Zona free hamster ova penetration test: fertility indicator of buck spermatozoa

An experiment was conducted to assess the sperm fertility of Sirohi bucks. Ejaculates from three bucks were collected through artificial vagina and evaluated microscopically at 37° C. After final washing with Biggers-Whitten Whittingham (BWW) medium, semen was kept in a CO2 incubator for 20 min (swim up). The hamster oocytes were recovered following superovulation and visualized on the basis of cumulus cell mass. The oocytes were washed in BWW medium and then denuded with 0.1% hyaluronidase, the cumulus-free oocytes were treated with 0.1% protease for 2–3 min for zona lysis. Oocytes and swim up motile spermatozoa were co-incubated at 37° C for 3 h. in already incubated BWW drop (50 μl). At the end of co-incubation, washing of oocyte sperm complexes was done with BWW medium and then kept in hypotonic KCl solution (50 mg/ml), fixed in 2.5% glutaraldehyde for 10 min and stained with 0.1μg/ml DAPI (4,6-di amidino 2-phenyl indole). The number of sperms penetrated into the oocytes was determined. The result of the study indicated that among the three bucks, buck No. 1 had higher penetration rate (100%) as compared to buck No. 2 (93.00%) and buck No 3 (86.66%).