DSM in Animal Nutrition & Health
Properties and Metabolism
Vitamin B12 is the largest and most complex of the vitamins (Illus. 10-1), and is synthesized only by microorganisms. A series of structurally related compounds possess vitamin B12 activity. In addition, there are structurally similar vitamin B12 antagonists, produced by bacteria such as Streptomyces griseus, E. Coli and Propionibacterium shermanii. All these compounds contain a cobalt atom stabilized within a corrin ring structure that is similar to the porphyrin ring of hemoglobin and the cytochromes (Ellenbogen and Cooper, 1991). Cyanocobalamin is the common term used to refer to the most active and stable supplemental form of vitamin B12. Tissue metabolism converts cyanocobalamin to the two primary coenzyme forms, adenosylcobalamin and methylcobalamin (Ellenbogen and Cooper, 1991).
Vitamin B12 is a dark-red, hygroscopic, needle-shaped crystal, freely soluble in water and alcohol but insoluble in acetone, chloroform and ether. Cyanocobalamin has a molecular weight of 1,355. Oxidizing and reducing agents and exposure to sunlight tend to destroy its activity. Losses of vitamin B12 during feed processing are usually not excessive because vitamin B12is stable at temperatures lower than 482°F (250°C). On average, 3% of dietary cobalt is converted to vitamin B12, and only 1% to 3% of the vitamin B12 synthesized in the rumen is absorbed from the small intestine (Girard, 1998a). Ruminal microflora also use dietary cobalt to produce different molecules, called analogs, closely related to cobalamin, but without biological activity for the host. Increasing dietary cobalt supply increases production of these analogs in the rumen at the expense of the biologically active forms of the vitamin. Most analogs are inactive for the host, but a few studies indicate that some analogs could have deleterious effects. In addition to cobalamin, the only biologically active molecule for the cow, seven analogs were identified in duodenal and ileal digesta (Girard et al., 2009). Although cobalamin was not the major form synthesized by ruminal microflora and, even if supplementary cyanocobalamin was extensively destroyed by ruminal microflora, based on calculations of apparent intestinal disappearance, cobalamin seems to be the major form absorbed in the small intestine. In ruminants, as in most species, the distal small intestine (ileum) is the primary absorptive site for vitamin B12. Substantial amounts of vitamin B12 are secreted into the duodenum and reabsorbed in the ileum, a process that is presumably subject to regulation based on the vitamin B12status of the animal. Absorption of vitamin B12 requires the presence of the intrinsic factor, a glycoprotein synthesized by the parietal cells of the gastric mucosa (McDowell, 2000). Pernicious anemia in humans is caused by a lack of the intrinsic factor. A second factor, transcobalamin II, has been identified in the intestinal villus (Quadros et al., 2000). Vitamin B12 absorption also occurs by diffusion, but only at very high concentrations (Ellenbogen and Cooper, 1991). In ruminants, vitamin B12 absorption requires the following: (a) adequate quantities of dietary cobalt, much of dietary vitamin B12 (e.g., cyanocobalamin) is destroyed by ruminal microflora (Girard et al., 2009); (b) a functional abomasum for digestion of food proteins and release of vitamin B12 and the production of intrinsic factor; (c) a functional pancreas (trypsin secretion) for protein digestion and release of bound vitamin B12; (d) a functional ileum. Intrinsic factor has been demonstrated in the cow but not the sheep. Factors that diminish vitamin B12 absorption include deficiencies of protein, iron and vitamin B6; thyroid removal/ hypothyroidism; and dietary tannic acid. Cobalamin analogs may compose up to 50% of the total plasma vitamin B12 in cattle, but are undetectable in sheep (Halpin et al., 1984).
Storage of vitamin B12 occurs principally in the liver. Other storage sites include the kidney, heart, spleen and brain. Andrews et al. (1960) reported that the proportion of liver cobalt that occurs as vitamin B12 varies with the cobalt status of the animal. In cattle or sheep grazing cobalt-adequate pastures, most of the cobalt in the liver is in the form of vitamin B12. However, in cobalt deficiency only about 33% of liver cobalt is in the form of vitamin B12. This indicates that during cobalt deficiency vitamin B12 is depleted more rapidly than other forms of cobalt in the liver.
Unlike the other water-soluble vitamins, vitamin B12 is stored in significant amounts in the liver and other tissues, providing a reserve in times of cobalt depletion. Kominato (1971) reported a tissue half-life of 32 days for vitamin B12, indicating considerable tissue storage. Cattle and sheep with normal liver stores can tolerate a cobalt-deficient diet for several months without showing vitamin B12 deficiency signs.
In ruminants, both cobalt and vitamin B12 are excreted primarily in the feces, with smaller amounts excreted in urine (Smith and Marston, 1970). Lactating cows fed normal diets excrete 86% to 87.5% of all excreted cobalt in the feces (mainly associated with bile acids), 0.9% to 1.0% in the urine, and 11.5% to 12.5% in milk.
Vitamin B12 is an essential part of several enzyme systems that carry out a number of basic metabolic functions. Most reactions involve transfer or synthesis of one-carbon units, such as methyl groups. Vitamin B12 is metabolically related to other essential nutrients, such as choline, methionine and folic acid. Although the most important tasks of vitamin B12 concern metabolism of nucleic acids and proteins, it also functions in metabolism of fats and carbohydrates. A summary of vitamin B12 functions include: (a) purine and pyrimidine synthesis; (b) transfer of methyl groups; (c) formation of proteins from amino acids; and (d) carbohydrate and fat metabolism. General functions of vitamin B12 are to promote red blood cell synthesis and to maintain nervous system integrity, which are functions noticeably affected in the deficient state (McDowell, 2000). The primary role of vitamin B12 is as an essential cofactor for the enzymes methionine synthase and methylmalonyl-CoA mutase (McDowell, 2000). Methionine synthase effects the transfer of a methyl group from folic acid (N5-methyl-tetrahydrofolate) to homocysteine, forming methionine. Therefore, a vitamin B12 deficiency reduces methionine supply and metabolic recycling of methyl groups. The latter effect interrupts the normal metabolism of folic acid and blocks its utilization. Thus, a vitamin B12 deficiency produces a secondary deficiency of folic acid, and a characteristic anemia (McDowell, 2000).
Under intensive dairy systems, methionine may be limiting in certain diets. Under such circumstances, increasing methionine supply through feeding rumen-protected methionine has the potential to augment milk protein, fat concentrations and yields in high-producing dairy cows, probably through increased protein synthesis (NRC, 2001). Methionine is not only an amino acid involved in the structure of proteins per se but it also plays a unique role as the initiating amino acid for the synthesis of proteins (Brosnan et al., 2007). Supply of methionine, because of its role as donor of preformed labile methyl groups, affects the needs for methylneogenesis (Bailey and Gregory, 1999) and consequently, for folic acid and vitamin B12.
The nature of the ruminant digestive system imposes a huge dependence on gluconeogenesis because very little glucose is being absorbed (Reynolds, 2006). In ruminants, the rate of gluconeogenesis increases with feed intake, and one of the major substrates for gluconeogenesis is propionate. Metabolic utilization of propionate after its absorption from the gastrointestinal tract is dependent on the transformation of propionate into succinyl-coenzyme A, requiring the successive actions of biotin- and vitamin B12-dependent enzymes. Propionate is first transformed into propionyl-CoA, which is carboxylated to methylmalonyl-CoA by a biotin-dependent enzyme, propionyl-CoA carboxylase. Methylmalonyl-CoA is then isomerized in succinyl-CoA under the action of the vitamin B12-dependent enzyme, methylmalonyl-CoA mutase. Succinyl-CoA finally enters into the Krebs cycle, from where it can be directed toward gluconeogenesis (Le Grusse and Watier, 1993; McDowell, 2000). Flavin and Ochoa (1957) established that for succinate production the following steps are involved:
Propionate + ATP + CoA→Propionyl-CoA
Propionyl-CoA + Coz + ATP →methylmalonyl-CoA (a)
Methylmalonyl-CoA (a) →methylmalonyl-CoA (b)
Methylmalonyl-CoA (b) →succinyl-CoA
Methylmalonyl-CoA (a) is an inactive isomer. Its active form, (b), is converted into succinyl-CoA by a methylmalonyl-CoA isomerase (methylmalonyl-CoA mutase) (fourth reaction above).
Methylmalonyl-CoA also arises from the metabolism of odd-chained fatty acids and certain amino acids (valine, isoleucine, methionine and threonine) (Girard, 1998a). The propionic acid pathway is of particular importance in ruminants because of their dependence on propionic acid as the primary glucose precursor, and the production of propionic acid in the rumen fermentation of dietary carbohydrates. As a result of the blockage in utilization of propionic acid, a vitamin B12 or cobalt deficiency in ruminants results in the accumulation of succinic acid in the rumen and methylmalonic acid in blood and urine (Kennedy et al., 1995).
The availability of vitamin B12 for methyl group transfer affects the supply and transfer rate of methyl groups in intermediary metabolism. Thus, vitamin B12 status affects the metabolism of choline and its derivatives, and the synthesis of the purines and pyrimidines, components of DNA and RNA. As a result, rapidly dividing cells, in particular the red blood cells, are sensitive to vitamin B12 status. The nervous system is also quite sensitive to the availability of vitamin B12, possibly due to its high rate of metabolism. Reduction in the rate of protein synthesis is thought to be a principal cause of the growth depression frequently observed in vitamin B12-deficient animals (Friesecke, 1980).
Vitamin B12 is metabolic essential for animal species studied, and vitamin B12 deficiency can be induced with the addition of high dietary levels of propionic acid. However, metabolism of propionic acid is of special interest in ruminant nutrition because large quantities are produced during carbohydrate fermentation in the rumen. Propionate production proceeds normally, but in cobalt or vitamin B12 deficiency, its rate of clearance from blood is depressed and methylmalonyl-CoA accumulates. This results in an increased urinary excretion of methylmalonic acid and also loss of appetite because impaired propionate metabolism leads to higher blood propionate levels, which are inversely correlated to voluntary feed intake (MacPherson, 1982).
An additional function of vitamin B12 relates to immune function. In mice, vitamin B12 deficiency was found to affect immunoglobulin production and cytokine levels (Funada et al., 2001). Cobalt deficient lambs had reduced concentrations of serum immunoglobulin G (IgG) and total protein (Fisher, 1991).
The origin of vitamin B12 in nature appears to be microbial synthesis. It is synthesized by many bacteria but apparently not by yeasts or by most fungi. There is little evidence that the vitamin is produced in tissues of higher plants or animals. A few reports have suggested limited B12 synthesis by a few plants, but in insignificant quantities in relation to animal requirements. Synthesis of this vitamin in the alimentary tract is of considerable importance for animals (McDowell, 2000; McDowell and Arthington, 2005). Vitamin B12 requirements are exceedingly small, measured in units of micrograms per kg of feed. The vitamin B12 requirement increases with higher rates of animal production and metabolism. The requirement is also affected by the relative intake of other nutrients. Excess dietary protein increases the vitamin B12 requirement. Interrelationships exist with dietary cobalt, choline, methionine and folic acid. Vitamin B12also appears to be affected by ascorbic acid metabolism (Scott et al., 1982).
Dietary cobalt and subsequent ruminal synthesis of vitamin B12 normally meet the requirement for vitamin B12 in ruminants. Under typical conditions, rumen synthesis of vitamin B12 would be functional by six to eight weeks of age, depending on intake of dry feed. Pre-ruminant calves, lambs and kids require supplemental vitamin B12. Vitamin B12requirements of the dairy calf are estimated between 0.34 and 0.68 µg per kg (0.15 to 0.31 µg per lb) body weight (NRC, 1989). On a dietary basis, the requirement of young dairy calves ranges from 20 to 40 µg per kg (9.1 to 18.2 µg per lb) of dry matter (Radostits and Bell, 1970).
The established dietary cobalt requirements for ruminants are 0.1 to 0.2 mg per kg (0.05 to 0.09 mg per lb) of diet. Precise estimates of minimum cobalt requirements are difficult because of the influence of variables such as seasonal changes in herbage cobalt concentrations, selective grazing and soil properties. Under grazing conditions, lambs are the most sensitive to cobalt deficiency, followed by mature sheep, calves, goats and mature cattle (Andrews, 1956).
Cobalt content of the diet is the primary factor affecting the synthesis of vitamin B12 by ruminal microflora. However, studies indicate that synthesis of vitamin B12 can be affected by other factors. High-concentrate diets can reduce the synthesis of vitamin B12 and also increase the production of vitamin B12 analogs, certain of which are antagonists of vitamin B12 (Sutton and Elliot, 1972). These natural analogs have little or no vitamin B12 activity.
Tiffany et al. (2006) recorded increased synthesis of vitamin B12 as the cobalt concentration of the diet increased from 0.10 to 1.0 mg per kg (0.045 to 0.455 mg per lb). Based upon performance and blood measures, the cobalt requirement for fattening cattle may be between 0.15 and 0.25 mg per kg (0.068 to 0.114 mg per lb) (Stangl et al., 2000a, b; Tiffany et al., 2003). However, ruminal synthesis of vitamin B12 was increased with dietary levels of 1.0 mg per kg (0.455 mg per lb) of cobalt (Tiffany et al., 2002).
Ruminants have higher vitamin B12 requirements per unit of body weight or diet than nonruminants, presumably because of the involvement of B12 in the metabolism of propionic acid. Experiments with sheep suggest an oral requirement for growing lambs of about 200 µg per day, about 10 times the reported oral requirement of other species per unit of food intake (Marston, 1970). The comparatively high cobalt requirement of ruminants arises partly from the low efficiency of production of vitamin B12 from cobalt by the rumen microflora and partly from the low efficiency of absorption of vitamin B12 (McDowell, 2000).
Grasslands containing 0.1 ppm cobalt will prevent deficiency symptoms, while levels of 0.05 to 0.07 ppm cobalt are deficient. Further evidence that 0.1 ppm cobalt in the diet will meet both the cobalt and the vitamin B12 requirements of sheep was provided by Mohammed (1983), who reported that ruminal concentrations of both vitamin B12 and propionic acid were maximized at 0.1 ppm dietary cobalt. However, cobalt concentration of 0.10 to 0.15 mg per kg (0.45 to 0.068 mg per lb) of dietary dry matter resulted in adequate vitamin B12 production to meet the requirements of ruminal microorganisms fed a high-concentrate diet in continuous-flow fermenters (Tiffany et al., 2006).
Tiffany and Spears (2005) found that ruminal vitamin B12 concentrations were greater and ruminal succinate concentrations were lower, in steers fed high-concentrate diets containing 0.19 mg of cobalt per kg (0.09 mg per lb) compared with those fed diets containing 0.04 or 0.09 mg cobalt per kg (0.018 or 0.041 mg per lb) of diet. Increasing dietary supply of cobalt from 0.19 to 0.93 mg per kg (0.086 to 0.423 mg per lb) of dry matter from parturition to 120 days of lactation had no effect on plasma concentrations of vitamin B12 or on milk production and milk component yields (Kincaid and Socha, 2007).
Feedstuffs of animal origin are generally good sources of vitamin B12 (Driskell et al., 2001). Liver and kidney are especially rich sources. Fermentation products sometimes contain vitamin B12. Among the richest sources are fermentation residues including activated sewage sludge and manure. However, these materials are also a source of anti-vitamin B12.
The origin of vitamin B12 in nature is bacterial synthesis. Yeast and fungi do not appear to synthesize vitamin B12. Unlike other B-complex vitamins, vitamin B12 (cobalamin) is synthesized almost exclusively by bacteria and is therefore present only in foods that have been bacterially fermented or are derived from animals that have obtained this vitamin from their gastrointestinal microflora or their diet. There is no convincing evidence that the vitamin is produced in the tissues of of animals. Microbial synthesis of vitamin B12 in the alimentary tract is of considerable importance to ruminants and species that practice coprophagy.
Evidence suggests that the amount and type of roughage in the diet affects the rumen synthesis of vitamin B12 even when cobalt is adequate. Restricted roughage rations have been reported to increase vitamin B12 levels in rumen fluid and serum, reduce milk and liver concentrations and increase urinary excretion compared to cows fed roughage ad libitum (Walker and Elliot, 1972). Vitamin B12 levels in the rumen of dairy heifers fed corn silage is 1.4 to 2.7 times higher than when fed chopped or pelleted hay (Dryden and Hartman, 1970).
Plant products are practically devoid of vitamin B12. Minute quantities of vitamin B12 may be synthesized by soil microorganisms, released into the soil, and subsequently absorbed by plants. Root nodules of certain legumes contain small quantities of vitamin B12. Certain species of seaweed and other algae contain appreciable quantities of vitamin B12, up to 1 µg per gram of solids, synthesized by adherent bacteria (Scott et al., 1982). Commercial sources of vitamin B12 are produced by fermentation and are available as cyanocobalamin.
Cobalt is the primary dietary precursor of vitamin B12 in the ruminant. Although dietary cobalt of confined livestock is usually adequate, cobalt and secondary vitamin B12 deficiency is a significant problem for grazing livestock in many areas of the world (McDowell, 2000; McDowell and Arthington, 2005). Concentration of cobalt in forages is affected by soil properties, plant species, stage of maturity, yield, pasture management and climate. Soil levels of less than 2 ppm cobalt is generally considered deficient for ruminants (Correa, 1957). Liming of soils increases the pH, reduces the uptake of cobalt by plants and may increase the severity of a deficiency. Plants grown on soil containing 15 ppm cobalt with neutral or slightly acid pH can contain more cobalt than plants grown with 40 ppm cobalt and an alkaline pH (Latteur, 1962). Heavy rainfall tends to leach cobalt from the topsoil. This problem is often aggravated by rapid growth of forage crops during the rainy season, further diluting the cobalt content of the diet. Plants have varying degrees of affinity for cobalt, some being able to concentrate the element much more than others. Legumes, for example, generally have a greater ability to concentrate cobalt than do grasses (Underwood, 1977). The nitrogen-fixing bacteria in the root nodules of legumes require cobalt. High dietary potassium levels were reported to induce a transient, six-week reduction in plasma vitamin B12 concentrations (Smit et al., 1999).
Vitamin B12 is produced by fermentation and is available commercially as cyanocobalamin. Vitamin B12 is only slightly sensitive to heat, oxygen, moisture and pH (Gadient, 1986). Vitamin B12 has good stability in premixes with or without minerals, regardless of the source of the minerals, and is little affected by pelleting (Scott, 1966; Verbeeck, 1975). However, Yamada et al. (2008) reported degradation of supplemental vitamin B12; the vitamin was affected by storage time, light exposure, temperature and vitamin C. It was determined that some vitamin B12 might have been converted into vitamin B12 analogues.
Lassiter et al. (1953) demonstrated vitamin B12 deficiency in calves less than six weeks old that received no dietary animal protein. Clinical signs characterizing the deficiency included poor appetite and growth, lacrimation, muscular weakness, demyelination of peripheral nerves and emaciation. Young lambs (up to two months of age), require vitamin B12 supplementation, especially with early-weaning programs (NRC, 1985). In vitamin B12-deficient lambs, there is a sharp decrease of vitamin B12concentrations in blood and liver prior to the appearance of gross deficiency signs such as anorexia, weight loss and a decrease in blood hemoglobin concentration. At necropsy, the body of a severely deficient animal is extremely emaciated, often with a total absence of body fat. Fatty liver, hemosiderized spleen and hypoplasia of the erythrogenic tissue of bone marrow are also characteristic (Filmer, 1933). The anemia in lambs in normocytic and normochromic, but the anemia is mild and is not responsible for the primary deficiency signs of cobalt deficiency. Anorexia and marasmus invariably precede any considerable degree of anemia. The first discernible response to feeding cobalt or parenteral vitamin B12is a rapid improvement in appetite and body weight. Metabolically, vitamin B12 deficiency is characterized by decreases in the activity of methylmalonyl-CoA mutase, with a resultant increase in plasma and urinary methylmalonic acid (MMA), and by decreased activity of methionine synthase with a concomitant rise in plasma homocysteine (Kennedy et al., 1992, 1995). The reduction in methionine synthase activity in turn reduces the availability of methylated compounds, including the methylated phospholipids (Kennedy et al., 1992). The latter effect may be a cause of nerve demyelination. A recent study has associated high blood concentrations of methylmalonic acid and homocysteine with symptoms of acute and chronic hepatitis in vitamin B12-deficient twin-lambs (Vellema et al., 1999). Brain lesions were also observed in vitamin B12 deficient lambs in that study (Vellema et al., 1999). In ruminating cattle and sheep, vitamin B12 deficiency is most often caused by a cobalt deficiency. The syndrome is also known as “white-liver disease,” and the signs mimic those of pre-ruminant vitamin B12 deficiency and are reversible by either cobalt or vitamin B12 supplementation (Kennedy et al., 1997; Ulvund and Pestalozzi, 1990). Vitamin B12-deficient lambs exhibit a reduced lymphocyte response to Mycobacterium paratuberculosis vaccination and higher fecal egg counts from nematode infection compared to lambs supplemented with adequate cobalt (Vellema et al., 1996). Likewise, Paterson and MacPherson (1990) reported that cattle fed a cobalt-deficient diet for 10 weeks or longer had a significant reduction in neutrophil killing of Candida albicans yeast and greater weight loss when infected experimentally with brown stomach worm (Ostertagia ostertagi). Cobalt deficiency in beef cattle resulted in reduced appetite and weight gain, reduced vitamin B12 status, decreased triiodothyronine in serum, reduced folate levels in liver and a significant increase in liver accumulation of iron and nickel (Stangl et al., 1999).
Liver vitamin B12 concentrations of 0.10 µg or less per gram wet weight are “clearly diagnostic of cobalt deficiency disease” (Underwood, 1979). Liver cobalt concentrations in the range of 0.05 to 0.07 mg per kg (ppm; dry basis) or below are critical levels indicating deficiency (McDowell, 1985). Normal concentrations of serum MMA are tentatively suggested as being less than 2 µmol per liter, while subclinical cobalt deficiency is indicated by serum MMA concentrations of 2 to 4 µmol per liter and outright cobalt deficiency by more than 4 µmol per liter of serum (Paterson and MacPherson, 1990). In sheep, Marca et al. (1996) reported blood vitamin B12 concentrations greater than 0.2 µg/ml, in agreement with previous deficiency studies. Plasma MMA concentrations remained below 2 µmol/liter, considered normal for sheep. Milk vitamin B12 concentrations were 10 µg/ml in colostrum and early milk and declined thereafter to 50% or less the original value by 21 days of lactation (Marca et al., 1996).
Other histological and biochemical signs of cobalt/vitamin B12 deficiency in ruminants include: cardiovascular lesions resembling arteriosclerosis in sheep (Mohammed and Lamand, 1986); accumulation of odd-number, branched-chain fatty acids in tissues (Kennedy et al., 1994); and accumulation of succinate in rumen fluid due to the blockage in propionic acid synthesis (Kennedy et al., 1996).
Vitamin B12 deficiency induced by exposure to nitrous oxide resulted in almost complete inhibition of methionine synthase activity in liver, heart and brain tissue of sheep (Xue et al., 1986). The results led the authors to conclude that methyl group transfer via methionine synthase is required for adequate supply of methionine and methylated compounds. Amino acid concentrations are altered by cobalt deficiency in cattle as described by Stangl et al. (1998). In that study, feeding a cobalt-deficient diet reduced appetite, growth and vitamin B12 status and significantly reduced plasma methionine (by 53%) as well as the concentrations of valine, leucine, isoleucine, threonine, arginine, tyrosine and taurine. Conversely, plasma serine and homocysteine were significantly increased 2.7 and 4.8 times, respectively. Liver amino acid content was not affected.
Girard and coworkers have begun to explore the effects of vitamin B12, folic acid and methionine metabolism in lactating dairy cows (Girard et al., 1998; Girard and Matte, 2005; Preynat et al., 2009a, b; 2010; Girard et al., 2009). After observing an inverse relationship between serum vitamin B12 and folate concentrations during the course of lactation, Girard (1998a) conducted an experiment in which primiparous heifers were fed a diet supplemented with both folic acid and methionine and injected with either vitamin B12 or a placebo. In this preliminary trial, vitamin B12supplementation tended to increase milk production (8.8%), milk solids production (12%) and milk fat yield (16%), suggesting that vitamin B12 status may affect the response of lactating cows to folic acid and methionine supplementation.
Separate previously findings (Girard and Matte, 1998; 2005; Girard et al., 2005) showed that, in early lactation, a positive response of milk production and milk component yields to supplementary folic acid was observed in cows with higher plasma concentrations of vitamin B12. It appears that when folic acid was given in combination with vitamin B12 metabolic efficiency was improved, as suggested by similar lactational performance and dry matter intake over cows fed folic acid supplements alone. The effects of folic acid and vitamin B12 on lactation performance were not mainly explained by methionine economy because of a more efficient methylneogenesis but were rather related to increased glucose availability and changes in methionine metabolism (Preynat et al., 2009, a, b; 2010). These findings support the hypothesis that, in early lactation, supply of vitamin B12 was not optimal and limited the lactation performance of the cows.
Cobalt deficiency has been reported to reduce lamb survival and increase susceptibility to parasitic infection in cattle and sheep (Ferguson et al., 1988; Suttle and Jones, 1989). Cobalt deficiency has been associated with photosensitization of lambs characterized by a swollen head (Hesselink and Vellema, 1990). The condition responded to two injections of vitamin B12 three weeks apart. Cobalt deficiency in pregnant ewes reduced lamb numbers and increased stillbirths and neonatal mortality (Fisher, 1991). Lambs born from cobalt-deficient ewes were slower to nurse, had reduced concentrations of serum immunoglobulin G (IgG) and total protein, lower serum vitamin B12 and elevated serum MMA concentrations compared to normal lambs (Fisher, 1991). In African goats, cobalt deficiency reduced serum vitamin B12 and erythrocyte counts, although growth rate was not significantly affected, suggesting that goats, like cattle, are more resistant to cobalt/vitamin B12 deficiency than sheep (Mburu et al., 1993). Goats depleted of cobalt and thus vitamin B12 exhibited irregular estrus cycles, macrocytic anemia, decreased plasma progesterone and luteinizing hormone, and elevated plasma corticosteroids compared to normal animals (Mgongo et al., 1984).
With the exception of phosphorus and copper, cobalt is the most common mineral deficiency for grazing livestock in tropical countries (McDowell et al., 1984; McDowell, 1992). Cobalt deficiency signs are nonspecific, and it is often difficult to distinguish between a cobalt deficiency and basic malnutrition or parasitization. Acute clinical signs of cobalt deficiency mimic those of vitamin B12 deficiency (Illus. 10-2 and 10-3).
Illustration 10-2: Cobalt (Vitamin B12) Deficiencyin Cattle.
Illustration 10-3: Cobalt (Vitamin B12) in Cattle.
Courtesy of L.R. McDowell, University of Florida
The incidence of cobalt deficiency can vary greatly from year to year, from an undetectable mild deficiency to an acute stage. Lee (1963) illustrated this variation in a 14-year experiment with sheep in southern Australia. Half the ewes, replacements and progeny received supplemental cobalt and remained healthy (in no particular order by years). The undosed half had the following performance for the 14 years: in 2 years, lambs were unthrifty, but there were no deaths; in 3 years, growth rate of the lambs was slightly retarded; in 4 years, 30% to 100% of the lamb crop was lost; in 5 years, the performance of the remaining stock was as good as that of dosed animals. The conclusion would be that if there was no benefit from cobalt supplementation in a particular year, there was no guarantee that in the following year that a moderate to severe cobalt deficiency would not occur (McDowell, 2000). McDowell and Arthington (2003) have presented an extensive review of cobalt nutrition.
Rumen bacteria synthesize approximately 2 to 3 mg vitamin B12 per day when provided with adequate dietary cobalt. Synthesis of competitive analogs of vitamin B12 has been reported with higher levels of grain in the diet (Walker and Elliot, 1972). Serum vitamin B12 is lower during late pregnancy and early lactation than during mid-lactation, although correlations with milk yield, grain consumption or blood glucose concentration were poor (Elliot et al., 1965). Peters and Elliot (1983) reported that vitamin B12 injections increased milk protein production, but not milk yield, solids or fat production in vitamin B12-deficient ewes during early lactation. Liver capacity for gluconeogenesis from labeled propionic acid was increased in vitamin B12-treated ewes. Croom et al. (1981) did not detect any effect of supplementary vitamin B12 on milk fat synthesis in dairy cows. The authors had hypothesized that accumulation of MMA inhibits milk fat synthesis by reducing activity of lipogenic enzymes. Daugherty et al. (1986) found no beneficial effect of vitamin B12injections on growth performance or propionate-metabolizing enzymes in the livers of feedlot lambs fed 80% concentrate diets with an ionophore. Girard et al. (1998) in a preliminary trial reported that primiparous dairy cows fed diets supplemented with both folic acid and methionine responded to vitamin B12 injections with increased milk production between 25 and 125 days of lactation. In further studies, supplemental vitamin B12 and folic acid were found to be beneficial for milk production and milk component yields for high producing dairy cows in early lactation (Girard and Matte, 1998; 2005; Girard et al., 2005; 2009; Preynat et al., 2009 a, b; 2010). Weekly intramuscular injections of vitamin B12 and folic acid increased milk concentration of vitamin B12 by 68% in commercial dairy herds (Duplessis et al., 2011). A glass (250 ml) of milk from supplemented cows provided 54% of the recommended daily allowance (2.4 µg) for adults. Primiparous and multiparous cows differed in their milk yield response to dietary cobalt supplementation (Kincaid et al., 2003). Primiparous cows secreted colostrum and milk with higher cobalt concentrations than did multiparous cows. Likewise, serum B12levels were higher in primiparous than multiparous cows.
Young ruminants require supplemental vitamin B12 prior to full rumen development. Milk is a good source of vitamin B12. The NRC (2001) suggests that milk replacer for dairy calves contain 0.11 mg of cobalt per kg (0.05 mg per lb).
Although dietary supplementation of cobalt is the normal means of meeting the vitamin B12 requirement of ruminants, parenteral administration is used to treat animals with apparent deficiency symptoms or the general appearance of malnutrition or poor health. Vitamin B12 is sometimes administered parenterally to incoming feedlot cattle as a prophylactic measure.
Intramuscular injection of vitamin B12, at the rate of 100 µg per week, or 150 µg every other week, produced a rapid remission of all signs of deficiency in lambs, and was equivalent to cobalt administered orally at the rate of 7 mg per week (Andrews and Anderson, 1954). For treatment of cobalt deficiency in cattle, intramuscular administration of vitamin B12 at 500 to 3,000 µg per head is recommended, which may be repeated weekly (Graham, 1991). Administration of intramuscular vitamin B12 to cobalt-deficient animals produces overnight improvement in appetite, whereas oral dosing with cobalt requires seven to 10 days to produce similar effects (MacPherson, 1982).
Many forages and concentrate feeds do not supply adequate (0.10-0.20 ppm) cobalt, and thus supplementation is required. Growth responses to supplemental cobalt have been demonstrated in steers fed finishing diets based on barley grain (Raun et al., 1968), and sorghum grain and silage (Morris and Gartner, 1967). More recently, Tiffany and Spears (2005) used fattening cattle to show an effect of grain source on ruminal synthesis of vitamin B12 with greater response to cobalt supplementation when corn was the grain source compared with barley.
Animals can tolerate large excesses of vitamin B12. Dietary levels of at least several hundred times the requirement are suggested as safe for the mouse (NRC, 1987). Vitamin B12 is reported to be toxic for some animal species with diets of around 5 mg per kg (2.3 mg per lb). Signs of toxicity are unclear, especially with many older reports, since results are likely confounded with toxic effects of fermentation residues, inadvertently included with vitamin B12 during manufacture (Leeson and Summers, 2001). Cobalt also has a low toxicity, with 10 mg per kg (4.5 mg per lb) the maximum dietary tolerable level for the common livestock species (NRC, 1980). Becker and Smith (1951) concluded that 150 mg per kg (68.2 mg per lb) on a dry diet basis, or 1,000 times normal levels, can be tolerated by sheep for many weeks without visible toxic effects. Characteristic signs of chronic cobalt toxicity in most species are reduced feed intake and body weight, emaciation, anemia, hyperchromenia, and increased liver cobalt (NRC, 1980). Cobalt toxicity in cattle is characterized by mild polycythemia; excessive urination, defecation and salivation; shortness of breath; and increased hemoglobin, red cell count and packed cell volume (NRC, 2000).