CRYOPRESERVATION OF EQUINE SPERMATOZOA

HISTORY:

Successful cryopreservation of semen (frozen semen) was first performed in the early 20th century with bull semen and is extensively used in the cattle industry today. The first pregnancy resulting from frozen stallion semen actually occurred in 1957 in the US, when spermatozoa were taken from a stallion's epididymis, frozen, thawed and inseminated into a mare. Interest has rapidly grown to transform equine semen cryopreservation throughout the world into a successful industry.

Equine semen is less tolerant of the freezing and thawing process than bull semen. Furthermore, not all stallion semen freezes alike; no single technique or magic formula exists to freeze all equine semen. Semen from some stallions fares better than others with certain freezing media. Some stallion's semen does not freeze well and may require extensive testing and laboratory procedures in attempt to preserve the spermatozoa. Furthermore, at different times of the year, and even at different collections, tolerance of the freezing and thawing process may show some variation in an individual stallion.

WHY FROZEN SEMEN?

ADVANTAGES:

The greatest advantage to the use of frozen semen probably belongs to the stallion owner. However, both mare and stallion owners have reasons to benefit from its availability. Several reasons include:

• Frozen semen is insurance against injury or death of the stallion.
• Allows international shipment of semen, thereby enhancing the equine gene pool in all countries.
• Horses from different hemispheres can be bred even during the stallions "off" season. This may represent an additional source of income to the stallion owner.
• The stallion's show schedule need not be interrupted for semen collection
• Overuse of the stallion is minimized since most freezing is performed after the breeding season. Furthermore, the use of frozen semen prevents low fertility due to heat stress in the summer months since semen can be preserved at a different time of year.
• Frozen semen may allow a stallion with behavioral problems to be gelded.
• More precise timing of insemination becomes possible because semen may be shipped many days prior to ovulation and thus, be available for insemination at the optimal time. This eliminates emergency collections and ensuing anxiety that semen may not arrive in time to cover ovulation.
• Delays in Customs for international shipment or due to airline difficulties do not affect the viability of the frozen semen.
• There is often a reduced cost for shipment of semen necessary for a single heat cycle. With fresh cooled semen, collection and shipment fees can run as high as $250 or greater/shipment and often more than one shipment may be requested for a single heat.

Finally, frozen semen offers the same advantages as fresh cooled transported semen in that it:

• Allows the ability to breed to any stallion regardless of distance.
• Eliminates the transportation and board fees to the mare owner at the the stud farm.
• Reduces the possibility of injury to both mare and stallion.
• Reduces management of the "difficult breeder", i.e. a mare that does not show heat or stand for a stallion.
• Reduces the chance of intrauterine infection since gross contamination is removed at the time of collection and antibiotics are added to the semen freezing media.
• Reduces the chance of infection to the stallion.

DISADVANTAGES:

There are some disadvantages to frozen semen use as well.

• There is remarkable variability in the ability of stallion spermatozoa to withstand the freezing and thawing process.
• There is considerable initial expense involved for the stallion owner. However, frozen semen is actually quite cost effective when compared over a breeding season to other methods of breeding.
• In some stallions, conception rates are lower with frozen semen compared to fresh cooled semen. With education, familiarity, and subsequent improvement of post-thaw insemination technique, this appears to be improving.
• Veterinary involvement may be more labor intensive, and thus, more costly for the mare owner.
• The number of capable inseminators continues to grow; however, it can be a source of frustration to both mare and stallion owners in finding an individual who is both willing and trained to use frozen semen.

PROCEDURE:

Semen collected into an artificial vagina is rapidly processed for cryopreservation in liquid nitrogen vapor at -196oC. It may be packaged in several different forms--straws (1/4 ml to 5ml), packets, ampules, etc. The 5 ml straws are generally considered more "user-friendly"; therefore, unless otherwise requested, semen is packaged and stored in these macrotubes. A test straw from each frozen batch is thawed, cultured for potential pathogenic bacteria, and examined for motility and velocity.

Straws that are processed at Honahlee, PC, available for commercial use, are estimated to contain a minimum of 200 million progressively motile spermatozoa and >25% motility unless otherwise stated. Straws that do not meet these requirements are available to the stallion owner for use in their own mares.

PREPARATION OF THE STALLION:

Dead spermatozoa accumulate in the reproductive tract of the stallion when he is not bred or collected every few days. Therefore, these dead sperm must be "flushed out" during the week before freezing to ensure optimal quality of the semen to be frozen. Also at this time, several cultures of the reproductive tract are taken, and examined for potential pathogenic growth in order to determine appropriate antibiotics for the freezing media. Basic semen evaluations are performed during this preparatory week in order to evaluate sperm number and motility. Lastly, this week allows stallions to become familiar with semen collection methods, thereby reducing the semen variability when collecting for a freeze.

Stallions must be tested for Equine Viral Arteritis (EVA) within a month of freezing or owners must furnish proof of vaccination following a negative titer.

Vaccinations for other diseases (Influenza, Rhinopneumonitis, Tetanus) are required before a stallion stays at Honahlee unless semen shipment to foreign countries precludes the use of these vaccines.

INSEMINATION:

Frozen semen, after rapid thawing, should be inseminated into a mare as close to ovulation as possible. Current research suggests that a mare should be inseminated within 12 hours (ideally within 6 hours) of ovulation for best pregnancy rates. It is often recommended that insemination takes place on either side of ovulation. Therefore, precise timing of ovulation is critical to success and usually requires intensive efforts on the veterinarian's part.

PREGNANCY:

With some stallions, conception rates are lower with frozen semen compared to fresh cooled semen. It is generally agreed that frozen semen does not maintain viability after thawing as long as cooled semen but there is a lack of objective data at this time to support this theory. Furthermore, a great deal of variation probably exists between stallions.

In recent years, however, several studies have been published that found no significant difference in foaling rates between mares that had been inseminated with extended semen immediately following collection, extended semen cooled for 24 hours, and frozen semen. With continuing improvement in the freezing techniques and media, as well as the familiarity of the inseminator with procedure, overall conception rates should continue to improve.

The optimal number of progressively motile spermatozoa necessary for pregnancy resulting from insemination with frozen semen has yet to be determined. One straw is recommended per insemination. However, the number of straws recommended/ heat cycle depends on the timing of ovulation. In very general terms, 3 straws will cover 1 heat. Again in very general terms, the average number of straws/pregnancy equals 6. Therefore, if 50 straws are available, a stallion owner might plan on covering 8-10 mares.

The number of acceptable straws produced by a stallion is determined by individual stallion variation. Most 1000 lb -1500 lb stallions produce between 10-20 straws per collection when collected on an every other day basis.

There is no concrete evidence as to the length of time that frozen semen can be stored in liquid nitrogen without losing viability upon thawing. It is generally accepted that, over time, the quality of semen will decrease by steady increments. However, foals conceived with semen stored for 20 years, have been born in the past few years.

Again, there is tremendous variability in pregnancy rates due not only to the stallion variability, mare fertility, and the level of expertise of the inseminator, but to many factors that are far beyond our control.

No guarantee is made concerning the fertilizing capacity of the frozen semen.

BREED REGISTRY:

It is the responsibility of the stallion and mare owners to determine the policies regarding the transport and use of frozen semen that have been set by individual breed registries.

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STORAGE:

Semen is stored in liquid nitrogen containers. This service is available at Honahlee or the semen can be stored with the stallion owner if a tank is available. These containers need to be "topped off" with liquid nitrogen at regular intervals or they run the risk of "running dry" and the sperm dying. There are several commercial outfits across the country that store animal semen and ship it as well.

Frozen semen intended for international shipment must be collected and stored at a USDA-approved facility. Honahlee meets these requirements.

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INTERNATIONAL SHIPMENT:

Frozen semen intended for international shipment must meet the requirements of the country to which it is being imported. Often, a quarantine period and specific protocol are required. It is the responsibility of the stallion owner to obtain a permit to import as well as ensure that all criteria are met for export of the semen. Plenty of time prior to freezing should be allotted for these procedures. The USDA/federal veterinarian can be quite helpful in determining the protocols of various countries.

Honahlee is a USDA-certified facility, approved for the collection and storage of frozen semen intended for international shipment.

COST:

In general, processed straws run between $35-$70/straw (total cost). Again, there is tremendous variability based on individual stallion variation. One can plan on spending between $2000-$3500 to produce 50 straws, depending on the number of progressively motile sperm produced by that stallion and the tolerance of the spermatozoa to freezing and thawing.

A breakdown of the cost follows:

HONAHLEE, PC:

Honahlee, PC is an equine facility located approximately 20 miles from the center of Portland, Oregon. The practice of veterinary medicine is limited exclusively to reproduction.

Dr. Lisa Metcalf owns and operates the facility. She received her Master's degree in Reproductive Endocrinology from UC-Davis and her DVM degree from The Virginia-Maryland Regional College of Veterinary Medicine. Dr. Metcalf completed her internship at the latter and and has since, divided her time between private practice, research in specific areas of reproduction, and teaching.