Reproductive performance in two organic sheep farming systems differing by the number of mating sessions in and out of the breeding season.

Hormonal treatments to control ovulation and the intensification of reproduction rhythm are usual strategies to increase ewe productivity. However, in organic farming the use of hormones is banned, making any increase of reproduction rhythm an inappropriate practice. This work tested if increasing the number of mating sessions per year could improve ewe annual productivity, while maintaining a non-intensive reproduction rhythm of one lambing per ewe per year. Reproductive performance was studied over five years in two organic sheep farming systems differing by the number of mating sessions (MS) per year, two (2MS) or four (4MS). The 4MS system included two mating sessions in the breeding season (September and November) and non-breeding season (April and June). The 2MS system included mating in November and April. Non-pregnant ewes were recycled one (2MS) or two times (4MS system) on consecutive mating sessions. Considering all mating attempts per ewe individually (n = 1366), the time elapsed from mating to lambing (159 ± 0.2 days), fertility (86%), prolificacy (166%) and productivity (143%) did not differ between the systems (P > 0.05). Fertility, prolificacy and productivity were higher in the breeding than the non-breeding season (P ≤ 0.05), and in adults than young ewes (P ≤ 0.05), without interaction with the systems (P > 0.05). In the non-breeding season, fertility, prolificacy and productivity did not relate to ewe cyclic status (P > 0.05), whereas the time elapsed from mating to lambing was shortened in cyclic ewes (P ≤ 0.05). Good ewe body condition at lambing and mating and dynamic gain in body condition from drying to mating improved productivity (P ≤ 0.05) by increasing fertility or prolificacy regardless of the system (P > 0.05). On a yearly scale, the number of lambed ewes was higher in the 4MS than the 2MS system (91% vs 85%; P ≤ 0.05) leading to higher ewe annual productivity (154% vs 141%; P ≤ 0.05). The percentage of ewes that were recycled on consecutive mating sessions was higher in the 4MS than the 2MS system (24% vs 18%; P ≤ 0.05), possibly explaining the 4MS increase of ewe annual productivity by extending mating opportunities for the ewes. The increase in the number of mating sessions per year is a hormone-free strategy suitable for organic sheep farming systems to improve annual ewe productivity on a basis of one lambing per ewe per year. This approach proved beneficial to compensate for lower fertility in the non-breeding season, especially for young ewes.

Chronic low BPS exposure through diet impairs in vitro embryo production parameters according to metabolic status in the ewe.

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 pick-up (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.

Discontinuity in the molecular neuroendocrine response to increasing daylengths in Ile-de-France ewes: Is transient Dio2 induction a key feature of circannual timing?

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.

The kisspeptin analog C6 is a possible alternative to PMSG (pregnant mare serum gonadotropin) for triggering synchronized and fertile ovulations in the Alpine goat.

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.

Seasonal breeding in mammals: From basic science to applications and back.

Seasonal breeding is a remarkable adaptive feature, which allows animals to coordinate physiological functions throughout the year. However, in the context of animal production, it becomes an undesirable complication, which needs to be circumvented. Therefore, eco-friendly methods based on photoperiodic treatments and the use of the male effect have been developed to control seasonal reproduction in small ruminants. In practice, such treatments are hardly used and hormonal treatments constitute the benchmark, but practicality of hormonal treatments comes at a high cost for human health and the environment. Here, we summarize our current understanding of the molecular and neuroendocrine mechanisms underlying seasonal breeding in small ruminants. We then move on to describe current methods to control reproduction and detail why such methods are not sustainable. Finally, using the neuropeptide kisspeptin as an example, we show that an improved understanding of the molecular and neuroendocrine mechanisms that underlie photoperiodism might help design novel strategies for the development of improved and sustainable breeding schemes.

Evaluation of hormone-free protocols based on the "male effect" for artificial insemination in lactating goats during seasonal anestrus.

Goat estrous and ovulatory responses to the "male effect" were characterized 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 optimize fertility. Adult bucks and does were exposed to artificially long days during winter and 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 estrus at the short cycle and 94% at the normal cycle. This second ovulation occurred within 48 hours of Day 6 and was the target for AI protocols. In protocol A (n = 79), goats were inseminated 12 hours after estrus detection from Day 5 to Day 9. Up to six AI times over 4 days were needed to inseminate goats in estrus. Forty-nine percent of the inseminated goats kidded. In protocol B (n = 145), estrus 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 estrus. Goats induced to ovulate by hormonal treatment were used as the control (n = 319). Fertility was lower in protocol B than in protocol C and controls (47% vs. 58% and 65% kidding; P ≤ 0.05), whereas this was higher in buck-marked goats than in unmarked ones (64% vs. 33%; P ≤ 0.05). In protocol B, fertility can increase (>60%) when only goats coming into estrus are inseminated. The best kidding rate (∼70%) was achieved when does were inseminated within 24 hours of the LH surge. Protocols involving insemination on Day 7 instead of Day 6.5 led to more goats being inseminated during this favorable time.

Progesterone improves the maturation of male-induced preovulatory follicles in anoestrous ewes.

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 of LHCGR and STAR in the granulosa and theca cells of the preovulatory follicles were higher in CIDR12 ewes than the control ewes. The expression of mRNA levels of CYP11A1 and CYP17A1 also 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 luteal outcome of anoestrus ewes induced to ovulate by the male effect is not related to the population of ovarian antral follicles before male exposure.

The ovarian status and its relationship with the response to the male effect were studied in Ile-de-France ewes entering anoestrus early (becoming anovulatory on January-February, n=13) or late (becoming anovulatory on March, n=13). The male effect was performed, in each group of ewes, at the beginning of the anoestrus season (March-April), approximately 35 days after ewes became anovulatory. Transrectal ultrasonography of ovaries was done at D-7, D-5, D-3 and D0 (ram introduction day) to examine the number and size of follicles ≥2mm, from D0 to D4 to analyze the ram-induced preovulatory follicles and at D14-D16 to identify luteal structures. Plasmatic progesterone level was assessed from D-7 to D14-16 to examine the ovulatory response to the male effect. Before ram introduction, the number of medium (3.5-4.4mm) and large (>4.4mm) follicles and the maximum follicle diameter were lower (p<0.05) in ewes entering anoestrus early than in ewes entering it late. The percentage of ewes developing a short cycle at the first ram-induced ovulation was higher in those starting anoestrus early (92% vs 31%; p<0.05); normal cycles were only observed in ewes entering anoestrus late (0% vs 54%; p<0.05). The time of the onset of anoestrus did not affect (p>0.05) the ram-induced preovulatory follicle characteristics; these parameters were similar (p>0.05) between ewes developing a short or a normal cycle. Results did not show any relationship between the ovarian status preceding male introduction and the growing dynamic of the ram-induced preovulatory follicles or the category of cycle (normal or short) displayed following ovulation. In conclusion, (1) the luteal outcome following the first ovulation induced by the male effect depends on the time of onset of seasonal anoestrus and (2) the number and size of follicles ≥2mm also depend on the time of onset of seasonal anoestrus but are not related to the luteal outcome following the first ovulation induced by the male effect.

Reproductive cycle of goats.

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.

High fertility using artificial insemination during deep anoestrus after induction and synchronisation of ovulatory activity by the "male effect" in lactating goats subjected to treatment with artificial long days and progestagens.

The response to the male effect was studied in two Saânen and two Alpine flocks over 5 consecutive years. Adult male and female goats were exposed to artificial long days (16h 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 12-24h or 0-12h after marking, respectively. Unmarked goats were inseminated along with group 2. In experiment 2 (n=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 group) 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 15h before and up to 4h 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 24h 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.

Highly synchronous and fertile reproductive activity induced by the male effect during deep anoestrus in lactating goats subjected to treatment with artificially long days followed by a natural photoperiod.

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<0.05). In conclusion, treatment of both males and females goats with artificially long days followed by a natural photoperiod is very effective in inducing highly synchronous and fertile reproductive activity via the male effect in the middle of seasonal anoestrus.

Cloning and seasonal secretion of the pancreatic lipase-related protein 2 present in goat seminal plasma.

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.