Sperm Thawing Protocol

Samples must be stored in liquid nitrogen (or liquid nitrogen vapor) until ready to thaw

Sperm Thawing

  1. 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).
  2. Remove from water and gently wipe straw dry (straw may be fractured, but sample is OK)
  3. Holding firmly, carefully cut off both sealed ends, beveling end with the 10ul sperm aliquot.
  4. Transfer only the thawed sperm suspension directly into a 500ul IVF drop (do not let larger buffer column contact media).
  5. Allow sperm to capacitate 40-60 minutes before adding oocytes.
  6. Continue with IVF as usual.

Reference

Conserving, Distributing and Managing Genetically Modified Mouse Lines by Sperm Cryopreservation G. Charles Ostermeier, Michael V. Wiles, Jane S. Farley, Robert A. Taft