97 CLONED CALF PRODUCTION BY AGGREGATION OF SOMATIC CELL\break NUCLEAR-TRANSFERRED EMBRYOS

Abstract

A low pregnancy rate and high frequency of abortion are observed in bovine somatic cell cloning. It is suggested that one of the factors is the low cell number of nuclear-transferred (NT) embryos at the blastocyst stage compared with that of in vivo-derived embryos. We reported that aggregation of bovine NT embryos can develop to blastocysts with cell numbers that are equivalent to in vivo-derived embryos (Akagi et al. 2005 Reprod. Fertil. Dev. 17, 162 abst). In this study, we examined the in vivo development of aggregates of NT embryos after embryo transfer (ET). Following culture in serum-starved medium for 5 to 7 days, fibroblast cells were used as donor cells for NT. NT was performed as previously described (Akagi et al. 2003 Mol. Reprod. Dev. 66, 264–272). Eight-cell stage embryos on Day 2 or 16- to 32-cell stage embryos on Day 4 were used for embryo aggregation after removal of the zona pellucida. Three NT embryos were placed into the depression in a drop of TCM-199 with 50 µg mL⁻¹ phytohemagglutinin for 20 min, and NT aggregates were then moved into the depression in a drop of IVD-101 (Akagi et al. 2005). On Day 7 after NT, ET was performed nonsurgically. Results are summarized in Table 1. Pregnancy rates obtained with the aggregated NT embryos tended to be high compared with the single NT embryos. All pregnant cows in the single NT embryos and 6 of 8 pregnant cows in the aggregated NT embryos were aborted within 90 days. One cow carrying an aggregate of 3 Day 2 NT embryos went to term, and one carrying an aggregate of 3 Day 4 NT embryos is still pregnant at more than 7 months. These results demonstrate that aggregates of 3 NT embryos can develop to term in cattle.

Table 1.  In vivo development of aggregated NT embryos