Effect of culture condition and cell-permeable superoxide dismutase on levels of reactive oxygen species (ROS) production in “in vitro” produced sheep embryos

This study was carried out to evaluate the pattern of reactive oxygen species (ROS) production variation during in vitro oocyte development and maturation and embryonic development in sheep, and to investigate whether embryo culture conditions and the presence of cell-permeable superoxide dismutase mimetic [Manganese (III) meso-tetrakis (4-benzoic acid) porphyrin (MnTBAP)] can influence the ROS production pattern. Oocytes at the germinal vesicle (GV), germinal vesicle breakdown (GVBD) and metaphase II (MII) stages and fertilized embryos at different stages of development (2, 4, 8 and 16-cell, morula, blastocyst and hatching) in two embryo culture media [modified synthetic oviductal fluid (mSOF) or tissue culture medium (TCM199)] were used for assessment of the ROS levels, using a 2,7-dichloro dihydro flourescein diacetate (DCHFDA) probe. A dose of 200 μM MnTBAP was added to the mSOF before, after or before/after embryo arrest (4th embryonic cell cycle) or to the TCM199 media throughout the culture period. ROS levels in immature and unfertilized oocytes were significantly lower, when compared to all fertilized embryonic stages (P < 0.05). Maximum ROS levels were detected in the morula stage embryos with TCM199 (77.9 ± 1.7) and for the 16-cell stage embryos in mSOF (75.2 ± 2.6). Blastocyst formation was concomitant with a sharp decrease in the ROS production level. The addition of MnTBAP did not significantly decrease the ROS levels and embryonic development. In conclusion it can be said that (i) fertilization triggers an increase in ROS levels, which peaked around the embryo arrest time window, (ii) embryo culture conditions do not confer significant changes in the ROS production pattern, accompanied by a subtle change in surge time, and (iii) MnTBAP does not affect the ROS levels and embryo yield rate.