Vitrification of mouse two-cell and blastocyst stage embryos in simplified closed system using either a hemi-straw or a hollow fiber device

Two-cell stage and blastocyst stage mouse embryos were equilibrated in a medium containing 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for 8–15 min. Vitrification was performed in a medium containing 0.5 M sucrose and either 15% EG + 15% DMSO, 17.5% EG + 17.5% DMSO, or 20% EG + 20% DMSO for 30 s. They were then placed either on a hemi-straw (HS) or a hollow fiber vitrification (HFV) device and vitrified by cooled air inside a 0.5-ml straw. In two-cell embryos, a 100% survival rate was obtained from all groups except the 20% HS group (P > .05). All vitrified two-cell groups showed similar rates of blastocyst development to that of fresh control group (P > .05), except 17.5% and 20% HFV groups, which were significantly lower than the other groups (P < .05). In the blastocyst embryos, the HFV groups were divided into two subgroups (non-collapsed; HFV-NC and collapsed; HFV-C blastocyst). Re-expansion rate in 15% HFV-NC, 17.5% HFV-NC, and 15% HFV-C groups was reduced (P < .05), whereas the rest were similar to control. In conclusion, we established a simplified, reliable, and closed system for HFV vitrification applying hemi-straw, which does not require skilled practitioners.     

Survival and developmental competence of bovine embryos at different developmental stages and separated blastomeres after vitrification in different solutions

Generating techniques to enhance the success of blastomere separation is important for bovine economy, because it increases the number of transferable embryos. This study aimed to identify the optimum cryoprotectants for the vitrification of bovine embryos and the separation of blastomeres at different stages. In experiment 1, expanded blastocysts were vitrified in two different vitrification solutions, either (1) ethylene glycol (EG) + propylene glycol (PG) or (2) EG. The survival rate of blastocysts in the EG + PG was higher than that of the EG. In experiment 2, intact two‐cell and eight‐cell stage embryos were vitrified in the same solutions used in experiment 1. The EG + PG produced more dead embryos than the EG (P < 0.05). In the EG, the rate of blastocyst formation was similar for the vitrified two‐ and eight‐cell embryos and the non‐vitrified ywo‐cell embryos. In experiment 3, separated blastomeres of two‐ and eight‐cell embryos were vitrified in EG. There was no difference in the rate of blastocyst formation and total number of cells between the two vitrified groups. In summary, at the blastocyst stage, EG + PG was superior, based on both survival rates and cell numbers; however, at the 2–8 cell stage, the use of EG alone was better than the other groups.     

Effect of medium additives during liquid storage on developmental competence of in vitro matured bovine oocytes

Our aim was to improve the developmental competence of bovine oocytes during their liquid storage by using additives. In vitro matured oocytes were stored for 20 h at 25°C in HEPES buffered TCM 199 medium (base medium). After storage, in vitro embryo development after in vitro fertilization was compared to those of non‐stored (control) ones. Addition of 10% (v/v) newborn calf serum or 10.27 mmol/L pyruvate alone to the base medium did not improve blastocyst formation rates in stored oocytes; however, their simultaneous addition significantly improved the rate compared with those stored in base medium (P < 0.05). Supplementation of the holding medium with dithiothreitol (DTT) at any concentrations did not improve embryo development from stored oocytes. Although supplementation with cyclosporine A (CsA) significantly reduced apoptosis and membrane damage rates during storage, it did not improve the developmental competence of oocytes. 1,2‐bis(2‐aminophenoxy) ethane N,N,N’,N’‐tetraacetic acid tetrakis‐acetoxymethyl ester and ruthenium red had no effect on oocyte apoptotic rates. Blastocyst formation rates in all stored groups remained significantly lower than that of the control. In conclusion, pyruvate and serum had a synergic effect to moderate the reduction of oocyte quality during storage, whereas mitochondrial membrane pore inhibitor CsA and the antioxidant DTT did not affect their developmental competence.