IntroductionMaternal nutrition during pregnancy critically influences offspring development and immune function. One-carbon metabolites (OCM) are epigenetic modifiers that may modulate antimicrobial peptide (AMP) expression, which is vital for innate immunity. This study investigated the effects of maternal nutrient restriction and OCM supplementation on mRNA expression of AMP in fetal and maternal lung, mammary gland, and small intestine of beef cattle.MethodsTwenty-nine crossbred Angus beef heifers were synchronized for estrus and artificially inseminated. They were assigned to one of four treatments in a 2 × 2 factorial design: nutritional plane [control (CON) vs. restricted (RES)] and OCM supplementation [without OCM (−OCM) or with OCM (+OCM)]. Heifers on the CON diet were fed to gain 0.45 kg/day, while RES heifers were fed to lose 0.23 kg/day. Treatments were applied from day 0 to 63 of gestation, after which all heifers were fed a common diet to gain 0.45 kg/day until day 161 of gestation, when samples were collected. Quantitative RT-qPCR was used to assess mRNA expression of AMP.ResultsNutritional plane had no effect (p ≥ 0.24) on mRNA expression of AMP in either the fetus or dams. However, the mRNA expression of cathelicidin5 (CATHL5; p = 0.07) and bovine neutrophil β-defensin5 (BNBD5; p = 0.07) in the fetal lung and mammary gland, respectively, was lower in the +OCM groups compared to the −OCM groups. In the maternal small intestine, the expression of enteric β-defensin (EBD) was lower (p = 0.01) in the +OCM groups compared to the −OCM groups. Additionally, in the maternal lung, there was a tendency (p = 0.06) for an interaction in CATHL5 mRNA expression, with the RES + OCM group showing greater expression compared to the CON + OCM (p = 0.07) and RES − OCM (p = 0.08) groups.DiscussionOur findings suggest that while restricted maternal nutrition did not affect mRNA expression of AMP, OCM supplementation modulated AMP expression in both fetal and maternal tissues. Further research is needed to elucidate the mechanisms underlying OCM’s impact on AMP expression.

Abstract In beef heifers, body weight (BW) gain and physiological stress are important considerations for reproductive outcomes and whole herd production efficiencies. The objective of this study was to determine if dietary one-carbon metabolite (OCM) supplementation in moderate BW loss pregnant heifers alters circulating total antioxidant capacity, superoxide dismutase, and glutathione peroxidase activity. In a replicated 2 × 2 factorial, Angus-cross heifers (n = 81) were stratified by BW to gain 0.45 kg/d (CON) or lose 0.23 kg/d (RES) and receive corn carrier with OCM supplements (+OCM) or without (−OCM). The OCM supplements consisted of vitamin B12 (20 mg) and folate (320 mg) injections weekly and dietary rumen-protected methionine (7.4 g/d) and choline (44.4 g/d) from breeding (d 0) to d 63 of gestation. The four treatment groups were: CON−OCM (n = 20), CON+OCM (n = 21), RES−OCM (n = 21), and RES+OCM (n = 19). Heifers underwent estrus synchronization and were bred via artificial insemination using female sexed semen to a single sire. Pregnancy was diagnosed and fetal sex was verified on d 35 of gestation. Blood was collected and assayed for total antioxidant capacity, superoxide dismutase, and glutathione peroxidase activity on d −2 (baseline), 35, and 63 of gestation for all three replicates. Data were analyzed using the MIXED procedure of SAS with repeated measures to determine the effect of gain, supplement, day, and their two-way and three-way interactions. The gain × supplement interaction remained in all models, but other interactions were removed if P > 0.10. Replicate and heifer BW were included in the model as covariates and covariance structure was determined by lowest AIC and BIC. Statistical significance was considered at P ≤ 0.05 and tendencies at 0.05 < P ≤ 0.10. There was no BW gain × supplement interaction, or main effect of OCM supplements in total antioxidants. There was a gain × day effect (P ≤ 0.01), in which total antioxidants were increased on d 35 and further increased on d 63 in CON compared with RES treatments. There was no gain × supplement interaction, or main effect of gain or OCM supplements in superoxide dismutase and glutathione peroxidase. However, both superoxide dismutase and glutathione peroxidase were decreased (P ≤ 0.01) on d 63 compared with d 0 and 35. These data suggest that moderate BW loss reduces total antioxidant capacity in pregnant beef heifers, which could affect redox homeostasis. However, OCM supplements did not affect total antioxidant capacity, superoxide dismutase, or glutathione peroxidase. These findings are somewhat surprising considering oxidative stress has been well characterized in nutrient-restricted pregnancies, and given the interconnection between energy metabolism and one-carbon metabolism. Future research is warranted to fully elucidate the therapeutic effects of one-carbon metabolites.

One-carbon metabolites (OCM) are metabolites and cofactors which include folate, vitamin B12, methionine, and choline that support methylation reactions. The objectives of this study were to investigate the effects of moderate changes in maternal body weight gain in combination with OCM supplementation during the first 63 d of gestation in beef cattle on (1) B12 and folate concentrations in maternal serum (2) folate cycle intermediates in maternal and fetal liver, allantoic fluid (ALF), and amniotic fluid (AMF) and (3) metabolites involved in one-carbon metabolism and related metabolic pathways in maternal and fetal liver. Heifers were either intake restricted (RES) and fed to lose 0.23 kg/d, or fed to gain 0.60 kg/d (CON). Supplemented (+ OCM) heifers were given B12 and folate injections weekly and fed rumen-protected methionine and choline daily, while non-supplemented (-OCM) heifers were given weekly saline injections. These two treatments were combined in a 2 × 2 factorial arrangement resulting in 4 treatments: CON-OCM, CON + OCM, RES-OCM, and RES + OCM. Samples of maternal serum, maternal and fetal liver, ALF, and AMF were collected at slaughter on day 63 of gestation. Restricted maternal nutrition most notably increased (./ ≤ 0.05) the concentration of vitamin B12 in maternal serum, 5,10-methylenetetrahydrofolate and 5,10-methenyltetrahydrofolate in maternal liver, and cystathionine in the fetal liver; conversely, maternal restriction decreased (P = 0.05) 5,10-methylenetetrahydrofolate concentration in fetal liver. Supplementing OCM increased (P ≤ 0.05) the concentrations of maternal serum B12, folate, and folate intermediates, ALF and AMF 5-methyltetrahydrofolate concentration, and altered (P ≤ 0.02) other maternal liver intermediates including S-adenosylmethionine, dimethylglycine, cystathionine Glutathione reduced, glutathione oxidized, taurine, serine, sarcosine, and pyridoxine. These data demonstrate that OCM supplementation was effective at increasing maternal OCM status. Furthermore, these data are similar to previously published literature where restricted maternal nutrition also affected maternal OCM status. Altering OCM status in both the dam and fetus could impact fetal developmental outcomes and production efficiencies. Lastly, these data demonstrate that fetal metabolite abundance is highly regulated, although the changes required to maintain homeostasis may program altered metabolism postnatally.

Maternal diet quality and quantity have significant impacts on both maternal and fetal health and development. The composition and function of the maternal gut microbiome is also significantly influenced by diet; however, little is known about the impact of gestational nutrient restriction on the bovine maternal microbiome during early gestation, which is a critical stage for maternal microbiome-mediated fetal programming to take place. The objective of the present study was to evaluate the impacts of diet restriction and one-carbon metabolite (OCM) supplementation during early gestation on maternal ruminal, vaginal, and blood microbiota in cattle. Thirty-three beef heifers (approx. 14 months old) were used in a 2 × 2 factorial experiment with main factors of target gain (control [CON]; targeted 0.45 kg/d gain vs restricted [RES]; targeted − 0.23 kg/d gain), and OCM supplementation (+ OCM vs − OCM; n = 8/treatment; except n = 9 for RES–OCM). Heifers were individually fed, starting treatment at breeding (d 0) and concluding at d 63 of gestation. Ruminal fluid and vaginal swabs were collected on d − 2, d 35, and d 63 (at necropsy) and whole blood was collected on d 63 (necropsy). Bacterial microbiota was assessed using 16S rRNA gene (V3–V4) sequencing. Overall ruminal microbiota structure was affected by gain, OCM, time, and their interactions. The RES heifers had greater microbial richness (observed ASVs) but neither Shannon nor Inverse Simpson diversity was significantly influenced by gain or OCM supplementation; however, on d 63, 34 bacterial genera showed differential abundance in the ruminal fluid, with 25 genera enriched in RES heifers as compared to CON heifers. In addition, the overall interaction network structure of the ruminal microbiota changed due to diet restriction. The vaginal microbiota community structure was influenced by gain and time. Overall microbial richness and diversity of the vaginal microbiota steadily increased as pregnancy progressed. The vaginal ecological network structure was distinctive between RES and CON heifers with genera-genera interactions being intensified in RES heifers. A relatively diverse bacterial community was detected in blood samples, and the composition of the blood microbiota differed from that of ruminal and vaginal microbiota. Restricted dietary intake during early gestation induced significant alterations in the ruminal microbiota which also extended to the vaginal microbiota. The composition of these two microbial communities was largely unaffected by OCM supplementation. Blood associated microbiota was largely distinctive from the ruminal and vaginal microbiota.

Abstract Maternal nutritional plane during pregnancy may leave lifelong impacts on offspring in beef cattle. Given that, we hypothesized that the supplementation of one-carbon metabolites (OCM) during pregnancy would mitigate the negative effects of maternal rate of gain through brain gene expression regulation in early developing bovine fetuses. The objective of this study was to evaluate the effects of OCM supplementation and maternal rate of gain on gene expression in the developing fetal brain in gestating beef heifers. Herein, Angus crossbred heifers (n = 72, body weight = 406 ± 33 kg) were randomly assigned to 2 × 2 factorial arrangement of treatments with two levels of maternal rate of gain (CON, 0.60 kg/d; and RES, -0.23 kg/d) each with or without OCM supplementation (+OCM, and -OCM). Heifers were fed individually and were bred using female-sexed semen from a single sire. The OCM supplementation included rumen-protected choline (44.4 g/d) and methionine (7.4 g/d) in corn carrier fed daily and weekly injections of folate (320 mg) and vitamin B12 (20 mg). The -OCM heifers received the corn carrier and saline injections. The treatments started at the time of breeding and continued until d 63 of gestation when pregnant heifers (n = 29) were slaughtered to collect fetal brain tissues for RNA sequencing. The fetal brain phenotypic data were analyzed using MIXED procedures of SAS, indicating that restricted nutrition affected the fetal right hemisphere weight (P < 0.05), and there was a strong tendency for overall fetal brain weight (0.05 < P < 0.1) to be affected as well. Brain transcriptomics data were analyzed using STAR aligner and a total of 177 differentially expressed genes (DEGs, FDR < 0.05) were identified across all treatment comparisons using DESeq2. Most of the DEGs were either isoforms of spliceosomal non-coding RNAs or micro-RNAs (miRNAs) associated with biological processes (FDR < 0.1) like mRNA processing, cellular nitrogen metabolism, lipid metabolism, and post-transcriptional gene silencing. The key miRNAs upregulated in the brain tissue of restricted fetuses without OCM supplementation, bta-mir-2375, bta-mir-876, and bta-mir-196a-1, were found to be associated with early embryogenesis and essential cellular regulatory processes. Conversely, in restricted fetuses with OCM supplementation, upregulated miRNAs such as bta-mir-216b, bta-mir-2285dd, and bta-mir-1298 were linked to early embryonic development, muscular development, and lipid metabolism in cattle. Both phenotypic and transcriptomics data here revealed that the maternal rate of gain altered fetal brain development, with differences seemingly compensated by OCM supplementation. USDA is an equal opportunity provider and employer.

Abstract One-carbon metabolite (OCM) supplementation in beef cattle has been shown to be a novel strategy that alters programming outcomes in tissues of the developing fetus. However, information regarding growth changes to the fetus during early gestation is limited. The objective of this study was to evaluate the effects of OCM supplementation in control-fed or nutrient-restricted pregnant heifers on fetal crown-rump length (CRL) during early gestation. We hypothesized that decreased nutrient availability in the maternal system would impact the growth of the fetus in utero and providing OCM supplementation would restore growth levels in a nutrient restricted environment. Angus crossbred heifers [n = 216; initial body weight (BW) = 365 ± 5.45 kg] across three replicates underwent estrus synchronization and bred via artificial insemination (AI) using female-sexed semen from a single sire. At breeding (d 0), heifers were assigned to treatments in a 2 × 2 factorial design, which included two levels of daily gain; control (CON; +0.45 kg/d gain) or restricted (RES; −0.23 kg/d gain) and supplementation of OCM [+OCM; rumen protected choline (44.4 g/d) and methionine (7.4 g/d) in a ground corn carrier and weekly injections of 320 mg folate and 20 mg vitamin B12] or no supplementation (-OCM; corn carrier and saline injections). Heifers were weighed weekly, and diets were adjusted on a dry matter basis of the total mixed ration to maintain targeted gain. Treatments were maintained through d 63 of gestation in pregnant heifers. Pregnancy diagnosis and fetal CRL was determined via transrectal ultrasonography on d 35. A total of 103 heifers were confirmed pregnant (replicate 1, n = 37; replicate 2, n = 32; replicate 3, n = 34). Data were analyzed using the MIXED procedures of SAS as a 2 × 2 factorial including the effects of two levels of gain, two levels of OCM treatment, replicate, and their two-way and three-way interactions. There were no three-way interactions between gain × treatment × replicate. However, there was a gain × treatment interaction (P ≤ 0.05), in which fetuses from RES heifers with and without OCM supplementation had reduced CRL compared with fetuses from CON heifers that did not receive OCM (14.15 ± 0.21 mm, 13.88 ± 0.21 mm, 14.84 ± 0.21 mm, respectively). Fetal CRL from OCM supplemented CON heifers was similar (P = 0.06) to all other treatments (14.27 ± 0.21 mm). In conclusion, fetuses from RES heifers exhibited a decreased CRL, and OCM supplementation did not mitigate these effects.

The objective of this study was to determine the dose-dependent response of one-carbon metabolite (OCM: methionine, choline, folate, and vitamin B-12) 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 +/- 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 B-12, and 80-mg folate injections at days 0 and 7, 1X: TMR, MET CHOL, 10-mg vitamin B-12, and 160-mg folate at days 0 and 7, and 2X: TMR, MET, CHOL, 20-mg vitamin B-12, 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 B-12 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 alpha-amylase were decreased (P <= 0.04) in the 0.5X treatment. Heifers on the 2X treatment had greater pancreatic alpha-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.

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.

Maternal nutrition is pivotal for proper fetal development, with one-carbon metabolites (OCM) playing a key role in fetal epigenetic programming through DNA and histone methylation. The study aimed to investigate the effects of nutrient restriction and OCM supplementation on fetal liver metabolomics in pregnant beef-heifers, focusing on metabolites and pathways associated with amino acid, vitamin and cofactor, carbohydrate, and energy metabolism at day 63 of gestation. Thirty-one crossbred Angus heifers were artificially inseminated and allocated to 4 nutritional treatments in a 2 x 2 factorial arrangement of treatments, with the 2 factors being dietary intake/rate of gain (control-diet [CON]; 0.60 kg/d ADG, vs. restricted-diet [RES]; -0.23 kg/d ADG) and OCM supplementation (supplemented [+OCM] vs. not supplemented [-OCM]). The resulting treatment groups-CON - OCM, CON + OCM, RES - OCM, and RES + OCM were maintained for 63 day post-breeding. Following this period, fetal liver tissues were collected and subjected to metabolomic analysis using UPLC-tandem mass-spectrometry. We identified 288 metabolites, with the majority (n = 54) being significantly influenced by the main effect of gain (P <= 0.05). Moreover, RES showed decreased abundances of most metabolites in pathways such as lysine metabolism; leucine, isoleucine, and valine metabolism; and tryptophan metabolism, compared to CON. Supplementation with OCM vs. no OCM supplementation, resulted in greater abundance of metabolites (P <= 0.05) affecting pathways associated with methionine, cysteine, S-adenosylmethionine and taurine metabolism; guanidino and acetamido metabolism; and nicotinate and nicotinamide metabolism. Notably, OCM supplementation with a moderate rate of gain increased the concentrations of ophthalmate, N-acetylglucosamine, and ascorbic-acid 3-sulfate, which are important for proper fetal development (P <= 0.05). Nutrient restriction reduced the majority of liver metabolites, while OCM supplementation increased a smaller number of metabolites. Thus, OCM supplementation may be protective of metabolite concentrations in key developmental pathways, which could potentially enhance fetal development under nutrient-restricted conditions. This research demonstrates that supplementing one-carbon metabolites during early gestation significantly counteracts some of the detrimental effects of nutrient restriction on fetal liver metabolism in beef heifers, providing a potential strategy to support fetal development and improve pregnancy outcomes under nutrient-restricted conditions. Maternal nutrition is crucial for pregnancy outcomes, influencing offspring health and productivity. Poor nutrition during pregnancy can lead to fetal growth restrictions, impacting liver development. Such changes can increase the risk of metabolic syndromes and predispose them to impaired immune function. In cattle, optimal nutrition during early pregnancy is essential for reproductive efficiency and herd health. This period is critical for developmental programming through epigenetic changes triggered by environmental or genetic factors. These modifications are heritable which are influenced by maternal diet and play a critical role in determining health outcomes post-birth, relying significantly on the availability of one-carbon metabolites (OCM) like methionine, choline, folate, and vitamin B12. Supplementing these nutrients during early gestation may counteract the negative effects of poor nutrition. This study explores the impact of OCM supplementation and dietary restrictions on the fetal liver metabolism in beef heifers during early gestation. Our findings showed that dietary restrictions decrease fetal liver metabolites, whereas OCM supplementation increases certain metabolites, indicating a compensatory effect to support fetal development under nutrient-restricted conditions. Highlighting the importance of maternal nutrition, our findings provide valuable insights for developing nutritional strategies to enhance livestock efficiency and inform dietary guidelines during pregnancy for better health outcomes.

We hypothesized that restricted maternal nutrition and supplementation of one-carbon metabolites (OCM; methionine, folate, choline, and vitamin B12) would affect placental vascular development during early pregnancy. A total of 43 cows were bred, and 32 heifers successfully became pregnant with female calves, leading to the formation of four treatment groups: CON - OCM (n = 8), CON + OCM (n = 7), RES - OCM (n = 9), and RES + OCM (n = 8). The experimental design was a 2 × 2 factorial, with main factors of dietary intake affecting average daily gain: control (CON; 0.6 kg/d ADG) and restricted (RES; -0.23 kg/d ADG); and OCM supplementation (+OCM) in which the heifers were supplemented with rumen-protected methionine (7.4 g/d) and choline (44.4 g/d) and received weekly injections of 320 mg of folate and 20 mg of vitamin B12, or received no supplementation (-OCM; corn carrier and saline injections). Heifers were individually fed and randomly assigned to treatment at breeding (day 0). Placentomes were collected on day 63 of gestation (0.225 of gestation). Fluorescent staining with CD31 and CD34 combined with image analysis was used to determine the vascularity of the placenta. Images were analyzed for capillary area density (CAD) and capillary number density (CND). Areas evaluated included fetal placental cotyledon (COT), maternal placental caruncle (CAR), whole placentome (CAR + COT), intercotyledonary fetal membranes (ICOT, or chorioallantois), intercaruncular endometrium (ICAR), and endometrial glands (EG). Data were analyzed with the GLM procedure of SAS, with heifer as the experimental unit and significance at P ≤ 0.05 and a tendency at P > 0.05 and P < 0.10. Though no gain × OCM interactions existed (P ≥ 0.10), OCM supplementation increased (P = 0.01) CAD of EG, whereas nutrient restriction tended (P < 0.10) to increase CAD of ICOT and CND of COT. Additionally, there was a gain × OCM interaction (P < 0.05) for CAD within the placentome and ICAR, such that RES reduced and supplementation of RES with OCM restored CAD. These results indicate that maternal rate of gain and OCM supplementation affected placental vascularization (capillary area and number density), which could affect placental function and thus the efficiency of nutrient transfer to the fetus during early gestation.

The objective of this study was to determine the dose of folate and vitamin B-12 in beef heifers fed rumen protected methionine and choline required to maintain increased B-12 levels and intermediates of the methionine-folate cycle in circulation. Angus heifers (n = 30; BW = 392.6 +/- 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 B-12, and 80 mg folate at day 0 and 7, 1X: TMR, MET CHOL, 10 mg vitamin B-12, and 160 mg folate at day 0 and 7, and 2X: TMR, MET, CHOL, 20 mg B-12, 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 B-12 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 B-12 and 320 mg folate once weekly to maintain increased concentrations of B-12 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. This study determined the optimal doses for vitamin B-12 (20 mg injected weekly) and folate (320 mg injected weekly) injection to beef heifers during the periconceptual period when rumen protected methionine and choline were fed at 0.08% of dry matter and 60 g/d, respectively. These doses set the basis upon which future studies can be based when feeding and injecting one-carbon metabolites. Lay Summary 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 B-12 and folate at 20 mg and 320 mg once per week, respectively resulted in sustained elevated concentrations of one-carbon metabolites.

When the developing embryo enters the uterus, before the placenta develops, the embryo receives nourishment from endometrially derived histotroph produced by the uterine glands that remain functional throughout gestation. Throughout pregnancy, this histotroph remains necessary for proper embryonic development. Maternal nutrition may affect histotroph composition resulting in altered embryo development. The objectives were to determine the effects of dietary intake on histotroph amino acid (AA) concentrations. Eighteen multiparous nonpregnant cows (initial BW = 597 kg) were housed at the North Dakota State University Animal Nutrition and Physiology Center (n = 6 cows/pen). Cows were individually fed a TMR once daily via a Calan feeding system and had ad libitum access to fresh water. Cows were randomly assigned to 1 of 2 treatments (n = 9/treatment): dietary intake designed to maintain body weight (CON) or dietary intake designed to lose body weight at a moderate rate (-0.7 kg/d; NEG), for 63 days. Slaughter group 1 contained 4 CON and 5 NEG cows, and group 2 contained 5 CON and 4 NEG cows. Cows were estrus-synchronized with the Co-Synch + CIDR protocol to ensure a common day of the estrous cycle at the time of slaughter (3 d after completion of the Co-Synch + CIDR protocol). When total uterine histotroph AA was calculated (luteal + non-luteal horns) and analyzed by treatment with group in the model, the differences were: CON > NEG for 1) Trp as % of essential AA, and 2) Ser as % of total AA (P = 0.04 and 0.05, respectively). Tendencies for total tract histotroph were observed for Phe and Trp as a % of total AA (P = 0.07 and 0.06, respectively) for CON > NEG. Luteal histotroph Ser as a % of total AA was greater (P = 0.05) for CON vs. NEG. When evaluating non-luteal histotroph, CON had greater (P < 0.05) Cys as a % of total AA compared with NEG cows. Non-luteal histotroph Arg concentration tended (P = 0.10) to be greater for NEG vs. CON. These results demonstrate that feed intake affects cow AA supply in the uterine histotroph, and these AA (Trp, Ser, Phe, Cys and Arg) may function in critical nutrient pathways, reproductive tract vascularization, and antioxidant production, which are necessary for optimal fetal development.

Vitamin B12 and folate are involved in the metabolic process known as one-carbon metabolism, which among other things, has a role in DNA, RNA, and protein methylation. This methylation connects one-carbon metabolism to epigenetics, which influences fetal development. Thus, an adequate supply of one-carbon metabolites (OCM) is important during fetal development. We hypothesized that differing planes of nutrition, along with supplementation of OCM, would impact concentrations of B12, folate, and folate intermediates in maternal liver, maternal serum, fetal liver, allantoic fluid (ALF), and amniotic fluid (AMF). Specifically, OCM supplementation would increase circulating and hepatic B12 and folate concentrations compared with non-supplemented heifers. Heifers (n = 72) were individually fed on one of two planes of nutrition: control (CON; 0.45 kg/d gain) or restricted (RES; -0.23 kg/d gain). To complete the 2 × 2 factorial arrangement, heifers were also given either OCM supplementation (10 g/d rumen-protected methionine + 60 g/d rumen-protected choline in the diet plus 20 mg B12 + 320 mg folic acid; weekly intramuscular injections) or no supplementation (grain carrier + saline injection). Thus, the final treatments were CON-OCM, CON+OCM, RES-OCM, and RES+OCM. Heifers were bred to a single sire via artificial insemination with sexed semen. Heifers pregnant with female calves (n = 31) were selected for slaughter on d 63. Samples of maternal liver, ALF, AMF, and fetal liver were collected at slaughter. Maternal serum was collected at breeding (d 0) and at slaughter. For B12 and folate concentration analysis, maternal serum was sent to IDEXX BioAnalytics (N. Grafton, MA). For analysis of folate intermediate concentrations in maternal and fetal liver, ALF, and AMF, samples were analyzed using LC-MS/MS. There were treatment × day interactions (P ≤ 0.05) for B12 and folate in maternal serum; on d 63, R+OCM was greater compared with C-OCM and R-OCM and all treatments at d 0 (Table 1). For folate intermediate concentrations, no plane of nutrition × supplement interactions (P ≥ 0.06) were found in fetal liver, ALF, or AMF; however, 5-methyl THF in ALF tended (P = 0.06) to be greater in R+OCM compared with all other treatments (Table 2). No interactions were found for concentrations in maternal liver when corrected for total liver weight; however, there was a supplement effect (P ≤ 0.05) where the concentrations of folic acid, dihydrofolate, tetrahydrofolate, 5-methyltetrahydrofolate, 5,10-methylenetetrahydrofolate and 5,10-methenyltetrahydrofolate were increased in +OCM compared with -OCM, and a plane of nutrition effect (P ≤ 0.05) with increased 5,10-methenyltetrahydrofolate and 5,10-methylenetetrahydrofolate concentrations in RES. We conclude that supplementing OCM increases concentrations of B12, folate and its intermediates in maternal liver and serum. USDA is an equal opportunity provider and employer.

Objective of our study were to investigate the impact of maternal nutrition during early pregnancy on the vascularity and proliferation of fetal intestine and whether supplementing one-carbon metabolites (OCM) could mitigate any changes arising from altered maternal nutrition. We used a 2 x 2 factorial arrangement of treatments with two levels of heifer gain: control (CON; 0.45 kg/d) and restricted (RES; -0.23 kg/d), with and without (+/- OCM) supplementation. Crossbred Angus beef heifers were estrous synchronized and bred via artificial insemination with single-sire female-sexed semen. Heifers were divided into four nutritional treatments (n = 8 ± 1 per treatment) at breeding. Targeted heifer gain (achieved by changing in intake of a common diet) 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. Treatments containing OCM were given rumen-protected methionine (10 g/d) and choline (60 g/d) in a fine-ground corn carrier administered daily, with weekly injections of folate (320 mg) and vitamin B12 (20 mg). The non-supplemented treatment (-OCM) received the corn carrier and saline injections. On day 161 of gestation heifers were harvested and samples were collected from fetal jejunum for analysis. Vascularity was assessed via measurement of capillary density using anti-CD31 and anti-CD34 fluorescent staining. Cell proliferation within intestinal tissues was assessed by KI-67 fluorescent staining. Images were analyzed using ImagePro-Premiere software. Maternal gain × OCM interactions were not present (P > 0.11). Fetuses from restricted dams had greater (P = 0.05) capillary area density in the villi compared with CON (5.48 vs. 4.72 ± 0.38%), while OCM had no effect. Crypt and total cell proliferation were greater (P < 0.05) in CON. Providing OCM reduced (P = 0.01) total intestinal cell proliferation. The observed increase in capillary density in the villi of fetuses from restricted dams may be a compensatory response to reduced maternal nutrient availability, aimed at preparing the offspring for increasing post-natal nutrient delivery. Conversely, the decreased proliferation in crypt cells appears to be a logical response to restricted nutrition and may indicate a slowing of the rate of cell division, which could have long-term consequences for intestinal development. Our results also demonstrate that maternal OCM can reduce total intestinal cell proliferation in the fetal intestine, regardless of maternal nutrition. One-carbon metabolites have crucial roles in DNA methylation and other cellular processes for proper cellular function and development. Observed decreases resulting from the provision of OCM may indicate enhanced energetic efficiency, reduced epithelial cell turnover, and/or reduce apoptotic events in the villa. Further studies are needed to determine the long-term effects of maternal OCM on fetal intestinal development and function. USDA is an equal opportunity provider and employer.

We hypothesized a low maternal plane of nutrition will impair fetal growth in early gestation and that supplementation of one-carbon metabolites (OCM) will rescue the growth restriction. The experimental design was a 2 × 2 factorial with 2 levels of gain, each with or without supplementation of OCM. Angus-cross heifers were bred with female-sexed semen from a single sire. At breeding (d 0), beef heifers were individually fed using a Calan gate system and assigned to treatment (n = 8 per treatment): Control intake (targeted: 0.45 kg/d, actual: 0.60 kg/d ADG) without OCM (CON-OCM), CON with OCM (CON+OCM), Restricted intake (targeted/actual: -0.23 kg/d ADG) without OCM (RES-OCM), or RES with OCM (RES+OCM). The OCM supplement consisted of ruminal-protected choline (60 g/d) and methionine (10 g/d) in a fine-ground corn carrier fed daily, and weekly injections of folate (320 mg) and vitamin B12 (10 mg). The -OCM heifers received the corn carrier and saline injections. Heifers were slaughtered and fetal tissues collected on d 63. Data were analyzed using the MIXED procedures of SAS. Fetal body weight tended (P = 0.07) to be lighter and stomach complex weight was lighter (P = 0.03) in RES than CON. When organ weights were normalized to total brain weight to assess allometric growth, OCM × gain interactions were observed in heart (P = 0.04), left longissimus dorsi (P = 0.04), and right hemisphere (P = 0.01) with CON+OCM greater than CON–OCM. The normalized left hemisphere trend to be greater in RES+OCM than RES-OCM (P = 0.08). These data suggest that OCM supplementation during early gestation increases allometric growth of fetal cardiac and skeletal muscle tissue. Day 63 is within the period of peak primary myogenesis in cattle, therefore differences in muscle growth during this time could have lasting implications in post-natal calf growth and performance.