by Sybil Leopold
Equine Research Centre
Guelph, Ontario, Canada
In the horse industry, the use of frozen semen still has a futuristic ring to it and many horse owners and breeders approach the subject with scepticism. Nevertheless, this procedure has a history that goes back to 1949, when a scientist in Cambridge, England discovered that bull sperm cells can survive freezing (cryopreservation). Only a year later researchers successfully froze stallion spermatozoa, and in 1957 the first pregnancy from frozen stallion sperm was reported in Guelph, Ontario.
For economic reasons most of the early research was focused on bull semen, but in the last 15 years the successful and growing use of frozen semen in bulls encouraged many to carry out further research into cryopreservation of equine semen. Initially the basic procedures for cryopreservation of bull spermatozoa were applied to stallion spermatozoa, but soon researchers realized that stallion spermatozoa are much more fragile and therefore more difficult to freeze and thaw than semen from other species. In the last 20 years scientists from several countries have been working at developing and improving the techniques used to successfully freeze stallion spermatozoa.
The use of artificial insemination with both fresh and frozen equine semen is increasing globally. In China, for example, from 1980 through 1985 more than 110,000 mares were inseminated with frozen semen. More recent data are available from Europe, where today frozen semen is widely used in the horse industry. Currently, frozen semen from over 200 German and Dutch warmblood stallions is available internationally and in 1993 over 2,400 Selle Francais mares were bred with frozen semen from 196 stallions, resulting in a pregnancy rate of 59%. In North America there are over a dozen facilities that offer freezing services and the number is increasing rapidly. Additionally there are numerous places that import and export frozen stallion semen.
Several advantages and disadvantages of artificial insemination with frozen equine semen have been noted, some of which are the following.
- Decreased stress and risk to mare and foal
- Decreased transport costs
Decreased stress and risk to mare and foal and the decreased transport costs are advantages that are applicable to the use of transported fresh semen. With frozen semen there is no limitation to the length of the transport. It can be shipped at any time, independent from the mare’s ovulation.
- Longer breeding season for the stallion
The stallion’s breeding season is no longer bound to that of the mare. The stallion can be collected and his ejaculates frozen all through the year.
- Breeding season during performance events
In the warmblood industry there are many stallions used as performance horses while they are breeding stallions.
- “Life insurance” for the stallion
A semen bank of a stallion can in some ways count as a genetic “life insurance”. If the horse is injured or dies, there is still the possibility of producing offspring for years to come.
- Opens large new international markets
With frozen semen, markets can be reached that were not accessible with natural breeding or transported fresh semen. So far there have been no limitations on the storage time of frozen semen.
- Individual freezing variability
The biggest disadvantage of frozen semen today is the individual freezing variability; stallions vary in fertility with frozen semen. About 20-30% of stallions with normal semen quality will produce semen that freezes extremely well, another 40% produce semen that freezes modestly well and approximately 30% of those stallions don’t freeze well at all.
- Techniques not standardized
As a consequence of this variability, there is currently no standardized method for freezing stallion semen. There are four proven extenders commonly used in the industry, but no one is necessarily better than another for all horses. Rather, individual stallions seem to prefer a specific formula.
- More intense mare management
After thawing, frozen semen has a shorter life span in the mare than fresh semen. Accurate pinpointing of the time of ovulation is very important for successful breeding with frozen semen. This results in more intensive mare management, such as ultrasound up to every eight hours to determine ovulation, or the use of hormonal induction of ovulation.
- Decreased conception rate
Conception rates of artificial insemination with frozen semen are lower than with fresh semen, although with a good freezing technique and well managed mares, conception rates of 60-65% can be reached.
Principles of freezing stallion semen
The process of freezing semen is called cryopreservation. Cryopreservation can be compared to hibernation. The metabolism of the sperm cells is lowered to the minimum they need to survive without losing their ability to fertilize an egg. During freezing and thawing the sperm cells must withstand a variety of stresses like exposure to cooling, ice crystal damage and dehydration associated with ice formation during freezing. To enable the spermatozoa to survive freezing and thawing, many factors have to be considered.
Spermatozoa have to be separated from the seminal plasma which mainly consists of the secretion products of the accessory sex glands. Sperm cells can be separated by centrifugation with an extender, for example the skim milk-glucose extender which is also used for transporting fresh semen. A different, less common way to separate the spermatozoa from the rest of the ejaculate is by using an open artificial vagina and collecting only the sperm rich fraction of the ejaculate which is ejected first.
For the actual freezing process spermatozoa have to be placed in an appropriate freezing extender, which commonly contains:
- energy source (sugar)
- protein source (egg yolk, milk)
- cryoprotectant (glycerol)
- electrolytes (buffers)
There are different packaging systems used for freezing stallion semen. Initially glass containers, plastic bags and aluminum containers, similar to toothpaste tubes, were used. For some years spermatozoa were frozen in little pellets which were prepared by placing small drops of extended semen into slight depressions in a block of solid carbon dioxide. These pellets were very hard to identify and transfer of spermatozoa from one depression to another was possible. Today the most commonly used packaging systems are either 5 ml or 0.5 ml plastic straws. These straws are either sealed with a little ball or a sealing powder that solidifies when it comes in contact with fluid.
Other important factors are the freezing and thawing rates which are different for each packaging system. For the plastic straws, a procedure called standard vapor technique is commonly used. For the initial freezing the straws are placed in liquid nitrogen vapor in a styrofoam box which cools them to -160°C at a fixed rate. After a defined time in the vapor the straws are plunged into the liquid nitrogen which instantly lowers the temperature to -196°C. Again some stallions freeze well with this method while others need different cooling rates which require the use of a computerized cooling unit. Thawing the sperm usually occurs by plunging the frozen straws into water of a defined temperature for a specific period of time. The temperature and time are different for each packaging system, and one of the keys to success is to follow the semen handling instructions carefully.
Example of a freezing process “step by step”
1. Collect gel-free ejaculate
The stallion is collected with an artificial vagina into a prewarmed receptacle to prevent cold shock of the spermatozoa. Commonly, the receptacle is equipped with a filter to remove the gel fraction of the ejaculate and debris coming from the penis. (The gel fraction originates from the seminal vesicle, one of the accessory sex glands.) Immediately after collection the ejaculate should be placed in an incubator at 37°C.
2. Evaluate motility and morphology
The quality of the ejaculate is evaluated under a microscope by checking the motility, which is a rough estimate of the percentage of viable sperm in the ejaculate, and morphology, which is simply whether the sperm cells have a normal structure.
3. Dilute with an extender and centrifuge
For the separation of the spermatozoa from the seminal plasma the semen is centrifuged. Prior to centrifugation the sample is diluted with an extender which makes the spermatozoa more resistant to stress factors like cold and the centrifugation forces.
Additional to separating the spermatozoa from the rest of the ejaculate, centrifugation also concentrates the spermatozoa in a smaller volume.
4. Estimate concentration
After centrifugation the concentrated sperm collects in a little pellet on the bottom of the test tube. The number of sperm is determined to calculate the number of straws that can be filled with a defined number of spermatozoa.
5. Dilute to desired concentration and fill straws
The concentrated sample is diluted with the appropriate freezing extender to the desired volume (according to the number of straws calculated) and filled into the straws. This can either be done manually with a syringe, or automatically with a mechanical unit that fills and seals the straws.
6. Initial freezing in liquid nitrogen vapor
The filled and sealed straws are placed on a freezing rack and immersed into liquid nitrogen vapor for the initial freezing. The time period the straws remain in the vapor is specific for each packaging system.
7. Rapid freezing to -196°C in liquid nitrogen
For the rapid freezing, the straws are then plunged into the liquid nitrogen which instantly drops the temperature to the storage temperature of -196°C. After the temperature of -196°C is reached the samples can be stored. So far there has been no limitation to the storage time of frozen semen in liquid nitrogen.