This study was carried out to investigate the effect of embryo culture medium and a permeable superoxide dismutase mimetic (MnTBAP) on developmental competence, ROS content and stage of developmental arrest in sheep embryos. Abattoir-derived sheep oocytes were matured and fertilized in vitro and then were cultured in either sub-optimal [tissue culture medium 199 (TCM199) +10% charcoal stripped serum (CSS)] or optimal sequential [synthetic oviductal fluid (SOF): SOF1 (day 0-3) and SOF2 (day3-8)] culture media 7 days. 200 µM MnTBAP was added before, after or both before and after day 3 in mSOF medium or throughout the culture period in TCM199. The ratios of zygotes cleaved and further developed to hatch blastocyst were recorded and chronological pattern of ROS alterations were determined using fluorimetric method of DCHFDA (2, 7-dichlorodihydroflourescein diacetate) probe.
Despite similar trend of development in SOF and TCM199 embryos until morula, percentages of blastocysts developed at days 5, 6 and 7 in SOF medium were significantly higher than did TCM199 [SOF: 12.5±4.7%, 41.8±3.9%, 51.8±3.1% and TCM199: 0.7%:±0.8, 10.7±2.8%, 16.1±1.7%, respetively]. Irrespective of MnTBAP addition time window, there was no significant effect of MnTBAP on developmental competence of SOF embryos. Irrespective of culture method, cleaved embryos had significantly higher ROS levels than immature oocytes.
which was a cell cycle later than that of SOF embryos (16-cells stage: 72.5±2.6%).
Blastocyst formation was concomitant with a sense decrease in ROS levels which was significant when compared with SOF morula embryos. MnTBAP significantly decreased ROS levels at maximum point levels of all culture systems.This study for the first time indicated that 1) sheep immature oocytes contain lowest levels of ROS which can be remained low during maturation but inevitably increased as fertilized embryos develop in vitro, 2) stage of ROS high rise is concomitant with the critical window of embryo developmental arrest, 3) culture method can induce subtle shift in ROS rise stage, and 4) MnTBAP can not be a suitable preamble SOD mimetic in sheep embryos, at least.