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.

One-carbon metabolite supplementation to nutrient-restricted beef heifers affects placental vascularity during early pregnancy.

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.

In cow­calf production, periods of poor forage availability or quality can result in nutrient restriction during pregnancy. Previous studies have shown that even moderate maternal feed restriction during pregnancy, including very early in pregnancy, has profound effects on fetal and placental development, potentially having lasting impacts on calf growth and body composition later in life. One-carbon metabolites (OCM) in the diet are biomolecules required for methylation reactions and participate in the regulation of gene expression. Our objective was to evaluate the effects of nutrient restriction and OCM supplementation (specifically methionine, choline, folate, and vitamin B12) on placental vascular development during early pregnancy. Proper placental vascular development is necessary for healthy pregnancy outcomes, reflected by normal birth weight and healthy offspring. Our results indicated that maternal rate of gain and OCM supplementation affect placental vascularization, which could affect placental function and thereby fetal development throughout gestation. In the context of beef cattle production, our study sheds light on strategies that could enhance placental vascular development during early pregnancy. However, it is essential to recognize the nuances in our data, highlighting the need for further research to fully comprehend these intricate processes.

Dataset of RNA-Seq transcriptome of the fetal liver at day 83 of gestation associated with periconceptual maternal nutrition in beef heifers.

Herein, we present a dataset based on the RNA-Seq analysis of liver tissue from bovine female fetuses at day 83 of gestation. The findings were reported in the main article, "Periconceptual maternal nutrition affects fetal liver programming of energy- and lipid-related genes" [1]. These data were generated to investigate the effects of periconceptual maternal vitamin and mineral supplementation and rates of body weight gain on the transcript abundance of genes associated with fetal hepatic metabolism and function. To this end, crossbred Angus beef heifers (n = 35) were randomly assigned to 1 of 4 treatments in a 2 × 2 factorial design. The main effects tested were vitamin and mineral supplementation (VTM or NoVTM - at least 71 days pre-breeding to day 83 of gestation) and rate of weight gain (low (LG - 0.28 kg/d) or moderate (MG - 0.79 kg/d) - from breeding to day 83). The fetal liver was collected on day 83 ± 0.27 of gestation. After total RNA isolation and quality control, strand-specific RNA libraries were prepared and sequenced on the Illumina® NovaSeq 6000 platform to generate paired-end 150-bp reads. After read mapping and counting, differential expression analysis was performed with edgeR. We identified 591 unique differentially expressed genes across all six vitamin-gain contrasts (FDR ≤ 0.1). To our knowledge, this is the first dataset investigating the fetal liver transcriptome in response to periconceptual maternal vitamin and mineral supplementation and/or the rate of weight gain. The data described in this article provides genes and molecular pathways differentially programming liver development and function.

Fetal Hepatic Lipidome Is More Greatly Affected by Maternal Rate of Gain Compared with Vitamin and Mineral Supplementation at day 83 of Gestation.

Herein, we evaluated the hepatic lipid metabolic profiles of bovine fetuses in response to maternal vitamin and mineral supplementation (VMSUP; supplemented (VTM) or not (NoVTM)) and two different rates of gain (GAIN; low gain (LG), 0.28 kg/d, or moderate gain (MG), 0.79 kg/d). Crossbred Angus heifers (n = 35; initial BW = 359.5 ± 7.1 kg) were randomly assigned to a 2 × 2 factorial arrangement, resulting in the following treatment combinations: NoVTM-LG (n = 9), NoVTM-MG (n = 9), VTM-LG (n = 9), and VTM-MG (n = 8). Heifers received their treatments until d 83 of gestation, when they were ovariohysterectomized. Fetuses were harvested and liver samples were analyzed via ultrahigh-performance liquid chromatography-tandem mass spectroscopy to characterize lipid profiles and abundances. We identified 374 biochemicals/metabolites belonging to 57 sub-pathways of the lipid metabolism super-pathway. The majority of the biochemicals/metabolites (n = 152) were significantly affected by the main effect of GAIN. Maternal moderate rates of gain resulted in greater abundances (p ≤ 0.0001) of ω-3 fatty acids (eicosapentaenoate, docosapentaenoate, and docosahexaenoate) and lower abundances (p ≤ 0.0001) of ω-6 fatty acids. Further, MG resulted in the accumulation of several diacylglycerols and depletion of the majority of the monoacylglycerols. Concentrations of nearly all acylcarnitines (p ≤ 0.03) were decreased in VTM-LG fetal livers compared to all other treatment combinations, indicating a greater rate of complete oxidation of fatty acids. Levels of secondary bile acids were impacted by VMSUP, being greater (p ≤ 0.0048) in NoVTM than in VTM fetal livers. Moreover, NoVTM combined with lower rate of gain resulted in greater concentrations of most secondary bile acid biochemicals/metabolites. These data indicate that maternal diet influenced and altered fetal hepatic lipid composition in the first trimester of gestation. Maternal body weight gain exerted a greater influence on fetal lipid profiles than vitamin and mineral supplementation. Specifically, lower rate of gain (0.28 kg/d) resulted in an increased abundance of the majority of the biochemicals/metabolites identified in this study.

Periconceptual Maternal Nutrition Affects Fetal Liver Programming of Energy- and Lipid-Related Genes.

During pregnancy, the fetus relies on the dam for its nutrient supply. Nutritional stimuli during fetal organ development can program hepatic metabolism and function. Herein, we investigated the role of vitamin and mineral supplementation (VTM or NoVTM-at least 71 days pre-breeding to day 83 of gestation) and rate of weight gain (low (LG) or moderate (MG)-from breeding to day 83) on the fetal liver transcriptome and the underlying biological pathways. Crossbred Angus beef heifers (n = 35) were randomly assigned to one of four treatments in a 2 × 2 factorial design (VTM_LG, VTM_MG, NoVTM_LG, and NoVTM_MG). Gene expression was measured with RNA-Seq in fetal livers collected on day 83 ± 0.27 of gestation. Our results show that vitamin and mineral supplementation and rate of weight gain led to the differential expression of hepatic genes in all treatments. We identified 591 unique differentially expressed genes across all six VTM-gain contrasts (FDR ≤ 0.1). Over-represented pathways were related to energy metabolism, including PPAR and PI3K-Akt signaling pathways, as well as lipid metabolism, mineral transport, and amino acid transport. Our findings suggest that periconceptual maternal nutrition affects fetal hepatic function through altered expression of energy- and lipid-related genes.

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.

Vitamin and Mineral Supplementation and Rate of Gain in Beef Heifers II: Effects on Concentration of Trace Minerals in Maternal Liver and Fetal Liver, Muscle, Allantoic, and Amniotic Fluids at Day 83 of Gestation.

We evaluated the effects of vitamin and mineral supplementation (from pre-breeding to day 83 of gestation) and two rates of gain (from breeding to day 83 of gestation) on trace mineral concentrations in maternal and fetal liver, fetal muscle, and allantoic (ALF) and amniotic (AMF) fluids. Crossbred Angus heifers (n = 35; BW = 359.5 ± 7.1 kg) were randomly assigned to one of two vitamin and mineral supplementation treatments (VMSUP; supplemented (VTM) vs. unsupplemented (NoVTM)). The VMSUP factor was initiated 71 to 148 d before artificial insemination (AI), allowing time for the mineral status of heifers to be altered in advance of breeding. The VTM supplement (113 g·heifer−1·d−1) provided macro and trace minerals and vitamins A, D, and E to meet 110% of the requirements specified by the NASEM, and the NoVTM supplement was a pelleted product fed at a 0.45 kg·heifer−1·day−1 with no added vitamin and mineral supplement. At AI, heifers were assigned to one of two rates of gain treatments (GAIN; low gain (LG) 0.28 kg/d or moderate gain (MG) 0.79 kg/d) within their respective VMSUP groups. On d 83 of gestation fetal liver, fetal muscle, ALF, and AMF were collected. Liver biopsies were performed prior to VMSUP factor initiation, at the time of AI, and at the time of ovariohysterectomy. Samples were analyzed for concentrations of Se, Cu, Zn, Mo, Mn, and Co. A VMSUP × GAIN × day interaction was present for Se and Cu (p < 0.01 and p = 0.02, respectively), with concentrations for heifers receiving VTM being greater at AI and tissue collection compared with heifers not receiving VTM (p < 0.01). A VMSUP × day interaction (p = 0.01) was present for Co, with greater (p < 0.01) concentrations for VTM than NoVTM at the time of breeding. VTM-MG heifers had greater concentrations of Mn than all other treatments (VMSUP × GAIN, p < 0.01). Mo was greater (p = 0.04) for MG than LG, while Zn concentrations decreased throughout the experiment (p < 0.01). Concentrations of Se (p < 0.01), Cu (p = 0.01), Mn (p = 0.04), and Co (p = 0.01) were greater in fetal liver from VTM than NoVTM. Mo (p ≤ 0.04) and Co (p < 0.01) were affected by GAIN, with greater concentrations in fetal liver from LG than MG. In fetal muscle, Se (p = 0.02) and Zn (p < 0.01) were greater for VTM than NoVTM. Additionally, Zn in fetal muscle was affected by GAIN (p < 0.01), with greater concentrations in LG than MG. The ALF in VTM heifers (p < 0.01) had greater Se and Co than NoVTM. In AMF, trace mineral concentrations were not affected (p ≥ 0.13) by VMSUP, GAIN, or their interaction. Collectively, these data suggest that maternal nutrition pre-breeding and in the first trimester of gestation affects fetal reserves of some trace minerals, which may have long-lasting impacts on offspring performance and health.

Vitamin and Mineral Supplementation and Rate of Weight Gain during the First Trimester of Gestation in Beef Heifers Alters the Fetal Liver Amino Acid, Carbohydrate, and Energy Profile at Day 83 of Gestation.

The objective of this study was to evaluate the effects of feeding heifers a vitamin and mineral supplement and targeting divergent rates of weight gain during early gestation on the fetal liver amino acid, carbohydrate, and energy profile at d 83 of gestation. Seventy-two crossbred Angus heifers were randomly assigned in a 2 × 2 factorial arrangement to one of four treatments comprising the main effects of vitamin and mineral supplementation (VTM or NOVTM) and feeding to achieve different rates of weight gain (low gain [LG] 0.28 kg/day vs. moderate gain [MG] 0.79 kg/day). Thirty-five gestating heifers with female fetuses were ovariohysterectomized on d 83 of gestation and fetal liver was collected and analyzed by reverse phase UPLC-tandem mass spectrometry with positive and negative ion mode electrospray ionization, as well as by hydrophilic interaction liquid chromatography UPLC-MS/MS with negative ion mode ESI for compounds of known identity. The Glycine, Serine, and Threonine metabolism pathway and the Leucine, Isoleucine, and Valine metabolism pathway had a greater total metabolite abundance in the liver of the NOVTM-LG group and least in the VTM-LG group (p < 0.01). Finally, both the TCA Cycle and Oxidative Phosphorylation pathways within the Energy Metabolism superpathway were differentially affected by the main effect of VTM, where the TCA cycle metabolites were greater (p = 0.04) in the NOVTM fetal livers and the Oxidative Phosphorylation biochemicals were greater (p = 0.02) in the fetal livers of the VTM supplemented heifers. These data demonstrate that the majority of metabolites that are affected by rate of weight gain or vitamin/mineral supplementation are decreased in heifers on a greater rate of weight gain or vitamin/mineral supplementation.

Vitamin and Mineral Supplementation and Rate of Gain in Beef Heifers I: Effects on Dam Hormonal and Metabolic Status, Fetal Tissue and Organ Mass, and Concentration of Glucose and Fructose in Fetal Fluids at d 83 of Gestation.

Thirty-five crossbred Angus heifers (initial BW = 359.5 ± 7.1 kg) were randomly assigned to a 2 × 2 factorial design to evaluate effects of vitamin and mineral supplementation [VMSUP; supplemented (VTM) vs. unsupplemented (NoVTM)] and different rates of gain [GAIN; low gain (LG), 0.28 kg/d, vs. moderate gain (MG), 0.79 kg/d] during the first 83 d of gestation on dam hormone and metabolic status, fetal tissue and organ mass, and concentration of glucose and fructose in fetal fluids. The VMSUP was initiated 71 to 148 d before artificial insemination (AI), allowing time for mineral status of heifers to be altered in advance of breeding. At AI heifers were assigned their GAIN treatment. Heifers received treatments until the time of ovariohysterectomy (d 83 ± 0.27 after AI). Throughout the experiment, serum samples were collected and analyzed for non-esterified fatty acids (NEFA), progesterone (P4), insulin, and insulin-like growth factor 1 (IGF-1). At ovariohysterectomy, gravid reproductive tracts were collected, measurements were taken, samples of allantoic (ALF) and amniotic (AMF) fluids were collected, and fetuses were dissected. By design, MG had greater ADG compared to LG (0.85 vs. 0.34 ± 0.04 kg/d, respectively; p < 0.01). Concentrations of NEFA were greater for LG than MG (p = 0.04) and were affected by a VMSUP × day interaction (p < 0.01), with greater concentrations for NoVTM on d 83. Insulin was greater for NoVTM than VTM (p = 0.01). A GAIN × day interaction (p < 0.01) was observed for IGF-1, with greater concentrations for MG on d 83. At d 83, P4 concentrations were greater for MG than LG (GAIN × day, p < 0.01), and MG had greater (p < 0.01) corpus luteum weights versus LG. Even though fetal BW was not affected (p ≥ 0.27), MG fetuses had heavier (p = 0.01) femurs than LG, and VTM fetuses had heavier (p = 0.05) livers than those from NoVTM. Additionally, fetal liver as a percentage of BW was greater in fetuses from VTM (P = 0.05; 3.96 ± 0.06% BW) than NoVTM (3.79 ± 0.06% BW), and from LG (p = 0.04; 3.96 ± 0.06% BW) than MG (3.78 ± 0.06% BW). A VMSUP × GAIN interaction was observed for fetal small intestinal weight (p = 0.03), with VTM-MG being heavier than VTM-LG. Therefore, replacement heifer nutrition during early gestation can alter the development of organs that are relevant for future offspring performance. These data imply that compensatory mechanisms are in place in the developing conceptus that can alter the growth rate of key metabolic organs possibly in an attempt to increase or decrease energy utilization.

DNA methylation dataset of bovine embryonic fibroblast cells treated with epigenetic modifiers and divergent energy supply.

Fetal programming is established early in life, likely through epigenetic mechanisms that control gene expression. Micronutrients can act as epigenetic modifiers (EM) by modulating the genome through mechanisms that include DNA methylation and post-translational modification of chromatin. Among the EM, methionine, choline, folate, and vitamin B12 have been suggested as key players of DNA methylation. However, the effects of supplementing these four EM, involved in the methionine folate cycle on DNA methylation, are still under investigation. This manuscript provides the genome-wide DNA methylation dataset (GSE180362) of bovine embryonic fibroblast cells exposed to different supplementation levels of glucose and methionine, choline, folate, and vitamin B12 (collectively named as Epigenetic Modifiers - EM). The DNA methylation was measured using MSP-I digestion and Reduced Representation Bisulfite Sequencing. Bioinformatics analyses included data quality control, read mapping, methylation calling, and differential methylation analyses. Supplementary file S1 and data analysis codes are within this article. To our knowledge, this is the first dataset investigating the effects of four EM in bovine embryonic fibroblast DNA methylation profiles. Furthermore, this data and its findings provide information on putative candidate genes responsive to DNA methylation due to EM supplementation.

Epigenetic Modifier Supplementation Improves Mitochondrial Respiration and Growth Rates and Alters DNA Methylation of Bovine Embryonic Fibroblast Cells Cultured in Divergent Energy Supply.

Epigenetic modifiers (EM; methionine, choline, folate, and vitamin B12) are important for early embryonic development due to their roles as methyl donors or cofactors in methylation reactions. Additionally, they are essential for the synthesis of nucleotides, polyamines, redox equivalents, and energy metabolites. Despite their importance, investigation into the supplementation of EM in ruminants has been limited to one or two epigenetic modifiers. Like all biochemical pathways, one-carbon metabolism needs to be stoichiometrically balanced. Thus, we investigated the effects of supplementing four EM encompassing the methionine-folate cycle on bovine embryonic fibroblast growth, mitochondrial function, and DNA methylation. We hypothesized that EM supplemented to embryonic fibroblasts cultured in divergent glucose media would increase mitochondrial respiration and cell growth rate and alter DNA methylation as reflected by changes in the gene expression of enzymes involved in methylation reactions, thereby improving the growth parameters beyond Control treated cells. Bovine embryonic fibroblast cells were cultured in Eagle's minimum essential medium with 1 g/L glucose (Low) or 4.5 g/L glucose (High). The control medium contained no additional OCM, whereas the treated media contained supplemented EM at 2.5, 5, and 10 times (×2.5, ×5, and ×10, respectively) the control media, except for methionine (limited to ×2). Therefore, the experimental design was a 2 (levels of glucose) × 4 (levels of EM) factorial arrangement of treatments. Cells were passaged three times in their respective treatment media before analysis for growth rate, cell proliferation, mitochondrial respiration, transcript abundance of methionine-folate cycle enzymes, and DNA methylation by reduced-representation bisulfite sequencing. Total cell growth was greatest in High ×10 and mitochondrial maximal respiration, and reserve capacity was greatest (p < 0.01) for High ×2.5 and ×10 compared with all other treatments. In Low cells, the total growth rate, mitochondrial maximal respiration, and reserve capacity increased quadratically to 2.5 and ×5 and decreased to control levels at ×10. The biological processes identified due to differential methylation included the positive regulation of GTPase activity, molecular function, protein modification processes, phosphorylation, and metabolic processes. These data are interpreted to imply that EM increased the growth rate and mitochondrial function beyond Control treated cells in both Low and High cells, which may be due to changes in the methylation of genes involved with growth and energy metabolism.

Supranutritional Maternal Organic Selenium Supplementation during Different Trimesters of Pregnancy Affects the Muscle Gene Transcriptome of Newborn Beef Calves in a Time-Dependent Manner.

Selenium (Se) is an essential micronutrient for growth and immune function in beef cattle. We previously showed that supranutritional maternal organic Se supplementation during late pregnancy improves immune function in their newborn calves; however, the effects of maternal organic Se-supplementation on fetal programming during different pregnancy stages have yet to be elucidated. Herein, we investigated the effects of supranutritional maternal organic Se-supplementation in different pregnancy trimesters on their beef calf's genome-wide transcriptome profiles. Within 12 to 48 h of birth, whole blood and Longissimus dorsi (LD) muscle biopsies were collected from calves born to 40 crossbred Angus cows that received, except for the control group (CTR), Se-yeast boluses (105 mg of Se/wk) during the first (TR1), second (TR2), or third (TR3) trimester of gestation. Whole-blood Se concentrations of newborn calves increased from CTR, TR1, TR2 to TR3, whereas muscle Se concentrations of newborn calves were only increased in TR3 group. We identified 3048 unique differentially expressed genes (DEGs) across all group comparisons (FDR ≤ 0.05 and |log2FC| ≥ 1.5). Furthermore, we predicted 237 unique transcription factors that putatively regulate the DEGs. Independent of supplementation trimester, supranutritional maternal organic Se supplementation downregulated genes involved in adaptive immunity in all trimesters. Dependent on supplementation trimester, genes involved in muscle development were upregulated by TR3 Se supplementation and downregulated by TR1 Se-supplementation, and genes involved in collagen formation were downregulated by TR2 Se-supplementation. Supranutritional maternal organic Se supplementation in the last trimester of pregnancy resulted in upregulation of myosin and actin filament associated genes, potentially allowing for optimal muscle function and contraction. Our findings suggest a beneficial effect of supranutritional maternal organic Se supplementation during late gestation on Se-status and muscle development and function of newborn calves.

Maternal Vitamin and Mineral Supplementation and Rate of Maternal Weight Gain Affects Placental Expression of Energy Metabolism and Transport-Related Genes.

Maternal nutrients are essential for proper fetal and placental development and function. However, the effects of vitamin and mineral supplementation under two rates of maternal weight gain on placental genome-wide gene expression have not been investigated so far. Furthermore, biological processes and pathways in the placenta that act in response to early maternal nutrition are yet to be elucidated. Herein, we examined the impact of maternal vitamin and mineral supplementation (from pre-breeding to day 83 post-breeding) and two rates of gain during the first 83 days of pregnancy on the gene expression of placental caruncles (CAR; maternal placenta) and cotyledons (COT; fetal placenta) of crossbred Angus beef heifers. We identified 267 unique differentially expressed genes (DEG). Among the DEGs from CAR, we identified ACAT2, SREBF2, and HMGCCS1 that underlie the cholesterol biosynthesis pathway. Furthermore, the transcription factors PAX2 and PAX8 were over-represented in biological processes related to kidney organogenesis. The DEGs from COT included SLC2A1, SLC2A3, SLC27A4, and INSIG1. Our over-representation analysis retrieved biological processes related to nutrient transport and ion homeostasis, whereas the pathways included insulin secretion, PPAR signaling, and biosynthesis of amino acids. Vitamin and mineral supplementation and rate of gain were associated with changes in gene expression, biological processes, and KEGG pathways in beef cattle placental tissues.

Vitamin and mineral supplementation and rate of gain during the first trimester of gestation affect concentrations of amino acids in maternal serum and allantoic fluid of beef heifers.

The objective of this study was to evaluate the effects of feeding vitamin and mineral (VTM) supplement and (or) rate of gain (GAIN) during early gestation on amino acid (AA) concentrations in allantoic fluid (ALF) and amniotic fluid (AMF) and maternal serum. Seventy-two crossbred Angus heifers (initial BW = 359.5 ± 7.1 kg) were randomly assigned to one of four treatments in a 2 × 2 factorial arrangement with main effects of VTM supplement (VTM or NoVTM) and rate of gain (GAIN; low gain [LG], 0.28 kg/d, vs. moderate gain [MG], 0.79 kg/d). The VTM treatment (113 g•heifer-1•d-1, provided macro and trace minerals and vitamins A, D, and E to meet 110% of the requirements specified by the NASEM in Nutrient requirements of beef cattle. Washington, DC: The National Academies Press. doi:10.17226/19014, 2016) was initiated 71 to 148 d before artificial insemination (AI). To complete the factorial arrangement of treatments, at breeding heifers were either maintained on the basal diet (LG), or received MG diet which was implemented by adding a protein/energy supplement to the LG diet. Thirty-five gestating heifers with female fetuses were ovariohysterectomized on d 83 of gestation and maternal serum, ALF, and AMF were collected. Samples were analyzed for concentrations of neutral AA: Ala, Asn, Cys, Gln, Gly, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val; cationic AA: Arg, His, and Lys; and anionic AA: Asp and Glu. In serum, a VTM × GAIN interaction (P = 0.02) was observed for Glu, with greater concentrations for VTM-LG than VTM-MG. Concentrations of serum Cys, Met, and Trp were greater (P ≤ 0.03) for MG than LG. In ALF, concentrations of Glu were affected by a VTM × GAIN interaction, where VTM-MG was greater (P < 0.01) than all other treatments. Further, ALF from VTM had increased (P ≤ 0.05) concentrations of His, Asp, and 12 of the 14 neutral AA; whereas GAIN affected concentrations of Arg, Cys, and Asp, with greater concentrations (P ≤ 0.05) in MG heifers. In AMF, AA concentrations were not affected (P ≥ 0.10) by VTM, GAIN, or their interaction. In conclusion, increased concentrations of AA in maternal serum and ALF of beef heifers were observed at d 83 of gestation in response to VTM supplementation and rate of gain of 0.79 kg/d, which raises important questions regarding the mechanisms responsible for AA uptake and balance between the maternal circulation and fetal fluid compartments.

Characterization of the Microbiota Associated With 12-Week-Old Bovine Fetuses Exposed to Divergent in utero Nutrition.

A recent study reported the existence of a diverse microbiota in 5-to-7-month-old calf fetuses, suggesting that colonization of the bovine gut with so-called "pioneer" microbiota may begin during mid-gestation. In the present study, we investigated 1) the presence of microbiota in bovine fetuses at early gestation (12 weeks), and 2) whether the fetal microbiota is influenced by the maternal rate of gain or dietary supplementation with vitamins and minerals (VTM) during early gestation. Amniotic and allantoic fluids, and intestinal and placental (cotyledon) tissue samples obtained from fetuses (n = 33) on day 83 of gestation were processed for the assessment of fetal microbiota using 16S rRNA gene sequencing. The sequencing results revealed that a diverse and complex microbial community was present in each of these fetal compartments evaluated. Allantoic and amniotic fluids, and fetal intestinal and placenta microbiota each had distinctly different (0.047 ≥ R 2 ≥ 0.019, P ≤ 0.031) microbial community structures. Allantoic fluid had a greater (P < 0.05) microbial richness (number of OTUs) (Mean 122) compared to amniotic fluid (84), intestine (63), and placenta (66). Microbial diversity (Shannon index) was similar for the intestinal and placental samples, and both were less diverse compared with fetal fluid microbiota (P < 0.05). Thirty-nine different archaeal and bacterial phyla were detected across all fetal samples, with Proteobacteria (55%), Firmicutes (16.2%), Acidobacteriota (13.6%), and Bacteroidota (5%) predominating. Among the 20 most relatively abundant bacterial genera, Acidovorax, Acinetobacter, Brucella, Corynebacterium, Enterococcus, Exiguobacterium, and Stenotrophomonas differed by fetal sample type (P < 0.05). A total of 55 taxa were shared among the four different microbial communities. qPCR of bacteria in the intestine and placenta samples as well as scanning electron microscopy imaging of fetal fluids provided additional evidence for the presence of a microbiota in these samples. Minor effects of maternal rate of gain and VTM supplementation, and their interactions on microbial richness and composition were detected. Overall, the results of this study indicate that colonization with pioneer microbiota may occur during early gestation in bovine fetuses, and that the maternal nutritional regime during gestation may influence the early fetal microbiota.

Influence of dietary fructose supplementation on visceral organ mass, carbohydrase activity, and mRNA expression of genes involved in small intestinal carbohydrate assimilation in neonatal calves.

The hypothesis of this experiment was that dietary fructose would influence visceral organ mass, carbohydrase activity, and mRNA expression of carbohydrases and nutrient transporters in the small intestine in neonatal calves. Therefore, our objective was to use the neonatal calf as a model to evaluate the effects of postruminal fructose supply on small intestinal carbohydrate assimilation. Ten calves (<7 d of age; 41.2 ± 1.46 kg of body weight) were fed milk replacer at 2.0% of body weight daily (816 ± 90.5 g/d; 272 ± 30.1 g/L; dry-matter basis) in 2 equal portions and assigned to the following dietary treatment groups: (1) milk replacer (control; n = 6) or (2) milk replacer + 2.2 g of fructose/kg of body weight (fructose; n = 4). Calves were fed dietary treatments for 28 d, with jugular blood sampled every 7 d before and after the morning feeding. Calves were slaughtered, and visceral weights were recorded. Postruminal carbohydrase activities were assayed. Quantitative real-time PCR was conducted for small intestinal mRNA expression of nutrient transporters [solute carrier family 2 member 5 (GLUT5), solute carrier family 2 member 2 (GLUT2), and solute carrier family 5 member 1 (SGLT1)], carbohydrases (lactase, maltase-glucoamylase, and sucrase-isomaltase), and ketohexokinase (KHK). Data were analyzed using MIXED procedures in SAS version 9.4 (SAS Institute Inc, Cary, NC). Dietary fructose supplementation decreased serum glucose concentration. Small intestinal mass was greater in calves supplemented with fructose. Dietary fructose supplementation did not influence pancreatic α-amylase, small intestinal isomaltase, or maltase activities. Sucrase activity was undetected in the small intestine. Dietary fructose supplementation increased small intestinal glucoamylase activity per gram of tissue by 30% and increased maltase-glucoamylase mRNA expression by 6.8-fold. Dietary fructose supplementation did not influence mRNA expression of GLUT5, SGLT1, GLUT2, or KHK. Dietary fructose supplementation increased small intestinal lactase mRNA expression by 3.1-fold. Sucrase-isomaltase mRNA expression in the small intestine decreased 5.1-fold with dietary fructose supplementation. Dietary fructose supplementation does not induce sucrase activity in neonatal calves; however, sucrase-isomaltase may be transcriptionally regulated by dietary fructose in neonatal calves. More research is needed to compare glucose and fructose at isocaloric intakes to examine effects of dietary fructose at equal metabolizable energy intake.

Fetal expression of genes related to metabolic function is impacted by supplementation of ground beef and sucrose during gestation in a swine model.

To determine the effects of maternal supplementation on the mRNA abundance of genes associated with metabolic function in fetal muscle and liver, pregnant sows (Landrace × Yorkshire; initial body weight (BW) 221.58 ± 33.26 kg; n = 21) fed a complete gestation diet (corn-soybean meal based diet, CSM) were randomly assigned to 1 of 4 isocaloric supplementation treatments: control (CON, 378 g/d CSM, n = 5), sucrose (SUGAR, 255 g/d crystalized sugar, n = 5), cooked ground beef (BEEF, 330 g/d n = 6), or BEEF + SUGAR (B+S, 165 g/d cooked ground beef and 129 g/d crystalized sugar, n = 5), from days 40 to 110 of gestation. Sows were euthanized on day 111 of gestation. Two male and 2 female fetuses of median BW were selected from each litter, and samples of the longissimus dorsi muscle and liver were collected. Relative transcript level was quantified via qPCR with HPRT1 as the reference gene for both muscle and liver samples. The following genes were selected and analyzed in the muscle: IGF1R, IGF2, IGF2R, GYS-1, IRS-1, INSR, SREBP-1C, and LEPR; while the following were analyzed in the liver: IGF2, IGF2R, FBFase, G6PC, PC, PCK1, FGF21, and LIPC. No effect of fetal sex by maternal treatment interaction was observed in mRNA abundance of any of the genes evaluated (P > 0.11). In muscle, the maternal nutritional treatment influenced (P = 0.02) IGF2 mRNA abundance, with B+S and SUGAR fetuses having lower abundance than CON, which was not different from BEEF. Additionally, SREBP-1 mRNA abundance was greater (P < 0.01) for B+S compared with CON, BEEF, or SUGAR fetuses; and females tended (P = 0.06) to have an increased abundance of SREBP-1 than males. In fetal liver, IGF2R mRNA abundance was greater (P = 0.01) for CON and BEEF than SUGAR and B+S; while FBPase mRNA abundance was greater (P = 0.03) for B+S compared with the other groups. In addition, maternal nutritional tended (P = 0.06) to influence LIPC mRNA abundance, with increased abundance in CON compared with SUGAR and B+S. These data indicate limited changes in transcript abundance due to substitution of supplemental sugar by ground beef during mid to late gestation. However, the differential expression of FBPase and SREBP-1c in response to the simultaneous supplementation of sucrose and ground beef warrants further investigations, since these genes may play important roles in determining the offspring susceptibility to metabolic diseases.

The effects of nutrient restriction on mRNA expression of endogenous retroviruses, interferon-tau, and pregnancy-specific protein-B during the establishment of pregnancy in beef heifers.

We hypothesize that syncytin-Rum1, bovine endogenous retrovirus-K1 (BERV-K1), pregnancy-specific protein-B (PSP-B), and interferon-τ (IFN-τ) will be influenced by maternal nutrient restriction and be differentially expressed during key stages (day 16, 34, and 50) of the establishment of gestation when fed to meet industry standards. Commercial crossbred heifers (n = 49) were maintained on a total mixed ration and supplemented with dried distillers grains with solubles. All heifers were subjected to 5-d CO-Synch + CIDR estrus synchronization protocol. Non-pregnant, non-bred control (NP-NB) heifers (n = 6) were ovariohysterectomized on day 16, and the remaining heifers were AI to a single Angus sire (day of breeding = day 0). On the day of breeding, heifers were randomly assigned to dietary treatments. One half were assigned to control treatment (CON) targeted to gain 0.45 kg/d, and the remaining half were assigned to restricted treatment (RES), which received 60% of control diets. Heifers were subjected to ovariohysterectomy on day 16, 34, or 50 of gestation. Utero-placental tissues were obtained from the uterine horn ipsilateral (P) and contralateral (NP) to the corpus luteum and separated into maternal caruncle (CAR), maternal endometrium, inter-caruncle, (ICAR), and fetal membrane (FM). There were no interactions between stage of gestation and nutritional treatment for syncytin-Rum1 or PSP-B (P > 0.22). Expression of BERV-K1 was influenced by a treatment × stage of gestation interaction (P = 0.03) in NP-CAR. On day 50, heifers fed the CON diet had greater BERV-K1 expression compared with CON heifers on day 16 and 34 and RES heifers at all sampling time points. There was a treatment × stage of gestation interaction (P < 0.01) for IFN-τ in FM tissue. On 16 d, mRNA expression of IFN-τ was greater (P < 0.01) compared with day 34 and 50 for both CON and RES heifers, but RES FM had greater (P < 0.01) IFN-τ expression compared with CON FM. In P-CAR, PSP-B expression increased (P < 0.01) by 18 000-fold on day 50 compared with NP-NB heifers. In P-ICAR, expression of syncytin-Rum1 in P-ICAR was greater (P = 0.01) on day 16 with a 14.14-fold increase compared with relative expression on day 34 and 50; whereas, PSP-B was increased (P < 0.01) on day 34 and 50 compared with day 16. In conclusion, 40% nutrient restriction had limited influence on mRNA of ERVs, PSP-B, and IFN-τ but stage of gestation differences reinforced the importance of these genes during the establishment of pregnancy.