RESUMO
Reproductive technologies are some of the key directions in the context of the need to preserve and select highly productive farmed animals in terms of economically useful traits. Improvements of the existing models of the in vitro oocyte maturation system help to solve the problem of low yield of porcine embryos at the final stages of preimplantation development. In the present study, a model of culture medium for gametes (NCSU-23 with 10 % homologous follicular fluid, 10 IU hCG and 10 IU eCG) modernized by the addition of 1·106 granulosa cells (GCs) per ml and 0.001 % of highly dispersed silica nanoparticles (HDSn) is proposed for use in the IVM and IVF technology of donor porcine oocytes. Analysis of the oocyte chromatin status by the Tarkowsky method and assessment of the level of destructive changes in chromatin (apoptosis, pyknosis) revealed a significant percentage increase in matured oocytes and a decrease in the proportion of granulosa cells with degenerated chromatin when using the original culture system. The positive effects of a joint addition of GCs and HDSn to the maturation system have made it possible to increase the indicators of the meiotic maturation and fertilization of oocytes. Optimal results of developmental competence of oocytes were achieved with the joint use of GCs and HDSn in the maturation system (the proportion of matured cells reached 89 %, the level of oocytes with chromosome degeneration was 12 %, 39 % of embryos reached the final stage of preimplantation development). The positive effect of HDSn on oocyte fertilization was accompanied by an abrupt decrease in destructive processes in GCs during culture in the presence of HDSn. The level of somatic cells with pyknotic nuclei was 32 % and the level of apoptosis (TUNELtest), 21 %, compared with the control (43 and 31 %, p <0.01, respectively). Thus, a high efficiency of the porcine oocyte maturation system in the joint culture of gametes with GCs and HDSn was revealed. It makes it possible to recommend a model of this culture medium at the IVM of female gametes of Sus scrofa domesticus for improving the quality of donor oocytes used in cell and genetic engineering.
RESUMO
The aim of the present study was to identify the inf luence of extra- (EOV) and intraovarian vitrif ication (IOV) on mitochondrial activity (MA) and chromatin state in porcine oocytes during maturation in vitro. During EOV porcine oocytes were exposed in cryoprotective solutions (CPS): CPS-1 - 0.7 M dimethyl sulfoxide (DMSO) + 0.9 M ethylene glycol (EG); CPS-2 - 1.4 M DMSO + 1.8 M EG; CPS-3 - 2.8 M DMSO + 3.6 M EG + 0.65 M trehalose. At IOV the ovarian fragments were exposed in CPS-1 - 7.5 % EG + 7.5 % DMSO, then in CPS-2 - 15 % EG, 15 % DMSO and 0.5 M sucrose. Straws with oocytes and ovarian fragments were plunged into LN2 and stored. For devitrif ication, the EOV oocytes were washed in solutions of 0.25, 0.19 and 0.125 M of trehalose, the IOV - in 0.5 and 0.25 Ð trehalose. Oocytes were cultured in NCSU-23 medium with 10 % f luid of follicles, follicular walls, hormones. 0.001 % of highly dispersed silica nanoparticles (ICP named after A.A. Chuyko of the NAS of Ukraine) were added to all media. The methods of fertilization and embryo culture are presented in the guidelines developed by us. MA and chromatin state were measured by MitoTracker Orange CMTMRos and the cytogenetic method. Signif icant differences in the level of oocytes with high-expanded cumulus between control and experimental vitrif ied groups (81 % versus 59 % and 52 %, respectively, p ≤ 0.001) were observed. The percentage of pyknotic cells in native oocytes was 19 %, EOV or IOV oocytes were 39 % and 49 %, respectively. After culture, the level of matured native oocytes was 86 %, 48 % EOV and 33 % IOV cells f inished the maturation ( p ≤ 0.001). Differences were also observed in the level of MA between groups treated by EOV and IOV (89.4 ± 7.5 µA and 149.2 ± 11.3 µA, respectively, p ≤ 0.05). For the f irst time, pre-implantation embryos were obtained from oocytes treated by IOV.
