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In mammals, MT1 and MT2 melatonin receptors are high affinity G protein-coupled receptors and are thought to be involved in the integration of the melatonin signaling throughout the brain and periphery. In the present study, we describe a new melatonin binding site, named MTx, with a peculiar pharmacological profile. This site had a low affinity for 2-[125I]-melatonin in saturation assays in hypothalamus and retina (pKD = 9.13 {plus minus} 0.05, Bmax = 1.12 {plus minus} 0.11 fmol/mg protein and pKD = 8.81 {plus minus} 0.50, Bmax = 7.65 {plus minus} 2.64 fmol/mg protein, respectively) and a very high affinity, in competition assays, for melatonin (pKi = 13.08 {plus minus} 0.18), and other endogenous compounds. Using autoradiography, we showed a preferential localization of the MTx in periventricular areas of the sheep brain, with a density 3 to 8 times higher than those observed for ovine MT1 In addition, using a set of well-characterized ligands, we showed that this site did not correspond to any of the following receptors: MT1, MT2, MT3 , D1, D2, noradrenergic, nor 5-HT2 Based on its affinity for melatonin, MTx did not seem to be implicated in the integration of cerebral melatonin concentration variations since they were saturating for MTx. Nevertheless, it remained of prime importance because of its periventricular distribution, in close contact with the CSF, and its peculiar pharmacological profile responding to both melatoninergic and serotoninergic compounds. Significance Statement Herein a putative new melatonin binding site is described in sheep brain parts in close contact with the 3rd ventricle. The characteristics of the pharmacological profile of this site is different from anything previously reported in the literature. The present work forms the basis of future full pharmacological characterization.

Les recherches sur la maîtrise de la reproduction des brebis et des chèvres ont abouti à des traitements hormonaux de synchronisation des chaleurs très largement utilisés. Leurs coûts sont supportés par la valeur ajoutée de la production laitière et le progrès génétique rapide permis par l’insémination artificielle associée à la synchronisation. Les éleveurs des pays en développement ne disposent pas des mêmes moyens financiers et des programmes de coopération conduits avec les chercheurs de ces pays portent sur des moyens moins coûteux de synchronisation, comme « l’effet mâle » qui ne nécessite que des manipulations de lots d’animaux, sans intervention hormonale. En France, l’effet mâle ne suscite d’intérêt que chez les éleveurs et professionnels ovins et caprins en Agriculture Biologique. Pendant plus de 20 ans, des programmes de coopération scientifique entre la France, la Tunisie et le Mexique ont permis de mieux connaître les mécanismes physiologiques de la réponse à l’effet mâle et les conditions de son utilisation. Ils ont produit de nombreuses publications et permis le développement de techniques durables de maîtrise de la reproduction, adoptées avec succès par les éleveurs tunisiens et mexicains. L’émergence récente de préoccupations environnementales a changé le point de vue des professionnels français, désormais plus soucieux de durabilité de leurs systèmes. Les informations accumulées lors de ces programmes internationaux de coopération sont maintenant utiles dans les systèmes d’élevage français et européens, pour accélérer la mise au point de nouvelles techniques alternatives à l’utilisation des traitements hormonaux.

The control of reproduction in goats is interesting for technical reasons (synchronization of kiddings, adjustment to forage availability or to economy), and for genetic reasons (identification and dissemination of improved genotypes). The use of short-light rhythms leads to markedly increased production of semen per buck and prevents occurrence of a 'resting' season. Recent identification of a bulbourethral lipase in goat spermatozoa opens new perspectives in sperm preservation. Light plus 'short day' treatments also allow induction of out-of-season oestrous cycles and ovulations leading to enhanced fertility. Repeated use of eCG provokes the production of antibodies, delays the timing of ovulation and causes a reduction in fertility after fixed-time artificial insemination. All steps of embryo production, freezing and transfer are now controlled and allow the attainment of satisfactory numbers of kids born per donor female, which are compatible with the development of the technique for exchanging genotypes between countries. In vitro production of embryos allows high development rates to be achieved after in vitro maturation and fertilization of oocytes, and will ensure the production of synchronous populations of one-cell zygotes at the stage required by new biotechnologies.

Melatonin, which is synthesized at night by the pineal gland, is present in the cerebrospinal fluid (CSF), but its entry site and its role in this compartment are not known. Using several approaches, we tested the hypothesis that melatonin enters the CSF through the pineal recess, an evagination of the third ventricle. CSF melatonin concentrations are higher near the pineal gland than in the anterior part of the third ventricle, and decrease markedly (80%) after sealing off the pineal recess. Moreover, ultrastructure and permeability analyses of the pineal-CSF interface showed that melatonin could reach the CSF either via delivery in situ by protruding pinealocytes that make direct contact with the CSF or via extracellular secretion and interstitial fluid draining into the ventricular lumen. These data indicate that melatonin in the CSF probably originates from a few pinealocytes of the basal part of the pineal gland neighbouring the pineal recess. Melatonin carried to the brain by the blood appears to be able to mediate the effects of photoperiod on reproduction, but it is unclear whether melatonin in CSF may fine-tune this response both in terms of timing and amplitude. It is critical to determine which pathway, blood or CSF, allows melatonin to reach its central targets more efficiently.

Erratum to: Continuous light after 2 months of long days stimulates ram testis volume and increases fertility in spring (vol 11, pg 1189, 2017) DOI: 10.1017/S1751731117002348 Clé UT: 000419147600025; International audience; Seasonal reproduction is one of the major biotechnical and economic constraints of sheep production in temperate latitudes. Treatments using extra light followed by melatonin implants have been used satisfactorily in open barns, farms and artificial insemination centres to produce out-of-season sexual activity in rams. The aim of the present study is to explore the possibility of replacing melatonin implants with continuous light (LL), which was recently shown to increase LH secretion similar to melatonin and/or pinealectomy. Four experiments during 4 consecutive years were conducted in ‘Ile-de-France’ rams. In each study, one group was systematically exposed to permanent light after a first photoperiodic treatment of 60 long days (LD-LL) during the winter and compared with various other control groups subjected either to a natural photoperiod or the classical LD-melatonin treatment. As expected, blood nocturnal melatonin secretion was suppressed by LL. In all four experiments, LL treatment produced a highly significant and robust increase in ram testicular volume in the spring compared with the testicular volume of control rams or of that of treated rams at the end of the LD. For the two experiments in which fertility was tested, fertility after hand-mating was significantly higher in LD-LL rams than in control rams (76% v. 64%). Therefore, permanent light after an LD treatment may be an interesting alternative to LD-melatonin treatment to induce out-of-season sexual activity in rams.

Melatonin receptors have been described to activate different G proteindependent signaling pathways, both in laboratory, heterologous, cellular models and in physiological conditions. Furthermore, the constitutive activity of G proteincoupled receptors has been shown to be key in physiological and pathological conditions. In the case of melatonin receptors, information is rather scare and concerns only MT1 receptors. In the present report, we show that the G proteincoupled melatonin receptors do have a constitutive, nonmelatonin-induced signaling activity using two cellular models of different origins, the Chinese hamster ovary cell line and Neuro2A, a neuroblastoma cell line. Furthermore, we show that this constitutive activity involves mainly Gi proteins, which is consistent with the common knowledge on the melatonin receptors. Importantly, we also describe, for the first time, inverse agonist properties for melatonin ligands. Although it is clear than more in-depth, biochemistry-based studies will be required to better understand by which pathway(s) the constitutively active melatonin receptors transfer melatonin information into intracellular biochemical events; our data open interesting perspectives for understanding the importance of the constitutive activity of melatonin receptors in physiological conditions.

The aim of the present study was to determine whether a treatment of 3 mo of artificial long days and daily contact with bucks can stimulate reproductive activity during the normal seasonal anoestrous in female goats, and whether such treatment modifies the onset of the normal breeding season. Thirty-nine adult, open does were assigned to two treatments of similar mean body weight (BW) and body condition score (BCS). One treatment (LD; n = 18) was housed in a light-proof building and exposed to long days (16 h of light/d) from 17 November to 5 February, and then exposed to the natural photoperiod in an open shed. The remaining females were housed in an open shed under natural photoperiod conditions throughout the experiment (control [C]: n = 21). Plasma samples for progesterone, BW and BCS were recorded every wk. Oestrous activity was checked daily using aproned bucks. Bucks were housed close to females in a separate barn from the onset of the experiment. Ovulation rate was determined by laparoscopy 7 d after positive identification of oestrus. The interaction of treatment by time for temporal concentrations patterns of progesterone concentrations indicated that luteal activity in LD does were greater (P < 0.001) than those of C does during the natural seasonal anoestrous season. None of the C does exhibited oestrous or luteal activity during the non-breeding season; whereas, 72% of LD does exhibited luteal activity only 33% of them showed oestrous activity and during this season (P < 0.01). Differences in resumption of the oestrous or luteal activity were not observed (P > 0.05) in the subsequent breeding season between treatments. In conclusion, 3 mo of exposing does to long days and daily contact with bucks during the breeding season appears to stimulate reproductive processes that normal would not occur during the anoestrous season. However, this treatment does not induce oestrus is adequate numbers of does to be of practical value. Finally, this treatment does not modify the onset of the subsequent natural breeding season. (C) 2011 Elsevier B.V. All rights reserved.

Robert Ortavant est décédé le 24 janvier 2011. Il fut l’un des pionniers de la physiologie de la reproduction des mammifères domestiques à l’INRA et en France. Tous ceux qui l’ont connu, se souviennent de sa puissance scientifique, de la rigueur de son raisonnement et de la précision qu’il développait dans l’expérimentation. Ils se rappellent également l’homme qu’il était avant tout, le soin qu’il apportait aux relations humaines, toujours soucieux de convaincre plutôt que d’imposer et de contribuer aux bonnes conditions matérielles de ceux qui étaient sous sa responsabilité. Quand il donnait sa confiance, elle était totale, et cela permettait à ses collaborateurs de travailler dans les meilleures conditions.

This study examined which neural mechanism (opioid, dopaminergic, or serotonergic system) is involved in the regulation of luteinizing hormone (LH) secretion, with and without nutritional modulation, at different times of the photoperiodic cycle. Goats were randomly distributed into two experimental groups that received either 1.1 (high group; n = 18) or 0.7 (low group; n = 18) times the nutritional maintenance requirements. The goats were exposed to alternations of 3 mo of long days and 3 mo of short days. Plasma LH concentrations were measured twice a week. The effects of intravenous injections of naloxone (endogenous opioid receptor antagonist), pimozide (dopaminergic2 receptor antagonist), and cyproheptadine (serotonin 5-hydroxytryptamine2 receptor antagonist) on LH secretion were assessed during challenges in three different photoperiodic situations: the onset of LH stimulation by short days (OnsetSD), the onset of LH inhibition by long days (OnsetLD), and during the LH inhibition by long days (LateL...

The objective of this work was to evaluate the reproductive performance of ewes exposed to a photoperiodic regimen consisting of continuous alternating 4-mo periods of long days (LD: 16 h of light/d) and short days (SHD: 8 h of light/d) in an accelerated lambing program of 3 lambings in 2 yr. A total of 211 prolific Rideau Arcott ewes were assigned to the photoperiodic treatment, whereas 37 ewes were maintained under natural annual variation in day length (control group). Ewes under the photoperiod regimen were divided into 4 subgroups (A, B, C, D). All these groups of ewes were exposed to the same light regimen, but the LD and SHD light sequences were staggered by 2 mo to permit the evaluation of the effect of time and season of mating on performance of the ewes treated with the photoperiod. The control ewes were treated with intravaginal sponges in the out-of-season breeding periods (conventional approach). Each group of ewes was studied over 3 reproductive cycles. Two groups of rams exposed to alternating 2-mo sequences of LD and SHD were used for mating. The short mean interval between ram introduction and conception for the groups exposed to artificial photoperiod (9.4 d) confirmed the effectiveness of the treatment to induce intense sexual activity. For the 12 breeding periods studied (8 in out-of-season and 4 in sexual season), fertility rate of the ewes treated with photoperiod, mated at various times of year, was 91.6%, which is comparable with the fertility normally seen in the natural breeding season. The number of lambs born/ewe remained constant across reproductive cycles and was greater in photoperiod-treated groups (2.81 vs. 2.27 for photoperiod and control groups, respectively; P = 0.0002). Groups exposed to photoperiod treatment obtained better fertility rate than the control group in out-of-season breeding (91.1 vs. 76.3%; P = 0.016). Ewes managed under the photoperiod regimen produced 1.38 lambings/yr and 69% of them lambed 3 times in 2 yr. Overall, the ewes in the photoperiodic treatment produced annually 3.78 lambs/ ewe. The reproductive performances achieved throughout the years indicate that the photoperiodic program tested, consisting of continuous alternating 4-mo periods of LD and SHD, allows control of the annual reproductive cycles in ewes.

The aim of this work was to determine whether treatments using photoperiod (long days) and/or exogenous melatonin are effective in enhancing reproductive activity during the natural anestrous season in Mediterranean bucks. Two experiments were carried out. In the first experiment, 19 adult bucks were allotted to 3 treatment groups. Fourteen bucks were exposed to 16 h of light/day (long day) for 98 days (late autumn-winter). At the end of the long day treatment, 7 bucks received 3 s.c. melatonin implants (LD-M group), and the other bucks remained a long day group (LD group, n =7). The control group (C1, n = 5), remained under natural photoperiocl. In the second experiment, 7 bucks received 3 s.c. melatonin implants (M group, n =7), while the other 5 bucks acted as the control group (C2, n = 5) during the spring equinox. During the natural sexual anestrous season (spring), the treated groups recorded higher plasma testosterone concentrations than the control groups. Reactivation of the natural reproductive season was delayed only in the LD-M group. All treated bucks recorded a higher mean semen volume, than the control groups (Exp 1: 1.16 +/- 0.04 mL, 1.16 +/- 0.04 mL, and 1.08 +/- 0.04 mL, for the LD-M, LD, and Cl groups. respectively P < 0.01; Exp 2:1.04 +/- 0.07 mL vs 0.77 +/- 0.05 mL, for M and C2 groups, respectively; P < 0.01). The percentage of bucks that ejaculated was modified by treatment in the first experiment (89 +/- 1.7%. 71 +/- 2.4%, and 88 +/- 1.9% for LD-M, LD, and Cl groups, respectively, P < 0.001). Results demonstrate that treating bucks with artificial long days and/or melatonin at Mediterranean latitudes can induce an increase in plasma testosterone concentration during the natural anestrous season. The long day treatment alone resulted in a less effective method of inducing adequate out-of-season buck sexual activity.

The objective of this study was to determine whether reproductive seasonality of local well-fed female goats from subtropical Mexico (26 degrees N) can be controlled by photoperiod. The control group (n = 12) remained in an open pen under natural photoperiod variations. The two experimental groups (n = 8 each) were placed in light-proof rooms and exposed for 2 yr to alternations of 3 months of long days and 3 months of short days. One group was first exposed to long days, Group 1, and the other one to short days, Group 2. Blood samples were obtained twice a week to determine ovulation status by progesterone plasma concentrations. Goats from the control group displayed a seasonality of ovulations. The mean (+/- SEM) dates of the onset and end of the ovulations were September 10 +/- 5 d and February 16 +/- 4 d, respectively. In contrast, in both experimental groups, ovulations were modified (P0.05), respectively, after the transfer from long to short days. In contrast, the timing of the cessation of ovulations after the transfer from short to long days differed (P

The authors investigated whether melatonin concentrations vary between the two jugular veins and whether absolute (nocturnal) or relative (nocturnal/diurnal ratio) plasma melatonin concentrations are associated with seasonal reproductive activity measured by oestrus or ovulatory activity in Payoya goats Thirty-two adult Payoya goats were penned under natural photoperiod. Oestrus activity was tested daily using aproned males-twice a week plasma was sampled for progesterone Melatonin plasma concentrations were studied at each equinox and solstice of the year in Jugular samples taken simultaneously by venipuncture Nocturnal and diurnal plasma melatonin concentrations from each jugular vein were assessed in 3 and 2 plasma samples per goat, respectively, taken at hourly intervals in each period No differences in melatonin concentrations between the two veins were observed. but there was a significant interaction (P < 0 001) between jugular vein and animal in nocturnal melatonin concentrations. There was no effect of sampling period on melatonin concentrations and the coefficient of correlation between sampling periods was very high The analyses performed indicated that neither absolute nor relative melatonin concentrations were related with the dates of onset or end of ovulatory/oestrus activity Therefore, we concluded that in goats (1) melatonin concentrations are highly variable between jugular veins in the same individual but not in the general population, (2) melatonin concentrations are highly repeatable for each individual, and (3) absolute and relative amplitudes of melatonin concentrations are not linked to the seasonal breeding activity in Mediterranean goats (C) 2010 Elsevier Inc. All rights reserved

In the sheep, photoperiod, through melatonin, and oestradiol negative-feedback are two major regulators of seasonal changes in luteinizing hormone (LH) and prolactin secretion. Melatonin and oestradiol act on dopamine neurons of the hypothalamus to modify the enzymatic activity of tyrosine hydroxylase (TH). To further understand how melatonin and oestradiol regulate TH activity, we have studied the level of TH mRNA by in situ hybridization with an homologous cDNA probe, in A12 and A15 dopamine neurons of four groups of ovariectomized ewes: long-day exposed ewes with or without subcutaneous oestradiol implants and short-day exposed ewes with or without oestradiol. Results were analysed in relation to the concentration of LH and prolactin in the peripheral circulation. In the A15 cell group, TH mRNA levels were elevated in the short-day, oestradiol-treated ewes compared to all other groups. In this group, the level of TH mRNA was elevated simultaneously with LH concentration. The low level of TH mRNA found in the long-day, oestradiol-treated ewes may indicate that the increase of TH enzymatic activity previously reported by this treatment is not caused by an increase of the level of enzyme. In the A12 cell group, the level of TH mRNA in both long-day and short-day oestradiol-treated ewes was significantly higher than in ewes without oestradiol replacement. Prolactin concentrations were not correlated with TH mRNA variations in the A12 cell group.

International audience; The pars tuberalis (PT) of the pituitary gland expresses a high density of melatonin (MEL) receptors and is believed to regulate seasonal physiology by decoding changes in nocturnal melatonin secretion. Circadian clock genes are known to be expressed in the PT in response to the decline (Per1) and onset (Cry1) of MEL secretion, but to date little is known of other molecular changes in this key MEL target site. To identify transcriptional pathways that may be involved in the diurnal and photoperiod-transduction mechanism, we performed a whole genome transcriptome analysis using PT RNA isolated from sheep culled at three time points over the 24-h cycle under either long or short photoperiods. Our results reveal 153 transcripts where expression differs between photoperiods at the light-dark transition and 54 transcripts where expression level was more globally altered by photoperiod (all time points combined). Cry1 induction at night was associated with up-regulation of genes coding for NeuroD1 (neurogenic differentiation factor 1), Pbef/Nampt( nicotinamide phosphoribosyltransferase), Hif1 alpha (hypoxia-inducible factor-1 alpha), and Kcnq5 (K+ channel) and down-regulation of Ror beta, a key clock gene regulator. Using in situ hybridization, we confirmed day-night differences in expression for Pbef/Nampt, NeuroD1, and Ror beta in the PT. Treatment of sheep with MEL increased PT expression for Cry1, Pbef/Nampt, NeuroD1, and Hif1 alpha, but not Kcnq5. Our data thus reveal a cluster of Cry1-associated genes that are acutely responsive to MEL and novel transcriptional pathways involved in MEL action in the PT. (Endocrinology 149: 5527-5539, 2008)

International audience; We developed a new technique of gene knockdown (KD) in a specific brain area of the ewe using an adeno-associated virus (AAV)-mediated short interfering RNA (siRNA) method to elucidate the importance of key factors of seasonal reproduction. Two 19-nucleotide sequences (TH1 or TH2) were chosen from the tyrosine hydroxylase (TH) gene. TH1, TH2 or a random sequence (TH3) was incorporated into an eGFP expressing AAV vector. Firstly, 5 mu l of AAV-TH1 or AAV-TH2 solutions (8-9 x 10(11) Vg/ml) were stereotaxically injected into one A 15 nucleus while the other received a control treatment. Ewes were killed after 15 or 75 days. The number of TH neurons was 49% and 36% lower on the AAV-TH1 treated side than on the control side 15 and 75 days post-injection, respectively. AAV-TH2 did not induce a significant variation in TH cell population. Finally, in order to increase the KD, two groups of ewes received 10 mu l of AAV-TH1 either in a bolus injection or in two 5 mu l inoculations carried out 2 weeks apart. Only ewes receiving a bolus injection showed a larger KD reaching 66% 2 months after inoculation. This method proved effective in reducing TH expression and will be further developed to understand cellular mechanisms driving seasonal functions. (c) 2008 Elsevier B.V. All rights reserved.

The present study examines the ovulatory activity of wild and domesticated ewes subjected to either a constant photoperiod of long days (16L:8D) or natural changes in daily photoperiod for 16 mo. The aim was to determine whether an endogenous reproductive rhythm controls seasonal reproductive activity in these sheep, and how the photoperiod might affect this. The effects of long-day photoperiods on long-term changes in prolactin and melatonin secretion were also evaluated. The two species showed changes in reproductive activity under the constant photoperiod conditions, suggesting the existence of an endogenous rhythm of reproduction. This rhythm was differently expressed in the two types of ewe (P < 0.05), with the domestic animals exhibiting much greater sensitivity to the effects of long days. A circannual rhythm of plasma prolactin concentration was also seen in both species and under both photoperiod conditions, although in both species the amplitude was always lower in the long-day animals (P < 0.01). The duration of the nocturnal melatonin plasma concentrations reflected the duration of darkness in both species and treatments. The peak melatonin concentration did not differ between seasons either under natural or long-day photoperiods.

Through the duration of its nocturnal secretion, melatonin is the primary transducer of photoperiodic information to the reproductive axis in seasonal breeders. In this review, we describe recent results supporting the hypothesis that melatonin, released by the pineal gland, reaches its central targets carried by cerebrospinal fluid (CSF), rather than blood. Firstly, melatonin is released in the CSF through a specific site in the pineal recess where protruding pinealocytes contact with the CSF. Consequently, melatonin is present in much higher concentrations (100 times), in the cerebrospinal fluid (CSF) than in the blood. Secondly, CSF melatonin diffuses in the brain tissues quickly and efficiently and thus reaches periventricular structures featuring 2-iodomelatonin binding sites. These data support the idea that CSF melatonin acts as a physiological signal for the brain, La mélatonine, par sa durée nocturne de sécrétion, traduit les effets de la photopériode sur la reproduction. Dans cette revue, nous décrivons des résultats récents qui étayent l’hypothèse selon laquelle la mélatonine est transportée de la glande pinéale vers ses cibles cérébrales par le liquide céphalorachidien (LCR), plutôt que par le sang. Premièrement, la mélatonine est libérée dans le LCR en un site spécifique dans le recessus pinéalien où des pinéalocytes sont en contact direct avec le LCR. En conséquence, la mélatonine est présente à des concentrations environ 100 fois plus élevées dans le LCR que dans le sang. Deuxièmement, la mélatonine du LCR est capable de diffuser dans les tissus cérébraux rapidement et efficacement et peut donc atteindre des structures périventriculaires dans lesquelles des sites de liaison à la 2-iodomélatonine sont présents. L’ensemble de ces données conforte l’idée que la mélatonine du LCR est un signal physiologique pour le cerveau., Malpaux Benoît, Legros Céline. Rôle du liquide céphalo-rachidien dans le transport de la mélatonine pinéalienne vers ses cibles centrales. In: Bulletin de l'Académie Vétérinaire de France tome 161 n°5, 2008. Séances thématiques : l’expérimentation animale en 2008. pp. 455-460.

Two experiments were conducted during the anestrous period to determine: (1) whether males rendered sexually active by exposure to artificial long days stimulate estrous activity of female goats under grazed conditions (Exp. 1); and (2) whether continuous presence of the buck is necessary to stimulate this estrous activity (Exp. 2). In Exp. 1, 2 groups of females (n = 20/group), one in confinement and another under grazing conditions, were exposed to 4 bucks subjected to natural photoperiod (2 males/group). Two other groups of females (n = 20/group), in confinement or grazing, were exposed to 4 males treated with artificial long days (2 males/group). All groups were exposed to males for 15 d. The percentage of does detected in estrus during these 15 d was greater (P < 0.001) in the 2 groups exposed to males sexually prepared by long days (confined, 95%; grazed, 90%) than in groups exposed to males in natural photoperiod (confined, 15%; grazed, 45%). Does in Exp. 2 were allowed to graze and were exposed continuously (n = 26) or discontinuously (from 1700 to 0900; n = 26) for 18 d to males that had been stimulated to enter the breeding season by exposure to long days. The proportion of does that displayed estrous behavior in 18 d did not differ (P = 0.55) between groups (96.2 and 92.3% for continuous and discontinuous groups, respectively). The results indicate that anestrous goats managed under grazing conditions can be stimulated to express estrus by joining with males previously exposed to artificial long days. Continuous presence of the male is not necessary for this male effect.

In the present study, we analyzed the results of two years of response to the male effect in seasonally anestrous goats to investigate whether the activation of female reproductive activity by the male effect is related to the body weight of the females. Seventy-nine adult female Mexican mixed breed goats were used. The anestrous females were exposed during 15 days to sexually active males, and were classified into three categories according to their mean body weight ±SD (42 ± 9 kg) (Light: ≤ 33 kg, n = 19; Medium: 34-50 kg, n = 46; Heavy: ≥ 51 kg, n = 14). More than 98% of the goats from the Medium and Heavy groups showed at least one estrus behavior within the first 15 days following the introduction of the bucks, versus only 63% of the females from the Light group (P < 0.01). The interval between the introduction of the males and the onset of estrus behavior was longer in the females of the Light and Medium groups (4.2 ± 0.8 and 3.3 ± 0.3 days) than in the females of the Heavy group (2.0 ± 0.2 days; P < 0.03). Also, body weight was negatively correlated with latency to first estrus (Spearman r = −0.57; P < 0.001). These results are in agreement with the hypothesis that the ability of anestrous goats to respond to the male effect is positively influenced by their body weight. seasonality / live weight / estrus induction / teasing / body condition / biostimulation / subtropics

Melatonin regulation of reproductive functions in sheep is mediated by action in the premammillary hypothalamus (PMH). The aim of this study was to identify the high-affinity melatonin-receptor subtypes expressed in this structure. To achieve this, we used reverse transcription-polymerase chain reaction (RT-PCR) and developed in situ hybridization techniques (ISH). By using RT-PCR, we detected a band corresponding to the MTNR1A melatonin-receptor cDNA in the PMH as well as in the pars tuberalis (PT). On the opposite, MTNR1B melatonin-receptor transcripts were not detected using degenerate primers in any of the structures considered, confirming the lack of expression of this receptor subtype in sheep. The expression of MTNR1A mRNA was further confirmed in the PMH by ISH with a 35S-labeled ovine MTNR1A riboprobe. We next investigated the variation in the expression of MTNR1A mRNA between the end of the day and the end of the night (absence and presence of melatonin, respectively). MTNR1A transcript expression was greater at the end of the night than at the end of the day in the PMH. In contrast, MTNR1A mRNA expression was lower at the end of the night than at the end of the day in the PT. No significant variation in the MTNR1A mRNA expression was observed in a more dorsal hypothalamic area. Overall, these results show that MTNR1A transcripts are expressed in the ovine PMH and that their expression follows a diurnal rhythm, which is different from the pattern of expression observed in the PT.

The nocturnal increase in circulating melatonin in vertebrates is regulated by the activity of arylalkylamine N -acetyltransferase (AANAT), the penultimate enzyme in the melatonin pathway (serotonin → N -acetylserotonin → melatonin). Large changes in activity are linked to cyclic AMP-dependent protein kinase-mediated phosphorylation of AANAT T31. Phosphorylation of T31 promotes binding of AANAT to the dimeric 14-3-3 protein, which activates AANAT by increasing arylalkylamine affinity. In the current study, a putative second AANAT cyclic AMP-dependent protein kinase phosphorylation site, S205, was found to be ≈55% phosphorylated at night, when T31 is ≈40% phosphorylated. These findings indicate that ovine AANAT is dual-phosphorylated. Moreover, light exposure at night decreases T31 and S205 phosphorylation, consistent with a regulatory role of both sites. AANAT peptides containing either T31 or S205 associate with 14-3-3ζ in a phosphorylation-dependent manner; binding through phosphorylated (p)T31 is stronger than that through pS205, consistent with the location of only pT31 in a mode I binding motif, one of two recognized high-affinity 14-3-3-binding motifs AANAT protein binds to 14-3-3ζ through pT31 or pS205. Two-site binding lowers the K m for arylalkylamine substrate to ≈30 μM. In contrast, single-site pS205 binding increases the K m to ≈1,200 μM. Accordingly, the switch from dual to single pS205 binding of AANAT to 14-3-3 changes the K m for substrates by ≈40-fold. pS205 seems to be part of a previously unrecognized 14-3-3-binding motif-pS/pT (X 1–2 )-COOH, referred to here as mode III.

The day- and night-time levels of plasma melatonin were measured in adult male and female highveld mole-rats, Cryptomys hottentotus pretoriae. This study aimed to assess whether melatonin secretion in this nocturnal, strictly subterranean but seasonally breeding rodent has a day-night rhythm and whether that rhythm is circadian and can be modified by photoperiod. In experiment 1, a day-night rhythm of plasma melatonin was found in all animals housed on a 12L:12D schedule, with significantly higher concentrations in the dark (D) compared with the light (L) phase. The increment of plasma melatonin concentration at night was the same on days 1 and 2 for animals in the control group and animals transferred to constant dark. The animals transferred to constant light substantially reduced the amplitude of the melatonin rhythm on day 2. This suggests that the endogenous melatonin rhythm in C. h. pretoriae has a circadian pattern, which can be synchronized by photoperiod and inhibited by exposure to light at night. In experiment 2, the concentration of plasma melatonin in animals kept under 14L:10D (long day, LD) conditions differed significantly from animals on 10L:14D (short day, SD). This finding supports the notion that C. h. pretoriae is sensitive to changes in day length.

International audience; To assess the genetic variability in both the nocturnal increase pattern of melatonin concentration and photoresponsiveness in coat changes, an experiment on 422 Rex rabbits (from 23 males) raised under a constant light programme from birth was performed. The animals were sampled at 12 weeks of age, according to 4 periods over a year. Blood samples were taken 7 times during the dark phase and up to 1 h after the lighting began. Maturity of the fur was assessed at pelting. Heritability estimates of blood melatonin concentration (0.42, 0.17 and 0.11 at mid-night, 13 and 15 h after lights-out respectively) and strong genetic correlations between fur maturity and melatonin levels at the end of the dark phase ($-0.64$) indicates that (i) the variability of the nocturnal pattern of melatonin levels is under genetic control and (ii) the duration of the nocturnal melatonin increase is a genetic component of photoresponsiveness in coat changes.

Sheep in temperate latitudes are seasonal breeders. In female sheep, ovarian activity decreases during the anestrous period due to modification of secretion of luteinizing hormone (LH). The seasonal changes in the hormonal LH pattern mainly reflect an increase in the brain responsiveness to the negative feedback exerted by estradiol during long days (LD) on the frequency of pulsatile LH secretion, under neurohormonal GnRH control. The resulting seasonal inhibition of LH secretion mainly involves the activation of dopaminergic systems by E2, which in turn inhibits the GnRH cells from the preoptico-hypothalamic structures. The increased responsiveness of the brain during LD could lead to increased expression of central E2 receptors. In addition, our study shows that steroid access to the brain could be modulated by photoperiodism, thus increasing the availability of steroids to the nervous structures during LD.

Subterranean mammals inhabit an environment that is normally devoid of light and are therefore deprived of photoperiodic information that can be used to time important life-history events. An assessment was made of whether melatonin secretion in a strictly subterranean rodent, the Damaraland mole-rat Cryptomys damarensis, can be modified by photoperiod. In experiment 1, a clear diurnal rhythm of melatonin secretion in animals housed under a neutral photoperiod (12L:12D) was observed, with significantly higher melatonin concentrations in the dark compared to the light phase. The same diurnal melatonin rhythm was found 1 day after animals were transferred to either continuous light or continuous dark, suggesting that a circadian rhythm was maintained under acute exposure to light and dark. In experiment 2, melatonin secretion was monitored in a long (14L:10D) and short day (10L:14D) photoperiod and was found to be modified by the photoperiodic change. We therefore suggest that the Damaraland mole-rat possesses a circadian melatonin rhythm that can be physiologically modulated in response to photoperiod.

Melatonin has a key role in the circadian rhythm relay to periphery organs. Melatonin exerts its multiple roles mainly through two seven transmembrane domain, G-coupled receptors, namely MT1 or MT2 receptors. A pharmacological characterization of these human cloned melatonin hMT1 and hMT2 receptors stably expressed in HEK-293 or CHO cells is presented using a 2-[125I]-iodo-melatonin binding assay and a [35S]-GTPgammaS functional assay. Both reference compounds and new chemically diverse ligands were evaluated. Binding affinities at each receptor were found to be comparable on either HEK-293 or CHO cell membranes. Novel non-selective or selective hMT1 and hMT2 ligands are described. The [35S]-GTPgammaS functional assay was used to define the functional activity of these compounds which included partial, full agonist and/or antagonist activity. None of the compounds acted as an inverse agonist. We report new types of selective antagonists, such as S 25567 and S 26131 for MT1 and S 24601 for MT2. These studies brought other new molecular tools such as the selective MT1 agonist, S 24268, as well as the non-selective antagonist, S 22153. Finally, we also discovered S 25150, the most potent melatonin receptor agonist, so far reported in the literature.

In the sheep, the pineal hormone melatonin displays nocturnal levels 20 times as high in the cerebrospinal fluid of the third ventricle as in the jugular blood. Moreover, in the pineal recess, the evagination of the third ventricle into the pineal stalk, the levels of melatonin in the cerebrospinal fluid are even higher than in the ventral part of the third ventricle. This finding suggests melatonin to be secreted directly from the pineal gland to the ventricular lumen of the pineal recess of this species. We have, therefore, studied the interface between the sheep pineal gland and the cerebrospinal fluid by light-, scanning-, and electron microscopy of the pineal recess, as well as the permeability of the interface by tracer injections into the third ventricle. First, we show that the classic ependymal lining of the third ventricle disappears in the superior part of the recess. In this area, bulging pinealocytes, displaying immunoreactivity for serotonin, directly appose the cerebrospinal fluid. This pineal-cerebrospinal fluid interface of the sheep is large compared with other species, especially rodent species. Intraventricular injections of horseradish peroxidase and fluorescein isothiocyanate showed that both these tracers could permeate from the pineal recess into the sheep pineal parenchyma. This permeation was due to the presence of gap and intermediate junctions connecting the pinealocytes apposing the ventricular lumen. Thus, our results show that endocrine cells in this specialized area of the ventricular system are in direct contact with the cerebrospinal fluid. This finding supports the physiological concept of a direct secretion of melatonin into the cerebrospinal fluid of the sheep pineal recess.

Two experiments were conducted to determine the response of Creole male goats treated with long days and melatonin implants, and the response of the anovulatory does to male effect using males treated only with artificially long days. All animals were allocated to open sheds. In Exp. 1, one group of males was under natural photoperiod (CG; n = 7); the second group was submitted to 2.5 mo of long days followed by the insertion of two s.c. melatonin implants (LD+MEL; n = 7); the third group was subjected only to 2.5 mo of long days (LD; n = 7). Testicular weight was measured every 2 wk. Plasma testosterone concentrations were determined weekly. A treatment x time interaction was detected (P < 0.001) for testicular weight and plasma testosterone concentration. In the LD+MEL and LD groups, testicular size and plasma testosterone levels varied in a similar way, but differed from those observed in CG (P < 0.001). In this latter group, testicular weight displayed seasonal variations and peaked in June, whereas in treated groups this peak occurred in March. In CG, testosterone varied in a seasonal manner and plasma concentrations increased in June and remained elevated throughout the study. In experimental groups, testosterone increased in February and peaked in March. In Exp. 2, one group of males was left under natural photoperiod (CG, n = 5) and the other one was submitted to 2.5 mo of artificially long days (LD, n = 4). On March 16, two control and two treated males were put in contact with 20 and 19 females, respectively. Sexual behavior of the bucks was observed during the 5 d following male introduction. Progesterone assays and estrous behavior were used to determine ovarian and behavioral responses of the females to teasing. The anogenital sniffing, nudging, and mount instances registered in LD-treated males were greater than those observed in CG (P < 0.05). Of the does exposed to CG, none ovulated and only two of 20 females displayed estrous behavior. All does in contact with LD-treated males ovulated and showed at least one estrous behavior during the 15 d following joining (P < 0.001). These results indicate that the sexual activity of male goats from subtropical latitudes can be induced using only artificially long days. In addition, males treated in this way are capable of stimulating sexual activity in anovulatory females by the male effect.

Sheep in temperate latitudes are seasonal breeders. Of the different seasonal cues, photoperiod is the most reliable parameter and is used by animals as an indication of the time of the year to synchronize endogenous annual rhythms of reproduction and physiology. The photoperiodic information is transduced into neuroendocrine changes through variations in melatonin secretion from the pineal gland. Melatonin triggers variations in the secretion of luteinizing hormone-releasing hormone, luteinizing hormone and follicle stimulating hormone (LHRH/LH/FSH) responsible for seasonal changes in reproductive activity. In female sheep, the seasonal changes in the hormonal LH pattern mainly reflect an increase in the negative feedback exerted by estradiol under long days on the frequency of pulsatile LH secretion. The resulting seasonal inhibition of LH secretion involves the activation of monoaminergic and especially dopaminergic systems by estradiol. Other types of physiological regulation subject to seasonal changes such as voluntary food intake (VFI), fat metabolism, body mass and pelage growth also occur in sheep, goats or related wild species. Several neuroendocrine intermediates seem to be shared by these different systems and may participate in their synchronization, providing the advantage that this helps mammalian species to adapt to their environment.

Studies in rodents and humans have suggested that the pineal gland and its secretory product melatonin play an important role in the modulation of the immune system. In this study, we tested the hypothesis of a difference in immune parameters between ewes with naturally high vs. low circulating melatonin. Thus, two comparable groups of 10 Ile-de-France sheep were selected from a large flock, for their naturally high and low plasma concentrations of melatonin. The mean plasma melatonin concentrations during daytime (09:00 hr) and nighttime (24:00 hr) were, respectively, 9 and 664 pg/mL (high group) and 5 and 169 pg/mL (low group; P

Seasonal anestrus in ewes results from an increase in response to the negative feedback action of estradiol (E2). This increase in the inhibitory effects of E2 is controlled by photoperiod and appears to be mediated, in part, by dopaminergic neurons in the retrochiasmatic area of the hypothalamus (A15 group). This study was designed to test the hypothesis that E2 increases multiunit electrical activity (MUA) in the A15 during inhibitory long days. MUA was monitored in the retrochiasmatic area of 14 ovariectomized ewes from 4 h before to 24 h after insertion of an E2-containing implant subcutaneously. In six of these ewes, MUA activity was also monitored before and after insertion of blank implants. Three of the 14 ewes were excluded from analysis because E2 failed to inhibit LH. When MUA was recorded within the A15, E2 produced a gradual increase in MUA that was sustained for 24 h. Blank implants failed to increase MUA in the A15 area, and E2 did not alter MUA if recording electrodes were outside the A15. These data demonstrate that E2 increases MUA in the A15 region of ewes and are consistent with the hypothesis that these neurons mediate E2 negative feedback during long photoperiods.

Two experiments were conducted to determine whether the failure of males to induce sexual activity in goats during seasonal anestrus is due to unresponsiveness of females to male stimulus or insufficient stimulation from males. In the first study, one group of males (sexually inactive, SI; n = 4) was kept under natural photoperiod while the other (sexually active, SA; n = 4) was subjected to 2.5 mo of long days (16L:8D) and received 2 s.c. implants of melatonin. Two mo later, 2 different flocks of anovulatory goats previously separated from bucks were exposed to either SI (n = 34) or SA (n = 40) bucks. Progesterone assays and estrous behavior were used to determine ovarian and behavioral responses of the females to teasing. Of the goats exposed to SI males, only 2 ovulated, and none showed estrous behavior during the 35 days of the study. In contrast, all females (40 of 40) in contact with SA males ovulated and showed at least one estrous behavior during the first 11 days following male introduct...

Melatonin has been implicated in several neurotropic effects, but few studies have investigated the bioavailability of melatonin in the brain. The discovery of periventricular sites of action adjacent to the third ventricle forced us to investigate the dynamics of cerebrospinal fluid (CSF) melatonin release and the source of this melatonin. Our first study demonstrated unequivocally that third ventricle CSF melatonin, like jugular plasma melatonin, accurately reflects the duration of the night and is rapidly suppressed by light. However, third ventricle CSF melatonin levels are 20-fold higher than nocturnal plasma concentrations. A further study showed that melatonin increased in plasma before third ventricle CSF, raising the possibility that melatonin is taken up from the blood after recirculation through the Galen vein. However, a final experiment suggested strongly that CSF melatonin is released directly into the third ventricle, as melatonin levels in the lateral ventricle were 7-fold lower than those in the third ventricle. Our study raises the possibility that there may be two compartments of melatonin affecting physiological functioning: the first in plasma acting on peripheral organs, and the second in the CSF affecting neurally mediated functions at a much higher concentration of this pineal indoleamine.

In ewes, photoperiod modulates LH release. The median eminence (ME) dopaminergic activity seems to be implicated in the inhibition of LH secretion by photoperiod. This study investigated the functional importance of ME dopaminergic activity for LH secretion inhibition in three inhibitory photoperiodic treatments: after 33 long days (LD) (LD1 treatment), after 72 LD (LD2 treatment), and after 34 short days. Using reverse microdialysis on three groups of seven ewes, a solution of α-methyl-paratyrosine [αMPT, an inhibitor of tyrosine hydroxylase (TH); 10 mm in Ringer’s lactate] was infused into the ME for 5 h, preceded by a 5-h control period during which only vehicle was infused, in each of the three photoperiodic treatments. αMPT dramatically decreased the 3,4-dihydroxyphenylacetic acid concentration, similarly in all three photoperiodic treatments, suggesting a similar inhibition of TH activity. In the LD1 treatment, αMPT significantly increased LH pulse frequency (+1.22 ± 0.46 pulse/5 h from control period, mean± sem, n = 9; P < 0.05) and mean concentration (+51 ± 28%; P < 0.001). In the other two photoperiodic treatments, αMPT had no significant effect on LH release. Thus, blockade of dopamine synthesis in the ME seems to stimulate LH secretion in early, but not long-term, inhibition by LD nor after the transition to short days. Therefore, dopaminergic activity of the ME seems to be critical for LH secretion inhibition in some photoperiodic inhibitory treatments but not in others.

Melatonin transduces the effect of day length on LH secretion by acting on the hypothalamus. However, the precise hypothalamic site is unknown. Two studies were undertaken to clarify where melatonin acts in the hypothalamus. Using autoradiographic methods, the hypothalami of 5 ewes were screened to determine whether specific regional densities in melatonin binding existed. A higher density of binding was observed in the premammillary area of the hypothalamus (PMH) (3- to 5-fold higher than the rest of the hypothalamus). This binding area is delimited rostrally by the infundibular recess, caudally by the mammillary bodies, dorsally by the fornix, and ventrally by the base of the brain; and it encompasses the premammillary and tuberomammillary nuclei. To test the functional importance of the identified area, 3 groups of animals received bilateral melatonin microimplants: 1) in the PMH (n = 11); 2) in the anterior/mediobasal hypothalamus (AH/MBH; n = 8); and 3) sham-operated animals received empty microimplants in the PMH (SHAM; n = 6). All ewes were ovariectomized and treated s.c. with a 20-mm SILASTIC brand capsule of estradiol and exposed to long days (16-h light, 8-h dark). At the end of the 80-day experiment, no animal of the SHAM group and only 2 of the 8 ewes of the AH/MBH group displayed a stimulation of LH secretion. In contrast, melatonin implanted in the PMH stimulated LH secretion in 10 of the 11 ewes on day 44.5 +/- 5.3 (mean +/- SEM). ANOVA revealed that the changes in LH secretion were not different between the SHAM and the AH/MBH groups but the PMH group differed from the other 2 groups (P < 0.0001). This study suggests that the PMH is an important target for melatonin to regulate reproductive activity.

In the present study we have identified a site of action of estradiol in the inhibition of LH secretion during anestrus in the ewe. In the first experiment, we studied six sites: the medial preoptic area, the lateral preoptic area, the ventromedial hypothalamus, the ventrolateral hypothalamus, the retrochiasmatic area (RCh), and the periventricular posterior hypothalamus. We compared the changes in parameters of pulsatile LH secretion (interpulse interval, mean nadir, mean amplitude, and mean area under curve) during three 6-h sampling periods: before and 30-36 h and 9 days after intracerebral implantation of crystalline estradiol. Animals that received estradiol in the RCh (n = 5) showed a significantly greater increase in both the intervals between pulses of LH (up 116%, p < 0.03) and the area under the curve (up 180%, p < 0.01) than any of the other groups of 7 animals. In the second experiment, implantation of estradiol in the RCh (n = 6) induced an increase in the intervals between pulses of LH (p < 0.03), whereas receiving an empty implant (n = 6) had no effect, showing that estradiol specifically induced increases in the intervals between pulses. Thus, estradiol appears to act in the RCh where the dopaminergic A15 nucleus, known to inhibit pulsatile LH release, is located.

Remerciements : INRA Institut National de la Recherche Agronomique, UMR PRC Physiologie de la Reproduction et des Comportements - Plate-forme Chirurgie et Imagerie pour la Recherche et l'Enseignement, Nouzilly, France; The pineal gland secretes melatonin that circulates in the blood and cerebrospinal fluid. We provide data that support the hypothesis that, in sheep and maybe in human, only the cerebrospinal fluid melatonin, and not the blood melatonin, can provide most of melatonin to the cerebral tissue in high concentrations, particularly in the periventricular area. The melatonin content of sheep brain, our chosen animal model, was found in significant concentration gradients oriented from the ventricle (close to the cerebrospinal fluid) to the cerebral tissue, with concentrations varying by a factor of 1 to 125. The highest concentrations were observed close to the ventricle wall, whereas the lowest concentrations were furthest from the ventricles (407.0 ± 71.5 pg/ml compared to 84.7 ± 5.2 pg/ml around the third ventricle). This concentration gradient was measured in brain tissue collected at mid-day and at the end of the night. Nocturnal concentrations were higher than daytime concentrations, reflecting the diurnal variation in the pineal gland. The concentration gradient was not detected when melatonin was delivered to the brain via the bloodstream. The diffusion of melatonin to cerebral tissues via cerebrospinal fluid was supported by in vivo scintigraphy and autoradiography. 2-[(123) I]-melatonin infused into the cerebrospinal fluid quickly and efficiently diffused into the brain tissues, whereas [(123) I]-iodine (control) was mostly washed away by the cerebrospinal fluid flow and [(123) I]-BSA remained mostly in the cerebrospinal fluid. Taken together, these data support a critical role of cerebrospinal fluid in providing the brain with melatonin.

One experiment was conducted to determine whether the treatment with artificial long days and exogenous melatonin can increase, by itself, luteinizing hormone (LH) secretion during spring (seasonal anoestrus) in Payoya goats and whether this treatment causes a variation in the reactivation of the LH secretion in the normal breeding season. The experiment started on 4 November and finished on 27 October. A total of 22 ovariectomised-oestradiol treated goats were used. Ten goats were exposed to long days (16 h of light/day) from 14 November to 20 February. On 20 February, they received one s.c. melatonin implant and were exposed to natural photoperiodic changes in an open shed. The control group was located in another open shed with any treatment during the whole experiment. For both groups, plasma samples were obtained twice a week for LH assay. The photoperiodic treatment induced an earlier fall of the LH secretion. However, after the melatonin treatment, a clear increase of LH secretion during the natural seasonal anoestrus in the treated group was observed, and no difference in the onset of the LH secretion at the normal breeding season was observed. We can conclude that, in Payoya goats, the photoperiodicmelatonin treatment is by itself able to induce a clear LH increase during the seasonal anoestrus and does not modify the reactivation of the LH secretion at the normal breeding season.

Remerciements : INRA, UMR PRC Plate-forme CIRE 37380 Nouzilly; Sheep are gregarious mammals with complex social interactions. As such, they are very sensitive to social isolation and constitute a relevant animal model to study specifically the biological consequences of social stress. We examined previously the behavioral and endocrine responses in ewes isolated socially in the familiar conspecific withdrawal model (FCW) and showed that stressful responses increased and maintenance behaviors decreased, confirming that social isolation is a strong stressor in sheep. Melatonin synchronizes seasonal and circadian rhythms; and several studies reported its implication in cognitive processes as emotion. Here we investigated its role in the modulation of social stressful responses. Firstly, we studied ewes in the FCW model during the day (characterized by low melatonin levels) and the night (characterized by high melatonin levels). We found lower stressful responses (significant lower levels of cortisol plasma, number of foot pawings, of circling attempts) during the night as compared to the day. To investigate whether these effects were due to melatonin or to darkness, we submitted ewes to FCW during the night with lights on, a condition that suppresses melatonin secretion. Ewes infused with melatonin under these conditions showed decreased stressful responses (significant lower levels cortisol plasma, number of vocalizations, time spent with the head out of the cage) as compared to ewes infused with saline. These findings demonstrate that melatonin diminishes the endocrine and behavioral impact of social isolation in ewes and support the idea that melatonin has a calming effect in socially stressful situations.

Two experiments were designed to determine whether gonadal steroids during the estrous cycle may modify the characteristics of the plasma melatonin rhythm. In the first experiment, 12 ovariectomized estradiol-treated ewes were used and exposed to constant short days. The experimental design was a latin square to distinguish between steroid treatments and individual effects on melatonin secretion. Twenty four hours before the bleeding period (hourly during 20 hr) and with a 1 week interval, animals were treated with a) additional subcutaneous estradiol implants, b) progesterone devices, or c) control. In the second experiment, nine ewes received a treatment combining fluorogestone acetate devices and pregnant mare serum gonadotrophin to induce synchronous ovulations. Samples for melatonin determination were obtained hourly for 13 hr at three stages of estrous cycle: follicular phase, early luteal phase, and late luteal phase. Ovarian activity was monitored by taking daily samples for progesterone analysis and ovulation rate was determined by laparoscopy. Duration and mean melatonin plasma concentrations of the elevation were calculated for each ewe and each night and analysed by latin square test (experiment 1) or ANOVA (experiment 2). Melatonin concentrations of elevation and duration of elevation were not significantly affected by hormonal treatments or by phase of estrous cycle. A strong individual effect was detected (P

This study was performed to test the hypothesis that a short-day-like melatonin treatment would induce simultaneous stimulations of LHRH and LH secretion. Simultaneous serial samples of jugular and portal blood to assay LH and LHRH, respectively, were carried out at three different periods according to the expected changes in LH secretion. Twenty-six ovariectomized ewes bearing a subcutaneous implant of estradiol and exposed to a long-day photoperiod (16L:8D) received s.c. implants of melatonin on Day 0. LHRH and LH pulsatile secretion was studied in the first group during inhibition of LH secretion by long days (on Day -1, n = 6). A second group (n = 9) was sampled on Day 39. Moreover, seven animals from this group were sampled on Day 46 to focus on the beginning of LH increase. The third group (n = 6) was sampled during maximal LH secretion (Day 74, n = 6). A fourth group (n = 5) composed of noncannulated animals was sampled for LH secretion only as a control for the effects of surgical procedures. On each occasion, blood was collected every 10 min for 6 h. Between Day -1 and Day 39, LHRH pulse frequency was low and did not differ. In the same animals, this value became 2.5-fold higher between Day 39 and Day 46. Finally, on Day 74, LHRH pulse frequency was about 6-fold higher than on Day -1.(ABSTRACT TRUNCATED AT 250 WORDS)

Remerciements : INRA, UMR PRC, Plate-Forme CIRE, 37380 Nouzilly; Cellular mechanisms induced by melatonin to synchronise seasonal reproduction in several species, including sheep, remain unclear. We sought to evaluate the scale and physiological significance of neural plasticity in order to explain the delay between the change of duration of melatonin secretion and the change of reproductive status following a transition from long days (LD, 16 h light/24 h) to short days (SD, 8 h light/24 h) and from SD to LD. Using Western blots in ovariectomised oestradiol-replaced ewes, we evaluated the content of the polysialylated form of neural cell adhesion molecule (PSA-NCAM), a plasticity marker, in the hypothalamus. From day 15 following a transition to SD, most hypothalamic areas showed a decrease of PSA-NCAM level that was particularly significant in the preoptic area (POA). Following a transition to LD, PSA-NCAM content increased at day 15 in most regions except in the premammillary hypothalamic area (PMH) in which a significant decrease was noted. The functional importance of PSA-NCAM variations for seasonal reproduction was assessed for the PMH and POA. PSA-NCAM was degraded by stereotaxic injections of endoneuraminidase N and luteinising hormone (LH) secretion was recorded in treated and control ewes. Degradation of PSA-NCAM in the PMH in SD-treated ewes failed to produce a significant effect on LH secretion, whereas a similar treatment in the POA before a transition to SD delayed activation of the gonadotroph axis in two-thirds of the ewes. Our results suggest that the photoperiod controls variations of the hypothalamic content of a plasticity marker and that these might be important for the regulation of seasonal reproduction, particularly in the POA.

In sheep, the seasonal patterns of reproductive activity are driven primarily by the annual photoperiodic cycle, but can also respond to other environmental factors, such as nutrition, yet little is known about the mechanisms underlying this interaction. This study was designed to define the interaction between photoperiodic and nutritional cues on seasonal patterns of ovarian activity, and to determine if there is a central interaction between these cues. Groups of Ile-de-France ewes were maintained in two nutritional states (restricted and well fed) under a simulated annual photoperiod of 8‐16 h of light per day over two breeding seasons. At the end of the first breeding season, half of the animals of each group were ovariectomized (OVX) and fitted subcutaneously with estradiol implants. Low nutritional status shortened the season of ovarian activity, determined from the pattern of progesterone concentrations, by modifying the timing of seasonal transitions between periods of ovarian activity and anestrus. The same results were observed for the seasonal rhythm of neuroendocrine activity, assessed in the OVX ewes, from the pattern of luteinizing hormone concentrations. These results were then confirmed for neuroendocrine activity induced by a photoperiodic treatment. We conclude that nutrition centrally modulates the interpretation of photoperiod to affect seasonal reproductive transitions. The mechanisms of this interaction are discussed in the paper. hypothalamus, metabolism, nutrition, ovary, photoperiod, seasonal reproduction, sheep

To adapt to seasonal variations in the environment, most mammalian species exhibit seasonal cycles in their physiology and behavior. Seasonal plasticity in the structure and function of the central nervous system contributes to the adaptation of this physiology in seasonal mammals. As part of these plasticity mechanisms, seasonal variations in proliferation rate and neuron production have been extensively studied in songbirds. In this report, we investigated whether this type of brain plasticity also occurs in sheep, a seasonal species, by assessing variations in cell proliferation in the sheep diencephalon. We administered the cell birth marker 5′-bromodeoxyuridine (BrdU) to adult female sheep in July and December, during long and short photoperiod, respectively. The BrdU incorporation was analyzed and quantified in the hypothalamus, a key center for neuroendocrine regulations, as well as in other structures involved in relaying neuroendocrine and sensory information, including the median eminence, the pars tuberalis of the pituitary gland, and the thalamus. In December, 2-fold and 6-fold increases in the number of BrdU+ nuclei were observed in the hypothalamus and thalamus, respectively, when compared with July. This variation is independent of the influence of peripheral gonadal estradiol variations. An inverse seasonal regulation of cell proliferation was observed in the pars tuberalis. In contrast, no seasonal variation in cell proliferation was seen in the subventricular zone of the lateral ventricle. Many of the newborn cells in the adult ovine hypothalamus and thalamus differentiate into neurons and glial cells, as assessed by the expression of neuronal (DCX, NeuN) and glial (GFAP, S100B) fate markers. In summary, we show that the estimated cell proliferation rates in the sheep hypothalamus, thalamus, and pars tuberalis are different between seasons. These variations are independent of the seasonal fluctuations of peripheral estradiol levels, unlike the results described in the brain nuclei involved in song control of avian species.

The aim of this study was to determine whether the male effect on reproductive performance in female Mediterranean goats could be enhanced if used in combination with long day and/or melatonin treatments. Thirty-two does were exposed to long days (16 h of light/day) for 95 days. At the end of this period, 16 received one melatonin implant (group LD-M) and 16 did not (group LD). Simultaneously, 16 does under natural photoperiod were implanted with melatonin (group M) or given no treatment (group C. n = 16). On April 14th, after 49 days of isolation from males, all does were exposed to bucks fitted with marking harnesses. Oestrous activity, as detected by visual observation, was recorded daily. Transrectal ultrasonography was used to record ovulation, and ovarian activity confirmed by determining plasma progesterone concentration. Oestrous activity, ovulation and fertility were greater in all treatment groups compared with group C (P < 0.05), but no differences in ovulation rate or litter size were observed (P > 0.05). Productivity (mean number of kids per female) of the LD-M and M groups was significantly greater than in group C (1.50 +/- 0.01 and 1.37 +/- 0.01 vs. 0.69 +/- 0.01 kids, respectively) (P < 0.05). Combining melatonin treatment with natural or artificial long days adds to the impact of the male effect.

The main melatonin receptors are two G-protein coupled receptors named MT(1) and MT(2). Having described the molecular pharmacology of the human versions of these receptors, we turned to two of the three species most useful in studying melatonin physiology: rat and sheep (a diurnal species used to understand the relationship between circadian rhythm and depression). We also employed previously used compounds to describe the mouse melatonin receptors; despite the early cloning of mouse receptors, few molecular pharmacology studies on these receptors exist. To our surprise, we detected no major differences between the data obtained from mice and those from other species.

Paradigms used so far to study the effects of social isolation in sheep confound the effects of social isolation with those of other stressors (e.g. new environment) and showed contradictory effects after multiple social isolations. We propose here to characterize and examine the repeatable effects of social isolation induced by the familiar conspecific withdrawal (FCW). This latter test consists of socially isolating the ewe by the removal of group mates from the room test for 3 h. Behavioral and endocrine responses of adult ovariectomized-estradiol implanted ewes were compared 90 min before and 90 min after FCW, which was applied three times every fourteen days. We observed that each FCW induced significant increases in plasma cortisol level, in the number of vocalizations, foot pawing, circling attempts and a significant decrease in time spent lying down. An increase in plasma cortisol levels and decrease in duration of maintenance behaviors were significantly lower after the third FCW than after the first one. These differences could be explained by higher plasma cortisol levels and lower duration of maintenance behaviors before the third FCW than before the first FCW suggesting an anticipation of the social isolation period. These data indicate that social isolation is sufficient to induce distress with stable stressful responses after multiple exposures to familiar conspecific withdrawal.