**2.4 Dose-response effect of Vn on sperm penetration after bovine IVF**

To reconfirm whether the Vn concentration (500 nM) applied in the study of Tanghe *et al.* (2004*b*) was the most appropriate, a preliminary experiment was conducted to assess the dose-response effect of Vn on sperm penetration after bovine IVF. *In vitro* matured COCs were randomly assigned to 6 different fertilization media (Tanghe *et al.* 2004*a*): fertilization medium supplemented with 0 nM, 10 nM, 100 nM, 250 nM, 500 nM and 1 µM Vn. The oocytes were co-incubated with sperm at a final concentration of 106 spermatozoa mL-1 for 20 h (39°C; 5% CO2). Prior to overnight fixation (2% paraformaldehyde – 2% glutaraldehyde in PBS) and staining with 10 µg mL-1 Hoechst 33342 for 10 min, the presumed zygotes were vortexed for 3 min to remove excess spermatozoa and/or cumulus cells. Zygotes were mounted in glycerol with 25 mg mL-1 DABCO and evaluated for sperm penetration using a Leica DMR fluorescence microscope.
