The present study was conducted to examine the generation of reactive oxygen species (ROS) during micromanipulation procedures in bovine somatic cell nuclear transfer (SCNT) embryos. Bovine enucleated oocytes were electrofused with donor cells, activated by a combination of Ca-ionophore and 6-dimethylaminopurine culture. Oocytes and embryos were stained in dichlorodihydrofluorescein diacetate or 3'-(p-hydroxyphenyl) fluorescein dye and the H2O2 or ˙OH radical levels were measured. In vitro fertilization (IVF) was performed for controls. The samples were examined with a fluorescent microscope, and fluorescence intensity was analyzed in each oocyte and embryo. The H2O2 and ˙OH radical levels of reconstituted oocytes were increased during manipulation (37.2~49.7 and 51.0~55.2 pixels, respectively) as compared to those of mature oocytes (p<0.05). During early in vitro culture, the ROS levels of SCNT embryos were significantly higher than those of IVF embryos (p<0.05). These results suggest that the cellular stress during micromanipulation procedures can generate the ROS in bovine SCNT embryos.

ABSTRACT   INTRODUCTION   MATERIALS AND METHODS    In Vitro Maturation of Oocytes    Culture of Somatic Cells    Nuclear Transfer    Electrofusion and Activation    In Vitro Fertilization (IVF)    In Vitro Culture of Embryos    Analysis of ROS Products    Statistical Analysis   RESULTS    ROS Generation Levels during Manipulation Procedures    ROS Generation Levels during Early In Vitro Culture of SCNT and IVF Embryos   DISCUSSIONS   REFERENCES

• In-Sun Hwang(College of Veterinary Medicine and Institute of Veterinary Science, Kangwon National University, Chuncheon 200-701, Korea)
• Hyo-Kyung Bae(College of Veterinary Medicine and Institute of Veterinary Science, Kangwon National University, Chuncheon 200-701, Korea)
• Choon-Keun Park(College of Animal Life Sciences, Kangwon National University, Chuncheon 200-701, Korea)
• Boo-Keun Yang(College of Animal Life Sciences, Kangwon National University, Chuncheon 200-701, Korea)
• Hee-Tae Cheong(College of Veterinary Medicine and Institute of Veterinary Science, Kangwon National University, Chuncheon 200-701, Korea) Corresponding author