Fluorescence probes

General reagents were purchased from Sigma (St. Louis, MO, EE UU.). Fluorescence probes were bought from Invitrogen (Carlsbad, CA, EE UU.). Stock solutions were prepared in deionized water (propidium iodide, 1.5 mM; Hoechst 33342, 9 mM; PNA FITC, 1 mg/ ml) or DMSO (YO PRO 1, Mitotracker deep red, Merocyanine 540, MitoSOX, CM H 2 DCFDA, 1 mM), and stored at -20 C. Porcicoll, adapted for boar semen, and its speciUc dilution buWer were supplied by Prof. J.M. Morrell at the Swedish University of Agricultural Sciences (SLU).

Animal handling was performed in accordance with Spanish Animal Protection Regulation, RD 53/2013, which conforms to European Union Regulation 2010/63. Seven males of Blanca-Celtibérica goat breed (age > 1.5 years) and five males of Manchega sheep breed (age > 1.5 years) were used. All males were maintained and managed at the Regional Center of Animal Selection and Reproduction (CERSYRA) located in Valdepeñas (Spain). The thawing procedure was conducted at laboratories from SaBio group in Albacete (Spain).
Chemicals were of reagent grade and purchased from Sigma (Madrid, Spain). Biladyl ® was purchased from Minitüb (Tiefenbach, Alemania). Fluorescence probes were purchased from Invitrogen (Barcelona, Spain), except for propidium iodide (PI) which was purchased from Sigma. Chromatographically purified acridine orange (AO) was purchased from Polysciences Inc. (Warrington, PA, USA).