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The objective of this study was to determine the effects of bovine nerve growth factor-β (NGF) on pre-ovulatory follicle vascular area, LH release, ovulation, and luteal function when administered systemically to heifers. Post-pubertal Holstein heifers (n = 12) received an intravaginal progesterone-releasing device (CIDR) and GnRH agonist (100 μg IM). The CIDR was removed 5 d later, and heifers were given dinoprost (25 mg IM) at CIDR removal and 24 h later, followed by a second dose of GnRH agonist 48 h later. Heifers were randomly assigned to treatments using a cross-over design. For example, heifers assigned to NGF (250 μg reconstituted in 12 mL PBS IM) in replicate 1 were assigned to control (12 mL PBS IM) in replicate 2. Transrectal ultrasonography was performed before treatment and repeated every 4 h up to 32 h to determine the pre-ovulatory follicle diameter, vascular area, and ovulation. Serum samples were obtained to assess LH concentrations during the periovulatory period and every 2 d post-ovulation for measuring progesterone concentrations. A subset of heifers had luteal biopsies performed on days 9 (n = 6 per treatment) and 14 (n = 6 per treatment) post-ovulation to count luteal cell numbers and measure relative mRNA abundance for steroidogenic and angiogenic enzymes and LH receptor. Treatment with NGF increased pre-ovulatory follicle diameter (P = 0.02) and serum LH concentrations (P = 0.03) but did not affect time to ovulation (P = 0.42). Heifers treated with NGF had increased serum progesterone concentrations in the subsequent luteal phase (P = 0.03), but no change in vascular area of the follicle (P = 0.16) or CL (P = 0.20). Heifers treated with NGF had a greater number of small luteal cells (P 0.01) and a tendency for increased LH receptor (LHR) mRNA abundance in the CL (P = 0.10). There was also increased steroidogenic acute regulatory protein (STAR; P = 0.05) and a tendency for increased cytochrome P450 family 11 (CYP11A1; P = 0.10) mRNA abundance in the CL of NGF-treated heifers. There was decreased prostaglandin E

Objectives were to test the hypothesis that nutritional extremes during prenatal development interact with postnatal nutrition during the juvenile period of heifers to impact KNDy neuron responsiveness to a neurokinin B (NKB) agonist (Senktide) during progesterone dominance. Heifers were selected from a larger population programmed nutritionally using a 3x2 factorial arrangement of pre- and post-natal diets. Bos indicus-influenced cows (n = 95) were fed to achieve body condition scores (BCS; 1-9 scale) of 3-3.5 (L; thin), 5.5-6 (M; moderate), or 7.5-8 (H; obese) by the onset of the third trimester and maintained thereafter. Heifer offspring were weaned at 3-3.5 mo of age and assigned to either a low- (L; 0.5 kg/day) or high-gain (H; 1 kg/day) diet until 8 mo of age, then fed a common diet until puberty. For the current experiment, heifers (n = 18; 6/group) representing LL, MH, and HH combinations were ovariectomized postpubertally (17.1 mo of age) and received estradiol (E2) replacement to maintain basal E2 concentrations at 2-4 pg/mL. To achieve progesterone dominance, 3 CIDR devices were placed intravaginally 6 days prior to the initiation of Senktide challenge. Heifers were sampled for a 4.5-h period and received intravenous injections of saline or Senktide (14.8 ug/kg I.V.) at 0, 90, and 180 min. Jugular blood samples were collected at 10-min intervals for radioimmunoassay of LH. Irrespective of Senktide treatment, heifers in the MH group had greater pulse amplitudes (P = 0.016) and mean concentrations (P < 0.0001) of LH compared with both the HH and LL groups. Senktide treatment increased (P = 0.003) concentrations of LH compared with saline in all groups; however, the response to Senktide was greatest in the MH group compared with HH and LL. Pre- and postnatal nutritional extremes appear to interact to impact KNDy neuron responsiveness to NKB receptor signaling in heifers.

Objectives were to test the hypothesis that pre- and post-natal nutrition in the bovine female, independently or interactively, affect age at puberty and functional characteristics of the estrous cycle of sexually mature offspring. Brangus and Braford (n = 97) beef cows bearing a female fetus were fed to achieve body condition scores of 7.5–8 (H, obese), 5.5–6 (M, moderate), or 3–3.5 (L, thin) by the start of the third trimester and maintained until parturition. Heifer offspring were weaned and fed to gain weight at either a high (H; 1 kg/day) or a low (L; 0.5 kg/day) rate between 4 and 8 months of age, then fed the same diet during a common feeding period until puberty, which resulted in compensatory growth of heifers in the L group. Heifers (n = 95) from the H postnatal diet reached puberty 2 months earlier (12 ± 0.4 months; P = 0.0002) than those from the L postnatal diet (14 ± 0.4 months). Estrous cycles of a subgroup of postpubertal heifers (n = 53) were synchronized to evaluate antral follicle count (AFC), rate of growth and size of the pre-ovulatory follicle, size of corpus luteum and ovary, endometrial thickness, and plasma concentrations of progesterone and estradiol-17β (E2). Although there was a trend for postnatal H heifers to have greater AFC and plasma concentrations of E2 compared to L heifers, neither pre- nor post-natal nutrition affected any other physiological or hormonal variables, including short-term fertility. Postnatal nutritional effects on pubertal age remained the dominant observed feature.

The aim of this study was to investigate the ovarian response of Brahman cows submitted to two different estrus synchronization protocols and to determine the estrus response and ovulation time. A total of 153 Brahman cows were randomly assigned to receive one of the following estrus synchronization protocols: 1) CIDR insert and an injection of prostaglandin F2α (25 mg, PGF) on Day -5 and CIDR removal with PGF on Day 0 (CIDR5; n = 77); 2) CIDR insert on Day -12 and an injection of PGF on Day -12, Day -5 and Day 0 upon CIDR removal (CIDR12; n = 76). Estrotect breeding indicator patches were placed on Day 0 and cows were artificially inseminated (AI) 12h after estrus detection; cows not detected in estrus after 96h received TAI coupled with 100 µg of GnRH. Ovarian dynamics were observed during the protocol and follicular diameter were measured every 12h after AI until ovulation. Cows synchronized with CIDR12 had increased follicular diameter (CIDR5, 10.19 ± 0.51 vs. CIDR12, 13.12 ± 0.52 mm; P = 0.01) and decreased serum concentrations of progesterone (CIDR5, 1.68 ± 0.12 vs. CIDR12, 0.81 ± 0.12 ng/ml; P < 0.01) at CIDR removal. The percentage of cows exhibiting estrus did not differ between treatments (CIDR5, 66.2 vs. CIDR12, 64.5%; P = 0.25). No difference was observed in ovulation rate (CIDR5, 94.1 vs. CIDR12, 94.1%; P = 0.6). Time of estrus expression and ovulation time after CIDR removal did not differ (CIDR5, 59.9 ± 2.3 vs. CIDR12, 61.5 ± 2.3h; P = 0.71 and CIDR5, 88.1 ± 2.6 vs. CIDR12, 90.9 ± 2.6h; P = 0.75; respectively). In conclusion, extended length of CIDR with additional PGF increased follicular diameter and decreased concentrations of progesterone but did not influence estrus response and ovulation time in Brahman cows.

A modification of the standard 5-day CO-Synch + CIDR procedure (5-day Bee Synch + CIDR; Bee Synch), developed for use in Bos indicus-influenced cows, utilizes the addition of prostaglandin F2α (PGF) on Day 0 of the protocol to eliminate mature corpora lutea (CL) and fixed-time AI (FTAI) at 66 h. Objectives were to test the hypothesis that elimination of GnRH on Day 0 (GnRH-1) does not impact significantly the synchronized development of a dominant follicle for presumptive FTAI. Seventy-one estrous cycling Brangus and Brahman x Hereford suckled cows were used in two replicates (35–36/replicate). Following stratification, cows were assigned randomly to a 2 × 3 factorial arrangement of treatments involving two truncated (no FTAI or GnRH-2) versions of Bee Synch (Bee Synch It and IIt), each begun 3, 7, and 10 days post-ovulation. Cows in Bee Synch It received 100 μg GnRH (GnRH-1), 25 mg PGF, and a CIDR on Day 0, whereas cows assigned to Bee Synch IIt received the same treatment but without GnRH-1. All cows received 50 mg PGF on Day 5 at CIDR removal. Synchronized new follicular wave emergence (NFWE; days 1–4) was observed in 68.6 and 38.9% of Bee Synch It and IIt, respectively (P = 0.01). This increased to 93.3% and 72.2%, respectively, if days 0–4 were considered. Inclusion of GnRH at CIDR insertion improved synchronized NFWE but size of the largest follicle at 66 h, the normal time of FTAI, did not differ due to treatment or day of the estrous cycle.

Telomeres are comprised of G-rich nucleotide sequences (5’-TTAGGG-3’) at the chromosome termini that are responsible for protecting chromosomes; however, attrition of these sequences has been observed due to aging and stress. The purpose of this study was to compare telomere length (TL) in control and prenatally stressed (PNS) calves. Mature Brahman cows inseminated to a single Brahman sire in 2018 were assigned to be either Control (n=35; not transported) or PNS (n=37; 2 h of transportation at 60, 80, 100, 120, and 140±5 d of gestation). Jugular blood samples were obtained by venipuncture from 10 Control (5 bulls and 5 heifers) and 10 PNS (5 bulls and 5 heifers) calves at 25±2 d of age. Peripheral leukocytes were isolated and genomic DNA was extracted utilizing silicone membrane spin column kits. The relative quantity of telomere products, which is proportional to the average TL, was determined by multiplex quantitative PCR analysis using the ratio of bovine telomere and β-globulin DNA (T/S ratio). An absolute standard of bovine telomere (1012–107 dilution series) and β-globulin (109-104 dilution series) genes was utilized to produce relative copy number. All samples were processed in triplicate and samples were included if the triplicate Cq difference was less than 0.25 cycles. Data were analyzed using ANOVA. TL differed by sex (P< 0.03; bulls 11,959±211; heifers 12,850±366) and treatment (P< 0.02; Control 12,895±278; PNS 11,913±303). There was not an interaction of sex and treatment (P >0.43) although TL of Control heifers (13,482±396) exceeded the TL of PNS bulls (11,609±348). These observations of fewer copies of telomere sequences suggest an influence of sex on TL within 1 month of age; and, that prenatal transportation stress may increase attrition of telomeres in cattle. Support: USDA NIFA Award No. 2019-67015-29573

Made available in DSpace on 2021-06-25T15:06:26Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-06-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) This study was conducted to evaluate the luteinizing hormone (LH) secretion pattern after gamma-aminobutyric acid (GABA(A)) antagonist to determine the effects of the GABAergic system on LH secretion during reproductive maturation in pre-pubertal Nellore heifers. Nellore heifers (n = 10) were administered a picrotoxin injection of 0.18 mg/kg, i.v. Blood samples were collected every 15 min for 3 h at different developmental stages (8, 10, 14 and 17 mo of age). Plasma concentrations of LH were quantified using an RIA (sensitivity of 0.04 ng/mL and CV of 15 %). There was an interaction between treatment and age (P = 0.034). Picrotoxin-treated heifers had lesser (P

Longitudinal studies of gene expression where sampling certain organ systems is precluded may become more feasible if a more accessible, representative biological sample could be identified. The objective of this study was to determine whether peripheral blood leukocytes (PBL) could be an effective surrogate for assessing gene expression differences within neuroendocrine tissues that regulate stress responses. Specifically, leukocyte samples and stress axis tissues, including the paraventricular region of the hypothalamus, anterior pituitary gland, adrenal cortex, and adrenal medulla were isolated from humanely slaughtered 5-yr-old Brahman cows (n = 8). RNA was extracted from each sample and gene expressions were calculated using bioinformatic approaches of RNA-Seq analysis. Differentially expressed genes (DEG) between adrenal axis tissue RNA and PBL RNA were identified using the edgeR Package from Bioconductor, R. More than 3,000 DEGs were identified in each tissue relative to the PBLs. Overall, higher gene expression levels were observed in the hypothalamus vs PBL, while the remaining tissues had reduced expression compared to PBL (Table 1). Pearson correlation values were calculated from the mean normalized gene count in the PBL and tissues for genes (n = 17) important for stress response and metabolism to assess PBL as a possible surrogate for gene expression analyses (Table 2). Only two genes, oxytocin and 11β-hydroxylase, were positively correlated (P < 0.05) between PBL and specific tissue gene expression. As typically observed among different cell types, gene expression differences exist between the PBL and various tissues of the HPA axis. Therefore, it remains to be determined if gene expression in the surrogate PBL will shift in a direction similar to that of specific adrenal axis tissue of mature Brahman cows exposed to stressors during key developmental periods.

Prenatal transportation stress (PNS) results in calves that are more temperamental and have greater circulating concentrations of cortisol compared to control calves. The objective of this experiment was to evaluate whether PNS alters the number of pituitary corticotrophs in mature Brahman cows. We hypothesized that the increased circulating cortisol concentrations previously characterized in this bovine model is associated with developmental changes in the anterior pituitary leading to an increased number of corticotrophs. Pregnant Brahman cows (n = 48) were transported in trailers for 2-hour periods at 60±5, 80±5, 100±5, 120±5, and 140±5 days of gestation. Non-transported pregnant cows (n = 48) were designated as the Control group. Control and PNS offspring heifers were managed together under the same environmental conditions. At approximately 5 yr of age, randomly selected non-pregnant cows (Control, n = 8; PNS, n = 6) were humanely harvested and the whole pituitaries were collected. Pituitaries were weighed, fixed in paraformaldehyde, serially dehydrated with graded ethanol, embedded in paraffin blocks, and cut into 5-μm sections. Immunohistochemistry was performed to detect cells expressing adrenocorticotropic hormone (ACTH) as a marker for corticotrophs. Three comparable sections from the midsagittal plane from each animal were processed using an ovine ACTH-specific antibody (Dr. A.F. Parlow, NIDDK). Five fields of view were analyzed per section (15 fields per animal). Anterior pituitary gland weight did not differ (P > 0.10) between groups (Control = 2.11 ± 0.12 g; PNS = 2.10 ± 0.15 g). The mean number of ACTH-positive cells between control (531±56 cells/section) and PNS cows (477±49 cells/section) also did not differ (P > 0.10). In conclusion, the number of pituitary corticotrophs in mature Brahman cows was not affected by prenatal transportation stress and is likely not a mechanism mediating the increased circulating cortisol concentrations seen in this bovine model of fetal programming.

Objectives were to test the hypotheses that nutritional extremes during prenatal development interact with postnatal nutrition during the juvenile period of heifers to impact 1) tonic secretion of gonadotropins, and 2) estradiol-17β (E2) negative and positive feedback responsiveness in adulthood. Heifers were selected from a larger population programmed nutritionally using a 3 x 2 factorial arrangement of pre- and postnatal diets. Beginning at 90 days of pregnancy, Bos indicus-influenced cows (n = 95) were fed to achieve body condition scores (BCS; 1–9 scale) of 3–3.5 (L; thin), 5.5–6 (M; moderate), or 7.5–8 (H; obese) by onset of the third trimester and maintained thereafter. Heifer offspring were weaned at 3–3.5 months of age and assigned to either a low- (L; 0.5 kg/day) or high-gain (H; 1.0 kg/day) diet until 8 mo. of age, then fed a common diet until puberty. Heifers (n = 18; 6/grp) representing HH, MH and LL combinations were ovariectomized postpubertally (17.1 mo. of age) and received E2 replacement. In Exp. 1, blood samples were collected at 10-min intervals to evaluate pulsatile secretion of LH and FSH for 5.5 hours. In Exp. 2, heifers received E2 (2.4 ug/kg I.M.), with blood sampling at 30-min to 1-h intervals for 30 h. Heifers in the MH (1.25 ± 0.11 ng/mL) group tended (P < 0.09) to have greater LH pulse amplitude compared to HH (0.91 ± 0.14 ng/mL) and LL (0.96 ± 0.09 ng/mL); otherwise, frequency, amplitude, and mean concentrations of LH and FSH did not differ among groups. Exogenous E2 (Exp. 2) suppressed (P < 0.0001) mean plasma concentrations of LH and FSH equally among groups, then stimulated equivalent surges of LH beginning at 14 ± 0.02 h, with only two FSH surges detected. Neither pre- nor postnatal nutrition affected E2 negative or positive feedback in this study.

Made available in DSpace on 2021-06-25T10:10:41Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) This study investigated the effects of growth rates and compensatory growth on puberty attainment in Nellore heifers. Nellore heifers (n = 120), weaned at 8 ± 0.75 mo of age, were blocked by sire and BW (180 ± 8.6 kg) and assigned randomly to receive 1 of 4 treatments over a 10-mo period. Treatments included ad libitum feeding (high gain, HG), feed intake to gain 0.6 kg/d (medium gain, MG), restricted feeding (0.2 kg/d) for 4 mo followed by ad libitum feeding for 6 mo (compensatory gain, CG), and alternating periods of ad libitum and restricted feeding for 2 mo each throughout the trial (alternated CG, ACG). Puberty was assessed weekly by transrectal ultrasonography. Blood samples were collected at 8, 11, and 18 mo of age and at puberty to determine circulating concentrations of leptin. At 18 mo of age, nonpubertal heifers were treated with a puberty induction protocol using an intravaginal progestin device. There was no treatment effect (P = 0.17) on the percentage of heifers pubertal by 18 mo of age (HG: 66, MG: 40, CG: 58, and ACG: 52%), BW at puberty, and age at puberty. However, HG heifers had higher ADG (P < 0.01), dry matter intake (P < 0.01), and leptin concentrations (P = 0.03) than heifers from other groups. The response to the puberty induction protocol was similar (P = 0.90) among treatments. Regarding sire effects (genetic effects), there was an effect (P = 0.03) on the percentage of heifers pubertal by 18 mo of age and a tendency (P = 0.07) of sire effect in response to the puberty induction protocol. Compensatory growth appears to be an effective managerial approach to decrease feeding costs and stimulate puberty in Nellore heifers. Department of Nutrition and Animal Production College of Veterinary and Animal Science University of São Paulo, Duque de Caxias North Avenue, n 225 Department of Animal Science Federal University of Amazonas, Macurany Avenue, Jacareacanga Department of Animal Science Texas A&M University, 2471 TAMU Department of Animal Science College of Agriculture “Luiz de Queiroz” University of São Paulo, Pádua Dias Avenue, n 11, PO Box 09 College of Veterinary Medicine São Paulo State University (UNESP) College of Veterinary Medicine São Paulo State University (UNESP)

Maternal undernutrition during pregnancy followed by ad libitum access to nutrients during postnatal life induces postnatal metabolic disruptions in multiple species. Therefore, an experiment was conducted to evaluate postnatal growth, metabolism, and development of beef heifers exposed to late gestation maternal nutrient restriction. Pregnancies were generated via transfer of in vitro embryos produced using X-bearing sperm from a single Angus sire. Pregnant dams were randomly assigned to receive either 100% (control; n = 9) or 70% (restricted; n = 9) of their total energy requirements from gestational day 158 to parturition. From post-natal day (PND) 301 until slaughter (PND485), heifers were individually fed ad libitum in a Calan gate facility. Calves from restricted dams were lighter than controls at birth (PPP>0.10). To assess pancreatic function, glucose tolerance tests were performed on PND315 and PND482 and a diet effect was seen with glucose area under the curve being greater (PPPP

Objectives were to test the hypothesis that nutritional extremes during pregnancy in the bovine female negatively affect phenotypic characteristics of the estrous cycle of sexually mature offspring, and to determine whether these effects interact with postnatal diets during the juvenile period. Beginning at 90 d of pregnancy, Brangus and Braford (n = 108) beef cows with a female fetus were fed to achieve body condition scores of 7.5–8 (H, obese), 5–5.5 (M, moderate) or 3–3.5 (L, very thin) by the start of the third trimester. Heifer offspring were weaned and fed to gain at either a high (H; 1 kg/d) or low (L; 0.5 kg/d) rate between 4 and 8 months of age, then fed a common growth diet until puberty. Estrous cycles of a subgroup (n = 53) were synchronized using 2 injections of prostaglandin F2α(PGF). Transrectal ultrasonography was employed during the follicular phase of two estrous cycles to evaluate antral follicle count(AFC), rate of growth and size of the ovulatory follicle, size of corpus luteum(CL), and endometrial thickness. Blood samples were collected daily for assay of plasma concentrations of progesterone(P4) and estradiol-17β(E2) by RIA. Preliminary data (n = 35) were analyzed using ANOVA and mixed model procedures. Least Squares Means estrous cycle length (20.9 ± 0.2 days), AFC (19.7 ± 1.0), follicular growth rate (0.81 ± 0.11), ovulatory follicle size (12.8 ± 0.2 mm), CL size (16.8 ± 0.6 mm), and endometrial thickness (14.6 ± 0.3 mm) did not differ among treatments. Heifers in postnatal H group tended (P < 0.1) to have a greater area (arbitrary units) under the E2 curve (12.6 ± 0.6) than postnatal L (11.3 ± 0.5) during the follicular phase. Normalized AUC for P4 was greater (P < 0.03) in L/H (56.94 ± 3.22) than both M/H (42.98 ± 3.61) and L/L (43.66 ± 3.22) groups. Despite marked pre- and postnatal nutritional contrasts, independent and interactive effects of nutritional treatments have been of minor significance or undetectable to date.

The objective of this experiment was to evaluate whether prenatal transportation stress (PNS) affects the weight of endocrine and immune tissues of calves. Mature Brahman cows inseminated to a single Brahman sire in 2018 were assigned to be either Control (n = 35; not transported) or PNS (n = 37; 2 h of transportation at 60, 80, 100, 120, and 140 ± 5 d of gestation). Of the calves born in 2019, 16 Control (8 bulls and 8 heifers) and 16 PNS (8 bulls and 8 heifers) calves were studied. Pen score, body weight, and blood samples were obtained from calves at 25 ± 2 d of age. At that time, calves were euthanized by barbiturate overdose in order to collect tissues (brain, pituitary and adrenal glands, spleen and thymus). Tissues were trimmed and weighed. Serum cortisol was determined by RIA. Data were analyzed using ANOVA, GLM, and CORR procedures of SAS with body weight at sample collection as a covariate. Whole pituitary weight tended (P = 0.08) to be greater in females compared to males, whereas anterior pituitary weight tended (P = 0.06) to be greater in PNS. Whole brain weight was greater (P < 0.01) in males. The interaction of treatment and sex on whole brain weight of PNS tended (P = 0.09) to fall between that of the Control females and Control males. Whole pituitary weight was positively correlated with total adrenal weight (r = 0.32; P = 0.08). Total thymus weight positively correlated with whole brain and total adrenal weight (r = 0.43 and 0.41, respectively; P < 0.05). Serum cortisol was negatively correlated with spleen weight and total immune tissue weight (r = -0.37 and -0.38, respectively; P < 0.05). Results suggest next steps should include a closer look at function of immune tissues by studying PNS effect on thymus response to immunization.

Background Prenatal testosterone (T) excess results in reproductive and metabolic perturbations in female sheep that closely recapitulate those seen in women with polycystic ovary syndrome (PCOS). At the neuroendocrine level, prenatal T-treated sheep manifest increased pituitary sensitivity to GnRH and subsequent LH hypersecretion. In this study, we investigated the early effects of gestational T-treatment on LH secretion and pituitary function in the female sheep fetus. Additionally, because prenatal T effects can be mediated via the androgen receptor or due to changes in insulin homeostasis, prenatal co-treatment with an androgen antagonist (flutamide) or an insulin sensitizer (rosiglitazone) were tested. Methods Pregnant sheep were treated from gestational day (GD) 30 to 90 with either: 1) vehicle (control); 2) T-propionate (~ 1.2 mg/kg); 3) T-propionate and flutamide (15 mg/kg/day); and 4) T-propionate and rosiglitazone (8 mg/day). At GD 90, LH concentrations were determined in the uterine artery (maternal) and umbilical artery (fetal), and female fetuses were euthanized. Pituitary glands were collected, weighed, and protein level of several key regulators of LH secretion was determined. Results Fetal pituitary weight was significantly reduced by prenatal T-treatment. Flutamide completely prevented the reduction in pituitary weight, while rosiglitazone only partially prevented this reduction. Prenatal T markedly reduced fetal LH concentrations and flutamide co-treatment partially restored LH to control levels. Prenatal T resulted in a marked reduction in LH-β protein level, which was associated with a reduction in GnRH receptor and estrogen receptor-α levels and an increase in androgen receptor. With the exception of androgen receptor, flutamide co-treatment completely prevented these alterations in the fetal pituitary, while rosiglitazone largely failed to prevent these changes. Prenatal T-treatment did not alter the protein levels of insulin receptor-β and activation (phosphorylation) of the insulin signaling pathways. Conclusions These findings demonstrate that prenatal T-treatment results in reduced fetal LH secretion, reduced fetal pituitary weight, and altered protein levels of several regulators of gonadotropin secretion. The observations that flutamide co-treatment prevented these changes suggest that programming during fetal development likely occurs via direct androgen actions.

This study was conducted to determine effects of pre-synchronization of ovulation timing among heifers and delayed fixed-time artificial insemination (TAI) with sex-sorted semen on proportion of heifers pregnant after TAI (PR/AI). Heifers were assigned to one of eight treatments: 1 and 2), 7-d CO-Synch + CIDR treatment regimen with administration of gonadotropin-releasing hormone and a CIDR insert on Day 0, prostaglandin F2α (PGF) at CIDR removal on Day 7, and TAI occurring 54 h later with conventionally processed (CTRL54-CNV) or sex-sorted semen (CTRL54-SEX); 3 and 4), same as CTRL54 but TAI delayed to 72 h with conventionally processed (CTRL72-CNV) or sex-sorted semen (CTRL72-SEX); 5 and 6), same as CTRL54 but additional administration of PGF on Day -7 and TAI with conventionally processed (PRE54-CNV) or sex-sorted semen (PRE54-SEX); 7 and 8), same as PRE54 treatments but TAI delayed to 72 h with conventionally processed (PRE72-CNV) or sex-sorted semen (PRE72-SEX). Proportion of heifers pregnant after TAI was greater (P ≤ 0.02) with conventionally processed semen compared with sex-sorted semen, yet PR/AI did not differ (P = 0.14) between heifers in PRE72-CNV and PRE72-SEX groups. There were greater PR/AI in the PRE72-SEX (P = 0.03) than CTRL54-SEX group (46.1 % and 36.9 %) and there was no difference (P = 0.31) in PR/AI between CTRL54-CNV and PRE72-SEX groups (50.4 % and 46.1 %). In conclusion, pre-synchronization of ovulation timing among heifers combined with delayed TAI resulted in increased PR/AI with sex-sorted semen compared with the 7-d CO-Synch+CIDR treatment regimen.

The aim of this study was to evaluate the effects of flunixin meglumine administration on pregnancy rates and luteal phase characteristics in bovine embryo recipients at the moment of embryo transfer. In experiment 1, in vitro produced embryos were transferred to 184 females divided as control and treated group (recipients treated with 1.1mg/kg flunixin meglumine). In experiment 2, 22 females were divided as control group; group 2 (animals submitted to a reproductive tract manipulation similar to an embryo transfer on the 7th day after estrous); and group 3 (females submitted to a manipulation and treatment with 1.1mg/kg flunixin meglumine). In experiment 1 no difference was observed between control and treated groups (40.2% and 44.6%, respectively) for pregnancy rates. In experiment 2 no difference was observed on the length of luteal phase between groups, however, animals in group 2 presented lower plasma progesterone concentrations than the control group and group 3. Therefore, we concluded that although the administration of flunixin meglumine at the moment of embryo transfer inhibited the reduction plasma progesterone concentrations, it was not effective in increasing pregnancy rates of bovine recipients. RESUMO O objetivo deste estudo foi avaliar os efeitos da administração de flunixina meglumina sobre as taxas de prenhez e características da fase lútea da receptora no momento da transferência de embriões em bovinos. No experimento 1, embriões produzidos in vitro foram transferidos para 184 fêmeas, divididas em grupos controle e tratado (tratados com 1,1mg/kg de flunixina meglumina). No experimento 2, 22 fêmeas foram divididas em grupo controle (n=7); grupo 2 (n=8; animais submetidos à manipulação do trato reprodutivo semelhante à transferência de embriões no sétimo dia pós-cio); e grupo 3 (n=7; fêmeas submetidas à manipulação e ao tratamento com 1,1mg/kg de flunixina meglumina). No experimento 1, não foi observada diferença nos grupos controle e tratado (40,2% e 44,6%, respectivamente) para as taxas de prenhez. No experimento 2, não houve diferença na extensão da fase lútea entre os grupos, entretanto os animais do grupo 2 apresentaram concentrações plasmáticas de progesterona mais baixas que o grupo controle e o grupo 3. Portanto, conclui-se que a administração de flunixina meglumina no momento da transferência de embriões inibiu a redução das concentrações plasmáticas de progesterona, no entanto não foi eficaz para aumentar as taxas de prenhez de receptoras em bovinos.

Results from previous studies indicate that maternal overnutrition during late gestation predisposes foals to metabolic disease, however, specific mechanisms resulting in disease remain unknown. Quarter Horse mares (n = 16), were randomly assigned to dietary treatments, beginning on gestational day 235, and consisted of a control group (CON- diet meeting nutrient requirement; n = 8) or an overfed diet (HIGH; n = 8) where mares received an additional 40 % above CON. On gestational days 285 and 315, an intravenous glucose tolerance test (FSIGTT) was conducted. Following parturition, foals were separated from the mare, prohibited from nursing, and an FSIGTT was conducted at 2 h postpartum. Foals were immediately euthanized and tissues preserved for analyses. There was no effect of treatment on foal BW (P = 0.50), pancreas weight (P = 0.60), or FSIGTT area under the curve for glucose (P = 0.80) and insulin (P = 0.70). Colocalization of α-amylase to isolate pancreatic islets of Langerhans indicated increased islet number and size in foals from HIGH mares (P0.01). Immunofluoresent analysis of insulin, glucagon, and somatostatin indicate no difference in intensity of staining (P0.10). Foals exposed to overnutrition during peak fetal growth had altered pancreatic islet development that may lead to adult-onset metabolic disease.

Embryonic mortality (EM) is a major factor limiting reproductive efficiency in cattle, and despite negative connotations related to reproductive performance, prostaglandin F2α (PGF2α) is capable of being released by the uterus by Day 30 of gestation. Therefore, the objective was to evaluate differences in PGF2α release after an oxytocin challenge between cows with high circulating concentrations of pregnancy-associated glycoproteins (PAGs) vs low PAG because of the association of increased PAG concentrations with pregnancy success. At Day 30 of gestation, pregnant cows were divided into oxytocin treatment (OT; n = 13) and control (CON; n = 12) groups. Treatment cows were further subdivided by circulating PAG concentration (high PAG, n = 7; and low PAG, n = 6). Blood samples were collected every 30 min beginning 1 h before oxytocin administration and continuing for 4 h. Prostaglandin F2α metabolite (PGFM), progesterone, estradiol-17β (E2), and PAG concentrations were quantified. The peak concentration of PGFM occurred 2 h after oxytocin injection in treatment animals and returned to baseline levels by 4 h. No correlations were observed between PAG and PGFM, progesterone, or E2 concentrations (P > 0.05). There was no difference in initial or final PGFM concentrations between groups (P > 0.05). Progesterone and E2 concentrations decreased in cows after treatment of oxytocin (P

Gestational nutrient restriction causes epigenetic and phenotypic changes that affect multiple physiological processes in the offspring. Gonadotropes, the cells in the anterior pituitary that secrete luteinizing hormone (LH) and follicle-stimulating hormone (FSH), are particularly sensitive to nutritional changes during fetal development. Our objective herein was to investigate the effects of gestational nutrient restriction on LH protein content and number of gonadotropes in the fetal bovine pituitary. We hypothesized that moderate nutrient restriction during mid to late gestation decreases pituitary LH production, which is associated with a reduced number of gonadotropes. Embryos were produced in vitro with X-bearing semen from a single sire then split to generate monozygotic twins. Each identical twin was transferred to a virgin dam yielding four sets of female twins. At gestational d 158, the dams were randomly assigned into two groups, one fed 100% NRC requirements (control) and the other fed 70% of NRC requirements (restricted) during the last trimester of gestation, ensuring each pair of twins had one twin in each group. At gestational d 265, the fetuses (n = 4/group) were euthanized by barbiturate overdose, and the pituitaries were collected. Western blots were performed using an ovine LH-specific antibody (Dr. A.F. Parlow, NIDDK). The total LH protein content in the pituitary tended to be decreased in the restricted fetuses compared to controls (P < 0.10). However, immunohistochemistry analysis of the pituitary did not reveal any significant changes in the total number of LH-positive cells (control = 460±23 cells/0.5 mm2; restricted = 496±45 cells/0.5 mm2, P = 0.58). In conclusion, while maternal nutrient restriction during gestation resulted in a trend of reduced LH content in the fetal pituitary, immunohistological findings suggest that these changes are likely related to the individual potential of each gonadotrope to produce LH, rather than alterations in cell differentiation during fetal development.

The primary objective of this investigation was to determine whether circadian clock genes were differentially expressed within or among bovine hypothalamic paraventricular nucleus (PVN), anterior pituitary gland (AP), adrenocortical (AC) and adrenomedullary (AM) tissues. The PVN, AP, AC, and AM were isolated from 5-yr-old Brahman cows (n = 8) harvested humanely at an abattoir between 0800-1100 h. Expression of target genes in each sample was evaluated via RNA-sequencing analyses. Gene counts were normalized using the trimmed mean of M values (TMM) method in the edgeR Package from Bioconductor, R. The normalized gene counts of genes important for circadian rhythm were statistically analyzed using the GLM Procedure of SAS. The genes analyzed were circadian locomotor output cycles protein kaput (CLOCK), cryptochrome circadian regulator 1 and 2 (CRY1 and CRY2), aryl hydrocarbon receptor nuclear translocator like (ARNTL), period circadian regulator 1 and 2 (PER1 and PER2), neuronal PAS domain protein 2 (NPAS2), and nuclear receptor subfamily 1 group D member 1 (NR1D1). Overall, relative expression profiles of clock genes differed (P < 0.01) within each tissue with PER1 having greater expression in all tissues (P < 0.01). Within the PVN expression of CLOCK, CRY1, ARNTL, and PER2 was less than that of CRY2, NPAS2, and NR1D1 (P < 0.01). In the AP, with the exception of PER1, no other clock gene differed in degree of expression. In the AC, expression of CLOCK and NPAS2 was greater than CRY1, ARNTL, PER2, and NR1D1 (P < 0.05), whereas CRY2 expression exceeded only CRY1 (P < 0.05). Within the AM, CLOCK and CRY2 expression was greater than CRY1 and ARNTL (P < 0.05). Overall, clock gene expression among tissues differed (P < 0.01) for each individual clock gene. The AC and AM had similar clock gene expression, except expression of CRY2 and PER2 was greater in AM (P < 0.05). The AC and AM had greater expression of CLOCK than the PVN and AP (P < 0.01), with PVN having greater expression than AP (P < 0.01). The AP had greater expression of NPAS2, followed by PVN, with the least expression in the AC and AM (P < 0.01). Both PVN and AP had greater CRY1 and NR1D1 expression than AC or AM (P < 0.01). The AP had greater PER1 expression than PVN, AC, and AM (P < 0.01), whereas PVN, AC, and AM had greater ARNTL expression than AP (P < 0.05). Both AP and AM had greater expression of PER2 than PVN or AC (P < 0.01). The PVN had greater expression of CRY2 than the AP, AC, and AM (P < 0.01). These results indicated that within each tissue the various clock genes were expressed in different quantities. Also, the clock genes were expressed differentially among the tissues of the bovine neuroendocrine adrenal system. Temporal relationships of these genes with the primary endocrine products of these tissues should be investigated to define the roles of peripheral clock genes in regulation of metabolism and health.

Puberty is a complex physiological process in females that requires maturation of the reproductive neuroendocrine system and subsequent initiation of high- frequency, episodic release of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH). Genetics and nutrition are two major factors controlling the timing of puberty in heifers. While nutrient restriction during the juvenile period delays puberty, accelerated rates of body weight gain during this period have been shown to facilitate pubertal development by programming hypothalamic centers that underlie the pubertal process. Among the different metabolic factors, leptin plays a critical role in conveying nutritional information to the neuroendocrine axis and controlling pubertal progression. Because GnRH neurons are devoid of the leptin receptor, leptin's effects on GnRH neurons must be relayed via an afferent neuronal network. Two neuronal populations located in the arcuate nucleus (ARC) that express the orexigenic peptide neuropeptide Y (NPY), and the anorexigenic peptide alpha melanocyte-stimulating hormone (αMSH), are key components of afferent pathways that convey inhibitory (NPY) and excitatory (αMSH) inputs to GnRH neurons. In addition, ARC neurons expressing kisspeptin, a potent stimulator of GnRH release, are also involved in the nutritional regulation of puberty. Our studies have demonstrated that increased planes of nutrition during juvenile development result in morphological and functional changes in hypothalamic pathways comprising NPY, proopiomelanocortin (POMC), and kisspeptin neurons. Changes included differential expression of NPY, POMC, and Kiss1 in the ARC, and plasticity in the axonal projections to GnRH and kisspeptin neurons. Additionally, increased rates of body weight gain also promoted changes in the pattern of DNA methylation, a key epigenetic mechanism for regulation of gene expression. Finally, our most recent findings suggest that maternal nutrition during gestation can also induce structural and functional changes in hypothalamic neurocircuitries that are likely to persist long after pubertal maturation and influence reproductive performance throughout adulthood in cattle.

In this study, we investigated whether decreased sensitivity to estradiol negative feedback is associated with reduced estrogen receptor α (ESR1) expression in kisspeptin neurons as ewe lambs approach puberty. Lambs were ovariectomized and received no implant (OVX) or an implant containing estradiol (OVX+E). In the middle arcuate nucleus (mARC), ESR1 messenger RNA (mRNA) was greater in OVX than OVX+E lambs but did not differ elsewhere. Post hoc analysis of luteinizing hormone (LH) secretion from OVX+E lambs revealed three patterns of LH pulsatility: low [1 to 2 pulses per 12 hours; low frequency (LF), n = 3], moderate [6 to 7 pulses per 12 hours; moderate frequency (MF), n = 6], and high [>10 pulses per 12 hours; high frequency (HF), n = 5]. The percentage of kisspeptin neurons containing ESR1 mRNA in the preoptic area did not differ among HF, MF, or LF groups. However, the percentage of kisspeptin neurons containing ESR1 mRNA in the mARC was greater in HF (57%) than in MF (36%) or LF (27%) lambs and did not differ from OVX (50%) lambs. A higher percentage of kisspeptin neurons contained ESR1 protein in all regions of the arcuate nucleus (ARC) in OVX compared with OVX+E lambs. There were no differences in ESR1 protein among the HF, MF, or LF groups in the preoptic area or ARC. Contrary to our hypothesis, increases in LH pulsatility were associated with enhanced ESR1 mRNA abundance in kisspeptin neurons in the ARC, and absence of estradiol increased the percentage of kisspeptin neurons containing ESR1 protein in the ARC. Therefore, changes in the expression of ESR1, particularly in kisspeptin neurons in the ARC, do not explain the pubertal escape from estradiol negative feedback in ewe lambs.

Three experiments were conducted to investigate the effects of the kisspeptin receptor agonist, 1-adamantanecarbonyl-RF-NH2 (RF9) on the secretion of gonadotropins, the role of estradiol-17β in modulating this response, and the metabolic half-life of RF9 in the mare. During the luteal phase of the estrous cycle (experiment 1), light horse mares were treated with sequential I.V. injections of RF9 (0.2 and 0.4 mg/kg) within a 1-hour interval to determine effects on coincident secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Dose-dependent increases (P

Using a previously established model for nutritional acceleration of puberty, beef heifers ( = 48; 1/2 Angus × 1/4 Hereford × 1/4 Brahman) were used in a replicated 2 × 2 factorial design to examine the effects of diet type (high forage [HF] vs. high concentrate [HC]) and rate of BW gain (low gain [LG], 0.45 kg/d, vs. high gain [HG], 0.91 kg/d) on key metabolic hormones and age at puberty. After weaning at 14 ± 1 wk of age, heifers were assigned randomly to be fed HC-HG, HC-LG, HF-HG, or HF-LG ( = 12/group) beginning at 4 mo of age for 14 wk. Heifers were then switched to a common growth diet until puberty. Average daily gain was greater ( < 0.04) during the dietary treatment phase in HG heifers (0.81 ± 0.06 kg/d) than in LG heifers (0.43 ± 0.06 kg/d), and there was no diet type × rate of gain interaction. Puberty was achieved at a younger age (54.5 ± 1.8 wk) in both HG groups than in LG groups (60.2 ± 1.9 wk; < 0.04), but dietary energy source (HC vs. HF) did not influence this variable. Moreover, mean BW at puberty did not differ by diet type or rate of gain during the dietary treatment phase. Nonetheless, heifers fed HC-HG exhibited a striking increase ( < 0.0001) in serum leptin beginning at 26 ± 1 wk of age and remained elevated ( < 0.01) throughout the remainder of the experimental feeding phase compared to all other treatments. However, serum leptin in HC-HG dropped precipitously when heifers were switched to the common growth diet and did not differ from that of other groups thereafter. Overall mean concentrations of serum glucose were greater ( < 0.006) in HG heifers than in LG during the dietary treatment phase, with serum insulin also greater ( < 0.04) in HG than in LG only during weeks 20, 22, and 30. Mean serum IGF-1 was not affected by dietary type or rate of BW gain. We speculate that failure of the marked increase in serum leptin observed in HC-HG heifers during the dietary treatment phase to further accelerate puberty compared to HF-HG occurred because of its abrupt decline at the onset of the common growth phase, thus attenuating the temporal cue for activation of the reproductive neuroendocrine system.

Nutrition during gestation and early postnatal life can program developmental changes within the reproductive neuroendocrine system that persist until adult life. Here we tested the hypotheses that maternal nutrition during the second and third trimester of gestation and dietary energy intake during the juvenile period interact to affect concentrations of neuropeptide Y (NPY) in the third ventricle cerebrospinal fluid (CSF) after fasting (basal) and in response to peripheral leptin administration in heifer offspring. Bos indicus-influenced beef heifers were developed using a 3 × 2 factorial arrangement of pre- and postnatal dietary treatments. Beginning at the onset of the second trimester of gestation, pregnant Braford and Brangus cows were fed to reach body condition scores of 7.5–8 (H, obese), 5–5.5 (M, moderate), or 3–3.5 (L; very thin) during the third trimester. Heifer offspring were weaned at ~3.5 mo of age and fed to gain at either a relatively high (H; 1 kg/d) or low (L, 0.5 kg/d) rate between 4–8 mo of age, then fed a common diet subsequently. A subgroup of pubertal heifers (n = 6/group) representing 3 of the maternal × postnatal groups (HH, MH, and LL) were ovariectomized and received estradiol replacement after puberty. Cannulas were placed surgically in the third ventricle. After at least a 2-wk recovery period, heifers were fasted for 54 h and CSF was collected every 30 min for 5.5 h, including a 30-min basal and 5-h treatment period involving 3 hourly IV injections of recombinant oleptin (0.2 µg/kg BW). Basal and post-leptin NPY concentrations in the CSF did not differ (P > 0.10) among LL, MH, and HH heifers. Treatment with leptin had no effect (P > 0.10) on NPY concentration in any group. Therefore, neither the interaction of pre- and postnatal nutrition nor exogenous leptin affected third ventricle NPY tone in this experimental model.

Efforts to understand biological functions and develop management schemes specific to Bos indicus-influenced cattle raised in tropical and subtropical environments are critical to meet the increasing global demand for protein. In the United States, B. indicus breeds are mostly used to generate B. indicus × B. taurus crosses with increased thermal and parasite tolerance, while retaining some productive characteristics of B. taurus cattle. Although crossbreeding represents a proven strategy to improve cattle adaptation almost immediately, research has also attempted to identify B. taurus genetics that can withstand subtropical and tropical climates. Reduced milk production and delayed reproductive maturation appear to be related with tropical adaptation of B. taurus breeds, as a means to conserve energy under stressful conditions and limited nutrition. Moreover, longevity may be the ultimate adaptation response to unfavorable environments, and retention of bulls and heifers from proven cows is the recommended strategy to improve longevity in B. indicus-influenced herds. Besides selection for longevity, other aspects should be considered when planning reproductive management in tropical and subtropical regions. Bos indicus and B. taurus breeds have multiple differences pertaining to reproductive function, including age at puberty, ovarian dynamics, and pregnancy development. Nutritional strategies such as the stair-step regimen, and use of exogenous progesterone (P4) inserts are options to hasten puberty attainment of late-maturing B. indicus-influenced heifers. Yet, limited pharmacological alternatives are available for reproductive management of B. indicus-influenced females in the United States, which rely on GnRH-based protocols not specifically designed to the reproductive function of B. indicus breeds. In contrast, hormonal protocols based on exogenous P4, estradiol esters, and equine chorionic gonadotropin are available for use in B. indicus females in South America. These include protocols tailored to prepubertal heifers, anestrous cows, and cycling nulliparous or parous females, which often yield pregnancy rates of 50% to fixed-time artificial insemination. The global dairy industry also faces similar challenges in increasing demand and production as the beef industry. Selection of cows capable of sustaining optimal milk yield, reproductive success, and health status in hot and humid conditions is essential for optimal dairy production in subtropical and tropical regions.

Puberty is a complex biological event that requires maturation of the reproductive neuroendocrine axis and subsequent initiation of high-frequency, episodic release of GnRH and LH. Nutrition is a critical factor affecting the neuroendocrine control of puberty. Although nutrient restriction during juvenile development delays puberty, elevated rates of body weight gain during this period facilitate pubertal maturation by programming hypothalamic centers that underlie the pubertal process. Recent findings suggest that maternal nutrition during gestation can also modulate the development of the fetal neuroendocrine axis, thus influencing puberty and subsequent reproductive function. Among the several metabolic signals, leptin plays a critical role in conveying metabolic information to the brain and, consequently, controlling puberty. The effects of leptin on GnRH secretion are mediated via an upstream neuronal network because GnRH neurons do not express the leptin receptor. Two neuronal populations located in the arcuate nucleus that express the orexigenic peptide neuropeptide Y (NPY), and the anorexigenic peptide alpha melanocyte-stimulating hormone (αMSH), are key components of the neurocircuitry that conveys inhibitory (NPY) and excitatory (αMSH) inputs to GnRH neurons. In addition, neurons in the arcuate nucleus that coexpress kisspeptin, neurokinin B, and dynorphin (termed KNDy neurons) are also involved in the metabolic control of puberty. Our studies in the bovine female demonstrate that increased planes of nutrition during juvenile development lead to organizational and functional changes in hypothalamic pathways comprising NPY, proopiomelanocortin (POMC, the precursor of αMSH), and kisspeptin neurons. Changes include alterations in the abundance of NPY, POMC, and Kiss1 mRNA and in plasticity of the neuronal projections to GnRH neurons. Our studies also indicate that epigenetic mechanisms, such as modifications in the DNA methylation pattern, are involved in this process. Finally, our most recent data demonstrate that maternal nutrition during gestation can also induce morphological and functional changes in the hypothalamic NPY system in the heifer offspring that are likely to persist long after birth. These organizational changes occurring during fetal development have the potential to not only impact puberty but also influence reproductive performance throughout adulthood in the bovine female.

Polycystic ovary syndrome (PCOS) is a complex disorder that results from a combination of multiple factors, including genetic, epigenetic, and environmental influences. Evidence from clinical and preclinical studies indicates that elevated intrauterine androgen levels increase the susceptibility of the female offspring to develop the PCOS phenotype. Additionally, early postnatal endocrine and metabolic imbalances may act as a “second-hit”, which, through activational effects, might unmask or amplify the modifications programmed prenatally, thus culminating in the development of adult disease. Animal models provide unparalleled resources to investigate the effects of prenatal exposure to androgen excess and to elucidate the etiology and progression of disease conditions associated with this occurrence, such as PCOS. In sheep, prenatal treatment with testosterone disrupts the developmental trajectory of the fetus, culminating in adult neuroendocrine, ovarian, and metabolic perturbations that closely resemble those seen in women with PCOS. Our longitudinal studies clearly demonstrate that prenatal exposure to testosterone excess affects both the reproductive and the metabolic systems, leading to a self-perpetuating cycle with defects in one system having an impact on the other. These observations in the sheep suggest that intervention strategies targeting multiple organ systems may be required to prevent the progression of developmentally programmed disorders.

Approximately 5 million beef heifers enter the U.S. cow herd annually, and their lifetime productivity is heavily-dependent upon their ability to attain puberty and produce a calf by 24 mo of age. However, a significant proportion of heifers within existing U.S. production systems fail to achieve these objectives. Therefore, approaches are needed that facilitate nutritional programming of the reproductive neuroendocrine axis, while minimizing feeding costs and optimizing the consistent establishment of puberty by 14–15 mo of age. Accelerated rates of BW gain during early calfhood, particularly between 4 and 9 mo of age, facilitate pubertal development by programming hypothalamic centers that regulate GnRH secretion. Among the different metabolic hormones, leptin plays a critical role in conveying nutritional information to the brain and controlling pubertal progression. Two hypothalamic neuronal populations that express the orexigenic peptide neuropeptide Y (NPY) and the anorexigenic peptide alpha melanocyte-stimulating hormone (αMSH) are key components of afferent pathways that convey inhibitory (NPY) and excitatory (αMSH) inputs to GnRH neurons. Our studies have demonstrated that short-term increases in dietary energy intake during juvenile development result in epigenetic, structural, and functional modifications in these hypothalamic pathways to promote high-frequency, episodic release of GnRH/LH. However, integrating the foundational knowledge of metabolic imprinting of the brain for early puberty with issues related to lifetime performance is complex. One approach has been to employ a novel stair-step nutritional regimen involving alternating periods of dietary energy-restriction and re-feeding during juvenile development. This approach is designed to support early onset of puberty by imprinting genomic, biochemical and morphologic alterations in the hypothalamus during key periods of brain development while optimizing other aspects of growth and performance. Finally, our recent findings suggest that maternal nutrition during gestation can also induce neuroendocrine changes that are likely to persist long after puberty and influence reproductive performance throughout adulthood in cattle.

Prenatal testosterone (T) treatment recapitulates the reproductive and metabolic phenotypes of polycystic ovary syndrome in female sheep. At the neuroendocrine level, prenatal T treatment results in disrupted steroid feedback on gonadotropin release, increased pituitary sensitivity to GnRH, and subsequent LH hypersecretion. Because prenatal T-treated sheep manifest functional hyperandrogenism and hyperinsulinemia, gonadal steroids and/or insulin may play a role in programming and/or maintaining these neuroendocrine defects. Here, we investigated the effects of prenatal and postnatal treatments with an androgen antagonist (flutamide [F]) or an insulin sensitizer (rosiglitazone [R]) on GnRH-stimulated LH secretion in prenatal T-treated sheep. As expected, prenatal T treatment increased the pituitary responsiveness to GnRH leading to LH hypersecretion. Neither prenatal interventions nor postnatal F treatment normalized the GnRH-stimulated LH secretion. Conversely, postnatal R treatment completely normalized the GnRH-stimulated LH secretion. At the tissue level, gestational T increased pituitary LHβ, androgen receptor, and insulin receptor-β, whereas it reduced estrogen receptor (ER)α protein levels. Although postnatal F normalized pituitary androgen receptor and insulin receptor-β, it failed to prevent an increase in LHβ expression. Contrarily, postnatal R treatment restored ERα and partially normalized LHβ pituitary levels. Immunohistochemical findings confirmed changes in pituitary ERα expression to be specific to gonadotropes. In conclusion, these findings indicate that increased pituitary responsiveness to GnRH in prenatal T-treated sheep is likely a function of reduced peripheral insulin sensitivity. Moreover, results suggest that restoration of ERα levels in the pituitary may be one mechanism by which R prevents GnRH-stimulated LH hypersecretion in this sheep model of polycystic ovary syndrome-like phenotype.

Polycystic ovary syndrome (PCOS) is a heterogeneous endocrine disorder with both reproductive and metabolic abnormalities affecting women of reproductive age. While the exact origin of PCOS is unknown, observations from clinical and animal studies suggest that maternal hyperandrogenism may be a contributing factor. Because women with PCOS manifest hyperandrogenism during pregnancy, changes in the gestational endocrine milieu may play a role in the vertical transmission of this syndrome. This review discusses the potential developmental origins of PCOS, the impact of maternal PCOS on the offspring’s health and contributions of the postnatal environment, capitalizing on findings from animal models that exhibit a PCOS-like phenotype. In addition, this review highlights the scarcity of data at early gestational stages in humans and the importance of animal experimentation to better understand the cellular and molecular mechanisms involved in the programming of adult diseases, therefore, helping identify therapeutic targets for preventive and treatment strategies.

Gestational testosterone (TS) excess, acting via both the androgenic and estrogenic pathways, advances puberty and disrupts the neuroendocrine estradiol (E2) feedback and periovulatory hormonal dynamics in female sheep. These prenatally programmed defects may be subject to postnatal modifications by continued organizational and/or activational effects of steroids. This study investigated (1) the organizational contribution of prenatal estrogen excess and (2) the impact of postnatal exposure to E2in modulating the effects of prenatal androgen excess (TS and dihydrotestosterone (DHT)) on puberty, neuroendocrine feedback mechanisms, and periovulatory hormonal dynamics in sheep. Pregnant Suffolk sheep were treated with TS, DHT, E2, or E2plus DHT (ED) from days 30 to 90 of gestation. A subset of the control (C), TS, and DHT female offspring received a constant-release E2implant postnatally. Findings revealed that (1) prenatal E2-treatment failed to reproduce the neuroendocrine disruptions predicted to be programmed by the estrogenic pathway and (2) prenatal E2D-treatment did not adequately replicate the reproductive neuroendocrine defects induced by prenatal TS excess. More importantly, continuous postnatal E2-treatment, while delaying the onset of puberty and reducing the inhibitory effects of E2on tonic luteinizing hormone (LH) release, failed to amplify the E2-positive feedback and periovulatory defects induced by prenatal TS-treatment. Our results indicate that disruptions in E2-positive feedback mechanisms and periovulatory gonadotropin secretion induced by prenatal TS-treatment are programmed predominantly during the prenatal life with postnatal exposure to E2excess not contributing further to these disruptions.

Accumulating evidence suggests that insults occurring during the perinatal period alter the developmental trajectory of the fetus/offspring leading to long-term detrimental outcomes that often culminate in adult pathologies. These perinatal insults include maternal/fetal disease states, nutritional deficits/excess, stress, lifestyle choices, exposure to environmental chemicals, and medical interventions. In addition to reviewing the various insults that contribute to developmental programming and the benefits of animal models in addressing underlying mechanisms, this review focuses on the commonalities in disease outcomes stemming from various insults, the convergence of mechanistic pathways via which various insults can lead to common outcomes, and identifies the knowledge gaps in the field and future directions.

We tested the hypotheses that in winter anovulatory mares (1) both chronic daily injections of estradiol-17β (E2) and subcutaneous E2 implants could enhance pituitary secretion of gonadotropins in response to continuous subcutaneous infusion of native gonadotropin-releasing hormone (GnRH); and (2) the secretory pattern of follicle-stimulating hormone (FSH) in response to continuous subcutaneous infusion of native GnRH is similar to that of luteinizing hormone (LH) but differs between mares that develop or fail to develop an estrogen-active, preovulatory follicle. In Experiment 1, 20 winter anovulatory mares (n = 5 per group) in early February received twice-daily injections of corn oil (control) or 5 mg of E2 with or without continuous subcutaneous treatment with native GnRH (100 μg/hr) or received GnRH only for up to 14 days. In Experiment 2, 24 winter anovulatory mares (n = 6 per group) were treated with a full-length (high dose) or quarter-length (low dose) E2 implant (Compudose) in combination with continuous GnRH infusion (100 μg/hr) for up to 28 days or served as sham controls. Mares developing 35-mm follicles were induced to ovulate with human chorionic gonadotropin. Mares not developing a 35-mm follicle within 14 days received a replacement 14-day GnRH pump. In Experiment 1, E2 enhanced the response to GnRH beginning on Day 3, with mean LH greater (P.001) in GnRH + E2 than in GnRH only and control mares. In Experiment 2, plasma E2 and estrone sulfate were increased in association with the development of a large (35 mm) follicle but did not increase in response to either E2 implant despite marked increases in uterine edema following their insertion. A sustained increase (P.0001) in plasma LH was observed in all GnRH-treated mares, but this effect was not modified by implant treatment. By Day 28, six of six GnRH, five of six GnRH + low E2, two of six GnRH + high E2, and zero of six control mares developed 35-mm follicles and were induced to ovulate. A marked increase (P.0001) in plasma FSH was observed within 24 hours in all GnRH-treated mares, returning to baseline by Day 4. In summary, twice-daily injection of 5 mg E2 enhanced pituitary secretion of LH in response to continuous administration of GnRH, but commercial E2 cattle implants failed to duplicate these effects. Continuous infusion of GnRH produced a differential but consistent pattern of FSH secretion (short-term increase) compared with LH (sustained increase). Differences in ovarian responses to GnRH treatment could not be explained by variation in gonadotropin secretion.

Prenatal exposure to excess steroids or steroid mimics can disrupt the normal developmental trajectory of organ systems, culminating in adult disease. The metabolic system is particularly susceptible to the deleterious effects of prenatal steroid excess. Studies in sheep demonstrate that prenatal exposure to excess native steroids or endocrine-disrupting chemicals with steroidogenic activity, such as bisphenol A, results in postnatal development of numerous cardiometabolic perturbations, including insulin resistance, increased adiposity, altered adipocyte size and distribution, and hypertension. The similarities in the phenotypic outcomes programmed by these different prenatal insults suggest that common mechanisms may be involved, and these may include hormonal imbalances (e.g., hyperandrogenism and hyperinsulinemia), oxidative stress, inflammation, lipotoxicity, and epigenetic alterations. Animal models, including the sheep, provide mechanistic insight into the metabolic repercussions associated with prenatal steroid exposure and represent valuable research tools in understanding human health and disease. Focusing on the sheep model, this review summarizes the cardiometabolic perturbations programmed by prenatal exposure to different native steroids and steroid mimics and discusses the potential mechanisms underlying the development of adverse outcomes.

Polycystic ovary syndrome (PCOS) is a heterogeneous syndrome characterized by abnormal reproductive cycles, irregular ovulation, and hyperandrogenism. This complex disorder has its origins both within and outside the hypothalamic-pituitary-ovarian axis. Cardio-metabolic factors, such as obesity and insulin resistance, contribute to the manifestation of the PCOS phenotype. Polycystic ovary syndrome is one of the most common endocrine disorders among women of reproductive age. Growing evidence suggested an association between reproductive and metabolic features of PCOS and exposure to endocrine-disrupting chemicals (EDC), such as bisphenol A. Further, the environmental obesogen tributyltin (TBT) was shown to induce reproductive, metabolic and cardiovascular abnormalities resembling those found in women and animal models of PCOS. However, the causal link between TBT exposure and PCOS development remains unclear. The objective of this review was to summarize the most recent research findings on the potential association between TBT exposure and development of PCOS-like features in animal models and humans.

Approximately 5 million beef heifers enter the U.S. cow herd annually and their lifetime productivity is heavily-dependent upon their ability to attain puberty and produce a calf by 24 mo of age. However, a significant proportion of heifers within existing U.S. production systems fail to achieve these goals, particularly in southern regions where Bos indicus-influenced cattle predominate. Therefore, approaches are needed that facilitate nutritional programming of the reproductive neuroendocrine axis, while minimizing feeding costs and optimizing the consistent establishment of puberty by 14–15 mo of age. Increased BW gain between 4 and 9 mo of age facilitates pubertal development by programming hypothalamic centers that regulate GnRH secretion. Among the different metabolic hormones, leptin plays a critical role in conveying nutritional information to the brain and controlling puberty. Two hypothalamic neuronal populations that express the orexigenic peptide neuropeptide Y (NPY) and the anorexigenic peptide alpha melanocyte-stimulating hormone (αMSH) are key components of afferent pathways that convey inhibitory (NPY) and excitatory (αMSH) inputs to GnRH neurons. Our studies have demonstrated that short-term increases in dietary energy intake during juvenile development result in epigenetic, structural, and functional modifications in these hypothalamic pathways to promote high-frequency, episodic release of GnRH/LH. However, integrating the foundational knowledge of metabolic imprinting of the brain for early puberty with issues related to lifetime performance is complex. One approach has been to employ a novel stair-step nutritional regimen involving alternating periods of dietary energy-restriction and re-feeding during juvenile development. This approach is designed to support early onset of puberty by imprinting functional alterations in the hypothalamus during key periods of brain development while optimizing other aspects of growth and performance. Finally, our recent findings suggest that maternal nutrition during gestation can also induce neuroendocrine changes that are likely to persist and influence reproductive performance throughout adulthood in cattle.

Nutrition during gestation and early postnatally can program developmental changes in the offspring that persist until adult life. Here we tested the hypotheses that maternal nutritional during the second and third trimesters of gestation 1) affects neonatal secretion of leptin in heifer offspring, and 2) interacts with dietary energy intake during the juvenile period to affect age at puberty and postpubertal pulsatile secretion of luteinizing hormone (LH) in heifers. Bos indicus-influenced beef cows (n = 108) in 3 replicates bearing heifer fetuses were assigned randomly to receive low (L), moderate (M), or high energy (H) diets to achieve body condition scores (BCS) of 3–3.5 (thin; n = 36), 5.5–6 (moderate; n = 36), or 7.5–8 (obese; n = 36) by the second trimester of gestation until calving. Heifer offspring were weaned at 3–3.5 months of age and assigned randomly to either a high- (H; 1 kg/day) or low-gain (L; 0.5 kg/day) diet for 5 months. Blood samples from a subgroup of 30 heifers (Exp. 1) from cows in each maternal treatment (n = 10/treatment) were collected once every 2–3 days for 2 weeks after birth. In Exp. 2, 18 heifers representing 3 of the maternal × postnatal groups (HH, MH and LL) were ovariectomized and received estradiol replacement (OVX+E) after puberty. Pulsatile secretion of LH was evaluated for 5.5 hours. Maternal nutrition did not affect postnatal circulating leptin. Based on two replicates (n = 61), postnatal diets had the greatest effect on age at puberty (L > H; P < 0.001), with a lesser maternal diet effect (P < 0.10), although LL > HH by 99 days. None of the characteristics of pulsatile LH secretion differed among treatments in OVX + E heifers. Dietary effects on age at puberty were not associated with hormonal characteristics evaluated in these initial studies.

Embryonic mortality (EM) is a major factor limiting pregnancy rates in cattle and occurs early (< day 28) or late (≥ day 28) during gestation. However, causes and mechanisms associated with late EM remain unknown. Despite negative connotations related to pregnancy, prostaglandin F2α (PGF) is capable of being released by the bovine uterus between days 28 and 32 of gestation. Therefore, the objective was to evaluate differences in PGF release between cows with high circulating concentrations of pregnancy associated glycoproteins (PAG) vs low PAG following an oxytocin (OT) challenge. Increased concentrations of PAG are associated with pregnancy success. At day 29 of gestation, pregnant cows were divided into high (n = 10) and low (n = 10) PAG groups and received either 100 I. U. of oxytocin intramuscularly (OT) or saline (control). Blood samples were collected every 30 minutes beginning 1 hour before and continuing for 4 hours after treatment. Prostaglandin F2α metabolite (PGFM), progesterone (P4), estradiol (E2) and PAG concentrations were determined. Peak concentration of PGFM occurred 2 hours after OT injection (average high PAG group peak: 345.6 ± 73.6 pg/mL; low PAG group peak: 326.4 ± 61.4 pg/mL, P > 0.05) and returned to baseline levels by 4 hours. No correlations were observed between PAG and PGFM concentrations (P > 0.05). There was no difference in initial or final PGFM concentrations between high or low PAG groups and control animals (P > 0.05). Furthermore, there was no difference in P4 or E2 concentration between treatment and control animals (P > 0.05) despite a luteolytic release of PGF2α. In summary, cows with high vs low PAG concentrations at day 30 of gestation have a similar PGFM response to OT challenge. This project supported by Agriculture and Food Research Initiative Competitive Grant no. 2017-67015-26457 from USDA-NIFA.

Prenatally testosterone (T)-treated sheep present metabolic disruptions similar to those seen in women with polycystic ovary syndrome. These females exhibit an increased ratio of small to large adipocytes, which may be the earliest event in the development of adult insulin resistance. Additionally, our longitudinal studies suggest the existence of a period of compensatory adaptation during development. This study tested whether 1) in utero cotreatment of prenatally T-treated sheep with androgen antagonist (flutamide) or insulin sensitizer (rosiglitazone) prevents juvenile insulin resistance and adult changes in adipocyte size; and 2) visceral adiposity and insulin sensitivity are both unaltered during early adulthood, confirming the predicted developmental trajectory in this animal model. Insulin sensitivity was tested during juvenile development and adipose tissue distribution, adipocyte size, and concentrations of adipokines were determined during early adulthood. Prenatal T-treated females manifested juvenile insulin resistance, which was prevented by prenatal rosiglitazone cotreatment. Neither visceral adiposity nor insulin sensitivity differed between groups during early adulthood. Prenatal T-treated sheep presented an increase in the relative proportion of small adipocytes, which was not substantially prevented by either prenatal intervention. A large effect size was observed for increased leptin concentrations in prenatal T-treated sheep compared with controls, which was prevented by prenatal rosiglitazone. In conclusion, gestational alterations in insulin-glucose homeostasis likely play a role in programming insulin resistance, but not adipocyte size distribution, in prenatal T-treated sheep. Furthermore, these results support the notion that a period of compensatory adaptation of the metabolic system to prenatal T exposure occurs between puberty and adulthood.

The timing of puberty and subsequent fertility in female mammals are dependent on the integration of metabolic signals by the hypothalamus. Pro-opiomelanocortin (POMC) neurones in the arcuate nucleus (ARC) comprise a critical metabolic-sensing pathway controlling the reproductive neuroendocrine axis. α-Melanocyte-stimulating hormone (αMSH), a product of the POMC gene, has excitatory effects on gonadotrophin-releasing hormone (GnRH) neurones and fibres containing αMSH project to GnRH and kisspeptin neurones. Because kisspeptin is a potent stimulator of GnRH release, αMSH may also stimulate GnRH secretion indirectly via kisspeptin neurones. In the present work, we report studies conducted in young female cattle (heifers) aiming to determine whether increased nutrient intake during the juvenile period (4-8 months of age), a strategy previously shown to advance puberty, alters POMC and KISS1 mRNA expression, as well as αMSH close contacts on GnRH and kisspeptin neurones. In Experiment 1, POMC mRNA expression, detected by in situ hybridisation, was greater (P