Single layer centrifugation improves the quality of fresh donkey semen and modifies the sperm ability to interact with polymorphonuclear neutrophils

This study sought to determine whether single layer centrifugation (SLC) of fresh donkey semen with Equicoll has any impact on sperm quality parameters and on the modulation of endometrial reaction following semen deposition using an in vitro model. Seventeen ejaculates from five jackasses were obtained using an artificial vagina and diluted in a skim-milk extender. Samples were either selected through SLC (Equicoll) or non-treated (control). Two experiments were performed. The first one consisted of incubating selected or non-selected spermatozoa at 38 °C for 180 min. Integrity and lipid disorder of sperm plasma membrane, mitochondrial membrane potential, and intracellular levels of calcium and reactive oxygen species were evaluated at 0, 60, 120, and 180 min. In the second experiment, polymorphonuclear neutrophils (PMN) isolated from jennies blood were mixed with selected and unselected spermatozoa. Interaction between spermatozoa and PMN was evaluated after 0, 60, 120, and 180 min of co-incubation at 38 °C. SLC-selection increased the proportions of spermatozoa with an intact plasma membrane and low lipid disorder, of spermatozoa with high mitochondrial membrane potential and with high calcium levels, and of progressively motile spermatozoa. In addition, selection through SLC augmented the proportion of phagocytosed spermatozoa, which supported the modulating role of seminal plasma proteins on sperm-PMN interaction. In conclusion, SLC of fresh donkey semen increases the proportions of functionally intact and motile spermatozoa, and appears to remove the seminal plasma proteins that inhibit sperm-PMN binding

Red-Light Irradiation of Horse Spermatozoa Increases Mitochondrial Activity and Motility through Changes in the Motile Sperm Subpopulation Structure

Previous studies in other mammalian species have shown that stimulation of semen with red-light increases sperm motility, mitochondrial activity, and fertilizing capacity. This study sought to determine whether red-light stimulation using a light emitting diode (LED) at 620-630 nm affects sperm motility and structure of motile subpopulations, sperm viability, mitochondrial activity, intracellular ATP levels, rate of O consumption and DNA integrity of horse spermatozoa. For this purpose, nine ejaculates were collected from nine different adult stallions. Upon collection, semen was diluted in Kenney extender, analyzed, its concentration was adjusted, and finally it was stimulated with red-light. In all cases, semen was packaged in 0.5-mL transparent straws, which were randomly divided into controls and 19 light-stimulation treatments; 6 consisted of a single exposure to red-light, and the other 13 involved irradiation with intervals of irradiation and darkness (light-dark-light). After irradiation, sperm motility was assessed using a Computerized Semen Analysis System (CASA). Flow cytometry was used to evaluate sperm viability, mitochondrial membrane potential and DNA fragmentation. Intracellular levels of ATP and O consumption rate were also determined. Specific red-light patterns were found to modify kinetics parameters (patterns: 4, 2-2-2, 3-3-3, 4-4-4, 5-1-5, and 5-5-5 min), the structure of motile sperm subpopulations (patterns: 2, 2-2-2, 3-3-3, and 4-1-4 min), mitochondrial membrane potential (patterns: 4, 3-3-3, 4-4-4, 5-1-5, 5-5-5, 15-5-15, and 15-15-15 min), intracellular ATP levels and the rate of O consumption (pattern: 4 min), without affecting sperm viability or DNA integrity. Since the increase in some kinematic parameters was concomitant with that of mitochondrial activity, intracellular ATP levels and O consumption rate, we suggest that the positive effect of light-irradiation on sperm motility is related to its impact upon mitochondrial activity. In conclusion, this study shows that red LED light stimulates motility and mitochondrial activity of horse sperm. Additional research is needed to address the impact of red-light irradiation on fertilizing ability and the mechanisms through which light exerts its effects

Specific activity of superoxide dismutase in stallion seminal plasma is related to sperm cryotolerance

While the removal of seminal plasma is a routine practice prior to equine sperm cryopreservation, this fluid contains the main source of antioxidant enzymes able to scavenge these reactive oxygen species. Therefore, stallion seminal plasma components may have an impact on ejaculate freezability. Against this background, this study was designed to investigate whether the activities of the main stallion seminal plasma antioxidant enzymes are related to sperm cryotolerance. With this purpose, 16 ejaculates were collected from 14 healthy stallions, and each ejaculate was split into two aliquots. The first one was used to evaluate the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and glutathione reductase (GSR) in seminal plasma. The second aliquot was extended and then processed for cryopreservation. Sperm motility and viability were evaluated before and after cryopreservation, and ejaculates were classified as of good (GFE) or poor freezability (PFE) based on total motile and viable spermatozoa at post-thaw. We observed that, while the specific activities of CAT, GPX, and GSR were similar between GFE and PFE, that of SOD was significantly (p < 0.05) higher in GFE than in PFE. We can thus conclude that, in stallions, the specific activity of SOD in the seminal plasma of a given ejaculate might be related to its freezability

The Effects of Red Light on Mammalian Sperm Rely upon the Color of the Straw and the Medium Used

Several studies have shown that the exposure of semen to red light improves sperm quality and fertilizing ability, which could improve the efficiency of assisted reproductive techniques with irradiated semen. However, despite being considered as possible sources of variation, the effects of the color of the container (straws) or the medium have not yet been evaluated. In this study, 13 ejaculates from different stallions were split into equal fractions, diluted either with Kenney or Equiplus extender, and subsequently packed into straws of five different colors. After storage at 4 °C for 24 h, the sperm were irradiated and different variables, including sperm motility, plasma membrane integrity, and mitochondrial membrane potential, were evaluated. Our results confirm that irradiation increases some motion characteristics and mitochondrial membrane potential without affecting sperm viability and demonstrate that the effects depend on the color of the straw and the extender used. Previous research has determined that irradiation of mammalian sperm with red light increases motility, mitochondrial activity, and fertilization capacity. In spite of this, no study has considered the potential influence of the color of the straw and the extender used. Therefore, this study tests the hypothesis that the response of mammalian sperm to red light is influenced by the color of the straw and the turbidity/composition of the extender. Using the horse as a model, 13 ejaculates from 13 stallions were split into two equal fractions, diluted with Kenney or Equiplus extender, and stored at 4 °C for 24 h. Thereafter, each diluted fraction was split into five equal aliquots and subsequently packed into 0.5-mL straws of red, blue, yellow, white, or transparent color. Straws were either nonirradiated (control) or irradiated with a light-dark-light pattern of 3-3-3 (i.e., light: 3 min, dark: 3 min; light: 3 min) prior to evaluating sperm motility, acrosome and plasma membrane integrity, mitochondrial membrane potential, and intracellular ROS and calcium levels. Our results showed that irradiation increased some motion variables, mitochondrial membrane potential, and intracellular ROS without affecting the integrities of the plasma membrane and acrosome. Remarkably, the extent of those changes varied with the color of the straw and the extender used; the effects of irradiation were more apparent when sperm were diluted with Equiplus extender and packed into red-colored straws or when samples were diluted with Kenney extender and packed into transparent straws. As the increase in sperm motility and intracellular ROS levels was parallel to that of mitochondrial activity, we suggest that the impact of red light on sperm function relies upon the specific rates of energy provided to the mitochondria, which, in turn, vary with the color of the straw and the turbidity/composition of the extender

Red LED Light Acts on the Mitochondrial Electron Chain of Donkey Sperm and Its Effects Depend on the Time of Exposure to Light

This work aimed to investigate how stimulation of donkey sperm with red LED light affects mitochondrial function. For this purpose, freshly diluted donkey semen was stimulated with red light for 1, 5, and 10 min, in the presence or absence of oligomycin A (Omy A), a specific inhibitor of mitochondrial ATP synthase, or FCCP, a specific disruptor of mitochondrial electron chain. The results obtained in the present study indicated that the effects of red LED light on fresh donkey sperm function are related to changes in mitochondria function. In effect, irradiation of donkey sperm resulted in an increase in mitochondrial membrane potential (MMP), the activity of cytochrome C oxidase and the rate of oxygen consumption. In addition, in the absence of oligomycin A and FCCP, light-stimulation augmented the average path velocity (VAP) and modified the structure of motile sperm subpopulations, increasing the fastest and most linear subpopulation. In contrast, the presence of either Omy A or FCCP abolished the aforementioned effects. Interestingly, our results also showed that the effects of red light depend on the exposure time applied, as indicated by the observed differences between irradiation protocols. In conclusion, our results suggest that exposing fresh donkey sperm to red light modulates the function of their mitochondria through affecting the activity of the electron chain. However, the extent of this effect depends on the irradiation pattern and does not exclude the existence of other mechanisms, such as those related to thermotaxis

Cryotolerance of stallion spermatozoa relies on aquaglyceroporins rather than orthodox aquaporins

Aquaporins (AQPs), a family of ubiquitous water channels divided into orthodox AQPs, aquaglyceroporins (GLPs), and superAQPs, are present in stallion spermatozoa. The aim of this study was to elucidate the functional relevance of each group of AQPs during stallion sperm cryopreservation through the use of three different inhibitors: acetazolamide (AC), phloretin (PHL) and propanediol (PDO). Sperm quality and function parameters were evaluated in the presence or absence of each inhibitor in fresh and frozen-thawed samples. In the presence of AC, different parameters were altered (p < 0.05), but not in a concentration-or time-depending manner. PHL was found to decrease sperm motility, viability, acrosome integrity, and the percentages of spermatozoa with low membrane lipid disorder, high mitochondrial membrane potential (MMP) and high intracellular levels of calcium and superoxides (p < 0.05). Finally, the sperm motility, viability, acrosome integrity, the percentages of spermatozoa with low membrane lipid disorder, high MMP and high intracellular calcium levels were higher (p < 0.05) in PDO treatments than in the control. The sperm response to AC, PHL and PDO indicates that GLPs, rather than orthodox AQPs, play a crucial role during stallion sperm cryopreservation. Furthermore, post-thaw sperm quality was higher in PDO treatments than in the control, suggesting that this molecule is a potential permeable cryoprotectant

How does seminal plasma protect mammalian spermatozoa? Species-linked variations within the genus Equus

El plasma seminal representa un important i actiu contribuent a la fertilitat dels mamífers. Entre els components del plasma seminal s’hi troben els antioxidants enzimàtics i no enzimàtics que modulen la producció d’espècies reactives d’oxigen. L’estrès oxidatiu, induït per un equilibri incorrecte entre els antioxidants i la producció de ROS, perjudica la funcionalitat espermàtica. D’aquesta manera, el plasma seminal representa el sistema de defensa més important quant a la prevenció dels danys que aquestes espècies d’oxigen provoquen en els espermatozoides. Tot i això, es sol recomanar l’eliminació del plasma seminal com a pas previ a la conservació dels espermatozoides, atès que la presència d’aquest fluid pot ser perjudicial per la longevitat dels gàmetes. En aquest context, l’objectiu principal d’aquesta Tesi Doctoral va ser determinar la importància dels enzims antioxidants del plasma seminal per l’estratègia reproductiva dels èquids. D’una banda, es va observar que les activitats de la superòxid dismutasa (SOD), la catalasa (CAT), la glutatió peroxidasa (GPX) i la glutatió reductasa (GSR) en el plasma seminal dels rucs eren molt més altes que en el dels cavalls. Curiosament, les activitats de SOD i GPX del plasma seminal van mostrar variacions estacionals en els rucs, però no en els cavalls. D’altra banda, i atès que s’està produint un augment de la demanda de semen congelat dels èquids i que cap estudi previ ha abordat la relació entre les activitats d’aquests quatre enzims i la capacitat dels espermatozoides de resistir a la criopreservació, els estudis segon i tercer van avaluar la relació entre les activitats dels enzims SOD, CAT, GPX i GSR, presents en el plasma seminal, i la criotolerància dels espermatozoides de ruc i cavall. Es va observar que l’activitat específica de la SOD en el plasma seminal està relacionada amb la criotolerància dels espermatozoides de rucs i cavall. Atès que estudis previs han demostrat que el plasma seminal modula la producció espermàtica de ROS en diferents espècies de mamífers, va estudiar si la presència del plasma seminal és capaç de controlar la generació espermàtica de ROS en condicions d’estrès oxidatiu induït pel peròxid d’hidrogen. Sorprenentment, es va observar que el contacte perllongat del plasma seminal amb els espermatozoides de ruc té un efecte limitat a l’hora de contrarestar l’estrès oxidatiu induït exògenament en aquests gàmetes. Finalment, la selecció d’espermatozoides, que comporta l’eliminació dels components del plasma seminal, s’està convertint en una tecnologia important dins l’àmbit de la reproducció assistida equina. Per tant, l’últim objectiu d’aquesta Tesi Doctoral va ser investigar els efectes de la centrifugació amb una sola fase/capa (SLC) mitjançant Equicoll sobre els paràmetres de qualitat del espermatozoides de ruc i les seves interaccions amb el tracte reproductor femení. Tot i que la selecció amb Equicoll va millorar els percentatges de viabilitat espermàtica després de la selecció, l’impacte sobre els paràmetres de motilitat va ser molt limitat. A més, l’extracció dels components del plasma seminal mitjançant Equicoll va augmentar la fagocitosi dels espermatozoides per part dels neutròfils, la qual cosa confirma que el plasma seminal modula la interacció entre els gàmetes masculins i aquestes cèl·lules immunitàries. En conclusió, aquesta Tesi Doctoral indica que, encara que les activitats dels antioxidants del plasma seminal difereixen entre cavalls i rucs, la SOD és, en ambdues espècies, un bon predictor de la capacitat dels espermatozoides de resistir a la criopreservació. A més, aquesta Tesi Doctoral també ha demostrat que el paper modulador del plasma seminal és més evident quant a la interacció entre els espermatozoides de burro i els neutròfils, que respecte a l’estrès oxidatiu induït en els gàmetes masculins d’aquesta espècie pel peròxid d’hidrogen.El plasma seminal representa un importante y activo contribuyente para la fertilidad de los mamíferos. Entre los componentes del plasma seminal se encuentran los antioxidantes enzimáticos y no enzimáticos que modulan la producción de especies reactivas de oxígeno. El estrés oxidativo, inducido por un equilibrio incorrecto entre los antioxidantes y la producción de ROS, perjudica las funciones del espermatozoide. De esta manera, el plasma seminal representa el sistema de defensa más importante capaz de prevenir los daños que dichas especies reactivas de oxígeno pueden provocar en los espermatozoides. A pesar de ello, se suele recomendar la eliminación del plasma seminal como paso previo a la conservación de los espermatozoides, ya que la presencia de este fluido puede ser perjudicial para la longevidad de los gametos. El objetivo principal de esta Tesis Doctoral fue determinar la importancia de los enzimas antioxidantes del plasma seminal en la estrategia reproductiva de los equinos. Se observó que las actividades de la superóxido dismutasa (SOD), la catalasa (CAT), la glutatión peroxidasa (GPX) y la glutatión reductasa (GSR) en el plasma seminal de burros eran mucho más altas que en el de caballos. Curiosamente, las actividades de SOD y GPX en el plasma seminal mostraron variaciones estacionales en los burros, pero no en los caballos. Por otro lado, debido a que la demanda de semen congelado en la industria equina está aumentando, y que ningún estudio previo abordó la relación entre las actividades de estos cuatro enzimas y la capacidad de los espermatozoides de resistir a la congelación, los estudios segundo y tercero evaluaron la relación entre las actividades de SOD, CAT, GPX y GSR en el plasma seminal fresco y la criotolerancia de los espermatozoides de burro y caballo. Se encontró que la actividad específica del SOD en el plasma seminal está relacionada con una mejor tolerancia de los espermatozoides a la congelación tanto en caballos como en burros. Basándose en estos resultados. Dado que otros estudios previos han observado que el plasma seminal modula la producción de ROS por parte de los espermatozoides en distintas especies de mamíferos, se estudió si la presencia de plasma seminal es capaz de controlar la generación espermática de ROS en condiciones de estrés oxidativo inducido por la adición de peróxido de hidrógeno. Sorprendentemente, se observó que el contacto prolongado de los espermatozoides de burro con el plasma seminal tiene un efecto compensatorio limitado en cuanto a los respuesta de aquéllos al estrés oxidativo inducido exógenamente. Por último, la selección de los espermatozoides, que comporta la eliminación de los componentes del plasma seminal, se está convirtiendo en una tecnología prominente en el ámbito de la reproducción asistida equina. Por consiguiente, se investigó los efectos de la centrifugación con una sola fase/capa (SLC) mediante Equicoll sobre los parámetros de calidad de los espermatozoides de burro y sus interacciones con el tracto reproductivo femenino. Aunque la selección con Equicoll mejoró los porcentajes de viabilidad espermática después de la selección, el impacto sobre los parámetros de motilidad fue mínimo. Además, la extracción de los componentes del plasma seminal mediante Equicoll aumentó la fagocitosis de los espermatozoides por los neutrófilos. En conclusión, aunque las actividades de los antioxidantes seminales difieren entre caballos y burros, la SOD parece ser, en ambas especies, un buen predictor de la capacidad de los espermatozoides en resistir a la congelación. Además, se demostró que el papel modulador del plasma seminal es más evidente respecto a la interacción entre los espermatozoides de burro y los neutrófilos, que en cuanto a la respuesta de aquéllos al estrés oxidativo inducido por el peróxido de hidrógeno.Seminal plasma represents an important and active contributor to mammalian fertility. Specific components of seminal plasma are adsorbed onto the surface of ejaculated spermatozoa while they pass through the male reproductive tract and provide sperm cells with the ability to reach and fertilize the oocyte. Among the components of seminal plasma, there are enzymatic and non-enzymatic antioxidants that modulate the production of reactive oxygen species (ROS), which are by-products generated during mitochondrial electron transport. Oxidative stress, which is induced by an incorrect balance between antioxidants and ROS production, impairs sperm function. In this context, seminal plasma represents the most important defense system able to prevent the damages that these species exert upon sperm cells. In spite of this, removal of seminal plasma is usually recommended prior to sperm storage, cooled or cryopreserved, as the presence of this fluid may be detrimental for sperm longevity. Against this background, the main aim of this Dissertation was to determine the relevance of the antioxidant enzymes present in seminal plasma for equine reproduction strategies. On the one hand, and as a first objective of this work, we observed much higher activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and glutathione reductase (GSR) in jackass seminal plasma than in that of stallions. Since equids are seasonal breeders, this study also investigated season influence on the antioxidant composition of seminal plasma. Interestingly, activities of SOD and GPX in seminal plasma showed seasonal variations in jackasses but not in stallions. On the other hand, because the demand of cryopreserved semen in the equine industry is increasing and no study had previously addressed whether the activities of these four enzymes are related to sperm cryotolerance, the second and third study evaluated the relationship between SOD, CAT, GPX and GSR activities in fresh seminal plasma and the sperm ability to withstand cryopreservation in these two species. It was found that the specific SOD activity in seminal plasma is related to sperm cryotolerance in both stallions and jackasses. Since seminal plasma has been reported to modulate ROS balance in several mammalian species, this Dissertation also investigated how the presence of seminal plasma modulates ROS generation by jackass spermatozoa under oxidative stress induced exogenously by hydrogen peroxide. With regard to these results, however, prolonged contact of seminal plasma was found to have limited effect against oxidative stress induced exogenously to jackass spermatozoa. Finally, sperm selection is getting prominent in equine assisted reproduction technologies and also leads to the removal of seminal plasma components. The last objective of this study aimed to investigate the effects of single layer centrifugation (SLC) with Equicoll on jackass sperm parameters and its interactions with the female reproductive tract. While SLC-Equicoll was found to increase the percentages of viable spermatozoa after selection, the impact on sperm motility parameters was marginal. In addition, removal of seminal plasma components through SLC-Equicoll increased sperm phagocytosis by polymorphonuclear neutrophils (PMNs), thereby confirming that seminal plasma modulates the interaction between sperm and PMNs. In conclusion, this Dissertation indicates that although the activities of seminal plasma antioxidants differ between stallions and jackasses, SOD appears to be a good predictor of sperm cryotolerance in these two species. In addition, this Dissertation has also demonstrated, for the first time, that the modulating role of seminal plasma with regard to the interaction between jackass spermatozoa and PMNs is more apparent than that observed when oxidative stress is induced by hydrogen peroxide

Improvement of cryopreservation protocol in both purebred horses including Spanish horses

There is a widely held belief that the semen of Purebred Spanish Horses (PRE) is of generally poorer quality than that of other breeds, and survives cryopreservation less well. To determine whether this is the case, sperm concentration, viability and morphological abnormalities were examined in a total 610 fresh ejaculates from 64 healthy PRE (N=47) and non-PRE stallions (N=17). Sperm concentration and viability were then re-examined after pre-freezing centrifugation, and once again after freezing-thawing. No differences were observed between the PRE and non-PRE stallions in terms of any sperm quality variable at any observation point. When considering all PRE and non-PRE samples together, differences in sperm viability were observed between fresh and fresh-centrifuged sperm viability (70.1±12.5% compared to 76.3±10.9%; p<0.01). After centrifugation the samples were also more homogeneous in terms of the total number of recovered sperm cells. Centrifugation also improved frozen-thawed sperm viability, reducing differences in sperm quality between individual stallions. For all centrifugations, a sperm:extender ratio of 1:5 was used. This would appear to provide better final results than those reported in the literature for the 1:1 ratio commonly used for PRE stallion sperm cryopreservation. In conclusion, obtained results show that the quality and frozen/thawed results of PRE stallion sperm are not lower than that of non-PRE breeds. In addition, using a 1:5 sperm:extender dilution ratio when selecting sperms by centrifugation prior to freezing, seems to provide better results than those usually reported when using a 1:1 ratio

Improvement of cryopreservation protocol in both purebred horses including Spanish horses

There is a widely held belief that the semen of Purebred Spanish Horses (PRE) is of generally poorer quality than that of other breeds, and survives cryopreservation less well. To determine whether this is the case, sperm concentration, viability and morphological abnormalities were examined in a total 610 fresh ejaculates from 64 healthy PRE (N=47) and non-PRE stallions (N=17). Sperm concentration and viability were then re-examined after pre-freezing centrifugation, and once again after freezing-thawing. No differences were observed between the PRE and non-PRE stallions in terms of any sperm quality variable at any observation point. When considering all PRE and non-PRE samples together, differences in sperm viability were observed between fresh and fresh-centrifuged sperm viability (70.1±12.5% compared to 76.3±10.9%; p<0.01). After centrifugation the samples were also more homogeneous in terms of the total number of recovered sperm cells. Centrifugation also improved frozen-thawed sperm viability, reducing differences in sperm quality between individual stallions. For all centrifugations, a sperm:extender ratio of 1:5 was used. This would appear to provide better final results than those reported in the literature for the 1:1 ratio commonly used for PRE stallion sperm cryopreservation. In conclusion, obtained results show that the quality and frozen/thawed results of PRE stallion sperm are not lower than that of non-PRE breeds. In addition, using a 1:5 sperm:extender dilution ratio when selecting sperms by centrifugation prior to freezing, seems to provide better results than those usually reported when using a 1:1 ratio

How does seminal plasma protect mammalian spermatozoa? Species-linked variations within the genus Equus /

Departament responsable de la tesi: Departament de Medicina i Cirurgia Animals.El plasma seminal representa un important i actiu contribuent a la fertilitat dels mamífers. Entre els components del plasma seminal s'hi troben els antioxidants enzimàtics i no enzimàtics que modulen la producció d'espècies reactives d'oxigen. L'estrès oxidatiu, induït per un equilibri incorrecte entre els antioxidants i la producció de ROS, perjudica la funcionalitat espermàtica. D'aquesta manera, el plasma seminal representa el sistema de defensa més important quant a la prevenció dels danys que aquestes espècies d'oxigen provoquen en els espermatozoides. Tot i això, es sol recomanar l'eliminació del plasma seminal com a pas previ a la conservació dels espermatozoides, atès que la presència d'aquest fluid pot ser perjudicial per la longevitat dels gàmetes. En aquest context, l'objectiu principal d'aquesta Tesi Doctoral va ser determinar la importància dels enzims antioxidants del plasma seminal per l'estratègia reproductiva dels èquids. D'una banda, es va observar que les activitats de la superòxid dismutasa (SOD), la catalasa (CAT), la glutatió peroxidasa (GPX) i la glutatió reductasa (GSR) en el plasma seminal dels rucs eren molt més altes que en el dels cavalls. Curiosament, les activitats de SOD i GPX del plasma seminal van mostrar variacions estacionals en els rucs, però no en els cavalls. D'altra banda, i atès que s'està produint un augment de la demanda de semen congelat dels èquids i que cap estudi previ ha abordat la relació entre les activitats d'aquests quatre enzims i la capacitat dels espermatozoides de resistir a la criopreservació, els estudis segon i tercer van avaluar la relació entre les activitats dels enzims SOD, CAT, GPX i GSR, presents en el plasma seminal, i la criotolerància dels espermatozoides de ruc i cavall. Es va observar que l'activitat específica de la SOD en el plasma seminal està relacionada amb la criotolerància dels espermatozoides de rucs i cavall. Atès que estudis previs han demostrat que el plasma seminal modula la producció espermàtica de ROS en diferents espècies de mamífers, va estudiar si la presència del plasma seminal és capaç de controlar la generació espermàtica de ROS en condicions d'estrès oxidatiu induït pel peròxid d'hidrogen. Sorprenentment, es va observar que el contacte perllongat del plasma seminal amb els espermatozoides de ruc té un efecte limitat a l'hora de contrarestar l'estrès oxidatiu induït exògenament en aquests gàmetes.Finalment, la selecció d'espermatozoides, que comporta l'eliminació dels components del plasma seminal, s'està convertint en una tecnologia important dins l'àmbit de la reproducció assistida equina. Per tant, l'últim objectiu d'aquesta Tesi Doctoral va ser investigar els efectes de la centrifugació amb una sola fase/capa (SLC) mitjançant Equicoll sobre els paràmetres de qualitat del espermatozoides de ruc i les seves interaccions amb el tracte reproductor femení. Tot i que la selecció amb Equicoll va millorar els percentatges de viabilitat espermàtica després de la selecció, l'impacte sobre els paràmetres de motilitat va ser molt limitat. A més, l'extracció dels components del plasma seminal mitjançant Equicoll va augmentar la fagocitosi dels espermatozoides per part dels neutròfils, la qual cosa confirma que el plasma seminal modula la interacció entre els gàmetes masculins i aquestes cèl·lules immunitàries.En conclusió, aquesta Tesi Doctoral indica que, encara que les activitats dels antioxidants del plasma seminal difereixen entre cavalls i rucs, la SOD és, en ambdues espècies, un bon predictor de la capacitat dels espermatozoides de resistir a la criopreservació. A més, aquesta Tesi Doctoral també ha demostrat que el paper modulador del plasma seminal és més evident quant a la interacció entre els espermatozoides de burro i els neutròfils, que respecte a l'estrès oxidatiu induït en els gàmetes masculins d'aquesta espècie pel peròxid d'hidrogen.El plasma seminal representa un importante y activo contribuyente para la fertilidad de los mamíferos. Entre los componentes del plasma seminal se encuentran los antioxidantes enzimáticos y no enzimáticos que modulan la producción de especies reactivas de oxígeno. El estrés oxidativo, inducido por un equilibrio incorrecto entre los antioxidantes y la producción de ROS, perjudica las funciones del espermatozoide. De esta manera, el plasma seminal representa el sistema de defensa más importante capaz de prevenir los daños que dichas especies reactivas de oxígeno pueden provocar en los espermatozoides. A pesar de ello, se suele recomendar la eliminación del plasma seminal como paso previo a la conservación de los espermatozoides, ya que la presencia de este fluido puede ser perjudicial para la longevidad de los gametos. El objetivo principal de esta Tesis Doctoral fue determinar la importancia de los enzimas antioxidantes del plasma seminal en la estrategia reproductiva de los equinos. Se observó que las actividades de la superóxido dismutasa (SOD), la catalasa (CAT), la glutatión peroxidasa (GPX) y la glutatión reductasa (GSR) en el plasma seminal de burros eran mucho más altas que en el de caballos. Curiosamente, las actividades de SOD y GPX en el plasma seminal mostraron variaciones estacionales en los burros, pero no en los caballos. Por otro lado, debido a que la demanda de semen congelado en la industria equina está aumentando, y que ningún estudio previo abordó la relación entre las actividades de estos cuatro enzimas y la capacidad de los espermatozoides de resistir a la congelación, los estudios segundo y tercero evaluaron la relación entre las actividades de SOD, CAT, GPX y GSR en el plasma seminal fresco y la criotolerancia de los espermatozoides de burro y caballo. Se encontró que la actividad específica del SOD en el plasma seminal está relacionada con una mejor tolerancia de los espermatozoides a la congelación tanto en caballos como en burros. Basándose en estos resultados. Dado que otros estudios previos han observado que el plasma seminal modula la producción de ROS por parte de los espermatozoides en distintas especies de mamíferos, se estudió si la presencia de plasma seminal es capaz de controlar la generación espermática de ROS en condiciones de estrés oxidativo inducido por la adición de peróxido de hidrógeno. Sorprendentemente, se observó que el contacto prolongado de los espermatozoides de burro con el plasma seminal tiene un efecto compensatorio limitado en cuanto a los respuesta de aquéllos al estrés oxidativo inducido exógenamente. Por último, la selección de los espermatozoides, que comporta la eliminación de los componentes del plasma seminal, se está convirtiendo en una tecnología prominente en el ámbito de la reproducción asistida equina. Por consiguiente, se investigó los efectos de la centrifugación con una sola fase/capa (SLC) mediante Equicoll sobre los parámetros de calidad de los espermatozoides de burro y sus interacciones con el tracto reproductivo femenino. Aunque la selección con Equicoll mejoró los porcentajes de viabilidad espermática después de la selección, el impacto sobre los parámetros de motilidad fue mínimo. Además, la extracción de los componentes del plasma seminal mediante Equicoll aumentó la fagocitosis de los espermatozoides por los neutrófilos. En conclusión, aunque las actividades de los antioxidantes seminales difieren entre caballos y burros, la SOD parece ser, en ambas especies, un buen predictor de la capacidad de los espermatozoides en resistir a la congelación. Además, se demostró que el papel modulador del plasma seminal es más evidente respecto a la interacción entre los espermatozoides de burro y los neutrófilos, que en cuanto a la respuesta de aquéllos al estrés oxidativo inducido por el peróxido de hidrógeno.Seminal plasma represents an important and active contributor to mammalian fertility. Specific components of seminal plasma are adsorbed onto the surface of ejaculated spermatozoa while they pass through the male reproductive tract and provide sperm cells with the ability to reach and fertilize the oocyte. Among the components of seminal plasma, there are enzymatic and non-enzymatic antioxidants that modulate the production of reactive oxygen species (ROS), which are by-products generated during mitochondrial electron transport. Oxidative stress, which is induced by an incorrect balance between antioxidants and ROS production, impairs sperm function. In this context, seminal plasma represents the most important defense system able to prevent the damages that these species exert upon sperm cells. In spite of this, removal of seminal plasma is usually recommended prior to sperm storage, cooled or cryopreserved, as the presence of this fluid may be detrimental for sperm longevity. Against this background, the main aim of this Dissertation was to determine the relevance of the antioxidant enzymes present in seminal plasma for equine reproduction strategies. On the one hand, and as a first objective of this work, we observed much higher activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and glutathione reductase (GSR) in jackass seminal plasma than in that of stallions. Since equids are seasonal breeders, this study also investigated season influence on the antioxidant composition of seminal plasma. Interestingly, activities of SOD and GPX in seminal plasma showed seasonal variations in jackasses but not in stallions. On the other hand, because the demand of cryopreserved semen in the equine industry is increasing and no study had previously addressed whether the activities of these four enzymes are related to sperm cryotolerance, the second and third study evaluated the relationship between SOD, CAT, GPX and GSR activities in fresh seminal plasma and the sperm ability to withstand cryopreservation in these two species. It was found that the specific SOD activity in seminal plasma is related to sperm cryotolerance in both stallions and jackasses. Since seminal plasma has been reported to modulate ROS balance in several mammalian species, this Dissertation also investigated how the presence of seminal plasma modulates ROS generation by jackass spermatozoa under oxidative stress induced exogenously by hydrogen peroxide. With regard to these results, however, prolonged contact of seminal plasma was found to have limited effect against oxidative stress induced exogenously to jackass spermatozoa. Finally, sperm selection is getting prominent in equine assisted reproduction technologies and also leads to the removal of seminal plasma components. The last objective of this study aimed to investigate the effects of single layer centrifugation (SLC) with Equicoll on jackass sperm parameters and its interactions with the female reproductive tract. While SLC-Equicoll was found to increase the percentages of viable spermatozoa after selection, the impact on sperm motility parameters was marginal. In addition, removal of seminal plasma components through SLC-Equicoll increased sperm phagocytosis by polymorphonuclear neutrophils (PMNs), thereby confirming that seminal plasma modulates the interaction between sperm and PMNs. In conclusion, this Dissertation indicates that although the activities of seminal plasma antioxidants differ between stallions and jackasses, SOD appears to be a good predictor of sperm cryotolerance in these two species. In addition, this Dissertation has also demonstrated, for the first time, that the modulating role of seminal plasma with regard to the interaction between jackass spermatozoa and PMNs is more apparent than that observed when oxidative stress is induced by hydrogen peroxide