264 EFFECT OF SEASON ON CRYOCAPACITATION OF BUFFALO (BUBALUS BUBALIS) SEMEN

Abstract

Buffalo are short-day breeders; at our latitudes, reproductive activity improves during autumn. Although extensive studies have been conducted on the female, seasonal variations were also reported on post-thaw motility and membrane integrity of buffalo sperm (Andrabi 2009 Reprod. Domest. Anim. 44, 552–569). It was reported that cryopreservation induces capacitation-like changes in buffalo spermatozoa, assessed by both chlortetracycline (CTC) fluorescent and protein tyrosine phosphorylation assays (Kadirvel et al. 2011 Theriogenology 75, 1630–1639; Elkhawagah et al. 2014 J. Buffalo Sci. 3, 3–11). The aim of this study was to evaluate the effect of season on cryocapacitation of buffalo semen. At least two ejaculates were collected from 4 bulls during 2 seasons with different daylength: spring (low season) and autumn (peak season). Each ejaculate was diluted at 37°C with BioXcell extender to a final concentration of 30 × 10⁶ spermatozoa per mL. After 4 h at 4°C, straws were frozen in an automated system. Immediately after thawing, sperm motility was evaluated by phase-contrast microscopy and viability, as well as capacitation status, were assessed by CTC fluorescent staining, as reported (Kadirvel et al. 2011 Theriogenology 75, 1630–1639). Briefly, sperm suspensions were first stained with 0.1 µg mL^–1 Hoechst 33258 for 2 min. Then, equal volumes of sperm suspension and CTC solution (750 mM CTC, 5 mM cysteine in 130 mM NaCl, and 20 mM Tris-HCl) were mixed at room temperature, and glutaraldehyde (12.5%) was added. Sperm suspensions were mounted on slides and stored at 4°C overnight (in the dark). Each sample was assessed twice under a microscope equipped with phase contrast and epifluorescent optics. At least 100 spermatozoa per slide were evaluated and classified into 3 CTC staining patterns: 1) uniform bright fluorescence over the entire head (uncapacitated spermatozoa, pattern F); 2) fluorescence-free band in the post-acrosomal region (capacitated spermatozoa, pattern B); and 3) dull fluorescence over the entire head, except for a thin punctuate band of fluorescence along the equatorial segment (acrosome-reacted spermatozoa, pattern AR). Data were analysed by chi-square. There were no differences in sperm viability between seasons (78.4 and 76.4%, respectively, in autumn and spring). However, post-thaw motility increased (P < 0.05) in autumn (60.0%) compared with spring (50.0%). The percentage of sperm displaying CTC pattern F increased in autumn compared with spring (40.5 and 27.3%, respectively; P < 0.01), whereas the percentage of sperm with both pattern B (57.9 and 66.6%, respectively; P < 0.01) and AR (1.6 and 6.1%, respectively; P < 0.01) decreased. The number of bulls and ejaculates used in this study was too low to draw definitive conclusions. However, these findings suggested that capacitation-like changes after sperm cryopreservation may be reduced during the favourable season in buffalo.