Protein tyrosine phosphorylation and zona binding ability of in vitro capacitated and cryopreserved buffalo spermatozoa

Many similarities between the changes associated with normal capacitation and cryocapacitation have been demonstrated. The present study was undertaken to determine whether similarities exist in the protein tyrosine phosphorylation pattern and zona binding ability between in vitro capacitated (heparin induced; 20 μg/ml) and frozen-thawed (cryocapacitated) buffalo spermatozoa. Semen from seven buffalo bulls (eight ejaculates each) was divided into two parts. Part I was used as fresh semen and part II was extended in Tris-egg yolk extender, equilibrated and frozen in liquid nitrogen. Localization of phosphotyrosine-containing protein was determined using an indirect immunoflourescence assay with anti-phosphotyrosine antibody. For zona binding assay, good quality oocytes collected by aspiration technique from fresh buffalo ovaries were used. The bound spermatozoa were stained with Hoechst 33342 dye and observed under fluorescent microscope. The results revealed sperm head associated protein tyrosine phosphorylation in both in vitro capacitated and frozen-thawed spermatozoa. In the zona binding assay, the mean number of bound spermatozoa was 90.6 ± 1.9 and 104.7 ± 2.2 in fresh semen after incubation in non capacitating media at 0 h and 3 h, respectively. But after incubation in capacitating media with heparin for 3 h, the mean number of spermatozoa attached to zona pellucida was 138.4 ± 2.6. The in vitro capacitated spermatozoa had significantly (P < 0.05) higher binding ability than that of fresh spermatozoa. After freezing and thawing, 2.5 fold reductions in the zona binding ability of cryopreserved spermatozoa was observed compared to in vitro capacitated spermatozoa. The binding ability of in vitro capacitated spermatozoa was significantly (P < 0.01) higher than that of frozen-thawed (cryocapacitated) spermatozoa. The study concluded that both in vitro capacitated and frozen-thawed (cryocapacitated) spermatozoa had similar immune-localization of tyrosine phosphorylated protein pattern, however, differed in the zona binding ability.