Effect of elevated Ca(2+) concentration in fusion/activation medium on the fusion and development of porcine fetal fibroblast nuclear transfer embryos.
The present study examined the effect of elevated Ca(2+) concentration in fusion/activation medium on the fusion and development of fetal fibroblast nuclear transfer (NT) porcine embryos. Frozen-thawed and serum starved fetal fibroblasts were transferred into the perivitelline space of enucleated oocytes. Cell fusion and activation were induced simultaneously with electric pulses in 0.3 M mannitol-based medium containing 0.1 or 1.0 mM CaCl(2). Some fused embryos were further activated 1 hr after the fusion treatment by exposure to an electric pulse. The NT embryos were cultured in vitro for 6 days. Fusion and blastocyst formation rates were significantly (P<0.05) increased by increasing the Ca(2+) concentration from 0.1 mM (67.1 and 6.3%) to 1.0 mM (84.7 and 15.8%). However, no difference in the number of cells in blastocysts was observed between the two groups. A higher percentage of blastocyst was also observed when control oocytes were parthenogenetically activated in the presence of elevated Ca(2+) (19.3% vs. 32.4%, P<0.05). When the reconstituted oocytes were fused in the medium containing 1.0 mM CaCl(2), increasing the number of pulses from 2 to 3 or an additional activation treatment did not enhance the blastocyst formation rate or cell number in blastocysts. These results demonstrate that increasing the Ca(2+) concentration in the fusion/activation medium can enhance the fusion and blastocyst formation rates of fetal fibroblast NT porcine embryos without an additional activation treatment.
Cheong
Hee-Tae
HT
Department of Animal Sciences, University of Missouri-Columbia, Columbia, Missouri 65211, USA.
Park
Kwang-Wook
KW
Im
Gi-Sun
GS
Lai
Liangxue
L
Sun
Qing-Yuan
QY
Day
Billy N
BN
Prather
Randall S
RS
D000818:Animals; D002118:Calcium; D002459:Cell Fusion; D019976:Cloning, Organism; D003470:Culture Media / Q000737:chemistry; D004624:Embryo Transfer; D005260:Female; D005347:Fibroblasts / Q000502:physiology* / Q000648:ultrastructure; D051379:Mice; D009865:Oocytes / Q000737:chemistry / Q000502:physiology; D013552:Swine / Q000196:embryology
D003470:Culture Media; D002118:Calcium