An evaluation of plasma homocysteine in the assessment of vitamin B12 status of pasture-fed sheep.

AIM: To assess the diagnostic potential of concentrations of homocysteine (Hcy) in plasma in relation to those of methylmalonic acid (MMA) and vitamin B12, as predictors of responsiveness of young sheep to supplementation with vitamin B12. METHODS: Eighty-two ewes grazing ryegrass-white clover pasture were used, 39 of which had been supplemented with a Co bullet and 43 unsupplemented. Thirty days after commencement of parturition their lambs (n=53 and 59, respectively) were randomly allocated into one of two treatments, in a 2 x 2 factorial design. Half of the lambs from each group of ewes received an injection of vitamin B12, while the remainder were controls. The trial commenced 31 October 2001 (Day 0), and continued until 01 May 2002 (Day 182). All lambs were weighed, and blood samples taken from 16 identified animals from each treatment group, at approximately monthly intervals. Changes in concentrations of Hcy, vitamin B12 and MMA in plasma, and liveweight gain (LWG) of the treatment groups were evaluated during the suckling (Days 0-89) and post-weaning (Days 90-182) periods. RESULTS: Mean LWG was 40% greater in supplemented than unsupplemented lambs. The concentrations of vitamin B12 and MMA in plasma in the unsupplemented lambs were in the deficient reference ranges of <170 pmol/L and >16 mumol/L, respectively. Mean monthly concentrations of Hcy in plasma ranged from 1.5 to 4.5 mumol/L but showed no pattern of response to vitamin B12 deficiency or supplementation. CONCLUSIONS: Measurement of the concentration of Hcy in plasma as a metabolic indicator of reduced methylation capability of sheep on typical pastures in New Zealand appeared to have little value in detection of vitamin B12 responsiveness, and was less sensitive than the concentration of the vitamin itself or the indicator of adenosyl-cobalamin deficiency, MMA, in plasma. The possibility that concentrations of Hcy in plasma remain low due to re-methylation of Hcy to methionine via the alternative betaine-choline rather than the vitamin B12-dependent methyl-tetrahydrofolate metabolic pathway is rejected, but the possibility is raised that high rates of trans-sulphuration of Hcy to cysteine in the gastrointestinal tract of grazing sheep could be responsible. CLINICAL RELEVANCE: The propionate-succinate pathway appears to be the first rate-limiting pathway in vitamin B12 deficiency, and the product of disruption of this pathway, increased MMA, is the most reliable indicator of metabolic abnormality in predicting responsiveness to supplementation.

Vitamin B12 absorption and metabolism in milk-fed lambs.

AIM: To quantify aspects of absorption and retention of vitamin B12 from milk in pre-ruminant lambs, and the possible effect of activation of dependent metabolic pathways (propionate-succinate) on vitamin B12 concentrations in tissues. METHODS: In Experiment 1, two groups of eight newborn lambs each received either milk substitute alone, or milk substitute with added propionate for 4 weeks. Half of the lambs in each group received vitamin B12 injections twice weekly. Blood and liver samples were taken on Day -1 and at the end of the trial, and blood was also collected twice weekly, for measurement in plasma of vitamin B12, methylmalonic acid (MMA), and blood-binder transcobalamin II/haptocorrin. The lambs were weighed at 0, 2 and 4 weeks. In Experiment 2, 12 milk-fed newborn lambs (n=2 or 3/group) were injected I/M with 0.2, 0.4, 0.8, 1.6 or 3.2 microg vitamin B12 and a further 12 were supplemented orally with 2, 4, 8, 16 or 32 microg vitamin B12, daily for 2 weeks. Three lambs remained as untreated controls. Blood and liver samples were taken at the start and end of the trial, and blood was also collected twice weekly, for measurement of vitamin B12 and MMA concentrations in plasma. The lambs were weighed on Days 0, 7 and 14. In Experiment 3, 14 lambs from Experiment 2 were used to repeat the highest I/M and oral rates of supplementation used in Experiment 2. Blood samples were taken at frequent intervals for 24 hours, and analysed for concentrations of vitamin B12 in plasma. RESULTS: Propionate supplementation was associated with increased concentrations of vitamin B12 in plasma in lambs supplemented with vitamin B12 but not in those not supplemented (p=0.047), but had no detectable effect on concentrations of vitamin B12 in liver (Experiment 1). Lambs with concentrations of vitamin B12 in plasma and liver in the marginal reference range were able to metabolise propionate and maintain normal concentrations of MMA at a rate of intake of propionate close to that which would depress appetite. Close to 100% of the vitamin B12 administered by I/M injection appeared in plasma in the first hour but 85% was removed from the circulation within 16 hours. The maximum liver concentration was achieved at 900 nmol/kg (=1,200 microg) fresh tissue. There were no significant differences in liveweight gain (LWG) regardless of treatment. CONCLUSIONS: Lambs with concentrations of vitamin B12 in plasma and liver in the marginal reference range were able to metabolise propionate and maintain normal concentrations of MMA in plasma at a rate of absorption of propionate close to that which would depress appetite. The coefficient of absorption of oral vitamin B12 in milk-fed lambs was low (<10%), and the ability of lambs to retain the vitamin even when tissues had physiologically low concentrations was poor. CLINICAL RELEVANCE: Pre-ruminant lambs had a low requirement for vitamin B12, and even at low tissue concentrations had poor ability to absorb and retain the vitamin. However, if supplementation is required, a method that provides a modest but continual supply may be most effective in protection against deficiency.

Protein supply influences the nutritional penalty associated with the development of immunity in lambs infected with Trichostrongylus colubriformis.

The influence of dietary protein supply on the nutritional penalty associated with the acquisition phase of the immune response to gastrointestinal nematodes in lambs was investigated. Groups of lambs were offered either a low-protein diet (L; 62 g metabolizable protein (MP)/kg dry matter (DM)) or high-protein diet (H; 95 g MP/kg DM) while being either infected with the equivalent of 2.000 L3 Trichostrongylus colubriformis/day (IF), similarly infected and concurrently immuno-suppressed with methylprednisolone acetate (ISIF), immuno-suppressed only (IS) or kept as uninfected controls (C). Body composition of all animals was measured on days -8 and 76 of infection using X-ray computed tomography. Temporal changes in serum phosphate and serum albumin concentrations, which provided an indicator of pathological damage, in addition to patterns of total daily nematode egg excretion and comparative worm burdens at slaughter indicated that a protective immune response was developed in H-IF, but not L-IF, H-ISIF or L-ISIF groups. Compared to their respective non-infected controls, the gross efficiency of use of metabolizable energy (ME) for net energy (NE) deposition in the carcass was reduced by 0.23 in H-IF (P < 0.05), 0.13 in H-ISIF (P > 0.05), 0.49 in L-IF (P < 0.01) and 0.23 in L-ISIF (P > 0.05). It is concluded that the reduction in ME utilization and reduced performance, which can be attributed to the immunological response, are lessened in animals offered a high-protein diet. Furthermore, evidence is presented to indicate a possible association between T. colubriformis L3 IgA antibody production and loss of performance in lambs infected with this nematode.

Survey of plasma and liver mineral concentrations and enzyme activities in ostriches (Struthio camelus) under farmed conditions in New Zealand.

AIM: To generate clinical biochemical data to aid diagnosis of suboptimal performance in ostriches farmed under pastoral systems in New Zealand. METHODS: Blood and liver samples were sought from three categories of bird, viz 3-12-week-old chicks, 7-12-month-old growers, and from breeding hens, managed under pastoral conditions on farms in several locations in New Zealand. Concentrations of the minerals Ca, PO4, Mg, Na, K, Cu and Zn, activities of the enzymes gamma-glutamyl transferase (GGT), creatine kinase (CK), lactate dehydrogenase (LDH) and aspartate aminotransferase (AST), and concentrations of total protein (TP) and uric acid and vitamin E in plasma and Se in whole blood were determined from four flocks of chicks, two flocks of growing and one flock of breeding birds. Concentrations of Cu, Fe and vitamin B12 were determined from liver samples from chicks and growing birds. Bodyweight was recorded at the times of blood-sampling, where this was feasible. RESULTS: It proved impossible to obtain blood samples by venepuncture of the radial vein from chicks <3 months of age. Plasma concentrations of minerals, with the exception of K, which were higher, were within the normal range observed in the literature for ratite species. Activities in plasma of AST and CK were almost double, and those of LDH similar, to those in the literature for ostriches. There was considerable variation in activities of the enzymes between farms, but no association with growth performance. Concentrations of Cu in plasma were lower and in liver were higher than would be expected in farmed ruminants. Though hindgut fermenters, the birds maintained concentrations of vitamin B12 in plasma and liver at least comparable to those observed in ruminants. CONCLUSIONS: Collection of blood samples by venepuncture via the wing in ostrich chicks younger than 3 months of age is difficult and unlikely to prove viable for routine diagnostic purposes. Clinical biochemistry data on mineral nutrients in ostriches farmed in New Zealand are similar to those in the literature from South Africa , but AST and CK are higher, and may be normal and a reflection of the high growth rates of the musculoskeletal system.

The effect of corticosteroid treatment on local immune responses, intake and performance in lambs infected with Teladorsagia circumcincta.

The nutritional cost of, and the sequential cellular changes associated with the developing immune response to the abomasal parasite Teladorsagia circumcincta were investigated using corticosteroid-induced immune-suppression. Six-month-old lambs with minimal nematode experience were either infected with 4000 L3 T. circumcincta per day (group IF), similarly infected and concurrently immune-suppressed with methylprednisolone acetate (group ISIF), immune-suppressed only (group IS) or remained as controls (group C). Food intake, faecal egg count (FEC) and antibody titres in plasma were recorded weekly, worm burden at necropsy on day 63 p.i. and body composition by X-ray computed tomography on days -2 and 62 p.i. Furthermore, sequential immunological changes at the site of parasite infestation in the abomasal mucosa were measured from serial biopsy tissue samples taken from additional animals that were fitted with an abomasal cannula and either infected with the same regime as IF animals above (group CnIF) or concurrently infected and immune-suppressed as above (group CnISIF). Corticosteroid treatment resulted in greater FECs (P<0.01) and worm burdens (P<0.01) in both ISIF and CnISIF compared with IF and CnIF sheep, respectively. Infection reduced feed intake by 17% between 14 and 28 days p.i. (P<0.05) and efficiency of energy utilisation by 20% (P=0.07) in IF animals but not in ISIF animals. Mast cells, globule leukocytes and IgA in tissue biopsy samples were elevated in CnIF from 42 days p.i., all of which were abrogated by corticosteroid treatment. The ability to regulate the worm population appeared to be associated with a rise in tissue IgA concentration and numbers of globule leucocytes (GL). The results support the hypothesis that a majority of the production losses that occur during infection of lambs with T. circumcincta in lambs are a consequence of the host immune response. These findings may have implications for regimes that promote the development of a strong host immune reaction to gastrointestinal parasites in lambs.

Does suckling offer protection to the lamb against Teladorsagia circumcincta infection?

This study tested the hypothesis that milk has a direct effect on the establishment of infection with Teladorsagia circumcincta, and provides information on the effects of suckling on resilience to infection in young lambs. Of 46 six-week-old twin-born lambs, one from each twin was allowed to continue suckling (S-) and its twin-weaned (W-) while both were concurrently infected with an average of either 0 (-0; n=7/group), 250 (-250; n=8/group) or 1000 (-1000; n=8/group) third stage infective larvae (L3) of T. circumcincta per day, providing six treatment groups. All groups grazed minimally contaminated pasture, and after 42 days were slaughtered for necropsy. Low pasture larval contamination was confirmed in W0 and S0 lambs by faecal egg counts (FEC) of less than 30 eggs per gram (EPG) and burdens of less than 140 worms. There was no difference in FEC between weaned and suckled lambs. Within infection regime, total worm burdens were 55-60% greater in the weaned compared with their suckled counterparts (P=0.05), and represented 27 and 17%, respectively, net establishment of larvae. The greater worm burdens of both groups of weaned animals, which compared with their suckled counterparts, and of those infected with 1000 compared with 250 larvae per day, were associated with shorter female adults that had fewer eggs in utero, perhaps indicating intra-worm population regulation, but highlighting the limitation of FEC in assessing nematode burdens of such young lambs. There was no effect of infection on live weight gain of either weaned or suckled groups and the possibility was raised that, in such young lambs, immune unresponsiveness may be responsible. The major benefit of continued milk consumption appears to lie more in providing nutrients for enhanced growth rather than in improving resilience of the lambs to infection.

The effect of vaccinating infection during pregnancy and dietary protein supply on the peri-parturient immune response of sheep to infection with Teladorsagia circumcincta and Trichostrongylus colubriformis larvae.

It is well established that dietary protein supply can influence the peri-parturient breakdown of immunity to nematode parasites but there is no information on the importance of exposure to nematode larvae during pregnancy for this response. We investigated this by exposing housed pregnant sheep, scanned as carrying two lambs, to a vaccinating infection with a trickle mixed infection of Teladorsagia circumcincta and Trichostrongylus colubriformis larvae (L3) or to no infection during weeks - 9 to - 4 relative to parturition. At the beginning of week - 3 all sheep were treated with anthelmintic to remove any vaccinating worm burden and from week - 2 to week +6 received a trickle challenge infection with the same nematodes. Within each vaccinating treatment there were two nutritional treatments (no. = 20 per subgroup) designed to provide 1.5 or 1.0 and 1.3 or 0.8 of metabolisable protein (MP) requirement during pregnancy and lactation, respectively. Five ewes were necropsied during weeks +1 and +3 to measure worm burdens and mucosal inflammatory cells and the remainder maintained until week +6. Serum levels of total, IgA and IgE antibodies against L3 antigen of each nematode were measured.Scanning errors and lamb losses resulted in some ewes carrying and/or rearing only one lamb. Numbers of lambs reared was therefore introduced as a treatment effect. Vaccinating infection delayed the peri-parturient rise in faecal egg count (FEC) by an average of 2 weeks but its effect on FEC during the first 6 weeks of lactation was smaller and less persistent than that of dietary MP supply and single- v. twin-suckling.Populations of both nematodes were lower in association with high MP supply, vaccination and single suckling. These changes were associated with increases in numbers of mucosal mast cells (MMC) as a result of both increased MP supply and vaccination. Evidence for a more rapid return of host ability to limit populations of the abdominal nematode T. circumcincta than of the intestinal nematode T. colubriformis was associated with fewer eosinophils and more globule leucocytes (GL) in abomasal than in intestinal tissue.None of the serum antibody isotypes was affected by dietary protein supply. Total and IgA antibodies were maintained by a current larval (vaccinating) intake. IgA titres, however, increased progressively during pregnancy, especially in twin-bearing ewes. IgE titres appeared to be sensitive primarily to the reproductive cycle itself, peaking around parturition.This work supports the conclusion that availability of MP supply influences the recruitment and activity of cells of the immune armoury of the gastro-intestinal tract to nematode parasites. The precise outcome may differ with site and/or nematode species.

Analysis of corrinoids in ovine tissues.

Corrinoids from various ovine tissue samples (liver, blood, small intestinal fluid and faeces) were analysed using a combination of high-performance liquid chromatography (HPLC) and a radioisotope dilution assay (RIDA) to estimate the distribution of corrinoids--the cobalamins hydroxocobalamin (OH-cbl), methylcobalamin (me-cbl) and 5'-deoxyadenosylcobalamin (ado-cbl), and cobalamin analogues--in these tissues. Samples were taken from either cobalt-deficient or cobalt-replete ewes, and ruminant and pre-ruminant lambs. In liver, ado-cbl predominated, followed by analogues, OH-cbl and me-cbl. Supplementation with either cobalt (ruminant) or vitamin B12 injections (pre-ruminant) increased the amount of ado-cbl and decreased analogues. In blood, OH-cbl predominated, followed by ado-cbl, analogues and me-cbl, respectively. In small intestinal fluid, the distribution from largest to smallest percentage was analogues, ado-cbl, OH-cbl and me-cbl. In faeces, analogues constituted the greatest proportion, followed by OH-cbl, ado-cbl and me-cbl, respectively. Owing to the small sample sizes only cautionary interpretations can be made. In contrast to humans, where me-cbl constitutes the highest proportion of corrinoids in plasma and ado-cbl in the liver, in sheep the amount of ado-cbl was consistently higher than me-cbl in all tissues. This may be due to the higher metabolic need of sheep for ado-cbl due to gluconeogenesis. Analogues and OH-cbl were found in each tissue, contrary to previous postulations. The much higher amount of vitamin B12 in small intestinal fluid compared with faeces indicates that a large proportion of the vitamin is absorbed by the gastro-intestinal tract.

Development of a method for the separation of corrinoids in ovine tissues by HPLC.

A method has been developed using a combination of high-performance liquid chromatography (HPLC) and a radioisotope dilution assay (RIDA) to routinely estimate the distribution of corrinoids (the cobalamins hydroxocobalamin, methylcobalamin and 5'-deoxyadenosylcobalamin, and cobalamin analogues) in liver, plasma, milk, intestinal fluid and faeces. Corrinoids were extracted with a sodium acetate buffer, separated by HPLC and quantified by RIDA. Recoveries of corrinoids were 29% for hydroxocobalamin, 50% for 5'-deoxyadenosylcobalamin and 64% for methylcobalamin. The method allows the routine analysis of many samples and maintains good standards of precision.

Studies using Teladorsagia circumcincta in an in vitro direct challenge method using abomasal tissue explants.

An in vitro direct challenge (IVDC) method in which abomasal tissue explants maintained at 37 degrees C in Hanks/Hepes solution (pH 7.6) in a high oxygen concentration (incubator gassed with pure oxygen and sealed prior to use) were-challenged with exsheathed third stage larvae of Teladorsagia circumcincta has been used to investigate the tissue association phase of larval establishment. Studies using naïve and recently challenged immune sheep and goat abomasal tissues have shown significant reductions (P < 0.05) in the numbers of tissue associated larvae in material from immune animals. The mechanisms that affect the tissue association process appear to be labile since more larvae were recovered from the tissue digests of previously infected sheep that had not been recently exposed to larval challenge in comparison to those that had been recently challenged (P < 0.05). The method has also been used to demonstrate the influence of protein nutrition on the establishment of larvae in abomasal tissues and region specific differences in the efficacy of exclusion mechanisms. The technique appears to have potential as a means of investigating the crucial first phase of the process of establishment.

A model of magnesium metabolism in young sheep: transactions between plasma, cerebrospinal fluid and bone.

An extension of the previously proposed model of Mg metabolism (Robson et al. 1997) has been developed to consider the transactions of Mg that are associated with cerebrospinal fluid (CSF) and bone. The representation of the CSF as a single MG compartment with uptake from the plasma described by Michaelis-Menten kinetics gives very good agreement with published experiments. Analysis of the available information on resorption of Mg from adult bone indicated that this process makes a negligible contribution to Mg homeostasis and can be omitted from the model.

The sequential analysis of local inflammatory cells during abomasal nematode infection in periparturient sheep.

A technique to take sequential tissue biopsy samples in multiparous, periparturient ewes from the abomasal mucosa is described, developed in parallel in Scotland and New Zealand. Samples were extracted via abomasal cannulae inserted into the wall of the abomasum and exteriorised through dorso-ventral laparotomy. Animals recovered quickly post-surgery, and tolerated the cannula and sampling without any adverse signs of pain or discomfort. The technique was deployed in two pilot studies to investigate the sequential mucosal inflammatory cell responses in well-defined parasitological models, during the periparturient relaxation of immunity in ewes infected with gastrointestinal nematodes and subjected to different feeding treatments. One experiment (Moredun Research Institute, Scotland) involved the infection of twin-bearing ewes with Teladorsagia circumcincta L3 either before, or after lambing. By feeding ewes with different levels of protein supplementation, preliminary data on the impact of nutrition on the eosinophil, mucosal mast cell and globule leucocyte responses during this period were investigated. A similar study was also performed at Lincoln University, New Zealand, to investigate these cell responses in sheep fed relatively high or low protein diets during pregnancy, and infected with a combined immunisation regime of T. circumcincta and Trichostrongylus colubriformis L3. These studies confirmed the phenomenon termed the periparturient relaxation in immunity (PPRI) where a transitory increase in faecal egg counts is observed during late pregnancy and lactation, and this effect was exacerbated during protein undernutrition. Although the number of animals was low in each experiment and the cell responses variable, the results together suggest a reduction in the number of mucosal mast cells and globule leucocyte during the PPRI when protein supply was restricted. The present paper thus describes a successful technique to monitor ovine mucosal cell populations during local immune responses in normal and pregnant sheep. It is envisaged that this technique will be a powerful adjunct to investigations into mucosal immune mechanisms and disease pathogenesis, and will be employed to confirm the influence of dietary protein on the local inflammatory cell responses during the PPRI.

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.

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.

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.

Liveweight gain and copper status of young deer treated or untreated with copper oxide wire particles on ten deer farms in Canterbury.

AIMS: To measure the liveweight response of young deer to copper supplementation on a range of deer farms in the Canterbury region of the South Island, New Zealand, and relate any response to plasma copper concentration. METHODS: On each of 10 farms throughout north and mid-Canterbury, young deer (4-7 months old) were allocated by liveweight to two groups of 45-50. The deer in one group each received 5 g copper oxide wire particles (COWP) at the start of the project (March-June 2000) and a further 10 g COWP 3 months later (+Cu group), while the other group received no copper supplementation (-Cu group). Liveweight (+/-1 kg) of all deer was recorded monthly. A 10 ml blood sample was taken from a sub-sample of 10 animals in each group at the beginning of the trial and from the same animals 3 and 6 months later for measurement of plasma copper concentration. RESULTS: There was no significant effect (p=0.96) of treatment with COWP on liveweight gain (overall mean liveweight gain = 169 g/day). This result was consistent for all farms and for both periods. Mean plasma copper concentration declined from 12.5 (SD 3.4) to 10.3 (SD 3.0) micromol/l in +Cu groups, and to a significantly lower level (p<0.001) of 8.6 (SD 3.2) in -Cu groups after 3 months, at which time 38% of -Cu animals were considered hypocupraemic (<8 micromol/l). At the end of the second 3-month period there was no significant treatment effect on plasma copper concentration, which averaged 9.4 (SD 2.3) micromol/l. Of the feed samples submitted (n=46), only 17% had a copper concentration <5.0 mg/kg dry matter (DM), 13% had a molybdenum concentration >1.0 mg/kg, and 21% had a sulphur content >3.5 g/kg. CONCLUSIONS: Under the conditions of these trials, no response in liveweight gain to copper supplementation was observed despite evidence of hypocupraemia in 38% of -Cu animals, which gained weight at similar rates to those that had adequate plasma copper levels. The extent of the hypocupraemia was either not sufficiently severe, or not maintained for a long enough period to cause copper deficiency resulting in reduced liveweight gain. No other signs of copper deficiency were evident. There is scope for deer farmers to reassess the need for copper supplementation in young deer.

Regional variations in the nematode worm populations of breeding ewes in New Zealand.

AIM: To determine whether the generic composition of nematode parasites in lactating ewes varies between geographic regions in New Zealand and whether age and fecundity of ewes affect nematode egg output. METHODS: Four ram breeding properties in each of the northern (NNI) and southern (SNI) North Island and the northern South Island (NSI) regions, together with six properties in the southern South Island (SSI) were investigated. Faecal samples were obtained on one occasion, 2-6 weeks after lambing, from at least 60 ewes on each property, stratified according to age and reproductive status. Nematode eggs were counted and coprocultures prepared to identify genera present. Information on the history of anthelmintic use on each property was also collected. RESULTS: Highest faecal egg counts (FEC) were observed from ewes in the NNI and SSI regions. Lambed hoggets and twin-rearing two-tooth ewes had higher FEC than their unlambed or single-lamb-rearing contemporaries, respectively. However, no significant effect of reproductive effort on FEC was demonstrated in mixed-aged ewes. Significant regional differences in generic composition of nematode parasites were observed: Teladorsagia spp (also referred to as Ostertagia spp) eggs made up 56% of total eggs shed from sheep in the SSI compared with14% in the NNI, and values for Haemonchus spp were 1%and 24% for these same regions, respectively. Trichostrongylus colubriformis and T. vitrinus together made up 7, 15, 13 and15% of eggs counted from sheep in the SSI, NSI, SNI and NNI, respectively. CONCLUSIONS: More comprehensive epidemiological information may suggest regional-specific approaches to the management of nematode parasites in ewes should be devised in the future.

Interactions between nutrition and gastrointestinal parasitism in sheep.

Effects of gastrointestinal nematode infection on metabolism and nutrient utilisation in sheep are reviewed. Infection induces protein deficiency by increasing the demand for amino acids in the alimentary tract while reducing supply through depression of appetite. Mechanisms through which improved protein nutrition could improve the performance of the host are then discussed. Opportunities for capitalising on such effects are limited by our rudimentary understanding of the cell-mediated immune response in gastrointestinal epithelial tissue. Both resistance of the animal to larval establishment and performance in the face of larval challenge can be enhanced by improved protein nutrition. However, enhanced immune responses may not necessarily be synonymous with improved productivity except at luxurious levels of protein intake, because of apparently competing demands for protein. Such levels of protein nutrition are difficult to achieve in pasture-based systems, because of the protein limiting role of the rumen. Work with proteinprotecting tannins to overcome this limitation is discussed. The much more limited evidence for effect of mineral nutrition, particularly copper (Cu), molybdenum (Mo), cobalt (Co) and phosphorus (P), on outcome of larval challenge is also reviewed.

Enhancing the profitability of pasture-based dairy production in the humid tropics through improved nutrition.

Dairy production in the tropics represents a major challenge, especially when reliant on grazed forages, because of the conflicting factors of a high nutritional demand to sustain lactation on the one hand, and the relatively low quality of tropical grasses and a stressful environment, on the other. This paper focuses primarily on those tropical situations where grazed pasture is the cheapest feed resource. Effective management of the pasture as well as the animals that graze it is required in order to maximize economic viability of the farming enterprise. Feed planning helps to ensure that pasture use is maximized, either directly by grazing or, where profitable, indirectly by cutting for conservation. High rates of pasture utilization at each grazing minimize pasture senescence and decay and ensure that pasture remains in a vegetative state of the highest possible nutritional quality. Total annual feed requirements of the grazing herd must therefore be matched as closely as possible to the total annual pasture production on the farm. Stocking rate is critical in this regard, and is the single most important determinant of productivity in pastoral farming. Periods of pasture surplus or deficit will inevitably arise, but can be minimized by matching the monthly feed requirements of the herd as closely as possible to the monthly feed production on the farm. Herd requirements can be influenced, for example, through altering calving patterns and drying off dates. Even with good pasture management practices, it is highly likely that tropical pasture quality will be low for part of the year. Utilization of low-quality forages can be improved through the appropriate use of feed supplements, and the key principles are discussed. Firstly, conditions for optimum fermentative digestion in the rumen must be promoted through adequate provision of fermentable energy and nitrogen as well as essential minerals, such as phosphorus and sulfur. Further supplementation with metabolizable energy or protein, depending on what limits animal production, may be profitable. Various practical supplementation strategies for tropical forages are discussed. Finally, some suggestions for future research and development for enhancing the profitability of pasture-based dairy production in the humid tropics are made.