Ovary cryopreservation protocol
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Cryopreservation Protocols Contents | |
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Sperm |
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Freezing
Remove ovaries from the fat pad and the bursa aseptically and place in a 10 X 35 mm disposable Petri dish containing 2 ml of M2 medium. Cut each ovary was in halves (unless it is unusually small).
Transfer the half ovaries from a single female into a cryotube (Nunc 1.8 cc) containing 200 ml of cryoprotectant solution (CPA) [1.5 M dimethylsulphoxide (DMSO, Sigma) in M2 medium supplemented with 10% fetal bovine serum (Sigma)].
Keep the sample in the CPA at 23 ?C (room temperature) for 10 minutes and then place the cryotubes on ice for approximately 45 minutes.
After the 45 minute equilibration, place the cryotubes in an ice/salt bath at - 6 ?C for 5 minutes.
Seed the surface of the medium inside the cryotube with a Pasteur pipette containing a small amount of frozen CPA in the tip that has been pre-cooled to -10 ?C. After seeding place the cryotubes in a controlled rate freezer (Cryomed 1010 with model 8024 freezing chamber, Forma Scientific) pre-equilibrated at -6 ?C.
Start the controller to cool at a rate of - 0.5 ?C per minute to -80 ?C. At that point the cryotubes can be transferred to liquid nitrogen storage.
Thawing
Thaw at room temperature (23 ?C) until ice is completely melted, and immediately after, remove the cryoprotectant and replace with 200 ml of M2 medium. Allow the tissue to rehydrate in fresh M2 medium for 10 minutes before surgery.
Ovary Transplantation
N.B. Recipient females must be histocompatible with the ovary donor.
Anesthetize the recipient with 0.2 ml/10 gr. body weight of 1.25% solution tribromo-ethanol (Aldrich) in Amylene hydrate (Aldrich) ("Avertin").
Aseptically, make a single transversal incision on the skin (hair removed) dorsally across the lumbar area to give access to the ovaries on both sides. Both host ovaries must be removed and a donor ovary half is then transplanted orthotopically into the right side bursa. Recipients can be mated with a fertile adult male 7 days after the surgery.
Sztein, J.M., Sweet, H., Farley, J.S., Mobraaten, L.E. 1998. Cryopreservation and orthotopic transplantation of mouse ovaries: new approach in gamete banking. Biol. Reprod. 58:1071-1074.
