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

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

Freezing of stallion semen: interactions among cooling treatments, semen extenders and stallions.

In the present study, the interactions among stallions, semen extenders and cooling treatments before stallion semen samples were frozen were studied. In Expt 1, the effects of four cooling treatments and three semen extenders were investigated (11 stallions x four split ejaculates), whereas in Expt 2, the effects of two semen extenders, two egg yolk concentrations and two glycerol concentrations were investigated (six stallions x five split ejaculates). Sperm motility after thawing was evaluated. In Expt 1, the extender x cooling treatment interaction was significant. Centrifugation and addition of glycerol at 22 degrees C, followed by filling the straws at 4 degrees C and immediate freezing had detrimental effects in INRA82 + 2% (v/v) egg yolk + 2.5% (v/v) glycerol semen extender and in Gent semen extender compared with the same processes in Kenney + 4% (v/v) egg yolk + 3.6% (v/v) glycerol semen extender. The other cooling treatments, including moderate cooling of semen to 4 degrees C before freezing, resulted in higher sperm motility and had similar effects among treatments. Semen samples from some stallions appeared to withstand freezing more effectively after some cooling treatments than other treatments. Some stallion spermatozoa demonstrated higher motility in Kenney semen extender, whereas other semen samples had the same motility in all extenders. In Expt 2, the stallion x extender interaction was not significant. Increasing the glycerol concentrations from 2.5 to 3.5% (v/v) enhanced sperm motility in semen extenders containing 4% (v/v) egg yolk (INRA82 and Kenney). Increasing the egg yolk concentrations from 2 to 4% (v/v) in extenders containing 3.1-3.6% (v/v) glycerol improved sperm motility in Kenney semen extender only. These experiments demonstrate that there are possible differences in the ability of spermatozoa from different stallions to withstand different cooling treatments, but not necessarily in their tolerance to different semen extenders.