THE FACTORS AFFECTING SPERM BINDING TO THE ZONA-PELLUCIDA IN THE HEMIZONA BINDING ASSAY

Sperm binding to the zona pellucida is a prerequisite for fertilizatio n. The hemizona binding assay (HZA) is commonly used to evaluate the z ona-binding capacity of spermatozoa. The present study reports three f actors that affect HZA. They were the base medium used, the protein so urce and the size of pipette used for removing loosely bound spermatoz oa during HZA. The number of spermatozoa bound on the hemizona was com pared 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 nu mber of bound spermatozoa when compared to Ham's F-IO (P < 0.0001, pai red t-test) and BSA (P < 0.05, paired t-test) respectively. Pipettes o f different diameters were used to study the effect of size in removin g loosely bound spermatozoa on hemizona, Data showed that the diameter of the pipette should be greater than or equal to 200 mm, in order no t to remove bound spermatozoa excessively. These results emphasize the importance of standardization of the protocol of the hemizona assay w orldwide to be able to compare results between different laboratories.