Improved developmental competence of cloned porcine embryos with different energy supplements and chemical activation

Gab Sang Lee, Hye Soo Kim, Sang Hwan Hyun, Dae Young Kim, So Hyun Lee, Dong Hyun Nam, Yeon Woo Jeong, Sue Kim, Sung Keun Kang, Byeong Chun Lee, Woo Suk Hwang

Research output: Contribution to journalArticlepeer-review

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 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 pg/ml cytochalasin B treatment at 2 hr after electric stimulus (Group D), and (3) 5 pg/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.

Original languageEnglish (US)
Pages (from-to)17-23
Number of pages7
JournalMolecular reproduction and development
Volume66
Issue number1
DOIs
StatePublished - Sep 1 2003
Externally publishedYes

Keywords

  • Cytochalasin B
  • Embryos
  • Lactate/pyruvate
  • Nuclear transfer
  • Porcine

ASJC Scopus subject areas

  • Genetics
  • Developmental Biology
  • Cell Biology

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