We have previously shown that the addition of Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) during in vitro oocyte maturation (IVM) can increase mitochondrial activity in cumulus-oocyte complexes (COCs) in a mouse model of human IVF. The present study was undertaken to determine if adding GM-CSF during IVM reduces the production of reactive oxygen species (ROS) in GM-CSF-treated oocytes. C57Bl6 x CBA F1 female mice aged 21-23 days were injected with 5IU of eCG and COCs aspirated from large antral follicles 46-48 h post-injection. Ten COCs were cultured per 50μL drop in bicarbonate-buffered α-MEM containing 3 mg/ml BSA, 1 mg/ml Fetuin and 5 mIU/mL FSH plus 0 or 10ng/ml of GM-CSF. 16 hours later matured COCs and denuded oocytes were incubated for 30 minutes in 2,7-dichlorodihydrofluorescein diacetate (CM-H2DCFD-FA; Life Technologies, California). After incubation denuded oocytes/ mature COCs were washed to remove excess stain and then mounted on a glass slide for imaging. Oocyte fluorescence intensity was measured at emission wavelengths of 500 nm and 529nm using The Cell Voyager CV1000 Confocal Scanner (Yokogawa, Japan). Z-stack imaging was done and analysed using Fiji Image J software. The experiments were replicated three times each, denuded oocytes and mature COC, 18-20 oocytes per group. Data were analysed using a univariate general linear model in SPSS. The addition of GM-CSF during IVM had no effect on ROS production in mature COCs, however, there was a significant decrease in ROS production in denuded oocytes (7565.3 + 733 vs 14449.5 + 1246; P < 0.05). In conclusion, we have shown that the addition of GM-CSF during IVM decreases ROS production which may contribute to the increase in mitochondrial activity in COCs as reported previously (an increase in mitochondrial activity reduces ROS levels)