Cycle In Sheep Research Articles

Unveiling the Role of IGF-I in Fertility: Effect of Long-Acting Bovine Somatotropin (bST) on Terminal Follicular Development and Fertility during an Annual Reproductive Cycle in Sheep.

The study aimed to assess the effect of long-acting bST treatment, in a dose that only increases IGF-I plasma concentrations, on ovarian and fertility markers of estrous synchronized ewes that were fed to keep their bodyweight. Three experiments were designed to evaluate this effect: in Experiment 1, 18 ewes were distributed in groups (bST 0, 30, 50 mg) to measure plasma IGF-I and insulin for 15 days; in Experiment 2, 92 ewes (5 replicates) in two groups (0 and 30 mg bST) were synchronized using a 6-day progesterone protocol during the breeding season to assess the effect of bST on follicular and luteal performances, estrous and ovulation, and fertility after mating. In Experiment 3, 50 ewes (3 replicates) were used to repeat the study before but during anestrus. Results indicate that 50 mg bST increased IGF-I and insulin plasma concentrations, but 30 mg bST only increased IGF-I concentrations; and that only during the breeding season did 30 mg bST increase the number of lambs born and the reproductive success of ovulatory-sized follicles compared to controls. This occurred without it affecting any other reproductive marker. In conclusion, 30 mg bST treatment may improve oocyte competence for fertility during the breeding season.

Association of faecal volatiles and steroids with behavioural expression during oestrous cycle of sheep (Ovis aries).

This study was investigated to determine the faecal volatiles, steroid hormones and their correlation with behavioural signs during oestrous cycle in sheep (Ovis aries). This experiment was monitored from pro-oestrous to met-oestrous phases for correlation of endocrine dependence biochemical constituents in faeces and blood to detection of oestrous biomarkers. Medroxyprogesterone acetate sponges were used for 8 days to bring uniformity of oestrus in sheep. Faeces were collected during different phases of cycle and subjected to determination of fatty acids, minerals, oestrogens and progesterone analysis. Likewise, blood samples were also obtained for enzymatic and no-enzymatic antioxidants. The results revealed that faecal progesterone and oestrogen levels significantly increased during pro-oestrous and oestrous phase, respectively (p < .05). The blood plasma enzymatic levels showed significant demarcation during the oestrous phase than other periods (p < .05). There were also reported marked variations in volatile fatty acids across different phases of the oestrous cycle. Interestingly, methyl octanoate, methyl cis-10 pentadecenoate and methyl heptadecanoate volatiles were found to be specific to oestrus period. In addition, methyl hexanoate, methyl palmitoleate and methyl cis-9 oleate were identified in met-oestrus, indicating their possible role to be an oestrous biomarker. It is concluded that the pattern of volatile compounds and faecal steroids, in association with behavioural patterns, could be measured as a non-invasive method of heat detection in sheep.

Comparative analysis of the hormone production and gene expression profiles in ovine uterus tissue during oestrus cycle synchronized using medroxyprogesterone acetate plus eCG and prostaglandin analogue

The combination of medroxyprogesterone acetate (MPA) and gonadotropin chorionic (eCG) has been widely used to synchronize oestrus cycle in sheep, but their effects on the gene expression in uterine tissue are yet to be elucidated. To evaluate the effect of MPA + eCG or prostaglandin analogue (PA) treatments on the rate of oestrus cycle synchronization, as well as further hormone production and gene expression profiles in uterine tissue, 14 Santa Inês ewes were randomly selected. The MPA + eCG group (n=7) received intravaginal insertion of MPA-impregnated sponges for 14 days and was administered 350 IU eCG on the day of sponge withdrawal. The PA group (n=7) was administered two doses of 100 μg of PA separated by 12 days. The ewes were assessed for the rate of oestrus cycle synchronization and the serum concentrations of progesterone (P4) and estradiol (E2). Additionally, the expression of estrogen receptor (ERα), progesterone receptor (P4R), and immunolocalization of interferon receptor (IFNAR1) in the uterine tissue samples collected 15th day post-mating were examined. The rate of oestrus cycle synchronization was 100% (n=7/7) and 57.14% (n=4/7) in the MPA + eCG and PA groups, respectively. Moreover, the MPA + eCG group exhibited higher serum concentration of P4 than the PA group (p &lt; 0.05). However, the E2 serum concentration did not differ between the two groups (p &gt; 0.05). The relative expression of P4R and ERα mRNA analyzed using real-time PCR and immunodetection of IFNAR1 were similar between the two groups tested (p &gt; 0.05). Conclusively, MPA + eCG treatment improved the rate of oestrus cycle synchronization and endogenous P4 production; however, it did not affect the expression of sex steroid receptors and IFNAR1 in uterine ovine tissue.

Expression and functional analysis of the Follistatin-like 3 (FSTL3) gene in the sheep ovary during the oestrous cycle.

Follistatin‐like 3 (FSTL3) is a regulator of cellular apoptosis and was previously identified via RNA‐Seq to be associated with follicular development in mammalian ovaries. However, the mechanism underlying the FSTL3 regulation of oestrus in sheep remained poorly understood. In this study, the oestrogen (E2) and progesterone (P4) concentrations in blood were detected, and the expression level and functional analysis of FSTL3 in the ovary were studied during the different reproductive stage in Aohan fine wool sheep (seasonal breeding breed in China). The concentrations of E2 and P4 at the anestrus were significantly lower compared to dioestrus, proestrus and oestrus stages. Higher expression levels of FSTL3 were observed in the sheep ovary, hypothalamus, and thyroid. During different reproductive stages, higher expression levels were found during the stages of dioestrus and proestrus, while lower levels were found during the oestrus and anestrus stages. Functional analysis of FSTL3 was performed in primary granulosa cells (GCs) of sheep. The concentration of E2 increased significantly after RNAi interference of FSTL3, while the P4 level decreased. FSTL3 can decrease P4 levels, which might be involved in mediating oestrous cycle in sheep.

Direct and correlated responses to selection for autumn lambing in sheep

BackgroundSeasonal reproduction limits productivity, flexibility, and profitability in commercial sheep production. Hormonal and (or) photoperiodic manipulation can be used to control estrous cycles in sheep and reduce limitations that are imposed by the seasonal anestrous but are often impractical or incompatible with the extensive management systems preferred for ruminant livestock. Thus, the current study investigated the use of selection to improve realized fertility (i.e., the proportion of ewes that lambed) following an out-of-season spring joining period (May and June) in a crossbred sheep population.ResultsOver 17 years, estimated breeding values (EBV) for fertility in selected (S) ewes increased by 0.175 (0.01 per year). The mean EBV for fertility of S ewes was greater than that of control ewes by year 10 (P = 0.02), and the fertility of adult (≥ 3 years old) ewes reached 0.88 ± 0.05 by year 17. Lambing began approximately 140 days after the introduction of rams, and 64% of the S ewes that lambed did so in the first 17 days of the potential lambing season, which indicated that most of the S ewes were cycling at the time of ram introduction and were not induced to cycle by the introduction of breeding males (i.e., the so-called “ram effect”). Animals in the S line had modest increases in body weight and scrotal circumference. A modest negative trend in the additive maternal effect on birth weight was observed but was reversed by additional selection on EBV for maternal birth weight. The heritability of litter size in autumn lambing was low (0.04) and could potentially limit the response to selection for this trait.ConclusionsSelection improved realized ewe fertility in out-of-season mating, with absolute increases of approximately 1% per year in the percentage of joined ewes that lambed in the autumn. Genetic antagonisms with other performance traits were generally small. A modest antagonism with maternal breeding values for birth weight was observed but it could be accommodated by selection on EBV for maternal birth weight. Our results support results of previous studies that indicate that these selected ewes had one of the shortest seasonal anestrous periods reported for temperate sheep breeds and that spring-lambing lactating ewes from the selection line were capable of relatively rapid rebreeding in the spring.

Combined effect of heat and nutritional stress on superovulation of Malpura ewes in a semi-arid region

Sheep reared in hot semi-arid environments are generally exposed to heat and nutritional stress in some seasons of the year, which affects both production and reproduction. To assess the effect of high ambient temperature and feed scarcity on superovulation, 16 adult Malpura ewes were randomly divided into two groups of 8 animals each. G1 (control) was kept under a shed and offered a maintenance diet, and G2 (combined stress) was subjected to both nutritional (30% less of maintenance diet) and heat (38–44 °C for 6 h/day) stress. Ewes were superovulated without estrus synchronization by a combination of single injection of 200 IU eCG and 8 injections of FSH (Folltropin-V) at 12-h intervals in tapering doses of 5 mg/kg body weight, starting from the day 7 of natural estrus. eCG was given with the first injection and PGF2α (10 mg) was given with the second last FSH injection. G2 increased respiration rate and rectal temperature (P < 0.01), and blood urea level (P < 0.05), whereas it decreased average daily gain, plasma T4 concentration (P < 0.01) and body weight (P < 0.05). Plasma estradiol level was lower (P < 0.05) in G2 ewes as compared to control (G1) ewes. However, the number of ewes showed a superovulatory response (88 vs 66% ewes ≥ 3 corpus luteum), ovulation rate (8.75 vs 5.88) and embryo production (5.5 vs 3.9) decreased, and the number of large follicles (anovulation) increased (1.0 vs 2.14) in G2 ewes. G2 had a comparable effect on the superovulatory response compared to control ewes although physiological changes occurred as an adaptive mechanism to stress. Therefore, the well-adapted cyclic sheep of the semi-arid region may be used for superovulation despite the stressful condition of heat exposure and nutritional insufficiency.

Expression and Functional Analysis of the BCL2-Associated Agonist of Cell Death (BAD) Gene in the Sheep Ovary During the Reproductive Cycle.

Most ewes in China are seasonally polyestrous with normal ovulatory cycles, which is controlled by photoperiod (length of the daily light phase). These ewes are estrous in the short-day season and anestrus in the long-day season and cannot mate during anestrus. Thus seasonal breeding limits both diversification and intensification of production. If sheep can estrus all round year, it can be mated twice per year, which can greatly improve the economic benefits. To change seasonal estrus at the genetic level and cultivating new sheep breeds, it is important to understand the molecular mechanisms of seasonal breeding trait in sheep. The BCL2-associated agonist of cell death (BAD) gene being a regulator of cellular apoptosis was identified by our previous RNA-Seq, which is associated with follicular development in mammalian ovaries. However, the mechanism how BAD can regulate estrus in sheep was poorly understood. In this study, we characterized ovine BAD, including full-length mRNA cloning and protein sequence prediction, as well as BAD expression profile in Small-tailed Han (STH) sheep. The highest expression levels of BAD were observed in sheep hypothalamus, lung, and pituitary, while the lowest expression was in liver. Functional analysis of BAD was performed in primary granulosa cells of sheep. The concentration of P4 was significantly increased after RNAi interference of BAD, while P4 level was shown to be opposite after BAD overexpression in vitro. It has been found that BAD can reduce progesterone levels by promoting ovarian GC apoptosis, which might be involved in regulating the estrus cycle in sheep.

Progesterone effects on extracellular vesicles in the sheep uterus.

Progesterone (P4) acts via the endometrium to promote conceptus growth and implantation for pregnancy establishment. Many cells release extracellular vesicles (EVs) that are membrane-bound vesicles of endosomal and plasma membrane origin. In sheep, endometrial-derived EVs were found to traffic to the conceptus trophectoderm. Thus, EVs are hypothesized to be an important mode of intercellular communication by transferring select RNAs, proteins, and lipids between the endometrium and conceptus. Electron microscopy analysis found that the endometrial luminal and glandular epithelia were the primary source of EVs in the uterus of cyclic sheep. Size exclusion chromatography and nanoparticle tracking analysis (NTA) found that total EV number in the uterine lumen increased from day 10 to 14 in cyclic sheep. Next, ewes were ovariectomized and hormone replaced to determine effects of P4 on the endometrium and EVs in the uterine lumen. Transcriptome analyses found that P4 regulated 1611 genes and nine miRNAs in the endometrium. Total EV number in the uterine lumen was increased by P4 treatment. Small RNA sequencing of EVs detected expression of 768 miRNAs and determined that P4 regulated seven of those miRNAs. These studies provide fundamental new information on how P4 influences endometrial function to regulate conceptus growth for pregnancy establishment in sheep.

Nutritional status affects the microRNA profile of the hypothalamus of female sheep.

Recent studies on the seasonal regulation of the oestrous cycle in sheep have focussed mainly on the responses to photoperiod. However, the brain systems that control reproductive activity also respond to nutritional inputs, although the molecular mechanisms involved are not completely understood. One possibility is that small, non-coding RNAs, such as micro-RNAs (miRNAs), have significant influence. In the present study, the amounts and characteristics of miRNAs in hypothalamus from oestrous and anestrous ewes, fed low- or high-nutrient diets, were compared using Illumina HiSeq sequencing technology. In total, 398 miRNAs, including 261 novel miRNAs, were identified in ewes with an enhanced nutritional status (HEN), whereas 384 miRNAs, including 247 novel miRNAs, were identified in the ewes with a lesser nutritional status (HAN). There were eight conserved and 140 novel miRNAs expressed differentially between the two libraries. Based on quantitative real-time polymerase chain reaction, six miRNAs were assessed to verify the accuracy of the library database. Moreover, the correlation between the miRNA target and several upstream and downstream genes in the oestrus-related pathways were also verified in hypothalamus nerve cells. According to the results, nutritional status plays an important role in oestrous regulation in sheep, and the hypothalamic processes and pathways induced by nutritional signals (folic acid and tyrosine) are different from those induced by photoperiodic regulation of oestrus. We have expanded the repertoire of sheep miRNAs that could contribute to the molecular mechanisms that regulate the initiation of oestrous cycles in anestrous ewes in response to the influence of nutritional status.

Influence of a device intravaginal to synchronization/induction of estrus and its reuse in sheep vaginal flora

The objective of this study was to characterize the vaginal bacterial flora and their antibiotic susceptibility in vitro after, application, and the reuse of an intravaginal progesterone device (CIDR) in controlling the estrous cycle in sheep. Thirty Texel ewe lambs were used, after carefully assessed on their health and body condition. Experimental groups were formed by new CIDR (CIDRn, n = 15) and CIDR reused (CIDRu, n = 15). Treatment with progesterone lasted 6 days. Sterile swabs were used for vaginal sample collection, a procedure performed previously of CIDR insertion and on the moment of CIDR removing. The predominant flora prior to insertion of intravaginal devices proved to be 93.3% (14/15) and 80.0% (12/15) of gram-positive agents, especially of the genera Bacillus sp. and Staphylococcus sp. in CIDRn CIDRu groups, respectively. Upon removal of the devices, however, it was observed that the emergence of gram-negative agents in 100% of the samples CIDRn, most of them belonging to the Enterobacteriaceae genus. In CIDRu group, there was a balance between negative- and gram-positive agents on the removal of the devices. Regarding to susceptibility, our findings revelated some degree of resistance in all samples. Therefore, our conclusions pointed out that intravaginal devices (CIDR), when employed in the first use, or reuse, for a period of 6 days, promote a change in vaginal flora of sheep, highlighting the predominance of gram-negative agents during its removal.

Luteal function during the estrous cycle in arginine-treated ewes fed different planes of nutrition.

Functions of corpus luteum (CL) are influenced by numerous factors including hormones, growth and angiogenic factors, nutritional plane and dietary supplements such as arginine (Arg), a semi-essential amino acid and precursor for proteins, polyamines and nitric oxide (NO). The aim of this study was to determine if Arg supplementation to ewes fed different planes of nutrition influences: (1) progesterone (P4) concentrations in serum and luteal tissue, (2) luteal vascularity, cell proliferation, endothelial NO synthase (eNOS) and receptor (R) soluble guanylate cyclase β protein and mRNA expression and (3) luteal mRNA expression for selected angiogenic factors during the estrous cycle. Ewes (n = 111) were categorized by weight and randomly assigned to one of three nutritional planes: maintenance control (C), overfed (2× C) and underfed (0.6× C) beginning 60 days prior to onset of estrus. After estrus synchronization, ewes from each nutritional plane were assigned randomly to one of two treatments: Arg or saline. Serum and CL were collected at the early, mid and late luteal phases. The results demonstrated that: (1) nutritional plane affected ovulation rates, luteal vascularity, cell proliferation and NOS3, GUCY1B3, vascular endothelial growth factor (VEGF) and VEGFR2 mRNA expression, (2) Arg affected luteal vascularity, cell proliferation and NOS3, GUCY1B3, VEGF and VEGFR2 mRNA expression and (3) luteal vascularity, cell proliferation and the VEGF and NO systems depend on the stage of the estrous cycle. These data indicate that plane of nutrition and/or Arg supplementation can alter vascularization and expression of selected angiogenic factors in luteal tissue during the estrous cycle in sheep.

The impact of diet and arginine supplementation on pancreatic mass, digestive enzyme activity, and insulin-containing cell cluster morphology during the estrous cycle in sheep

To determine the effect of feed intake and arginine treatment during different stages of the estrous cycle on pancreatic mass, digestive enzyme activity, and histological measurements, ewes (n = 120) were randomly allocated to 1 of 3 dietary groups; control (CON; 2.14-Mcal metabolizable energy/kg), underfed (UF; 0.6 × CON), or overfed (OF; 2 × CON) over 2 yr. Estrus was synchronized using a controlled internal drug release device for 14 d. At controlled internal drug release withdrawal, ewes from each dietary group were assigned to 1 of 2 treatments; Arg (L-Arg HCl, 155-μmol/kg BW) or Sal (approximately 10-mL saline). Treatments were administered 3 times daily via jugular catheter and continued until slaughter on d (day) 5 and 10 of the second estrus cycle (early luteal phase, n = 41 and mid-luteal phase, n = 39; yr 1) and d 15 of the first estrus cycle (late luteal phase, n = 40; yr 2). A blood sample collected from jugular catheters for serum insulin analysis before slaughter. The pancreas was then removed, trimmed of mesentery and fat, weighed, and a sample snap-frozen until enzyme analysis. Additional pancreatic samples were fixed in 10% formalin solution for histological examination of size and distribution of insulin-containing cell clusters. Data were analyzed as a completely randomized design with a factorial arrangement of treatments. Diet, treatment, and diet × treatment were blocked by yr and included in the model with initial BW used as a covariate. Day of the estrous cycle was initially included in the model but later removed as no effects (P > 0.10) were observed for any pancreatic variables tested. Overfed ewes had the greatest (P < 0.001) change in BW, final BW, change in BCS, and final BCS. A diet × treatment interaction was observed for change in BW and final BW (P ≤ 0.004). Overfed and CON had increased (P < 0.001) pancreas weight (g) compared with UF ewes. Protein concentration (g/pancreas) was the lowest (P < 0.001) in UF ewes, whereas protein content (mg/kg BW) was greater (P = 0.03) in UF than OF ewes. Activity of α-amylase (U/g, kU/pancreas, U/kg of BW, and U/g protein) and trypsin (U/pancreas) was greater (P ≤ 0.003) in OF than UF ewes. Serum insulin was the greatest (P < 0.001) in OF ewes. No effects were observed for pancreatic insulin-containing cell clusters. This study demonstrated that plane of nutrition affected several measurements of pancreatic function; however, the dosage of Arg used did not influence pancreatic function.

FIELD EVALUATION OF ESTRUS SYNCHRONIZATION AND PREGNANCY DETECTION USING ULTRASONOGRAPHY TO THE LOCAL AWASSI EWES FOR SHEEP OWNERS IN KANAAN DISTRICT- DIYALA PROVINCE

The objective of this study was to carry out an on-farm experiment in sheep owner farms in Kanaan district for the period from 7th Feb. 2015 to 27th Mar. 2015 and to identify the unconventional methods in controlling the reproduction in sheep and increase their productivity using 45 local Awassi ewes, randomly selected from three sheep owners (15 ewes / owner). Treatment group (10 ewes/ owner) were treated with a vaginal sponges containing 20 mg fluorogestone acetate (FGA) inserted for 10 days, followed by I.M. injection of 500 IU equine chorionic gonadotropin (eCG), while 5 ewes / owner were regarded as control group without any treatment. Estrus was observed for all ewes for 5 days post withdrawal of sponges. Ewes that showed estrus were mated by rams in their flocks, thereafter, pregnancy detection was conducted at days 35-37 post- mating using transabdominal ultrasonography. A questionnaire involved 20 sheep owners and a demonstrative field day was conducted for owners contributed in the experiment, and others from the region of study to explain the importance and results of the experiment. The results showed the appearance of estrus in all ewes 100% within three days after the withdrawal of sponges. Pregnancy rate of 85.71% at days 35-37 of mating. The questionnaire showed there is an urgent need of knowledge for owners to find out the importance of controlling the reproductive cycle in sheep using vaginal sponges. Also, there were a correlation between the need of knowledge for sheep owners with (educational level for most of them, herd sizes, income from breeding, training courses and other activities in the fields of breeding and reproduction of sheep). In conclusion, the technology of estrus synchronization has an important role to improve reproductive performance of sheep and pregnancy could be detected through transabdominal ultrasonography at 35-37 days with high accuracy as well as there is great desire for the most owner to conduct estrus synchronization technique and to take care their birth of herds simultaneously.

Ewe reproduction: stimulation and synchronisation

Farmers are looking to breed sheep earlier in the year to take advantage of higher meat prices for finished lambs, and to breed replacement ewe lambs that can lamb that same year. To do this, the oestrus cycle of sheep must be manipulated so that the sheep are able to breed in the summer. This article details how this can be done, but the method chosen must be tailored to the farm. The benefit of stimulating the ovulation is that this helps to synchronise the oestrus periods meaning that lambing periods are short, helping to save costs to the farmer.

Extracellular Vesicles Originate from the Conceptus and Uterus During Early Pregnancy in Sheep.

Cells release diverse types of membrane-bound vesicles of endosomal and plasma membrane origin, termed exosomes and microvesicles, respectively. Extracellular vesicles (EVs) represent an important mode of intercellular communication by transferring select RNAs, proteins, and lipids between cells. The present studies tested the hypothesis that the elongating ovine conceptus and uterus produces EVs that mediate conceptus-maternal interactions during early pregnancy. In Study 1, EVs were purified from uterine luminal fluid of Day 14 cyclic sheep. The EVs were fluorescently labeled with PKH67 dye and infused into the uterine lumen of pregnant sheep for 6 days using an osmotic pump. On Day 14, labeled EVs were observed in the conceptus trophectoderm and uterine epithelia, but not in the uterine stroma or myometrium. In Study 2, Day 14 conceptuses were cultured ex vivo for 24 h and found to release EVs into the culture medium. Proteomics analysis of the Day 14 conceptus-derived EVs identified 231 proteins that were enriched for extracellular space and several protein classes, including proteases, protease inhibitors, chaperones and chaperonins. RNA sequencing of Day 14 conceptus-derived EVs detected expression of 512 mRNAs. The top-expressed genes were overrepresented in ribosomal functions and components. Isolated EVs from conceptuses were fluorescently labeled with PKH67 and infused into the uterine lumen of cyclic sheep for 6 days using an osmotic pump. On Day 14, labeled EVs were observed in the uterine epithelia, but not in the uterine stroma or myometrium. Labeled EVs were not observed in the ovary or in other maternal tissues. These studies support the ideas that EVs emanate from both the conceptus trophectoderm and uterine epithelia, and are involved in intercellular communication between those cells during the establishment of pregnancy in sheep.

178 IS PROGESTERONE THE DETERMINING REGULATORY FACTOR BEHIND OVULATION RATE IN EWES?

Ovarian antral follicles in the ewe grow in an orderly succession, producing 3–4 waves per oestrous cycle. In prolific sheep, some large antral follicles from the second-last wave of the oestrous cycle are added to the ovulatory follicles emerging just before oestrus to give a higher ovulation rate; it is feasible that regression of these follicles is prevented by an increase in serum concentrations of FSH and/or LH pulsatility at pro-oestrus. Prolific sheep tend to have a shorter luteal phase than non-prolific breeds and there is a great deal of evidence that luteal progesterone (P4), in addition to regulating LH release, may govern the secretion of FSH heralding the emergence of follicular waves. The specific purpose of the present experiments was to determine whether or not extending the duration of the luteal phase would alter the ovulation rate in prolific sheep. In both studies, exogenous P4 (7.5 mg ewe–1 IM) was administered on Days 11 and 12 (Day 0 = ovulation) in moderately prolific Rideau Arcott x Polled Dorset (Exp. 1, n = 8) and highly prolific Olkuska ewes (Exp. 2, n = 7), while the equinumerous groups of animals served as controls (CTR). Transrectal ovarian ultrasonography was performed daily and jugular blood samples were drawn twice a day from Day 9 until ovulation. All single-time point observations were compared between groups by Student t-test. Progesterone injections resulted in uniform increments in serum P4 levels in all animals allocated to the treatment (TRT) groups. However, the mean duration of the interovulatory interval did not differ (P &gt; 0.05) between TRT and CTR groups of ewes in both experiments. The mean (± s.e.m.) ovulation rate was 1.6 ± 0.2 v. 3.2 ± 0.4 (Exp. 1; P &lt; 0.001) and 3.2 ± 0.8 v. 4.0 ± 1.0 (Exp. 2; P &lt; 0.05) in TRT v. CTR ewes, respectively. There were no differences in terms of the timing of penultimate and final wave emergence between the two subsets of animals studied in either experiment. The number/percentage of ovulating follicles from the penultimate wave of the interovulatory interval studied was 0.25 ± 0.16 v. 1.75 ± 0.45 (P &lt; 0.01)/25.0 ± 16.4% v. 75.0 ± 16.4% (P &lt; 0.05) in Exp. 1 and 0.50 ± 0.30 v. 1.60 ± 0.40 (P &lt; 0.05)/13.8 ± 9.0% v. 53.4 ± 16.7% (P &lt; 0.05) in Exp. 2, in TRT v. CTR animals, respectively. In summary, administration of P4 at the end of diestrus reduced the incidence of ovulations from the penultimate wave of the oestrous cycle in moderately and highly prolific strains of sheep. Therefore, progesterone appears to be a key endocrine signal governing the ovulation rate in cyclic sheep, presumably by acting directly at the level of the ovary. The present results may pave a way to devising a simple and inexpensive method of controlling lamb productivity in commercial flocks of sheep and fertility in other polyovulatory species.

The effects of diet and arginine treatment on serum metabolites and selected hormones during the estrous cycle in sheep

The aim of this study was to determine the effects of diet and arginine (Arg) treatment on serum concentrations of selected metabolites and metabolic and reproductive hormones in nonpregnant ewes. Sixty days before the onset of estrus (Day 0), Rambouillet ewes were randomly assigned to one of three dietary groups: maintenance control (C; N = 16; 100% National Research Council requirements), overfed (O; N = 16; 2 × C), or underfed (U; N = 16, 0.6 × C) to achieve and maintain three different body conditions during their estrous cycle(s). At Day 0, ewes from each nutritional group were randomly assigned to receive one of two treatments: saline (Sal) or Arg (L-Arg-HCl; 155 μmol Arg per kg of body weight [BW]; intravenous), which was administered three times per day for 21 or 26 days. Blood samples were collected on Days 0, 6, 10, 12, 16, 21, and 26 of Sal or Arg treatment for evaluation of Arg, nitric oxide metabolite, cholesterol, glucose, insulin, insulin-like growth factor 1, leptin, and progesterone. For a time-response trial, blood samples were collected at 0, 1, 2, 4, and 7 hours after Sal or Arg treatment at the mid-luteal phase to determine serum Arg concentrations. During the 11-week study, C maintained body weight, O gained 9.6 ± 0.7 kg, and U lost 13.9 ± 0.1 kg. Overall, serum concentrations of Arg, glucose, insulin, insulin-like growth factor 1, leptin, and progesterone were greater (P < 0.05) in O ewes than C and/or U ewes and were not affected by Arg treatment. Serum Arg concentration increased at 1 and 2 hours and decreased to basal level at 4 and 7 hours after Arg treatment. These data reinforce the importance of diet in regulation of metabolic and endocrine functions, and demonstrated that the dose and duration of Arg treatment used in this study does not alter serum metabolites or hormones in nonpregnant ewes of various nutritional planes.

Short oestrous cycles in sheep during anoestrus involve defects in progesterone biosynthesis and luteal neovascularisation

Anoestrous ewes can be induced to ovulate by the socio-sexual, 'ram effect'. However, in some ewes, the induced ovulation is followed by an abnormally short luteal phase causing a so-called 'short cycle'. The defect responsible for this luteal dysfunction has not been identified. In this study, we investigated ovarian and uterine factors implicated in male-induced short cycles in anoestrous ewes using a combined endocrine and molecular strategy. Before ovulation, we were able to detect a moderate loss of thecal expression of steroid acute regulatory protein (STAR) in ewes that had not received progesterone priming (which prevents short cycles). At and following ovulation, we were able to identify a significant loss of expression of genes coding key proteins involved in the biosynthesis of progesterone (STAR, CYP11A1 and HSD3B1 (HSD3B)) as well as genes coding proteins critical for vascular development during early luteal development (VEGFA and KDR (VEGFR2)), suggesting dysfunction in at least two pathways critical for normal luteal function. Furthermore, these changes were associated with a significant reduction of progesterone production and luteal weight. Additionally, we cast doubt on the proposed uterus-mediated effect of prostaglandin F2α (PGF2α) as a cause of short cycles by demonstrating the dysregulation of luteal expression of the PGF receptor, which mediates the luteal effects of PGF2α, and by finding no significant changes in the circulating concentrations of PGFM, the principal metabolite of PGF2α in ewes with short cycles. This study is the first of its kind to examine concurrently the endocrine and molecular events in the follicular and early luteal stages of the short cycle.

Increase in ovine plasma cortisol at oestrus and its relation with the metabolic status during the sexual cycle in sheep

Metabolic status is an important factor modulating reproductive function in ruminants. Evidence from our group has led to the concept that reproductive hormones, in turn, were capable of modulating metabolic responses. In the present study, cortisol plasma concentrations have been determined in the peri-oestrus of cyclic ewes fed half or all of their maintenance requirements, and related to concentrations of insulin, insulin-like growth factor 1 (IGF-1), leptin, glucose and non-esterified fatty acids (NEFA). Cortisol concentrations experienced a six-fold rise at oestrus, independently of the level of nutrition. Moreover, they were positively correlated with glucose and IGF-1 concentrations. It is suggested that the cortisol increase at oestrus could be due to oestrogenic action in the adrenal cortex, and that it could explain some of the metabolic fluctuations observed before.

Amounts of an estrogen receptor β isoform increased in the theca of preovulatory follicles of sheep

Determination of the specific roles of the estrogen receptor (ER) forms in reproductive processes of different species remains incomplete. In the present experiment, cellular localization and changes in relative amounts of the ERα and ERβ in late developing ovarian follicles, oviduct, and uterus were determined during the follicular phase of the estrous cycle in sheep. Ewes in mid-luteal phase were treated with prostaglandin F2α (PG) to induce luteolysis and control the onset of the follicular phase. The oviducts, uterus, and the ovaries were collected at 0 (ewes not treated with PG), 4, 18, and 36h after PG treatment (early, mid, and late follicular phase, respectively) and processed to evaluate the ERs using immunohistochemical (IHC) procedures. The ERα was localized to nuclei of granulosa cells of late developing follicles and most cells of the oviduct and uterus. The ERβ was detected only in ovarian follicles using two antibodies directed to different regions of the ERβ. Western immunoblotting demonstrated that the antibody directed against the N-terminal region of the ERβ detected one isoform (approximately 53kDa) whereas the antibody directed against the C-terminus detected two ERβ isoforms (approximately 53kDa and 59kDa). Western and IHC results combined indicated presence of the 59kDa ERβ in granulosa cells and the 53kDa ERβ in both granulosa and theca cells. Relative amounts (immunostaining intensity) of the ERα increased (P<.05) in granulosa cells of preovulatory follicles and in the isthmian muscularis of the oviduct at the late follicular phase. Amounts of the ERα in the mucosal epithelium of the oviductal regions (isthmus, ampulla, and infundibulum), and in various uterine cell types (glandular and luminal epithelia, endometrial stromal cells, and myometrium) did not change (P>.05) throughout the follicular phase. A major increase (four-fold) in expression of the 53kDa ERβ in the theca and a less pronounced increase in the granulosa occurred at the late follicular phase. The ERα is broadly expressed in reproductive organs of sheep and is upregulated only in few cell types during the late follicular phase. Immunoreactive ERβ was detected only in the ovary. Important estrogen actions in theca cells during preovulatory follicular development likely occur in association with a major increase in expression of an ERβ isoform.