Centrifugation and addition of glycerol at 22 °C instead of 4 °C improve post-thaw motility and fertility of stallion spermatozoa

Abstract

The aims of this study were to evaluate the effects of cooling rate to 4 °C and temperature at the time of centrifugation/glycerol-addition (freezing extender: INRA82 + 2% egg yolk + 2.5% glycerol) on postcentrifugation recovery rate, post-thaw motility and per-cycle fertility. When centrifugation/glycerol-addition was performed at 4 °C (14 ejaculates), a moderate cooling rate (37 °C to 4 °C in 1 h) resulted in higher post-thaw motility (45%) than when using a slow cooling rate (37 °C to 4 °C in 4 h) (39%; P<0.05). When centrifugation/glycerol-addition was performed at 22 °C (37 °C to 22 °C in 10 min) (10 ejaculates), post-thaw motility was lower when spermatozoa were frozen directly from 22 °C (23%) than when spermatozoa were cooled to 4 °C (22 °C to 4 °C in 1 h) before freezing (47%; P<0.0001). When centrifugation/glycerol-addition was performed at 22 °C (before cooling at a moderate rate), as opposed to 4 °C (after cooling at a moderate rate), a significant improvement of 1) recovery of spermatozoa after centrifugation (P<0,0001), 2) postthaw motility of spermatozoa at thawing (40% vs 36% (n≤291 ejaculates/group), P<0.0001) and 3) per-cycle fertility (56% vs 42% (n≥190 cycles/group), P<0.01) was observed. In conclusion, centrifugation/glycerol-addition at 22 °C followed by cooling to 4 °C at a moderate rate results in an improvement of post-thaw motility, spermatozoa recovery rate and per cycle fertility.