Aim:The present study was designed to evaluate the effect of supplementation of glutamine and replacement of Tris-egg yolk (TE) based buffer with defatted cow milk on the spermatozoa quality after equilibration and thawing.Materials and Methods:Semen was collected from five Bhadawari bulls biweekly, and a total of 30 ejaculates were taken. The semen ejaculates were pooled and divided into three equal parts. The pooled semen was diluted by TE based extender (control), TE + glutamine (8 mM) (T1) and 50% TE + 50% deffated cow milk + glutamine (8 mM) (T2). At two stages viz. after equilibration and after 12 h of cryopreservation (thawed samples), progressive motility, percent live, and percent acrosomal damage of the spermatozoa was assessed.Results:Supplementation of glutamine improved (p<0.05) the spermatozoa quality with respect to the progressive motility, percent live and acrosomal damage both post-equilibration and post-thaw. T2 improved (p<0.05) the spermatozoa quality as compared to control, however; it was less (p<0.05) effective as compared T1 both post-equilibration and post-thaw.Conclusion:From the results of present study it can be concluded that glutamine supplementation was effective in maintaining post-equilibration and post-thaw spermatozoa quality whereas defatted cow milk was not as effective as TE based buffer in the extender in improving the spermatozoa quality.