Optimization of in vitro bovine embryo production: effect of duration of maturation,length of gamete co-incubation,sperm concentration and sire

The aim of these experiments was to investigate the effect of duration of IVM, duration of gamete co-incubation, and of sperm dose on the development of bovine embryos in vitro. In addition, the speed of sperm penetration of six bulls of known differing in vivo and in vitro fertility was examined. In Experiment 1, following IVM for 16, 20, 24, 28 or 32 h, cumulus oocyte complexes (COCs) were inseminated with 1 x 10(6) spermatozoa/ml. After 24 h co-incubation, presumptive zygotes were denuded and placed in droplets of synthetic oviduct fluid (SOF). In Experiment 2, following IVM and IVF, presumptive zygotes were removed from fertilization wells at 1, 5, 10, 15 or 20 h post insemination and placed in culture as described above. In Experiment 3, following IVM, COCs were inseminated with sperm doses ranging from 0.01 x 10(6) to 1 x 10(6) spermatozoa/ml. Following co-incubation for 24 h, presumptive zygotes were placed in culture as described above. In Experiment 4, following IVM, oocytes were inseminated with sperm from six bulls of known differing field fertility. To assess the rate of sperm penetration, oocytes were subsequently fixed every 3 h (up to 18 h) following IVF. Based on the results of Experiment 4, in Experiment 5, following IVM for 12, 18 or 24 h, COCs were inseminated with sperm from two sires with markedly different penetration speeds. After 24 h co-incubation, presumptive zygotes were denuded and placed in culture. The main findings from this study are that (1) the optimal duration of maturation of bovine oocytes in vitro to maximize blastocyst yield is 24 h, (2) sperm-oocyte co-incubation for 10 h is sufficient to ensure maximal blastocyst yields, (3) sperm concentrations of 0.25 x 10(6) and 0.5 x 10(6) spermatozoa/ml yielded significantly more blastocysts than any other concentration within the range of 0.01 x 10(6) 1 x 10(6) spermatozoa/ml, (4) there are marked differences in the kinetics of sperm penetration between sires and this may be a useful predictor of field fertility, and (5) the inferior development associated with slower penetration rates may in part be overcome by carrying out IVF at a time when the actual penetration is most likely to coincide with the completion of maturation.