Development of cloned embryos from adult rabbit fibroblasts: Effect of activation treatment and donor cell preparation

This research was to study the in vitro and in vivo development of cloned e mbryos derived from adult rabbit fibroblasts following various activation p rotocols. Effects of serum starvation and passage number of donor cells on the efficiency of cloning were also examined. In experiment I, oocytes were activated either by electric pulses or by electric pulses followed by cult ure with 6-dimethylaminopurin (DMAP). For experiment II, the best activatio n protocol from experiment I was employed for cloning using adult rabbit fi broblasts that were cultured for 0-15 passages. In experiment III, the effe ct of serum starvation of the donor cells on cloning was examined. Finally, in experiment IV, embryo transfers were conducted. These experiments showe d that combined electrical pulse and DMAP treatment resulted in superior pa rthenogenetic blastocyst development (up to 29%), and that activation of th e cytoplast before versus after fusion was not different in supporting the in vitro development of nuclear transferred embryos (16%-18% blastocysts), Adult fibroblasts derived from nonpassaged cells were less capable of devel oping into blastocysts than passaged cells (6% vs. 17%), Serum starvation o f donor cells improved cleavage (up to 71%) but did not improve blastocyst development (13%), and no progeny was obtained, irrespective of the treatme nt, Cell-cycle analysis of adult rabbit fibroblast cells showed that passag e 6 and 12 cells were more likely to be in G(0)/G(1) than passage 0 cells, which agrees with the improved embryo development in the passaged-cell grou ps.