Canine oocytes require an extended period of culture (72 h) in vitro for nuclear maturation to the metaphase II stage, which also results in high degeneration. Canine cumulus oocyte complexes were isolated by slicing from ovaries collected after ovariohysterectomy and cultured in serum-free synthetic oviductal fluid incubated at low (5%) or high (20%) oxygen levels. Changes in oocyte nuclear maturation rates, H2O2 levels within the oocytes and mRNAs of reactive oxygen species inhibitory genes superoxide dismutase 1 and 2 (SOD1 and 2), glutathione reductase (GSR), glutathione peroxidase (GPX1), and catalase (CAT) were quantified. Higher meiotic resumption from germinal vesicle breakdown up to MII was observed in low O2 (41.8±13.1%) compared to high O2 (15.8±8.2%) (P=0.014) after 52 h of culture (n=112). Extension of the culture period up to 84 h at low O2 (n=457 oocytes) produced the highest meiotic resumption at 72 h (64.1±6.0%; P=0.008), compared with 52 h. Oocytes (n=110) cultured in high O2 contained higher levels of peroxidase measured using the 20, 70-dichlorodihydrofluorescein diacetate fluorescence assay after 72 h of culture compared with low O2 (P=0.004). High O2-cultured oocytes also showed higher amounts of SOD1, SOD2, GSR, GPX1, and CAT mRNA. Vitamin E in high oxygen level was able to decrease degeneration (P=0.008) but had no improving effect on percentage of oocytes in MII. These results for the first time showed that low oxygen gas composition improves nuclear maturation rates and alleviates the oxidative stress for canine oocytes during in vitro maturation.