title: Free-radical production after post-thaw incubation of ram spermatozoa is related to decreased in vivo fertility.
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 2h, 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 2h 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 2h of incubation in SOF could be a useful procedure for predicting fertility of ram spermatozoa.
authors.affiliation: SaBio IREC (CSIC - UCLM - JCCM), Campus Universitario s.n. 02071 Albacete, Spain.
authors.forename: Enrique
authors.initials: E
authors.lastname: Del Olmo
authors.affiliation: SaBio IREC (CSIC - UCLM - JCCM), Campus Universitario s.n. 02071 Albacete, Spain.
authors.forename: Alfonso
authors.initials: A
authors.lastname: Bisbal
authors.affiliation: SaBio IREC (CSIC - UCLM - JCCM), Campus Universitario s.n. 02071 Albacete, Spain.
authors.forename: Olga
authors.initials: O
authors.lastname: García-Álvarez
authors.affiliation: SaBio IREC (CSIC - UCLM - JCCM), Campus Universitario s.n. 02071 Albacete, Spain.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Maroto-Morales
authors.affiliation: Regional Center of Animal Selection and Reproduction (CERSYRA) JCCM, 13300 Valdepeñas, Spain.
authors.forename: Manuel
authors.initials: M
authors.lastname: Ramón
authors.affiliation: Regional Center of Animal Selection and Reproduction (CERSYRA) JCCM, 13300 Valdepeñas, Spain.
authors.forename: Pilar
authors.initials: P
authors.lastname: Jiménez-Rabadán
authors.affiliation: SaBio IREC (CSIC - UCLM - JCCM), Campus Universitario s.n. 02071 Albacete, Spain.
authors.forename: Luis
authors.initials: L
authors.lastname: Anel-López
authors.affiliation: SaBio IREC (CSIC - UCLM - JCCM), Campus Universitario s.n. 02071 Albacete, Spain.
authors.forename: Ana J
authors.initials: AJ
authors.lastname: Soler
authors.affiliation: SaBio IREC (CSIC - UCLM - JCCM), Campus Universitario s.n. 02071 Albacete, Spain.
authors.forename: J Julián
authors.initials: JJ
authors.lastname: Garde
authors.affiliation: SaBio IREC (CSIC - UCLM - JCCM), Campus Universitario s.n. 02071 Albacete, Spain.
authors.forename: María R
authors.initials: MR
authors.lastname: Fernández-Santos
mesh_terms: D000818:Animals; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary; D005260:Female; D005298:Fertility / Q000502:physiology*; D005434:Flow Cytometry; D020287:In Situ Nick-End Labeling; D008297:Male; D011247:Pregnancy; D018873:Pregnancy Rate; D017382:Reactive Oxygen Species / Q000378:metabolism*; D055101:Semen Analysis; D012662:Semen Preservation / Q000379:methods / Q000662:veterinary*; D012756:Sheep; D013081:Sperm Motility / Q000502:physiology*; D013094:Spermatozoa / Q000378:metabolism*
chemical_list: D017382:Reactive Oxygen Species