Methods and approaches to estimate B vitamin status in dairy cows: Knowledge, gaps and advances.

Clinical symptoms of B vitamin deficiency are rarely observed in ruminants because these vitamins are synthesized by the rumen microbiota. However, over the last decades, numerous reports of beneficial effects on production and metabolic efficiency of dairy cows have been published supporting that, under some conditions, B vitamin subclinical deficiency is present in these animals. Due to their roles as coenzymes or cofactors in major metabolic pathways, an adequate supply in B vitamins is critical to optimize metabolic efficiency. Nowadays, taking into account the growing interest for the Smart Farming concept, fulfilling ruminant requirements for B vitamins according to their physiological stage under different feeding management cannot be neglected. In dairy cows, B vitamin supply is greatly dependent of the activity of the ruminal microbiota. Indeed, the amount of vitamins reaching the small intestine is dependent of the utilization of the vitamins provided by the diet and their synthesis by the microorganisms present in the rumen. The two major challenges faced to determine B vitamin status of ruminants are the difficulty to estimate B vitamin supply due to the lack of knowledge on factors driving the fate of B vitamins in the digestive tract, especially in the rumen, and the choice and thresholds of biomarkers reflecting adequately the animal status. The present paper aims to present the actual state of knowledge on the methodological approaches used to estimate B vitamin supply and status of ruminant and to point out future research orientations.

Combined biotin, folic acid, and vitamin B12 supplementation given during the transition period to dairy cows: Part I. Effects on lactation performance, energy and protein metabolism, and hormones.

Biotin (B8), folates (B9), and vitamin B12 (B12) are involved and interrelated in several metabolic reactions related to energy and protein metabolism. We hypothesized that a low supply of one of the latter vitamins during the transition period would impair metabolic status. The purpose of this study was to evaluate the effect of B8 supplementation on the response of lactation performance and selected energy and protein metabolites and hormones to a combined supplementation of B9 and B12 given to periparturient dairy cows, from d -21 to 21 relative to calving. A total of 32 multiparous Holstein cows housed in tie stalls were randomly assigned, according to their previous 305-d milk yield, to 8 incomplete blocks of 4 treatments: (1) a 2-mL weekly i.m. injection of saline (0.9% NaCl; B8-/B9B12-); (2) 20 mg/d of dietary B8 (unprotected from ruminal degradation) and 2-mL weekly i.m. injection of 0.9% NaCl (B8+/B9B12-); (3) 2.6 g/d of dietary B9 (unprotected) and 2-mL weekly i.m. injection of 10 mg of B12 (B8-/B9B12+); and (4) 20 mg/d of dietary B8, 2.6 g/d of dietary B9, and weekly i.m. injection of 10 mg of B12 (B8+/B9B12+) in a 2 × 2 factorial arrangement. Milk yield and dry matter intake were obtained daily and milk components weekly. Blood samples were taken weekly from d -21 to calving and 3 times per week from calving to 21 d following parturition. Prepartum plasma concentrations of glucose, insulin, nonesterified fatty acids (NEFA), ß-hydroxybutyrate (BHB), and adiponectin were unaffected by treatments. Biotin, B9, and B12 supplements increased their respective concentrations in plasma and milk. Cows fed the B8 supplement tended to have lower dry matter intake, but only cows in B8+/B9B12- had greater plasma concentrations of NEFA compared with B8-/B9B12-. Milk and total solid yields were greater by 13.5 and 13.9%, respectively, for B8-/B9B12+ [45.5 (standard error, SE: 1.8) and 5.81 (0.22) kg/d, respectively] compared with B8-/B9B12- [40.1 (1.9) and 5.10 (0.23) kg/d, respectively], but these effects were suppressed when combined with the B8 supplement. Cows in the B8-/B9B12+ group had decreased plasma insulin and tended to have increased NEFA concentrations, but postpartum plasma concentrations of glucose, BHB, leptin, and adiponectin were not affected. These cows also mobilized more body fat reserves, as suggested by a tendency to increased plasma NEFA and more milk total solids compared with B8-/B9B12- cows. However, plasma concentrations of BHB and adiponectin were similar among treatments. This suggests that the B9 and B12 supplements enhanced efficiency of energy metabolism in early lactation cows. Folic acid and B12 supplementation increased postpartum plasma Cys and homocysteine concentrations but did not affect plasma Met concentration, suggesting an upregulation of the transsulfuration pathway. In summary, our results showed that, under the current experimental conditions, increasing B8 supply did not improve responses to the B9 and B12 supplementation.

Combined biotin, folic acid, and vitamin B12 supplementation given during the transition period to dairy cows: Part II. Effects on energy balance and fatty acid composition of colostrum and milk.

Biotin (B8), folate (B9), and vitamin B12 (B12) are involved in several metabolic reactions related to energy metabolism. We hypothesized that a low supply of one of these vitamins during the transition period would impair metabolic status. This study was undertaken to assess the interaction between B8 supplement and a supplementation of B9 and B12 regarding body weight (BW) change, dry matter intake, energy balance, and fatty acid (FA) compositions of colostrum and milk fat from d -21 to 21 relative to calving. Thirty-two multiparous Holstein cows housed in tie stalls were randomly assigned, according to their previous 305-d milk yield, to 8 incomplete blocks in 4 treatments: (1) a 2-mL weekly i.m. injection of saline (0.9% NaCl; B8-/B9B12-); (2) 20 mg/d of dietary B8 (unprotected from ruminal degradation) and 2-mL weekly i.m. injection of 0.9% NaCl (B8+/B9B12-); (3) 2.6 g/d of dietary B9 (unprotected) and 2-mL weekly i.m. injection of 10 mg of B12 (B8-/B9B12+); (4) 20 mg/d of dietary B8, 2.6 g/d of dietary B9, and 2-mL weekly i.m. injection of 10 mg of B12 (B8+/B9B12+) in a 2 × 2 factorial arrangement. Colostrum was sampled at first milking. and milk samples were collected weekly on 2 consecutive milkings and analyzed for FA composition. Body condition score and BW were recorded every week throughout the trial. Within the first 21 d of lactation, B8-/B9B12+ cows had an increased milk yield by 13.5% [45.5 (standard error, SE: 1.8) kg/d] compared with B8-/B9B12- cows [40.1 (SE: 1.9)], whereas B8 supplement had no effect. Even though body condition score was not affected by treatment, B8-/B9B12+ cows had greater BW loss by 24 kg, suggesting higher mobilization of body reserves. Accordingly, milk de novo FA decreased and preformed FA concentration increased in B8-/B9B12+ cows compared with B8-/B9B12- cows. In addition, cows in the B8+/B9B12- group had decreased milk de novo FA and increased preformed FA concentration compared with B8-/B9B12- cows. Treatment had no effect on colostrum preformed FA concentration. Supplemental B8 decreased concentrations of ruminal biohydrogenation intermediates and odd- and branched-chain FA in colostrum and milk fat. Moreover, postpartum dry matter intake for B8+ cows tended to be lower by 1.6 kg/d. These results could indicate ruminal perturbation caused by the B8 supplement, which was not protected from rumen degradation. Under the conditions of the current study, in contrast to B8+/B9B12- cows, B8-/B9B12+ cows produced more milk without increasing dry matter intake, although these cows had greater body fat mobilization in early lactation as suggested by the FA profile and BW loss.

Meta-analysis of apparent ruminal synthesis and postruminal flow of B vitamins in dairy cows.

As milk production has significantly increased over the past decade(s), existing estimates of the B-vitamin needs of the modern dairy cow are currently being reconsidered, as suboptimal B-vitamin supply may affect metabolic efficiency. At the same time, however, "true" (i.e., biologically active forms, excluding nonfunctional analogs) B-vitamin supply also cannot be adequately estimated by dietary intake, as the rumen microbiota has been shown to play a significant role in synthesis and utilization of B vitamins. Given their complex impact on the metabolism of dairy cows, incorporating these key nutrients into the next generation of mathematical models could help to better predict animal production and performance. Therefore, the purpose of this study was to generate hypotheses of regulation in the absence of supplemental B vitamins by creating empirical models, through a meta-analysis, to describe true B-vitamin supply to the cow (postruminal flow, PRF) and apparent ruminal synthesis (ARS). The database used for this meta-analysis consisted of 340 individual cow observations from 15 studies with 16 experiments, where diet and postruminal digesta samples were (post hoc) analyzed for content of B vitamins (B1, B2, B3, B6, B9, B12). Equations of univariate and multivariate linear form were considered. Models describing ARS considered dry matter intake (DMI, kg/d), B-vitamin dietary concentration [mg/kg of dry matter (DM)] and rumen-level variables such as rumen digestible neutral detergent fiber (NDF) and starch (g/kg of DM), total volatile fatty acids (VFA, mM), acetate, propionate, butyrate, and valerate molar proportions (% of VFA), mean pH, and fractional rates of degradation of NDF and starch (%/h). Models describing PRF considered dietary-level driving variables such as DMI, B-vitamin dietary concentration (mg/kg of DM), starch and crude protein (g/kg of DM) and forage NDF (g/kg of DM). Equations developed were required to contain all significant slope parameters and contained no significant collinearity between driving variables. Concordance correlation coefficient was used to evaluate the models on the developmental data set due to data scarcity. Overall, modeling ARS yielded better-performing models compared with modeling PRF, and DMI was included in all prediction equations as a scalar variable. The B-vitamin dietary concentration had a negative effect on the ARS of B1, B2, B3, and B6 but increased the PRF of B2 and B9. The rumen digestible NDF concentration had a negative effect on the ARS of B2, B3, and B6, whereas rumen digestible starch concentration had a negative effect on the ARS of B1 and a positive effect on the ARS of B9. In the best prediction models, the dietary starch increased PRF of B1, B2, and B9 but decreased PRF of B12. The equations developed may be used to better understand the effect of diet and ruminal environment on the true supply of B vitamins to the dairy cow and stimulate the development of better-defined requirements in the future.

Response to a glucose tolerance test in early-lactation Holstein cows receiving a supplementation of biotin, folic acid, and vitamin B12.

The aim of the study was to evaluate glucose and insulin metabolism of cows receiving a supplementation of biotin (B8), folic acid (B9), and vitamin B12 (B12) during the transition period. According to a 2 × 2 factorial arrangement, 32 cows were randomly assigned to 9 incomplete blocks according to their previous 305-d milk yield. Within each block, cows were randomly assigned to 1 of the following levels of biotin from -27 to 28 d relative to the parturition: (1) no biotin supplement (B8-) or (2) 20 mg/d of dietary biotin (B8+). Within each level of biotin, the cows received either (1) 2-mL weekly intramuscular injections of saline 0.9% NaCl (B9B12-) or (2) 2.6 g/d of dietary folic acid and 2-mL weekly intramuscular injections of 10 mg of vitamin B12 (B9B12+). An intravenous glucose tolerance test was performed at 25 d in milk. Baseline plasma glucagon, glucose, and nonesterified fatty acid concentrations did not differ among treatments. For B9B12+ cows, baseline plasma insulin concentration and maximal glucose concentration after glucose administration were greater when also combined with biotin compared with no biotin combination, whereas there was no effect in B9B12- cows. There was no treatment effect on time to reach half-maximal glucose and insulin concentrations, glucose positive incremental area under the curve, and glucose and insulin clearance rates. Regarding insulin results, maximal plasma concentration and positive incremental area under the curve were respectively 51 and 74% greater for cows receiving the B8 supplement than for cows who did not. Moreover, plasma nonesterified fatty acid concentration nadir tended to be reached later for B8 cows. Insulin peak was reached earlier for cows in the group B9B12+ than cows in B9B12-, regardless of B8 supplementation. Under the current conditions, our results suggested that cows receiving a B8 supplement had a reduced insulin sensitivity in early lactation. Insulin response was faster for B9B12+ cows, but this was not translated into further improvements following the glucose administration challenge.

Technical note: Extrapolation of hepatic glycogen concentration of the whole organ by performing a liver biopsy.

Glycogen, a complex polysaccharide, is the form of storage of glucose in mammals that can be released rapidly when needed. Recent studies have mainly reported hepatic glycogen concentration for early-lactating cows, when the energy demand is higher than the energy supply from dry matter intake, driving the cow to use the energy stored as hepatic glycogen. Generally, liver samples are obtained through percutaneous needle biopsies in the right lobe of the liver. Our objective was to analyze the variation of glycogen concentration in the livers of Holstein and Jersey cows among different liver locations representing all lobes, to evaluate whether samples obtained by liver biopsies are representative of the whole organ. Liver from 10 culled lactating cows (5 Holstein and 5 Jersey cows) from 30 to 113 mo of age at slaughter were obtained. Each liver was sampled no more than 3 h after death on the following sites: 3 sites in the right lobe (1 to 3), 2 in the diaphragmatic surface of the left lobe (4 and 5), 3 in the visceral surface of the left lobe (6 to 8), 1 in the quadrate lobe (9), and 1 in the caudate lobe (10). Samples were snap frozen in liquid N2 and were then analyzed for glucose concentration after conversion of glycogen to glucose using amyloglucosidase (EC 3.2.1.3). Glycogen results are reported as grams of glucose per 100 g of wet weight of liver (i.e., percent of wet weight of liver). Liver weights averaged 5.1 [standard deviation (SD) 1.2, minimum 3.3, maximum 6.2] kg for Holstein and 6.0 (SD 1.8, minimum 4.7, maximum 8.9) kg for Jersey cows. Holstein cows [1.31, standard error of the mean (SEM) 0.05% of wet weight] had greater liver glycogen concentration than did Jersey cows (0.75, SEM 0.05% of wet weight). No significant difference was noted among the 10 liver locations regarding glycogen concentration and averaged, for both breeds, 1.03% of wet weight (SEM 0.10). These results suggest that, in dairy cows, percutaneous needle liver biopsy in the right lobe is an accurate technique to fairly extrapolate glycogen concentration of the whole organ.

Short communication: Potential prediction of vitamin B12 concentration based on mid-infrared spectral data using Holstein Dairy Herd Improvement milk samples.

The purpose of this study was (1) to predict the quantitative concentration of vitamin B12 in milk using mid-infrared (MIR) spectrometry, and (2) to evaluate the potential of MIR spectra to discriminate different clusters of records based on their B12 concentration. Milk samples were collected from 4,340 Holstein cows between 3 and 592 d in milk and located in 100 herds. Samples were taken using in-line milk meters and divided into 2 aliquots: one for MIR spectrometry and the other for B12 concentration reference analyses by radioassay. Analyses were performed on 311 selected spectral wavelengths. A partial least squares regression model was built to quantify B12 concentration. Discriminant analysis was used to isolate B12 concentration clusters. A B12 concentration threshold was set at 442 ng/dL, because this represents the cutoff value for a 250-mL glass of milk to fulfill 46% of the daily vitamin B12 recommended dietary allowance for individuals 14 yr or older. For each analysis, records coming from two-thirds of herds were used to calibrate prediction equations, and the remaining records (one-third of herds for validation) were used to assess the prediction performance. In the case of discriminant analysis, validation sets were divided into evaluation sets (one-third of herds) to obtain alternate probability cutoffs and in test sets (two-thirds of herds) to validate equations. Spectral and B12 concentration outliers were identified by calculating standardized Mahalanobis distance and with a residual analysis, respectively (n = 3,154). Regarding quantitative B12 concentration, cross-validation and validation coefficients of determination averaged 0.51 and 0.46, respectively, which are relatively low, which would limit the potential use of the developed quantitative equations. In addition, root mean square errors of prediction of cross validation and validation sets averaged 88.9 and 94.7 ng/dL, respectively. Area under the receiver operating characteristic curve of test sets averaged 0.81 based on the 442 ng/dL threshold, which could be considered to represent good accuracy of classification. However, the false discovery rate averaged 36%. In summary, models predicting quantitative B12 concentration had low cross-validation and validation coefficients of determination, limiting their use, but the proposed discriminant models could be used to identify milk samples with naturally high B12.

Cross-sectional study of the effect of diet composition on plasma folate and vitamin B12 concentrations in Holstein cows in the United States and Canada.

The objective of this cross-sectional study was to assess the variability of plasma folate and vitamin B12 concentrations in lactating Holstein cows across the United States and Canada. We also evaluated the effect of diet composition and cow characteristics on folate and vitamin B12 plasma vitamin concentrations. A total of 22 and 24 US and Canadian dairy herds were enrolled, totaling 427 and 476 cows at 10 to 197 days in milk across all US and Canadian herds, respectively. Blood samples were taken to analyze plasma folate and vitamin B12 concentrations, and ingredients of the diet were collected to determine nutrient composition. To reduce the number of interdependent variables in the analysis of the association of diet composition with plasma vitamin concentrations, we conducted a principal component analysis. Plasma folate concentrations were lower for US cows [13.4 ng/mL, 95% confidence interval (CI): 12.7-14.2] than for Canadian cows (14.5 ng/mL, 95% CI: 13.7-15.2), and the opposite was observed for plasma vitamin B12 concentrations (US 206 pg/mL, 95% CI: 192-221; Canada 170 pg/mL, 95% CI: 159-181). The highest plasma concentrations of both vitamins were observed in the Northwest region of the United States (Oregon and Washington). Cows in California had the lowest plasma folate concentrations, and cows in Québec and New York State had the lowest plasma vitamin B12 concentrations. Plasma folate concentrations were higher for multiparous than for primiparous cows and plasma vitamin B12 concentrations progressively increased from parity 1 to 3 and higher. For both studied vitamins, plasma concentrations were lower at 0 to 55 than at 56 to 200 days in milk. Of 3 principal components, the one associated with dietary carbohydrates was significantly correlated with plasma folate and vitamin B12 concentrations. Indeed, plasma folate concentrations decreased with dietary fiber concentrations (i.e., neutral and acid detergent fibers and lignin) and increased with dietary nonfiber carbohydrate concentrations. We obtained the opposite results for plasma vitamin B12 concentrations. Both multivariable models explained 41% (pseudo-R2) of the variation in plasma folate and vitamin B12 concentrations. Information gathered in this study is the first step toward determining sources of variation in plasma folate and vitamin B12 concentrations, as well as the vitamin status of cows.

Symposium review: One-carbon metabolism and methyl donor nutrition in the dairy cow.

The present review focuses on methyl donor metabolism and nutrition in the periparturient and lactating dairy cow. Methyl donors are involved in one-carbon metabolism, which includes the folate and Met cycles. These cycles work in unison to support lipid, nucleotide, and protein synthesis, as well as methylation reactions and the maintenance of redox status. A key feature of one-carbon metabolism is the multi-step conversion of tetrahydrofolate to 5-methyltetrahyrofolate. Homocysteine and 5-methyltetrahyrofolate are utilized by vitamin B12-dependent Met synthase to couple the folate and Met cycles and generate Met. Methionine may also be remethylated from choline-derived betaine under the action of betaine hydroxymethyltransferase. Regardless, Met is converted within the Met cycle to S-adenosylmethionine, which is universally utilized in methyl-group transfer reactions including the synthesis of phosphatidylcholine. Homocysteine may also enter the transsulfuration pathway to generate glutathione or taurine for scavenging of reactive oxygen metabolites. In the transition cow, a high demand exists for compounds with a labile methyl group. Limited methyl group supply may contribute to inadequate hepatic phosphatidylcholine synthesis and hepatic triglyceride export, systemic oxidative stress, and compromised milk production. To minimize the perils associated with methyl donor deficiency, the peripartum cow relies on de novo methylneogenesis from tetrahydrofolate. In addition, dietary supplementation of rumen-protected folic acid, vitamin B12, Met, choline, and betaine are potential nutritional approaches to target one-carbon pools and improve methyl donor balance in transition cows. Such strategies have merit considering research demonstrating their ability to improve milk production efficiency, milk protein synthesis, hepatic health, and immune response. This review aims to summarize the current understanding of folic acid, vitamin B12, Met, choline, and betaine utilization in the dairy cow. Methyl donor co-supplementation, fatty acid feeding strategies that may optimize methyl donor supplementation efficacy, and potential epigenetic mechanisms are also considered.

Glucose and insulin responses to an intravenous glucose tolerance test administered to feed-restricted dairy cows receiving folic acid and vitamin B12 supplements.

The present experiment was conducted to determine whether, during periods of negative energy balance, the increase in glucose availability, despite similar DMI and greater milk production, induced by a combined supplement of folic acid and vitamin B12 was related to effects of insulin on metabolism. Sixteen multiparous Holstein cows averaging 45 days in milk (standard deviation: 3) were assigned to 8 blocks of 2 animals each according to their milk production (45 kg/d; standard deviation: 6) during the week preceding the beginning of the experiment. Within each block, they received weekly intramuscular injections of either saline (CON) or folic acid and vitamin B12 (VIT) during 5 consecutive weeks. During the last week, the cows were fed 75% of their ad libitum intake during 4 d. Blood samples were taken the morning before starting the feed restriction and on the third day of feed restriction. On the fourth day of feed restriction, the daily meal was not served and an intravenous glucose tolerance test was performed. During the 4 wk preceding the feed restriction, milk production and DMI were not affected by treatments. During the feed restriction, the vitamin supplement tended to decrease milk fat concentration and increase milk concentration of lactose. Plasma concentrations of homocysteine, Ile, Leu, Val, and branched-chain AA increased in VIT cows during the restriction but not in CON cows. During the glucose tolerance test, insulin peak height was lower and insulin incremental positive area under the curve tended to be lower for VIT than for CON [83 (95% confidence interval, CI: 64-108) vs. 123 (95% CI: 84-180) µg·180 min/L, respectively]. Free fatty acid nadir was reached earlier for VIT than for CON [34 (95% CI: 26-43) vs. 46 (95% CI: 31-57) min, respectively]. Glucose area under the curve, clearance rate and peak height, insulin time to reach the peak and clearance rate, and free fatty acid nadir did not differ between VIT and CON. The reduction in insulin release during a glucose tolerance test without changes in glucose clearance rate or area under the curve suggests that the vitamin supplement improved insulin sensitivity in feed-restricted lactating dairy cows.

Short communication: Relationships among plasma and milk vitamin B12, plasma free fatty acids, and blood ß-hydroxybutyrate concentrations in early lactation dairy cows.

This study was undertaken to evaluate the relationship between plasma and milk concentrations of vitamin B12 as well as the relationship between plasma or milk concentrations of vitamin B12 and plasma concentration of free fatty acids (FFA) or blood concentration of ß-hydroxybutyrate (BHB) of early lactating Ayrshire (AY) and Holstein (HO) cows. A total of 44 dairy herds (7 AY and 37 HO herds) and 62 AY (21 in first, 19 in second, and 22 in third and more lactations) and 228 HO (51 in first, 74 in second, and 103 in third and more lactation) cows between 3 and 40 d in milk were involved in the study. Hand-stripped milk samples and blood samples were taken 6 h after the morning milking. Milk and plasma samples were analyzed for vitamin B12 concentration and plasma samples were analyzed for FFA concentration. A handheld device was used for blood BHB concentration determination. Thresholds for elevated plasma FFA concentration and hyperketonemia were set at ≥0.70 and ≥1.2 mmol/L, respectively. Vitamin B12 concentration in milk of AY primiparous cows [2,557 (1,995-3,276) pg/mL] was lower than in milk from HO primiparous cows [3,876 (3,356-4,478) pg/mL], whereas no difference was observed among other parities and breeds. Regardless of breeds, plasma concentration of vitamin B12 of first and second parities was lower than plasma concentration of third and more lactation cows. Milk vitamin B12 concentration was positively correlated with plasma vitamin B12 concentration, but the relationship was stronger for AY (ρ averaging 0.63) than for HO cows (ρ averaging 0.36). For AY and HO breeds, a significant relationship between milk or plasma vitamin B12 concentrations and plasma FFA concentration (ρ between 0.29 and 0.59) was observed. Moreover, cows with elevated plasma FFA concentration had greater milk and plasma vitamin B12 concentrations than cows with normal plasma FFA concentration. No relationship between vitamin B12 concentration in milk or plasma and blood BHB concentration and hyperketonemia was noted. In summary, milk is not well correlated with plasma vitamin B12 concentration for HO. It could be hypothesized that elevated plasma concentration of FFA could have a negative effect on the use of vitamin B12 by cow cells, which increases the concentration of the vitamin in plasma and its secretion in milk.

Particle size and endosperm type of dry corn grain altered duodenal flow of B vitamins in lactating dairy cows.

The objective of the experiment was to determine if factors such as endosperm type (floury vs. vitreous) and particle size (fine vs. medium) of dry corn grain, known to affect starch digestibility in the rumen, modify apparent ruminal synthesis and duodenal flow of B vitamins in lactating dairy cows. Eight lactating multiparous Holstein cows equipped with rumen and duodenal cannulas were assigned randomly to a treatment sequence according to a 2 × 2 factorial arrangement in duplicate 4 × 4 Latin square design experiment. Duration of each experimental period was 21 d. When expressed per unit of dry matter intake (DMI), floury treatments increased duodenal flow and apparent ruminal synthesis of niacin and folates but tended to increase apparent degradation of thiamin in the rumen. Duodenal flow of thiamin, riboflavin, niacin, folates, and vitamin B12, expressed per unit of DMI, decreased with an increase in particle size. Similarly, apparent degradation of thiamin and riboflavin was greater and apparent synthesis of niacin, folates, and vitamin B12 was reduced when cows were fed coarser dry corn grain particles. Neither endosperm type nor particle size had an effect on duodenal flow and apparent ruminal synthesis of vitamin B6. Apparent ruminal syntheses, expressed per unit of DMI, of all studied B vitamins but thiamin were negatively correlated with apparent ruminal digestibility of neutral detergent fiber. Duodenal flow of microbial N was positively correlated with apparent ruminal synthesis of riboflavin, niacin, vitamin B6, and folates. Under the conditions of the present experiment, except for thiamin, the effects of factors increasing starch digestibility of dry corn grain in the rumen on the amounts of B vitamins available for absorption by the dairy cow seem to be mediated through differences on ruminal digestibility of neutral detergent fiber and, to a lesser extent, on duodenal microbial N flow.

Effects of intramuscular injections of folic acid, vitamin B12, or both, on lactational performance and energy status of multiparous dairy cows.

The purpose of this experiment was to gain understanding on changes in energy partitioning when folic acid and vitamin B12 supplements, alone or combined, were given by weekly intramuscular injections from 3 wk before the expected calving date until 7 wk postpartum. Twenty-four multiparous cows were assigned to 6 blocks of 4 cows each according to previous 305-d lactation yield to either 0 or 320 mg of folic acid and 0 or 10 mg of vitamin B12 in a 2 × 2 factorial arrangement. Plasma concentration of folates was increased by folic acid supplement, and this increase was greater with the combined supplement. Vitamin B12 supplement increased plasma concentration of vitamin B12. Even though postpartum energy balance was similar among treatments, postpartum body condition score was higher for cows receiving folic acid supplement compared with cows that did not. Milk yield of cows receiving folic acid supplement reached a plateau earlier than for cows that did not. Fat and protein, as well as total solid concentrations and yields, were unaffected by treatments. Postpartum plasma concentrations of glucose and insulin were higher and postpartum plasma concentration of nonesterified fatty acids was lower for cows that received weekly folic acid supplement compared with cows that did not. Plasma concentration of methylmalonic acid was low and unaffected by treatments, suggesting that vitamin B12 supply was not limiting, even for unsupplemented cows. Postpartum plasma concentrations of Cys, His, Phe, and Tyr were increased, whereas plasma concentration of Gly was decreased, by folic acid supplement. In the present study, supplementary folic acid altered energy partitioning in early lactation as suggested by similar milk total solid yield and postpartum energy balance, lower plasma nonesterified fatty acid concentration and body condition score losses, and higher plasma glucose and insulin concentrations for cows receiving folic acid supplement compared with cows that did not.

Short communication: Apparent ruminal synthesis of B vitamins in lactating dairy cows fed Saccharomyces cerevisiae fermentation product.

Apparent ruminal synthesis and duodenal flow of thiamin, riboflavin, niacin, vitamin B6, folates, and vitamin B12 were evaluated in an experiment using 15 ruminally and duodenally cannulated lactating Holstein cows fed a basal diet, according to a crossover design, supplemented or not with 56 g/d of Saccharomyces cerevisiae fermentation product. Duration of the treatment period was 28 d. The basal ration had 28% neutral detergent fber, 30% starch and 16.5% crude protein; forages were corn silage (67% of forage dry matter) and alfalfa silage (33% of forage dry matter). Concentrations of B vitamins were analyzed in feed and duodenal digesta. Apparent ruminal synthesis of each B vitamin was calculated as the duodenal flow minus the intake. Under the present experimental conditions, a dietary supplement of Saccharomyces cerevisiae fermentation product had no effect apparent synthesis of B vitamins in the rumen or on the amounts of these vitamins reaching the duodenum and available for absorption by the dairy cow.

Whole-body propionate and glucose metabolism of multiparous dairy cows receiving folic acid and vitamin B12 supplements.

This study was undertaken to evaluate the effect of supplementation of folic acid and vitamin B12 on glucose and propionate metabolism. Twenty-four multiparous cows were assigned according to a complete block design in a 2 × 2 factorial arrangement to one of the following treatments: (1) saline 0.9% NaCl, (2) 320 mg of folic acid, (3) 10 mg of vitamin B12, or (4) 320 mg of folic acid and 10 mg of vitamin B12. Intramuscular injections were given weekly from 3 wk before the expected calving date until 9 wk postpartum. At 63 d in milk, d-[6,6-2H2]-glucose (16.5 mmol/h; jugular vein) and [1-13C]-sodium propionate (13.9 mmol/h; ruminal vein) were simultaneously infused for 4 h; blood samples were collected from 2 to 4 h of the infusion period. Liver biopsies were carried out the following day. Supplements of folic acid and vitamin B12 respectively increased folate and vitamin B12 concentrations, both in milk and liver. Although dry matter intake was unaffected by treatments, milk and milk lactose yields tended to be lower by 5.0 and by 0.25 kg/d, respectively, for cows receiving the folic acid supplement. Plasma ß-hydroxybutyrate concentration with the folic acid supplement followed the same tendency. Hepatic gene expression of methylmalonyl-CoA mutase and S-adenosylhomocysteine hydrolase was higher for cows receiving the combined folic acid and vitamin B12 supplement compared with cows receiving only the supplement of folic acid, whereas no treatment effect was noted for cows not receiving the folic acid supplement. Whole-body glucose rate of appearance and the proportion of whole-body glucose rate of appearance secreted in milk lactose decreased by 229 g/d and 5%, respectively, for animals receiving the folic acid supplement, concomitant with the lower milk lactose synthesis in these cows, indicating that supplementary folic acid may alter energy partitioning in cows. The absence of treatment effect on plasma concentrations of methylmalonic acid as well as on the proportion of glucose synthesized from propionate, averaging 60%, supports the fact that vitamin B12 supply was sufficient in control cows in the current study. Our results suggest that the folic acid supplement reduced glucose-derived lactose synthesis by redirecting glucose for other metabolic activity in the mammary gland or in other tissues.

Short communication: Effect of fatty acid supplements on apparent ruminal synthesis of B vitamins in lactating dairy cows.

The effect of fat supplements (FS) providing different proportions of saturated (SFA) and unsaturated (UFA) fatty acids on supply, apparent ruminal synthesis (ARS), and duodenal flow (DF) of some B vitamins (thiamine, riboflavin, niacin, vitamin B6, folates, and vitamin B12) were evaluated in an experiment using 8 ruminally and duodenally cannulated lactating Holstein cows. The experiment was a replicated 4 × 4 Latin square design with 21-d treatment periods. The 4 treatments were a control diet without fatty acid supplement and 3 diets with 2.5% additional fatty acids from supplements containing (1) SFA, (2) an intermediate mixture of SFA and UFA, or (3) UFA. All diets were served as a total mixed ration once daily at 115% of the expected intake. B-vitamin concentrations were analyzed in feed and duodenal digesta. Apparent ruminal synthesis of each B vitamin was calculated as the DF minus the intake. B-vitamin concentrations were similar among the 4 treatments; consequently, daily intake of the vitamins followed the same pattern as dry matter intake. Adding FS decreased B-vitamin intakes (except vitamin B12), as did increasing the proportion of UFA. Riboflavin and niacin DF and ARS, expressed as total daily amount or per unit of dry matter intake, were not affected by FS, but increasing the proportion of UFA decreased riboflavin and niacin DF and ARS. Fat supplements decreased DF of vitamin B6, expressed either as total daily amount or per unit of dry matter intake. No treatment effects were observed on total daily folate DF and ARS. However, when expressed per unit of dry matter intake, folate DF and ARS were greater when cows were fed FS and they increased linearly with the proportion of UFA in the supplement. Inclusion of fat supplements into the dairy cow diet had a limited effect on the fate of most B vitamins in the rumen although increasing the proportion of UFA in the FS linearly decreased apparent synthesis of riboflavin and niacin in the rumen and the amounts of these vitamins reaching the small intestine.

Apparent ruminal synthesis of B vitamins in lactating dairy cows fed diets with different forage-to-concentrate ratios.

Effects of the forage-to-concentrate ratio on apparent ruminal synthesis of thiamine, riboflavin, niacin, vitamin B6, folates, and vitamin B12 were evaluated in an experiment using 14 ruminally and duodenally cannulated Holstein cows. The experiment was a crossover design with two 15-d treatment periods and a 14-d preliminary period in which cows were fed a diet intermediate in composition between the treatment diets. Treatments were diets containing low-forage (44.8% forage, 32.8% starch, 24.4% neutral detergent fiber) or high-forage (61.4% forage, 22.5% starch, 30.7% neutral detergent fiber) concentrations. Both diets were formulated with different proportions of the same ingredients. Concentrations of B vitamins were analyzed in feed and duodenal digesta. Apparent ruminal synthesis of each B vitamin was calculated as the duodenal flow minus the intake. The high-forage diet had the highest concentrations of riboflavin, niacin, vitamin B6, and folates, whereas the low-forage diet had the highest thiamine concentration. Vitamin B12 in the diets was under the level of detection. Consequently, despite a reduction in dry matter intake when the cows were fed the high-forage diet, increasing dietary forage concentration increased or tended to increase intakes of riboflavin, niacin, and vitamin B6 but reduced thiamine and folate intakes. Increasing dietary forage concentration reduced apparent ruminal degradation of thiamine and apparent ruminal synthesis of riboflavin, niacin, and folates and increased ruminal degradation of vitamin B6, but had no effect on ruminal synthesis of vitamin B12. As a consequence, increasing the forage-to-concentrate ratio had no effect on the amounts of thiamine, riboflavin, and vitamin B12 reaching the small intestine but decreased the amounts of niacin, vitamin B6, and folates available for absorption. Apparent ruminal syntheses of riboflavin, niacin, folates, and vitamin B12 were correlated positively with the amount of starch digested in the rumen and duodenal flow of microbial N, whereas these correlations were negative for thiamine. Apparent ruminal syntheses of thiamine and vitamin B6 were negatively correlated with their respective intakes, whereas folate intake was positively correlated with its synthesis in the rumen.

Short communication: Factors affecting vitamin B12 concentration in milk of commercial dairy herds: An exploratory study.

Only bacteria can synthesize vitamin B12, and this requires adequate Co supply. The natural source of vitamin B12 in human diets comes from animal products, especially those from ruminants. This study aimed to describe variability regarding vitamin B12 concentration in milk among and within commercial dairy herds in early lactation. A secondary objective was to explore potential causes for this variability such as genetic variation and diet characteristics. In total, 399 dairy cows (135 primiparous and 264 multiparous; 386 Holstein and 13 Jersey cows) in 15 commercial herds were involved. Milk samples were taken at 27.4±4.1 and 55.4±4.1d in milk. Neither parity (primiparous vs. multiparous) nor sampling time affected milk concentrations of vitamin B12. Nevertheless, vitamin B12 concentration in milk was highly variable among and within dairy herds. The lowest vitamin B12 concentration in milk of cows was observed in the Jersey herd. Among herds, vitamin B12 concentration in milk ranged from 2,309 to 3,878 pg/mL; one glass (250mL) of milk from those herds would provide between 23 and 40% of the vitamin B12 recommended daily allowance. Among individual cows, however, this provision varied between 16 and 57% of the recommendation. In spite of the limited size of the studied population, the heritability value was 0.23, suggesting that genetic selection could modify milk vitamin B12 concentration. We observed a positive relationship between milk vitamin B12 concentration and dietary acid detergent fiber content and a negative relationship between milk concentration of vitamin B12 and dietary crude protein content.

Choline absorption and evaluation of bioavailability markers when supplementing choline to lactating dairy cows.

The metabolites of choline have a central role in many mammalian biological processes, and choline supplementation to the periparturient dairy cow improves hepatic lipid metabolism. However, variability in responses to choline supplementation has highlighted a lack of understanding of choline absorption in the lactating dairy cow. Our objective was to determine net choline absorption by measuring net portal fluxes of choline and choline metabolites in cows receiving either dietary supplements of rumen-protected choline (RPC) or abomasal delivery of choline (ADC). We also evaluated markers for choline bioavailability by examining relationships between net portal absorption of choline and choline metabolites in plasma and milk. Five late-lactation Holstein cows were used in a 5×5 Latin square design, with 5-d treatment periods and a 2-d interval between periods. Treatments were (1) control (0g/d of choline), (2) 12.5g/d of choline fed as RPC, (3) 25g/d of choline fed as RPC, (4) 12.5g/d of choline provided as ADC, and (5) 25g/d of choline provided as ADC. At the end of each 5-d period, milk was sampled and 9 blood samples were collected simultaneously from an artery and portal vein at 30-min intervals. Plasma, milk, and feed ingredient concentrations of acetylcholine, betaine, free choline, glycerophosphocholine, lysophosphatidylcholine, phosphatidylcholine, phosphocholine, and sphingomyelin were quantified by hydrophilic interaction liquid chromatography-tandem mass spectrometry. With an increasing dose of ADC, the net portal flux of free choline increased and regression analysis indicated 61% net absorption of the infused dose. Among the choline metabolites, only concentrations of betaine, free choline, and phosphocholine increased in both arterial plasma (3.9, 1.9, and 0.4 times, respectively) and milk (2.5, 1.4, and 1.0 times, respectively) with 25g/d of ADC relative to the control. For RPC, the net portal flux of free choline was low relative to ADC (13%), which was similar to the relative difference observed in the concentrations and yields of milk free choline and betaine (averaged 21%). When evaluating markers for choline bioavailability, betaine was the leading candidate. Betaine in plasma and milk (alone or in combination with phosphocholine) was strongly associated with net free choline portal flux (coefficient of determination ranging from 0.64 to 0.79). In summary, free choline supply to the lactating dairy cow increases only specific choline metabolites in plasma and milk, which can be potential markers for choline bioavailability.

Particle length of silages affects apparent ruminal synthesis of B vitamins in lactating dairy cows.

Effects of particle length of silages on apparent ruminal synthesis (ARS) and postruminal supply of B vitamins were evaluated in 2 feeding trials. Diets containing alfalfa (trial 1) or orchardgrass (trial 2) silages, chopped to either 19mm (long cut, LC) or 10mm (short cut, SC) theoretical particle length, as the sole forage were offered to ruminally and duodenally cannulated lactating Holstein cows in crossover design experiments. Forages chopped to a theoretical particle length of 19 and 10mm had mean particles sizes of 14.1 and 8.1mm, respectively, in trial 1, and 15.3 and 11.3mm, respectively, in trial 2. Trial 1 was conducted with 13 multiparous cows in two 19-d treatment periods; both diets contained approximately 20% forage neutral detergent fiber (NDF), 25% total NDF, and forage-to-concentrate ratios were approximately 47:53. Trial 2 was conducted with 15 cows in two 18-d treatment periods; both diets contained approximately 23% forage NDF, 28% total NDF, and had a forage-to-concentrate ratio of 50:50. Thiamine, riboflavin, niacin, vitamin B6, folates, and vitamin B12 were measured in feed and duodenal content. Daily ARS was calculated as the duodenal flow minus the intake. In trial 1, daily intake of individual B vitamins was increased with the LC diet, but ARS of thiamine, riboflavin, vitamin B6, and folates was reduced. In trial 2, except for folates, intakes of the other B vitamins were decreased with the LC diets, whereas ARS of riboflavin, niacin, and vitamin B6 was increased. Daily ARS of thiamine, riboflavin, niacin, and vitamin B6 were correlated negatively with their intake, suggesting that ruminal bacteria reduced their synthesis when dietary supply increased. Microbial activity could have also reduced degradation of thiamine, riboflavin, and niacin, which is supported by (1) the negative correlation between ARS of these vitamins and ruminal pH or microbial N duodenal flow; and (2) the positive correlation between ARS and ruminal concentrations of volatile fatty acids. Folate ARS followed the opposite correlation pattern. Nevertheless, in spite of differences in intake and ARS, with both forages, decreasing particle length of silages had limited effects on the amounts of B vitamins reaching the sites of absorption in the small intestine of dairy cows.