Advanced reproductive technologies such as cooled semen, frozen semen, embryo transfer and gamete inter fallopian tube transfer (GIFT) have given horse owners choices and freedom. Mares can be bred at home with semen collected from stallions that live anywhere in North America, Europe or Australasia. Stallions can compete during the breeding season while mares are bred with previously frozen semen. Embryos can be collected from performance mares between competitions. The genetics of valuable infertile mares can be maintained through GIFT, a procedure that involves the aspiration of eggs from ovarian follicles from the donor mare. The eggs are then placed in a fertile recipient mare’s oviduct after which she is inseminated with semen. But as with all freedoms, these technologies come with a price. To be successful, a higher level of veterinary expertise is needed, mares need to be examined more often, pregnancy rates are lower and the costs are higher. Equine semen does not tolerate cooling, freezing and the manipulation needed for processing as well as other species. So, more money will be spent to obtain somewhat lower pregnancy rates then that seen with natural breeding or breeding with fresh semen by artificial insemination.
The following article discusses the logistics of using cooled semen, frozen semen and embryo transfer in order to provide a better understanding of each procedure.
History of Artificial Insemination (AI)
The first record of successful AI was in 1780 when an Italian scientist named Spallanzani bred a dog with freshly collected semen and later delivered three puppies from that mating. However, there was little selective breeding practiced and no shortage of willing male dogs so little notice was taken of the discovery.
In 1803 Spallanzani placed dog semen in snow and recorded it becoming motionless. Later the semen was warmed, and the sperm began to move again. Early in this century, AI in horses and cows using fresh, diluted semen became widely practiced in Russia and Japan. Fresh, cooled semen saw increased use in cattle in Denmark and the United States in the 1930s. Semen extenders or diluters, which allowed semen to be cooled and used within 24 hours, were developed. The driving force behind the development of AI was the desire to make wider use of better sires.
Artificial insemination using frozen semen has not enjoyed a lot of acceptance or success in the horse industry. In the early 1980s a commercial shipping container specifically designed for shipping horse semen became available (The Hamilton Equitainer). Since then, a number of disposable shipping containers have become available. The American Quarter Horse Association (AQHA) approved the use of cooled shipped semen in 1997. Given that AQHA is by far the biggest breed registry in the US, their approval of the process will likely be a major boost to the use of cooled shipped semen.
The Ins and Outs of Breeding Mares With Cooled Semen
To breed mares successfully with cooled semen all parties involved, mare owner, stallion manager and veterinarians, need to cooperate when coordinating the semen shipments with the timing of the mare’s ovulation. Before shipping semen, the attending veterinarian or a representative for the veterinarian should clarify several points with the stallion manager.
- The cost of stallion collection
- The cost of preparing the semen for shipment, the number of collections provided gratis (if any), the cost of shipping semen tanks by air, and when and how the semen tanks must be returned
- The days of the week the stallion is collected
- Times during the breeding season when the stallion will not be available
- The number of days notice that the stallion manager needs before the semen shipment
- The latest time one can call to obtain semen (for example-one must call by 9am to receive semen by the next day)
- The longevity of the semen-does it live in the tank for 12, 24 or 36 hours
- First-cycle conception rate of the stallion
- The method of air transport used (sameday air or overnight shipment
- Number of times the mare can be bred if she does not conceive (is the contract limited to 1, 2 or 3 years)
- The breed registry requirements, and the number and timing of post-insemination clinical (pregnancy) examinations must be established.
First cycle conception rates tend to be slightly lower with shipped semen than with natural breeding or when using artificial insemination with a stallion housed at the same facility as the mare. Also, breeding management is more intensive and veterinary cost are higher. Mares need to be examined daily when in heat and bred within 24 hours of ovulation. Stabling a mare at a facility, such as a veterinary clinic or farm where the veterinarian visits daily, saves money on veterinary travel fees. Furthermore, these facilities have a stallion to tease the mare to determine when she is in heat, thereby, limiting the number of times that she will need to be examined.
Pregnancy rates are highest when mares are bred within the 24 hours before ovulation using semen of high fertility. The quality of the semen is of paramount importance: stallions of low fertility usually have much lower conception rates than those with high inherent fertility. In addition, the handling of the semen is critical; failure to prepare it correctly as well as poor subsequent handling at the mare end can make the process very disappointing. Timing of the breeding with the ovulation can be difficult especially if the stallion is collected only 3 times a week.
Ovulation can be induced with drugs such as hCG or Ovuplant, however the window from injection of the drug to ovulation varies. Mares may ovulate as quickly as 24 hours, as late as 48 hours after administration of hCG or they may not respond at all. The window from injection of Ovuplant to ovulation is tighter than that of hCG with most mares ovulating between 42 and 48 hours, however, it costs about 2.5 times more than hCG. In either case it is extremely helpful if you know the specific idiosyncrasies of your mare’s estrous cycle, especially the number of days she is in heat and the size of the follicle that she ovulates.
There are standards that the semen needs to meet to be considered of adequate quality. A dose of semen should contain a minimum of 500 million progressively motile sperm with at least 30% of the sperm being progressively motile. Each time the mare is bred with cooled semen, it should be examined carefully after it has been warmed for a minimum of 3 minutes. If it is of poor quality the stallion manager or veterinarian for the stallion should be notified.
After insemination, the reproductive tract of the mare should be examined daily until she ovulates. If she does not ovulate within 24 hours she should be bred a second time.
Advantages and Disadvantages of Cooled Shipped Semen
The advantages of using cooled shipped semen include:
- Location of stallion is of little consequence.
- Avoids the cost, stress and danger of shipment for the mare and foal.
- Avoids mare care costs at breeding facilities.
- Avoids the risk of exposure to contagious disease at the breeding and boarding facility for both mare and foal.
- Increases ability to select stallion with desired pedigree and/or performance traits.
The disadvantages of using of cooled shipped semen include:
- Some stallions having acceptable fertility using fresh semen will not maintain that level of fertility when their semen is cooled and shipped.
- An increased number of sperm are required for good pregnancy rates when compared to AI using fresh semen.
- Mare management must be top notch for an acceptable pregnancy rate.
- Cost for equipment, supplies, semen, semen transport and veterinary costs can offset any savings.
Costs associated with breeding a mare will vary widely but include:
- The cost of collecting, extending and shipping the semen.
- The cost of teasing and/or altering the mare’s heat cycle.
- Veterinary costs for palpation or ultrasound exam and breeding.
- Drug costs.
Breed Registry Restrictions and Interstate Shipping Regulations
Not every breed organization allows registry of foals that result from breeding using cooled shipped semen. Always check with your breed registry regarding restrictions on the use of cooled shipped semen.
Interstate shipment regulations do not exist in some instances, are ignored in others, and are enforced in yet others. Generally, it is the responsibility of the shipper to see that any interstate shipment regulations such as health tests are met.
Fertility Results Obtained Using Cooled Shipped Semen
Fertility rates in various research trials and on the farm range from 0 percent to 70 percent pregnancy per heat cycle. This wide variation in results points out the importance of management and quality control in successful breeding using cooled shipped semen. Generally, pregnancy rates for a given stallion will run 5 percent to 10 percent lower with shipped semen when compared to fresh semen. Remember that not all fertile stallions’ semen survive the cooling process well. Before signing a breeding contract, be sure the stallion you select has a history of success in cooled, shipped semen breeding.
Mare Management
All arrangements should be made with the stallion owner prior to the breeding season. Contracts should be signed and a plan of action developed as to when in the breeding season you will attempt to breed your mare. Planning and preparation are key to a successful mating using cooled shipped semen.
Selecting a fertile mare is a good first step in mare management. It’s just not a good idea to use a mare with a questionable or unknown breeding history for breeding with cooled shipped semen. The mare should be cycling normally, based on regular teasing, and free of health problems. Vaccinations and dewormings should be current.
In order to achieve an acceptable pregnancy rate (more than 50 percent per cycle), the mare should be bred as near to ovulation as possible. Once the mare is found to be in heat by teasing, the mare should be palpated or examined with ultrasound equipment daily or every other day. Once a follicle is found, its size is monitored until its diameter reaches at least 35 mm. At this point the mare is given an injection of the hormone Human Chorionic gonadatropin (HCG) and the semen is ordered.
The injection should result in ovulation in 36 to 48 hours. If the semen is ordered at the time of the HCG injection, it should arrive in about 24 hours or just before ovulation. This is a good time to breed the mare.
Stallion Management
It is important to remember that not all stallions work equally well as producers of semen and not all extenders (diluters) work equally well on all stallions. Successful experience with stallions and extenders is the best way to determine what will or will not work.
Within a few minutes of collection, the semen should be mixed with warm semen extender. A variety of extenders are available but all contain water and nutrients for the sperm cells. Also, chemicals in the extenders keep the acidity of the ejaculate within an acceptable range. Finally, most extenders contain antibiotics to limit the growth of bacteria during storage. A list of sources of horse semen extenders is provided in Table 1.
The volume of extender to be added varies with the number of sperm cells in the ejaculate. Sperm cells can be counted using several systems. This process is best done with each collection but can be done periodically. Semen extenders are added so that the final solution contains 25 to 50 million cells per cc. The percent alive can be estimated using a light microscope.
The number of sperm necessary for acceptable pregnancy is about 500 million live cells. Experience will show what percentage of the stallion’s sperm are still alive 24 hours after collection and cooling. Typically, about one-half of a stallion’s sperm will still be alive at 24 hours. Therefore, at least one billion cells are shipped in a volume of 20 to 150 cc of extended semen.
An example of the calculations used follows:
- Volume = 50 cc
- Concentration = 150 million/cc
- Percent alive at 24 hours = 50%
- Total sperm = 50 x 150 = 7,500,000,000 cells in 50 cc
- 7,500,000,000 ) 37,500,000 = 200 cc of extender to add
- Add 200 cc extender = 37,500,000 cells/cc
- Need 500,000,000 alive at 24 hours, so send 1 billion total cells
- 1,000,000,000 ) 37,500,000 = 26.6 cc.
Calculations often are not done each collection and sending more than the minimum is OK.
Perhaps the most common shipping container used is the Hamilton-Thorne Equitainer. These units cost between $200-$300 but are reusable, durable and proven. Disposable shipping containers are less costly ($25.00) but keep semen at refrigerator temperature for shorter periods of time.
Whatever the system used, it is important to follow the manufacturer’s instructions. After packaging, the semen is shipped by reputable carrier so that it arrives at the mare’s farm in 24 hours.
With cooled semen, Insemination of the mare is generally done immediately after the container is opened. The semen is not warmed before use. Double inseminations at 24-hour intervals don’t seem to improve pregnancy rates.
Breeding with cooled shipped semen allows mares to be bred to distant stallions. However, to be successful, attention to detail is a must. There are many ways to do things wrong and only one way to get it right.
Trials and Tribulations of Breeding With Frozen Semen
Breeding mares with frozen/thawed semen often results in a significantly lower first-cycle pregnancy rate than when mares are bred with either fresh or fresh, cooled semen. Pregnancy rates of 0 to 70% /estrous cycle have been reported with an average pregnancy rate/cycle of 35 to 40%. The highest conception rates are achieved by using a full dose of frozen semen from stallions of known fertility in young mares of known fertility. Mares are examined every 6 to 8 hours, given either hCG or Ovuplant and inseminated during the period from 12 hours before ovulation until 6 hours after ovulation.
Breeding with frozen semen can be confusing for the horse owner because there are no standards concerning size of the straw semen, thawing rate and temperature, number of straws/dose, and how the semen is sold, ie by the dose or by the straw. If a half milliliter straw is used, a dose consists of 4 to 8 straws. However, semen may be sold by the straw and not by the dose. A mare owner may attempt to breed the mare with less than what is considered a complete dose of semen. This often times leads to the mare owner being disappointed and the veterinarian frustrated as the mare does not conceive. Points that must be clarified before a mare is attempted to be bred with frozen semen are as follows:
- What is the first cycle conception rate of the stallion when breeding with the frozen semen
- What is the cost of semen
- What constitutes a dose- number of straws
- What is the size of the straws
- Will there be directions with the semen on how to thaw the straws
- Does your veterinarian have nitrogen tanks to store the semen
- What is the rental fee for the dry shipper and how long can it be rented
- How frequently can your veterinarian examine the mare
- What paperwork will be included and what does the breed registry require
Mares can be managed in a variety of ways during estrus depending on the facilities, semen quality and availability of the veterinarian. One management method is to examine the reproductive tract every 24 hours during estrus until the dominant follicle reaches 30 to 35 mm in diameter (35 mm for a warmblood mare; 30 mm for an Arabian, Morgan or Paso Fino mare). Either hCG or Ovuplant is given at that time. The reproductive tract is then examined by ultrasonography at 24, 30, and 36 hours after hormone treatment. If, at any of these times, the mare has ovulated, she should be inseminated immediately. If the follicle is still present 36 hours after hormone treatment, the mare should be inseminated at that time. Twelve hours after insemination, the reproductive tract should be reexamined. If the mare has not ovulated, she should continue to be examined until ovulation at which time she is inseminated again. This regime reduces the period of intensive monitoring and, with a maximum of 2 inseminations, it should be effective for most mares. The system may be modified to accommodate an individual mare’s estrous cycle and the rate of follicle development.
If only one dose of semen is available or the owner wishes to use only one dose of semen per cycle, the management must be more intensive. In this scenario the mare must be examined every 6 to 8 hours beginning 24 hours after she is given hormone treatment until she has ovulated. Once ovulation is detected, the mare is bred immediately. Pregnancy rates may be lower and the costs higher when only one dose is used for a cycle.
Some mares may develop a prolonged inflammatory reaction to the semen because there is no seminal plasma in the frozen semen (if the seminal plasma is included, motility of sperm is poor semen post thaw). Therefore, many veterinarians routinely perform a uterine lavage twelve hours after breeding in an attempt to clear any remaining inflammatory by-products induced from the breeding. If a mare develops a prolonged inflammatory reaction after she is bred twice with frozen semen from the same stallion and she does not conceive, it is advised that she be bred with either fresh semen or fresh cooled semen as both contain seminal plasma.
There are no clear cut recommendations on what is the recommended dose. Doses vary from stallion to stallion, on the method of freezing the semen, on the country of origin and on the individual freezing the semen. Doses range from 100 to 500 million progressively motile sperm with a dose of 200 to 250 million being the average. Most investigators suggest that there be a minimum of 25% progressively motile sperm after thawing and warming to 37C for at least 3 minutes. Motility of the semen after thawing is not always correlated with pregnancy rates and reliance on it as an indicator of fertility must be cautioned.
The Joys of Embryo Transfer
Embryo transfer (ET), an advanced reproductive technology, has several potential uses in equine reproduction. ET can be used to increase the annual reproductive rates in mares. For example, some breed associations will allow multiple foals to be registered annually per donor mare. Young mares (2 years old) can have offspring through ET where otherwise this might not be a good idea. Females in use for shows, training or exhibition can have offspring without being removed from their primary use. Last, less fertile mares — particularly those with uterine problems — can have foals.
Embryo transfer has become a relatively commonplace procedure in the equine industry, especially with the establishment of larger recipient herds throughout the country. Success rates vary depending on the fertility of the stallion and mare and on the synchronization of the recipient mare. Many individuals in the business feel that the quality of the recipient mare and how she is managed is the most important factor in achieving high pregnancy rates. Mean pregnancy rates/cycle if both the mare and stallion are fertile are 50-60%. Rates drop precipitously from there if either horse is subfertile or if the recipient quality is poor. The average cost of a foal produced from an embryo transfer if both the stallion and mare are fertile, will range from $5500 – $7500. Therefore, if a mare owner is to break even, the foal needs to be worth a minimum of $10,000 to $12,000 as a weanling.
There are a number of large well managed recipient herds through the country. The mare owner needs to discuss options with their veterinarian and then contact the recipient facility that they wish to work with in the future. A contract will be sent detailing the specific costs, what will be covered such as number of attempts, when the pregnant recipient needs to be picked up, if the recipient can be returned and the price that will be paid to the mare owner for returning the recipient. Some facilities include 3 embryo flush attempts in their fee after which time the mare owner pays for each attempt. Once a contract is signed a holding fee, usually $1500, needs to be paid. The remainder of the fee is paid ($2500-$3500) when the mare is declared pregnant.
Breed Registry Regulations
Before opting to use ET, consider that it is a very technical, expensive and time consuming process with a fairly low success rate. Additionally, breed registries place limitations on the use of ET to produce registered offspring. For example, the Tennessee Walking Horse Breeders and Exhibitors Association Corporate Bylaws contain a rule that governs embryo transfer for Walking Horses. The rule is as follows: “Registration of foals resulting from embryo transfers will follow the same standard requirements for registration, except that multiple foal registrations from a donor mare during an eleven month period will be accepted.
Blood typing of donor mare, stallion and foal is required. Blood typing will be the sole responsibility of the person(s) sending registration and entirely at his expense. Any foal that does not meet the blood type comparison will be forever barred from registration.”
Each horse breed registry has different rules and requirements for ET registration. The Clydesdale Breeders of the USA requires genetic testing to verify parentage, and two foals per mare per year may be registered. The American Quarter Horse Association will allow the registration of ET foals from a mare provided certain documentation has been provided to AQHA. Moreover, additional fees are often charged for registration of ET foals.
The Process of Embryo Transfer
The technique is relatively simple, however, one must strictly adhere to management guidelines. The donor is bred using proper veterinarian management. The mare should be palpated every 12 hours until ovulation after she is bred to accurately determine the time of ovulation. If the mare owner prefers to use their own recipient mares, it is best (highest success) to monitor ovulation in recipient three mares for every transfer attempt. Three mares are needed because there is wide variation in the length of estrus, in the rate of follicular development and in the timing of ovulation after either hCG or Ovuplant is given. It is preferred that the recipient ovulates 12 to 48 hours after the donor.
Embryos are collected from the mare by uterine lavage using specialized fluids eight days after the mare has ovulated. The fluids are collected from the mare through tubing that is attached to a special cup containing a filter. The holes in the filter are very small and will not allow passage of the embryo through it. The fluids that remain in the cup are searched using a dissecting microscope. Once an embryo is identified, it is aged and graded for quality and stage of development. It is then removed from the wash fluids, placed in a dish containing transfer media and washed a number of times. The recipient is then brought up, and readied for the transfer. The majority of embryos are now placed into the recipient by a non-surgical technique. The embryo is put into an embryo insemination gun which the operator carefully inserts into the cervix and then discharges the embryo into the uterus.
For shipping, horse embryos have been stored in Hamm’s F10 tissue culture fluid with 10 percent fetal calf serum added. A mixture of 5 percent carbon dioxide, 5 percent oxygen and 90 percent nitrogen is bubbled through the fluid before filtering. Embryos can be placed in this fluid and shipped in a controlled shipping container with no reduction in pregnancy rate if the embryo is transferred within 24 hours.
Conclusions
Reproductive technologies have given horse owners and veterinarians many options. These techniques have improved and widened genetic pools. Using them is exciting for all involved. However, methodologies must be strictly adhered to and owners must do their homework before they begin if they are to be successful.
