The World Health Organization estimates that 15 million babies are born preterm each year, with one newborn dying every 40 seconds. Understanding the mechanisms that initiate normal parturition is critical to identifying disruptions associated with preterm birth. In sheep, activation of the fetal hypothalamic-pituitary-adrenal axis, resulting in cortisol secretion, is believed to be the initial trigger for labor. We previously established a sheep model in which estradiol (E2) administration reliably induces parturition, enabling controlled investigation of labor mechanisms. In this study, we used this model to investigate the transcriptomic effects of E2 on fetal organ maturation. Pregnant ewes at late gestation (days 139–142) were randomly assigned to an E2-treated (n = 6; 4 Silastic® implants × 50 mg each) or a control (n = 6; 4 empty implants) group. Implants were inserted subcutaneously in the axillary region, and ewes were euthanized 26 h post-treatment for tissue and blood collection. Samples of the fetal hypothalamus, pituitary, adrenal gland, and lungs were collected for RNA sequencing. Estradiol concentration was assessed in maternal systemic blood, umbilical vein, and fetal vein samples. Maternal E2 levels increased five-fold in treated ewes (p ≤ 0.01), while umbilical and fetal E2 levels were not affected relative to controls. Differentially expressed genes (DEGs) (p ≤ 0.05; |log2FC| ≥ 1) were identified in all fetal tissues: 4 (hypothalamus), 240 (pituitary), 5 (adrenal), and 2 (lungs). These findings suggest that maternal E2 indirectly influences fetal organ gene expression, particularly in the pituitary, potentially contributing to fetal organ maturation and preparedness for birth. (Supported by USDA 2021-67015-34277)

Abstract Despite growing evidence linking maternal nutrition to placental development, the specific effects of mid-gestation supplementation remain unclear, even though this period marks the peak of placental growth and vascular expansion essential for fetal development. The current study evaluated the effects of mid-gestation protein and energy supplementation on placental development in beef heifers. We hypothesized that maternal supplementation would enhance placental growth and vascularization, potentially supporting fetal development and long-term offspring productivity. Angus based heifers (n = 44; initial body weight (BW) 339.7 ± 33.0 kg.), were bred via artificial insemination using male-sexed semen from a single sire, and at d 90 of gestation were randomly assigned to receive either: 1) a forage-based control diet fed for target gains of 0.28 kg/d (CON; n = 22); or 2) the forage-based diet with the addition of a protein and energy supplement to target 0.79 kg/d (SUPP; n = 22) through d 186 of gestation. All heifers were then managed as a single group after d 186 through calving. Immediately upon expulsion of the placenta, samples of cotyledon (CON = 18; SUPP = 18) were collected and preserved in neutral buffered formalin. Tissues were subsequently stained using immunohistochemistry for key indicators of vascularization (using CD 31/34) and cell proliferation (using Ki-67), with data used to calculate capillary area density (CAD), capillary number density (CND), Ki-67 positivity ratio (PR), and spatial cell density (SCD). All statistical analyses were performed using the General Linear Model (GLM) procedure of SAS 9.4 with individual animal as the experimental unit. Data are reported as least squares mean ± standard error of the mean. Significance was considered at P ≤ 0.05, and tendencies were recognized at 0.05 < P ≤ 0.10. Results showed that CAD tended to be greater (P = 0.07) in SUPP compared with CON, indicating enhanced placental vascular development and potential for improved nutrient transfer to the fetus. Conversely, CND was reduced (P = 0.02) in the SUPP, suggesting a more efficient network of fewer, but larger and more functional, capillaries. No significant differences were present between treatments for PR or SCD (P ≥ 0.51), indicating that overall cellular proliferation and tissue cellularity were not affected by maternal supplementation. These findings suggest that mid-gestation protein and energy supplementation can enhance placental vascularity, which is critical for fetal development, particularly by promoting vascular efficiency rather than increasing cellular proliferation. The development of a more advanced and efficient vascular network may improve nutrient and oxygen delivery to the fetus, potentially supporting greater calf growth and birth weights during the neonatal period.

Precise coordination of parturition and fetal organ maturation is critical for neonatal survival. In sheep, a fetal cortisol surge, triggered by hypothalamic-pituitary-adrenal (HPA) axis activation, drives organ maturation and placental estradiol (E2) synthesis to promote labor, but the upstream signals initiating this cascade remain unclear. We tested whether maternal E2 administration at physiological levels alters fetal endocrine and pulmonary maturation through transcriptional regulation. Pregnant ewes (139-142 days gestation) received Silastic implants containing E2 (200 mg; n = 6) or empty controls (n = 6). Fetal hypothalamus, pituitary, adrenal, and lung tissues were collected 26 h post treatment for RNA sequencing; lung samples also underwent immunohistochemistry, and maternal, fetal, and umbilical plasma were analyzed for hormones. Maternal E2 increased approximately fourfold (P < 0.01), whereas fetal and umbilical E2 remained unchanged. Transcriptomic responses were strongest in the pituitary (914 differentially expressed genes, DEGs), followed by lung (150), adrenal (16), and hypothalamus (6) (FDR ≤ 0.1, |log2FC| ≥ 1). Pituitary responses included pro-opiomelanocortin (POMC) downregulation and enrichment of neurogenesis, axon guidance, and cell adhesion pathways. The hypothalamus showed enrichment for hormone secretion, neuroendocrine regulation, and estrogen signaling. In the lungs, ERα-positive nuclei increased (P = 0.04), whereas surfactant proteins A and C were unchanged. These findings demonstrate that maternal E2, without altering fetal circulating E2, induces tissue-specific fetal transcriptomic changes consistent with endocrine and pulmonary maturation. Maternal E2 may therefore contribute to fetal readiness for birth through indirect mechanisms acting independently of, or in concert with, fetal cortisol.NEW & NOTEWORTHY Successful birth requires precise coordination between fetal organ maturation and the hormonal signals that trigger labor. Using a sheep model, we show that maternal estradiol (E2) administered at physiological levels induces broad transcriptomic changes in fetal endocrine and pulmonary tissues without altering fetal circulating E2 levels. These findings highlight a previously unrecognized role for maternal estrogen in modulating fetal development, suggesting alternative or complementary pathways to cortisol in preparing the fetus for birth.

Introduction Maternal nutrition during early gestation is associated with metabolic and immunological adaptations that may influence postnatal growth, with possible long-term and multigenerational effects.Methods This study evaluated how the rate of maternal body weight gain in F0 dams during early gestation affects gene expression in the skeletal muscle of F1 offspring and their F2 fetuses. A subset of 16 crossbred Angus heifers (F0) were assigned to a low gain (LG; 0.28 kg/day) or moderate gain (MG; 0.79 kg/day) treatment during the first 84 days of gestation.Results and discussion F1 heifers from the LG group showed upregulation of PCK2, ARG2, PHGDH, PSAT1, PSPH, and ASNS, associated with the biological processes cytochrome complex assembly, mitochondrion organization, mitochondrial gene expression, and mitochondrial transport, relative to the MG group, suggesting greater metabolic plasticity and possible compensatory adjustments related to satellite cell function and postnatal muscle hypertrophy. Conversely, the upregulation of TNFAIP3, ITGB2, EOGT, BOLA-NC1, and C4A in F1 heifers from the MG group, associated with the pathways cell adhesion molecules (CAMs), antigen processing and presentation, and cytokine-cytokine receptor interaction, may be related to the activation of local immunomodulatory mechanisms and to the preservation of skeletal muscle structural integrity. At the multigenerational level, the modulation of ITGB2, BOLA-NC1, C4A, and TNFAIP3 in F1 and F2 progeny from the MG group, associated with enrichment of the CAMs pathway and consistent negative regulation of thermogenesis, oxidative phosphorylation, and ribosome pathways relative to the LG group, together with the positive regulation of bta-miR-133a-1 in the LG progeny, points to the involvement of epigenetic mechanisms in the coordination of persistent metabolicand myogenic adjustments across generations. Moreover, the maternal rate of body weight gain during early gestation is associated with metabolic changes that support postnatal hypertrophic growth and with local immunological modulation in skeletal muscle, with multigenerational molecular outcomes.

Uterine glands provide critical nutrients during pregnancy, but uterine gland number is fixed shortly after birth. Thus, we sought to determine if maternal immune stimulation decreases uterine gland density in offspring. Pregnant sows were treated on day 77 of gestation with lipopolysaccharide (LPS), vaccine (VAC), or saline (CON). Postnatal gilts were sampled from each group. Reproductive tracts were collected from gilts pre-puberty (LPS, n = 4; VAC, n = 6; CON, n = 6) and post-puberty (LPS, n = 3; VAC, n = 5; CON, n = 7). Uteri were weighed. Cross sections were collected from four non-sequential mid-horn segments, fixed, embedded, and sectioned. Sections were stained using hematoxylin and eosin, imaged, and assessed for the number of deep and superficial uterine glands per area (density). Data were analyzed by ANOVA for the fixed effect of treatment. Statistical significance was considered at P ≤ 0.05. In the pre-pubertal phase, there were no differences in deep or superficial uterine gland density among treatment groups. However, uterine weight tended to be reduced in gilts from the VAC group compared with the CON and LPS groups. In the post-pubertal phase, there were no differences in deep or superficial uterine gland density among treatment groups. However, uterine weight was increased in the VAC and LPS groups compared with the CON group. Gestational immune stimulation does not affect uterine gland density in offspring; however, it does alter uterine weight, and thus affects the total number of uterine glands, and perhaps secretory capacity.

Infertility is a major barrier to reproductive efficiency in livestock. Early pregnancy is a critical window for placental vascular development, which is essential for embryonic survival and fetal growth. This study evaluated the individual and combined effects of embryo and ewe breed on pregnancy establishment and placental vascularization in sheep. A total of 85 embryo transfers (ET) were performed using straight-bred and reciprocal combinations of Rambouillet (Rambo) and Romanov (Romo) ewes. Pregnancy was assessed on day 24 of gestation. Straight-bred groups showed the greatest pregnancy rates: Rambo × Rambo (31.3%) and Romo × Romo (26.7%). The reciprocal Rambo × Romo group had the lowest pregnancy rate (11.8%), while Romo × Rambo was intermediate (22.7%), indicating a tendency for breed-dependent effects (P=0.10). To assess vascular development, uterine cross-sections were collected at slaughter on day 25 and stained with CD31 and CD34 to quantify capillary area density (CAD) and capillary number density (CND) in the caruncle (CAR), fetal membrane (FM), and inter-caruncular (ICAR) regions. compared to the other groups, Rambo × Romo had increased CAD in CAR and ICAR regions (P<0.01), while Romo × Romo had reduced CND in CAR (P<0.05), indicating differences in vascular development due to embryo and ewe breed. These vascular differences may underlie reduced pregnancy success after ET. This study highlights a link between fertility and early placental vascularization, offering insight into mechanisms impacting pregnancy maintenance. Understanding these effects could lead to breeding strategies to improve reproductive outcomes in sheep.

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

The objective of this study was to determine if moderate changes in rate of body weight (BW) gain and dietary one-carbon metabolite (OCM) supplementation in pregnant heifers alters maternal and fetal morphometrics. In three experiments, Angus-cross heifers (n = 81) were stratified by BW into a 2 × 2 factorial arrangement of treatments: fed to gain 0.45 kg/d (CON) or lose 0.23 kg/d (RES) BW and to receive corn carrier with OCM supplement (+OCM) or without (-OCM). The OCM supplement consisted of vitamin B12 (20 mg/wk) and folate (320 mg/wk) injections and dietary rumen-protected methionine (7.4 g/d) and choline (44.4 g/d) from breeding (d 0) to d 63 of gestation. Heifers underwent estrus synchronization and were bred to a single sire via artificial insemination using female-sexed semen. In experiment 1, heifers (n = 31) were slaughtered on d 63 of gestation (early gestation). In experiment 2, heifers (n = 29) were slaughtered on d 160 of gestation (mid-gestation) and in experiment 3, heifers (n = 30) were slaughtered on d 250 of gestation (late gestation). Data were analyzed using the MIXED procedure of SAS to determine the effect of heifer gain, supplement, and their interaction. Statistical significance was considered at P ≤ 0.05. In all experiments, final BW, gain, and average daily gain were reduced (P < 0.01) in RES compared with CON heifers by experimental design. In experiment 1, maternal pancreas weight was reduced (P = 0.04) in CON-OCM, RES-OCM, and RES+OCM compared with CON+OCM. Fetal left cerebrum weight was reduced (P < 0.01) in CON+OCM and RES-OCM compared with RES+OCM, while CON-OCM was intermediate. Fetal right longissimus dorsi by brain weight was reduced (P = 0.03) in RES+OCM compared with CON+OCM, while CON-OCM and RES-OCM were intermediate. In experiment 2, there were no gain × supplement effects on maternal morphometrics. However, fetal mammary and fetal mammary by brain weight were reduced (P = 0.02) in CON+OCM compared with CON-OCM, while RES+OCM and RES-OCM were intermediate. In experiment 3, maternal kidney weight was reduced (P = 0.03) in CON+OCM and RES-OCM compared with CON-OCM, while RES+OCM was intermediate. Furthermore, empty uterine weight was reduced (P = 0.05) in CON+OCM and RES+OCM compared with CON-OCM, while RES-OCM was intermediate. There were no gain × supplement effects in fetal morphometrics. However, fetal mammary tissue adjusted by brain weight tended to be reduced (P = 0.08) in CON-OCM compared with CON+OCM, while RES+OCM and RES-OCM were intermediate. These data indicate our dietary restriction model was successful in causing moderate BW loss in pregnant heifers during early gestation. Furthermore, moderate BW loss in early gestation reduces maternal and fetal organ weights throughout gestation, and OCM supplementation did not substantially mitigate these effects.

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.

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 aim of our study was to examine how maternal nutrition during early pregnancy affects the glucose transporter proteins (GLUT1 and GLUT3) in the fetal intestine and to determine whether supplementing with one-carbon metabolites (OCM) could alleviate any changes caused by altered maternal nutrition. We employed a 2 × 2 factorial design with two nutritional planes: control (CON; 0.45 kg/d) and restricted (RES; -0.23 kg/d), along with or without OCM supplementation (+/- OCM). Crossbred Angus heifers were synchronized for estrus and bred via artificial insemination using single-sire female-sexed semen. They were assigned to one of four nutritional treatment groups (n = 8 ± 1 per group) at breeding. Targeted weight gain and OCM supplementation were administered from day 0 to day 63 of gestation, after which all heifers were fed a common diet aimed at a 0.45 kg/d gain. OCM-supplemented treatments received daily rumen-protected methionine (7.4 g/d), choline (44.4 g/d) in a corn carrier, and weekly injections of folate (320 mg) and vitamin B12 (20 mg). Non-supplemented groups received only the corn carrier and saline injections. On day 161 of gestation, heifers were harvested, and fetal jejunum samples were collected for analysis. GLUT1 and GLUT3 expressions were assessed via anti-GLUT1 and anti-GLUT3 fluorescent staining, and the images were analyzed with ImagePro-Premiere software. Maternal OCM supplementation reduced (P = 0.003) the abundance of GLUT1 in fetal villi. Maternal nutritional plane and OCM tended (P = 0.09) to interact in the crypt, with the CON+OCM group showing lower staining intensity than the CON-OCM (P = 0.0009), RES-OCM (P = 0.0003), and RES+OCM (P = 0.0072) groups for GLUT1. Total (villi and crypt combined) GLUT1 staining intensity was lower (P = 0.003) in the +OCM groups compared to -OCM groups. Fetuses from restricted dams tended (P = 0.08) to have greater GLUT3 abundance, with RES groups showing 19% greater abundance than CON groups. In crypts, the staining intensity of GLUT3 was significantly reduced by OCM (P < 0.0001), with +OCM groups showing 39% lower staining intensity compared to -OCM groups. No interaction (P = 0.43) was found between nutrition and OCM for total GLUT3. These findings demonstrate that OCM impacts the abundance of glucose transporters in the fetal intestine. The reduction in GLUT1 in +OCM groups may be due to the role of OCM in enhancing metabolic efficiency in future calves. Conversely, the upregulation of GLUT3 abundance in fetuses from restricted dams could reflect a compensatory response to ensure the readiness for sufficient glucose uptake at birth. Additional research is required to understand the long-term impact of maternal OCM on fetal development. USDA is an equal opportunity provider and employer.

Maternal periconceptual nutrition affects the growth trajectory of developing fetuses by modulating gene expression. The regulatory mechanisms and their role in fetal development remain underexplored in livestock models. Herein, we investigated the effects of maternal rate of body weight (BW) gain during early gestation on the DNA methylation, microRNA profiles, and their interaction with the hepatic gene expression in female fetuses. At breeding, 36 crossbred beef heifers (∼13 months of age) were randomly assigned to a nutritional plane to gain Low (0.28 kg/day; LG, n = 18) or Moderate (0.79 kg/day; MG, n = 18) BW through the first 83 days of gestation. A subset of pregnant heifers (n = 17) was selected, and fetal liver samples were collected on day 83 of gestation for DNA methylation and miRNA-Sequencing. After data quality control, miRDeep2 and Bismark tools were used to analyze miRNA and methylation data, respectively. The bta-miR-206 was the only differentially expressed miRNA (FDR = 0.02). Eight differentially methylated genes were identified (DMGs, FDR < 0.1). The over-represented pathways and biological processes (adj. p < 0.05) for bta-miR-206 targeted genes were associated with embryonic development, energy metabolism, and mineral transport, whereas the DMGs regulated anatomical structural development and transcriptional regulation. Our results show that key genes involved with liver metabolism, tissue structure, and function were regulated by DNA methylation and the miR-206. However, further investigation is warranted to determine physiological responses and long-term consequences on animal performance.

The objective of this study was to investigate the effects of nutritional planes and one-carbon metabolite (OCM) supplementation during early pregnancy on the abundance of phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) in the maternal liver at day 260 of gestation. A 2 x 2 factorial design was used with two nutritional planes; control (CON; 0.45 kg/d) and restricted (RES; -0.23 kg/d), with or without OCM supplementation (+/- OCM). Crossbred Angus beef heifers were synchronized for estrus and bred via artificial insemination using single-sire female-sexed semen. At breeding, heifers were randomly assigned to one of four dietary treatments (n = 8 ± 1 per group). Nutritional treatments were administered from day 0 to day 63 of gestation, after which all heifers were provided a common diet (0.45 kg/d gain). OCM-supplemented groups received daily rumen-protected methionine (7.4 g/d), choline (44.4 g/d) in a corn carrier, and weekly injections of folate (320 mg) and vitamin B12 (20 mg), while non-supplemented groups received only the corn carrier and saline injections. Maternal liver samples were collected at day 260 of gestation and analyzed via anti-phosphorylated p38 MAPK immunofluorescence, with image analysis performed using ImagePro-Premiere software. There was no nutritional plane x OCM interaction (P = 0.28), and p-p38 MAPK abundance did not differ (P = 0.13) between nutritional planes. However, heifers receiving OCM supplementation had a 54.75% higher (P = 0.02) p-p38 MAPK density per cell compared to non-supplemented heifers. These findings suggest that OCM may play a critical role in modulating cellular signaling pathways, particularly p38 MAPK, which is involved in stress responses, inflammation, metabolic regulation, cellular proliferation and differentiation. The increased activation of p38 MAPK in OCM-supplemented heifers may reflect improved hepatic function or adaptive responses to the physiological demands of pregnancy, potentially supporting maternal metabolic adaptation and fetal development. Further research is warranted to explore the downstream effects of enhanced p38 MAPK signaling and its broader impact on pregnancy outcomes. USDA is an equal opportunity provider and employer.

The initiation of parturition in sheep is influenced by multiple factors, with estrogens recognized as key contributors. However, the specific effects of estrogens in regulating the timing of delivery, fetal organ maturation, and neonatal adaptation to extrauterine life remain unclear. This study aimed to clarify the role of estradiol in the timing of parturition and neonatal outcomes in periparturient Rambouillet ewes by evaluating the relationship between maternal systemic estradiol and progesterone levels, the timing of delivery, and lamb birth weight, vigor, cortisol levels, mortality, and postnatal weight gain until weaning. Pregnant Rambouillet ewes were randomly assigned to 1 of the 2 groups between gestational days 139 and 142: the E group (n = 13), which received 6 Silastic implants containing 50 mg of estradiol each (300 mg total per ewe), or the C group (n = 12), which received 6 empty Silastic implants. The implants were inserted subcutaneously in the axillary space and removed 2 d postpartum. Ewes were allowed to deliver naturally. Maternal blood samples were collected from the jugular vein at 5 timepoints: - 1 d (pretreatment), + 8 h and + 24 h (post-treatment), day of birth (Birth), and + 2 d postpartum (PP), for analysis of E2 and P4 concentrations. Lamb blood samples were collected from the jugular vein on the day of birth and at + 2 d (PP) to determine cortisol concentrations, and lamb weight gain was monitored biweekly until weaning. The results demonstrated that estradiol treatment shortened the time to parturition (P < 0.008) without altering maternal progesterone concentrations. Importantly, estradiol treatment had no significant effects on lamb birth weight, vigor, cortisol concentrations, or weight gain until weaning. This research demonstrates that E2 induces parturition in sheep, without a withdrawal in systemic P4, supporting the possibility of functional progesterone withdrawal, and highlights the potential use of E2 silastic implants for reproductive management, including the synchronization of parturition in livestock.

Maternal nutrition during pregnancy influences fetal development; however, the regulatory markers of fetal programming across different gestational phases remain underexplored in livestock models. Herein, we investigated the regulatory role of long non-coding RNAs (lncRNAs) on fetal liver gene expression, the impacts of maternal vitamin and mineral supplementation, and the rate of maternal body weight gain during the periconceptual period. To this end, crossbred Angus heifers (n=31) were randomly assigned to a 2×2 factorial design to evaluate the main effects of the rate of weight gain (low gain [LG, avg. daily gain of 0.28 kg/day] vs. moderate gain [MG, avg. daily gain of 0.79 kg/day]) and vitamins and minerals supplementation (VTM vs. NoVTM). On day 83±0.27 of gestation, fetuses were collected for morphometric measurements, and fetal liver was collected for transcriptomic and mineral analyses. The maternal diet significantly affected fetal liver development and mineral reserves. Using an RNA-Seq approach, we identified 320 unique differentially expressed genes (DEGs) across all six comparisons (FDR <0.05). Furthermore, lncRNAs were predicted through the FEELnc pipeline, revealing 99 unique differentially expressed lncRNAs (DELs). The over-represented pathways and biological processes (BPs) were associated with energy metabolism, Wnt signaling, CoA carboxylase activity, and fatty acid metabolism. The DEL-regulated BPs were associated with metal ion transport, pyrimidine metabolism, and classical energy metabolism-related glycolytic, gluconeogenic, and TCA cycle pathways. Our findings suggest that lncRNAs regulate mineral homeostasis- and energy metabolism-related gene networks in the fetal liver in response to early maternal nutrition.

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.