Rescue In Vitro Fertilization Method for Legacy Stock of Cryopreserved Spermatozoa

Materials and Equipment

  1. Legacy stock of cryopreserved spermatozoa
  2. Female mice superovulated with PMSG and hCG
  3. FERTIUP® (Preincubation medium: PM; COSMO BIO)
  5. mHTF
  6. Water bath maintained at 37℃
  7. Float for thawing
  8. 1.5mL tube (Quality Scientific Plastics 1.5ml Graduated Microcentrifuge Tube with Flat Top Cap, Natural Cat.No. 509-GRD-Q)
  9. Centrifuge
  10. Micropipettes
  11. Plastic dishes (35mm x 10mm Cat.No.430588; CORNING)
  12. Humidified incubator (37℃, 5% CO2, 95% air)


Preparation of the Float for Thawing

  1. Using styrofoam and a 50 mL plastic centrifuge tube, make a float as shown in the diagram below.

Preparation for Thawing

  1. Prepare a water bath to 37°C.

  2. Pour water (37°C) into the 50mL plastic centrifuge tube section of the styrofoam/centrifuge tube assembly, and float it in a water bath.

  3. 30 minutes before thawing a frozen sperm sample, put 1 drop (100μL/drop) of FERTIUP®(PM) into a dish and cover it with liquid paraffin. Place the dish in an incubator (37°C, 5% CO2 in air).

  4. 10 minutes before collecting oocytes, put 1 drop (200μL/drop) of CARD MEDIUM into a dish and cover it with liquid paraffin. Place the dish in an incubator (37°C, 5% CO2 in air).
    Note: There are three different methods of preparing CARD MEDIUM, depending on whether in vitro fertilization will be carried out using fresh, frozen-thawed or cold-temperature transported spermatozoa.
    Please refer to the CARD MEDIUM instruction manual.

  5. Put 4 drops (80μL / drop) of mHTF into a dish and cover them with liquid paraffin. Place the dish in an incubator (37°C, 5% CO2 in air) for at least 30 minutes.

Collection and Preincubation of Oocytes

  1. Sacrifice female mice 15-17 hours after an hCG injection and remove the oviducts.

  2. Using fine, sharp needles, release between 6 and 20 masses of cumulus-oocytes-complexes (COCs) into a drop of CARD MEDIUM (200μL) (Oocyte dish), and preincubate the dish for 60 minutes.
    Note: Be sure to carry out all operations, from sacrificing the female and removing her oviducts to introducing the COCs into a drop of CARD MEDIUM, in the shortest time possible (within 30 seconds). Moreover, when carrying out this process alone, do not sacrifice multiple mice at once; instead, sacrifice one mouse and swiftly remove its oviducts before moving on to the next mouse.

Thawing the Mouse Spermatozoa

  1. Remove a frozen sperm sample from the liquid nitrogen. If the sperm sample is stored in a cryotube, open the cap and discard any liquid nitrogen in the tube. Immerse the sample in a water bath maintained at 37°C (using a styrofoam or the styrofoam/centrifuge tube assembly) for 10 minutes.

  2. Transfer the sperm suspension from the cryotube or the straw into a 1.5mL tube. Slowly add 1.2mL of mHTF kept at 37°C to the tube, and centrifuge it at 300g at room temperature for 5 minutes.

  3. After centrifugation, remove as much supernatant as possible, and add 70μL of FERTIUP®(PM) kept at 37°C into the tube (the final volume is approx.100μL).

  4. After pipetting gently, transfer all of the contents in the tube into the 100μL drop of FERTIUP®(PM) (Sperm dish). Place the dish in an incubator (37°C, 5% CO2 in air) for 30 minutes.


  1. Using a tip, suck up the preincubated COCs with a minimum amount of medium from the drop of CARD MEDIUM (Oocyte dish). Then, release them into the drop of sperm suspension (Sperm dish), and incubate it in an incubator (37°C, 5% CO2 in air).

  2. After incubating for 3 hours, wash the oocytes 3 times in fresh mHTF (80μL) in a washing dish.

  3. 6 hours after insemination, observe them in the third drop of mHTF and remove any parthenogenetic oocytes which have only one pronucleus.

  4. After culturing the oocytes overnight, transfer the obtained 2-cell stage embryos only to the fourth drop of mHTF. These embryos can now be vitrified or transferred.


  1. Nakagata N., Takeo T., Fukumoto K., Haruguchi Y., Kondo T., Takeshita Y., Nakamuta Y., Umeno T., and Tsuchiyama S. 2014. Rescue In Vitro Fertilization Method for Legacy Stock of Frozen Mouse Sperm. J Reprod Dev. 60(2): 168-171

Update history

  1. Updated : 20 December, 2013
  2. Modified : 6 February, 2014
    A reference was added.