Genotyping of rams based on melatonin receptor 1A gene polymorphisms: a tool in sire selection?

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.

MEK signalling pathway is required for hypoblast specification and migration in ovine.

In brief: MEK signalling pathway is required for hypoblast differentiation in mouse embryos, but its role in ungulate embryos remains controversial. This paper demonstrates that MEK is required for hypoblast specification in the inner cell mass of the ovine blastocyst and that it plays a role during the hypoblast migration occurring following blastocyst hatching. Abstract: Early embryo development requires the differentiation of three cell lineages in two differentiation events. The second lineage specification differentiates the inner cell mass into epiblast, which will form the proper fetus, and hypoblast, which together with the trophectoderm will form the extraembryonic membranes and the fetal part of the placenta. MEK signalling pathway is required for hypoblast differentiation in mouse embryos, but its role in ungulate embryos remains controversial. The aim of this work was to analyse the role of MEK signalling on hypoblast specification at the blastocyst stage and on hypoblast migration during post-hatching stages in vitro in the ovine species. Using well-characterized and reliable lineage markers, and different MEK inhibitor concentrations, we demonstrate that MEK signalling pathway is required for hypoblast specification in the inner cell mass of the ovine blastocyst, and that it plays a role during the hypoblast migration occurring following blastocyst hatching. These results show that the role of MEK signalling pathway on hypoblast specification is conserved in phylogenetically distant mammals.

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.

Effect of melatonin and nitric oxide on capacitation and apoptotic changes induced by epidermal growth factor in ram sperm.

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 Analysis as a Predictive Tool of Ram Sperm Quality.

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.

Sperm Behavior and Response to Melatonin under Capacitating Conditions in Three Sheep Breeds Subject to the Equatorial Photoperiod.

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.

Effect of Body Condition Score, Treatment Period and Month of the Previous Lambing on the Reproductive Resumption of Melatonin-Treated Sarda Breed Sheep during Spring.

Stakeholders place great emphasis upon rationalizing the management and rearing techniques which are utilized within sheep farms. The present study aimed to investigate factors which may improve the reproductive performance of melatonin-treated Sardinian sheep via a series of three trials. The first trial (n = 100) investigated the effect of melatonin treatment alongside body condition score (BCS), the second trial (n = 150) investigated the effect of treatment alongside the date of treatment (treatment period) and the third trial (n = 150) investigated the effect of treatment alongside the previous lambing of the ewes. The findings indicated that melatonin is an effective tool for anticipating and improving the reproductive activity of in Sarda breed sheep during the springtime. Furthermore, to obtain optional results, melatonin implantation should be conducted in April, in ewes that have a BCS of >2.5 and that have passed their third month of lactation.

NADPH Oxidase 5 and Melatonin: Involvement in Ram Sperm Capacitation.

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 (O2 ⋅-), 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 O2 ⋅- 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.

Vasectomy and Photoperiodic Regimen Modify the Protein Profile, Hormonal Content and Antioxidant Enzymes Activity of Ram Seminal Plasma.

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.

Melatonin membrane receptors MT1 and MT2 are expressed in ram spermatozoa from non-seasonal breeds.

In mammals, many melatonin biological functions are mediated through its interaction with the membrane receptors MT1 and MT2. We have previously reported their presence in ram spermatozoa from males located in temperate climates, but there is no information on their presence in spermatozoa from rams in areas with an equatorial photoperiod (12L:12D). Thus, we have investigated the existence and cellular distribution of melatonin receptors in spermatozoa from three sheep breeds in Colombia (Colombian Creole, Hampshire, and Romney Marsh) during dry and rainy seasons, using indirect immunofluorescence and western blot. Our results indicated the presence of melatonin receptors in spermatozoa from these rams, and that their distribution differs from that previously found in spermatozoa from rams in temperate climates. Moreover, two new immunotypes of MT2 were identified: type N, with staining only in the neck, and type E with a band of immunofluorescence in the upper part of the post-acrosome and the apical edge. Likewise, differences between breeds and climate seasons were detected for both receptors. However, densitometry analysis of western blot bands only revealed differences between seasons in the Creole rams for MT1 and the Romney Marsh rams for MT2, whereas differences between breeds were only detected for MT2. It could be inferred that melatonin receptors in rams subjected to an equatorial photoperiod might be more closely related to sperm quality than seasonal control. Therefore, the presence of these receptors suggests that melatonin could be a useful tool to increase the fertility of rams located in tropical or equatorial climates.

Does Melatonin Exert Its Effect on Ram Sperm Capacitation Through Nitric Oxide Synthase Regulation?

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.

Presence of melatonin-catabolizing non-specific enzymes myeloperoxidase and indoleamine 2,3-dioxygenase in the ram reproductive tract.

The melatonin catabolism is very complex and not completely understood. Melatonin can be metabolized by free radical interaction, but also pseudo-enzymatically or by enzymatic pathways. We have previously detected the existence of melatonin-synthesizing enzymes and melatonin receptors MT1 and MT2 in the ram reproductive tract; thus, in order to start to elucidate melatonin catabolism in these organs, we have investigated the presence of the melatonin-catabolizing enzymes indoleamine 2,3-dioxygenase (IDO, both IDO1 and IDO2 isoforms) and myeloperoxidase (MPO) in testis, epididymis and accessory glands. Gene expression analyses by real-time PCR showed the presence of MPO, IDO1 and IDO2 in all the organs of the ram reproductive tract and revealed that MPO is the main melatonin-catabolizing enzyme, which is mainly expressed in the testis and the bulbourethral glands (p < .05). These results were further corroborated by immunohistochemical staining, and by Western blot. Likewise, MPO was also evidenced in epididymal and ejaculated spermatozoa by indirect immunofluorescence and Western blot. In conclusion, melatonin-catabolizing enzymes MPO, IDO1 and IDO2 are expressed in the ram reproductive tract, and MPO is the most expressed one, mainly in the testis and the bulbourethral glands. The presented results warrant further studies on the function of these enzymes and their melatonin-metabolizing activity.

Efecto de la adición de antioxidantes en los diluyentes para la preservación de semen ovino / Effect of adding antioxidants in diluents for the preservation of sheep semen / Efeito da adição de antioxidantes nos diluentes para preservação de sêmen ovino

Resumen El objetivo del presente estudio fue evaluar el efecto sobre la calidad espermática de tres substancias antioxidantes (50 µM de trolox/108 espermatozoides, 50 µg de catalasa/ml de eyaculado y cisteamina 5mM) en una base de diluyente comercial Triladyl™, bajo condiciones de refrigeración y congelación/descongelación en semen ovino. Se congeló semen de seis machos adultos en una base del diluyente comercial Triladyl™. El eyaculado de cada macho fue puesto en cuatro diferentes alícuotas: una para control, y a las demás se les adicionó con la base del diluyente 50 µM de trolox/108 espermatozoides, 50 µg de catalasa/ml de eyaculado y cisteamina 5 mM. La calidad espermática precongelación (5 °C) y poscongelación se evaluó con la ayuda de un sistema de análisis computarizado (IVOS II®), con lo cual se eliminó la subjetividad a la prueba. Se observó que 50 µM de trolox/108 SPZ y 50 µg de catalasa/ml tienen la capacidad de mantener valores (p < 0,05) superiores de motilidad total (64,26 ± 0,82 y 55,54 ± 0,85) y motilidad progresiva (47,26 ± 0,75 y 44,32 ± 1,13) en condiciones de precongelación, y para motilidad total (67,60 ± 1,91 y 63,47 ± 3,40), motilidad progresiva (50,87 ± 2,58 y 38,88 ± 2,31) y viabilidad (66,97 ± 2,25 y 61,16 ± 4,31) en poscongelación, comparados con los tratamientos control o la adición de 5 mM de cisteamina. La adición de 50 µM de trolox/108 espermatozoides y de 50 µg de catalasa/ml de semen mejora la motilidad total y progresiva del semen ovino refrigerado a 5 °C y la viabilidad en semen congelado/descongelado.

Abstract The present study aimed to evaluate the effect on sperm quality of three antioxidant substances (50 μM of trolox/108 spermatozoa, 50 μg of catalase/ml of ejaculate, and 5mM cysteamine) in a Triladyl™ commercial diluent base, under conditions of refrigeration and freezing/thawing in sheep semen. Semen from six adult males was frozen in a commercial Triladyl™ diluent base. The ejaculate of each male was placed in four different aliquots: one for control, and the other three with additional 50 μM of trolox/108 spermatozoa, 50 μg of catalase/ml of ejaculate, and 5 mM cysteamine, respectively, in the diluent base. Pre-freezing (5 °C) and post-freezing sperm quality was evaluated using a computerized analysis system (IVOS II®), which eliminated subjectivity during the test. It was observed that 50 μM of trolox/108 SPZ and 50 μg of catalase/ml are able to maintain higher values (p < 0.05) of total motility (64.26 ± 0.82 and 55.54 ± 0.85 ) and progressive motility (47.26 ± 0.75 and 44.32 ± 1.13) under pre-freezing conditions, and of total motility (67.60 ± 1.91 and 63.47 ± 3.40), progressive motility (50.87 ± 2.58 and 38.88 ± 2.31), and viability (66.97 ± 2.25 and 61.16 ± 4.31) in the post-freezing period, compared to control treatments or the addition of 5 mM of cysteamine. The addition of 50 μM of trolox/108 spermatozoa and 50 μg of catalase/ml of semen improves the total and progressive motility in ovine semen refrigerated at 5 °C, as well as viability in frozen/thawed semen.

Resumo O objetivo do presente estudo foi avaliar o efeito sobre a qualidade espermática de três substancias antioxidantes (50 µM de trolox/108 espermatozoides, 50 µg de catalasa/ml de ejaculado e cisteamina 5mM) numa base de diluente comercial Triladyl™, sob condições de refrigeração e congelamento/descongelamento em sêmen ovino. Sêmen de seis machos adultos foi congelado numa base do diluente comercial Triladyl™. O ejaculado de cada macho foi posto em quatro diferentes alíquotas: uma para controlo e nas outras foi adicionada a base do diluente 50 µM de trolox/108 espermatozoides, 50µg de catalasa/ml de ejaculado e cisteamina 5mM. A qualidade espermática precongelação (5 °C) e pós-congelação avaliou-se com ajuda de um sistema de análise computadorizado (IVOS II®), eliminando assim a subjetividade ao teste. Observou-se que 50 µM de trolox/108 SPZ e 50 µg de catalasa/ml têm a capacidade de manter valores (p < 0,05) superiores de motilidade total (64,26 ± 0,82 e 55,54 ± 0,85) e motilidade progressiva (47,26 ± 0,75 e 44,32 ± 1,13) em condições de pre-congelação, e para motilidade total (67,60 ± 1,91 y 63,47 ± 3,40), motilidade progressiva (50,87 ± 2,58 e 38,88 ± 2,31) e viabilidade (66,97 ± 2,25 e 61,16 ± 4,31) em pós-congelação, comparados com os tratamentos controle ou a adição< de 5 mM de cisteamina. A adição de 50 µM de trolox/108 espermatozoides e 50 µg de catalasa/ml de sêmen melhora a motilidade total e progressiva do sêmen ovino refrigerado a 5 °C e a viabilidade em sêmen congelado/descongelado.

Changes in melatonin concentrations in seminal plasma are not correlated with testosterone or antioxidant enzyme activity when rams are located in areas with an equatorial photoperiod.

In temperate climates, photoperiod and melatonin regulate ram reproduction, modulating hormonal secretions, sperm quality, and seminal plasma composition. Information on the effect of an equatorial photoperiod (12L:12D) on ram reproduction, however, is scarce, and no data on hormonal concentrations and antioxidant enzyme activity in seminal plasma have been reported. Thus, the variation was investigated of melatonin and its relationship with testosterone and antioxidant enzyme activity in the seminal plasma of three sheep breeds in Colombia, when there was a consistent photoperiod during two dry and two rainy seasons per year. Semen was collected once a week from 12 mature rams (four of each breed: Colombian Creole, Hampshire, and Romney Marsh). Seminal plasma was obtained by centrifugation. The concentration of melatonin and testosterone were quantified along with the enzymatic activity of glutathione peroxidase (GPx), glutathione reductase (GRD), and catalase (CAT). Correlation analyses between melatonin and testosterone concentrations or enzymatic activity were also performed. Melatonin concentration was affected by season (P < 0.05) but not breed, with lesser concentrations in the first rainy season. Testosterone concentration, however, was affected by breed and season, with greater concentrations (P < 0.01) in the Hampshire and Romney Marsh rams during the second dry season. Regarding antioxidant enzyme activity, there was only seasonal variation in GPx activity (P < 0.05). When correlation analyses were used for data assessments, there was a negative correlation between melatonin and testosterone concentrations in Hampshire rams. In conclusion, melatonin concentrations in seminal plasma of rams that were located in an area with an equatorial photoperiod was affected by the climatological season but there was no positive correlation with testosterone concentration or antioxidant enzyme activity.

Evaluación de los parámetros de calidad seminal y cinemática espermática en tres razas ovinas de lana en condiciones de trópico alto colombiano / Evaluation of semen quality parameters and sperm kinematics in three wool sheep breeds in conditions of high tropics in Colombia / Avaliação dos parâmetros de qualidade seminal e cinemática espermática em três raças ovinas de lã em condições de trópico alto colombiano

Resumen El objetivo del presente estudio fue evaluar el efecto del eyaculado, la raza y las colectas sobre los parámetros de calidad seminal y cinemática en 3 tipos raciales ovinos de lana bajo condiciones de trópico alto colombiano. Se colectó semen de 12 machos de la raza criollo, romney marsh y hampshire de 2 a 5 años de edad, con vagina artificial, durante 4 colectas con un tiempo de abstinencia de 3 d. Se realizó el análisis de la motilidad con ayuda de un sistema computarizado de análisis seminal (CASA) en combinación con una tinción de fluorescencia Hoeschst 33259 para el estudio de la viabilidad y la morfología. Se realizó un análisis de medidas repetidas en el tiempo con el paquete estadístico SAS, empleando el método Proc Mixed. No hubo diferencias significativas (p > 0,05) en el porcentaje de motilidad total y motilidad progresiva para el primer y segundo eyaculado, pero sí hubo diferencias (p < 0,05) para la concentración, volumen y viabilidad; el primer eyaculado fue significativamente superior al segundo, independiente de la raza y la colecta. Las variables de cinemática espermática relacionadas con el movimiento progresivo y la velocidad (ALH, VAP, LIN) no difieren entre eyaculados, pero sí hay un efecto racial (p > 0,05) en el que la raza criolla es superior en estas variables, lo que podría sugerir que las células espermáticas de esta raza tienen la capacidad de recorrer mayor distancia en menor tiempo, comparado con los espermatozoides de las otras dos razas, en las condiciones del presente experimento.

Abstract This study aimed to evaluate the effect of the ejaculate, race, and sperm collections on semen quality and kinematic parameters in three wool sheep breeds in conditions of high tropics in Colombia. Semen was collected from 12 males of Creole, Romney marsh, and Hampshire sheep breeds ranging from 2 to 5 years of age, with artificial vagina, during 4 collections with a time of abstinence of 3 days. Motility analysis was performed with the help of a computer-assisted semen analysis (CASA) in combination with a fluorescent Hoechst 33259 stain for the study of viability and morphology. An analysis of measures repeated over time was performed with the SAS statistical package, using the Proc Mixed method. There were no significant differences (p > 0.05) in the percentage of total motility and progressive motility for the first and second ejaculates, but there were differences (p < 0.05) for concentration, volume, and viability; the first ejaculate was significantly superior to the second, independent of breed and collection. Sperm kinematic variables related to progressive movement and speed (ALH, VAP, LIN) do not differ between ejaculates, but there is a racial effect (p > 0.05), since the Creole breed is superior in all these variables, which could suggest that the sperm cells of this breed have the ability to travel greater distance in less time, compared with sperm from the other two breeds, under the conditions of the present experiment.

Resumo O objetivo deste estudo foi avaliar o efeito da ejaculação, a raça e as coletas sobre os parâmetros de qualidade seminal e cinemática em 3 tipos raciais ovinos de lã sob condições de trópico alto colombiano. Foram colhidas amostras de sêmen de 12 machos da raça crioulo, romney marsh e hampshire de 2 a 5 anos de idade, com vagina artificial, durante 4 coletas com um tempo de abstinência de 3 d. Realizou-se a análise da motilidade com ajuda de um sistema computarizado de análise seminal (CASA) em combinação com uma tinção de florescência Hoeschst 33259 para o estudo da viabilidade e a morfologia. Realizou-se um análise de medidas repetidas no tempo com o paquete estadístico SAS, empregando o método Proc Mixed. Não houve diferenças significativas (p > 0,05) na porcentagem de motilidade total e motilidade progressiva para a primeira e segunda ejaculação, mas, sim houve diferenças (p < 0,05) para a concentração, volume e viabilidade; a primeira ejaculação foi significativamente superior ao segundo, independente da raça e da coleta. As variáveis de cinemática espermática relacionadas com o movimento progressivo e velocidade (ALH, VAP, LIN) não diferem entre as ejaculações, mas sim há um efeito racial (p > 0,05) no que a raça crioula é superior em estas variáveis, o que poderia sugerir que as células espermáticas desta raça têm a capacidade de percorrer maior distância em menor tempo, comparado com os espermatozoides das outras dois raças, nas condições desta experiência.

Melatonin MT1 and MT2 Receptors in the Ram Reproductive Tract.

Some melatonin functions in mammals are exerted through MT1 and MT2 receptors. However, there are no reports of their presence in the reproductive tract of the ram, a seasonal species. Thus, we have investigated their existence in the ram testis, epididymis, accessory glands and ductus deferens. Real-time polymerase chain reaction (qPCR) revealed higher levels of m-RNA for both receptors in the testis, ampulla, seminal vesicles, and vas deferens, than in the other organs of the reproductive tract (p < 0.05). Western blot analyses showed protein bands compatible with the MT1 in the testis and cauda epididymis, and for the MT2 in the cauda epididymis and deferent duct. Immunohistochemistry analyses revealed the presence of MT1 receptors in spermatogonias, spermatocytes, and spermatids, and MT2 receptors in the newly-formed spermatozoa in the testis, whereas both receptors were located in the epithelial cells of the ampulla, seminal vesicles, and ductus deferens. Indirect immunofluorescence showed significant differences in the immunolocation of both receptors in spermatozoa during their transit in the epididymis. In conclusion, it was demonstrated that melatonin receptors are present in the ram reproductive tract. These results open the way for new studies on the molecular mechanism of melatonin and the biological significance of its receptors.