Production of cloned calves by combination treatment of both donor cells and early cloned embryos with 5-aza-2/-deoxycytidine and trichostatin A

Abstract

We previously reported that treatment of both donor cells and early cloned embryos with a combination of 0.01 μM 5-aza-2/-Deoxycytidine (5-aza-dC) and 0.05 μM trichostatin A (TSA) significantly improved development of cloned bovine embryos in vitro. In the present study, we investigated the effect of this combination treatment on the in vivo development potency and postnatal survivability of cloned calves. Blastocysts (77 and 82 blastocysts derived from untreated (control) and treated groups, respectively) were individually transferred to recipient cows. Relative to the control group, the combination treatment of both donor cells and early embryos with 5-aza-dC and TSA dramatically increased the cleavage rate (49.2 vs 63.6%, P < 0.05) at 24 h of culture, and blastocyst development rate on Days 6 and 7 of culture (18.8 vs 33.9% and 27.1 vs 38.5% respectively, P < 0.05). Although pregnancy rate did not differ 40 d after transfer, it was lower in the treated than control group 90 d after transfer (7.8 vs 29.3%, P < 0.05). In the control group, there were three calves born to 77 recipients (only two survived beyond 60 d), whereas in the treated group, 17 calves were born to 82 recipients, and 11 survived beyond 60 d. In conclusion, a combination treatment of donor cells and early cloned embryos with 5-aza-dC and TSA significantly enhanced development of somatic cell cloned bovine embryos in vivo; cloning efficiency (number of surviving calves at 60 d of birth/number of recipient cows) was increased from 2.6 to 13.4%.