Efecto de la centrifugación sobre la membrana plasmática y el ADN de espermatozoides bovinos

Authors

  • Rodrigo A. Urrego
  • Andrea Ríos
  • Martha Olivera A.
  • Omar Camargo

DOI:

https://doi.org/10.17533/udea.rccp.324253

Keywords:

bovine spermatozoa, DNA fragmentation, membrane integrity

Abstract

Resumen

El objetivo de este estudio fue determinar si la integridad de la membrana plasmática y el ADN de los espermatozoides pueden ser afectados por la centrifugación realizada en el proceso de lavado y selección. Los espermatozoides fueron sometidos a diferentes tiempos de centrifugación (10, 30 y 45 min); se utilizó un control negativo con espermatozoides no centrifugados y un control positivo con espermatozoides sometidos a estrés oxidativo con peróxido de hidrógeno (H2O2) (200 μM). Para evaluar la integridad de la membrana se utilizó la prueba hipoosmótica (HOST) y para evaluar la fragmentación del ADN se utilizó el ensayo cometa: El análisis estadístico se realizó mediante la prueba de Fisher. La centrifugación durante 10, 30 y 45 min, presentó un efecto estadísticamente significativo sobre el daño en el ADN, respecto de los espermatozoides no centrifugados (p < 0.05). Además, se observó diferencia estadística significa (p < 0.05) entre los espermatozoides centrifugados a diferentes tiempos con respecto al control positivo realizado con H2O2 (200 µM). La comparación de medias no indicó diferencia estadística significativa entre los espermatozoides no centrifugados y los centrifugados por un periodo de 10 y 30 min (p > 0.05) en la reacción positiva a la prueba HOST, lo cual sí sucedió (p < 0.05) al comparar los no centrifugados y los centrifugados por 45 min. El control positivo realizado con H2O2 presentó diferencia significativa (p < 0.05) con el resto de los tratamientos. En conclusión, los resultados del presente trabajo sugieren que la centrifugación de los espermatozoides durante 10, 30 ó 45 min a 700 x g para la realización del gradiente diferencial de Percoll, tiene un efecto deletéreo sobre el ADN de los espermatozoides bovinos y que la centrifugación por 45 min además de causar daño en el ADN produce pérdida de la integridad de la membrana plasmática.

Palabras clave: espermatozoides bovinos, fragmentación de ADN, integridad de la membrana.

Summary

The aim of this study was to evaluate the effects of different times of centrifugation on plasma membrane integrity and DNA of bovine sperm cells, by means of the hyposmotic test (HOST) and the comet assay, respectively. The sperm cells were centrifuged at 700 x g for 10, 30 or 45 min. No-centrifuged thawed semen served as negative control whereas hydrogen peroxide (H2O2) (200 mM) treated sperm cells were used as a positive control. The results indicate that while the integrity of the plasma membrane was not affected by centrifugation, bovine sperm DNA was damaged independently of centrifugation times. Significant differences between negative control and treatments were found (p < 0.05) and in the same way, between treatments and positive control, where the damage for oxidative stress was greater. These results indicate that centrifugation could be detrimental for in vitro bovine embryo production. Additionally, some grade of DNA fragmentation in not centrifuged sperm cells (negative control) was registered, suggesting that DNA of bovine sperm cells could be affected by other factors, probably freezing procedure.

Key words: bovine spermatozoa, DNA fragmentation, membrane integrity.

Resumo

O objetivo de este estudo foi determinar se a integridade da membrana plasmática e o DNA dos espermatozóides podem ser afetados pela centrifugação realizada no processo de lavado e seleção. Os espermatozóides foram submetidos a diferentes tempos de centrifugação (10, 30 e 45 min); foi utilizado um controle negativo com espermatozóides não centrifugados e um controle positivo com espermatozóides submetidos a estresse oxidativo com peróxido de hidrogênio (H2O2) (200 μM). Para avaliar a integridade da membrana foi utilizado o teste hipoosmótica (HOST) e para avaliar a fragmentação do DNA Fo utilizado o ensaio cometa: A análise estatística se realizou mediante o teste de Fisher. A centrifugação durante 10, 30 e 45 min, apresentou um efeito estatisticamente significativo sobre o dano no DNA, em relação aos espermatozóides não centrifugados (p < 0.05). Além disto, observou-se diferença estatisticamente significativa (p < 0.05) entre os espermatozóides centrifugados a diferentes tempos em relação ao controle positivo realizado com H2O2 (200 µM). A comparação de medias não detectou diferença estatisticamente significativa entre os espermatozóides não centrifugados e os centrifugados por um período de 10 e 30 min (p > 0.05) na reação positiva à prova HOST, o qual ocorreu ao comparar os não centrifugados e os centrifugados por 45 min (p < 0,05). O controle positivo realizado com H2O2 apresentou diferença significativa (p < 0.05) quando comparado contra os outros tratamentos. Em conclusão, os resultados do presente trabalho sugerem que a centrifugação dos espermatozóides durante 10, 30 ou 45 min a 700 x g para realização do gradiente diferencial de percoll, têm um efeito deletério sobre o DNA dos espermatozóides bovinos e que a centrifugação por 45 min além de causar dano no DNA produz perda da integridade da membrana plasmática.

Palavras chave: espermatozóides bovinos, fragmentação do DNA, integridade da membrana.

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Published

2009-07-08

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Urrego, R. A., Ríos, A., Olivera A., M., & Camargo, O. (2009). Efecto de la centrifugación sobre la membrana plasmática y el ADN de espermatozoides bovinos. Revista Colombiana De Ciencias Pecuarias, 21(1), 8. https://doi.org/10.17533/udea.rccp.324253

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