This material is from the 4th edition of The Zebrafish Book. The 5th edition is available in print and within the ZFIN Protocol Wiki.


Freezing Sperm

(Source: C. Walker and G. Streisinger)
Sperm may be frozen and thawed later for in vitro fertilization. This method is well suited for "backing up" genetic stocks against accidental loss or for reducing the number of tanks used to house different lines.
To Freeze sperm:
1. Anesthetize and prepare the male as described in the section on Embryo Production By In Vitro Fertilization.
2. Collect sperm in a labeled micro glass capillary, being careful to exclude bubbles (i.e. use very little suction).
3. Mix sperm thoroughly with a 5x volume of sperm freezing medium (see below or RECIPES, Chapter 10) being careful to avoid air bubbles.
4. Draw the mixture into the capillary, transfer the capillary into a 7 ml screw cap tube, cap the tube, and put it into a 15 ml plastic conical centrifuge tube. Quickly plunge the whole works into crushed dry ice for 20 minutes. This configuration provides the proper freezing rate.
Figure 4. Container for freezing sperm

5. After 20 minutes, remove the capillary from the screw cap tube and immerse in liquid nitrogen.
6. The capillary can be stored for years open ended in a test tube on a wand in a liquid nitrogen freezer. NOTE: avoid condensation as much as possible keep everything dry work fast

Sperm freezing recipes:

Ginzburg Fish Ringers

Note: The order of addition is important to prevent precipitation

Freezing Medium

Note: This order is important to prevent precipitation of milk
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