Roscovitine treatment improves synchronization of donor cell cycle in G0/G1 stage and in vitro development of handmade cloned buffalo (Bubalus bubalis) embryos

Abstract

This study investigated the effects of serum-starvation, total confluence, and roscovitine treatment on cell-cycle synchronization of buffalo ear skin fibroblasts to the G0/G1stage and on the developmental competence of cloned embryos. Serum starvation of total confluence cultures for 24 h had a higher (p<0.05) proportion of cells at G0/G1stage (94.4%) compared with serum starved cyclic and nonstarved confluent cultures (76.8 and 86.0%, respectively), whereas differences between cyclic cells with or without serum starvation were not significant. The proportion of cells at G0/G1 was higher (p<0.05) with 20 and 30 μM roscovitine treatment than that with 10 μM (94.4, 96.4, and 86.6%, respectively), which was similar to that for total confluence (86.0%). MTT assay showed that cell viability decreased as dose of roscovitine increased. The blastocyst rate was significantly higher (p<0.05) when nuclear transfer embryos were reconstructed using donors cells from total confluence, confluence serum starved, and roscovitine-treated (20 and 30 μM) groups (48.8, 48.9, 57.9, and 62.9%, respectively) compared to nontreated cyclic cells (20.2%). However, the cleavage rate and total cell number of cloned embryos were similar for all the groups. The number of ICM cells was improved by 30 μM roscovitine treatment (45.25±2.34). The cryosurvival rate of blastocysts derived from cells synchronized with 20 or 30 μM roscovitine was higher compared to that for total confluence group (33.6, 37.8 vs. 23.8%). In conclusion, treatment with 30 μM roscovitine is optimal for harvesting G0/G1stage cells for producing high quality cloned buffalo embryos, and that it is better than serum-starvation or total confluence for cell synchronization.