Freezing Sperm

(Source: C. Walker and G. Streisinger from Zebrafish Book 5th Edition)

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 (see page 2.14).
2.   Collect sperm in a labeled 10 μl 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.
a.   A sim­ple way to do this is to mark the capil­lary at the end of the sperm drop­let and mea­sure the amount of sperm in mm.
b.  Make a second mark on the cap­il­lary at 6x mm.
c.   Draw freezing medi­um into the capillary with the sperm up to the sec­ond mark.
d.  Carefully expel most of the sperm mixture onto a clean watch glass and draw in and out 2 times to mix sperm and freezing medi­um.  Work quickly, being careful not to intro­duce 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.
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:

Ginsburg Fish Ringers:

6.5 g NaCl
0.25 g KCl
0.3 g CaCl₂ (0.4 g CaCl₂- 2H₂O)
Add ddH₂O to almost 1 liter
0.2 g NaHCO₃
Add ddH₂O to 1 liter
Note:The order of addition is important to prevent precipitation

Sperm Freezing Medium:

9 ml Ginsburg Fish Ringers
1 ml Methanol
1.5 g Carnation Powdered Skim Milk
 Note:  This order is important to prevent
precipitation of milk