**2.6 Effect of Vn on sperm-zona binding**

*In vitro* matured COCs were denuded by vortexing and randomly assigned to two groups (4 replicates). The first group was fertilized under control conditions (in standard fertilization medium), the second group in the presence of 500 nM Vn. The oocytes were co-incubated with sperm at a concentration of 105 sp mL-1. This sperm concentration was previously determined in order to allow unambiguous counting of the number of spermatozoa bound to the ZP. Each group consisted of 77 to 100 oocytes. After 20 h of co-incubation, the oocytes were washed 3 times to remove loosely attached spermatozoa, and subsequently fixed and stained with Hoechst 33342. Per presumed zygote the number of spermatozoa bound to the ZP was determined.
