Effects of extender and equilibration time on post-thaw motility and membrane integrity of cryopreserved Gyr bull semen evaluated by CASA and flow cytometry

The objectives of the present study were to investigate the effects of three equilibration times (0, 2, and 4 h) and two extenders (TRIS or Bioxcell®) for cryopreservation of bull semen. Semen from 12 Gyr bulls was cryopreserved using an automated freezing machine. There were significant interactions between equilibration times and extenders for sperm motility and membrane integrity. The control treatment (0 h equilibration) had the lowest values (P < 0.05) for total (MOT) and progressive motilities (PROG), and percentage of sperm with intact plasma and acrosomal membranes (IPIA), with no significant differences between extenders. Extender TRIS had greater cryoprotective action than Bioxcell®, with greater MOT, PROG, IPIA at 2 and 4 h, as well as the lowest proportion of damaged plasma membrane (DPM, 72.2% vs. 85.8%) for all times. Equilibration for 4 h yielded the most desirable (P < 0.05) for MOT, PROG, and IPIA, and the least DPM percentage (86.5, 78.0, and 72.6% for 0, 2, and 4 h, respectively). Overall, the combination of TRIS and 4 h of equilibration was the most desirable semen cryopreservation method, with greatest MOT, PROG, and IPIA (TRIS-T4 = 26.8%; BIO-T4 = 18.3%) and the least DPM. In conclusion, based on objective analyses, equilibration during cryopreservation was essential for maintaining motility and integrity of sperm membranes; equilibration for 4 h yielded the greatest sperm survival, independent of the extender used.