Given the high economic value of some stallions and the willingness to breed horses based more on their competitive potential than on their breeding soundness, many resources and expensive tests are sometimes used to evaluate breeding soundness, and especially to assess sperm function in those stallions proven subfertile after their first breeding season or displaying potential defects after a routine breeding soundness examination (BSE). Moreover, the extent of a BSE may also be determined by the intended use of the stallion regarding factors such as expected mare book size, or whether the stallion will be used for natural service or in an artificial insemination program. Additional tests may be required if the stallion semen is to be cool-transported and/or frozen-thawed.
Classically, a complete BSE of the stallion has comprised a history, general physical examination, examination of the external and internal genitalia, culture swabs of the urethra and penis, assessment of libido and breeding ability, and collection and evaluation of at least two ejaculates collected 1 hour apart.
Regardless of the circumstances of a particular stallion, the first step should always be attaining an accurate history that should include prior breeding results and/or BSEs, previous use, nutrition, vaccination status, and disease history. Racing stallions or those in training may have been receiving anabolic steroids or other drugs. Therefore, stallions recently retired from a performance career may be evaluated again in 3–6 months if they fail the initial BSE.
During the physical examination, the stallion should be evaluated for general body condition and the presence of any conditions that might interfere with breeding (ie, lameness or back problems). Genetically inherited defects, including parrot mouth and cataracts, render the stallion unfit for breeding. Blindness, lameness or ataxia, penile paralysis, or other defects that prevent the stallion from breeding also render it unsatisfactory as a breeding prospect. Recent evidence supports cryptorchidism as a potentially inherited defect in horses, thus rendering the stallion an unsatisfactory prospective breeder. Common laboratory tests such as Coggins test, complete blood count, serum chemistry, and urinalysis can be performed to support the general health of a stallion.
Scrotal palpation should be performed and may be done after the first ejaculation when the stallion may be more relaxed. The scrotum should be thin and elastic with a distinct neck. The scrotum is normally pendulous; however, it may be voluntarily drawn towards to the body during examination. The testes with their respective epididymides should be freely movable within the scrotum. The testes should be firm, resilient, and homogeneous on palpation. The epididymides run dorsally along the testes, in a craniocaudal orientation, with the tail most caudad. The tail of the epididymis is easily palpable, whereas the body and head of the epididymis tend to blend with the dorsal surface of the testis. The caudal ligament of the epididymis is palpable as a small (1–2 cm) nodule adjacent to the tail of the epididymis and serves as a landmark to determine testicular orientation within the scrotum. Rotation of one testis 180° (the tail of the epididymis being palpated on the cranial aspect of the testis; also called torsion of the spermatic cord) is common and has no clinical importance in healthy stallions.
Ultrasonography is also commonly used to evaluate scrotal contents and to take individual measurements of each testis. A portable console fitted with a linear 5 MHz transducer typically used for reproductive evaluation of mares can be used. The testes should have a homogeneous echoic appearance; a cross or longitudinal section of a central vein that runs along the longitudinal aspect of the testis starting at the cranial pole is a hallmark of the normal equine testis and should not be confused with pathology. The tail of the epididymis and spermatic cord display a homogeneous “cheesecloth-like” appearance; asymmetric dilations of the vessels of the spermatic cord may be consistent with varicose veins and may or may not affect blood flow and testis function.
Testicular size is highly correlated with daily sperm output (DSO). Total scrotal width has been typically used to assess testicular size using a blunt caliper across both testes pulled together to the bottom of the scrotum. This should measure > 8 cm (preferably > 9 cm) for a stallion to be considered satisfactory. Nowadays, individual testis measurements obtained via ultrasonography are considered more accurate as way to estimate expected DSO. The width (W), height (H), and length (L) of each testis can be used to calculate testis volume using the formula: testis volume (TV) = (W × H × L) × 0.5233 for each testis, and total testes volume (TTV) is the sum of the TV of each testis. Then, DSO can be estimated using the formula: DSO (109) = (0.024 × TTV) − 0.76.
Courtesy of Dr. Sylvia Bedford-Guaus.
Courtesy of Dr. Sylvia Bedford-Guaus.
The penis is usually examined in the tumescent state while it is washed before the first semen collection. The penis can vary in size with no effect on fertility. It should be freely extensible from the sheath without lesions. Particular attention should be given to the fossa glandis and urethral process during washing. Lesions within the fossa glandis can lead to hemospermia. Common penile lesions include those induced by trauma (ie, during breeding or semen collection) as well as those produced by equine coital exanthema, Habronema spp infection, squamous cell carcinoma, sarcoids, and papillomas.
Courtesy of Dr. Sylvia Bedford-Guaus.
Courtesy of Dr. Sylvia Bedford-Guaus.
Of the internal genitalia of the stallion, the ampullae, vesicular glands, and lobes of the prostate gland are palpable per rectum. The vesicular glands are difficult to palpate unless the stallion is first teased to a mare in estrus to stimulate filling of the glandular lumina with fluid. The bulbourethral glands are covered by muscle, making it impossible to palpate their structure.
Transrectal ultrasonography can be used to more thoroughly evaluate each of the accessory sex glands and pelvic urethra. Because of the danger inherent in adequately restraining a stallion, some veterinarians perform a transrectal examination only if deemed necessary because of an abnormal finding on the remainder of the examination, such as blood or pus in the ejaculate. When performing palpation per rectum on a stallion, the internal inguinal rings should also be palpated to determine their size and presence of any abnormalities. They are felt as flaps of peritoneum that form pockets in the abdominal lining at the 3 o’clock and 9 o’clock locations at the entrance of the pelvis.
Evaluation of sexual behavior and breeding ability is of crucial importance as it will determine the ability of a given stallion to deliver an ejaculate to a mare or artificial vagina. Libido can be evaluated by exposing a stallion to a mare in estrus. A stallion with good libido will immediately show interest as evidenced by restlessness, pawing, vocalization, and precopulatory activity characterized by sniffing and licking at the mare's genitalia, as well as exhibiting the flehmen response (curling of the upper lip to stimulate the vomeronasal organ). Once the stallion mounts a mare or phantom for semen collection, the ability to normally thrust and ejaculate can also be assessed. A stallion with normal libido will usually display 3–5 strong copulatory thrusts before ejaculation. Each ejaculatory jet (5–7) can be evidenced by feeling the corresponding pulses at the base of the penis as well as by the "tail flagging" (the tail moves up and down with each jet).
Semen is collected from the stallion using an artificial vagina (AV) filled with water at ~50°C (~122°F), which typically cools to 42°–45°C (107.6°–113°F) by the time of collection; some stallions might be accustomed to higher AV temperatures (50°–55ºC [122°–131°F]) for adequate stimulation; however, the internal temperature of the AV should not exceed 55ºC (131°F) at the time of semen collection.
The stallion is teased to an estrous (or ovariectomized) mare; once an erection is achieved, the penis is washed with warm water and blotted dry with a paper towel. If the penile skin is suspected to harbor potentially transmissible bacteria (eg, Klebsiella pneumoniae or Pseudomonas aeruginosa), a culture swab of the sheath and fossa glandis should be obtained before the penile area is washed and rinsed for semen collection. Smegma can fill the dorsal urethral diverticulum, located dorsal to the urethral process, and harden to form a “bean.” This can cause irritation and swelling and should be removed. After the penis is washed and dried, a swab sample of the distal urethra is obtained. The stallion is then allowed to mount the mare or phantom (breeding dummy), and the semen is collected by diverting the penis into the AV. A second swab sample is taken from the distal urethra immediately after ejaculation. The stallion should be given 1 hour of rest before the second ejaculate is collected.
The culture results of the urethral swab samples can be difficult to interpret. Prewash penile cultures usually show moderate to heavy growth of mixed bacteria. Pre-ejaculate urethral swabs also may reveal growth of a mixed bacterial population. Growth of Pseudomonas or Klebsiella may indicate that the penis has been colonized by these organisms; in the US these are the only bacteria (besides Taylorella equigenitalis, the cause of contagious equine metritis) that, in some cases, may be passed to mares and cause endometritis. The postejaculate urethral swab should produce less bacterial growth, because the urethra has been “washed” by the ejaculate. High numbers of bacteria, especially of a single species, on the postejaculate swab may indicate infection of the internal genitalia, most commonly the urethra and/or seminal vesicles.
The ejaculate should be evaluated for gross appearance, volume, sperm concentration, sperm motility, percentage of morphologically normal sperm, and percentages of specific spermatozoal morphologic abnormalities. The ejaculate should be free of pus (pyospermia), urine (urospermia), or blood (hemospermia). The normal ejaculate may contain gel, a viscous, clear to cloudy material, that originates from the seminal vesicles and forms the third and last fraction of the ejaculate. Because it is ejected last, the gel fraction can be removed by having an in-line filter at the mouth of the collection bottle in the AV (preferred), or alternatively, by pouring the ejaculate through a milk filter once in the laboratory.
The analysis of the semen is done on the gel-free (sperm-rich) fraction. Concentration may be determined using a hemocytometer or a properly calibrated photometric instrument. Several instruments specially designed for this purpose are commercially available. Sperm motility and morphology evaluations are performed as described for the bull; however, the sperm concentration is much lower in stallions (typically 100–400 million/mL), so only individual sperm motility is evaluated. Assessment of motility should be performed with both raw semen and semen diluted with a good quality extender. After initial extension, a high percentage of sperm may exhibit a circular motility pattern which should resolve after 5–10 minutes of equilibration in the extender. Semen should be warmed to 35°–37°C (95°–98.6°F) before assessing spermatozoal motility.
Notably, conventional measures of sperm quality correlate only moderately with fertility, and variations in the percentages of motile/progressively motile and morphologically normal sperm account for only 20% of the total variation in fertility. Given the limitations of standard sperm tests in predicting fertility or identifying subfertile stallions, other sperm tests have been evaluated and may be applied for stallion BSEs performed in referral centers. Although not necessarily providing a better correlation with fertility, CASA offers a more objective and complete assessment of sperm motion characteristics. As in bulls, certain changes in different motion parameters, namely increases in curvilinear velocity and decreases in linearity and straightness, correlate with the acquisition of hyperactivated motility, which has been recently characterized in stallions. In addition, computer-assisted sperm head morphometry (shape) assessment has been suggested as a useful adjunctive test to predict stallion fertility.
The percentage of morphologically normal sperm is evaluated under 1000× magnification using a formalin-fixed wet mount (phase contrast) or stained smear (light microscopy). At least 100 cells should be counted and the percentage of distinct abnormalities recorded. These include head abnormalities, detached heads, cytoplasmic droplets (proximal and distal), bent midpieces, hairpin bents, and coiled tails, among the most frequent abnormalities. Abaxial midpiece insertion is considered normal in the stallion.
Membrane integrity can be easily evaluated via eosin-nigrosin staining (as in bulls). The hypoosmotic swelling test evaluates the ability of sperm to swell to establish an osmotic equilibrium with a hypoosmotic surrounding medium, and may also hold some value for sperm membrane evaluation; however, a direct correlation with fertility has not been established. Fluorescent probes such as rhodamine 123 or JC-1 in conjunction with flow cytometry have also been used to evaluate mitochondrial potential and hence metabolic integrity. In addition, the combination of these with other fluorescent stains offers the advantage of concomitantly evaluating viability and chromatin integrity in a large number of sperm. In this regard, the sperm structure chromatin assay, which assesses sperm chromatin stability, has been thoroughly researched in stallions and is moderately correlated with fertility. It is a good adjunctive test for stallions in which routine laboratory test results do not correlate with subfertility or infertility (see Ancillary Sperm Tests).
Finally, true tests of sperm function (ie, ability to fertilize an oocyte) have been restricted because of the inability to successfully capacitate stallion sperm in vitro or to perform in vitro fertilization in this species. The acrosomal responsiveness assay has been reported as a method to further evaluate sperm function in stallions. That is the ability of the sperm acrosome to react when challenged in vitro with calcium ionophore. This is useful because a subset of stallions may display normal sperm motility, morphology, and chromatin quality while still being sub- or in-fertile. In some of these, the only abnormality found is the inability of their sperm to undergo acrosomal exocytosis under laboratory conditions.
In addition to assessing sperm quality, once two ejaculates have been collected, the total number of spermatozoa in each is calculated as volume (gel-free) × concentration. The total number of sperm in the second ejaculate of two collected 1 hour apart is considered a rough estimate of the daily sperm output for that stallion (ie, ejaculates are considered to be representative if the second ejaculate contains about half the number of sperm as the first). The second ejaculate should have about the same, or a slightly higher, percentage of morphologically normal sperm, as well as the same or better motility than the first.
If sperm numbers or quality differ from this guideline, then either prolonged sperm storage in the excurrent ducts has occurred, or one of the ejaculates was not complete, and a third ejaculate should be obtained. The third ejaculate should have about half as many sperm as the second ejaculate, and the same or better morphology and motility. A third ejaculate may also be collected if the sperm evaluation does not appear to agree with the total scrotal width (eg, high sperm numbers from a stallion with small testes) or the calculated daily sperm output.
Prolonged storage of sperm in the excurrent ducts after prolonged sexual rest results in high numbers in the initial ejaculate; however, these sperm may have poor motility and morphology. Some stallions with extreme sperm storage require daily collection for 7–10 days before representative ejaculates are obtained (sperm evaluation results are consistent on successive collections). In extreme cases, sperm may accumulate in the ampullae and inspissate, causing blockage of the ductus deferentes. This may result in no sperm or few sperm, typically with detached heads, present in the ejaculate. Multiple attempts at collection may be necessary before the blockage is cleared. Relief of the blockage is typically evident by the ejaculation of very large numbers of dead sperm with detached heads.
In cases of severe ampullary blockage or in stallions with ejaculatory problems, attempts at semen collection might initially produce only clear seminal fluid. In such instances, it might be warranted to differentiate between these problems and azoospermia (ie, lack of sperm production) before successive semen collection attempts. For this purpose, collected seminal fluid can be submitted for determination of alkaline phosphatase levels. Because alkaline phosphatase concentrations are high in epididymal fluid, a value < 100 U/L is consistent with blockage or ejaculation failure, whereas a value > 1,000 U/L is consistent with collection of epididymal fluid and thus true azoospermia.
In a satisfactory potential breeding stallion, sperm numbers after > 5 days of sexual rest should be ≥ 8–10 billion in the first ejaculate and ≥ 4 billion in the second ejaculate. Total spermatozoal motility should be ≥ 65%, and progressive motility ≥ 50%. At least 50% of the sperm should be morphologically normal. A stallion is considered satisfactory if it produces at least 1 billion progressively motile and morphologically normal sperm in the second (or third) ejaculate of two (or three) collected 1 hour apart.
Stallions are classified as “satisfactory,” “questionable,” or “unsatisfactory” potential breeders based on the results of the above detailed examination. However, classification can be somewhat subjective, and an excellent finding in one category can balance a marginal finding in another. Satisfactory potential breeders should achieve a seasonal pregnancy rate of > 80% when bred to 50 mares by natural breeding or to 120 mares by artificial insemination under normal management conditions. Questionable potential breeders may experience difficulty in doing the above. Typically, stallions are placed in this category if a problem is detected that might resolve over time with or without treatment; thus, a recheck is recommended within 6–12 months. Unsatisfactory breeders have problems that may profoundly decrease their fertility or have undesirable heritable traits that may be transmissible to their offspring.
Some stallions may be used to inseminate a percentage of mares with cooled-transported semen. Under these circumstances, longevity of spermatozoal motility should be tested using commercial semen containers before a decision is rendered regarding fertility potential. Additionally, in horses, pregnancy rates with frozen-thawed semen are not only suboptimal but highly stallion-dependent. Therefore, a test freeze-thaw is essential before using or commercializing frozen semen from a given stallion. Notably, adequate post-thaw sperm motility and morphology is not always correlated to good fertility results even under optimal breeding management conditions.
