Samples must be stored in liquid nitrogen (or liquid nitrogen vapor) until ready to thaw
- Using forceps, grasp straw and hold in air for ~10 seconds then gradually lower into 37* water bath (10ul sperm fraction first, slowly lower until buffer fraction is in water) until ice disappears (~10 seconds).
- Remove from water and gently wipe straw dry (straw may be fractured, but sample is OK)
- Holding firmly, carefully cut off both sealed ends, beveling end with the 10ul sperm aliquot.
- Transfer only the thawed sperm suspension directly into a 500ul IVF drop (do not let larger buffer column contact media).
- Allow sperm to capacitate 40-60 minutes before adding oocytes.
- Continue with IVF as usual. ( http://cryo.jax.org/ivf.html )
Conserving, Distributing and Managing Genetically Modified Mouse Lines by Sperm Cryopreservation G. Charles Ostermeier, Michael V. Wiles, Jane S. Farley, Robert A. Taft