Assessment of Reproductive Problems in the Male Dog

Introduction
Normal Male Functional Morphology
Spermatogenesis
Intersexuality
Brucellosis and Herpesvirus Concerns
Breeding Soundness Exam (BSE)
History and Physical Examination
Semen Evaluation and Sperm Analyses
Seminal Cytology, Alkaline Phosphatase, and Semen Cultures
Brucella Testing
Ultrasonography of Testis and Prostate
Testis and Testicular Disease Evaluated by Ultrasound
Prostate and Prostate Disease Evaluated by Ultrasound
Serum Testosterone and other Endocrine Testing
Testis Biopsy
Analysis of Results of Semen Evaluation

Introduction

Dog breeding is a multimillion-dollar industry worldwide. Routine reproductive evaluations of male dogs are often requested by breeders to confirm fertility prior to purchase or sale, to check daily sperm output (DSO) particularly in older stud dogs, to assess the potential of a stud dog as a provider of semen for frozen-semen or chilled-semen artificial insemination (AI) or to address problems of apparent infertility and/or testicular abnormalities. Such reproductive evaluations rely on appropriate and successful semen collection and accurate assessment of semen.

Lack of breeding success is often multifactorial. Thus, a comprehensive reproductive exam or breeding soundness evaluation (BSE) will include examination of the genitalia, testis measurement, palpation of the testes and prostate, and semen evaluation as well as ultrasonography of the testes and prostate in some cases. Testing for sexually transmitted disease, especially Brucellosis, is a critical part of a BSE. The following review is based on the teaching, clinical and research experiences of the authors and of others, as well as previously published reviews and clinical and research reports [1-19].

Normal Male Dog Functional Morphology

Age at puberty versus sexual maturity

Nine months is the commonly accepted age for puberty in the male dog, often coincidental with the age when male urination behavior occurs, but likely varies within and across breeds. Sperm first appear in the ejaculate at about 7 to 9 months of age [1,2].

Testes

The testes lie horizontally in the scrotum. They should be symmetrical, and on palpation should feel like "hard boiled eggs" and freely movable within the scrotum.

Epididymides

The epididymis lies dorsal to each testis. The epididymides of the left and right testes should be of similar size and shape. The head of the epididymis is at the cranial pole and the tail at the caudal pole of each testis.

Vas deferens

The vas lies medial to the testis, courses cranially through the inguinal ring and ends in the dorsal prostate.

Prostate

The prostate is the major accessory sex gland in the dog. Its purpose is to produce prostatic fluid as a transport and support medium for sperm. With experience, it is possible to assess the size, consistency and surface texture of the prostate gland by digital palpation per rectum. In young dogs, is found on the pelvic floor, close to the pelvic brim. In older dogs may be over the pelvic brim. It is normally bi-lobed, symmetrical, non painful, no lumps and of homogenous consistency.

Penis

The penis should be examined when it is fully erect (Fig. 1). It has an os penis that extends much of the length of the non-erect penis and thus the non-erect penis is somewhat rigid and non-flexible. The erectile tissues of the penis require venous engorgement for normal penile erection. The tip of the os penis should extend to within 1 - 2 cm of the tip of the glans penis. The bulbus glandis will be most obvious when the penis is fully erect.

Figure 1. The erect or partially-erect (as seen here) penis should be reasonably turgid along its full length and carefully examined for the absence of contusions, lacerations or puncture wound. Hemorrhage, local pain, irritation or signs of inflammation should be absent.

Prepuce

The prepuce should cover the entire non-erect penis. Interior surface should have appearance of normal mucous membrane, although the normal surface may have small lymphatic follicles. On occasion, a mucopurulent or mucoid discharge may be present within the prepuce of normal dogs.

Spermatogenesis

Daily sperm production, the number of potentially fertile sperm produced each day by the testes, is an expression of how well spermatogenesis is occurring. An estimated 15 - 19 million sperm are produced per day per gram of testicular tissue. This relative daily sperm production is independent of breed, body weight, testicular weight, or season. Thus, total daily sperm production depends on total grams of testis, which in turn is correlated to testis size and body weight, and is greater in larger breeds than in smaller breeds. Daily sperm production can be estimated by measuring total scrotal width [2,3] that can be best measured by the use of calipers (Fig 2).

Figure 2. Measurement of total scrotal width. Using a cranial approach the testicles are forced down by downward pressure of the thumb and forefinger. The calipers should be snugly fitted against the scrotal skin at the widest point. Two to three repeated measurements are made to the nearest millimeter and averaged.

The extragonadal sperm reserve is the total sperm contained within the epididymides and vasa deferentia. Studies in dogs ejaculating once daily or more or les frequently demonstrate that the number of sperm in the extragonadal sperm reserve is influenced by the daily sperm production and by the frequency of ejaculation. In particular, the number of sperm present within the cauda of the epididymis is considerably depleted after a single ejaculation [3].

The sperm output in an ejaculate is influenced by age, size of the testicles, frequency of ejaculation, sexual rest and degree of sexual stimulation [2-4]. The interval of abstinence is an important piece of information to obtain when conducting a BSE. In a dog ejaculating regularly, the number of sperm in the cauda of the epididymis is reduced by 25% relative to the number found in the male that has not ejaculated during the previous 7 days [3]. Therefore, if the interval from last ejaculation to examination is less than 5 - 7 days, fewer sperm are ejaculated than would be following sexual rest.

Intersexuality

When the process of sexual differentiation goes wrong during fetal development, intersexuality may occur. Intersexuality refers to an incompatibility between genetic (XX or XY) and gonadal sex (ovary or testis) or phenotypic (genitalia) sex. There are several forms of inter-sex states relevant to the male dog examination including the following. These are discussed in detail in the IVIS review by Meyers-Wallen [5].

1. True Hermaphroditism. In this condition, gonadal tissue of both sexes is present either as separate organs (one ovary and one testis) or within the same gonad resulting in one or two ovotestes. The external genitalia are usually ambiguous.
2. XX Sex Reversal. This is sometimes called inherited male pseudohermaphroditism. Dogs with this condition have a 78, XX chromosome constitution and some testicular tissue in one or both gonads. They are either XX true hermaphrodites, having at least one ovotestis, or XX males, having bilateral testes. The degree of phenotypic masculinization is associated to the amount of testicular tissue present.
3. Persistent Müllerian Duct Syndrome (PMD). PMD has been best described in some miniature Schnauzer dogs. These animals are genetically XY or XXY, have testes (often cryptorchid), and have development of mesonephric duct (Wolffian) and urogenital sinus-derived structures. Thus, the external genitalia are normal male. However, the paramesonephric (Müllerian) duct structures fail to regress, resulting in a uterus being present. The proposed mechanism is a lack of MIH (Müllerian Inhibiting Hormone) production. Pyometra and Sertoli cell tumors have been described in these animals.

Brucellosis and Herpesvirus Concerns

Brucellosis

Canine brucellosis is a zoonotic disease. The etiologic agent is Brucella canis. Transmission occurs primarily via ingestion, secondarily by venereal or congenital routes. The disease causes death in fetuses and neonates, infertility and persistent vaginal discharges in bitches, and orchitis and epididymitis in male dogs. The prepatent period is 1 to 3 months. Bacteremia with no fever may persist for 6 - 12 months. This bacterium eludes the immune system by persisting inside macrophages and PMNs. In male dogs the testes and the epididymides are the organs most commonly infected, causing orchitis, epididymitis, scrotal irritation, poor semen quality and infertility [6,7]. Diagnosis. For serological testing, the dog must be free of antibiotics for 4 weeks before testing. Testing methods and results are considered in the BSE section, below. Even in countries in which brucella is uncommon or rare, failure to test for brucella as part of a BSE exam could be considered negligent.

Herpesvirus

Canine herpesvirus-1 (CHV-1) is responsible for causing a fatal hemorrhagic disease in newborn puppies. The virus causes papulovesicular genital lesions, and can also cause embryonic resorption, abortion and stillbirth. The oronasal and venereal transmission are considered as the main routes of infection, but fetuses can also be infected in utero. In males, mating experience was found to have an impact on CHV-1 antibody titers. CHV-1 antibody titers may be affected by many factors, both on an environmental and host level. Therefore, interpretation of the serological status requires caution [7].

The Breeding Soundness Evaluation (BSE)

A BSE in a dog should include (1) the medical and reproductive history, (2) a general and reproductive physical examination, and (3) semen collection for semen and sperm analyses. In addition, (4) culture of semen for microorganisms should be done where appropriate. Furthermore, (5) testing for Brucella canis should be considered as mandatory if the animal is to be used in breeding. If the equipment is available, (6) ultrasound of the testis and (7) ultrasound of the prostate can be useful diagnostically. Some evaluations may benefit from (8) serum testosterone assay(s) or other endocrine testing. Finally, (9) a biopsy of the testis might be considered. These procedures are considered in detail below.

1. Medical and Reproductive History

The Complete History will ascertain the reason for requesting the present BSE. The Complete pertinent past medical history should collect information on immunization status, medications including parasiticides, feed regimen, supplements, and laboratory data. The reproductive history should include the following: age at first use as a stud; history of infertility in related dogs, male or female; breeding management and other details of how the breedings were timed with each bitch; any observations on stud dogs libido; number of pups sired per litter.

Breeding management records. Ideally, a record can be made of every breeding: bitch, bitch age, dates of matings or AI, outcome, whelping dates, number of pups born, pups weaned. It is very useful to write the breeding history in chronological order with the number of breedings per bitch (dates of individual matings vs., whether matings were considered to be "inside ties" versus "outside ties", AI with fresh, cooled or frozen semen). "Inside tie" is jargon for a coital lock following intromission and ejaculation and is reasonably confirmatory that there was an ejaculation. An "outside tie" is jargon for a possible mating based on observed mounting, pelvic thrusting and apparent intromission, but without a copulatory lock. The potential fertility of such "outside ties", if any, is suspect and remains to be demonstrated.

The history should also include the results of any previous semen evaluations or DSO determinations, the dates and result of any prior brucella testing.

Specific effort should be made to determine any past or present signs of urogenital disease. Is there any history of stranguria or urinary tract infection?

2. General and Reproductive Physical Examination

General Exam

The general physical examination should include examination for diseases or conditions that are inherited e.g., cryptorchidism, hip dysplasia.

Clinical Pathology

CBC and blood chemistry should be performed as part of a complete medical examination. Urinalysis following cystocentesis, should be considered on an as-needed basis.

Reproductive System Exam

This should include an examination of the testes, epididymides, spermatic cords, prostate, penis and prepuce.

Testes

Palpation should confirm that both testes are present in the scrotum and they are of normal size and consistency. The scrotum should be checked for dermatitis and erythema. If scrotal swelling is present, consider the following differentials: inguinal hernia, torsion of the spermatic cord, testicular neoplasia, orchitis, periorchitis, hydrocele, hematocele and scrotal abscess. Other disorders of the canine testes include acquired testicular degeneration or atrophy (Fig 3a, Fig. 3b) and idiopathic testicular hypoplasia.

Figure 3a. Scrotum of a dog with testicular atrophy of the left testicle due to torsion of the spermatic cord. Refer to text on torsion of the spermatic cord. The enlarged compensatory right testicle is seen in this caudal view. Using a cranial approach the right testicle was forced down by downward pressure of the thumb and forefinger.

Figure 3b. Dog with testicular atrophy of the left testicle. The measurement of the compensatory right testicle was 3.8 cm.

Figure 4. Semen collection using the open paper cup method, Prostatic fluid is being collected into a single paper cup after the sperm-rich fraction was collected into another cup previously stacked within this cup. Note that the penis has been deflected 180 degrees laterally due to the dog stepping over the hand of the collector, and that the dorsal aspect remains up. This is the same as the normal flexion (not torsion) of the penis during the copulatory lock typically observed at mating. Note that the collectors hand maintains a firm grip behind (proximal to) the fully exposed bulbus. Note the surface blood vessels. Because no lubricant is used, it is important to avoid any unnecessary rubbing of or trauma to the vessels, especially the fragile engorged vessels near the tip of the glans penis.

Epididymides

The epididymis should be palpated for irregularities in shape, size or consistency, e.g., sperm granuloma. If gentle palpation elicits a pain reflex, consider the possibility of acute epididymitis.

Spermatic Cords and Vasa deferentia

The spermatic cord or the vas deferens should be examined from tail of the epididymis to the inguinal ring. An abnormally large inguinal ring orifice predisposes to inguinal hernia.

Prostate

The prostate should be examined by simultaneous abdominal and rectal palpation. The size, shape, symmetry, consistency, and presence of pain should be noted. Ultrasonography of the prostate should also be part of the routine examination whenever possible. (Please see "Ultrasonography", below).

Penis and Prepuce

With animals of questionable hygiene, the owner should be instructed to wash and cleanse the genitals one day before the examination, if semen collection is planned. Cleaning solutions should not be applied near the time of semen collection for fear they will interfere will the viability of the ejaculate. Examine the prepuce before inducing an erection for semen collection as well as again during erection and semen collection. Check the prepuce for inflammation, foreign bodies and excessive or abnormal discharge. The erect penis can be examined during and following semen collection. The erect penis should be turgid along its full length. Consider the following disorders when examining the penis and prepuce: congenital abnormalities, diphalia, penile frenulum, hypospadias, phimosis, paraphimosis, urethral prolapse, fracture of the os penis, penile trauma, balanoposthitis, persistent erection, penile preputial neoplasia. Consider if there is any evidence of stranguria or urinary tract infection, or any urethral discharge from the non-erect penis.

3. Semen Collection and Analysis

Consideration should be given to when the dog was last collected as well as possible recent pairings, breedings and ejaculations, as these might affect results of semen collection and evaluation.

Collection can be accomplished using either clean hands or clean or sterile gloves. In either case, make sure there is no contamination of the genitalia or collection vessel with disinfectant solution, glove power, or other foreign materials.

The prepuce should be gently pushed behind the bulbus glandis. The penis should be encircled with the fingers behind the bulbus glandis and slight pressures exerted; some dogs require some manipulation back and forth, most do not. A failure to obtain an erection and pelvic thrusting should be countered by exertion of addition circumferential pressure by tightening the grip of the thumb and forefinger. Be prepared to allow the dog to step one leg over the collector’s arm and hand at any time during the procedure. The semen is collected into a clean 50 ml urine-sample or other type paper cup, a 50-ml vial or into a latex collection cone with an attached graduated centrifuge tube. As an alternative, some people prefer to use a disposable baby bottle liner. If the 50-ml vial is used, the rim of the vial should be encircled with the index finger and thumb to preventing cutting the penis. Using a set of stacked paper cups allows easy changing from one to another to separate the second and third semen fractions. Collect the first clear fraction and the second sperm rich fraction together. The third fraction is clear and is a product of the prostate. The presence of a bitch in estrus facilitates a good erection and collection.

If an ejaculate appears to be substandard, consider retaining the dog for several hours for purposes of a second collection, possibly under improved circumstances (without owners present, in a quiet area, and/or in the presence of a submissive or estrus bitch).

Before allowing the dog to be released, wait until the penis is flaccid and confirm that the prepuce is not retained reflected behind the bulbus but has returned to the normal position The semen should be evaluated for volume, color, and sperm motility, concentration, as well as seminal plasma pH, cytology and alkaline phosphatase, as outlined below.

Semen Evaluation in Relation to Normal Semen Parameters

The ejaculate or sperm rich fraction should be characterized in terms of volume and color, sperm motility and percentage of progressively motile, sperm concentration and percentage of morphological normal sperm, calculated total sperm number, pH, cytology and presence of WBCs, and alkaline phosphatase activity. The results in turn should be compared to values for normal ejaculates. The normal values for many semen parameters are summarized in Table 1, Table 2, Table 3.

Volume and Color

Volume is estimated based on transfer to or collection into a graduated tube. The volume varies from 2.5 to over 80 ml depending on the amount of prostatic fluid collected. The first and second fraction or the second fraction alone should be recorded. The color of the semen should be evaluated. A yellow color may represent urine in the ejaculate and a red or brown indicates the presence of fresh red blood cells or autolized red blood cells due to either damage of blood vessels of the penis or prostatic disease.

Sperm Motility

The motility should be analyzed immediately. For a normal dog, progressive motile sperm should be > 70%. A drop of semen is placed on a warmed microscopic slide, covered with a warmed cover slip, and observed with a 20 X or 40 X objective of a phase contrast microscope equipped with a stage warmer. Dog semen does not cold shock if temperatures are kept above 21° C, i.e., room temperature. Computer assisted analysis of sperm motility if available, will give an objective evaluation of sperm motion dynamics. Visual estimation of sperm motility involves counting 10 sperm per field in 4 - 5 different fields of the slide. It should include total sperm motility (% sperm showing any kind of motility), progressive sperm motility (% sperm moving rapidly and progressively forward in a straight line), and sperm velocity (on a scale 0 - 4). For example, a motility value-set of "85/80 (4)" would indicate that 85% of sperm were motile and 80% of sperm were progressively motile, exhibiting a rapid and linear movement.

Percentage of Morphologically Normal Sperm

Make a smear of a sperm rich fraction, air dry and stain with eosin nigrosin (Fig. 5) or Diff-Quick (Fig. 6, Fig. 7, Fig. 8, Fig. 9, Fig. 10, Fig. 11). With Diff-Quick, the slide has to be immersed in each solution for 5 minutes. It may be preferable to place 1 - 2 drops of semen in 1 ml buffered formol saline in an Eppendorf tube and evaluate the sperm as a wet mount by phase contrast microscopy (Fig. 12, Fig. 13, Fig. 14, Fig. 15, Fig. 16, Fig. 17, Fig. 18, Fig. 19, Fig. 20, Fig. 21). Normal males should have at least 80% normal sperm.

Figure 5. Smear of a sperm rich fraction, air dried and stained with eosin-nigrosin. This slide has areas of low and high concentration of sperm.

Figure 6. Smear of a sperm rich fraction, air dried and stained with Diff-Quick.

Figure 7. Normal sperm. Smear of a sperm rich fraction, air dried and stained with Diff-Quick.

Figure 8. Bent sperm tail. Smear of a sperm rich fraction, air dried and stained with Diff-Quick.

Figure 9. Terminal coiled sperm tail. Smear of a sperm rich fraction, air dried and stained with Diff-Quick.

Figure 10. Coiled sperm tails. Smear of a sperm rich fraction, air dried and stained with Diff-Quick.

Figure 11. Detached sperm head. Smear of a sperm rich fraction, air dried and stained with Diff-Quick.

Figure 12. Normal sperm. Unstained sperm cells seen by differential interference contrast microcopy.

Figure 13. Bent sperm tail. Unstained sperm cells seen by differential interference contrast microcopy.

Figure 14. Bent sperm tail. Unstained sperm cells seen by differential interference contrast microcopy.

Figure 15. Bent sperm tail. Unstained sperm cells seen by differential interference contrast microcopy.

Figure 16. Bent sperm midpiece. Unstained sperm cells seen by differential interference contrast microcopy.

Figure 17. Coiled sperm tail. Unstained sperm cells seen by differential interference contrast microcopy.

Figure 18. Coiled sperm tail. Unstained sperm cells seen by differential interference contrast microcopy.

Figure 19. Coiled and bent sperm tails. Unstained sperm cells seen by differential interference contrast microcopy.

Figure 20. Coiled sperm tails. Unstained sperm cells seen by differential interference contrast microcopy.

Figure 21. Coiled tails. Unstained sperm cells seen by differential interference contrast microcopy.

Sperm Concentrations

Dilute the semen in a unopette (Becton-Dickinson, Rutherford, NJ) 1:100 and count the large central square of a hemacytometer. The number of sperm counted x 106 is the concentration. For the total number of sperm multiply concentration x volume. The total number of sperm for a small dog should be 400 x 106. Large dogs should yield an ejaculate with around 1.4 x 109 sperm [2].

Seminal Plasma pH

Normal semen pH values in the dog range from 6.3 - 7.0 and the range for prostatic fluid is 6.0 - 7.4 pH units [8]. The pH should be measured as soon as possible after collection and could be a useful parameter for antibiotic selection in case of infection.

Seminal Cytology, Alkaline Phosphatase, and Semen Cultures

Cytology

Cytology of the sperm rich fraction and the prostatic fluid rich fraction should be done separately. This can be accomplished for each by preparing the samples with the Cytospin® technique. Alternatively, 0.5 ml fractions could be spun at 120 g for 7 minutes, smearing the sediment on a slide and staining with Diff-Quick®. Some non-degenerating leucocytes are common in normal dogs. Increase numbers of degenerating PMN’s are an indication to culture.

Alkaline Phosphatase (ALP)

In normal semen samples ALP is typically > 5,000- U/L [8]. ALP is produced in the epididymis and is an excellent marker of ductal blockage. If ALP < 1,000 IU/L, either the epididymis is blocked or a complete ejaculate was not obtained. Male dogs with true azoospermia, due to causes other than tubular blockage, usually have ALP concentrations > 5,000 U/L.

Semen Cultures

Semen culture, where appropriate, should consider the need to avoid contamination or introduction of foreign chemicals during preparation of the dog for examination, semen collection and semen handling. When collecting specimen for cultures the prepuce and tip of the penis should be washed with sterile saline. Vials should be sterile. Culture is commonly for aerobic bacteria since anaerobes are not usually found. Port-A-Cul® (Becton, Dickinson, Sparks MD, USA) transport media is usually recommended for transportation to the laboratory. Greater than 10,000 cfu aerobic bacteria per ml of semen indicate infection. If anaerobes are suspected, the lab should be contacted to suggest the transport media. Mycoplasmas should be considered normal flora. However, a large number may cause problems, and mycoplasma has been suggested as a cause of canine infertility based on findings in other species.

5. Testing for Brucella canis (Brucellosis Testing)

A brucella test should be performed. Note that routine B. canis testing every 6 months is recommended if the dog is used for breeding more than five bitches annually.

Initial testing typically utilizes a rapid slide agglutination test (RSAT) or a tube agglutination test (TAT). These tests are sensitive but not specific. The RSAT is not quantitative. All positives should be confirmed by a second, more specific test such as gel immunodiffusion testing or culture and direct identification of B. canis. Agar gel immunodiffusion test (AGID) - This test targets cytoplasmic antigens and cell wall antigens. Culture testing is time consuming but also definitive. Consider blood, lymph node aspirates, and any obvious preputial discharge as material for culture.

Chronically infected animals are only intermittently bacteremic and have intermittent decreases in titers and therefore may test negative falsely with any test. Three negative tests at monthly intervals are required to call a suspicious animal truly negative. Treatments and management. Castration should be performed in infected animals that are not euthanized because gonadectomized animals shed fewer organisms. Consider euthanasia, especially in kennel situations. Otherwise isolation from other dogs, especially breeding animals is important. Antibiotic therapy may decrease titers but is not a cure.

Ultrasonography of the Testis and Prostate

Imaging of the testes and prostate (Fig. 22-43) provides assessment of the anatomy and parenchyma and related lymph nodes. When reproductive disease is present, ultrasound guided aspiration for culture, cytology and biopsy specimens can be obtained safely for a more definitive diagnosis. Pathology of the prostate includes benign prostatic hyperplasia, prostatic and paraprostatic cysts, infection and inflammation including acute and chronic prostatitis, and prostatic neoplasia. Ultrasound can be helpful in the differential diagnosis of prostate disease.

6. Testis, and Testicular Disease and Abnormalities Evaluated by Ultrasound

Testes

The normal testis is echogenic with a homogenous texture. The parietal and visceral tunics produce a thin hyperechoic peripheral echo. The tail of the epididymis is less echoic than the testis. In contrast, the head and body of the epididymis have similar echogenicity to the testis. The mediastinum testis, on a mid sagittal plane scan, is seen as an echogenic linear central area, and on a mid transverse scan plane as a central focal echo (Fig. 22a, Fig. 22b).

Figure 22a. Normal scrotal sonogram in longitudinal plane from a 6-year-old Newfoundland with acute bacterial prostatitis. The testis (approximately 2.5 cm thick, oval and located in the top half of this image) is echogenic with a homogenous echo texture. The parietal and visceral tunics produce a thin hyperechoic peripheral echo. The mediastinum testis (MT), on a mid sagittal scan plane as shown here, is seen as a central echoic line. The tail of the epididymis (not seen in this view) is less echoic than the testis. In contrast, the head (also not seen) and body of the epididymis (seen as a 0.5 cm thick band slightly darker than, and immediately below, the testis in this image) have similar echogenicity to the testis.

Figure 22b. Normal scrotal sonogram in the transverse plane in a 6 year old Newfoundland with acute bacterial prostatitis. The large homogeneous echoic oval structure is the testis. Note that the mediastinum testis seen on a mid transverse scan plane as shown here appears as a central echoic dot. Below the testis is the body of the epididymis.

Figure 23a. Sonogram of a unilateral cryptorchid one year old Yorkshire Terrier with an intra-abdominal testis. This image of the peritoneal cavity shows the testis as a 2 x 0.6 cm structure immediately beneath the body wall, appearing as a darker oval structure in the top center of the image, with a central linear echo indicating the normal mediastinum. Such a linear echogenic mediastinum testis in a spherical or ovoid structure within the abdomen is a marker for positive recognition of the retained testis. Unilateral cryptorchidism is more common than bilateral cryptorchidism, with the right testis being retained more often. Also, see Fig. 23b.

Figure 23b. Sonogram of the same unilateral cryptorchid one year old Yorkshire Terrier with an intra-abdominal testis, here shown in oblique section scan plane in the top center of the image. The mediastinum testis appears as a hyperechoic horizontal line in the testis at the top center of the image. The testis (seen as a 1 x 0.6 cm structure in the top-center of the image, immediately beneath the body wall) was located next to the small intestine (seen in cross section as a 1 cm diameter oval hyperechoic lumen casting an acoustic shadow immediately to the right of the testis), and the aorta and vena cava (seen as two anechoic oval structures immediately below the testis in this image).

Orchitis

Testis and/or epidydimal inflammation may result in an enlarged (and often painful) scrotum, and the tissues may appear enlarged upon palpation or ultrasound (Fig. 24a, Fig. 24b).

Figure 24a. Epididymitis. Scrotal sonogram of a 12-year-old mixed breed dog with epididymitis. The testis and tail of the epididymis are seen in longitudinal view. The testis, located on the left side of the image is normal in appearance except for the convex caudal contour. That contour is the result of compression caused by the greatly enlarged tail of the epididymis. The latter is the round structure near the right side of the image.

Figure 24b. Epididymitis. Sonogram of the body of the epididymis, in longitudinal view, of a 12- year-old mixed breed dog with epididymitis. The epididymis is greatly enlarged because of a generalized thickening of the spermatic duct. The body of the epididymis extends across the image from left to right. The electronic calipers show a epididymal diameter of 2.3 cm. The serpentine structure of the diffusely thick spermatic cord is inferred by the hypo echoic edge shadows that form dark jail-bar like stripes across the image of the body of the epididymis.

Torsion

Torsion of the spermatic cord is a rare condition in the dog. Reports from the literature show a mean age of dogs at the time of diagnosis to be close to 6 years without breed predisposition [9]. It is typically associated with a neoplastic, abdominally-retained testis, due to instability of the suspensory tissue allowing the testis to rotate more freely compared to the descended testis. Non-neoplastic testes are enlarged after torsion because venous occlusion, edema, and inflammation. This scenario was suspected in a 10- year-old Labrador dog presenting with a history that one of the testis had visibly swollen up 3 - 4 months previous to presentation, but shrank down subsequently within 2 months. There was a concern over the declining poor sperm quality and an apparent asymmetry of the testes. The dog had been used as a breeder, and had two scrotal testes and he had (previously and subsequently) sired several litters. No acute onset of abdominal pain, stiff gate, vomiting, lethargy, dysuria, hematuria or pyrexia, were reported (please see Fig. 3a and Fig. 3b).

Testicular Tumors

The most common testicular tumors of dogs are Sertoli cell tumors, interstitial cell tumors and seminomas. The ultrasonographic appearance of each type of testicular tumor is variable and there is no characteristic or diagnostic appearance for any of the tumor types. In addition, mixed testicular tumors can occur, adding to the complexity of the diagnosis. Hormone profiles, especially the testosterone-estradiol ratio (see Section 8, below), may assist in the evaluation and diagnosis of these tumors. Feminization occurs in most but not all dogs with Sertoli cell tumors, and the changes appear to be more the result of reduced testosterone and a resulting reduced testosterone-estradiol ratio than of extent that estradiol might be elevated. Feminization may also be observed in some dogs with Leydig cell tumors. Whether unilateral or bilateral, testis tumors are best managed by orchiectomy.

Cryptorchidism

Ultrasound is useful to determine the location of the testis when cryptorchid testis is suspected. Likewise, ultrasound is also useful to evaluate for a caudal abdominal mass when neoplastic cryptorchid testis is suspected. Cryptorchidism is discussed in detail in the chapter by M. Memon and A. Tibary. (see chapter Canine and Feline Cryptorchidism)

7. Prostate and Prostate Disease Evaluated by Ultrasound

Normal Prostate Gland

The prostate surrounds the pelvic urethra, beginning at the level of the neck of the urinary bladder. The normal prostate of a young to middle age intact dog is homogenous, medium to fine echotexture, and most commonly with moderate echogenicity (Fig. 25, Fig. 26). The prostate of neutered (orchidectomized) dogs is atrophied and less echoic than in the intact dog (Fig. 27, Fig. 28, Fig. 29, Fig. 30).

Figure 25. Sonogram of a normal prostate from a middle age intact dog. The image is made in sagittal plane with cranial to the left. The prostate is the oval structure oriented diagonally in the top right portion of the image, with the prostatic urethra running length-wise through it and appearing as an hypoechoic element running diagonally from lower right to upper left within the prostate. The prostate of the intact male dog is obviously larger (in this case 4.0 x 3.2 cm) than that of the neutered dog, and is more echogenic that in the neutered dog.

Figure 26. Sonogram of a normal prostate from a middle age intact dog. Image is made in transverse plane (cross-section). The bi-lobed prostate that appears in the center of the image measures 3.3 x 3.0 cm. Note the subtle variation in prostate echogenicity. The mid-portion of the prostate is more echoic because of collagen associated with the ductal tissue. The ventral and dorsal portions of the prostate are less echoic. These regions of the gland are the secretory glandular tissue. The urethra and its acoustic shadow (vertically oriented line) separate the right and left lobes are visible as an hypoechoic, vertically oriented line.

Figure 27. Sonogram of a normal prostate from a middle age neutered dog. The prostate is just right of center in this image and appears in the sagittal plane with craniad being to the left. The prostate of neutered dogs, as seen here, is much smaller (in this case 1.2 x 1.3 cm) than in the intact dog. The prostatic parenchyma is homogeneous, has fine echotexture, and is less echoic than adjacent fat. Immediately cranial to the prostate is the anechoic, triangular shaped urinary bladder. Immediately caudal to the prostate is the pelvic portion of urethra, which is here 0.7 cm thick.

Figure 28. Sonogram of a normal prostate in a middle age neutered dog with the round prostate seen in transverse plane in the lower center of the image. The prostate is small (1.3 x 1.2 cm), round, and less echoic compared to surrounding fat.

Figure 29. Sonogram a normal prostate of a middle age neutered dog with an incidentally observed atypical orientation of the bladder. The image is in the sagittal plane with cranial to the left. The urinary bladder has an atypical round shape at the bladder neck resulting in the bladder bulging caudo-ventral to the prostate.

Figure 30. Sonogram of a normal prostate of a neutered dog with an incidental atypical orientation of the bladder. The prostate image is in the transverse plane and shows the bladder ventral to (and imaged above) the 1 cm prostate. The colon (a 2 cm hyperechoic curved structure) is located in its usual position, dorsal to (and imaged below) the prostate.

Benign Prostatic Hyperplasia

This disease involves an increase in epithelial cell number as well as epithelial cell size, but the increase in numbers is more marked. It is the most common canine prostatic disease, with almost 100% of intact dogs developing histological evidence of prostatic hyperplasia with aging. Hyperplasia is associated with an altered androgen to estrogen ratio, and requires the presence of the testis. Prostate enlargement can be detected by ultrasound examination (Fig. 31, Fig. 32) and it can also be detected by radiography (Fig. 33). Accurately made measurements of prostate size can be determined using ultrasound and electronic calipers to measure length, height/depth and width. Maximum length, height and width for normal prostates in intact dogs have been published as reference values by Ruel, Barthez, et al [15].

Dehydrotestosterone formed within the gland serves as the main hormonal mediator of the actions of testosterone. Castration is the treatment of choice for BPH. The prostate decreases in size by 75% by the 9th week of castration. Medical management is sometimes accomplished using finasteride, an inhibitor of 5-a-reductase that thereby blocks conversion of testosterone to dehydrotestosterone [19]. This experimental use of the human medication (Proscar) in dogs involves a regimen of 0.1 to 1mg/Kg/day, which can cause a decrease in the prostate up to 70% within 12 weeks after the beginning of treatment. Optimum length of treatment is unknown, and continued management may require continuous or repeated treatment. The prostate size returns to near-pretreatment values by 8 weeks after the drug is discontinued.

Figure 31. Sonogram showing benign prostatic hyperplasia in a middle age dog, with prostate imaged in the sagittal plane. The enlarged 8 cm x 5.3 cm prostate is the oval structure that occupies most of the image. The urethra runs length-wise through the prostate; the triangular shaped neck of the urinary bladder is located cranial to (here, to the left of) the prostate. Throughout the hyperechoic prostatic parenchyma are innumerable tiny 1 - 2 mm anechoic cysts.

Figure 32. Sonogram showing benign prostatic hyperplasia in a middle age intact dog. The image of the prostate is in the transverse plane. The prostate is enlarged (6.3 x 5 cm), has an asymmetrical, bi-lobed shape, and occupies the majority of the image. Numerous 1 - 2 mm anechoic cysts can be seen multifocally throughout the prostate.

Figure 33. Lateral radiograph of the caudal abdomen of an intact dog demonstrating substantial prostatomegaly of nonspecific pathogenesis. The prostate is the round soft-tissue structure ventral to the colon and immediately cranial to the pubic bone. The urinary bladder is the oval soft tissue structure ventral to the colon and immediately cranial to the enlarged prostate. At the ventral aspect, the margins of the urinary bladder and the prostate are demarcated by a small triangle of fat seen as a darker gray triangle.

Prostatic and Paraprostatic Cysts

In dogs, cysts are typically classified into (1) cysts associated with prostatic hypertrophy or squamous metaplasia, (2) true retention cysts within the prostatic parenchyma (Fig. 34, Fig. 36 and Fig. 37) and (3) paraprostatic cysts outside the prostatic parenchyma. Prostatic cysts may be an incidental finding upon ultrasound examination of the male urogenital tract and are often asymptomatic. Intra- prostatic cysts may be the result of a prostatitis (see below) or a response to the dysplasia that occurs in prostatic adenocarcinoma (see below and Fig. 45). Sonographically, intra-parenchymal prostatic cysts appear as discrete anechoic or hypoechoic foci of variable shape. Cysts cannot be reliably distinguished from abscesses associated with prostatitis (Fig. 40, Fig. 43) based on ultrasound appearance alone. Prostatic carcinomas may also contain fluid cavities, and these tend to appear more irregular or more complex than retention cysts or abscesses. Paraprostatic cysts originate from remnants of the Müllerian ducts or as extensions of the prostate lobe. Sonographically there are distinguished from retention cysts because of their extra-parenchymal location. Almost invariably, paraprostatic cysts have the appearance of a "second urinary bladder" because cranially they have a rounded shape and caudally they have a tapered neck. Their distinct wall has a variable thickness and is occasionally mineralized. Laboratory examination of prostatic cyst fluid is not a common procedure, and there is the risk of inducing a needle tract infected with bacteria. If the dog is symptomatic and visible prostatic cyst(s) is the primary finding, aspiration, examination and culture of prostatic cyst fluid may provide useful information. Associated signs can include tenderness, lethargy, dysuria, hematuria secondary to cyst hemorrhage, and straining to defecation secondary to compression of the rectum by a large cyst. In one study [14], the fluid obtained from 42% of prostatic cyst cases was found to contain aerobic bacteria. Medical therapy of prostatic cysts has not been extensively reported. Corrective modalities could include cyst drainage by aspiration, cyst resection, marsupialization, and partial prostatectomy.

Figure 34. Prostatic retention cyst. Sonogram showing the prostate of a 7-year-old male Border Collie whose chief problem was tetraparasis caused by spinal cord fibrocartilagenous embolus. He also had fever and urinary tract infection (E. coli). Cytology of prostatic cyst fluid showed inflammation with no bacteria (culture negative). Fever resolved with antibiotics. The prostate is enlarged (with 10 cm maximum diameter) and cranially (left side of image) it contains a large (6.5 cm) hypoechoic fluid cavity lesion, which distorts the shape of the prostate and seen as a bulge in the dorsal contour of the prostate. Based on the cytology, negative culture and resolution of signs, the fluid cavity was considered a retention cyst.

Figure 35. Sonogram showing the prostate of the same 7 year old male Border Collie presenting with tetraparasis and large prostatic cyst as reviewed in Fig. 34. The image of the prostate is in the transverse plane caudal to the large cyst. The prostate has an asymmetric bi-lobed shape, with the right lobe (left side of image) enlarged (4 x 6 cm); this was the lobe containing the large cyst. Additionally the left lobe contains several smaller (< 1 cm) anechoic ovoid cysts. The prostate is well defined from the surrounding tissues.

Figure 36. Sonogram showing the prostate of a 6-year-old male German shepherd with tenesmus, stranguria, and hematuria. The prostate is imaged in the transverse plane with the left side of the dog located to the right side of the image. The prostate is at the upper limit of normal size (7 cm x 4.5 cm), has a normal, bi-lobed shape with the left lobe containing an irregular shaped hypoechoic fluid cavity lesion measuring 3 x 2 cm. The rest of the prostate had normal echogenicity and shape. Cyst fluid was serosanguinous and cytology with moderate number of RBC, macrophages, of lymphocytes, few neutrophils, and no bacteria. Dog was treated with enrofloxacin and finasteride. A recheck-ultrasound 2 months later showed decreased size of prostate (3.7 x 3.2 x 4.5) and the cyst.

Figure 37. Sonogram showing the prostate of the 6 year old male German shepherd with tenesmus, stranguria, and hematuria shown in Fig. 36. The prostate is imaged in the para-sagittal plane with cranial to the left. An irregular shaped, 4 cm long, anechoic prostatic cyst is shown dorsally in the prostate. The prostate is bordered cranially (to the left) by the anechoic urinary bladder, dorsally (lower aspect of image) by a hyperechoic line that is the colon, caudally (right) by the pubic bone shadow, and ventrally (upper aspect of image) by the abdominal body wall.

Figure 38. Sonogram of the prostate (longitudinal view) from a 6-year-old male German Shepherd dog at 2 months after treatment with enrofloxacin and finasteride. Treatment resulted in decreased size of the prostate (3.7 x 3.2 x 4.5) and cyst.

Figure 39. Sonogram of the prostate (transverse view) from a 6-year-old male German Shepherd dog at 2 months after treatment with enrofloxacin and finasteride. The treatment resulted in decreased size of the prostate (3.7 x 3.2 x 4.5) and cyst.

Prostate Infection and Inflammation - (Acute and Chronic Prostatitis)

Prostatitis (Fig. 40, Fig. 41) is an inflammatory disease of the prostate gland usually due to bacterial infections (Fig. 40, Fig. 41, Fig. 42). If prostatitis is present, hematuria and/or hemaspermia may be seen. Cytology of the last fraction of semen may show large numbers of white blood cells. Hematuria alone without inflammatory cells is more indicative of benign prostatic hyperplasia or trauma to the urethra and penis. Dogs with prostatic abscess (Fig. 42 and Fig. 43) may have fever and abdominal pain and sometimes peritonitis or septic shock subsequent to the rupture of an abscess. Culture of the 3rd fraction or urine obtained by cystocentesis is indicated. Gram Negative colony counts above 105 are suggestive of infection. Treatments with antibiotics need to have high lipid solubility.

Figure 40. Sonogram of the prostate (transverse view) of a six-year-old male Newfoundland dog with prostatitis. The dog presented with fever, lethargy, pyuria, bacturia (E. coli), and elevated white blood count with a left-shift. These clinical problems resolved with antibiotic treatment and castration. The prostate (6 x 4.7 x 6.6 cm) is at the upper limit of normal size [2] for an intact male, giant-breed dog of 6 years of age. Dorsally, the prostate has a poorly margined, hypoechoic region (arrow). It does not contain a distinct fluid cavity.

Figure 41. Sonogram of the prostate of a six-year-old male Newfoundland with prostatitis (longitudinal view). Prostate (6 x 4.7 x 6.6 cm) is at the upper limit of normal for giant-breed dogs. Dorsally, the prostate has a poorly margined, heteroechoic to hypoechoic region (arrows).

Figure 42. Sonogram of the prostate (longitudinal view) of a 6 year old intact male Australian cattle dog that presented with fever, lethargy, stranguria, hemolytic anemia, and thyrombocytopenia resulting from a prostatic abscess. The prostate has an irregular shape and a distinct hypoechoic cavity. See Fig 43, The abscess was resolved via surgical drainage.

Figure 43. Prostatic abscess secondary to prostatitis. The image is a sonogram of the prostate (transverse view) of the same cattle dog shown in Fig. 42. The prostate appears as a bi-lobed, weakly echogenic structure (with hyperechoic margins) in the center of the image. The approx. 3 cm abscess is the irregular, semi-oval shaped, well marginated, hypoechoic area within the left portion of the prostate image, and its diameter is marked diagonally (X---X) by the electronic calipers.

Prostatic Neoplasia

The most common neoplasm of the canine prostate is the adenocarcinoma. In prostatic adenocarcinoma (Fig. 44a, Fig. 44b, Fig. 45a, Fig. 45b, Fig. 45c, Fig. 46), the gland is typically enlarged and irregular in shape with an heterogeneous echotexture. Metastasis to the lumbar vertebrae Fig. 44a and Fig. 44b, regional lymph nodes (Fig. 45c), or lungs is common (Fig. 44a and Fig. 44b). Calcification is not uncommon (Fig. 46). When prostate calcification is detected it is highly associated with prostatic carcinoma (Fig. 44a, Fig. 44b, Fig. 45a, Fig. 45b, Fig. 46). In radiographs, irregular bone formation on the ventral aspect of the lumbar vertebrae and/ or the ilium in the presence of an enlarged, mineralized prostate indicates metastasis (Fig. 44a and Fig. 44b). The majority of prostatic neoplasms are malignant. On ultrasound examination the neoplasitic prostate appears hyperechoic or heteroechoic. Calcification is a common sign of carcinoma. Euthanasia is often recommended. Medical management can be palliative.

Figure 44a. Lateral radiograph of the caudal abdomen of a 10 year old neutered male Labrador Retriever dog with lumbar pain, pelvic limb ataxia and paresis resulting from prostatic adenocarcinoma. The prostate (arrows in annotated image 44b) is too large to be normal for a neutered male dog and it is faintly mineralized. It displaces the hypoechoic colon ventrally. There is an obviously mineralized sub lumbar mass (arrow-heads in 44b). The ventral aspect of lumbar vertebrae 4 - 7, and the ilium, have irregular bone formation. These findings are pathognomonic for regional metastasis.

Figure 44b. Lateral radiograph of the caudal abdomen of a 10 year old neutered male Labrador Retriever dog with lumbar pain, pelvic limb ataxia and paresis resulting from prostatic adenocarcinoma. The prostate (arrows) is too large to be normal for a neutered male dog and it is faintly mineralized. It displaces the hypoechoic colon ventrally. There is an obviously mineralized sub lumbar mass (arrow heads). The ventral aspect of lumbar vertebrae 4 - 7, and the ilium, have irregular bone formation. These findings are pathognomonic for regional metastasis.

Figure 45a. Prostatic carcinoma. Sonogram of the prostate, (in longitudinal view) of a 5 year old neutered Keeshond dog with prostatic carcinoma. The caudal aspect of the bladder is seen as a triangular anechoic (black) area at the left of the image, and appears normal. The prostate, seen as the round structure in the middle of the image, is enlarged (3.0 x 2.3 cm) compared to that expected in a neutered male. It is abnormally heteroechoic with several very hypoechoic (dark grey) regions that may be lesions with fluid-filled cavities. These contrast with the irregularly margined, intensely-hyperechoic (white) regions that may represent dystrophic mineralization characteristic of prostatic carcinoma.

Figure 45b. Prostatic carcinoma. Sonogram of the prostate (seen in transverse plane) of the same 5 year old neutered Keeshond with prostatic carcinoma. The prostate, the oval structure in the middle of the image, is enlarged (3.1 x 3.2 cm) and abnormally heteroechoic with anechoic (dark) fluid containing regions and intensely hyperechoic regions of dystrophic mineralization.

Figure 45c. Prostatic carcinoma. Sonogram of the aortic trifurcation, right-medial iliac lymph node, and urinary bladder (at top left of image in longitudinal view) of a 5 year old neutered male Keeshond. The dog had a chief complaint of inappropriate urination indoors. Antibiotic treatment failed to resolve the problem. The medial iliac lymph node, the round structure in the middle of the image, is mildly enlarged (2.2 x 1.3 cm) since it has a diameter greater than that of the aorta. The lymph node has a normal smooth contour and normal homogenous echogenicity. This mild enlargement may represent either a reactive lymph node or metastasis of prostatic carcinoma to a regional lymph node. The lymph node is recognized by its location immediately lateral to the aorta at the point where the aorta branches into the external iliac artery. These blood vessels form the fork-shaped, anechoic (dark) structures deep to (and imaged below) the lymph node. The normal urinary bladder is located ventral to (and imaged above) the lymph node.

Figure 46. Sonogram of the prostate (longitudinal view) of an 8 year old, intact Rottweiler with prostate calcification. The prostate, the oval structure occupying most of the image, is enlarged (8.5 x 5.5 cm), extending cranially to surround the neck of the urinary bladder. The bladder is the anechoic, triangular-shaped structure to the left side of the image. Importantly, the prostate contains calcification - the multifocal, intensely hyperechoic regions that cast acoustic shadows. Calcification is frequently associated with carcinoma.

8. Serum Testosterone and other Endocrine Testing

These might include one or more of the following: serum testosterone assay; thyroid evaluation; prolactin assay; adrenal disease testing; gonadotophin-challenge testis (testosterone) response tests; and testicular biopsy.

Serum or Plasma Testosterone (and other Reproductive Hormones)

Testosterone (T) is secreted in a pulsatile fashion, with peaks 3 - 8 h apart. Normal T concentrations ranged from 0.1 to 4 ng/ml, and averaged about 2 or 3 ng/ml, in recent studies, using assays validated for canine serum [16,18], but higher values can be encountered. T levels do not appear to change greatly with age in dogs. Estradiol concentrations range from 8 to 32 pg/ml and average 18 pg/ml, and tend to decrease with age [17]. In dogs with Sertoli cell tumors, mean estradiol is elevated (29 pg/ml) and mean testosterone is reduced (0.1 ng/ml). However, in Sertoli cell tumor dogs in one study [16], the range in values overlapped those of normal dogs for both estradiol (14 - 48 pg/ml) and testosterone (0.03 - 0.77 ng/ml). The testosterone to estradiol ratios also overlapped between normal and Sertoli cell tumor affected dogs, but it is often elevated in the affected dogs [16]. Serum LH and FSH are both secreted in a pulsatile manner, and thus normal concentrations are variable. Mean concentrations can vary among laboratories due to differences in reference hormones standards. In castrate animals, T is low or non-detectable, and LH and FSH typically are elevated due to loss of testosterone and estradiol negative feedback effects on the pituitary. LH and FSH are typically reduced in dogs with Sertoli cell tumors because of enhanced estradiol negative feedback effects and the FSH-reducing effects of elevated inhibin secretion [17]. Hormone concentrations in dogs with seminomas are similar to those in normal dogs because these tumors are not endocrinologically active.

Thyroid Evaluation

Hypothyroidism may be the cause of infertility in some dogs as it is in some males of other species. However, many hypothyroid dogs have normal fertility. Any cause-effect relationship is poorly understood. In some animals it is possible that the lack of negative feedback of thyroid hormone on pituitary TSH secretion may also result in excess secretion of prolactin. Prolactin has profertility effects at low levels and anitfertility effects at abnormally high concentrations in males of several species. Dogs have not been studied in this regard. Thyroid testing is usually readily available and of reasonable expense and merits consideration in any cases of obvious infertility.

Prolactin Assays

These may be worth considering when readily available. Persistent hyperprolactinemia, if documented, might be expected to contribute to reproductive as well as other abnormalities such as mammary hyperplasia, galactorrhea, and/or abnormal hair growth or shedding.

Adrenal Disease Evaluation

Hyperadrenocorticism with abnormally elevated plasma cortisol concentrations can have reproductive as well as metabolic and systemic effects, including suppression of gonadotrophin secretion. Some forms of adrenal disease may involve a hypersecretion of androgen that can similarly suppress LH and FSH secretion by negative feedback on pituitary gonadotrophs. Exogenous glucocorticoid and exogenous androgen treatments can have similar antifertility effects.

Testis Stimulation Tests

In cases of abnormally low concentrations of testosterone, it may be important to evaluate the hypoandrogenism based on repeated sampling every 4 - 5 h for a full day, since testosterone secretion is normally pulsatile with 4 - 8 pulses occurring at more or less equal intervals each day. A response test can be conducted using a challenge injection of hCG (human chorionic gonadotrophin) as a substitute for LH the normal endogenous stimulus for pulses of testosterone secretion, or using a challenge injection of GnRH (gonadotrophin releasing hormone) the hypothalamic peptide that normally stimulates LH secretion. Purified hCG and synthetic, native GnRH are both readily available. Pre and post samples are obtained and assayed for testosterone content. In healthy dogs hCG doses of 4 - 10 IU/kg administered intramuscularly (IM) are expected to result in a large increase (> 4.5 ng/ml) in testosterone within 1 - 2 h of injection. IM GnRH doses of 1-2 ug/kg release maximal amounts of LH within 15 min and results in elevated testosterone at 60 min in normal dogs.

9. Testis Biopsy

The procedure has been conducted in a variety of ways. The most common involves a wedge biopsy through an incision in the tunic albuginea [11]. Bouins fixative is typically recommended for reproductive pathology. The consulting pathology lab should be contacted as regards tissue handling and fixative before the procedure is performed. Fine-needle aspiration and core collection of testicular tissue has also been preformed [10].

10. Analysis of Results of Semen Evaluation

The BSE semen parameters in a stud with apparent infertility or decreased fertility, may be classified overall as being in one of three different categories: (a) normal semen parameters; (b) abnormal semen parameters including aspermia; azoospermia, oligospermia and teratospermia; (3) retrograde ejaculation.

a. Normal Semen Parameters

Normal sperm parameters, with no obvious physical cause of infertility. This may be due to female infertility or to problems with breeding management such as poorly timed matings. If leucocytes are present in the ejaculate, culture of the prostatic fluid is indicated.

b. Abnormal Sperm Parameters

Abnormal semen parameters can result for a variety of reproductive and non-reproductive medical problems including the following:

• endocrine disease such as hypothyroidism or adrenal disorders, especially hyperglucocorticoidism
• infectious disease of the reproductive and/or urinary tract
• febrile illness
• exposure to drugs, toxin, environmental steroids, pesticides or other agents that can alter spermatogenesis or sperm maturation
• inflammation of scrotum, torsion of the spermatic cord, trauma, testicular tumors or other pathological insults to the testis of reproductive tract
• infections such as B. canis.

These medical problems can result in ejaculate, semen or sperm abnormalities of the following types, reviewed below: aspermia, azoospermia, leucospermia, teratozoospermia, asthenozoospermia , necrozoospermia, oligozoospermia, hematospermia, and sperm agglutination.

Aspermia

No ejaculate. This can occur under a variety of circumstances:

• failure of orgasm and ejaculation due to libido or neural deficit
• failure or blockage of prostatic fluid secretion such that the sperm rich fraction is not transported to the exterior
• failure of prostatic fluid secretion and small, unnoticed and uncollected pre-sperm and sperm rich fractions. A repeated attempt at semen collection should be attempted 6 or more hours later.

Azoospermia

No sperm in the ejaculate. There are three possible causes of azoospermia: (1) the testes are not producing sperm; (2) spermatozoa are being produced but there is an epididymal blockage; (3) the testes are producing sperm but only an incomplete ejaculate is obtained. If ALP is < 1,000IU/L, the epididymis is blocked or a complete ejaculate was not obtained. Repeat collection is warranted. A better and more representative ejaculate may be collected in the presence of a docile or estrus bitch, and/or in a more private locale and in the absence of other people including the owners. An azoospermic dog should be submitted to careful palpation and ultrasound examination of the epididymis and spermatic cords to detect possible tract abnormalities.

Leucospermia

Leukocytes in the ejaculate. This requires further consideration of prostate, epidydimal or testicular bacterial infection. Semen culture is also recommended. The following conditions are also encountered and merit follow up beginning with careful ultrasound examination and one of more supplementary tests (see above). Semen culture is also recommended.

Teratozoospermia

Where there are too many morphologically abnormal forms. Normal male dogs should have 80% or greater of morphological normal spermatozoa. Age and breed are not considered a factor. Seasonal effect, with decrease in the % morphological normal spermatozoa is a possibility. Teratozoospermia may be caused by testicular tumors, orchitis, fever, prostatitis, abstinence or overuse and obesity with increase of intrascrotal temperature. Fucosidosis a lysosomal storage disease causes retention of cytoplasmic droplets. Treatment may include unilateral orchidectomy. Even if treatment is successful, semen quality is unlikely to improve earlier then 62 days after resolution of the problem, the time required for sperm to be produced and delivered into the ejaculate.

Asthenozoospermia

Characterized by absence of sperm motility. Causes include those of teratozoospermia, as well as contaminated collection equipment and immotile cilia syndrome. Residues from manufacture latex, and a number of water-soluble lubricants may decrease progressive motility of sperm. Extension of semen in appropriate extender may be beneficial.

Necrozoospermia

In which cases all sperm in the ejaculate are dead. Necrospermia, if hormonally dependent may be possible to reverse [12].

Oligozoospermia

Where there are too few sperm in the ejaculate. A sperm count that yields less than 200 million/ejaculate in dogs weighing greater than 4.5 kg is considered abnormal. Seasonal oligozoospermia may occur in the summer. Sertoli cell tumors, prostate disease, orchitis and epididymitis, drugs, e.g. anabolic steroids, may cause decreased spermatogenesis. Treatment strategies may include, orchidectomy, or medical therapy with GnRH or hCG.

Hematospermia

Blood in the ejaculate occurs most commonly secondary to benign prostatic hypertophy or due to trauma during collection or copulation. Extending the semen pellet after centrifugation in a nutrient media may enhance longevity of sperm.

Sperm Agglutination

Autoimmune orchitis is responsible for spontaneous infertility in dogs. In addition, sperm agglutination has been demonstrated in dogs infected chronically with B. canis. Agglutinated sperm have impaired motility. Dogs with B. canis should be castrated.

Testis Biopsy

A supplementary procedure used in some cases of azoospermia is testicular sampling for biopsy. This procedure can show whether or not spermatogenesis is present, and identifies instances of testicular degeneration, spermatogenetic arrest and/or intra-testicular inflammation. It is an invasive procedure but determines whether testis is producing sperm. In general, testicular biopsy is recommended only in azoospermic or oligospermic dogs and only after less invasive tests have been performed including semen evaluation, testis diameter measurements, and assays of serum concentrations of gonadotropin and testosterone.

c. Retrograde Ejaculation

This involves the ejaculation of some or all of the entire sperm-rich second fraction of the seminal fluid into the urinary bladder. It may be a chronic problem for a dog, and a cause of reduced fertility or infertility, or it may be acute and intermittent, and the reason for a poor ejaculate to be collected in an otherwise fertile dog. Note that the normal route of elimination of sperm in the absence of ejaculation is the bladder. In suspect animals, one can catheterize and empty the bladder. Then an ejaculate is collected and the bladder is again catheterized and the post ejaculation urine is examined for sperm. Treatment includes administrations of alpha-adrenergic agonists: phenylpropanolamine, (3 mg/kg per os twice daily) or pseudoephedrine (4 mg/kg per os three times daily or 1 to 3 hours before attempted breeding or semen collection).