Effects of fetuin on zona pellucida hardening and fertilizability of equine oocytes matured in vitro

In vitro fertilization (IVF) has had poor success in the horse, a situation related to low rates of sperm penetration through the zona pellucida (ZP). Zona pellucida hardening (ZPH) is seen in mouse and rat oocytes cultured i n serum-free medium. The hardened ZP is refractory to sperm penetration. Fe tuin, a component of fetal calf serum, inhibits ZPH and allows normal ferti lization rates in oocytes cultured in the absence of serum. We evaluated wh ether fetuin is present in horse serum and follicular fluid (FF) and whethe r fetuin could inhibit ZPH in equine oocytes matured in vitro, thus increas ing sperm penetration during IVF. The presence of fetuin in equine serum an d FF was confirmed by immunoblotting. Oocytes submitted to in vitro maturat ion (IVM) in medium containing fetuin were used for ZPH assay or IVF. Intra cytoplasmic sperm injection (ICSI) was carried out as a control procedure, The presence of fetuin during IVM did not affect the rate of maturation to metaphase II. Maturation of oocytes in the presence of fetuin reduced ZPH i n a dose-dependent manner. After both IVF and ICSI, there was no significan t difference in oocyte fertilization between fetuin-treated and untreated o ocytes. The fertilization rate was significantly higher after ICSI than aft er IVF, both in fetuin-treated and in untreated oocytes. In conclusion, fet uin reduced ZPH in equine oocytes but did not improve sperm penetration dur ing IVF. This implies that, in the horse, "spontaneous" ZPH is unlikely to be the major factor responsible for inhibiting sperm penetration in vitro.