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

Abstract

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.