A reproductive evaluation, often termed a breeding soundness examination, may be performed to: 1) estimate future reproductive potential of a stallion, 2) provide a routine evaluation prior to purchase, or 3) determine cause(s) of poor reproductive performance. The breeding soundness examination should encompass general health of the horse, behavior, mating ability, genital examination, semen evaluation, and other parameters. In most cases, a breeding soundness examination can be performed in one day. It is generally recommended that a stallion be evaluated after a period of sexual rest. In addition to the standard breeding soundness examination, a test cool, and/or test freeze should be performed if a stallion is to be used in a cooled-transported semen or a frozen semen breeding program.
Note: A single diagnostic test is often not an absolute or accurate predictor of the fertility of a stallion. Therefore, in many cases a combination of sperm function tests are used to improve the reliability of fertility estimation. Flow cytometry has become an integral technique to rapidly evaluate multiple sperm parameters on hundreds or thousands of sperm cells using fluorescent stains. Other tests, such as ultrasound and endoscopy are used to determine if pathology is present when clinical signs of scrotal swelling or hemospermia are observed.
Each stallion should be properly and positively identified prior to evaluation. Registration name and number, age, and breed are recorded. Coat color and markings, lip tattoos, microchip information (if used), body brands, hair whorls, permanent scars, and any other identifying features should all be noted. A digital photograph may be valuable as part of the permanent medical record. The goal is to be able to accurately connect the results of a breeding soundness examination to a specific stallion at a later date if necessary.
A complete breeding history should be obtained, as past breeding results (conception and foaling rates) are the best indicators of a stallion’s previous fertility level. The number and type of mares bred per season (i.e. maiden, barren, and foaling mares) and the breeding method used (i.e., hand breeding, pasture breeding, or artificial insemination with fresh, cooled, or frozen semen) are recorded. Results of previous reproductive evaluations, medical conditions, vaccination history (especially vaccination against equine arteritis virus), and other significant information are noted. It is also important to record the intended future use of the stallion and the number of mares anticipated for the next season, as it will be important for breeding management recommendations.
A general physical examination should be performed on each stallion. To be considered fertile, a stallion must be able to mount a mare in estrus, insert his penis into the vagina, thrust appropriately, and ejaculate completely. Musculoskeletal abnormalities that may interfere with mating ability, such as back, hock, or lameness issues, or neurologic problems should be identified along with other medical conditions. Additional medical tests may be warranted in some situations. Any evidence of heritable or congenital conditions should be reported. Serum of stallions should be tested for presence of antibodies against equine arteritis virus, the agent that causes equine viral arteritis. In general, all stallions should be tested for natural exposure to the equine arteritis virus and then vaccinated.
External Genitalia Examination
The penis, prepuce, sheath, scrotum, and scrotal contents are routinely evaluated during a breeding soundness examination. Inspection of the penis and prepuce is often performed after the stallion has either “dropped” his penis or gained an erection after teasing a mare in estrus. The penis should be of normal size, shape, and be free of lesions. Palpation of the testes and epididymides are often performed after semen collection, as the stallion will be less excited and will allow for a more complete and safe examination. There should be two testes fully descended into the scrotum. Each testis should be smooth, slightly turgid, and resilient on palpation, and freely movable within the scrotum. The tail of the epididymis should be easily palpated at the caudal pole of each testis. The spermatic cord should be identified on the cranial dorsal aspect of each testis.
Testes of mature stallions are normally capable of producing approximately 18-20 million sperm per gram of testicular tissue per day. Since testicular weight cannot be obtained in a live, intact stallion, daily sperm production potential is estimated from total testicular volume. Measurement of testicular size is performed to estimate potential daily sperm production.
Two types of testicular measurements may be obtained. Total scrotal width can be determined with scrotal calipers. The testes are gently and evenly held in the ventral scrotum as the calipers are used to determine the widest measurement across both testes. Total scrotal width is correlated with both testis weight and daily sperm production. The minimum acceptable total scrotal width for light horse stallions is 8.0 cm. Actual minimal total scrotal width is age dependent (8.1 cm for 2-3 year olds, 8.5 cm for 4-6 year olds, and 9.5 cm for stallions >7 years old). It should be noted that determination of testicular volume is considered to be a more accurate method of predicting sperm production potential than measurement of total scrotal width.
Alternatively, the length, width and height of each individual testis can be measured using either scrotal calipers or ultrasound and the data recorded in centimeters. These measurements are used to calculate testicular volume and subsequently estimate potential daily sperm production.
Testicular volume (in cubic centimeters, cm3) of each testis can be determined using the following formula: Testicular Volume = 0.5233 x length (cm) x width (cm) x height (cm)
The calculated volume of each testis is added together to obtain the total testicular volume (in milliliters). Expected daily sperm production (in billions of sperm per day) can subsequently be estimated using the following formula: Expected Daily Sperm Production = (0.024 x total testicular volume) – 1.26
Spermatogenic efficiency can then be determined by comparing the actual number of sperm produced each day (daily sperm output) with the calculated daily sperm production rate based on testicular volume. Stallions with an actual daily sperm output that is less than the predicted daily sperm production based on testicular volume should be evaluated for disease or dysfunction of the testes, epididymides, ductus deferens or accessory sex glands. For example, a stallion with a total testicular volume of 400 cm3 should have a daily sperm output of approximately 5-11 billion. A problem may be suspected if his actual production is less than 5 billion spermatozoa per day.
Ultrasound examination of the testes and other scrotal contents may also be performed in stallions to identify lesions within the testes or characterize fluid within the scrotum.
Internal Genitalia Examination
Examination of the internal genitalia of a stallion per rectum is not always performed as part of a reproductive evaluation, unless indicated by an abnormal history, physical examination findings, or abnormal semen parameters. Risk of injury to the stallion and personnel often outweigh potential benefits during a routine evaluation. In the event that a stallion is to be palpated per rectum, the attending veterinarian may opt to sedate the horse and administer a medication to relax the musculature of the caudal rectum (Buscopan®). It would also be preferable to perform the examination with the stallion restrained in stocks.
Venereal Disease Evaluation
Microbiological culture swabs are routinely collected as part of a complete breeding soundness examination. Swab samples to be collected prior to washing the stallion and collection of semen include the urethral fossa, urethra, shaft of the penis and prepuce. After semen collection, a second swab from the urethra is obtained and the semen itself is often cultured. The post-ejaculate urethral swab is considered to be similar to a culture of the semen. A positive post-ejaculate urethral culture suggests an infection of the urethra, accessory sex glands or epididymis.
The two main potentially pathogenic bacteria cultured from stallions are Pseudomonas aeruginosa and Klebsiella pneumoniae. Other microbial organisms may be cultured and are often considered normal nonpathogenic bacterial flora. To determine if the organism cultured is a true pathogen, mares bred by the stallion should also be cultured. If the mares do not become pregnant and the same organism is subsequently cultured from the uterus, the organism may be considered a significant pathogen. Conversely, if mares bred to a stallion that has a positive culture for Pseudomonas or Klebsiella become pregnant and do not become infected, the organism should be considered to be a non-pathogenic bacterium.
Taylorella equigenitalis, the bacterial organism causing contagious equine metritis, and the viral agents of equine infectious anemia, coital exanthema (equine herpesvirus-3), and equine viral arteritis are also potentially venereally transmitted. It is now common to initially screen stallions for exposure to equine arteritis virus by serologic testing for the presence of antibodies in a blood sample. Unvaccinated stallions with antibodies against equine arteritis virus (i.e. seropositive) are subsequently tested for the presence of virus in semen to determine if the horse is a chronic carrier or shedder of the virus.
Libido and Mating Ability
Libido when teased to a mare in estrus and ability to gain and maintain an erection are recorded. Subsequently, the ability to mount a mare or breeding phantom, insert the penis into the mare or artificial vagina, thrust, and ability to ejaculate completely are all noted.
Techniques and equipment for collection of semen is presented in another chapter. In a breeding soundness evaluation, semen is often collected in an artificial vagina twice, one hour apart. It is anticipated that 1) the total number of spermatozoa on the second ejaculate of most horses will be approximately one-half that of the first ejaculate, 2) the volume of the two ejaculates will be approximately equal, 3) the pH should remain the same or go up slightly, and 4) the spermatozoal motility should stay the same or increase.
Semen should be evaluated immediately after collection. Semen should only come in contact with clean materials that are maintained at 35° to 38° C (95° to 100° F). The total elapsed time from semen collection until the semen has been evaluated should be no longer than 10 to 15 minutes. If a delay in processing is unavoidable, the raw semen should be mixed with warm semen extender in order to protect the sperm cells until the evaluation can be completed. The most widely utilized semen extender is comprised of non-fat skim milk solids, glucose and an appropriate antibiotic.
Assessment of each ejaculate should include: 1) a gross evaluation of semen quality, 2) determination of semen pH, 3) determination of the volume of gel-free semen, 4) determination of sperm concentration, 5) calculation of the total number of sperm in the ejaculate (volume x concentration), 6) estimation of the percentage of sperm that are progressively motile, 7) determination of the percentage of morphologically normal spermatozoa in the ejaculate, and finally, 8) calculation of the number of progressively motile, morphologically normal sperm in the ejaculate.
Gross Evaluation of Semen Quality
The gel fraction of the semen, produced by the seminal vesicles, is removed using an in-line filter or by aspiration into a syringe and discarded without further evaluation. The gel-free semen is transferred into a warm graduated cylinder and initially evaluated for color and consistency. Color changes may be associated with the presence of debris, blood, urine, or purulent material (i.e. infection of the reproductive tract). Clarity or consistency of the sample can often be used as an initial assessment of sperm concentration.
A pH meter should ideally be used to determine the pH of raw semen. The normal pH of equine semen ranges from 7.2 to 7.7. The pH of normal stallion semen can be affected by season, frequency of ejaculation, and sperm concentration. Inflammation of the reproductive tract or contamination of the ejaculate with soap or urine can lead to an abnormally high pH.
The volume (ml) of gel-free semen is determined using a graduated cylinder. An accurate assessment of volume is critical for determination of total number of spermatozoa in an ejaculate. Volume of the ejaculate may be increased following excessive teasing of a stallion to a mare in estrus prior to collection. However, the concentration of sperm per ml is reduced, and as a consequence the overall number of sperm in the ejaculate is not affected.
Concentration of spermatozoa per ml in the ejaculate may be manually counted using a hemocytometer, estimated using a commercial calibrated spectrophotometer (Densimeter) or measured using a commercial cell counter (NucleoCounter). An accurate determination of sperm concentration is a critical parameter in the calculation of the total number of sperm in an ejaculate.
An accurate evaluation of sperm motility is one of the most important components of the breeding soundness examination. Unfortunately, sperm motility is not always highly correlated with fertility of a stallion. The motility of raw, gel-free semen may be difficult to evaluate accurately since the concentration is often too high to examine individual cells and sperm cells in raw semen tend to clump or agglutinate. Consequently, raw semen is usually diluted with warm extender to aid in the evaluation of sperm motility.
One drop of extended semen is placed onto each end of a clean glass slide and covered with a clean coverslip. The quality of the examination is enhanced if the glass slides and coverslips are maintained at 37° C using slide warmer. After each slide is prepared, it is examined microscopically at a magnification of 200X to 400X. The most accurate evaluations are performed using a phase-contrast microscope equipped with a heated stage warmer maintained at 37° C.
Generally, two motility estimates are determined when examining a semen sample, the percentage of sperm that are moving in any manner is recorded as the total motility, and the percentage of sperm that are moving in a forward manner is recorded as the progressive motility. Several computer-assisted semen analysis machines are commercially available that provide automated objective estimates of total motility, progressive motility, sperm velocity, and other motion characteristics. Computer-assisted semen analysis units provide rapid unbiased motility estimates of large numbers of sperm
Longevity of sperm motility is used as a crude indicator of ejaculate quality. Longevity can be determined by evaluation of motion characteristics over time for raw and extended semen samples maintained at either room temperature (20° to 25° C; 68° to 77° F) or cooled to refrigerator temperature (4° to 6° C; 39° to 43° F).
Examination of sperm morphology (or physical characteristics of individual spermatozoa) is an important component of a complete Breeding Soundness Evaluation. An eosin-nigrosin stain is a simple, one-step vital dye and is one of the most common stains used to evaluate sperm morphology. A drop of stain (approximately 50 µl) is placed adjacent to a similar sized drop of raw semen on one end of a glass slide. The stain and semen are mixed and then smeared along the length of the slide using a cover slip or another glass slide. The test slide is allowed to dry and then examined under oil immersion at 1000x magnification using a standard light microscope.
Morphological characteristics are evaluated for a minimum of 200 spermatozoa in each of two slides and the results averaged. The number of normal sperm and the number of sperm with specific morphologic defects are recorded. The most common defects observed in stallion spermatozoa are abnormal head, detached head, abnormal/broken neck, abnormal midpiece, proximal droplet, distal droplets, coiled tail, and kinked tail. Studies have reported that fertility in stallions is positively correlated with the percentage of morphologically normal sperm and inversely correlated with the percentage of sperm with abnormal heads, proximal droplets and abnormal midpieces.
Eosin-nigrosin has also been used as a ‘live-dead’ stain. Live sperm, with intact plasma membranes do not take up eosin and will appear white against a dark background, while dead sperm with damaged plasma membranes will take up eosin and appear red, pink or light purple against the dark nigrosin background (i.e. “red is dead”). In most stallions, the percentage of live cells as determined by vital dye exclusion is highly correlated with sperm motility. A discrepancy may suggest that a population of live immotile sperm is present in an ejaculate.
An alternative to the eosin-nigrosin stain for sperm morphology evaluation is to preserve semen in buffered formol-saline, prepare a wet mount slide, and examine the semen sample using a phase-contrast microscope or a differential interference contrast microscope.
Individual stallions may have semen with high progressive motility and acceptable pregnancy rates using fresh semen, but low pregnancy rates in a cooled-transported semen program. It is advantageous to test semen for motility characteristics following cooling. Semen is collected and aliquots are extended in two or more extenders and cooled in an appropriate passive cooling system designed for equine semen. The samples are evaluated for total and progressive motility at 24 and 48 hours after cooling. A test cool can identify an optimal extender for a given stallion and identify stallions whose semen does not cool well. Adjustments in breeding management procedures may allow some stallions with poor cooling characteristics to still be used successfully in a transported semen program. However, ultimately the only true test of fertility for a stallion in a cooled-transported semen program is pregnancy rates in mares bred.
Cryopreservation may dramatically alter motility characteristics and fertility potential of sperm from some stallions. It is usually not possible to predict ahead of time which stallions will freeze well and which stallions will not freeze adequately. Consequently, a test freeze is routinely recommended to determine which semen extender is optimal and the post-thaw motility characteristics for each extender. As with the test cool, semen is collected and aliquots are mixed with several different freezing extenders. Several straws of each sperm-extender combination are frozen and later thawed and evaluated. If it is determined that semen from the stallion has adequate post-thaw motility, subsequent freezes will be performed in the best extender identified in the test freeze. As noted previously, motility of semen after thawing is not necessarily correlated with future pregnancy rates. Some stallions have very good post thaw semen motility, but very poor pregnancy rates in mares bred with the frozen semen.
The presence of an adequate number of motile spermatozoa in an ejaculate is no guarantee of fertility. If results of a standard breeding soundness examination do not identify a cause of subfertility or infertility, additional diagnostic tests may be indicated.