Abstract
The objective of this study was to investigate the formation of bovine embryonic stem (ES) like colonies according to different stage (morula or blastocyst) and derivation (in vivo or in vitro fertilization) of bovine embryos. The zona pellucidae of morula stage embryos were removed manually by stainless steel blade (BIO-CUT, FEATHER), and blastomeres were disaggregated by trypsin. Thereafter, blastomeres were seeded onto mitomycin C-treated mouse embryonic fibroblasts feeder layers cultivated in DMEM containing 20%FBS, glucose, sodium bicarbonate, non-essential amino acid and β-mercaptoethanol. On the other hand, inner cell masses (ICM) were isolated from blastocyst stage embryos by BIO-CUT and seeded onto the same medium as described above. At 7 day after seeding, the formation rate of ES like colonies were determined. When morula stage embryos were used, ES like colony was not observed neither in vivo nor in vitro. In contrast,when blastocyst stage embryos were used, the formation rate of ES like colonies in vivo and in vitro were 16.0% and 17.1% respectively. There was no significant difference between these 2. In conclusion, our experiment demonstrated that blastocyst stage embryos were suitable for obtaining ES like colonies on our culture method.
