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 evaluate the zone-binding capacity of spermatozoa. The present study reports three factors that affect HZA. They were the base medium used, the protein source and the size of pipette used for removing loosely bound spermatozoa during HZA. The number of spermatozoa bound on the hemizona was compared between (1) Earle's balanced salt solution (EBSS) and Ham's F-10, and (2) between human serum and bovine serum albumin (BSA). Results indicated that EBSS and human serum both significantly increase the number of bound spermatozoa when compared to Ham's F-10 (P < 0.0001, paired t-test) and BSA (P < 0.05, paired t-test) respectively. Pipettes of different diameters were used to study the effect of size in removing loosely bound spermatozoa on hemizona. Data showed that the diameter of the pipette should be >/=200 mm, in order not to remove bound spermatozoa excessively. These results emphasize the importance of standardization of the protocol of the hemizona assay worldwide to be able to compare results between different laboratories.