RESEARCH ARTICLE Molecular Reproduction & Development 9999:1–11 (2009) Method Q1 of Oocyte Activation Affects Cloning Efficiency in Pigs KRISTIN M. WHITWORTH, 1 RONGFENG LI, 1 LEE D. SPATE, 1 DAVID M. WAX, 1 AUGUST RIEKE, 1 JEFFREY J. WHYTE, 2 GAURISHANKAR MANANDHAR, 1 MIRIAM SUTOVSKY, 1 JONATHAN A. GREEN, 1 PETER SUTOVSKY, 1,3 AND RANDALL PRATHER 1 * 1 Division of Animal Sciences, University of Missouri, Columbia, Missouri 2 Department of Biomedical Sciences, Columbia, Missouri 3 Departments of Obstetrics, Gynecology and Women’s Health, Columbia, Missouri SUMMARY The following experiments compared the efficiency of three fusion/activation protocols following somatic cell nuclear transfer (SCNT) with porcine somatic cells transfected with enhanced green fluorescent protein driven by the chicken b-actin/ rabbit b-globin hybrid promoter (pCAGG-EGFP). The three protocols included electrical fusion/activation (NT1), electrical fusion/activation followed by treatment with a reversible proteasomal inhibitor MG132 (NT2) and electrical fusion in low Ca 2þ followed by chemical activation with thimerosal/dithiothreitol (NT3). Data were collected at Days 6, 12, 14, 30, and 114 of gestation. Fusion rates, blastocyst-stage mean cell numbers, recovery rates, and pregnancy rates were calculated and compared between protocols. Fusion rates were significantly higher for NT1 and NT2 compared to NT3 (P < 0.05). There was no significant difference in mean nuclear number. Pregnancy rate for NT2 was 100% (n ¼ 19) at all stages collected and was significantly higher than NT1 (71.4%, n ¼ 28; P < 0.05), but was not significantly higher than NT3 (82.6%, n ¼ 23; P < 0.15). Recovery rates were calculated based on the number of embryos, conceptuses, fetuses, or piglets present at the time of collection, divided by the number of embryos transferred to the recipient gilts. Recovery rates between the three groups were not significantly different at any of the stages collected (P > 0.05). All fusion/activation treatments produced live, pCAGG- EGFP positive piglets from SCNT. Treatment with MG132 after fusion/activation of reconstructed porcine embryos was the most effective method when comparing the overall pregnancy rates. The beneficial effect of NT2 protocol may be due to the stimulation of proteasomes that infiltrate donor cell nucleus shortly after nuclear transfer. ß 2008 Wiley-Liss, Inc. Mol. Reprod. Dev. 9999: 1–11, 2009. ß 2008 Wiley-Liss, Inc. Received 31 July 2008; Accepted 2 November 2008 * Corresponding author: Division of Animal Sciences E125 ASRC University of Missouri Columbia, MO 65211. E-mail: pratherr@missouri.edu Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mrd.20987 INTRODUCTION Nuclear transfer efficiency in pigs and other large animal species is low and sometimes results in pigs with low birth weights and physical abnormalities (Carter et al., 2002; Estrada et al., 2007). Increasing the efficiency of cloning would be highly beneficial, as genetically modified cloned animals are being used as models for human disease (Rogers et al., 2008) and as potential sources of organs for organ transplantation (Kolber-Simonds et al., 2006), and could potentially be used to improve agriculture production (Prather et al., 2008). The primary objective of this study was to compare somatic cell nuclear transfer (SCNT) efficiencies of three different fusion/activation methods. A secondary objective was to create a line of transgenic swine that expressed enhanced green fluorescent protein (EGFP) ß 2008 WILEY-LISS, INC. MRD08-0184:R1ð20987Þ