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Collection and Transport at Cold Temperature of Cauda Epididymis

 Freezing Medium and Culture Medium for Mouse Reproductive Engineering

  CARD Cold Transport Kit

    Reproductive Engineering Manuals

Materials and equipment

1. Male mouse (over 12 weeks old)

2. Anesthetic

3. Hot Plate (37°C)

4. Fine scissors

5. Pair of watchmaker's #5 forceps

6. Wound clip (Autoclip 9mm; Clay Adams 427631) and clip applicator (Mik-Ron Autoclip Applier; Clay Adams 427630)

7. Temperature data logger (Thermochron iButton Cat.No.DS1921G; Maxim Integrated Products)

8. Cold storage solution for cauda epididymides (Cat. no. KYD-007-EX )

9. CARD Cold Transport Kit (Cat. no. KYD-006-EX)

    Thermos bottle (Cat.No.JMK-501; Thermos K.K.)
    Paper box (in which a 0.2mL tube can stand)
    Cotton wool
    Cold packs (small and large)
    Polystyrene foam transport box (KARUX KC-3)

Both the CARD Cold Transport Kit and the Cold storage solution for cauda epididymides must be precooled to 4-8°C before use.

Procedure

Collection of Cauda Epididymis

1. Anesthetize a male mouse.

2. Make a small incision in the scrotum of the mouse and expel a cauda epididymis.

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3. Cut the vas deferens and the corpus epididymis, and collect the cauda epididymis.

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[Removing one Cauda Epididymis from an Anesthetized Male]

* The collection of epididymis with leaving a long length of vas deference will prevent the leakage of sperm in the preservation solution. However, there is no problem when a small amount of sperm was leaked in the medium.

4. Push the testis back into the abdomen and close the wound using a wound clip.

5. Keep the mouse warm on a 37°C warming plate until the mouse recovers from the effects of the anesthesia.

Comment: 1 week after the operation, the male mouse can be used for mating with a female mouse.

Packing and Transport of Cauda Epididymis

The items to be used when packing the cauda epididymis should be kept at 4-8°C until just before use. Moreover, packing procedures should be completed as quickly as possible to prevent the cauda epididymis and the packing items from warming up.

1. Put the removed cauda epididymis into the 0.2mL tube containing preservation solution.

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2. Place the tube containing the cauda epididymis, a temperature data logger and a piece of cotton wool in the paper box.

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3. Insert the paper box containing the cauda epididymis into a thermos bottle.

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4. Insert two cold packs (small) into the thermos bottle.

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5. Close the bottle cap.

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6. Place a cold pack (large) at the bottom of a foam transport box, then put the thermos bottle on top of it.

7. Pack one cold pack (large) on either side of the bottle, then set a further pack (large) on top and close the lid.

Note: Take care not to place the paper box upside down.
Note: It is only possible to place the thermos bottle in the center of the foam transport box and not the actual bottom, because the length of the thermos bottle is the same as that of the inner length of the foam transport box. This is to protect the thermos bottle during shipping.

8. Seal the lid of the foam transport box using packing tape.

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9. Keep the foam transport box in the refrigerator until a courier comes to pick it up.

10.Send the samples via a regular courier service.

Note: The sample must be transferred at a refrigerated temperature. Please ask the courier service directly about conditions during transport.
Comment: Epididymal sperm at cold temperature maintain fertilizing ability for up to 72 hours.

References

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.