Effect of duration of сycloheximide and 6‐dimethylaminopurine (6‐DMAP) treatments on development competence of cloned embryos in cattle

Abstract

Cycloheximide and 6‐dimethylaminopurine (6‐DMAP) are widely used in protocols of somatic cell nuclear transfer (SCNT) for inhibition of maturation promoting factor (MPF) activity in SCNT-oocytes in the post-activation period of their culture. Nevertheless, you should remember that these agents have a wide range of activity and can conflict with other cell processes. Therefore, a definition of the optimal period of culture of SCNT-oocytes with the previously mentioned inhibitors may help to prevent the undesirable negative consequences. In this research the effects of cycloheximide (10 µg/ml) and 6‐DMAP (2mM) treatments duration (3.0, 4.0 or 5.0 h) on the reprogramming of somatic nuclear was estimated by the cleavage and blastocyst rates, and by the total cell number and a level of apoptotic cell in the obtained cloned blastocysts. The cleavage rate did not differ between the experimental groups, varying from 63.7 to 77.0 %. Also, there was not found an effect of treatment duration of the investigated factors on the development of activated SCNT-oocytes before blastocyst stage. For 3-hr treatment, the blastocyst rate was 19.6 ± 1.8%. The prolonged up to 4 and 5 hours duration did not change this rate. Meanwhile, we found out the effect of culture duration with сycloheximide and 6‐DMAP on quality of cloned embryos. In case of 3-hr treatment, the total cell number in cloned blastocyst was 58.8 ± 2.4. With prolongation of duration up to 4 hours the result was growing up to 76.6 ± 1.4 (p<0.05), but prolongation up to 5 hours reduced the total cell number in blastocyst as compared to that in 4-hr group (p<0.05). The apoptosis rate had no difference between the treated groups and had its variety between 5.4 to 7.0 %. Our date indicate that efficiency of bovine cloned embryo production depends on duration of 6-DMAP and cycloheximide treatment of the SCNT-oocytes in the post-activated period of their culture. The optimal duration according to the described protocol of SCNT for the best quality of embryos is 4 hours.