One of the most valuable tools a stallion owner or manager can use to assess reproductive health is a breeding soundness examination (BSE). A routine BSE provides insight into a stallion’s reproductive status and helps determine how many mares he can reasonably breed in a season and if the stallion is a candidate for cooled or frozen semen breeding. An annual BSE allows managers to adjust book size, monitor changes, and identify problems prior to fertility issues. Additionally, a BSE may be a necessary component of insurance coverage on a breeding stallion and is a strongly recommended component of the prepurchase examination performed when a stallion is a current or future breeding prospect.
How to perform a stallion breeding soundness exam
There are multiple components to a full BSE. Initially, a complete physical examination is performed including evaluation of body condition, soundness, and neurological health; it’s important to look for any pain or instability that may interfere with mounting or ejaculation. Reproductive examination starts with reproductive history. A detailed breeding history is often overlooked but provides essential context on age and prior breeding performance, methods of breeding (live cover, fresh, cooled, frozen), prior pregnancy and foaling rates, and prior pathologies such as infections or known fertility issues.
The collection process begins with exposure of the stallion to a tease mare for assessment of sexual behavior, evaluation of external genitalia anatomy, and health including the stallion’s ability to achieve and maintain erection. A stallion is expected to show prompt interest in the tease mare, achieve erection quickly, and mount the mare or breeding phantom as directed. When stimulated, ejaculation is expected within one or two mounts. Delays may indicate pain, behavioral issues, problems with the environment or equipment preparation. While protocols vary and are influenced by the individual stallion, the goal is to assess daily sperm output (DSO) and overall reproductive health as an indicator of fertility.
- Frequent collections (variable – potentially daily for five to 10 days or every other day for three weeks) provide the most accurate DSO.
- Alternative protocol: Two ejaculates collected one hour apart after sexual rest. The second ejaculate should contain about 50% of the first if ejaculation is complete.
Semen collection methods
Prior to collection, all equipment that the ejaculate will contact should be stored in a clean environment and warmed to body temperature prior to use. Many facilities have an incubator for the collection bottle, filters, and lubricant used during collection. An artificial vagina (AV) should be prepared for collection. Following every collection, the AV should be washed, disinfected, and rinsed. The AV must be dry prior to next collection to avoid water contact with sperm.
An artificial vagina (AV) is often used to collect a stallion for semen analysis and processing. This may require a mount mare or breeding phantom for the stallion to comfortably mount. Generally, the stallion and handler will enter the breeding area and introduce the stallion to a mare in estrus to evaluate his sexual behavior and achieve erection. The stallion is ideally washed prior to collection. A technician will rinse the penis and urethral sinus with warm water and allow to dry. Teasing may be repeated if necessary. Upon mounting, the penis is deflected into the AV by the collector. It is critical that the AV is warm (44-50°C) and of the appropriate pressure for the individual stallion adjusted at the time of collection. The AV should also have enough nonspermicidal lubricant applied to reduce friction during collection. Too much lubricant should be avoided to reduce excess running into the collection bottle.
Collection tools and terms:
- Mount mare: Mare displaying estrus, teasin`g, or an ovariectomized mare. Exogenous estrogen can be used to augment sexual behavior but is often not necessary.
- Breeding phantom: A consistent, stable, and safe mount.
- A condom is recommended for stallions that refuse to breed the AV or back off prior to ejaculation. Typically, these are stallions that have been used exclusively for natural service.
- Vagina aspiration is used following natural mating. It is not recommended for BSE evaluation due to the inability to assess all parameters and contamination from the mare’s reproductive tract.
- Ground collection can be considered when medical conditions prevent a stallion from mounting.
- Pharmacological induction of ejaculation can be considered when other attempts fail or if a stallion is unable to mount for medical reasons. Not recommended for BSE evaluation due to the inability to assess all parameters.
Testicular evaluation
Dependent on stallion demeanor, testicular evaluation can be performed prior to or after semen collection, allowing sedation. Palpation and ultrasonography can be utilized to evaluate testicular size, shape, and consistency. Measurement of testicular size volume (via calipers or ultrasound) is important to record any abnormalities and calculate daily sperm output (DSO).
Testicular measurements help estimate DSO and could be tracked during breeding season.
- The length (L), width (W), and height (H) of the testicles can be used to calculate the volume of each testicle using the equation: 0.523 (L × W × H) = Testicular Volume.
- The stallion’s daily sperm output (DSO) can then be predicted by the equation: Total Testicular Volume × 0.024 – 0.76 = DSO.
Semen evaluation
Gross evaluation
The first step is to separate the gel fraction and visualize the sperm-rich component of the ejaculate: raw semen. The gel is a normal component of the ejaculate produced by accessory sex glands. It is removed to allow semen to be analyzed. If a semen sample is highly viscous, additional filtration to remove gel can be useful. Next, record the color and consistency of the semen. Raw semen should be gray or white in color dependent on sperm concentration. Sometimes semen contains urine, blood, debris and/or pus that cause a change in color or consistency. Any abnormalities may highlight the need for additional testing.
The pH of the raw semen may also be recorded at this stage. The normal pH of stallion semen is 7.2-7.7. Abnormally high pH can be associated with urine contamination or infection.
Volume
The gel-free volume of the ejaculate is often measured using a graduated cylinder or conical tube. Again, any equipment used should be sterile and warmed to body temperature (37°C) prior to raw semen exposure. Another method to accurately measure volume is to weigh the semen. The container used for collection can be preweighed and subtracted from the total weight after collection to establish raw semen weight. This is then converted to volume: 1gram = 1milliliter.
Concentration
The sperm concentration is often expressed as millions/milliliter (e.g., 300 x 106/mL) and measured accurately using calibrated devices such as the hemocytometer, densimeter, or Nucleocounter®. The volume and concentration are multiplied to calculate total sperm count, recorded in billions. For example, a raw semen volume of 20 milliliters, concentration of 300 x106/mL would equate to total sperm count of six billion (6.0 x109) for that collection.
Motility
Semen is evaluated under a heated microscope stage to assess total and progressive movement. The raw semen should be diluted in extender to a concentration of 25 million per milliliter. This dilution allows visualization of individual sperm motility. Motility should be estimated once the sperm has been in the extender for 5-10 minutes and maintained at 37°C. A computer-assisted sperm analysis (CASA) machine is frequently used to track and record individual sperm cell motility parameters. It should be noted that variability does exist between technicians and individual CASA instrument settings. While motility parameters are often stated when giving an evaluation of semen quality, it is a single test in the entire process and represents only one component of sperm function. While correlations exist, sperm motility parameters alone cannot predict fertility.
Morphology
Sperm are examined using a specialized stain and/or under a specialized microscope to determine the percentage of normal versus abnormal cells. Generally, 100-200 sperm cells are counted and the number of normal sperm and sperm with abnormalities of the head, midpiece, and tail recorded as a percentage of the total sperm counted. Sperm with multiple abnormalities are recorded to have both but represent a single cell in the total, thus the percentages don’t always add up to 100%. Abnormal cell populations are associated with various causes of subfertility. Often, additional testing, monitoring, or management strategies are necessary if a critical percentage of sperm cells are determined abnormal.
Bacteriology
Bacteria are naturally present in most organs of the body in a balanced microbiome. The penis and prepuce in the stallion provide an ideal environment to cultivate bacteria. Hygiene during semen collection thus becomes important to ensure bacterial exposure in the semen is limited. This may include washing the penis and prepuce with water prior to collection. Soap and detergents should be avoided as they may disrupt the natural microbiome and allow opportunistic bacterial overgrowth and infection. To rule out infection as a cause of subfertility, cultures may be taken from semen and reproductive anatomy (prepuce/penis, urethral sinus, urethra, semen) to identify potential sexually transmitted pathogens. Cytology is an additional quick diagnostic to determine if semen is contaminated with bacteria or if an infection is present. If bacteria are identified, breeding strategies can be adjusted to reduce risk of infection to the mare.
Advanced and additional testing
In addition to the routine BSE, we recommend that a stallion scheduled to collect and ship cooled semen undergo a chilled semen evaluation. Not all extenders are created equally, and not all stallions will chill equally well in every extender. Semen can be collected and divided into breeding doses using different extenders available. The semen is chilled as normal and evaluated every 12-24 hours to determine the extender providing the best environment for sperm longevity.
For unexplained fertility problems, specialized tests may be performed beyond the scope of a routine BSE. These tests may include a longevity analysis, hypo-osmotic swelling test (HOST), live-dead staining, and flow cytometry fluorescent analysis of individual components of the sperm including acrosomal function, mitochondrial function, and chromatin structure.
While a BSE cannot predict exact fertility rates, the exam helps identify reproductive problems, guide breeding management decisions including appropriate mare book size, and track changes in reproductive health and success over time. It is a valuable procedure for anyone who owns and manages a breeding stallion.
To schedule a breeding soundness exam for your stallion, email the Equine Reproduction Laboratory at [email protected].