Fertilization and early embryology: The factors affecting sperm binding to the zona pellucida in the hemizona binding assay

Sperm binding to the zona pellucida is a prerequisite for fertilization.The hemizona binding assay (HZA) is commonly used to evaluatethe zona-binding capacity of spermatozoa. The present studyreports three factors that affect HZA. They were the base mediumused, the protein source and the size of pipette used for removingloosely bound spermatozoa during HZA. The number of spermatozoabound on the hemizona was compared between (1) Earle's balancedsalt solution (EBSS) and Ham's F-10, and (2) between human serumand bovine serum albumin (BSA). Results indicated that EBSSand human serum both significantly increase the number of boundspermatozoa when compared to Ham's F-10 (P < 0.0001, pairedt-test) and BSA (P < 0.05, paired t-test) respectively. Pipettesof different diameters were used to study the effect of sizein removing loosely bound spermatozoa on hemizona. Data showedthat the diameter of the pipette should be 200 mm, in ordernot to remove bound spermatozoa excessively. These results emphasizethe importance of standardization of the protocol of the hemizonaassay worldwide to be able to compare results between differentlaboratories.