THE EFFECT OF EXTENDING RAM SPERM BEFORE AND AFTER CRYOPRESERVATION ON THEIR VIABILITY AND VELOCITY

Abstract

The current study aimed to assess the effect of adding cryopreserving extender (soy-bean lecithin-SBLE), reduced glutathione (GSH), and seminal plasma (SP) before and after thawing on viability and velocity of cryopreserved ram sperm in liquid nitrogen. Fresh ejaculates (Ovchepolska pramenka rams, n=10) were collected and pooled. One portion was extended up to 50 million/ml with SBLE (control C-a), SBLE and GSH 5 mM (E1-a), SBLE and SP 20 vol% (E2-a), and SBLE, GSH 5 mM and SP 20 vol% (E3-a), respectively. The second portion was extended with SBLE to 100 million/ml. Both portions were cryopreserved in liquid nitrogen. Following thawing, the second portion was extended in the same manner to 50 million/ml and was separated into C-b, E1-b, E2-b, and E3-b, respectively. Each group was sampled in ten replicates immediately following thawing. Thawed samples were analyzed for viability (Hancock-2 stain), and velocity (Hamilton Thorne, USA). Each sample included at least 200 cells and the results were expressed in percent values (mean±SEM). Normality (Kolmogorov) and variance comparison (factorial-ANOVA) were performed in Statistica 8 with a significance level p<0.05. E2-a (57.58% ±2.40) and E3-a (56.94% ±1.85) yielded significantly higher viability compared to the C-a (40.73 ±1.53). There were no significant differences between C-b (50.00% ±2.33), E1-b (43.61% ±1.37), E2-b (49.16% ±1.50), and E3-b (48.50% ±1.85). In conclusion, the addition of SBLE, GSH, and SP prior vs after cryopreservation has a significant effect on thawed ram sperm viability and velocity.