Refrigeration of donor cells in preparation for bovine somatic nuclear transfer

In mammals, preparation of donor cells for somatic nuclear transfer is very important because the character of the donor cell directly affects the eff iciency and outcome of transfer. The protocols used most commonly for donor preparation are (i) disaggregating cells from fresh tissue 1-2 h before mi cromanipulation or (ii) trypsinizing cultured cells temporarily, after spec ial treatments for 3-8 days (for example, serum starvation). In this study, a new simple protocol was designed, whereby the donor cells (cumulus cells ) used in bovine somatic nuclear transfer were refrigerated. in brief, cult ured cells at 80-100% confluency were detached using trypsin, washed by cen trifugation, aliquoted into different vials and refrigerated at 4 degreesC. The density of viable cells was decreased after day 1 of refrigeration; ho wever, the rate of decrease tended to slow down with increasing duration of refrigeration. Cells refrigerated for 15 days were seeded at a density of 5 X 10(4) ml(-1) and reached 70% confluency after day 2 of culture. Most ce lls had the normal number of chromosomes (2n = 60). Cells chilled at 4 degr eesC for different durations were removed from refrigeration and immediatel y subjected to micromanipulation. The in vitro development of reconstructed embryos (fusion rates, cleavage rates, morula and blastocyst rates) indica ted that there were no significant differences among treatment groups regar dless of the duration of refrigeration (0-2 weeks) of the donor cells. Reco nstructed embryos were transferred into the uteri of recipient cows. No sig nificant differences were observed in established early pregnancies between embryos derived from the non-refrigerated donor cells and those derived fr om refrigerated donor cells. This study indicates that refrigeration of don or cells for 1-2 weeks is a feasible protocol for preparing donor cells for bovine somatic nuclear transfer, and does not compromise development in vi tro and early development in vivo.