Samples must be stored in liquid nitrogen (or liquid nitrogen vapor) until ready to thaw
- Using forceps, grasp straw and hold in air for 40 seconds then plunge into room temperature water until ice disappears.
- Remove from water and gently wipe straw dry.
- Holding firmly cut off the sealed end midway though air column at mid-point of PVA plug.
- Push contents out of straw with wire rod or plastic plunger into room temperature 35 mm dish. Do not let plug fall into drop. (a straightened out paperclip can be used for this step)
- Wait 3 to 5 minutes, embryos will shrink considerably due to sucrose diluent and will settle on the bottom of the dish.
- Wash embryos in a drop of M2.
- Transfer to a new drop of M2 and wait 3 to 5 minutes.
- Transfer to equilibrated KSOM under oil and incubate until ready to transfer to oviducts of d0.5 pc pseudo-pregnant female.
High survival of mouse embryos after rapid freezing and thawing inside plastic straws with 1-2 propanediol as cryoprotectant, 1984, J. P. Renard *, C. Babinet