B12 concentrations of the treated ewes were increased at least 70% during gestation. Foetal liver Vitamin B12 concentrations were increased 270%. Over the lactation, ewe serum and milk Vitamin B12 concentrations were increased at least 200% and 44%, respectively. The liver Vitamin B12 stores of the newborn lambs from Vitamin B12-treated ewes were depleted within 58 days. There were no significant differences in the serum Vitamin B12 concentrations of suckling lambs from Vitamin B12-treated and untreated ewes. CONCLUSION: Ewes with a high Vitamin B12 status will ensure an adequate supply of Vitamin B12 to their lambs for at least the first 30 days of life. CLINICAL SIGNIFICANCE: In flocks grazing Co-deficient pastures, treating ewes with a long-acting Vitamin B12 supplement at mating will prevent Vitamin B12 (Co) deficiency in ewes, as well as their lambs, until they can be treated at tailing at 4-6 weeks of age.