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Received: Revised: Accepted: Published online: July 28, 2018 November 06, 2018 November 09, 2018 January 28, 2019 This study evaluates the integrity of the plasma membrane (PM), and the mitochondrial transmembrane potential and their relationship with the motility and kinematic parameters of buffalo spermatozoa frozen in pellets and straws. Ejaculates (n=20) from eight Bulgarian Murrah buffaloes were frozen in pellets (Nagaze-Niva; medium GH22L) and in straws (Cassou; medium Triladyl) at final sperm concentration 80×10/ml. The comparative CASA analysis of the motility and kinematic parameters of spermatozoa showed significant differences in the rapid motility and in the VCL values in favor of pellets vs straws (P<0.05). The molecular changes in PM phospholipid asymmetry (assessed by double staining Annexin V-Cy3 Apoptosis Detection Kit) were higher after freezing – thawing, when compared to the fresh semen (P<0.05). No significant differences were observed in the number of live and functional spermatozoa with intact PM (6CF+/Ann V Cy3-) and the number of apoptotic spermatozoa (6CF+/Ann V Cy3+) between the two freezing technologies. The percentage of spermatozoa with well preserved and functioning mitochondria visualized by Rhodamine123 (Rh123) in the fresh semen was higher, when compared to spermatozoa after cryopreservation (P<0.05). An increase in the percentage of cells with nonfunctional mitochondria (Rh123or Rh123+) was observed after cryopreservation using both freezing technologies. In conclusion, the freezing of buffalo sperm in pellets, using GH22L protective medium, preserves high sperm motility, kinematics, PM integrity, and mitochondrial transmembrane potential, which is a guarantee of good fertilization capacity of the spermatozoa. ©2018 PVJ. All rights reserved

Changes in the Phospholipid Asymmetry of Buffalo Sperm Plasma Membrane in Relation to Cryopreservation Technology