Sychronization of donor cell cycle by roscovitine treatment in bovine [Bos taurus] nuclear transfer

The aim of this study was to determine the effect of donor cells treated by roscovitine (R) (cyclin dependent kinase inhibitor) on the developmental competence of embryos after nuclear transfer and the caving rate following transfer and to compare with those by serum starvation (SS). Donor cells of succession, which were derived from cumulus oocphorus collected by ovum pick up were cultured in the medium of Dulbecco's minimum essential medium (DMEM) with 0.5% fetal calf serum (FCS) for 4-7 days (SS), or with 10% FCS containing 15 muM R for 24 hours. These donor cells were fused with enucleated oocytes and fused reconstructed oocytes were activated, followed by incubation in CR1aa medium with 10 mu g/ml cycloheximide (C) and 2.5 mu g/ml cytochalasin D for 1 hour. Embryos were transferred to CR1aa medium containig C for 4 hours. They were cultured in the CR1aa medium with bovine serum albumin or FCS for 7 days. The first cleavage rate of reconstructed embryos and the developmental rate to the blastocyst stage and the number of cells of Day 7 blastocysts were examined. To assess the ability to continue development to full term, 5 nuclear transfer embryos derived from adult granulosa cells treated with R were transferred to 5 recipients. (1) There was no significant difference (P0.05) in the rates of fusion, first cleavage of reconstructed embryos and the developmental rate to the blastocyst stage between R and SS treatments. (2) Each of 5 nuclear-transferred embryos was transferred to 5 recipients, and 3 recipients were confirmed to be pregnant. One was aborted at early gestation stage and finally 2 apparently normal female calves were delivered, and their birth weights were within the normal range of Tajima strain of Japanese Black cattle. (3) These results demonstrated that R treatment is as useful as SS treatment for synchronizing the cell cycle of donor cells in bovine nuclear transfer.