Bovine follicular dynamics, oocyte recovery, and development of oocytes microinjected with a green fluorescent protein construct

The present study was carried out to 1) evaluate the viability of in vitro fertilized zygotes after microinjection of DNA, 2) assess the influence of oocyte quality upon the development rate of embryos when injected with DNA, and 3) determine the integration frequency of green fluorescent protein DNA into microinjected embryos. Oocytes were aspirated from ovaries of nine nonlactating Holsteins and were categorized into grades A, B, C, and D. At 16 h after in vitro fertilization, approximately half of the pronuclear stage presumptive zygotes were classified as having 1 pronucleus or 2 pronuclei, and they were microinjected with DNA constructs. A potential predictor of DNA integration frequency at d 10 was assessment of the incidence of green fluorescing embryos. The proportion of cleaved embryos that developed to morulae or blastocysts was not different between groups with 1 pronucleus injected (45%), 1 pronucleus uninjected (64%), or 2 pronuclei injected (49%). However, the development of morulae or blastocysts was higher in the group with 2 pronuclei uninjected (69%). The overall developmental score of green fluorescent protein-positive embryos was higher for grade A oocytes (1.3 +/- 0.1) than for grade B (0.8 +/- 0.1), C (0.6 =/- 0.1), or D (0.3 +/- 0.1) oocytes. The results show that production of transgenic bovine blastocysts can occur from the microinjection of a presumptive zygote having only one visible pronucleus. Initial oocyte quality is an important factor in selection of oocytes suitable for microinjection of DNA and for preimplantation development to produce bovine transgenic embryos.