Comparison of Conventional Freezing and Vitrification with Dimethylformamide and Ethylene Glycol for Cryopreservation of Ovine Embryos

The aim of this work was to evaluate the efficiency of the cryoprotectants dimethylformamide and ethylene glycol for cryopreservation of ovine embryos using vitrification and conventional freezing. The recovered embryos were distributed randomly in three treatment groups: Gr. 1: conventional freezing (n = 44), Gr. 2: vitrification with ethylene glycol (n = 39) and Gr. 3: vitrification with dimethylformamide (n = 38). Quality of fresh embryos in control group as well as of frozen and vitrified embryos was examined by three methodologies: staining with propidium iodide and Hoechst 33258 and evaluation under fluorescent microscopy, evaluation of re‐expansion and hatching rates after culture, and determination of apoptotic index with TUNEL technique. It was established that re‐expansion rate in all treatment groups was similar. In the same time, hatching rates were higher in Gr. 1 (40.5%) and Gr. 2 (35.3%) in comparison with Gr. 3 (15.5%, p < 0.05). The number of dead cells in vitrified embryos of Gr. 2 and Gr. 3 was higher (42.6 ± 26.2 and 63.2 ± 34.65, respectively) in comparison with Gr. 1 (conventional freezing, 10.1 ± 8.5, p < 0.05). Embryos vitrified with dimethylformamide included the same quality of apoptotic cells that Gr. 1 (conventional freezing) and fresh embryos. In conclusion, the dimethylformamide and ethylene glycol used as cryoprotectant to vitrify ovine embryos, in the concentrations and exposition time tested in this work, were not as efficient as the conventional freezing for cryopreservation of ovine embryos Thus, the conventional freezing with ethylene glycol was the most efficient method to cryopreserve ovine embryos in comparison with vitrification.     

Viability of OPS Vitrified Sheep Embryos After Direct Transfer

The aim of this study was to evaluate the viability in the effect of open pulled straw (OPS) vitrification procedure of sheep embryos after direct transference. Embryos were produced in vivo and cryopreserved in slow freezing or OPS vitrification. The survival rates of cryopreserved embryos were compared to non-frozen standard pattern. In a first set of experiments, embryos at morula and blastocyst stages were dived in ethylene glycol (1.5 M) and frozen in an automatic freezer. After being thawed, they were directly or indirectly transferred to ewes recipient. A second group of embryos were drawn into OPS and plunged into liquid nitrogen after being exposed at room temperature for 1 min and 45 s in 10% EG plus 10% dimethyl sulphoxide (DMSO), then again for 30 s in 20% EG + 20% DMSO + 0.5 M sucrose. After being warmed, embryos were also directly transferred using a French mini straw as the catheter for the transplantation process or after in vitro dilution of cryoprotectants (two-step-process). No significant difference was observed among fresh, frozen or vitrified embryos on pregnancy rate (50.0%, 38.6% and 55.8%). However, when we evaluated only the direct transference, the pregnancy rate of OPS vitrified embryos was higher than that of frozen embryos (57.1% vs 34.8%) (p = 0.07). In addition, vitrified morulae had a higher pregnancy rate than the one with frozen embryos (64.0% vs 38.9%) (p = 0.07). Finally, our results indicate that OPS vitrification technique in association with direct transference improves the viability of sheep embryos with potential applications to field conditions.