Effects of culturing time before electrical fusion and chemical activation on constructed sheep embryos.

In order to compare the effects of culturing time before electrical fusion and chemical activation on the fusion and development of the constructed embryo, embryos were reconstructed by subzonal injecting the sheep (Bos gaurus) somatic cells into matured sheep oocytes in vitro. Results showed that culturing for 1 h before electrical fusion, there were no significant differences in cleavage, morulas and blastulas rates among the groups(P 0.05), however, the integrity rate of couplets cultured for 2 h before activation (95.81%) was significantly higher than control (74.64%)(P 0.05); under the condition of culturing for 2 h before chemical activation, there were no remarkable differences in the rate of fusion, integrity and the blastulas (P 0.05), but the rate of cleavage and morula which were cultured for 1h before activation (76.88% and 40.63%) were dramatically higher than control(64.48%and 21.50%) (P 0.05). When developed into morula or blastulas in commercial G1/G2 sequential medium, the embryos were transferred into 41 recipients, only one finally was developed into a fetus but aborted when it was 104 day old in vivo. Then microsatellites DNA analysis with 5 pairs of primers was carried out on the fibroblast of aborted fetus, donor cells, the fibroblast of recipient sheep and one control chosen occasionally. Results indicated that the bands had polymorphism after 10% Ago-Gel electrophoresis of PCR products and silver staining, and the fingerprint of cloned fetus were completely the same as those of donor cells, while definitely different from those of recipient sheep and the control, and the gene of cloned fetus came from donor cells and the fetus was a cloned sheep.