title: A separate procedure of fusion and activation in an ear fibroblast nuclear transfer program improves preimplantation development of bovine reconstituted oocytes.
abstract: This study was conducted to examine whether preimplantation development of bovine ("HanWoo," Bos Taurus corcanae) oocytes reconstituted with ear fibroblasts could be improved by a modified procedure of fusion and activation. In Experiment 1, enucleated oocytes were reconstituted with ear fibroblast by a combined procedure of electric fusion and activation at either 24 or 28 hours after IVM. The 28 hours reconstitution yielded more blastocysts (4% vs. 21%, P = 0.0025) and higher ratio of blastocysts per 2-cell embryos (0.05 vs. 0.25, P = 0.003) than the 24 hours. In Experiment 2, enucleated oocytes were reconstituted by one of the three fusion and activation protocols; 1) a combined procedure of electric fusion and activation at 28 hours after IVM, 2) a combined procedure of electric fusion and chemical activation at 28 hours, and 3) a separate procedure of electric fusion at 24 hours and chemical activation at 28 hours. When compared two combined procedures, chemical activation with 5 microM ionomycin for 4 minutes did not promote embryo development and significantly reduced the fusion rate (42% vs. 53%, P = 0.0395). However, significant (P < 0.0113) increases in the development to 2-cell (90% vs. 70 to 74%) and blastocyst (47% vs. 7 to 13%) stages and in the ratio of blastocysts per 2-cell embryos (0.52 vs. 0.11 to 0.18) were obtained by a separate procedure of electric fusion and chemical activation than by the combined procedures. This separate protocol did not reduce the fusion rate compared with the combined procedures (58%). In conclusion, improved development of oocytes reconstituted with ear fibroblasts was achieved by undergoing a separate procedure of electric fusion and chemical activation 4 hours apart.
authors.affiliation: Department of Theriogenology, College of Veterinary Medicine, Seoul National University, Korea.
authors.forename: S J
authors.initials: SJ
authors.lastname: Shin
authors.forename: B C
authors.initials: BC
authors.lastname: Lee
authors.forename: J I
authors.initials: JI
authors.lastname: Park
authors.forename: J M
authors.initials: JM
authors.lastname: Lim
authors.forename: W S
authors.initials: WS
authors.lastname: Hwang
mesh_terms: D000818:Animals; D002417:Cattle; D002459:Cell Fusion; D002469:Cell Separation / Q000379:methods / Q000662:veterinary*; D019976:Cloning, Organism / Q000662:veterinary*; D004423:Ear; D004558:Electric Stimulation; D047108:Embryonic Development; D005260:Female; D005347:Fibroblasts / Q000166:cytology*; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000166:cytology* / Q000254:growth & development; D011247:Pregnancy