Procedure for Freezing Semen
Option 2: This option differs from option 1 in that it does not require cooling prior to freezing.
- Initial evaluation of semen: concentration, motility, morphology
- Dilute semen 1:1 with room temperature CO-1, BGM3, IMV centrifugation buffer, Minitube M-1 or other clear medium.
- Transfer 10-13 ml diluted semen to individual 15 ml conical tubes or use larger 50 ml tubes if desired.
- Place 25-50 µl percoll, IMV or Minitube cushion solutions at bottom of 15 ml tubes or 0.5 ml in 50 ml tubes.
- Centrifuge at 1000 X g for 15 - 20 minutes
- Discard supernatant, remove any remaining cushion solution with a pipette but be careful to not remove the sperm pellet.
- Pool sperm pellets, take 10 µl of pooled sperm pellet and add to 90 µl of centrifugation medium. Determine concentration and multiply by 10 to get concentration of sperm pellet. If desired, motility can be checked in the diluted sample. Motility should be greater than 50% at this point.
- Resuspend sperm to 250 x 10^6/ ml with a freezing extender that does not require cooling such as Minitube's CryoGuard complete.
- Package in 0.5 ml straws (French straws) and seal with powder or beads (minitube).
- Freeze and store as described for Option 1.
