The assessment of stallion fertility is a common cause for veterinary consulting in private practice. Due to the economic implications that reduced stallion fertility could have on the success of a breeding operation, a complete and thorough examination of the stallion reproductive health or breeding soundness becomes necessary. For this purpose, the Society for Theriogenology (SFT) has established a series of guidelines for the clinical evaluation of stallion fertility [1]. This manual considers several parameters that a stallion must meet to be considered as a “satisfactory breeding prospect.”

• Have the physical capacity to copulate and deliver semen into the mare’s reproductive tract or an artificial vagina (AV).
  
• Possess good libido.
  
• Have a pair of normal testes, consistent with their capacity for producing sperm.
  
• Test negative for venereal pathogens, such as Klebsiella pneumoniae, Pseudomonas aeruginosa, Taylorella equigenitalis, equine herpes virus‐3, equine arteritis virus, and dourine.
  
• Ejaculate at least 1 billion progressively motile, morphologically normal spermatozoa in the second of two ejaculates collected one hour apart, preceded by one week of reproductive rest.

However, when the manual was published, most reproductive programs were focused on natural cover, and few clinical trials about the effect of cooling or cryopreservation on sperm quality and pregnancy rates were available. Currently, there is a whole body of knowledge related to the effects that storage procedures (cooling or cryopreservation) have on the spermatozoa [2, 3]. Thus, new methods for assessing those changes in the sperm have become available in clinical practice and research laboratories.

For these reasons, the evaluation of semen quality is important not only when establishing stallion fertility potential but also for determining whether the semen of a stallion could withstand the stresses related to the cooling or freezing process in programs of artificial insemination with cooled or frozen semen, as well as to determine the quality of a breeding dose before or after insemination. This chapter focuses on the common methods for stallion semen evaluation that practitioners should be familiar with, for assessing sperm quality and stallion fertility.

17.1 Semen Collection and Handling

Amongst the methods reported for stallion semen collection, the use of an AV is the most common and practical for veterinary practitioners. Many types of AVs are commercially available, the most common being the Missouri‐type and Colorado‐type AVs in North America, the Hanoverian type in Europe, and the Botucatu type in Brazil. Other methods reported for semen collection in stallions include the collection of dismount samples from the mare’s vagina in Thoroughbred breeding programs or chemically induced ejaculation [4].

Before semen collection, the practitioner must prepare the collection equipment and the laboratory for sperm evaluation, taking care to keep all the plastic equipment, AV, collection bottles, and covers at 37 °C. Most laboratories and breeding farms use disposable plastic liners within the AV to reduce the probability of cross‐contamination of bacteria present in the smegma and stallion external genitalia. A nonspermicidal lubricating jelly must be used for the AV. Some commercially available brands of nonspermicidal lube in North America include Priority Care (First Priority, Inc., Elgin, IL), HR Lubricating Jelly (HR Pharmaceuticals, Inc., York, PA), Their Gel (Aptech, Inc., Manhattan, KS) and Clarity AI Lubricating Jelly (Aurora Pharmaceuticals, North Field, MN). Before semen collection, the stallion’s penis must be washed with warm water (37–40 °C) and dried using paper towels, with emphasis on the glans penis, glans fossa, and fossa urethralis. This is particularly useful to reduce the amount of smegma and epithelial cells which can contaminate the ejaculate and reduce sperm longevity.

The semen is usually collected into plastic bottles, which are equipped with plastic liners for receiving the ejaculate and nylon micromesh filters to exclude the gel fraction from the ejaculate. It is recommended to protect the semen receptacle from direct sunlight and temperature changes by using a thermal cover, and to transport the ejaculate to the laboratory as quickly as possible, to avoid artefactual changes to the sperm quality and subsequently the sample’s interpretation of quality.

17.2 Macroscopic and Physicochemical Analysis of the Ejaculate

After removing the gel fraction and placing the ejaculate into an incubator at 37 °C, the semen must be measured to determine its volume. Usually, the ejaculate volume is measured with prewarmed, sterile graduated cylinders. However, the most practical and accurate method for volume measurement is based on weighing the ejaculate on a scale, assuming an equivalence of 1 g to 1 mL. As a general procedure, the ejaculate should be split into two parts. One small aliquot can be kept as is (raw semen) and left inside an incubator, whilst the other part is mixed with at least an equal volume of prewarmed semen extender (1:1 dilution, v:v) and allowed to equilibrate to room temperature. This last aliquot will be used for estimation of sperm longevity after cooled storage (see Section 17.4). The ejaculate color must also be observed, being an indirect measure of sperm concentration. With higher sperm concentration, the ejaculate color tends to be more opaque‐white, whilst with lower sperm concentration, the ejaculate will have a translucent or clear appearance.

Estimation of ejaculate color can be particularly useful when cases of urospermia or hemospermia are suspected. Urospermia and hemospermia are amongst the most commonly reported pathologies that can affect sperm motility and subsequently stallion fertility [5, 6]. This is mainly due to marked changes in semen pH and osmolarity (urospermia) or the presence of white blood cells which can impart oxidative stress to the spermatozoa (hemospermia). Although these pathologies cannot be ruled out just by assessing ejaculate color, marked changes of the color from yellow to greenish, or from pink to reddish are commonly seen in those cases. Normal pH and osmolarity of stallion semen have been reported as 7.2–7.8 and 300–334 mOsm/kg, respectively, within one hour of ejaculation [7, 8]. Some laboratories perform pH and osmolarity analysis of the ejaculate but in clinical practice, these procedures are only performed when pathologies such as urospermia or reproductive tract infections are suspected.

Although precise measurement of semen pH and osmolarity is usually obtained by using high‐cost equipment, and the use of pH paper is not recommended due to the inaccuracy of the obtained results, some published research suggest that other diagnostic aids such as the measurement of creatinine and urea levels or the use of strip‐paper kits for detection of metabolites (Azostix^®, Siemens Healthcare Diagnostics, Tarrytown, NY) are particularly useful when cases of urine contamination are suspected [9, 10].

A relevant chemical marker of the stallion ejaculate that could be used in clinical practice for sperm quality assessment is the determination of alkaline phosphatase (AP) levels, particularly to differentiate between azoospermia (lack of sperm in the ejaculate), excurrent duct blockages (spermiostasis) and ejaculatory failure. AP is an enzyme which is active in several tissues, including testes and epididymides, and its presence in high concentrations in stallion ejaculatory fluid is associated with normal ejaculation (AP >5000 IU/L) [11]. When no spermatozoa are observed in the ejaculate and high levels of AP are obtained from the semen, azoospermia should be considered as the cause whilst when no spermatozoa are observed in the ejaculate and low levels of AP are obtained, spermiostasis (“plugged ampullae”) or retrograde ejaculation should be considered as differential diagnosis [12].

17.3 Routine Microscopic Analysis of Semen

17.3.1 Sperm Concentration

Determination of total sperm numbers in the ejaculate is fundamental to classify the reproductive potential of the stud, as well as to adequately prepare a breeding dose for artificial insemination, or to determine if a breeding dose contains sufficient sperm numbers. For this purpose, the total volume of the gel‐free ejaculate must be multiplied by the sperm concentration, usually expressed in millions per mL (1 × 10⁶ spermatozoa/mL).

Two methods are the most frequently reported in laboratories and breeding farms for analysis of sperm concentration: direct observation using hemocytometers and indirect determination using spectrophotometer‐based counters. Several types of hemocytometers are commercially available, the most commonly used being the Improved Neubauer hemocytometer (Figure 17.1a), and the Makler chamber (Sefi Medical Instruments, Israel) (Figure 17.1b). A protocol commonly used in the authors’ practice for estimation of sperm concentration using the hemocytometer is described in Box 17.1. However, despite the hemocytometer is considered the gold standard for sperm enumeration, and the analysis being relatively inexpensive and easy to conduct, it has become unpopular due to the time that analysis takes (~5–7 minutes) and the variation in results between technicians [13].

In the last 20 years, the hemocytometer has been replaced in breeding farms and private practice by semiautomated cell counters or spectrophotometers. Commercially available spectrophotometers are marketed as SpermaCue (Minitube, Tiefenbach, Germany) (Figure 17.2), Accuread (IMV Technologies, L’Aigle, France) and the Densimeter or “blue box” (Animal Reproduction Systems, Chino, CA) (Figure 17.3). These devices measure the changes in the opacity of a given fluid (i.e., semen) in comparison with a “zeroed” control (commonly buffered formalin saline); changes in fluid opacity between the control sample and the semen are assumed to be due to the presence of sperm.

Although these analyzers can offer estimations of sperm numbers in less than two minutes, some disadvantages with their use have been reported. For instance, as these devices measure the sperm concentration indirectly based on the opacity of the sample, they cannot be used for analysis of semen samples containing opaque extenders (milk or egg yolk‐based extenders). Likewise, erroneous results can be obtained when ejaculates contaminated with cellular debris and smegma are analyzed. Lastly, their capacity to accurately estimate sperm concentration in an ejaculate is dependent on an optimal range of sperm concentration; thus, overestimation of sperm numbers is commonly seen with diluted (<100 × 10⁶ sperm/mL) or highly concentrated (>300 × 10⁶ sperm/mL) ejaculates [14].

More recently, an automated fluorescent‐based cell counter, the NucleoCounter^® SP‐100™ (ChemoMetec A/S, Allerød, Denmark) (Figure 17.4), has been validated for estimation of sperm concentration in different domestic animal species, including the stallion [15]. This device uses disposable cassettes containing a fluorescent dye, propidium iodide, which crosses through the sperm plasma membrane and intercalates with DNA, generating a red‐staining pattern over the entire sperm head. For the assessment of sperm concentration using the NucleoCounter SP‐100, the sample must be diluted (usually 1:100 to 1:200 when raw semen is analyzed) with a detergent solution that permeabilizes all sperm membranes, allowing the propidium iodide to gain access to the sperm DNA. Studies have demonstrated a high statistical agreement level between the results obtained with the hemocytometer, the NucleoCounter, and flow cytometers in semen from boars, bulls, and stallions [15, 16]; thus, the use of the NucleoCounter has been widely accepted in research laboratories, breeding farms, and private clinics worldwide. Other automated methods for assessment of sperm concentration in equine ejaculates have also been reported, such as the use of computer‐assisted sperm analyzers (CASA) coupled with fluorescent applications (Hamilton‐Thorne IVOS–IDENT™) [17].

17.3.2 Sperm Motility

Estimation of sperm motility (or motion characteristics) is a fundamental test for the assessment of sperm quality and stallion fertility. Studies conducted in the late 1980s to early 1990s suggested that of all the conventional tests which can be performed to assess sperm quality, sperm progressive motility was the most correlated with stallion fertility [18]. Thus, the assessment of sperm progressive motility became a landmark when assessing semen quality and stallion fertility. Based on what the breeding industry expected at that time, threshold values of sperm progressive motility higher than 60% in freshly ejaculated semen or 30% in cooled semen (after 24–48 hours) and frozen/thawed semen were established.

However, practitioners must be aware that some stallions exhibiting “low” progressive motility in freshly ejaculated semen might be just as fertile as stallions with high progressive motility, when other sperm quality characteristics such as sperm total motility, plasma membrane intactness or sperm morphology are normal. In fact, recent studies have shown that sperm total motility might be more associated with stallion percycle pregnancy rates and seasonal pregnancy rates, using both fresh (natural cover programs) and cooled‐stored semen, than sperm progressive motility [19, 20]. Of particular interest is that semen from stallions with normal fertility (i.e., per cycle pregnancy rates >60%) may display a high incidence of sperm with circular movement, usually considered as abnormal in other species such as ruminants. This can be attributed to an abaxial attachment of the midpiece to the sperm head (see Section 17.3.3), as well as being caused by cooling or freezing procedures.

Under field conditions, sperm motility is commonly evaluated in both raw and diluted (extended) semen. Ideally, the semen sample should be evaluated using a phase‐contrast microscope coupled to a warming stage. This is done to avoid temperature fluctuations in the sample that could induce an artefactual reduction on observed sperm motility. The use of phase‐contrast optics instead of conventional light microscopy is desirable due to the inability to distinguish immotile spermatozoa, yielding false “high” motility estimates. Sperm motility is considerably susceptible to different environmental factors, such as excessive presence of lubricants, urine contamination, pH, and osmolarity unbalances, or low ambient temperature.

Commonly, sperm motility is assessed by placing a drop of raw semen onto a microscope slide and covering it with a coverslip. However, using this method, the practitioner cannot distinguish between individual motility patterns very well. Likewise, this method is commonly associated with overestimation of sperm motility, due to the accumulation of high quantities of spermatozoa in different planes of the vision field. Therefore, it is recommended to dilute the raw semen with an appropriate extender to a sperm concentration approximately between 25 and 30 × 10⁶ sperm/mL, and then incubate it for at least 10 minutes at 37 °C before analysis (Box 17.2). With this concentration, the evaluator can assess sperm total and progressive motility in a more objective way, using either low‐ or dry‐high power objectives (20–40×). When assessing sperm motility, the practitioner ideally determines the percentages of sperm total motility (spermatozoa displaying any form of movement), sperm progressive motility (spermatozoa displaying movement that follows a straight‐line pattern), and sperm velocity classified on an arbitrary scale from 0 (static sperm) to 5 (fast‐moving spermatozoa).

The use of computer systems for the assessment of sperm motion characteristics has become common in research laboratories and some veterinary hospitals. These systems, commonly termed CASA, are composed of a microscope with negative phase‐contrast objectives coupled to a built‐in stage warmer and a real‐time video camera, and attached to a computer which displays the sperm observed through the microscope. The computer software uses algorithms to track each individual sperm based on their head size and movement, and expresses them in sperm velocity and displacement indices. These indices are used to determine the percentage of motile sperm and progressively motile sperm, as well as several sperm velocity indices. Commercially available CASA systems used widely in both clinical and research scenarios include CEROS and IVOS (Hamilton‐Thorne Biosciences, Beverly, MA), SpermVision, AndroVision (Minitube, Tiefenbach, Germany), and ISAS (ProISER, Valencia, Spain).