Jessica G. Syring

Background: Mitochondria are essential for fetal development, regulating energy metabolism and metabolic programming. This study examined how maternal nutrition and one-carbon metabolite (OCM) supplementation during early gestation affect mitochondrial function in fetal liver and muscle at day 161 of gestation in beef heifers. Methods: Twenty-nine crossbred Angus heifers were assigned to one of four treatments in a 2 × 2 factorial design: control (CON; 0.45 kg/day ADG) or restricted gain (RES; −0.23 kg/day), with or without OCM supplementation. Treatments were applied from breeding to day 63 of gestation, after which all heifers received a common diet. Fetal liver and muscle tissues were collected at day 161. Mitochondrial respiration (Seahorse assay), mtDNA copy number (qPCR), and mitochondria-related gene expression (RNA-seq) were assessed. Results: In fetal liver, state 3 respiration was highest in CON + OCM, while state 4o respiration was lowest in RES + OCM (p ≤ 0.05). mtDNA copy number was greater in RES and +OCM groups. In fetal muscle, mtDNA copy number was influenced by gain, but respiration was unaffected. Transcriptomic analysis revealed more mitochondria-related differentially expressed genes (mtDEGs) in fetal muscle than liver (90% versus 10% of total mtDEG), with most genes downregulated in the RES and +OCM groups compared to the CON and −OCM groups (FDR ≤ 0.10). Conclusions: OCM supplementation enhanced mitochondrial respiration and biogenesis in fetal liver, likely via post-translational mechanisms. In contrast, fetal muscle showed downregulation of mitochondria-related genes without functional changes, indicating transcriptional reprogramming with potential effects on later metabolic function. These results underscore early gestation as a critical window for OCM-based nutritional interventions to improve metabolic outcomes in livestock.

The small intestine, particularly the jejunum, is a key site for nutrient absorption and metabolic activity in mature and growing ruminants

Background: Maternal nutrition during early gestation induces metabolic adaptations that support maternal health and fetal development. This study evaluated the effects of maternal one-carbon metabolite (OCM: methionine, choline, folate, and vitamin B₁₂) supplementation and restricted rates of maternal gain on the hepatic lipid profiles of dams and fetuses at day 63 of gestation. Methods: Thirty-one crossbred Angus heifers were inseminated and assigned to a 2 × 2 factorial design with two factors: maternal dietary intake (control [CON]; 0.60 kg/day average daily gain [ADG] vs. restricted [RES]; −0.23 kg/day ADG) and OCM supplementation (supplemented [+OCM] vs. not supplemented [−OCM]). The four resulting groups (CON − OCM, CON + OCM, RES − OCM, RES + OCM) were maintained for 63 days post-breeding. Maternal and fetal liver samples were collected, and lipidomic profiling was performed using ultra-performance liquid chromatography–tandem mass-spectrometry. Results: In maternal liver, 485 lipid metabolites were detected, with 243 differing significantly in maternal gain. RES heifers showed increased levels (p ≤ 0.05) of acylcarnitines, plasmalogens, lysoplasmalogens, glycosphingolipids, and sphingomyelins. Additionally, RES combined with OCM supplementation led to the accumulation of secondary bile acids and a depletion of monoacylglycerols (p ≤ 0.05) in maternal liver. In fetal liver, 487 lipid metabolites were detected, but treatment effects were minimal. Conclusions: Maternal rate of gain significantly influenced hepatic lipid metabolism in the maternal liver, while fetal liver lipid profiles remained relatively unaffected. These findings underscore the significant role of dietary intake/rate of gain compared with OCM supplementation in modulating hepatic lipid metabolism and highlight the maternal liver’s metabolic adaptations during early pregnancy.

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.

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.