Effect of butylated hydroxytoluene on capacitation like changes in spermatozoa of Hariana bull during cryopreservation of semen

Abstract

The experiment was planned to analyse the effect of butylated hydroxytoluene (BHT) supplementation as an antioxidant on capacitation like changes in spermatozoa of Hariana bull during cryopreservation. A total 24 ejaculates were collected bi-weekly by artificial vagina method from four Hariana bulls. The ejaculates fulfilling the minimum criteria as laid down for cryopreservation were further processed. The ejaculated samples were extended upto 80 million spermatozoa/mL in a Glycerolated Egg Yolk Tris (GEYT) Extender. Different groups of diluted semen were prepared having different concentration of BHT. One part was left as such (Control) while other two parts contain 0.5 and 1.0 mM BHT were considered Group 1 (G1) and Group 2 (G2), respectively. Following the process of extension and equilibration (4 h), the semen was frozen in liquid nitrogen vapour in French mini straw (20 million spermatozoa/straw) using biological freezer. Following freezing and storage in LN2 for 24 h, thawing was performed at 37°C for 45 sec for all the cryopreserved straws. Chlortetracycline (CTC) assay was conducted to evaluate capacitation like changes in samples at initial stage of dilution, equilibration and following thawing. BHT was found to prevent capacitation like changes at all the stages of semen cryopreservation process. Inclusion of 1.0 mM was found to be effective in preventing capacitation like changes in Hariana bull spermatozoa.