title: Improved developmental competence of cloned porcine embryos with different energy supplements and chemical activation.
abstract: The present study investigated the effect of lactate/pyruvate supplement in culture medium and of chemical activation after electric stimulus on in vitro development of porcine somatic cell nuclear transfer (SCNT) embryos. In vitro matured gilt oocytes were enucleated, reconstructed with fetal fibroblasts, and simultaneously fused/activated using a single pulse of 2.0 kV/cm for 30 microsec. In Experiment 1, reconstructed embryos were cultured in North Carolina State University (NCSU)-23 medium supplemented with either 5.5 mM glucose (Group A) or lactate (5.0 mM)/pyruvate (0.5 mM) (Group B). Compared to Group A, cleavage rate (64% vs. 47%) was higher and more blastocysts developed in Group B (17% vs. 6% at Day 6, 21% vs.11% at Day 7). Experiment 2, embryos reconstructed by electric stimulus (2.0 kV/cm for 30 microsec) were subjected to three activation protocols: (1) no chemical activation (Group C), (2) 7.5 microg/ml cytochalasin B treatment at 2 hr after electric stimulus (Group D), and (3) 5 microg/ml 6-dimethylaminopurine (Group E) treatment at 2 hr after electric stimulus. The reconstructed embryos were cultured for 7 days in NCSU-23 medium supplemented with lactate (5.0 mM)/pyruvate (0.5 mM). The rates of blastocyst formation on Day 6 and Day 7 in Group C (17 and 20%, respectively) or Group D (15, 20%, respectively) were higher than in Group E (9 and 12%, respectively). The percentage of two pseudo-pronucleus (PPN) formations in Group D (88%) was significantly higher than in Group C (71%) and Group E (72%). Mean cell numbers of blastocysts in Group D (63.4 +/- 15.8) were higher than in Group C (43.9 +/- 16.5) and Group E (32.9 +/- 17.9), due to increased trophectoderm (TE) cell numbers. Our results indicate that supplementing NCSU-23 medium with lactate/pyruvate and exposure of cytochalasin B after electrical stimulus can improve in vitro developmental competence of porcine SCNT embryos.
authors.affiliation: College of Veterinary Medicine, Seoul National University, Seoul, Korea.
authors.forename: Gab-Sang
authors.initials: GS
authors.lastname: Lee
authors.forename: Hye-Soo
authors.initials: HS
authors.lastname: Kim
authors.forename: Sang-Hwan
authors.initials: SH
authors.lastname: Hyun
authors.forename: Dae-Young
authors.initials: DY
authors.lastname: Kim
authors.forename: So-Hyun
authors.initials: SH
authors.lastname: Lee
authors.forename: Dong-Hyun
authors.initials: DH
authors.lastname: Nam
authors.forename: Yeon-Woo
authors.initials: YW
authors.lastname: Jeong
authors.forename: Sue
authors.initials: S
authors.lastname: Kim
authors.forename: Sung-Keun
authors.initials: SK
authors.lastname: Kang
authors.forename: Byeong-Chun
authors.initials: BC
authors.lastname: Lee
authors.forename: Woo-Suk
authors.initials: WS
authors.lastname: Hwang
mesh_terms: D000818:Animals; D019976:Cloning, Organism / Q000379:methods*; D003470:Culture Media; D004558:Electric Stimulation; D005947:Glucose / Q000378:metabolism; D019344:Lactic Acid / Q000378:metabolism; D019289:Pyruvic Acid / Q000378:metabolism; D013552:Swine / Q000235:genetics* / Q000378:metabolism
chemical_list: D003470:Culture Media; D019344:Lactic Acid; D019289:Pyruvic Acid; D005947:Glucose