Analysis of actinomycin D treated cattle oocytes and their use for somatic cell nuclear transfer

The present work aimed to evaluate the transcription and replication inhibitor, actinomycin D, for oocyte chemical enucleation. Cattle oocytes matured in vitro were treated with actinomycin D according to the following treatments: T1, control; T2 = 1.0 μg/ml for 16 h; T3 = 1.0 μg/ml for 14 h; T4 = 2.5 μg/ml for 14 h; T5 = 5.0 μg/ml for 14 h. The oocytes were denuded and activated during 24–26 h of maturation. Oocytes were fixed to determine the maturation status and for chromosome morphology evaluation. Furthermore, oocytes treated with actinomycin D were used for somatic cell nuclear transfer (SCNT). Parthenogenetic and SCNT embryos were fixed to evaluate the percentage of apoptotic nuclei by the TUNEL assay. The maturation (T1 = 90.4%; T2 = 82.3%; T3 = 79.1%; T4 = 83.4%; T5 = 74.7%), cleavage (T1 = 68.9%; T2 = 46.0%; T3 = 49.7%; T4 = 33.4%; T5 = 29.3%) and blastocyst rate at D8 (T1 = 41.1%; T2 = 1.8%; T3 = 1.3%; T4 = 0.9%; T5 = 0.0%) after actinomycin D treatment were significantly different. There was a significant chromosome uncoiling when treated with greater concentrations (2.5 and 5.0 μg/ml). After SCNT, the cleavage rate (61.3%) was similar to the actinomycin D-treated control group (61.3%) and less than the non-treated control (70.2%), although the blastocyst rate was greater in the SCNT group (11.8%) comparing with the treated control (3.6%) and less than the untreated control (38.0%). Treated parthenogenetic embryos had more apoptotic cells than the parthenogenetic controls (24.2% compared with 4.8%). However, the SCNT group using treated cytoplasts was similar from the SCNT control (9.3 compared with 13.0%). Actinomycin D treatment was efficient in blocking embryonic development. Moreover, it was possible to obtain reconstructed embryos that possess an apoptotic cell index indistinguishable from controls.