Effect of cooling rate and equilibration time on pre-freeze and post-thaw survival of buck sperm

Abstract

Survival of buck sperm is affected due to duration and temperature of stages of refrigerated or frozen storage. This study investigated interactive effect of cooling rates (moderate; MC and rapid cooling; RC); and equilibration times (0, 2, 4 and 8h) on survival before freezing at 4°C and post-thaw quality of buck sperm. Semen was collected (three Beetal bucks; replicates=6), pooled and diluted with Tris-citrate extender. Pooled semen samples were subjected to either RC (−2.2°C/min) or MC (−0.3°C/min) from 37°C to 4°C in separate aliquots and further equilibrated at 4°C for 8h. Semen was frozen using standard procedure after completion of each equilibration period i.e. 0, 2, 4 and 8h. Semen was evaluated for motility, viability, plasma membrane integrity (PMI) and normal apical ridge (NAR) before freezing and after thawing. The survival time (time for survival above threshold limit i.e. 60%) at 4°C, of motility and PMI was observed 5 and 6h respectively in RC group while >8h in MC group. Rate of decline (slope) in motility and viability was higher (P<0.05) in RC overtime during equilibration at 4°C while PMI and NAR declined at equal rate in both cooling groups. Post-thaw motility and NAR were higher (P<0.05) in MC when equilibrated for 2–8h while viability and PMI of RC was observed equal to MC group. In conclusion, survival of buck sperm is higher when cooled with moderate rate. However, RC can maintain post-thaw sperm viability and PMI equal to MC when equilibrated for 2–8h. The methods should be explored to maintain motility and NAR during rapid cooling of buck sperm.