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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), β-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.
It is well recognized that dairy cows must cope with major challenges before calving, at parturition, and at the onset of lactation. In addition to the final rapid growth of the fetus and the decrease in intake capacity at the end of gestation, colostrogenesis begins, which increases energy requirements (
). Moreover, after calving, the energy demand required for milk production exceeds the energy supply obtained from feed intake, and this is often accompanied by insulin resistance as a homeorhetic mechanism coupled with body protein and fat mobilization (
). Elevation of plasma concentration of nonesterified fatty acids (NEFA) from body fat mobilization is normal up to a certain point around this challenging period; exceeding the critical point can lead to detrimental effects on milk production, health, and reproduction (
Associations of elevated nonesterified fatty acids and β-hydroxybutyrate concentrations with early lactation reproductive performance and milk production in transition dairy cattle in the northeastern United States.
Evaluation of nonesterified fatty acids and β-hydroxybutyrate in transition dairy cattle in the northeastern United States: Critical thresholds for prediction of clinical diseases.
Among nutritional strategies to enhance metabolic efficiency in early lactation, supplementary folic acid (B9, the synthetic form of folates) and vitamin B12 (B12) can be used (
Milk production and composition, and body measurements of dairy cows receiving intramuscular injections of folic acid and vitamin B12 in commercial dairy herds.
). Indeed, it has been shown that giving this supplement from 3 wk before calving until 8 or 16 wk after calving increased milk production without increasing DMI and plasma NEFA concentration (
). Another study reported that this supplement, while maintaining similar milk yield and DMI among treatments, decreased NEFA plasma concentration and increased plasma concentrations of insulin and glucose compared with cows not receiving the supplement (
did not report any difference. The former hypothesized that the combined vitamin supplement increased tissue B8 utilization. Biotin acts as a coenzyme for 4 carboxylases (
). Among them, propionyl-CoA carboxylase with its coenzyme B8 transforms propionyl-CoA into methylmalonyl-CoA and the latter is transformed into succinyl-CoA by methylmalonyl-CoA mutase coupled with its coenzyme B12 for its entry into the Krebs cycle (
reported that expression of the methylmalonyl-CoA mutase gene was greater in cows receiving a supplement of B9 and B12 than in control cows or cows receiving only a B12 supplement. Moreover, B8 is a coenzyme of pyruvate carboxylase, which forms oxaloacetate from pyruvate, a key step in gluconeogenesis (
). In addition to the role of B12 as a coenzyme of methylmalonyl-CoA mutase as previously mentioned, B12 also plays a crucial role in one-carbon pool metabolism. Indeed, 5-methyl-tetrahydrofolate, the methylated form of B9, transfers its methyl group to homocysteine (Hcy) to form Met, requiring methionine synthase and B12 as a coenzyme (
). Then, Met could be used in protein synthesis or, requiring ATP, be transformed in S-adenosylmethionine (SAM), the latter being the major methyl donor in mammals. Lactation is known to increase the need for methyl groups (
Given that B8, B9, and B12 have interrelated roles in the metabolism as described above, we hypothesized that a low status of one of these during the transition period would impair metabolic status. For instance,
concluded that low plasma B9 concentration in early lactating cows restrained the responses to B12 supplementation. To our knowledge, no study has assessed whether B8 status in early lactation could affect the use of B9 and B12 supplements. Indeed, experiments have mainly focused on the effect of combined supplements of 2 vitamins in early lactation: either a combined supplement of B9 and B12 (
Effect of dietary supplements of biotin, intramuscular injections of vitamin B12, or both on postpartum lactation performance in multiparous dairy cows.
). Thus, this trial was undertaken to assess whether B8 supply during the transition period; that is, from d −21 to 21 relative to calving, has an impact on the effects of a supplement of B9 and B12 on energy and protein metabolism observed in previous studies. In this article, we present milk performance, residual feed intake (RFI), and concentrations of plasma vitamins, energy-related metabolites, hormones, and AA for cows receiving or not a supplementation of B8, B9, and B12, or those 3 vitamins given together. The companion article (
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.
) focuses on colostrum and milk fatty acid composition, BW, BCS, DMI, and the energy balance of those cows.
MATERIALS AND METHODS
The experiment protocol was approved by the Institutional Committee for Animal Care of the Sherbrooke Research and Development Center (internal approval number 509). All procedures were conducted according to the code of practice of the
This study, held at the dairy herd of Agriculture and Agri-Food Canada Research Centre (Sherbrooke, QC, Canada), was conducted in 2 periods: (1) 17 Holstein dairy cows [8 entering their second lactation and 9 entering their third or more lactation (mean: 3.8, minimum: 3 and maximum: 6)] were involved from December 2016 to June 2017, and (2) 15 Holstein dairy cows [6 entering their second lactation and 9 entering their third and more lactation (mean: 3.8, minimum: 3 and maximum: 6)] were enrolled from September 2017 to April 2018. At enrollment, cows had a BCS of 3.00 [standard deviation (SD): 0.44, minimum: 2.25, and maximum: 4.00], according to a 1-to-5 scale (
) and a BW of 752 (SD: 71, minimum: 622, and maximum: 894) kg. To join the study, cows were required to have completed at least one lactation at the time of the enrollment and to have a previous 305-d milk production >9,500 kg, which was the average milk production of Holstein cows in 2016 in the province of Québec, Canada (
). Previous 305-d lactation milk yield averaged 10,957 kg (SE: 105, minimum: 8,952 kg, maximum: 13,090 kg) and did not differ among treatments (P > 0.60). One cow had a milk yield below the threshold of 9,500 kg due to cow availability at the barn. Sample size was determined according to previous studies (
). Cows were assigned to 8 incomplete blocks based on their previous 305-d milk production; within each block, they were randomly assigned to 1 of the 4 following treatments according to a 2 × 2 factorial arrangement: (1) 2-mL weekly i.m. injections of saline (0.9% NaCl; B8−/B9B12−); (2) 20 mg/d of dietary B8 (Sigma-Aldrich) and 2-mL weekly i.m. injections of saline 0.9% NaCl (B8+/B9B12−); (3) 2.6 g/d of dietary B9 (pteroylmonoglutamic acid, Sigma-Aldrich) and 2-mL weekly i.m. injections of 10 mg of B12 (cyanocobalamin, 5,000 µg/mL, Vétoquinol; B8−/B9B12+); and (4) 20 mg/d of dietary B8, 2.6 g/d of dietary B9, and weekly i.m. injections of 10 mg of B12 (B8+/B9B12+). The doses of vitamins were chosen according to the trials of
). Due to the nature of treatments (dietary or i.m. injections and color of the injected substances), administration of treatments was not blinded. Between ort removal and TMR distribution, dietary supplements of vitamins, which were unprotected from ruminal degradation, were mixed with 200 g of fine-ground corn and offered daily to the cows with a handful of corn silage to ensure that the animal ate the entire quantity of the vitamin supplement. The targeted beginning time of the treatment was 21 d prepartum and the treatment period lasted until 21 d postpartum.
Cows were milked twice daily at 0730 and 1930 h and housed in tie stalls with 17 h of light per day, from 0530 to 2230 h. The same close-up and postpartum rations were individually offered to the animals regardless of treatment (Table 1). To ensure that the supplement and ration were eaten by the proper cows, one stall was left empty between 2 cows and separators were used to divide troughs. Before calving, TMR was distributed twice a day, at 0830 and 1300 h, and after parturition, once a day at 0830 h. Feeds were pushed toward cows several times during the day. Refusals were discarded daily at 0800 h. If necessary, feed offered was adjusted to allow 10% refusals. Cows had ad libitum access to water. Table 2 shows major ingredient composition from periods 1 and 2. Differences in major components existed between dry and lactation hay from the 2 periods. However, percentages of dietary inclusion were adjusted every week to keep the diet isoenergetic and isonitrogenous for the duration of the trial. Two weeks before the expected calving date, 10 mL of selenium and vitamin E were injected i.m. (5 mg/mL selenium and 50 mg/mL δ-α tocopheryl acetate; Merck Canada). Three days before expected calving date, cows were transferred into a calving pen and moved in their regular stall after first colostrum milking (±3 h following calving). Calf management has been reported elsewhere (
Effect of maternal biotin, folic acid, and vitamin B12 supplementation before parturition on colostral and Holstein calf plasma concentrations in those vitamins.
On a DM basis, close-up mineral contained per kilogram: 63 g of Ca, 44 g of P, 99 g of NaCl, 162 g of Mg, −350 mEq of DCAD, 1,210 mg of Cu, 3,307 mg of Mn, 4,463 mg of Zn, 49 mg of Se, 38 mg of Co, 681,430 IU of vitamin A, 184,554 IU of vitamin D, and 12,219 IU of vitamin E; lactation mineral contained per kilogram: 93 g of Ca, 49 g of P, 111 g of Na, 82 g of Cl, 11 g of K, 16 g of S, 55 g of Mg, 524 mg of Cu, 1,660 mg of Mn, 2,968 mg of Zn, 20 mg of Se, 21 mg of Co, 447,811 IU of vitamin A, 56,671 IU of vitamin D, and 2,777 IU of vitamin E.
Calculated using NRC (2001) model according to precalving (DMI of 13.9 kg/d and BW of 772 kg) and postcalving (DMI of 22.0 kg/d, BW of 706 kg with milk yield of 40.1 kg/d at 4.53% of fat and 3.61% of CP) B8−/B9B12− (control treatment; no B-vitamin supplementation) cow inputs.
Calculated using NRC (2001) model according to precalving (DMI of 13.9 kg/d and BW of 772 kg) and postcalving (DMI of 22.0 kg/d, BW of 706 kg with milk yield of 40.1 kg/d at 4.53% of fat and 3.61% of CP) B8−/B9B12− (control treatment; no B-vitamin supplementation) cow inputs.
Calculated using NRC (2001) model according to precalving (DMI of 13.9 kg/d and BW of 772 kg) and postcalving (DMI of 22.0 kg/d, BW of 706 kg with milk yield of 40.1 kg/d at 4.53% of fat and 3.61% of CP) B8−/B9B12− (control treatment; no B-vitamin supplementation) cow inputs.
According to NRC (2001) using DMI of cows that did not receive vitamin supplements (B8−/B9B12−; DMI of 13.9 and 22.0 kg/d at precalving and postcalving, respectively).
Mcal/d
21.1
36.1
Ca
0.91
1.00
P
0.32
0.42
K
1.27
1.43
Mg
0.27
0.29
Folates, mg/kg of DM
0.57
0.50
Co, mg/kg of DM
0.69
0.55
Co, mg/kg of DM from mineral and vitamin premix
0.49
0.36
1 On a DM basis, close-up mineral contained per kilogram: 63 g of Ca, 44 g of P, 99 g of NaCl, 162 g of Mg, −350 mEq of DCAD, 1,210 mg of Cu, 3,307 mg of Mn, 4,463 mg of Zn, 49 mg of Se, 38 mg of Co, 681,430 IU of vitamin A, 184,554 IU of vitamin D, and 12,219 IU of vitamin E; lactation mineral contained per kilogram: 93 g of Ca, 49 g of P, 111 g of Na, 82 g of Cl, 11 g of K, 16 g of S, 55 g of Mg, 524 mg of Cu, 1,660 mg of Mn, 2,968 mg of Zn, 20 mg of Se, 21 mg of Co, 447,811 IU of vitamin A, 56,671 IU of vitamin D, and 2,777 IU of vitamin E.
model according to precalving (DMI of 13.9 kg/d and BW of 772 kg) and postcalving (DMI of 22.0 kg/d, BW of 706 kg with milk yield of 40.1 kg/d at 4.53% of fat and 3.61% of CP) B8−/B9B12− (control treatment; no B-vitamin supplementation) cow inputs.
3 NDF with the inclusion of heat-stable α-amylase.
Table 2Average major nutrient composition (% of DM unless otherwise specified) of some ingredients of the ration for the 2 study periods (SD shown in parentheses)
Figure 1Theoretical timeline of treatment administration and data collection from d −21 to 21 relative to calving. Biotin (20 mg/d) and folic acid (2.6 g/d) supplements were given daily and vitamin B12 (10 mg) or saline (0.9% NaCl) i.m. injections were given weekly. Intake and milk yield were measured daily, blood sampling was taken weekly before parturition and 3 times per week after calving, and milk samples were collected weekly.
, CP, ADF, NDF, Ca, P, and K (Agri-Analyze Agricultural Laboratory, Sherbrooke, QC, Canada). According to these analyses, quantities of ingredient inclusion were adjusted weekly. In addition, all ingredients in the close-up and lactation TMR were sampled every week throughout the experimental period and stored at −20°C until wet chemistry analyses. These samples were thawed, dried at 55°C for 48 h using a forced-air oven, and then ground through a 1-mm sieve (Thomas Scientific), pooled by month, and analyzed by wet chemistry for CP, ADF, NDF with the inclusion of heat-stable α-amylase, and minerals (SGS Canada, Guelph, ON, Canada) as described by
Effect of maternal biotin, folic acid, and vitamin B12 supplementation before parturition on colostral and Holstein calf plasma concentrations in those vitamins.
using a commercial microbiological microtiter plate test (Vita-Fast Folic Acid; R-Biopharm Inc.). The interassay coefficient of variation (CV) was <5%. Diet compositions were reconstituted according to the percentages of inclusion on a DM basis and the nutrient composition of each ingredient. Dry matter intake was calculated daily by subtracting the refusals from the TMR quantity as fed and then multiplied by the DM of the TMR.
Blood
Blood samples were collected weekly before calving on Friday and 3 times per week, on Monday, Wednesday, and Friday, within the first 3 wk of lactation at 1315 h from the coccygeal vein by venipuncture using a Vacutainer system (Becton, Dickinson and Co.). The first blood collection, at enrollment, was performed before giving any vitamin supplements and hence could be considered pretreatment data. Over the whole experimental period, blood sampling was done before injecting B12 and approximately 6 h after dietary vitamin supplement ingestion. Tubes with EDTA (Becton, Dickinson and Co.) were used for plasma B8, B9, B12, NEFA, and BHB concentration analyses, and tubes with heparin (Becton, Dickinson and Co.) were used for plasma glucose, insulin, leptin, adiponectin, methylmalonic acid (MMA), urea, and AA concentration analyses. Tubes were centrifuged within 30 min of collection for 15 min at 2,400 × g and 4°C. Plasma samples were aliquoted in 2-mL microcentrifuge tubes (Eppendorf Canada Ltd.) and stored at −20°C until analysis, except tubes for MMA and AA analyses, which were stored at −80°C. For AA analysis, 1.0 g of cow plasma mixed with 0.2 g of an internal standard of AA labeled with stable isotopes (CDN Isotopes Inc. and Cambridge Isotope Laboratories Inc.), with concentrations previously reported (
), were weighed in microcentrifuge tubes (Eppendorf Canada Ltd.) before freezing.
Plasma B8 concentrations were analyzed in duplicate according to the manufacturer instructions (IDK Biotin ELISA, Immundiagnostik AG). Plasma B9 and B12 concentrations were determined in duplicate by RIA using a commercial kit (SimulTRAC B12/FOLATE-S, MP Biomedicals). The interassay CV were 2.6, 4.4, and 3.5% for B8, B9, and B12 analyses, respectively.
Commercial kits were used to analyze plasma concentrations of glucose [Glucose (Tinder) assay, Genzyme Diagnostics P.E.I. Inc.], BHB (β-hydroxybutyrate reagent set, Pointe Scientific Inc.), NEFA [HR Series NEFA-HR(2), Wako Chemicals USA Inc.], insulin (Mercodia Bovine Insulin ELISA, Mercodia AB), and urea (QuantiChrom Urea Assay Kit, BioAssay Systems).
Leptin concentration in plasma was analyzed according to
using an indirect competitive ELISA for bovine adiponectin. The interassay CV were 11.5 and 9.5%, whereas the intraassay CV were 7.5 and 9.0% for leptin and adiponectin analyses, respectively. Leptin-to-adiponectin ratio was calculated as per
, and measurements were made using GC-MS in electron ionization mode (model CG7890B-MSHunter 5977A, Agilent Technologies). Plasma MMA concentration was also analyzed by isotopic dilution using GC-MS in electron ionization mode (model CG7890B-MSHunter 5977A, Agilent Technologies) as described by
Effects of intramuscular injections of vitamin B12 on lactation performance of dairy cows fed dietary supplements of folic acid and rumen-protected methionine.
. Measurements were done using m/z ions 119.0 and 122.0.
Milk
Milk yields were recorded at each milking from the calibrated milking machine device. Milk samples were collected weekly using inline milk meters for 2 consecutive milkings. For each milk sample, 2 subsamples were taken: one was preserved with bronopol and immediately sent to the DHIA laboratory (Lactanet, Sainte-Anne-de-Bellevue, QC, Canada) for analyses and one was aliquoted into several tubes and stored at −20°C until analyses. Milk fat, protein, and lactose concentrations were analyzed by mid-infrared reflectance spectrometry (MilkoScan FT 6000, Foss) at the Lactanet laboratory. Milk concentrations of B8 were analyzed in duplicate using a commercial kit (IDK Biotin ELISA, Immundiagnostik AG). Milk concentrations of B9 and B12 were analyzed in duplicate by RIA as described by
using a commercial kit (SimulTRAC B12/FOLATE-S, MP Biomedicals). The interassay CV were 3.1, 4.0, and 4.2% for B8, B9, and B12 analyses, respectively.
Calculations and Statistical Analyses
Daily milk component concentrations were obtained by the following equation: {[milk component of evening milking (%) × milk yield of evening milking (kg)] + [milk component of morning milking (%) × milk yield of morning milking (kg)]}/daily milk yield (kg). Daily milk component yields were calculated by summing the multiplication of the component percentages with the respective milk yield of the evening and morning milkings. Energy-corrected milk was calculated based on the following equation: ECM (kg/d) = [(0.0929 × milk fat %) + (0.0547 × milk protein %) + (0.0395 × milk lactose %)] × [daily milk yield (kg)/0.749] (
Data before and after parturition were separately analyzed for each variable according to a 2 × 2 factorial arrangement in a randomized incomplete block design. Two levels of B8 (0 or 20 mg/d) and 2 levels of B9 with B12 (0 or 2.6 g of B9/d and 10 mg of B12/wk) were considered in the following model:
Yijkl = µ + Vi + βj + δijk + Tl + VTil + εijkl,
in which Yijkl is the studied variable results for the kth cow of the ith treatment during lth week within the jth block, µ is the overall mean, Vi is the treatment effect, βj is the block effect (from 1 to 8), δijk is the error a, Tl is the week effect, VTil is the interaction treatment × week, and εijkl is the residual error. The block effect in the model considered that the study was performed during 2 periods as cows involved in the first period were not assigned to the same block number as cows in the second period. Within the whole study, there were 6 blocks of 4 cows from each treatment, 1 block with 3 cows (1 cow missing in B8+/B9B12−), and 1 block with 5 cows (1 extra cow in B8−/B9B12+).
Milk and component yields; milk component percentages; RFI; postcalving DMI; plasma concentrations of B8, B9, B12, glucose, insulin, NEFA, BHB, leptin, adiponectin, MMA, urea, and AA; and leptin-to-adiponectin ratio were subsequently entered as dependent variables in Proc MIXED of SAS (version 9.4,
) considering repeated measures of weekly collection separately for data before and after calving. As sample times were evenly spaced, both before (3 time points) and after calving (9 time points for plasma concentrations of glucose, insulin, NEFA, BHB, and urea and 3 time points for the remaining variables), the following 8 covariance structures were tested: compound symmetry, heterogeneous compound symmetry, first-order autoregressive, heterogeneous first-order autoregressive, Toeplitz, heterogeneous Toeplitz, first-order ante dependence, and unstructured. For each dependent variable studied, the covariance structure leading to the smallest fit statistics was chosen. Degrees of freedom were adjusted according to the Kenward-Roger method. Residuals of all models were visually evaluated for the assumption of normality and homoscedasticity. Plasma concentrations of pre- and postpartum NEFA, insulin, B9 and B12, postpartum BHB, and milk concentration and yield of B9 disregarded normality, and data were therefore log-transformed. Least squares means and 95% confidence intervals (95% CI) from models in which the dependent variable was log-transformed were back-transformed using the ex calculation, where x represents the log-transformed data, and presented as geometric means and 95% CI. For uniformity, untransformed data are presented as least squares means and 95% CI in tables. For each studied variable, the INFLUENCE option in the model statement was used to detect and delete influential data as per the method of
. For each dependent variable from blood samples, pretreatment measurements (i.e., results from blood collection before the beginning of treatments) were considered as covariates in models if the latter was significant (P ≤ 0.05) to account for possible biological difference not related to the treatments, or otherwise not included. To simplify the text, treatment abbreviations (i.e., B8−/B9B12−, B8+/B9B12−, B8−/B9B12+, and B8+/B9B12+) or individual supplement abbreviations (i.e., B8−, B8+, B9B12−, and B9B12+) will be used in the following sections. According to the factorial arrangement used, statistical comparisons were main B8 effect (B8−/B9B12− and B8−/B9B12+ vs. B8+/B9B12− and B8+/B9B12+), main B9B12 effect (B8−/B9B12− and B8−/B9B12− vs. B8−/B9B12+ and B8+/B9B12+), and the B8 × B9B12 interaction. When an interaction was significant, the SLICE option of the LSMEANS statement was used to help its interpretation, allowing us to compare the simple effects of B8 or B9B12 within each level of the other treatment; that is, for each level of B8 supplementation (B8− and B8+) comparing the response to the B9B12 supplement or, inversely, comparing the response to the B8 supplement within each level of B9B12 supplement (B9B12− and B9B12+); P-values of each simple effect are reported in the Results section. Results were considered significant if P ≤ 0.05 and a tendency if 0.05 < P ≤ 0.10. Blocks were incomplete because of cows' availability in the herd and because 2 cows in B8+/B9B12− treatment were culled at calving due to locomotion and disease problems unrelated to the treatments. After calving, 3 cows had a retained placenta, 1 had metritis, 1 had a displaced abomasum, and 1 had milk fever. They were treated according to veterinarian recommendations and the disease incidences were not related to the treatments.
RESULTS
Pretreatment Vitamin Status of Cows
The study began, on average, 26 (SD: 8, range: 5–39, P > 0.17) d before parturition. One cow in the B8−/B9B12− group, not receiving any vitamin supplement, calved 5 d following the start of her enrollment. All cows in the 3 vitamin supplement groups received at least 18 d of supplementation before calving. Plasma concentration of B8 averaged 1.73 (SE: 0.1) ng/mL before vitamin supplementation and did not differ among treatments (P ≥ 0.20). Plasma concentration of B9 was higher for B9B12+ groups [19.8 (95% CI: 17.3–22.7) ng/mL] compared with B9B12− groups [14.8 (95% CI: 12.9–16.9) ng/mL; B9B12 effect, P = 0.005]; no difference in plasma concentration of B9 was noted in the B8 groups (B8 effect; P = 0.77). Plasma concentration of B12 did not differ among treatments and averaged 260 (SD: 97) pg/mL (P ≥ 0.32). Plasma MMA concentration did not differ among treatments (P ≥ 0.49) and averaged 0.75 (SE: 0.08) µM.
Lactation Performance
During the first 21 d of lactation, milk yield was greater by 13.5%, lactose yield by 19.8%, and TS yield by 13.9%, and ECM by 14.9% in the B8−/B9B12+ group compared with the B8−/B9B12− group (SLICE test, P ≤ 0.05), although no treatment effect was noted for B8+ cows (SLICE test, P ≤ 0.82; B8 × B9B12 interaction, P ≤ 0.08; Table 3). Supplementary B8 tended to decrease DMI by 1.6 kg/d (B8 effect, P = 0.09; Table 3). Residual feed intake tended to be lower for B8−/B9B12+ cows than for B8−/B9B12− cows (SLICE test, P = 0.10), whereas no difference was noted for B8+ cows (SLICE test, P = 0.41; B8 × B9B12 interaction, P = 0.09). No treatment effect was observed for milk fat and CP yields or milk fat and TS concentrations (P ≥ 0.11). Milk CP concentrations were lower in B8−/B9B12+ cows than in B8+/B9B12+ cows (SLICE test, P = 0.03), and tended to be lower in B8+/B9B12− cows than in B8−/B9B12− cows (SLICE test, P = 0.06; B8 × B9B12 interaction, P = 0.006). Milk lactose concentration and yield were lower for the B8+/B9B12+ group than for the B8−/B9B12+ group (SLICE test, P ≤ 0.001), whereas there was no effect for B9B12− groups (SLICE test, P ≤ 0.33; B8 × B9B12 interaction, P ≤ 0.04).
Table 3Milk yield and composition during the first 21 d postcalving of multiparous Holstein cows receiving supplementation of biotin (B8), folic acid (B9), and vitamin B12
Treatments are defined as follows: B8−/B9B12− = 2-mL weekly i.m. injection of saline (0.9% NaCl); B8+/B9B12− = 20 mg/d of dietary biotin and 2-mL weekly i.m. injection of 0.9% NaCl; B8−/B9B12+ = 2.6 g/d of dietary folic acid and 2-mL weekly i.m. injection of 10 mg of vitamin B12; B8+/B9B12+ = 20 mg/d of dietary biotin, 2.6 g/d of dietary folic acid, and 2-mL weekly i.m. injection of 10 mg/wk of vitamin B12. Treatments were given from d −26 to 21 relative to parturition. Unless otherwise noted, least squares means and 95% CI are presented.
Statistical comparisons: main B8 effect (B8−/B9B12− and B8−/B9B12+ vs. B8+/B9B12− and B8+/B9B12+); main B9B12 effect (B8−/B9B12− and B8−/B9B12− vs. B8−/B9B12+ and B8+/B9B12+); B8 × B9B12 (interaction effect between each simple effect level of B8 and B9B12).
1 Treatments are defined as follows: B8−/B9B12− = 2-mL weekly i.m. injection of saline (0.9% NaCl); B8+/B9B12− = 20 mg/d of dietary biotin and 2-mL weekly i.m. injection of 0.9% NaCl; B8−/B9B12+ = 2.6 g/d of dietary folic acid and 2-mL weekly i.m. injection of 10 mg of vitamin B12; B8+/B9B12+ = 20 mg/d of dietary biotin, 2.6 g/d of dietary folic acid, and 2-mL weekly i.m. injection of 10 mg/wk of vitamin B12. Treatments were given from d −26 to 21 relative to parturition. Unless otherwise noted, least squares means and 95% CI are presented.
2 Statistical comparisons: main B8 effect (B8−/B9B12− and B8−/B9B12+ vs. B8+/B9B12− and B8+/B9B12+); main B9B12 effect (B8−/B9B12− and B8−/B9B12− vs. B8−/B9B12+ and B8+/B9B12+); B8 × B9B12 (interaction effect between each simple effect level of B8 and B9B12).
B Vitamins in Milk and Plasma and MMA Plasma Concentrations
As expected, milk B8, B9, and B12 yields and concentrations increased with their respective supplementation (P < 0.0001; Table 3). Biotin supplement increased milk B8 concentrations to a higher extent in the first week of lactation, with the increment averaging 1,742, 788, and 692% in wk 1, 2, and 3 of lactation, respectively (B8 × week interaction, P = 0.003). Supplementation of B9B12 increased milk B9 concentration by 97, 84, and 66% at 1, 2, and 3 wk postpartum, respectively (B9B12 × week interaction, P = 0.04).
Biotin supplementation increased plasma concentration of B8 by 237 and 111% before and after calving, respectively (B8 effect, P < 0.0001; Table 4). Prepartum and postpartum plasma B8 concentrations were stable for B8− cows (SLICE test, P > 0.32), whereas prepartum plasma B8 concentration increased from 5.6 to 6.4 ng/mL between −21 and −7 DIM, and postpartum plasma B8 concentration decreased from 3.7 to 2.7 ng/mL between 7 and 21 DIM for B8+ cows (SLICE test, P < 0.0001; B8 × week interaction, P < 0.02). Prepartum and postpartum plasma B9 concentrations increased by 1,023 and 811%, and plasma B12 concentration increased by 75 and 76%, respectively, with B9B12 supplementation (B9B12 effect, P ≤ 0.006; Table 4). Plasma B9 concentration did not differ among experimental weeks for B9B12− cows (SLICE test, P = 0.66), whereas it decreased by 4 and 14% from week −3 to −1 and from wk 1 to 3, respectively, relative to parturition for B9B12+ cows (SLICE test, P = 0.002; B9B12 × week interaction, P ≤ 0.05). Plasma B12 concentration means by treatment are depicted in Figure 2. Between d −14 and −7 relative to calving, plasma B12 concentration was not different for B8− cows (SLICE test, P > 0.14), whereas it was higher for B8+/B9B12+ cows compared with B8+/B9B12− cows (SLICE test, P < 0.008; B8 × B9B12 × week interaction, P = 0.05; Figure 2). Following parturition, B8+/B9B12+ cows had greater plasma B12 concentration than B8−/B9B12+ cows (SLICE test, P = 0.001), whereas there was no difference between B8−/B9B12− and B8+/B9B12− cows (SLICE test, P = 0.95; B8 × B9B12 interaction, P = 0.02; Table 4 and Figure 2).
Table 4Plasma concentrations of biotin, folates, vitamin B12, methylmalonic acid, and energy-related metabolites before and after parturition for multiparous Holstein cows receiving supplementation of biotin (B8), folic acid (B9), and vitamin B12
Treatments are defined as follows: B8−/B9B12− = 2-mL weekly i.m. injection of saline (0.9% NaCl); B8+/B9B12− = 20 mg/d of dietary biotin and 2-mL weekly i.m. injection of 0.9% NaCl; B8−/B9B12+ = 2.6 g/d of dietary folic acid and 2-mL weekly i.m. injection of 10 mg of vitamin B12; B8+/B9B12+ = 20 mg/d of dietary biotin, 2.6 g/d of dietary folic acid, and 2-mL weekly i.m. injection of 10 mg/wk of vitamin B12. Treatments were given from d −26 to 21 relative to parturition. Unless otherwise noted, least squares means and 95% CI are presented, and blood samples were taken once per week.
Statistical comparisons: main B8 effect (B8−/B9B12− and B8−/B9B12+ vs. B8+/B9B12− and B8+/B9B12+); main B9B12 effect (B8−/B9B12− and B8−/B9B12− vs. B8−/B9B12+ and B8+/B9B12+); B8 × B9B12 (the interaction effect between each simple effect levels of B8 and B9B12).
1 Treatments are defined as follows: B8−/B9B12− = 2-mL weekly i.m. injection of saline (0.9% NaCl); B8+/B9B12− = 20 mg/d of dietary biotin and 2-mL weekly i.m. injection of 0.9% NaCl; B8−/B9B12+ = 2.6 g/d of dietary folic acid and 2-mL weekly i.m. injection of 10 mg of vitamin B12; B8+/B9B12+ = 20 mg/d of dietary biotin, 2.6 g/d of dietary folic acid, and 2-mL weekly i.m. injection of 10 mg/wk of vitamin B12. Treatments were given from d −26 to 21 relative to parturition. Unless otherwise noted, least squares means and 95% CI are presented, and blood samples were taken once per week.
2 Statistical comparisons: main B8 effect (B8−/B9B12− and B8−/B9B12+ vs. B8+/B9B12− and B8+/B9B12+); main B9B12 effect (B8−/B9B12− and B8−/B9B12− vs. B8−/B9B12+ and B8+/B9B12+); B8 × B9B12 (the interaction effect between each simple effect levels of B8 and B9B12).
3 Week effect, P < 0.07.
4 B8 × week interaction, P < 0.09.
5 B8 × B9B12 × week interaction, P < 0.08.
6 Geometric mean and 95% CI for log-transformed data computed as ex, where x represents the log-transformed data.
Figure 2Plasma vitamin B12 concentration for multiparous Holstein cows receiving either 2-mL weekly i.m. injection of saline (0.9% NaCl; B8−/B9B12−; n = 8), 20 mg/d of dietary biotin and 2-mL weekly i.m. injection of 0.9% NaCl (B8+/B9B12−; n = 7), 2.6 g/d of dietary folic acid and 2-mL weekly i.m. injection of 10 mg of vitamin B12 (B8−/B9B12+; n = 9), or 20 mg/d of dietary biotin, 2.6 g/d of dietary folic acid, and weekly i.m. injection of 10 mg/wk of vitamin B12 (B8+/B9B12+; n = 8). Treatments were given from d −26 to 21 relative to the parturition. Error bars represent 95% CI. Before calving: B8 × B9B12 × week interaction, P = 0.05; after calving: B8 × B9B12 interaction, P = 0.02.
Prepartum plasma MMA concentration did not differ among treatments (P ≥ 0.76) and averaged 0.57 (SE: 0.03) µM (Table 4). However, during the first 21 d of lactation, B8 supplement decreased plasma MMA concentration [0.47 (95% CI: 0.42–0.52 µM) vs. 0.54 (95% CI: 0.49–0.59 µM); B8 effect, P = 0.05; Table 4]. Supplementary B9B12 increased postpartum plasma MMA concentration over time from 0.40 (95% CI: 0.36–0.45) µM at 7 d of lactation to 0.61 (95% CI: 0.53–0.70) µM at 21 DIM (SLICE test, P < 0.0001), whereas it was stable in B9B12− groups (SLICE test, P = 0.11; B9B12 × week interaction, P = 0.06).
Glucose, Insulin, NEFA, BHB, Leptin, and Adiponectin Plasma Concentrations, and Leptin-to-Adiponectin Ratio
Prepartum plasma concentrations of glucose [3.73 (95% CI: 3.68–3.78) mM], insulin [32.1 (95% CI: 28.0–36.2) µIU/mL], NEFA [128 (95% CI: 115–141) µM], and BHB [0.81 (95% CI: 0.77–0.84) mM] did not differ among treatments (P ≥ 0.11; Table 4). After parturition, plasma concentrations of glucose and BHB were not different among treatments (P ≥ 0.12). For B8− cows, B9B12 supplementation decreased postpartum plasma concentration of insulin by 1.9 µIU/mL (SLICE test, P = 0.03), whereas the difference was not significant for B8+ cows (SLICE test, P = 0.16; B8 × B9B12 interaction, P = 0.02). Overall, plasma NEFA decreased from 379 (95% CI: 323–444) to 191 (95% CI: 163–224) µM over the first 21 d of lactation (week effect, P < 0.0001). Nonesterified FA concentration after parturition was greater in the B8+/B9B12− group compared with the B8−/B9B12− (SLICE test, P = 0.02), but this metabolite did not differ for B9B12+ cows (SLICE test, P = 0.48; B8 × B9B12 interaction, P = 0.03). In addition, postcalving plasma NEFA concentration tended to be greater in the B8−/B9B12+ group compared with B8−/B9B12− (SLICE test, P = 0.09), although no significant effect was noted for B8+ cows (SLICE test, P = 0.13; B8 × B9B12 interaction, P = 0.03). At sampling times d −14 and −7 relative to calving, B8 supplementation decreased plasma leptin concentration when no B9B12 supplement was provided (SLICE test, P ≤ 0.07), whereas there was no significant effect for B9B12+ cows (SLICE test, P ≥ 0.88; B8 × B9B12 × week interaction, P = 0.03). No treatment effect was observed on postpartum plasma leptin concentration (P ≥ 0.33). Plasma adiponectin concentration did not differ among treatments throughout the experimental period (P ≥ 0.15; Table 4), but differed with sampling time (week effect, P ≤ 0.01). Indeed, plasma adiponectin concentrations averaged from 26.89 to 22.87 and from 24.02 to 21.96 µg/mL during the pre- and postpartum periods, respectively. There was no treatment effect on leptin-to-adiponectin ratio (P ≥ 0.18).
Urea and AA
Prepartum plasma urea concentrations tended to be lower for B8+ cows (B8 effect, P = 0.08), whereas prepartum and postpartum plasma concentrations of urea tended to be (B9B12 effect, P = 0.09) or were increased (B9B12 effect, P = 0.007) by the supplement of B9B12 (Table 5).
Table 5Plasma concentrations of urea and AA before and after parturition for multiparous Holstein cows receiving supplementation of biotin (B8), folic acid (B9), and vitamin B12
Treatments are defined as follows: B8−/B9B12− = 2-mL weekly i.m. injection of saline (0.9% NaCl); B8+/B9B12− = 20 mg/d of dietary biotin and 2-mL weekly i.m. injection of 0.9% NaCl; B8−/B9B12+ = 2.6 g/d of dietary folic acid and 2-mL weekly i.m. injection of 10 mg of vitamin B12; B8+/B9B12+ = 20 mg/d of dietary biotin, 2.6 g/d of dietary folic acid, and 2-mL weekly i.m. injection of 10 mg/wk of vitamin B12. Treatments were given from d −26 to 21 relative to parturition. Least squares means and 95% CI are presented.
Statistical comparisons: main B8 effect (B8−/B9B12− and B8−/B9B12+ vs. B8+/B9B12− and B8+/B9B12+); main B9B12 effect (B8−/B9B12− and B8−/B9B12− vs. B8−/B9B12+ and B8+/B9B12+); B8 × B9B12 (interaction effect between each simple effect levels of B8 and B9B12).
1 Treatments are defined as follows: B8−/B9B12− = 2-mL weekly i.m. injection of saline (0.9% NaCl); B8+/B9B12− = 20 mg/d of dietary biotin and 2-mL weekly i.m. injection of 0.9% NaCl; B8−/B9B12+ = 2.6 g/d of dietary folic acid and 2-mL weekly i.m. injection of 10 mg of vitamin B12; B8+/B9B12+ = 20 mg/d of dietary biotin, 2.6 g/d of dietary folic acid, and 2-mL weekly i.m. injection of 10 mg/wk of vitamin B12. Treatments were given from d −26 to 21 relative to parturition. Least squares means and 95% CI are presented.
2 Statistical comparisons: main B8 effect (B8−/B9B12− and B8−/B9B12+ vs. B8+/B9B12− and B8+/B9B12+); main B9B12 effect (B8−/B9B12− and B8−/B9B12− vs. B8−/B9B12+ and B8+/B9B12+); B8 × B9B12 (interaction effect between each simple effect levels of B8 and B9B12).
3 Week effect, P < 0.10.
4 B9B12 × week interaction, P < 0.10.
5 B8 × B9B12 × week interaction, P < 0.08.
6 B8 × week interaction, P = 0.02.
7 BCAA (branched-chain AA) = Ile + Leu + Val.
8 EAA = Arg + His + Ile + Leu + Lys + Met + Phe + Thr + Trp + Val.
9 NEAA = Ala + Asn + Asp + Cys + Gln + Glu + Gly + Pro + Ser + Tyr.
Supplementary B8 decreased prepartum plasma concentrations of Ala, Asn, Gly, Pro, Ser, Trp, and NEAA, and postpartum plasma concentrations of Ala and Tyr (B8 effect, P ≤ 0.10; Table 5). At d −14 relative to calving, B8 supplementation decreased plasma Gly, Ser, Thr, and total AA concentrations for B9B12+ cows (SLICE test, P ≤ 0.05), whereas no effect was observed for B9B12− cows (SLICE test, P ≥ 0.12; B8 × B9B12 × week interaction, P ≤ 0.08). Biotin supplement increased postpartum plasma concentration of Gly for B9B12− cows (SLICE test, P = 0.04), whereas no effect was noted in B8− cows (SLICE test, P = 0.41; B8 × B9B12 interaction, P = 0.04). After calving, supplement of B9B12 tended to increase plasma concentration of NEAA in B8− cows (SLICE test, P = 0.06), whereas no effect was noted for B9B12− cows (SLICE test, P = 0.27; B8 × B9B12 interaction, P = 0.04).
Before parturition, B9B12 supplement increased Hcy plasma concentration in B8− cows (SLICE test, P = 0.03), whereas there was no difference for B8+ cows (SLICE test, P = 0.25; B8 × B9B12 interaction, P = 0.02). Supplements of B9B12 increased postpartum plasma Hcy concentration (B9B12 effect, P = 0.03). Similarly, B9B12 supplementation tended to increase postcalving Cys concentration by 9% (B9B12 effect, P = 0.08). Plasma Met concentration, however, did not differ among treatments before or after calving (P ≥ 0.11). At 14 d after calving, B9B12 supplements increased plasma concentrations of Ile and Leu (SLICE test, P ≤ 0.07), whereas no effect was noted for other sampling times (SLICE test, P ≥ 0.44; B9B12 × week interaction, P ≤ 0.07).
DISCUSSION
At d −26 relative to calving, before the beginning of treatments, plasma B8 concentration was lower by about 20% compared with values reported by
14 d before parturition. Before supplementation began, plasma B9 and B12 concentrations, respectively, were greater by 100% and 7% than in the study of
. Moreover, before giving any treatment, plasma B9 concentration was higher for B9B12+ cows. This finding is hard to explain because treatments were randomly assigned. These comparisons show that vitamin status before supplementation differs among studies, which should be kept in mind while interpreting results.
As expected, milk B8, B9, and B12 yields and concentrations and plasma B8, B9, and B12 concentrations increased with their respective supplementation, as observed in other trials (
Effects of supplements of folic acid, vitamin B12, and rumen-protected methionine on whole-body metabolism of methionine and glucose in lactating dairy cows.
). The larger increment of plasma B9 concentration (>140 ng/mL) following the dietary B9 supplement observed in the present study compared with results from
, reporting an increment of approximately 10 ng/mL, could be explained by the time of blood sampling relative to ingestion of the supplement: 6 versus 24 h, respectively. Indeed,
observed that the highest plasma B9 concentration, after a dietary supplement of B9, was reached 5 to 6 h following ingestion and decreased thereafter until 24 h after ingestion. Postpartum plasma MMA concentration for control cows was slightly higher (0.52 vs. 0.43 µM) in the current study than in
. Similarly, postpartum plasma B12 concentration of cows receiving no supplementation was slightly lower in the current study than previously reported by
). Indeed, MMA accumulates in blood when L-methylmalonyl-CoA mutase activity is impaired. This enzyme transforms methylmalonyl-CoA into succinyl-CoA for its entry into the Krebs cycle and this step requires B12 as a coenzyme (
). In our study, postpartum plasma MMA concentration was lower for B8+ cows, irrespective of B12 supplementation, even though there is no known link between the activity of methylmalonyl-CoA mutase and B8 status. However, it is noteworthy that B8 acts upstream as a coenzyme for propionyl-CoA carboxylase to convert propionyl-CoA into methylmalonyl-CoA (
). Two hypotheses could therefore explain our results: (1) the lower DMI by 1.6 kg/d for B8+ cows compared with B8− cows decreased ruminal fermentation, hence less propionate was available for its transformation through the above pathway to enter the Krebs cycle, reducing MMA accumulation; and (2) the milk FA profile of B8+ cows showed that this supplement altered ruminal function (
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.
Glucose and insulin responses to an intravenous glucose tolerance test administered to feed-restricted dairy cows receiving folic acid and vitamin B12 supplements.
observed that plasma MMA concentration decreased with a decrease in DMI.
Within the first 21 DIM, milk and TS yields and ECM of B8−/B9B12+ cows were the highest among treatments. Milk yield was 13.5% higher and RFI was 52.5% lower for B8−/B9B12+ cows than for B8−/B9B12− cows, meaning that feed efficiency was greater for B8−/B9B12+ cows. In the absence of B8 supplementation, B9B12 supplements did not affect DMI of cows and averaged 21.9 kg of DM/d during the first 21 d of lactation.
Effects of supplements of folic acid, vitamin B12, and rumen-protected methionine on whole-body metabolism of methionine and glucose in lactating dairy cows.
also reported increased milk yield in early lactating cows receiving a B9 supplement, alone or combined with B12, without an effect on DMI. In a meta-analysis,
did not observe a significant effect. In the current study, milk yield of B8+/B9B12− cows was numerically greater by 3.2 kg compared with B8−/B9B12− cows. This increase was similar to that previously reported, although not significant. Moreover, RFI was 74.1% lower in B8+/B9B12− cows than in B8−/B9B12− cows, suggesting better feed efficiency for the former group. For several variables, such as milk yield and milk fat, CP, lactose, and TS yields, results from B8+/B9B12+ cows were surprisingly similar to those of B8−/B9B12− cows. Given the positive effect of the B9B12 supplementation on milk and TS yields, we expected that cows fed B8 in addition to B9B12 supplementation would have similar, or even higher, milk production performance. The present results suggest that the dietary B8 supplement impeded the action of the B9B12 supplementation. Supporting this hypothesis, after calving, plasma B12 concentration was greater for B8+/B9B12+ than for B8−/B9B12+, with similar milk B12 yields between 14 and 21 DIM. As explained earlier, postpartum plasma MMA concentration was lower for B8+ cows, suggesting that the use of B12 by cells differed depending on the B8 supply. The postpartum DMI decline with supplementary B8 can also explain the results obtained.
Effect of dietary supplements of biotin, intramuscular injections of vitamin B12, or both on postpartum lactation performance in multiparous dairy cows.
did not observe a difference in plasma B12 concentration for cows receiving a supplement of B12 alone or combined with a dietary supplement of B8 starting 21 d before the expected calving date from that at 57 d postcalving. However, they only reported plasma B12 concentration at 57 d postpartum. To our knowledge, this is the first study reporting the effect of combined B8 and B9B12 supplementation during the transition period. Colostrum and milk FA profiles of cows involved in the current study revealed that B8 supplementation probably altered rumen function (
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.
concluded that a dietary supplement of B8 altered ruminal fermentation, which might suggest that the DMI decline in B8+ cows could be linked to the possible altered ruminal function with this supplementation. Meta-analyses of 11 studies concluded that B8 supplement increased DMI (
), but this finding is not in line with the current results.
One major challenge that cows must cope with at the onset of lactation is that the energy demand for milk production exceeds the energy intake. It is now well accepted that dairy cows have a homeorhetic mechanism for prioritizing milk production (
). Indeed, in early lactation, a cow's metabolic profile undergoes changes toward lower plasma insulin and glucose concentrations as well as greater plasma NEFA and BHB concentrations (
The role of exogenous insulin in the complex of hepatic lipidosis and ketosis associated with insulin resistance phenomenon in postpartum dairy cattle.
The role of exogenous insulin in the complex of hepatic lipidosis and ketosis associated with insulin resistance phenomenon in postpartum dairy cattle.
). Hence, lowering plasma insulin concentration in early lactation advantages glucose uptake by the insulin-independent mammary gland cells to support lactose synthesis (
reported that low plasma insulin concentration at the onset of lactation was associated with higher milk production. In the current study, the lowest plasma concentration of insulin was observed for dairy cows producing the highest quantity of milk within the first 21 d of lactation; that is, in B8−/B9B12+ cows. The lower plasma concentration of insulin for these cows coupled with plasma glucose concentration similar to that of control cows could suggest that insulin responsiveness was better in these cows (
Glucose and insulin responses to an intravenous glucose tolerance test administered to feed-restricted dairy cows receiving folic acid and vitamin B12 supplements.
following an intravenous glucose tolerance test for B9B12+ cows. Following an intravenous glucose tolerance test conducted at 25 DIM on the cows of the present experiment,
suggested that the insulin response was improved for B9B12+ cows. Even though B8−/B9B12+ cows produced 13.5% more milk than B8−/B9B12− cows, they only tended to have greater plasma NEFA concentration than control cows. In the companion paper, we showed that these cows lost more BW (5.3% of BW for B8−/B9B12+ and 2.1% of BW for B8−/B9B12−) within the first 21 DIM (
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.
). In opposition to the latter results, B8+/B9B12− cows had greater plasma NEFA concentration and BW loss than B8−/B9B12− cows, combined with a nonsignificant milk yield increase and a trend for a decreased DMI.
did not report increased plasma NEFA concentrations following B8 supplementation in early lactation. This difference might be caused by several factors, including different herd management and cow genetics, as milk yield, DMI, and plasma B8 concentration were greater and plasma NEFA concentration was lower for B8−/B9B12− in the current experiment compared with these 2 previously published studies. Even though no B8 effect was observed for postpartum plasma insulin concentration in the present experiment,
concluded that B8 supplement reduced insulin sensitivity at 25 DIM for the same cows as in the current study. These results suggest a better metabolic adaptation in the early postpartum period for B8−/B9B12+ cows than for B8+/B9B12− cows. The similar plasma BHB concentrations among treatments suggest that, even though B8−/B9B12+ cows tended to have higher plasma NEFA concentration and mobilized more body reserves, as suggested by higher BW loss than B8−/B9B12− cows, this probably did not translate into a liver impairment limiting the capacity of the cow liver to oxidize FA (
). These observations suggest that, even with the increased milk yield, plasma concentration of NEFA was not excessive in B8−/B9B12+ cows and that plasma NEFA and BHB concentrations were within the normal range of the adaptation to negative energy balance in early lactation (
Adiponectin and leptin are adipokines almost solely synthesized and secreted by adipose tissue. Adiponectin is known for its insulin-sensitizing functions, as its circulating concentrations decrease during the peripartum period in dairy cows (
concluded that plasma adiponectin concentration was not affected by plasma leptin and insulin concentrations in early lactation. This could explain why the plasma concentration of adiponectin did not differ among treatments, even though changes in plasma insulin concentration were observed. We expected that supplementation would affect plasma adiponectin concentration based on studies performed in human patients with type 2 diabetes showing increased adiponectin with B12 supplementation, alone or in combination with B9 (
). It is possible that the effect of B12 reported in that study apply to the tested situation of chronic pathologies related to human metabolic syndrome. Although there are some similarities in cows undergoing transition, such as insulin resistance, the metabolic and endocrine situation in cows differs by the transient and highly dynamic endocrine and metabolic changes that are limited to relatively short times.
To our knowledge, an increase in plasma urea concentration with B9B12 supplements has not been reported before. Indeed, previous studies have reported no significant effect of this supplement on plasma urea concentration (
). Urea is mainly produced by the liver either from ammonia produced by the gut, for the most part by rumen fermentation, or by deamination and catabolism of AA (
observed no effect of dietary B9 supplement on rumen fermentation, we could hypothesize that the greater plasma urea concentration in B9B12+ cows was caused by an increased AA utilization as energy substrates to support the greater milk production for those cows (
concluded that, in early lactating cows, AA contribution to net hepatic glucose release is minor and most plasma AA concentrations were not different for cows receiving B9B12 supplement or not, and NEAA concentrations decreased only in B8+/B9B12+ cows. Thus, the current result on plasma urea concentration is hard to explain.
In mammals, Met has fundamental roles in protein synthesis and transmethylation reactions (
). Indeed, the latter role involves the transformation of Met into SAM, requiring 1 ATP, which is the major methyl donor source. After giving its methyl group, SAM is converted into S-adenosylhomocysteine and then into Hcy. Subsequently, Hcy can either be remethylated into Met, requiring 5-methyltetrahydrofolate and methionine synthase with its coenzyme B12, or be transformed to Cys through the transsulfuration pathway (
reported a regulatory mechanism to adapt to excess Met supply in rats. They observed an increase in cystathionine synthase activity in liver of rats fed increased amounts of Met. This enzyme is involved in the transformation of Hcy to cystathionine, which is then transformed into Cys. In the current study, plasma Met concentrations were higher than (
) previously reported values, which could indicate no severe limitation in Met supply. We can hypothesize that, in the present experiment, under a B9B12 supplementation, Hcy remethylation into Met was not increased and then the transsulfuration pathway was favored, as suggested by
. The transsulfuration pathway might have been upregulated with B9B12 supplementation, as suggested by the increased postpartum plasma concentrations of Hcy and Cys.
also observed increased plasma concentrations of Cys and Hcy combined with stable plasma Met concentration for cows receiving a supplement of B9, alone or combined with B12 within the first 7 wk of lactation; these effects were already present during the first 3 wk of lactation (
). Interestingly, cystathionine β-synthase activity, the enzyme transforming Hcy into cystathionine in the transsulfuration pathway, is promoted by an oxidant status to increase the synthesis of antioxidants, such as glutathione and taurine (
), and a supplement of B9 and B12 changed the expression of genes related to tissue repair as well as resorption of inflammation in hepatic tissue of dairy cows during early lactation (
). Further studies are needed to evaluate whether the upregulation of the transsulfuration pathway following B9B12 supplementation in dairy cows was a means to regulate oxidative stress and inflammation in early lactation by increasing glutathione and taurine syntheses.
stressed the variability in response to B9 and B12 supplementation. As discussed above, vitamin statuses of cows at the beginning of the current trial were different from those previously reported in similar studies (
concluded that the response to the vitamin supplementation was linked to the vitamin statuses of the animals. Moreover, a study involving 15 commercial dairy herds showed a variable effect of a B9B12 supplementation among farms (
Milk production and composition, and body measurements of dairy cows receiving intramuscular injections of folic acid and vitamin B12 in commercial dairy herds.
), probably caused by different cow vitamin status and herd management. The fact that the current experiment took place in 2 periods could have increased the variability. For instance, dry and lactation hay nutrient composition varied between the 2 periods, although diets were adjusted to be isoenergetic and isonitrogenous throughout the trial. Another limitation is the fact that the health and reproduction performance of cows could not have been monitored beyond the duration of the study. Under the current state of knowledge, it is not possible to calculate vitamin supplies from ruminal bacteria of cows under different management, which impedes our ability to evaluate whether B-vitamin supplementation before supplementation is needed. Future efforts should be made to evaluate B-vitamin supplies under different herd management and housing systems.
CONCLUSIONS
Milk and TS yields were greater for B8−/B9B12+ cows within the first 21 d of lactation. When coupled with supplemental B8, the B9B12 supplement no longer had a positive effect on milk performance. The enhanced milk performance was associated with a lower plasma insulin concentration and a trend to a greater plasma concentration of NEFA. Nevertheless, plasma concentrations of glucose, BHB, leptin, and adiponectin did not differ among treatments, suggesting that body fat mobilization was not excessive and was probably not detrimental for cow health. Supplemental B9B12 favored the transsulfuration pathway rather than the methylation pathway, possibly because the Met supply was sufficient, relative to some other EAA or energy supply, or as a way to regulate oxidative stress. Under the current conditions, increasing B8 supply had no beneficial effect on milk performance when cows received a supplement of B9B12.
ACKNOWLEDGMENTS
This project was publicly supported by Agriculture and Agri-Food Canada (AAFC, Ottawa, ON, Canada). The authors are grateful to Valérie Beaudet, Véronique Roy, Liette Veilleux, Jasmin Brochu, Jocelyne Renaud, Mario Léonard, Camélia Marchand (Agriculture and Agri-Food Canada, Sherbrooke, QC, Canada), Lya Blais, Myriam Beauregard (Université de Sherbrooke, Sherbrooke, QC, Canada), William Poisson (Université Laval, Québec, QC, Canada), and Thomas Blees (University of Bonn, Bonn, Germany) for technical assistance. We greatly acknowledge the help of Marie-Ève Bouchard, Étienne Viens, and the barn staff for animal care throughout the study (Agriculture and Agri-Food Canada, Sherbrooke, QC, Canada) and Mario Gauthier (NutriPartenaire, Saint-Apollinaire, QC, Canada) for ration formulation. The authors have not stated any conflicts of interest.
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Shaver R.D.
Effects of cobalt supplementation and vitamin B12 injections on lactation performance and metabolism of Holstein dairy cows.
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.
Effect of maternal biotin, folic acid, and vitamin B12 supplementation before parturition on colostral and Holstein calf plasma concentrations in those vitamins.
Milk production and composition, and body measurements of dairy cows receiving intramuscular injections of folic acid and vitamin B12 in commercial dairy herds.
Effects of intramuscular injections of vitamin B12 on lactation performance of dairy cows fed dietary supplements of folic acid and rumen-protected methionine.
Glucose and insulin responses to an intravenous glucose tolerance test administered to feed-restricted dairy cows receiving folic acid and vitamin B12 supplements.
The role of exogenous insulin in the complex of hepatic lipidosis and ketosis associated with insulin resistance phenomenon in postpartum dairy cattle.
Associations of elevated nonesterified fatty acids and β-hydroxybutyrate concentrations with early lactation reproductive performance and milk production in transition dairy cattle in the northeastern United States.
Evaluation of nonesterified fatty acids and β-hydroxybutyrate in transition dairy cattle in the northeastern United States: Critical thresholds for prediction of clinical diseases.
Effects of supplements of folic acid, vitamin B12, and rumen-protected methionine on whole-body metabolism of methionine and glucose in lactating dairy cows.
Effect of dietary supplements of biotin, intramuscular injections of vitamin B12, or both on postpartum lactation performance in multiparous dairy cows.
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.
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