Effect of nobiletin in the in-vitro development and vitrification of mice embryo

Reactive oxygen species (ROS) are molecules produced during the normal functioning of cells and can be beneficial for some metabolic processes. However, excessive ROS production can lead to oxidative stress in cells and trigger apoptosis. Excessive ROS production during in vitro culture of embryos and after vitrification can cause peroxidation of cell lipids, mitochondrial dysfunction, seriously threatening cellular viability. Antioxidants play an important role in reducing this stress rendering them harmless. Thus, they protect cells by preventing oxidative stress. Nobiletin is a polymethoxylated flavonoid isolated from citrus fruits. While there is literature on the antioxidant effect of nobiletin in many in vitro experiments, there is no study on mouse embryo development and freezing. In this study, the antioxidant effect of nobiletin was investigated in three steps. First, a toxicity test was performed on in vitro embryonic cells. Based on the toxicity test results, the doses of 25 µM, 50 µM and 75 µM nobiletin were added to the embryo culture medium of single-cell mouse embryos and in vitro embryo development and embryo quality were evaluated. In the last step, frozen embryos were thawed and the viability of the embryos was examined in media supplemented with nobiletin. Embryo development rates were significantly lower in the Nob25 (58.15%), Nob50 (42.10%) and Nob75 (48.98%) dose groups than in the control group (67.19%) (p<0.05). In terms of the mean number of blastocyst cells, the control group (53.61) was significantly higher than the other groups (DMSO: 45.98, Nob25 49.74; Nob50: 44.57, Nob75: 45.82). Although the post-thawing viability rates of embryos cultured in nobiletin media before and after vitrification were highest in the Nob75 group (61.9%), the difference with the other groups (Control: 53.2%, DMSO: 54.2%, Nob25: 42.9%, Nob50: 38.1%) was not significant. In this thesis, we tried to establish a protocol with a new antioxidant that can be used in in vitro culture for in vitro development and long-term storage of embryos. However, the results of the study show that the use of nobiletin as an antioxidant in in vitro culture of single-cell mouse embryos did'nt produce the expected effect.