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International audience; Bisphenol A (BPA), an endocrine disruptor, has been replaced by structural analogues including bisphenol S (BPS). BPA and BPS exhibited similar effects regarding reproductive functions. Moreover, metabolic status and lipid metabolism are related to female fertility and could worsen BPS effects. The objective was to determine BPS in vivo effects on folliculogenesis and embryo production after chronic exposure through diet, and the influence of metabolic status in adult ewes. Sixty primiparous 2.5 year-old ewes, undergoing a restricted or well fed diet, were exposed to BPS (0, 4 or 50 µg/kg/day) for at least three months. After hormonal oestrus synchronisation and ovarian stimulation, ewes were subjected to ovum pickup (OPU) procedures to collect immature oocytes, that underwent in vitro maturation, fertilisation and embryo production. Body weight, body condition score and plasma glucose were higher in well-fed compared to restricted ewes, while plasma NEFA was lower during the 4-5 months after the beginning of the diets. Plasma progesterone levels increased on day 5 before OPU session in well-fed compared to restricted ewes. No effect of BPS dose was observed on follicle population, plasma AMH levels and embryo production numbers and rates. However, a significant diet x BPS dose interaction was reported for cleaved embryos, > 4-cell embryos, blastocyst and early blastocyst numbers, and plasma triiodothyronine levels. Our study showed that a contrasted diet did not affect follicle population nor embryo production in adult ewes but could affect the quality and progesterone secretion of the corpus luteum. Chronic low BPS exposure had no effect on follicular population and oocyte competence. Nevertheless, the significant diet x dose interactions observed on embryo production suggest that BPS effect is modulated by metabolic status. Further studies are required to assess the risk of BPS exposure for public reproductive health.

La maîtrise de la saisonnalité de la reproduction est un enjeu pour les filières ovines et caprines. Dans le contexte socio-économique actuel, l’essor de pratiques alternatives aux traitements hormonaux d’induction et de synchronisation des chaleurs et des ovulations est nécessaire en Agriculture Conventionnelle (AC) et de nouvelles perspectives d’évolution sont attendues en Agriculture Biologique (AB). À partir d’enquêtes réalisées auprès de 97 intervenants en élevage et 298 éleveurs ovins et caprins en AB et en AC, nous avons dressé un état des lieux des pratiques de gestion de la reproduction mises en œuvre dans les principaux bassins de production en France. Puis, nous avons évalué l’acceptabilité des acteurs vis-à-vis de deux outils innovants associés à la gestion de la reproduction : l’utilisation de phéromones impliquées dans l’effet mâle (comme alternative aux traitements hormonaux pour l’induction et la synchronisation des ovulations et des chaleurs) et la détection automatisée des chaleurs (pour optimiser la mise à la reproduction par IA ou pour la lutte en main). D’après les enquêtes, les pratiques de dessaisonnement de la reproduction impliquant les traitements lumineux, le recours à la mélatonine et les lactations longues sont quasi exclusivement mises en œuvre par les producteurs caprins. Les traitements hormonaux pour l’induction et la synchronisation des chaleurs et la mélatonine sont interdits par le cahier des charges en AB et donc exclusivement utilisés en AC. Au contraire, l’effet mâle est pratiqué dans l’ensemble des filières. La détection des chaleurs n’est mise en œuvre que par les éleveurs caprins et ovins laitiers. Enfin, le flushing est surtout pratiqué en filière ovine. L’utilisation potentielle des phéromones pour induire et synchroniser les chaleurs intéresse à la fois les intervenants et les éleveurs. En AB, cette technique soulève cependant des questions quant au respect du cahier des charges. L’accueil réservé aux détecteurs automatisés de chaleurs est plus mitigé. Si leur intérêt pour l’organisation du travail et la facilitation des IA a été exprimé par les éleveurs, ces derniers ont soulevé une inquiétude quant à l’inadéquation par rapport aux conduites d’élevage au pâturage a été soulevée. Si certains éleveurs sont prêts à acquérir les deux types de dispositifs, ils sont en attente des résultats concernant l’efficacité, le coût et la praticité des outils avant d’envisager de s’équiper.

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.

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.

The aim of this work was to purify, identify, and characterize the component of goat bulbourethral gland secretion (BUS) responsible for goat sperm deterioration in skim milk extender. BUS extracts promote a decrease in the percentage of motile spermatozoa, deterioration in the quality of movement, breakage of acrosomes, and cellular death of goat spermatozoa diluted in skim milk. A 55- to 60-kDa monomeric glycoprotein (BUSgp60) was purified by cation-exchange, concanavalin A, and heparin-affinity chromatography and was identified as the only BUS component responsible for the deterioration of spermatozoa in milk. The BUSgp60 was shown to display triacylglycerol hydrolase activity, and partial sequences (37 amino acids in all) exhibited 50-70% homology with sequences of various types of pancreatic lipases (PLs), especially PL-related protein 2 (PL-RP2). In addition, porcine PL produced deterioration of goat sperm properties in milk as effectively as BUS and BUSgp60. These results support the preliminary identification of the goat BUSgp60 as a bulbourethral lipase belonging to the PL-RP2 family. Since seminal plasma also contains factors favorable for sperm survival, it is envisaged that, for the best preservation of goat semen, specific inhibitors of this family of lipase could be added into milk-based extenders without eliminating seminal plasma.

The control of reproduction is necessary to optimise the productivity of livestock production systems. The regulations of organic farming do not impose specific reproductive practices among those that are already in use except that some of them are not authorized. Whatever the animal species, the success of reproduction in organic systems mainly relies on the choice of adapted breeds. Because sheep and goats are well-known seasonal breeders the major issue is to overcome this limitation of reproductive possibilities. In these organic farming systems the reproduction is possible all year round thanks to the manipulation of daylight duration without using melatonin, however up to now these treatments are not developed. The male effect is another method to overcome seasonal blockade am that allows synchronizing lambing and kidding without any hormonal treatments. This method is however rather difficult to apply in all situations. Artificial insemination (AI) is the main way to benefit from selection's progress. In conventional systems At is performed a fixed time after hormonal synchronization of ovulations without necessity to detect oestrus. AI, which is authorized on natural oestrus, in not really used in organic flocks because they are spread over several days. Synchronization of oestrus with the male effect is however promising way to perform AI and allow organic breeders to benefit from selection schemes. In conventional swine production systems the classical practice is to drive sows into a 7 batches system at 3 weeks interval. The productive cycle duration is of 21 weeks including 28 days of suckling. In the European regulation of organic farming systems, the piglet suck ling period should not be shorter than 40 days. Practically the weaning will occur at 42 days with a 8 batches system. Hence the whole productive cycle of the sow will be lengthened up to 23 weeks. In organic system, like in conventional systems, weaning is the main method to synchronize sows oestrus within a given batch. The overall efficiency of organic production is however reduced because no hormonal treatment is allowed either to synchronize gilts or to transfer open sows into another band and those that may show oestrus during lactation. In all pig production systems AI is performed after oestrus detection. In cattle, AI is generally performed after detection of natural oestrus. Reproduction is mainly performed through AI in dairy cows ani with bull mating in beef cows. Thus the fact that hormonal treatments are not allowed in organic systems do not penalise them Furthermore reproduction is possible all year round except that it is performed according to productive goals and local constraints. The development of natural methods for the control of reproduction in organic livestock systems implies numerous research approache and multidisciplinary programs, often common to different species. INRA is strongly involved in the study of hormone-free methods ani their adaptation to breeding constraints., La maîtrise de la reproduction contribue à optimiser la productivité de l’exploitation. La réglementation de la production biologique n’impose pas de conduite de reproduction spécifique, mais limite les pratiques autorisées parmi l’ensemble des stratégies disponibles pour les éleveurs. Les conséquences de ces limitations sont plus ou moins pénalisantes selon l’espèce animale considérée. Ainsi, l’interdiction des traitements hormonaux représente le principal obstacle pour la maîtrise de la reproduction en agriculture biologique (AB) chez les petits ruminants et les porcins. La maîtrise de la saisonnalité de la reproduction est un objectif pour la plupart des éleveurs ovins et caprins. Dans ces deux espèces, la reproduction est possible tout au long de l’anoestrus saisonnier grâce à des traitements lumineux sans recours à la mélatonine, mais des difficultés de mise en œuvre font qu’ils sont aujourd’hui peu utilisés en AB. En élevage conventionnel ovin et caprin, l’insémination artificielle (IA) est réalisée à un moment fixe après synchronisation hormonale des ovulations et sans détection d’oestrus. En AB, l’IA est autorisée sur chaleurs naturelles : cela implique la pratique systématique de la détection des chaleurs sur plusieurs jours pour identifier les femelles à inséminer. Pour cette raison, l’IA est très peu pratiquée en AB chez ces deux espèces. La synchronisation des ovulations par effet mâle est une alternative prometteuse pour simplifier les protocoles d’IA, facilitant l’accès des éleveurs AB aux schémas de sélection et donc la généralisation de cette pratique. La conduite de reproduction la plus courante en élevage de porc conventionnel est dite en 7 bandes. La durée du cycle d’une bande est de 21 semaines dont 4 semaines d’allaitement. En AB, le règlement européen impose l’augmentation du temps d’allaitement des porcelets à 40 j minimum. En pratique, le sevrage a lieu à 42 j avec une conduite en 8 bandes, le cycle de reproduction s’étend donc à 23 semaines. En AB comme en conventionnel, le sevrage est la principale méthode utilisée pour la synchronisation de l’oestrus des truies dans leur bande. L’efficacité de la conduite en bandes est toutefois pénalisée en AB par l’interdiction des traitements hormonaux, utilisés fréquemment en conventionnel pour intégrer les cochettes dans les bandes, pour recycler des femelles décalées ou pour prévenir des oestrus de lactation. Cependant, l’interdiction de l’utilisation d’hormones ne pénalise pas la pratique de l’IA car elle est réalisée sur oestrus naturels synchronisés par le sevrage chez la majorité des producteurs en AB et en élevage conventionnel. Chez les bovins, l’insémination est pratiquée de manière générale sur chaleurs naturelles. Lors de la mise à la reproduction, la mise en place de la semence est réalisée par insémination artificielle (majoritaire en élevage laitier) ou par monte naturelle (majoritaire en élevage allaitant). L’interdiction des traitements hormonaux d’induction et de synchronisation des chaleurs n’est donc pas pénalisante en AB par rapport à l’élevage conventionnel. La mise à la reproduction est possible toute l’année, avec des contraintes qui diffèrent selon les objectifs de production. Les voies de recherche pour le développement de méthodes naturelles de maîtrise de la reproduction utilisables en AB sont très diverses, multidisciplinaires et souvent communes aux différentes espèces de mammifères d’élevage. L’INRA contribue fortement à l’amélioration des méthodes existantes et à leur adaptation aux contraintes d’élevage. Il s’implique également dans la conception et la validation de nouvelles approches de maîtrise de la reproduction sans hormones. Ce développement ne pourra connaître un véritable essor que dans le cadre d’une relation étroite entre la recherche finalisée et les professionnels.

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Le contrôle de la reproduction caprine est intéressant pour des raisons techniques (synchronisation des mise bas, ajustements aux disponibilités fourragères ou aux contraintes économiques), et pour des raisons génétiques (identification et dissémination des génotypes améliorateurs). L’utilisation de rythmes photopériodiques accélérés conduit à une augmentation très importante des doses produites pour l’insémination artificielle (IA) et évite l’apparition d’une contre-saison sexuelle. L’identification récente d’une lipase bulbo-uréthrale dans le sperme de bouc offre de nouvelles perspectives pour la conservation de la semence. Chez la chèvre, les traitements " lumière + jours courts " permettent également l’induction de cycles oestriens et ovulatoires à contre-saison, ce qui conduit à une amélioration de la fertilité des troupeaux. En revanche, l’utilisation répétée de eCG (autrefois appelée PMSG) provoque l’apparition d’anticorps, retarde le moment de l’ovulation et induit une baisse de fertilité après IA, lorsqu’elle est réalisée après un délai fixe après la fin du traitement. Toutes les étapes de la production d’embryons, de leur congélation et de leur transfert sont maintenant maîtrisées et permettent d’atteindre des taux satisfaisants de chevreaux nés par femelle donneuse, qui sont compatibles avec le développement de la technique dans le but d’échanger du matériel génétique entre les pays. La production in vitro d’embryons permet des taux élevés de développement après maturation et fécondation in vitro des ovocytes et pourrait permettre l’obtention de populations synchrones de zygotes au stade unicellulaire, nécessaire à l’application des biotechnologies., The control of goat reproduction is interesting for technical reasons (synchronisation of kidding, adaptation to forage availability or to economy), and for genetic reasons (identification and dissemination of improved genotypes). The use of short-light rhythms leads to dramatically increased production of AI doses per buck and prevents the occurrence of a "resting" season. Recent identification of a bulbo-urethral Lipase In goat sperm opens new perspectives in sperm preservation. Light+"short days" treatments also allow the induction of out-of-season estrous cycles and ovulations leading to enhanced fertility. Repeated use of eCG provokes the production of antibodies, delays the timing of ovulation and causes a reduction in fertility after fixed-time artificial insemination. All steps of embryo production, freezing and transfer are now controlled and allow the attainment of satisfactory rates of kids born per donor female, which are compatible with the development of the technique for exchanging genotypes between countries. In vitro production of embryos allows high development rates to be achieved after in vitro maturation and fertilisation of oocytes, and will ensure the production of synchronous populations of one-cell zygotes at the stage required by new biotechnologies.

The aim of the present study was to elucidate the mode of action of goat bulbourethral lipase (BUSgp60 lipase) previously identified as responsible for the deterioration of goat sperm viability in skimmed milk-based extenders. Milk fractions were purified by micro- and ultrafiltration and characterized by SDS-PAGE, thin layer chromatography, triglyceride quantitative analysis and by their ability to potentiate the lipase and the sperm-deteriorating activity of the bulbourethral lipase. Components in both the phosphocaseinate and soluble whey protein fractions enhanced the lipase activity of BUSgp60 but only the phosphocaseinate fraction, which contains triglycerides, promoted deterioration of spermatozoa in the presence of bulbourethral gland secretion. These data suggest that the sperm-deteriorating effect of bulbourethral gland secretion is due to the catalysis of triglyceride hydrolysis, and that proteins increase this activity. BUSgp60 hydrolysed milk triglycerides and triolein very effectively, and its lipase activity was enhanced by several highly purified milk proteins. The major cis-unsaturated fatty acid from milk (oleic acid) but not the major saturated fatty acid (palmitic acid) exhibited dose-dependent detrimental effects on goat spermatozoa. Therefore, the catalysis of oleic acid formation from residual milk triglycerides by BUSgp60 appears responsible for the deterioration of goat spermatozoa when unwashed semen is diluted in skimmed milk-based extenders. The precise mechanism of action of oleic acid remains to be elucidated but the drawbacks of washing buck semen might be avoided by inhibiting BUSgp60 or by depriving it of substrate.