IMPROVEMENT OF ELECTRICAL ACTIVATION PROTOCOL FOR PORCINE OOCYTES

Authors: ZHU, JIE - ROSLIN INST, EDINBURGH; TELFER, E - UNIV OF EDINBURGH, UK; FLETCHER, J - ROSLIN INST, EDINBURGH,UK; SPRINGBETT, A - ROSLIN INST, EDINBURGH,UK; Dobrinsky, John; DESOUSA, P - ROSLIN INST,EDINBURGH, UK; WILMUT, IAN - ROSLIN INST,EDINBURGH, UK

Submitted to: Biology of Reproduction
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/22/2001
Publication Date: N/A
Citation: N/A

Interpretive Summary: The capacity of artificial stimuli to activate oocytes and elicit development is essential to the success of animal cloning by nuclear transfer. Published activation systems are not efficient for the pig oocyte. We investigated the efficiency of electrical activation of pig oocytes, including: effects of amino acids in oocyte maturation medium; effect of oocyte age at activation, applied voltage field strength, pulse number and duration. Parthenogenetic blastocysts produced by improved protocol were karyotyped to assess potential for development in vivo. Addition of amino acids in maturation medium improved developmental competence of activated oocytes. Development was dependent on interactions between oocyte age and applied voltage field strength, voltage strength and pulse number, and pulse number and duration. Near 84% of parthenogenetic blastocysts yielded by this protocol were diploid, implying potential for further in vivo development. We developed a simple and reliable method for activation of porcine oocytes that can produce developmentally competent parthenogenetic embryos. This methodology is essential for the activation of embryonic development after reconstruction of embryos during somatic cell nuclear transfer in the production of cloned pigs. Without this improved oocyte activation technology, cloned embryos will not develop further.

Technical Abstract: Factors influencing pig oocyte activation by electrical stimulation were evaluated by their effect on the development of parthenogenetic embryos to the blastocyst stage, in order to establish an effective activation protocol for pig nuclear transfer. This included: (1) a comparison of the effect of epidermal growth factor and amino acids in maturation medium, (2) an investigation of interactions between oocyte age, applied voltage field strength, electrical pulse number and pulse duration, and (3) a karyotype analysis of the parthenogenetic blastocysts yielded by an optimised 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 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 3 x 80 msec 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.