Freezing Protocols

Cryopreservation of Mouse Spermatozoa

Materials:

A. 1.8ml cryotubes (Nunc Cat No 363401K).
B. Cryotube rack.
C. Eppendorf centrifuge 5415C and 1.5ml Eppendorf tubes.
D. 35mm culture dishes, (e.g. Falcon 3001).
E. Deep polystyrene box with lid suitable for holding liquid nitrogen.
F. Hot block held at 37°C.
G. Sexually mature male mice at least 8 weeks old, (preferably not recently mated).

Cryoprotective Agent (CPA):

A. Composition = 18% raffinose, (Sigma R-7630) 3% skim milk, (Difco; Cat No 232100).

B. Place 9ml Sigma H2O (W-1503) in a screw top 15ml Falcon tube (2097) and equilibrate to 60oC in a 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 Eppendorfs and centrifuge at 14,000 rpm for 10 min. Tip off supernatant and millipore (0.45 µm) filter into cryotubes. Store at -20oC in 1.0ml aliquots.

Cryopreservation Method:

A. Prepare the cooling apparatus. Place a platform, (for example, the insert from a Gilson yellow tip box), into the polystyrene box. This acts 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 vapour. Replace the lid on the polystyrene box and allow it to fill with vapour. Replenish the liquid nitrogen as necessary during the freezing session, but do not allow the level to rise above the platform.

B. Thaw one aliquot of cryoprotectant solution for each male mouse and bring to 37°C in the incubator or hot block. Mix by inversion if there is any precipitation.

C. Pipette 1.1ml CPA into a small Falcon dish (3001) on the hot block at 37oC. Dissect the vasa deferentia and cauda epididymides from the mouse and clean off all fat and blood. This is best achieved by placing the organs on a tissue and examining them under a microscope. Using watchmaker's forceps mince the cauda and squeeze sperm gently out of the vas. To disperse the sperm, shake the dish gently for ~ 30 seconds and then incubate for 10 minutes on a hot plate at 37oC.

D. Keeping the dish at an angle, remove the epididymal and vas tissue from the suspension by scraping them to one side of the dish with the tip of a Gilson. Using a wide bore pipette, aliquot 100µl into each of 8 cryotubes. Replace the screw cap and tighten to seal the cryotube.

E. Place the cryotubes in the liquid nitrogen vapour phase in the pre-cooled freezing apparatus and leave for 10 min.

F. Plunge cryotubes into liquid nitrogen. Store in a liquid nitrogen refrigerator until required.

Thawing method:

A. Using forceps, hold the cryotube in air for 30 sec, and then thaw rapidly by placing in a 37°C water bath. Take special care that the tube has not filled with liquid nitrogen before plunging into the water bath, (such tubes may explode). If liquid nitrogen is present in the tube, wait for it to evaporate and escape first.

B. Gently agitate the cryotube to mix the sperm. Using a wide bore pipette, take 5-10µl aliquots and pipette gently into 200µl fertilisation drops. There should be enough sperm for 5 x 200µl drops (5 dishes). Do not pipette the sperm up and down in the fertilisation drop

References

Okoyama, M., Isogai, S., Saga, M., Hamada, H., and Ogawa, S. (1990). J. Fert. Implant. (Tokyo), 7, 116.

Nakagata, N., and Takeshima, T. (1992). Theriogenology, 37, 1283.

Sztein, J. M., Farley, J. S., Young, A. F., and Mobraaten, L.E. (1997). Cryobiology, 35, 46.

Huffstadt, U. & Balling, R. Personal communication (1997).

Sztein, J. M., Farley, J. S., & Young, A. F. Personal communication (1997).

Wood, M. J., & Whittingham, D. G. Personal communication (1997).

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