Abstract :
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 MMnTBAP
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.
