Effect of equilibration times, freezing, and thawing rates on post-thaw quality of buffalo (Bubalus bubalis) bull spermatozoa

The effects of equilibration times (E1, 2 h; E2, 4 h; E3, 6 h), freezing rates (FR1, manual, 5 cm above liquid nitrogen (LN2 ) for 10 min, plunging in LN2 ; FR2, programmable ultra-fast, holding at +4 °C for 2 min, from 4 to -10 °C at -10 °C/min, from -10 to -20 °C at -15 °C/min, from -20 to -120 °C at -60 °C/min, holding at -120 °C for 30 sec, plunging in LN2 ), and thawing rates (T1, 37 °C for 30 sec; T2, 50 °C for 15 sec; T3, 70 °C for 7 sec) were evaluated on quality of buffalo bull spermatozoa. Progressive motility (%), rapid velocity (%), average path velocity (VAP, μm/s), straight line velocity (VSL, μm/s), and mitochondrial transmembrane potential (%) were higher (p < 0.05) with E2, FR2, and T3 compared to other groups. Sperm curved line velocity (VCL, μm/s) was higher (p < 0.05) with E2 and FR2 compared to other groups. Sperm straightness (%) and linearity (LIN, %) were higher (p < 0.05) with E2 compared to other groups. Sperm LIN was affected (p < 0.05) with T3 compared to other groups. Supravital-plasma membrane integrity (%), viability and acrosome integrity (%) of spermatozoa were higher (p < 0.05) with E2 and FR2 compared to other groups. Sperm DNA integrity (%) was higher (p < 0.05) with FR2 and T1 compared to other groups. We concluded that inclusion of 4 h-equilibration time, programmable ultra-fast freezing rate, and rapid thawing at 70 °C for 7 sec in cryopreservation protocol improves the post-thaw quality of buffalo bull spermatozoa.