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To stabilise and improve spermatozoa cryopreservation results and in vitro fertilization rates for laboratory mice

Cryopreservation of Mouse Spermatozoa in FERTIUP®

 Freezing Medium and Culture Medium for Mouse Reproductive Engineering


    Reproductive Engineering Manuals

Materials and Equipment

1. Male mice (>12 weeks old)

2. Micro-spring scissors (5mm blade)

3. Pair of watchmaker's #5 forceps

4. FERTIUP Mouse Sperm Cryoprotectant: CPA (Cat. #KYD-001-EX)

5. HTF (Cat. #CSR-R-B070)
* It can be used as the same as mHTF (CaCl2 modified HTF) 

6. Plastic dish (35mm X 10mm Cat. #430588; CORNING)

7. Pipette tips

8. 0.25mL PETG cotton-plugged Sperm Straws (Cat. no. KYD-S020X10)

9. Micropipettes

10. Straw connector (Cat. #KYD-S025)

11. Impulse sealer

12. Freezing canister (Cat. #KYD-S018)

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 siliconetube, 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 (0.25mL French 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 culture 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.
* The drop of CPA should be warmed at 37 degrees for several minutes before 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°C 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 HTF,
  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 HTF into the straw prevents the straw from floating on the surface of liquid nitrogen.
This is because the HTF 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

・ Transfer the straw containing sperm suspension into a triangular cassette.
・ Set the triangular cassette in a precooled canister.
・ 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: 1283-1291.

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): 546-551.

6. 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.

7. Takeo T., and Nakagata N. 2010. Combination medium of cryoprotective agents containing L-glutamine and methyl-

To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.