In recent years, the use of supplementary vitamin B12 in sheep to improve fertility and weight gain has increased. This use has extended to areas that do not have cobalt deficient soils and where deficiency of vitamin B12 in sheep is not suspected such as the Wagga Wagga LHPA district. This paper reviews the evidence for the beneficial use of supplementary vitamin B12 in sheep throughout Australia and especially in Wagga Wagga.
Cobalt is deficient in many well drained soils of diverse geological origin (Mitchell et al., 2007) and leads to vitamin B12 deficiency in sheep grazed on these soils. In Australia B12 deficiency in sheep has been recorded in South Australia, Western Australia, and in the granite soils in New England (Judson and Babidge, 2002; Watt et al, 2012). Vitamin B12 supplementation was reported to have a profound benefit on live weight gain in deficient lambs (Grace et al., 2003). Whilst cobalt deficiency has not been recognised in soils or animals in Wagga Wagga, there are local claims that supplementing with vitamin B12 improves sheep productivity (Johnson, 2012). Current vitamin B12 supplements on the market boast improved growth rates and well being, (Watt et al., 2012) however the extent of these products benefits on increasing live weight and / or fertility in sheep that are not deficient in vitamin B12 is not clear. Whilst there is evidence that supplementing sheep deficit in vitamin B12 improves productivity, determining an accurate method to diagnose responsiveness to supplementation remains difficult. Further, the supposed production benefits of supplementation require cost benefit analysis taking into account the increased cost of supplemented products (Johnson, 2012).
Sheep and cattle require cobalt for the synthesis of vitamin B12 (cobalamin) by microorganisms in the reticulorumen (Grace et al., 2003; Mitchell et al., 2007). From 5-10% of the vitamin B12 produced is then absorbed and transported to the liver and other tissues in association with transcobalamins (blood proteins) (Grace et al., 2003). Vitamin B12 acts as a cofactor for two enzymes, one methylmalonyl-coenzyme A (CoA) mutase (MMA) is involved in energy metabolism in the liver. The other, methionine synthase is used in the general metabolism of methionine and folate (Mitchell et al., 2007). MMA is required in ruminant animals for gluconeogenesis converting propionate acid via methylmalonic acid to succinic acid to be metabolised to glucose via the tricarboxylic cycle (Grace et al., 2003). This results in less than 15% of circulating glucose needing to be obtained directly from the diet. The efficiency of production of vitamin B12 from cobalt is however, affected by the nature of the diet (Smith and Marston, 1970; Grace et al., 2003).
Toxicity from excess vitamin B12 supplementation has not been observed (Gruner et al., 2009), with excess cobalt largely excreted in faeces (Smith and Marston, 1970). No adverse signs were reported when vitamin B12 (3.2µg) was administered by IM injection to newborn lambs twice weekly for 4 weeks. Vitamin B12 concentrations in plasma and liver taken before supplementation and after a month assessed as similar to control lambs. The addition of vitamin B12 to these lambs did not appear to have the potential to increase metabolic efficiency and is likely to have increased excretion of the vitamin (Gruner et al., 2009).
Deficiency of vitamin B12 can lead to a reduction in neuronal transmission, glucose synthesis and alter the secretion of associated metabolic hormones such as insulin (Mitchell et al., 2007). Vitamin B12 is directly involved in the synthesis of neurotransmitters affecting myelination (Mitchell et al., 2007). A study undertaken by Mitchell et al (2007) observed lambs born to cobalt deficient ewes were slower to stand and suckle than their supplemented counterparts. Impairment of glucose synthesis also affects insulin production and subsequent hypothalamic-pituitary-ovarian axis, which has been attributed to reduced fertility in ewes (Mitchell et al., 2007).
The range of clinical symptoms due to vitamin B12 deficiency includes loss of appetite, suppressed growth rates in young lambs after weaning, anaemia, reduced wool production, and watery ocular discharge (Grace et al., 2003; Grace, Knowles, and West, 2006; Johnson, 2012). Anecdotal reports also suggest faster healing of supplemented lambs post mulesing, and flystrike reduction (Johnson, 2012). There also appears to be different clinical presentations dependent on the age and sex of sheep. It has been hypothesised that wethers are more sensitive to vitamin B12 levels then ewes lambs (Judson and Babidge, 2002), whilst adult ewes were reported to appear clinically normal despite inadequate circulating vitamin B12 (Mitchell et al., 2007).
Vitamin B12 supplementation has been thought to affect the number of corpora lutea produced in superovulated ewes, gestational length, and behavioural characteristics of lambs. In a study by Mitchell et al (2007) involving ewes from cobalt deficient farmland (0.06mg cobalt per kg dry matter) were supplemented (n=17) with an intraruminal cobalt bolus (3g cobalt oxide administered 30 days before embryo recovery) or not compensated for the dietary deficit (n=16). All ewes prior to the study were considered to be cobalt deficient with plasma vitamin B12 levels < 328pmol/L. Rumen boluses increased serum vitamin B12 levels (182 and 1288 pmol/L in un-supplemented and supplemented ewes respectively) at time of ovum recovery. Further the number of corpora lutea (9.9 and 14.4 p<0.05) and the gestational length was shorter in ewes that had received a cobalt bolus (147.8 and 146.2 days embryos p =0.061). It is postulated that lower ovulation rates seen in deficient ewes reflected a reduction in ovarian follicular recruitment in response to reduced glucose synthesis and insulin secretion.
Due to the nature of the above study, differences observed in the cobalt status of the ewe impacted the first 6 days of embryonic life, and this implies a critical window in embryonic development. Lambs from cobalt supplemented ewes were active and were recorded as spending more time interacting with their dams. Fisher and Macpherson (1991) also reported that lambs born to ewes low in vitamin B12 were slower to stand and suckle and had higher morbidity and mortality than supplemented counterparts. However, in Fisher and Macpherson's study (1991) ewes were supplemented in the third trimester indicating a potential role for vitamin B12 supplementation throughout gestation.
Cobalt supplementation has shown positive effects on the rumen microbial population and rumen fermentation in calves and heifers (Wang et al., 2010) and it has been postulated to increase live weight in lambs. In a study undertaken by Grace et al (2003) mixed sexed lambs grazing on low cobalt pasture (0.6mg Co/Kg DM) and deficient in vitamin B12 (serum vitamin B12 < 220pmol/L) were supplemented (n=30) with 3 mg of microencapsulated vitamin B12 delivered by intramuscular injection (IM) at 4-6 weeks of age (marking). This was compared to a control group that were not supplemented (n=47). Two months later (weaning) a significant live weight advantage of 5.5kg was observed.
These results were not consistent with those of Gruner et al (2004a) who saw varying responses to cobalt supplementation in lambs. The study conducted by Gruner et al (2004a) examined five properties with lambs on pasture below 0.07µg/g (0.07mg Co/kg DM). Replacement ewe lambs (age unspecified) were supplemented with either a 10g cobalt bullet (n=10), a short or long acting IM injection of vitamin B12 (1mg hydroxycobalamin) (n=10), or remain as a control (n=10). On three out of the five properties, cobalt supplementation regardless of method administered did not show a difference in live weight (measured at monthly intervals for 5 months) to those left un-treated. On the other two properties in this study, there was a 25% reduction in live weight seen in the un-supplemented lambs. Regardless of the property all supplementation methods significantly increased liver vitamin B12 concentrations (<0.05) as diagnosed by liver biopsy, serum vitamin B12 and MMA to control lambs. Serum MMA concentrations appeared more indicative of responsiveness, however differences observed between the two studies may be reflective of the relative sensitivities of wethers and ewes (Judson and Babidge, 2002; Gruner et al., 2004a; Gruner et al., 2004b). The differing studies show the limitations of laboratory testing in predicting responsiveness to vitamin B12 supplementation.
The affects of supplementation have also been described in suckling lambs. In a study supplementing new born lambs (sex unspecified) with milk replacer (n=4), milk with oral vitamin B12 supplement (1,000 µg hydroxycobalamin) (n=4), milk with added proprionate (n=4), or milk with proprionate and twice weekly IM vitamin B12 injection (1,000µg hydroxycobalamin) (n=4), no significant difference in live weight was shown (Gruner et al., 2009). The lambs were not considered to be predisposed to cobalt deficiency with milk replacer containing 3µg/L vitamin B12. Supplementation with oral proprionate or injectible vitamin B12 was able to increase serum vitamin B12 (measured twice weekly), however supplementation with proprionate alone impaired feed intake. Further close to 100% of the vitamin B12 administered by IM injection appeared in the plasma in the first hour but 85% of the supplement was removed from the circulation within 16 hours and no detectable effects on concentration of vitamin B12 remained in the liver. Lower concentrations of vitamin B12 were observed in blood from orally supplemented lambs compared to IM supplemented lambs. This is likely to be due to the time for absorption and incomplete absorption of oral supplementation. Orally supplemented vitamin B12 must pass through the gastrointestinal tract, be taken into the ileal entrocyte, and move to the liver before appearing in systemic blood. Gruner et al (2009) calculated that the absorption of vitamin B12 from orally supplemented milk fed lambs and weaned ruminant lambs was approximately 7% and 3-24% respectively.
It is anticipated that the requirement of vitamin B12 for gluconeogenesis is low in suckling lambs, as they are able to directly source glucose from milk (Gruner et al., 2004b). Therefore, it is presumed that serum vitamin B12 analysis is not a reliable test for deficiency in suckling lambs. Vitamin B12 can cross the placenta however the main transfer from mother to offspring is via colostrum (Mitchell et al., 2007). Milk is not a particularly rich source of vitamin B12 for suckling lambs even from supplemented ewes, and typically contains <5µg vitamin B12/L while the estimated requirement is 100-300µg/day (Grace, Knowles, and West, 2006). During the suckling period, the physiology and biochemistry of the digestive tract changes form that of a monogastric to a ruminant (Gruner et al., 2004). At 8-10 weeks of age, 50% of the lamb's energy is derived from pasture. At the same time, there is a corresponding decrease in dependency on lactose as a source of glucose and energy and an increase in proprionic acid production from rumen fermentation (Gruner et al., 2004). These studies strongly suggest that vitamin B12 requirements of lambs vary with age and weaning status.
Typically, to prevent deficiency 2 mg of vitamin B12 is given to lambs at marking followed by a cobalt pellet orally at weaning for long term prevention (Judson and Babidge, 2002). It is assumed that the effective period of protection of the injection is 2-3 months but it may be as short as 3-6 weeks (Judson and Babidge, 2002). Single injections have been shown to alleviate metabolic dysfunction for 28-51 days and prevent growth rate depression for at least 112 days in deficient lambs (Judson and Babidge, 2002).
Cobalt has been co-administered with other supplements to ascertain its combined effects. Wang et al. (2010) investigated combined cobalt and copper supplementation (n=20). It was found that cobalt and copper supplementation whilst having no influence on vitamin B12 status was able to enhance nutrient utilisation through increasing fat and fibre digestibility as measured by faecal analysis. The addition 0.3mg/Kg cobalt and 20mg/kg DM of copper are above the National Research Council of the United States requirements and are suggested by Wang et al (2010) to utilise low quality forages and hay compared to different supplementation regimes. It is proposed that supplementation aids folate production by utilising folacin derivatives produced by methionine synthase. The study did not however show a corresponding increase in plasma glucose or serum vitamin B12. Weight analysis, and control groups were not used to ascertain the direct effect of combination and individual treatments.
Edmonstone and Curran (2012) investigated three treatment regimens of varying vaccination, mineral and vitamin supplementation on the growth rate and financial returns of feedlot lambs. The trial consisted of 300 cross bred (sex unspecified) feedlot lambs receiving oral cobalt, injectible vitamin B12, or control (n=100). Before the trial commenced 12 animals from each treatment group had blood taken to determine adequate vitamin B12 levels (serum vitamin B12 >700pmol/L). No significant difference in weight gains were recorded between the groups over the month long study. Vitamin B12 levels measured at the conclusion of the study were also adequate (serum vitamin B12 > 1000pmol/L), with the significance of the different serum levels not calculated.
Using the data obtained from the above study, sheep were considered as small or large with the division made at 38.3kgs. Small sheep had a significantly greater (p=0.04) weight gain when supplemented with injectible vitamin B12 (n=20) than small sheep receiving oral supplementation (n=30), marginally better then control (p=0.10). Whilst larger sheep benefited (p=0.09) from oral supplementation (n=70) compared to injectible (n=80), but were not different to those not supplemented (n=80). It was therefore proposed that smaller sheep may have had subclinical deficiency of vitamin B12 or an underdeveloped rumen inadequate to produce vitamin B12 from cobalt, whilst larger sheep had a more developed rumen and were able to utilise cobalt to produce vitamin B12. It should be noted that when the trial re-examined the differences between small and large sheep the number of animals in each group decreased and sample sizes were too small to test whether a component of a treatment was important. The study does suggest however that different sized sheep may benefit from different supplemental regimes, but did not consider that the different sized sheep may be reflective of differing ages.
There is inconsistency in the definition of cobalt deficiency in soils and in serum vitamin B12 and serum MMA levels in sheep. Grace et al (2003) defining serum vitamin B12 deficiency <220pmol/L, whilst Mitchell et al (2007) defined levels <328pmol/L, and reports by Gruner et al (2004b) of studies using <355pmol/L. Grazing soils defined as low in cobalt by Mitchell <0.06mg Co/Kg DM while Gruner et al (2004) defined <0.07mgCo/KG DM. Current methods for assessment include pasture cobalt levels, multiple serum vitamin B12 levels and serum MMA. Grace, Knowles, and West (2006) however, reported a total dietary deficiency for supplemented sheep of <80µg.Kg DM. The inconsistency of responses reported may in part be due to differing reporting levels. However in a study conducted by Gruner et al (2004b) growth responses to vitamin B12 supplementation were observed on less than half of the properties in deficient areas using the same criteria. This lack of response may have been due to problems with the performance of the laboratory test.
Serum vitamin B12 samples have been acknowledged to be difficult to handle, with varying cut off levels postulated and multiple samples potentially required. Serum vitamin B12 samples are light sensitive and reported to artificially increase in fasting sheep (Johnson, 2012). Further, Grace et al (2003) indicated the substantial variation of 25% when samples were taken from the same sheep advocating that at least two blood samples should be taken 30 days apart from 10 sheep to diagnose low vitamin B12 in a flock. It has also been found that sheep on a low cobalt diet alter their microbial fermentation to succinate rather than propionate for glucose synthesis and levels of vitamin B12 may be lower than previously expected (Gruner et al., 2004a). Gruner et al (2004b) advocated the cut-off for serum vitamin B12 inadequacy of <200pmol/L (down from the previous <355pmol/L). Gruner et al (2004b) additionally noticed that high responsiveness to supplementation was observed when the same mean had a high standard deviation indicating a few animals caused the elevation. The standard deviation may be used to aid the diagnosis of a flock that would be responsive to supplementation.
MMA and liver concentrations have been postulated as a more accurate measure of vitamin B12 deficiency. MMA accumulates in serum because of incomplete metabolism of absorbed propionate from carbohydrate fermentation (Gruner et al., 2004b). >5mmol/L of MMA (in serum) has been suggested to indicate vitamin B12 deficiency, however in a study undertaken by Gruner et al (2004a) a corresponding weight response did not occur in weaned pasture fed lambs until MMA concentrations were in excess of 13mmol/L. Liver concentrations have been measured and are likely to be indicative of ongoing vitamin B12 suppression. Rumen development in lambs compromises the ability to perform liver biopsies and precludes its use as a functional alternative to blood sampling (Gruner et al., 2009)
Cost benefit analysis is required to ascertain if the weight gain from supplementing vitamin B12 is matched by the increase cost of administration, and that of alternative products to increase weight. Edmonstone and Curran (2012) attempted a cost-benefit analysis in a feedlot trial considering the value of sheep live weight at $3 per kg. The smaller sheep in the trial given vitamin B12 supplementation gained a mean of 2.12kgs over those not supplemented. Over the 70 head of smaller sheep, this equated to an increase of 148.4kgs live weight ($445.20). Further analysis of the best treatment over the least effective treatment valued at $4.34 a head. However, the value of the products given was not provided so the cost of producing the additional live weight could not be calculated from the study report.
There are several preparations of vitamin B12 supplementation on the market including cobalt boluses, combined short and long acting injections, oral drenches, and combined supplementation with vaccination. Johnson (2012) estimated the addition of vitamin B12 to a six antigen vaccination for sheep to be 95% more for 21c a dose. Other authors have proposed using soil to increase cobalt levels. In a study by Grace (2006), the concentration of cobalt in the soil was 3 times higher than that found in pasture. Drenching lambs with soil prevented cobalt deficiency and elevated vitamin B12 levels in the liver and serum. It was noted in the study that other supplementation such as lucerne pellets where also able to provide adequate vitamin B12 concentration in the tissue for health and growth, however soil was able to provide extra cobalt for the synthesis of vitamin B12 (Grace, 2006). Grace (2006) postulated that soil drenching may offer a novel approach to vitamin B12 supplementation.
Cost benefit analysis has also be undertaken in trials using Glanvac 6 B12® (Pfizer Animal Health) compared to Glanvac6® (Pfizer Animal Health) without added B12. Two trials have been undertaken in lambs not considered vitamin B12 deficient (Johnson, 2012; Morton, 2012) with both studies not reporting a significant difference in live weight. However, if a 0.5kg weight increase is gained, using Johnson's (2012) approximation an additional 21c per dose an approximate profit of $1.29 can be seen in sheep that appeared to have adequate vitamin B12. This is compared to a profit of $16.29 with live weight gains of 5.5kgs reported by Grace et al (2003) in deficient flocks. The increase in weights and profits however need to be weighed against the cost of other strategies such as additional feeding.
There is strong evidence that supplementing cobalt or vitamin B12 to deficient lambs results in an improved weight gain (Grace et al., 2003). There are difficulties in identifying a deficient flock and predicting benefits of supplementation based on pre-treatment laboratory analysis. Different studies report on deficit or marginal vitamin B12 status, using differing cut off levels. It is proposed from the studies presented that altering the current level of vitamin B12 in serum to less than 200pmol/L (down from 355pmol/L) may more accurately represent deficiency of vitamin B12 in non-suckling lambs. Vitamin B12 levels should be cross referenced to serum MMA levels between 9-14mmol/L (Gruner et al., 2004a) and repeat testing a month later currently appearing the most thorough diagnostic aid. Due to the cost of testing, it may be more cost-effective to supplement vitamin B12 and monitor clinical signs.
The ability of supplements to increase weight in suckling lambs and to improve fertility and nutrient utilisation requires further investigation. Quality of the diet has a major influence on the pattern of fermentation in the rumen such that propionate as a proportion of total volatile fatty acid production can decrease from 40% to 20% as feed digestibility falls from 80% to 60% (Gruner et al., 2004a). The possible role of interfering factors requiring more detailed and controlled research (Gruner et al., 2004a).
Other notable confounding factors on vitamin B12 trials have included different sex, weight and age groups. It has been hypothesised that wethers are more sensitive to vitamin B12 levels then ewe lambs (Judson and Babidge, 2002). Further Edmonstone and Curran (2012) observed differences in live weight responsiveness between small and large sheep. It would be advisable for other studies to take heed of these observations.
Vitamin and mineral supplementation are likely to remain key areas of interest in both human and animal medicine. Producers in Wagga Wagga LHPA need to be mindful that the scientific backing of products and sound cost benefit analysis are often lacking prior to products being marketed. There is currently difficulty in defining cobalt deficiency on farms and predicting benefits from supplementation based on laboratory diagnostics (Gruner 2004a). Further product testing may be warranted for producers.