Add time:09/02/2019         Source:sciencedirect.com

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.

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