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Abstract 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.

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.

Context Several polymorphisms in the melatonin receptor 1A gene (MTNR1A ) have been related to reproductive performance in ovine. Aims To investigate the effect of the Rsa I and Mnl I polymorphisms on ram seminal quality. Methods Eighteen Rasa Aragonesa rams were genotyped for the Rsa I (C/C, C/T, T/T) and Mnl I (G/G, G/A, A/A) allelic variants of the MTNR1A gene. Individual ejaculates were analysed once a month throughout the whole year. Sperm motility, morphology, membrane integrity, levels of reactive oxygen species (ROS), phosphatidylserine (PS) inversion, DNA fragmentation and capacitation status were assessed. The effect of the season and polymorphisms on seminal quality was evaluated by mixed ANOVA. Key results Both polymorphisms had an effect on membrane integrity and viable spermatozoa with low levels of ROS and without PS translocation, and Rsa I also on motile and DNA-intact spermatozoa. An interaction between both polymorphisms was found, pointing to a negative effect on seminal quality of carrying the T or A allele in homozygosity. Differences were higher in the reproductive than in the non-reproductive season. Conclusions Mutations substituting C by T and G by A at Rsa I and Mnl I polymorphic sites, respectively, in the MTNR1A gene in rams could decrease the seminal quality. Implications Genotyping of rams based on melatonin receptor 1A could be a powerful tool in sire selection.

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.

Sperm orientation mechanisms, such as chemotaxis, are essential for the sperm to reach the oocyte and fertilize it. Melatonin is secreted by the cumulus cells and is also present in the follicular fluid in mammals. The presence of membrane receptors for melatonin in ram spermatozoa, and its proven involvement in the sperm functionality, may suggest a possible role in the guided movement towards the oocyte. Hence, the objective of the present work is to study the in vitro potential chemotactic action of melatonin on ram spermatozoa, analysing the influence of the season (breeding and non-breeding) and the sperm capacitation state. The first experimental approach consisted in the inclusion of melatonin in the upper layer of a swim-up selection method. During the non-breeding season, the presence of melatonin at 100 pM and 1 μM concentrations significantly increased the cell recovery rate, and induced changes in the sperm location of the MT 2 melatonin receptor, compared with the standard swim-up. Moreover, the selected sperm population with 100 pM melatonin presented a higher percentage of capacitated spermatozoa. The greater recovery rate obtained with melatonin could be due to the stimulation of sperm movement in random directions, i.e., a chemokinetic effect, or due to a guided movement (chemotaxis) towards the gradient of the melatonin. To elucidate this issue, together with the study of the influence of the sperm capacitation status, we performed a second experimental approach which consisted in the use of chemotaxis chambers and an open-source software (Open-CASA) that analyses the sperm trajectories towards the hormone gradient and calculates a chemotaxis index (SL index). There was a significant difference between the SL index in the presence of 1 μM melatonin and the control without hormone. This effect was only observed in capacitated spermatozoa with cAMP-elevating agents (Cap-CK samples) obtained during the non-breeding season. These results would point to an in vitro chemotactic effect of melatonin on ram spermatozoa, although chemokinesis cannot be ruled out. Nonetheless, the inclusion of this hormone in the swim-up procedure could enhance the sperm recovery rate., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Context: Apart from the canonical cAMP-PKA pathway, ram sperm capacitation can be achieved by the MAPK ERK1/2 signalling cascade, activated by epidermal growth factor (EGF)., Aims: This study aims to investigate the effect of melatonin and nitric oxide (NO·) on capacitation and apoptotic-like changes in EGF-capacitated ram spermatozoa., Methods: In vitro capacitation was induced by EGF in the absence or presence of melatonin (100pM or 1μM). Also, a NO· precursor, L-arginine, or a NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), were added to capacitation media to study the interaction of NO· and melatonin during EGF-capacitation. Sperm functionality parameters (motility, viability, capacitation state), apoptotic markers (caspase activation and DNA damage), NO· levels, and phosphorylated c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (assessed by Western blot), were evaluated in swim-up and capacitated samples with EGF., Key Results: NO· levels and the apoptotic-related markers were raised after EGF incubation. Melatonin had a bimodal role on sperm EGF-capacitation, preventing it at high concentration and promoting acrosome reaction at low concentration, but neither of the two concentrations prevented the increase in apoptotic-like markers or NO· levels. However, melatonin at 1μM prevented the activation of JNK., Conclusions: NO· metabolism does not seem to modulate the apoptosis-like events in ram spermatozoa. Melatonin at 1μM prevents ram sperm capacitation induced by EGF independently from nitric oxide metabolism, and it could be exerted by limiting the JNK mitogen-activated protein kinase (MAPK) activation., Implications: This study improvesour understanding of the biochemical mechanisms involved in sperm capacitation, and ultimately, fertility.

Testicular ultrasound is a non-invasive technique that could be very useful for predicting ram seminal quality. Recent software developments allow macroscopic and microscopic evaluation of testicular parenchyma. Thus, the aim of this study was to evaluate the testicular echotexture using ultrasound-video analysis and investigate its possible correlation with semen quality. Nine rams were evaluated for one year using a portable ultrasound scanner and the echotexture was analyzed with ECOTEXT ® software. The number of black (Ec1), white (Ec2), and grey pixels (Ec3), tubular density (TD), lumen area (LA), and lumen diameter (LD) were analyzed. Semen was collected by an artificial vagina the same day and the sperm concentration, morphology, motility, viability, phosphatidylserine (PS) translocation, reactive-oxygen-species (ROS) levels, DNA damage and capacitation state were evaluated. Ec2 and Ec3 correlated positively with "bad quality" sperm parameters (the percentage of spermatozoa with high ROS levels, with PS translocation and proximal cytoplasmic droplets), and negatively with motility. In contrast, TD and LA showed a positive correlation with "good quality" parameters (motility or normal morphology) and a negative correlation with spermatozoa with high ROS levels, with DNA fragmentation, and proximal or distal cytoplasmic droplets. Thus, echotexture analysis by ultrasound-video analysis could be a valuable tool for assessing ram fertility.

In this study, we demonstrated that, in seasonal Mediterranean ovine breeds, supplementing the TALP medium with cAMP-elevating agents (the cocktail medium) is effective for achieving ram sperm capacitation, and that melatonin is able to regulate this phenomenon. We investigated the behavior under capacitating conditions using the TALP and cocktail mediums, and the response to melatonin, of spermatozoa from three sheep breeds (Colombian Creole, Romney Marsh, and Hampshire) subject to the equatorial photoperiod, during the dry and the rainy seasons. The cocktail medium was able to induce sperm capacitation, assayed by chlortetracycline staining and phosphotyrosine levels, to a greater extent than TALP, without a higher loss of viability (membrane integrity and viable spermatozoa without phosphatidylserine (PS) translocation). The addition of melatonin at 100 pM or 1 µM in the cocktail medium partially prevented the decrease in viability without PS translocation and the increase in capacitated spermatozoa from all breeds, with no significant effect on phosphotyrosine levels. Differences between breeds and seasons were evidenced. This study shows that melatonin is able to exert direct effects on spermatozoa in ovine breeds under equatorial photoperiod conditions, as it does in seasonal breeds located in temperate regions.

The prediction of the fertilizing ability of a seminal dose continues to be a primary aim in the field of artificial insemination (AI). To achieve this goal, in this study we have included the evaluation of some non-conventional sperm quality markers. A total of 3,906 ewes from 52 different farms were inseminated with 357 refrigerated seminal doses obtained from 45 mature Rasa Aragonesa rams. The same samples were used for sperm quality analysis including membrane integrity, capacitation status, oxygen consumption and apoptotic-like markers such as phosphatidylserine translocation (PS), plasmalemma disorganization/mitochondrial membrane potential, caspase activation and DNA damage. Seminal doses from the breeding (B) season presented higher percentages of intact membrane (IM), non permeant (NP) membrane with high mitochondrial membrane potential (ΔΨm) and IM without PS translocation spermatozoa than those from the non-breeding (NB) season. Therefore, we can conclude that there were less spermatozoa showing apoptotic-like features in the seminal doses from the B than the NB season, although these differences did not affect field fertility. Only the percentage of intact membrane, non-capacitated (IM-NC) spermatozoa showed a significant correlation with in vivo fertility ( P = 0.005) and fecundity ( P = 0.007) values obtained after cervical AI when all data were evaluated. When the data were sorted by season and distance to the farms where AI was performed, the correlation between the percentage of IM-NC spermatozoa and reproductive parameters increased in the NB season and progressively with remoteness from the farms. Some other sperm parameters, like NP with high ΔΨm, IM sperm without active caspases and DNA-intact spermatozoa, also showed significant correlations with the reproductive parameters in the sorted data. Moreover, the increment in both the percentage of IM-NC and DNA-intact spermatozoa would increase the probability of obtaining a fertility higher than the mean (>52%), as revealed by a multiple logistic regression analysis. In conclusion, we have identified two seminal markers-the percentage of intact membrane, non-capacitated spermatozoa, and DNA intact spermatozoa-which could be used as a test to discard males in AI programs, which is highly important from an economic point of view and can contribute to achieving satisfactory fertility rates., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mendoza, Casao, Domingo, Quintín, Laviña, Fantova, Cebrián-Pérez, Muiño-Blanco and Pérez-Pe.)

Reactive oxygen species (ROS) play an essential role in mammalian sperm capacitation. NADPH oxidase 5 (NOX5) has been described as the main source of ROS production in some mammalian spermatozoa, such as human and equine. On the other hand, melatonin can decrease cellular ROS levels and regulates NOX activity in somatic cells. Therefore, the objectives of this work were (1) to identify NOX5 in ram spermatozoa and analyze its possible changes during in vitro capacitation and (2) to investigate the effect of melatonin on NOX5 expression and localization and on superoxide levels in capacitated ram spermatozoa. Protein bands associated with NOX5 were detected by Western blot analysis. Likewise, indirect immunofluorescence (IIF) revealed six different immunotypes for NOX5, which varied throughout in vitro capacitation. Superoxide (O 2 ⋅- ), evaluated by DHE/Yo-Pro-1, rose after in vitro capacitation and in the presence of the calcium ionophore A23187 but decreased in the presence of the NOX inhibitor GKT136901. GKT also reduced the percentage of capacitated and acrosome-reacted spermatozoa that had increased during incubation in capacitating conditions. The presence of melatonin at micromolar concentrations avoided the increment in O 2 ⋅- and the changes in NOX5 immunotypes provoked by capacitation. In conclusion, NOX5 is present in ram spermatozoa and the changes in its distribution, associated with sperm capacitation, can be prevented by melatonin. To this extent, it could imply that melatonin exerts its antioxidant role, at least in part, by modulating NOX5 activity during ram sperm capacitation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Miguel-Jiménez, Pina-Beltrán, Gimeno-Martos, Carvajal-Serna, Casao and Pérez-Pe.)

This work aimed to determine the contribution of the testis and epididymis and the effect of the photoperiodic regimen on ram seminal plasma (SP). Semen was collected from 15 mature rams located in an equatorial (Colombian Creole and Romney Marsh, eight intact and two vasectomized) or a temperate climate (Rasa Aragonesa, three intact and two vasectomized). SP proteins were analyzed by Bradford, SDS-PAGE and difference gel electrophoresis (DIGE). Melatonin and testosterone concentrations were quantified by ELISA, and activity of glutathione peroxidase (GPx), glutathione reductase (GRD), and catalase by enzymatic assays. Vasectomy increased protein concentration and the intensity of high molecular weight bands ( p < 0.001), with no differences between breeds. DIGE revealed the absence of six proteins in vasectomized rams: angiotensin-converting enzyme, lactotransferrin, phosphoglycerate kinase, sorbitol dehydrogenase, epididymal secretory glutathione peroxidase and epididymal secretory protein E1. Vasectomy also decreased melatonin concentrations in seasonal rams, and testosterone in all of them ( p < 0.001), but did not affect antioxidant enzyme activity. Equatorial rams showed lower melatonin and testosterone concentration ( p < 0.01) and catalase, but higher GPx activity ( p < 0.05). In conclusion, vasectomy modifies the protein profile and hormonal content of ram seminal plasma, whereas the exposure to a constant photoperiod affects hormonal concentration and antioxidant enzymes activity.

Computer assisted sperm analysis (CASA) systems can reduce errors occurring in manual analysis. However, commercial CASA systems are frequently not applicable at the forefront of challenging research endeavors. The development of open source software may offer important solutions for researchers working in related areas. Here, we present an example of this, with the development of three new modules for the OpenCASA software (hosted at Github). The first is the Chemotactic Sperm Accumulation Module , a powerful tool for studying sperm chemotactic behavior, analyzing the sperm accumulation in the direct vicinity of the stimuli. This module was validated by comparing fish sperm accumulation, with or without the influence of an attractant. The analysis clearly indicated cell accumulation in the treatment group, while the distribution of sperm was random in the control group. The second is the Sperm Functionality Module, based on the ability to recognize five sperm subpopulations according to their fluorescence patterns associated with the plasma membrane and acrosomal status. The last module is the Sperm Concentration Module, which expands the utilities of OpenCASA. These last two modules were validated, using bull sperm, by comparing them with visual counting by an observer. A high level of correlation was achieved in almost all the data, and a good agreement between both methods was obtained. With these newly developed modules, OpenCASA is consolidated as a powerful free and open-source tool that allows different aspects of sperm quality to be evaluated, with many potential applications for researchers.

Nitric oxide (NO·), synthesized from L-arginine by nitric oxide synthase (NOS), is involved in sperm functionality. NOS isoforms have been detected in spermatozoa from different species, and an increment in NOS activity during capacitation has been reported. This work aims to determine the presence and localization of NOS isoforms in ram spermatozoa and analyse their possible changes during in vitro capacitation. Likewise, we investigated the effect of melatonin on the expression and localization of NOS and NO· levels in capacitated ram spermatozoa. Western blot analysis revealed protein bands associated with neuronal NOS (nNOS) and epithelial NOS (eNOS) but not with inducible NOS (iNOS). However, the three isoforms were detected by indirect immunofluorescence (IFI), and their immunotypes varied over in vitro capacitation with cAMP-elevating agents. NO· levels (evaluated by DAF-2-DA/PI staining) increased after in vitro capacitation, and the presence of L-arginine in the capacitating medium raised NO· production and enhanced the acrosome reaction. Incubation in capacitating conditions with a high-cAMP medium with melatonin modified the NOS distribution evaluated by IFI, but no differences in Western blotting were observed. Melatonin did not alter NO· levels in capacitating conditions, so we could infer that its role in ram sperm capacitation would not be mediated through NO· metabolism.

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.