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The study was designed to investigate the effect of cholesterol supplementation in extender on post-thaw quality of cryopreserved Nili-Ravi buffalo bull spermatozoa. Semen was collected from three Nili-Ravi buffalo bulls with artificial vagina (42°C) for five weeks ie., two ejaculates/ week and bull (replicate; n=30). Two consecutive ejaculates from each bull were mixed and processed for initial evaluation, semen from each bull was split into four aliquots and diluted (37°C) in tris-citric acid extender having cholesterol 0.0 (control), 5.0, 10.0 and 20.0 ng/mL Diluted semen was cooled to 4°C in 2 h and equilibrated for 4 hours at 4°C. Cooled semen was filled in 0.5 ml French straws at 4°C, kept on liquid nitrogen vapours for 10 min. and plunged in liquid nitrogen for storage. Frozen semen was thawed after 24 h at 37°C for 30 seconds. Sperm progressive motility, plasma membrane integrity and viability were higher (P≤0.05) in extender containing 5.0 ng/mL of cholesterol. However, cholesterol did not provide any significant benefit for chromatin integrity of buffalo spermatozoa. In conclusion, cholesterol supplementation in extender at a concentration of 5.0 ng/mL improved the post-thaw quality of cryopreserved Nili-Ravi buffalo bull spermatozoa.