Vitrification of In Vitro Derived Ovine Blastocysts by High Osmolality Solution

Background: At present, optimal cryopreservation protocol for livestock species is use of high osmolality vitrification solution following high cooling and warming rates. This technique sup-ports the emergence of a glassy state and inhibits ice crystals growth. Recently, Ficoll PM70 as a non-penetrating cryopro-tectant (CP), a high molecular weight sucrose-polymer, has im-proved blastocyst cryopreservation outcomes. In this study, we aimed to optimize vitrification protocol with Ficoll for ovine blastocysts in terms of re-expansion rate.Materials and Methods: Expanded blastocysts derived from in vitro fertilization in ovine species were used for assessing the efficiency of vitrification with Ficoll™. Briefly, blastocysts were put in equilibrium solution (ES) (8% EG + 8% DMSO or 20% EG + 20% DMSO) for 6 minutes and then embryos were held in vitrification solution (VS) (40%EG+20% DMSO+1%Ficoll or 16%EG+16% DMSO+10%Ficoll or 40%EG+20% DMSO+10%Ficoll or 16%EG+16% DMSO+1%Ficoll) for 35 seconds, and subsequently were immersed in liquid nitrogen. During warming procedure, embryos were incubated in 1 and 0.5 mol/l sucrose solution for 1 and 3 minutes, respectively. Fi-nally, embryos were washed in PBS- containing 20% FBS, and cultured for 48 hours in synthetic oviductal fluid (SOF) medium under mineral oil at 38.5°C, 5% CO2, 5% O2 and maximum humidified air. The percentage of re-expansion rate in various treatment groups was assessed and analyzed statistically with one-way ANOVA test.Results: Re-expansion rate was significantly (P<0.05) higher in ES: 8% EG+ 8% DMSO, VS: 16%EG+16% DMSO+1%Ficoll group (91.12 ± 2.41%) in compare to other groups (19.45 ± 3.09%, 55 ± 3.33% and 29.17 ± 3.53 %, respectively).Conclusion: It seems that high percentage of Ficoll, EG and DMSO (10, 40, 20, respectively) were not an acceptable combi-nation for the vitrification solution, while their low percentage (1, 16, 16, respectively) were the best. Using high osmolality vitrification solution for cryopreservation of embryos in animal facilities and biomedical laboratories should be optimized for other stages of embryos or in other species.

Keywords:

Blastocyst , Vitrification , Ficoll , Ovine , Re-Expan-sion Rate