Untangling the placentome gene network of beef heifers in early gestation.

The cotyledon and caruncle tissues provide a functional bridge between the fetus and the dam. However, the relationship between these tissues and the transcriptomic profile that underlies the tissue functions remains elusive. Herein we investigate the expression profile of cotyledon and caruncle from nulliparous beef heifers carrying female fetuses at day 83 of pregnancy to identify changes occurring across tissues that contribute to placental function and their tissue-specific roles. We identified 2654 differentially expressed genes [padj ≤ 0.05, abs(log2FC) ≥ 1], including nutrient transporters and paternally imprinted genes. We found key regulators of tissue function and differentiation, including FOXO4, GATA2, GATA3, and HAND1, rewired between the tissues. Finally, we shed light on the over-represented pathways related to immune tolerance, tissue differentiation and remodeling. Our findings highlighted the intricate and coordinated cross-talk between fetal-maternal tissues. They provided evidence of a fine-tuned gene regulatory network underlying pregnancy and tissue-specific function in the bovine placenta.

Angiopoietin expression in ovine corpora lutea during the luteal phase: Effects of nutrition, arginine and follicle stimulating hormone.

The aim of this study was to evaluate angiopoietin (ANGPT) 1 and 2, and tyrosine-protein kinase receptor 2 (TIE2) expression in the corpora lutea (CL) of FSH-treated, or non-treated sheep administered arginine (Arg) or vehicle (saline, Sal), and fed a control (C), excess (O) or restricted (U) diet. Ewes from each dietary group were treated with Arg or Sal (experiment 1), and with FSH (experiment 2). Luteal tissues were collected at the early-, mid- and/or late-luteal phases of the estrous cycle. Protein and mRNA expression was determined using immunohistochemistry followed by image analysis, and quantitative RT-PCR, respectively. The results demonstrated that ANGPT1 and TIE2 proteins were localized to luteal capillaries and endothelial cells of larger blood vessels, and ANGPT2 was localized to tunica media of larger blood vessels. TIE2 protein was also present in luteal cells. In experiment 1, ANGPT1 protein expression was greater in O than C during early- and mid-luteal phases, and was greatest during late-luteal phase, less at the mid- and least at the early-luteal phase; 2) TIE2 protein expression was greatest at the mid-, less at the early- and least at the late-luteal phase; 3) ANGPT1 and 2 mRNA expression was greater at the mid- and late- than the early-luteal phase, and TIE2 mRNA expression was greatest at the late-, less at the mid- and least at the early-luteal phase. The ANGPT1/2 ratio was less at the early- than mid- or late-luteal phases. In experiment 2, ANGPT1 protein expression was greater in O during the mid-luteal phase than in other groups, and was greater at the mid- than early-luteal phase. TIE2 protein expression was highest at the mid-, less at the early- and least during the late-luteal phase. ANGPT1 and 2, and TIE2 mRNA expression was higher at the mid- than the early-luteal phase. During mid-luteal phase, ANGPT1 mRNA expression was greater in C than O and U, ANGPT2 was greatest in C, less in O and least in U, and TIE2 mRNA expression was greater in C than O and U. The ANGPT1/2 ratio was higher in U than in any other group. Comparison of FSH vs. Sal treatment effects (experiment 2 vs. experiment 1) demonstrated that FSH affected ANGPT1 and/or -2, and TIE2 protein and mRNA expression depending on luteal phase and/or diet. Thus, expression of ANGPTs and TIE2 in the CL changes during the luteal lifespan, indicating their involvement in luteal vascular formation, stabilization and degradation. Moreover, this study has demonstrated that plane of nutrition and/or FSH treatment affect the ANGPT system, and may alter luteal vascularity and luteal function in sheep.

Divergent planes of nutrition in mature rams influences body composition, hormone and metabolite concentrations, and offspring birth measurements, but not semen characteristics or offspring growth.

Objectives of this experiment were to characterize the effects of ram plane of nutrition on body composition, concentrations of hormones and metabolites, sperm characteristics, and offspring outcomes. Mature Rambouillet rams (n = 24, BW = 82.9 ±â€…2.63 kg) were individually housed and randomly assigned to either a positive (POS; n = 8), maintenance (MAINT; n = 8), or negative (NEG; n = 8) plane of nutrition for an 84-day feeding period. Rams were fed a common diet, with daily feed allocations adjusted weekly based on body weight (BW) to achieve the targeted weight gain or loss (approximately 12% of initial BW). On 0, 28, 56, and 84-d, body condition score (BCS) and scrotal circumference (SC) were recorded, and blood and semen were collected. Following the feeding period, rams were placed in pens with 10 ewes each for a 28-d breeding period. Ewes were managed similarly throughout gestation and body weight and measurements were recorded at birth and weaning. Data were analyzed as repeated measures in time where appropriate with the mixed procedure of SAS, and individual ram was the experimental unit for all analysis. Ram BW was influenced by a treatment × day interaction (P < 0.001), with POS (0.12 ±â€…0.01 kg) having greater daily weight change than MAINT (0.1 ±â€…0.01 kg), which was greater than NEG (-0.12 ±â€…0.01 kg). Ram BCS and SC were influenced by treatment × day interactions (P ≤ 0.01), being similar on day 0 but POS being greater than NEG by day 56. Concentrations of triiodothyronine (T3) and T3:T4 ratio exhibited treatment × day interactions (P ≤ 0.02), as POS had greater values than NEG by day 84 (P ≤ 0.02). Concentration of insulin-like growth factor-1 was greater in POS than MAINT and NEG (P ≤ 0.02), and non-esterified fatty acids and thyroxine (T4) were influenced by a day effect (P ≤ 0.01), but testosterone was unaffected (P ≥ 0.09). Minimal differences in semen volume, sperm concentration, motility, or morphology were observed among treatments (P ≥ 0.31). A similar proportion of ewes bred by rams in the respective treatments lambed and weaned lambs (P ≥ 0.54). Birth weight, chest circumference, and shoulder-hip length were greater (P ≤ 0.05) in NEG lambs compared with POS and MAINT; however, no differences were detected in weaning weight and weaning body measurements (P ≥ 0.40). Findings suggest paternal nutrition during the period of sperm development may influence offspring outcomes, potentially as a result of in-utero programming of paternal origin.

This study was conducted to evaluate whether ram nutrition during the spermatogenesis impacts their body composition, concentrations of circulating hormones and metabolites, semen characteristics, fertility, and subsequent offspring growth and development. Rams were managed on treatments to gain, lose, or maintain body weight over an 84-day period. The changes in ram body weight that were imposed by our treatments resulted in changes in body condition score, scrotal circumference, and concentrations of several metabolic hormones, including thyroid hormones and insulin-like growth factor-1. However, no differences in sperm concentration or motility were observed. After the 84-d feeding period, rams were placed with ewes for a 28-d breeding period and ewes were monitored throughout gestation, lambing, and until weaning of the resulting lambs. Although no differences in ewe pregnancy rates were observed after the breeding period, lamb birth weight and body measurements were greater in rams that lost weight during spermatogenesis. Thereafter, body weight and growth performance of offspring were similar among sire treatments, but continued evaluation of offspring throughout the postnatal period is necessary. These findings indicate that paternal nutrition during spermatogenesis can impact offspring outcomes, potentially through epigenetic alterations to the sperm and subsequent in-utero programming of paternal origin.

Supplementing vitamins and minerals to beef heifers during gestation: impacts on mineral status in the dam and offspring, and growth and physiological responses of female offspring from birth to puberty.

We evaluated the effects of feeding a vitamin and mineral supplement to nulliparous beef heifers throughout gestation on the mineral status of the dam, calf, placenta, and colostrum; offspring growth performance; and physiological responses of offspring raised as replacement heifers. Angus-based heifers (n = 31, initial body weight [BW] = 412.5 ±â€…53.68 kg) were adapted to an individual feeding system for 14 d, estrus synchronized and bred with female-sexed semen. Heifers were ranked by BW and randomly assigned to receive either a basal diet (CON; n = 14) or the basal diet plus 113 g heifer-1 d-1 of the vitamin and mineral supplement (VTM; n = 17). Targeted BW gains for both treatments was 0.45 kg heifer-1 d-1. Liver biopsies were obtained from dams at breeding, days 84 and 180 of gestation. At calving, liver biopsies were taken from dams and calves; colostrum, placenta, and blood samples were collected; and calf body measurements were recorded. After calving, all cow-calf pairs received a common diet through weaning, and F1 heifer calves were managed similarly after weaning. Offspring growth performance, feeding behavior, blood metabolites, and hormones were evaluated from birth through 15 mo of age. Data were analyzed using the MIXED procedure in SAS with repeated measures where appropriate. Hepatic concentrations of Se decreased in VTM dams (P ≤ 0.05) from day 84 to calving, while concentrations of Cu decreased in VTM and CON (P ≤ 0.05) from day 84 to calving. Calf liver concentrations of Se, Cu, Zn, and Co at birth were greater for VTM than CON (P ≤ 0.05), but calf birth BW and body measurements were not different (P = 0.45). Placental Se, colostrum quantity, total Se, Cu, Zn, and Mn in colostrum were greater (P ≤ 0.04) in VTM dams than CON. Finally, offspring from VTM dams were heavier than CON (P < 0.0001) from weaning through 15 mo of age. These results were coupled with greater (P ≤ 0.04) blood glucose at birth, decreased (P ≤ 0.05) blood urea nitrogen at pasture turn out and weaning, and altered feeding behaviors in VTM offspring compared with CON. Maternal gestational vitamin and mineral supplementation enhanced mineral status in dams and F1 progeny, augmented postnatal offspring growth and blood metabolites. Consequently, in utero vitamin and mineral supplementation may exert programming outcomes on the performance and productivity of females raised as herd replacements and should be considered when developing diets for gestating cows and heifers.

Great variation exists in management decisions to offer a vitamin and mineral supplement to cow­calf herds in the Northern Great Plains. Decisions to supplement (or not) vitamins/minerals during critical periods of fetal development may have lasting postnatal impacts on the offspring; however, there is a lack of reports focusing on the long-term offspring outcomes. Our objectives were to determine the impacts of supplementing vitamins/minerals during gestation in beef heifers on mineral status in the dam, calf, placenta, and colostrum; offspring postnatal performance and feeding behavior; blood metabolite and endocrine profiles; and puberty attainment in heifer calves. We observed enhanced hepatic mineral status in heifers receiving supplemental vitamins/minerals during pregnancy, at calving, and in their neonatal calves compared with non-supplemented cohorts. Calves born to supplemented dams had improved measures of growth during postnatal development, increased concentrations of key blood metabolites, and differences in body measurements and carcass ultrasound traits at post-weaning evaluation. These results suggest that fetal nutritional environment is pivotal for the long-term growth and success of the offspring. We hypothesize that fetal programming outcomes on the offspring in this experiment may have the potential to affect the subsequent generation of beef calves.

Effects of rate of body weight gain during the first trimester of gestation on beef heifer and offspring performance, concentrations of hormones and metabolites, and response to vaccination.

Our study objectives were to evaluate the effects of divergent rates of body weight (BW) gain during early gestation in beef heifers on F0 performance, metabolic and endocrine status, colostrum immunoglobulins, and subsequent F1 calf characteristics, growth performance, concentrations of hormones and metabolites, and response to vaccination. Angus-based heifers (n = 100; BW = 369 ±â€…2.5 kg) were adapted to individual feeding for 14 d and bred using artificial insemination with female-sexed semen. Heifers were ranked by BW and assigned to either a basal diet targeting 0.28 kg/d gain (low [LG], n = 50) or the basal diet plus an energy/protein supplement targeting 0.79 kg/d gain (moderate gain [MG], n = 50) until day 84 of gestation. Dam BW and blood samples were collected at 6 time points during gestation; body composition was evaluated on days -10 and 84; and fetal measurements were taken on days 42, 63, and 84. At calving (LG, n = 23; MG, n = 23), dam and calf BW were recorded; and colostrum, calf body measurements, and blood samples were collected. Cow-calf pairs were managed on a common diet from calving to weaning, followed by a common postnatal development period for all F1 female offspring. Growth performance, hormone and metabolite profiles, feeding behavior, and reproductive performance were assessed from birth to prebreeding in F1 heifers. Offspring were vaccinated against respiratory disease and bovine viral diarrhea pathogens on days 62.3 ±â€…4.13 and 220.3 ±â€…4.13 postcalving. By design, MG dams were heavier (P < 0.0001) than LG on day 84, and the BW advantage persisted until subsequent weaning of F1 calves. Concentrations of serum IGF-1 and glucose were increased throughout gestation (P < 0.001) in MG dams, whereas concentrations of NEFA were decreased (P < 0.001) in LG dams. Calves from MG dams were 2.14 kg heavier (P = 0.03) and had larger chest circumference (P = 0.04) at birth compared with LG cohorts. Heifers from MG dams continued to have greater (P ≤ 0.03) BW gain and feed efficiency during the development period, but no differences were observed (P ≥ 0.13) in body composition, concentrations of hormones and metabolites, feeding behavior, puberty attainment, and response to vaccination in F1 offspring. Hence, early gestation rate of gain impacted BW and concentrations of glucose and IGF-1 throughout gestation in the F0 dam, resulting in altered F1 calf BW and measurements at birth and increased gain and efficiency during the development period.

Generally, beef heifers are managed on grazing pastures during early gestation, which are subject to fluctuations in forage quantity and quality. Variations in the nutrients available to the dam can impact the developing offspring during early gestation. Providing energy/protein supplements to grazing cattle is a method to ensure nutrient requirements are being met and to enhance the rate of gain. This study modeled the effects of pasture supplementation in beef heifers during early gestation to determine whether 2 rates of body weight (BW) gain alter maternal body composition and concentrations of hormones and metabolites, as well as changes to postnatal characteristics of the subsequent F1 generation heifer calves. The rate of gain affected the heifer's BW, body composition, and concentrations of key metabolites and hormones, which likely altered the nutritional environment experienced by the fetus. Subsequently, F1 offspring from supplemented dams had greater morphometric characteristics at birth and had greater BW gain, feed efficiency, and eating rate during the postweaning development period. However, body composition, concentrations of hormones and metabolites, other feeding behaviors, puberty attainment, and response to vaccination of offspring were not affected. Further research is warranted to investigate how the early gestational rate of BW gain impacts key metabolic organs and mechanisms involved in transferring programming outcomes to subsequent generations.

Rate of body weight gain during early gestation in F0 beef heifers has effects that extend multigenerationally to the F2 fetuses.

Our aim was to investigate the effects of maternal (F0) body weight (BW) gain during the first 84 d of gestation on body composition, ovarian reserve, and hormonal and metabolic parameters of breeding-age F1 heifers, as well as the BW and morphometry of F2 fetuses. The study also evaluated the effect of maternal BW gain (F0) on the mRNA relative abundance of the small intestine of both F1 heifers and F2 fetuses. Crossbred Angus heifers (F0; n = 100) were managed to gain 0.20 kg/d (low gain [LG], n = 50) or 0.75 kg/d (moderate gain [MG], n = 50) for the first 84 d of gestation. Subsequently, F0 dams were managed on a common forage-based diet for the rest of gestation until the weaning of the F1 offspring. At 15 mo of age, a subset of F1 heifers was randomly selected for the current experiment (n = 8 LG and n = 8 MG). Heifers were bred via artificial insemination (AI; day 0), then harvested on day 84 of gestation. On days -10, 42, and 84, BW was recorded, and blood was collected and analyzed for concentrations of glucose, non-esterified fatty acids, progesterone, insulin, and insulin-like growth factor-1. The weight of F1 carcasses, organs, gravid uteri, and F2 fetuses and organs were recorded at harvest. Visible follicles were counted on F1 ovaries at harvest, and histology was used to count microscopic follicles. Liver and jejunal samples from F1 heifers were collected to measure tissue oxygen consumption and jejunal samples from F1 heifers and F2 fetuses were collected for mRNA relative abundance analysis. BW of F1 heifers from MG dams tended to be 12 kg greater (P = 0.06) than for F1 heifers from LG dams. Concentrations of glucose were greater (P = 0.03) in F1 heifers from the MG group, with no differences in other blood metabolites or follicular populations (P ≥ 0.16). Interestingly, mammary glands were heavier (P = 0.05), and placentas and body depth tended to be heavier and greater, respectively (P ≤ 0.10), for F2 fetuses from F0 LG heifers. Oxygen consumption in the liver and jejunum, as well as mRNA relative abundance in the jejunum of F1 heifers, were not affected by F0 rate of gain (P ≥ 0.16). However, the NDUFC1, SDHA, UQCR1, and PPARG genes were upregulated (P ≤ 0.05) in the jejunum of F2 fetuses from the LG group. In conclusion, BW gain of F0 heifers during early gestation exerts subtle effects on pre-breeding BW and blood metabolites in F1 offspring, with impacts present in F2 placenta, mammary gland, and intestine.

Early gestation is a period of rapid development of the placenta and fetal organs. In some species, impacts of nutrition during gestation persist across generations. Thus, adequate nutrition is crucial for the offspring's future productivity and reproductive success. This study aimed to assess the impact of 2 rates of body weight gain (0.20 and 0.75 kg/d) in F0 dams during early gestation on the productive and reproductive outcomes of F1 progeny and the development of F2 fetuses. The results indicate that a moderate rate of body weight gain during early gestation did not affect the development of ovarian follicles in F1 offspring but increased the body weight of F1 heifers in the pre-breeding period and concentrations of blood glucose during gestation. There were tendencies of F0 nutrition to impact placental weight, F2 mammary gland weight, and intestinal mRNA relative abundance, which could have implications on nutrient supply to the developing fetus and on future lactation and metabolism. These findings highlight the complexity of multigenerational interactions in the context of maternal nutrition and provide valuable insights to optimize productivity and reproductive performance in livestock systems.

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.

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.

Plane of nutrition and FSH-induced superovulation affect the expression of steroid hormone receptors and growth factors in caruncular tissue of non-pregnant sheep.

Implantation is a critical step in the establishment of pregnancy and an important part of embryo-maternal contact. Uterine receptivity can be affected by changes in body condition and the maternal endocrine milieu, including those caused by the use of exogenous gonadotropins in controlled ovarian hyperstimulation to induce the development of multiple follicles. This study demonstrates the effects of FSH-mediated ovarian hyperstimulation on the caruncles of ewes under various feeding regimes. Sheep were classified into 3 categories: control fed (CF), overfed (OF), or underfed (UF). In each group, animals were superovulated with FSH or injected with a saline solution (non-treated control). Uterine caruncles were collected at the early (d 5) and mid-luteal phase (d 10) of the estrous cycle. The transcript levels of steroid hormone receptors (ESR1, ESR2, PGR) and growth factors (IGF1, IGF2, VEGFA) were investigated and their expression localized by immunohistochemical staining. As for the main findings, day of the estrous cycle affected expression of ESR1, IGF1 and IGF2, but not of ESR2, PGR and VEGFA; both feeding and superovulation had modulatory effects, with feeding (UF/OF) stimulating expression of all genes studied, and superovulation altering expression of some genes, eg IGF1, PGR and ESR1 and ESR2, in CF animals. Similarly, feeding (UF/OF) altered responsiveness to superovulation for PGR on d 5 and ESR1/ESR2 on d 5 and/or 10. Our data emphasize possible effects of dietary and/or hormonal stimuli on uterine physiology, which may affect pregnancy outcomes by disrupting uterine functionality.

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 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.

Effects of feeding 60% dried corn distillers grains plus solubles or the equivalent sulfur as CaSO4 on performance and reproductive traits of yearling Angus bulls.

The objectives of this study were to investigate the effects of feeding 60% dried corn distillers grains plus solubles (DDGS) or the equivalent sulfur as calcium sulfate (CaSO4) on semen quality and performance characteristics in yearling bulls. Thirty-six half-sibling Angus bulls [291 ± 8.5 d; initial body weight (BW) = 320 ± 2.7 kg] were assigned to one of three diets: 1) 60% concentrate as corn (CON; S = 0.18%; n = 12); 2) 60% DDGS replacing corn (60DDGS; S = 0.55% DM; n = 12); 3) CON + equivalent sulfur of 60DDGS added as CaSO4 (SULF; S = 0.54%; n = 12). Bulls were fed for 112 d to target an average daily gain (ADG) of 1.6 kg/d. Blood samples were collected on d 0, 56, and 112, and evaluated for testosterone, thyroxine, triiodothyronine (T3) and glutathione peroxidase (GPx) activity. Ruminal H2S was measured on d 0, 14, and 42. Scrotal circumference and semen were collected on d 0, 28, 56, 84, and 112 to evaluate sperm characteristics and GPx activity in seminal plasma. A computer assisted semen analysis was used to evaluate kinematic profiles in motile and progressive sperm throughout the study. Data were analyzed as repeated measures using MIXED procedures of SAS. No differences (P ≥ 0.14) were observed for final BW, ADG, or scrotal circumference; however, SULF tended (P = 0.07) to have reduced gain:feed compared with CON, with 60DDGS being intermediate. Concentrations of ruminal H2S on d 42 were greatest (P < 0.01) for SULF. Increased ejaculate volume was observed for 60DDGS and CON (P < 0.01) compared with SULF. For motile populations of sperm, velocity on an average path (VAP) and curvilinear velocity (VCL) were reduced (P ≤ 0.02) for SULF compared with CON, with 60DDGS being intermediate. In progressively motile sperm throughout the study, VAP and VSL were reduced (P ≤ 0.05) in 60DDGS and SULF compared to CON. For VCL, SULF was reduced (P ≤ 0.02) compared with CON, with 60DDGS being intermediate. In serum, concentrations of T3 were reduced (P = 0.009) in 60DDGS compared with CON or SULF. A treatment by day interaction (P = 0.03) was observed for seminal plasma GPx. At d 56, GPx activity was greater (P = 0.03) for 60DDGS compared with CON, with SULF intermediate; and at d 112, 60DDGS had the greatest (P ≤ 0.02) GPx activity. Therefore, feeding 60% DDGS to developing bulls altered semen kinematics, T3 concentrations, and GPx activity leading to the conclusion that these differences may not be solely dependent on concentrations of dietary sulfur.

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.

Effects of feeding bulls dried corn distiller's grains plus solubles or CaSO4 on mineral and metabolite concentrations in serum and seminal plasma.

Yearling Angus bulls (n = 36) were assigned one of three diets: 1) 60 % concentrate as corn (CON, 0.2 % S, 13.4 % CP; n = 12); 2) 60 % dried corn distiller's grains plus solubles (60DDGS 0.5 % S, 22.0 % CP; n = 12); 3) CON diet + equivalent sulfur of 60DDGS as CaSO4 (SULF, 0.5 % S, 13.9 % CP; n = 12) to evaluate effects of feeding 60 % DDGS or sulfur as CaSO4 on mineral and metabolite concentrations in serum and seminal plasma. Treatment × day interactions (P < 0.03) were observed for serum Cu, Se, and Mo. For Cu at d 112, lesser (P < 0.01) concentrations were observed in bulls fed the 60DDGS compared to SULF and CON diets. There were greater (P < 0.01) concentrations of Se at d 112 in bulls fed 60DDGS than CON and SULF diets. Concentrations of Mo were greater at d 56 and 112 (P < 0.01) in bulls fed CON compared to SULF and 60DDGS diets. In seminal plasma, there were treatment × day interactions (P < 0.02) for Cu and Mo. For Cu, at d 112, there was a lesser (P < 0.01) concentration in the bulls fed SULF compared to CON and 60DDGS diets. For Mo, there was a greater (P < 0.01) concentration in bulls fed the CON than 60DDGS and SULF diets at d 56 and 112. Changes in mineral and metabolite concentrations may have effects on bull reproductive performance when there is a relatively greater dietary sulfur content.

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.

Nuclear and membrane progesterone receptors expression in placenta from early to late pregnancy in sheep: Effects of restricted nutrition and realimentation.

Nutrient restriction and/or realimentation may affect several placental functions, such as expression of selected regulatory factors, blood flow and other processes in sheep and other species. To determine the effects of the plane of nutrition, nulliparous white face ewes (6-8 months) carrying singletons on day 50 of gestation were randomly assigned to two dietary treatments receiving 100% of National Research Council recommendations (control; C) or 60% of C (restricted; R). Two groups remained on C or R diets from day 50 until day 130. From day 90-130 another group of C fed ewes was switched to the R diet, and another group of R fed ewes was switched to the C diet. This resulted in 7 groups (n = 5-6 ewes/group): C (day 50, 90 and 130), R (day 90 and 130), CR (day 130) and RC (day 130). At these time points, placental tissues were collected for the evaluation of progesterone receptor (PGR) protein expression (whole tissue), and mRNA expression in maternal (caruncular, CAR) and fetal (cotyledon, COT) (separated tissues). Data were statistically analyzed using analysis of variance (SAS 9.4). Protein for PGRAB and PGRB isoforms was detected using immunohistochemistry in all placental tissues, but the pattern of expression differed depending on pregnancy stage and placental compartment (e.g., CAR vs COT). PGRAB protein expression, quantified using image analysis, was greater (P < 0.04) on day 50 than 90 or 130, and was not affected by plane of nutrition. In CAR and COT, PGRAB mRNA expression was greater (P < 0.05) on day 50 than 90 or 130. PGRB mRNA expression was greater (P < 0.03) in CAR on day 50 than 90 and 130, and was greatest (P < 0.02) in COT on day 50, less on day 130, and least on day 90. For the membrane progesterone receptors, PAQR7 (membrane PGR alpha) mRNA expression was greater (P < 0.05) on days 50 and 90 than 130 in CAR, and greater (P < 0.01) on days 50 than 90 and 130 in COT; PAQR8 (membrane PGR beta) was similar throughout pregnancy in CAR and COT, and PAQR5 (membrane PGR gamma) was greatest (P < 0.0001) on day 130 in COT, but similar throughout pregnancy in CAR. Plane of nutrition affected (P < 0.05) mRNA expression for all genes in CAR and COT throughout pregnancy. These data indicate that expression of PGR in ovine placenta is dependent on stage of pregnancy and plane of nutrition in sheep. The mechanisms of how diet and stage of pregnancy influences placental PGR expression and function remains to be elucidated.

Effects of plane of nutrition and arginine on ovarian follicles in non-pregnant sheep: Cell proliferation, and expression of endothelial nitric oxide and its receptor.

The aim of this study was to investigate the role of the nitric oxide (NO) system in ovarian function, by determining if arginine (Arg) supplementation impacts follicle number, cell proliferation, and expression of the NO system members in nutritionally compromised ewes. Ewes were randomly assigned into maintenance (C, 100% requirements), excess (O; 2xC), or restricted (U; 0.6xC) diets 8 weeks prior to Arg treatment. Ewes were individually fed twice daily with pelleted diets. Ewes from each nutritional group were randomly assigned to one of two treatments: saline or Arg, which was initiated on day 0 of the estrous cycle and administered 3 times per day. Ovaries were collected at the early-luteal, mid-luteal and late-luteal/follicular phases of the estrous cycle to determine 1) the number of surface follicles, 2) follicle cell proliferation marked by Ki67 protein expression, and 3) expression of endothelial nitric oxide (eNOS; NOS3) and soluble guanylyl cyclase beta (sGC; GUCY1B3) protein and mRNA in granulosa (G) and theca (T) layers using immunohistochemistry followed by image analysis and qPCR, respectively. During nutritional treatment, C maintained body weight, O gained 6±1.2 kg, and U lost 14±1.3 kg. Our data show that: 1) Ki67 was expressed in all ovarian compartments, eNOS protein was detected in blood vessels of T and stroma, and sGC protein was detected in T cells, and blood vessels of T layer and other ovarian compartments; 2) plane of nutrition affected the number of surface follicles, and thus folliculogenesis, cell proliferation in the T layer, eNOS and sGC protein expression in T, and NOS3 and GUCY1B3 mRNA expression in G; 3) Arg treatment affected cell proliferation in G and T, eNOS and sGC protein expression in T, mRNA expression of NOS3 in T in all groups, and GUCY1B3 in G depending on the stage of the estrous cycle; and 4) G and T cell proliferation, and expression of eNOS and sGC protein in T was affected by the stage of the estrous cycle. Our data demonstrated that plane of nutrition and Arg are involved in the regulation of follicular functions in non-pregnant sheep.

Placental development during early pregnancy in sheep: nuclear estrogen and progesterone receptor mRNA expression in the utero-placental compartments.

Sex steroid hormones are major regulators of uterine and placental growth and functions, as well as many other biological processes. To examine the mRNA expression of nuclear estrogen (ESR1 and 2) and progesterone (PGRAB and B) receptors in different compartments of the uterus and placenta, tissues were collected in experiment 1 on days 16, 20, and 28 after natural mating (NAT) and on day 10 after estrus (nonpregnant controls [NP]); and in experiment 2 on day 22 of NAT, and pregnancies established after transfer of embryos generated through mating of FSH-treated ewes (NAT-ET), in vitro fertilization (IVF), or in vitro activation (parthenotes). In experiment 1, ESR1 expression in endometrial stroma (ES), endometrial glands (EGs), and myometrial blood vessels (MBVs), ESR2 in endometrial blood vessels (EBV), PGRAB in ES, and PGRB in ES, EG, and MBV was greater in pregnant than NP ewes depending on the day of pregnancy. The day of pregnancy affected the expression of ESR1 in MBV, ESR2 in EBV and MBV, and PGRAB in ES. In experiment 2, ESR1, PGRAB, and PGRB in EG, but not in other compartments, was greater in NAT-ET than NAT, and PGRB was greater for NAT-ET than IVF. These data demonstrate that ESR and PGR expression differ in pregnant versus NP ewes in selected compartments and was affected by pregnancy stage or embryo origin in selected utero-placental compartments. Thus, sex steroid hormone mRNA expression is differentially regulated in a spatiotemporal manner in the uterus and placenta and is affected by the application of assisted reproductive technology in sheep.

TCR signaling and environment affect vasoactive intestinal peptide receptor-1 (VPAC-1) expression in primary mouse CD4 T cells.

Strict regulation of T cell function is imperative to control adaptive immunity, and dysregulation of T cell activation can contribute to infectious and autoimmune diseases. Vasoactive intestinal peptide receptor-1 (VPAC-1), an anti-inflammatory G-protein coupled receptor, has been reported to be downregulated during T cell activation. However, the regulatory mechanisms controlling the expression of VPAC-1 in T cells are not well understood. Therefore, mouse splenic CD4 T cells were treated in complete media+/-anti-CD3 for 24h, total RNA isolated and VPAC-1 levels measured by qPCR. Surprisingly, we discovered that T cells incubated in complete media steadily upregulated VPAC-1 mRNA levels over time (24h). Importantly, CD4 T cells isolated from blood also showed elevated VPAC-1 expression compared to splenic T cells. Collectively, these data support that the vascular environment positively influences VPAC-1 mRNA expression that is negatively regulated by TCR signaling. This research was supported by a national service award (1KO1 DK064828) to G.D., the Center for Protease Research (2P20RR015566), and INBRE (P20 RR016741).