Multiple generational bovine embryo cloning.

Repeated cycles (multiple generations) of nuclear transfer procedures have the potential of producing a large number of identical offspring from the micromanipulation of one mammalian embryo. This paper outlines the differences in nuclear transfer procedure efficiencies and developmental rates for the various stages and generations of embryos. We report the production of 54 genetically identical embryos that developed to the morula stage from one parent embryo. When a limited number of nuclear transfer embryos (clones) were transferred to recipient females, first, second, and third generation calves were born. Clones cultured for 5 days in the sheep oviduct had a greater number of cells (28.1 +/- 1.7) than clones cultured for 4 days (17.8 +/- 1.7). However, fusion rates were lower for Day 5 clones (Day 5, 57% vs. Day 4, 68%; p < 0.001) used as donors for the next generation of cloning. Different generations of clones also varied in fusion rates. Fusion rates generally declined with each additional generation and ranged from 66% for the parent-donor embryos to 52% for second generation donors (p < 0.01). Developmental rates were highest for first and third generation donors (20% and 19%) and lowest for second generation donors (10%; p < 0.001). Further understanding of the factors that are conducive to high developmental rates and nuclear reprogramming may make it possible to produce an unlimited number of genetically identical animals.