In vitro fertilization (IVF) in pigs is still considered sub-optimal, due to the variable occurrence of polyspermy, variability mainly related to sperm differences. The present study was conducted in an attempt to increase the efficiency of the in vitro production of porcine embryos by optimizing the in vitro fertilization (IVF) protocol for individual males, with regard to the composition of the fertilization medium (experiments 1 and 2) and the length of gamete co-incubation time (experiment 3). A total of 5,943 COC's were in vitro matured (IVM) and inseminated with frozen-thawed spermatozoa from 2 boars (A and B). Experiment 1 determined the effect of additives caffeine (2mM), hyaluronic acid (HA; [0.5mg/mL]) and adenosine (10 microM), alone or in combination, to the IVF-medium during sperm-oocyte co-incubation. Experiment 2 tested the addition of various HA (0, 0.5, 1.0 and 1.5mg/ml) and adenosine (0, 10, 20 and 40 microM) concentrations in the fertilization medium; while experiment 3 investigated the effect of two periods of sperm-oocyte co-incubation (10 min or 6h). In the case of 10 min sperm-oocyte co-incubation, oocytes with attaching spermatozoa were further cultured in IVF-medium containing no spermatozoa until the 6h of insemination was completed. Presumptive zygotes were cultured in embryo culture medium for 12-15 h to assess fertilization parameters. In experiment 1, only caffeine significantly influenced the outcome of fertilization, albeit being a clearly boar-dependent effect. In experiment 2, similar boar differences were seen for HA supplementation while presence of exogenous adenosine did not influence fertilization parameters in either boar. The results of experiment 3 demonstrated that a short co-incubation time significantly (P<0.001) increased penetration rate and mean number of spermatozoa per oocyte (74.9+/-3.9% versus 62.7+/-3.9% and 1.5+/-3.2 versus 1.3+/-3.5 for 10 min or 6h, respectively), but reduced mono-spermy (P<0.001, 57.9+/-2.5% versus 70.0+/-2.8%) when boar A was used. However, such effects were not seen with boar B, in which sperm-oocyte co-incubation time did not affect the efficiency of fertilization. In view of the present results, a preliminary screening for each individual male is required to select optimal conditions for IVF.