Production of cloned calves following nuclear transfer with cultured adultmural granulosa cells

Adult somatic cell nuclear transfer was used to determine the totipotent po tential of cultured mural granulosa cells, obtained from a Friesian dairy c ow of high genetic merit. Nuclei were exposed to oocyte cytoplasm for prolo nged periods by electrically fusing quiescent cultured cells to enucleated metaphase II cytoplasts 4-6 h before activation (fusion before activation [ FBA] treatment). Additionally, some first-generation morulae were recloned by fusing blastomeres to S-phase cytoplasts. A significantly higher proport ion of fused embryos developed in vitro to grade 1-2 blastocysts on Day 7 w ith FBA(27.5 +/- 2.5%) than with recloning (13.0 +/- 3.6%; p < 0.05). After the transfer of 100 blastocysts from the FBA treatment, survival rates on Days 60, 100, 180, and term were 45%, 21%, 17%, and 10%, respectively. Ten heifer calves were delivered by elective cesarean section; all have survive d. After the transfer of 16 recloned blastocysts, embryo survival on Day 60 was 38%; however, no fetuses survived to Day 100, DNA analyses confirmed t hat the calves are all genetically identical to the donor cow. It is sugges ted that the losses throughout gestation may in part be due to placental dy sfunction at specific stages. The next advance in this technology will be t o introduce specific genetic modifications of biomedical or agricultural in terest.