Exogenous Cholesterol Prevents Cryocapacitation-like Changes, Membrane Fluidity, and Enhances in Vitro Fertility in Bubaline Spermatozoa

A study was conducted to determine the optimum dosage of the exogenous cholesterol-loaded cyclodextrins (CLC) to get maximum cryoprotection for bubaline spermatozoa. In the present study, 120 × 10 spermatozoa were incubated in 2, 3 and 4 mg of CLC as grouped as Gr II, III and IV, respectively, and sperm progressive motility, intracellular Ca , capacitation status by protein tyrosine phosphorylation (PTP) assay and zona binding per cent (ZBP) and cleavage rate (CR) of the cryopreserved buffalo spermatozoa by in vitro fertility assay were assessed in comparison with an untreated control group (Gr I). Results revealed that there was a significant (p < .05) linear decrease in percentage of sperm population with higher intracellular Ca and percentage of sperm population with medium or high capacitated by PTP in CLC treated from 2 to 3 mg and then increased to 4 mg/120 × 10 spermatozoa whereas sperm progressive motility, percentage of sperm population with low capacitated, ZBP and CR were increased significantly (p < .05) in sperm population treated from 2 to 3 mg CLC and then decreased to 4 mg/120 × 10 spermatozoa. The study has clearly indicated that CLC at 3 mg/120 × 10 spermatozoa has maximum beneficial effects in protection of sperm progressive motility, membrane fluidity (low intracellular Ca ); prevention of cryocapacitation (low capacitation pattern in immunolocalization) and enhancement of in vitro ZBP and CR. Post-thaw motility of the CLC-treated sperm has shown positively significant (p < .05) correlation with sperm population with low intracellular Ca , low capacitated sperm population, ZBP and CR, whereas it was negatively (p < .05) correlated with sperm population with high intracellular Ca , medium or high capacitated sperm. The present study has revealed for the first time that incubation of spermatozoa with CLC of higher dose (>3 mg/120 × 10 spermatozoa) had adverse effects on sperm cryopreservation, although incubation of sperm with 3 mg/120 million prior to processing had minimised the freezing-thawing-associated damages in bubaline species.