It has been more than 40 years since Canadian investigators reported the birth of the first foal from the insemination of a mare using frozen stallion semen. Since then, the technology of semen freezing has evolved tremendously. On a yearly basis, thousands of mares are inseminated with frozen semen around the world. Although the technology for freezing stallion semen has improved dramatically, pregnancy rates of mares bred with frozen semen average around 50%. These results lead us to believe that research for optimizing the efficiency and fertility of frozen semen has come to a halt in the last 30 years. However, one has to look at averages with a certain degree of caution since these numbers sometimes can be misleading.

It is well accepted that although an average pregnancy rate per cycle with frozen semen is around 30-40% when properly used, there is a great deal of variation, and it is not uncommon to have per cycle pregnancy rates ranging between 0-70%. In addition to this variation, there is a lack of standardization in the information that is available regarding insemination freezing and thawing protocols, as well as semen handling procedures.

Factors Affecting Fertility Of Frozen Semen

In order to maximize the efficiency of the use of equine frozen semen and to establish a successful insemination program with frozen semen, one has to try and control the factors that determine the success. The three major factors that should be considered are 1) the stallion and the quality of his semen after thawing; 2) age and status, as well as reproductive history, of the mares; and 3) the reproductive management of the mares and the handling of the frozen semen.

There are thousands of foals born every year using frozen-thawed semen. Although the technology is still far from optimal, it is gaining wider acceptance in the industry. An increasing number of stallion and mare owners are taking advantage of the benefits of using frozen semen. Perhaps one of the most important reasons why frozen equine semen is still far from optimal is the great variability in each stallion's semen to tolerate the freezing and thawing process. It is thought that only 25% of the stallions will have pregnancy rates comparable to fresh semen or natural cover when their frozen semen is inseminated into healthy mares at the proper time. Although the other 75% of the stallions will produce suboptimal pregnancy rates on a per cycle basis, pregnancy rates at the end of the season with some but not all of these stallions can be normal (more than 80%). But the number of breeding cycles needed to achieve this rate is often too high and too expensive for the mare owner.

It is still uncertain why there is such a variation in stallion's semen to tolerate the freezing and thawing process. It is thought that among others, constituents of the seminal plasma, or molecules on the sperm itself, are responsible for this variation. Stallion owners also must realize that, unfortunately, the selection criteria for stallions rarely, if ever, involve semen quality or fertility. Therefore, breeding to stallions with poor semen quality potentially could affect the semen quality of his offspring. It is well accepted that there are sire lines known for poor semen quality as well as others known for good quality. There also are sire lines known for good frozen semen quality.

Previous work done by the author has shown that this inherent stallion variability is one of the most important factors in determining the pregnancy rate with frozen-thawed semen. Because sperm from individual males respond differently to the freezing and thawing process, several investigators and laboratories have tried numerous systems to try and improve the survival of equine sperm after thawing.

Recent research showed that one of the most important aspects affecting the quality of frozen stallion semen is the quality of the raw ejaculate. The technique for semen processing (i.e., the extender type) is perhaps not as important. However, if the quality of the raw ejaculate is an important factor in determining the post-thaw quality, it would seem quite obvious that no stallion should be collected for freezing while he is sexually rested. Collection of at least two or three ejaculates prior to the first freezing should be done to maximize the quality of the raw semen. This procedure should be done even when the semen looks good under the microscope.

The protocol for processing semen for cryopreservation (freezing) involves 1) the collection and evaluation of gel-free semen; 2) the dilution of the ejaculate and centrifugation; 3) the removal of the supernatant or excess fluid; 4) the addition of a freezing extender that contains the products necessary to protect the sperm during freezing; and 5) the final packaging, freezing, and evaluation of the semen. For every step in the process, there are a number of alternatives that can be used.

Recent investigations have revealed that freezing semen also affects several other parameters involved in the fertilization process. We know that spermatozoa that have been frozen have an impaired ability to reach the oviduct (site of fertilization). Fewer sperm reach the site of fertilization or perhaps take a longer time to reach it. In addition, it is now clear that sperm that have been frozen and thawed have a complex change in their carbohydrate composition around the membranes; therefore, those sperm cells also have a decreased capacity to attach to the fallopian tube, where they acquire their capacity to penetrate the egg.

Furthermore, sperm cells that are frozen and thawed have changes in their level of calcium, which is an important factor during the time that sperm cells need to prepare for fertilization. All of these changes that happen to sperm during the freezing and thawing process affect the ability of the mare to form a sperm reservoir that would otherwise normally happen when mares are bred by natural cover or with fresh semen. These also are some of the reasons why it is very important that frozen-thawed semen be inseminated very close to the time of ovulation (less than 12 hours) to maximize fertility. However, this involves more labor for both veterinarians and mare owners.

Although it has been suggested that differences in the composition of the extender will improve the post-thaw motility of certain stallions, recent experimental data have not supported this concept. However, it is well accepted that the raw semen from stallions which are considered good freezers have lower volumes, higher sperm concentrations, and higher sperm motility compared to the semen from the those classified as poor freezers.

Utilization Of Frozen Semen

The utilization of frozen semen includes, among other factors, the thawing, the evaluation, and the timing of the insemination. The thawing of semen is dependent on the type of package in which the semen was frozen. Semen that is packaged in 0.5 ml straws is generally thawed at 37° Centigrade for a minimum of 30 seconds. Some laboratories recommend thawing of 0.5 ml straws at 75° Centigrade for seven seconds. Semen that is frozen in 2.5, 4, or 5 ml straws is generally thawed at 50° Centigrade for 45 seconds.

Post-thawing evaluation generally is done by evaluating the post-thaw motility of the sperm. It also is becoming common to evaluate the longevity of motility. It is thought that frozen-thawed semen having better longevity tends to mean better pregnancy rates. Unfortunately, motility of frozen-thawed semen, besides being a fairly subjective measure of quality, is a very poor predictor of fertility.

Although there are no standards, criteria required for semen to be used commercially seem to require a minimum of 25-30% motility and more than 600 million total sperm per insemination dose. There is no information regarding the minimum number of progressively motile sperm per dose that are needed to maximize fertility.

There is a great deal of inconsistency between laboratories in recommendations for timing of insemination. In a survey conducted by the author, 14 of 21 laboratories processing semen for commercial purposes recommended that mares should be bred with one dose per cycle. Of these 14 laboratories, 12 recommended breeding before ovulation, preferably within 12 hours. Only two laboratories recommended breeding only once within six to 12 hours post ovulation. Seven of 21 laboratories recommended breeding the mares pre- and post-ovulation. There were only two laboratories that recommended inseminating the mare every 24 hours after the mare had been in heat for at least three days.

In the author's experience, fertility of mares which are bred only once after ovulation is reduced to about 30% per cycle compared to 60% for those bred only once before or before and after of ovulation. Availability of semen is an important factor in determining the timing and number of doses inseminated. Mare owners are encouraged to purchase frozen semen for two breedings per cycle.

Mare Fertility And Management

Per cycle pregnancy rates for mares inseminated with frozen semen range between 30-60%. It is not uncommon to have pregnancy results ranging between less than 10% per cycle and perhaps the same figure for the entire breeding season, up to 70% per cycle. Some of the factors that affect the pregnancy rates of frozen semen have been analyzed by the author over the past five years. These data, generated from the analysis of more than 200 mares bred with nearly 400 doses of semen, indicated that on the average, 1.5 doses of semen were used per cycle in all mares, and it took about 1.7 cycles per pregnancy. Mares were examined daily during the first days of their heat period, and at least twice daily when close to ovulation. Every attempt was made to breed the mares within 12 hours prior to ovulation. Mares which did not ovulate after the first insemination were rebred with a second dose as soon as ovulation was detected, if semen were available.

Besides the wide range of fertility rates inherent to the stallion, mare status also is a major factor determining the pregnancy rates with frozen semen. Mares were allocated into one of four groups: a) young maiden mares (less than seven years old); b) older maiden mares (more than eight years old); c) barren mares; and d) mares with foal at foot. The average per cycle pregnancy rate was 67.3%, 34%, 50.7%, and 50.9% for each of these groups, respectively. Mares which had been treated for uterine infections during the cycle prior to the insemination had a reduced first cycle pregnancy rate of 43% compared to an average of 50.5%.

It is suggested that mares which are going to be bred with frozen semen should be carefully and routinely examined at short intervals and bred only once close to and prior to ovulation. To determine the ideal breeding time, routine ultrasound examinations should be performed to check uterine and follicular echotexture. Furthermore, to time the insemination more accurately and to minimize the number of breeding doses, the use of ovulatory inducing agents such as Ovuplant or HCG is highly recommend.

In addition to depositing the semen at the appropriate time, an examination post-ovulation is a crucial component of the insemination process. This examination helps the veterinarian confirm that the mare has ovulated, as well as helps him or her detect possible fluid accumulation in the uterus. This is particularly important in the old maiden group and in those mares susceptible to uterine infections.

The perception that some mares accumulate excessive amounts of fluid due to an allergic reaction has been recently addressed by Dutch, Finish, and American investigators. Their data indicated that fluid accumulation in the uterus post-insemination is a normal process, is not an allergic reaction, and is not dependent on the type of extender or any of its components (such as egg yolk). Small amounts of uterine fluid can be detected on most mares shortly after breeding. This small amount of fluid is drained through the cervix a few hours later in the normal mare. Excess fluid accumulation is perhaps the result of poor cervical relaxation in the old maiden mare, or poor uterine contractility in mares susceptible to uterine infections. Mares which are in these categories should be checked routinely and a uterine lavage performed to drain this excess fluid, particularly when bred with frozen semen.


Individuals involved in artificial insemination with frozen semen often are presented with semen accompanied without instructions, or with an array of different instructions with respect to type of package, thawing, and timing of insemination. The lack of consensus among laboratories processing semen invariably places the industry in a precarious position and makes the technique lose credibility among some of the users.

Individuals purchasing frozen semen should inquire about the previous fertility of the stallion with frozen semen. The fact that some stallions have very good conception rates with natural breeding and/or shipped semen does not guarantee their fertility with frozen semen. Mare owners also are encouraged to ask their veterinarian to evaluate the semen immediately after thawing and prior to insemination, and to determine parameters such as motility, sperm concentration, timing of insemination, number of doses used, type of package, etc.

Gathering this type of information from large numbers of inseminations will be the only way to standardize some of the basic parameters to increase the pregnancy rates. Higher pregnancy rates per cycle will be the best way to promote the use of frozen semen in the equine industry.

Note: The Society for Theriogenology and the Reproduction committee of the American Association of Equine Practitioners have designed a form to retrieve and analyze some of the basic parameters regarding the use of frozen stallion semen. Individuals interested in participating in this worldwide project are encouraged to contact the author for further information at or 604/530-6386.

About the Author

Juan Samper, DVM, MSc, PhD, Dipl. ACT

Juan C. Samper, DVM, MSc, PhD, Dipl. ACT, practices in British Columbia specializing in reproduction.

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