Extending, Storing and Shipping Stallion Semen

Considering that it takes some powers of persuasion to obtain a semen specimen from a stallion, it is judicious to handle the sample in a manner that does not cause excessive damage to the spermatozoa. Over the last century, many common semen handling techniques unknowingly abused the prized sperm.

Spooning, which is the recovery of the semen from the floor of the mare's vagina, usually occurred by using a metal spoon washed in soap and water. Fragile equine sperm cells cannot survive contact with cold metal surfaces, nor can they tolerate electrolytes in their environment. Any solution or surface that is a good conductor of electricity (i.e., a metal spoon) is not good for sperm. Soap film is lethal and millions of sperm can be killed simply by coming in contact with the surface of a spoon or container rinsed well in tap water.

Only distilled water, free of any electrolytes, should be used to rinse an object coming in contact with the spermatozoa. It is also better to use glass or plastic containers, tubes, and spoons. These minor changes will prevent the assassination of more than half of the sperm cells by equipment with lethal surfaces.

Antiquated techniques are not the only way a sperm can end up in a hostile environment-a fact I learned in a research setting. As I was busily conducting stallion semen experiments at Texas A & M in the mid 1960’s, I noticed that another researcher, working in the same lab seemed very curious about my activities. After several weeks of quiet interest, this gentleman from the Middle East approached me and indicated that he had been doing research on bull semen for some time but had never worked with stallion sperm. He further elaborated that the people in control in his country had a keen interest in good-blooded, swift horses and he thought it would be to his advantage to broaden his knowledge to include the manipulation of the male horse gamete.

I agreed that this sounded like a worthy project for him and he asked me to bring him a semen sample from a particular black racehorse stallion. I tried to brush off the request by saying that I would the next time I collected this horse, but since it was the dead of winter, it would be a while before a sample could be obtained. I hoped that by then the bull semen researcher would forget about the whole thing.

Unfortunately, he was much more persistent than I anticipated. Every time he saw me, he asked when I would bring him the sample. Finally, I realized I was going to have to make good on my offer or go back on my word. So, during a great “blue norther” with the chill factor hovering around -40, I trooped out to collect the black stallion.

After procuring the sample, I protected it against the cold like it was a newborn infant. As I waited in the lab to be treated for frostbite I handed the sample over to the anxiously waiting researcher. He walked over to his work station and gently poured the raw semen into a goblet full of bull semen extender, promptly killing fifteen billion sperm cells. (Bull semen is preserved for storage and increased in volume by an extender that contains sodium citrate, an electrolyte. While bull semen can withstand this electrolytic buffer, stallion semen cannot.)

The sperm cells of the horse are among the most fragile sperm cells of any species when it comes to survival outside the animal (in-vitro environments). This is especially true when it comes to drastic changes in temperature-an obstacle that is difficult to avoid when collecting semen in the middle of winter. In its natural environment, sperm is kept at approximately 96 degrees Fahrenheit - a temperature that is much warmer than the atmospheric temperature during much of the breeding season.
Care must be taken to have all contact surfaces warmed to near 100 degrees Fahrenheit. Keeping the bottles, pipettes, syringes, and extender media in an incubator will reduce the possibility of death due to cold shock, the term used to describe hypothermia in sperm cells. Overexposure to low temperatures causes the male gamete to develop a characteristic appearance: The tail bends backward in an arc toward the head.

This causes the live cell to swim in a circular motion (imagine a rowboat being propelled by one oar). This deviant movement prevents the cold-shocked sperm from being able to compete with the straight forward moving "streakers" in the race to fertilize the egg. Therefore, a cold-shocked sample decreases the fertilizing capability of an artificial insemination procedure and makes it more difficult to accurately evaluate the fertility of a horse.
Another environmental hazard that needs to be controlled in order to have a viable sample is ultraviolet light. Place a semen sample in a clear container in direct sunlight and within minutes the mass slaughter of sperm cells will begin. To counter this problem, semen should be collected in containers that have ultraviolet ray filters and then quickly moved into work areas devoid of ultraviolet light.
The next two lethal conditions concern only reproductive physiologists trying to protect the semen sample over a period of time. As the ejaculate sits around waiting to be processed, it becomes its own worst enemy. By-products of cell metabolism change the acidity of the environment. The waste produced by billions of cells becomes life threatening to all the cells in the beaker - a concept we are becoming increasingly aware of in our own habitat.
Also as time lapses, the bacteria naturally found in this nutritive mix of sperm and seminal fluids begin to multiply at an ever-increasing rate, further adding metabolites that reduce the pH (a measure of the acidity or alkalinity of a solution), making the media more acidic. These bacteria are also in competition for the same nutrients as the sperm cells. Together these two factors, left unchecked, will eventually lead to the destruction of the fragile sperm.
For ejaculates to be kept for more than half an hour, counteractive measures to these and other detrimental conditions should be taken. The first line of defense is to cool the temperature of the semen to slow the metabolic rate. To do this and prevent cold shock, the change in temperature should be regulated to drop at a rate of about 1 degree Fahrenheit per minute until the solution reaches refrigerator temperature which is about 39 degrees Fahrenheit).
One way to do this is to take the semen which is in a baby bottle or a baby bottle liner and place it in a container of warm water. The water needs to be the same temperature as the semen. Place this unit in the refrigerator. The water bath will slow the cooling rate of the semen to about the correct level.
Cold shock can be further reduced by increasing the volume of the sample through the use of a prepared solution (but not bull extender!). The sperm should be placed in the appropriate extender medium before cooling. The correct procedure is to warm the extender and the ejaculate to the same temperature. Mix and then cool the extended semen at the rate given above.
The extender medium can also be designed to help control the deleterious effects of the acid and bacteria. The addition of proteins to the solution will create a mild buffering of the acid. Antibiotics in the extender will help limit the replication of the bacteria.
Remember that time determines which factor is most lethal to sperm cells. Initially, acidity is the biggest problem counteracted by the proteins in the extender. After 24 hours, however, significant sperm death can be attributed to the build-up in bacteria populations. Samples that need to be kept long than 24 hours may need to be inoculated with antibiotics.
There are other reasons to have more pathogen-free samples. For example, some mares seem to have little resistance to pathogens found in semen. For these mares, it may be desirable to use artificial insemination techniques with semen incubated in an antibiotic extender to retard the growth of these negative bacteria.
Unfortunately, however, our ever-fragile equine sperm cells also find these antibiotics detrimental to their health. So the extender must balance the positive effect of the antibiotics against the bacteria versus the negative effects of the drug on the sperm cells themselves. Concentration, therefore, becomes critical and the concept "if a little is good, a lot is better" will turn life-givers into warriors of death.
Nutrition for the sperm cells and obtaining the proper osmolarity of the liquid are two other considerations in the development of a proper equine extender media. For this reason, glucose water is the standard ingredient in most equine extender recipes.

This article is an excerpt from a section in The Stallion: A Breeding Guide For Owners and Handlers by Jim McCall PhD.