OXIDATIVE STRESS AND LIPID PEROXIDATION IN BOVINE IN VITRO-PRODUCED EMBRYOS WITH DIFFERENT DEVELOPMENTAL SPEEDS

In vitro-produced embryos are less viable than their in vivo counterparts. It is known that the developmental speed is a reliable marker of embryo viability. One of the major factors impairing in vitro embryo development is oxidative stress. The aim of the study was to evaluate oxidative stress and lipid peroxidation in bovine in vitro-produced embryos that reached different developmental stages at the end of culture. Abattoir-derived oocytes were matured in vitro in TCM-199 with 15% bovine serum, 0.5mugmL(-1) of FSH, 5mugmL(-1) of LH, 0.8mM L-glutamine and 50mgmL(-1) of gentamicin. Mature cumulus-oocyte complexes (COC) were fertilized in Tyrode's modified medium, supplemented by 5.3 SImL(-1) of heparin, 30muM penicillamine, 15muM hypotaurine, 1muM epinephrine and 1% of bovine serum. Both in vitro maturation and IVF were carried out at 39degreesC and 5% CO(2) in air. After 20 to 22h of gamete co-incubation, presumptive zygotes were denuded and cultured in SOF for 7 days at 39degreesC under humidified air with 5% CO(2), 7% O(2) and 88% N(2) in air. At the end of culture, embryos were assessed according to the stage of development as tight morulae (TM), early blastocysts (eBl), blastocysts (Bl), expanded blastocysts (XBl) and hatched blastocysts (HBl). For each stage of development, an average of 20 embryos were used to determine manganese superoxide dismutase (MnSOD) activity and levels of nitric oxide (NO(2)(-)) and thiobarbituric acid-reactive substances (TBARS). The SOD activity was determined by a colourimetric method (Caraglia M et al. 2011 Cell Death Dis. 2, 150, doi:10.1038/cddis.2011.34) whereas NO(2)(-) and TBARS were measured by a spectrophotometric method (Balestrieri et al. 2011 J. Cell. Physiol. doi:10.1002/jcp.22874). Data were analysed by t-test. Greater (P<0.05) MnSOD activity was observed in faster developing embryos (i.e. XBl and HBl) compared with slower ones (i.e. TM, eBl and Bl; 0.460.04, 0.460.03, 0.140.01, 1.660.01 and 3.260.3Umug(-1) of protein, in TM, eBl, Bl, XBl and HBl, respectively). At the same time, XBl and HBl showed the lowest NO(2)(-) levels. However, NO(2)(-) values were lower in TM compared with eBl and Bl (0.040.002, 0.070.005, 0.060.003, 0.010.002 and 0.010.001nMmug(-1) of protein, in TM, eBl, Bl, XBl and HBl, respectively). Similarly to NO(2)(-), TBARS levels were lower in XBl and HBl compared with the other stages (0.00590.002, 0.0090.003, 0.0060.002, 0.0010.0001 and 0.00090.0002muMmug(-1) of protein, in TM, eBl, Bl, XBl and HBl, respectively). In conclusion, these results clearly indicate developmental stage-dependent changes in MnSOD activity and levels of NO(2)(-) and TBARS, suggesting that oxidative stress and lipid peroxidation are reduced in faster developing embryos.