title: Method of oocyte activation affects cloning efficiency in pigs.
abstract: 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 beta-actin/rabbit beta-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(2+) 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 = 19) at all stages collected and was significantly higher than NT1 (71.4%, n = 28; P < 0.05), but was not significantly higher than NT3 (82.6%, n = 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.
authors.affiliation: Division of Animal Sciences, University of Missouri, Columbia, Missouri 65211, USA.
authors.forename: Kristin M
authors.initials: KM
authors.lastname: Whitworth
authors.forename: Rongfeng
authors.initials: R
authors.lastname: Li
authors.forename: Lee D
authors.initials: LD
authors.lastname: Spate
authors.forename: David M
authors.initials: DM
authors.lastname: Wax
authors.forename: August
authors.initials: A
authors.lastname: Rieke
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Whyte
authors.forename: Gaurishankar
authors.initials: G
authors.lastname: Manandhar
authors.forename: Miriam
authors.initials: M
authors.lastname: Sutovsky
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Green
authors.forename: Peter
authors.initials: P
authors.lastname: Sutovsky
authors.forename: Randall S
authors.initials: RS
authors.lastname: Prather
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D001755:Blastocyst; D002118:Calcium / Q000494:pharmacology; D002467:Cell Nucleus; D065150:Cellular Reprogramming; D019976:Cloning, Organism / Q000379:methods*; D004558:Electric Stimulation; D004624:Embryo Transfer; D005260:Female; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007976:Leupeptins / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000187:drug effects / Q000502:physiology*; D011247:Pregnancy; D018873:Pregnancy Rate; D013552:Swine / Q000235:genetics / Q000502:physiology*; D013849:Thimerosal / Q000494:pharmacology; D015053:Zygote / Q000378:metabolism
chemical_list: D007976:Leupeptins; D049452:Green Fluorescent Proteins; D013849:Thimerosal; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; D002118:Calcium