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

This study assessed in vitro and in vivo developmental ability of bovine embryo-derived clones after one, four or seven rounds of nuclear transfer. Initial donor embryo production and all subsequent cultures were performed in vitro. Donor clonal embryo lines were vitrified and warmed either once (first 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 different between first, fourth and seventh generation clones. However, development to morulae and blastocysts decreased significantly as the number of recycling rounds increased (24.8, 15.1 and 13.6% for first, fourth and seventh generation, respectively). In addition, the proportion of blastocysts compared to morulae decreased, indicating slower developmental speed in later generation clones. After transfer of 16, 25 and 7 clones to 7, 11 and 2 recipients (first, fourth and seventh generation, respectively) initial pregnancy 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 rounds of nuclear recycling. Whether it is caused by intrinsic factors associated with the genome modification and reprogramming as such, or by external factors such as prolonged in vitro culture period or the effects of vitrification, remains to be determined.