Cryopreservation of Mouse Spermatozoa

After the cryopreservation, this page has the detailed procedures for the IVF using the cryopreserved spermatozoa.

Materials and Equipment

  1. Male mice (over 12 weeks old)
  2. Micro-spring scissors (5mm blade)
  3. Pair of watchmaker's #5 forceps
  4. FERTIUP® (Cryoprotectant: CPA; COSMO BIO)
  5. mHTF
  6. Plastic dish (35mm x 10mm Cat.No.430588; CORNING)
  7. Pipette tips
  8. Straws (PETG sperm straw 0.25mL, Cat.No.010261; Cryo Bio System or Mini Straws, Cat.No.005565; IMV Technologies)
  9. Micropipettes
  10. Straw connector
  11. Impulse sealer
  12. Freezing canister
  13. Cryobiological container
  14. Hot plate


Preparing the Freezing Canister

  1. Insert a piece of styrofoam tightly into the bottom of the syringe.

  2. Heat seal the mouth of the syringe tip.

  3. Fix the syringe to the acrylic bar.

Preparing a Straw Connector

  1. Using a 1mL syringe, a 3-way stopcock, a piece of silicone tube, a plastic tube and a silicone cap, make a straw connector as shown in the diagram below.

  2. To use the straw connector, cut away the cotton plug from a straw , then attach the straw to the silicone cap at the end of the connector.

Preparing Sperm Suspension

  1. Make a drop of 60μL of FERTIUP® (CPA) on a 35mm plastic dish and cover it with liquid paraffin.

  2. Add a 60μL aliquot of the same solution to the drop (final volume: 120μL) to make a tall, semispherical drop.
    Keep the dish on a hot plate at 37℃ until use.

  3. Sacrifice a male mouse (>12 weeks-old) via cervical dislocation and remove the two cauda epididymides aseptically.

  4. Place the cauda epididymides on a piece of filter paper and completely remove any fat and blood under a microscope.

  5. Transfer the cauda epididymides into the drop of FERTIUP® (CPA) and use a pair of watchmaker's #5 forceps and micro-spring scissors to make 5 or 6 incisions in the epididymides.

  6. Place the dish on a hot plate at 37℃ for 3 minutes. During this time, rotate the dish every minute to disperse sperm from the organs in the FERTIUP® (CPA).

    [Cutting the Epididymides and Preparing Sperm Suspension]

Preparing Freezing Straw Containing Sperm Suspension

  1. Connect a straw to a straw connector.

  2. Carefully aspirate the contents into the straw in following order:
    a. 100μL of mHTF,
    b. 15mm of air,
    c. 10μL of the sperm suspension,
    d. Another 15 mm of air.

  3. Seal both sides of the straw using an impulse sealer.
    Comment: Loading 100μL of mHTF into the straw prevents the straw from floating on the surface of liquid nitrogen.
    This is because the mHTF acts as a weight that forces the straw to sink into the liquid nitrogen.

  4. Create 10 samples per mouse in the same manner as described above.

Sperm Freezing using a Cryobiological Container

  1. Put the samples into a freezing canister and float them on liquid nitrogen in a cryobiological container.

  2. After 10 minutes, quickly immerse the freezing canister into the liquid nitrogen.

    [Freezing the Straws]

  3. Take out the freezing canister filled with liquid nitrogen, and transfer the straws into a triangular cassette to store them in a liquid nitrogen tank.

Sperm Freezing using a Dry Shipper

  1. Transfer the straw containing sperm suspension into a triangular cassette.

  2. Set the triangular cassette in a precooled canister.

  3. Return the triangular cassette to the canister in the dry shipper and leave it there for 10 minutes.
    Comment: Sperm freezing using a dry shipper can be used for the transport of cryopreserved sperm.


  1. Nakagata N., and Takeshima T. 1992. High fertilizing ability of mouse spermatozoa diluted slowly after cryopreservation. Theriogenol. 37: 1283-129.
  2. Nakagata N., Ueda S., Yamanouchi K., Okamoto K., Matsuda Y., Tsuchiya T., Nishimura M., Oda S., Koyasu K., Azuma S., and Toyoda Y. 1995. Cryopreservation of wild mouse spermatozoa. Theriogenol. 43: 635-643.
  3. Nakagata N. 1996. Use of cryopreservation techniques of embryos and spermatozoa for production of transgenic (Tg) mice and for maintenance of Tg mouse lines. Lab. Anim. Sci. 46: 236-238.
  4. Okamoto M., Nakagata N., Ueda O., Kamada N., and Suzuki H. 1998. Cryopreservation of gene disrupted mouse spermatozoa. J. Mamm. Ova. Res. 15: 77-80.
  5. Takeo T., Hoshii T., Kondo Y., Toyodome H., Arima H., Yamamura KI., Irie T., and Nakagata N. 2008. Methyl-beta-cyclodextrin improves fertilizing ability of C57BL/6 mouse sperm after freezing and thawing by facilitating cholesterol efflux from the cells. Biol Reprod. 78(3): 546-51.
  6. Takeo T., and Nakagata N. 2010. Combination medium of cryoprotective agents containing L-glutamine and methyl-β-cyclodextrin in a preincubation medium yields a high fertilization rate for cryopreserved C57BL/6J mouse sperm. Lab. Anim. 44(2): 132-7.
  7. Nakagata N. 2011. Cryopreservation of mouse spermatozoa and in vitro fertilization. Methods Mol Biol. 693: 57-73.
  8. Nakagawa Y., Fukumoto K., Kondo T., Koga M., Takeshita Y., Nakamuta Y., Sakaguchi M., Haruguchi Y., Tsuchiyama S., Kaneko T., and Nakagata N. 2009. Fertilization ability of C57BL/6J mouse spermatozoa frozen in a dry shipper. Exp. Anim., 58(3) Suppl: 297.

Update history

  1. Updated : 11 May, 2011