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Correction de GPR50 is the mammalian ortholog of Mel1c: Evidence of rapid evolution in mammals, BMC Evolutionary Biology20088:105, DOI: 10.1186/1471-2148-8-105; International audience; The melatonin receptor subfamily contains three members Mel1a, Mel1b and Mel1c, found in all vertebrates except for Mel1c which is found only in fish, Xenopus species and the chicken. Another receptor, the melatonin related receptor known as GPR50, found exclusively in mammals and later identified as a member of the melatonin receptor subfamily because of its identity to the three melatonin receptors despite its absence of affinity for melatonin. The aim of this study was to describe the evolutionary relationships between GPR50 and the three other members of the melatonin receptor subfamily.

Mouflon and domestic Manchega sheep differ in the timing of their reproductive season under natural photoperiod (NP) conditions. This study examines whether they also differ in their reproductive responses to artificial photoperiod cues. For this, mouflons (n = 24) and ewes (n = 24) were exposed between the winter and summer solstices to artificial long days (LD: 16 h light/day), to short days (SD) simulated via the use of melatonin implants, or to NP conditions (controls), and their ovulatory activity monitored. The effects of these treatments on annual changes in prolactin concentration were also recorded. In the LD mouflon ewes, the offset and onset (7 March 5 and 2 October +/- 4, respectively) of cyclic ovulatory activity occurred earlier (P < 0.001) than in the NP animals (26 April +/- 6 and 20 October +/- 2, respectively), but no differences were seen (P > 0.05) between the SD and NP mouflon ewes in either the onset of anoestrus (12 May +/- 6 and 26 April +/- 6, respectively) or the onset of subsequent ovulatory activity (13 October +/- 8 and 20 October +/- 2, respectively); however the duration of the anoestrus period was significantly reduced in the SD. In LD Manchega ewes, the onset of anoestrus was advanced (2 February +/- 5 vs 15 March +/- 11), but ovulatory activity started at the same time as in NP Manchega ewes (16 July +/- 4 vs 5 July +/- 8). In the SD Manchega ewes, two animals showed continuous cyclic ovulatory activity over the course of the experiment while the remainder entered anoestrus two months later (16 May +/- 6, P < 0.001) than their NP counterparts. In these SD ewes, the onset of cyclic ovarian activity was very variable. An annual rhythm of plasma prolactin concentration was seen in both the mouflon and Manchega ewes under all three photoperiod conditions. However, the amplitudes of the changes seen in prolactin concentration were smaller in both the LD and SD animals than in the corresponding NP animals (P < 0.001). In conclusion, the results show that these two types of Mediterranean sheep differ in their ovulatory response when subjected to artificial photoperiods. The results also suggest that refractoriness to SDs may be the most important physiological mechanism regulating the onset of anoestrus in highly seasonal breeds, but not in less seasonal breeds.

In the ewe, the inhibition of LH secretion during long days results from increased negative feedback by estradiol. This effect depends on aminergic systems, mainly in the lateral retrochiasmatic area, a region that includes the dopaminergic A15 nucleus. Recently we demonstrated that the inhibition of LH secretion by estradiol under long days is accompanied by an increase in extracellular levels of amine metabolites (3,4-dihydroxyphenylacetic acid [DOPAC], homovanillic acid [HVA], and 5-hydroxyindolacetic acid [5-HIAA]) in the lateral retrochiasmatic area of the hypothalamus. The increase in catecholamine metabolite levels could be due to stimulation of tyrosine hydroxylase (TH) activity in the catecholaminergic systems of this region. In the present study, we tested this hypothesis by incorporating in vivo measurement of TH activity in the brain into our microdialysis model. We used this approach to first examine the effect of estradiol on TH activity in both the lateral retrochiasmatic area and the caudate nucleus during long days (experiment 1). Two dialysis sessions were carried out on each of six ewes: one after a 10day estradiol treatment (s.c. implant) and the other after 10 days without estradiol treatment. Estradiol treatment reduced pulsatile LH secretion without affecting the secretion of prolactin. The steroid also significantly increased TH activity in the lateral retrochiasmatic area as assessed by the formation of L-3-4 dihydroxyphenylalanine (L-DOPA). There was no variation in the TH activity of the catecholaminergic systems of the caudate nucleus, suggesting that the effect of estradiol is specifically hypothalamic. In experiment 2, we estimated the relative contribution to the accumulation of L-DOPA in the lateral retrochiasmatic area by dopamine (DA) synthesis in the dopaminergic neurons and noradrenaline (NA) synthesis in the noradrenergic afferents of the A15 nucleus. This study also allowed us to test whether DOPAC and HVA molecules released in this medium originate from DA metabolism in dopaminergic cells of the A15 nucleus or in its noradrenergic afferents. We monitored TH activity as well as amine metabolite concentrations in the lateral retrochiasmatic area after a partial destruction of noradrenergic nerves in the brain using intracerebroventricular (i.c.v.) injections of nomifensine followed by 6-hydroxydopamine (6-OHDA) injections. We compared the responses in four experimental ewes with those in four control ewes receiving a single i.c.v. injection of nomfifensine only. Decreases in extracellular concentrations of 4-hydroxy-3-methoxyphenylethyleneglycol (MHPG) in the left lateral retrochiasmatic area and the tissue content of NA in the contralateral structure confirmed the effectiveness of the lesion of the noradrenergic endings. By contrast, the lack of any modification of the tissue content of DA was evidence of the integrity of dopaminergic cells of the A15 nucleus. These studies show that extracellular L-DOPA, DOPAC, and HVA originate from dopaminergic cells of the A15 nucleus and not from its noradrenergic afferents. The long-day inhibition of LH secretion by estradiol results from a stimulation of TH activity of the A15 nucleus dopaminergic neurons.

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.

Melatonin receptors have been described to activate different G protein-dependent signaling pathways, both in laboratory, heterologous, cellular models and in physiological conditions. Furthermore, the constitutive activity of G protein-coupled 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 protein-coupled 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.

This research examines which neural mechanisms among the endogenous opioid, dopaminergic, serotonergic and excitatory amino acid systems are involved in the stimulation of LH secretion by melatonin implantation and their modulation by nutritional level. Female goats were distributed to two experimental groups that received either 1.1 (group H;n=24) or 0.7 (group L;n=24) times their nutritional maintenance requirements. Half of each group was implanted with melatonin after a long-day period. Plasma LH concentrations were measured twice per week. The effects of i.v. injections of naloxone, pimozide, cyproheptadine andN-methyl-d,l-aspartate (NMDA) on LH secretion were assessed the day before melatonin implantation and again on days 30 and 45. The functioning of all but the dopaminergic systems was clearly modified by the level of nutrition, melatonin implantation and time elapsed since implantation. Thirty days after implantation, naloxone increased LH concentrations irrespective of the level of nutrition (PPPPP

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.

This paper reports the influence of nutrition on the photoperiodic control of luteinizing hormone (LH) secretion in female Mediterranean goats (i.e., goats from the Mediterranean area in general). Ovariectomized, oestradiol-treated goats were subjected to two consecutive intervals of 3 months of long days followed by 3 months of short days (group LDSD, N=20), or vice versa (group SDLD, N=20). The LDSD and SDLD does were also randomly assigned to one of two nutrition groups that received either 1.1 (H group, N=10) or 0.7 (L group, N=10) times their maintenance requirements. Live weight and body condition score were determined weekly and LH concentrations twice per week. To establish the pulsatility of secretion of LH, three periods of intensive sampling were undertaken. Melatonin was determined after a period of 45 short or long days. All photoperiod/nutrition groups showed large variations in LH concentrations according to photoperiod, with nutrition having a significant effect (P

Background It is now widely accepted that AMP-activated protein kinase (AMPK) is a critical regulator of energy homeostasis. Recently, it has been shown to regulate circadian clocks. In seasonal breeding species such as sheep, the circadian clock controls the secretion of an endogenous rhythm of melatonin and, as a consequence, is probably involved in the generation of seasonal rhythms of reproduction. Considering this, we identified the presence of the subunits of AMPK in different hypothalamic nuclei involved in the pre- and post-pineal pathways that control seasonality of reproduction in the ewe and we investigated if the intracerebroventricular (i.c.v.) injection of two activators of AMPK, metformin and AICAR, affected the circadian rhythm of melatonin in ewes that were housed in constant darkness. In parallel the secretion of insulin was monitored as a peripheral metabolic marker. We also investigated the effects of i.c.v. AICAR on the phosphorylation of AMPK and acetyl-CoA carboxylase (ACC), a downstream target of AMPK, in brain structures along the photoneuroendocrine pathway to the pineal gland. Results All the subunits of AMPK that we studied were identified in all brain areas that were dissected but with some differences in their level of expression among structures. Metformin and AICAR both reduced (p < 0.001 and p < 0.01 respectively) the amplitude of the circadian rhythm of melatonin secretion independently of insulin secretion. The i.c.v. injection of AICAR only tended (p = 0.1) to increase the levels of phosphorylated AMPK in the paraventricular nucleus but significantly increased the levels of phosphorylated ACC in the paraventricular nucleus (p < 0.001) and in the pineal gland (p < 0.05). Conclusions Taken together, these results suggest a potential role for AMPK on the secretion of melatonin probably acting trough the paraventricular nucleus and/or directly in the pineal gland. We conclude that AMPK may act as a metabolic cue to modulate the rhythm of melatonin secretion.

The objective was to determine whether refractoriness to short and long days were involved in the end and onset of the breeding season, respectively, in goats adapted to subtropical latitudes. Ovariectomized does given a subcutaneous implant constantly releasing estradiol-17 beta (OVX+E) were used in two experiments. Plasma LH concentrations were determined twice weekly. In Experiment 1, the control group remained in an open-shed pen (natural day length and ambient temperature). Two experimental groups were placed in light-proof buildings (with natural temperature variations). One group was exposed to natural simulated increasing days (winter to spring), whereas the other was exposed to a winter solstice photoperiod (10 h of light) from December 21 to April 28. In Experiment 2, the control group remained under natural day length and ambient temperature. One experimental group was exposed to natural simulated decreasing days (summer to autumn), whereas the other group was exposed to a summer solstice photoperiod (14 h of light) from June 21 to October 20. In Experiment 1, the breeding season was not prolonged in does maintained in the winter solstice day length. Mean dates of decrease in LH secretion (end of the breeding season) did not differ significantly between does exposed to natural (February 3 +/- 5 d) or natural simulated photoperiod (January 26 +/- 14 d) and those exposed to constant short days of winter solstice (February 4 +/- 10 d). In Experiment 2, the onset of the breeding season was not delayed in does maintained in the summer solstice day length. Mean dates of increase in LH secretion (onset of the breeding season) did not differ significantly between does exposed to natural (September 7 +/- 8 d) or natural simulated photoperiod (September 18 +/- 10 d) and those exposed to constant long days photoperiod of summer solstice (September 24 +/- 4 d). In goats adapted to a subtropical environment, we concluded that: I) the end of breeding season was due to refractoriness to short days, and not the inhibitory effect of increasing day length; and 2) the onset of the breeding season was due to refractoriness to long days, and not a stimulatory effect of decreasing day length.

Spontaneous ovulatory activity (SOA) in spring has been used to study the out-of-season breeding ability of Merinos d’Arles (MA) ewes. Within this breed, an association was found between more intense seasonality and genotype 2/2 at a MnlI restriction site (allele 2 for its absence v. 1 for its presence) in Exon II of the MT1 receptor gene. This study was designed to ascertain whether this association results in a direct effect of the MT1 genotype on the expression of seasonality in MA ewes. In the first year of the study, genotyping of 314 MA ewes at locus MnlI was carried out and resulted in frequencies of 43.0%, 44.9% and 12.1% for genotypes 1/1, 1/2 and 2/2, respectively. The SOA of these ewes was determined in early April of two consecutive years by assaying plasma progesterone concentrations in two blood samples taken 9 days apart. Groups of 30 ewes of each homozygous genotype (1/1 and 2/2) were identified from this population and their SOA was followed by taking blood samples at regular intervals between January and mid-April of the second and third year of the study. In the second year, groups of ewes were managed together on rangelands, whereas in the third year each group was split into two subgroups given differential feed levels. The results clearly showed that genotype had no significant effect on SOA during the 2- to 3-month period preceding the introduction of rams for spring mating. In the second year of the study, in which the experimental procedure allowed a fair comparison of the fertility of ewes in spring mating, fertility was similar for both genotypes. The reciprocity of the association was not demonstrated and the MnlI polymorphic site could not be used as a genetic marker of selection for out-of-season breeding ability, at least not in the MA breed. The percentage of cycling ewes significantly decreased between January and April, and older ewes (5 or 6 years old depending on the year of the study) were more cyclic than younger ones (2 and 3 years old, respectively). The differential feeding level of ewes from early February did not significantly affect their SOA during the time period studied in the third year of the study.

One experiment was conducted to determine whether the treatment with artificial long days and exogenous melatonin can induce reproductive activity during spring (seasonal anoestrus) in Mediterranean goats that are in daily contact with bucks and whether this treatment causes a variation in the reactivation of the reproductive activity in the normal breeding season. The experiment started on 4 November 2005 and finished on 27 October 2006. Thirty-four adult and barren does were used, distributed into two groups balanced according to their live weight (LW) and body condition score (BCS). Seventeen females were exposed to long days (16 h of light/day) from 14 November 2005 to 20 February 2006. On 20 February, they received one s.c. melatonin implant (LD-M group) and were exposed to natural photoperiodic changes in an open shed. The other females during the experiment were placed in an open shed under natural photoperiod and remained as the control group (C group). The C and LD-M groups were keeping in contact with males during the whole experiment. During the experiment, the LW, BCS and plasma progesterone concentrations were measured weekly, oestrous activity was tested daily using entire aproned bucks, and ovulation rate was evaluated by laparoscopy 7 days after positive identification of the oestrus. A clear treatment–time interaction was observed for plasma progesterone concentrations ( P,0.001), with a period of high progesterone concentrations during the natural seasonal anoestrus in the LD-M group. Although 94.1% of females in the LD-M group presented ovarian activity during this period, no female in group C did. Resumption of ovarian activity in the subsequent natural breeding season was 2 weeks later in the LD-M group in comparison with group C ( P,0.05). We can conclude that in Mediterranean goat breeding systems, when females are in daily contact with bucks, the treatment with 3 months of long days and melatonin implant at the end of the light photoperiodic treatment can induce ovarian and oestrous activity during the seasonal anoestrus. Finally, this treatment causes a short delay in the subsequent reactivation of ovarian activity in the natural breeding season.

Experiments were designed to localize the central sites of action of melatonin involved in the control of seasonal reproduction. Ewes were exposed to long days and received microimplants of melatonin in the preoptic area (n = 9), anterior hypothalamus (n = 4), dorsolateral hypothalamus (n = 4), or mediobasal hypothalamus (n = 12). The effects of implants were determined by comparison with control ewes (untreated or sham-operated, n = 10) and with ewes treated with an s.c. implant of melatonin (n = 8) or ewes subjected to short days (n = 8). All ewes were ovariectomized and treated s.c. with a silastic capsule of estradiol. Melatonin released in the preoptic area as well as in the anterior and lateral hypothalamus did not cause any difference as compared with the controls (no stimulation of LH secretion and no inhibition of prolactin secretion). In contrast, melatonin implanted in the mediobasal hypothalamus caused an increase in LH secretion in 7 of the 12 ewes on Day 53.0 +/- 4.2 after implantation (mean +/- SEM). Their response was not different compared with that of ewes treated s.c. with melatonin or exposed to short days either in terms of timing (Day 56.3 +/- 6.2 and 59.5 +/- 3.1, respectively, for controls) or of amplitude of the LH response. Similarly, melatonin caused only a reduction of prolactin secretion in the mediobasal, s.c., and short-day groups. It is concluded that the mediobasal hypothalamus or the surrounding tissues could be the sites of action of melatonin involved in the control of seasonal reproduction.

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 (dopaminergic(2) receptor antagonist), and cyproheptadine (serotonin 5-hydroxytryptamine(2) 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 (LateLD). The role of the different neural systems was clearly modified by the level of nutrition. In the low-nutrition group, only naloxone increased LH concentrations during onsetLD (P0.05). However, in the high-nutrition group, naloxone increased the concentration and pulsatility of LH (P0.05) in onsetSD and onsetLD. Pimozide increased LH concentration and pulsatility (P0.05) in onsetLD and LH concentration in lateLD (P0.001). Finally, cyproheptadine significantly increased LH concentration at all three times (P0.001). These results provide evidence that all three systems are involved in the inhibition of LH release in onsetLD, and that the opioid and serotonin mechanisms are involved during the onsetSD that were enhanced by a high plane of nutrition.

Two experiments were designed to determine if melatonin concentrations differ between jugular veins. In a first experiment, blood was collected continuously every 30 min from each jugular during 12-h from 6 ewes. In a second experiment, 100 ewes were sampled twice at night simultaneously from the two jugular veins. In both experiments, mean melatonin concentrations were similar between right and left jugulars. However, within individuals, large differences were observed between the two sides (p < 0.001). This difference was stable over time and the higher side varies among individuals. The concentrations of prolactin and oxytocin, measured in the same samples did not show such differences. This observation raises the question of the origin of this phenomenon. Moreover, it has important implications for studies in which melatonin concentrations need to be assessed accurately. Indeed, a reliable quantitative assessment of melatonin production by the pineal gland requires sampling from both jugular veins. (C) 2009 Elsevier Ltd. All rights reserved.

This study examines the patterns of melatonin secretion during postnatal development in wild (mouflon; n = 7) and domestic (Spanish Merino; n = 6) female lambs under their natural photoperiod conditions. The aim was to determine whether these types of sheep which differ in the timing of puberty, also differ in the establishment of daily melatonin secretory rhythms and/or in the nocturnal plasma melatonin secretion. In addition, the time when the lambs first reached nocturnal levels of melatonin similar to adults was also evaluated. For this purpose, the melatonin secretion in lambs was compared with those of their mothers. A day/night (D/N) difference in plasma melatonin concentration was noticed as early as 1 day after birth in the Merino female lambs (D: 5.9 +/- 1.0 pg/ml compared to N: 22.0 +/- 3.3 pg/ml; p < 0.05), and by 1 week after birth in the mouflon female lambs (D: 4.9 +/- 0.3 pg/ml compared to N: 56.9 +/- 15.3 pg/ml; p < 0.05). An effect of the genotype (p < 0.05) and age (p < 0.01) was detected on mean nocturnal plasma melatonin concentrations, which were lower in the Merino than in the mouflon lambs. Night-time plasma concentration of melatonin was also high in mouflon than in Merino mothers (p < 0.05). No differences were detected between the wild genotype and the domestic one in the time when the lambs first reached nocturnal levels of melatonin similar to those of their mothers. (C) 2009 Elsevier B.V. All rights reserved.

The study was conducted on two consecutive years to determine whether ovulatory activity can be induced in anovulatory goats by exposing them to sexually active bucks for 4, 8, 12 or 16 h per day during 15 consecutive days. In experiment 1, females remained continuously in the experimental pens where they were in contact with males. One group remained isolated from males (controls) and four other groups were exposed to sexually active males for 4, 8, 12 or 16 h per day. In experiment 2, females were taken away to "resting" pens free of male odours between the periods of contact with bucks. They were allocated to 5 groups as in experiment 1. Ovulations were determined by progesterone plasma levels and transrectal ultrasonography. Pregnancy was determined by abdominal ultrasonography. In both experiments, more than 90% of females exposed to the bucks had at least one ovulation during the whole experiment whereas only 11 or 0% (experiments 1 and 2, respectively) did so in the control group (P0.05). In both experiments, pregnancy rates were not affected by the daily duration of contact with males (P>0.05). To conclude, 4 h of daily contact with sexually active males is sufficient to stimulate ovulatory activity in anovulatory goats and this effect is not due to the presence of olfactory cues from the males remaining in the pens. (C) 2010 Elsevier Inc. All rights reserved.

Photoperiodic treatments and reproduction in farm animals. Farm animal productions are frequently tied to the seasons, which explain the seasonal variations in availability of fresh animal products (meat, milk, cheese and eggs). Among farmed species, fishes and birds are generally considered as more directly sensitive to external factors (mainly temperature in fish and photoperiod in birds). Artificial photoperiodic treatments are widely used in numerous species including mammals ; they consist in providing extra-light during natural short days (chickens, turkeys, guinea fowl, sheep and goats), or in the administration of melatonin during long days (goats, sheep). These measures help adjust the breeding season to the requirements of the breeder and/ or cancel completely seasonal variations of sperm production in semen production centres (mammals) as well as in poultry and fish farms. Additionally, pure light treatments (without melatonin), especially when there is outside access, are non invasive and preserve the animals’ integrity and welfare., Les productions issues d’animaux d’élevage sont fréquemment rythmées sur les saisons, avec pour conséquence une disponibilité saisonnière en produits frais sur les marchés (viandes, lait et fromages, oeufs). Parmi les espèces d’intérêt en production animale, les poissons et les oiseaux sont généralement considérés comme plus directement sensibles aux facteurs externes, essentiellement les variations de la température pour les poissons et de la durée de la phase claire du jour, ou photopériode, pour les oiseaux. Chez de nombreuses espèces incluant des mammifères, nous utilisons largement des traitements photopériodiques artificiels qui consistent en un éclairement supplémentaire pendant les jours courts naturels (chez le poulet, la dinde, la pintade, le mouton et la chèvre) ou en l’administration de mélatonine pendant les jours longs (chez le mouton et la chèvre) ; ils permettent d’ajuster la période de reproduction aux souhaits de l’éleveur et/ ou d’abolir complètement les variations saisonnières de la production spermatique dans les centres producteurs de semence destinée à l’insémination artificielle (mammifères), et dans les élevages (volailles, poissons). Par ailleurs, des traitements photopériodiques «purs» (sans mélatonine), en particulier lorsqu’ils sont appliqués en bâtiments ouverts, sont non invasifs et donc respectent l’intégrité et le bien-être des animaux., Chemineau Philippe, Malpaux Benoît, Brillard Jean-Pierre, Fostier Alexis. Traitements photopériodiques et reproduction chez les animaux d'élevage. In: Bulletin de l'Académie Vétérinaire de France tome 163 n°1, 2010. pp. 19-26.

Remerciements : Plate-forme CIRE : Chirurgie et Imagerie pour la Recherche et l'Enseignement, INRA UMR PRC 0085, Centre Val de Loire; Kisspeptin (Kiss) is a key regulator of reproductive function in both prepubertal and adult mammals. Its expression appears to vary throughout the year in seasonal species. We aimed to determine the impact of a change of photoperiod on the size of Kiss neuronal populations found in the preoptic area (POA) and arcuate nucleus (ARC) of the ewe brain. Using immunocytochemistry, we first examined the proportion of neurones expressing Kiss, using HuC/D as a neuronal marker, at different time-points after transition from long days (LD; 16 : 8 h light/dark cycle) to short days (SD; 8 : 16 h light/dark cycle). Luteinising hormone (LH) secretion was measured in ovariectomised oestradiol replaced ewes from the month preceding the transition to SD until the sacrifice of the animals at days 0, 45 and 112 from this photoperiodic transition. High LH levels were only observed in animals killed at day 112. The number of Kiss neurones/mm(2) doubled in the caudal ARC at day 112. The percentage of neurones showing Kiss immunoreactivity increased significantly in both the POA and ARC in the day 112 group. In a second experiment, ewes kept in LD received an i.c.v. injection of colchicine 20 h before sacrifice. Colchicine treatment increased the number and the percentage of neurones with Kiss in both the POA and caudal ARC. The data obtained suggest that the increase in Kiss neurones detected in the POA and caudal ARC after transition to SD stemmed from an increase in Kiss synthesis. This up-regulation of Kiss content under the shorter day condition appears to be a late event within the cascade activated by a longer secretion of melatonin, which is a critical factor in switching gonadotrophin-releasing hormone secretion to a breeding season profile.

Photoperiod, metabolism and reproduction : advantage of the equine model. Equidae are born in the spring. This is due to the fact that mares go through a period of anovulation from October to the end of April. For breeders, bringing forward the first ovulation presents an enormous advantage, as it increases the number of usable cycles in the mare and increases the actual age of the offspring at the time of sports performance or slaughter. In mares under our latitudes, the first ovulation can be moved to its earliest by using 14.5 h of daily light for 35 days, beginning around the winter solstice. This treatment is robust, as repeated accidents do not affect its outcome. The administration of melatonin to pony mares during winter ovarian inactivity, either daily in the evening or with a subcutaneous implant, suppresses the photostimulating effect of long days (LD). On the other hand, the use of this implant around the summer solstice brings forward the first ovulation, as in photostimulated mares, thereby confirming the existence of an annual endogenous rhythm. These implants inserted under the same conditions in non-pregnant mares do not induced ovarian inactivity. Therefore, the role of melatonin seems limited to the timing of winter inactivity. In thin but healthy mares subjected to the natural photoperiod, winter inactivity is systematic and long lasting. This inhibition, induced by the feed restriction, masks the effect of the photostimulation. On the other hand, 60% of fat mares have cycles all year long, and in the remaining 40%, the period of inactivity is very short, missing only one or two cycles. Increasing the feed in thin mares in autumn or winter seems to have no effect on the date of the first annual ovulation. Nutrition is the main cause of ovarian inactivity in winter, but it does not play a role in the resumption of activity in the spring. The mechanism inhibiting the cycles in autumn results from an interaction between short days and a negative energy balance. This nutrition-photoperiod interaction seems to be applicable to numerous species in temperate areas., Chez les Équidés, les mises-bas ont lieu au printemps, conséquence d'une période sans ovulation commençant vers octobre pour s'achever fin avril. Pour les éleveurs, une avance de la première ovulation est capitale car elle augmente d'une part, le nombre de cycles utilisables et d'autre part, l'âge réel au moment de l'épreuve sportive ou de l'abattage. Chez les juments, l'avance maximale de la date de la première ovulation annuelle, sous nos latitudes, est obtenue avec un éclairement quotidien de 14h30, commençant vers le solstice d'hiver et pouvant être interrompu au bout de 35 jours longs (JL). Ce traitement est robuste : des accidents répétés n'altèrent pas son effet. L'administration de mélatonine à des ponettes en inactivité hivernale, chaque soir ou sous forme d'implants sous-cutanés, supprime l'effet photostimulant des JL. Par contre, la pose des implants, aux alentours du solstice d'été, avance la première ovulation de l’année suivante comme chez des juments photostimulées, démontrant ainsi l'existence d'un rythme endogène annuel. Ces implants posés dans les mêmes conditions chez des juments non gravides n'induisent pas l'inactivité. Le rôle de la mélatonine semble donc limité au centrage du moment de la phase d'inactivité. Chez des animaux maigres, en bonne santé, sous photopériode naturelle, l'inactivité est systématique et durable. Cette inhibition, liée à la sous-alimentation masque l'effet de la photostimulation. Par contre, 60 % des juments grasses cyclent toute l'année, les 40 % restants présentent une inactivité très courte correspondant à l'absence d’un ou deux cycles. Une réalimentation automnale ou hivernale des juments amaigries ne semble pas avoir d’effet sur la date de la première ovulation annuelle. La nutrition est la principale cause de l'arrêt hivernal de la cyclicité mais n'intervient pas sur sa reprise printanière. Le mécanisme qui induit à l'automne l’arrêt de la cyclicité est dû à l'interaction entre des jours courts et une balance énergétique négative. Cette interaction nutrition-photopériode est semble-t-il généralisable à de nombreuses espèces des zones tempérées., Guillaume Daniel, Salazar-Ortiz Juan, Menassol Jean-Baptiste, Malpaux Benoît, Chemineau Philippe. Photopériode, métabolisme et reproduction : intérêt du modèle équin. In: Bulletin de l'Académie Vétérinaire de France tome 163 n°1, 2010. pp. 5-18.

Breeds of sheep and goats from temperate latitudes exhibit seasonal variations of breeding activity which are controlled by annual photoperiodic changes. Short days (SD) stimulate sexual activity, but prolonged exposure results in refractoriness to short days and subsequent cessation of reproductive activity. Refractoriness can be broken by exposing the animals to long days (LD), thus alternations between LD and SD are essential for the photoperiodic control of seasonal reproduction. Light pulses during the dark period can mimic a long day (‘LD’) and melatonin treatments can mimic a short day. For out-of-season control of sexual activity, treatments using the succession ‘LD’-decreasing days or ‘LD’-melatonin were very effective in advancing puberty in young rams in which sperm production was increased, permitting these animals to be submitted earlier for progeny testing, using artificial insemination (AI). In adult rams, such treatments also caused an important increase in testicular weight and sperm production in the spring. In the female goat, the succession ‘LD’-melatonin treatment efficiently induces and maintains oestrous and ovulatory activities in spring, leading to high fecundity after natural mating. This treatment so far appears to be less effective in the seasonal sheep breeds of Northern Europe than in goats. However, melatonin alone can be used after the end of May to advance the breeding season and to increase fecundity. Induction of permanent reproductive activity in rams and bucks was made possible by the observation that monthly alternations between LD and SD (short light cycles) abolished seasonality of behavioural and spermatogenic activities. These males could be used all the year round to produce a high number of AI doses without variations in sperm quality and with no variation in fertility. Short light cycles can be used in open barns by alternating ‘LD’ and melatonin. In contrast, in the ewe, such short light cycles were unable to abolish seasonality of ovulatory activity. Knowledge of the different effects of photoperiod on neuroendocrine pathways and the reproductive activity in sheep and goats has therefore allowed us to successfully apply light treatments to control seasonal reproductive activity in field conditions and in males raised in AI centres.

Background Sheep are seasonal breeders. The key factor governing seasonal changes in the reproductive activity of the ewe is increased negative feedback of estradiol at the level of the hypothalamus under long-day conditions. It has previously been demonstrated that when gonadotropin secretions are inhibited during long days, there is a higher concentration of estradiol in the cerebrospinal fluid (CSF) than during short days. This suggests an involvement of the CSF and choroid plexus in the neuroendocrine regulatory loop, but the mechanisms underlying this phenomenon remain unknown. One possible explanation of this difference in hormonal content is an effect of concentration or dilution caused by variations in CSF secretion rate. The aim of this study was thus to investigate changes in the CSF turnover rate related to light-dark cycles. Methods The turnover rate of the CSF was estimated by measuring the time taken for the recovery of intraventricular pressure (IVP) after removal of a moderate volume (0.5 to 2 ml) of CSF (slope in mmHg/min). The turnover rate was estimated three times in the same group of sheep: during a natural period of decreasing day-length corresponding to the initial period when gonadotropin activity is stimulated (SG1), during a long-day inhibitory period (IG), and finally during a short-day stimulatory period (SG2). Results The time taken and the speed of recovery of initial IVP differed between groups: 8 min 30 sec, 0.63 ± 0.07 mmHg/min(SG1), 11 min 1 sec, 0.38 ± 0.06 mmHg/min (IG) and 9 min 0 sec, 0.72 ± 0.15 mmHg/min (SG2). Time changes of IVP differed between groups (ANOVA, p < 0.005, SG1 different from IG, p < 0.05). The turnover rate in SG2: 183.16 ± 23.82 μl/min was not significantly different from SG1: 169. 23 ± 51.58 μl/min (Mann-Whitney test, p = 0.41), but was significantly different from IG: 71.33 ± 16.59 μl/min (p = 0.016). Conclusion This study shows that the turnover rate of CSF in ewes changes according to the light-dark cycle; it is increased during short day periods and reduced in long day periods. This phenomenon could account for differences in hormonal concentrations in the CSF in this seasonal species.

In sheep, as in many vertebrates, the seasonal pattern of reproduction is timed by the annual photoperiodic cycle, characterized by seasonal changes in the day length. The photoperiodic information is translated into a circadian profile of melatonin secretion. After multiple neuronal relays (within the hypothalamus), melatonin affects gonadotrophin-releasing hormone (GnRH) secretion, which in turn controls ovarian cyclicity. The pattern of GnRH secretion is mirrored by that of luteinizing hormone (LH) secretion, whose plasmatic level can be easily measured. We addressed the question of whether there exists an endogenous circannual rhythm in a tropical sheep (Blackbelly) population that exhibits clear seasonal ovarian activity when ewes are subject to temperate latitudes. We based our analysis on LH time series collected in the course of 3 years from ewes subject to a constant photoperiodic regime. Owing to intra- and interanimal variability and unequal sampling times, the existence of an endogenous rhythm is not straightforward. We have used time–frequency signal processing methods, and especially the smooth pseudo-Wigner–Ville distribution, to extract possible hidden rhythms from the data. To further investigate the low-frequency (LF) and high-frequency (HF) components of the signals, we have designed a simple mathematical model of the LH plasmatic level accounting for the effect of experimental sampling times. The model enables us to (i) confirm the existence of an endogenous circannual rhythm as detected by the LF signal component, (ii) investigate the action mechanism of the photoperiod on the pulsatile pattern of LH secretion (control of the interpulse interval), and (iii) conclude that the HF component is mainly due to the experimental sampling protocol.

International audience; This work was designed to determine whether melatonin treatment at the spring equinox can induce reproductive activity in goats without separation from males (separation being the normal practice in Spanish farming systems) and whether this treatment modifies the onset of the natural breeding season. Twenty-nine entire does were distributed into two groups (Group M, n = 14; Group C, n = 15). A third group of ovariectomized, estradiol-treated goats (OVX group, n = 5) was used to study the effect of melatonin on reproductive activity. On March 18, Groups M and OVX received a subcutaneous melatonin implant. In entire females, estrus was tested daily using entire aproned males, and ovulation rate was assessed after identification of estrus. Plasma progesterone in entire goats, plasma luteinizing hormone (LH) in the OVX group, and live weight and body condition score for all animals were recorded once a week. In entire goats, a clear treatment by time interaction was observed for progesterone concentrations (P < 0.001), with a period of high progesterone concentrations during the natural seasonal anestrus in Group M. A similar period of high LH concentrations was observed in the OVX group. Whereas all females of Group M presented ovarian activity during this period, no female of Group C did. The resumption of the natural breeding season was retarded in Group M in comparison with that in Group C (P < 0.05). We can conclude that in Mediterranean goats, melatonin implants can induce reproductive activity without separation from males, and it causes a small retardation in the reactivation of reproductive activity in the natural breeding season. (C) 2009 Elsevier Inc. All rights reserved.

The objective of this study was to determine the importance of male sexual behavior in stimulating LH secretion in anovulatory female goats. Two groups of females (n=10 per group) were each exposed to a buck in sexual rest and submitted to natural daylength. In one group, the buck was awake, whereas in the other group, it was sedated to prevent its sexual behavior. Two other groups of goats (n=10 per group) were exposed to sexually active bucks that had been exposed to 2.5 months of long days. In one group, the buck was awake, and in the other group, it was sedated. LH secretion was determined every 15 min from 4 h before introducing the bucks to 8 h after, then every 15 min again from 20 to 24 h after introducing the bucks. The bucks submitted to natural daylength did not stimulate LH secretion (P0.05), whether they were sedated or not. In contrast, both the awake and the sedated light-treated bucks induced an increase (P0.05) of LH pulsatility in the first 4 h following their introduction. However, pulsatility remained elevated until 24 h in the females exposed to the light-treated awake buck, whereas in the group with the light-treated sedated buck, pulsatility diminished (P0.05) after the first 4 h of stimulation by the buck. In conclusion, the sexual behavior of males contributes to the maintenance of a high LH pulsatility up to 24 h after introduction into a group of anovulatory goats.

Les diverses productions issues d’animaux d’élevage sont fréquemment saisonnières, et par là même leur disponibilité en produits frais sur les marchés (viandes, lait et fromages, oeufs). Ceci relève, en partie, de la décision de l’éleveur qui vise à commercialiser ses produits à la période la plus favorable. Cette saisonnalité peut être aussi imposée par l’accès à des ressources alimentaires saisonnières, comme chez les herbivores, ou par des contraintes biologiques spécifiques, comme celle de la température de l’eau pour les poissons. Les variations saisonnières de la disponibilité en produits animaux sont aussi la conséquence de contraintes résultant d’une saison de reproduction plus ou moins marquée des animaux d’élevage qu’il s’agisse de poissons, d’oiseaux ou de mammifères. Sous des latitudes moyennes et hautes, la plupart des animaux d’élevage, comme leurs « cousins » sauvages, donnent en effet naissance à leurs petits en fin d’hiver et/ou au début du printemps, période souvent la plus favorable pour la survie des jeunes et la mise en place d’une nouvelle génération. En conséquence, la plupart des espèces manifestent des variations saisonnières de la fréquence d’ovulation (présence ou absence d’ovulations chez les mammifères et les poissons ; variations ou suppression des taux de ponte chez les oiseaux), de la qualité des gamètes (variations des taux de fécondation et de survie embryonnaire), mais aussi du comportement sexuel. Parmi les espèces d’intérêt en production animale, les oiseaux et les poissons sont généralement considérés comme plus directement sensibles aux facteurs externes (majoritairement température, mais aussi photopériode, pour les poissons ; et photopériode pour les oiseaux). Cependant, chez de nombreuses espèces incluant des mammifères, il est intéressant de constater que des traitements photopériodiques artificiels consistant en un éclairement supplémentaire pendant les jours courts naturels (chez le poulet, la dinde, la pintade, le mouton et la chèvre) ou de la mélatonine pendant les jours longs (chez le mouton et la chèvre), sont utilisés largement pour ajuster la période de reproduction aux souhaits de l’éleveur et/ou pour abolir complètement les variations saisonnières de la production spermatique dans les centres producteurs de semence destinée à l’insémination artificielle (mammifères), et dans les élevages (volaille, poissons). Par ailleurs, des traitements photopériodiques « purs » (sans mélatonine), en particulier lorsqu’ils sont appliqués en bâtiments ouverts, sont non invasifs et donc respectent pleinement le bien-être animal., A large majority of farm animals express seasonal variations in their production traits, thus inducing seasonal availability of fresh derived animal products (meat, milk, cheese and eggs). This pattern is in part the consequence of the farmer's objective to market his/her products during the most economically favourable period. It may also be imposed by the season-dependent access to feed resources, as in ruminants, or by the specific requirements derived from adaptation to environmental conditions such as water temperature in fish. But seasonal variations in animal products are also the consequence of constraints resulting from the occurrence of a more or less marked seasonal reproductive season in most farm animal species including fish, poultry and mammals. Like their wild counterparts, at mid and high latitudes, most farm animals normally give birth at the end of winter-early spring, the most favourable period for the progeny to survive and thus promote the next generation. As a consequence, most species show seasonal variations in their ovulation frequency (mammals and fish: presence or absence of ovulation; birds: variations or suppression of laying rates), spermatogenic activity (from moderate to complete absence of sperm production), gamete quality (variations in fertilisation rates and embryo survival), and also sexual behaviour. Among species of interest for animal production, fishes and birds are generally considered as more directly sensitive to external factors (mainly temperature in fish, photoperiod in birds). In all species, it is therefore advisable that artificial photoperiodic treatments consisting of extra-light during natural short days (in chickens, turkeys, guinea fowl, sheep and goats) or melatonin during long days (in goats, sheep) be extensively used to either adjust the breeding season to the animal producer's needs and/or to completely overcome seasonal variations of sperm production in artificial insemination centres (mammals) and breeder flock operations (poultry, fish farming). Pure light treatments (without melatonin), especially when applied in open barns, could be considered as non invasive ones that fully respect animal welfare.

International audience; Two experiments were conducted to determine if nutritional supplementation improved ovulation and pregnancy rates in female goats managed under grazing conditions and submitted to the male effect. In Experiment 1, one group of does did not receive nutritional supplementation, while the other group was supplemented daily for 7 days starting at the time when the males were introduced to the females. The ovulation rate at the second male-induced ovulation was greater(P < 0.05) in supplemented (2.0 +/- 0.1) than in non-supplemented (1.6 +/- 0.1) does. For Experiment 2, female goats were supplemented for 0, 7,14 or 28 days, starting 9 days following buck introduction. The proportion of does that were pregnant in the group supplemented for 28 days was greater (P < 0.05) than in the non-supplemented group. but did not differ from 14-day and the 7-day supplemented groups. The proportion of pregnant does was greater (P < 0.05) in the group supplemented for 14 days compared to the group supplemented for 7 days and the non-supplemented group. These latter two groups did not differ (P > 0.05). In conclusion, feed supplementation for 7 days, starting at the time when males were introduced increased ovulation rate and feed supplementation for 14 or 28 days starting 9 days after males were introduced improved pregnancy rates in goats managed under grazing conditions and exposed to males. (C) 2009 Elsevier BY. All rights reserved.

International audience; Intracellular signaling events are often organized around PDZ (PSD-95/Drosophila Disc large/ZO-1 homology) domain-containing scaffolding proteins. The ubiquitously expressed multi-PDZ protein MUPP1, which is composed of 13 PDZ domains, has been shown to interact with multiple viral and cellular proteins and to play important roles in receptor targeting and trafficking. In this study, we show that MUPP1 binds to the G protein-coupled MT 1 melatonin receptor and directly regulates its G i-dependent signal transduction. Structural determinants involved in this interaction are the PDZ10 domain of MUPP1 and the valine of the canonical class III PDZ domain binding motif DSV of the MT 1 carboxyl terminus. This high affinity interaction (K d ϳ 4 nM), which is independent of MT 1 activation, occurs in the ovine pars tuberalis of the pituitary expressing both proteins endogenously. Although the disruption of the MT 1 /MUPP1 interaction has no effect on the subcel-lular localization, trafficking, or degradation of MT 1 , it destabilizes the interaction between MT 1 and G i and abolishes G i-mediated signaling of MT 1. Our findings highlight a previously unappreciated role of PDZ proteins in promoting G protein coupling to receptors.

International audience; To determine the effect of body condition on the characteristics of the breeding season in female goats from subtropical Mexico, does in either greater- or lesser-body condition (n = 20/group) were monitored for the expression of estrous behavior, ovulation and ovulation rate between June and April. The commencement of estrus and ovulation occurred earlier (P < 0.05) in does in greater than lesser body condition. The cessation of estrus and ovulation was later (P < 0.05) in female goats with greater than those with lesser body condition. Does in greater body condition had more (P < 0.001) normal estrous cycles than those in lesser body condition. As consequence, does from the greater body condition group had fewer short (P < 0.001) or long (P < 0.05) estrous cycles than those of in the lesser body condition group. The ovulation rate was greater (P < 0.01) in the greater (1.9 +/- 0.1) than lesser body condition does (1.6 +/- 0.11). In conclusion, female goats in lesser body condition have a shorter breeding season, more abnormal estrous cycles, and fewer ovulations than does in greater body condition. (c) 2008 Elsevier B.V. All rights reserved.

International audience; 5-Hydroxyoxindole has been identified as a urinary metabolite of indole, which is produced from tryptophane via the tryptophanase activity of gut bacteria. We have demonstrated recently that 5-hydroxyoxindole is an endogenous compound in blood and tissues of mammals, including humans. To date, 5-hydroxyoxindole's role is unknown. The aim of this study was to compare 5-hydroxyoxindole levels in plasma and cerebrospinal fluid (CSF) during day-night and seasonal changes, as a common approach to pilot physiological characterization of any compound. Simultaneous blood and CSF sampling was performed in the ewe, because its size allows collection in quantities suitable for 5hydroxyoxindole assay (HPLC-ED) in awake animals, without obvious physiological or behavioral disturbance. 5-Hydroxyoxindole concentration was quite stable in plasma (2-6 nM range), whereas, in CSF, it displayed marked day-night and photoperiodic variations (4-116 nM range). 5-Hydroxyoxindole levels in CSF were twofold higher at night than during the day and at least one order of magnitude higher during the long compared with the short photoperiod. These day/night and photoperiodic variations persisted after pinealectomy, indicating that 5-hydroxyoxindole rhythms in CSF are independent of melatonin formation. In conclusion, high levels of 5-hydroxyoxindole in the CSF during long photoperiod and its daily modulation suggest physiological involvement of 5-hydroxyoxindole in rhythmic adjustments in the brain, independently of the pineal gland.

International audience; The aim of this study was to determine whether the seasonal change in food availability provides a predictive cue that synchronizes the breeding season in goats adapted to the subtropical conditions of Northern Mexico. Groups of multiparous intact (n = 7) and ovatiectomized does bearing a subcutaneous implant constantly releasing estradiol-17 beta (OVX+E; n = 12) were allocated in two pens and received a constant amount of feed. Another group of OVX+E does (n = 10) was incorporated into a flock raised under natural grazing conditions with seasonal fluctuations in food availability. Blood samples were taken twice weekly. Ovulation of intact goats was inferred from plasma progesterone levels. In OVX+E does plasma levels of LH were measured. Intact does displayed seasonal variations in ovulation and the breeding season lasted from September to February. OVX+E goats displayed large seasonal variations in LH secretion regardless of whether they were fed constantly indoors or kept under natural grazing conditions (P < 0.0001). The mean date of increase in LH secretion varied between years (P < 0.0001) but did not differ between groups (naturally grazed: August 13 +/- 10 days; constantly fed: August 26 +/- 10 days). In contrast, the seasonal decline in LH secretion differed between groups: it was consistently delayed in constantly fed animals compared to those feeding naturally (naturally grazed: January 28 +/- 4 days; constantly fed: February 24 +/- 6 days, group effect, P < 0.01). These results show that local female goats from subtropical Mexico display large reproductive seasonality independently of food availability. (C) 2008 Published by Elsevier Inc.

International audience; In the eel, a deficit in gonadotrophin-releasing hormone (GnRH) and a strong dopaminergic (DA) inhibition are responsible for the blockade of gonad development if silver eels are prevented from their reproductive migration. Environmental factors that eels encounter during their oceanic reproductive migration are thought to play an important role in the stimulation of eel pubertal development. We investigated the potential role of melatonin, a known mediator of the effects of external factors on reproductive function in vertebrates. We demonstrated that a long-term melatonin treatment increased brain tyrosine hydroxylase (TH, the rate limiting enzyme of DA synthesis) mRNA expression in a region-dependent way. Melatonin stimulated the dopaminergic system of the preoptic area, which is involved in the inhibitory control of gonadotrophin [luteinising hormone (LH) and follicle-stimulating hormone (FSH)] synthesis and release. Moreover, we showed that the increased TH expression appeared to be consistent with melatonin binding site distribution as shown by 2[(125)I]-melatonin labelling studies. On the other hand, melatonin had no effects on the two eel native forms of GnRH (mGnRH and cGnRH-II) mRNA expression. Concerning the pituitary-gonad axis, we showed that melatonin treatment decreased both gonadotrophin beta-subunit (LHbeta, FSHbeta) mRNA expression and reduced sexual steroid (11-ketotestosterone, oestradiol) plasma levels. This indicates that melatonin treatment had a negative effect on eel reproductive function. To our knowledge, the results of the present study provide the first evidence that melatonin enhances TH expression in specific brain regions in a non-mammalian species. By this mechanism melatonin could represent one pathway by which environmental factors could modulate reproductive function in the eel.

Background The melatonin receptor subfamily contains three members Mel1a, Mel1b and Mel1c, found in all vertebrates except for Mel1c which is found only in fish, Xenopus species and the chicken. Another receptor, the melatonin related receptor known as GPR50, found exclusively in mammals and later identified as a member of the melatonin receptor subfamily because of its identity to the three melatonin receptors despite its absence of affinity for melatonin. The aim of this study was to describe the evolutionary relationships between GPR50 and the three other members of the melatonin receptor subfamily. Results Using an in silico approach, we demonstrated that GPR50 is the ortholog of the high affinity Mel1c receptor. It was necessary to also study the synteny of this gene to reach this conclusion because classical mathematical models that estimate orthology and build phylogenetic trees were not sufficient. The receptor has been deeply remodelled through evolution by the mutation of numerous amino acids and by the addition of a long C-terminal tail. These alterations have modified its affinity for melatonin and probably affected its interactions with the other two known melatonin receptors MT1 and MT2 that are encoded by Mel1a and Mel1b genes respectively. Evolutionary studies provided evidence that the GPR50 group evolved under different selective pressure as compared to the orthologous groups Me11 a, b, and c. Conclusion This study demonstrated that there are only three members in the melatonin receptor subfamily with one of them (Me11c) undergoing rapid evolution from fishes and birds to mammals. Further studies are necessary to investigate the physiological roles of this receptor.

In this study we investigated the distribution of melatonergic receptors in the tree shrew brain. The psychosocial stress in the tree shrew is a validated model of depression with disturbance of circadian rhythms.Given the role of melatonin in the modulation of circadian rhythms, we determined by autoradiography the distribution of binding sites of 2-[125I]-MLT, a ligand for the two melatonergic receptors MT1 and MT2, in the tree shrew brain focusing mostly on the suprachiasmatic nucleus (SCN), the biological clock involved in resynchronization of circadian rhythms. We also analyzed the distribution of 2-[125I]-MLT in other brain areas involved in regulation of mood.Specific binding of 2-[125I]-MLT was found in the SCN. In addition, several structures in the tree shrew brain were labeled, among them the pars tuberalis, the cerebellum, structures of the hippocampal formation and dopaminergic areas such as the caudate putamen and nucleus accumbens, providing other potential targets of psychosocial stress in the tree shrew, in addition to the SCN.The demonstration of melatonergic receptors in these brain areas supports their probable involvement in the behavioural, neuroendocrine and circadian rhythms disturbances observed in the psychosocial stress model of depression in the tree shrew.

In the ewe, seasonal anestrus results from an increased responsiveness of the hypothalamus to the negative feedback of estradiol (E2) on the gonadotropic axis under long-day conditions. However, this seasonal effect could also depend upon variable uptake of steroids by the brain. The aim of the present experiment was to compare the concentration of E2 in the blood plasma and in the cerebrospinal fluid (CSF) from the third ventricle in groups of ovariectomized, estradiol treated ewes maintained under short day (SD) or long day (LD) conditions and to study the involvement of the pineal gland in this photoperiodic regulation. Pinealectomized and sham-operated ewes were equipped with an intracerebral cannula to sample the CSF. The plasma E2 concentrations showed no difference between LD and SD in sham-operated and pinealectomized animals. In contrast, in the CSF, E2 concentration was higher in the LD than the SD group, and pinealectomy suppressed this effect of photoperiod. Concomitantly, the stimulatory effect of SD on luteinizing hormone levels observed in sham-operated ewes was abolished by pinealectomy. The results demonstrate that LD increases the E2 concentration in the CSF by a mechanism involving the pineal gland.

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This chapter provides an overview of the physiological processes that allow mammals to breed at the optimal time of year given the annual changes in environmental factors of their habitat. Reproductive activity in many mammalian populations shows dramatic seasonal variations with certain species oscillating between periods of reproductive activity and quiescence. The time of the year when animals are reproductively active varies from species to species and depends mainly on the length of gestation. In some mammals, in addition to timing fertilization, environmental cues also time the implantation of the embryo after an obligatory diapause of the blastocysts such as European badger, mink, and roe deer. Photoperiod is the main environmental parameter that controls reproduction and photoperiodic species are divided into two main categories depending on how photoperiod controls the annual rhythms and on the endogenous nature of the annual changes. Circannual species display endogenously generated rhythms, and photoperiod synchronizes these internally generated rhythms (referred to as type II rhythms). Noncircannual species do not display endogenous rhythms and are at least partially dependent on the environment for the generation of the seasonal cycle.

In long-day breeders like horses, the length of nocturnal melatonin secretion is the main messenger of photoperiod. Previous studies have shown that the nocturnal jugular melatonin concentration is lower in horses, than in mules but is unknown in donkeys. The aim of this study was to estimate the inter-animal variability of plasma melatonin concentration in domestic mares and to compare this concentration with those observed in domestic jennies and in their hybrid mules. In the autumn, blood samples were collected at 22 h, 23 h, 0 h and 1 h during 2 nights at 3 weeks intervals, in 110 pony mares, 10 jennies and 6 mules maintained under natural photoperiod. Melatonin was assayed by a validated RIAmethod. The statistical analysis of themeasures was done with a specific unbalanced analysis of variance model. The effect of species and individuals (nested under species) was highly significant. The mean melatonin concentration was 24 pg.mL−1 in mares and was significantly lower than in jennies and in mules which were 90 pg.mL−1 and 169 pg.mL−1 respectively. The melatonin plasma concentration was higher in jennies than in mares. These results suggest that the melatonin concentration is genetically determined.

A study was performed to determine if complete separation between sexes is a pre-requisite to stimulate sexual activity of anovulatory female goats by male exposure in mid-anestrus when reproductive activity is greatly inhibited. The response to the male was studied in anovulatory goats which had either been in contact with bucks before the stimulation (n = 22; CONT), or totally isolated from bucks (n = 22; ISOL). At the time of male exposure, the bucks from the CONT groups were removed and females of both groups (CONT and ISOL) were divided into two subgroups (n = 11) exposed to novel bucks (n = 1 per subgroup), either in sexual rest (sexually inactive; SI) or in which sexual activity had been induced by a photoperiodic treatment (sexually active; SA). The sexual behavior of the SA bucks during the first 5 days of stimulation was greater than that of SI bucks (P < 0.01). Regardless of whether females had been (82%) or not (91%) exposed to bucks before teasing, most goats stimulated by SA males displayed estrous behavior and ovulation in the first 15 days following stimulation, whereas no female from the two subgroups exposed to SI bucks showed estrous behavior and only one of them ovulated (P < 0.02). These results indicate that previous isolation of seasonally anovulatory goats from bucks is not required to stimulate their reproductive activity by the male effect, if sexually active bucks are used.

This study compares morphological changes, performed with real-time transrectal ultrasonography, of testis and accessory glands and seasonal endocrine changes in a wild (Mouflon) and a domesticated (Spanish Merino) breed of sheep. In Mouflons, the maximum plasma testosterone concentrations, testicular diameter and vesicular gland size occurred synchronously during autumn (P < 0.001). In Merino rams the highest circulating levels of testosterone (P < 0.05) and maximum testicular diameter (P < 0.001) occurred during summer, with no seasonal variations in vesicular and bulbourethral glands. The seasonal changes in plasma concentrations of prolactin were not correlated with annual variations in testicular and glandular size in neither wild nor domestic species. No differences were observed between both species for the seasonal pattern of prolactin secretion and mean amplitude of melatonin. Wide differences in reproductive patterns between wild and domestic types of rams do not appear to be attributed to seasonal changes in prolactin and melatonin secretion. © 2005 Elsevier Inc. All rights reserved.

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.

International audience; The aim of this study was to determine whether the reproductive seasonality of local male goats from subtropical Mexico (26° N) is controlled by photoperiod. The control group (n = 7) remained in an open shed under natural daylight. The two experimental groups (n = 6 each) were placed in light-proof buildings and exposed for 2 years (yr) to alternations of 3 months (mo) of long days and 3 mo of short days. One group was first exposed to long days and the other one to short days. Body and testicular weights were determined every 2 wk. Blood samples were obtained weekly to determine testosterone plasma concentrations. In the control group, the body weight exhibited variations (P < 0.0001) and it increased during the non-breeding season. In both treated groups, long days stimulated weight gain and short days inhibited it (P < 0.0001). In the control group, testicular weight displayed variations (P < 0.0001), and high values were registered in June. In the treated groups, a testicular weight reduction occurred 6-9 mo after the onset of the study. Afterwards, the changes in testicular size varied according to daylength (P < 0.01). The pattern of plasma testosterone concentration in the control group varied over the study (P < 0.0001) and the levels were higher from May-June to November. In both treated groups, the changes in testosterone secretion occurred according to photoperiod changes (P < 0.0001). Short days enhanced testosterone secretion one photoperiodic cycle after the onset of the study and long days inhibited it. Local male goats from subtropical Mexico are sensitive to photoperiodic changes and this environmental cue may control the timing of the breeding season in natural conditions.

The Natal mole-rat, Cryptomys hottentotus natalensis, rarely, if ever, is exposed to external light cues because it occurs in completely sealed tunnel systems. As a result, their classical visual system is regressed, and therefore, their circadian system is expected proportionally to be expanded. Locomotor activity was investigated under a number of different photic regimes. Nine of the 12 mole-rats exhibited endogenous circadian rhythms of locomotor activity under constant darkness, with a mean free run period of 24.13 h (range 23.93-24.13 h), with these animals entrained to a light-dark cycle (12 L:12 D). Because C. hottentotus natalensis are able to entrain their locomotor activity to an external light source, light must reach the suprachiasmatic nucleus (SCN), suggesting a functional circadian clock. A clear day-night rhythm of melatonin secretion in animals housed under a neutral photoperiod (12 L:12 D) was observed, with higher melatonin concentrations in the dark compared with the light phase. The rhythm was maintained after the animals were transferred to either continuous light (LL) or dark (DD), suggesting that the endogenous rhythm was maintained under acute exposure to light and dark. However, under DD, the rhythm appeared to shift slightly, potentially as a result of the rhythm free running. These results show that C. hottentotus natalensis has endogenous rhythms of both locomotor activity and melatonin secretion, which are modulated by light.

The hormone melatonin is synthesised and secreted from the pineal gland in darkness and triggers the daily and seasonal timing of various physiological and behavioural processes. The Mashona mole-rat, Cryptomys darlingi, lives in subterranean burrows that are completely sealed and is therefore rarely, if ever, exposed to light under natural conditions. Hence, this species is of particular interest for studies on rhythms of melatonin secretion. We investigated how plasma melatonin concentrations of the Mashona mole-rat responded to exposure to a long-term standard photoperiod of 12 h light, 12 h dark (12:12 LD), constant light (LL) and constant dark (DD). In addition, we examined whether plasma melatonin concentration was coupled to locomotor activity. Mashona mole-rats displayed rhythms of plasma melatonin concentration that appeared entrained to the standard LD photoperiod, suggesting that the mole-rat is capable of perceiving and entraining to this photic zeitgeber. Furthermore, under chronic constant lighting conditions (DD, LL), circadian rhythms in plasma melatonin concentration were observed, suggesting the possible existence of an endogenous rhythm. Light suppressed melatonin secretion, but constant light did not abolish the rhythm of plasma melatonin concentration. Between active and non-active animals, no difference in plasma melatonin concentration was found for any of the sequential photoperiods (LD1 DD, LD2, LL), tentatively suggesting that the rhythm of melatonin secretion is uncoupled from that of locomotor activity.

Reproductive seasonality is observed in some breeds originating from or adapted to subtropical latitudes. In ‘photoperiodic flexible breeds’, such as Australian cashmere goats, the annual breeding season can be manipulated through nutrition, whereas in ‘photoperiodic rigid breeds’, such as Creole goats from subtropical Mexico, sexual activity can be controlled by altering the photoperiod. In males from the latter breed, artificial long days, whether or not accompanied by the administration of melatonin, stimulate sexual activity during the non-breeding season. These treated males are able to induce the sexual activity of anoestrous females through the male effect under intensive or extensive conditions. Photoperiodic treatments and the male effect can be easily integrated into different breeding management systems in subtropical latitudes.

Este estudio se efectuó para determinar si la separación previa de los dos sexos es necesaria para estimular la actividad sexual de las cabras anéstricas mediante el efecto macho. Se utilizaron 50 hembras Criollas del subtrópico mexicano (26°23’ N y 104°47’ O), que estuvieron en contacto permanente con dos machos cabríos desde diciembre. El 15 de marzo se formaron dos grupos (n = 25 cada uno, un macho por grupo). El 16 de marzo los dos machos fueron retirados e inmediatamente un grupo de cabras fue puesto en contacto con otros tres machos en reposo sexual, mientras que el otro fue expuesto a tres machos tratados con 2.5 meses de días largos para estimular su actividad sexual. Las frecuencias de automarcajes con orina, olfateos ano-genitales, aproximaciones, intentos de montas y montas de los machos testigo durante los primeros cinco días después de su introducción, fueron inferiores que las de los tratados (P 0.05). En los primeros diez días de estimulación, más de 95% de las hembras en contacto con los machos testigo y tratados, mostraron al menos un estro (P 0.05). Estos resultados indican que la separación previa de los dos sexos no es necesaria para estimular la actividad sexual de las cabras sometidas al efecto macho, al menos cuando el anestro es moderado.

The variations of the pharmacological properties of melatonin receptors between different mammalian species in transfected cell lines have been poorly investigated. In the present study, melatonin analogues have been used to characterize the pharmacology of the recombinant ovine melatonin receptor (oMT1) expressed in CHO cell lines and the native oMT1 from the pars tuberalis (PT). Studies with selective ligands on native and transfected oMT1 showed similar properties for binding affinities [r2(PT/CHO) = 0.85]. The affinities and the functional activities of these ligands were compared with the human receptors (hMT1 or hMT2) expressed in CHO cells as well. The oMT1 and hMT1 receptors had similar pharmacological profiles (r2=0.82). Nevertheless, some of the selective compounds at the human receptor presented a reduced affinity at the ovine receptor. Furthermore, some compounds showed marked different functional activities at oMT1 vs. hMT1 receptors. Our findings demonstrated differences in the pharmacological properties of melatonin receptors in ovine and human species.

We investigated if absolute (nocturnal) or relative (nocturnal/diurnal ratio) plasma melatonin concentrations were associated with the seasonal ovulatory activity in Ile-de-France ewes. Ninety-six and 121 ewes in two different groups of the same flock were used to determine the potential existence of a relationship between melatonin concentrations at the summer and winter sol- stices, and the dates of onset and offset of the ovulatory activity, respectively. The dates of the first and last ovulations were estimated by assaying progesterone in plasma samples taken once weekly. Mean ± SEM (1) plasma melatonin concentrations at the summer solstice and at the winter solstice were 302.4 ± 19.4 and 412.0 ± 18.7 pg·mL -1 , respectively, (2) date of onset of the breeding season 29 Jul. ± 1.6 days, (3) date of offset of the breeding season 24 Jan. ± 2.2 days. In spite of a large vari- ability in the different traits studied here, the analyses of correlation and regression indicated that neither the absolute nor relative melatonin concentrations were significantly related with the dates of onset or offset of ovulatory activity. Therefore, we concluded that absolute or relative plasma melatonin concentrations are not linked to the seasonal breeding activity, in Ile-de-France ewes. sheep / melatonin / reproduction / seasonality / genetics

International audience; Seasonal changes in ovulatory activity, assessed by the measurement of plasma progesterone and plasma prolactin concentrations were monitored in 10 Spanish ibex females captured in the National Wildlife Reserve of Sierra Nevada (South Spain, 37° N). Five of the 10 female ibex showed ovulatory activity with a mean ($\pm$ s.e.m.) duration of the oestrous cycle of 19.4 $\pm$ 1 days (range: 17-23 days). On average, the five cyclic females weighed 28 $\pm$ 0.9 kg. Progesterone cycles occurred only in animals older than 4 years of age. Ovulatory activity extended from December to January. The duration of the breeding season was 43.2 $\pm$ 7.7 days. Ibex females younger than 4 years of age had a body weight lower ($P < 0.01$) than that of adults and none of them displayed ovulations. Plasma concentrations of prolactin levels were significantly affected by season ($P < 0.05$), following a trend that was roughly parallel to daylength. The highest values occurred in the spring (119.7 $\pm$ 21.4 ng$\cdot$mL$^{-1}$) and the summer (139.3 $\pm$ 19.8 ng$\cdot$mL$^{-1}$), the lowest values in the autumn (26 $\pm$ 7.4ng$\cdot$mL$^{-1}$) and in the winter (19.7 $\pm$ 3.2 ng$\cdot$mL$^{-1}$). Our results showed a very restricted breeding season, despite the fact that the Spanish ibex originates from and lives in a temperate latitude, revealing a remarkably good adaptation to the harsh climatic and nutritional conditions of their mountainous habitat.

En los caprinos locales del norte de México, en particular los de la Comarca Lagunera (26°N), existe una estacionalidad reproductiva. En los machos el periodo de reposo sexual ocurre de enero a abril, mientras que en las hembras, el periodo de anestro sucede de marzo a agosto. En ambos sexos, esta estacionalidad es provocada por las variaciones de la duración del día. Los días cortos estimulan la actividad sexual y los días largos la inhiben. Asimismo, la exposición a 2.5 meses de días largos por sí sola o seguida por la aplicación de dos implantes subcutáneos de melatonina, estimulan la actividad sexual de los machos de febrero a abril. En las hembras, el efecto macho es capaz de inducir el estro y la ovulación durante el anestro estacional, solamente si se utilizan machos inducidos a una intensa actividad sexual mediante un tratamiento fotoperiódico. Los machos tratados con días largos, seguidos o no de la aplicación de melatonina, inducen la actividad sexual de 100% de las hembras en anestro, mientras que los machos testigos, en reposo sexual, inducen solamente alrededor de 10% de éstas. Estos resultados indican que la respuesta de las cabras al efecto macho depende de la intensidad de la actividad sexual de los machos.