Effects of microinjection-related treatments on the subsequent development of in vitro-produced bovine oocytes.

The effects of gene injection-related handling on the subsequent development of in vitro-produced bovine oocytes were studied. In Experiment 1, centrifuged oocytes were stored in an injection chamber for 30 min on a warm (+39 degrees C) stage at 18, 22, 26 or 30 h post insemination. In Experiment 2, centrifuged oocytes were stored for 60, 90 or 120 min on a warm stage, while in Experiment 3 they were stored for 60 min on a warm or cool (+22 degrees C) stage. In Experiment 4, the centrifuged zygotes were injected with buffer either into the pronucleus or cytoplasm. Development to morulae and blastocysts at Day 7 was monitored. The results indicate that handling of oocytes either very early (18 h post insemination) or very late (30 h post insemination) significantly reduced development (P<0.05). The duration of the storage or the temperature during storage did not have any significant effect on the development of the embryos. Development (counted from the cleaved oval, however, was significantly lower (P<0.01) in pronucleus-injected than in cytoplasm-injected or control embryos (27.7, 45.5 and 44.0% morulae and blastocysts, respectively). The conclusion of this study is that the main reason for decreased development of pronucleus-injected, in vitro-produced bovine zygotes is the pronucleus-injection itself rather than injection-related handling or the overall damage caused by zygote piercing.