Impact of B-vitamin supply on major metabolic pathways of lactating dairy cows C. L. Girard and J. J. Matte

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Agriculture and Agri-Food Canada, Dairy and Swine Research and Development Centre, Sherbrooke, Quebec, Canada J1M 1Z3 (e-mail: [email protected]). Received 6 July 2005, accepted 12 October 2005. Girard, C. L. and Matte, J. J. 2006. Impact of B-vitamin supply on major metabolic pathways of lactating dairy cows. Can. J. Anim. Sci. 86: 213–220. Knowledge of the major nutrient requirements of dairy cows has increased substantially during the past decades. Little is known, however, about the importance of the roles played by B vitamins. Since most of those vitamins act as essential cofactors in energy, protein and lipid metabolism, it is likely that as milk yield increases, the demand for these cofactors also increases. The supply of B vitamins from dietary sources and synthesis by the ruminal microflora is generally sufficient to avoid deficiency symptoms, but could be insufficient for optimizing metabolic efficiency, production, composition and the nutritional quality of milk in high-producing dairy cows. Results from recent experiments show how the supply of three B vitamins — folic acid, biotin and vitamin B12 — affects major metabolic pathways. Supplementary biotin has frequently been reported to increase milk yield but has a limited effect on milk composition. Folic acid supplements have been found to increase milk and milk protein yields in multiparous cows without affecting dry matter intake when vitamin B12 supply was adequate. An insufficient vitamin B12 supply blocked those effects but they can be restored through vitamin B12 supplementation. Supplemental vitamin B12 and biotin increased milk and milk protein yields without changing dry matter intake. Vitamin B12 utilization by tissues increased in cows fed supplementary folic acid simultaneously; plasma glucose also increased in these cows but plasma biotin decreased. From these findings, it appears that, in high-producing dairy cows, especially in early lactation, the strong competition for nutrients that occurs between gluconeogenesis, methylneogenesis and protein synthesis increases the amount of folic acid, vitamin B12 and biotin required to maintain metabolic efficiency, especially when the nutrient supply is limited. These observations emphasize the need to review the paradigm according to which B-vitamin supply by ruminal microflora cannot be limiting in dairy cow. Key words: Dairy cow, B vitamins, folic acid, vitamin B12, biotin, lactation, metabolism Girard, C. L. et Matte, J. J. 2006. Impact des apports en vitamines du complexe B sur les principales voies métaboliques pendant la lactation des vaches laitières. Can. J. Anim. Sci. 86: 213–220. Les connaissances sur les besoins des vaches laitières en nutriments majeurs ont augmentées substantiellement au cours des dernières décennies. Cependant, on connaît encore peu l’importance des rôles des vitamines B. La plupart de ces vitamines sont des cofacteurs essentiels dans les métabolismes énergétiques, protéiques et lipidiques, il est donc probable que la demande pour ces cofacteurs augmente avec la production laitière. Les apports en vitamines B provenant de l’alimentation et de la synthèse par la microflore du rumen suffisent généralement à éviter les symptômes de déficience mais sans nécessairement permettre d’optimiser l’efficacité métabolique, la production, la composition et la qualité nutritionnelle du lait chez les vaches hautes productrices. Les résultats d’expériences récentes mettent en évidence l’impact sur les voies métaboliques majeures des apports en trois vitamines B, l’acide folique, la biotine et la vitamine B12. Plusieurs études rapportent une augmentation de la production laitière mais des effets limités sur la composition du lait suite à une supplémentation en biotine. Des suppléments d’acide folique augmentent les quantités de lait et de protéines du lait produites par des vaches multipares sans modifier la consommation de matière sèche lorsque les apports en vitamine B12 sont adéquats. Un apport insuffisant de vitamine B12 bloque ces effets lesquels sont restaurés par un supplément de vitamine B12. Des suppléments de vitamine B12 et de biotine augmentent les quantités de lait et de protéines du lait produites sans augmenter la consommation de matière sèche. L’utilisation de la vitamine B12 par les tissus est augmentée chez les vaches recevant simultanément des suppléments d’acide folique; le glucose plasmatique de ces vaches est aussi augmenté alors que la concentration plasmatique de biotine est diminuée. En conclusion, ces résultats indiquent que, chez les vaches laitières hautes productrices, spécialement en début lactation, la très forte compétition entre la gluconéogénèse, la méthylnéogénèse et la synthèse protéique pour les nutriments augmente la demande pour l’acide folique, la vitamine B12 et la biotine afin de maintenir l’efficacité métabolique, ceci étant d’autant plus marqué que les quantités de nutriments disponibles sont limitées. Ces observations mettent l’emphase sur le besoin de revoir le paradigme selon lequel les apports en vitamines B via leur synthèse par la microflore du rumen ne peuvent être limitants chez la vache laitière. Mots clés: Vache laitière, vitamines B, acide folique, vitamine B12, biotine, lactation, métabolisme

high levels of B vitamins, even if the animal’s diet provided very small amounts of those vitamins. Furthermore, over the years since the discovery of B vitamins, it appears that true deficiency of these vitamins is rare in animals with a functional rumen. It is generally accepted that B-vitamin requirements can be met through synthesis by ruminal bacteria and dietary

Approximately 80 years ago, Bechdel et al. (1928) demonstrated that bacteria present in the rumen of a cow produced Presented at the Canadian Society of Animal Science Symposium “Vitamin Nutrition of Livestock Animals” held in Cincinnati, Ohio, USA, in July 2005. 213

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sources that escape from the rumen (National Research Council 2001). Consequently, very little research effort has been directed at defining dairy cow requirements for B vitamins. Over the past 50 years, however, milk and milk component yields have increased dramatically. It is likely that the B-vitamin requirements of high-producing dairy cows have likewise increased and that ruminal synthesis alone is not sufficient to meet these new needs. This hypothesis is supported by studies that have reported beneficial effects from supplementation with thiamin (Shaver and Bal 2000), niacin (Fronk and Schultz 1979; Riddell et al. 1981), biotin (Zimmerly and Weiss 2001), folic acid (Girard et al. 1995; Girard and Matte 1998) and vitamin B12 (Girard and Matte 2005). Even if B vitamins are frequently considered as an entity, it should be borne in mind that this nomenclature has no chemical or metabolic significance. The basis for this classification is purely historical and ignores the heterogeneity of the chemical structure and metabolic roles of these substances. Although there are many interactions among B vitamins, the present paper will focus on three B vitamins sharing common metabolic pathways, biotin, folic acid, vitamin B12, and their impact on lactating dairy cow performance and metabolism. MECHANISMS OF ACTION AND ENZYMES INVOLVED Biotin Along with folic acid and S-adenosylmethionine, biotin plays a role in intermediary metabolism in the transfer of one-carbon units. In particular, biotin participates in the transfer of the most oxidized form of one-carbon units, namely carbon dioxide (McMahon 2002). Biotin is the coenzyme for four carboxylases, which catalyze the incorporation of CO2 into different substrates: (1) pyruvate carboxylase catalyzes the carboxylation of pyruvate, resulting from the degradation of glucose and amino acids (serine, cysteine and alanine), to form oxaloacetate for glucose synthesis or the acetyl-CoA shuttle; (2) propionyl-CoA carboxylase converts propionyl-CoA, generated by the degradation of odd-numbered chain fatty acids, propionate or amino acids (valine, isoleucine, methionine, threonine) to methylmalonylCoA, which will enter Krebs cycle as succinyl-CoA; (3) β-methylcrotonyl-CoA carboxylase catalyzes the carboxylation of β-methylcrotonyl-CoA, resulting from leucine catabolism, to β-methylglutaconyl-CoA, which is in turn transformed into acetyl-CoA; (4) acetyl-CoA carboxylase catalyzes the incorporation of CO2 into acetyl-CoA to form malonyl-CoA for fatty acid synthesis. These enzymes are of major importance for the metabolism of glucose, fatty acids and some amino acids (Fig. 1). Folic Acid In mammals folic acid has the single important biochemical function of accepting and releasing one-carbon units (Choi

and Mason 2000). This role is essential for: 1) synthesis of purines and pyrimidines and (2) de novo synthesis of methyl groups for the formation of the primary methylating agent, S-adenosylmethionine (Fig. 2) (Bailey and Gregory 1999). Many enzymes are folic acid-dependent, among them: (1) formiminoglutamate transferase catalyzes the transformation of formiminoglutamic acid, a product of histidine degradation, to glutamic acid; (2) polyenzyme complex, for the degradation of glycine; (3) serine hydroxymethyltransferase catalyzes the reversible transformation of serine into glycine and is the major source of one-carbon units in mammals; (4) methionine synthase, for the irreversible methylation of homocysteine to methionine; (5) glycinamide-ribose transformylase and aminodazolecarboxamide-ribose transformylase, for purine ring formation; (6) thymidylate synthase, for the synthesis of thymidylic acid from desoxyuridylic acid. Vitamin B12 The three vitamin B12-dependent enzymes are involved in two reactions: transmethylation (transfer of a methyl group) and isomerization (structural modifications of a molecule) reactions. These enzymes are: (1) methionine synthase, described previously, for which vitamin B12 serves as an intermediary transporter of methyl groups between methyltetrahydrofolate (methylated form of folates) and homocysteine. This enzyme is the link between metabolism of folic acid and vitamin B12; (2) methylmalonyl-CoA mutase for the transformation of methylmalonyl-CoA to succinyl-CoA, through the degradation of odd-numbered-chain fatty acids, propionate and some amino acids (valine, isoleucine, methionine, threonine). This enzyme is the link between biotin and vitamin B12 metabolism (Fig. 1); (3) leucine mutase for the isomerization of L-α-leucine to L-β-leucine. The metabolic importance of this enzyme has not been characterized but it has been identified in rat liver and kidney, sheep and monkey liver and human leukocytes (Schneider and Stroi´nski 1987). IMPACT OF SUPPLEMENTAL BIOTIN AND FOLIC ACID ON DAIRY COW PERFORMANCE Biotin Although most scientific publications on the effect of biotin supplementation for dairy cows have focused on hoof health, the present paper examines the effects of biotin on dairy cow performance and metabolism. Higuchi et al. (2003) observed changes in serum concentrations of biotin during lactation, indicating that the difference between the supply and the demand for biotin varied in dairy cows. The serum concentration of biotin was higher in mid-lactation than in early lactation or the dry period. However, because the authors found an inverse correlation between the biotin concentrations in serum and in milk, they concluded that changes in serum concentrations could be

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Fig. 1. Simplified metabolic pathways involving biotin (B8)- and vitamin B12-dependent enzymes. (1) pyruvate carboxylase; (2) propionylCoA carboxylase; (3) β-methylcrotonyl-CoA carboxylase; (4) acetyl-CoA carboxylase; (5) methylmalonyl-CoA mutase.

Fig. 2. Roles of folic acid in DNA and methylation cycles. B12(1), vitamin B12-dependent enzyme, methionine synthase; DHF, dihydrofolate; THF, tetrahydrofolate, 5-CH3-THF, methyltetrahydrofolate; 5, 10-CH2-THF, methylenetetrahydrofolate; 10-CHOTHF, formyltetrahydrofolate; dUMP, desoxyuridylic acid; dTMP, thymidylic acid.

explained by the amount of biotin secreted in milk although they did not report the total amount of biotin secreted in milk daily. On the other hand, Zimmerly and Weiss (2001) observed that, from 30 to 100 d of lactation, plasma and milk concentrations of biotin are positively correlated in cows fed different levels of biotin supplementation. Moreover, other studies have shown that dairy cows could in fact benefit from biotin supplementation, most of those studies used a daily dietary supplementation of 20 mg of

biotin. Table 1 shows the effects of biotin supplementation on the production performance of dairy cows in studies specifically designed for that purpose (Bonomi et al. 1996; Zimmerly and Weiss 2001; Majee et al. 2003; Rosendo et al. 2004) and in studies focusing on foot lesions (Cooke and Brumby 1982; Midla et al. 1998; Fitzgerald et al. 2000; Bergsten et al. 2003). In summary, supplementary biotin was found to increase milk yield in five out of eight studies. It had a very limited effect on milk composition but biotin

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Table 1. Responses of lactational performance to dietary supplementation with biotinz

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Dry matter intake Cooke and Brumby (1982) Midla et al. (1998)y Bonomi et al. (1996) Fitzgerald et al. (2000)x Zimmerly and Weiss (2001) Majee et al. (2003)w Bergsten et al. (2003)y Rosendo et al. (2004)

ND ND ND ND = + ND =

Milk yield

Milk fat content

Milk protein content

Milk lactose content

Milk fat yield

Milk protein yield

= + + = + + + =

= ND + – = = ND =

= ND + = = = ND +

ND ND = ND ND + ND ND

ND ND ND ND = = + =

ND ND ND ND + + ND =

zND, not determined; =, no effect; +, increase; –, decrease. yCalculated 305-d mature equivalent milk production. xDaily milk production and composition of bulk milk samples wMilk lactose yield was also increased.

from 10 supplemented and 10 control herds were compared.

supplements increased milk component yields in some studies. For example, biotin increased the milk protein yield in two out of three studies. The mode of action of biotin on production performance remains to be elucidated. Clearly, the production effect is not exclusively due to an improvement in hoof health because the positive effects on performance become apparent shortly after supplementation begins and a long time before any effect is seen on hoof horn quality. Milligan et al. (1967) demonstrated that, in vitro, a lack of biotin decreased cellulose digestion and volatile fatty acid production by rumen microflora. However, in vivo, supplementary biotin did not change total tract organic matter, dry matter or NDF digestibility (Majee et al. 2003) nor did it affect volatile fatty acid concentration in ruminal fluid (Zimmerly and Weiss 2001). Biotin supplements increased plasma glucose in two (Bonomi et al. 1996; Rosendo et al. 2004) out of four studies (Bonomi et al. 1996; Zimmerly and Weiss 2001; Majee et al. 2003; Rosendo et al. 2004). Only Rosendo et al. (2004) observed lower plasma concentrations of non-esterified fatty acids during the first 4 wk of lactation, along with a decrease in liver concentrations of total lipids and triacylglycerols, indicating reduced mobilization of body reserves in biotin-supplemented cows. However, as McMahon (2002) points out, biotin is a poorly understood vitamin; its role probably extends well beyond its essential function in carboxylation reactions and there is now evidence that it participates in controlling gene expression. The recent review by Zempleni (2005) emphasizes the roles of biotin in cell signalling, which control the expression of genes and chromatin structure, which plays a role in cell proliferation, gene silencing and cellular responses to DNA repair. Folic Acid Girard et al. (1995) reported that intramuscular injections of folic acid (160 mg of pteroylmonoglutamic acid) given weekly to primiparous and multiparous dairy cows from day 45 of gestation to 6 wk after calving increased milk production and milk protein content from day 45 of gestation to drying off. Data before parturition were reported according to the number of weeks of lactation achieved to correct for cows that required repeated breeding. After calving, injections of folic acid had no effect on milk production or on milk protein content in

primiparous cows, whereas they increased milk protein content in cows in their second or greater lactation. Dietary supplements of folic acid (0, 2 or 4 mg kg–1 of body weight) given daily from 4 wk before expected calving until day 305 of lactation decreased milk production in primiparous cows during the first 100 d of lactation although this effect was no longer significant over a 305-d lactation period. By contrast, in multiparous cows, supplementary folic acid boosted milk production, with the largest effect being observed between 100 and 200 d of lactation. Whereas dietary supplements of folic acid did not change milk composition in multiparous cows, milk component yields exhibited the same upward trend as milk yield (Girard and Matte 1998). In a study undertaken to elucidate the interactions between daily dietary supplements of folic acid (0, 3 and 6 mg kg–1 of body weight) and rumen-protected methionine and the effects on the lactational performance of multiparous cows during a 305-d lactation period, total milk production and milk component yields were not changed by the folic acid and/or rumen-protected methionine supplementation (Girard et al. 2005). Supplemental folic acid decreased milk urea and increased casein concentrations in the milk of cows that received no supplementary methionine, and the effect increased as lactation progressed. Folic acid supplementation had the opposite effect in cows that were fed rumenprotected methionine, increasing milk urea while decreasing milk casein concentrations. In all three experiments, supplementary folic acid had no effect on the dry matter intake of cows. One observation that stands out from those experiments is that cows that did not respond to folic acid supplementation had low serum concentrations of vitamin B12. Girard and Matte (1999) observed low levels of serum vitamin B12 in early lactation, more so in primiparous than in multiparous cows. In contrast, in that same experiment, folic acid supplementation increased the milk and milk protein yields of multiparous cows but not primiparous cows (Girard and Matte 1998). The lowest serum concentration of vitamin B12 was observed in early lactation at a time when the serum concentrations of folates peaked, with this increase being especially noticeable in cows fed dietary supplements of folic acid. Later, after 8 to 12 wk of lactation, serum vitamin B12 levels increased whereas serum concentrations of folates fell in the supplemented cows. Girard et al. (2005)

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found identical patterns for serum concentrations of folates and vitamin B12 although the vitamin B12 levels were lower than previously reported for multiparous cows and comparable to those of primiparous cows (Girard and Matte 1999). Moreover, serum clearance of folic acid following an intravenous bolus of folic acid was also slower in early lactation than later in lactation (Girard et al. 2005). Despite the fact that in both experiments, the dietary supply of cobalt was above the current NRC requirement (2001) and recommendations for beef cattle (Stangl et al. 2000), serum vitamin B12 concentrations in cows that were not responsive to folic acid supplements were under 200 pg mL–1 in early lactation. By comparison, Stangl et al. (2000) observed plasma vitamin B12 concentrations of approximately 108 pg mL–1 in growing cattle fed a cobalt-deficient diet and 271 pg mL–1 in those fed a cobalt adequate-diet. Low serum concentrations of vitamin B12 have frequently been observed in dairy cows during early lactation (Elliot et al. 1965; Mykkänen and Korpela 1981). Walker and Elliot (1972) observed the opposite pattern, serum vitamin B12 increasing from 4 wk before the expected time of calving until 12 wk of lactation but decreasing after 16 wk of lactation. However, in this last experiment, the method used was sensitive not only to the biologically active forms of vitamin B12, but also to at least two analogues of the vitamin. A lack of vitamin B12 in early lactation could reduce utilization of supplementary folic acid by the cow’s tissues, given that folic acid becomes “trapped” in the serum under its methylated form, 5-methyl-tetrahydrofolate. In fact, a lack of vitamin B12 inhibits methionine and S-adenosylmethionine synthesis. All available one-carbon units are diverted to the synthesis of 5-methyl-tetrahydrofolate. This reaction is irreversible, and demethylation through the regeneration of methionine is blocked by the lack of vitamin B12. Purine and DNA synthesis are therefore deprived of one-carbon units, the proliferation of rapidly dividing cells is slowed and protein incorporation of methionine is reduced to permit more urgent methylation functions. The accumulation of 5-methyl-tetrahydrofolate leads to a lack of folates at the cell level. This explains the identical symptoms of anaemia seen in humans suffering from severe folic acid or vitamin B12 deficiency (Bässler 1997). Vitamin B12 might be a limiting factor for the action of folic acid in early lactation; this hypothesis is supported by production data (Girard and Matte 1998; Girard et al. 2005). RELATIONSHIP BETWEEN METABOLISM OF FOLATES AND VITAMIN B12 In early lactation, in the absence of a folic acid supplementation, weekly (10 mg cyanocobalamin) or bi-weekly (150 mg hydroxocobalamin) intramuscular injections of vitamin B12 had no effect on milk production or milk fat content or yield (Elliot et al. 1979; Croom et al. 1981). By contrast, in a study in which primiparous cows were fed daily supplements of folic acid (4 mg kg–1 of body weight) and rumen-protected methionine (to bring the estimated supply of methionine to 2.2% of metabolizable protein), weekly intramuscular injec-

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tions of vitamin B12 (10 mg cyanocobalamin) from weeks 4 to 18 of lactation tended to increase milk yield from 28.5 to 31.1 kg d–1 and increased energy-corrected milk as well as milk yields of solids, fat and lactose. Supplementary vitamin B12 had no effect on dry matter intake or milk composition. Furthermore, the finding that packed cell volume and blood hemoglobin increased and serum methylmalonic acid decreased in cows that received vitamin B12 injections supports the hypothesis that vitamin B12 supply is suboptimal in early lactation and that this limits the lactational performance of dairy cows (Girard and Matte 2005). In a factorial experiment, multiparous cows were fed supplementary folic acid (2.6 g d–1) and/or vitamin B12 (500 mg d–1) from 3 wk before expected calving until week 8 of lactation. Dry matter intake was similar in all the treatments. Supplemental folic acid increased significantly milk production and milk fat and protein yields. Supplementary vitamin B12 significantly increased milk concentrations of protein (Graulet et al. unpublished). Plasma and liver concentrations of folates and vitamin B12 were significantly increased by the corresponding dietary supplements. Nevertheless, the increases in plasma concentrations of folates and vitamin B12 along with liver vitamin B12 levels following ingestion of vitamin supplements were smaller in cows fed the two vitamins simultaneously than in cows receiving only one of the vitamins, even if similar amounts were supplied. These observations support the hypothesis that vitamin B12 utilization, even more so by extrahepatic tissues, is increased in cows that are given the two vitamin supplements simultaneously (Graulet et al. unpublished). RELATIONSHIP BETWEEN METABOLISM OF BIOTIN, VITAMIN B12 AND FOLIC ACID In dairy cows, the roles of only two vitamin B12-dependent enzymes have been described so far. Methionine synthase, described earlier in this paper, is a cystosomal enzyme which plays an essential role in transferring one-carbon units from the methylated form of folic acid to homocysteine, to regenerate methionine and tetrahydrofolate. Methylmalonyl-CoA mutase, a mitochondrial enzyme, transforms methylmalonyl-CoA into succinyl-CoA. This is where the interaction with biotin metabolism comes into play. Methylmalonyl-CoA results from the degradation of odd-chain fatty acids, some amino acids (valine, isoleucine, methionine, threonine) and propionate. In brief, propionate is first transformed into propionyl-CoA; then through the action of propionyl-CoA carboxylase, a biotin-dependent enzyme, it receives an additional carbon and becomes methylmalonyl-CoA. Under the action of methylmalonylCoA mutase, a vitamin B12-dependent enzyme, the CO-SCoA group is transferred from one carbon to another on the molecule, forming succinyl-CoA, which will enter the Krebs cycle (Fig. 1; Le Grusse and Watier 1993; McDowell 2000). Vitamin B12 deficiency causes an accumulation of methylmalonic acid, which can disrupt glucose and glutamic acid metabolism (Combs 1998). Therefore, it is likely that this metabolic pathway plays an important role in the energy metabolism of dairy cows.

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The high-producing dairy cow requires a large supply of energy and glucose. Huge amounts of glucose are required by the lactating mammary gland to synthesize lactose, the primary osmotic controller of milk volume (Overton 1998). A cow producing 40 kg of milk per day requires approximately 3 kg of glucose per day (Elliot 1979). The nature of the ruminant digestive system imposes a huge dependence on gluconeogenesis, as very little glucose is absorbed. Unlike the situation in non-ruminants, in ruminants, the rate of gluconeogenesis increases with feed intake (Hocquette and Bauchart 1999) and the major substrates for gluconeogenesis are propionate, glucogenic amino acids and lactate. Lactate metabolism is closely related to propionate metabolism because, in addition to lactate produced in the rumen, lactate is formed during the catabolism of glucose by peripheral tissues or the degradation of propionate by the ruminal epithelium (Overton 1998). Seal et al. (1992) reported that 83% of propionate was transformed into glucose in steers, making up about 53% of whole-body glucose. Propionate metabolism likely increases ruminant requirements for biotin and vitamin B12 as compared with monogastric animals. The other major source of glucose is the gluconeogenic amino acids absorbed from the gastrointestinal tract and supplied by substantial degradation of skeletal muscle protein in early postpartum (Overton 1998). Therefore, in early lactation, amino acid requirements are high, owing to their role as substrates for gluconeogenesis and the enormous requirements for milk protein synthesis. Moreover, lactation increases the demand for methylated compounds (synthesis of milk choline, creatine, creatinine and carnitine) and for methionine to support milk protein synthesis (Xue and Snoswell 1985). Significant amounts of free choline, lipid choline, creatine and creatinine are secreted in milk (Xue and Snoswell 1985). The large amount of creatine that is irremediably lost in urine and has to be replaced entirely by de novo synthesis represents another appreciable loss of methyl groups (Snoswell and Xue 1987). De novo synthesis of methyl groups, which is clearly of paramount importance, occurs through the action of methionine synthase; this ubiquitous enzyme mediates the transfer of a methyl group from the folate cofactor 5-methyl-tetrahydrofolate to homocysteine to form methionine (Snoswell and Xue 1987). Ruminants have considerable capacity to generate the methyl group of 5-methyl-tetrahydrofolate through folate metabolism of one-carbon units, for which serine, glycine, histidine and formate are fairly abundant sources. Gluconeogenic precursors, such as glycine and serine, are the primary sources for de novo synthesis of methyl groups (Armentano 1994). Moreover, de novo synthesis of serine utilizing glycerophosphate as a substrate allows for the transfer of one-carbon units from the 3-carbon glucose precursor pool to the methyl group pool (Emmanuel and Kennelly 1984; Armentano 1994). During early lactation, the demand for methyl groups is high and there is a concomitant increase in pressure to synthesize glucose in order to support lactose output and provide energy. At that time, competition between gluconeogenesis and methylneogenesis is likely to create a shortage of precursors for de novo synthesis of methylated compounds as well as decrease amino acid supply for protein synthesis. These reactions

emphasize the interactions between metabolic reactions relying on folic acid, vitamin B12 and biotin-dependent enzymes. Supplements of rumen-protected choline given in early lactation are likely to decrease the pressure of competition between these reactions by reducing the demand for methylneogenesis. This aspect is covered in detail in the reviews of Pinotti et al. (2002) and Baldi and Pinotti (2005). Girard et al. (unpublished data) conducted an experiment using four cows (118 ± 5.6 d of lactation) in a double 2 × 2 Latin square, in which the cows received either no vitamin supplementation or daily dietary supplements of biotin (20 mg) and vitamin B12 (500 mg). They found that the combined supplement of biotin and vitamin B12 increased milk production by 1.1 kg d–1 from 30.5 to 31.6 kg d–1 and milk protein yield from 1.04 to 1.07 kg d–1 without affecting dry matter intake (P ≤ 0.05). Besides increasing milk secretion of the supplemented vitamins, combined supplementation increased the amount of folates secreted daily in milk and decreased arterial plasma concentrations of urea but had no effect on plasma concentrations of glucose (P ≤ 0.05). Combined supplements of biotin and vitamin B12 seem to improve the efficiency of nitrogen metabolism, although the exact mechanism of action remains to be elucidated. Findings from the experiment described at the end of the previous section investigating the effects of supplemental folic acid and/or vitamin B12 in multiparous cows fed a low-methionine diet from 3 wk prepartum to 8 wk postpartum indicate that vitamin B12 utilization was increased in cows that received supplementary folic acid simultaneously but biotin status also seemed to be affected (Graulet et al., unpublished). In cows that received no folic acid supplements, plasma biotin was increased but plasma glucose was unchanged by the vitamin B12 supplementation. Meanwhile, in cows that received dietary supplements of folic acid, plasma biotin decreased but plasma glucose increased with supplementary B12 (interaction folic acid × vitamin B12, P ≤ 0.03 and P ≤ 0.1 for glucose and biotin, respectively). Changes in plasma concentrations only reflect differences between the supply and tissue demands. In the experiment concerned, all cows received the same basal diet; hence, the nutrient supply should be similar among the treatments and differences in plasma concentrations are likely to reflect changes in tissue utilization. Therefore, in cows fed supplementary folic acid, it seems that tissue utilization of biotin was increased by vitamin B12 supplementation and resulted in increased formation of glucose. It is possible that dietary supplements of vitamins may have modified ruminal fermentations and the cows’ nutrient supply. Nonetheless, the results seem to indicate that supplementary vitamin B12 acts on the two vitamin B12-dependent metabolic pathways. Neither the interrelationships among the three B vitamins nor the effect that a suboptimal supply has on the major metabolic pathways are well understood. However, the literature clearly shows that these metabolic pathways are closely linked and that the supply of biotin, folic acid and vitamin B12 affects metabolic efficiency. This is made more obvious by the fact that dry matter intake was not altered in most studies that reported effects from supplementary vitamins on milk performance and composition.

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GIRARD AND MATTE — B VITAMINS IN RUMINAL FRACTIONS OF DAIRY COWS

CONCLUSION In a dairy production context in most Western countries, research is no longer oriented towards increasing production but instead focuses on meeting consumers’ demands for safe and high-quality dairy products that are produced in a manner that is respectful of the environment and animal wellbeing. Improving metabolic efficiency is an approach that helps to reduce the environmental impacts of dairy production while taking into account dairy cow welfare. The present review highlights the fact that B vitamins, especially biotin, folic acid and vitamin B12 can influence metabolic efficiency through their role as facilitators (coenzyme, cofactor) of enzymatic reactions. However, research on these micronutrients is still in its infancy, having being put on hold for decades under the premise that B vitamin supply is not limiting because they are synthesized by ruminal microflora. Future research should focus on dietary factors modifying ruminal synthesis of B vitamins as well as the effects of nutritional and management practices on tissue demand for B vitamins and the effects of B-vitamin supply on metabolic efficiency. The mechanisms of action of dietary supplements of B vitamins should also be elucidated, in order to dissociate their effects on ruminal microflora and fermentation products from their direct effect on the cow post-absorptive metabolism. The major problems to overcome are that the literature on the subject is scarce and, as for the methodological methods, they have to be adapted mostly from research in humans. Armentano, L. E. 1994. Impact of metabolism by extragastrointestinal tissues on secretory rate of milk proteins. J. Dairy Sci. 77: 2809–2820. Bailey, L. B. and Gregory III, J. F. 1999. Folate metabolism and requirements. J. Nutr. 129: 779–782. Baldi, A. and Pinotti, L. 2006. Choline metabolism in high-producing dairy cows: Metabolic and nutritional basis. Can. J. Anim. Sci. 86: 207–212. Bässler, K. H. 1997. Enzymatic effects of folic acid and vitamin B12. Int. J. Vit. Nutr. Res. 67: 385–388. Bechdel, S. I., Honeywell, H. E., Dutcher, R. A. and Knutsen, M. H. 1928. Synthesis of vitamin B in the rumen of the cow. J. Biol. Chem. 80: 231–238. Bergsten, C., Greenough, P. R., Gay, J. M., Seymour, W. M. and Gay, C. C. 2003. Effects of biotin supplementation on performance and claw lesions on a commercial dairy farm. J. Dairy Sci. 86: 3953–3962. Bonomi, A., Quarantelli, A., Sabbioni, A. and Superchi, P. 1996. L’integrazione delle razioni per le bovine da latte con biotina in forma rumino-protetta. Effeti sull’efficienza produttiva e riporduttiva (contributo sperimentale). [Dairy cattle ration integration with rumen-protected biotin. Effects on production and reproductive efficiency (experimental contribution)]. La Rivista di Scienza dell’Alimentazione.25: 49–68. Choi, S.-W. and Mason, J. B. 2000. Folate and carcinogenesis: an integrated scheme. J. Nutr. 130: 129–132. Combs, G. F., Jr. 1998. The vitamins. Fundamental aspects in nutrition and health. 2nd ed. Academic Press, San Diego, CA. 618 pp. Cooke, B. C. and Brumby, P. E. 1982. Biotin – a dairy herd feeding trial. Pages 21–26 in Proceedings of the Roche Vitamin Symposium, London, UK. 1982 Nov. 11.

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