40 CHOLESTEROL SUPPLEMENTATION REDUCES CRYOCAPACITATION DAMAGES IN BUFFALO (BUBALUS BUBALIS) SPERM

Buffalo sperm are more sensitive to cryopreservation and thawing-induced damages than cattle sperm, thus resulting in lower fertilizing ability. Cryopreservation induces considerable capacitation-like changes in buffalo sperm (Elkhawagah et al. 2014 J. Buffalo Sci. 3, 3–11). It is known that specific lipid mixtures, including cholesterol, stabilise and protect spermatozoa from freeze-thaw damage (Rajoriya et al. 2014 Vet. World. 7, 702–706). Therefore, the aim of this study was to investigate the effect of cholesterol loaded cyclodextrins (CLC) before cryopreservation on the capacitation status of buffalo frozen sperm. Four ejaculates from 4 bulls were selected, split in 3 aliquots, and diluted at 37°C with BULLXcell extender, containing 0 (control), 1.5, and 3 mg mL–1 CLC, to a final concentration of 30 × 106 sperm mL–1. Cyclodextrin was loaded with cholesterol as described earlier (Purdy and Graham 2004 Cryobiology 48(1), 36–45). The aliquots were frozen according to standard procedures. At thawing, sperm motility was evaluated by phase contrast microscopy, and viability and capacitation status were evaluated by Hoechst 33258/CTC. Briefly, Percoll separated spermatozoa were incubated with 1% (wt/vol) Hoechst 33258 in a protein-free medium. After centrifugation (900 × g, 5 min), the pellet was resuspended in CTC staining solution (750 mM CTC, 5 mM cysteine in 130 mM NaCl, and 20 mM Tris acid, pH 7.8), fixed with glutaraldehyde (12.5% vol/vol), and placed on a slide. At least 100 sperm per slide were analysed and classified into 1 of 3 CTC staining patterns: pattern F, with fluorescence over the entire sperm head (noncapacitated); pattern B, with a fluorescence-free band in the postacrosomal region (capacitated); pattern AR, with no acrosome and a thin band of fluorescence along the equatorial segment (acrosome reacted). Data were analysed by ANOVA. No differences among control, 1.5, and 3 mg mL–1 CLC-treated groups were recorded in both sperm motility (66.5 ± 5.6, 68.8 ± 4.8, and 68.8 ± 4.8, respectively) and viability (86.5 ± 1.9, 87.6 ± 1.5, 88.4 ± 2.3, respectively). The most interesting result arising was the strong reduction of sperm cryocapacitation observed when the extender was supplemented with CLC, as shown by the increased percentage of sperm displaying pattern F (28.6 ± 3.3, 61.5 ± 1.7, and 47.6 ± 4.3 in control, 1.5, and 3 mg mL–1 CLC groups, respectively; P < 0.01) and the decreased percentage of sperm displaying pattern B (69.6 ± 3.4, 37.8 ± 1.5, and 51.3 ± 4.7 in control, 1.5, and 3 mg mL–1 CLC groups, respectively; P < 0.01) compared with the control. The maximum beneficial effect on semen cryopreservation was recorded with the dose of 1.5 mg mL–1 CLC. However, no differences were detected in pattern AR that remained low in all groups (1.8 ± 0.7, 0.8 ± 0.4, and 1.1 ± 0.6 in the control, 1.5, and 3 mg mL–1 CLC groups, respectively). In conclusion, it was demonstrated that treating buffalo sperm with cholesterol before cryopreservation strongly decreases cryocapacitation damages, likely by stabilising sperm membrane, hence improving sperm quality.