The aim of this study was to investigate the effect of the duration of oocyte in vitro maturation (IVM) and gamete co-incubation on the in vitro embryo (IVEP) production efficiency in River buffalo. In Experiment 1, abattoir-derived cumulus oocyte complexes were fixed at 0, 3, 6, 9, 12, 15, 18, 21 and 24 h after the start of in vitro maturation to study the kinetics of nuclear maturation. In Experiment 2, cumulus oocyte complexes were fertilized in vitro following in vitro maturation for 18, 21, 24, 27 or 30 h. After 20 h of gamete co-incubation, presumptive zygotes were denuded and cultured in vitro in synthetic oviduct fluid (SOF) medium. In Experiment 3, following in vitro maturation and fertilization, presumptive zygotes were removed from fertilization drops at 8, 12, 16 and 20 h post-insemination (pi) and placed in culture as described above. Representative samples of oocytes were fixed at 4, 8, 12, 16 and 20 h to evaluate the sperm penetration rate and the incidence of polyspermy at different co-incubation times. The main conclusions of the study are that: (1) the majority of buffalo oocytes accomplish nuclear maturation between 21 and 24 h after the start of in vitro maturation; (2) both cleavage and blastocyst rates linearly decrease with increasing duration of in vitro maturation (from 18 to 30 h); (3) sperm-oocyte incubation for at least 16 h is required for maximum blastocyst yields.