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Improved developmental competence of cloned porcine embryos with different energy supplements and chemical activation
Author(s) -
Lee GabSang,
Kim HyeSoo,
Hyun SangHwan,
Kim DaeYoung,
Lee SoHyun,
Nam DongHyun,
Jeong YeonWoo,
Kim Sue,
Kang SungKeun,
Lee ByeongChun,
Hwang WooSuk
Publication year - 2003
Publication title -
molecular reproduction and development
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.745
H-Index - 105
eISSN - 1098-2795
pISSN - 1040-452X
DOI - 10.1002/mrd.10329
Subject(s) - blastocyst , cytochalasin b , biology , somatic cell nuclear transfer , andrology , embryo , embryo culture , oocyte , embryogenesis , in vitro , biochemistry , medicine , endocrinology , microbiology and biotechnology
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 μsec. 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 μsec) were subjected to three activation protocols: (1) no chemical activation (Group C), (2) 7.5 μg/ml cytochalasin B treatment at 2 hr after electric stimulus (Group D), and (3) 5 μg/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. Mol. Reprod. Dev. 66: 17–23, 2003. © 2003 Wiley‐Liss, Inc.