Differentially expressed transcripts study during pregnancy and postpartum anestrus of yak (Bos grunniens).

Background: Yak is the main livestock species in the plateau area, and its reproductive performance is low, usually two years or three years. A very few of yaks recover within a certain period of time after delivery and smoothly enter the next estrous cycle, while most of them enter the postpartum anestrus and show no estrus performance. However, the key biological factors and influencing mechanisms that cause postpartum anestrus in yaks are not clear. Objective: To study the expression of differential transcripts in ovaries of yak during pregnancy and postpartum anestrus. Methods: Each three yaks in pregnancy and anestrus under natural grazing conditions in Haiyan County, Qinghai Province were selected and slaughtered, and their ovaries were collected and sent to Biomarker Technologies. Oxford Nanopore Technologies single-molecule real-time electrical signal sequencing technology was used to perform full-length transcriptome sequencing. Astalavista software was used to identify the types of alternative splicing events in yak estrus and pregnancy, and TAPIS pipeline was used to identify alternative polyadenylation. Results: The results showed that there were 1751 differentially expressed transcripts (DETs) between pregnancy and anestrus in yak, of which 808 were upregulated and 943 were downregulated. GO analysis showed that the biological processes of DETs were mainly reproductive, reproductive and rhythmic processes. KEGG analysis showed that the DET cell junction-related adhesion junction protein (ß-catenin) and amino terminal kinase (JNK) were involved in FAs (local adhesion). Phosphatidylinositol-3-kinase (PI3K) is involved in the PI3K/AKT/mTOR signaling pathway. Circadian rhythm output cycle failure (Clock) and brain and muscle tissue aromatic hydrocarbon receptor nuclear transporter-like protein 1 (Bmal1) are involved in circadian rhythm signaling pathway. Conclusion: This study found that ß-catenin, JNK, PI3K, Clock and Bmal1 were closely related to postpartum anestrus in yak.

Sexually active male goats stimulate the endocrine and sexual activities of other males in seasonal sexual rest through the "buck-to-buck effect".

Male goats rendered sexually active by exposure to a photoperiodic treatment are more efficient than untreated goats in stimulating LH secretion and ovulation in seasonally anestrous goats. This phenomenon is called the "male effect." Here, we determined whether sexually active bucks are able to stimulate the endocrine and sexual activities of other bucks in seasonal sexual rest through the phenomenon that we called the "buck-to-buck effect." We used bucks rendered sexually active (SA) during sexual rest by exposure to 2.5 mo of artificial long days (16 h of light per d) and untreated, sexually inactive (SI) bucks. In Experiment 1, we determined the short-term (21 d) LH and testosterone responses of sexually inactive bucks joined with a SA or SI buck. In Experiment 2, we determined the long-term (60 d) testosterone and sexual behavior responses of sexually inactive bucks joined with 2 SA or SI bucks. In Experiment 3, we determined the efficacy of bucks initially exposed to the buck-to-buck effect, the SABB bucks, to thereafter induce a "classical" male effect in seasonally anestrous goats. In Experiments 1 and 2, there was an interaction between time and groups in LH and testosterone plasma concentrations (P < 0.01). In Experiment 1, plasma LH concentrations were greater in bucks joined with a SA buck than in those joined with an SI buck (P < 0.05). In Experiments 1 and 2, testosterone concentrations were greater in bucks joined with SA bucks than in those joined with SI bucks (P < 0.05). In addition, in Experiment 2, the sexually inactive bucks joined with SA bucks displayed more nudging than those joined with SI bucks (P < 0.001). In Experiment 3, kidding rates did not differ between females joined with SA (34 of 40: 85%) or SABB bucks (32 of 40: 80%; P > 0.05). We concluded that the endocrine and sexual activities of bucks during sexual rest can be stimulated by SA bucks. In addition, SABB bucks are able to stimulate the reproductive activity of seasonally anestrous goats.

Regulation of uterine function during estrous cycle, anestrus phase and pregnancy by steroids in red deer (Cervus elaphus L.).

Steroid synthesis and production in ruminant uterus is not obvious, especially in seasonally reproduced. We compared steroid production by investigating enzymes involved in red deer uterine steroid metabolism in reproductive seasons. Blood and uteri (endometrium and myometrium) were collected post mortem from hinds on 4th day (N = 8), 13th day of the cycle (N = 8), anestrus (N = 8) and pregnancy (N = 8). The expression of cytochrome P450 aromatase (P450), 3 -beta-hydroxysteroid dehydrogenase (3ß-HSD), 17 -beta-hydroxysteroid dehydrogenase (17ß-HSD), aldo-keto reductase family 1 C1 (AKR1C1), estrogen receptor alpha (ERα), and progesterone receptors (PRs), were analyzed using real-time-PCR and Western Blotting. Plasma samples were assayed for 17-beta-estradiol (E2), progesterone (P4), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone (T4) concentrations by EIA. Hinds at the beginning of the estrous cycle, mainly in endometrium, were characterized by a high mRNA expression of 3ß-HSD, AKR1C1, PRs and ERα, contrary to the expression in myometrium during pregnancy (P < 0.05). For P4, E2, and FSH, concentration was the highest during the 13th day of the estrous cycle (P < 0.05). Uterine steroid production and output in hinds as a representative seasonally reproduced ruminant occurred mainly during the estrous cycle and sustained in anestrus.

Heat shock protein HSP90 immunoexpression in equine endometrium during oestrus, dioestrus and anoestrus.

Heat shock proteins play a crucial role in cellular development, proliferation, differentiation and apoptosis. Heat shock protein 90 (HSP90) has been localised in the human endometrium, where its immunoexpression changes during the menstrual cycle. Similar studies have not been done for the equid species, so the present study aimed to describe endometrial HSP90 immunoexpression in mare endometrium. Endometrial biopsies were formalin-fixed and paraffin-embedded, and sections were stained with haematoxylin-eosin in preparation for HSP90 immunohistochemistry. Immunostaining and morphometric analyses were performed on the epithelial lining, endometrial glands and connective stroma during oestrus, dioestrus phase and anoestrus period (n = 7 per phase or period). Immunoexpression was localised in the basal region of the epithelial cells lining the lumen. Immunoexpression was greater during oestrus than during either dioestrus or anoestrus. During anoestrus, there was little immunostaining in the endometrium, suggesting that HSP90 is involved in the functional modulation of sex steroid receptors in cyclic mares. Indeed, the function of HSP90 as a chaperone in the folding of proteins, such as steroid receptors, might explain the greater intensity of immunostaining during the oestrus and dioestrus phases, compared the anoestrus period. We conclude that, in the mare, HSP90 plays a role in endometrial function and that further studies are needed to test whether it is important in pathological conditions as endometritis.

Association of taurine with ovarian follicular steroids and postpartum anestrus condition in Murrah buffaloes.

Taurine is an abundant intracellular beta-amino acid majorly synthesized in the liver and transported through plasma. In mammals, taurine was reported to be involved in various physiological functions, including the enhancement of testosterone levels, the major estradiol precursor. Therefore, we hypothesize that taurine levels are associated with ovarian follicular steroids as well as with a reproductive problem called postpartum anestrus (PPA) in dairy buffaloes. To understand the taurine levels and its possible role in buffalo ovarian follicles, a correlation was established among taurine, estradiol, and testosterone levels in the ovarian follicular fluid. For this purpose, buffalo ovaries were obtained from the slaughterhouse, and follicular fluid samples were collected from small (<4 mm), medium (4-8 mm) and large (>8 mm) follicles. Taurine and steroid levels in the follicular fluid were analyzed by TLC and ELISA, respectively. Taurine and testosterone levels were significantly (P < 0.05) higher in the follicular fluid of small and medium follicles than large follicles, whereas the estradiol levels were significantly (P < 0.001) higher in the large follicles. Thus, taurine showed a positive correlation (r = 0.75) with testosterone and a negative correlation (r = -0.77) with estradiol in buffalo follicular fluid, indicating its possible role in testosterone function during follicular development. Interestingly, significantly (P < 0.001) lower plasma taurine levels in PPA (n = 50) than normal cyclic (n = 50) buffaloes represented its association with PPA. Therefore, our present study recommends the need for future nutrition studies on taurine supplementation to PPA buffaloes.

Body condition score, parity, shelter cleanliness and male proximity: Highly associated non-genetic factors with post-partum anestrus in Murrah buffalo in field conditions.

Postpartum anestrus (PPA) is a major reproductive problem in buffalo. Although both genetic and non-genetic factors influence the incidence of PPA, identifying associated non-genetic factors would be helpful for effective management of this problem. No systematic study, however, has been conducted to identify the association of non-genetic factors with PPA in buffalo, especially in field conditions. Data for 39 variables affecting postpartum anestrus interval (PPAI), health, nutrition, management and environmental status of the animals were collected on 575 buffalo from 15 villages. Data were analyzed using a general linear model considering PPAI as a dependent variable and the remaining 38 non-genetic parameters as independent variables. The results from the analysis indicated body condition score (BCS) variables, such as pelvic girdle score (P <  0.0001) and girth (P <  0.05), parity (P < 0.0001), shelter cleanliness (P <  0.001), male proximity (P <  0.005), amount and frequency of concentrate feeding during the early postpartum period (P <  0.05), general feeding frequency in a day (P <  0.05) and the treatments with oxytocin at the time of milking to facilitate milk release from the udder (P <  0.05) were associated with PPAI in Murrah buffalo. Plotting the PPAI residuals indicated the possibility of other unexplained factors, including genetics, that could also affect the differences in values for this variables in Murrah buffalo. In conclusion, a greater understanding of associated non-genetic factors with PPAI would help farmers in conducting proper managemental practices for early induction of reproductive cycles following parturition in Murrah buffalo.

First proteomic analysis of diestrus and anestrus canine oocytes at the germinal vesicle reveals candidate proteins involved in oocyte meiotic competence.

In domestic dogs, oocyte maturation rates are low and the percentage of oocytes that remain in the stage of germinal vesicle (GV) regardless of culture conditions is high. The present study was conducted to characterize the proteome of canine oocyte at the germinal vesicle stage using label-free mass spectrometry. Ovaries were collected from 415 adult domestic dogs and oocytes were divided anestrus and diestrus group. Protein lysates were subjected to quantitative proteomic analysis to identify differentially expressed proteins in different status reproductive. All runs for each sample were performed on an Easy nLC1000 nano-LC chromatograph system directly connected to a quadrupole-type Orbitrap mass spectrometer. For identification of peptides and proteins, raw data of the spectra were loaded into MaxQuant software version 1.5.2.8. Proteomic data were analysed according to gene ontology and a protein-protein interaction network. 312 proteins were identified and grouped according to their biological processes, molecular functions and cellular component. Forty-six differentially expressed proteins among diestrus and control group were associated with at least one GO term in the biological process database. Several proteins involved in the cell cycle, fertilization, regulation of transcription and signalling pathways that are essential for the full development of oocytes and fertilization were expressed. This study identified proteins that were absent, and more or less expressed in different status reproductive. These differentially expressed proteins revealed a framework of molecular reorganization within a GV that renders its competency. This knowledge will enable the identification of target competence biomarkers and thus the establishment of more adequate means of cultivation to improve the M-I and II indexes in this species and also to better understand the physiology of the domestic dog, promoting the development of new reproduction biotechniques.

Systematic review and meta-analysis of the efficacy of reproductive management practices used to induce resumption of ovarian cyclical activity in anestrous does.

Reproductive management practices that use hormones, sociosexual biostimulation, nutritional management, or abiotic factors are used to induce the resumption of reproduction in anestrous does. However, their overall efficacy remains uncertain; therefore, the identification of evidence-based management recommendations to manipulate anestrous in goats is important. Electronic databases were searched to retrieve reports on studies using interventions based on hormonal, sociosexual, nutritional, and abiotic factors. Only experimental studies in which a group of anestrous does was treated and compared against an untreated group were included. Estrus, ovulation, and pregnancy were primary outcomes, whereas the onset of estrus after treatment, the ovulation rate, and the number of anovulatory days were secondary outcomes. Odds ratio (OR) and mean differences were used to synthesize pooled data, and random effects models were used to calculate them. Seventy studies involving 3974 goats met the inclusion criteria. Unclear risk of bias for random sequence generation and allocation concealment predominated across studies. Pooled data for hormonal, sociosexual, and abiotic interventions showed a significant, though variable, increase in estrus (OR range 7.15-144.80), ovulation (OR range 6.08-56.95), and pregnancy (OR range 3.94-30.8). Hormonal treatments significantly reduced the onset of estrus, whereas abiotic interventions failed to reduce the number of anovulatory days. Secondary outcomes were not assessed in trials using sociosexual approaches. Finally, except for pregnancy, no significant efficacy was observed for studies using nutritional management. In conclusion, reproductive management practices using sociosexual approaches showed the highest efficacy for restoring reproductive activity in anestrous does.

Ovarian response is not affected by the stage of seasonal anestrus or breed of goats when using a progesterone injection plus human chorionic gonadotropin-based protocol.

The aim of this study was to evaluate the effect of the stage of seasonal anestrus and breed on ovarian response in non-estrous cycling goats using a progesterone (P4) injection plus human chorionic gonadotropin (hCG)-based protocol. In Experiment 1, non-estrous cycling local Mexican goats were treated with 20 mg of P4 plus 100 IU of hCG injections 24 h apart during April (early anestrus, n = 13) or June (late anestrus, n = 12). The estrous response, interval from hCG-to-estrus, and interval to ovulation were not affected by season (P > 0.05). In addition, the size of the follicle from which ovulation occurred and the size of the corpus luteum were not different between the two stages of seasonal anestrus (P > 0.05). In Experiment 2, the estrous response was compared between multiparous non-estrous cycling local Mexican (n = 18) and Alpine (n = 19) goats in which stage of the estrous cycle was synchronized using the same P4+hCG protocol as in Experiment 1. Neither the onset of estrus nor the time of ovulation differed between breeds, and the estrus-to-ovulation interval also was similar for both breeds (P > 0.05). The diameter of the dominant follicle at the time of ovulation was similar between local and Alpine goats (P > 0.05). In addition, the pregnancy rate was not different for both local and Alpine goats (P > 0.05). In conclusion, results of this study indicate that the stage of seasonal anestrus or breed do not modify estrous and ovarian response in non-estrous cyclic goats synchronized with a P4 injection plus hCG-based protocol.

Sexually active bucks are a critical social cue that activates the gonadotrope axis and early puberty onset in does.

In rodents, early exposure to adult male is well known to induce an early puberty in females (Vandenbergh effect). This phenomenon has been less studied in other mammals. In goats, despite our extensive knowledge about the "male-effect" phenomenon in adults (i.e. ovulation induced by the introduction of the male during the anestrous), there are few data on the consequences of an early exposure of females to males. Here, we evaluated the puberty onset of young alpine goats when raised since weaning with intact bucks (INT), with castrated bucks (CAS) or isolated from bucks (ISOL). The INT group had the first ovulation 1.5 month before the two other groups. Despite the earlier puberty the INT group of females had normal and regular ovarian cycles. Morphological study of the genital tract showed that at 6 months, uterus of INT goats was 40% heavier than CAS and ISOL goats. Moreover, INT females had a myometrium significantly thicker and INT was the only group having corpora lutea. In our study, INT females were pubescent in the month following the entry of bucks into the breeding season, suggesting that only sexually active bucks provide the signal responsible for puberty acceleration. By removing direct contact with the bucks, we showed that somatosensory interactions were dispensable for an early puberty induction. Finally, no difference in the GnRH network (fiber density and number of synaptic appositions) can be detected between pubescent and non-pubescent females, suggesting that the male stimulations triggering puberty onset act probably on upstream neuronal networks, potentially on kisspeptin neurons.

[The molecular mechanism of sheep seasonal breeding and artificial regulatory techniques for estrus and mating in anestrus].

Seasonal breeding is an important factor limiting sheep production efficiency. Detailed analysis on the molecular mechanisms of seasonal breeding is the premise for improving estrus and mating rate of sheep during anestrus. Recent research showed that under long-photoperiod and short-photoperiod conditions, a series of changes in signaling molecules and cell morphology could be observed in ovine seasonal reproduction pathway. Based on the molecular mechanisms of seasonal reproduction, several technologies or methods for inducing estrus and mating of ewes in anestrus have been developed. In this review, photoperiod-induced changes in signaling molecules and cell morphology in pituitary and hypothalamic tissue are first summarized in terms of the molecular mechanisms and characteristics of seasonal reproduction. The application effect, advantages and disadvantages for applying these technologies for inducing estrus and mating of ewes in anestrus are then discussed, thereby providing the critical insights in identifying a new technology, which is environmentally friendly and efficient, to improve breeding rate in anestrus.

Monitoring changes in back fat thickness and its effect on the restoration of ovarian activity and fertility in Bos indicus cows.

With the objective of testing the hypothesis if animals with a stable layer of body fat (FAT) during the peripartum have a better chance of becoming pregnant after calving, fifty-nine multiparous Brahman cows in their last trimester of pregnancy were used. Animals averaged four parturitions and were stocked at a rate of 1.25 animal units per hectare and divided into two groups depending on the time postpartum (dpp) that the intravaginal releasing device CIDR was inserted; Group 1 (<30 dpp; n = 30) received the implant at 25.2 ± 4.21 and withdrawn 9 days later. Group 2 (≥30 dpp; n = 29) received the CIDR at 38.41 ± 5.8. Animals were AI at detected oestrus until 170 dpp and calculated as pregnant at first service or requiring more than one service (1s and >1s), not pregnant but cycling (not pregnant) and those not cycling at all (anestrus). The FAT measurements were taken twice each month from the last trimester of gestation until 96 dpp. The onset of ovarian activity was monitored through blood levels of progesterone (P4) at days 14 and 9 prior to CIDR insertion and days 10, 13, 30 and 33 after CIDR withdrawal. Animals pregnant did not have any major changes in their fat thickness. In contrast, cows pregnant in the group ≥30 dpp had changes in their FAT homoeostasis, and pregnant animals in the 1s and >1s groups did not show differences in dorsal back fat in the last trimester of pregnancy and early postpartum. In contrast, animals not pregnant and in anestrus FAT values decreased considerably after parturition. Overall, fertility was 49%, but 18% of all the animals remained anestrus losing FAT. Thus, animals with adequate metabolic conditions will have a better chance of pregnancy regardless of the time postpartum when the reproductive programme starts.

Effects of body condition score (BCS) on steroid- and eicosanoid-metabolizing enzyme activity in various mare tissues during winter anoestrus.

The objective of this study was to determine the activity of steroid- and eicosanoid-metabolizing enzymes in horses with varying BCSs. The BCSs of twenty non-pregnant, anoestrous mares were determined prior to euthanasia, and tissue samples were collected from the liver, kidney, adrenal gland, ovary and endometrium. Cytochrome P450 1A (CYP1A), 2C (CYP2C), 3A (CYP3A) and uridine 5'-diphospho-glucuronosyltransferase (UGT) activities were determined using luminogenic substrates. The MIXED procedure of SAS was used to test the effect of BCS on enzyme activity and differences between tissues. Activity of CYP1A in adrenals was increased (p ≤ .05) in BCS 5 versus BCSs 4 and 6. Activity of CYP1A in the liver was increased (p = .05) in BCS 4 versus BCSs 5 and 6. Activity of CYP1A was 100-fold greater (p < .0001) in the liver than in the adrenal, ovary and kidney. Activity of CYP2C was 100-fold greater (p < .0001) in the liver than in the adrenal, ovary and endometrium. Activity of CYP3A was only detectable in the liver. Activity of UGT in the kidney was decreased (p = .02) in BCS 4 versus BCSs 5 and 6. Activity of UGT was threefold greater (p < .0001) in the liver than in the kidney, whereas activity of UGT was ninefold greater (p < .0001) in the kidney than in the ovary and endometrium. In general, BCS did not alter the activity of steroid- and eicosanoid-metabolizing enzymes in horses. However, tissue differences in these enzymes indicated abundant hepatic metabolism in horses, which is similar to other livestock species.

Non-invasive hormonal characterization of the ovarian cycle, pregnancy, and seasonal anestrus of the female addra gazelle (Nanger dama ruficollis).

Non-invasive fecal hormone metabolite monitoring was used to characterize the estrous cycle, pregnancy, and seasonal anestrus of the critically endangered addra gazelle (Nanger dama ruficollis). With less than 250 animals remaining in the wild and ∼168 individuals managed in captivity, it is crucial to maintain sustainable populations. Progestogen and estrogen profiles were obtained from analysis of fecal samples collected approximately every other day, within varying intervals, over the course of 7 years (n = 8 adult females). Average estrous cycle length was 19.5 ± 0.4 days (range, 14-26 days), with a luteal phase length of 14.6 ± 1.2 days (range, 10-16 days), and an inter-luteal period of 5.2 ± 1.4 days (range, 2-7 days). Mean gestation length for six pregnancies was 200.7 ± 0.4 days (range, 200-202 days). Fecal progestogens increased at 12 weeks of gestation and remained elevated until parturition. Addra gazelle females exhibited a period of seasonal anestrus with consistently low progestogen concentrations and no cyclic activity from about September to March. Analysis of reproductive and climate records demonstrated a peak in U.S. births that coincided with maximal rainfall in the native habitat of the addra gazelle. Results show that estrous cycle, luteal phase, and inter-luteal phase lengths in addra are similar to those observed in other gazelle species, however, to our knowledge, this is the first study to demonstrate seasonal anestrus in the Nanger genus.

Body condition and stage of seasonal anestrus interact to determine the ovulatory response after male biostimulation in anovulatory Criollo × Nubian goats.

The effect of goat nutritional condition on the response to biostimulation with sexually active males during different stages of anestrus was determined. Fifty-eight Criollo × Nubian females on high and low body mass index (BMI) diets were used. Each BMI group was divided into two for biostimulation with sexually active males during May (mid-anestrus) or July (transition period). Ovulatory responses to biostimulation were characterized from serum progesterone, as well as the delay for response (first and second ovulations followed by a normal length luteal phase, O-WNLP). The percentage of goats showing one O-WNLP was greater in the high BMI group than in the low BMI group and greater during the transition period than in the mid-anestrus. However, the interaction between factors revealed that the difference between BMI groups was only significant in the transition period and the difference between stages was only significant in goats with high BMI. Occurrence of a second O-WNLP tended to be greater in the high BMI group than in the low BMI group. Response delay was shorter in the transition period than in mid-anestrus. In conclusion, female nutritional status interacting with the stage of anestrus determined the ovulatory response to male biostimulation in crossbred Criollo goats.

Fifteen minutes of daily contact with sexually active male induces ovulation but delays its timing in seasonally anestrous goats.

The present experiment was conducted to determine (1) whether the sexually active bucks are able to stimulate the ovulatory activity of seasonal anestrous goats when the daily contact is reduced to 15 min/day during 15 days and (2) the exact ovulatory activity in anestrous goats exposed to bucks for 2 hours or less per day during 15 days. One group of goats (n = 15) was isolated from bucks. The other six groups (n = 15 each) were exposed to sexually active bucks (n = 1 each) for 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, or 24 hours during 15 days. Goats with plasma concentrations of progesterone greater than 0.5 ng/mL were considered to have ovulated. More than 93% of females exposed to bucks ovulated throughout the experiment regardless of the duration of contact with males, whereas none of them ovulated in the isolated group (P < 0.0001). The proportions of females that ovulated at least once did not differ among groups as well as the proportions of goats that displayed normal or short ovulatory cycles. The interval between the introduction of males and the first ovulation did not differ among groups of goats in contact with bucks for 15 minutes, 30 minutes, 1 hour, or 2 hours. However, in these groups, this ovulation occurred about 2 days later than in females in contact with males during 4 or 24 hours (6.3 vs. 4.4 days; P < 0.05). We conclude that 15 minutes of daily contact with sexually active buck is sufficient to stimulate the ovulatory activity in seasonally anestrous goats, but the first ovulation is delayed in goats exposed to males for 2 h/day or less during 15 days in comparison with those in contact with males for 4 or 24 h/day.

Identification and profiling of microRNAs from ovary of estrous Kazakh sheep induced by nutritional status in the anestrous season.

Estrous regulation in sheep has an important role in the sheep industry in improving production of meat and wool. It has been reported that an enhanced nutritional status can induce estrus resulting in an end of the anestrous season earlier than occurs in ewes with a lesser nutritional status. However, the endocrine and physiological mechanisms that induce the increased incidence of estrus remains unclear. In the present study, the differences in amounts and characteristics of miRNAs in ewes at estrus or during the anestrous season were screened by using the Illumina HiSeq sequencing technology. In total, 294 miRNAs, including 174 novel miRNA candidates, were identified in ewes with an enhanced nutritional status (OEN) through assessment of the OEN library for this group and 307 miRNAs including 186 novel miRNA candidates were identified in the ewes with a lesser nutritional status (OAN) through assessing the OAN library, among which there were nine conserved and 104 novel miRNAs in differential amounts between the two libraries. Based on poly (A) q-PCR, six miRNAs were assessed to verify the accuracy of the library database. Furthermore, the family of the known miRNAs, the target genes and related pathways were also analyzed. The results indicated that the nutritional status had important roles in estrous regulation in sheep. The PLA2G4D can directly regulate ovarian follicle development, or indirectly influence leptin secretion involved in the regulation of the reproductive endocrine and physiological systems during the anestrous season. The identification of significantly different miRNAs expanded the repertoire of sheep miRNAs that have been examined and could contribute to further studies on the molecular mechanism of regulation of initiation of estrous cycles in previously anestrous ewes as influenced by different nutritional status.

A high level of male sexual activity is necessary for the activation of the medial preoptic area and the arcuate nucleus during the 'male effect' in anestrous goats.

In small ungulates such as sheep or goats, the introduction of a male among a group of anovulatory females during the anestrus season leads to the reactivation of the gonadotrope axis and ovulation, a phenomenon known as the 'male effect'. In goats, our previous studies have demonstrated the importance of male sexual activity for an efficient reactivation of the gonadotrope axis assessed through ovulation and blood LH pulsatility. In the present experiment, we assessed whether the level of male sexual activity would also induce differential activation of two brain regions of key importance for the reactivation of GnRH activity, namely the medial preoptic area and the hypothalamic arcuate nucleus. In both structures, we observed a differential activation of Fos in females, depending on the level of buck sexual activity. Indeed, goats unexposed to males showed low levels of expression of Fos while those exposed to sexually inactive bucks showed an intermediate level of Fos expression. Finally, the highest level of Fos expression was found in females exposed to sexually active males. However, and contrary to our initial hypothesis, we were not able to find any specific activation of kisspeptin cells in the arcuate nucleus following the introduction of highly sexually active males. As a whole, these results demonstrate that the level of male sexual activity is a key factor to stimulate brain regions involved in the control of the gonadotrope axis in the context of the male effect in goats.

The "Ram Effect": A "Non-Classical" Mechanism for Inducing LH Surges in Sheep.

During spring sheep do not normally ovulate but exposure to a ram can induce ovulation. In some ewes an LH surge is induced immediately after exposure to a ram thus raising questions about the control of this precocious LH surge. Our first aim was to determine the plasma concentrations of oestradiol (E2) E2 in anoestrous ewes before and after the "ram effect" in ewes that had a "precocious" LH surge (starting within 6 hours), a "normal" surge (between 6 and 28h) and "late¼ surge (not detected by 56h). In another experiment we tested if a small increase in circulating E2 could induce an LH surge in anoestrus ewes. The concentration of E2 significantly was not different at the time of ram introduction among ewes with the three types of LH surge. "Precocious" LH surges were not preceded by a large increase in E2 unlike "normal" surges and small elevations of circulating E2 alone were unable to induce LH surges. These results show that the "precocious" LH surge was not the result of E2 positive feedback. Our second aim was to test if noradrenaline (NA) is involved in the LH response to the "ram effect". Using double labelling for Fos and tyrosine hydroxylase (TH) we showed that exposure of anoestrous ewes to a ram induced a higher density of cells positive for both in the A1 nucleus and the Locus Coeruleus complex compared to unstimulated controls. Finally, the administration by retrodialysis into the preoptic area, of NA increased the proportion of ewes with an LH response to ram odor whereas treatment with the α1 antagonist Prazosin decreased the LH pulse frequency and amplitude induced by a sexually active ram. Collectively these results suggest that in anoestrous ewes NA is involved in ram-induced LH secretion as observed in other induced ovulators.