Differential effect of melatonin on ram spermatozoa depending on the allelic variant of the RsaI polymorphism of the MTR1A gene, incubation medium and season.

Context The Rsa I polymorphism of the melatonin receptor MTNR1A gene affects seasonal reproduction in sheep, but its effect on ram spermatozoa and their response to melatonin is unknown. Aims This study aims to evaluate whether Rsa I polymorphism of the MTNR1A gene influences the response of ram spermatozoa to in vitro added melatonin. Methods Spermatozoa from rams carrying different Rsa I allelic variants were incubated with melatonin in a TALP medium or a capacitation-triggering medium during the reproductive and non-reproductive seasons. After incubation, sperm motility, membrane integrity, mitochondria activity, oxidative damage, apoptotic markers and capacitation status were assessed. Key results In the reproductive season, the T/T genotype was related to some adverse effects of melatonin when spermatozoa were incubated in TALP medium, whereas the C/C genotype was linked with adverse effects when the hormone was added in a capacitation-triggering medium. The decapacitating effect of melatonin on spermatozoa was also different depending on genotype. Conclusions The melatonin effect on spermatozoa from rams carrying different Rsa I genotypes differed depending on the season and the medium. Implications The knowledge of the Rsa I allelic variant of the MTNR1A gene of rams could be helpful when carrying out in vitro reproductive techniques in the ovine species.

Hair from sexually active bucks strongly activates olfactory sensory inputs but fails to trigger early first ovulation in prepubescent does.

Early exposure of does to sexually active bucks triggers early puberty onset correlating with neuroendocrine changes. However, the sensory pathways that are stimulated by the male are still unknown. Here, we assessed whether responses to olfactory stimuli are modulated by social experience (exposure to males or not) and/or endocrine status (prepubescent or pubescent). We used a calcium imaging approach on goat sensory cells from the main olfactory epithelium (MOE) and the vomeronasal organ (VNO). For both cell types, we observed robust responses to active male hair in females under three physiological conditions: prepubescent females isolated from males (ISOL PrePub), pubescent females exposed to males (INT Pub) and isolated females (ISOL Pub). Response analysis showed overall greater proportion of responses to buck hair in ISOL PrePub. We hypothesized that females would be more responsive to active buck hair during the prepubertal period, with numerous responses perhaps originating from immature neurons. We also observed a greater proportion of mature olfactory neurons in the MOE and VNO of INT Pub females suggesting that male exposure can induce plastic changes on olfactory cell function and organization. To determine whether stimulation by male odor can advance puberty, we exposed prepubescent does to active buck hair (ODOR). In both ODOR and females isolated from males (ISOL) groups, puberty was reached one month after females exposed to intact bucks (INT), suggesting that olfactory stimulation is not sufficient to trigger puberty.

Male-induced early puberty correlates with the maturation of arcuate nucleus kisspeptin neurons in does.

In goats, early exposure of spring-born females to sexually active bucks induces an early puberty onset assessed by the first ovulation. This effect is found when females are continuously exposed well before the male breeding season starting in September. The first aim of this study was to evaluate whether a shortened exposure of females to males could also lead to early puberty. We assessed the onset of puberty in Alpine does isolated from bucks (ISOL), exposed to wethers (CAS), exposed to intact bucks from the end of June (INT1), or mid-August (INT2). Intact bucks became sexually active in mid-September. At the beginning of October, 100% of INT1 and 90% of INT2 exposed does ovulated, in contrast to the ISOL (0%) and CAS (20%) groups. This demonstrated that contact with males that become sexually active is the main factor prompting precocious puberty in females. Furthermore, a reduced male exposure during a short window before the breeding season is sufficient to induce this phenomenon. The second aim was to investigate the neuroendocrine changes induced by male exposure. We found a significant increase in kisspeptin immunoreactivity (fiber density and number of cell bodies) in the caudal part of the arcuate nucleus of INT1 and INT2 exposed females. Thus, our results suggest that sensory stimuli from sexually active bucks (e.g., chemosignals) may trigger an early maturation of the ARC kisspeptin neuronal network leading to gonadotropin-releasing hormone secretion and first ovulation.

Improvement of the Seminal Characteristics in Rams Using Agri-Food By-Products Rich in Phytomelatonin.

The aim of this study was to evaluate the effect of a phytomelatonin-rich diet, including by-products from the food industry, on ram sperm quality and seminal plasma composition. Melatonin content in several by-products before and after in vitro ruminal and abomasal digestion was determined by HPLC-ESI-MS/MS. Finally, 20% of a mix of grape pulp with pomegranate and tomato pomaces was included in the rams' diet, constituting the phytomelatonin-rich diet. Feeding the rams with this diet resulted in an increase in seminal plasma melatonin levels compared with the control group (commercial diet) in the third month of the study. In addition, percentages higher than those in the control group of morphologically normal viable spermatozoa with a low content of reactive oxygen species were observed from the second month onwards. However, the antioxidant effect does not seem to be exerted through the modulation of the antioxidant enzymes since the analysis of the activities of catalase, glutathione reductase and glutathione peroxidase in seminal plasma revealed no significant differences between the two experimental groups. In conclusion, this study reveals, for the first time, that a phytomelatonin-rich diet can improve seminal characteristics in rams.

Efficiency of Artificial Insemination at Natural Estrus in Organic Churra Ewes.

Hormonal treatments used in the artificial insemination (AI) of sheep can cause several physiological problems that can affect negatively fertility and animal health; however, AI protocols based on the detection of natural estrus offer a more sustainable option and can achieve high fertility. In this study, an AI protocol at natural estrus in organic Churra sheep was performed. In the first phase (AI protocol development), 125 ewes were exocervically inseminated, and their fertility was assessed based on the following factors: number of AI, physiological state, body condition, estrus detection-AI interval, and vaginal fluids in cervix. That protocol was repeated for six consecutive years. In all individuals, fertilities based on the timing of insemination after estrus detection were very high. Lactating ewes produced better results than did dry ewes, which was probably because of the better feeding of the former. In addition, double insemination increased the fertility of ewes whose estrus was detected within 16 h of onset. Body condition and amount of vaginal fluid were correlated with fertility. Exocervical inseminations at natural estrus can produce acceptable fertility and prolificity in Churra ewes.

Influence of Mediterranean climate and lunar calendar on milk production in Lacaune breed ewes.

Environmental factors affect daily milk production in dairy animals. The aim of this study was to quantify the effects of environmental factors, specifically mean temperature (°C), relative humidity (%), temperature-humidity index (THI), solar radiation (°), pluvial precipitation (mm) and lunar calendar (full moon, waxing quarter, waning quarter, new moon), on milk production (kg/d). The analysis was based on 96,195 morning and evening milking records documented on 109 consecutive days, from 869 Lacaune ewes. Ewes were housed in groups of 174 individuals. The analysis was performed in two independent procedures, a Pearson correlation analysis and a multivariate analysis of the ewe's interrelationships, which was based on the total variance estimate and a Varimax-rotated factorial analysis. Milk yield (kg/d) was significantly (p < 0.05) negatively correlated with mean temperature (-0.24), relative humidity (-0.16), THI (-0.24), and radiation (-0.18), which suggests that the higher these environmental factors, the lower the milk yield. Lunar calendar had a significant (p < 0.01) effect on milk production yield; specifically, yields were higher on the full moon and new moon (2.25 ± 0.05 kg/day) than they were on the crescent or waning moon (2.17 ± 0.05 kg/day). In conclusion, ewes that had been exposed to higher mean temperature, relative humidity, THI and solar radiation had the lowest milk yield, and milk yields are highest on full and new moons. The results of this work may be helpful in making predictions for milk production in Lacaune ewes in the Mediterranean region throughout the year.

Milk Production of Lacaune Sheep with Different Degrees of Crossing with Manchega Sheep in a Commercial Flock in Spain.

The objective of the present study was to evaluate the effect of the grade of crossbreeding (Lacaune x Manchega) and environmental factors on milk production in a commercial flock in Spain. A total of 5769 milk production records of sheep with different degrees of purity of the Lacaune breed crossed with Manchega were used as follows: 100% Lacaune (n = 2960), 7/8 Lacaune (n = 502), 13/16 Lacaune (n = 306), 3/4 (n = 1288), 5/8 Lacaune (n = 441) and 1/2 Lacaune: Manchega (n = 272). Additional available information included the number of parity (1 to 8), litter size (single or multiple), and the season of the year of lambing (spring, summer, autumn and winter). A mixed model was used to evaluate the level of crossbreeding and environmental factors on milk production. The 100% Lacaune sheep presented the highest milk production with respect to the F1 Lacaune x Manchega sheep (p < 0.01), showing that as the degree of gene absorption increases with the Manchega breed, it presents lower milk yield. The 100%, 13/16, and 3/4 Lacaune genotypes had the highest milk yields with respect to the 1/2 Lacaune/Manchega breed (p < 0.001). The Lacaune registered on average 181.1 L in a period adjusted to 160 days of lactation (1.13 L/ day). Likewise, the parity number, litter size, and season of lambing effects showed significant differences (p < 0.01). It was concluded that 13/16 and 3/4 Lacaune/Manchega ewes presented the highest milk yields with respect to the other crosses.

Light-induced sexually active rams provoke LH preovulatory surges and enhances LH concentrations in ewes after progestagen treatment.

The effect of the introduction of sexually active rams in spring on LH secretion in ewes after progestagen treatment has been studied. Two rams were induced into a sexually active state by exposure to 2 months of long days (16 h of light/d) from 1 January (SAR), and another 2 rams were exposed to the natural photoperiod, so that they were not sexually activated in spring (control; C). At the end of the long-day period, rams were returned to natural photoperiod conditions. Fifteen ewes synchronized in estrus by intravaginal sponges were assigned to three groups at sponge withdrawal (hour 0): SAR (n = 5), exposed to SAR rams; C (n = 5), exposed to C rams, and ISO (n = 5), kept isolated from rams. Twenty-four hours after pessary removal (hour 0), rams were introduced into the SAR and C groups. Three SAR ewes presented preovulatory LH surges; the proportion was significantly (P < 0.05) higher in this group (3/5) than in the others (C: 0/5, ISO: 0/5). SAR introduction induced a more marked (P < 0.05) increase in mean LH plasma concentrations (before rams: 1.34 ± 0.19; after rams: 6.94 ± 2.66 ng/ml) than C (before: 0.96 ± 0.29; after: 3.60 ± 1.44) or ISO (before: 1.26 ± 0.42; after: 2.14 ± 1.36) groups, and significantly (P < 0.05) higher plasma LH levels after ram introduction. In conclusion, only light-treated sexually activated rams induced LH preovulatory surges in ewes in the seasonal anestrus, when ewes are synchronized with progestagen treatment in the absence of eCG.

Exposure to Photoperiod-Melatonin-Induced, Sexually-Activated Rams after Weaning Advances the Resumption of Sexual Activity in Post-Partum Mediterranean Ewes Lambing in January.

This study was aimed to determine whether the presence of sexually stimulated rams by photoperiodic and melatonin treatments can advance the resumption of post-partum sexual activity in Mediterranean ewes lambing in January and weaned at the end of the breeding season at 41°N, in March. Rams were exposed to two months of long days (16 h light/day) and given three melatonin implants at the end of the long days (sexually-activated rams; SAR). Control rams (CR) were exposed to the natural photoperiod. Thirty-six ewes weaned on 25 February were assigned to one of two groups. From 1 March to 30 June, one group was housed with four SAR males (SAR-treated; n = 18), and the other group (CR-treated; n = 18) was housed with four unstimulated rams. Ovulation was assessed once per week based on plasma progesterone concentrations. Estrus was monitored daily by marks left on ewes by rams' harnesses. SAR-treated ewes had a shorter (p < 0.01) weaning-first estrus interval than CR-treated ewes (61 ± 17 days vs. 102 ± 47 days; mean date of first estrus after weaning on April 26 and June 6, respectively). The proportion of the ewes ovulating in April or May was higher (p < 0.05) in the SAR-treated group than in the CR-treated group. SAR-treated ewes resumed estrous activity sooner than CR-treated ewes such that, in April, May, and June, the proportion of females that exhibited estrus was higher (p < 0.01) in the SAR-treated group (72%, 89%, and 100%, respectively) than in the CR-treated group (17%, 44%, and 61%, respectively). In conclusion, the introduction at weaning of sexually activated rams advanced the resumption of estrous activity in ewes in spring. The practical implications of this work could be important in ewes adapted for intensive production and accelerated lambing systems.

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.