P-32: Effect of Culture Condition and Cell-Permeable Superoxide Dismutase on Reactive Oxygen Species of In vitro Sheep Embryos

Background: This study was carried out to evaluate the pattern of reactive oxygen species (ROS) variations during in vitro oocyte maturation and embryo development in sheep, and to investigate whether embryo culture condition and the presence of cell-permeable superoxide dismutase mimetic [Manganese (III) meso-tetrakis (4-benzoic acid) porphyrin (MnTBAP)] can influence ROS pattern. Materials and Methods: Oocytes at germinal vesicle (GV), germinal vesicle breakdown (GVBD) and metaphase II (MII) stages and IVF-derived embryos at different stages of development (2, 4, 8 and 16-cells, morula, blastocyst and hatching) in modified synthetic oviductal fluid (mSOF) or tissue culture medium (TCM) were used for assessment of reactive oxygen species (ROS) level using 2, 7-dichloro dihydro flourescein diacetate (DCHFDA) probe. A dose of 200 µM MnTBAP was added to mSOF before, after or before/after embryo arrest (4th cell cycle) or to TCM199 throughout all culture period. Results: The ROS level in immature and unfertilized oocytes were significantly lowest compared to all fertilized embryo stages (p<0.05). Maximum ROS level was detected at morula stage cultured in TCM199 (77.9 ± 1.7) and at 16-cells stage embryos cultured in mSOF (75.2 ± 2.6). Blastocyst formation was concomitant with a sharp decrease in ROS level. The addition of MnTBAP did not significantly decreased ROS level and embryo development. Conclusion: 1. Fertilization triggers increase in ROS level which peaked around embryo arrest time window. 2. Embryo culture condition does not confer significant change in ROS pattern unless a subtle modulation of surge time point and 3. MnTBAP does not affect ROS level and embryo yield rate.