THE BITCH

The average age at the time of puberty in bitches is 9 to 10 months, and the range is 6 to 24 months of age. The interval from the beginning of one cycle to the beginning of the next, or the interestrous interval, varies from 4 to 12 months and averages 7 months. Therefore bitches have only one or two cycles per year. The interestrous interval is extremely variable within individual bitches, more so than it is among bitches. Because of this variability, the past interestrous interval does not accurately predict the next cycle in an individual bitch. Although a few bitches are very consistent, in most there is more than a month’s variation from cycle to cycle. The interestrous interval is not influenced by pregnancy or the photoperiod, although breeds such as the Basenji cycle only once each year, indicating a possible effect of the photoperiod in some individuals.

The estrous cycle in the bitch is divided into four components: proestrus, estrus, diestrus, and anestrus. Proestrus and estrus together are often referred to as heat or season. Together they constitute the follicular phase of the reproductive cycle. The luteal phase of the cycle is referred to as diestrus. The canine estrous cycle is distinctly different from that of other domestic species in several regards. These include the very long anestrus (months as opposed to days or weeks), the long proestrus and estrus (days to weeks as opposed to hours or days), the fact that the corpora luteal lifespan is independent of the presence (or absence) of pregnancy, ovulation of an immature oocyte, and long viability (days as opposed to hours) of oocyte and sperm within the female tract.

Proestrus

Proestrus is considered to begin when vulvar swelling and a sanguineous discharge are first observed. It ends when the bitch allows copulation. The average duration of proestrus is 9 days, and the range is 3 to 17 days. Attractiveness and receptivity to male dogs gradually increase throughout proestrus. The factors that end anestrus and initiate a new follicular phase in the bitch are poorly understood. Throughout anestrus follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are secreted concomitantly, in a pulsatile pattern. The FSH pulses are of lesser magnitude but longer duration than are the LH pulses. Basal concentrations of FSH increase as anestrus progresses, whereas basal LH concentrations are unchanged. The increase in FSH concentrations is considered crucial to initiate ovarian follicular development and the onset of proestrus. The developing ovarian follicles are 1.5 to 5 mm in diameter. They produce estrogens, the most important of which is estradiol 17-β. Estradiol causes the vulvar swelling, vaginal edema and cornification, and uterine bleeding that is recognized by a serosanguineous vulvar discharge. Estradiol serum concentrations gradually increase during early proestrus. They increase sharply just before the preovulatory LH surge and rapidly decline to basal levels thereafter (Fig. 56-1).

FIG 56-1 The canine estrous cycle.

(From Morrow DA, editor: Current therapy in theriogenology, ed 2, Philadelphia, 1986, WB Saunders.)

Under the influence of estrogen the vaginal epithelial cells proliferate and mature (cornification). The stratified squamous epithelium increases in thickness from a few cell layers in anestrus to 20 to 30 cell layers in late proestrus. The degree of estrogenic influence and therefore the stage of the estrous cycle with respect to the serum estrogen concentration can be monitored by vaginal cytology (see p. 891).

Estrus

Behavioral estrus is characterized by acceptance of mating. The bitch’s feet are firmly planted to allow the male to mount—hence the term standing heat (Fig. 56-2). The tail is deviated to the side to allow intromission; this behavior has been referred to as flagging. Stroking the perineum may occasionally elicit flagging, which would be an indication that the bitch is in estrus. The average duration of estrus is 9 days, and the range is 3 to 21 days. The swollen vulva is less turgid than during proestrus. The vulvar discharge of estrus is usually less bloody than that of proestrus, but normal bitches often have a sanguineous discharge throughout both. Therefore changes in the gross appearance of the discharge are not necessarily indicators of the transition from proestrus to estrus.

FIG 56-2 Canine breeding behavior. A, Flagging and standing. B, The postcoital tie.

The preovulatory follicles have reached 3 to 8 mm in diameter. The increase in estradiol concentrations during proestrus, via positive feedback to the hypothalamus, initiates the LH surge, which in turn causes ovulation and the subsequent formation of corpora lutea (CL) and progesterone secretion by the ovary (Fig. 56-1). In the bitch the initial increase in progesterone secretion coincides with the LH surge. Although the onset of behavioral estrus usually occurs within a day or two of the LH surge, behavioral estrus may occur as early as 4 days before or as late as 6 days after the LH surge. Therefore the day on which a bitch allows copulation is not closely associated with the LH surge or ovulation.

In most bitches ovulation occurs within 48 hours of the LH surge (the range is 0 to 96 hours). Ovulation from both ovaries is apparently completed within 24 hours. Primary oocytes (prophase I) are ovulated and resume meiosis during tubal transport. By 2 to 3 days after ovulation, oocytes have matured (metaphase II) and fertilization can occur. Mature oocytes have a fertile life of 2 to 4 days, perhaps longer. The time during which mature oocytes are available for fertilization has been referred to as the fertile period. Although semen is initially deposited in the cranial vagina during copulation, the large volume of prostatic fluid and the postcoital tie force semen through the cervix (see Fig. 56-2). For that reason dogs are considered among the species having intrauterine semen deposition during natural mating. Fertilization occurs in the uterine tubes. Sperm transport is enhanced by vaginal and uterine contractions that spontaneously occur with natural mating but not during artificial insemination. Freshly ejaculated canine sperm bind to the uterine crypts and glands and to the distal uterine tube, which serves as the sperm reservoir. The sperm reservoir maintains sperm viability between insemination and ovulation, regulates sperm capacitation to synchronize sperm function with ovulation, and controls sperm transport in the uterine tube. Canine sperm remain capable of fertilization in the female tract for 3 to 4 days and occasionally for as long as 6 days. Some sperm can be found in the female tract up to 11 days. The end of the fertile period is thought to be due to oocyte aging, but changes in the cervix and uterine tube environment also play a role.

THE QUEEN

Female cats are seasonally polyestrous. Cyclicity is controlled by the photoperiod, which must be approximately 12 to 14 hours of light with an intensity of 50 foot-candles. Melatonin appears to be the signal of photoperiod in domestic cats. Cats exposed to natural light usually cease cycling during short days of winter, whereas cats in equatorial photoperiods or maintained under artificial light often cycle throughout the year. It has been shown that maintaining 14 : 10 to 16 : 8 hour light : dark schedules maximizes the number of cycling queens in the colony. In the presence of adequate light, sexual maturity and the first estrous cycle normally occur at 6 to 9 months of age, with a range of 5 to 12 months. Unlike bitches, which ovulate spontaneously, queens are induced to ovulate by coital stimulation of the vagina. In addition to coitus-induced ovulation, many domestic cats also have cycles in which spontaneous ovulation occurs.

The follicular phase of the cycle is characterized by increasing serum concentrations of estradiol 17-β associated with the onset of proestrus and estrus. Because there is negligible vulvar swelling or discharge in queens compared to bitches, proestrus and estrus are usually recognized by behavioral changes. When it is observed, the vulvar discharge is a clear fluid. Proestrus is characterized by rubbing, treading with the rear feet, vocalization, and decreasing hostility toward the male, although queens will continue to strike at the tom. Proestrus may be so short as to be unrecognized, but more typically it lasts 1 to 2 days.

Estrus is characterized by increased vocalization, rolling, lordosis, holding the tail to one side, and allowing copulation. The characteristic estrual posture can sometimes be elicited by stroking the perineum (Fig. 56-3). Tremors of the body or tail may also be seen. The cytologic appearance of exfoliated vaginal epithelial cells during the estrous cycle is similar to that of bitches, except that red blood cells are much less common. The duration of estrus among queens is quite variable but averages 5 to 8 days. Its duration is not influenced by copulation. Anovulatory cycles occur every 2 to 3 weeks (average 18 days with 12 hours of light) as long as light is adequate. There may or may not be a short interestrous period of a few days.

FIG 56-3 Estrual posture of the queen.

Ovulation occurs as a result of a neuroendocrine reflex that is initiated by the mechanical stimulation of sensory receptors in the vagina and cervix. This sensory input causes a surge of LH to be released from the pituitary gland (Fig. 56-4), which in turn causes ovulation. A high level of estradiol is also required for ovulation. The precise intensity of the copulatory stimulation necessary to induce the LH surge is unknown but varies among queens. The frequency of coital stimulation is apparently the single most important determinant of ovulation in cats. A single copulation induces the LH surge necessary for ovulation in approximately 50% of cats, whereas more than 90% of normal domestic shorthair cats ovulate if bred 3 times daily for the first 3 days of estrus. The day of estrus on which mating occurs and the duration of estrus have no apparent effect on ovulation, except insofar as the concentration of estradiol varies. Once the LH surge occurs, hormonal responses to additional copulatory stimuli are diminished. Ovulation occurs approximately 48 hours after the LH surge. Although cats continue to be referred to as induced ovulators, it is also clear that many cats (35% to 60%) also ovulate spontaneously, in the absence of coital stimulation or direct physical contact with other cats.

FIG 56-4 Hypothalamic-pituitary-gonadal axis. ABP, Androgen-binding protein; DHT, dihydrotestosterone; E, estrogen; FSH, follicle-stimulating hormone; GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone; P, progesterone; PRL, prolactin; T, testosterone.

After intromission and ejaculation, the queen emits a characteristic scream that signals to the male to dismount. Despite willing acceptance of copulation moments before, queens will attack the tom at this time. Because cats often prefer seclusion, breeding may not be witnessed by the owner. The queen’s scream may be the only evidence that mating has occurred. The queen then begins frenzied rolling and grooms her perineum for several minutes and aggressively rebuffs the male. When this “after-reaction” subsides, the queen allows another mating, by either the same tom or another one. Mating frequency is greatest during the first 2 hours (average of five copulations per hour), after which the frequency decreases to about one copulation per hour for the next 3 days. To ensure adequate copulatory stimulation to induce ovulation, three breedings per day for the first 3 days of estrus are recommended. Semen is deposited in the vagina during copulation. The cervix and uterotubal junction are barriers to sperm transport in the cat. The cervix is open on the first day of estrus in both ovulatory and nonovulatory cycles. It is closed when estradiol concentrations fall and when progesterone concentrations rise. As with the bitch, the queen’s uterine contractions during mating promote sperm transport. The uterotubal junction and uterine crypts are sperm reservoirs before ovulation, and the tubal isthmus is the reservoir near ovulation.

Because of the territorial nature of cats, especially males, the queen should be brought to the stud. The two should be placed together for short periods so that their behavior can be observed. In this way the manager can be confident that matings have occurred; conversely, the cats can be separated if fighting occurs. This supervised mating scheme may be the best way to optimize conception, but it is labor intensive. Some managers prefer to house the queen and tom together and allow mating to occur ad libitum, without direct observation. In some large breeding colonies, harem, rather than individual, mating schemes are used. In the harem scheme, one or two toms are housed with several queens. Even though both toms have equal access to the queens, the dominant male usually does most of the breeding. This method is the least labor intensive but has the disadvantages of unknown breeding dates, unknown paternity if more than one tom is involved, and delayed recognition of subfertility in individual animals.

After ovulation the follicles luteinize and produce progesterone. This is the luteal phase of the cycle. Serum concentrations of progesterone rise 24 to 48 hours after ovulation and peak 25 to 30 days later. As with bitches, luteal progesterone is necessary for the maintenance of pregnancy. The corpora lutea continue to produce progesterone throughout the approximately 65-day gestation (Fig. 56-5), with the serum concentrations gradually declining during the second half of pregnancy. Contrary to what was previously thought, the feline placenta either does not secrete progesterone or does so in amounts insufficient to maintain pregnancy. Serum concentrations of estradiol increase in late pregnancy in cats. Although estrous behavior has been observed in pregnant queens, true superfetation has not been proved.

FIG 56-5 The feline estrous cycle.

There apparently are pregnancy-specific luteotropic hormones from the feline placenta or pituitary that control the life span of the corpus luteum. After the nonfertile induction of ovulation (i.e., when the animal is not pregnant), the corpora lutea persist for about 30 to 40 days. The next cycle may begin any time thereafter, usually within 10 days. In one colony the average interestrous interval was 61 days in queens that were bred but did not conceive, whereas the average interestrous interval for nonbred, anovulatory queens was 22 days. Serum concentrations of progesterone were not determined in those cats.

Litters typically consist of two to five kittens. Queens usually do not resume cycling while they are nursing a litter. Estrous behavior is usually evident 2 to 3 weeks after weaning, although this is quite variable. The postpartum estrus is shorter in duration and less fertile than others. Litter size and neonatal survival are best for queens age 1 to 5 years, provided that first parity occurs before 3 years of age. Litter size and neonatal survival usually improve after the first parity. However, if the first parity occurs after 3 years of age, litter size and neonatal survival usually remain poor. Reproductive performance declines after 6 years of age. Because of decreased fertility, decreased litter size, increased neonatal losses, and the increased prevalence of other illnesses in older queens, most should be retired from breeding after 8 years of age.

VAGINAL CYTOLOGY

The importance of exfoliative vaginal cytology in breeding management and in the evaluation of females with reproductive disorders cannot be overemphasized. Vaginal cytology is used to determine the stage of the estrous cycle, determine breeding and whelping (see Chapter 58) dates, and identify the nature of certain abnormal processes within the reproductive tract (see Chapter 57). Specimens may be obtained with a moistened, cotton-tipped swab or by flushing and aspirating a small volume of saline solution from the vagina. Specimens can be stained with any number of commercially available stains, including Wright’s, Wright-Giemsa, modified Wright-Giemsa (Diff-Quik; Baxter Scientific), trichrome, or new methylene blue. The number and morphologic characteristics of vaginal epithelial cells are evaluated. The preparations are also examined for the presence of other material, such as bacteria, white blood cells, red blood cells, mucus, cellular debris, endometrial cells, or neoplastic cells.

The vaginal epithelium changes dramatically under the influence of estrogen, a process known as cornification. During early proestrus the noncornified parabasal and intermediate vaginal epithelial cells are the predominant cells (more than 80%). As proestrus progresses, the population of exfoliated cells gradually matures; parabasal and intermediate cells disappear as superficial (cornified) cells increase in number. At the end of proestrus superficial and anuclear squamous cells account for 70% to 80% of the epithelial cells. White blood cells decrease in number. Extracellular bacteria may be present throughout proestrus and estrus (Fig. 56-6).

FIG. 56-6 Vaginal cytology of estrus. A, parabasal and intermediate cells. B, Intermediate and superficial cells. C, Anuclear squamous cells.

A predominance of superficial cells, an absence of neutrophils, and a clear background characterize vaginal cytologic specimens obtained during estrus. During estrus 90% or more of the epithelial cells are superficial and anuclear squamous cells. White blood cells are normally absent during estrus. Red blood cells and extracellular bacteria are often present (see Fig. 56-6). The beginning of diestrus is marked by an abrupt change in vaginal cytology. Diestrus is characterized by a sudden reduction in the number of superficial cells and the reappearance of intermediate cells, neutrophils, and background debris. On the first day of cytologic diestrus, parabasal and intermediate cells outnumber the superficial and anuclear squamous cells. Sheets of intermediate cells are also often observed. White blood cells return in high numbers during the first day or two of diestrus. Red blood cells and bacteria disappear. The initial dramatic change in cytologic appearance is followed by a gradual change to the anestrual cytologic appearance. On the basis of the examination of only a single cytologic specimen, proestrus cannot necessarily be distinguished from diestrus. The vaginal cytology of anestrus is quite acellular; it contains primarily parabasal cells and a few small intermediate epithelial cells. The transition from proestrus, through estrus, and into diestrus is usually adequately monitored by cytologic studies done every 2 or 3 days.

VAGINOSCOPY

Vaginoscopy is useful for evaluating animals with lower urinary tract signs or urinary incontinence, vulvar discharge, infertility, and anatomic abnormalities; for determining the nature and extent of lesions within the vestibule and vagina; and for identifying the stage of the estrous cycle. Samples for cytologic, microbiologic, and histopathologic studies can easily be obtained through the endoscope. Assuming that the clinician has access to the proper equipment, laser surgery can also be performed. Complications resulting from vaginoscopy, which include hemorrhage, laceration, and introduction of infection, are uncommon with proper technique. Endoscopic findings are assessed by comparing them to the normal anatomic features of the vagina, often in conjunction with vaginal cytology. The endoscopic appearance varies tremendously with the stage of the estrous cycle.

The canine vagina is quite long. In Beagles, for example, it measures 10 to 14 cm in length and 1.5 cm in diameter, whereas in Newfoundlands the length may be up to 29 cm. The endoscopic equipment must be of the appropriate size for the particular female. Proctoscopes and cystoscopes designed for human pediatric or adult patients or flexible fiberoptic endoscopic equipment of appropriate diameter can be used. Pediatric anoscopes or veterinary otoscopes may be narrow enough for use in queens and small bitches but are too short for examination of the cranial vagina and cervix.

In bitches that are in heat, vaginoscopy is usually performed with the animal awake and standing and without sedation or anesthesia unless a biopsy is planned. Anesthesia is necessary for vaginoscopy in queens, small bitches, and puppies and when cystoscopes with saline infusion to distend the vagina will be used. The perineum is inspected and cleansed. The endoscope is then lubricated with warm saline solution or with sterile, water-soluble lubricant. The clitoris and clitoral fossa must be avoided. Therefore the endoscope is passed in a dorsal direction through the dorsal commissure of the vulva. There will be increased resistance at the narrow vestibulovaginal junction (Fig. 56-7) in all but estrual bitches. It is especially narrow in prepubertal and neutered animals. The angle of the speculum is adjusted to be more parallel with the spine after it passes through the vestibulovaginal junction.

FIG 56-7 Vaginoscopy demonstrating the vestibulovaginal junction in a 1-year-old, spayed female retriever with lympho-nodular urethritis causing persistent pollakiuria.

During proestrus the longitudinal folds of the vagina are edematous, round, and smooth. As new folds develop, the vaginal lumen becomes filled with folds. A clear, bright-red fluid is seen in the vaginal lumen, sometimes in large amounts. As estrus approaches, the vaginal folds become lower and wrinkled. During estrus the folds appear sharp, angular, and crinkled. The mucosa is pale, and the vaginal lumen is wide. There is less luminal fluid than there is during proestrus. This fluid is clear and usually straw colored; however, it may continue to be bright red throughout estrus.

During diestrus (the luteal phase) the vaginal folds are low, round, and soft. The folds in the cranial vagina have a characteristic rosette appearance and may be mistaken for the cervix. Clear or opalescent mucus is present in the vaginal lumen during diestrus. The vaginal mucosa has streaks of hyperemia. During anestrus and in neutered bitches, the vaginal folds are low and round and do not fill the lumen. There is a thin mucous coating that gives the mucosa a translucent, pink-red appearance. In these animals the mucous membranes are thin and easily traumatized. Pinpoint submucosal hemorrhages may develop in response to seemingly gentle contact with the endoscope. During anestrus and in neutered animals there is usually some resistance to the passage of the endoscope unless the instrument is very well lubricated.

In bitches one of the vaginal folds, known as the dorsal median postcervical fold, is often mistaken for the cervix. This fold extends from the caudal-dorsal edge of the vaginal portion of the cervix along the dorsal midline and eventually blends into lesser folds of the vagina. It is composed of longitudinal and oblique smooth muscle bundles and irregularly arranged collagen. Unlike other folds of the vagina, the dorsal median fold has no elastic fibers. In Beagle-size bitches, this fold is 15 to 42 mm long and 2 to 10 mm wide, compared with the average vaginal length in the same bitches of 158 ± 30 mm. The lumen of the cranial vagina in this area is quite narrow. Because of its length, location, and inelastic nature, the dorsal median postcervical fold often prevents visualization and catheterization of the canine cervix.

The vaginal portion of the cervix is tubular, with small furrows radiating from the os, which give it the appearance of a star or rosette. The cervical os is not obviously “open,” even if fluid is seen flowing through it, except during the puerperium. The vaginal lumen around the cervix and the cranial aspect of the dorsal median postcervical fold is quite narrow, and except during estrus the use of small-diameter (0.5 cm) instruments is usually necessary to visualize the cervix. The narrow pericervical vaginal lumen with the dorsal median postcervical fold and the rosette appearance of the cranial vagina can be confused with the cervix.

VAGINAL BACTERIAL CULTURES

Bacterial infections of the reproductive tract are relatively common. Bacterial culture is indicated for the evaluation of many reproductive disorders, including infertility, vulvar discharge, pyometra, metritis, abortion, and stillbirth. Because the uterus is usually sterile, except in some bitches during proestrus and estrus, the interpretation of uterine culture results is relatively straightforward. Unfortunately, because of the difficulty in catheterizing the cervix in the bitch or queen, uterine samples are usually obtained only during laparotomy. Vaginal cultures are usually performed in lieu of uterine cultures. To minimize contamination from the vestibule and caudal vagina, samples for bacterial culture should be obtained from the cranial vagina using a guarded culture swab (e.g., those manufactured by Kalayjian Industries and Nasco) or through a sterile speculum.

The canine vagina has normal bacterial florae, which are listed in Box 56-1. Only 2% of 826 specimens obtained from intact bitches were negative for bacterial growth (Bjurström et al., 1992), whereas 23% of 66 specimens from queens were negative (Ström-Holst et al., 2003). In order of reported frequency, the most commonly isolated organisms from bitches are Pasteurella, Streptococci, and Escherichia coli. With the exception of Mycoplasma, anaerobic organisms are much less commonly isolated than aerobic. Cultures from bitches usually yield mixed populations of bacteria; however, in Bjurström’s study 18% were a growth of only one organism. The florae vary within and among individuals and throughout the cycle. The normal florae of the feline vagina are similarly diverse. E. coli, Staphylococcus, and Streptococus canis are the most common organisms recovered from queens. Unlike the situation in bitches, a single organism (most often E. coli) is isolated from 41% of cats. Anaerobic organisms are uncommon in queens. Even in normal bitches and queens, organisms may be recovered in large numbers.

Most of the organisms that make up the normal vaginal florae are also potential pathogens. Several studies have shown that there are no differences among the bacterial isolates from normal fertile bitches, infertile bitches, and bitches with evidence of genital disease. Isolation of opportunistic pathogens from the vagina is therefore not proof of infection. Thus the results of vaginal cultures and the potential role of the isolated organisms in the pathogenesis of the clinical signs must be interpreted cautiously. Brucella canis (see Chapter 58) is always considered a pathogen, even in the absence of clinical signs. The role of Mycoplasma spp. and Ureaplasma spp. in reproductive disorders in cats and dogs remains unclear.

VIROLOGY

Viral diseases may cause reproductive problems by directly affecting reproductive organs or because of the systemic illness they cause in the pregnant female. Respiratory disease and neonatal death are the most common manifestations of canine and feline herpes infection. Canine herpes virus also causes apparent failure to conceive; abortion; and, less commonly, genital lesions. Rarely, vesicular lesions may be found on the mucosa of the vestibule or prepuce of infected dogs. The most important route of transmission is oronasal contact with infected secretions. Transplacental and venereal transmission are much less important. The virus may be isolated from nasal, conjunctival, tracheal, vaginal, or preputial scrapings for 2 to 3 weeks after acute infection. Thereafter virus isolation and polymerase chain reaction (PCR) are usually negative. Because canine herpesvirus is poorly immunogenic, virus-neutralizing antibodies are present in small amounts for short periods. The finding of any detectable titer in the presence of compatible clinical signs is therefore considered significant. Fetal and neonatal necropsy findings are generalized, multifocal hemorrhages in kidney, lung, and liver and necrotic foci with intranuclear inclusion bodies. In queens panleukopenia, calici virus, feline infectious peritonitis, and feline leukemia virus infection are reported to be potential causes of infertility, abortion, and neonatal death.

ASSESSMENT OF REPRODUCTIVE HORMONES

Measurement of serum concentrations of reproductive hormones can be useful in evaluating animals with suspected or known reproductive disorders. The reproductive hormones are released in cyclic, episodic, or pulsatile manners; therefore the results of a single determination often are not diagnostic because the phase of the cyclic release at the time of sample collection is unknown. For that reason, repetitive determinations performed over the course of hours, days, or weeks, or provocative testing, may be necessary. Most hormone assays, such as radioimmunoassays (RIA), chemiluminescent, and enzyme-linked immunosorbent assays (ELISA), depend on immunologic reactions. Errors can result if antibodies or antigens in homologous assay systems are not species specific and if species-specific interference with antibody binding occurs in heterologous systems. For these reasons, it is critical that each laboratory validate its procedures and determine reference ranges for each species and each hormone to be tested.

Estradiol

Estradiol-17β is the main estrogen in circulation. The primary source of estradiol in sexually intact females is the ovarian follicle. In both males and females estradiol is also derived in peripheral tissue by the aromatization of testosterone and androstenedione. In sexually intact males the testis produces small amounts of estradiol, but this accounts for only about 20% of estradiol production in dogs. The majority is derived from aromatization of circulating androgens, testosterone and androstenedione. Androstenedione is of adrenal origin. Typical mean serum estradiol concentrations in the bitch are 5 to 10 pg/ml during anestrus, 10 to 20 pg/ml during early proestrus, and 50 to 100 pg/ml during late proestrus. Estradiol concentrations decline through estrus (see Fig. 56-1). During estrus in queens, estradiol is also typically >25 pg/ml to above 50 pg/ml. It returns to basal levels of <15 pg/ml in between cycles and during the seasonal anestrus.

Unfortunately, estradiol concentrations are often at or below the limits of detection of the assays used by many commercial endocrine laboratories. Estradiol concentrations also fluctuate widely and rapidly, and the high concentrations that occur during proestrus may be detectable for only a day or two. Deficiencies in circulating concentrations of estradiol are rarely documented in dogs and cats. Pathologic increases in estradiol production, such as those that occur in animals with ovarian follicular cysts or Sertoli cell tumors, may still be less than the detectable limits of many assays. For these reasons the measurement of estradiol concentrations often does not yield diagnostic results. A simple, accurate means of gauging estrogenic activity in the female is to evaluate vaginal epithelial cells for signs of cornification (see Fig. 56-6). All things considered, vaginal cytology is often preferable to determination of serum concentrations of estradiol in females. The preputial epithelium is also responsive to estrogen, exhibiting changes similar to those of the vaginal epithelium (Fig. 56-8). The paraneoplastic syndromes associated with excessive estrogen in dogs include alopecia, gynecomastia, pendulous prepuce, and bone marrow suppression. In bitches and queens cystic follicles in intact or remnant ovaries may continuously produce estradiol and cause persistent signs of heat and, much less commonly, alopecia. Assessing estradiol or its influence on vaginal epithelium is indicated for determining the stage of the estrus cycle for breeding management and for evaluating females suspected of having an ovarian remnant after being spayed. Finding cornification of vaginal epithelium or very high estradiol concentrations in a supposedly spayed queen or bitch that is displaying characteristic physical or behavioral signs of heat would be consistent with a diagnosis of ovarian remnant. Finding very high estradiol concentrations or the influence of estradiol on vaginal or preputial epithelium in an animal displaying estrogen-induced paraneoplastic syndromes justifies a search for a gonadal source (estrogen-producing testicular tumor, cystic ovarian follicles) or an exogenous source of estrogen. These would be far more likely than an adrenal source of estrogen in species other than the ferret.

FIG 56-8 A, Vaginal cytology from an 18-month old cat with an ovarian remnant after being spayed 1 year ago. Estrus cycles began a month ago. B, Preputial cytology from a 10-year-old, male retriever with an estrogen-producing interstitial cell tumor.

DIAGNOSTIC IMAGING

Radiology and ultrasonography are useful for evaluating the ovaries, uterine wall, and intrauterine contents; confirming pregnancy; and assessing fetal viability. The normal uterus and ovaries in a nonpregnant animal are not detected by routine abdominal radiography (see Fig. 56-9). During normal anestrus they may be difficult to identify by ultrasonography. Increased size and density and an abnormal shape of the uterus may be detected by either technique. Ultrasonography can be used to evaluate the uterine wall and the intrauterine contents. Ultrasonography may also help identify ovarian remnants, ovarian cysts in animals with persistent estrus and hyperestrogenism (follicular cysts), or persistent anestrus (nonfunctional or luteal cysts). It may be able to identify ovarian neoplasia as well. In males diagnostic imaging is very helpful in evaluating the prostate and testes (see Chapters 61 and 62). Negative findings with diagnostic imaging do not necessarily exclude disease in the reproductive tract, especially in females.

FIG 56-9 A, Sonogram of canine gestational sac (arrows) at 29 days. Scale is in centimeters. B, Sonogram of canine pyometra showing thickened uterine wall (W) and lumen distended with fluid (F). C, Radiograph of feline pyometra showing fluid-filled uterus (arrows). D, Radiograph of mummified fetus. E, Sonogram of 1.8 × 1.2-cm ovary with corpora lutea in a normal 3-year-old Weimaraner 30 days after estrus. The serum progesterone concentration was 64 nmol/L. LK, Left kidney.

(A Courtesy Dr. Tom Bell, East Lansing, Mich.)

Because of the difficulty involved in catheterizing the cervix, contrast studies of the uterus and uterine tubes (i.e., hysterosalpingography) are rarely done in bitches and queens. During estrus contrast material deposited in the cranial vagina may enter the uterus and provide a hysterogram, but at other stages of the cycle the cervix is normally closed. Positive-contrast vaginography, using a Foley catheter and a water-soluble contrast agent (e.g., diatrizoates such as Renografin®), is easily performed, but general anesthesia is necessary. Vaginography can be considered if vaginoscopy fails to clearly identify strictures, anatomic defects, masses, or foreign material in the vagina.

KARYOTYPING

Some intersex conditions and developmental abnormalities of the reproductive tract may be associated with chromo somal anomalies (e.g., XXX, XO). These animals are usually seen because of abnormal external genitalia, infertility, or persistent anestrus. Karyotype analysis can be performed if a congenital rather than an acquired cause is suspected and if routine diagnostic tests have failed to identify the cause of the reproductive dysfunction. Cells from any tissue can theoretically be used for chromosomal analysis, but lymphocytes from heparinized blood samples are the usual specimen. (e.g., University of Minnesota Veterinary Cytogenetics Laboratory, Department of Veterinary Pathobiology).

LAPAROSCOPY AND CELIOTOMY

Exploratory celiotomy is often the most cost-effective way to diagnose and treat intersex animals. In all other circumstances, however, diagnostic laparoscopy or exploratory celiotomy should not be done until a noninvasive diagnostic evaluation of the bitch or queen with a reproductive disorder has been completed. Laparoscopy and celiotomy allow gross visualization of the reproductive tract, bacterial culture of the uterine lumen, and full-thickness biopsy of the uterus. The patency of the uterine horn and uterine tubes might be determined by infusion of sterile saline solution, using the techniques developed for in vitro fertilization and embryo transfer. Laparoscopy and celiotomy are best performed during anestrus to fully appreciate persistent pathologic changes in the uterus.

FAILURE TO CYCLE

There are two subcategories of animals with persistent anestrus. Primary anestrus refers to females 24 months of age or older that have never cycled. Secondary anestrus applies to females that have previously cycled but are no longer doing so. An animal that has never cycled may be a normal prepubertal animal younger than 24 months of age, may be experiencing “silent” heats, may have a congenital gonadal or chromosomal anomaly, or may have a concurrent disorder that is preventing estrous cycles. Exposure to light may be inadequate to initiate and maintain cyclicity in queens with persistent anestrus. Gonadal dysfunction, concurrent metabolic disorders or medications, and advancing age should be considered in females with secondary anestrus.

Diagnostic tests for persistent anestrus are usually delayed until a female is 2 years of age because of the probability that she is a normal prepubertal animal. Some veterinarians believe that an initial undetected or “silent” first heat cycle is common in bitches. If so, this could explain why some young bitches appear to have persistent anestrus. Unobserved or silent heats may be detected retrospectively by measuring the serum progesterone concentration. If the concentration is greater than basal anestrus levels (>2 ng/ml, or >6.4 nmol/L) in a bitch, a cycle has occurred within the previous 60 to 90 days. The finding of high serum concentrations of progesterone in a supposedly anestrous queen indicates that unobserved estrus has occurred and also that either unobserved mating or spontaneous ovulation occurred within the past 30 to 40 days. Clinical signs of false pregnancy (see Chapter 58) would also indicate that an undetected cycle occurred approximately 60 days earlier. Silent cycles could be detected prospectively by examining vaginal cytology every 1 to 2 weeks. Noncycling females should be housed with cycling females whenever possible because the pheromones from cycling females may induce noncycling females to cycle. Queens should be exposed to at least 12 hours of light for at least 2 months before further testing is done.

Persistent anestrus may result from suppression of function of the hypothalamic-pituitary-ovarian axis. Hypothyroidism, exogenous glucocorticoid therapy, and concurrent metabolic disease are commonly reported but rarely confirmed causes in bitches. Thyroid function is assessed by measuring serum concentrations of the thyroid hormones and canine thyroid-stimulating hormone (cTSH; see Chapter 51). The role of hypothyroidism in infertility in the bitch has not yet been thoroughly evaluated. Exogenous glucocorticoids are commonly administered to animals and cause many alterations in reproductive function, including prolonged anestrus and abortion. The history should be reviewed to determine if the animal could have received glucocorticoid treatment. In mature bitches increased serum alkaline phosphatase activity in conjunction with relatively normal alanine aminotransferase activity is suggestive of supraphysiologic amounts of glucocorticoids. If there is still doubt about excess endogenous or exogenous glucocorticoids, adrenocortical function can be assessed with an adrenocorticotropic hormone (ACTH) stimulation test. The presence of other concurrent metabolic disease is determined with a CBC, serum biochemistry panel, and urinalysis.

Persistent anestrus may also result from a primary abnormality anywhere within the hypothalamic-pituitary-gonadal axis, including intersex conditions, ovarian dysgenesis, progesterone-secreting luteal cysts, or ovarian tumor. It may also result from previous ovariohysterectomy. Females with ovarian dysgenesis or that have undergone oophorectomy are expected to have chronically increased serum concentrations of LH, which can be measured. Serum progesterone concentrations can be determined to assess functional luteal cysts. The functional status of the hypothalamic-pituitary-ovarian axis can be evaluated by measuring serum LH and progesterone concentrations before and after GnRH administration. Ultrasonographic evaluation of the ovaries may identify ovarian abnormalities such as cysts or neoplasia. On close inspection, many apparently female intersex animals have detectable anatomic abnormalities of the clitoris, vestibule, and/or vagina that result from exposure to androgens. A GnRH stimulation or human chorionic gonadotropin (hCG) test, done to assess the serum concentrations of testosterone, could be used to demonstrate the presence of testicular tissue. Because protocols vary among laboratories, the laboratory should be consulted for dosages and sampling times. Karyotyping can also be performed, although intersex animals may have normal karyotypes. Abnormal karyotypes have been found in bitches and queens with ovarian dysgenesis.

Induction of estrus may be tried in otherwise normal, healthy females if other diagnostic tests have failed to identify the cause of persistent anestrus. Exploratory celiotomy or laparoscopy, done to assess the gross appearance of the reproductive tract and to obtain biopsy specimens of the internal genitalia, should be considered only after all noninvasive diagnostic methods have been tried.

PROLONGED INTERESTROUS INTERVAL

Interestrous intervals of greater than 12 months in bitches and greater than 1 month in cycling queens are usually considered abnormal, although long interestrous intervals may also be a normal breed variation, as seen in the Basenji, Tibetan Mastiff, and Dingo dogs, which often cycle only once a year. Many of the causes of persistent anestrus, such as glucocorticoid administration in bitches and inadequate photoperiods in queens, may also cause a prolonged interestrous interval. Pregnancy, pseudopregnancy, and early embryonic death are causes of prolonged interestrous intervals in queens but not in bitches. This difference is because the CL life span in bitches is 60 to 70 days, irrespective of pregnancy status. Lack of hiding places and irregular feeding times have been shown to disrupt normal cycles in cats (Pelican, 2006). Prolonged interestrous intervals may also occur with increasing age or may signify an underlying disorder. Silent or unobserved heats should also be considered. The diagnostic workup in animals with prolonged interestrous intervals should include a thorough review of the estrus identification techniques used by the owner, identification of medications being administered to the animal, assessment of the overall metabolic health of the animal (i.e., CBC, biochemistry panel, urinalysis), and an evaluation of thyroid gland and adrenocortical function in bitches.

SHORT INTERESTROUS INTERVAL

Abnormally short interestrous intervals of less than 4 months are occasionally seen in bitches and are usually associated with infertility. Infertility in these animals presumably results from implantation failure because the endometrium has not recovered from the previous cycle, a process that takes 120 to 150 days, although ovulation failure may be involved. In some breeds, most notably the German Shepherd Dog, and in some individual animals, an interestrous interval of 4 to 4.5 months may be normal and may not interfere with fertility. Cystic ovarian follicles might cause frequent cycling (i.e., short interestrous interval) but most commonly are associated with persistent estrus. The administration of gonadotropins, prostaglandin F_2α, prolactin antagonists, or estrogen can artificially shorten the interestrous interval. In most bitches, however, the cause of short interestrous intervals is not discovered.

A short interestrous interval must be differentiated from a split heat cycle in bitches. Split heats are characterized by normal proestrus that stops abruptly before progressing to estrus. Two to 4 weeks later, proestrus begins again and progresses through normal, fertile estrus. Split heats are a normal phenomenon that can occur in any bitch during any estrus. Split heats are seen most often in pubertal bitches that have normal proestrus and estrus during subsequent cycles. Rarely do split heats occur repeatedly in an individual bitch. Split heats do not cause infertility, except in the sense that the initial proestrus frustrates breeding management.

Additional diagnostic tests are often not performed in bitches with confirmed short interestrous intervals, although ovarian ultrasound would be reasonable. Another diagnostic consideration would be to monitor the changes in serum progesterone concentrations during estrus and diestrus to assess whether the short cycles may be related to ovulation failure. Administration of an androgen such as mibolerone or methyltestosterone to prevent estrus for at least 6 months has been considered, but there is little published evidence of efficacy. Even though estrus can easily be delayed with androgen treatment, affected bitches usually remain subfertile. Interrupting the short cycle by administering a progestin during proestrus has enabled 10 previously infertile bitches to conceive on the next cycle (Wanke, 2006). Previous interestrous intervals for the bitches were 2 to 4 months (mean 3.2 months). Treatment with megestrol acetate (2 mg/kg, orally) or clormadinone acetate (0.5 mg/kg, orally) for 8 days, beginning within the first 3 days of proestrus, stopped the cycle before ovulation. Progesterone concentrations remained at basal levels. The next cycles occurred 1.5 to 3.5 months (mean 2.7 months) after treatment and were fertile. Although subsequent cycles were not discussed in this report, breeding on the first estrus occurring after the discontinuation of therapy has previously been recommended because short interestrous intervals frequently resume. The role of genetics in this problem is not known.

ABNORMAL PROESTRUS AND ESTRUS

The most common abnormalities of proestrus and estrus are refusal to allow mating, prolonged estrus, and abnormally short estrus. Females that are not in estrus refuse mating. An occasional bitch or queen exhibits partner preference by refusing to mate with one male but readily mating with another. Inexperienced and timid females may also be reluctant to breed. In bitches physical abnormalities of the vulva or vagina are common causes of refusal to mate (see Chapter 57). Physical abnormalities include vaginal strictures; congenital defects in the vulva and vagina; vaginal hyperplasia/prolapse; and, rarely, vaginal neoplasia.

Vaginal cytologic studies should be performed to identify the present stage of the cycle (see Fig. 56-6). As just mentioned, females that are not in estrus will not accept mating. Digital palpation of the vulva, vestibule, and vagina in animals of adequate size can identify vaginal prolapse and most vaginal strictures and congenital defects. Vaginoscopy should be performed if digital palpation fails to identify a cause for the refusal to allow mating.

Vaginal strictures that are identified during anestrus should always be palpated again during estrus to determine their actual significance. Annular vaginal strictures are usually located immediately cranial to the external urethral orifice, at the anatomic junction between the vestibule and the vagina. The vestibulovaginal junction is normally the narrowest part of the posterior tract (see Fig. 56-7). During anestrus this normal narrowing may be mistaken for an annular stricture. The diameter of the vestibulovaginal junction normally increases significantly during proestrus and estrus, making differentiation from a true stricture easy at this stage of the cycle. Strictures of the vulva or vestibule usually do not change as dramatically during estrus. Similarly, normal vaginal examination findings during anestrus do not exclude the possibility of vaginal hyperplasia/prolapse, which occurs only at times of estrogenic stimulation, as a potential cause for reluctance to mate. Artificial insemination can be used to breed otherwise normal estrual females that refuse to mate as well as those with vaginal hyperplasia/prolapse. With the exception of vaginal hyperplasia/prolapse, physical abnormalities should be surgically corrected if the female is to remain in the breeding program. Surgery is best performed during anestrus. The heritability of congenital vaginal and vulvar anomalies is unknown.

Prolonged or Persistent Estrus

Although proestrus and estrus each last an average of 9 days, proestrus lasting as long as 17 days and estrus lasting 21 days have been observed in normal, fertile bitches. Understandably, many owners become concerned if a season (proestrus plus estrus) lasts longer than 3 weeks. Nevertheless, a season is not considered abnormally long in bitches until it reaches 35 to 40 days. In queens estrus lasting longer than 16 days is considered abnormal. This must not be confused with the normal, multiple cycles that occur in queens.

Prolonged proestrus/estrus is usually caused by functional follicular cysts (Fig. 56-11), which occur in intact ovaries and also in ovarian remnants in spayed bitches and queens. Ovarian neoplasia and exogenous estrogen administration may also cause persistent signs of estrus. Vaginal cytology should be performed to confirm that estrogenic stimulation is present and thus could reasonably be considered the cause of the behavioral and physical signs. Usually, the diagnosis of ovarian follicular cysts is based on the historic, physical, vaginal cytologic, and ultrasound findings. Serum concentrations of estrogen could also be determined. Because spontaneous regression of follicular cysts may occur, watchful waiting for 2 to 4 weeks is often the initial therapeutic approach. If clinical signs do not promptly resolve, treatment is indicated. Induction of ovulation can be attempted using GnRH (Cystorelin®; 2.2 μg/kg, adminstered intramuscularly q24h for 3 days); however, the results have been variable. If mature follicles are present and induced to ovulate, signs of estrus should resolve in 5 to 7 days. The cysts can be manually ruptured via laparoscopy or celiotomy. In cases of unilateral ovarian cysts, unilateral oophorectomy can be performed. Ovariohysterectomy should be considered for those females that fail to respond promptly to medical management for cystic ovaries because the prognosis for fertility is guarded and continued estrogenic stimulation may be harmful to the uterus and bone marrow. Ovarian neoplasia is uncommon in bitches and queens. Surgical excision is the treatment of choice. If exposure to exogenous estrogenic drugs is the cause of persistent signs of estrus, it should be discontinued.

FIG 56-11 Cystic ovaries and uterus with cystic endometrial hyperplasia from a 2-year-old Mastiff in heat for 12 weeks.

NORMAL CYCLES

Infertility in a female otherwise normal in all aspects of the reproductive cycle may result from improper breeding management; infertility in the male; abnormalities in the ovary, uterine tubes, uterus, or vagina; early embryonic death; or advancing age. A history of false pregnancy occurring after previous cycles strongly suggests that the hypothalamic-pituitary-gonadal axis was intact during those cycles. Therefore the investigation should initially focus elsewhere. Conception rates and litter size are greatest and neonatal mortality is lowest in bitches (Beagles) between 2 and 3.5 years of age. Reproductive performance in queens is best between 1 and 6 years of age. After 5 years of age in Beagles and 6 years of age in queens, conception rates and litter size decline and neonatal mortality begins to increase. Because of this age-related decrease in fertility, an extensive diagnostic evaluation of older females may not be warranted.

The most common causes of infertility in females with normal estrous cycles are improper timing of insemination and poor semen quality. Because male fertility can be so easily evaluated (see Chapter 60), the male should be evaluated before an extensive diagnostic evaluation of the female is undertaken. A solid history that the male sired litters that were born shortly before and shortly after mating with the bitch in question would provide good circumstantial evidence against male infertility. Semen evaluation would provide information about the male’s current status. The process of freezing and thawing canine semen substantially decreases its quality. Because the post-thaw life span may be only 24 hours and because its ability to transverse cervical mucus is so diminished, pregnancy rates using frozen semen are very poor unless ovulation timing and intrauterine, not intravaginal, insemination are used. Although the effects of chilling semen are far less deleterious, freshly ejaculated semen retains the best quality.

In well-managed colonies of normal dogs, conception rates of better than 90% are expected. A thorough history concerning breeding management, particularly how the owner determines when to breed, is imperative. Canine breeding management is discussed on p. 887. Feline breeding management is discussed on p. 889. A common practice for dog breeders is to simply begin breeding on a predetermined day after the onset of proestrus (commonly day 10) and continue breeding every other day for as long as the bitch is receptive. This method works well for normal bitches that have a very typical cycle. However, if a particular bitch has a short proestrus, for example, day 10 may actually be at the end of estrus rather than at the beginning. In queens the frequency of mating during estrus is a more important determinant of ovulation, and thus conception, than is the specific day of the cycle on which mating occurs.

Vaginal cytology can be used to identify estrus. Obtaining the specimen for cytologic evaluation may induce ovulation in some estrous queens, but this is apparently not a common occurrence. Monitoring serum concentrations of LH or progesterone can be used to determine ovulation and the fertile period in bitches. It has been shown in bitches in artificial insemination (AI) programs that two inseminations, done 48 hours apart during the fertile period, improve pregnancy rates and increases the number of pups per litter compared with only one insemination. If the first insemination happens to occur late in the fertile period, the second is done 24 hours later. Unlike the situation in queens, breeding a bitch several times during the same day appears to offer no advantage over breeding a single time on a given day. Intrauterine insemination with fresh, chilled, or frozen-thawed semen increases pregnancy rates over vaginal insemination. Also, the mean litter size was larger with intrauterine AI than with vaginal AI.

After the female has been bred using optimal protocols and semen of excellent quality, it should be examined 20 to 30 days later to determine whether pregnancy has occurred. Pregnancy can be diagnosed on the basis of abdominal palpation, ultrasonographic findings, or by finding high serum concentrations of relaxin. Ultrasonography should be performed in animals found not pregnant because the ovaries and uterus can be evaluated for a potential cause. If the female is not pregnant, the serum progesterone concentration should be measured to determine if ovulation occurred. Low serum progesterone concentrations (less than 2 to 5 ng/ml or approximately 6.4 to 16 nmol/L) suggest ovulation failure or premature luteolysis. The cause of ovulation failure may be an ovarian abnormality or, in the case of queens, inadequate coital stimulation. Premature luteolysis, or failure of the corpora lutea to maintain progesterone production, results in fetal resorption with no outward clinical signs when it occurs before day 35 of gestation. Premature luteolysis is rarely documented in bitches or queens; however, to differentiate ovulation failure from premature luteolysis, serum progesterone concentrations are serially determined using quantitative methods (e.g., RIA) from the time of proestrus through diestrus. Progesterone concentrations that never exceed 8 ng/ml (approximately 25 nmol/L) suggest ovulation failure, whereas premature luteolysis is reflected by a more rapid or earlier decline than normal from high postovulatory concentrations of progesterone. Hypothalamic or pituitary dysfunction would be unlikely causes of ovulation failure in the female that is otherwise cycling normally. More likely, hypothalamic or pituitary malfunction would be manifest as abnormal cycles.

When a female with normal cycles is known to have been bred appropriately during estrus to a male that is known to be fertile and when ovulation has been confirmed by the finding of elevated serum progesterone concentrations during diestrus, the hypothalamic-pituitary-gonadal axis is considered intact (see Fig. 56-4). Abnormalities in the vagina, uterus, uterine tubes, placenta, or the conceptus itself are then likely to be the source of the infertility. The diagnostic approach should begin with a review of the history to identify potential causes of early embryonic death. Special attention should be paid to infectious disease and medications administered to the female. Early embryonic death is difficult to confirm in bitches and queens, but in queens a prolonged interestrous interval provides a clue. Attempts to confirm pregnancy by ultrasound or measuring serum concentrations of relaxin can be done as early as day 14 to 20, although false-negative results are understandably common at that stage.

Infectious agents are an important cause of early embryonic death. Although many agents are capable of causing placentitis or fetal death, B. canis in bitches and calici and herpes viruses in queens are the foremost such agents. In lieu of cultures of specimens from the uterine lumen, cultures of the cranial vagina should be performed. Some clinicians recommend that cultures be obtained during estrus because the cervix is open at that time and fluid in the cranial vagina may have originated from the uterus. Bacterial infections should be treated appropriately before breeding. The uterus may be incapable of supporting implantation or pregnancy because of disorders such as bacterial endometritis or cystic endometrial hyperplasia. Ultrasound of the reproductive tract is indicated. Vaginal lesions can easily be excluded as the cause of infertility by vaginoscopy and vaginal cytology. The potential teratogenic or abortifacient effects of medications should always be considered. Many commonly used medications, such as glucocorticoids and certain antibiotics, also cause embryonic death. In addition, some congenital fetal anomalies cause early embryonic death because they are incompatible with continued survival.

The presence of antisperm antibodies in the female has not yet been documented as a cause of infertility in dogs or cats. However, were they to occur, breeding with a different male could circumvent the problem. Finally, exploratory celiotomy can be performed during anestrus to visualize the reproductive tract, assess the patency of the uterus and uterine tubes, obtain uterine specimens for culture, and procure full-thickness uterine biopsy specimens for histologic assessment.

SURGICAL METHODS

In the United States and Canada ovariohysterectomy and castration are the most common methods of population control for dogs and cats. They are permanent and relatively expensive, invasive procedures. Ovariohysterectomy can be accomplished by midline or flank approaches or via laparoscopy. Laparoscopy has also been used for castration of cryptorchid testicles. Some clinicians have recommended ovariectomy over ovariohysterectomy, although this has not been widely accepted among veterinarians in the United States. Ovariohysterectomy has traditionally been recommended at 5 to 8 months of age, just before the animal reaches puberty. Doing so dramatically reduces the risk that the animal will develop mammary cancer in the future, in addition to preventing estrus and unwanted pregnancy. To reduce the number of unwanted (i.e., relinquished by their owners) and stray animals euthanized at animal shelters, many shelters mandate surgical sterilization as part of the adoption agreement. Because postadoption compliance with sterilization agreements has universally been poor, preadoption “early spay-neuter” policies at 6 to 8 weeks of age have been advocated. Safe and effective anesthetic and surgical techniques have been developed. Several studies have shown that the physical and behavioral traits of animals neutered at 7 weeks of age are the same as those in animals neutered at the more conventional age of 7 months. However, there is an increased rate of urinary incontinence in female dogs spayed at 6 to 8 weeks of age compared with those spayed at 6 to 8 months. Whereas the risks associated with early spay-neuter of male dogs and of both male and female cats are minimal, it may be prudent to delay spaying female dogs until they are older. Gonadectomy at any age results in decreased metabolic rate and decreased caloric requirements, irrespective of any change in physical activity. It has also been reported that food intake actually increases after neutering in male and female cats fed ad libitum. Neutering has been shown to cause hyperleptinemia in male cats, but not female cats, after neutering. Unless caloric intake is diminished to match the changed metabolic rate, animals will gain weight after gonadectomy. Gonadectomized animals have delayed physeal closure, and less developed genitalia and secondary sex characteristics than do age-matched, sexually intact controls. Tubal ligation and vasectomy can be used to prevent pregnancy in dogs and cats without circumventing the physical and behavioral changes associated with sexual maturation. Owners may or may not consider this desirable.

NON-SURGICAL METHODS FOR CONTRACEPTION OR STERILIZATION

Less expensive, permanent, nonsurgical methods of sterilization would be ideal for preadoption programs at humane societies and animal shelters and for large-scale application in trap-neuter-release programs to control feral populations. The injection of sclerosing agents into the testis, epididymis, or ductus deferens has been investigated in dogs and cats. These agents have included various concentrations of zinc, formalin, chlorhexidine in DMSO, ethanol, silver nitrate, potassium permagnate, Freund’s Complete Adjuvant, BCG, methallibure, dexamethasone, metopiron, niridazol, αchlorohydrine, and danazol. Treated animals may remain fertile for 6 to 8 weeks after treatment. Some animals never become sterile, whereas in others the effects are transient. There appears to be some species variation as well because intratesticular 70% glycerol was ineffective in dogs, whereas it has consistently resulted in azoospermia in monkeys, rabbits, rats, and hamsters. Some studies have reported minimal signs of discomfort. Others have reported swelling, scrotal ulceration and mutilation, local granulomatous reactions, vomiting, diarrhea, leukocytosis, and lethargy. A zinc gluconate solution (Neutersol®; Addison Biological Laboratory) for intratesticular injection is marketed for chemical castration of puppies between 3 and 10 months of age. At this time there is no published evidence that the use of sclerosing agents has resulted in lifelong sterility in pet dogs or cats or that there has been an impact on control of feral populations.

The concept of immunizing animals against GnRH, LH, LH receptors, sperm antigens, and the zona pellucida of oocytes has appeal for both permanent sterilization and temporary contraception. Because GnRH and LH control gonadal function, blocking their effects would theoretically suppress estrus cycles and ovulation in females, spermatogenesis in males, and sexual behavior in males and females. These effects might be maintained permanently or temporarily, depending on the duration of adequate antibody titers. GnRH is highly conserved across species and therefore is poorly immunogenic unless conjugated with other molecules. A GnRH vaccine (Improvac®; CSL Animal Health) is available in Australia for use in mares. An antigonadotropin releasing factor (GnRF) product (Canine Gonadotropin Releasing Factor Immunotherapeutic®; Pfizer) is marketed in the United States for treatment of benign prostatic hyperplasia in dogs. Although not marketed for these effects, it does cause the testes to shrink and serum testosterone concentrations to decline, both of which would be deleterious to spermatogenesis. GnRH has also been conjugated with cytotoxic agents designed to destroy the GnRH receptors. Although this markedly suppressed reproductive activity in peripubertal male dogs, the response was variable. Induction of antibodies against LH and LH receptors results in impaired reproduction; however, the responses have been so variable that a consistent vaccination protocol has not been established for dogs or cats. Bitches and queens have resumed normal cycling after antibody titers declined. Attempts to immunize animals against sperm antigen have not had satisfactory results for sterilization or contraception. The zona pellucida (ZP) is the acellular coating around ova, and vaccination with porcine ZP has been found to consistently produce high antiZP titers and prevent conception in bitches. Significant ovarian abnormalities also develop, including ovarian cysts of various types and prolonged proestrus/estrus. Vaccination of queens against ZP has not resulted in contraception. An antiporcine ZP vaccine (SpayVac®; ImmunoVaccine Technologies, Nova Scotia) is commercially available for immunocontraception in some captive wild species.

Intravaginal spermacides have not yet been developed for use in bitches or queens. An intravaginal mechanical barrier was marketed for use in bitches, but the failure rate was high. An intrauterine contraceptive device is available for bitches (Biotumer, Argentina). Although it was quite effective in a small clinical trial (Volpe, 2001), it is somewhat impractical because of the difficulty in transcervical placement. High intensity ultrasound suppresses spermatogenesis when applied to the testes and causes luminal occlusion when applied to the epididymides and ductus deferentia in dogs and cats. However, skin burns occur in approximately 20% of treated animals. Bisdiamines are amebicidal drugs that have been found to cause spermatogenic arrest in all species studied thus far, including dogs and cats. They have generated great interest for contraception in zoo animals because, when administered daily in food, they cause spermatogenic arrest before the spermatid stage but spermatogonia are preserved. Therefore the effects are reversible. Side effects in dogs and men have been only slight weight loss and slight decrease in red blood cell count. In cats there was no change in red blood cell counts, but testosterone concentrations declined during treatment. The drug must be administered daily to be effective. Females must not be exposed to bisdiamines because they are highly teratogenic.

CONTRACEPTION

Contraception may be defined as a reversible method of blocking fertility. Contraception is particularly desirable for animals that eventually will be bred but for which estrus will interfere with their work (e.g., racing, hunting) or show career. Progestins and androgens can inhibit the release or synthesis of gonadotropins in dogs and cats and thereby prevent estrus. GnRH analogues reversibly inhibit reproductive function in males and females by downregulating LH and FSH receptors, thereby suppressing the pituitary-gonadal axis.

THE QUEEN

The photoperiod can be manipulated to induce estrus in queens. Continuous exposure to 12 to 14 hours of light of at least 50 foot-candles and exposure to 10 to 12 hours of dark per day causes normal, mature queens to begin cycling within 4 to 8 weeks. Housing anestrous queens with cycling queens also helps to induce estrus. If the hormonal induction of estrus is to be attempted, the queen should first be exposed for several months to a minimum of 12 hours of light per 24 hours. Induction of ovulation may be indicated for cycling queens in AI or IVF/ET programs or to induce pseudopregnancy, which would temporarily prolong the interestrous interval when frequent cycles are troubling the owners. The intramuscular administration of 250 IU of hCG or 25 μg of GnRH (Cystorelin; Ceva) on the first 2 days of estrus is recommended, although a single 25-μg dose of GnRH is known to cause an LH surge in normal cats. Probing the vagina with a smooth rod such as a thermometer or with a cotton swab four to eight times, at 5- to 20-minute intervals, may also stimulate an LH surge in normal cycling queens. The probing need last only 2 to 5 seconds.

Estrus induction has primarily involved using domestic cats as models for AI and IVF/ET in endangered wild felids. The most common hormones used to induce estrus (i.e., folliculogenesis) in cats are porcine FSH and equine chorionic gonadotropin (eCG). A disadvantage of pFSH is the need for daily administration until the onset of estrus. Because of its longer half-life, a single injection of eCG (100 IU, intramuscularly) is often sufficient. Ovulation is then induced with LH (hCG; 75, 100, or 250 IU, adminis-tered intramuscularly or intravenously, depending on the formulation), or mating with a tomcat. A commonly used protocol for artificial insemination regimens is eCG followed by hCG 80 to 84 hours later. Protocols using GnRH for estrus induction have also been successful, but to date, pregnancy rates have been lower. Although there is much variation in response to estrus induction protocols among feline species, a common problem is hyperstimulation of the ovaries. This has resulted in excessive or prolonged estrogen production, premature or prolonged progesterone production, or premature luteolysis, all of which disrupt rather than enhance reproduction. It has been shown in cattle that follicular cysts develop following luteal phases with inadequate progesterone levels. Therefore the use of progestins before gonadotropins to help prevent ovarian hyperstimulation is being investigated (Pelican, 2006).

THE BITCH

Many drugs have been used to induce estrus—in other words, to shorten the interestrual interval—in bitches. Prostaglandin F_2α, which is used to treat pyometra, causes luteolysis and decreased progesterone production by the CL. This shortens the normally long diestrous period and thereby shortens the interestrual interval by 1 to 2 months.

The mechanisms by which the dopamine agonists cabergoline and bromocriptine induce estrus are multifactorial. One mechanism is by suppression of prolactin, which is luteotropic. Suppressing luteal function decreases progesterone, shortens diestrus, and increases LH pulsatility in bitches. However, this is not the sole explanation because metergoline, a seratonin antagonist that also decreases prolactin, does not induce estrus in bitches. Furthermore, even at low doses that do not suppress prolactin nor shorten diestrus, bromocriptine significantly shortens the interestrous interval. Bromocriptine (Lactafal®, Eurovet), given orally q12h at doses of 5, 20 and 50 μg, beginning in mid-diestrus until the onset of the next cycle, resulted in mean interestrual intervals of 136 ± 16 days, 96 ± 6 days, and 92 ± 11 days, respectively, compared with untreated controls’ mean interval of 216 ± 9 days (Beijerink et al., 2003). The higher doses suppressed prolactin and progesterone, whereas the low dose did not. Other researchers have reported that some bromocriptine-induced cycles are not fertile, despite hormonal variations similar to those in normal cycles. Cabergoline (Galastop®, Ceva Vetem; Dostinex®, Pfizer) at a dosage of 5 μg/kg orally, q24h, through the first 2 days of proestrus has induced fertile estrus in bitches with primary and secondary anestrus. The duration of treatment ranged from 4 to 34 days, with a mean of 16 days. Other researchers have suggested that continuing treatment through the first 4 to 5 days of proestrus is important for success.

GnRH agonists, administered as slow-release implants, induce estrus in bitches. The effectiveness of estrus induction and the fertility of the induced cycle are dose dependent and age dependent and vary according to the stage of the cycle during which the drug is administered and perhaps the anatomic location of the implant. These variations are apparently the result of competing upregulation and downregulation of LH release. The GnRH agonist deslorelin has been studied for both estrus induction and estrus suppression in bitches. High doses induced fertile estrus, but abortions occurred during the ensuing diestrus. Lower doses of 1.05 mg or 2.1 mg deslorelin (Ovuplant®, Fort Dodge) implanted in Beagle bitches beneath the vestibular submucosa in the ventral commissure of the vulva reliably induced proestrus within 3 to 5 days of administration, and the LH surge occurred 9 to 17 days later (Volkmann et al., 2006). All the bitches implanted during anestrus ovulated, and 69% became pregnant when bred to the same dogs as were the untreated controls. All the untreated controls also ovulated, and 67% became pregnant. However, although bitches implanted during diestrus also came into estrus and experienced an LH surge, only 69% ovulated and the pregnancy rate was only 15%. Furthermore, 15% of those treated in diestrus developed pyometra during the diestrus after the induced estrus.

Induction of ovulation might be indicated for cycling bitches in which ovulation is failing to occur or bitches with follicular ovarian cysts. Potential agents include hCG (22 IU/kg, intramuscularly) and GnRH (50 to 100 μg, intramuscularly; or 2.2 μg/kg, intramuscularly GnRH; Cystorelin®, Ceva). It has been recommended that hCG and GnRH be given on the first day of estrus, as determined by the behavior of the bitch and vaginal cytology. Success is confirmed by the finding of a serum progesterone concentration of greater than 8 ng/ml in early diestrus. For the treatment of follicular cystic ovarian disease in the bitch, 2.2 μg/kg of GnRH is administered intramuscularly for 3 days. However, the results of this treatment have been disappointing.