Evaluation of estrous synchronization protocols on ewe reproductive efficiency and profitability.

A 2-year experiment was conducted to determine the effect of a single injection of prostaglandin after initiation of the breeding season on ewe estrous synchronization. Rambouillet ewes (n = 101; Year 1 = 52; Year 2 = 49) assigned to one of three treatments: untreated (CON); 12-d CIDR insert (CIDR); or 1 injection of prostaglandin at d 2.5 (1 PG) after rams were placed with ewes. Rams were placed with ewes at the time of CIDR removal (d 0) and remained with ewes during a 35-d breeding season. Both the CIDR- (94%) and 1 PG (73.5%) treatment groups had a larger number (P ≤ 0.01) of ewes bred in the first 5 d of the breeding season compared to ewes of the control (33%) group. As expected, CIDR-treated ewes had a shorter time to mating, than 1 PG-treated ewes and ewes of the control group had a longer interval to mating than both CIDR- and 1 PG-treated ewes (P ≤ 0.01). The number of lambs born per ewe and kg of lamb weaned per ewe was not different (P ≥ 0.31) among treatment groups. Additionally, there was no difference (P =  0.78) in net profit per ewe among treatment groups. Based on these data, utilizing a single injection of PG 2.5 d after initiation of the breeding season resulted in similar pregnancy rates at d 5 of the breeding season when compared with CIDR-treated ewes indicating the potential utilization of the 1PG protocol in a confinement setting as a viable method for estrous synchronization.

ASAS-SSR Triennial Reproduction Symposium: Looking Back and Moving Forward-How Reproductive Physiology has Evolved: WNTs role in bovine folliculogenesis and estrogen production.

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. ß-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.

Canonical WNT signaling inhibits follicle stimulating hormone mediated steroidogenesis in primary cultures of rat granulosa cells.

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.

Evaluation of steroidogenic capacity after follicle stimulating hormone stimulation in bovine granulosa cells of Revalor 200® implanted heifers.

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.

Effects of supplementary selenium source on the blood parameters in beef cows and their nursing calves.

Over 2 years, 32 beef cows nursing calves in southwest Arkansas were randomly selected from a herd of 120 that were managed in six groups and were assigned to six 5.1-ha bermudagrass (Cynodon dactylon [L.] Pers.) pastures. Treatments were assigned to pastures (two pastures/treatment) and cows had ad libitum access to one of three free-choice minerals: (1) no supplemental selenium (Se), (2) 26 mg of supplemental Se from sodium selenite per kilogram, and (3) 26 mg of supplemental Se from seleno-yeast per kilogram (designed mineral intake = 113 g/cow daily). Data were analyzed using a mixed model; year and pasture were the random effects and treatment was the fixed effect. At the beginning of the calving and breeding seasons, cows supplemented with Se had greater (P < 0.01) whole blood Se concentration (WBSe) and glutathione peroxidase activities (GSH-Px) than cows receiving no supplemental Se; cows fed seleno-yeast had greater (P ≤ 0.05) WBSe than cows fed sodium selenite, but GSH-Px did not differ (P ≥ 0.25) between the two sources. At birth and near peak lactation (late May), calves from cows supplemented with Se had greater (P < 0.01) WBSe than calves from cows fed no Se and calves from cows fed seleno-yeast had greater (P ≤ 0.01) WBSe and GSH-Px than calves from cows fed sodium selenite. Thyroxine (T4), triiodothyronine (T3), and the T4:T3 ratio in calves did not differ among treatments (P ≥ 0.35). At birth, insulin-like growth factor 1 (IGF-1) was greater (P = 0.02) in calves nursing cows with no supplemental Se than in ones with supplemental Se; in calves nursing cows with supplemental sodium selenite, IGF-1 did not differ (P = 0.96) from ones offered supplemental seleno-yeast. Selenium supplementation of gestating beef cows benefited cows and calves by increasing WBSe and GSH-Px. The use of seleno-yeast as a Se supplement compared to sodium selenite increased the WBSe of both cows and their calves without affecting the T4 to T3 conversion or IGF-1 concentrations.

Validation of a fecal glucocorticoid metabolite assay to assess stress in the budgerigar (Melopsittacus undulatus).

The budgerigar (Melopsittacus undulatus) is a small parrot native to Australia that is commonly held in zoos, laboratories, and private homes. Assessment of budgerigar stress levels would aid welfare monitoring and improve our understanding of their biology. Analyzing fecal glucocorticoid metabolites provides a noninvasive method to measure stress levels in birds. For this method to be reliable, the antibody to be used in an immunoassay must be carefully selected for each species, and validation must be performed. A common limitation in many existing assays is the inability to accurately detect variable fecal glucocorticoid metabolites in minute quantities of feces, requiring small samples to be combined. We have developed a double antibody radioimmunoassay protocol based on a commercially available (125) I-corticosterone radioimmunoassay kit for use in detecting fecal glucocorticoid metabolites in small quantities (<20 mg) of budgerigar droppings. The assay was validated pharmacologically with an adrenocorticotropic hormone challenge and with oral administration of corticosterone. Our validation has demonstrated our assay is both sensitive and a reliable approach to noninvasive monitoring of stress in budgerigars.

Effects of exogenous melatonin and tryptophan on fecal shedding of E. coli O157:H7 in cattle.

Fecal prevalence of Escherichia coli O157 in ruminants is highest in the summer decreasing to very low levels in the winter. We hypothesize that this seasonal variation is a result of physiological responses within the host animal to changing day-length. To determine the effects of melatonin (MEL) on fecal shedding of E. coli O157:H7 in cattle, eight crossbred beef steers identified as shedding E. coli O157:H7, were allotted to treatment: control or MEL (0.5 mg/kg body weight (BW); 1x) administered orally daily for 7 days. After a 5-day period of no treatment, a second MEL dose (5.0 mg/kg BW; 10x) was administered daily for 4 days. Fecal samples were collected daily for qualification of E. coli O157:H7. No differences (P > 0.10) were observed in the percentage of E. coli O157:H7 positive fecal samples in steers receiving the 1x MEL dose, however the 10x dose decreased (P = 0.05) the percentage of fecal samples E. coli O157:H7 positive. Serum MEL concentrations were higher in the 1x, but not 10x, treated animals compared to control animals. Although it is difficult to explain, this may be a result of decreasing day-length increasing serum melatonin concentrations that may have masked any treatment effect on serum melatonin. In a second similar experiment, a second group of cattle (heifers and steers) were administered tryptophan (TRP) over a 17-day experimental period (5 g/head/day for 10 days followed by 10 g/head/day for 7 days). Tryptophan had no effect (P > 0.20) on the percentage of fecal samples positive for E. coli O157. Serum TRP (P < 0.05), but not MEL (P > 0.20), concentrations were elevated in TRP-treated animals. The decrease in the number of positive fecal samples observed in the first experiment, may be related to gastrointestinal MEL, affected by the 10x, but not 1x MEL dose.

Influence of exogenous triiodothyronine (T3) on fecal shedding of Escherichia coli O157 in cattle.

Fecal prevalence of Escherichia coli O157 in ruminants is highest in the summer months and decreases to low or undetectable levels in the winter. We hypothesize that the seasonal variation of this pathogen is a result of physiological responses within the host animal to changing day length. The thyroid is an endocrine gland known to respond to changing day length. Two experiments were conducted to determine if a hyperthyroid status would initiate fecal shedding of E. coli O157 in cattle during the winter when shedding is virtually nonexistent (winter experiment) or influence cattle actively shedding E. coli O157 (summer experiment). Yearling cattle were group-penned under dry-lot conditions, adjusted to a high concentrate ration, and randomly assigned to treatment: control (1 mL corn oil injected s.c. daily) or triiodothyronine (T(3); 1.5 mg suspended in corn oil injected s.c daily). Cattle were individually processed daily for collection of fecal and blood samples. Treatment with exogenous T(3) produced a significant change in serum thyroid hormone concentrations indicative of a hyperthyroid status in both experiments. No differences (P>0.10) were observed in fecal shedding of E. coli O157 in the winter experiment. In the summer experiment, fecal shedding of E. coli O157 was decreased (P=0.05) by administration of T(3) during the treatment period (days 1-10), tended to be lower (P=0.08) during the following 7-day period of no treatment, and was lower (P=0.01) when examined across the entire experimental period. Results of this research indicate that the thyroid or its hormones may be involved in the seasonal shedding patterns of E. coli O157 in cattle.

Influencia de la condición corporal y la bioestimulación sobre la eficacia reproductiva en vacas de raza Simmental en agostadero / Influence of body condition and biostimulation on the reproductive efficiency in Simmental beef cows grazing

En este experimento se evaluó el efecto de la condición corporal al parto y la bioestimulación con toros, sobre la eficiencia reproductiva mediante la duración del anestro posparto y la tasa de preñez al sincronizar estros. Se utilizaron 73 vacas de raza Simmental, manejadas en agostadero. Los tratamientos se asignaron bajo un diseño completamente al azar, con arreglo factorial de 2 x 2, con covarianza. Los factores fueron: Condición corporal alta o baja (CCA o CCB), con o sin bioestimulación con toros (CT o ST). Los tratamientos (t) fueron: t1, CCA-CT; t2; CCA-ST; t3, CCB-CT; y t4, CCB-ST. Se analizó progesterona (P4) en suero sanguíneo por técnica RIA, para determinar el inicio de la actividad cíclica. Se palparon ovarios para detectar el primer cuerpo lúteo posparto (CL). La actividad cíclica inició a las semanas seis, ocho, 11 y 15 posparto (P < 0.01), para vacas del t1 al t4, respectivamente. Las vacas se sincronizaron con norgestomet más valerato de estradiol para iniciar el empadre con IA. La preñez al primer servicio por inseminación artificial (IA) fue de 46 por ciento y 24 por ciento (P < 0.05), para vacas con CCA y CCB, respectivamente; 46 por ciento y 25 por ciento (P < 0.05), para vacas CT y ST; y de 63 por ciento, 30 por ciento, 28 por ciento y 19 por ciento (P < 0.05), para los tratamientos uno al cuatro, respectivamente. La preñez en empadre de 60 días fue de 84 por ciento, 70 por ciento, 61 por ciento y 44 por ciento (P < 0.05), para el t1 al t4, respectivamente. En conclusión, la duración del anestro posparto se redujo a 62 días en vacas con moderada condición corporal (CC ü 5.5) y bioestimuladas con toros inmediatamente después del parto. La tasa de preñez mejoró al primer servicio (IA) en 44 por ciento y en empadre de 60 días en 40 por ciento.