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

Gabsang 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 journalArticle

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

Fingerprint

Pyruvic Acid
Mental Competency
Lactic Acid
Blastocyst
Swine
Embryonic Structures
Cytochalasin B
Embryo Transfer
Cell Count
Oocytes
Culture Media
Fibroblasts
Glucose
Therapeutics
In Vitro Techniques

Keywords

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

ASJC Scopus subject areas

  • Genetics
  • Developmental Biology
  • Cell Biology

Cite this

Improved developmental competence of cloned porcine embryos with different energy supplements and chemical activation. / Lee, Gabsang; Kim, Hye Soo; Hyun, Sang Hwan; Kim, Dae Young; Lee, So Hyun; Nam, Dong Hyun; Jeong, Yeon Woo; Kim, Sue; Kang, Sung Keun; Lee, Byeong Chun; Hwang, Woo Suk.

In: Molecular Reproduction and Development, Vol. 66, No. 1, 01.09.2003, p. 17-23.

Research output: Contribution to journalArticle

Lee, G, Kim, HS, Hyun, SH, Kim, DY, Lee, SH, Nam, DH, Jeong, YW, Kim, S, Kang, SK, Lee, BC & Hwang, WS 2003, 'Improved developmental competence of cloned porcine embryos with different energy supplements and chemical activation', Molecular Reproduction and Development, vol. 66, no. 1, pp. 17-23. https://doi.org/10.1002/mrd.10329
Lee, Gabsang ; Kim, Hye Soo ; Hyun, Sang Hwan ; Kim, Dae Young ; Lee, So Hyun ; Nam, Dong Hyun ; Jeong, Yeon Woo ; Kim, Sue ; Kang, Sung Keun ; Lee, Byeong Chun ; Hwang, Woo Suk. / Improved developmental competence of cloned porcine embryos with different energy supplements and chemical activation. In: Molecular Reproduction and Development. 2003 ; Vol. 66, No. 1. pp. 17-23.
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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.",
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AU - Lee, Gabsang

AU - Kim, Hye Soo

AU - Hyun, Sang Hwan

AU - Kim, Dae Young

AU - Lee, So Hyun

AU - Nam, Dong Hyun

AU - Jeong, Yeon Woo

AU - Kim, Sue

AU - Kang, Sung Keun

AU - Lee, Byeong Chun

AU - Hwang, Woo Suk

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N2 - 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.

AB - 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.

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