Analysis of cryoresistance of cumulus of Ovis Arias oocytes after intra- or extra-ovarian vitrification

Abstract

The aim of this study was to identify the effects of highly dispersed silica nanoparticles (HDSns) on the cryoresistance of Ovis Aries cumulus of oocytes in conditions of intra-(IOV) or extra-(EOV) ovarian vitrification.Materials and methods. Cumulus-oocyte complexes (COC) intended for EOV were treated with solutions of cryoprotectants (CPA) prepared on TC-199 with 10% FBS (fetal bovine serum): CPA-1-0.7 M dimethyl sulfoxide (DMSO) + 0.9 M ethylene glycol (EG);CPA-2-1.4 M DMSO + 1.8 M EG; CPA -3- 2.8 M DMSO + 3.6 M EG + 0.65 M trehalose. The COC was gradually exposed for 30 seconds in CPA-1, 30 seconds in CPA-2, and 20 seconds in CPA-3. At the IOV, the ovaries of the animals were divided into fragments and placed in sterile gauze containers in CPAs prepared on the basis of Dulbecco (PBS) with 20% FBS: CPA-1 - 7.5% EG + 7.5% DMSO (25 min), then in CPA-2 - 15% EG+15% DMSO + 0.5 M sucrose (15 min). 0.001% HDSns (A.A. Chuiko ICP of the National Academy of Sciences of Ukraine) were added to the CPAs and media for thawing of the experimental groups. The straws with the biomaterial were stored in liquid nitrogen for at least 24 hours. The COCs were removed from the straws after thawing and placed successively at 3 min: in a 0.25 M solution of trehalose in TC-199 with 10% FBS at 37°C, then in 0.19 M, and finally in 0.125 M trehalose. COCs from the thawed fragments were sequentially treated with 0.5 M (1 min) and 0.25 M (5 min) solutions of sucrose in PBS with 20% FBS. The final washing of all COCs was carried out in TC-199 with 10% FBS.Results. The results of morphological (the character of oocyte-cumulus communications) and cytological monitoring (the status of cumulus cells chromatin) revealed a positive effect of 0.001% HDSns on the parameters of cryoresistance of COC both at EOV and IOV. The proportion of cells with compact cumulus in the group of EOV of gametes with HDSns significantly exceeded that in their absence (69 % vs. 51 %, P<0.05). A similar trend was shown in IOV of oocytes (35 % vs. 20 %, P<0.05). The level of apoptotic (TUNEL-test) cells in the group of EOV (44 % vs. 32 %, P<0.05) and IOV (72 % vs. 51 %, P<0.001) of gametes sharply decreased when HDSns were added to cryoprotective media, and the proportion of pyknotic cells decreased during EOV (from 31 % to 21 %, P<0.05) and IOV (from 58 % to 41 %, P<0.05). In general, the obtained results indicate the expediency of using 0.001 % HDSns in the protocols of IOV and EOV of female gametes OVIS ARIAS.