Partial deoxygenation of extender improves sperm quality, reduces lipid peroxidation and reactive oxygen species during cryopreservation of buffalo (Bubalus bubalis) semen

Abstract

The present study was designed to investigate the effect of partial deoxygenation of extender on sperm quality, lipid peroxidation (LPO) and reactive oxygen species (ROS) in buffalo (Bubalus bubalis) during cryopreservation of semen. Semen extender was prepared freshly and split into three sub-extenders [Extender I: control (non-deoxygenated), Extender II (partially deoxygenated by using LN2 flushing) and Extender III (partially deoxygenated mechanically by vacuum pump)]. Amounts of dissolved oxygen (DO) were determined in all the three extenders and also in post-thaw semen. Ejaculates with mass motility of ≥3+ and individual progressive motility of 70% or greater were collected from Murrah buffalo bulls and utilized in the study. Each semen sample was divided into Groups I (diluted with Extender I), II (diluted with Extender II) and III (diluted Extender III) with a maximum of 60 × 106 sperm/mL. French mini straws (0.25 mL) were filled with the extended semen samples, sealed with polyvinyl alcohol powder, kept for 3 h at 5 °C for equilibration and then stored in an automatic programmable freezer until the temperature of straws reached −145 °C followed by plunging the straws into liquid nitrogen (–196 °C). Semen samples were evaluated at pre-freeze and post-thaw stages for various variables [sperm motility, live sperm count, acrosomal integrity, hypo-osmotic swelling (HOS) response, LPO and ROS concentrations]. The mean DO was less (P < 0.05) in Extender II as compared to I and III. The DO was less (P < 0.05) in Group II (semen extended with Extender II) as compared with III (semen extended with Extender III) and I (semen extended with Extender I). The percentages for sperm motility, viability and intact acrosomes (PIA) were greater (P < 0.05) in Groups II and III as compared to the control group at the pre-freeze stage, while at the post-thaw stage, percentages of sperm motility, viability, PIA and HOS response were greater (P < 0.05) in Group II as compared with the control group and Group III. Pre-freeze HOS response (%) was greater (P < 0.05) in Group II as compared with the control and Group III. At the pre-freeze stage, sperm LPO and ROS were less (P < 0.05) in Groups II and III as compared with the control and at post-thaw stage, spermatic LPO and ROS concentrations were less (P < 0.05) in Group II than in the control group and Group III. In conclusion, partial deoxygenation of extender improves sperm quality, reduces sperm LPO and ROS concentrations in buffalo during cryopreservation. Partial deoxygenation of the extender with LN2 flushing may be one of the ways for improving quality and fertility of frozen-thawed buffalo sperm.