Cryopreservation induces partially irreversible damage to sperm membrane as a result of membrane destabilization.  The membrane damage after freezing might be linked to the modification of the membrane fluidity before freezing. Methyl β cyclodextrin (mβCD) was found to selectively extract cholesterol from the plasma membrane and therefore, increased membrane fluidity.  The present study aimed at investigating the effect of adding mβCD to the Tris-based diluent on the freezability and in vitro fertilizing potentials of buffalo spermatozoa. Buffalo semen was collected, evaluated and extended in Tris-based extender supplemented with different concentrations of mβCD (experiment 1) or with cholesterol-3-sulfate and/or mβCD (experiment 2).  Semen was examined post-thawing to evaluate their freezability, cholesterol content and in vitro fertilizing potentials. The present results revealed that, addition of mβCD to the semen diluent before freezing improved significantly the freezability and fertilizing potentials in a dose dependent trend.  Addition of 5 mg/ml mβCD increased significantly (P<0.01) post-thawing motility, viability index and maintained the acrosomal integrity (57.50±2.50%, 157.50±8.29 and 12.00±0.81%, respectively) compared to the control semen (41.25±1.25%, 105.55±2.88 and 18.25±2.46%, respectively) and increased significantly (P<0.05)thedevelopment to morula and blastocyst stage (24.44% and 15.56%, respectively) compared to the control semen (6.38 % and 2.13%, respectively).  Moreover, 5 mg/ml mβCD could delete the drastic effect of cholesterol-3-sulfate on the buffalo semen freezability and in vitro fertilizing potentials.
In conclusion, addition of methyl β cyclodextrin to the semen diluent prior to freezing is an effective method that increases the cryosurvival and in vitro fertilizing potentials of buffalo spermatozoa.