Breeding behaviour at puberty is not instinctive; it has been demonstrated that 61% of young male dogs will demonstrate abnormal behaviours such as attempting to mount the bitch’s head instead of hindquarters (Beach, 1968).

The normal male will investigate the female and, if she stands and is not hostile, will attempt to mount. The amount of time before the male mounts and the number of mounts he attempts before intromission occurs are variable and are not associated with fertility. The male dog clasps his forelimbs just in front of the bitch’s hind limbs and thrusts vigorously for several minutes. Spermatozoa are deposited into the vagina at that time. As the male’s penis continues to engorge, it becomes too large for withdrawal from the vagina and vulva, forming the copulatory lock, or tie. The male, still with his penis caught within the vulvar lips, will step one hind limb over the bitch’s back, twisting the penis in a horizontal plane such that the bitch and stud are standing facing opposite directions. During this time, vaginal contractions and pulses of prostatic fluid help move spermatozoa cranially in the bitch’s reproductive tract. The average duration of the copulatory lock is about 15 min.

Underlying factors

As in many species, puberty occurs when males reach about 80% of adult body weight. Because of the great variation in size of domestic dog breeds, there is a great variation in expected age of puberty onset; there is a positive correlation between age at puberty onset and expected weight at adulthood. Toy and small breed dogs may be sexually mature as early as 4–6 months of age, while large and giant breed dogs may not reach sexual maturity until 18–24 months of age (Beaver, 1977; Root Kustritz, 2010a).

Fertility and determination of breeding soundness

History and pre-breeding testing

When dogs are presented for breeding soundness examination, questions that should be asked include: history of breeding attempts, if any; time since last used for breeding or semen collection; any traumas or surgery, especially to the scrotum, prepuce or penis; history of febrile or systemic disease; drugs or other chemicals administered; brucellosis testing; general historical information, including diet and dietary supplements, and working history. Questions also may be asked about the sire’s semen quality; some semen characteristics, such as high sperm motility and low sperm output, have been reported to be highly heritable (England et al., 2010).

Any internal or external insult causing increased intrascrotal temperature may lead to morphological abnormalities of spermatozoa and decreased number of spermatozoa, even if no change of the testis is obvious on palpation. This includes febrile disease, hyperthermia and any direct insult to the scrotum, including frostbite. Drugs implicated in poor semen quality in male dogs include glucocorticoids, anabolic steroids, antifungal agents (including ketoconazole) and chemotherapeutic agents (Johnston et al., 2001a).

Genetic testing takes two forms in stud dogs. One is evaluation for hereditary disease that might preclude use of the animal for breeding. Specific genetic disorders of concern and tests available vary by breed and are best determined by accessing national breed club health information. For example, the national breed clubs of all registered breeds in the American Kennel Club in the USA are required to provide current information about diseases of concern and availability of testing.

Genetic testing also may be used to track the use of a given stud dog historically. Cheek swab samples can be collected easily from stud dogs and genetic information stored to permit parentage testing and create a database to better define the incidence of hereditary diseases if information from offspring is submitted to an open registry.

The other test that should be performed in all male dogs before use at stud is for Brucella canis, the causative agent of canine brucellosis (Hollett, 2006). Prevalence of this organism is higher among roaming dog populations than in pet populations and is increasing in some parts of the world, possibly because of increased movement of dogs between and within countries. B. canis preferentially grows in tissues of the reproductive tract, including the prostate, testes and epididymides (Serikawa et al., 1984). The organism is shed in body fluids, including the urine and semen. Transmission is through ingestion or inhalation, but venereal transmission may also occur. Because the disease is caused by a bacterium, culture is a definitive diagnostic test (Boeri et al., 2008). There are, however, concerns about cultural tests, which include: difficulty in appropriate sampling; overgrowth by normal flora in areas from which samples are drawn, necessitating multiple simultaneous cultures to enhance accuracy; cost; and potential exposure of laboratory personnel to this zoonotic organism. Serological tests include agglutination tests, agar gel immunodiffusion (AGID) tests, enzymelinked immunosorbent assays (ELISA) and polymerase chain reaction (PCR) tests. At present, agglutination testing is best done for screening, with all positives double checked by AGID testing. When PCR tests become commercially available, they will become the screening tests of choice (Root Kustritz, 2009). PCR testing on semen samples can be performed with exquisite sensitivity and specificity (Keid et al., 2009).

Physical examination findings

PROSTATE. The prostate is the only accessory sex gland in male dogs. It is palpable on transrectal examination as a bilobed, symmetrical structure. It encircles the neck of the urinary bladder and may fall forward into the abdomen with increasing size and weight. If it is not palpable per rectum after this occurs, ability to palpate may be increased by use of the free hand to push the caudal abdominal contents towards the gloved finger in the rectum, or by having an assistant ‘wheelbarrow’ the dog up on to its hind legs (Plate 6). If the prostate is normal, the dog should not exhibit signs of pain on transrectal palpation.

If the prostate is symmetrically enlarged with age, this provides evidence of benign prostatic hypertrophy/hyperplasia (BPH), which may or may not be associated with changes in semen quality (O’Shea, 1962; Zirkin and Strandberg, 1984; Read and Bryden, 1995). If the dog exhibits signs of pain on palpation of the prostate, acute prostatitis may be present; with chronic prostatitis, dogs rarely show signs of pain.

GENERAL PHYSICAL EXAMINATION. A complete physical examination is recommended. This may include specific testing, such as testing for ophthalmic disorders through the US Canine Eye Registry Foundation (CERF), and cardiac or hip evaluation for submission to the US Orthopedic Foundation for Animals (OFA), or other registries, as proof of worthiness of the dog as a candidate for breeding. For determination of breeding soundness, complete physical examination may allow the detection of signs of systemic diseases or causes of poor libido due to the pain from arthritis or other conditions, and signs of specific conditions such as brucellosis. Obesity has been shown to be associated with poor semen quality in some species due to increased intrascrotal fat and the subsequent increase in intrascrotal temperature, but this has not been reported in dogs. Some people attribute the association of poor semen quality with obesity as a sign of hypothyroidism, although experimental induction of hypothyroidism has not been associated with a decline in semen quality in dogs (Johnson et al., 1999).

Semen collection and libido

Electroejaculation is not routinely used in domestic dogs (Kutzler, 2005), in which manual ejaculation is the method of choice. Semen collection should take place on a non-slip surface. Some practitioners use a consistent rug or room, both to prevent injury and as a training aid. Presence of a teaser bitch, especially if she is in oestrus, increases the number of spermatozoa in the ejaculate (Traas and Root Kustritz, 2004). Other techniques for increasing the number of spermatozoa in the ejaculate include administration of prostaglandin F2alpha (PGF2alpha; 0.1 mg/kg subcut 10–15 min before collection) or gonadotrophinreleasing hormone (GnRH) (1–2 µg/kg IM 60 min before collection) (Purswell and Wilcke, 1993; Root Kustritz and Hess, 2007). These techniques are synergistic; best results are with administration of PGF2alpha in the presence of an oestrous teaser bitch. The teaser bitch should be muzzled or otherwise restrained so that she will not turn on the male dog.

Some male dogs are hesitant to breed bitches and rarely show investigative behaviour such as licking or sniffing at urine spots, or flehmen. Established causes of poor libido in male dogs include:

• Lack of appropriate stimulation. This may include bitches being presented before they are in oestrus or at optimal breeding time. The pheromone produced by bitches that identifies them as being in oestrous is methyl-p-hydroxybenzoate (Goodwin et al., 1979). A commercial product containing this compound has been marketed for enhancement of male libido.

• Negative breeding experience. This may include threatened or real injury from an aggressive bitch, injury to the penis during mating, or constant discipline by humans for mounting behaviour outside breeding. Some dogs that fail to show normal breeding behaviour as a result of anxiety may respond to use of dog appeasing pheromone (DAP ^® – Dog Appeasing Pheromone; CEVA Santé Animale, Intervet/Schering Plough Animal Health Canada Inc, Kirkland, Quebec), a compound that originates from the intramammary sebaceous glands of lactating bitches and has been demonstrated to reduce anxiety in adult dogs (Mills et al., 2006). Melatonin has been shown to increase sexual behaviour in some laboratory animal species, but has not been evaluated in dogs (Brotto and Gorzalka, 2000).

• Pain when attempting to mount or ejaculate. This may be due to prostate disease, or to spinal or rear limb disorders.

• Mate preference. Some dominant male dogs will breed with the dominant bitch only or with females of their own breed.

• Improper age. The dog is too young or too old.

• Abnormalities of sexual differentiation. This includes hermaphroditism and pseudohermaphroditism, persistent Müllerian duct syndrome and segmental epididymal aplasia (Majeed, 1974; Brown et al., 1976; Hare, 1976; Marshall et al., 1982; Meyers-Wallen and Patterson, 1989; Batista et al., 1998).

Hypothesized causes for poor libido in animals include hyperprolactinaemia, which is associated with poor libido in human males and females, and hypothyroidism. Hyperprolactinaemia is not a demonstrated disorder in dogs, and researchers have also been unable to demonstrate change in libido with experimental induction of hypothyroidism in dogs (Johnson et al., 1999). Low serum testosterone concentration has never been reported as a cause of poor libido in animals, although some males will show more interest in females when treated with testosterone. Routine treatment with testosterone is not recommended; remember that testosterone must be secreted in pulses for the normal production of spermatozoa to occur and if a dog is periodically provided with a large amount of testosterone, that pulsatile release is disrupted and negative feedback exerted on the pituitary, stopping the secretion of luteinizing hormone (LH) and subsequent secretion of testosterone for an unpredictable length of time.

Semen evaluation

GENERAL INFORMATION. There is little information in the veterinary literature linking specifics of semen evaluation to fertility in dogs. One author suggests that one can only make accurate predictions based on canine semen evaluation findings if semen quality is either very good or very poor (Peña Martínez, 2004). Ejaculated spermatozoa have generally not yet undergone capacitation and so do not exhibit the same functions as they would in a bitch’s reproductive tract. Spermatozoa attain motility while moving through the epididymis, so testicular or epididymal aspirates will yield immature spermatozoa. Results of the tests that may be performed as described below are affected by sample collection technique, time from sample collection to evaluation, equipment used, skill of the investigator and other factors (Rui et al., 1986; WHO, 1999; Verstegen et al., 2006). Human semen collection is well described. The recommendations below may reflect some of those of the World Health Organization (WHO), which regularly updates a handbook describing quality control measures for human andrology laboratories (WHO, 1999). However, the procedures outlined here are for the evaluation of ejaculated canine spermatozoa (Root Kustritz, 2007).

Canine semen is evaluated at room temperature. Excessive attempts to warm the semen may decrease its quality (Bartlett, 1962; Chatterjee et al., 1976; Threlfall, 2003). The different components that are involved in the manual evaluation of semen are outlined below. Computer assisted sperm analysis (CASA) systems are also available and generate repeatable and accurate results with standardized settings (Schäfer-Somi and Aurich, 2007); they are very useful for some of the evaluations that need to be made (as noted below). Procedures for the use of CASA systems vary with the equipment used.

VOLUME. Volume is not in and of itself an indicator of semen quality because it is dependent on the operator who collected the semen. If more of the third, or prostatic, fraction is collected, the sample will be larger in volume and more dilute. Volume should be recorded before any samples are withdrawn as that value will be used to calculate total number of spermatozoa in the ejaculate.

PH. Measurement of pH was done traditionally to help clinicians choose an appropriate antibiotic that could penetrate and be trapped within the prostate. Normal pH in non-fractionated canine semen is reported to vary from pH 6.4 to 6.8 (Bartlett, 1962; Chatterjee et al., 1976; Daiwadnya et al., 1995). The inherent inaccuracy of pH measurement with pH paper and the emergence of antibiotics that ionize at multiple pH values have decreased the value of pH testing of canine semen.

PERCENTAGE PROGRESSIVE MOTILITY