Sperm subpopulations, buffalo semen, sperm kinematics, cryopreservation, computer assisted sperm analyzer.

Abstract

Abstract:This study aimed to identify and characterize the sperm subpopulations existing in water buffalo semen using a computer assisted sperm analyzer (CASA), as well as assess the effects of cryopreservation on the sperm subpopulation structure and evaluate bull variability. The semen of eight Bulgarian Murrah bulls was collected by four times in an interval of one week each. The semen was cryopreserved following a standard protocol and sperm kinematics was assessed. Clustering methods were applied to individual sperms, forming two significantly different (P < 0.05) subpopulations (P 1 and P 2 ). Subpopulation P 1 represents those spermatozoa that moved most rapidly and progressively (46.29%), and subpopulation P 2 includes spermatozoa with relatively low velocity or poorly motile but with high progressiveness (53.41%). There was a decline on the population of P 1 sperms from fresh (52.52%), pre-freeze (45.73%) to post-thaw (35.17%) stages and significant difference on the sperm kinematics between P 1 and P 2 . A significant decline in the values of distance, velocity and amplitude of lateral head (ALH) parameters were observed at post-thaw stage, while an increase was observed on trajectory and beat cross frequency (BCF) kinematics. Values of sperm kinematics were also significantly different (P < 0.05) among all bulls. The frequency distribution of spermatozoa on both subpopulations P 1 and P 2 was quite similar for all bulls in pre-freeze and post-thaw stages, but with significant (P < 0.05) variability on fresh stage. Bulls with the highest maintained frequency of P 1 sperms are denoted as good freezer bulls. In sum, kinematic characterization of water buffalo sperm and clustering into subpopulation enable to identify bulls that are more resistant to cryopreservation and production of quality semen for genetic propagation.