Browsing by Subject "Semen"

Now showing 1 - 15 of 15
  • Thumbnail Image
    Publication
    Open Access
    Calidad seminal del pez piracanjuba (Brycon orbignyanus) post-descongelación
    (Murcia: Universidad de Murcia, Servicio de Publicaciones, 2001) Murgas, L. D. S.; Gualhanone, A.; Silva, M.O.B; Mello, C.B; Freitas, R.T.F.; Zangeronimo, M.G.; Facultad de Veterinaria
  • Thumbnail Image
    Publication
    Open Access
    Determinantes asociados a la variabilidad de la calidad seminal :un estudio de seguimiento
    (Universidad de Murcia, 2019-01-09) Pérez Palazón, Consuelo; Mendiola Olivares, Jaime; Torres Cantero, Alberto Manuel; Escuela Internacional de Doctorado
    INTRODUCCIÓN: Múltiples factores podrían ejercen un impacto potencial sobre la calidad seminal humana, y de hecho los determinantes de la variabilidad de la calidad seminal son aún ampliamente desconocidos. Los hábitos de vida (sexuales, dieta, ejercicio físico, estrés, tabaco, alcohol, cafeína, etc.) y un marcador de exposición prenatal a andrógenos [distancia anogenital, (AGD)] podrían ser factores importantes y que podrían estar involucrados en esta cuestión. Por tanto, el objetivo general de este trabajo fue describir y analizar los factores asociados a la variabilidad de la calidad seminal humana. MATERIAL Y MÉTODO: Se llevó a cabo un estudio prospectivo y de seguimiento realizando medidas repetidas de análisis de calidad seminal en 19 varones voluntarios sanos durante un año. La obtención de las muestras seminales fue aproximadamente cada 4-6 semanas. Siguiendo las directrices de la OMS, se evaluaron el volumen, concentración, recuento total, movilidad y morfología espermática y la fragmentación del ADN espermático. Se tomaron dos tipos de mediciones de AGD, desde el ano hasta la base posterior del escroto (AGDAS) y hasta la inserción cefálica del pene (AGDAP) en cada individuo. Los sujetos cumplimentaron encuestas epidemiológicas sobre hábitos de vida y exposiciones ambientales en cada una de las entrevistas. Se calculó el porcentaje de coeficiente de variación (CV) intraindividual (%CVi) y el CV interindividual (%CVe) para los parámetros seminales y la fragmentación del ADN espermático, y se examinaron sus diferencias estadísticas con respecto a sus respuestas a los factores estudiados. La asociación entre la AGD y los parámetros seminales se estudió utilizando tres herramientas estadísticas: a) coeficiente de variación general (CV) y coeficiente de variación intraindividual (CVi); b) modelos lineales generales para medidas repetidas; y c) modelos mixtos de efectos fijos. RESULTADOS: La mayor variabilidad intraindividual se observó en el recuento total y la concentración espermática (CVi=74,5% y 65,6% respectivamente), seguidos de la morfología (CVi=41,2%) y el volumen de eyaculado (CVi=30,1%) y por último en la movilidad (CVi=14,6%). Un patrón similar se observó para la variabilidad interindividual, aunque los CVe fueron muy superiores a los CVi. Se obtuvieron resultados comparables al considerar los hábitos de vida. En general, los CVi y los CVe fueron más bajos en sujetos que practicaban habitualmente ejercicio ligero o moderado, o en consumidores no habituales de vino, cerveza o café. El CVi y CVe para el porcentaje de fragmentación espermática fueron 47,2% y 262,9% respectivamente. Los CV fueron significativamente distintos para todas las variables estudiadas, a excepción de la exposición a tóxicos ambientales (similar CV) y el ejercicio físico ligero (similar CVi). Dicho índice también se relacionó positivamente con el número de horas empleadas en actividades sedentarias (p-valor = 0,05). Las mediciones de AGD se asociaron con la variabilidad de los parámetros seminales. Los varones con una AGDAP acortada presentaron una mayor variabilidad intraindividual en la concentración, recuento total y la morfología espermática. Sin embargo, se observó una mayor variabilidad en la movilidad espermática total (progresiva y no progresiva) en varones con una AGDAS alargada. Además, una AGDAS acortada se asoció con una variabilidad intraindividual menor en la movilidad espermática total. CONCLUSIONES: Nuestros resultados sugieren que un único análisis seminal podría considerarse más fiable o consistente si se practica habitualmente ejercicio ligero o moderado, o no está presente un consumo habitual de vino, cerveza o café. No obstante, la práctica de ejercicio intenso o moderado/intenso, la exposición a tóxicos o el estrés psicológico, son factores que podrían predecir una medida más constante de la fragmentación del ADN espermático. La AGD podría ser útil para determinar la variabilidad intraindividual en los parámetros seminales.
  • Thumbnail Image
    Publication
    Open Access
    Epidemiología nutricional y calidad seminal. Análisis de la composición de la dieta y su asociación con la calidad espermática.
    (2013-10-03) Cutillas Tolín, Ana; Torres Cantero, Alberto Manuel; López Espín, José Juan; Mendiola Olivares, Jaime; Mínguez Alarcón, Lidia; Facultad de Medicina
    Antecedentes/ Objetivos Desde los últimos cincuenta años la calidad seminal ha disminuido de forma progresiva. Nuestro objetivo es investigar si la calidad seminal en hombres jóvenes está asociada con la dieta. Material y Métodos Este estudio transversal comprende a 215 jóvenes universitarios de Murcia. Se evaluó la dieta a través de un cuestionario de frecuencia alimentaria. Se identificaron patrones dietéticos mediante análisis factorial, de clústers e índices de calidad de la dieta. Se estudió su relación con la calidad seminal. Resultados Un patrón dietético “mediterráneo” se asocia con mejores recuentos espermáticos y de espermatozoides móviles. No se observó una asociación lineal entre la calidad seminal y los índices aMED y AHEI. La movilidad espermática total estaba relacionada positivamente con altas ingestas de los ácidos grasos α-linolénico (ALA) y esteárico. Conclusión La calidad seminal de la población joven y sana podría verse influenciada por los patrones de ingesta alimentaria. Palabras clave: ácidos grasos, calidad seminal, dieta, dieta Mediterránea, índice AHEI, índice aMED, ingesta alimentaria, patrones dietéticos. Abstract Introduction Over the past fifty years there have been evidences of a decline in semen quality. This study investigates whether semen quality in young men is associated with diet. Materials and Methods This cross sectional study included 215 university students from Murcia. Diet was assessed through a food frequency questionnaire. We identified dietary patterns using factor and cluster analysis and diet quality indexes. We evaluated its relationship with semen quality. Results “Mediterranean” pattern was associated with higher sperm counts and mobile spermatozoa counts. A lineal association between aMED and AHEI indexes was not observed. Total motility was related to higher intakes of fatty acids α-linolenic (ALA) and stearic. Conclusion The semen quality in healthy young population could be influenced by patterns of dietary intake Keywords: aMED index, AHEI index, dietary intake, dietary patterns, fatty acids, Mediterranean diet, semen quality.
  • Thumbnail Image
    Publication
    Open Access
    Estudio sobre la calidad seminal en jóvenes universitarios de la Región de Murcia
    (2015-11-03) Sarabia Cos, Laura; Torres Cantero, Alberto Manuel; Mendiola Olivares, Jaime; Arense Gonzalo, Julián Jesús; Facultad de Medicina
    Diversos estudios muestran una disminución en la concentración espermática en las últimas décadas, aunque en este hecho se han observado diferencias geográficas importantes. El descenso de la concentración espermática se ha atribuido a exposiciones a tóxicos y contaminantes ambientales, estilos de vida o ciertos factores nutricionales pero que, en gran medida, son aún desconocidos, permanecen inexplorados o no han sido suficientemente estudiados. Entre 2010 y 2011 se llevó a cabo un estudio transversal en jóvenes universitarios de la Región de Murcia (n=215) con el propósito de estudiar su calidad seminal. Los participantes proporcionaron una muestra seminal, se les realizó un examen andrológico y cumplimentaron cuestionarios epidemiológicos sobre hábitos de vida, incluido un cuestionario de frecuencia alimentaria. El análisis de los parámetros seminales se realizó siguiendo las recomendaciones de la OMS (2010) y también se analizó la fragmentación del ADN espermático mediante el método de dispersión de la cromatina espermática (SCD). A partir de los datos de este estudio, en la presente tesis doctoral se profundiza en el estudio de la calidad seminal en jóvenes varones, abordando el problema desde distintos aspectos. En el primer trabajo se estudió la asociación entre la ingesta dietaria de nutrientes antioxidantes y la calidad seminal en dichos varones. Los resultados indicaron una asociación positiva entre el consumo de distintos nutrientes antioxidantes (criptoxantina, vitamina C, licopeno y b-caroteno) y el recuento total de espermatozoides móviles. El volumen seminal también aumentó con una mayor ingesta de vitamina C, licopeno y b-caroteno. En conclusión, nuestro estudio sugirió que algunos parámetros espermáticos podrían ser sensibles a la ingesta de nutrientes antioxidantes, y que la recomendación actual de ingesta de vitamina C podría ser insuficiente para alcanzar el máximo beneficio en términos de calidad seminal. En el segundo trabajo, el objetivo fue examinar si la calidad seminal había variado durante la última década entre los jóvenes del sureste español. Para llevar a cabo este trabajo se utilizaron los datos obtenidos en un estudio anterior realizado en Almería entre 2001 y 2002. Los datos del estudio de Almería se incluyeron en un análisis de tendencias junto con los datos relativos a los jóvenes estudiantes murcianos. Se utilizó modelos de regresión lineal múltiple para analizar en la población combinada un efecto de cohorte de nacimiento durante el período de estudio 2001-2011. Nuestros resultados indicaron que el recuento total y la concentración espermática podrían haber disminuido en los jóvenes del Sureste español durante la última década, mostrando una tendencia temporal adversa en dichos parámetros. En el sur de España recientemente ha tenido lugar un aumento de la industrialización, y con ello, un aumento del riesgo de posibles exposiciones potencialmente adversas que podrían afectar a distintos parámetros reproductivos masculinos. En el tercer trabajo se estudió la fragmentación del ADN espermático en los jóvenes varones. Dicha fragmentación podría estar relacionada con procesos de estrés oxidativos y comprometer la calidad seminal. Por una parte, se describieron y analizaron los índices de fragmentación del ADN espermático (SDF) de las muestras tras la eyaculación, lo que se denominó como SDF basal. Y por otro lado, se estudió la dinámica de la fragmentación del ADN espermático, es decir, el incremento de la fragmentación del ADN con el tiempo tras la eyaculación. Para estudiar la dinámica se incubaron las muestras a 37ºC durante 2.5, 17 y 24 horas y se calculó la tasa de fragmentación del ADN espermático (rate SDF; rSDF). Los resultados obtenidos indicaron unos valores medios de SDF basal relativamente altos comparados con otros estudios publicados. La rSDF fue mayor durante las primeras horas tras la eyaculación. Por otra parte, las muestras con SDF basal superior a 30% presentaron una rSDF mayor durante las primeras horas de incubación comparadas con las muestras con niveles basales inferiores a 15%. Summary In the last decades several studies have shown a decrease in human sperm concentrations. In spite of this fact, there have been significant geographical differences. The decline in sperm count has been attributed to exposure to environmental toxins and pollutants, lifestyle or certain nutritional factors which are still largely unknown, unexplored or have been understudied. Between 2010 and 2011 we conducted a cross-sectional study among university students in the Region of Murcia (n = 215) in order to study their sperm quality. Participants provided a semen sample, underwent an andrological examination, and filled out epidemiological questionnaires on lifestyle and a food frequency questionnaire. The seminal analysis was performed following the WHO guidelines (2010) and sperm DNA fragmentation was also analyzed by the sperm chromatin dispersion method (SCD). From all this data, the current thesis explores the semen quality in young males, approaching the problem from different perspectives. In the first study, the association between dietary intake of antioxidant nutrients and semen quality was studied. The results indicated a positive association between the consumption of various antioxidant nutrients (cryptoxanthin, vitamin C, lycopene and b-carotene) and total motile sperm count. The seminal volume also increased with a higher intake of vitamin C, lycopene and b-carotene. In conclusion, our study suggested that some sperm parameters could be sensitive to the intake of antioxidant nutrients, and that the current recommended intake of vitamin C may be insufficient to achieve the maximum benefit in terms of semen quality. In the second study, the aim was to examine whether semen quality has changed among Spanish young men in the last decade. To carry out this study we used data obtained in a previous study in Almeria between 2001 and 2002. This data was added to our current population in order to analyze the sperm quality trend in Southern Spain. Multiple linear regression models were used to analyze the combined effect of population birth cohort during the study´s period 2001-2011. Our results indicated that the total count and sperm concentration may have decline in young Spanish men over the last decade, showing an adverse temporal trend in these parameters. Southern Spain has recently gone through a growing innovation and industrialization in many areas, and with this, an increased risk of potential adverse exposures, which might affect reproductive parameters in men. In the third study, the sperm DNA fragmentation was studied in young men. Such fragmentation could be related to oxidative stress and compromise semen quality. On the one hand, sperm DNA fragmentation (SDF) of the samples after ejaculation was analyzed, which was named as basal SDF. On the other hand, the dynamics of sperm DNA fragmentation, that is the increase of DNA fragmentation after ejaculation time was studied. To study the dynamics, samples were incubated at 37°C for 2.5, 17 and 24 hours and the rate of sperm DNA fragmentation (SDF rate; rSDF) was calculated. The results showed average values of basal SDF relatively high compared with other published studies. The rSDF was higher during the first hours after ejaculation. Besides, the samples with basal SDF higher to 30% showed an elevated rSDF during the first hours of incubation compared with the samples with basal levels lower than 15 %.
  • Thumbnail Image
    Publication
    Open Access
    Evaluación de la viabilidad y motilidad espermática dosis seminales caprinas para el desarrollo de un modelo de contaminación experimental con mycoplasma spp
    (2012) Gómez-Martín, A.; Vieira, L.A; De Ondiz, A.; Paterna, A.; Gadea Mateos, Joaquín; De la Fe, C.
    En los últimos años se ha detectado en diversos centros de inseminación artificial la presencia de machos caprinos en los que se han aislado Mycoplasma agalactiae y Mycoplasma mycoides subsp. capri en muestras de semen, sin que presenten ningún síntoma clínico asociado. La capacidad de las bacterias del género Mycoplasma spp. para producir efectos perjudiciales en la calidad espermática es un hecho constatado en diversas especies animales, incluido el hombre. Además, el riesgo de una posible transmisión venérea y afectación de la calidad espermática podría comprometer los programas de mejora genética caprinos fundamentados en la inseminación artificial. El objetivo del presente trabajo es desarrollar un modelo experimental para el estudio del efecto de la contaminación seminal con Mycoplasma spp. en ganado caprino. Para ello, evaluamos la viabilidad y motilidad espermática de dosis seminales, tras dos horas de incubación a 37 ºC en muestras previamente transportadas bajo dos condiciones de temperatura (4-5 °C y 15-16 ºC). Posteriormente se estudió el efecto de la adición del medio PPLO, en el que se preparan los inóculos de Mycoplasma spp., sobre la calidad espermática. El transporte de dosis seminales a 4 ºC ofrece mejores resultados de motilidad total en semen incubado a 37 ºC durante 120 minutos. El medio PPLO no ejerció efecto significativo sobre los porcentajes de espermatozoides vivos o mótiles totales a lo largo de 150 minutos de incubación. Con dicho medio, se pueden obtener valores de viabilidad y motilidad (total y progresiva) espermática superiores al 50% después de 150 y 60 minutos de incubación, respectivamente. En conjunto, este modelo permitiría el desarrollo de contaminaciones experimentales de dosis seminales caprinas con Mycoplasma spp. con el fin de evaluar su efecto sobre la viabilidad y motilidad espermática.
  • Thumbnail Image
    Publication
    Open Access
    Expresión proteómica del plasma seminal del toro criollo San Martinero y su relación con la fertilidad y desarrollo embrionario in vitro en condiciones del trópico bajo colombiano.
    (2013-10-09) Barajas Pardo, Diana Patricia; Coy Fuster, Pilar; Facultad de Veterinaria
    La producción animal en un país tropical como Colombia se ve limitada, entre otras cosas, por factores ambientales adversos, como temperatura, humedad relativa y radiación solar altas a lo largo del año. En los sistemas de producción bovina colombiana existen razas criollas, como la San Martinero, que ha demostrado los mejores índices reproductivos (fertilidad, natalidad, intervalo entre partos, etc.). Para esclarecer el porqué de la mayor fertilidad, la presente investigación se realiza con el propósito de estudiar proteínas del plasma seminal y su asociación con indicadores de calidad espermática pre- (volumen seminal, concentración, motilidad, viabilidad, morfología, índice de calidad espermática (ICE) y espermatozoides congelables) y post-descongelación (motilidad, viabilidad, morfología e ICE), fertilidad y producción in vitro de blastocistos de esta raza criolla en la época lluviosa comparada con la época seca y con la raza Cebú Brahman. Para la investigación se seleccionaron al azar 7 toros reproductores de la raza San Martinero (SM) y 7 de la raza Cebú Brahman. Los ejemplares se mantuvieron bajo pastoreo en Brachiaria decumbens en el Centro de Investigaciones la Libertad de Corpoica. La evaluación por época se realizó en SM tanto en período lluvioso como de sequía y la evaluación entre razas (SM y Cebú) únicamente se realizó en lluvias. Los muestreos y análisis seminales por toro en fresco se realizaron tres veces y post descongelación 5 veces para evaluación de calidad espermática, fecundación y producción in vitro de blastocistos. El análisis de la información se basó en Modelos mixtos con medidas repetidas en el tiempo y en ecuaciones de predicción y análisis de componentes principales. Los resultados del estudio permitieron la generación de los primeros mapas referenciales uni y bidimensionales de la raza San Martinero y de la Cebú Brahman caracterizados bajo las condiciones climáticas del trópico bajo colombiano. Los resultados obtenidos de los toros SM para la época de lluvias en términos de indicadores de calidad espermática, fecundación in vitro y efecto en el desarrollo embrionario vistos en forma integrada permiten predecir la fertilidad in vivo. Los valores de estos indicadores no generaron grandes cambios entre la época seca o lluviosa, pero sí superaron a los indicadores de eficiencia reproductiva de toros reproductores de la raza Cebú Brahman. Los cambios de las variables estudiadas que determinan la calidad espermática de los toros por efecto de las proteínas plasmáticas son respuesta de la participación en forma de clúster que integra, bajo un modelo de predicción, proteínas con y sin cambios significativos y no en forma independiente. En este estudio reconocimos algunos modelos cuya asociación explican en gran forma el comportamiento de las variables de calidad seminal (volumen seminal, concentración, motilidad, viabilidad y morfología espermáticas), del ICE y de la producción de espermatozoides congelables, en función de la raza, la época climática, o de acuerdo a si se trata de semen fresco o descongelado. La investigación realizada permitió identificar que el punto de proteína A2301, de de peso molecular 16.74 kDa y pI 4.78, se asocia con los toros seleccionados como de mayor fertilidad, mientras que los puntos A4113 y A4119, de pesos moleculares 15.37 y 15.49 kDa y pI de 5.38 y 5.15, respectivamente, se relacionaron con los toros de baja fertilidad. Los resultados encontrados de la evaluación de calidad espermática del toro criollo SM y de participación de las proteínas del plasma seminal estudiados entre época y comparado con la raza Cebú Brahman muestran la superioridad del bovino Criollo y señalan a la proteómica como una gran herramienta para la selección de toros. Animal production in a tropical country like Colombia is constrained, among other things, by adverse environmental factors such as temperature, relative humidity and high solar radiation throughout the year. In cattle production systems Colombian landraces exists, such as San Martinero, which has shown the best reproductive rates (fertility, birth, calving interval, etc..). To clarify the reason for its increased fertility, this research was done with the purpose of studying seminal plasma proteins and their association with indicators of sperm quality pre- (volume, concentration, motility, viability, morphology, sperm quality index (ICE) and freezable sperm) and post-thaw (motility, viability, morphology and ICE), fertility and in vitro production of blastocysts in this race Creole during the rainy season compared to the dry season and to Zebu Brahman semen. Seven San Martinero (SM) bulls and 7 Zebu Brahman were randomly selected for the research. The specimens were kept under grazing with Brachiaria decumbens in the Research Center Corpoica “La Libertad”. The evaluation was conducted in SM both in the rainy and drought seasons and evaluation between races (SM and Zebu) only took place in the rainy season. Sampling and quality assessment for bull fresh semen were performed three times; for post-thaw sperm, fertilization and in vitro blastocyst production, the evaluations were 5 times repeated. The data analysis was based on mixed models with repeated measures over time and predictive equations and principal component analysis. The results of the study allowed the first generation and two-dimensional reference maps of seminal plasma proteins of San Martinero and Zebu Brahman races characterized under tropical climatic conditions in Colombia. The results of the SM bulls for the rainy season in terms of indicators of sperm quality, fertilization and in vitro embryo development, showed in an integrated way, let predict the in vivo fertility. The values of these indicators did not produce major changes between the dry and rainy season, but outscored the reproductive efficiency indicators of breeding bulls of Brahman Zebu. The changes of the studied variables that determine the sperm quality of the bulls by the effect of the plasma proteins are the response for their participation in a cluster manner and not in an independently manner, that integrates, under a prediction model, proteins with and without significant changes. In this study, some models were recognized whose association explains the behavior of variables that account for semen quality (seminal volume, concentration, motility, viability and sperm morphology), SQI and sperm production, depending on the race, the climatic season, or according to whether it was fresh or frozen-thawed sperm. The investigation identified the protein point A2301, with molecular weight of 16.74 kDa and pI 4.78, associated with bulls selected as those with higher fertility, while points A4113 and A4119, with molecular weight 15.49 15.37 kDa and pI 5.38 and 5.15, respectively, were associated with low fertility bulls. The results of the evaluation of native SM bull sperm quality and participation of seminal plasma proteins studied between seasons, and compared with Zebu Brahman cattle, show the superiority of San Martinero and point to proteomics as a great tool for selecting of bulls.
  • Thumbnail Image
    Publication
    Open Access
    Extracellular vesicles isolated from porcine seminal plasma exhibit different tetraspanin expression profiles
    (2019-08-09) Barranco, Isabel; Padilla, Lorena; Parrilla, Inmaculada; Alvarez-Barrientos, Alberto; Pérez-Patiño, Cristina; Peña, Fernando; Martínez, Emilio A; Rodriguez-Martínez, Heriberto; Roca, Jordi; Roca, Jordi; Medicina y Cirugía Animal
    Seminal extracellular vesicles (EVs) include exosomes (ø 40-120 nm) and microvesicles (MVs, ø 120-1000 nm), which would be involved in multiple functional reproductive roles. The study aimed to establish which EV subtypes are present in pig semen, using a high-resolution flow cytometer to explore differences in their tetraspanin expression profile. The EVs were isolated from 12 pig ejaculates using serial ultracentrifugation and characterized by dynamic light scattering and electron microscopy for size and morphology as well as for tetraspanin expression using flow cytometry with Carboxyfluorescein succinimidyl ester (CFSE) and antibodies against CD9, CD63 and CD81. Pig semen contained a heterogeneous EV-population regarding size and morphology. Flow cytometric analysis demonstrated that the proportion of EVs expressing CD63 and CD9 was higher in MVs (P < 0.001 and P < 0.05, respectively) than in exosomes, while the opposite was true for CD81; higher (P < 0.001) in exosomes than in MVs. In conclusion, (1) the new generation of flow cytometers are able to accurately identify EVs and to gate them in two size-different populations named exosomes and MVs. (2) Tetraspanins CD9, CD63 and CD81 are present in both seminal EVs, albeit with exosomes and MVs differing in expression profiles, suggesting dissimilar cargo and binding affinity.
  • Thumbnail Image
    Publication
    Open Access
    Extracellular vesicles would be involved in the release and delivery of seminal TGF-β isoforms in pigs
    (Frontiers Media, 2023-02-10) Padilla, Lorena; Barranco, Isabel; Parra, Ana; Parrilla, Inmaculada; Rodríguez Martínez, Heriberto; Lucas, Xiomara; Roca, Jordi; Martínez Hernández, Jesús; Pastor García, Luis Miguel; Medicina y Cirugía Animal
    Introduction: pig seminal plasma (SP) is rich in active forms of all three isoforms (1-3) of transforming growth factor β (TGF-β), a chemokine modulatory of the immune environment in the female genital tract once semen is delivered during mating or artificial insemination (AI). The present study aimed to examine how TGF-βs are secreted by the epithelium of the male reproductive tract and how they are transported in semen, emphasizing the interplay with seminal extracellular vesicles (sEVs). Methods: Source of TGF-βs was examined by immunohistochemistry in testis, epididymis, and accessory sex glands, by immunocytochemistry in ejaculated spermatozoa, and by Luminex xMAP® technology in SP and sEVs retrieved from healthy, fertile male pigs used as breeders in AI programs. Results: All three TGF-β isoforms were expressed in all reproductive tissues explored and would be released into ductal lumen either in soluble form or associated with sEVs. Ejaculated spermatozoa expressed all three TGF-β isoforms, both inside and outside, probably the outer one associated with membrane-bound sEVs. The results confirmed that pig SP contains all three TGF-β isoforms and demonstrated that a substantial portion of them is associated with sEVs. Discussion: Seminal EVs would be involved in the cellular secretion of the active forms of seminal TGF-β isoforms and in their safe transport from the male to the female reproductive tract.
  • Thumbnail Image
    Publication
    Open Access
    High total antioxidant capacity of the porcine seminal plasma (SP-TAC) relates to sperm survival and fertility
    (2015-12-21) Barranco, Isabel; Tvarijonaviciute, Asta; Perez-Patiño, Cristina; Parrilla, Inmaculada; Cerón, Jose J; Martínez, Emilio A; Rodriguez-Martinez, Heriberto; Roca, Jordi; Medicina y Cirugía Animal
    The study attempted to clarify the role of total antioxidant capacity of seminal plasma (SP-TAC) on boar sperm survival and fertility after artificial insemination (AI). SP-TAC differed (P < 0.001) among boars (n° = 15) and, to a lesser degree, among ejaculates within male (4 ejaculates/boar). SP-TAC also differed (P < 0.001) among ejaculate fractions (43 ejaculates and 3 fractions per ejaculate), of which the sperm-peak portion of the sperm rich ejaculate fraction (SRF) had the highest SP-TAC. SP-TAC was not correlated with sperm quality (motility and viability) or functionality (intracellular ROS generation and lipid peroxidation) of liquid AI-semen samples stored at 17 °C for 72 h (90 AI-samples), but the decline in sperm quality was larger (P < 0.05) in ejaculates with low, compared with high SP-TAC (hierarchically grouped). The SP-TAC differences among ejaculate portions agree with sperm cryosurvival rates (14 ejaculates from 7 boars), showing sperm from sperm-peak portion better (P < 0.01) post-thaw quality and functionality than those from the entire ejaculate (mainly post-SRF). Boars (n° = 18) with high SP-TAC (hierarchically grouped) had higher (P < 0.05) fertility outcomes (5,546 AI-sows) than those with low SP-TAC. Measurement of SP-TAC ought to be a discriminative tool to prognosis fertility in breeding boars.
  • Thumbnail Image
    Publication
    Open Access
    Immunophenotype profile by flow cytometry reveals different subtypes of extracellular vesicles in porcine seminal plasma
    (2024-01-23) Barranco, Isabel; Alvarez-Barrientos, Alberto; Parra, Ana; Martinez-Diaz, Pablo; Lucas, Xiomara; Roca, Jordi; Medicina y Cirugía Animal
    Background: Porcine seminal plasma (SP) is endowed with a heterogeneous population of extracellular vesicles (sEVs). This study evaluated the immunophenotypic profile by high-sensitivity flow cytometry of eight sEV subpopulations isolated according to their size (small [S-sEVs] and large [L-sEVs]) from four different SP sources, namely three ejaculate fractions (the first 10 mL of the sperm rich fraction [SRF-P1], the remaining SRF [SRF-P2], and the post-SRF [PSRF]) and entire ejaculate (EE). Methods: Seminal EVs were isolated using a size exclusion chromatography-based protocol from six SP pools (five ejaculates/pool) of each SP source and characterized using complementary approaches including total protein (BCA™assay), particle size distribution (dynamic light scattering), morphology (transmission electron microscopy), and purity (albumin by Western blot). Expression of CD9, CD63, CD81, CD44 and HSP90β was analyzed in all sEV subpopulations by high-sensitivity flow cytometry according to MIFlowCyt-EV guidelines, including an accurate calibration, controls, and discrimination by CFSE-labelling. Results: Each sEV subpopulation exhibited a specific immunophenotypic profile. The percentage of sEVs positive for CD9, CD63, CD81 and HSP90β differed between S- and L-sEVs (P < 0.0001). Specifically, the percentage of sEVs positive for CD9 and CD63 was higher and that for CD81 was lower in S- than L-sEVs in the four SP sources. However, the percentage of HSP90β-positive sEVs was lower in S-sEVs than L-sEVs in the SRF-P1 and EE samples. The percentage of sEVs positive for CD9, CD63, and CD44 also differed among the four SP sources (P < 0.0001), being highest in PSRF samples. Notably, virtually all sEV subpopulations expressed CD44 (range: 88.04-98.50%). Conclusions: This study demonstrated the utility of high-sensitivity flow cytometry for sEV immunophenotyping, allowing the identification of distinct sEV subpopulations that may have different cellular origin, cargo, functions, and target cells.
  • Thumbnail Image
    Publication
    Restricted
    Improvement of boar sperm cryosurvival by using single-layer colloid centrifugation prior freezing
    (Elsevier, 2012-09-15) Martínez Alborcia, M. J.; Morrell, Jane M.; Parrilla Riera, Inmaculada; Barranco Cascales, Isabel; Vázquez Autón, José María; Martínez García, Emilio; Roca Aleu, Jorge; Medicina y Cirugía Animal
    The aim of this experimental study was to evaluate the effectiveness of sperm selection using single-layer centrifugation (SLC) prior to freezing on the sperm cryosurvival of boar ejaculates. Twenty-four sperm rich ejaculate fractions (SREF), collected from 24 boars (one per boar), were divided into two groups according to their initial semen traits: standard (n = 15) and substandard (n = 9). Semen samples from each SREF were split in two aliquots, one remained untreated (control samples) and the other was single-layer centrifuged (500 g for 20 min) using 15 mL of Androcoll-P Large (SLC samples). The yield of total, motile (assessed by CASA) and viable (cytometrically evaluated after staining with H-42, propidium iodide (PI) and FITC-PNA) sperm after SLC was higher (P < 0.05) in standard than substandard semen samples. The semen samples were cryopreserved using a standard 0.5-mL straw freezing protocol. Post-thaw sperm motility and viability (assessed at 30 and 150 min post-thawing) were higher (P < 0.05) in SLC than in control samples, regardless of the initial semen traits of the ejaculates. Additionally, thawed spermatozoa from SLC samples were more resistant (P < 0.05) to lipid peroxidation (BIOXYTECH MDA-586 Assay Kit) than those from control samples, regardless of the initial semen traits of the ejaculates. The SLC-treatment also influenced the functionality of thawed spermatozoa undergoing an in vitro capacitation process. The percentage of viable sperm showing high membrane fluidity (assessed with merocyanine 540) was lower (P < 0.05) in the SLC than in the control samples, regardless of the initial semen traits of the ejaculates. Thawed viable spermatozoa of SLC samples generated less (P < 0.05) reactive oxygen species (assessed with CM-H(2)DCFDA) than those of control samples in the substandard ejaculates. These findings indicate that the sperm selection before freezing using SLC improves the freezability of boar sperm.
  • Thumbnail Image
    Publication
    Open Access
    Metabolite Profiling of Pig Seminal Plasma Identifies Potential Biomarkers for Sperm Resilience to Liquid Preservation
    (Frontiers Media, 2021-05-28) Mateo-Otero, Yentel; Fernández-López, Pol; Ribas-Maynou, Jordi; Roca, Jordi; Miró, Jordi; Yeste, Marc; Barranco, Isabel; Medicina y Cirugía Animal
    Metabolomic approaches allow the study of downstream gene expression events since metabolites are considered as the products of cell signaling pathways. For this reason, many studies in humans have already been conducted to determine the influence of the metabolites present in seminal plasma (SP) on sperm physiology, and to identify putative biomarkers. However, in livestock species, these relationships are yet to be uncovered. Thus, the present study aimed to explore: (i) if concentrations of metabolites in pig SP are related to sperm quality and functionality, and (ii) if they could predict the sperm resilience to liquid storage at 17°C. To this end, 28 ejaculates were individually collected and split into three aliquots: one was used for SP analysis through nuclear magnetic resonance (NMR) spectroscopy; another served for the evaluation of sperm concentration and morphology; and the last one was utilized to determine sperm functionality parameters using computer-assisted sperm analysis (CASA) and flow cytometry after 0 h and 72 h of liquid-storage at 17°C. NMR analysis allowed the identification and quantification of 23 metabolites present in pig SP which, except for fumarate, were not observed to follow a breed-dependent behavior. Moreover, specific relationships between metabolites and sperm variables were identified: (i) glutamate, methanol, trimethylamine N-oxide, carnitine, and isoleucine were seen to be related to some sperm quality and functionality parameters evaluated immediately after semen collection; (ii) leucine, hypotaurine, carnitine and isoleucine were found to be associated to the sperm ability to withstand liquid storage; and (iii) Bayesian multiple regression models allowed the identification of metabolite patterns for specific sperm parameters at both 0 h and 72 h. The identification of these relationships opens up the possibility of further investigating these metabolites as potential sperm functional biomarkers.
  • Thumbnail Image
    Publication
    Open Access
    Post-Thaw quality of spermatozoa frozen with three different extenders in the Murciano Granadina goat breed.
    (MDPI, 2023-01-16) Galián, S.; Peinado, B.; Almela, L.; Poto, A.; Ruiz, S.; Fisiología
    In caprine species, the semen quality of thawed samples still needs to be improved in order for this type of semen to provide an acceptable pregnancy rate when it is used for artificial insemination. It is therefore essential to have an adequate freezing protocol and extender for this species. By adapting freezing protocols that have proven successful in other species, such as pigs, together with the development of an extender that protects the sperm cells from damage caused by the freezing and thawing processes, and taking into account its composition in nutrients and cell-membrane-stabilizing substances, it has been possible, at least in vitro, to achieve high sperm quality in buck semen. In vivo studies of the fertilizing capacity of the semen cryopreserved in this way will be the next step to verify the effectiveness of the extender and semen-freezing protocol described in this work.
  • Thumbnail Image
    Publication
    Open Access
    Seminal plasma mitigates the adverse effect of uterine fluid on boar spermatozoa
    (Elsevier, 2019-09-15) Luongo, Chiara; Abril Sánchez, Silvia; Hernández Cifre, José G.; García Vázquez, Francisco A.; Química Física
    After natural or artificial insemination, spermatozoa start their journey within the uterus to reach the site of fertilization, but only few of them attain this goal. Part of this spermatozoa loss happens in the uterus, in which uterine fluid (UF) seems to be involved. It is known from other species that UF provokes damage to spermatozoa, which is avoided when seminal plasma (SP) is present. Therefore, the aim of the present study was to evaluate the effect of UF on the quality of ejaculated (previously contacted with SP) and epididymal (without previous contact with SP) boar spermatozoa analyzing motility, kinetic parameters, viability and acrosome integrity in the presence or absence of SP over time. Three experimental groups were evaluated in each source of spermatozoa (ejaculated and epididymal): 1) Control: spermatozoa with 20% of SP; 2) UF: spermatozoa with 20% of UF; and 3) UF-SP: spermatozoa with 20% of SP and 20% of UF. Total and progressive motility, kinetic parameters (VCL, VSL, VAP, LIN, STR, WOB, and BCF), viability and acrosome damage were analyzed at 15, 60, 120 and 180 min after incubation. Total and progressive motility decreased when ejaculated spermatozoa were incubated in UF in contrast to control and UF-SP groups (p < 0.0007), with no differences between control and UF-SP. The VCL decreased in the UF group compared to the control and UF-SP groups in ejaculated spermatozoa (p = 0.0002). The VSL, VAP, LIN and STR kinetic parameters were greater when ejaculated spermatozoa were incubated in the UF-SP group than in the UF group (all: p < 0.02). Acrosome damage increased in ejaculated and epididymal spermatozoa incubated in the UF group compared to the control and UF-SP groups (both: p < 0.0001). Also, the viability of epididymal spermatozoa decreased in the UF group, while it did not change in the control and UF-SP groups (p = 0.0004). The rest of the parameters in either ejaculated or epididymal spermatozoa did not differ between experimental groups, except for WOB when epididymal spermatozoa were used (UF-SP higher than the control group, with UF being similar for both; p = 0.03). In conclusion, both ejaculated and epididymal spermatozoa are affected by UF, suggesting a negative effect on their quality. This negative effect is reduced by the presence of SP, improving the spermatozoa functionality, preserving motility, viability and acrosome integrity.
  • Thumbnail Image
    Publication
    Open Access
    The Proteome of Large or Small Extracellular Vesicles in Pig Seminal Plasma Differs, Defining Sources and Biological Functions
    (Elsevier -- American Society for Biochemistry and Molecular Biology, 2023-04) Barranco, Isabel; Sánchez-López, Christian M; Bucci, Diego; Alvarez-Barrientos, Alberto; Rodriguez-Martinez, Heriberto; Marcilla, Antonio; Roca, Jordi; Medicina y Cirugía Animal
    Seminal plasma contains many morphologically heterogeneous extracellular vesicles (sEVs). These are sequentially released by cells of the testis, epididymis, and accessory sex glands and involved in male and female reproductive processes. This study aimed to define in depth sEV subsets isolated by ultrafiltration and size exclusion chromatography, decode their proteomic profiles using liquid chromatography-tandem mass spectrometry, and quantify identified proteins using sequential window acquisition of all theoretical mass spectra. The sEV subsets were defined as large (L-EVs) or small (S-EVs) by their protein concentration, morphology, size distribution, and EV-specific protein markers and purity. Liquid chromatography-tandem mass spectrometry identified a total of 1034 proteins, 737 of them quantified by SWATH in S-EVs, L-EVs, and non-EVs-enriched samples (18-20 size exclusion chromatography-eluted fractions). The differential expression analysis revealed 197 differentially abundant proteins between both EV subsets, S-EVs and L-EVs, and 37 and 199 between S-EVs and L-EVs versus non-EVs-enriched samples, respectively. The gene ontology enrichment analysis of differentially abundant proteins suggested, based on the type of protein detected, that S-EVs could be mainly released through an apocrine blebbing pathway and be involved in modulating the immune environment of the female reproductive tract as well as during sperm-oocyte interaction. In contrast, L-EVs could be released by fusion of multivesicular bodies with the plasma membrane becoming involved in sperm physiological processes, such as capacitation and avoidance of oxidative stress. In conclusion, this study provides a procedure capable of isolating subsets of EVs from pig seminal plasma with a high degree of purity and shows differences in the proteomic profile between EV subsets, indicating different sources and biological functions for the sEVs.