Development and apoptosis of pre-implantation porcine nuclear transfer embryos activated with different combination of chemicals

Artificial activation of oocytes is a pre-requisite for successful cloning by nuclear transfer (NT). This study investigated effect of different combination of activation chemicals such as electric pulse (E), thimerosal (Thi) + dithiothreitol (DTT), 6-dimethylaminopurine (6-DMAP), or cycloheximide (CH) on the developmental ability and the frequency of apoptosis of porcine NT embryos during the culture in vitro. NT embryos activated with chemicals showed significantly higher developmental rate to blastocyst stage compared to embryos activated with E alone (21.5%-26.6% vs. 15.7%, respectively). Of chemicals, Thi + DTT supported higher development to blastocyst stage as compared to 6-DMAP or CH (26.6% vs. 21.5%-23.4%, respectively). Apoptosis of NT embryos were analyzed by using a terminal deoxynucleatidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) assay. The onset of apoptosis of embryos activated E alone was on Day 4, whereas embryos activated with chemicals showed apoptosis on Day 3 post-activation NT embryos exposed to chemicals for activation had higher frequency of apoptosis compared to that of embryos exposed to E alone from Day 3 to Day 7 during the culture. In conclusion, this study shows that chemical activation after fusion could increase not only the developmental ability of porcine NT embryos but also the mean cell number with an increased ratio of inner cell mass (ICM) to trophectoderm (TE) cells. However, the chemical activation also could increase the frequency of apoptosis and induced apoptosis earlier in porcine NT embryos.