11 Applied Andrology in Horses Barry A. Ball* Reproductive evaluation of the stallion is a reasonably common procedure in equine veterinary practice, and the techniques to evaluate potential fertility of the stallion have improved considerably over the past several years. The evaluation typically includes physical examination, semen collection and evaluation, examination of the internal and external genitalia, and some evaluation of mating behaviour and libido. Ancillary diagnostic tests, such as endoscopy or ultrasonography of the reproductive tract may be used to provide additional information. Genetic testing, endocrine evaluation and diagnostic testing for specific infectious diseases are increasingly common as part of reproductive evaluation of the stallion. Ultimately, however, the evaluation of the stallion can only provide an estimate of stallion fertility. In many cases, reproductive evaluation is more effective in determining potential sub-fertile stallions than in accurately identifying future stallion fertility, which may be influenced by a number of factors that are not considered as part of the routine reproductive evaluation. Reproductive evaluation of the stallion attempts to provide an estimate of a stallion’s future fertility based on evaluation of history, physical examination, semen evaluation and other diagnostic procedures. Many other factors, such as management, have a large impact on stallion fertility, and the fertility of a stallion may change over time. Therefore, the reproductive evaluation is in many cases an attempt to provide an estimate of a stallion’s potential fertility and should not be interpreted as an absolute measure of a particular stallion’s fertility. The typical reproductive evaluation – or breeding soundness evaluation (BSE) – is composed of the following components: • history; •general physical examination, including examination of the external and internal genitalia; •evaluation of libido and mating behaviour; •semen collection and evaluation; and •ancillary diagnostic procedures. All of the components in the list above are covered in the first part of this chapter. The second part of the chapter is devoted to other specific topics in andrology that are related to a stallion’s breeding soundness, and expand on some of the subjects covered in the first half. These include the following: oxidative stress in normal and abnormal functioning of equine spermatozoa; endocrinological evaluation of prospective and active breeding stallions; testicular biopsy; diseases of the scrotum and testis; diseases of the scrotum, tunica vaginalis and spermatic cord; diseases of the excurrent duct system; diseases of the accessory sex glands; diseases of the penis and prepuce; behavioural dysfunction; and ejaculatory dysfunction. A detailed history is one of the most important aspects of the BSE but may be difficult to obtain. On many occasions, the owner or agent may not have direct knowledge of the stallion’s past use or fertility and, in some cases, may be unwilling to provide a detailed history. It is also important to be aware of the reason for the evaluation being made. Emphasis on different portions of the examination may differ between a stallion that is being evaluated for infertility and one on which a pre-purchase examination is being made. It is important that the clinician positively identifies the stallion that is examined. In addition to the signalment (recording of peculiar, appropriate, or characteristic marks), this should include lip tattoos, photographs, and markings or scars or microchip numbers. These should become part of the permanent record that is kept with each BSE. It may become important later to be able to refer to the record and positively identify the stallion that was examined on a particular date. The history should include prior and intended future use of the stallion, as well as establishing previous ownership. The owner or agent may be able to provide little useful information if the stallion has recently been acquired. General health should be queried, including past illnesses or injuries, previous or current medication, type of housing, feeding programme and routine health maintenance. Therapy with steroids or gonadotrophins is of particular concern. The history should attempt to detail past breeding performance of the stallion. This should include the number of years at stud, size of the stallion’s book, frequency of use, type of breeding management (natural cover versus artificial insemination), and the date the stallion was last bred or semen was collected. If the stallion has been used for breeding, the history should elicit breeding shed behaviour and management techniques that may influence libido. If possible, fertility should be expressed as services per conception (Kenney et al., 1971), or as per cycle conception rate (calculated as the number of mares that were detected pregnant/number of cycles mated or inseminated) (Love, 2003). The detected incidence of embryonic or fetal loss should be recorded as well, along with any other unusual occurrences, such as the number of mares noted with endometritis or shortened oestrus cycles after mating. The distribution of the stallion’s book (maiden versus foaling versus barren) should also be noted. Reproductive management of the mare band, such as teasing method, ovulation detection, hormonal therapy, vaccination programme and method of pregnancy detection should be discussed. The status of the stallion relative to equine infectious anaemia, equine viral arteritis and contagious equine metritis (CEM) should be noted. The incidence of possible genetic defects, such as cryptorchidism or parrot mouth, in the stallion’s progeny should also be recorded (Kenney et al., 1983). A general physical examination should be included in the routine BSE of the stallion. The purpose of this examination is to identify defects that might possibly be of genetic origin and that will adversely affect the ability of the stallion to serve. In particular, the visual, cardiopulmonary and locomotor systems should receive special attention in the stallion. Evaluation of the penis, prepuce and urethral process should be done when the stallion’s penis is first washed for semen collection. The relative size of the erect penis should be assessed. The skin of the penis and prepuce should be carefully examined for lesions such as habronemiasis or neoplasia (e.g. squamous cell carcinoma), and for scars indicating prior trauma or infection with equine herpesvirus 3 (which causes coital exanthema). The fossa glandis surrounding the urethral process should be examined for accumulations of smegma ‘bean’. Evaluation of the scrotum and testes is best conducted after semen collection when the stallion is usually more tractable. The scrotal skin should be thin and pliable. The number, size, orientation and consistency of both testes should be noted. The stallion’s testes normally are positioned horizontally within the scrotum with the tail of the epididymis oriented caudally. The length, width and height of each testis should be measured. The testes should be roughly symmetrical in size and consistency. They should not be overly firm or soft (the normal consistency approximates that of the fleshy portion of the hand at the base of the thumb with the thumb extended). The testes should be free within the scrotum (Plate 19). The epididymis originates at the head located on the craniodorso-lateral pole of the testis and continues along the dorso-lateral aspect of the testis as the thin body. The epididymis terminates caudally as the prominent tail. A remnant of the gubernaculum, known as the scrotal ligament, can often be palpated as a firm structure attached to the tail of the epididymis of the stallion. The spermatic cord should be examined from its origin at the cranio-dorsal pole of the testis until it enters the external inguinal ring. Rotation of the testis may occasionally be noted with up to an 180° rotation noted without apparent effects. If the testis is rotated more than 180°, vascular compromise with clinical signs may occur secondarily to torsion. Total scrotal width is used as one estimate of testis mass in the stallion (Gebauer et al., 1974). Testis mass, in turn, is correlated with daily sperm production and output (DSO) in the stallion. Total scrotal width and DSO increase with age, though the relationship between total scrotal width and DSO appears less valid in aged stallions; in one study, the correlation between total scrotal width and DSO was 0.55 (Thompson et al., 1979). Total scrotal width is measured by placing one hand above the testes to position both testes in the ventral aspect of the scrotum and then measuring the widest portion across both testes. The accuracy of the measurement is increased by the use of calipers and by taking the average of three measurements. Stallions with a total scrotal width of less than 8 cm should be strongly suspected of testicular hypoplasia or degeneration (Kenney et al., 1983; Pickett et al., 1987). The shape of the testes can influence the relationship between total scrotal width and testis mass; therefore, measurements of length, width and thickness of individual testes are often included in the BSE. A more accurate determination of testis volume can be made based upon ultrasonographic determination of testis width, height and length: volume = 0.52 × height × width × length and DSO can be estimated as: DSO (billions) = (0.024 × volume) – 1.26 (Love et al., 1991) Comparison of projected DSO based upon testis volume with measured DSO based upon semen collection provides a useful means to assess the efficiency of spermatogenesis in the stallion. When large disparities occur between estimated DSO based upon measured testis volume and actual DSO based upon semen collection, this suggests that efficiency of spermatogenesis may be reduced (Blanchard et al., 2001). As with the external genitalia, examination of the internal genitalia of the stallion is best conducted after semen collection. Restraint of the stallion and protection of the examiner are important considerations, because most stallions are not accustomed to examination per rectum. Usually, with a slow careful approach, most will tolerate the examination. The internal genitalia of the stallion include the bulbourethral gland, pelvic urethra, prostate gland, vesicular glands and ampullae. The paired bulbourethral glands lie at the root of the penis at the ischial arch and are not palpable. The pelvic urethra is the best landmark for palpation of the internal genitalia, and is identified as a cylindrical object lying on the floor of the pelvis with a diameter of 3 to 4 cm. At the cranial extent of the pelvic urethra, the prostate gland is palpated as a firm, 2 × 4 cm glandular structure, with lobes located on either side of the urethra. The ampullae are located along the midline just cranial to the prostate as muscular ducts approximately 1 to 2 cm in diameter and 10 to 20 cm in length. The vesicular glands are sac-like structures located lateral to the ampullae that are often difficult to palpate unless they are filled as a result of sexual stimulation. The internal inguinal rings are palpated just off midline approximately 10 cm cranio-ventral to the pelvic brim. Microbiological culture of the stallion is frequently included in the BSE (Kenney et al., 1983; Pickett et al., 1989). It is important to realize, however, that the penis and prepuce of the stallion have a number of bacteria isolated that represent ‘normal’ microflora, although some may also represent potential pathogens. Cultures are typically taken from the urethra after the penis is washed with water and cotton, and again immediately after semen collection, by passing a culturette up the distal urethra. Pre-ejaculatory cultures should be taken after the stallion has been teased so that some pre-ejaculatory fluid is present. Cultures are also taken from the prepuce and semen. Semen cultures require that a sterile semen receptacle be used. Swabs are held in transport media and are submitted for routine aerobic culture. Interpretation of the microbiological portion of the examination should be done carefully. The organisms most commonly associated with venereal transmission in the stallion include Taylorella equigenitalis (the causative agent of CEM), Pseudomonas aeruginosa, Klebsiella pneumoniae (particularly capsule types 1 and 5) and, rarely, haemolytic streptococci and Escherichia coli (Pickett et al., 1989). Because all of these organisms (except for T. equigenitalis) may also be isolated from stallions without fertility problems, interpretation of the microbiological findings requires consideration of other findings from the BSE. The organisms are considered as potential venereal pathogens if recovered in moderate or heavy growth in pure culture from several ejaculates, or if associated with increased leucocytes in the semen, or with an increased incidence of post-coital endometritis in mares bred to that stallion. The source of these organisms is typically urethritis, although vesiculitis, ampullitis or epididymitis are rarely associated with these isolates. The ability of the stallion to respond to a mare in oestrus, achieve erection, mount, seek the vulva or artificial vagina, thrust and ejaculate are critical to his use in a natural service or artificial insemination (AI) programme (McDonnell, 1986). Therefore, careful observation of the stallion’s sexual behaviour during the BSE is important. Reaction time (interval from presentation of the mare until erection) and number of mounts per ejaculate should be recorded. The agility of the stallion in mounting the mare, seeking the vulva or artificial vagina (AV), thrusting and dismounting the mare are subjectively assessed. Evidence of pain or reluctance to mount may indicate musculoskeletal abnormalities that may impair the stallion’s breeding performance. Overly aggressive behaviour towards the handlers or mare should be noted. Aggression and libido are not synonymous in the stallion. Interpretation of sexual behaviour and mating ability is somewhat subjective. Age, experience of the stallion and season can affect reaction time and libido. Young, inexperienced stallions require careful, patient handling to successfully collect semen and not unduly disturb the development of normal sexual behaviour. Experience (learning) plays an important role in the sexual behaviour of the stallion. Stallions may become conditioned to respond to events that are not normally related to sexual behaviour, such as phantom mares, handlers, the presence of the AV, etc. Likewise, the stallion may have impaired sexual behaviour owing to negative conditioning associated with breeding-related injuries or excessive discipline in a sexual context. The number of ejaculates examined from a stallion will determine the reliability of the estimates made of semen parameters. Authors differ as to the number of ejaculates that should be examined as part of the routine stallion BSE. Currently, the Society for Theriogenology guidelines recommend a minimum of two ejaculates collected an hour apart if they meet the criteria of representativeness (Kenney et al., 1983). The two ejaculates are considered representative if volumes of the ejaculates are similar, and the second ejaculate contains approximately half the number of spermatozoa as the first ejaculate, and has comparable or increased sperm motility. If the two ejaculates do not meet these criteria, the examiner should consider that the collections are not representative. For example, stallions with prolonged sexual rest before evaluation may have much higher numbers of spermatozoa in the first ejaculate than in the second. During the breeding season, it is recommended that the two ejaculates be collected during the regular breeding or collection schedule of the stallion. Other investigations have recommended that five daily ejaculates be assessed in order to provide reliable estimates of semen parameters (Rousset et al., 1987), and an average of 4.7 days was required to stabilize extragonadal sperm reserves after periods of prolonged sexual rest (Thompson et al., 2004). In some cases, semen may be collected from stallions for at least 5 to 7 days to deplete the extragonadal sperm reserves and to provide an estimate of DSO (Gebauer et al., 1974; Thompson et al., 2004). Practical considerations tend to limit these types of examinations to very valuable stallions, or to those in which numbers of motile, morphologically normal spermatozoa are subnormal or questionable with the standard two collections made an hour apart. Evaluation of semen for the BSE should be conducted after collection with the AV (see next section on semen evaluation). Other methods of collection, such as the condom, provide an inferior sample, particularly from the microbiological standpoint. Care should be taken to protect the sample from temperature shock, light and excessive agitation/oxygenation, which can adversely affect spermatozoa. All materials that contact the semen should be clean, dry and warmed to body temperature (35 to 37°C) (Kenney et al., 1983). Immediately after collection, the sample is evaluated for colour, clarity and foreign debris. Normal stallion semen has a skimmed milk appearance that depends on the concentration of spermatozoa present. The volume of the ejaculate is recorded and gel, if present, is removed by filtration through a milk filter or by aspiration. The volume of gel and gel-free semen is also recorded. Filtration also acts to remove any gross debris present in the ejaculate. It should be noted that appreciable numbers of spermatozoa may be lost in the collection equipment and filters (up to 25%), but this number is typically not accounted for in the routine BSE. Immediately after removal of the gel, aliquots of semen are removed for the assessment of motility, sperm concentration, morphology and pH (Kenney et al., 1971). Subsamples of semen should be removed after mixing the semen by gently swirling the sample, because spermatozoa sediment. Care should be used in handling the sample, such that all pipettes are warmed and clean and no cross-contamination of the sample with chemical fixatives, such as formol-buffered saline (BFS), occurs. Initial estimates of motility and pH should be made within 5 min of collection. Subsamples for concentration and morphological investigations should be taken soon after collection so as to reduce the occurrence of agglutination of spermatozoa in raw semen and also to reduce the morphological artefacts that may occur with time. Alkaline phosphatase (ALP) activity in seminal fluid from the stallion is relatively high, with a reported range of 1640–48,700 IU/l (Turner and McDonnell, 2003). This activity appears to be derived primarily from epididymal secretions, with contributions from the testis. ALP activity can be a useful marker in cases of azoospermia, to help confirm ejaculation and the contribution of epididymal fluids. The activity is low (<90 IU/l) in pre-seminal fluid and in cases of bilateral obstructive azoospermia secondary to ampullar blockage (Turner and McDonnell, 2003). It may be useful to establish reference values for seminal plasma from normal stallions if ALP activity is to be used for the evaluation of azoospermia. The pH of raw semen should be measured with a pH meter soon after collection. The normal pH of raw semen ranges from 7.2 to 7.7, with a slight increase in pH between the first and second ejaculates (Kenney et al., 1983). An elevated pH may indicate incomplete ejaculation (pre-ejaculatory fluids have a pH of 7.8 to 8.2), urine contamination, inflammation within the genital tract or equipment contamination with soap. Samples that are incubated for a period of time after collection tend to have a lower pH as a result of the accumulation of metabolic by-products (lactic acid). The osmolality of seminal plasma varies considerably in the stallion. In one study of five ejaculates each from ten stallions, osmolality was 336 ± 10.5 mOsm (± SEM; B.A. Ball, unpublished data). Elevations of osmolality can be used to diagnose urine contamination of semen samples (Griggers et al., 2001). Alternatively, excessive use of water-soluble lubricants based on sodium methylcellu-lose for the lubrication of AVs during semen collection may result in contamination of the semen sample and elevation of measured osmolality (Devireddy et al., 2002). This increased osmolality may, in turn, adversely affect both sperm motility and freezability. Urine contamination of semen (urospermia) can be detected in some ejaculates by a gross change of colour or odour, or by the presence of urine crystals on microscopic examination (Fig. 11.1). In other cases, urospermia may be more difficult to detect and requires the use of assays for urea or creatinine. The use of rapid tests for urea nitrogen (Azostix) allowed the detection of urospermia (Althouse et al., 1989); a colour change (yellow to green) was noted within 10 s in ejaculates that had urea nitrogen concentrations greater than 39 mg/dl. Alternatively, measurement of creatinine concentrations in semen can be used for more specific determination of urine contamination; concentrations >2.0 mg/dl are indicative of urine contamination (Dascanio and Witonsky, 2005). The normal equine spermatozoon differs from that of other large domestic animals in several respects. Distinguishing characteristics include abaxial attachment of the midpiece, asymmetry of the head, small acrosomal volume and small head size. The percentage of normal sperm in the stallion appears to be lower than that of other domestic animals, with most studies citing between 50 and 60% normal spermatozoa (Dowsett and Pattie, 1982; Jasko et al., 1990; Kenney et al., 1995). Based on the current Society for Theriogenology guidelines, only the total number of normal spermatozoa is considered; the differential distribution of abnormal spermatozoa is not considered. In a recent publication, per cycle pregnancy rates for 88 stallions were correlated with sperm morphological parameters (Love, 2011a). There was a moderate correlation between the percentage of normal sperm and per cycle pregnancy rate (r = 0.42), whereas morphological defects, including abnormal heads (r = –0.22), proximal droplets (r = –0.34), abnormal midpieces (r = –0.30) and coiled tails (r = –0.35) had a moderate-to-weak negative correlation with per cycle pregnancy rates (Love, 2011a). Although the percentage of normal spermatozoa is ultimately used for the BSE, the data above suggest that consideration of the type of morphological defect present may be useful in the interpretation of sperm morphology. The method used to fix and stain spermatozoa can influence the morphological artefacts encountered in assessing stallion sperm (Hurtgen and Johnson, 1982). Cold shock of sperm before fixation and staining can lead to artefactual changes in the acrosome and mid-piece. Ideally, raw semen should be fixed (1:10) in BFS immediately after collection. The evaluation of wet-mount specimens with phase-contrast microscopy or differential interference contrast microscopy (DIC) (≥1000× magnification) provides the best assessment of morphology, particularly of the acrosome, midpiece and cytoplasmic droplets. If phase contrast or DIC microscopy is not available, then eosin–nigrosin stained smears are probably the next best alternative for the evaluation of sperm morphology. Eosin–nigrosin stain is mixed with an equal volume of raw semen and smeared on to a glass slide. Note that stain, semen and slides should be at 35–37°C to avoid inducing artefacts in sperm morphology. While eosin–nigrosin staining has been used to assess live/dead ratios in semen, this test is not highly repeatable under field conditions, and the stain is best used as a counterstain to assess morphology. The assessment of sperm morphology with either wet mounts of BFS specimens or eosin–nigrosin smears should be conducted on a good-quality microscope under oil immersion (≥1000×). A minimum of 100 (preferably 200) cells should be counted and classified. Eosin–nigrosin stained smears can be held indefinitely, and BFS-fixed samples can be held for extended periods in well-sealed containers. Neither BFS-fixed samples nor eosin–nigrosin stained smears are suitable for assessing types of cells other than spermatozoa, such as inflammatory cells, red blood cells or spermatocytes in a semen sample. Air-dried smears of semen can be stained with a routine blood stain such as Wright’s (Diff-Quik), Giemsa or new methylene blue to observe somatic cells. Inflammatory cells should be noted only rarely (less than 1 per 5–10 high power fields) in normal stallions. Round cells (spermatids, spermatocytes and spermatogonia) should also be present only infrequently (<1–2% of all cells) in ejaculates from normal stallions (Plate 20). The product of the volume of gel-free semen and the sperm concentration/ml is used to assess the total number of spermatozoa in the ejaculate. There are several methods used to assess the concentration of spermatozoa. These include the use of counting chambers (the haemocytometer), spectrophotometry, electronic particle (Coulter) counters, flow cytometery and image-based particle counters (the NucleoCounter SP100®). The haemocytometer method is the least expensive and most time-consuming way to determine the concentration of spermatozoa. With this method, an aliquot of well-mixed raw semen is diluted (1:100), mixed again and then used to fill a standard Neubauer-ruled haemocytometer. The chamber is allowed to sit for 5 min to allow all sperm to settle to the same plane of focus. The chamber is then examined with either a phase contrast microscope or with a bright-field microscope adjusted for maximum contrast. Spectrophotometric methods are quicker than this and provide acceptable accuracy if properly calibrated. A standard spectrophotometer can be used after constructing a standard curve determined from duplicate haemocytometer counts of semen. There are also a number of commercially available units to determine sperm concentration in stallion semen based upon optical density. Electronic particle counters (Coulter) and flow cytometers can be used to determine sperm concentration, but the cost of obtaining, operating and maintaining this equipment precludes its use for routine semen evaluation. More recently, an image-based method for the determination of sperm concentration has been introduced commercially (Johansson et al., 2008). This unit allows the determination of both sperm number and sperm viability, based upon the use of propidium iodide (PI), a fluorescent, DNA-binding dye. Reliable estimation of the motility of stallion sperm requires that all materials that contact the semen are clean, dry and warmed to 35 to 37°C. The initial estimation of motility should be made within 5 min of collection in both raw semen and extended semen. Raw stallion semen tends to agglutinate with both head-to-head agglutination and agglutination to the microscope slide. However, estimates of gross motility in raw semen may be useful for identifying potential technical problems with the extender or possible adverse effects of the extender on a particular semen sample. The repeatability of estimates of sperm motility is generally better with extended than with raw semen (62 versus 41%) (Rousset et al., 1987). For these reasons, both the author and colleagues estimate motility in both raw and extended semen, but rely more heavily on estimates based on extended semen. The estimation of motility includes both total motility (TM) and progressive motility (PM). TM is an estimation of the percentage of sperm that show any movement, while PM includes only those sperm that are actively moving forward or are moving in large circular paths. Ideally, motility should be evaluated using a standard dilution (25 × 106/ml) and volume of semen for microscopy. This can typically be accomplished by diluting raw semen with a skimmed milk–glucose extender at a ratio of >1:5. Aliquots of semen for evaluation should be taken after gently mixing the semen by swirling the container, because sperm sediment as the sample is stationary. Extender, microscope slides, coverslips, and pipettes should be pre-warmed on a warming tray or in an incubator. A small drop (6–10 μl) of extended semen is placed on a slide, coverslipped and examined with a microscope. Phase contrast microscopy with a heated microscope stage is ideal for motility assessment. If phase contrast is not available, the microscope condenser should be lowered and the iris diaphragm of the bright-field microscope closed to enhance contrast of the specimen. The examiner should be careful not to move too close to the edge of coverslip, where drying of the specimen occurs rapidly, and estimates of TM and PM should be made after examining several fields. Extended semen from some stallions may demonstrate a high proportion of sperm with circular motility immediately after dilution. In these stallions, incubation for a period of 5 to 10 min may be required before accurate estimates of progressive motility can be obtained. The evaluation of motility should be performed quickly, particularly if the microscope stage is not heated. If necessary, new slides should be made rather than continuing to look at repeated fields on the same slide. Samples in which there are no motile sperm should alert the examiner to possible contamination of the semen with spermicidal compounds, such as alcohol or soaps used to clean the AV or the glassware. Computer assisted sperm analysis (CASA) systems have been available for many years as a method to provide more objective analysis of sperm motility parameters. These systems provide a host of kinematic data regarding sperm motion and velocity parameters and offer the ability to obtain more repeatable estimates of sperm motility than do subjective estimates obtained using only a microscope. In most cases though, data obtained from CASA does not substantially improve estimates of fertility of a particular sample compared with subjective estimates obtained using a good-quality microscope and heated microscope stage. Although the concept of PM has long been a part of the routine BSE, its repeatability across different examiners is not good, and the variation in measurement of TM is typically less (Love, 2011b). When sperm motility parameters determined by CASA were compared based upon per cycle pregnancy rates in stallions (91% versus 56% versus 32% per cycle pregnancy rate), the parameters of TM and PM, and of path velocity and progressive velocity were significantly lower for stallions with low seasonal pregnancy rates (Love, 2011a). Not surprisingly, many of the measured motility parameters were highly correlated, including PM and TM and mean progressive velocity, as determined by CASA (Love, 2011a). Correlation coefficients between TM and PM and per cycle pregnancy rates were 0.59 and 0.52, respectively (Love, 2011a). Transmission electron microscopy (TEM) of equine spermatozoa can provide detailed resolution of the ultrastructure of ejaculated sperm, including defects in the plasma or acrosomal membrane, mitochondria and axoneme, as well as showing chromatin condensation (Veeramachaneni et al., 2006). There are a number of in vitro assays that have been used to infer different functional compartments of equine sperm, including the plasma membrane, acrosome, mitochondria and a variety of sperm-specific proteins, but there are relatively little data that associate these in vitro assays with stallion fertility. The relative lack of information relating various sperm function tests with fertility in the stallion limits the ultimate use of many of these assays in clinical applications. None the less, a considerable body of information on the normal function of equine spermatozoa has accumulated through such studies, and may ultimately be useful in accurately predicting the fertility of equine semen based solely upon in vitro assays. Varner (2008) provides an excellent overview of many of these assays as applied to equine sperm. A number of fluorescence probes have been used to assess different functional compartments of sperm. Integrity of the plasma membrane can be assessed by the ability of membrane-impermeant, DNA-binding dyes (including PI, ethidium and others) to stain nuclear DNA and thereby assess sperm viability. Likewise, a number of cationic fluorescent probes (rhodamine 123 and derivatives) are preferentially taken up by mitochondria and may be useful for detecting mitochondrial membrane potential. One probe, JC-1, has the advantage of differentially labelling mitochondria with low versus high membrane potential, thereby providing quantitative information on mitochondrial function (Plate 21) (Gravance et al., 2000). Other probes can be used to detect oxidative damage to the sperm membrane (e.g. C-11-Bodipy 581/591) (Ball and Vo, 2002) or the generation of reactive oxygen species during sperm metabolism (Ball et al., 2001b; Sabeur and Ball, 2006; Burnaugh et al., 2007; Ball, 2008). Other changes, such as alterations in sperm membrane structure (Thomas et al., 2006) and apoptosis (Brum et al., 2008) have also been characterized for equine sperm. Many of these fluorescent probes can be detected using fluorescence microscopy; however, more accurate quantitation can be achieved using flow cytometry, which offers the opportunity to assess a relatively large number of sperm (104) quickly. The sperm acrosome, the membrane-bound vesicle covering the rostral sperm head, contains enzymes that are important in the process of sperm penetration through the zona pellucida of the oocyte during fertilization. The acrosome can be damaged during freezing and thawing, and failure of normal acrosomal exocytosis has been identified as a cause of infertility in stallions (Bosard et al., 2005). The acrosome of equine sperm is relatively small and difficult to image using standard microscopy; detection of acrosomal exocytosis is typically based upon either TEM or other specialized staining techniques. Fluoresceinated lectins, such as peanut agglutinin (PNA), have been used to preferentially label the acrosomal membrane and thereby improve detection of the equine acrosome (see Plate 22, which shows equine sperm stained with fluoresceinated pea lectin). FITC (fluorescein isothiocyanate)-PNA is typically combined with a viability probe (PI) to differentiate sperm that have undergone a true acrosome reaction from those that have undergone cell death with subsequent loss of the acrosome. Alternatively, the equine acrosome can be identified with bright-field microscopy after staining with Commassie blue, although this methodology does not allow the simultaneous detection of sperm viability (Brum et al., 2006). Changes in the susceptibility of sperm chromatin (which is composed of highly compacted DNA and nuclear proteins) to damage, and thereby the relationship of sperm chromatin structure with fertility, has been known for several years in a variety of animals, including the stallion (Evenson et al., 2002). To measure such changes in sperm chromatin structure, sperm are exposed to acid to denature chromatin, and then stained with a dye (acridine orange) that differentially stains single- versus double-stranded DNA as a means to detect DNA strand breaks. Stained sperm are examined by flow cytometry and the amount of DNA fragmentation is determined (Evenson et al., 2002). Changes in the stability of sperm chromatin have been associated with changes in stallion fertility (Love and Kenney, 1998). In most cases, the aetiology of differences in susceptibility to the denaturation of sperm chromatin are unknown; however, thermal trauma to the testis may disrupt normal spermatogenesis, with alterations in sperm chromatin stability detected following acute thermal injury to the testis (Love and Kenney, 1999). Other assays have been used to assess changes in sperm chromatin or DNA, including single-cell gel electrophoresis (the comet assay; Fig. 11.2) (Baumber et al., 2002b) and the TdT (terminal deoxynucleotidyl transferase)-mediated-dUTP nick end labelling (TUNEL) assay (Brum et al., 2008); no data are presently available to relate the outcomes of these assays to fertility in the stallion. Evaluation of the scrotum, testis, epididymis and spermatic cord by B-mode ultrasonography has become a routine part of reproductive evaluation of the stallion (Pozor, 2005), and the normal ultrasonographic anatomy of the stallions’ reproductive tract has been described (Little and Woods, 1987; Love, 1992; Ginther, 1995). In addition, Doppler ultrasonography has been used for the evaluation of both the internal and external genitalia of the stallion (Pozor and McDonnell, 2004; Ginther, 2007; Pozor, 2007). The ultrasonographic appearance of the normal equine testicular parenchyma is homogeneous with the exception of the central vein of the testis (Fig. 11.3) (Love, 1992). Changes that may be detected within the testis parenchyma by ultrasound include the presence of tumours, parenchymal oedema, increased echogenicity of the testis parenchyma associated with late-stage testicular degeneration and haematomas or abscesses within the testis parenchyma. Testicular tumours are relatively rare in stallions, although seminomas, Sertoli cell tumours, Leydig cell tumours, teratomas and mast cell tumours, among others, have been reported (McEntee, 1990; Brown et al., 2008; Edwards, 2008). Seminomas and Leydig cell tumours occur most often in aged stallions and may be present in either normally descended or cryptorchid testes. Teratomas are more often reported in retained testes (McEntee, 1990). The ultrasonographic appearance of these tumours varies, and relatively few reports have characterized their appearance in the stallion testis. In one report, the ultrasonographic appearance of a seminoma was characterized as diffusely hypoechoic with heterogeneous areas of increased echogenicity (Fig. 11.4) (Beck et al., 2001). Seminomas can metastasize and examination of the spermatic cord and abdominal cavity may be useful for detecting metastatic disease (Fig. 11.5). In another report, a testicular mastocytoma (mast cell tumour) appeared as a heterogenic flocculent mass within the testis (Brown et al., 2008). Routine examinations of the testes of the stallion are likely to yield better diagnostic information on the frequency of testis neoplasia in stallions, along with an earlier detection during the course of the disease. Fig. 11.3. Ultrasonogram of a normal testis showing the central vein. Fluid accumulations in the vaginal tunic (or tunica vaginalis, the serous covering of the testis), such as hydrocele (an accumulation of serous fluids) and haematocele (an accumulation of blood), are readily detected by ultrasonographic examination of the scrotum and testis in the stallion. Hydrocele can accompany scrotal oedema or may result because of transfer of peritoneal fluid from the abdomen to the vaginal cavity. Hydrocele may also occur during periods of high ambient temperature and resolve as environmental temperatures moderate (Varner et al., 1991). Such fluid accumulations are typically characterized by an anechoic fluid without evidence of increased cellularity or fibrin. In contrast, haematocele (Fig. 11.6) is characterized by the accumulation of blood in the vaginal cavity often associated with trauma to the scrotum or possibly with haemoperitoneum. Ultrasonographically, haematocele is characterized by the presence of increased echoic debris within the tunics, including the presence of fibrin strands. Scrotal ultrasonography can be very useful in examining the stallion with acute scrotal enlargement (Morresey, 2007). Acute torsion of the spermatic cord, with accompanying testicular oedema, congestion and ischaemia may result in ultrasonographic signs, including increased oedema (reduced echogenicity of the testis parenchyma) (Fig. 11.7), along with evidence of reduced blood flow in the spermatic cord (Pozor, 2007). Inguinal or scrotal hernias typically involve passage of a loop of small intestine through the vaginal ring into the inguinal or scrotal portion of the vaginal tunics. Ultrasound provides a ready method to identify the presence of intestine within the scrotum and greatly facilitates accurate diagnosis. In addition to torsion, other lesions of the spermatic cord include varicoceles, which represent a venous engorgement or enlargement of the veins of the pampiniform plexus. Although common in human males, varicoceles appear to be rare in the stallion (Varner et al., 1991). The presence of a grossly enlarged venous plexus can be detected by both B-mode and Doppler ultrasonography in the region of the spermatic cord (Pozor, 2005). In human males, the presence of varicocele is associated with increased abnormalities in the spermiogram; anecdotal evidence suggests that stallions with varicocele may also have a suppression of semen quality. Fig. 11.4. Ultrasonogram of a seminoma in a stallion showing a mixed echoic and hypoechoic pattern. The epididymis can be imaged along its entire length by transcutaneous ultrasonography. Epididymal cysts have been detected ultrasonographically in the caput epididymis of the stallion, and anecdotal evidence suggests that these cysts may be associated with ejaculatory problems (Pozor, 2005). The origin of these cysts has not been defined, but they may be derived from blind-ended efferent ductules that fail to fuse in formation of the epididymal duct, or possibly represent cysts of the appendix epididymis, a remnant of the mesonephric duct (McEntee, 1990; Love, 1992). These cysts may be noted also in stallions in which no decline in semen parameters has been noted; however, rupture of blind-ending efferent ductules can also lead to sperm granuloma formation in the caput epididymis, possibly resulting in obstructive lesions of the epididymal duct. Epididymitis is relatively uncommon in the stallion, but most often involves either unilateral or bilateral disease of the cauda epididymis. These lesions are typically characterized by the presence of epididymal enlargement, along with enlargement of the epididymal lumen (Fig. 11.8). In the acute phase, epididymitis may be accompanied by painful enlargement of the epididymis, also with the presence of pyospermia (or leucocytospermia, a condition in which there is an unusually high number of white blood cells in the semen). As the condition becomes chronic, there may be a reduction in pain associated with the lesion. Acute trauma to the penis, often associated with breeding or collection injuries, may result in haematomas of the corpus cavernosum, which may result in abnormal erection (Hyland and Church, 1995). Such injuries may be characterized ultrasonographically by the appearance of an increased echogenicity (Fig. 11.9) within the corpus cavernosum, accompanied by penile deviation (Fig. 11.10). Kicks to the inguinal region of the stallion may result in penile, or more often preputial, injuries, with haematomas forming in the large venous plexus located dorsal to the prepuce; again, ultrasound imaging may aid the detection and characterization of these lesions. The internal genitalia of the stallion are readily imaged by transrectal ultrasonography, and changes in the appearance of the accessory sex glands before, during and after ejaculation have been characterized (Weber et al., 1990; Ginther, 1995). Lesions of the terminal portion of the ductus deferens (ampulla) appear to be the most commonly identified abnormality of the internal genitalia of the stallion. Some stallions appear to have an abnormal retention of spermatozoa within the excurrent duct system, with an accumulation of spermatozoa (Varner et al., 2000). Retained spermatozoa appear to undergo degenerative changes within the excurrent duct system, and ejaculates from these stallions may be characterized by a high sperm concentration (>500 million/ml), low motility and a high percentage of detached heads (Fig. 11.11). In some cases, sperm and epithelial cell debris may form casts that appear within the ejaculate (Fig. 11.12). Occasionally, spermatozoa may form obstructive plugs that are retained within the distal ductus deferens, resulting in unilateral or bilateral obstruction of the ductus deferens. In these stallions, transrectal ultrasonography of the accessory glands may reveal a dilation of one or both ampullar lumen due to obstruction (Fig. 11.13). In most cases, these lesions are readily identified by transrectal ultrasonography. Fig. 11.10. Marked penile deviation in stallion at the time of ejaculation. Fig. 11.11. Photomicrograph of a semen sample with a high percentage of detached heads. In addition to obstructive lesions of the ampulla, segmental aplasia of the terminal portion of the ductus deferens has been described in the stallion, associated with an apparent failure of fusion of the mesonephric ducts and the urogenital sinus during development (Estrada et al., 2003). Segmental aplasia of the ductus deferens characterized by the presence of dilated terminal cysts within the ampulla near the pelvic urethra has been observed by Ball and colleagues. Although rare, aplasia of different regions of the excurrent duct of the stallion has been reported and should be considered in cases of infertility associated with azoospermia. Cystic structures associated with the internal genitalia of the stallion have been described based upon their ultrasonographic appearance. One of these, the uterus masculinis, represents a remnant of the paramesonephric duct in the male, which is located in the urogenital fold between the two ampullae of the ductus deferens as they enter the pelvic urethra at the colliculus seminalis (Plate 23). In some stallions, the uterus masculinis may be detected ultrasonographically as a hypoechoic cystic structure lying between the ampullae. While uterus masculinis is frequently found in normal stallions, cystic distension of this structure has been associated with ejaculatory problems by some authors (Pozor, 2005). Urethral cysts have also been identified during ultrasonographic examination of the pelvic urethra of the stallion, though the significance of these lesions remains undetermined (Pozor, 2005). Inflammation of the vesicular glands in the stallion is uncommon, but seminal vesiculitis can present an important source of inflammatory cells in the ejaculate when it occurs. The gross appearance of a semen sample may show a large amount of flocculent debris with evidence of mild haemorrhage (Plate 24). Changes in the ultrasonographic appearance of the vesicular glands may not be detected in vesiculitis but, in some cases, the fluid content of the vesicular glands has been noted to change from a normal anechoic fluid to a more hyperechoic fluid, along with a detectable thickening of the wall of the vesicular gland (Fig. 11.14). Phase contrast microscopy of a semen sample collected from a stallion with vesiculitis showed sperm and many somatic cells (Plate 25), which were identified after Wright’s staining as neutrophils. Ultrasonography can be a useful adjunct to identify testes retained within the inguinal canal or abdomen of the cryptorchid stallion (Jann and Rains, 1990; Schambourg et al., 2006). Identification of the retained testis within the inguinal canal is relatively straight forward using ultrasound, although localization of the abdominal testis by transrectal ultrasonography can require more patience and experience (Jann and Rains, 1990; Schambourg et al., 2006). A technique for the identification of abdominal testes by a systematic transabdominal ultrasound examination with a relatively high sensitivity and specificity has been described (Jann and Rains, 1990; Schambourg et al., 2006). Urethroscopy of the stallion is most useful for delineating lesions of the urethra associated with bacterial urethritis or haemospermia (the occurrence of blood in the ejaculate) (Sullins et al., 1988). A 100 cm flexible video endoscope is adequate for imaging the length of the urethra into the pelvis. Lesions identified during endoscopy of the urethra include urethritis, strictures of the urethra, varicosities and urethral rents. Urethral rents that communicate with the corpus spongiosum may result in haemospermia and are typically located near the ischial arch. Within the pelvic urethra, the termination of the ductus deferens and the openings of the vesicular glands can be imaged dorsally at the colliculus seminalis. The openings of the vesicular glands can be cannulated with a small (3–5 French) polyethylene catheter to facilitate sampling of the content of the vesicular gland. The openings of the ampullae, however, are not readily cannulated via an endoscopic approach. Karyotypic analysis is frequently useful in examining individuals whose sexual phenotype is uncertain (i.e. intersex conditions). Such an analysis may also be useful in some undiagnosed cases of infertility or subfertility in stallions. Autosomal abnormalities and sex-chromosome mosaics (i.e. 63,XO/64,XY) have been described as causes of reduced fertility or infertility in stallions (Kenney et al., 1991). More frequent application of cytogenetic studies is likely to reveal other abnormalities that are associated with reduced fertility in the stallion. Completion of genomic sequencing for the horse, along with an improved understanding of the molecular mechanisms responsible for successful reproduction, offers the opportunity for an improved understanding of the genetics of stallion fertility (Leeb, 2007). As an example of such application, equine cysteine-rich secretory protein (CRISP3) is a major secretory protein in seminal plasma, which has three non-synonymous single nucleotide polymorphisms (SNPs) that are associated with fertility in the stallion (Hamann et al., 2007). Future studies will likely provide an improved understanding of the genetics of stallion fertility, through the identification either of candidate genes or of markers related to fertility (Giesecke et al., 2010). A number of genetic diseases have been characterized for the horse, including several defects in which the specific genetic mutation underlying the disease have been characterized. A growing number of these diseases have genetic tests that are available to screen for such mutations. Depending upon the breed of stallion being examined, these tests will become increasingly important in the elimination of potential genetic diseases from the breeding population. Some of the diseases characterized to date for which specific mutations have been identified include those listed in Table 11.1. A final recommendation on breeding capabilities based on the result of the BSE of the stallion requires consideration of all aspects of the examination. Because many factors in addition to the stallion itself potentially affect its breeding performance, the BSE does not provide a precise measure of the future fertility of a given stallion and may have more utility in the identification of potentially sub-fertile stallions. Based upon current knowledge, stallions that have poor sperm quality are more likely to have reduced fertility, though the converse may not be true (Love, 2011b). Stallions with normal sperm quality may have associated good fertility but other factors, such as management, may adversely affect this fertility, thereby reducing the predictive basis of sperm quality for fertility (Love, 2011b). Table 11.1. Genetic diseases in horses caused by specific mutations.
University of Kentucky, Lexington, Kentucky, USA
Introduction
Stallion Reproductive Evaluation
History
Physical Examination of the Stallion
Bacteriological Evaluation
Evaluation of Sexual Behaviour and Mating Ability
Semen Collection and Handling
Semen Evaluation
Biochemical analysis of seminal fluids
Evaluation of spermatozoa
Sperm concentration
Sperm motility
Ultrastructure of sperm
Sperm function testing
Evaluation of sperm chromatin
Ancillary Diagnostic Procedures
Ultrasonographic and endoscopic examination of the stallion’s reproductive organs1
Ultrasonography
Lesions of scrotum, epididymis and testis
Evaluation of penis and prepuce
Evaluation of internal genitalia
Evaluation of retained testes
Endoscopy
Genetic testing
Interpretation of Findings of the BSE
Genetic disease | Abbreviation | Breed association (if any) |
Hereditary equine regional dermal asthenia | HERDA | American Quarter Horse |
Hyperkalemic periodic paralysis | HYPP | American Quarter Horse |
Glycogen branching enzyme deficiency | GBED | American Quarter Horse |
Junctional epidermolysis bullosa | JEB | Belgian Draft |
Polysaccharide storage myopathy | PSSM | – |
Severe combined immunodeficiency | SCID | Arabian |
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