Зголемување на криопрезервацискиот капацитет на ејакулатни медиуми за овчеполска праменка со употреба на семинална плазма и глутатион

Abstract

The complementary effect of protein and antioxidant components of the seminal plasma in regulating oxidative radical concentration was the basic concept of investigating the cryopreserving potential of tris-based (tris-hydorymethyl-aminomethane) media (tris-CM) supplemented with reduced glutathione (GSH) and homologous-seminal-plasma (HSP) in aim of achieving higher cryopreserving (CP potential of ovchepolska pramenka spermatozoa. The aims of this research were: 1. To assess the CP success of ovchepolska pramenka spermatozoa extended in HSP/GSH supplemented tris-CM; 2. To assess the CP success of ovchepolska pramenka spermatozoa extended prior- or following-CP; 3. To assess the optimizing effect of incubation time on HSP/GSH supplemented tris-CM. Single ejaculates were acquired from ten rams (ovchepolska pramenka) with artificial vagina method. Fresh ejaculates were pooled in single volume which was divided in aliqotes-samples (N=240). Four tris-CM were prepared: 1. (С) - HSP and GSH free; 2. (Е1) - with GSH (5 mM); 3. (Е2) - with HSP (20 %); and (Е3) - with GSH (5 mM) and HSP (20 %). One half of the samples were extended prior- (a-CT), and the other following-CP (b-CT). After the CP in liquid nitrogen, the samples were placed in incubation chamber (37°С) and were assessed at 0- and 3-hours incubation. The CP success was assessed according to the degree of retaining structural integrity, morphology (Hancock-2) and motility (CASA – Hamilton Thorne). Interpretation was performed by Pearson’s χ2 test of independence, which was evaluated according to degrees of freedom, probability levels (p < α) and minimal χ2 value, determined algorithmically G*Power v.3.1). E3 in a-CT method at 0-hour incubation had the highest proportion of cells with intact structural integrity and morphology (57.16 %), and retained trajectory linearity of motile cells. C in a-CT method at 0-hour incubation had the highest proportion of cells with deteriorated integrity and morphology (6.82 %) as well as highly deviated trajectory of the motile cells. In b-CT method at 0-hour incubation, there were no significant discrepancies for spermatozoa categories with intact structural integrity in C (50.05 %), E2 (48.95 %), and E3 (48.25 %). Despite this, C had high deviation of trajectory linearity. E3 samples of a-CT at 0-hour incubation had higher proportion of spermatozoa with intact structural integrity (57.16 %) in relation to E3 samples of b-CT method (48.25 %). It was conclusive that the combined supplementation of GSH (5 mM) and HSP (20 %) in tris-CP media used for extending ejaculates prior CP, resulted in significantly higher CP capacity for ram spermatozoa of ovchepolska pramenka compared to other tris-CP media which contained GS (5 mM) or HSP (20 %). Post-thaw incubation time of 3-hours has increased the number of spermatozoa with damaged structural integrity, affected morphology or hyper-activated motility, regardless of the CP-media or extending method.