Oviduct fluid increases the time required for digestion of the zona pellucida (ZP) by protelolytic enzymes (ZP hardening). This effect has been associated to the levels of monospermy after fertilization in vitro (IVF) in the pig and cow but the possible existence of a directly proportional relation between hardening and monospermy remains unknown. To investigate whether variations in the hardening of different OFs are correlated to variations in the levels of monospermy after IVF, porcine oocytes were incubated with three batches of OF known to produce different ZP hardening effects (3, 7 min and 25 min, respectively). After IVF, the monospermy levels were 0%, 14.58 ± 5.14% and 35.14 ± 7.95%, respectively. These results could partially explain the lack of polyspermy found during in vivo fertilization in pigs (with a hardened oviductal ZP) compared with the levels found during in vitro fertilization (with no hardened ZP). Using the bovine model, OF was fractionated by heparin affinity chromatography and the hardening effect on ZP was tested for each fraction obtained from a linear gradient of NaCl concentration. The highest effect was obtained with the fraction eluted with 0.4 M of NaCl. Fractions with high or low level effects were processed by on-chip electrophoresis and HPLC-MS/MS. The list of potential proteins responsible for this effect includes OVGP1 and members of HSP and PDI families.