Semen Handling 102

This article was first published in the Lancaster Farmer

Cryogenically frozen reproductive material can last for one thousand years when stored correctly! However, improper handling techniques can cause frozen semen and embryos to decrease in quality. Work done by Professor F. Neal Schrick from the University of Tennessee suggests that semen that has been handled by breeders and stored on the farm is four times more likely to be graded unacceptable at thawing than semen that has been handled only by the facility that froze the semen. This is because of the mishandling that happens to the semen on the farm. This article was written to help educate producers on handling frozen reproductive material so they do not fall into this cycle. If you haven't read Semen Tank 101 , please refer to it for a more introductory article!

The Freezing Process

The freezing process is done by exposing semen and embryos to cryoprotectants, and then they are slowly cooled to a low temperature. When freezing these reproductive materials, removing the intracellular water and replacing it with a cryoprotectant is imperative. When the intracellular water is exposed to cold temperatures, it creates large, sharp crystals. These sharp ice crystals can damage the cellular organelles, cell membranes, or even chromosomes! So, instead, the embryos and semen to be stored are put into a solution with a cryoprotectant, like glycerol, prior to freezing. When the cryoprotectant is frozen correctly, it creates a glass-like ice that causes little to no damage to the reproductive material cells. So, using osmosis, the intracellular water is swapped with the cryoprotectant, allowing reproduction professionals to freeze the reproductive material safely.

Once the exchange of the intracellular water and cryoprotectant occurs, the materials are moved on to the freezing process. The freezing process is slow-cooled, meaning the reproductive materials are cooled slowly to freezing temperatures. For example, embryos are initially at room temperature and then are slowly dropped to -35 degrees Celsius before being plunged into liquid nitrogen at -196 degrees Celsius. Once the reproductive materials are frozen under -130 degrees Celsius, re-freezing should not occur as it causes recrystallization. Recrystallization, in a simple sense, is when the small ice crystals created by the initial freezing are thawed and then rearranged, causing larger ice crystals. The ice structure mentioned is volatile when it is at a temperature above -80°C, even at an exposure time of a couple of seconds. This process of forming larger ice crystals damages the quality of stored semen and embryos.

According to Brad Stroud, DVM, the severity of this damage depends on how long and how high. That means how long the frozen reproductive material was exposed to a temperature higher than -130 °C and how high that temperature was. Due to this, how frozen reproductive material is handled directly corresponds to the quality of the semen and embryos after thawing.

Handling Material Inside the Tank

Even handling the reproductive material when moving it around the tank itself or looking inside the tank can damage the quality. This could be when people are organizing, taking inventory of tanks for shipment, or just for routine management. One of the biggest mistakes with handling semen is taking the canister up too far into the neck of the tank. Within the neck of the tank, there is the most significant variation in temperature. It can range from almost room temperature to -130°C. As stated in the above paragraph, that sort of temperature variation can greatly affect the quality of your reproductive materials. The frost line on a tank serves as the "baseline" for when the temperature gets too high. Most of the time, the temperature above the frost line barely reaches freezing temperatures. When holding a canister up to the frost line, ensure the tops of the canes are at least half an inch below the line. This ensures that most of the semen straws can be kept below -80°C.

In addition to keeping the materials below the frost line, the “eight-second rule” established by Dr. Brad Stroud must be used. The “eight-second rule” refers to the time that a canister can be safely pulled up into the neck of the tank below the frost line. When pulling up a canister to inspect a cane tab, ensure that you hold it up for less than eight seconds, then let it drop back under the liquid nitrogen.

One of the easiest ways to preserve the quality of frozen reproductive materials and limit the amount of handling is to keep proper records. Understanding which code corresponds to a specific sire or dam can be helpful in the long run. When buying semen or embryos from a reputable source, they should be able to offer you a sire or dam code that corresponds to the label on the cane tab. Keeping records handy when performing artificial inseminations (AI) can not only limit the amount of handling of frozen reproductive materials, but it can also make the whole process more streamlined and easier for the AI technician. In addition to code records, keeping an up-to-date record of cane locations within the tank is very helpful. Knowing where specific semen or embryos are located in the tank can limit the amount of exposure to these materials. As a good rule of thumb, knowing what canister everything is in, how many straws are in each cane, and what each code or color means makes a huge difference in terms of handling.

Handling Material Outside the Tank

There are always going to be instances where the frozen reproductive material must be moved out of the tank. With proper handling, the quality of those materials may be maintained. However, with improper handling of materials, especially outside of a tank, the probability of damaging that material increases exponentially. 

When moving canes between tanks, the semen or embryos should be moved as quickly as possible. To help with this, place the two tanks side-by-side when moving semen or embryos between tanks. The time that the cane is exposed to the air should be as short as possible, for example, 1 to 2 seconds. This minimizes the damage to frozen materials and the effect on the quality

When directly handling materials, no skin should contact the straw or goblet. When skin encounters frozen reproductive materials, it causes the temperature to rise and the quality to be severely impacted. So, when moving canes, ensure only the cane tabs are touched, or use tweezers to move materials. Movements are best left to reproduction professionals, especially when "breaking a straw". Taking a straw out of a goblet and moving it to another cane with bare hands is a very efficient way to decrease the quality of your semen or embryos.

Conclusion

When faced with poor conception rates, people usually turn to causes such as improper synchronization and nutritional factors. That could be the case; however, poor-quality semen or embryos could also be a contributing factor. Just remember, when handling frozen reproductive materials, every degree and second counts!

Resources

Schrick F. N., Saxton A. M., Stroud B. K. (2006) 369 Assessment of Semen Quality for Predicting Recovery of Viable Embryos in Superovulated Cattle.  Reproduction, Fertility and Development 18, 292-292

Stroud, B. (2012, December 3). Consequences of Mishandling Frozen Semen and Embryos. Applied Reproductive Strategies in Beef Cattle, 191-203.