Development of bovine embryo-derived clones after increasing rounds of nuclear recycling

This study assessed in vitro and in vivo developmental ability of bovine em bryo-derived clones after one, four or seven rounds of nuclear transfer. In itial donor embryo production and all subsequent cultures were performed in vitro. Donor clonal embryo lines were vitrified and warmed either once (fi rst generation), twice (third generation) or three times (sixth generation) before the final round of cloning. No differences were observed in fusion, cleavage and development rates to the 16-cell stage between the first six cloning generations. Likewise, neither the fusion nor cleavage rates were d ifferent between first, fourth and seventh generation clones. However, deve lopment to morulae and blastocysts decreased significantly as the number of recycling rounds increased (24.8, 15.1 and 13.6% for first, fourth and sev enth generation, respectively). In addition, the proportion of blastocysts compared to morulae decreased, indicating slower developmental speed in lat er generation clones. After transfer of 16, 25 and 7 clones to 7, 11 and 2 recipients (first, fourth and seventh generation, respectively) initial pre gnancy rates of 57, 27 and 0% were obtained. Final rates of calves to term were 25 and 4% per transferred clone for first and fourth generation clones , respectively. These results indicate greatly reduced in vitro and in vivo developmental capacity of bovine embryo-derived clones after several round s of nuclear recycling. Whether it is caused by intrinsic factors associate d with the genome modification and reprogramming as such, or by external fa ctors such as prolonged in vitro culture period or the effects of vitrifica tion, remains to be determined. (C) 2001 Wiley-Liss, Inc.