Effect of Adding Equex STM Paste and BHT to Different Extenders on Viability, Plasma Membrane and Dna Integrity of German Shepherd Dog Spermatozoa During Freezing and Thawing Methods in Egypt

Abstract

Many factors influencing semen quality through all steps of cryopreservation such as semen processing, the combination of extender, cooling rate during the freezing procedure and the thawing technique. The present study was conducted to evaluate the potential cryoprotective effect of Equex STM paste and / or butylated hydroxytoluene (BHT) occlusion in Tris and milk based extenders, freezing according to 2 methods, thawing at 2 regimes, and the interactions between these treatments, on the post-thaw semen quality. A total number of 80 ejaculates was collected from 15 German shepherd stud dogs. Semen samples was frozen in two different extenders, Tris (TCF) and milk (SMG) based extenders, with or without 0.5% Equex STM paste and / or 1mM BHT compared with commercial bovine tris based semen extender (BULLXcell ™ , IMV technologies, Franc) using 2 freezing protocols (4 and 6 cm) above LN 2 and 2 thawing rates (45 o C for 30 sec. and 55 o C for 5 sec.). Post-thaw motility, viability, plasma membrane integrity in terms of post-thaw live spermatozoa, acrosomal defects, acrosome reaction and rate of increase in activity of extracellular AST, ALT and ALP enzymes as well as assessment of sperm DNA integrity using comet assay were recorded. The results clarified that addition of Equex STM paste and / or BHT to the TCF and SMG extenders significantly (P<0.01) improved post-thaw motility, viability, plasma membrane integrity and reduced acrosomal defects, acrosomal reaction, release of extracellular AST, ALT and ALP enzymes and DNA damage. The best post-thaw semen qualities was observed when freezing in a Styrofoam box above 6 cm highest above LN 2 and thawing at 55 o C for 5 sec. the superior results were obtained with Tris based extenders than milk based extenders. It is concluded that fortification of 0.5% Equex STM paste and / or 1mM BHT in Tris based extenders freezing above 6 cm highest above LN 2 and thawing at 55 o C for 5 sec. can improve the post-thaw dog semen motility, viability and plasma membrane integrity. Also, the commercial bovine extender BULLXcell™ is useful for cryopreservation of dog semen.