Prognostic Value of Various Spermatological Attributes as Predictors of Zona Binding and Zona Penetration of Buffalo (Bubalus bubalis) Semen

Twenty‐four ejaculates from six (four ejaculates each) Surti buffalo bulls aged 4–8 years were used to assess various attributes of spermatozoa influencing the zona‐binding and zona‐penetration tests. Ejaculates from each bulls were subjected to in vitro sperm‐‐zona binding and sperm‐‐zona penetration tests (four replicates per bull) using immature buffalo oocytes. The average number of spermatozoa bound per oocyte was 27.79 ± 5.90. The average number of spermatozoa penetrated per oocyte was 3.35 ± 0.64. The average number of zona‐bound and ‐penetrated spermatozoa differed significantly between animals. Significant difference (p < 0.05) was observed between the plasmalemma integrity as assessed by eosin‐‐nigrosin stain and hypo‐osmotic swelling (HOS) test. Furthermore, the percentage of cells positive for the HOS test, i.e. functional membrane integrity (51.25 ± 2.32) was significantly (p < 0.05) higher than hypo‐osmotic swelling‐Giemsa (HOS‐G) test, i.e. the subpopulation of spermatozoa positive for functional membrane and acrosomal integrities (42.87 ± 4.56). The HOS test had significant correlations with plasmalemma integrity as measured by the vital stain, eosin‐‐nigrosin (r = 0.85, p < 0.05). The HOS‐G test also had significant correlation with plasmalemma integrity measured by vital stains such as eosin‐‐nigrosin (r = 0.90, p < 0.05) and fluorogenic stains [carboxyfluorescein diacetate (CFDA) and propidium iodide (PI); r = 0.92, p < 0.01] and HOS test (r = 0.93), acrosomal integrity (r = 0.86, p < 0.05) and mitochondrial membrane potential (r = 0.99, p < 0.01). The plasmalemma integrity (fluorogenic stain), functional membrane integrity (HOS test), subpopulation of spermatozoa positive for functional membrane and acrosomal integrities (HOS‐G test) and mitochondrial membrane potential had significant (p < 0.05) correlation with sperm zona binding and penetration. The present study indicates that these parameters could represent important determinants of sperm quality influencing zona binding and penetration.