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Beta-catenin (CTNNB1), a key component of wingless-type mouse mammary tumor virus integration site family (WNT) signaling, participates in follicle stimulated hormone-mediated regulation of estrogen (E2) production. The purpose of these studies was to determine if CTNNB1's contribution to FSH-mediated steroidogenesis in primary rat granulosa cells was due in part to extracellular stimulation of the canonical WNT signaling pathway. To achieve this purpose, primary cultures of rat granulosa cells were exposed to vehicle or a canonical member of the WNT signaling pathway, WNT3A, before co-culture and in the presence or absence of FSH for 24 h. Activation of the canonical WNT signaling pathway was determined by dose-dependent induction of Axin2 mRNA expression and stimulation of the CTNNB1/T cell factor promoter-reporter TOPflash. WNT pathway induction was demonstrated at doses of 50 and 500 ng/mL of WNT3A. Granulosa cells treated with WNT3A in combination with FSH had enhanced CTNNB1/T cell factor transcriptional activity above cells treated with WNT3A alone. Steroidogenic enzymes and ovarian differentiation factor mRNAs were quantified via quantitative PCR. Expression of steroidogenic enzyme mRNAs aromatase (Cyp19a1), P450 side chain cleavage (Cyp11a1), and steroidogenic acute regulatory protein (Star) were increased following FSH treatment. Co-incubation of WNT3A and FSH reduced the ability of FSH to stimulate steroidogenic enzymes and subsequent E2 and progesterone (P4) production. Concomitant activation of FSH and WNT pathways results in marked reduction of ovarian differentiation factors, LH receptor (Lhcgr) and inhibin-alpha (Inha). Therefore, WNT inhibits FSH target genes and steroid production associated with maturation and differentiation of the ovarian follicle.

Background： Heifers not used as breeding stock are often implanted with steroids to increase growth efficiency thereby altering hormone profiles and potentially changing the environment in which ovarian follicles develop. Because bovine granulosa cell culture is a commonly used technique and often bovine ovaries are collected from abattoirs with no record of implant status, the objective of this study was to determine if the presence of an implant during bovine granulosa cell development impacts follicle stimulating hormone-regulated steroidogenic enzyme expression. Paired ovaries were collected from 16 feedlot heifers subjected to 1 of 3 treatments： non-implanted （n = 5）, Revalor 200 for 28 d （n = 5）, or Revalor 200 for 84 d （n = 6）. Small follicle （1 to 5 mm） granulosa cells were isolated from each pair and incubated with phosphate buffered saline （n = 16） or 100 ng/mL follicle stimulating hormone （n = 16） for 24 h. Results： Granulosa cells of implanted heifers treated with follicle stimulating hormone produced medium concentrations of progesterone similar （P = 0.22） to non-implanted heifers, while medium estradiol concentrations were increased （P 〈 0.10） at 28 and 84 d compared to non-implanted heifers indicating efficacy of treatment. Additionally, real-time PCR analysis in response to follicle stimulating hormone treatment demonstrated a decrease in steroidogenic acute regulatory protein （P = 0.05） mRNA expression in heifers implanted for 84 d and an increase in P450 side chain cleavage mRNA in granulosa cells of heifers implanted for 28 （P 〈 0.10） or 84 d （P 〈 0.05） compared to non-implanted females. However, no difference in expression of 3-beta-hydroxysteroid dehydrogenase （P= 0.57） and aromatase （P = 0.23） were demonstrated in implanted or non-implanted heifers. Conclusions： These results indicate follicles which develop in the presence of high concentrations of androgenic and estrogenic steroids via an implant tend to demonstrate an altered capacity to respond to follicle stimulating hormone stimulation. Thus, efforts should be made to avoid the use of implanted heifers to study steroidogenesis in small follicle granulosa cell culture systems.

Sensor technologies can identify modified animal activity indicating changes in health status. This study investigated sheep behavior before and after illness caused by mold-contaminated feed using tri-axial accelerometers. Ten ewes were fitted with HerdDogg biometric accelerometers. Five ewes were concurrently fitted with Axivity AX3 accelerometers. The flock was exposed to mold-contaminated feed following an unexpected ration change, and observed symptomatic ewes were treated with a veterinarian-directed protocol. Accelerometer data were evaluated 4 days before exposure (d −4 to −1); the day of ration change (d 0); and 4 days post exposure (d 1 to 4). Herddogg activity index correlated to the variability of minimum and standard deviation of motion intensity monitored by the Axivity accelerometer. Herddogg activity index was lower (p < 0.05) during the mornings (0800 to 1100 h) of days 2 to 4 and the evening of day 1 than days −4 to 0. Symptomatic ewes had lower activity levels in the morning and higher levels at night. After accounting for symptoms, activity levels during days 1 to 4 were lower (p < 0.05) than days −4 to 0 the morning after exposure. Results suggest real-time or near-real time accelerometers have potential to detect illness in ewes.

Abstract The objective of this study was to evaluate the impacts of stress challenge duration on metabolic, hematologic, and rumen fermentation parameters of beef steers. Thirty steers (416 ± 19 kg) were used in a randomized complete block design with 2 blocks and 3 treatments. Treatments were intravenous injection of (1) saline at 0, 24, 48, and 72 h (n = 10; control); (2) corticotropin-releasing hormone (CRH) and vasopressin (VP) at 0 h and saline at 24, 48, and 72 h (n = 10; acute); or (3) CRH and VP at 0, 24, 48, and 72 h (n = 10; chronic). Serum samples were collected at various time points for analysis of serum chemistry and nonesterified fatty acids (NEFA). Whole blood was collected for analysis of complete blood count, and ruminal fluid was collected via oral lavage to evaluate volatile fatty acid (VFA) composition. Serum cortisol was greater (treatment × hour; P ≤ 0.01) for cattle receiving acute and chronic than control at 1 h (P < 0.01) and greater for chronic than acute and control at 25, 26, 49, 50, 73, and 74 h (P < 0.01). Similarly, there was a treatment × hour interaction (P < 0.01) for serum glucose concentrations such that cattle receiving acute and chronic had greater glucose at 1 h than control (P < 0.01), and cattle receiving chronic had greater glucose at 25, 49, 73, and 74 h than acute and control (P ≤ 0.04). Serum insulin concentrations were greater (treatment × hour P < 0.01) in chronic and acute than control at 1 h (P < 0.01) and greater for chronic compared to acute and control at 25, 49, and 73 h (P ≤ 0.01). Serum NEFA tended (P = 0.09) to be greater in stressed cattle compared to control. There was a treatment × hour interaction (P = 0.003) for total white blood cell count such that chronic had greater concentration than control at 72 h (P < 0.01). Conversely, monocyte concentration was less (treatment × hour interaction P < 0.01) for chronic than acute and control at 144 h (P < 0.01) and eosinophil concentration was greater (treatment × hour interaction P = 0.02) for chronic than control steers at 48 h (P = 0.02) and greater for chronic than acute at 72 and 144 h (P ≤ 0.03). Minimal differences were observed in VFA concentrations with the exception of acetate (treatment × hour interaction P = 0.05). These results demonstrate that administration of CRH and VP affects complete blood count and serum chemistry, and longer duration of treatment exposure prolongs the physiological responses to a stress challenge. Exposing cattle to acute and chronic stress influences biological indicators of nutritional and immunological status. Lay Summary In livestock, exposure to stress initiates a series of biological signaling cascades that influence the animal’s nutritional and immunological status. The duration of time an animal is exposed to stress may influence the magnitude of the response and determine if the resulting changes are beneficial or detrimental. Our study evaluated if biological responses to a stress challenge model using exogenous hormones are impacted differently if cattle are exposed to short-term (acute), long-term (chronic), or negative control (no stress hormones) treatments. Cattle that received a stress challenge had increased cortisol, glucose, and insulin concentrations. The stress challenge also influenced total white blood cell, monocyte, and eosinophil concentrations. Rumen fermentation end products were minimally influenced by stress treatment, but stress reduced the liquid passage rate. Therefore, the administration of stress hormones influenced biological indicators of nutritional and immunological function of growing steers. Additionally, administration of a chronic stress challenge resulted in longer alteration of some variables compared to steers that were acutely stressed.

In 1953, Sir Peter Medawar first recognized the allogeneic properties of a developing conceptus and rationalized that an \"immune-tolerant\" physiological state must exist during pregnancy. Early theories speculated that the conceptus evaded the maternal immune system completely, but 40 yr after Medawar's observations, Wegmann proposed that the maternal immune system shifts the cytokine profile away from inflammatory cytokine production when an embryo is present. The economic consequences and production losses of subfertile animals have been well documented in studies evaluating calving distribution. Despite advances in understanding infertility or subfertility, few technologies exist to identify subfertile animals or improve fertility beyond hormonal intervention associated with synchronization protocols. Work in rodents and some livestock species indicates that the uterine immune cell population shifts dramatically after copulation and these early immune-modulated events establish a receptive uterine environment. Clearly, as evident in embryo transfer, the presence of a conceptus is sufficient to establish communication for pregnancy establishment but does not rule out the importance of other physiological events to prime the maternal immune system prior to blastocyst arrival in the uterus. In support of this concept, work in our laboratory and by others has demonstrated that autologous intra-uterine transfer of peripheral immune cells prior to embryo transfer can increase pregnancy rates and accelerate conceptus development in women and cattle. Understanding aberrant immune regulation in subfertile animals may provide markers for subfertility or targets for clinical intervention to enhance fertility, particularly when using reproductive technologies.

Appreciation of mechanisms that affect steroidogenesis is critical to identifying compromising signals that may decrease reproductive efficiency. Follicle maturation and steroidogenesis requires coordinated actions from the pituitary gonadotropins and local ovarian signaling molecules. β-Catenin (CTNNB1), the lynchpin molecule of canonical wingless-type mouse mammary tumor virus integration site (WNT) signaling, is required for maximal gonadotropin stimulation of steroid production from granulosa (GC) and luteal cells. WNTs are locally secreted glycoproteins involved in ovarian development and folliculogenesis. In cultured bovine GC, WNT2 and AKT mRNAs and CTNNB1 protein increase after FSH stimulation. Likewise, CTNNB1 protein is greater in large antral follicles with high intrafollicular estradiol concentrations, suggesting the hormonal milieu responsible for increased estradiol content modulates CTNNB1 accumulation. In addition, concurrent treatment of FSH and WNT3A in GC results in reduced steroidogenic enzymes and ovarian differentiation factors. It is likely that FSH regulation of WNT signaling establishes a negative feedback loop to ensure CTNNB1 remains controlled. To explore the mechanism resulting in this inhibitory effect, AKT pathway modulators were utilized and unveiled a requirement for AKT activity in FSH-mediated CTNNB1 accumulation. Cells treated with FSH, IGF-1, and IGF-1 + FSH had increased CTNNB1 protein accumulation compared with controls. Similarly, estradiol medium concentrations increased in treated cells compared with non-treated controls, while co-treatment of FSH and IGF-1 with the AKT inhibitor LY294002 reduced CTNNB1 and estradiol production. Subsequent studies evaluated whether FSH regulation of CTNNB1 occurs through a specific phosphorylation event. In bovine GC, phosphorylation of CTNNB1 at Ser-552 was demonstrated in FSH-treated cells, whereas IGF-1 treatment did not phosphorylate CTNNB1 Ser-552. Data indicate that in cattle phosphorylation on CTNNB1 Ser-552 is a protein kinase A (PKA) dependent, protein kinase B (AKT) independent event. Data suggest that CTNNB1 regulated by AKT is a fundamental component of FSH-induced estrogen production. However, AKT's role in estradiol synthesis does not appear to be through phosphorylation of CTNNB1 Ser-552. The complex interplay between FSH and ovarian WNT/CTNNB1 signaling is key to regulation of follicle maturation and steroidogenesis.