The effect of Equex STM® in freezing media on post thaw motility, viability and DNA integrity of frozen-thawed ram spermatozoa.

Abstract

In this study we investigated the effect of Equex STM® on quality and in-vitro survival of ram spermatozoa frozen in Tris egg yolk based extender. Ejaculates from 6 crossbreed rams were frozen according to the standard procedure after two step dilution with Tris-egg yolk extender (1). The second extender, added to the semen at 5°C, contained 14% of glycerol and was supplemented with detergent 0.75% Equex STM® (group OEP) or contained no detergent (control group). After thawing the samples were incubated in a water bath at 37°C and analysis were performed 10 minutes, 6, 12 and 24 hours later. Motility and the viability (Viadent®) of the semen were analysed with Hamilton Thorne Biosciences, Version 12.3 and membrane integrity with HOS (hypoosmotic swelling test). DNA fragmentation (DFI %) of F/T spermatozoa was analyzed 10 minutes and 3 hours after thawing using sperm chromatin structure assay (SCSATM). The sperm membrane integrity was analysed 15 minutes and 3 hours after thawing by Sybr-14/PI test. Percentage of motile spermatozoa was significantly higher in OEP group in comparison to control group at 0, 6, 12 and 24 h (P<0.001). Viability of spermatozoa was significantly higher (P<0.001) in OEP compared to control group in all analysed times after thawing. Percentage of HOS positive spermatozoa was significantly higher in OEP compared to control group respectively for 0 (P=0.001), 6 (P=<0.001), 12 and 24 h (P=0.002) after thawing.