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Vertebrate reproductive science and technology
RESEARCH ARTICLE

Free-radical production after post-thaw incubation of ram spermatozoa is related to decreased in vivo fertility

Enrique Del Olmo A , Alfonso Bisbal A , Olga García-Álvarez A , Alejandro Maroto-Morales A , Manuel Ramón B , Pilar Jiménez-Rabadán B , Luis Anel-López A , Ana J. Soler A , J. Julián Garde A and María R. Fernández-Santos A C
+ Author Affiliations
- Author Affiliations

A SaBio IREC (CSIC – UCLM – JCCM), Campus Universitario s.n. 02071 Albacete, Spain.

B Regional Center of Animal Selection and Reproduction (CERSYRA) JCCM, 13300 Valdepeñas, Spain.

C Corresponding author. Email: MRocio.Fernandez@uclm.es

Reproduction, Fertility and Development 27(8) 1187-1196 https://doi.org/10.1071/RD14043
Submitted: 6 February 2014  Accepted: 27 April 2014   Published: 19 June 2014

Abstract

The aim of the present study was to evaluate the effect of sperm reactive oxygen species (ROS) production and DNA changes on male fertility. For that purpose, six rams with significantly different pregnancy rates were used; these were classified as having high fertility, i.e. 59.4% average pregnancy rate, or low fertility, i.e. 23.1% average pregnancy rate. Sperm quality was assessed after a two-step process of sample thawing followed by an incubation of 2 h, either in the freezing extender (37°C) or after dilution in synthetic oviductal fluid (SOF; 38°C, 5%CO2). Sperm viability (YO-PRO-1), ROS production (5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein acetyl ester (CM-H2DCFDA)) and undamaged chromatin (sperm chromatin structure assay, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling, chromomycin A3) were evaluated by flow cytometry. Although no significant differences in sperm viability were observed, our results showed increased ROS production during incubation in the freezing extender as well as in SOF medium. Comparison between fertility groups showed significant differences in ROS production after 2 h of incubation for the two treatments. Regarding DNA integrity, our results showed no significant differences either between treatments and incubation times or fertility groups. Linear regression analysis showed that ROS production determined by CM-H2DCFDA was a good indicator parameter for in vivo male fertility of SOF-incubated samples, yielding a fair correlation between both parameters (r = –0.92). These results indicate that detection of ROS production by CM-H2DCFDA and flow cytometry after 2 h of incubation in SOF could be a useful procedure for predicting fertility of ram spermatozoa.

Additional keywords: artificial insemination, flow cytometry, fluorescein (CM-H2DCFDA), frozen semen.


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