Abstract: The cryopreservation technique adversely affects the metabolism, motility and membrane integrity of the spermatozoa, thereby decreasing the fertility potential of the processed semen sample. In current study, we investigated the effect of cryopreservation on buffalo sperm apoptosis at five different stages of cryopreservation. We employed a combination of fluorescence-based microscopic as well as flow cytometric assays and assessed the quality of spermatozoa undergoing apoptosis. Semen ejaculates from Murrah buffalo bulls were collected and cryopreserved using standard protocol. Annexin-V/Propidium Iodide (AN/PI) assay was used to detect the externalization of phosphatidylserine (PS) across the plasma membrane of buffalo spermatozoa, a marker to monitor the early apoptosis. Four different sperm subpopulations were identified: viable, early apoptotic, late apoptotic, and necrotic spermatozoa. Cooling at 4 °C and freeze-thawing significantly decreased the motility percent, plasma membrane integrity (P<0.01), the percentage of viable spermatozoa (P < 0.001), and dramatically increased the percentage of late apoptotic spermatozoa (P < 0.01), but not of early apoptotic spermatozoa and necrotic spermatozoa. Moreover, frozen thawed semen showed an increase in PS translocation index (P < 0.05) as compared to that of fresh semen. Several new classes of AN+ sperms were identified where Annexin-V stained the buffalo spermatozoa in head, midpiece and tail. Our results suggests that cooling and freeze-thawing steps during cryo-preservation result in apoptosis in buffalo spermatozoa, which might be responsible for the decreased fertilizing potential of cryopreserved spermatozoa. Efforts are therefore required to minimize cryodamage and maximize the fertility of cryopreserved buffalo spermatozoa by using various cryoprotectants.

Keywords: apoptosis; spermatozoa; cryopreservation; flow cytometry; phosphatidylserine externalization

| DOI: 10.17148/IARJSET.2018.568