Dehesa Comunidad:
http://hdl.handle.net/10662/6860
2024-03-28T14:23:42ZSelected Metabolites Found in Equine Oviductal Fluid do not Modify the Parameters Associated to Capacitation of the Frozen-thawed Equine Spermatozoa "In Vitro"
http://hdl.handle.net/10662/17171
Títulos: Selected Metabolites Found in Equine Oviductal Fluid do not Modify the Parameters Associated to Capacitation of the Frozen-thawed Equine Spermatozoa "In Vitro"
Autores/as: Fernández Hernández, Pablo; García Marín, Luis Jesús; Bragado González, María Julia; Domingo, Andrés; González Fernández, Lauro; Macías García, Beatriz
Resumen: In the horse, a repeatable protocol for in vitro fertilization has not been developed, possibly due to incomplete sperm capacitation. We have previously identified the metabolites present in equine oviductal fluid (OF). We aimed to test the effects of different metabolites found in equine oviductal fluid on quality parameters of frozen-thawed spermatozoa. Different concentrations of myoinositol (5–25 mM), lactate (6–60 mM), glycine (0.1–5 mM), β-alanine (1–6 mM), and histamine (0.05–0.4 mM) were added independently to modified Whitten's medium (pH = 7.25). Thawed equine spermatozoa (three stallions, one ejaculate per stallion, n = 3) were incubated for 2 hours at 37˚C in presence of the selected metabolites. After sperm incubation, total motility (TM), and progressive motility (PM) were evaluated by computer-assisted sperm analysis. Viability (SYBR-14+/PI−), mitochondrial membrane potential (ΔΨm) (JC-1), acrosome reaction (PNA+/PI−) and reactive oxygen species (ROS) production (CellRox+/PI−), were evaluated by flow cytometry. Protein tyrosine phosphorylation (PY) was evaluated by indirect immunofluorescence. Our results show that the addition of the metabolites at the dosages tested does not exert any effect on the sperm parameters analyzed. More research is needed to ascertain if metabolite addition at the dosages found in the equine OF exerts any remarkable effect on in vitro equine sperm capacitation.; En el caballo, no se ha desarrollado un protocolo repetible para la fertilización in vitro, posiblemente debido a una capacitación incompleta de los espermatozoides. Hemos identificado previamente los metabolitos presentes en el líquido oviductal equino (OF). Nuestro objetivo fue probar los efectos de diferentes metabolitos encontrados en el líquido oviductal equino sobre los parámetros de calidad de los espermatozoides congelados y descongelados. Se agregaron independientemente diferentes concentraciones de mioinositol (5 a 25 mM), lactato (6 a 60 mM), glicina (0,1 a 5 mM), β-alanina (1 a 6 mM) e histamina (0,05 a 0,4 mM). Medio de Whitten (pH = 7,25). Los espermatozoides equinos descongelados (tres sementales, un eyaculado por semental, n = 3) se incubaron durante 2 horas a 37 °C en presencia de los metabolitos seleccionados. Después de la incubación de los espermatozoides, se evaluaron la motilidad total (TM) y la motilidad progresiva (PM) mediante análisis de espermatozoides asistido por computadora. La viabilidad (SYBR-14+/PI−), el potencial de membrana mitocondrial (ΔΨm) (JC-1), la reacción del acrosoma (PNA+/PI−) y la producción de especies reactivas de oxígeno (ROS) (CellRox+/PI−), se evaluaron por flujo citometría La fosforilación de proteína tirosina (PY) se evaluó por inmunofluorescencia indirecta. Nuestros resultados muestran que la adición de los metabolitos a las dosis ensayadas no ejerce ningún efecto sobre los parámetros espermáticos analizados. Se necesita más investigación para determinar si la adición de metabolitos en las dosis encontradas en el OF equino ejerce algún efecto notable sobre la capacitación in vitro del esperma equino.
Descripción: Financiación de acceso abierto gracias al acuerdo CRUE-CSIC con Elsevier.2022-01-01T00:00:00ZExpanded equine cumulus-oocyte complexes exhibit higher meiotic competence and lower glucose consumption than compact cumulus-oocyte complexes
http://hdl.handle.net/10662/6856
Títulos: Expanded equine cumulus-oocyte complexes exhibit higher meiotic competence and lower glucose consumption than compact cumulus-oocyte complexes
Autores/as: González Fernández, Lauro; Sánchez-Calabuig, M.J.; Alves, M.G.; Oliveira, P.F.; Macedo, S.; Gutiérrez-Adán, A.; Rocha, Afonso Duarte dos Reis; Macías García, Beatriz
Resumen: Equine cumulus-oocyte complexes (COCs) are classified as compact (cCOC) or expanded (eCOC) and vary in their meiotic competence. This divergence could be related to different glucose metabolism. To test this hypothesis eCOCs, cCOCs, and expanded or compact mural granulosa cells (EC and CC respectively) were matured in vitro for 30 hours and the maturation rate, glucose metabolism, and expression of genes involved in glucose transport, glycolysis, apoptosis and meiotic competence were determined. Significant differences were found between eCOCs and cCOCs maturation rates (50% vs. 21.7 %; n = 192 and 46 respectively, p < 0.001), glucose consumption (1.8 ± 0.5 vs. 27.9 ± 5.9 nmol/COC; mean ± SEM), pyruvate production (0.1 ± 0.0 vs. 2.4 ± 0.8 nmol/COC; mean ± SEM) and lactate production (4.7 ± 1.3 vs. 64.1 ± 20.6 nmol/COC; mean ± SEM) respectively (p < 0.05). Moreover, similar glucose consumption was observed for EC and CC. Hyaluronan binding protein (TNFAIP6) expression was increased in eCOCs and EC, solute carrier family 2 (facilitated glucose transporter) member 1 (SLC2A1) was increased in eCOCs, while glycolysis-related enzymes and solute carrier family 2 (facilitated glucose transporter) member 3 (SLC2A3) expression did not vary between COCs or mural granulosa cell type. Our data demonstrate that metabolic and genomic differences exist between eCOCs and cCOCs and mural granulosa cells in the horse.
Descripción: Publicado en: Reproduction, Fertility and Development 30(2) 297-306 https://doi.org/10.1071/RD16441 Submitted: 4 November 2016 Accepted: 6 June 2017 Published: 6 July 20172018-01-18T00:00:00ZHSP90 maintains boar spermatozoa motility and mitochondrial membrane potential during heat stress
http://hdl.handle.net/10662/6748
Títulos: HSP90 maintains boar spermatozoa motility and mitochondrial membrane potential during heat stress
Autores/as: Calle Guisado, Violeta; Bragado González, María Julia; García Marín, Luis Jesús; González Fernández, Lauro
Resumen: Heat Shock Proteins (HSP) is a family of proteins that protects cells from high temperatures. The present work aimed to elucidate the role that HSP90 exerts on boar sperm incubated under heat stress conditions on viability, total motility (TM), progressive motility (PM), acrosome status, mitochondrial membrane potential and plasma membrane lipid organization. Sperm were incubated in non-capacitating conditions (Tyrode’s basal medium or TBM) for 3, 8 and 24 h or in capacitating conditions (Tyrode’s complete medium or TCM) for 4 h at 38.5 °C or 40 °C (Heat stress) in the presence or absence of 5 or 20 μM of 17-AAG, a specific HSP90 inhibitor. Sperm viability was not affected by the presence of 17-AAG in any condition tested compared with its own control (at the same temperature and incubation time). In non-capacitating conditions TM (22.7 ± 4.1 vs. 1.9 ± 1.1; % mean ± SEM), PM (3.1 ± 0.9 vs. 0) and high mitochondrial membrane potential (19.5 ± 2.2 vs. 11.8 ± 0.8) decreased significantly in sperm incubated at 40 °C for 24 h in the presence of 20 μM 17-AAG (control vs. 20 μM 17-AAG, respectively; p < 0.05). In sperm incubated at 38.5 °C only a mild decrease in TM was observed (48.7 ± 3.1 vs. 32.1 ± 4.8; control vs. 20 μM 17-AAG, respectively; p < 0.05). However, under capacitating conditions none of the sperm parameters studied were affected by 17-AAG after 4 h of incubation. These results demonstrate for the first time the role of HSP90 in the maintenance of boar sperm motility and mitochondrial membrane potential during prolonged heat stress in non-capacitating conditions.
Descripción: Publicado en: Animal Reproduction Science 187 (2017) 13–19 https://doi.org/10.1016/j.anireprosci.2017.09.0092017-12-29T00:00:00ZFisiología celular y calidad seminal durante la conservación del semen porcino refrigerado
http://hdl.handle.net/10662/799
Títulos: Fisiología celular y calidad seminal durante la conservación del semen porcino refrigerado
Autores/as: Martín Hidalgo, David
Resumen: La Inseminación Artificial con semen diluido refrigerado es una de las tecnologías de la reproducción más utilizadas en el ganado porcino. Para un máximo aprovechamiento del eyaculado y para que los espermatozoides conserven su capacidad fecundante durante varios días los eyaculados son diluidos con diluyentes comerciales, obteniéndose dosis seminales para inseminación. En la presente Tesis Doctoral hemos evaluado la capacidad de conservación de dosis seminales de dos diluyentes, MRA y X-Cell, en dos razas, Duroc e Ibérica, concluyendo que la raza es un factor de variación más en la capacidad del diluyente para la conservación de la motilidad espermática a largo plazo. Pese a ser diluidas en un medio adecuado, las dosis seminales van perdiendo durante la conservación la capacidad fecundante, lo que podría ser debido a la susceptibilidad de los espermatozoides porcinos a la oxidación. Por este motivo estudiamos el efecto de dos antioxidantes, melatonina y resveratrol, en la conservación. Nuestros resultados desaconsejan el uso de estos dos antioxidantes para mejorar la conservación de dosis seminales porcinas durante 7 días a 17ºC. Adicionalmente, estudiamos la implicación de una quinasa reguladora del metabolismo, AMPK, en la conservación. Hemos descrito por primera vez que la actividad de AMPK (fosforilación en Thr172 de la AMPK) se incrementa en función del tiempo de conservación de los espermatozoides porcinos a 17ºC. Finalmente, nuestros resultados indican que la actividad de la AMPK es esencial en el mantenimiento de la calidad de las dosis seminales conservadas varios días a 17ºC.; Currently, one of the most used techniques in the field of porcine reproduction is the artificial insemination with refrigerated semen. In order to optimize the maximum use of an ejaculate and to preserve spermatozoa fertilizing capacity, ejaculates are diluted in commercial extenders to produce seminal doses. In this Doctoral Thesis we have evaluated the ability of two extenders, MRA and X-Cell, to preserve seminal doses from two breeds, Duroc and Iberic. Our conclusion is that the ability of a diluent to preserve spermatozoa motility for long time is influenced by the breed. It is known that seminal doses time-dependently lose their fertilizing capacity, likely due to the susceptibility of boar spermatozoa to oxidation. For this reason, we have studied the effect of two antioxidants, melatonin and resveratrol, during seminal dosis conservation. Our results do not support/advice the use of any of these two antioxidants to improve the preservation of boar seminal doses during 7 days at a 17ºC. Additionally, we have studied the implication of a kinase that regulates metabolism, AMPK, during semen conservation. We have described for the first time that AMPK activity (evaluated as phosphorylation at residue Thr172) (PThr172-AMPK) increases over the time of boar spermatozoa preservation at 17ºC. Finally, our results indicate that a proper function of AMPK activity is necessary/required for the maintenance of the quality of seminal doses preserved for long term at 17ºC.2013-12-19T00:00:00Z