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 outline s the differences in nuclear transfer procedure efficiencies and devel opmental rates for the various stages and generations of embryos. We r eport the production of 54 genetically identical embryos that develope d to the morula stage from one parent embryo. When a limited number of nuclear transfer embryos (clones) were transferred to recipient femal es, first, second, and third generation calves were born. Clones cultu red 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, fus ion 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 g enerations of clones also varied in fusion rates. Fusion rates general ly declined with each additional generation and ranged from 66% for th e parent-donor embryos to 52% for second generation donors (P < 0.01). Developmental rates were highest for first and third generation donor s (20% and 19% ) and lowest for second generation donors (10%; p < 0.0 01 ). 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.