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Bovine inner cell mass (ICM) cells were used as donor nuclei in nuclear transfer procedures to determine their totipotency. ICMs were isolated by immunosurgery from expanded Day 7-9 blastocysts that had been produced in vitro. Each individual ICM cell was transferred into an enucleated oocyte. Oocytes were checked for enucleation with Hoechst dye to ensure that all DNA was removed, thus eliminating the possibility of parthenogenetic development. ICM cell-oocyte units were fused by a brief electrical pulse (110 V DC, 15 microseconds in a 500-microns chamber), and the resulting zygotes were placed into CR1 bovine embryo culture medium supplemented with amino acids and fetal calf serum. The nuclear transfer embryos were scored for development to the blastocyst stage on Day 7 (day of fusion = 0). A total of 948 nuclear transfers were completed in 25 trials. In 12 of the trials, development to the blastocyst stage (5%, 30 of 629) was observed. This resulted in an overall developmental rate of 3% for all trials. Twenty-six of the ICM-derived blastocysts were transferred. The initial pregnancy rate at 30 days was 23% with six pregnant recipients. Two pregnancies were lost after 60 days, and four calves were born, two of which were stillborn. These results demonstrated that nucleic of ICM cells from expanded bovine blastocysts were pluripotent, if not in fact totipotent, since these nuclei after nuclear transfer to enucleated oocytes could direct embryonic and fetal development resulting in live offspring. |
