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

Abstract

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. Mol. Reprod. Dev. 58:384–389, 2001. © 2001 Wiley‐Liss, Inc.

Production of Nuclear Transfer Horse Embryos by Piezo-Driven Injection of Somatic Cell Nuclei and Activation with Stallion Sperm Cytosolic Extract1

“…was supported by grant HD38381 from the National Institutes of Health. than those achieved when cloning with embryo-derived blastomeres in the same species (86%-99%) [10,14]. The reported percentages of reconstruction with somatic cells by electrofusion in the pig are 59%-81% [15,16] and in sheep are 63%-85% [1,17].…”

A Comparison of Established and New Approaches in Ovine and Bovine Nuclear Transfer

“…Nevertheless, if the availability of oocytes is not a limitation, fast enucleation without the need of UV-confirmation facilitates speedy and efficient production of reconstructed embryos, thus compensating for the low peroocyte efficiency. Again, the overall performance of the culture system dictates the subsequent development rates, but blastocyst development rates between 19% and 23% have been achieved (Peura et al, 1998(Peura et al, , 1999(Peura et al, , 2001.…”

“…To reconstruct the original volume, two cytoplasts were fused with one blastomere, using alternating current to establish proper alignment and cell-to-cell contact. Reconstructed embryos were then cultured in drops, and offspring were produced after transfer of blastocysts to recipients (Peura et al, 2001). As the method did not require the use of micromanipulators, an entirely new way for performing nuclear transfer had been developed. Unfortunately, the introduction of this nuclear transfer procedure coincided with the sharp decrease of interest in embryonic cell cloning, and the sudden rise of the newly discovered somatic cell cloning.…”

Production of Cloned Cattle from In Vitro Systems

“…However, the low number of cells that could be obtained from these early embryos precluded their use in large-scale cloning efforts [8,9]. Expansion of embryonic stem (ES) cells in culture, particularly inner mass cells, was pursued as a means of generating a large number of cells that could be genetically altered [10][11][12].…”