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KMID : 1023620120360020011
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Reproductive and Developmental Biology
2012 Volume.36 No. 2 p.11 ~ p.120
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Inhibition of Reactive Oxygen Species Generation by Antioxidant Treatments during Bovine Somatic Cell Nuclear Transfer
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Bae Hyo-Kyung
Kim Ji-Ye
Hwang In-Sun
Yang Boo-Keun
Park Choon-Keun
Cheong Hee-Tae
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Abstract
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This study was conducted to examine the optimal concentration and treatment time of antioxidants for inhibition of the ROS generation in bovine somatic cell nuclear transfer (SCNT) embryos. Bovine oocytes were activated parthenogenetically, during which oocytes were treated with various antioxidants to determine the optimal concentrations and kind of antioxidants. Determined antioxidants were applied to oocytes during in vitro maturation (IVM) and/or SCNT procedures. Finally, antioxidant-treated SCNT embryos were compared with in vitro fertilized (IVF) embryos. H₂O₂ levels were analyzed in embryos at 20 h of activation, fusion or insemination by staining of embryos in 10㎛ 2'7'-dichlorodihydrofluorescein diacetate (H2DCFDA) dye, followed by fluorescence microscopy. H₂O₂ levels of parthenogenetic embryos were significantly lower in 25㎛ β-mercaptoethanol (β-ME), 50㎛ L-ascorbic acid (Vit. C), and 50㎛ L-glutathione (GSH) treatment groups than each control group (24.0±1.5 vs 39.0±1.1, 29.7±1.0 vs 37.0±1.2, and 32.9±0.8 vs 36.3±0.8 pixels/embryo, p<0.05). There were no differences among above concentration of antioxidants in direct comparison (33.6±0.9~35.2±1.1 pixels/embryo). Thus, an antioxidant of 50㎛ Vit. C was selected for SCNT. H₂O₂ levels of bovine SCNT embryos were significantly lower in embryos treated with Vit. C during only SCNT procedure (26.4±1.1 pixels/embryo, p<0.05) than the treatment group during IVM (29.9±1.1 pixels/embryo) and non-treated control (34.3±1.0 pixels/embryo). Moreover, H₂O₂ level of SCNT embryos treated with Vit. C during SCNT procedure was similar to that of IVF embryos. These results suggest that the antioxidant treatment during SCNT procedures can reduce the ROS generation level of SCNT bovine embryos.
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KEYWORD
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Somatic cell nuclear transfer, Reactive oxygen species, Antioxidants, Cattle
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