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Exposure of anoestrous ewes to rams induces an increase in LH secretion, eventually leading to ovulation. This technique therefore is an effective, low-cost and hormone-free way of mating sheep outside the breeding season. However, the use of this technique is limited by the variability of the ewes’ responses. In this study, our objective was to understand more completely the origins of this variability and to determine the relative roles of breed, the point in time during anoestrus and the depth of anoestrus on the response to the ‘ram effect’. In the first experiment, the pattern of anoestrus on the basis of the concentration of progesterone determined weekly, was determined in four breeds including two less seasonal (Mérinos d'Arles and Romane), one highly seasonal (Mouton Vendéen) and one intermediate (Île-de-France) breeds. Anoestrus was longer and deeper in Mouton Vendéen and Île-de-France than in Romane or Mérinos d'Arles. In the second experiment, we used the same four breeds and tested their hypophyseal response to a challenge with a single dose of 75 ng gonadotrophin-releasing hormone (GnRH) in early, mid and late anoestrus, and then we examined their endocrine and ovarian responses to the ‘ram effect’. Most (97%) ewes responded to GnRH and most (93%) showed a short-term increase in LH pulsatility following the ‘ram effect’. The responses in both cases were higher in females that went on to ovulate, suggesting that the magnitude of the hypophyseal response to a GnRH challenge could be a predictor of the response to the ‘ram effect’. As previously observed, the best ovarian response was in Mérinos d'Arles at the end of anoestrus. However, there was no relationship between the proportion of females in the flock showing spontaneous ovulation and the response to the ‘ram effect’ of anoestrous ewes from the same flock.

Au cours des cinquante dernières années, des progrès considérables dans les performances d’élevage des espèces domestiques ont été obtenus grâce à une meilleure connaissance acquise dans le domaine de la physiologie, en particulier en ce qui concerne la nutrition, la croissance, la reproduction et la lactation, et qui s’est conjuguée aux progrès génétiques accomplis dans les différentes espèces animales dans un contexte de meilleure maîtrise sanitaire des troupeaux et de maîtrise plus fine des systèmes d’élevage en place. Le recours aux substances hormonales pour mieux maîtriser les fonctions de reproduction et de croissance a pris une place déterminante dans ce progrès. Néanmoins, une tendance profonde consistant à restreindre leur utilisation à certains moments critiques de la vie de l’animal est apparue en Europe ces quinze dernières années, obligeant à rechercher des alternatives par le biais d’une meilleure maîtrise alimentaire, génétique et des systèmes d’élevage, ceci afin de garantir aux consommateurs une meilleure qualité définie a priori des produits animaux. Les fonctions physiologiques qui offrent des possibilités d’un contrôle hormonal exogène, les substances hormonales utilisables et les spéculations animales concernées sont présentées tour à tour. Un éclaircissement sur les risques toxicologiques que présentent ces substances, le contrôle et la traçabilité de leur utilisation et les réglementations en vigueur sont ensuite rappelés. Enfin, sont évoquées les recherches qui permettront de faire progresser sensiblement la question de l’utilisation des hormones en élevage dans un contexte sociétal renouvelé.

Chez les caprins, comme dans la plupart des espèces, l’expression du comportement sexuel dépend à la fois de facteurs internes notament le taux d’hormones stéroides, et externes. Chez le bouc, le niveau d’activité sexuelle fluctue au cours de l’année en liaison avec le taux de testostérone. Cependant, cette dépendance est plus limitée que dans d’autres espèces et peut être modulée par l’environnement social : présence d’un congénère, exposition régulière à des femelles réceptives, etc. Le comportement sexuel de la chèvre, comme celui de la brebis, dépend de la présence, cyclique, de progestérone et d’oestradiol. Le seuil de sensibilité à ces hormones est peu différent de celui des ovins. Comme chez les ovins, la progestérone possède à la fois un effet inhibiteur, à l’origine des traitements de synchronisation de l’oestrus, et un rôle facilitateur, celui-ci étant moins net que chez les ovins. Les chèvres comme les boucs sont très sensibles à l’effet de l’environnement social qui peut -être utilisé comme moyen de manipuler le cycle reproductif (effet mâle pendant la période d’anoestrus par exemple).

Exposure to a ram during spring stimulates luteinizing hormone (LH) secretion and can induce ovulation in sexually quiescent ewes ("ram effect"). Kisspeptin (Kiss) present in the arcuate nucleus (ARC) and the preoptic area (POA) is a potent stimulators of LH secretion. Our aim was to investigate whether Kiss neurons mediate the increase in LH secretion during the ram effect. With double immunofluorescent detection, we identified Kiss neurons (Kiss IR) activated (Fos IR) by exposure to a ram for 2 hours (M2) or 12 hours (M12) or to ewes for 2 hours (C). The density of cells Kiss + Fos IR and the proportion of Kiss IR cells that were also Fos IR cells were higher in M2 and M12 than in C in ARC (P < 0.002) and POA (P < 0.02). In ARC, these parameters were also higher in M12 than in M2 (P < 0.02 and P < 0.05). Kiss antagonist (P234 10-6M) administered by retrodialysis in POA for 3 hours at the time of introduction of the ram reduced the amplitude of the male-induced increase in LH concentration compared with solvent (P < 0.02). In ARC, P234 had a more limited effect (P < 0.038 1 hour after P234) but pulse frequency increased less than after solvent (P = 0.07). In contrast, Kiss antagonist (P271 10-4M) infused in ARC but not POA 6 to 18 hours after introduction of the ram prevented the LH surge in the ewe (0/6 vs 4/5 and 4/6 in C). These results suggest that both populations of Kiss neurons are involved in the ram-induced pulsatile LH secretion and in the LH surge., (Copyright © 2017 Endocrine Society.)

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 "late» 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.

Sheep are seasonal breeders, but exposure of anoestrus ewes to rams results in a rapid increase in luteinizing hormone (LH) secretion, eventually leading to surge in LH. Although LH secretion is known to be under the control of many neurotransmitters, noradrenaline (NA) is of particular importance for the LH surge in induced ovulators, although little is known about its role in LH secretion induced by males in spontaneous ovulators. To address this question, anoestrus ewes fitted with guide-tubes in the medial preoptic area (MPOA) or the ventromedial hypothalamus were subjected to microdialysis and blood sampling every 15 min for an hour before and 2 h after exposure to rams, and the concentrations of LH, monoamine and amino acid transmitters were measured. In ewes implanted in the posterior MPOA that responded to the ram by an increase in LH pulses, NA concentrations changed after exposure to the ram (P<0.018) and were higher at 15 (P<0.054) and 45 min (P<0.03) after male introduction than before. By contrast, no change in NA could be detected in ewes implanted in the same region, but not responding to the ram, or in those showing increased LH pulsatility, but implanted in the anterior MPOA or in the ventromedial hypothalamus. No changes were observed in other neurotransmitters or when the ewes were exposed to male odour alone. These results suggest that NA release in the posterior MPOA is selectively involved in the triggering of LH secretion by rams in anoestrus ewes.

The proportion of anoestrous ewes ovulating after exposure to a sexually active ram is variable mainly due to whether an LH surge is induced. The aim of this study was to determine the role of oestradiol (E2) in the ram-induced LH surge. In one study, we measured the plasma concentrations of E2 in ewes of different breeds before and after the 'ram effect' and related these patterns to the presence and latency of the LH surge, while another compared ovarian responses with the 'ram effect' following exposure to rams for 2 or 12 h. In all ewes, the concentration of E2 increased 2-4 h after rams were introduced and remained elevated for 14.5 ± 0.86 h. The quantity of E2 secreted before the LH surge varied among breeds as did the mean concentration of E2. The granulosa cells of IF ewes collected after 12 h exposure to rams secreted more E2 and progesterone and had higher levels of StAR than the 2 h group but in MV ewes there was no differences between these groups for any of these parameters. These results demonstrate that the LH surge induced by the rams is a result of increased E2 secretion associated with increased levels of STAR in granulosa cells and that these responses varied among breeds. The results suggest that the variable occurrence of a LH surge and ovulation may be the result of variable ovarian responses to the 'ram effect' and insensitivity of the hypothalamus to the E2-positive feedback signal., (© 2015 Society for Reproduction and Fertility.)

Reproduction in mammals is controlled by the hypothalamo-pituitary-gonadal (HPG) axis under the influence of external and internal factors such as photoperiod, stress, nutrition, and social interactions. Sheep are seasonal breeders and stop mating when day length is increasing (anestrus). However, interactions with a sexually active ram during this period can override the steroid negative feedback responsible for the anoestrus state, stimulate luteinizing hormone (LH) secretion and eventually reinstate cyclicity. This is known as the "ram effect" and research into the mechanisms underlying it is shedding new light on HPG axis regulation. The first step in the ram effect is increased LH pulsatile secretion in anestrus ewes exposed to a sexually active male or only to its fleece, the latter finding indicating a "pheromone-like" effect. Estradiol secretion increases in all ewes and this eventually induces a LH surge and ovulation, just as during the breeding season. An exception is a minority of ewes that exhibit a precocious LH surge (within 4 h) with no prior increase in estradiol. The main olfactory system and the cortical nucleus of the amygdala are critical brain structures in mediating the ram effect since it is blocked by their inactivation. Sexual experience is also important since activation (increased c-fos expression) in these and other regions is greatly reduced in sexually naïve ewes. In adult ewes kisspeptin neurons in both arcuate and preoptic regions and some preoptic GnRH neurons are activated 2 h after exposure to a ram. Exposure to rams also activates noradrenergic neurons in the locus coeruleus and A1 nucleus and increased noradrenalin release occurs in the posterior preoptic area. Pharmacological modulation of this system modifies LH secretion in response to the male or his odor. Together these results show that the ram effect can be a fruitful model to promote both a better understanding of the neural and hormonal regulation of the HPG axis in general and also the specific mechanisms by which male cues can overcome negative steroid feedback and trigger LH release and ovulatory cycles.

In sheep, the 'ram effect' induces out-of-season fertility and good nutrition increases prolificacy. This experiment determined if fatness or short-term nutritional supplementation modified the response to the 'ram effect'. A group of 48 Île-de-France ewes were fed diets that produced groups with body-condition scores (BCS) of >3.0 and <2.0. Within each BCS group animals were supplemented daily with 500g of lupins from Day -5 to Day 0 (ram introduction) resulting in four groups: low BCS, supplemented (n=7) and non-supplemented (n=8) and high BCS, supplemented (n=12) and non-supplemented (n=11). The blood concentrations of glucose and insulin and the LH response to gonadotrophin-releasing hormone (GnRH) were determined. After the 'ram effect' the pattern of LH pulsatility, the LH surge and ovarian responses were analysed. Low BCS ewes had lower glucose and insulin (P<0.001) and supplementation increased both (P≤0.001). The increase in LH induced by GnRH was reduced in low BCS ewes (P=0.015) but it was not affected by supplementation. Similarly, LH pulsatility was reduced in low BCS ewes (P<0.05). The LH surge and ovarian cyclicity were not affected but the follow-up cycle was delayed (P=0.034) and progesterone was reduced (P=0.029) in low BCS ewes. There was an effect of BCS on ovulation rate (P<0.05). These results show that the BCS can modify the response to the 'ram effect' and that supplementation has little effect on this response.

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

Using Fos, as a marker, we analysed the brain structures of rams, with different libidos or sexual preferences that had been activated by contact with males or females. Ile de France rams aged from 1.5 to 7 years were used. Fos immunoreactivity (Fos IR) was analysed in rams with high (HL) or low libido (LL) after 90 min of direct contact with females (HL DirF n=7 or LL DirF n=7) or in rams of high libido having indirect contact through a fence, with females (HL IndF n=6) or males (HL IndM n=5) and finally, in males who preferred other males as partners by indirect contact through a fence with males (MO IndM n=4). Direct or indirect contact with a preferred sexual partner (LL DirF, HL Dir F, HL IndF, MO IndM) induced the appearance of Fos-IR cells in several diencephalic and cortical structures. Conversely, indirect contact with males did not induce Fos-IR in males interested in females (HL IndM). In the medial preoptic area (MPOA), the paraventricular nucleus and the medial bed nucleus of the stria terminalis the cell density of Fos IR cells was higher in HL Dir F than in LL DirF suggesting involvement in sexual motivation whereas only the MPOA seemed involved the consummatory component of sexual behaviour (Fos IR density HL DirF>HL IndF). The enthorinal cortex was the only structure specifically activated by males attracted to other males (Fos IR density MO IndM>HL IndM) whereas Fos IR density did not differ between the HL IndF and HL IndM groups., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Objective: Gonadotropin-inhibitory hormone (GnIH)-3 is a neuropeptide that plays a major role in the regulation of reproduction and feeding in mammals., Materials and Methods: We measured endocrine and behavioural parameters of reproduction in sheep, and sexual behaviour in sheep, mice and cynomolgus monkeys. In addition, GnIH gene expression (in situ hybridization) was examined in ewes, and effects of GnIH-3 on food intake and energy expenditure were measured in various species. GnIH-3 was infused (i.v.) into ewes after an i.m. injection of estradiol benzoate to determine whether the peptide blocks the surge in luteinizing hormone (LH) secretion., Results: GnIH gene expression was reduced in the preovulatory period in ewes. Infusion (i.v.) of GnIH-3 blocked the estrogen-induced LH surge (in ewes). Intracerebroventricular infusion had no effect on female or male sexual behaviour in each of the three species, but increased food intake. There were no effects on energy expenditure in sheep or rats. GnIH increased fos protein (immunohistochemistry) was seen in orexigenic neurons (in sheep and rats), but also in anorexigenic neurons (in sheep)., Conclusions: GnIH-3 reduces reproductive hormone levels and increases food intake in mammals without reducing energy expenditure. There is minimal effect on reproductive behaviour. The dual effect on reproduction and feeding suggests that GnIH-3 provides a molecular switch between these two functions. Blockade of the positive feedback effect of estrogen with parenteral infusion indicates that this peptide may have utility as a blocker of reproductive function in mammals., (Copyright © 2012 S. Karger AG, Basel.)

Recently, one Quantitative Trait Locus (QTL) of female fertility located on Bos Taurus chromosome 3 (BTA3), QTL-F-Fert-BTA3, has been identified in Holstein breed. It is implied in the success rate after the first AI (AI1) in cow. The failure of pregnancy can be due to several factors involved in the different steps of the reproductive process. The aim of our study was to finely phenotype heifers and primiparous cows selected for their haplotype at the QTL-F-Fert-BTA3. We specifically studied the ovarian follicular dynamic and several fertility parameters. Females carrying the favourable haplotype "fertil+" or unfavourable haplotype "fertil-" were monitored by transrectal ultrasonography during their cycle before the first AI (AI1). Follicular dynamic was similar between the two groups. However, the length of the estrus cycle was shorter in heifers than in primiparous cows and two-wave cycles were shorter than three-wave cycles, regardless of the age and the haplotype. The concentration of plasma anti-Müllerian hormone was correlated with the number of small antral follicles. It was higher in heifers than in primiparous cows, independently of their haplotype. The success rate at the AI1 was significantly higher in "fertil+" than in "fertil-" primiparous cows, 35 d after the AI1 (70% vs 39%). In both haplotypes, pregnancy failure occurred mainly before 21 d after AI1. The commencement of luteal activity after calving was significantly earlier in "fertil+" than in "fertil-" primiparous cows. Calving-AI1 and calving-calving intervals were similar between "fertil+" and "fertil-" primiparous cows. Taken together, "fertil+" and "fertil-" primiparous cows present a difference in the success rate after AI1 that is not explained by variations of ovarian dynamics., (Copyright © 2011 Elsevier Inc. All rights reserved.)

In seasonally anoestrous ewes of many breeds, the introduction of rams triggers an increase in gonadotrophin secretion that induces ovulation, a phenomenon known as the 'ram effect'. The ram effect is a practical method for mating ewes outside the natural breeding season, and also can provide synchronised lambing, but the variability of the response, especially in young animals, reduces its potential for widespread application. The aim of our study was to assess two factors that are thought to contribute to the variability in young ewes: temperament and sexual experience. We used anovulatory ewes from a flock that had been genetically selected for 'calm' or 'nervous' temperament and compared the endocrine and ovarian responses to the ram effect in four groups (each n=15): 'calm' and parous (3-6 years old); 'calm' and nulliparous (2 years old); 'nervous' and parous; and 'nervous' and nulliparous. Parous ewes, independently of their temperament, exhibited a faster endocrine response and a higher proportion of females cycling after ram introduction than nulliparous ewes. 'Nervous' ewes exhibited a higher proportion of females cycling after ram introduction than calm ewes, but only in the nulliparous group. We conclude that temperament exerts little influence on the response to the ram effect in sexually experienced ewes, and that females of 'nervous' temperament appear to respond better when sexually 'naive'. Both sexual experience and temperament need to be taken into consideration when flock management involves the ram effect. Finally, some ewes were cyclic at ram introduction, yet exhibited an increase in LH secretion even in the presence of high concentrations of progesterone. The mechanism by which the inhibitory effect of progesterone on LH secretion was bypassed needs to be clarified., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Exposure of anoestrous ewes to rams induces oestrous cycles (the 'ram effect'). This response is poor in young-sexually naive females, possibly because they lack sexual experience and are sensitive to stress. Firstly, we assessed the conditions required for the acquisition of sexual experience. We exposed naive females at 5 or 9 months of age to rams and subsequently assessed their response at 1 year of age, to the ram effect. Unexpectedly, 2 weeks pre-exposure at 5 months and under some conditions at 9 months, inhibited the ram effect at 1 year of age, suggesting that early contact with rams was stressful and had long-term negative effects on reproduction. Consequently, we assessed the combined effect of stress and sexual experience on responses to the ram effect. At 9 months of age, we pre-exposed naive females using sexually active rams to achieve 'positive' (voluntary contact) or 'negative' (forced contact) experiences, and assessed responses to the ram effect at a year of age. In parallel, we subjected naive females to stressful conditions at the time of the male effect. During pre-exposure, females with 'negative' contact exhibited more vigilance and stress cues and had higher levels of cortisol than 'positive' contact ewes. Regardless of pre-exposure or stress, the response to the ram effect at a year of age was inhibited. These experiments confirm that contact with sexual partners can be stressful to young ewes and highlight the need to consider their emotional state when conditioning them to the presence of rams and when managing breeding programs., ((c) 2009 Elsevier Inc. All rights reserved.)

Exposure of anestrous females to a ram or his odour induces rapid secretion of LH that can lead to ovulation. This response is mediated by the main olfactory system (MOS) in sexually experienced ewes. The accessory olfactory system (AOS) has a minor, but unclear, role in this response. In sexually naive ewes, male odour is less effective than in experienced ewes, but the neural pathway involved is not known. In our experiment, we investigated the brain regions activated by the male or his odour in young and sexually naive anestrous ewes using immunohistochemistry for Fos alone, Fos double-labeled with Gonadotropin-Releasing Hormone (GnRH) or with Tyrosine Hydroxylase (TH). Ram odour caused neural activation in the main olfactory bulb (MOB) and not the AOS. Exposure to a male induced significant activation in the MOB, the cortical and medial nuclei of the amygdala and the ventromedial hypothalamus but not in cortical areas, or in GnRH or TH positive neurons. These results confirm the predominant role of the MOS in the detection of olfactory signals in sheep and underline the importance of learning processes.

Similarities as well as differences across species in the control of sexual behavior are helping to fully understand the subtle relations between physiology and eco-ethological constraints and how the brain integrates such information. We will illustrate this with sexual behavior in domestic ruminants and especially ewes. Females of these species like humans, but unlike rodents, have a long luteal phase. A prolonged exposure to progesterone (Pg) before the preovulatory estradiol rise is necessary for estrous behavior to be displayed. Estradiol action and receptor localization is very similar to that observed in other species. But not too surprisingly, the role of Pg is rather different with a priming effect not observed in rodents. However, as in rodents, Pg also has an inhibitory effect, is necessary for the display of proceptivity and is responsible for the timing of the different periovulatory events. These steroids act on the central nervous system in similar areas across mammalian species to regulate estrous behavior. Steroid fluctuations during the estrous cycle cause changes in catecholaminergic activity in the hypothalamus. Interestingly, these neurotransmitters seem to have very similar effects in ewes and rats as illustrated by the norepinephrine rise after male-female interactions observed in both species. Similar comparisons can be made regarding the action of some neuropeptides, including oxytocin and GnRH, and more integrative processes like sexual differentiation and modulation of reproduction by social interactions. Data on sheep, goats and cows will be compared with those of rodents.

For a better understanding of the mechanisms that lead to the preovulatory GnRH/LH surge and estrus behavior, the minimum estradiol (E) requirements (dose and duration) to induce each of these events were determined and compared between two breeds of ewes having either single (Ile de France) or multiple (Romanov) ovulations. The ewes were initially studied during a natural estrus cycle, and were then ovariectomized and run through successive artificial estrus cycles. For these artificial cycles the duration and amplitude of the follucular phase E increase were manipulated by E implants. Under all conditions, the onset of estrus behavior was similar in the two breeds, although its duration was longer in Romanov ewes. While a moderate E signal (6 cm for 12 h) induced an LH surge in 10/10 Ile de France ewes, a larger E signal (12 cm for 12 h) was minimally effective in Romanov ewes (4/10). Additional studies revealed that a small E signal (3 cm for 6 h) induced full estrus behavior in all Romanov ewes but was completely ineffective in Ile de France animals (0/10). Higher doses and mostly longer durations of the E signal (12 cm for 24 h) were required to induce a surge in all the Romanov ewes. These results demonstrate a clear difference in the E requirement for the induction of estrus behavior and the LH surge between breeds of ewes that have different ovulation rates. These data provide compelling evidence that, in one breed, the neuronal systems that regulate both events require different estrogen signals.

During the non-breeding season, anestrous ewes do not experience ovarian cycles but exposure to a ram or its odor results in the activation of the luteinizing hormone secretion leading to ovulation. The aim of our work was to identify the neural pathways involved in this phenomenon. Using Fos immunocytochemistry, we examined the brain areas activated by the male or its fleece, in comparison with ewes exposed to the female fleece or the testing room (control group). In comparison with the control group, the male or its odor significantly increases Fos neuronal expression in the main and accessory olfactory bulbs, anterior olfactory nucleus, cortical and basal amygdala, dentate gyrus, ventromedial nucleus of the hypothalamus, piriform and orbitofrontal cortices. The main olfactory bulb, the cortical amygdala and the dentate gyrus are specifically more activated by the male odor than the female odor. Using a procedure of double labeling for Fos and gonadotropin-releasing hormone, we also compared the number of gonadotropin-releasing hormone neurons activated in the four groups of females. The male or its odor significantly increases the number and the proportion of gonadotropin-releasing hormone cells expressing Fos-immunoreactivity in the preoptic area and the organum vasculosum of the lamina terminalis, whereas no such induction of Fos-immunoreactivity was found in gonadotropin-releasing hormone neurons of ewes exposed to the female odor or the testing room. These findings emphasize the role of the main olfactory system in the detection and the integration of the ram odor, and also suggest the participation of the accessory olfactory system. Numerous structures widely distributed seem involved in the processing of the male olfactory cue to reach the gonadotropin-releasing hormone neurons.

In sheep, exposure of seasonally anestrous females to the male or its fleece results in activation of luteinizing hormone (LH) secretion and synchronized ovulation. The study of the neural pathways involved in this phenomenon, commonly named "male effect", show that the main olfactory system plays a critical role in the detection and the integration of the male odor. The accessory olfactory system participates in the perception of the ram odor but does not seem necessary for the endocrine response. According to the hypothesis that the neuroanatomical differences between the two olfactory systems could be associated with different functional roles, we investigated the importance of sexual experience and learning processes in the male effect. Our results showed that female responses depend on previous sexual experience. We also demonstrated that the LH response to male odor could result from an associative learning process. The aim of the present report was to summarize our current knowledge concerning the "male effect" and in particular to clarify the role of sexual experience and learning in the processes involved in this effect.

In sheep and goats, exposure of seasonally anestrous females to sexually active males results in activation of luteinizing hormone (LH) secretion and synchronized ovulation. This phenomenon is named "the male effect" and seems to constitute a major factor in the control of reproductive events. This effect depends mostly on olfactory cues and is largely mimicked by exposure to male fleece only. In sheep, preventing the vomeronasal organ (VNO) from functioning does not affect the female responses to male odor suggesting that, unlike in rodents, the accessory olfactory system does not play the major role in the perception of this pheromonal cue. Female responses also seem to depend on previous experience, an effect that is not common for pheromones and renders this model of special interest. The aim of the present report is to summarize our current knowledge concerning the "male effect" and in particular to clarify the respective roles of the two olfactory systems in the processes involved in this effect.

Exposure of anestrous ewes to a ram or its odor results in the activation of the luteinizing hormone (LH) secretion leading to reinstatement of cyclicity in most females. Sexual experience and learning have been suggested as important factors to explain the variability of the female responses. In experiment 1, we compared the behavioral and endocrine responses of four groups of anestrous females that differed in age (young or adult) and previous exposure to males [naive (no exposure) or experienced (courtship behavior for young and numerous mating for adults)]. Age did not seem to affect the LH response to males or their odor. In contrast, sexual experience was a critical factor: the proportion of females exhibiting an LH response to male odor was significantly higher in experienced than in naive ewes. Sexual experience affected the response to male odor, but did not have an effect on responses to the male himself. A second experiment investigated whether the LH response to male odor could result from an associative learning process. Accordingly, we tested the effectiveness of a conditioned stimulus (lavender odor) previously associated with the male, in inducing the endocrine response. The results indicate that the odor of lavender activated LH secretion only in ewes that have been previously exposed to scented males. This demonstrates that ewes are able to learn the association between a neutral odor and their sexual partner.

In the ewe, the mediobasal hypothalamus (MBH) is the primary central site for estradiol to generate the preovulatory GnRH/LH surges and sexual behavior. This area contains numerous neurons expressing the estradiol receptor alpha, distributed in the ventromedial nucleus (VMN) and the infundibular nucleus (IN). A large proportion of these neurons express somatostatin, making this neuropeptide a potential candidate for transmission of the estradiol signal to the GnRH neurons located in the preoptic area. We tested this hypothesis using ovariectomized ewes that had been subjected to an artificial estrous cycle. In the first experiment, 22 h after progesterone removal, ewes received estradiol (treated ewes) or empty implants (control ewes) for 4 h and then were killed. Using in situ hybridization, we showed that this short estradiol treatment increased the somatostatin mRNA amount by about 50% in the VMN and 42% in the IN. In the second experiment, preovulatory estradiol signal was replaced by somatostatin intracerebroventricular (ICV) administration. This treatment abolished LH pulsatility and dramatically decreased the mean basal level of LH secretion while it did not affect the mean plasma GH concentration. We demonstrated that an increase in somatostatin mRNA occurs at the time of the negative feedback effect of estradiol on LH secretion during the early stage of the GnRH surge induction. As ICV somatostatin administration inhibits the pulsatile LH secretion by acting on the central nervous system, we suggest that somatostatin synthesized in the MBH could be involved in the estradiol negative feedback before the onset of the preovulatory surge.

Our aim was to study the role of the olfactory amygdala (medial and cortical nuclei) and the ventromedial nucleus of the hypothalamus (VMN) in the ability of the male odour or live males to induce a release of luteinizing hormone in anoestrus ewes. To achieve this, we temporarily blocked the activity of these structures by localized retrodialysis administration of the anaesthetic lidocaine. The effect of ram odour on the secretion of luteinizing hormone was completely blocked by inactivation of the cortical nucleus of the amygdala. In contrast, inactivation of part of the accessory olfactory system (the medial nucleus of the amygdala or the VMN) had no effect. In the presence of the male, lidocaine never impaired the endocrine response of the ewes. These results show that modulation of reproduction by the sexual partner even through pheromonal cues does not occur via the direct circuit of the accessory system. On the contrary, the cortical nucleus of the amygdala is absolutely necessary for the treatment of and/or the response to the male olfactory signal but this structure can be bypassed when other sensory cues are available.

We assessed the role of learning in the expression of female sexual behavior and evaluated the relative importance of age versus experience. Two studies were conducted with ovariectomized ewes submitted to steroid treatment that mimicked an estrus cycle. We compared behavioral (experiments 1 and 2), neurochemical (experiment 1), and endocrine (experiment 2) responses of sexually naive young and adult ewes versus sexually experienced adults when exposed to males. In a third study, we compared their performance in an instrumental learning test and the extent to which it was affected by stress. These experiments showed that proceptivity is affected both by age and sexual experience. In experiment 1 only experienced adults were proceptive and displayed an increase in hypothalamic norepinephrine. By the second estrus cycle (experiment 2) naive adults performed similarly to experienced adults but proceptive behavior was still inferior in young ewes. Receptivity was also different between groups but affected more by age than by sexual experience. All ewes mated during the first interaction with a male, although males' latency to ejaculation was shorter with experienced females than naive adults or naive young. Young ewes found food as readily as adults in experiment 3 but were more affected by stress. Together, these experiments show that both experience and age influence sexual activity and that sensitivity to stress may also be involved. This may contribute to the deficient reproductive performance that is often observed in young female mammals.

In sheep, the mediobasal hypothalamus (MBH) has been shown to be the primary central site of estradiol (E2) action that induces both the preovulatory surge and sexual behaviour. However, the nature of the neurotransmitters or neuromodulators synthesized in the MBH during E2 stimulation remains to be clearly defined. After the cloning of the ovine cDNA sequences and using in situ hybridization, hypothalamic proopiomelanocortin (POMC), and preproneuropeptide Y (preproNPY) mRNA expression was studied in ovariectomized ewes that received a sequential treatment of progesterone and E2. As we showed that an exposition to E2 only for 4h well in advance on the LH surge onset is sufficient to induce the preovulatory surge and estrous behaviour, mRNA expression was evaluated in ewes treated with 6x30-mm E2 implants (experimental group) or with empty implants (control group) and slaughtered 4h after the start of the E2 treatment. Our results demonstrate that this short E2 treatment significantly decreased both the mean number of silver grains per POMC-containing cell (35%) and the mean number of POMC-cells (38%) in the ovine infundibular nucleus, whereas the treatment had no effect on preproNPY mRNA expression. These observations suggest that a reduction of POMC gene transcription could participate to the early neural mechanism of E2 feedback.

In sheep, essentially all the neurokinin B (NKB) neurones of the infundibular nucleus express oestradiol receptor alpha, and analysis of female and male brains has revealed an exceptionally marked female-dominant sex difference in the numbers of NKB neurones in the infundibular nucleus. This neuronal population is located in an oestradiol-sensitive brain area involved in the control of gonadotropin-releasing hormone (GnRH) secretion and oestrous behaviour, but its physiological role is poorly documented. The aim of the present study was to analyse NKB mRNA expression at a crucial time when the steroid has stimulated the pathways leading to the induction of these two events. After cloning a specific ovine NKB antisense riboprobe, we examined the effects of a short oestradiol treatment (4 h subcutaneously) on the expression of NKB mRNA in the caudal part of the infundibular nucleus of progesterone-primed ovariectomized ewes. We demonstrated that oestradiol decreased both the level of NKB mRNA expression (34%) and the number of cells containing NKB mRNA (43%). Oestradiol acts strongly on these NKB cells in the short term. We suggest that this early change in NKB mRNA expression during the preovulatory period might be involved in the control of the induction of GnRH secretion or oestrous behaviour.

Dopamine has been implicated in the control of sexual behaviour, but its role seems quite complex and controversial. The aim of the present experiments was to investigate the effects of dopamine (DA) acting on D2 receptors in the mediobasal hypothalamus (MBH) on sexual behaviour in female sheep. To achieve this, the D2 agonist, quinpirole, was administered bilaterally via microdialysis probes into the MBH of ovariectomized ewes either before or after oestradiol (E2) administration. Quinpirole (100 ng/ml) infused for 6 h just before E2 hastened the onset of oestrus behaviour and the luteinizing hormone surge, whereas the same treatment given 6-12 h or 18-21 h after E2 decreased the intensity of sexual receptivity without affecting LH or prolactin secretion. We then tested the hypothesis that E2 stimulates the onset of oestrus partly by decreasing DA activation of D2 receptors. In this case the D2 antagonists pimozide or spiperone (100 ng/ml) were infused into the MBH via microdialysis probes for 11 h in the absence of E2 administration. A significant number of ewes showed induction of receptivity with both antagonists, although its intensity was significantly lower than that induced by E2. These treatments generally did not significantly alter extracellular concentrations of monoamines or aminoacids although quinpirole modulated the ability of sexual interactions to increase noradrenaline release. These experiments show that DA acts via D2 receptors in the MBH to control female sexual behaviour in a biphasic manner: the onset of sexual motivation and receptivity requiring an initial increase in activation followed by a decrease. This dual action could explain some of the controversies concerning DA action on sexual behaviour.

Preovulatory GnRH secretion in ewes, measured in portal blood and cerebrospinal fluid, starts at the time of the LH surge, approximately 4 h after the onset of estrous behavior, and lasts as long as receptivity (36-48 h), which is much longer than the LH surge. This study tested the hypothesis that the extended GnRH secretion is involved in the maintenance of receptive behavior, prolonging the initial triggering effect of E2. Ovariectomized ewes were subjected to artificial estrous cycles and infused intracerebroventricularly either with a water soluble GnRH antagonist (Teverelix, Exp 1 and 2) or GnRH (Exp 3 and 4) after preovulatory E2 challenges of various intensity. The GnRH antagonist infused for 20 h (0.5 mg/ml, flow rate 3 microl/min) following a treatment with 2 x 30-mm E2 implants for 24 h (Exp 1) significantly reduced receptivity 36-48 h post E2 compared with vehicle infusion. By contrast, when the GnRH antagonist was infused with E2 implants still present (Exp 2: E2 for 48 h, GnRH antagonist given 24-44 h after E2 insertion, n = 14) receptivity was not affected. Administration of GnRH (0.5 mg/ml, flow rate 3 microl/min) when receptivity began to decline (Exp 3: 30-48 h after a 6-h 2 x 30-mm E2 implants n = 12) resulted in significantly higher receptivity scores at 48 and 52 h post E2 in GnRH treated animals compared with vehicle treated. GnRH infusion of ewes under subthreshold E2 treatment (Exp 4: GnRH 6-24 h after implantation of 1 x 30-mm E2 for 3 h, n = 12 in a cross-over design) significantly increased their receptivity compared with vehicle administration at 18 and 24 h post E2 insertion, but receptivity remained lower than when induced by high doses of E2. Our results demonstrate for the first time that GnRH is involved in the control of receptivity in a ruminant species and suggest that in the cycling ewe the sustained preovulatory GnRH secretion plays a physiological role in extending the duration of estrous behavior. They also indicate that it is possible to dissociate a direct effect of E2 on estrous behavior from its effect via stimulation of GnRH secretion.

Although a neural site of action for estradiol in inducing a LH surge via a surge of GnRH is now well established in sheep, the precise target(s) for estrogen within the brain is unknown. To address this issue, two experiments were conducted during the breeding season using an artificial model of the follicular phase. In the first experiment, bilateral 17beta-estradiol microimplants were positioned in either the medial preoptic area (MPOA) or the mediobasal hypothalamus (MBH), and LH secretion was monitored. An initial negative feedback inhibition of LH secretion was observed in ewes that had estradiol microimplants located in the MPOA (6 of 6 ewes) or caudal MBH in the vicinity of the arcuate nucleus (4 of 4). In contrast, a normal LH surge was only found in animals bearing estradiol microimplants in the MBH (5 of 10). Detailed analysis of estradiol microimplant location with respect to the estrogen receptor-alpha-immunoreactive cells of the hypothalamus revealed that 4 of the 5 ewes exhibiting a LH surge had microimplants located bilaterally within or adjacent to the area of estrogen receptor-expressing cells of the ventromedial nucleus. Two of these ewes exhibited a LH surge without showing any form of estrogen negative feedback. In the second experiment, we used the technique of hypophyseal portal blood collection to monitor GnRH secretion directly at the time of the LH surge induced by estradiol delivered either centrally or peripherally. Central estradiol implants induced the GnRH surge. The duration and mean plasma concentration of GnRH during the surge were not different between animals given peripheral or central MBH estradiol implants. Cholesterol-filled MBH microimplants did not evoke a GnRH surge. We conclude that the ventromedial nucleus is the primary site of action for estradiol in stimulating the preovulatory GnRH surge of the ewe, whereas the MPOA and possibly the caudal MBH are sites at which estrogen can act to inhibit LH secretion. These data provide evidence for the sites within the ovine hypothalamus responsible for mediating the bimodal influence of estradiol on GnRH secretion and suggest that different, and possibly independent, neuronal cell populations are responsible for the negative and positive feedback actions of estradiol.

Monoamines are widely distributed in the brain and are involved in arousal and motivational processes as well as motor activity and neuroendocrine control. Interactions between these central monoaminergic systems and steroid hormones play a major role in the integration of reproductive behaviour and gonadal function. This review describes the development of understanding of the relationship between steroids and monoamines, particularly in the control of sexual behaviour.

Differential activation of neural substrates was investigated in female sheep exposed to a male when they were in oestrus, and sexually receptive and attracted to males, as opposed to anoestrus when they were not. Changes in neuronal activation were visualized in ovariectomized, hormone-treated ewes by quantifying changes in cellular expression of c-fos messenger RNA by in situ hybridization histochemistry. Results showed that, while oestrus induction had no significant effects on c-fos expression per se, a 5-min exposure to a male significantly increased it in a number of primary and association cortical regions (the mitral and granule cell layers of the olfactory bulb, visual, somatosensory, orbitofrontal, piriform, cingulate and temporal cortices), the limbic system (CA1 region of the hippocampus, subiculum, lateral septum, lateral and basolateral amygdala, bed nucleus of the stria terminalis) and hypothalamus (mediobasal hypothalamus, medial preoptic area and paraventricular nucleus) as well as the nucleus accumbens and mediodorsal thalamus. Intromissions did not contribute significantly to these c-fos changes however. In anoestrus females, exposure to a male only produced a small significant increase in c-fos messenger RNA expression in the temporal cortex inspite of receiving similar amounts of visual and olfactory cues from him and a number of mating attempts. These results clearly demonstrate that changes in sexual motivation markedly alter the neural processing of sensory cues from males. They also show that the hormonal induction of sexual attraction to males cues and the resultant stimulation of sexual behaviour is due not only to altered responsiveness of oestrogen-sensitive brain regions involved in mediating behavioural responses towards the male, but also to changes in primary and secondary/tertiary somatosensory, olfactory and visual processing regions which relay sensory information to them.

During behavioural oestrus female sheep, like females of many species, become both attracted to and sexually receptive towards males, whereas at other times they will avoid them. The mediobasal hypothalamus is the main site for the feedback action of sex steroids to induce sexual behaviour in the sheep and in previous studies we have shown that noradrenaline and serotonin are released in this region during sexual interactions with males. The current study investigated whether such changes are specific to interactions with males and if visual or olfactory cues or somatosensory stimulation during mating are critical. In vivo microdialysis sampling was carried out in the mediobasal hypothalamus of ovariectomized ewes submitted to artificial oestrous cycles. Release of monoamines and amino acid transmitters was first measured in animals during and after oestrus when they were exposed to interactions with either males or females or presentation of food. Noradrenaline concentrations only increased significantly when the females were in oestrus and interacted with males irrespective of whether intromissions were permitted. Females were then exposed to visual (faces) or odour (a home pen) cues from males or to the males themselves. Slide images of male faces increased concentrations of amines, glutamate and GABA during early oestrus, when females spent most time looking at them. During late oestrus noradrenaline, glutamate and GABA concentrations also increased in response to the male faces but no transmitter changes were seen during the luteal phase or at any time where the females were exposed to female faces, or inverted male faces. Exposure to male odour produced a lower increase in noradrenaline concentrations when females were in early oestrus but marked increases 20 and 30 min after exposure to male odours in late oestrus. No other transmitters were affected. Exposure to a male and mating with him when females were in early or late oestrus produced increased noradrenaline concentrations similar to those seen with face stimuli alone although other neurotransmitters were unaffected. These results show that noradrenaline, and to a lesser extent dopamine, serotonin, glutamate and GABA release in the mediobasal hypothalamus, can be modulated specifically in the oestrous female by sensory information coming from the male during oestrus. The differential effects of male cues during early and late oestrus suggest their involvement in (i) proceptive or anticipatory sexual responses shown by the female to male cues, and (ii) receptive sexual responses, and suggest that the mediobasal hypothalamus plays a key role in the integration of hormonal action on sexual motivation and processing of sensory information during oestrus.

In female sheep, progesterone blocks the induction by oestradiol of both sexual behaviour and the pre-ovulatory surges of gonadotrophin releasing hormone (GnRH) and luteinising hormone (LH). However, the central sites of action of progesterone remain poorly defined, so we attempted to locate them by implanting progesterone intracerebrally in ovariectomised ewes treated with exogenous steroids to induce oestrous behaviour and the LH surge. Single bilateral implants or a double bilateral implants filled with progesterone or cholesterol were placed in the ventromedial hypothalamus (VMH) or the preoptic area (POA). Control ewes were not implanted. To determine the inhibitory capacity of the central progesterone implants, ewes received an injection (i.m.) of 8 micrograms or 16 micrograms of oestradiol. The single bilateral implants of progesterone failed to block oestrous behaviour and the LH surge induced by 8 micrograms of oestradiol. Double bilateral progesterone implants in the VMH blocked the sexual behaviour (P < 0.05) and the LH surge (P < 0.05), but implants in the POA blocked only sexual receptivity (P < 0.05). No changes were observed after central implantation of cholesterol. Our results support the hypothesis that progesterone acts centrally in the VMH and the POA to inhibit the induction of LH surge and sexual behaviour by oestradiol.

The preovulatory surge of LH, or positive feedback response to oestradiol, is related to the steroid's direct stimulating action on the pituitary gland, but we have demonstrated that, in sheep, the central nervous system plays an essential role in its action: a preovulatory GnRH surge. Since GnRH neurons appear to contain few, if any receptors for oestradiol the question is raised: Where in the central nervous system does oestradiol act to stimulate GnRH secretion? A series of experiments were conducted with a combination of techniques for sampling pituitary portal blood and stereotaxic brain micro-implantations of oestradiol. Castrated Ile-de-France breed ewes were treated during the pseudo follicular phase of successive artificial cycles, receiving either peripheral oestradiol implants or brain implants containing oestradiol or cholesterol. Administration of oestradiol implants into the ventromedial region of the hypothalamus induced a preovulatory GnRH surge in 5 out of 10 animals. The interval between estradiol and the GnRH surge was comparable with that observed in animals treated with a peripheral implant although the amplitude of the surge was smaller. No GnRH surges were observed in animals treated with cholesterol. Finally, there was an inverse correlation between the response intensity (amplitude of the GnRH/LH surges) and the distance separating implants from cells in the region carrying oestradiol receptors. These results show that the mediobasal hypothalamus is a site of action for the oestradiol-induced preovulatory GnRH surge in the ewe. Immunohistochemical tests should identify the nature of the cells forming the relay for the steroid action.

Oestrus behaviour and the luteinizing hormone (LH) surge are induced in ovariectomized ewes by oestradiol (E2) after a period of progesterone priming with a low level of E2 (Pge2) and we have previously shown that these effects are primarily mediated through their action on the mediobasal hypothalamus (MBH). The aim of the present study was to assess what neurochemical changes in the MBH are induced by these steroids that might mediate their action on oestrus behaviour and LH release. Eight ovariectomized ewes were implanted with microdialysis probes in the MBH and submitted to three artificial cycles, so that they exhibited either both oestrus behaviour and an LH surge (Pge2 + E2), an LH surge alone (E2 alone) or neither oestrus behaviour nor an LH surge (Pge2 alone). Microdialysis and blood samples were collected every 30 min from 4 h before the end of Pge2 treatment until the end of oestrus. Behavioural tests with a ram were made to assess receptivity. Dopamine (DA) levels were found to increase significantly at the termination of Pge2 treatment after both Pge2 + E2 and Pge2 treatments. When the ewes received E2 after a Pge2 + low estradiol priming (Pge2 + E2), DA levels decreased 16 h later (4 h after E2) whereas they did not change after E2 or Pge2 alone. By contrast, serotonin (5HT) levels did not change significantly during the first 24 h but then increased when ewes received E2 alone and decreased when they were treated with Pge2 + E2. gamma-Aminobutyric acid (GABA) concentrations decreased significantly at the beginning of the sampling period after all treatments but this decrease lasted longer after Pge2 + E2 and was most pronounced at the beginning of receptivity. No significant long term effects of these steroid treatments were found on noradrenaline (NA), aspartate, glutamate, glycine and taurine levels. However, E2 administration was followed during the next few hours by a significant increase in glycine and to a smaller extent in glutamate and GABA. More importantly, when ewes were treated with Pge2 + E2, NA levels increased significantly following the behavioural interactions with a ram when the ewes were sexually receptive. In contrast to this, DA levels only increased during interactions with the ram when the ewes were not receptive. 5HT levels increased after tests where the ewe was either receptive or unreceptive to the male. GABA, aspartate and glycine levels increased in the sample just preceding the test and then decreased during it.(ABSTRACT TRUNCATED AT 400 WORDS)

Oestrogen plays a key role in the regulation of the endocrine and behavioural events associated with the oestrous cycle. It is important, therefore, to know the location of neurones receptive to this steroid and to know whether their distribution varies with the oestrous cycle. We have undertaken experiments to identify the location of oestrogen receptors (ER) within the preoptico-hypothalamic continuum of ovariectomized ewes submitted to a variety of different hormone replacement regimes which mimic the different stages of the oestrous cycle. We used a monoclonal antibody to ER and detected receptors with immunohistological methods in the non-vascular part of the organum vasculosum of the lamina terminalis, the lateral septum, the medial preoptic area, the supraoptic, suprachiasmatic and arcuate (ARC) nuclei, the ventromedial hypothalamus (HVM) and in the region close to the mamillari recess. ER neurones were scarce or absent from the anterior hypothalamus and the paraventricular nucleus. The density of ER staining in the HVM, but in no other localization, was found to be higher, and in a more lateral position, during the induced luteal phase (progesterone treatment) than during the follicular phase (7 days of progesterone treatment followed by oestradiol) or in the ovariectomized female. In all areas studied, except for the ARC, the apparent surface area of the nucleus in ER immunoreactive cells varied with hormonal treatment. These data, and especially those in the HVM, contribute towards our understanding of how steroids may act in the ovine to control sexual behaviour.

Dose-response relationships have been suggested for the role of estradiol (E2) in the induction of both the LH surge and estrous behavior in the ewe. However, it is not always clear whether it is the amount of E2 or the length of time that E2 is present that is important. Furthermore, in previous studies, the intensity of sexual behavior has rarely been studied quantitatively. Our aims in this work were to analyze in more detail the effects of two injected doses of estradiol benzoate (25 or 50 micrograms) for the induction of both the LH surge and estrous behavior (experiment I) and to dissociate the roles of quantity and duration of presence of E2 as administered by implants (experiment II). Increasing the dose of E2 in both experiment I and experiment II decreased the latency to onset of both the LH surge and the receptivity. The duration of E2 presence marginally affected the latency of both responses and the duration of the LH surge. By contrast, the duration of receptivity depended largely on the duration of E2 presence (experiment II: r = 0.569, P < 0.01). This result explains the dose-duration effect mentioned previously in the literature and found in experiment I. The maximum response increased with dose in both experiment I and experiment II for receptivity and only in experiment I for the LH surge. We conclude that, at estrus, E2 acts in a similar fashion to induce receptive behavior and the LH surge but controls the maximum levels and the duration of these endocrine and behavioral responses differently.

In vivo microdialysis and retrodialysis were used to investigate the role of oxytocin (OXY) release in the mediobasal hypothalamus (MBH) of the ewe in the control of sexual receptivity. Initial experiments showed that OXY release was significantly increased in ovariectomized animals treated with progesterone and oestradiol when they were sexually receptive towards males and received intromissions. No such increases were seen during tests where the ewes were receptive but the males were prevented from achieving intromission. By contrast, OXY release was significantly reduced in tests where the ewes were not receptive to the male. In a second experiment artificial vaginocervical stimulation (VCS) was found to significantly increase OXY release when the animals were treated with oestradiol and this effect was potentiated by progesterone priming. OXY release in the MBH was not significantly altered by VCS in the presence of progesterone priming alone. Plasma OXY concentrations were significantly increased by VCS following all three hormone treatments but no one treatment was significantly more effective than another. Noradrenaline release in the MBH was only significantly increased following VCS when progesterone priming was given before oestradiol treatment. No effects of VCS on release of GABA, glutamate or dopamine were seen but their basal concentrations were significantly increased by the combined steroid treatment compared to oestradiol alone. In a third experiment it was found that OXY (10 microM) infused bilaterally into the MBH of receptive ewes, by retrodialysis, significantly decreased sexual receptivity and increased the release of noradrenaline and GABA.(ABSTRACT TRUNCATED AT 250 WORDS)

In a series of experiments using a quantitative method for measuring receptivity and implants that allowed rapid and controlled changes in the blood concentrations of oestradiol-17 beta and progesterone, we have re-examined the roles of these steroids in the induction of sexual behaviour and the LH surge in ovariectomized ewes. Progesterone priming was found to increase the proportion of ewes showing oestrus, reduce the latency to the onset of oestrus, and increase the 'intensity' of the behaviour as measured by the receptivity index, but it did not affect the size of the LH surge. Progesterone was able to facilitate the expression of oestrus even when it was withdrawn 8 days before oestrogen treatment, suggesting that it exerts its effect by restoring the sensitivity of the oestrogen-refractory animal to oestrogen. When it was present at the time oestrogen was administered, progesterone inhibited the stimulatory effect of oestrogen, but this effect disappeared as soon as the progesterone was withdrawn. Thereafter, expression of both the behavioural and endocrine responses was delayed by 24-30 h. These data show that the timing of the preovulatory behavioural and endocrine events is determined primarily by the time of progesterone withdrawal. The amount of oestradiol and the timing of any rise in concentration serve only to modulate these effects.

When estrous behavior is induced in ovariectomized ewes by subjecting them to progestagen priming followed by a dose of estrogen large enough to guarantee estrus in all animals, an abnormally long period of estrus in induced, suggesting that the regime of steroid replacement needs modification. Using quantitative tests for proceptivity and receptivity, we studied the patterns of sexual behavior of intact ewes and then attempted to reproduce them in the same animals after they had been ovariectomized. We used various combinations of exogenous estrogen, androgen, and progestagen and compared the behavioral responses with an endocrine response, the preovulatory surge of luteinizing hormone (LH). In intact ewes, sexual behavior and the LH surge were closely synchronized and their characteristics differed slightly between the middle and the end of the breeding season. Proceptive behavior was not greatly affected by the frequency of tests, but the duration of receptivity was significantly reduced by frequent testing. In ovariectomized ewes, we found that: (a) progesterone priming is essential for normal patterns of receptive and proceptive behavior, and for synchronizing the behavioral and endocrine responses to estrogen; (b) androgens do not play a major role in the control of either receptive or proceptive behavior; and (c) the inclusion of a low dose of estrogen with the progestagen in the 'priming' regime improves the responses to estradiol-17 beta. Under these conditions, the timing, intensity and duration of the behavior are very close to those observed in the same ewes when they were intact and cycling spontaneously.

Adult ewes were studied after prenatal treatment with androgen. Although seven of the eight androgenized ewes (ANDR) when intact presented signs of cyclic ovarian activity at least once, none of them showed cyclic female receptivity when intact or after ovariectomy and progesterone (P) + estradiol (E2) treatments, whereas all ten control (CONT) ewes did. Receptivity also was induced in CONT, but not in ANDR, ewes by long term E2 or testosterone (T) treatments. Increases in LH levels, with latency and duration similar to the LH surges of the CONT ewes, were observed in six (P + low E2 cycle) and five (P + high E2 cycle) of the eight ANDR ewes. The maximum LH level was lower in the ANDR than in the CONT ewes after high E2 treatment (p less than 0.002). Progesterone had a clear inhibitory effect on the induction of the LH surge by E2 only in the CONT ewes. Male-type mounting and nudging were observed more often in the ANDR than in the CONT ewes when intact (p less than 0.05). However, there was no facilitation of male sexual behaviour induced by long term E2 or T treatment in the ANDR compared to the CONT ewes. Progesterone inhibited male behaviour induced by T in the CONT but not in the ANDR ewes. This study shows that prenatal androgens defeminize LH secretion and sexual behaviour of ewes in a dissociated manner. It also suggests that prenatal androgen does not really masculinize the females but decreases their sensitivity to the inhibitory effect of P, and thus extends the "juvenile state" in which male-like patterns appear independently of the hormone milieu.

All motor patterns of male sexual behaviour can be observed in young lambs from the first days of the life. They considerably increase on weeks 2 to 5 when they decrease dramatically and remain stable up to over 6 months of age. This evolution has no direct relation with that of the physiological events. The adult female has a complete bisexual potentiality. The development of the sexual behaviour during ontogeny appears as a continuous process rather than to be limited to the period of puberty or sexual differentiation.

Oestrous cycles were induced in seasonally anoestrous ewes by introducing rams into the flock and giving to the ewes one intramuscular injection of 20 mg progesterone. At the second ovulation the onset of oestrus and the preovulatory surge of luteinizing hormone (LH) were recorded. It was found that the LH surge began in significantly more ewes during the night (79%) than during the day (21%). The onset of oestrus tended to follow a similar pattern. This temporal pattern was not related to the time of ram introduction, but may be the result of daily rhythms in ovarian activity. Furthermore, a preferred period for the LH surge indicates a preferred period for ovulation and this may be important in deciding when to begin artificial insemination.

Herds of grazing mammals characteristically produce precocial offspring in synchrony, and it is therefore important for the mother to form a rapid recognition of her own offspring to distinguish them from others. In sheep, the ewe forms such a selective bond with her lamb within 2-4 hours of parturition, a bond which is primarily dependent on olfactory sensory recognition. Here we report that the neuronal mechanism whereby the olfactory "imprint" is made is dependent on the centrifugal noradrenergic projections to the olfactory bulbs. Lesioning of this neural pathway prevents the ewe from forming a selective bond with her own lamb, enabling her to adopt alien lambs.

The effects of treating captive talapoin monkeys acutely (twice daily for 7 days) with naltrexone hydrochloride (0.25 mg 0.5 mg and 1 mg/kg intramuscular injections twice daily), naloxone hydrochloride (0.5 mg/kg IM twice daily) and sulpiride (1.5 mg/kg IM twice daily) was studied in social pairs and singly caged animals. The behaviour of social pairs and endocrine changes in all treated monkeys were monitored before, during and after withdrawal of the course of drug treatment. Naltrexone and naloxone, but not sulpiride, significant increased grooming and grooming invitations while aggressive behaviour, self grooming, scratching and general locomotor activity were unaffected. There was an overall increase in LH, testosterone and cortisol in plasma samples taken 60 mins after opiate receptor blockade. Prolactin was unchanged but increased dramatically in animals treated with sulpiride. No significant endocrine changes were observed to precede the increased grooming behaviour which opiate receptor blockade induced. The behavioural changes reported for this primate support the view that positive affect arising from social bonds may be mediated by cerebral endorphin containing systems.

Adult ovariectomized ewes, daily injected with testosterone propionate (2 to 22 mg), showed male-like sexual behaviour, never seen in untreated females, but very similar to that in normal rams. After a few days of latency (3 days minimum), this behaviour gradually appeared and increased in frequency until a plateau was reached. The time-course in which the plateau was reached depended on the androgen dose and on previous experience with male sexual behaviour. Some male-like behaviour might have persisted after the end of the treatment. The results have been discussed in terms of possible mechanisms of action on the central nervous system.

The re-introduction of rams after a period of separation was used to stimulate LH secretion and induce ovulation in seasonally anovulatory ewes maintained under natural photoperiod. In 2 experiments, the rams were introduced in the morning or the evening to test for diurnal variations in responsiveness to the treatment. In the first experiment, with Romanov ewes, the ram-induced increase in tonic LH secretion was significantly earlier in the ewes treated (N = 6) at 07:30 h (mean +/- s.e.m. delay to first pulse: 20 +/- 6 min) than in those (N = 5) treated at 19:30 h (66 +/- 15 min; P = 0.006). The pulse interval after the ram effect was significantly shorter in ewes that subsequently ovulated (120 +/- 10 min) than in ewes that did not ovulate (288 +/- 108 min; P = 0.043). There was a significant decline in pulse amplitude from 6.7 +/- 1.2 to 3.4 +/- 0.6 ng/ml (both groups combined) after the introduction of rams (P = 0.040). Of the 11 ewes, 7 subsequently ovulated and a preovulatory LH surge was observed in 6 of these 30-36 h after ram introduction. In the second experiment, with seasonally anoestrous Préalpes-du-Sud ewes, the effect of the timing of the introduction of rams on the periovulatory events was tested. The delay to the onsets of oestrus and the LH surge was not affected, but the ovulation rate was higher after ram introduction in the morning (1.42) than in the evening (1.14). In the 12-h period before the introduction of the rams in the first experiment, there was a difference between the groups in the secretion of LH, but the existence of diurnal rhythms in the concentrations of LH or FSH were not confirmed in a later study in which 7 ewes were sampled every 20 min for 36 h. In contrast, there was a distinct diurnal variation in the secretion of prolactin, with the highest values being recorded at night and the lowest around midday (P = 0.025). The rise and fall in prolactin values did not appear to coincide with dawn or dusk.(ABSTRACT TRUNCATED AT 400 WORDS)

Ovariectomized adult Ewes daily treated with I/M injections of testosterone propionate (10 mg/day) or oestradiol benzoate (200 microgram/day) show male-like sexual behaviour and simultaneously a permanent female receptivity. Additional daily treatment with 100 mg of progresterone results in a rapid decrease in male sexual responses both when Ewes are treated with testosterone and with oestradiol. No effect is observed on the female receptivity. Cyproterone acetate (100 mg/day) given to Ewes in addition to daily injection of testosterone propionate (10 mg/day) has effects similar to those of progesterone. These results show that progesterone has antiandrogen and antioestrogen properties and that this hormone administered at high doses is able to inhibit male activity.

Ovariectomized Ewes, treated daily with 100 micrograms of oestradiol benzoate (OB) show simultaneously female and male sexual behaviour. Male sexual behaviour is never seen in intact females. When given simultaneous with OB, progesterone prevents the action of the oestrogen. When given before the start of oestrogen treatment, progesterone also inhibits the effects of OB on male sexual behaviour for several weeks. By contrast, progesterone pretreatment has both inhibitory and facilitatory effect on the induction of female sexual behaviour. At first, it delays the appearance of female behaviour but then increases the response to oestrogen. These results suggest that progesterone could be involved in the dissociation between male and female behaviour normally seen in Sheep.