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In Vitro Fertilization using Epididymal Sperm Transported at Cold Temperature

 Freezing Medium and Culture Medium for Mouse Reproductive Engineering


   Reproductive Engineering Manuals

Materials and equipment

1.Cauda epididymis transported at cold temperature

2. FERTIUP Preincubation medium: PM (Cat. #KYD-002-EX)

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

4. Liquid Paraffin

5. Micropipettes

6. Plastic dishes (35mm X 10mm Cat.No.430588; CORNING)

7. Fine scissors

8. Pair of watchmaker's #5 forceps

9. Filter paper

10. Humidified incubator (37°C, 5% CO2, 95% air)


Collection of Cauda Epididymis

1.Put 1 drop (100µL / drop) of FERTIUP(PM) into a dish and cover it with liquid paraffin 30 minutes before collection of epididymal sperm transported at cold temperature, and place the dish in an incubator (37°C, 5% CO2 in air).


2.Remove the 0.2mL tube containing the sample from the foam transport box.

3. Open the tube, pick up the cauda epididymis and wipe away any preservation solution using filter paper.


4.Wash the cauda epididymis in each of the three drops of mHTF in a washing dish. After washing, wipe away any excess mHTF using filter paper.
Comment: To make a washing dish, put 3 drops (about 100µL / drop) of mHTF into a dish without liquid paraffin just before use.KYD_image_3.JPG

5.Place the cauda epididymis in the sperm dish containing liquid paraffin. Epididymal spermatozoa transported at cold temperature can be utilized for in vitro fertilization in the same manner as fresh spermatozoa.
(Please refer to the chapter of In Vitro Fertilization)

Note: If you find it difficult to release sperm from the cauda epididymis, make one more incision in cauda epididymis to release more sperm.

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.


1. Takeo T., Tsutsumi A., Omaru T., Fukumoto K., Haruguchi Y., Kondo T., Nakamuta Y., Takeshita Y., Matsunaga H., Tsuchiyama S., Sakoh K., Nakao S., Yoshimoto H., Shimizu N., and Nakagata N. 2012.
Establishment of a transport system for mouse epididymal sperm at refrigerated temperatures. Cryobiology. 65(3): 163-168.

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