Enriching membrane cholesterol improves stability and cryosurvival of buffalo spermatozoa

Buffalo spermatozoa are comparatively more susceptible to freezing hazards than cattle spermatozoa. In recent times incubation of spermatozoa with cholesterol-loaded-cyclodextrins (CLC) has shown improvements in semen quality in several species. Therefore, this study was undertaken to evaluate the incubation level of CLC at which maximum benefit is derived for the buffalo spermatozoa. For the study, 120 million spermatozoa were incubated in 2, 3 and 4 mg/mL of CLC (Gr II, III and IV, respectively) and cholesterol and phospholipids content, their ratio, flow cytometric evaluation of plasma membrane integrity (PMI), plasma membrane fluidity and extent of cryoinjury (Chlortetracycline, CTC assay) were compared with an untreated control (Gr I). Additionally the ability of cholesterol-loaded-spermatozoa to undergo induced acrosome reaction (IAR) using ionophore calcium (A23187) was evaluated in frozen–thaw samples. Data show a significant and linear increase (CV = 0.88) in cholesterol content of spermatozoa in Gr II, III and IV and a significant decrease in phospholipids content at frozen–thaw stage in Gr IV than Gr III spermatozoa. The study revealed a significant improvement in PMI and significant reduction in plasma membrane fluidity and cryoinjury of CLC treated spermatozoa at progressive stages in three groups compared to control. Nevertheless, spermatozoa of Gr II, III and IV were significantly less responsive to ionophore calcium (A23187) than Gr I. This study shows for the first time that incubation of buffalo bull spermatozoa with CLC (3 mg/120 × 106) prior to processing permits greater numbers of sperm to survive cryopreservation while allowing spermatozoa to capacitate and the acrosome to react to AR inducer ionophore calcium (A23187).