Synergistic effects of glutathione and β-mercaptoethanol treatment during in vitro maturation of porcine oocytes on early embryonic development in a culture system supplemented with L-cysteine.
Various methods have been used to remove reactive oxygen species (ROS) generated from in vitro culture (IVC) conditions that can cause cell injury or death, including the application of low oxygen (O(2)) tension and the addition of antioxidants. The beneficial effects of antioxidants and O(2) tension on IVC of porcine embryos, however, are controversial among researchers. In this study, we sought to determine the effects and optimal concentrations of antioxidants for the development of porcine embryos in an IVC system. Specifically, we examined the synergistic effects of antioxidants on development to the blastocyst stage in a culture system supplemented with L-cysteine during IVM. Of the antioxidants tested (melatonin, glutathione (GSH), β-mercaptoethanol (β-ME), N-acetylcysteine (NAC) and dithiothreitol (DTT)), addition of GSH (1 mM) or β-ME (25 µM) significantly increased development to the blastocyst stage compared with the controls without antioxidant treatment (22.2 ± 4.2% for 1 mM GSH, 25.9 ± 2.2% for 25 µM β-ME and 12-13% for the control, P<0.05). In addition, the mean cell number per blastocyst was increased by approximately 1.7-fold in the presence of GSH or β -ME. These GSH- and β-ME-induced increases in development to the blastocyst stage and total cell number, however, were not mimicked by melatonin, NAC or DTT, all of which are ROS scavengers. The combination of GSH or β-ME with L-cysteine significantly reduced high O(2) tension-induced ROS production (P<0.05). These results suggest that a combination of 1 mM GSH or 25 µM β-ME with 1 mM L-cysteine could be used for production of high quality porcine blastocysts in IVC systems.
Choe
Changyong
C
Republic of Korea Animal Genetic Resources Station, National Institute of Animal Science, RDA, Namwon, Korea.
Shin
Yong-Won
YW
Kim
Eun-Jin
EJ
Cho
Sang-Rae
SR
Kim
Hyun-Jong
HJ
Choi
Sun-Ho
SH
Han
Man-Hye
MH
Han
Jaehee
J
Son
Dong-Soo
DS
Kang
Dawon
D
D000822:Animal Husbandry; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D001755:Blastocyst / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D002452:Cell Count; D003545:Cysteine / Q000378:metabolism*; D004357:Drug Synergism; D004477:Ectogenesis / Q000187:drug effects*; D046149:Embryo Culture Techniques / Q000662:veterinary*; D005260:Female; D005307:Fertilization in Vitro / Q000379:methods / Q000662:veterinary; D005978:Glutathione / Q000494:pharmacology; D008297:Male; D008623:Mercaptoethanol / Q000494:pharmacology; D009865:Oocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D009994:Osmolar Concentration; D018384:Oxidative Stress / Q000187:drug effects; D010100:Oxygen / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D034421:Sus scrofa / Q000196:embryology* / Q000378:metabolism
D000975:Antioxidants; D017382:Reactive Oxygen Species; D008623:Mercaptoethanol; D005978:Glutathione; D003545:Cysteine; D010100:Oxygen