Assessment of competence for the development of Bos taurus oocytes after intraor extraovarial vitrification
The purpose of the studies was to compare the functioning of mitochondria in Bos taurus oocytes subjected to extra- and intraovarial vitrification, as well as to assess their ability to mature and fertilize in vitro. We analyzed the functional state of mitochondria and chromatin status in native, extra- and intraovarially vitrified oocytes. The average number of oocytes in a group was 31-40. The experiment was repeated 3 times. To obtain embryos, we used 462 oocytes. The average number of oocytes in a group was 44-54. The experiment was repeated 3 times. During extraovarial vitrification, oocytes were gradually treated with cryoprotectants (CP) based on T-199 medium with the addition of 10% fetal bovine serum (FBS): 30 s in CP-1 - 0.7 M dimethyl sulfoxide (DMSO) + 0.9 M ethylene glycol (EG); 30 s in CP-2 - 1.4 M DMSO + 1.8 M EG; 20 s in CP-3 - 2.8 M DMSO + 3.6 M EG + 0.65 M trehalose. During intraovarial vitrification, ovarian fragments of15 x 20 mm were kept in CP based on Dulbecco’s phosphate-buffered saline with the addition of 20% of FBS: CP-1 - 7.5% EG + 7.5% DMSO (25 min), then in CP-2 - 15% EG + 15% DMSO + 0.5 M sucrose (15 min). CP solutions and defrosting media contained 0.001% of highly dispersed silica nanoparticles. According to the fluorescence intensity of Mito Tracker Orange CMTMRos, the functional activity of mitochondria of extraovarially vitrified oocytes was significantly higher than that of intraovarially vitrified oocytes - (411.0 ± 17.8) uA and (198.0 ± 10.5) uA, respectively. The share of cells crushed after extraovarial vitrification was 52%. For intraovarial vitrification, it was significantly lower - 32%. Significant differences in the yield of embryos at the stages of late blastocyst morula from devitrified oocytes (11% and 6%, respectively) and the proportion of degenerated embryos (24% and 29%, respectively) were not detected.