CHAPTER 5 Techniques for Artificial Insemination
Although artificial insemination (AI) in horses can be done with semen collected at the farm for immediate use, maximization of this technology is achieved when mares are bred at considerable distances from where the stallion stands. To achieve this, semen either can be shipped in its liquid state after passive cooling and deposited in the mare several hours (12–36 hours) after collection or can be processed for freezing and maintained in liquid nitrogen until its use several weeks or years later. This chapter will discuss semen collection, handling and processing of cooled shipped and frozen semen, and the factors that affect the success of AI programs.
Breeding stallions that are going to be used for AI should have a semen evaluation performed prior to each breeding season. Because it is virtually impossible to improve the inherent quality of semen from a stallion, it is important for the mare owners as well as the stud farm to be aware of the stallion’s semen quality. A veterinarian working for clients that decide to stand a stallion or stallions for AI has the responsibility of overseeing that the clients are properly set up for what they are offering. This preparation includes proper stimulus for the stallion such as, if necessary, the availability of a mare in heat at all times during the breeding season, availability of shipping containers, and a laboratory for the proper evaluation and processing of the semen. It is also important to inform the stallion owner that a proper semen evaluation should be performed to determine semen quality and microbiologic status of the semen including equine viral arteritis.
Stallions can be trained to achieve an erection by conditioning them to certain routines or by exposure to certain objects or animals associated with breeding. A stallion can be stimulated to achieve an erection by exposure to a mare in natural estrus, an ovariectomized mare that has been injected with estrogens, or a phantom or dummy mare where he is normally collected. In addition, stallions can be manually stimulated to drop their penis and then massaging the glans will elicit a full erection and pelvic thrusting movements. The stallion when collected with a properly prepared artificial vagina (AV) will follow a similar pattern of thrusting to that of natural mating.
To ejaculate into an AV it is not necessary for the stallion to jump a mare or a dummy. Collection on the ground can be achieved by teasing the stallion in cross ties, in an open area, or in a stall. Once the stallion has achieved a full erection, his penis is washed with clean water, dried and inserted into a properly prepared AV. The stallion in many instances will vocalize and move forward until the pelvic thrusts start at which time he might lift his front legs slightly or arch his back and have all four limbs on the ground until ejaculation. Most stallions with good libido can be easily trained to ejaculate on the ground.
It is the belief of many horse owners whose stallion(s) breed only by natural cover that a number of stallions cannot be collected artificially. However in the author’s experience most stallions can be collected with an AV. Several models of AVs are available on the market, and although each has advantages and disadvantages, each operator should choose a model based on stallion suitability and ease of use. All AVs work under the same principle, which consists of a double rubber liner, usually latex, that will provide space to form a water jacket that at the adequate temperature and pressure will stimulate the stallion to ejaculate. Contrary to what would be expected, the temperature in the water jacket to stimulate a stallion to ejaculate is not body temperature but in general should be at least 42 to 43° C. In addition, older stallions often require temperatures of over 45° C to be properly stimulated. Furthermore, proper lubrication with a nonspermicidal water-soluble lubricant and the proper pressure are critical for adequate stallion stimulation. For stallions that have difficulty ejaculating into the AV, use of hot towels at the base of the penis to provide additional stimulation has been successful.
Although the author prefers the Missouri model AV due to its light weight and convenience of assembly and cleaning, there are other alternatives for semen collection. Condoms provide an alternative for obtaining a semen sample, particularly from stallions that are reluctant to ejaculate in an AV. Other methods of semen collection include the use of pharmacologic agents. These methods to induce ejaculation involve the use of pharmacologic regimens to either reduce the ejaculatory threshold in stallions or to induce extracopular ejaculation. Reduction of the ejaculatory threshold can be achieved with the use of diazepam 0.05 mg/kg IV or imipramine 500 to 1000 mg orally 15 to 30 minutes prior to breeding. Pharmacologic induction of ejaculation is achieved by slow IV injection of xylazine (0.66 mg/kg) with or without prior administration of 2.2 mg/kg of imipramine IV. In general, pharmacologic induction of ejaculation occurs within a few minutes of the injection and the success rate is about 30 to 50%. It must be pointed out that this method does not always result in ejaculation from the same stallion. Therefore, it is difficult to program breedings if pharmacologic ejaculation is the only method available for semen collection.
Because many stallions breed a reduced number of mares and therefore ejaculate only a few times during the breeding season, it is important to identify stallions that are “sperm accumulators.” These stallions in general have large testicles, and if sperm is not collected on a regular basis, they have the tendency to accumulate sperm in their reproductive tract. The accumulated sperm tend to have a lower motility, reduced longevity, and poorer morphology. Therefore, when such a stallion is offered for AI it is important to collect him several times prior to breeding mares, shipping, or processing for freezing. The number of ejaculates necessary to collect to improve the semen quality will vary among stallions. Two consecutive ejaculates of similar quality should indicate that the stallion has voided the accumulated sperm.
Raw semen is very fragile and should not be exposed to direct sunlight or sudden temperature changes. Once the stallion ejaculates, the semen should come into contact with a clean and warm container to prevent cold shock. The semen is filtered in line during ejaculation or filtered immediately after collection with nylon mesh or milk filter paper. This procedure is done to remove all extraneous particles and the gel fraction. Immediately after collection the raw semen should be placed in an incubator at 37° C prior to extension. The volume and color of the ejaculate should be recorded. In addition, the other physical characteristics of the ejaculate such as sperm movement, sperm concentration, and sperm morphology should be assessed from a small aliquot (1–2 ml) of raw semen that is removed prior to the addition of any diluents or extender. Once semen is diluted with a prewarmed extender it should be immediately removed from the incubator and cooled down to room temperature (20° C). An accurate assessment of the percentage of progressively motile sperm can only be done when the semen has been diluted to 25 million to 50 million sperm per milliliter.
The processing of semen should be done according to the period of time that the semen is expected to remain viable. It is recommended to always place the semen in an appropriate extender even if it is going to be inseminated within a few minutes. There are several reasons why semen should be extended within a few (<5 minutes) after collection. A proper extender buffers pH changes of the raw semen, maintains the osmolality, provides energy and protein sources for sperm metabolism and membrane stabilization during thermal changes, reduces the detrimental effects of the seminal plasma, and provides antibacterial properties through the antibiotics. Semen can be preserved for varying periods of time. However, the state and the processing technique will be determined by the expected longevity of a given ejaculate. Fresh extended semen can be kept at 20° C, avoiding exposure to direct sunlight, without losing its fertilizing ability. If semen is intended to last 12 hours or more, either because the stallion will not be available or because the semen will be shipped, it should be slowly cooled and maintained at 4 to 8° C. If semen is to be stored longer than 72 hours, freezing is recommended.
It is extremely important to ensure that the extenders that are going to be in contact with the sperm are isothermic with the semen. In other words, extender that will be added to raw semen should be prewarmed. However, extender added to semen that has been cooled to room temperature should be also at room temperature. Failure to do so induces cold shock to the sperm, resulting in irreversible reduction of the progressive motility and membrane damage reducing the fertilizing potential.
Semen that is collected and used immediately or up to 6 hours after collection does not necessarily have to be cooled and in most cases can be diluted with prewarmed extender at ratios of 1 : 1 to 1:3, depending on raw semen concentration and ejaculate volume. Immediately after extension, the semen is removed from the incubator and can be cooled to room temperature (15–20° C) without loss of its fertilizing potential.
AI using cooled semen is still gaining popularity among breeders since more breed registries are accepting registrations of foals resulting from the use of this technology. Although there is a tremendous increase in its use there is also a tremendous variability in the results achieved. In order to minimize the variability in the results it is important that the handling and processing of the semen be standardized.
The semen handling and processing factors that could have the biggest impact of the quality of an insemination dose are (1) cooling rates and containers, (2) dilution rates, (3) and the insemination process.
Although there are several extenders that can be used for preserving semen in its liquid state it appears that most researchers and clinicians favor the use of nonfat, dried milk solids and glucose or the Kenney extender. This is perhaps due to its ease of preparation and handling, as well as its clarity making it easy to evaluate sperm motility.
Spermatozoa suspended in an extender could be rapidly cooled from 37° to 20° C, but require a slow cooling rate of −0.5° to −0.1° C per minute between 20° and 5° C to maximize spermatozoal viability. If sperm are cooled slowly, once they have reached 4° to 8° C, this temperature storage is superior to storage at 20° C. It appears that the ideal storage temperature range for maintaining motility and fertility of cooled equine semen preserved for more than 12 hours in a Kenney type extender is between 4° and 6° C. However, Magistrini and associates found that a storage temperature of 15° C was superior to 10° or 4° C for maintaining spermatozoal motility when the extender does not contain milk solids. The current industry standard for preparing semen for shipping is to pack 1 × 109 progressively motile sperm in a nonfat dried milk (NFDM) glucose extender containing antibiotics, so that the final sperm concentration is 25 to 50 × 106 sperm/ml. Semen is slowly cooled, maintained, and stored at 4° to 8° C.
There are several containers currently available commercially that are designed for cooling and transport of equine semen. The Equitainer is the most widely used container for passive cooling and transporting of equine semen. Less expensive, disposable semen-transport containers are also available, such as the Equine Express or Expect-A-Foal. These containers are also passive-cooling transport devices, which provide variable rates of cooling. However, the big difference between the Equitainer and other semen transport systems is the effect of environmental temperature on the rate at which the semen is cooled or their ability to maintain their internal temperature. A study conducted to evaluate the effect of storage containers and environmental temperature suggested that sperm motility is adequately maintained in most commercial equine semen transport containers when the outside temperature is between 22° C and 37° C. However, environmental temperature of −20° C for 6 hours resulted in the reduction of progressive motility in most of the disposable containers. This study suggested that the Equitainer was a better container to isolate the semen from the effect of environmental temperatures.