Get ready for the breeding season: reproductive evaluation of the stallion

By Patrick McCue, DVM, PhD, Diplomate American College of Theriogenologists
Colorado State University

The 2019 equine breeding season is almost here. One way to help ensure a successful start is to have a reproductive evaluation performed on your stallion(s) prior to the onset of the season. The goals of the reproductive evaluation would be:
Confirm that the stallion has adequate libido, can physically mount a mare or breeding phantom, ejaculates normally, has adequate semen parameters, etc.
Confirm that the stallion does not have any significant reproductive abnormalities or identify reproductive issues that may reduce fertility potential
Acquire data that may be helpful in development and/or implementation of a breeding management plan tailored to each stallion
Specific components of the stallion evaluation are detailed below. In some instances collection and evaluation of a single ejaculate may be sufficient. In other cases, a more traditional reproductive evaluation of two ejaculates collected approximately one hour apart may be optimal. Finally, some stallions accumulate large numbers of spermatozoa in their reproductive tract and multiple repeated collections over several days may be necessary to interpret the stallion’s daily sperm output after the ‘clean-out collections’ have been performed.


Each stallion should be properly and positively identified prior to evaluation.  Registration name and number, age, and breed should be recorded. Coat color and markings, lip tattoos, microchip information (if present), brands, hair whorls, permanent scars, and all other identifying features should be noted. A series of digital photographs (left side, right side and head) may be valuable as part of the permanent medical record.   

Reproductive History

A complete breeding history should be obtained, as past breeding results (pregnancy rates per cycle and per season and foaling rates) are the best indicators of a stallion’s previous fertility level. The number and types of mares bred per season (i.e. maiden, barren, open and foaling mares) and the breeding method used (i.e., hand breeding, pasture breeding or artificial insemination with fresh, cooled or frozen semen) should be recorded. 

Results of previous reproductive evaluations, medical conditions, vaccination history (especially vaccination against equine arteritis virus), and other significant information should be noted. It is also important to record the intended use of the stallion and the number of mares anticipated for the next season, as it will be important for breeding management recommendations. 

Physical Examination

A general physical examination should be performed. To be considered fertile, a stallion must be able to mount a mare in estrus, insert his penis into the vagina or artificial 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. Any evidence of heritable or congenital conditions should also be reported. A blood sample should be collected and serum tested for presence of antibodies against equine arteritis virus (EAV) unless the stallion has previously been vaccinated.

It is recommended that all stallions be vaccinated against EAV on an annual basis. Stallions to be vaccinated against EAV for the first time should have a blood sample collected and evaluated for the presence of antibodies. Seronegative stallions should be vaccinated and documentation of pre-vaccination EAV antibody status and vaccination history retained. Seropositive stallions without a history of vaccination should have semen tested to determine if the stallion is shedding virus in his semen.
Examination of External Genitalia

The penis, prepuce, sheath, scrotum, and scrotal contents should all be evaluated. Inspection of the penis and prepuce is often performed after the stallion has either ‘dropped’ his penis or gained an erection subsequent to teasing a mare in estrus. The penis should be of normal size, shape and be free of lesions. 

Palpation of the testes and epididymides may be performed after semen collection, as the stallion will be less excited which will usually 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 located at the caudal pole of each testis. The spermatic cord should be identified on the cranial dorsal aspect of each testis.

Estimation of Daily Sperm Production

Testes of mature stallions are normally capable of producing approximately 18 to 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 (TSW) 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 testis size, testis weight and consequently daily sperm production potential. 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.

A more accurate method of determining potential sperm production is by measurement of total testicular volume (in cm3). Testicular volume (TV) is calculated by measuring the length, width and height of each individual testis using ultrasound and then applying the measurements into a formula for calculating the volume of an ellipsoid.  Adding the volume of each testis together will yield total testicular volume, which can subsequently be used to estimate potential daily sperm production.

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.

Ultrasound Examination of the Reproductive Tract

Ultrasound examination may be performed on the testes and other scrotal contents (i.e. epididymides, spermatic cord, etc.) to detect abnormalities or characterize fluid present within the vaginal cavity of the scrotum. Normal testis should be uniformly echogenic and the central vein or mediastinum may be observed on ultrasound as a hyperechogenic structure located just off the middle of each testis. Understanding normal ultrasonographic architecture of the normal testis is helpful when examining a stallion with low fertility, low libido, low sperm production, an enlarged testis, enlarged scrotum or when attempting to locate a cryptorchid testis.

There is normally a very small volume of non-echogenic fluid present within the vaginal cavity of the scrotum (i.e. between the scrotal wall and the testes). The fluid may or may not be readily visible on ultrasound examination. An increased volume of non-echogenic fluid is suggestive of a hydrocele, while the presence of echogenic fluid is suggestive of a hematocele or, potentially, an inflammatory/infectious condition.

The spermatic cord and epididymides may also be imaged by ultrasound. The spermatic cord runs from the dorsal-cranial aspect of the testis proximally upward into and through the inguinal canal. Ultrasonographically, a cross-sectional image of the spermatic cord shows a variable pattern of echogenicity due to the presence of various tissue types from the external cremaster muscle to the highly vascular pampiniform plexus.

The head and body of the epididymis are challenging to image with ultrasound. However, the tail of the epididymis should be readily visible adjacent to the caudal pole of each testis. On ultrasound, the epididymal tail has a ‘Swiss Cheese’ appearance due to the highly coiled tubular structure filled with fluid and spermatozoa.

Examination of Internal Genitalia

Transrectal palpation and ultrasound examination of the internal genitalia of a stallion per rectum is not always performed as part of a routine reproductive evaluation, unless indicated by an abnormal history, physical examination findings, or abnormal semen parameters. Examples of clinical situations in which transrectal palpation and ultrasonography of the reproductive tract of a stallion may be indicated include:

§ Hemospermia
§ Presence of white blood cells (WBC) in the semen
§ Azoospermia or oligospermia

Evaluation for Bacterial Infection of the Reproductive Tract

Microbiological culture swabs are routinely collected as part of a complete stallion breeding soundness evaluation. Swab samples to be collected prior to washing the stallion and collection of semen include the shaft of the penis, urethral fossa, pre-ejaculatory urethra and post-ejaculatory urethra. Additional cultures may be collected from the prepuce prior to ejaculation and the actual semen sample after collection. In most instances the post-ejaculate urethral swab is considered to be similar to a culture of the semen. 

A positive post-ejaculate urethra or semen culture suggests an infection of the urethra, accessory sex glands, epididymides or testes. The most common venereally transmitted bacteria in stallions are the Gram-negative organisms Pseudomonas aeruginosa and Klebsiella pneumoniae. Another important bacterial pathogen that may be transmitted in semen is Taylorella equigenitalis, the causative organism of contagious equine metritis (CEM).

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 also noted.

Semen Evaluation

Semen is often collected in an artificial vagina twice, approximately one hour apart as part of a stallion breeding soundness evaluation. Assessment of each ejaculate should include:

   § Gross evaluation of semen appearance
   § Gel-free volume
   § Sperm concentration
   § Total and progressive sperm motility
   § Sperm morphology
   § Calculation of the total number of sperm in the ejaculate
   § Calculation of the number of progressively motile, morphologically normal sperm in the ejaculate.

Additional tests may include:

   § pH of the semen
   § Osmolality of the semen

It is anticipated that: 1) total number of spermatozoa on the second ejaculate of most horses will be approximately one-half that of the first ejaculate, 2) volume of the two ejaculates will be approximately equal, 3) pH should remain the same or go up slightly, and 4) spermatozoal motility should stay the same or increase. Semen parameters of the second ejaculate are commonly used as an estimate of daily sperm output (DSO). The actual determination of daily sperm output is made by collection and evaluation of semen samples over a 5 to 7 day period.

Gross Appearance of the Semen

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 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). 

Normal stallion semen is white to slightly off-white in color. Brown to gray discoloration is suggestive of contamination with dirt or debris.Yellow discoloration is suggestive of urine contamination (urospermia) and red discoloration is suggestive of blood contamination (hemospermia).

Evaluation of Semen pH 

Ideally a pH meter should be used to determine the pH of raw semen. pH paper is not accurate and therefore not informative. 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.  

Semen Volume

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 increase following excessive teasing of a stallion to a mare in estrus prior to collection. However, in this situation the concentration of sperm per ml is usually reduced, and as a consequence the overall number of sperm in the ejaculate is not significantly affected.

Sperm Concentration

Concentration of spermatozoa per ml in the ejaculate may be manually counted using a hemocytometer or estimated using a commercial calibrated spectrophotometer (i.e. Densimeter; Figure 1). 
Figure 1. Estimation of stallion sperm concentration using a spectrophotometer (Densimeter Model 590B; Animal Reproduction Systems). The Densimeter can accurately estimate sperm concentration in raw un-extended semen, but cannot be used when semen is diluted into an opaque extender.

Sperm Motility

An accurate evaluation of sperm motility is one of the most important components of a semen evaluation. The motility of raw (i.e. unextended), gel-free semen may be difficult to evaluate accurately since the sperm concentration is often too high to examine individual sperm motility and equine sperm in raw semen tend to clump or agglutinate. Consequently, raw semen is usually diluted to a concentration of 25 to 50 million sperm per ml with warm extender to aid in the evaluation of total and progressive motility. Two motility parameters are determined when examining a stallion semen sample:
Total motility refers to the percentage of sperm that exhibit any type of movement, and include sperm moving in a straight line, circling, and /or twitching
Progressive motility refers to sperm that exhibit forward motility across the microscope field or are moving in a large circular path
Normal fertile stallions should have a minimum of 60 % progressively motile spermatozoa.

Motility of stallion spermatozoa may be estimated by standard visual assessment (i.e. manually) or by a computer assisted sperm analysis (CASA) system. Visual assessment of sperm motility is usually performed at 200 to 400x (i.e. a 20x or 40x objective x 10x eyepiece). A small drop of extended semen is placed on a war clean glass slide and covered with a warm clean coverslip. The slide is then placed on the stage of a good quality light microscope, preferably equipped with a stage warmer. Estimates of total and progressive motility are evaluated after evaluation of multiple visual fields.

Sperm Longevity

Evaluation of sperm survival over time or longevity of sperm motility is used as another 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) or cooled to refrigerator temperature (4 to 6° C).

Sperm Morphology 

Sperm morphology can be determined after staining with an eosin-nigrosin stain, which has also been used as a ‘live-dead’ stain.  An alternative is to preserve semen in a buffered formalin-saline solution, prepare a wet mount slide, and examine the semen sample using a phase-contrast microscope or a differential interference contrast (DIC) microscope. A fertile stallion should have a minimum of 60 % morphologically normal spermatozoa in each ejaculate. Morphologic evaluation of stallion sperm should include the following categories:

   § Normal sperm
   § Abnormal acrosome
   § Abnormal head
   § Detached head
   § Proximal droplets
   § Distal droplets
   § Abnormal mid-piece
   § Bent/coiled tails

The presence of other cells, such as germ cells, white blood cells, red blood cells, etc. should also be recorded. 

AncillaryTests or Procedures

If results of a reproductive evaluation do not identify a cause of subfertility or infertility, or if one of the standard examination procedures is abnormal, additional diagnostic tests may be indicated (Table 1). 

Table 1.  Diagnostic tests that may be performed in addition to the standard tests in a stallion breeding soundness evaluation.
Test Indication
Sperm Chromatin Structure Assay (SCSA) Evaluate structural integrity of sperm chromatin by determining the susceptibility of sperm DNA to denaturation in vitro
Fluorescent Stains Evaluation of plasma membrane integrity (5-CFDA and PI), viability (SYBR-14), acrosomal integrity (PSA) or mitochondrial function (JC-1 and rhodamine 123)
Hypo-osmotic Swelling Test Evaluation to determine if sperm plasma membrane is intact and osmotically active
Sperm Capacitation Test Determine the percentage of sperm that are capacitated and able to undergo the acrosome reaction (CTC assay and merocyanine 540)
Acrosome Reaction Tests Determine the percentage of acrosome-intact and acrosome reacted sperm in untreated semen (FITC-PNA) and to evaluate the ability of spermatozoa to undergo the acrosome reaction in vitro (calcium ionophore A23187)
Progesterone Receptor Exposure Determine the proportion of sperm with progesterone receptors and therefore capable of zona binding and ZP-mediated acrosome reaction
Sperm-Zona Binding Assay Evaluate the ability of sperm to bind to the zona pellucida of an oocyte
Glass wool-sephadex filtration Identify the percentage of sperm that are capacitated or have plasma membrane or acrosome damage
Transmission Electron Microscopy Evaluation of ultrastructure detail of spermatozoa and to evaluate acrosome status of sperm
Chromosome Analysis Evaluate numeric or structural changes in chromosomes
Testicular Biopsy Histologic evaluation of testicular parenchyma
Hormone Analysis (baseline and provocative endocrine tests) Evaluate pituitary and gonadal endocrine function
Ultrasound Evaluation of the accessory sex glands, penis, testes and scrotum
Alkaline Phosphatase Test Evaluation of ejaculatory function in azoospermic stallions
Urethral Endoscopy Evaluate urethral integrity in stallions with hemospermia
Daily Sperm Output. Daily sperm output can be determined after collection of a stallion once per day for a minimum of 5 to 7 days to deplete the extragonadal sperm reserves (epididymis and ductus deferens). Thereafter, the total number of sperm collected each day will be relatively constant and represents the number of sperm produced each day. Determination of daily sperm output can be used to estimate the number of mares that may be bred per day and therefore the number of mares that may be booked to a stallion for a season.

Interpretation of the results of a stallion reproductive evaluation should take into account the number of mares to be bred and the type of breeding program to be utilized (i.e. live cover or insemination with fresh, cooled or frozen semen). Accepted values for semen analysis are:
A minimum of 60% progressively motile spermatozoa and 60% morphologically normal spermatozoa in each ejaculate
A minimum of 1.0 billion progressively motile, morphologically normal spermatozoa in the second of two ejaculates collected one hour apart after sexual rest for one week at any time of the year.
However, the presence of an ‘adequate number’ of motile spermatozoa in an ejaculate is no guarantee of fertility. If the stallion experienced a significant medical condition (i.e. fever, colic) recently, it may be recommended to re-evaluate him at least 60 days after the conclusion of the illness before a final assessment is made.  

If the reproductive evaluation identifies a stallion with a potential reproductive problem, management or therapeutic programs can be instituted to optimize future fertility. It is important to emphasize that the final criteria upon which to assess stallion fertility are ultimately the conception and foaling rates in mares bred.

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