The effect of residual pellet volume on cryoprotectant concentration and post-thaw motility of stallion sperm

Abstract

Standard protocols for cryopreservation of stallion sperm involve centrifugation to remove plasma and concentrate ejaculates. Typically, semen is diluted 1:1 or higher to a concentration of 50 to 100 × 106/ml and centrifuged on a cushion. Most supernatant and cushion is aspirated leaving 5-10% of the original volume of diluted semen along with the sperm pellet. Pellets are resuspended in freezing diluent containing cryoprotectant to 200 × 106 sperm/ml. Due to the extreme variability of sperm concentration between ejaculates, the volume of resuspended sperm pellet and supernatant may contribute significantly to the final volume of the extended semen resulting in variability in the final percent of cryoprotectant (CPA) in the extended semen. To determine the degree of variability and the effect of the final CPA concentration on retention of sperm motility we determined final CPA concentration in extended semen of 129 ejaculates from 16 different stallions. Our hypothesis was that ejaculates cryopreserved with CPA concentration significantly lower than that present in the freezing diluent would have lower cryosurvival. Pellet volume was determined by weight and used to calculate the final CPA concentration prior to freezing for each ejaculate. The final CPA concentration was expressed as percentage of the CPA concentration in the original freezing extender. For the 129 ejaculates, the mean final CPA concentration aspercentage of original was 69.5%, median 73.7%, min 39.2% and max 87.8%. Final CPA concentration 1.3 - 5.1%. Sperm motility was analyzed by CASA for fresh extended and post-thaw samples. Thawed samples were diluted in centrifugation media and incubated (37C for 30 minutes) prior to analysis. For each sample a minimum of 5 fields and 400 motile sperm were analyzed (40 frames at 60 frames/sec); progressive cells defined as (VAP > 50 mic/sec and STR > 75%). Cryosurvival was expressed as percentage change in motility between pre-freeze and post-thaw values. Ejaculates were divided into 5 groups based on final CPA concentration as percentage of the original extender amount (G1 = 40-50%, G2 = 50-60%, G3 = 60-70%, G4 = 70-80%, G5 = 80-90%). Mean values for change in motility were compared between groups using ANOVA with multiple comparisons. Decrease in percent total motility (mean,sd) was less (p<0.05) in G5 (22.8,11.0) compared to all other groups G4 (31.3,11.3), G3 (31.2,13.1), G2 (29.5,8.8), G1 (30.9,10.8). Decrease in progressive motility was less (p<0.05) in G4 (8.5,14.8) and G5 (2.7,10.5) vs G1(17.8,8.7), G5 vs G2 (13.6,10.9) and G5 vs G3 (12.9,14.3). These data suggest that current protocols for cryopreservation of stallion semen may result in inadequate CPA concentration in extended semen of many ejaculates. Further experiments are underway to determine critical thresholds.