A series of experiments were designed to study factors affecting the ability of pig spermatozoa to fertilize in vitro and the rate of development of pig oocytes fertilized in vitro.In experiment 1, the effect of different capacitation promoters on the ability of spermatozoa to fertilize pig oocytes in vitro was studied. Fresh pig ejaculated spermatozoa were preincubated in m-TCM199 without bovine serum albumin and supplemented in 4 groups with either heparin (50 μg/ml), inophone A23187 (1μM), caffeine (5mM) or without any promoter for 10 to 30 min at 35 to 37°C. Pig oocytes (n=759) were matured at 38 to 39°C for 32 to 36 h (Theriogenology 37:733-739, 1992). Preincubated spermatozoa and matured oocytes were transferred into drops of m-TCM199 supplemented with caffeine (5 mM) for IVF incubation for 8 to 9 h at 38 to 39°C. After IVF, the highest (36%) cleavage rate (2- to 4-cell stage) was obtained by the pig spermatozoa treated with heparin.In experiment 2, in vitro insemination time and frequency were studied. Oocytes, (n=699) matured in vitro, were fertilized in vitro using fresh ejaculated spermatozoa at different insemination times. This experiment demonstrated that multiple inseminations were more effective in increasing IVF rate of pig oocytes. After IVF, the highest (34.5%) cleavage rate (2- to 4-cell stage) was obtained after 2 inseminations. Spermatozoa were treated with heparin, added to drops of m-TCM199 supplemented with caffeine and containing oocytes for 3 to 4 h. Then, oocytes were transferred into fresh drops of the same medium and the fresh spermatozoa treated with heparin were added for another 3 to 4 h.In experiment 3, the most suitable temperature for IVM and IVF of pig oocytes was studied. Oocytes (n=480) were randomly incubated at either 37.5 ± 0.5, 38.5 ± 0.5, 39.5 ± 0.5 or 40.5 ± 0.5 °C for IVM and IVF. After IVF, the highest (50.8%) cleavage rate (2- to 4-cell stage) was obtained in the 39.5 ± 0.5 °C group. At this temperature, the development and cleavage rate of oocytes fertilized in vitro was near to the normal developmental rate of oocytes in vivo and more rapid than that below 39°C.Experiment 4 was designed to validate improvements in the IVM and IVF system for porcine oocytes. Oocytes (n=318) were matured and fertilized in vitro by the optimum methods achieved above. Of the 63.3% of oocytes developing to 2- to 4-cell stage, 40 to 48 h post-IVF, 37.6% developed to morula stage by 100 to 120 h post-IVF. Another 227 oocytes, fertilized in vitro, were transferred into 2 recipient gilts and 17.8% and 15.8% developed to morula and hatched-blastocysts, respectively, by 120 to 160 h of in vivo incubation.