Butylated hydroxytoluene (BHT) improved semen quality and sperm DNA of frozen-thawed Arabian stallions preserved in modified INRA-82 extender.

Abstract: Alpha-tocopherol is one of the non-enzymatic lipophilic antioxidants. Butylated hydroxytoluene (BHT) is a synthetic analog with similar modes of action in protecting the cryopreserved sperms. Objective: This study hypothesized that a certain concentration of any antioxidant is suitable for improving the post-thaw semen quality of stallions. Methods: To determine the optimum BHT concentration, a synthetic antioxidant similar to vitamin E in potency and scavenging oxidative stress power in concentrations of 0.0, 0.25, 0.50, 1.0, 2.0, and 4.0 mM/ml were added to semen extender. The post-thaw sperm progressive motility at 0, 1 h, 2 h, and 3 h, the sperm viability index, the plasma membrane integrity tested by the hypo-osmotic swelling test (HOST), the acrosome integrity, non-fragmented DNA, % of DNA in the comet head, % of DNA in the comet tail, comet tail length, and comet tail moment were compared. Results: According to our hypothesis, 1.0 mM BHT was the most suitable concentration that preserved the highest (P < 0.0001) post-thaw sperm progressive motility at 0.0, 1 h, 2 h, 3 h., the highest viability index (P < 0.0001), plasma membrane and acrosome integrities (P < 0.0001),non-fragmented DNA (P < 0.001), and % of DNA in the comet head (P < 0.001), the lowest DNA % in the comet tail (P < 0.001) and comet tail moment (P < 0.0001), and the shortest (P < 0.0001) comet tail length. Conclusions: The concentrations of BHT around 1.0 mM/ml (0.5, 2.0mM/ml) proved better post-thaw semen characteristics, but increasing BHT concentrations from >2.0mM/ml to 4.0 mM/ml started to deteriorate semen quality than non-supplemented control. The optimum BHT concentration for modified INRA-82 is 1.0 mM/ml.