Modernization of the cryoprotective medium compoud for intraovarian vitrification of female gametes of Sus scrofa domesticus

Abstract

Aim. Comprehensive analysis of the morphofunctional state of somatic (cumulus) and germ cells (oocytes) of Sus scrofa domesticus subjected to intraovarian vitrification using silicon dimethylglycerolate (SDMG) are presented.Materials and methods. Fragments of porcine ovaries (FsPO) 15×20 mm in size were gradually kept in cryoprotective agents (CPA) prepared in phosphate-buffered saline (PBS) with 20% fetal bovine serum (FBS): 25 min. in CPA-1 [7.5 % EG (ethylene glycol) with 7.5 % DMSO (dimethyl sulfoxide)] and 15 min. in CPA-2 (15 % EG with 15 % DMSO and 0.5 M sucrose). The composition of the CPA-2 in experimental groups was modified by addition of SDMG (at concentrations of 2 %, 6 %, or 10 %). FsPO were stored in liquid nitrogen. FsPO were devitrified by exposure 1 minute in solution 1 (80 % PBS, 20 % FBS, 0.5 mol/l sucrose) and 5 minutes in solution 2 (80 % PBS, 0.25 mol/l sucrose). The following indicators of cryoresistance of devitrified cumulus-oocyte complexes (COCs) were analyzed: degree of cumulus cells expansion; oocyte morphology and the functional status of lipidome in female gametes (fluorescence intensity of Nile red /lipid droplets complex - FILDs).Results. The addition of SDMG into cryoprotective media reduced the level of denuded oocytes after vitrification. The level of gamete with different degree of cumulus cells expansion (low, medium, high) in the experimental group with 10 % SDMG tended to indicators in the group of native cells. The level of native oocytes with the signs of morphological degeneration (7.7%) had no significant differences with the level of intraovarian vitrified gametes with 10 % SDMG (11 %). The proportion of native oocytes with low FILDs (38.9 %) exceeded the level of oocytes with the above indicator in vitrified oocytes of the control (16.5 %) group and in experimental groups of cells with the addition of 6 % SDMG (4.8 %) and 10 % SDMG (11.8 %, P<0.001).Conclusion. In general, comprehensive monitoring of indicators cryoresistance of COCs in Sus scrofa domesticus subjected to intraovarian vitrification revealed the cryoprotective properties of SDMG. The effects were dose-dependent and were expressed in the stabilization of oocyte-cumulus communication, a decrease in the level of oocytes with the signs of morphological degeneration, and features of the lipidome functioning in intraovarian vitrified female gametes using SDMG at various concentrations.