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This is the first detailed report on the response of buffalo spermatozoa to low temperatures during freezing. The study determined the critical temperature zone for buffalo spermatozoa and developed a suitable freezing rate for this species. Semen from four Nili-Ravi buffalo bulls diluted in Tris-citric acid was frozen in a programmable freezer. Motion characteristics, plasma membrane integrity and acrosome morphology were determined at +4, 0, -5, -10, -20, -30, -40, -50, -80 and -196 degrees C by removing semen straws from the freezer at exactly these temperatures and rewarming them at 37 degrees C. The first statistical decline in sperm motility and lateral head displacement was observed at -40 degrees C. For all other parameters, there was biphasic decline: for curvilinear velocity, at 0 degrees C and -50 degrees C; and for plasma membrane integrity and acrosome morphology, at -30 degrees C and -50 degrees C. In a second series of experiments, buffalo spermatozoa were frozen using slow (-10 degrees C min(-1)), medium (-20 degrees C min(-1)) or fast (-30 degrees C min(-1)) freezing rates, between -10 degrees C and -80 degrees C. Freezing of buffalo spermatozoa at a rate of -30 degrees C min(-1) yielded higher post-thaw motion characteristics, plasma membrane integrity and normal acrosomes. In conclusion, different sperm characteristics respond differently at low temperatures and the freezing of buffalo spermatozoa at a higher rate ensures higher post-thaw semen quality.

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