Cryopreservation of Mouse Spermatozoa using a Cryotube

The original method for cryopreservation of mouse spermatozoa was developed using straws. However, cryotubes may also be used for cryopreservation.

Materials and Equipment

  1. Male mice (over 12 weeks old)
  2. Superovulated female mice
  3. Micro-spring scissors (5mm blade)
  4. FERTIUP® (Cryoprotectant: CPA; COSMO BIO)
  5. FERTIUP® (Preincubation medium: PM; COSMO BIO)
  7. Plastic dishes (35mm x 10mm Cat.No.430588; CORNING)
  8. Pipette tips (QSP pipette tip Cat.No.114 Thermo scientific)
  9. Micropipettes
  10. Cryotubes (We recommend Cryogenic Vials Cat.No.MS-4501W; Sumitomo Bakelite, Japan. Alternatively, you could use 366656; NUNC.)
  11. Vial canes
  12. Styrofoam box
  13. Pieces of styrofoam (15mm thick)
  14. Liquid nitrogen
  15. Liquid nitrogen tank
  16. Water bath maintained at 37℃
  17. Humidified incubator (37℃, 5% CO2, 95% air)


Preparing Sperm Suspension

  1. Prepare the sperm suspension in the same manner as you would for sperm suspension in straws.
    Refer to this page for further information.

Cryopreservation of Mouse Spermatozoa

  1. Transfer 35μL of sperm suspension into cryotubes, and set the cryotubes on a cane.

  2. Pour liquid nitrogen into a styrofoam box. Float two pieces of styrofoam (15mm thick) on the surface of the liquid nitrogen, and place the cane holding the cryotubes on the stryrofoam pieces.
    Comment: Keep the cryotubes 15mm above from the surface of the liquid nitrogen.

  3. Close the lid of the styrofoam box and leave for 10 minutes.

  4. Open the lid and transfer the cane into a liquid nitrogen tank immediately.

    [Freezing the Tubes]

Preparation for Thawing

  1. Collect oocytes in a drop of CARD MEDIUM and incubate them for 30-60 minutes.
    Refer to this page for further information.

  2. 30 minutes before thawing a cryotube, place 1 drop (90μL / drop) of FERTIUP®(PM) into a dish, cover it with liquid paraffin, and place it in an incubator (37℃, 5% CO2 in air).

Thawing the Mouse Spermatozoa in a Cryotube

  1. Remove a cryotube from the liquid nitrogen tank, open the cap and discard the liquid nitrogen inside.

  2. Close the cap and place the cryotube in a water bath maintained at 37℃. Leave the cryotube in the water bath for 10 minutes.

  3. After 10 minutes, transfer 10μL of the sperm suspension from the cryotube into the drop of FERTIUP®(PM) (sperm dish).
    Comment: 1 cryotube can provide enough sperm for 3 sperm dishes .

  4. Keep the dish in an incubator (37℃, 5% CO2 in air) for 30 minutes.

  5. Using the collected oocytes and a sperm dish, inseminate in the same manner as used for frozen spermatozoa.

Update history

  1. Updated : 3 September, 2015
  2. Modified : 7 March, 2016
    The recommended pipette tip for insemination was changed, because the old one was discontinued.
    Old one: Cat.No.3520; Thermo SCIENTIFIC
    New one: QSP pipette tip Cat.No.114 Thermo scientific