263 ADDING RESVERATROL TO THE EXTENDER AFFECTS PROTEIN TYROSINE PHOSPHORYLATION IN BUFFALO SPERM

Abstract

Cryopreservation induces remarkable capacitation- like changes in buffalo sperm (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 resveratrol, a natural phytoalexin with antioxidant properties, on capacitation status of frozen-thawed buffalo sperm, assessed by protein tyrosine phosphorylation assay. Three ejaculates from four bulls were used for the trial. Each ejaculate was split into two equal aliquots and diluted at 37°C with BioXcell extender containing no supplement (control) or 50 µM resveratrol, 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, sperm viability by Trypan Blue/Giemsa staining and localization of phosphotyrosine proteins by indirect immunofluorescence, as described Kadirvel et al. (2011 Theriogenology 75, 1630–1639). Briefly, after thawing, semen was centrifuged (300 × g, 10 min), fixed in 2% formaldehyde for 1 h at 4°C, and sperm pellets were incubated overnight at 4°C in modified phosphate buffer saline containing 2% BSA. After centrifugation, sperm pellets were resuspended, diluted 1 : 10 in mPBS, smeared onto slides, air-dried, and permeabilized with absolute ethanol for 5 min. Then, spermatozoa were incubated with rabbit anti-phosphotyrosine primary antibody for 1 h at room temperature in a humid chamber. Slides were incubated with secondary antibody, FITC conjugated goat anti-rabbit IgG, for 1 h in a dark humid chamber at room temperature and mounted with 90% glycerol. A total of 100 spermatozoa were screened per slide and classified as described (Luño et al. 2013 Reproduction 146, 315–324): pattern A: uniform fluorescence over the entire acrosome (low capacitation level); pattern E: signal in the equatorial segment (medium capacitation level); and pattern EA: fluorescence at both equatorial and acrosomal areas (high capacitation level). Data were analysed by chi-square. There were no significant differences between control and treated groups for sperm motility (50.0 and 55.0%, respectively) or viability (77.4 and 72.9%). The percentage of sperm cells that did not exhibit fluorescence was very low (2.4 and 4.3% in the control and resveratrol groups, respectively). In resveratrol-treated group, pattern E was higher than the control (4.9 and 2.0%; P < 0.01). More interestingly, in the resveratrol-treated group, an increased percentage of sperm with pattern A (79.6 and 52.5%) and a decreased percentage of sperm with pattern EA (12.2 and 43.1%) were recorded. Based on decreased sperm with a high capacitation level (EA pattern) and increased sperm with low capacitation level (A pattern) at thawing, we concluded that adding resveratrol to semen extender before cryopreservation of buffalo was beneficial.