. Enriching membrane cholesterol improves stability and cryosurvival of buffalo spermatozoa.

Abstract

tBuffalo spermatozoa are comparatively more susceptible to freezing hazards thancattle 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 ben-efit is derived for the buffalo spermatozoa. For the study, 120 million spermatozoa wereincubated in 2, 3 and 4 mg/mL of CLC (Gr II, III and IV, respectively) and cholesterol and phos-pholipids 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 decreasein phospholipids content at frozen–thaw stage in Gr IV than Gr III spermatozoa. The studyrevealed a significant improvement in PMI and significant reduction in plasma membranefluidity and cryoinjury of CLC treated spermatozoa at progressive stages in three groupscompared to control. Nevertheless, spermatozoa of Gr II, III and IV were significantly lessresponsive to ionophore calcium (A23187) than Gr I. This study shows for the first timethat incubation of buffalo bull spermatozoa with CLC (3 mg/120 × 106) prior to processingpermits greater numbers of sperm to survive cryopreservation while allowing spermatozoato capacitate and the acrosome to react to AR inducer ionophore calcium (A23187).