Looking to breed with chilled semen? There are many benefits but unfortunately, not all stallions produce sperm that survive the cooling process. Here, the American Association of Equine Practitioners outlines the process of breeding horses with cooled semen.
An evaluation of the viability of a stallion’s sperm after cooling is an important quality control measure. Stallions should be tested before the breeding season to determine if sperm motility will be acceptable when mares are bred with cooled transported semen.
Management of the mare should be precise and intense in order to reduce expenses and maximize fertility associated with breeding with cooled, transported semen. Semen must be ordered so that it arrives at a proper time for insemination, preferably within 12-24 hours before ovulation. Following are recommendations for testing stallion semen for the ability to survive cooling, processing and shipping of semen, and management of the mare for breeding with cooled transported semen.
Not all of the breed registries allow registration of foals produced by artificial insemination and, of those that do, not all permit the use of preserved or transported semen. Several breed organizations sanction the use of liquid transported semen. To ensure full compliance with the breed registry involved, the specific breed registry should be contacted before becoming involved in breeding with transported semen so all requirements are met to permit registration of any foals produced.
Fertility trials using cool-stored equine semen have yielded pregnancy rates varying from 0% to 70% per cycle, emphasizing that there is great variability among stallions in the ability of their sperm to maintain fertilizing capacity during cooling/storage. Breeding with semen cooled for 48 hours at 5°C usually reduces pregnancy rates to about half that achieved with fresh semen. Since the pregnancy rate per cycle achieved by breeding with semen cooled for 24 hours at 5°C typically averages 60 to 70%, one should expect near-normal pregnancy rates when semen is used for breeding after short term (œ24 hours) storage, provided semen quality is good following this cooling period, and mare management has been optimal.
Factors influencing fertility
Numerous factors influence pregnancy rates achieved when mares are bred with cooled stallion semen. Some of the factors identified as influencing the fertility of cooled stallion semen in controlled experiments include the number of sperm inseminated, frequency of insemination, the concentration of sperm in the extender, the type of extender and antibiotic(s) used, the cooling rate of extended sperm, storage duration and storage temperature, stallion variability in the response of sperm to cooling, and inherent fertility of the stallion and mare.
If the quality of fresh stallion semen is poor, or if fertility achieved by breeding with fresh semen is poor, it is highly unlikely that successful results can be obtained by breeding with cooled transported semen. Other factors which undoubtedly impact fertility using this technology include whether or not the semen is collected and handled properly, the raw semen is promptly and accurately diluted in a suitable prewarmed extender, the semen is properly packaged for passive cooling and shipment, the insemination is properly timed in relation to ovulation, and proper technique is utilized during insemination. Sperm are very sensitive to many environmental factors, including temperature, light, physical trauma, and a variety of chemicals. Any factor that impacts the ability of sperm to resist environmentally-induced damage will adversely affect fertility achieved when using cooled transported semen for breeding.
Can it survive cooling?
It is recommended that stallions are tested before the breeding season to determine if the sperm will survive the cooling process. The practitioner is referred to the Society for Theriogenology manual on examination of the stallion for breeding soundness for procedural considerations (Journal of the Society for Theriogenology; Volume IX, Theriogenology and the Equine: Part II – The Stallion; August, 1983). Collection of several ejaculates may be required to stabilize extragonadal sperm reserves and improve semen quality, thereby maximizing the chances sperm will survive the cooling process. If sperm quality is acceptable after 24 hours of cooling, it is reasonable to assume the stallion’s semen will survive the cooling process and the stallion can be recommended for breeding with cooled transported semen. Equally important, an estimation can be made of the total number of sperm that will need to be processed in order to provide an insemination dose of cooled semen that should maximize pregnancy rates.
Preservation of semen begins with the actual collection process. Accurate assessment of semen quality relies heavily on proper semen collection techniques. Ejaculated semen is very susceptible to environmental influences, so mishandling semen samples before evaluation negates their value for representing the ability of a stallion’s sperm to survive the cooling process. Semen collection techniques and procedures utilized to ensure that sperm quality is optimized should be meticulously followed when collecting stallion semen.
Following collection, semen should be processed in a careful and efficient manner. Briefly, the gel is separated from the gel-free fraction of the ejaculate, and the volume of filtered gel-free semen is measured. Sperm concentration of the gel-free semen is determined. The total sperm number in the ejaculate is calculated by multiplying sperm concentration by volume of gel-free semen. A small portion of the gel-free semen is diluted in a suitable prewarmed extender prior to estimating progressive sperm motility using a microscope equipped with a warming stage (a phase-contrast microscope provides optimal viewing of sperm). Warmed (37°C) nonfat dry skim milk-glucose extender may be used since it supports sperm motility and does not interfere with microscopic visualization of the sperm. To standardize the sperm motility testing protocol, all semen samples should be diluted to a specific concentration with extender before analysis. If the quality of fresh stallion semen is poor, it is highly unlikely that successful results can be obtained by breeding with preserved semen.
The remainder of the semen should be mixed with a prewarmed (37°C) extender within 2-15 minutes after ejaculation in preparation for cooling. A final concentration of 25-50 million sperm per ml in extended semen generally maximizes sperm survivability in vitro. The semen should be diluted with extender at a ratio of at least 1 part semen to 4 parts extender. If this dilution results in a sperm concentration ‹ 25 million sperm per ml, the semen may be centrifuged to concentrate sperm. The semen is first mixed with extender at a ratio of 1-2 parts extender to 1 part semen, and then is centrifuged in 50-ml round-bottom centrifuge tubes at 500 x g for 10 minutes. The supernatant is removed and the sperm pellet is resuspended in additional extender. If seminal plasma is removed by centrifugation, the sperm can be resuspended to a concentration of 50-100 million sperm/ml without adversely affecting motility. The extended semen must contain a minimum of 5% seminal plasma to prevent reduction in longevity of sperm motility. Extended semen should not be left for a prolonged period in an incubator (37°C) because extensive cellular death will occur within a few hours if sperm are maintained at this temperature.
Determination of insemination dose
The most accurate method for determining the insemination dose required to transport for breeding is to conduct semen cooling trials for the individual stallion. The semen is diluted in an appropriate extender(s) as described above, and the semen is cooled for 24 hours. The cooled semen sample is gently remixed after this cooling period, and an aliquot is warmed to 37°C. Sperm motility is evaluated after 15 minutes of warming, and the percentage progressively motile sperm obtained is used to calculate the number of progressively motile sperm so that a minimum of 500 million progressively motile sperm will be present after 24 hours of cooling. For example, if after 24 hours of cooling the progressive sperm motility is 50%, 1 billion total sperm would need to be prepared for shipment to ensure an insemination dose of 500 million progressively motile sperm was available for breeding the mare.
The number of normal motile sperm in an insemination dose appears to be more critical to fertility than the volume of the inseminate. Although smaller or larger volumes can be used successfully, typical insemination volumes for cooled transported equine semen range from 20 to 120 ml. Many practitioners prefer to limit insemination volume to about 60 ml. Insemination volumes as large as 120 ml of cooled semen have been found to have no adverse effect on the fertility of pony mares as long as a sufficient number of progressively motile sperm are placed in the uterus.
Some practitioners believe large insemination volumes should be avoided when breeding mares susceptible to uterine infection. Uterine inflammation commonly follows breeding in the horse, and it is likely that uterine inflammatory response in such mares is at least partly due to neutrophil migration into the uterus as a result of sperm chemotactic stimuli (sperm themselves have been shown to stimulate an influx of neutrophils into the uterus). In this regard, it appears that more concentrated semen produces a greater influx of neutrophils into the uterus. Therefore, if chilled semen is properly diluted (i.e., to 25 to 50 million sperm per ml), the inflammatory response may be lessened.
To reduce time and expense involved in breeding a mare with cooled transported semen, the practitioner should strive to minimize the number of shipments required for breeding. If pregnancy does not result from breeding during one estrous cycle, expenses are multiplied as additional breeding(s) will be necessary. If breeding during two estrous cycles does not result in pregnancy, a thorough breeding soundness examination (if not already performed) may be necessary to determine if the mare is at fault. If semen quality is suspect, particularly if it cannot be corrected, breeding to another stallion may be an option for consideration. To avoid the expense of repeat breedings, mare owners should try to select a stallion that has been proven to achieve good pregnancy rates with cooled semen. If the stallion has not been used previously for breeding with cooled semen, they should attempt to use a stallion that is known to have acceptable semen quality after being cooled to optimize fertility. However, this information may not always be available, particularly if a stallion’s semen is being offered for cooled transported breeding for the first time.
The practitioner should play a primary role in maintaining quality control (semen quality, insemination dose, timing of breeding, etc.) when horses are bred with transported cooled semen. To be successful, stallion owners and managers, mare owners, and veterinarians involved in stallion and mare management must work together as a team to optimize the chance of conception from each breeding.
Pre breeding preparation of the mare
Before the onset of the breeding season, the mare should be examined to detect reproductive abnormalities. Underlying problems that might contribute to infertility, such as poor body condition or endometritis, should be corrected.
Prediction or detection of ovulation
When the mare is in estrus, the cervix becomes progressively more relaxed and the uterus becomes softer and more edematous as follicle size increases and ovulation approaches. Once a follicle reaches 35-50 mm in diameter, the ovulatory follicle(s) soften(s) detectably on palpation per rectum. Using transrectal ultrasonography, the cross-sectional size and shape of follicles and the echogenicity of the follicular fluid can be used to aid in prediction of ovulation. Within 24 hours prior to ovulation, the shape of most follicles tends to change from spherical to a conical or “pear” shape, and the follicular wall may become “scalloped” or thickened in appearance. The apex of the conical follicle will be located at the ovulation fossa. The echogenicity of the follicle changes from a predominantly anechoic appearance to a heterogenous echotexture as ovulation occurs and luteal development begins. The developing corpus luteum often contains a small anechoic center resulting from retained follicular fluid or blood.
Hormones to control ovulation
When cryopreserved semen is used for breeding, or when breeding to certain subfertile stallions, the fertilizable lifespan of sperm may be reduced, requiring breeding closer to the time of ovulation. To optimize fertility, hormones can be administered to increase the probability that ovulation will occur near the time of breeding with cooled semen. The onset of estrus can be controlled in cycling mares by the administration of progestogens and/or prostaglandins.
Human chorionic Gonadotropin (hCG) or a GnRH analog can be administered to reliably induce ovulation. Administration of hCG (1500-3300 units, i.v., or i.m.) when a 3-35 mm in diameter follicle is present in mares in estrus will induce ovulation in a majority of mares within 36-48 hours. If the day of estrus is unknown, and the follicle is larger than 35 mm in diameter, the interval to ovulation is less precise and may occur earlier than expected after hCG administration. Some practitioners believe hCG will more predictably induce ovulation at a precise time if endometrial edema (as detected by ultrasound examination) is maximal at the time of administration.
The stallion manager should be consulted to make sure semen will be available for breeding when the mare is expected to be in estrus. Human chorionic gonadotropin (hCG) can also be used to improve synchrony between the arrival of the shipped semen and the time of ovulation. When a mare is in estrus, daily or even twice daily examinations are performed to monitor the size of developing follicles. Typically, once follicular diameter reaches 35 mm, hCG may be administered after it is confirmed that transported semen can be obtained and will arrive the next day. The semen generally arrives 24 to 36 hours later in time for breeding just before ovulation.
Timing and frequency
Since the viability of stallion semen may be shortened by cooling, many practitioners recommend breeding as near to ovulation as is feasible to optimize pregnancy rates. While satisfactory pregnancy rates have been reported in mares inseminated after ovulation with transported cooled semen, critical study of the optimal time for breeding in relation to ovulation with cooled semen has not been done.
Breeding the mare with cooled semen
When the cooled semen arrives for insemination of the mare, current recommendations are to:
Prepare the mare for breeding. The mare should be adequately restrained with her tail wrapped and held to the side. The perineal area is thoroughly scrubbed and rinsed, paying particular attention to the vulva. Any dirt or fecal material within the caudal vestibule should be removed during the washing process to prevent contamination of the anterior reproductive tract during insemination. Two to three scrubs with a non-irritating soap (e.g., Ivory Soap) or a surgical scrub are recommended, followed by thorough rinsing to eliminate residual soap that may be spermicidal or irritating to mucous membranes. The perineal and vulvar area should be thoroughly dried prior to breeding.
Open the shipping container and confirm the identity of the stallion using the accompanying form. Remove the chilled semen, gently mix it, aspirate the semen into a syringe, and attach an insemination pipette. Sterile non-toxic disposable equipment is recommended for AI procedures. Syringes with non-spermicidal, plastic plungers are preferable for AI since rubber plungers may possess spermicidal properties.
Inseminate the semen into the uterus of the mare. To inseminate the mare, a sterile shoulder-length plastic sleeve is first placed over the arm used for insemination. The tip of a 20 to 22-inch insemination pipette is then positioned in the cupped hand and a small amount of sterile, non-spermicidal lubricant is applied to the back of the hand. The covered hand and insemination pipette are passed into the cranial vaginal vault where the index finger identifies and penetrates the cervix. The insemination pipette is then advanced through the cervix to the mid-body of the uterus. A syringe containing extended semen is attached to the insemination pipette and the semen is slowly deposited into the uterine lumen. An alternate, but equally satisfactory, method of insemination is to pass the insemination pipette through the cervix into the body of the uterus using a lighted speculum preplaced in the vagina.
A small sample of the extended semen should be kept and warmed to 37°C, and sperm motility should be assessed and recorded for quality control purposes. If sperm motility is poor, inquiries can be made in an effort to determine if this was an unexpected problem. It is sometimes helpful to prepare a specimen for assessment of sperm morphology. A high percentage of morphologically abnormal sperm would be expected to yield a low precentage of progressively motile sperm following cooling. If sperm motility is consistently poor and the mare fails to conceive on repeated breedings, it is possible the stallion is incapable of producing sperm which survive the cooling/transportation process. Alternatively, use of a different semen extender or dilution ratio may prove beneficial to semen harvested from that particular stallion.
Overseas workers often recommend warming the cooled semen to body temperature prior to insemination of the mare. In the US, the cooled semen is usually inseminated as soon as possible after opening the shipping container. Since no detrimental effects on pregnancy rate have yet been determined by inseminating mares with cooled semen, at present we see no reason to prewarm the cooled semen prior to insemination unless a cream-gel extender is used. This extender forms a gel at refrigerated temperature, so it must be warmed before insemination. The packaged sample can be placed in an incubator or water bath preset at 37-38°C. It is important to avoid contamination of the extended semen with water when warming the sample in a water bath.
It has become common practice for many stallion owners/managers to prepare two bags (insemination doses) of extended semen for shipment — one to be used for initial insemination upon arrival, and one to be held for insemination again the next day. For many stallions, the longer the semen is held at refrigerated temperature, the poorer sperm motility becomes. However, if semen is to be held for breeding again the next day, precautions should be taken to maintain it at 4-6°C until the time of the second insemination. Once a shipping container is opened for the first breeding, the temperature of the semen left in the container is likely to rise above 10°C, causing premature sperm death. The remaining semen dose can be immediately placed in an insulated box within a refrigerator (4-6°C) — until it is to be opened for breeding the next day. This would be especially critical for semen cooled in some “disposable” type shipping containers which may only maintain semen at < 10°C for 27.5 to 33.5 hours.
Post breeding treatment for mares with intrauterine fluid accumulation
If a given mare needs treatment after breeding that includes evacuation of uterine fluid or debris, we recommend either post-breeding uterine lavage or oxytocin treatment. Treatment as early as 4 hours after breeding does not appear to affect fertility. Treatment can also be performed after ovulation is detected. Intrauterine treatment more than 2 days after ovulation should be avoided since it may impair CL function, resulting in an early return to estrus with subsequent loss of pregnancy if the breeding was successful.
Examination for pregnancy
We recommend the mare bred with transported cooled semen be examined for pregnancy using transrectal ultrasound at 14-15 days postovulation. This enables early detection of a pregnancy that is normal for stage of development, allows early detection of twins before they become fixed in the uterus, and provides time for rebreeding during the same estrus if the mare failed to become pregnant.
This article, originally published in February 2008, has been reprinted with permission from the American Association of Equine Practitioners. Headquartered in Lexington, Kentucky, the AAEP was founded in 1954 as a non-profit organization dedicated to the health and welfare of the horse. AAEP reaches more than 5 million horse owners through its 6500 members worldwide and is actively involved in ethics issues, practice management, research and continuing education in the equine veterinary profession and horse industry.