Cryopreservation of Mouse Spermatozoa
Materials:
- Cryoprotectant solution
- 1.8ml Nunc cryotubes
- Cryotube rack
- 1.5ml Eppendorf tubes.
- 35mm Petri dishes
- Deep polystyrene box with lid suitable for holding liquid nitrogen
- Small Dewar flask of liquid nitrogen
- CO 2 incubator 37 0C, 5% CO 2
- Hot block at 37 0C
- Sexually mature male mice at least 8 weeks old (preferably not recently mated but a proven breeder).
Cryoprotective Agent (CPA)
Place 9ml Embryo H 2O in screw top 15ml Falcon tube and equilibrate to 60 0C in water bath.
Add 1.8g Raffinose and dissolve by gentle inversion. Add 0.3g skim milk and dissolve by gentle inversion. Make up to 10ml if necessary.
Aliquot into 1.5ml eppendorf tubes and centrifuge at 14,000 rpm for 10min.
Tip off supernatant and Millipore filter (0.45 um) into cryotubes. Store at -20 0C (1.1 ml aliquots).
Cryopreservation Method:
- Prepare the cooling apparatus. Place a platform into the polystyrene box. This act as a support for the cryotube rack. Carefully pour liquid nitrogen into the polystyrene box to just cover the platform. Place a cryotube rack on top of the platform so that it is suspended in liquid nitrogen vapor. Replenish the liquid nitrogen as necessary during the freezing session, but do not allow the level to rise above the platform.
- Thaw one aliquot of CPA solution for each male mouse and bring to 37 0C in the incubator or hot block. Mix by inversion if there is any precipitation.
- Pipette 1.1ml CPA into 35mm Petri dish on the hot block at 37 0C.
- Dissect the vas deferens and cauda epididymes from the mouse and clean off all fat and blood. This is best schieved by placing the organs on a tissue and examining them under a microscope. Using Watchmaker’s forceps, mince the caudae and squeeze the sperm gently out from the vas. To disperse the sperm, tap and shake the dish gently for ~30sec. Place in a CO 2 incubator at 37 0C for 10 min., resting the sperm at the angle on the lid.
- Keeping the dish at an angle, remove the epididymal and vas tissue from the suspension by scraping them to one side of the dish. Aliquot 100ul into each 10 cryotubes. Replace the screw cap and tighten to seal the cryotube.
- Place the cryotubes into the pre-cooled freezing apparatus and leave for 10 min.
- Remove and plunge into liquid nitrogen. Store in a liquid nitrogen refrigerator until required.
- Record all relevant data concerning the strain, genotype and storage details.