Improved developmental competence of cloned porcine embryos with different energy supplements and chemical activation.
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.
Lee
Gab-Sang
GS
College of Veterinary Medicine, Seoul National University, Seoul, Korea.
Kim
Hye-Soo
HS
Hyun
Sang-Hwan
SH
Kim
Dae-Young
DY
Lee
So-Hyun
SH
Nam
Dong-Hyun
DH
Jeong
Yeon-Woo
YW
Kim
Sue
S
Kang
Sung-Keun
SK
Lee
Byeong-Chun
BC
Hwang
Woo-Suk
WS
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
D003470:Culture Media; D019344:Lactic Acid; D019289:Pyruvic Acid; D005947:Glucose