Disrupted one-carbon metabolism in heifers negatively affects their health and physiology.

The objective of this study was to determine the dose-dependent response of one-carbon metabolite (OCM: methionine, choline, folate, and vitamin B12) supplementation on heifer dry matter intake on fixed gain, organ mass, hematology, cytokine concentration, pancreatic and jejunal enzyme activity, and muscle hydrogen peroxide production. Angus heifers (n = 30; body weight [BW] = 392.6 ±â€…12.6 kg) were individually fed and assigned to one of five treatments: 0XNEG: total mixed ration (TMR) and saline injections at days 0 and 7 of the estrous cycle, 0XPOS: TMR, rumen-protected methionine (MET) fed at 0.08% of the diet dry matter, rumen-protected choline (CHOL) fed at 60 g/d, and saline injections at days 0 and 7, 0.5X: TMR, MET, CHOL, 5-mg B12, and 80-mg folate injections at days 0 and 7, 1X: TMR, MET CHOL, 10-mg vitamin B12, and 160-mg folate at days 0 and 7, and 2X: TMR, MET, CHOL, 20-mg vitamin B12, and 320-mg folate at days 0 and 7. All heifers were estrus synchronized but not bred, and blood samples were collected on days 0, 7, and at slaughter (day 14) during which tissues were collected. By design, heifer ADG did not differ (P = 0.96). Spleen weight and uterine weight were affected cubically (P = 0.03) decreasing from 0XPOS to 0.5X. Ovarian weight decreased linearly (P < 0.01) with increasing folate and B12 injection. Hemoglobin and hematocrit percentage were decreased (P < 0.01) in the 0.5X treatment compared with all other treatments. Plasma glucose, histotroph protein, and pancreatic α-amylase were decreased (P ≤ 0.04) in the 0.5X treatment. Heifers on the 2X treatment had greater pancreatic α-amylase compared with 0XNEG and 0.5X treatment. Interleukin-6 in plasma tended (P = 0.08) to be greater in the 0XPOS heifers compared with all other treatments. Lastly, 0XPOS-treated heifers had reduced (P ≤ 0.07) hydrogen peroxide production in muscle compared with 0XNEG heifers. These data imply that while certain doses of OCM do not improve whole animal physiology, OCM supplementation doses that disrupt one-carbon metabolism, such as that of the 0.5X treatment, can induce a negative systemic response that results in negative effects in both the dam and the conceptus during early gestation. Therefore, it is necessary to simultaneously establish an optimal OCM dose that increases circulating concentrations for use by the dam and the conceptus, while avoiding potential negative side effects of a disruptive OCM, to evaluate the long-term impacts of OCM supplementation of offspring programming.

The feeding of one-carbon metabolites (including methionine and B vitamins) has been shown to improve fetal growth and milk production in species such as mice, sheep, and dairy cattle. Extending this to beef cattle around the time of breeding is a growing area of research. Our group previously determined that one-carbon metabolite supplementation to beef heifers altered the abundance of circulating methionine-folate cycle intermediates in a dose-dependent manner. Therefore, we aimed to determine a whole-body response to one-carbon metabolite supplementation in heifers by measuring the effects on specific physiological systems as well as a total systemic response. We determined that treatments that negatively altered the methionine-folate cycle yielded a fundamental negative whole-body response to supplementation.

Influence of Maternal Nutrition and One-Carbon Metabolites Supplementation during Early Pregnancy on Bovine Fetal Small Intestine Vascularity and Cell Proliferation.

To investigate the effects of nutrient restriction and one-carbon metabolite (OCM) supplementation (folate, vitamin B12, methionine, and choline) on fetal small intestine weight, vascularity, and cell proliferation, 29 (n = 7 ± 1 per treatment) crossbred Angus beef heifers (436 ± 42 kg) were estrous synchronized and conceived by artificial insemination with female sexed semen from a single sire. Then, they were allotted randomly to one of four treatments in a 2 × 2 factorial arrangement with the main factors of nutritional plane [control (CON) vs. restricted feed intake (RES)] and OCM supplementation [without OCM (-OCM) or with OCM (+OCM)]. Heifers receiving the CON level of intake were fed to target an average daily gain of 0.45 kg/day, which would allow them to reach 80% of mature BW by calving. Heifers receiving the RES level of intake were fed to lose 0.23 kg/heifer daily, which mimics observed production responses in heifers that experience a diet and environment change during early gestation. Targeted heifer gain and OCM treatments were administered from d 0 to 63 of gestation, and then all heifers were fed a common diet targeting 0.45 kg/d gain until d 161 of gestation, when heifers were slaughtered, and fetal jejunum was collected. Gain had no effect (p = 0.17) on the fetal small intestinal weight. However, OCM treatments (p = 0.02) displayed less weight compared to the -OCM groups. Capillary area density was increased in fetal jejunal villi of RES - OCM (p = 0.02). Vascular endothelial growth factor receptor 2 (VEGFR2) positivity ratio tended to be greater (p = 0.08) in villi and was less in the crypts (p = 0.02) of the RES + OCM group. Cell proliferation decreased (p = 0.02) in villi and crypts of fetal jejunal tissue from heifers fed the RES + OCM treatment compared with all groups and CON - OCM, respectively. Spatial cell density increased in RES - OCM compared with CON + OCM (p = 0.05). Combined, these data show OCM supplementation can increase expression of VEGFR2 in jejunal villi, which will promote maintenance of the microvascular beds, while at the same time decreasing small intestine weight and crypt cell proliferation.

Concentrations of vitamin B12 and folate in maternal serum and fetal fluids, metabolite interrelationships, and hepatic transcript abundance of key folate and methionine cycle genes: the impacts of maternal nutrition during the first 50 d of gestation.

Adequate maternal nutrition is key for proper fetal development and epigenetic programming. One-carbon metabolites (OCM), including vitamin B12, folate, choline, and methionine, play a role in epigenetic mechanisms associated with developmental programming. This study investigated the presence of B12 and folate in maternal serum, allantoic fluid (ALF), and amniotic fluid (AMF), as well as how those concentrations in all three fluids correlate to the concentrations of methionine-folate cycle intermediates in heifers receiving either a control (CON) or restricted (RES) diet for the first 50 d of gestation and fetal hepatic gene expression for methionine-folate cycle enzymes. Angus cross heifers (n = 43) were estrus synchronized, bred via artificial insemination with semen from a single sire, and randomly assigned to one of two nutrition treatments (CON = 20, RES = 23). Heifers were ovariohysterectomized on either day 16 (n = 14), 34 (n = 15), or 50 of gestation (n = 14), where samples of maternal serum (n = 42), ALF (n = 29), and AMF (n = 11) were collected and analyzed for concentrations of folate and B12. Concentrations of B12 and folate in ALF were greater (P < 0.05) in RES compared to CON. For ALF, folate concentrations were also greater (P < 0.01) on day 34 compared to day 50. There was a significant (P = 0.04) nutrition × fluid interaction for B12 concentrations where concentrations were greatest in restricted ALF, intermediate in control ALF, and lowest in CON and RES serum and AMF. Folate concentrations were greatest (P < 0.01) in ALF, intermediate in serum, and lowest in AMF. Additionally, positive correlations (P < 0.05) were found between ALF and AMF folate concentrations and AMF concentrations of methionine, serine, and glycine. Negative correlations (P < 0.05) between AMF folate and serum homocysteine were also observed. Both positive and negative correlations (P < 0.05) depending on the fluid evaluated were found between B12 and methionine, serine, and glycine concentrations. There was a downregulation (P = 0.05) of dihydrofolate reductase and upregulation (P = 0.03) of arginine methyltransferase 7 gene expression in RES fetal liver samples compared with CON fetal liver on day 50. Combined, these data show restricted maternal nutrition results in increased B12 and folate concentrations present in fetal fluids, and increased expression of genes for enzymes within one-carbon metabolism.

When pregnant cattle have restricted access to feed or specific nutrients, calf development can be affected, and the degree of impairment depends, at least partially, on timing, duration, and severity of the limitations. A biochemical pathway present in cells that can be affected by limited nutrition is one-carbon metabolism. This pathway is related to epigenetics, which regulates gene expression or the turning on and off of genes. Two important vitamins in one-carbon metabolism are vitamins B12 and folate. By understanding the amounts of those vitamins available to the developing calf, we can gain better insight into the regulation and potential avenues of improvement of calf growth and development. In this study, we found a nutrient restricted maternal diet increased the amount of B12 and folate in calf allantoic and amniotic fluids. We also found that folate and B12 were correlated to the presence of other nutrients in serum, allantoic fluid, and amniotic fluid. In addition, we found that a protein methylating gene in one-carbon metabolism had increased expression in calves from heifers receiving limited nutrition. This study is an important step in understanding how the nutrients available to a pregnant heifer during gestation affects nutrients available to the conceptus.

One-carbon metabolite supplementation to heifers for the first 14 d of the estrous cycle alters the plasma and hepatic one-carbon metabolite pool and methionine-folate cycle enzyme transcript abundance in a dose-dependent manner.

The objective of this study was to determine the dose of folate and vitamin B12 in beef heifers fed rumen protected methionine and choline required to maintain increased B12 levels and intermediates of the methionine-folate cycle in circulation. Angus heifers (n = 30; BW = 392.6 ±â€…12.6 kg) were individually fed and assigned to one of five treatments: 0XNEG: Total mixed ration (TMR) and saline injections at day 0 and 7 of the estrous cycle, 0XPOS: TMR, rumen protected methionine (MET) fed at 0.08% of the diet DM, rumen protected choline (CHOL) fed at 60 g/d, and saline injections at day 0 and 7, 0.5X: TMR, MET, CHOL, 5 mg B12, and 80 mg folate at day 0 and 7, 1X: TMR, MET CHOL, 10 mg vitamin B12, and 160 mg folate at day 0 and 7, and 2X: TMR, MET, CHOL, 20 mg B12, and 320 mg folate at day 0 and 7. All heifers were estrus synchronized but not bred, and blood was collected on day 0, 2, 5, 7, 9, 12, and 14 of a synchronized estrous cycle. Heifers were slaughtered on day 14 of the estrous cycle for liver collection. Serum B12 concentrations were greater in the 0.5X, 1X, and 2X, compared with 0XNEG and 0XPOS on all days after treatment initiation (P < 0.0001). Serum folate concentrations were greater for the 2X treatment at day 5, 7, and 9 of the cycle compared with all other treatments (P ≤ 0.05). There were no differences (P ≥ 0.19) in hepatic methionine-cycle or choline analyte concentrations by treatment. Concentrations of hepatic folate cycle intermediates were always greater (P ≤ 0.04) in the 2X treatment compared with the 0XNEG and 0XPOS heifers. Serum methionine was greater (P = 0.04) in the 0.5X and 2X heifers compared with 0XNEG, and S-adenosylhomocysteine (SAH) tended (P = 0.06) to be greater in the 0.5X heifers and the S-adenosylmethionine (SAM):SAH ratio was decreased (P = 0.05) in the 0.5X treatment compared with the 0XNEG, 0XPOS, and 2X heifers. The hepatic transcript abundance of MAT2A and MAT2B were decreased (P ≤ 0.02) in the 0.5X heifers compared with the 0XNEG, 0XPOS, and 2X heifers. These data support that beef heifers fed rumen protected methionine and choline require 20 mg B12 and 320 mg folate once weekly to maintain increased concentrations of B12 and folate in serum. Furthermore, these data demonstrate that not all supplementation levels are equal in providing positive responses, and that some levels, such as the 0.5X, may result in a stoichiometric imbalance in the one-carbon metabolism pathway that results in a decreased SAM:SAH ratio.

The strategic inclusion of one-carbon metabolites, which include vitamins and minerals that are found in human prenatal vitamins, to beef cattle feeding and management protocols during the periconceptual period (the time around breeding) is a novel concept. Therefore, this study aimed to identify the feeding and injection doses of one-carbon metabolites in beef heifers to maintain increased circulating concentrations of one-carbon metabolites for use as a model from which other studies could base their treatments on. We determined that daily feeding of methionine and choline at 0.08% of dry matter and 60 g/d, respectively, and administration of vitamin B12 and folate at 20 mg and 320 mg once per week, respectively resulted in sustained elevated concentrations of one-carbon metabolites.