Colder Than Cold

A few months ago I wrote about the lack of male bovines on dairy farms due to the use of artificial insemination (A.I.) .  What I failed to talk about in that article was what makes A.I. possible.  What makes A.I. possible is the ability to freeze bovine semen, store it at super cold temperatures, then thaw and use it. 

As you know, water expands as it freezes into ice.  When this process occurs, sharp ice crystals form.  Sperm, like most cells, contains a large percentage of water.  If you attempted to preserve sperm by freezing it in your freezer, it would no longer be motile (able to move) upon thawing.  This would be caused by the ice crystals that formed during the freezing process and destroyed the sperm cells.  In order to end up with motile sperm after thawing, a controlled freezing process is required.  Sperm are very cold sensitive.  They are prone to something known as “cold shock”, which can quickly kill them.  They are happiest at 93-95 degrees F, which is slightly below body temperature.  The problem is that they don’t survive for more than a day or two at this temperature making storage and distribution a bit difficult. 

Photo Credit Trevor Beaudry

This being said, we need to freeze the sperm, but they don’t survive the process very well.  What are we to do?  The first step is to extend the sperm with something that will provide energy for the sperm as well as protect them during the cooling and freezing process.  The two most commonly used substances are: (1) the yolk from hen’s eggs and (2) pasteurized whole milk. 

After yolk or milk, and some other ingredients are added, the sperm are cooled to about 39 degrees F over a couple of hours.  This cooling process helps to greatly reduce the metabolic rate of the sperm.  After the sperm has been cooled, glycerol is added before the final freezing process.  Glycerol is a cryoprotective agent that protects the sperm during the final freezing process.  The mechanism by which it offers protection is unknown, but the protection itself is known to work quite well.  It is added to pre-cooled sperm because it has been found to damage sperm when added at higher temperatures.  After the addition of glycerol, the mixture is allowed to equilibrate for a couple hours.  During this time, it is packaged in preparation for the freezing process.  It is packaged in 0.5 ml plastic straws, which can be easily stored, used, and distributed, along with offering a high surface to volume ratio.

Examples of the straws sperm is stored in.  Photo credit Trevor Beaudry

The final step in the preservation process involves rapid freezing.  For this process, an extremely cold substance is required.  What can you think of that is exceedingly cold and can be found in many laboratories?  If anyone said liquid nitrogen, then you would be correct!  With a temperature of -320 degrees F, liquid nitrogen is perfect for the task of making something very cold very fast.  It’s so cold that if you touch it, it burns!  A common method for freezing sperm is the vapor method.  Using this method, the straws containing the sperm are placed on racks 4cm above the surface of the liquid nitrogen for 10 minutes.  After the 10 minutes are up, the straws are plunged into the liquid nitrogen completing the freezing process.  The initial vapor cooling stage is important to help reduce the damage the freezing process has on the sperm.

After the freezing process is complete, the straws of frozen sperm are stored in insulated tanks containing liquid nitrogen.  The sperm can then be safely stored for long periods of time and be distributed worldwide.  This enables a bull that lives in Wisconsin to impregnate a cow that lives in New Zealand without either animal ever leaving their home!

References

M.I. Baracaldo , A.D. Barth and W. Bertrand.  Steps for Freezing Bovine Semen: From Semen Collection to the Liquid Nitrogen Tank.  http://www.uesc.br/cursos/pos_graduacao/mestrado/animal/bibliografia2007/baracaldo_et_at_2007.pdf.  11/22/2013

John E. Parks.  Processing and Handling Bull Semen for Artificial Insemination
- Don't Add Insult to Injury!  http://www.ansci.cornell.edu/pdfs/bullsemen.pdf  11/22/2013