| The potential use of nuclear transfer (NT) with embryonic stem (ES) cells for transgenic animal production provides a unique opportunity to increase efficiencies, decrease costs and provide stable gene integration. However, no live animals have been produced by this methodology and only one live pig has been produced by NT alone. Therefore, the primary goal of this thesis was to obtain in vitro development of nuclear transfer embryos constructed from porcine ES cells. The objectives were: (1) to develop an efficient system to culture one-cell porcine embryos to the blastocyst stage, (2) to determine an efficient method for activation of porcine in vivo matured oocytes to obtain pronuclear cytoplasm, and (3) to determine the appropriate stage of cytoplast for in vitro development of NT embryos.; Results from embryo culture experiments indicated: (1) bovine serum albumin (BSA) supplementation improved developmental rates of four- and eight-cell Meishan embryos to the blastocyst stage, (2) fetal bovine serum supplementation improved hatching rates of later stage Meishan embryos, (3) fatty acid-free BSA did not improve developmental rates of early stage embryos, (4) more Meishan than Yorkshire one-cell embryos developed to the eight-cell and compact morula stages following 96 hours of culture, (5) more Yorkshire than Meishan two-cell embryos developed to the eight-cell stage following 96 hours of culture and (6) modified Whitten's medium +1.5% BSA was the most efficient medium for culture of one-cell embryos to the blastocyst stage. Results from activation and culture of in vivo matured oocytes indicated: (1) ethanol had no effect on activation rates, (2) cold shock, sham enucleation, electroactivation and electroactivation + culture with cycloheximide improved activation rates, and (3) electroactivation + culture with cycloheximide was the most efficient treatment for production of pronuclear stage cytoplasm. Results from NT experiments indicated that neither enucleated, unactivated oocytes nor enucleated, activated oocytes promoted development of NT embryos constructed from porcine ES cells beyond the four-cell stage. However, enucleated zygotes supported development of NT embryos beyond the four-cell stage to the compact morula stage. These results provide preliminary evidence for in vitro development of NT embryos produced from porcine ES cells. |
