Role of melatonin on embryo viability in sheep.

Melatonin is a natural hormone synthesised in the pineal gland, the activity of which is regulated by day-night perception and dictates seasonal rhythms in reproduction in ovine species. Exogenous melatonin, administered via subcutaneous implants, is used to prolong the breeding season of ewes and can increase the proportion of pregnant ewes (fertility rate) and litter size. The increased proportion of ewes that become pregnant and the number of lambs born per lambing among melatonin-treated sheep may be caused by increased embryo survival, through enhanced luteal function, reduced antiluteolytic mechanisms, or improved embryo quality. This review focuses on the effects of melatonin on embryo viability and summarises the processes by which this hormone affects the ovary, follicle, oocyte, corpus luteum and embryo. Moreover, the effects of melatonin on the mechanisms of invivo maternal recognition of pregnancy in sheep and the protective action that it appears to have on the invitro procedures that are used to obtain healthy embryos are reviewed.

Failure to establish and maintain a pregnancy in undernourished recipient ewes is associated with a poor endocrine milieu in the early luteal phase.

Embryos from undernourished and control donor ewes were transferred to undernourished and control recipient ewes. Progesterone and metabolic hormones were investigated in recipient ewes to determine their association with pregnancy success. Forty-five donor and 52 recipient Rasa Aragonesa ewes were fed 1.5 (control group; donor n=20; recipient n=25) or 0.5 (low group; donor n=25; recipient n=27) times the daily requirements for maintenance from the onset of estrous synchronization treatment to embryo collection and transfer. The embryos were collected 7days after the onset of estrus (day 0), and two good-quality embryos were transferred into each recipient ewe. The percentage of pregnant ewes on day 18 and 40 did not differ between the two groups, although the recipient undernourished ewes tended to have greater late embryonic mortality (from days 18-40) than the control recipient ewes (P=0.11). No effect of the nutrition of the donor was found. Recipients that became pregnant had a higher ovulation rate than non-pregnant ewes (P=0.02). Undernourished ewes had lower plasma insulin concentrations than control ewes (P=0.03), and those that suffered late embryo mortality (from days 18-40) tended to have lower insulin and progesterone concentrations than their counterparts that remained pregnant (P=0.06 and P=0.07, respectively). In this study, pregnancy in control and undernourished recipient ewes was not associated with the origin of the embryo (undernourished and control donors). In conclusion, failure to establish and maintain a pregnancy was associated with lower progesterone and insulin levels one week after estrus in recipient ewes.

Improving the environment for weaned piglets using polypropylene fabrics above the animals in cold periods.

This study aimed at evaluating the use of polypropylene fabrics in weaned pig facilities (5-10 weeks of age) during the winter period to improve thermal environment and energy saving for heating. Two experiments were conducted to validate the effects of fabrics (F) compared to control (C) in three 2-week periods using natural ventilation (assay 1, 2013) and forced ventilation (assay 2, 2014). Air temperature was greater in F than in C compartments in both years, particularly during the first 2-week periods (2 °C of mean difference). Natural ventilation was not enough to maintain relative humidity levels below 70 % at the end of the postweaning period (9-10 weeks of age) in both groups (F and C), whereas forced ventilation allowed controlling daily mean relative humidity levels <60 %. About 12-26 % of the radiant heat was transmitted through the fabrics cover, depending on the wavelength. There were no differences on growth performance of piglets in the two compartments in both years. The use of polypropylene fabrics was associated with a significant electric energy saving for heating during the first (data available only in 2014) and second 2-week period in both years. In conclusion, polypropylene fabrics may be an interesting tool to provide optimal environmental conditions for weaned piglets in winter, especially during the two first weeks after weaning. Their transmittance properties allow trapping infrared emission produced by the piglets and heating, avoiding heat losses through the roof, and therefore saving heating energy.

The effect of exogenous melatonin during the non-reproductive season on the seminal plasma hormonal profile and the antioxidant defence system of Rasa Aragonesa rams.

The aim of this study was to analyze the effect of melatonin implants, during the non-reproductive season, on the content of melatonin, testosterone and 17-ß estradiol levels, and the antioxidant enzymes glutathione peroxidase, glutathione reductase, superoxide dismutase and catalase of seminal plasma in Rasa Aragonesa rams. Five rams were implanted with melatonin, and four others were used as a control group. Seminal plasma was separated from ejaculates collected one week before melatonin treatment until 21 weeks after melatonin placement, and the hormonal levels and the antioxidant enzyme activity were determined. Exogenous melatonin treatment significantly (P<0.05) increased the levels of endogenous melatonin in seminal plasma immediately, and this effect lasted for 14 weeks. Testosterone and 17-ß estradiol levels significantly (P<0.05) increased four and eight weeks after melatonin treatment, respectively. As regards the antioxidant enzymes, melatonin treatment significantly increased (P<0.05) glutathione peroxidase and glutathione reductase activity only, and had no effect on superoxide dismutase and catalase. Therefore, melatonin treatment during the non-breeding season modifies the seminal plasma hormonal profile and some antioxidant enzyme activity in Rasa Aragonesa rams.

Identification and immunolocalisation of melatonin MT(1) and MT(2) receptors in Rasa Aragonesa ram spermatozoa.

The reproductive seasonality of sheep suggests that melatonin receptors may be present in ram spermatozoa. The present study confirms the presence of melatonin MT(1) and MT(2) receptors. The MT(1) receptor was detected using immunocytochemistry, with four sperm subpopulations identified based on the following labelling patterns: (1) one small subpopulation with labelling over the entire head and tail; (2) one of two main subpopulations that exhibited reactivity at the equatorial, post-acrosomal, neck and tail regions; (3) another main subpopulation with equatorial and tail labelling only; and (4) a subpopulation in which staining was detected only in the tail. Immunocytochemistry revealed the presence of the melatonin MT(2) receptor, with intense staining on the acrosome, post-acrosomal region and neck and tail regions of all cells, but not in the equatorial region. Western blot identification of ram protein extracts revealed a 39-kDa band compatible with both MT(1) and MT(2) receptors, a 75-kDa band compatible with MT(1)/MT(2) heterodimerisation, a 32-kDa band compatible with MT(1) receptor activation and a double band of 45-55 kDa that is compatible with MT(2) receptor homodimerisation or heterodimerisation with other G-proteins. In conclusion, we provide evidence of the presence of MT(1) and MT(2) receptors in ram spermatozoa, although the biochemical pathway triggered by these receptors and their function in terms of fertility remains to be elucidated.

Pharmaceutical control of reproduction in sheep and goats.

Small ruminant species such as sheep and goats are short-day breeders, which is a crucial factor affecting the offer of lambs and kids throughout the year. An appropriate management of reproduction allows ewes and does to breed in the spring to increase the supply of product to the marketplace on a year-round basis. Pharmaceutical control of reproduction is possible, usually through administration of hormones or analogues related to the natural estrous cycle, such as progesterone, prostaglandins, and/or melatonin.

Seasonal variations of melatonin in ram seminal plasma are correlated to those of testosterone and antioxidant enzymes.

BACKGROUND: Some breeds of sheep are highly seasonal in terms of reproductive capability, and these changes are regulated by photoperiod and melatonin secretion. These changes affect the reproductive performance of rams, impairing semen quality and modifying hormonal profiles. Also, the antioxidant defence systems seem to be modulated by melatonin secretion, and shows seasonal variations. The aim of this study was to investigate the presence of melatonin and testosterone in ram seminal plasma and their variations between the breeding and non-breeding seasons. In addition, we analyzed the possible correlations between these hormones and the antioxidant enzyme defence system activity. METHODS: Seminal plasma from nine Rasa Aragonesa rams were collected for one year, and their levels of melatonin, testosterone, superoxide dismutase (SOD), glutathione reductase (GRD), glutathione peroxidase (GPX) and catalase (CAT) were measured. RESULTS: All samples presented measurable quantities of hormones and antioxidant enzymes. Both hormones showed monthly variations, with a decrease after the winter solstice and a rise after the summer solstice that reached the maximum levels in October-November, and a marked seasonal variation (P < 0.01) with higher levels in the breeding season. The yearly pattern of GRD and catalase was close to that of melatonin, and GRD showed a significant seasonal variation (P < 0.01) with a higher activity during the breeding season. Linear regression analysis between the studied hormones and antioxidant enzymes showed a significant correlation between melatonin and testosterone, GRD, SOD and catalase. CONCLUSIONS: These results show the presence of melatonin and testosterone in ram seminal plasma, and that both hormones have seasonal variations, and support the idea that seasonal variations of fertility in the ram involve interplay between melatonin and the antioxidant defence system.

Melatonin prevents capacitation and apoptotic-like changes of ram spermatozoa and increases fertility rate.

We recently demonstrated the presence of melatonin in ram seminal plasma and differences in its concentration in this fluid between the breeding and nonbreeding season. In this study, we investigate the hypothesis that in vitro treatment with melatonin affects ram sperm quality, and that this is reflected in the in vitro fertilization (IVF) results. Semen from nine rams was collected during the nonreproductive season and treated with 1 mum, 10 nm and 100 pm melatonin. Samples were incubated at 39 degrees C and 5% CO2, and motility, viability, capacitation status and phosphatidylserine (PS) translocation were assessed before and after melatonin addition, either 1 or 3 hr of incubation. Fertility rate of the melatonin-treated samples was determined by means of IVF. Although melatonin failed to affect both sperm kinematic parameters and viability, the exposure of ram spermatozoa to melatonin has a direct effect, decreasing capacitation and PS translocation at 1 mum, and increasing short-term capacitation at 100 pm, which caused an increased oocyte fertilization rate following IVF. Furthermore, cleavage rate of oocytes fertilized with 100 pm melatonin-treated spermatozoa was higher than that with 1 mum melatonin and control samples (P < 0.1). These results prove that melatonin has a direct effect on ram spermatozoa in the nonreproductive season, which can be explained, at least in part, by the melatonin capacity as a reactive oxygen species scavenger and antioxidant. These findings might help to select the optimal experimental conditions for IVF and to improve sperm preservation protocols.

The effect of nutrition on the seasonality of reproduction in ewes.

The beneficial effects of nutrition on reproduction in sheep have been described, particularly on ovulation rate. However, the relationships between nutrition and reproductive seasonality are not well known. This review will deal with the effects of body fat or food intake on sexual and hypothalamic/pituitary activity in sheep, mainly focused on Mediterranean genotypes. Although only severe malnutrition can significantly extend the length of the seasonal anestrous period, the level of fat reserves can play a significant role on reproductive seasonality delaying the onset of seasonal anoestrus, particularly on the Mediterranean environment. The effect of overfeeding on LH secretion has also been reported, specially at short term. Several experimental approaches have elucidated that both high body fat and food intake are able to modify the sensitivity of the hypothalamus to oestradiol negative feedback during seasonal anoestrus, with those effects being associated to a reduced amount of NPY mRNA and to an increase of plasma insulin, glucose and leptin concentrations, particularly in the late scenario. However, the highest receptivity to nutritional stimulation in terms of increasing LH occurs when ewes are subjected to a photoperiodic state of early anoestrus or late breeding season rather than under a photoperiod characteristic of the end of anoestrus or the beginning of the breeding season.

The effect of undernutrition on the establishment of pregnancy in the ewe.

The relationship between nutrition and reproduction in sheep has been the subject of research in several international groups. This review will particularly focus on the effects of undernutrition on the potential causes of reproductive failure including abnormalities of the ovum or the embryo, luteal inadequacy and failure of the supply of progesterone to the uterus, or the mechanisms involved in maternal recognition of pregnancy. The level of nutrition and peripheral progesterone concentrations are inversely related, and increased rates of embryo loss, associated with higher progesterone concentrations in ewes with low levels of nutrition have been reported. Undernutrition may act through changes in the distribution of progesterone in the endometrium. Thus, lower endometrial levels on day 5 of the cycle in ewes fed half of their maintenance requirements have been observed, providing a link between the known role of progesterone in embryo survival by the modulation of uterine function and the higher embryo losses found in undernourished ewes. The evidence of an effect of maternal nutrition on IFNtau secretion from the conceptus and of PGF2alpha production from the uterus is presented. Moreover, undernutrition provokes a reduction in the sensitivity of the endometrium to progesterone that may affect embryo survival. Finally, a state of undernutrition induces changes in the endometrial sensitivity to steroid hormones at early stages of pregnancy that could adversely alter uterine environment to the detriment of embryo survival.

Effect of exogenous melatonin on in vivo and in vitro prostaglandin secretion in Rasa Aragonesa ewes.

The effect of exogenous melatonin on prostaglandin secretion was measured on Rasa Aragonesa ewes. Fourteen ewes received an 18 mg melatonin implant (M+) on 10 April and were compared with 13 control animals (without implants M-). Twenty days later, intravaginal pessaries were inserted in all animals to induce a synchronized oestrus (day 0). On day 14, ewes were injected, i.v., with 0.5 IU oxytocin. Plasma 15-ketodihydro-PGF(2alpha) (PGFM) concentrations were measured to assess uterine secretory responsiveness to oxytocin. After euthanasia, pieces of endometrium were collected to determine progesterone content and PGE(2) and PGF(2alpha) secretion in vitro, in the presence or absence of either 20 microg/ml recombinant ovine interferon-tau (roIFNt) or 1 nmol/l oxytocin in the medium. Endometrial progesterone content was similar in the two treatments (M+: 50.25+/-17.34 ng/mg tissue, M-: 43.08+/-11.21 ng/mg tissue). M+ ewes that responded to oxytocin had significantly higher plasma PGFM concentrations between 10 and 80 min after oxytocin administration, a higher mean PGFM peak (P<0.001), higher plasma PGFM levels after the challenge (P<0.05) and higher plasma progesterone concentrations (P<0.01) than control ewes. In the in vitro experiment, M+ and M- control samples secreted similar amounts of PGE(2). The presence of roIFNtau and oxytocin only stimulated PGE(2) production (P<0.05) in M- tissues. Control M+ tissues secreted higher amounts of PGF(2alpha) (P=0.07) and PGF(2alpha) secretion was significantly (P<0.01) stimulated by roIFNtau. Oxytocin produced this effect only in M- samples (P<0.01). In conclusion, although previous studies have demonstrated a positive effect of melatonin on lamb production, PGF(2alpha) secretion is higher in vitro and the PGE(2):PGF(2alpha) ratio is unfavourable in response to IFNtau, which could affect embryo survival. Whether or not these mechanisms are similar in pregnant ewes remains to be elucidated.

The role of nutrition in the regulation of LH secretion during anestrus by the serotoninergic and dopaminergic systems in Mediterranean ewes treated with melatonin.

The steroid-dependent inhibition of LH secretion involves dopaminergic and serotoninergic systems but it is unclear how the plane of nutrition affects this inhibition during anestrus in melatonin treated ewes. Melatonin implants (18 mg) were inserted (Day 0) into ovariectomized, estradiol treated adult Rasa Aragonesa ewes on a high (H; n = 8) or low energy diet (L; n = 6) which were applied in early anestrus (Day 29-57) and late anestrus (Day 90-104). Cyproheptadine (0.1 mg/ kg), a serotoninergic (SHT2) receptor antagonist, was administered in early and late anestrus (Day 50 and 107) followed by pimozide (0.08 mg/kg), a dopaminergic2 receptor antagonist (Day 57 and 114). The H ewes had significantly higher LH concentrations (P < 0.05) before cyproheptadine treatment in early anestrus. The H and L ewes responded in a similar way to the antagonists in both early and late anestrus, except for L ewes who had a higher LH pulse amplitude after pimozide treatment in both periods (P < 0.05). During early anestrus, cyproheptadine tended to increase (P = 0.06) LH pulse frequency in L ewes and LH concentrations in H ewes. The LH secretion also increased in L ewes after pimozide administration during early anestrus (P < 0.05 for mean LH concentrations and LH pulse frequency and amplitude). However, pimozide dramatically increased LH secretion during late anestrus (Day 114) irrespective of the plane of nutrition (P = 0.06-0.08 for LH pulse frequency and P < 0.05 for LH concentrations and pulse amplitude). In melatonin treated Mediterranean ewes, the plane of nutrition appeared to modify the effect of dopaminergic and serotoninergic systems on the steroid-dependent inhibition of LH secretion throughout anestrus.