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Improvement of an Electrical Activation Protocol for Porcine Oocytes¹

^a Department of Gene Expression and Development, Roslin Institute, Roslin, Midlothian EH25 9PS, United Kingdom ^b Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh EH9 3JR, United Kingdom ^c Germplasm and Gamete Physiology Laboratory, U.S. Department of Agriculture, Beltsville, Maryland 20705

Factors influencing pig oocyte activation by electrical stimulation were evaluated by their effect on the development of parthenogenetic embryos to the blastocyst stage to establish an effective activation protocol for pig nuclear transfer. This evaluation included 1) a comparison of the effect of epidermal growth factor and amino acids in maturation medium, 2) an investigation of interactions among oocyte age, applied voltage field strength, electrical pulse number, and pulse duration, and 3) a karyotype analysis of the parthenogenetic blastocysts yielded by an optimized protocol based on an in vitro system of oocyte maturation and embryo culture. In the first study, addition of amino acids in maturation medium was beneficial for the developmental competence of activated oocytes. In the second study, the developmental response of activated oocytes was dependent on interactions between oocyte age at activation and applied voltage field strength, voltage field strength and pulse number, and pulse number and duration. The formation of parthenogenetic blastocysts was optimal when activation was at 44 h of maturation using three 80-µsec consecutive pulses of 1.0 kV/cm DC. Approximately 84% of parthenogenetic blastocysts yielded by this protocol were diploid, implying a potential for further in vivo development.

First decision: 25 July 2001.

¹ These experiments were funded by Roslin Bio-Med and subsequently by Geron Bio-Med.

² Correspondence. FAX: 44 0131 527 4493; ian.wilmut{at}bbsrc.ac.uk

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