Sperm membrane cholesterol influences cryodamage during cryopreservation. The present study was carried out to evaluate the effect of varying cholesterol levels in Tris based extenders on the freezability of sexually healthy Malabari buck semen. A total of 48 ejaculates from two adults healthy sexually healthy Malabari bucks were utilized for the study. The collected and pooled ejaculates were divided into four groups with Group I serving as Control - I, Group II and III were treated with 1 mg and 2 mg of cholesterol-loaded-cyclodextrin (CLC)/120 × 10(6) spermatozoa, respectively, and Group IV, treated with 1 mg methyl-β-cyclodextrin (MβCD) served as Control - II. Manual freezing was carried out to cryopreserve the treated and control spermatozoa. Treatment of semen samples with CLC resulted in improved maintenance of sperm motility at pre-freeze and post-thaw stages of cryopreservation without affecting hypo-osmotic swelling response. Treatment of semen with 1 mg of CLC/120 × 10(6) spermatozoa was observed to be better than treatment with 2 mg of CLC/120 × 10(6) spermatozoa. In general, MβCD treatment was found to result in significantly lower sperm characteristics than those of Control - I and CLC treatment at pre-feeze and post-thaw stages and when incubated up to 4 h. Cholesterol treatment of sexually healthy Malabari buck semen was found to hold promise for improving cryopreservability of spermatozoa.