105 ALTERING SEMEN EXTENDER AND GLYCEROL CONCENTRATION TO OPTIMIZE RESULTS AFTER CRYOPRESERVATION OF EQUINE SPERMATOZOA

Abstract

After cryopreservation, spermatozoa from many stallions may have a lower capacity to fertilize an oocyte than fresh or cooled semen. The aim of this study was to evaluate a standard panel of semen extenders and varied concentrations of the cryoprotective agent (glycerol) to optimize sperm survival rates after cryopreservation. Semen was collected from Quarter Horse stallions (n = 3) from March to May 2006 (6 collections per stallion). Semen was filtered immediately after collection, and sample volume and sperm concentration were measured. A drop of raw semen was placed on two prewarmed slides to estimate the percentage of progressively motile sperm. The semen sample was then diluted to 100 × 10⁶ spermatozoa/mL with a dried skim milk glucose extender (EZ Mixin Original Formula; ARS, Chino, CA, USA) or a chemically defined, milk-free diluent (INRA 96; IMV, Maple Grove, MN, USA). After 1 h of slow cooling and equilibration to 4°C, semen samples were centrifuged for 10 min at 400g. A defined volume of supernatant was removed, so that a concentration of 1000 × 10⁶ spermatozoa/mL was obtained after resuspension of the sperm pellet. A 150-µL aliquot of semen was then added to specified quantities of the same semen extender used after semen collection and cryopreservation medium (Cryoguard^®; Minitube, Verona, WI, USA) to obtain final glycerol concentrations of 2, 3, and 4%. This also gave a concentration of 100 × 10⁶ spermatozoa/mL. After equilibration for 1 h at 4°C, spermatozoa were loaded into 0.5-mL straws and frozen in liquid nitrogen vapor. After 10 min, straws were plunged into liquid nitrogen. Semen was thawed at 37°C for 30 s and evaluated as prior to cryopreservation. Mean total semen volumes were 56, 11, and 60 mL in the 3 stallions. Their respective mean sperm concentrations were 124 × 10⁶, 505 × 10⁶, and 161 × 10⁶ sperm/mL, respectively. Mean percentages of progressively motile sperm prior to cryopreservation were 64, 89, and 72%, respectively. With the paired Student's t-test, percentages of progressively motile sperm after cryopreservation were evaluated with respect to semen extender and concentration of glycerol used. Mean overall progressive motility of spermatozoa after cryopreservation differed significantly between the two extenders and was 46% for INRA 96 and 35% for EZ Mixin OF (P < 0.001). Using EZ Mixin OF as semen extender, the best mean post-thaw progressive motility was achieved with 4% glycerol (39%) and differed significantly from that with 2% glycerol (32%; P < 0.01). When INRA 96 was used (49% for 4%, and 42% for 2% glycerol), there was no difference. This results provide evidence that, during freezing of equine spermatozoa, there is a significant effect of the semen extender and the concentration of the cryoprotectant on post-thaw sperm motility. We therefore suggest mini-freezing trials prior to freezing large numbers of sperm to find the semen extender and glycerol concentration that provides optimal survival rates.