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International audience; In temperate regions goat's reproduction is seasonal. To obtain year-round breeding, hormonal treatments are currently applied. These treatments usually combine a progesterone analog with the pregnant mare serum gonadotropin (PMSG). However, their use has significant ethical and environmental drawbacks. Therefore, alternative methods to manage reproduction are needed. The discovery that in mammals the neuropeptide kisspeptin is a major positive regulator of hypothalamo-pituitary gonadal axis offered an attractive alternative strategy to control reproduction. We have previously designed a kisspeptin analog, called C6, which offers pharmacological advantages over endogenous kisspeptin. These include a longer lasting effect and enhanced activity following intramuscular injection. In the present work, we evaluated C6 effect on LH and FSH plasma concentrations in the Alpine goat breed and tested whether C6 could replace PMSG to trigger ovulation. An intramuscular injection of C6 (15 nmol/doe) given 24 hours after the end of progestogen treatment induced a surge-like peak of both LH and FSH. This was followed by an increase of progesterone, a hallmark of ovulation induction and corpus luteus formation. These results were obtained at three different time of the year: during the breeding season, the non-breeding season and at the onset of the breeding season. Furthermore, we compared the efficacy of C6 and PMSG to induce fertile ovulations when these treatments are given at the onset of the breeding season and are followed by artificial insemination. The results of this first attempt were extremely promising with gestation rates of 45% and 64% for C6 and PMSG respectively. Pending optimization of the treatment procedure in order to improve efficacy, kisspeptin analogs could be the long sought-after alternative to PMSG.

This is the peer reviewed version of the following article: Dardente, H., Lomet, D., Chesneau, D., Pellicer-Rubio, M.-T. & Hazlerigg, D. (2019). Discontinuity in the molecular neuroendocrine response to increasing daylengths in Ile‐de‐France ewes: Is transient Dio2 induction a key feature of circannual timing? Journal of Neuroendocrinology, 31(8), e 12775, which has been published in final form at https://doi.org/10.1111/jne.12775. . This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. In mammals, melatonin is responsible for the synchronisation of seasonal cycles to the solar year. Melatonin is secreted by the pineal gland with a profile reflecting the duration of the night and acts via the pituitary pars tuberalis (PT), which in turn modulates hypothalamic thyroid hormone status via seasonal changes in the production of locally‐acting thyrotrophin. Recently, we demonstrated that, in the Soay sheep, photoperiodic induction of Tshb expression and consequent downstream hypothalamic changes occur over a narrow range of photoperiods between 12 and 14 hours in duration. In the present study, we aimed to extend our molecular characterisation of this pathway, based on transcriptomic analysis of photoperiodic changes in the pituitary and hypothalamus of ovariectomised, oestradiol‐implanted Ile‐de‐France ewes. We demonstrate that photoperiodic treatments applied before the winter solstice elicit two distinctive modes of accelerated reproductive switch off compared to ewes held on a simulated natural photoperiod, with shut‐down occurring markedly faster on photoperiods of 13 hours or more than on photoperiods of 12 hours and less. This pattern of response was reflected in gene expression profiles of photoperiodically sensitive markers, both in the PT (Tshb, Fam150b, Vmo1, Ezh2 and Suv39H2) and in tanycytes (Tmem252 and Dct). Unexpectedly, the expression of Dio2 in tanycytes did not show any noticeable increase in expression with lengthening photoperiods. Finally, the expression of Kiss1, the key activator of gonadotrophin‐releasing hormone release, was proportionately decreased by lengthening photoperiods, in a pattern that correlated strongly with gonadotrophin suppression. These data show that stepwise increases in photoperiod lead to graded molecular responses at the level of the PT, a progressive suppression of Kiss1 in the hypothalamic arcuate nucleus and luteinising hormone/follicle‐stimulating hormone release by the pituitary, despite apparently unchanged Dio2 expression in tanycytes. We hypothesise that this apparent discontinuity in the seasonal neuroendocrine response illustrates the transient nature of the thyroid hormone‐mediated response to long days in the control of circannual timing.

International audience; This study was designed to determine the effect of melatonin from continuous slow-release implants on (i) the mRNA expression for the lipopolysaccharide (LPS) receptor complex, which includes toll-like receptor 4 (TLR4), myeloid differentiation-2 factor (MD-2) and cluster of differentiation-14 (CD14) in the choroid plexus (CP) and (ii) on the plasma concentration of LPS binding protein (LBP) in ewes subjected or not to a systemic inflammatory stimulus (intravenous injection of LPS). Experiments were conducted in ovariectomized, oestradiol-implanted ewes during seasonal anoestrus (May/June). In experiment 1, the ewes were divided into four groups: sham-implanted and placebo-treated (C/C), melatonin-implanted and placebo-treated (M/C, Melovine 18 mg), sham-implanted and LPS-treated for 3 h (C/LPS, 400 ng/kg of body mass) and melatonin-implanted and LPS-treated for 3 h (M/LPS). In experiment 2, the ovariectomized ewes were melatonin-implanted or sham-implanted and treated with LPS for 6 h (M/LPS and C/LPS, respectively). Plasma melatonin concentrations reached 4.5 ± 1.5 pg/ml in the C/C group, 151.4 ± 56.4 pg/ml in the M/C group, 7.8 ± 4.3 pg/ml in the C/LPS group and 240.6 ± 93.0 pg/ml in the M/LPS group one month after melatonin or sham implantation. Plasma cortisol concentrations remained at basal level throughout the experiment in the control ewes (C/C and M/C), whereas in the LPS-treated ewes, mean cortisol concentrations increased from 13.7 ± 2.4 ng/ml to 92.0 ± 10.2 ng/ml and from 15.4 ± 5.9 ng/ml to 100.0 ± 10.7 ng/ml in the C/LPS and M/LPS groups, respectively. The LPS treatment significantly (p ≤ 0.05) increased the mRNA expression for TLR4 and CD14 but had no effect on the MD-2 mRNA expression in the C/LPS and M/LPS groups. Plasma LBP concentration was not affected within 3 h of the LPS-treatment (experiment 1) but increased significantly at 4–6 h after LPS-treatment in the C/LPS and M/LPS groups (experiment 2). Melatonin from the slow-release implants did not change the mRNA expression of all the examined genes or the plasma LPB concentration. These results allow us to infer that melatonin implantation does not disturb the CP potential to detect the specific microbial components derived from gram-negative bacteria.

Goats are spontaneously ovulating, polyoestrous animals. Oestrous cycles in goats are reviewed in this paper with a view to clarifying interactions between cyclical changes in tissues, hormones and behaviour. Reproduction in goats is described as seasonal; the onset and length of the breeding season is dependent on various factors such as latitude, climate, breed, physiological stage, presence of the male, breeding system and specifically photoperiod. In temperate regions, reproduction in goats is described as seasonal with breeding period in the fall and winter and important differences in seasonality between breeds and locations. In tropical regions, goats are considered continuous breeders; however, restricted food availability often causes prolonged anoestrous and anovulatory periods and reduced fertility and prolificacy. Different strategies of breeding management have been developed to meet the supply needs and expectations of consumers, since both meat and milk industries are subjected to growing demands for year-round production. Hormonal treatments, to synchronize oestrus and ovulation in combination with artificial insemination (AI) or natural mating, allow out-of-season breeding and the grouping of the kidding period. Photoperiodic treatments coupled with buck effect now allow hormone-free synchronization of ovulation but fertility results after AI are still behind those of hormonal treatments. The latter techniques are still under study and will help meeting the emerging social demand of reducing the use of hormones for the management of breeding systems.

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Remerciements : Plate-Forme Phénotypage Endocrinologie, INRA, UMR PRC, 37380 Nouzilly, Centre Val de Loire; Goat oestrous and ovulatory response to the ‘male effect’ was characterised to determine the time range over which fertile ovulations occur after buck exposure. The results were used to explore the efficacy of different hormone-free artificial insemination (AI) protocols aimed at diminishing the number of inseminations needed to optimise fertility. Adult bucks and does were exposed to artificially long days during winter, then exposed to a natural photoperiod before buck exposure (Day 0). Most goats (>70%) ovulated twice, developing a short cycle followed by a normal cycle over 13 days after buck exposure. Among them, 21% were in oestrus at the short cycle and 94% at the normal cycle. This second ovulation occurred within 48 h of Day 6 and was the target for AI protocols. In protocol A (n=79), goats were inseminated 12h after oestrus detection from Day 5 to Day 9. Up to six AI times over four days were needed to inseminate goats in oestrus. Forty-nine percent of the inseminated goats kidded. In protocol B (n=145), oestrus detection started on Day 5. The earlier (group 1) and later (group 2) buck-marked goats received one single insemination at fixed times on Days 6.5 or 7 and 8, respectively; unmarked goats (group 3) were inseminated along with group 2. In protocol C (n=153), goats were inseminated twice on Days 6.5 or 7 and 8 without needing to detect oestrus. Goats induced to ovulate by hormonal treatment were used as the control (n=319). Fertility was lower in protocol B than C and control (47 vs. 58 vs. 65% kidding; p≤0.05), whereas this was higher in buck-marked goats than unmarked ones (64 vs. 33%; p≤0.05). In protocol B, fertility can increase (>60%) when only goats coming into oestrus are inseminated. The best kidding rate (∼70%) was achieved when does were inseminated within 24h of the LH surge. Protocols involving insemination on Day 7 instead of Day 6.5 led to more goats being inseminated during this favourable time.

The first ovulation induced by male effect in sheep during seasonal anoestrus usually results in the development of a short cycle that can be avoided by progesterone priming before ram introduction. In elucidating the involvement of the hypothalamic–pituitary–gonadal axis in the occurrence of short cycles, the effects of progesterone and the time of anoestrus on the development of male-induced preovulatory follicles were investigated in anoestrous ewes using morphological, endocrine and molecular approaches. Ewes were primed with progesterone for 2 (CIDR2) or 12 days (CIDR12) and untreated ewes used as controls during early (April) and late (June) anoestrus. The duration of follicular growth and the lifespan of the male-induced preovulatory follicles were prolonged by ∼1.6 days in CIDR12 ewes compared with the controls. These changes were accompanied by a delay in the preovulatory LH and FSH surges and ovulation. Intra-follicular oestradiol concentration and mRNA levels ofLHCGRandSTARin the granulosa and theca cells of the preovulatory follicles were higher in CIDR12 ewes than the control ewes. The expression of mRNA levels ofCYP11A1andCYP17A1also increased in theca cells of CIDR12 ewes. CIDR2 ewes gave intermediate results. Moreover, ewes ovulated earlier in June than in April, without changes in the duration of follicular growth, but these effects were unrelated to the lifespan of corpus luteum. Our results give the first evidence supporting the positive effect of progesterone priming on the completion of growth and maturation of preovulatory follicles induced by male effect in seasonal anoestrous ewes, thereby preventing short cycles.

International audience; The response to the male effect was studied in two Saanen and two Alpine flocks over 5 consecutive years. Adult male and female goats were exposed to artificial long days (16 h light and 8h darkness, 16L:8D)in open barns for approximately 3 months (between December 1 and April 15) followed by a natural photoperiod. Goats were treated for 11 days with fluorogestone acetate (FGA) or progesterone (CIDR) immediately before joining. Bucks carrying marking harnesses with adapted aprons joined females 49-63 days after the end of the long-day treatment (between April 30 and June 5) and were left with them for 5 days. In experiment 1 (n = 142), FGA- and CIDR-treated goats were inseminated at a time based on the detection of oestrus. Two insemination groups were distinguished by the occurrence of marking over a 48-h period. Earlier (group 1) and later (group 2) buck-marked goats received one single insemination 12-24 In or 0-12 h after marking, respectively. Unmarked goats were inseminated along with group 2. In experiment 2 (it = 344), FGA-treated goats were inseminated 52 and 70 h (52 h:70 h group) or 52 and 75 h (52 h:75 h group) after joining. In experiment 3 (n = 285), FGA-treated goats were inseminated 52 h (1-AI group) or 52 and 75 h(2-AI g-roup) after joining. In all experiments, an external control group given the "classical" insemination program was analysed. Over the 5-year period, 92% of the goats exhibited an LH surge during days 1-4 after joining and 98% of them ovulated. Eighty-seven percent of the LH surges detected in milk occurred during the 33-57 h interval after joining, indicating that ovulation took place around 45-69 h. In experiment 1, 96% of the goats were marked 22-70 h after joining. Kidding rate (KR; 78%) was similar between insemination groups and between FGA- and CIDR-treated goats (p > 0.05). Most of the goats (95%) were inseminated during the interval between 15 h before and up to 4 h after ovulation. KR was not affected by the time between detection of marking and insemination or between insemination and ovulation (p > 0.05). In experiment 2, KR (75%) was similar in both insemination groups (P > 0.05). In experiment 3, KR was higher (p < 0.05) in the 1-AI (71%) than the 2-AI group (57%). In all experiments, KR of the control group (68-73%) was similar to that achieved in goats induced to ovulate by the male effect. Prolificity (2.1 +/- 0.7) was not affected by any of the factors examined (p > 0.05). In conclusion, high fertility can be achieved during anoestrus when 1 or 2 inseminations are performed over a 24 h period, determined by oestrus or by the introduction of the buck, if light-treated goats receive 11-day FGA or CIDR treatment and are then induced to ovulate by the male effect. (C) 2007 Elsevier B.V. All rights reserved.

The response to the male effect was studied in two flocks of Saanen and three of Alpine goats during deep anoestrus in three consecutive years. Males and females were subjected to artificially long days for about 3 months (between December 4 and April 1) followed by a natural photoperiod. Bucks joined goats 42-63 days after the end of the long days treatment (between April 20 and June 3) and fertilisation was ensured by natural mating. In experiment 1 (n=248), female goats were treated or untreated with melatonin at the end of the long days treatment and treated or untreated for 11 days with fluorogestone acetate (FGA) before teasing. The males received melatonin implants. In experiment 2 (n=337), the factor studied was the association or non-association of the 11-day FGA treatment. Neither males nor females received melatonin implants. In experiment 3 (n=180), goats were treated for 11 days with FGA or with natural progesterone (CIDR). Neither males nor females received melatonin implants. In experiment 1, among the non-cycling goats (n=218), 99% ovulated and 81% kidded at 161+/-8 days after joining. Ninety-two percent of FGA-treated goats displayed an LH surge at 65+/-11h after teasing. Melatonin treatment did not affect any parameter but FGA advanced the kidding date. In experiment 2, 94% of the goats ovulated and 87% kidded. A major peak of conception was observed on days 3 and 8 after joining in FGA-treated and untreated goats, respectively. Among the FGA-treated goats, 83% displayed an LH surge. Over all flocks, most of the LH surges occurred over a 24-36 h interval, but the surge was initiated at different times in different flocks (36, 48 or 60 h after joining). FGA treatment did not influence the results, except for advancement of births of about 5 days. Differences among flocks were highly significant. In experiment 3, 94% of the goats displayed the LH surge, 93% ovulated and 68% kidded. Significant differences were found among flocks, but not between the FGA and CIDR groups. Bucks marked 85% of the goats 24-72 h after joining. The time interval between the detection of marked goats and detection of the LH surge depended on the time of marking (r=-0.62; p

The storage of frozen semen for artificial insemination is usually performed in the presence of egg yolk or skimmed milk as protective agents. In goats, the use of skimmed milk extenders requires, however, that most of the seminal plasma is removed before dilution of spermatozoa because it is deleterious for their survival. It has been previously demonstrated that a lipase (BUSgp60) secreted by the accessory bulbourethral gland was responsible for the cellular death of goat spermatozoa, through the lipolysis of residual milk lipids and the release of toxic free fatty acids. This lipase was purified from the whole seminal plasma of goat and was found to display both lipase and phospholipase A activities, this latter activity representing the main phospholipase activity detected in goat seminal plasma. Based on its N-terminal amino acid sequence, identical to that of BUSgP60 purified from bulbourethral gland secretion, and the design of degenerated oligonucleotides, the lipase was cloned from total mRNA isolated from bulbourethral gland. DNA sequencing confirmed it was the goat pancreatic-lipase-related protein 2 (GoPLRP2). The physiological role of GoPLRP2 is still unknown but this enzyme might be associated with the reproductive activity of goats. A significant increase in lipase secretion was observed every year in August and the level of lipase activity in the semen remained high till December, i.e., during the breeding season. A parallel increase in the plasmatic levels of testosterone suggested that GoPLRP2 expression might be regulated by sexual hormones. The lipase activity level measured in goat seminal plasma, which could reach 1000 U/ml during the breeding season, was one of the highest lipase activity measured in natural sources, including gastric and pancreatic juices.