Freezing Semen

The technology that allows us to freeze a stallion's semen for future use has opened up a world of opportunities. Not only does it allow the stallion's DNA to be available to mare owners around the globe, but it enables a stallion to stand at stud while he continues his performance career. It also means reduced transport costs and less stress for mares (and their foals-at-foot). And with frozen semen, there is no limit to the length of the transport time, nor any worry about the container arriving at the appropriate time in the mare's cycle. The use of extenders means that a single ejaculate can be used to inseminate a number of mares. Stallion semen can be collected, and the ejaculate frozen, at any time of year, and straws of frozen semen can be thawed and put to good use within seconds.


KAREN BRIGGS

Semen must be kept from cold shock, so the vial used for collection has a warm water bath inserted. However, because water is toxic to sperm, the water is contained in a plastic sleeve and seals are double checked.

Many stallion owners now are looking at freezing semen as genetic "life insurance" for their valuable animals. Should the horse die, or be injured so that breeding is impossible, his frozen store of semen can insure that he can produce offspring for years to come. So far, no one has discovered any limitation on the length of time that semen can remain in the deep freeze. However, the storage temperature must be exceedingly low (i.e., -196� Centigrade).

All in all, frozen semen offers an impressive list of advantages. So it's not surprising that stallion owners are becoming increasingly interested in what's involved in freezing semen.

Freezing semen isn't for every stallion owner, of course. The lab equipment required is elaborate and expensive, so it's prohibitive for many small operations. A number of large breeding farms are finding it a reasonable investment, and other options also exist for small breeders. For example, Gencor, a company headed by Pat Meyers, DVM, and Yassin Ramadan, will travel to your farm to supervise the collection and freezing of your stallion's semen, and provide tips on handling the straws and inseminating your mares. Larger farms sometimes offer collection services for other stallion owners. One case in point is Ontario's Glengate Farms, a large Standardbred operation, which has made a tidy side business of freezing the semen of visiting Percheron, warmblood, and Thoroughbred stallions. Whatever way you accomplish it, it's helpful to understand the mechanics of the process, particularly as the technology becomes more accessible, and (eventually) more affordable.

On Sept. 20, the Equine Research Centre (ERC) in Guelph, Ontario, hosted the second of two sold-out seminars for stallion owners: a hands-on, do-it-yourself look at the process of collecting, preparing, extending, freezing, and thawing semen, conducted by leaders in research and in the industry, including the ERC's Reproduction Research Associate and frozen semen expert Debra Ottier; Peter Penner, director of reproductive technology at Semex Alliance; Dr. Steven Lorton, of Minitube Canada (manufacturer of equipment and supplies for freezing semen); Pat Meyers, DVM, partner in Gencor and a reproductive specialist; and Doug Nash, stallion manager at Glengate Farms. By the end of the day, the participants had examined sperm for motility, learned how to handle liquid nitrogen safely, calculated thawing times, and been walked through the entire process from stallion to straw. Here are some of the highlights.

First Things First

Collection is the point where the whole process begins. For this, the stallion must be accustomed to the use of a phantom mare and an artificial vagina, or AV. There are three main varieties of AVs on the market: the Colorado, the Hanover, and the Missouri.

The Missouri system, favored by the team at the Equine Research Centre, is described by Reproductive Research Associate Ottier as "cumbersome," but providing good results. Inside its leather casing is a double latex liner with a valve. Warm water (45-50� Celsius) fills the space between the liners in order to provide the correct temperature and snugness to encourage ejaculation by the stallion. A disposable plastic liner, lightly greased with a non-spermicidal lubricant, is inserted into the latex liner until an equal length of the liner protrudes from each end of the AV; it's important to make sure there are no wrinkles or twists in the liner that could impede breeding or tear the plastic during the collection process.

At the cone-shaped far end of the AV, a plastic semen-collecting bottle is securely attached, with a locking ring, to the inner plastic liner. Within the bottle is a filter that strains out the gel portion of the ejaculate from the sperm-rich portion; depending on the stallion's ejaculate, single or double filters can be used. The bottle and liner, which protrude from the end of the leather casing, are covered with an insulated drawstring bag, designed to help keep the temperature of the ejaculate constant once it's collected. (Any sudden temperature change is potentially spermicidal, and that is one of the major problems lab technicians face when freezing semen.)

Making sure the semen is not contaminated also is crucial to the success of the process, and so stallions should be meticulously washed before every collection. Ivory soap and warm water generally are recommended, and because both soap and water are spermicidal, the penis must be thoroughly rinsed and dried before the stallion is allowed to approach the phantom mare. Most stallions learn to enjoy this process, and they will wait patiently while their handlers finish the ablutions.

The stallion should be collected immediately after washing, again to keep the risk of contamination to a minimum. Collection usually requires two people, one to stand at the stallion's head, and the other to guide the penis into the AV from the left-hand side of the phantom. During collection, the AV is held horizontally, but when ejaculation occurs (usually signaled by the stallion "flagging" his tail), the back of the AV should be lowered slightly to allow the semen to run into the collecting bottle.

Once the stallion's semen has been collected, 50-75% of the warm water is carefully drained from the latex sleeve of the AV, thus reducing the interior pressure so any remaining semen drains into the collecting bottle. Then it's on to the lab to prepare the sample for freezing.

In The Lab

Because any kind of temperature shock (sudden temperature change) can do irreversible damage to sperm, it's important to have all your equipment pre-warmed to about 37� Celsius, and to work quickly. First, the collecting bottle is disconnected from the plastic liner and the gel filter quickly removed from the sample. The filtration apparatus is very good at separating out the gel (sperm-poor) fraction of the ejaculate, according to Ottier, but if it is not removed quickly, some gel will seep into the sperm-rich portion you are trying to save.

Before you think about freezing semen, it's important to establish the quality of the sample. The success (pregnancy) rate of frozen semen often is significantly lower than that of cooled or fresh semen, starting with the best possible sample you can get helps ensure a good pregnancy rate. A drop or two of the ejaculate, mixed with a trisodium citrate solution or centrifuge extender (collected with a pre-warmed pipette and placed on a pre-warmed slide) and examined under the microscope at 37� Celsius will give you an idea of the percent motility of the sperm and their morphology (how many of the sperm are abnormally shaped and thus not viable--it is not unusual for about 10-25% of equine sperm in a given sample to look or behave abnormally). Look also for any foreign matter (a sign that the sample was contaminated) or white blood cells (pus cells) that could indicate the stallion is suffering from a reproductive tract infection. Sperm motility is easily assessed by using a phase-contrast microscope, under 200X magnification. Sperm morphology is generally evaluated at 1000X magnification.

A sperm count per milliliter can be determined by a number of different methods, such as with a "SpermaCue" photometer specifically calibrated for horses. A drop of the semen sample in the photometer reservoir will yield an estimated count in the millions, with 100-180 million per ml being average (with a range of 20 to an amazing 600 million per milliliter).

Once the sample has been determined to be good, with at least 60-70% estimated progressive motility (sperm that are alive and moving in a progressive direction, not spinning in tight circles), the sample is centrifuged so that the sperm concentrate at the bottom of a large test tube. (Some studies have shown that anything above 500xg
g-force for 12 minutes is detrimental to sperm.) The result will be a largely sperm-free seminal fraction at the top of the test tube, which can be gently drawn off with a pipette and discarded, and a "pellet" of sperm in the bottom. It's beneficial to the freezing process to leave between 5-20% of the seminal fluid in the bottom of the tube; the pipette can be used to break up the pellet very gently and mix it with the fluid to result in a concentrated semen sample. (If the pellet doesn't break up easily, it's likely that the speed of your centrifuge was too high.)

To this is added one or more antibiotics, a standard precaution to protect mares from possible infection, although Peter Penner, a long-time researcher in freezing techniques at Semex Alliance, noted that there is still considerable controversy over what antibiotics work best. He suggests that you consult with your veterinarian as to what antibiotics are most appropriate.

Finally, the ejaculate is diluted or mixed with a specialized semen extender in preparation for freezing. Extenders serve a dual purpose, helping both to increase the volume of the ejaculate so as to serve more mares, and provide nutrients and fertility preservation to the sperm cells. An ideal extender, says Penner, will provide repeatable results, will be transparent enough to provide some clarity for evaluation of the sperm, and will help protect the sperm from the detrimental effects of freezing. There is a variety of extender recipes that have been used for horse semen over the years, and each has its proponents; most are some combination of sugar, lactose solution or skim milk, trisodium citrate, glycerol, glycine, and fresh egg yolk. While you can become an expert at mixing up a complicated batch of extender if you are so inclined, the easiest and most error-proof method for most small breeders is to buy a commercially prepared extender, ready to go.

Calculating how much extender to use is based on the concentration of sperm multiplied by your original sample volume, multiplied by the estimated percentage of live, progressively motile sperm. All of this is divided by 200 million, the number of live sperm needed for each 0.5 ml straw of frozen semen.

The extender is carefully mixed with the concentrated semen with a pipette (taking care not to add any air bubbles or physically damage the sperm). Then it's time to fill the straws.

Semen generally is frozen in labeled plastic straws, each of which holds half a milliliter of the sample (larger straws also are commonly used). The small straws (0.5 ml) come equipped with a tiny cotton-and-collagen plug at one end; it helps form a seal so that the semen doesn't leak out. To aid straw filling, each straw can be attached, one by one, to the nozzle of a small portable vacuum pump, and inserted into the test tube of extended semen plus extender; the vacuum pump "fills" each straw in a matter of a second or two. A small air bubble is added to each straw to enable some expansion when it is frozen. Then the opposite end of the straw is sealed, either with a small metal or glass ball, or with a polyvinyl powder that is pounded gently into the ends of the straw and dipped in a 25� Celsius water bath to make a plug. Finally, any excess powder is wiped off the straws, and they are placed on a stainless steel rack, ready for freezing.

Into The Deep Freeze

Freezing semen straws isn't as simple as just popping them in your chest freezer along with the apple pie filling; they must first be gradually cooled to avoid temperature shock and prolong the life of the sperm. Specially designed, small, computerized refrigerators can be programmed to take the straws down to five degrees Celsius at a rate of half a degree per minute. At this rate, it usually takes about 1 1/2 hours to complete the cooling process.

Liquid nitrogen is used to freeze the cooled straws of semen by any of several methods ranging from simple to elaborate, depending on the quantity you wish to freeze. Whatever the method, it's important to remember that liquid nitrogen is hazardous stuff. Not only should you take great care not to get it on your skin, but since it will displace oxygen in the atmosphere (and in your lungs), it should only be used in a well-ventilated area.

When semen freezes, ice crystals will form in the liquid surrounding the sperm. The sperm cells become confined into spaces or channels between the growing blocks of ice, but the fact that the solution is frozen does not mean that it is completely stable and unchanging. Ice patterns within the frozen specimen can change considerably (this is called "migratory recrystallization"). Some sperm cells will, inevitably, not survive the process; the most common problem is a rupture of the cell membrane. The glycerol used in many extenders is fairly effective in protecting the sperm cells during the freezing process; sugar has also been used, but does not seem to give results that are quite as good.

Ottier uses the Standard Vapor Technique to freeze 0.5 ml straws in the lab at the Equine Research Centre. This method involves placing liquid nitrogen in a Styrofoam cooler box to a level of a few centimeters, then carefully placing the stainless steel rack of cooled straws not into the liquid, but into the vapor above it, about five centimeters from the liquid surface, where the temperature is approximately -120� C. The straws are allowed to cool in the vapor for 10 minutes, with the box lid closed; then the box is opened, a pair of forceps is dipped into the liquid nitrogen to chill them, and the straws are, one by one, grasped at the cotton-plug end and plunged into the liquid. Once the straw hits the liquid nitrogen, its temperature is instantly lowered to -198� C. Once this temperature has been achieved, the samples can be stored for an indefinite period of time. (Ottier recommends a minimum of seven days of storage prior to using sraws for evaluation or insemination of mares.)

This same process can be carried out on a larger scale with an automatic unit, of course, and farms which are routinely freezing semen from several stallions might find such a machine a reasonable investment.

Frozen straws can be stored in a cryogenic storage container designed to keep the temperature constant. These come in various shapes and sizes depending on the volume of straws you wish to store, and they have interior canisters that can hold up to 220 of the 0.5 ml straws. Time is of the essence when you are transferring straws from the freezing container to the storage facility; it's best to move the straws in goblets chilled to the same temperature (-196�C) and to keep the straws exposed to room temperature for three seconds or less. The same rule holds true if you are examining or removing straws from storage--keep the interior canister lifted no higher than the "frost line" in the neck of the container, and if you don't find what you are looking for in three to five seconds, lower the canister back into the bottom of the tank for at least 30 seconds before lifting it again. While it's a good idea to keep opening the containers to a minimum, you also must keep an eye on the level of liquid nitrogen in the tank; if it gets below 12 cm, it's time to refill.

...And Out Again

Thawing frozen semen, fortunately, is a simpler procedure than freezing it. Thawing can be done by most horse owners and veterinarians, provided they make the correct preparations beforehand. Naturally, you don't want to thaw the straws until you have a mare who is ready to breed; palpation and ultrasound techniques will confirm her reproductive status and will help prevent wasting precious straws of semen.

Early in the history of freezing stallion semen, it was thought that a slow thaw with ice water would achieve the best results, but it turns out the opposite is true. Thawing frozen semen rapidly allows for better acrosomal retention (fewer ruptured cell membranes) and a higher overall count of viable sperm. In a lab situation, Ottier often thaws semen by plunging the straws into a 55�C water bath for five seconds, then transferring them to 35�C water until the temperature of the straws stabilizes at somewhere under 37�C (anything over that will tend to overheat and destroy the sperm). In an on-farm situation, however, she says that the 35� water bath alone might be easier to work with.

"It's a little slower, but there is a lot more margin for error," she notes, "and at 35�C you know the semen isn't going to get too warm. You can leave the straws in the water bath for 10-15 minutes, if necessary, without running the risk of damage."

Ordinary thermometers, she adds, are notoriously inaccurate and should not be relied on for this process. Instead, she recommends the use of a battery-powered electric thermometer with a clip-on lead.

Once the straws are thawed, it's best to breed your mare as soon as possible. Remove the straws from the water bath and give them a quick shake with a flick of your wrist to move the air bubble to the end of the straw. Then snip off the ends with a sharp pair of scissors and allow your veterinarian to inseminate your mare artificially. Keep in mind that it usually takes more than one straw to do the job--depending on the concentration and estimated motility of the sperm in your original sample. You might use two, five, or even ten straws to achieve the concentration you want in the mare's reproductive tract. Your veterinarian will have some specific preferences as to the numbers of sperm introduced to achieve a successful pregnancy.

Success Rates And Variables

While the technology of freezing semen is becoming more accessible, there is still much we don't know about the way in which equine semen reacts to the freezing process. It's widely known in the industry, for example, that some stallions' semen freezes better than the semen of other stallions, and that the semen of some stallions just cannot be frozen successfully, even if the stallions have been very successful in settling mares through live cover or other avenues. The reasons behind this are as yet unclear, and only experimentation will tell you whether you are lucky enough to have a stallion whose semen freezes well.

Fortunately, we do understand many of the factors that can determine the success of an individual sample. In addition to errors in processing the semen, including temperature shock (which is probably the most common problem), poor results can result from defects in the semen itself. Semen production can be influenced by nutrition, environment, genetics, stress levels, disease, and even the ambient temperature and time of year. There are ways to compensate for a sample with problems; for example, if there is poor motility, a larger insemination dose sometimes will "make up" for those non-viable sperm. Likewise, if there are abnormally shaped sperm, using more straws to inseminate the mare might help balance the equation. However, you can't compensate for other, more serious problems caused by genetics, serious disease, injury, or a host of unknown factors. These include more serious shape abnormalities, such as "craters" or vacuoles in the sperm heads, indicating damaged or lost DNA.

It is a good idea to collect a stallion's sperm for several consecutive days to remove the epididymal sperm before collection for freezing purposes.

It's important to remember, too, that freezing equine semen is a fairly recent innovation, still in its infancy compared to the work that has been done in other livestock species, especially cattle. This, coupled with the naturally low conception rate of horses, means that freezing semen will probably never be foolproof. There will always be room for further research, improvement, and innovation--which is as it should be.

About the Author

Karen Briggs

Karen Briggs is the author of six books, including the recently updated Understanding Equine Nutrition as well as Understanding The Pony, both published by Eclipse Press. She's written a few thousand articles on subjects ranging from guttural pouch infections to how to compost your manure. She is also a Canadian certified riding coach, an equine nutritionist, and works in media relations for the harness racing industry. She lives with her band of off-the-track Thoroughbreds on a farm near Guelph, Ontario, and dabbles in eventing.

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