We tried to develop new vitrification solutions based on the original EFS solution, PB1 medium containing ethylene glycol, Ficoll, and sucrose. The solution was first developed for the vitrification of mouse morulae and has proven effective for mouse embryos at various developmental stages, as well as for embryos of many mammalian species.
In the present study, original EFS solutions (renamed EFSa) and modified EFS solutions (named EFSc) were used. EFSa solutions (EFS20a, EFS30a, EFS40a, and EFS50a) were composed by mixing ethylene glycol (20%, 30%, 40%, and 50%, v/v) and an FSa solution (80%, 70%, 60%, and 50%, v/v, respectively). The FSa solution was PB1 medium containing 30% (w/ v) Ficoll PM-70 (average molecular weight, Mr 70 000; GE Healthcare, BioSciences AB, Uppsala, Sweden) plus 0.5 M sucrose.
EFS20a was for the pretreatment of embryos, and the other solutions were for vitrification. For near-equilibrium vitrification, we composed EFSc solutions by increasing the osmolality. EFSc solutions (EFS30c, EFS35c, EFS40c, and EFS50c) were composed by mixing ethylene glycol (30%, 35%, 40%, and 50%, v/v) and an FSc solution (70%, 65%, 60%, and 50%, v/v, respectively). The FSc solution was PB1 medium containing 30% (w/v) Ficoll PM-70 plus a higher concentration (1.5 M) of sucrose to promote dehydration of the cytoplasm before cooling. All of the EFSc solutions were for the vitrification of embryos. The osmolality of EFS solutions is given in Table 1. http://buy-asthma-inhalers-online.com/proventil-inhaler-100-mcg-albuterol.html
Vitrification of Embryos
Embryos were vitrified by a two-step method reported previously. Embryos were manipulated in a room at 25°C 6 1°C. A 0.25-ml plastic straw (IMV, L’Aigle, France) was loaded with ~65 mm of PB1 medium containing 0.5 M sucrose (S-PB1 medium), ~15 mm of air, ~3 mm of EFS solution, ~4 mm of air, and ~13 mm of EFS solution. A total of 5-12 embryos were pretreated with EFS20a at 25°C by being suspended and washed in the solution for permeation by ethylene glycol and dehydration by sucrose. After 2 min of pretreatment, the embryos were transferred into the larger (~13-mm) column of vitrification solution (EFS30a, EFS40a, EFS30c, EFS35c, or EFS40c) for further dehydration, using a fine pipette with a minimal volume of EFS20a.
The straw was sealed with a heat sealer. After the embryos were exposed to the vitrification solution at 25°C for 60 sec, the straw was placed horizontally on a Styrofoam boat (thickness, 1.5 cm) floated on LN2 in a Dewar flask (inner diameter, 140 mm; temperature on the boat, approximately —150°C) and was left there for 3 min or more before the straw was immersed in LN2. The average rate of cooling between 25°C and — 100°C was;300°C/min, which was determined by recording the change in temperature of the EFS solution in the straw using a thermocouple during cooling in LN2 gas. A total of 5-10 straw samples were vitrified for each experiment, and the experiment was replicated five to eight times except for the experiment for embryo transfer, which was replicated three times with 16-28 embryos for each experiment.