Insemination of Frozen Semen

Procedure for Freezing Semen

Option 1: Below we provide you with a procedure for freezing equine semen that utilizes an extender that requires cooling prior to freezing. The procedure can be adapted for use in a modest lab but we will describe the procedure we use. One important aspect though is we use a cold room in place of a refrigerator. It is difficult to fill, seal and place the straws on a rack in a refrigerator without warming the semen.

Initial evaluation of semen: concentration, motility, morphology
Dilute semen 1:1 with room temperature CO-1, BGM3, IMV centrifugation buffer, Minitube M-1 or other clear medium.

Transfer 10-13 ml diluted semen to individual 15 ml conical tubes or use larger 50 ml tubes if desired.
Place 25-50 µl percoll, IMV or Minitube cushion solutions at bottom of 15 ml tubes or 0.5 ml in 50 ml tubes.
Centrifuge at 1000 X g for 15 - 20 minutes
Discard supernatant, remove any remaining cushion solution with a pipette but be careful to not remove the sperm pellet.
Pool sperm pellets, take 10 µl of pooled sperm pellet and add to 90 µl of centrifugation medium. Determine concentration and multiply by 10 to get concentration of sperm pellet. If desired, motility can be checked in the diluted sample. Motility should be greater than 50% at this point.

Resuspend sperm to 250 x 10^6 / ml with a freezing extender such as CO-5, IMV freezing extender or other extender that requires cooling.
Cool semen to 5°C over 2 hr
1. Place container of semen in a beaker with 200 ml of room temperature water.
2. Move beaker into a cold room or refrigerator at 5°C (40°F)
3. Requires 2 hours to cool to 5°C.
If the cryoprotectant is not yet added do so at this time. For the IMV extender we mix glycerol 1:1 with the extender, cool to 5°C and then add 80 µl to each ml of extended semen to give a 4% glycerol solution.
Package in 0.5 ml straws (French straws) and seal with powder or beads (Minitube: be sure to follow recommended procedure carefully as beads can explode out of the straw).
Place straws on a rack such as the one in the image below:

A homemade rack can be substituted but the straws should be 3 cm above the bottom of the legs. We have made racks out of hardware cloth, test tube racks or similar items that are made of metal. They need to be able to withstand liquid nitrogen temperatures.
Suspend a screen in a styrofoam ice chest that is big enough to fit your straw rack. Fill the ice chest with liquid nitrogen to the level of your screen. Place the rack of straws on the screen (shown below) and place the lid on the ice chest. The rack will suspend the straws 3 cm above the liquid nitrogen. The semen will take 15 minutes to freeze. When finished, place the straws in liquid nitrogen and load into canes. Be sure to keep canes and straws under liquid nitrogen.
Store the canes under liquid nitrogen in a semen storage tank.