History
General history and specific reproductive history are relevant, including diet, medications, prior diseases, evidence of endocrine dysfunction, and kennel demographics including disease prevalence. Reproductive history including preputial discharge, preputial mass, scrotal redness, pain or swelling, signs of feminization, and pain on urination or defecation. Breeding history including interest in estrus bitches, libido, details of the last mating including frequency and number of breedings, natural or artificial insemination, and the diagnostic tools used to manage breeding. Conception history to include date of the last litter sired, conception rate (females pregnant/females bred), occurrence of abortion in females bred to this male, average litter size, and the occurrence of hereditary defects in offspring. Clinical pathology including results and dates of prior endocrinology, haematology, biochemistry, serology, and semen evaluations. Kennel management including nutrition and housing and travelling.
Examination
General physical examination: body condition, weight, skin and hair coat, evidence of endocrine abnormalities are relevant.
The canine testes are ovoid and aligned dorsocaudal within the scrotum. The scrotal skin is thin, may be pigmented and covered with hair, and has sweat glands and little subcutaneous fat. The normal dog has two scrotal testes which are bilaterally symmetrical and have a smooth, homogenous, elastic consistency. Testicular size may be recorded by measuring scrotal width with calipers or direct testicular measurement by ultrasonography. Testis weight is highly correlated with sperm production. Testis weight is also related to body weight, testis size, and scrotal width. The epididymis, vas deferens, and spermatic cords should be palpated for evidence of abnormalities. The head of the epididymis lies dorsocranial, the body runs down the medial surface of the testis, and the tail of the epididymis is caudal.
The penis of the dog has muscular, cavernosus, and bony components. The glans penis consists of the bulbus glandis containing erectile tissue surrounding the midpoint of the urethra and os penis; and the pars longa glandis with erectile tissue dorsally and laterally. The os penis reaches from the bulbus glandis almost to the tip of the glans. The penis and prepuce should be palpated in situ, the penis should then be extruded and, together with the internal lamina of the prepuce, examined for evidence of inflammation, discharge, trauma, foreign body, or masses. The examination may be repeated on the erect penis at the time of semen collection. The os penis is palpated through the non-erect penis for fractures or congenital anomalies. Evidence of existing or previous urinary tract disease should be noted.
The prostate gland is the only accessory sex organ in the dog. It lies on the cranial pelvic floor and surrounds the urethra just caudal to the neck of the bladder. It is symmetrical, globular, and divided into two halves by a median septum. Changes in size, consistency, and symmetry are detected by rectal palpation.
Clinical Pathology
Hematology CBC, biochemistry profile, and urine analysis will aid in detection of primary reproductive tract diseases as well as underlying systemic illness.
Serology
Elsewhere, a Brucella canis titer is performed every six months in an active breeding dog. This disease is exotic to Australia and New Zealand.
Endocrinology
Disorders of the adrenal and thyroid glands have been linked to infertility in other species, although the relationship between hypothyroidism and subfertility has not been well established in the dog. Diagnosis of abnormal adrenal or thyroid function is contingent upon holistic assessment of gland function rather than resting hormone levels.
Semen Collection
Semen is best collected in the presence of an estrous bitch with manual stimulation after 5 days of sexual rest (Foote 1964). Some morphologic abnormalities will increase in the first ejaculate after prolonged sexual rest and a second ejaculate will be more representative (Purswell et al. 2010). Semen collection in the presence of an oestrus bitch is successful in most dogs and results in increased total sperm number (Root Kustritz, Hess 2007). Some dogs may ejaculate in the presence of a non-estrous bitch or in the absence of a bitch, particularly when trained to the procedure. In the absence of a bitch, use of the pheromone methyl-p-hydroxybenzoate or swabs soaked in estrous vaginal fluid may act as a stimulus. A right-handed collector approaches the right side of the dog. The penis is gently held within the prepuce until penile engorgement begins. The dog's penis is then extruded from the prepuce and pressure applied with the forefinger and thumb to the penis on the proximal surface of bulbus glandis. Ejaculation is induced by pressure on the bulbus glandis and the rostral aspect of the glans penis rather than by masturbation. Care is taken to avoid hemorrhage from superficial vessels caused by vigorous manipulation. The pre-sperm fraction is scant and clear and is rapidly followed by the cloudy, sperm-rich fraction. Pelvic thrusting will accompany the first and possibly the second fraction. The third fraction contains prostatic fluid. It is clear and voluminous. The dog may 'step over' the arm of the operator during ejaculation of the third fraction. The collection vessel may be changed between fractions for separate collection of each. The dog should lose the erection and retract the penis within the prepuce within 5 to 10 minutes. A male can be collected a second time within minutes after the first collection. There is a very marginal increase in semen motility and morphology in the second ejaculate of dogs collected twice after a period of sexual rest. Semen collected by electroejaculation in anaesthetised dogs may or may not be the representative of naturally ejaculated semen.
Semen Handling
Semen collection apparatus and the collected sample should be held at 37°C until processing is complete. The presence of potential spermicidal agents such as detergents, some gels, lubricants, latex, dirt, and bacteria should be avoided. Failure to collect an adequate sample may be caused by poor technique, inexperience of the dog, unfamiliar surroundings, excessive disturbance in the collection area, lack of an estrous bitch, or pain associated with reproductive or musculoskeletal lesions.
Semen Evaluation
Semen should be evaluated immediately. The evaluated ejaculate is fractionated. Volume is recorded for calculation of total numbers of spermatozoa. The volume of prostate fluid collected is variable and not related to fertility. Colour is cloudy to milky white. Abnormal colour may occur with urine contamination or the presence of red or white blood cells. Semen pH is in the range of 6.5–7.0 and is useful for selecting antibiotics with which to treat prostatitis (Olson 1984).
A drop of raw semen is added to a pre-warmed glass slide, coverslipped, and evaluated by light or phase contrast microscopy for total and progressive motility, spermatozoal agglutination, and the presence of debris at 100 to 400X magnification. Progressive motility is the proportion of sperm showing linear, progressive forward motion. Concentrated ejaculates may be diluted for estimation of progressive motility by addition of phosphate-buffered isotonic saline or extender. A normal ejaculate has > 70% total and 80% progressive motility.
Total numbers are calculated by multiplying the concentration of spermatozoa (calculated from a sample of known dilution in a hemocytometer or Makler chamber, counted twice) with volume. Concentration is usually 50–1000 million spermatozoa per ml of semen. Total semen numbers are dependent on testicular weight, which is influenced by body weight. A 20- to 50-kg dog will produce 300 million to 2 billion spermatozoa. Spectrophotometers can approximate sperm numbers, e.g., the SpermaCue (Minitube) which is limited in accuracy to a range of 400 to 700 million/ml and is not calibrated for prostatic fluid.
Spermatozoal morphology should be examined by staining smears with modified Wright's-Giemsa or nigrosin-eosin stain. Thin smears should be stained for five minutes in each of the Wright's-Giemsa reagents. This stain has the advantage of staining somatic cells in addition to spermatozoa. Alternatively, one drop of nigrosin-eosin is mixed with one drop of semen and a spreader slide is used to make an even smear of stained cells; or phase contrast microscopy of a sample fixed in formol-buffered saline.
The proportions of spermatozoa with normal and abnormal morphology are recorded. Abnormal spermatozoa may be recorded as specific defects or by the classifications major/minor (indicating the proposed effect of the defect on fertility) or primary/secondary (indicating the suspected site of origin of the defect). Abnormal head shape, nuclear vacuolations, abnormalities of midpieces or tail, midpiece structural abnormalities, coiling of the midpiece, and proximal cytoplasmic droplets are primary abnormalities. Secondary abnormalities include distal cytoplasmic droplets, detached heads, simple bent tails, and loosened or detached acrosomes. A normal ejaculate has > 80% morphologically normal cells with no more than 10% primary abnormalities and no more than 20% secondary abnormalities, although the specific relationship of morphologic abnormalities to fertility is poorly documented.
Cytologic examination of an air-dried smear stained with Wright's-Giemsa allows identification and quantification of epithelial and inflammatory cells and erythrocytes. A Gram stain will identify bacteria. Cells in the ejaculate may originate from the testes, excurrent ducts (epididymis, vas deferens, urethra), urinary bladder, prostate, urethra, and penile surface. High numbers of white cells or phagocytosis of bacteria may be associated with infections.
Semen Culture
Quantitative aerobic bacterial culture can be performed on the fractionated ejaculate. Gram-positive bacteria are more likely to be urethral contaminants, while Gram-negative bacteria are more likely to represent infection. The role of anaerobes in male genital tract infection is thought to be minor. Semen should be transported in Ames transport medium for the isolation of Ureaplasma or Mycoplasma species, though the association of these organisms with reproductive tract pathology is tenuous and they are recovered in normal fertile dogs. A pure culture may be interpreted as suspicious if isolated in association with cytology and clinical signs indicative of infection.
Diagnostic Endocrinology
Testosterone is essential for normal libido and spermatogenesis; however, levels vary in the normal dog due to the pulsatile nature of secretion from 0.1–5 ng/ml (0.35–17 nmol/l). Concentrations in castrates are usually < 0.015 ng/ml (0.05 nmol/l). Testosterone-secreting ability is better measured by stimulation testing. Gonadotropin secretion is episodic, so measurements of FSH or LH should be made three times at 20-minute intervals. For example, FSH has been used as an indicator of spermatogenesis. Inhibin concentrations are reduced in the presence of testicular lesions, and FSH consequently rises. Concentrations in normal dogs range from 20–130 ng/ml (69–451 nmol/l), while concentrations > 250 ng/ml (867 nmol/l) might indicate testicular damage. Peripheral LH concentration may fluctuate from 0.5–10 ng/ml.
Semen Alkaline Phosphatase
Concentration of semen ALP is used as a marker for epididymal contribution to the ejaculate and is useful in distinguishing azoospermia from an incomplete ejaculate. The enzyme originates in the epididymis and is present at 5,000–40,000 IU/l. Ejaculates with AP concentrations < 5,000 IU/l are incomplete or may be delivered by dogs with bilateral outflow obstruction distal to the epididymis.
Spermatozoal Ultrastructure
Scanning or transmission electron microscopy may be performed on spermatozoa if ultrastructural defects are suspected.
Aspiration of Spermatozoa from the Epididymis
May be indicated if the dog has a palpable abnormality affecting one or both epididymides or if the ejaculate is azoospermic. Aspiration may indicate the presence of obstructive lesions of the outflow tract, neoplasia, sperm granuloma, and epididymitis. In the standing or sedated dog, the scrotal skin over the tail of the epididymis is prepared aseptically, and a 22- to 25-gauge needle is used to aspirate epididymal contents. Microscopic examination of the aspirate indicates the presence of spermatozoa, inflammatory cells, or bacteria. Extravasation of sperm during the procedure may induce sperm granuloma.
Testicular Fine-Needle Aspirate Biopsy and Testicular Biopsy
Fine-needle aspirate biopsy (FNAB) for testicular cytology is useful in diagnosis of azoospermia and differentiating neoplastic and inflammatory conditions. For azoospermia, FNA can be used to differentiate obstruction, spermatogenic arrest, or complete absence of spermatogenesis. Diagnosis of neoplasia can be made with a homogeneous neoplastic cell population; however, the cell type may not be identifiable. Aspiration does not always result in an adequate sample for diagnosis. In addition, diagnosis requires an ability to identify spermatogenic cell types. Testicular aspiration is contraindicated in acute inflammation. Testicular FNA is not as sensitive and specific as biopsy; however, it is less invasive, quick, and inexpensive. Biopsy will provide information regarding the etiology and severity of a testicular lesion and aid in prognosis. Biopsy is indicated in animals with oligospermia and azoospermia and in those with high proportions of primary morphologic abnormalities in the ejaculate. Under anaesthesia, the scrotum is prepared aseptically, and tissue may be obtained by fine-needle aspiration, split-needle biopsy, or wedge resection. Sequelae such as hematoma, adhesions, inflammation, and local autoimmune reactions are more likely with the more invasive techniques. Tissues samples may be cultured before being fixed in Zenker's or Bouin's solution for histologic examination. Analysis includes the presence of germ cells and Sertoli cells; proliferation and maturation of the germinal epithelium; presence of spermatozoa in seminiferous tubule lumens; and presence of histologically normal Leydig cells. Quantitative measures such as germ cell to Sertoli cell ratio, seminiferous tubule diameter, and identification of maturational stage of germ cells may allow more accurate interpretation of the severity of a testicular lesion.
Pelvic and Abdominal Radiography and Ultrasonography
Sonography may be used to provide additional data in animals with scrotal testicular and epididymal lesions and tumors, retained testes, testicular torsion, inguinal hernia, and diseases of the prostate gland. Ultrasound can also be used for guided-needle biopsies and aspirates of the prostate gland. Survey radiographs are indicated for prostate disease.
Additional Diagnostic Aids
Dogs with testicular hypoplasia or aplasia, abnormal external phenotype, or other signs consistent with intersex conditions should be karyotyped. Numerous additional tests exist for spermatozoal structure and function. These include tests of membrane integrity, acrosomal structure and integrity, binding ability to oocytes and oviductal cells, penetrating ability of oocytes, and computer-assisted analysis of patterns of motility and morphology. Few of these parameters have been associated with fertility in the dog. Molecular and genomic methods are becoming available for determining the genetic basis of reproductive disease.
References
1. Foote RH. The influence of frequency of semen collection, fractionation of the ejaculate, and dilution rate on the survival of stored dog sperm. Cornell Vet. 1964;54:89–97.
2. Root Kustritz MV, Hess M. Effect of administration of prostaglandin F2alpha or presence of an estrous teaser bitch on characteristics of the canine ejaculate. Theriogenol. 2007;67:255–258.
3. Olson PSN. Disorders of the canine prostate gland. Proc Soc for Theriogenol. 1994:46–60.
4. Purswell BJ, Althouse GC, Root Kustritz MV. Guidelines for using the canine breeding soundness evaluation form. Clin Theriogenol. 2010;2:51–59.