Infertility in the Bitch
Infertility in the bitch refers to the inability to conceive and produce viable offspring.  Most of the bitches presented to reproductive practice for infertility are normal, their failure to conceive is instead due to breeding management or stud dog problems.  Considerable variation exists within the normal canine reproductive cycle and breeders commonly interpret such variation as an indication that an abnormality exists.  The clinician must differentiate bitches having normal estrus cycles with unexpected patterns from those with true abnormalities.  Detection of individual variation within the normal range of events in a fertile bitch can be crucial to providing effective counseling concerning breeding management.  Additionally, evaluation of the estrus cycle for actual abnormalities is an important component in assessing the apparently infertile bitch.  Variations from normal estrus cycle events in the bitch can sometimes then be traced to specific ovarian disorders.

The complete history of the bitch should be obtained and it is important to be thorough, including not only the reproductive and breeding management history, but also the general health history.  A thorough physical examination should be completed before beginning a focused evaluation of the reproductive system.  A minimum database consisiting of a complete blood count, serum biochemistry profile, urinalysis (with culture if indicated), and Brucella canis screening should be evaluated for evidence of systemic disease.  Vaccination and deworming history and heartworm status should also be assessed.

A solid understanding of the bitch reproductive cycle, described below, is essential when evaluating a breeding bitch for infertility.

Puberty:
Normal puberty can occur at 6-24 months, and larger dogs tend to reach puberty at an older age than smaller dogs.  The first estrus can be irregular and split heats are common, reflecting a period of folliculogenesis without ovulation occuring.  Between 2 weeks and 2 months puberty is rare in the bitch and that which is a consequence of a nutritional etiology has a poor prognosis for future fertility.  The first estrus cycle can sometimes be hastened by housing  the bitch with a kennelmate that is in proestrus, due to the pheromone effect; medical indication can be attempted in an otherwise normal bitch.

The interestrus interval:
Interestrus consists of diestrus, typically lasting 45-60 days, and anestrus, which is variable, although usually between a 90 and 150 days duration.  Thus, the average duration of interestrus is 7 months.  German Shepherd bitches average about 6.5 months interestrus, Dachshunds average about 8.3 months interestrus, and in general larger dogs tend to have shorter interestus intervals than small dogs.  Variation in the interestrus interval is not abnormal.  Interestrus tends to become longer as bitches age beyond  8 years.  Seasonality may exist in the bitch, with cycles more common in the summer and winter.

The estrus cycle:
The stages of the canine estrus cycle are anestrus, proestrus, estrus, and diestrus.  Anestrus is a time of mandatory endometrial repair after progesterone has exerted a proliferative effect during diestrus for the preceding 45-60 days.  Fertility is low if at least a 90 day anestrus (or a 150 interestrus interval) is not attained, because the uterus has not repaired enough from diestrus to subsequently establish and maintain pregnancy. 

During anestrus the male shows no sexual interest in the female.  the female shows no sexual interest in the male.  The vulva is not swollen or edematous.  Vaginal cytology is hypocellular with noncornified parabasals.  The vaginal wall is very thin and appears pale on vaginoscopic examination.  Progesterone is at baseline concentration (<1 ng/ml).  Note that even spayed bitches have basal levels of progesterone, probably of adrenal origin.  Prolactin secretion by the pituitary may promote anestrus, because prolactin inhibitors can be used to terminate anestrus.  The termination of anestrus is associated with an increased pulse frequency of LH and increased FSH late in anestrus.

Proestrus averages 9 days in duration, but may last 3-17 days.  Males are interested in the female but females show no interest in the males.  The vulva is swollen because of estrogen secretion by the growing follicles.  There is serosanguinous discharge from the vulva that results from diapedesis of erythrocytes through uterine vessels.  Vaginoscopically, the vaginal mucosa appears smooth because estrogen causes edema.  LH pulses have an increased frequency and FSH is suppressed.  Estrogen originates from the growing follicles and peaks at the end of  proestrus.  It is actually the decline in estrogen at the end of proestrus that causes the bitch to show clinical signs of estrus (receptivity).  Interestingly, testosterone is relatively high at the end of proestrus, perhaps resulting from a "backup" in estrogen synthesis.  This may result in some masculine behavior by the bitch, such as mounting other dogs.  The proestrus vaginal cytology demonstrates an increase in cornification of approximately 10%/day until about 100% of the cells are cornified.  Erythrocytes and a bloody vulval discharge are variably present and even if there is a bloody discharge apparent grossly, erythrocyes may not appear on vaginal cytology.  Visualized vaginoscopically, the vaginal mucosa is glistening with rounded edges to the mucosa because of cellular edema.

Estrus averages 9 days in duration, but can be as short as 3 days or as long as 21 days.  The male and female are both interested in each other.  The bitch will "flag" her tail, as a sign of receptivity.  the estrus behavior results from the estrogen that peaked during proestrus abruptly declining while progesterone is rising.  The estrual vaginal cytology is very cellular and greater than 90% of the cells are cornified.  Most of the cells are anuclear.  Neutrophils are absent because the hyperplastic vaginal wall is too thick for them to cross the mucosa.  Erythrocytes are often absent.  The backround of the smear becomes very clean.  The cells may slough off as sheets as the end of estrus approaches.  Vaginal vaginoscopy reveals the vaginal epithelium with a wrinkled appearance owing to the acute fall in estrogen concentrations which results in dehydration of the mucosa.

The LH peaks about 24-48 hours into estrus and is triggered by the estrogen peak during proestrus.  Th LH peak causes ovulation of the folicles.  The LH peak has a very short duration and it must be assayed every day in order to detect it.  Ovulation occurs 24-48 hours after LH peak (day 3-4 of estrus) and takes about 24 hours for completion.  The fertile period of breeding is generally recommended as the 3rd through the 5th-6th day after the LH surge, covered by breeding every other day of estrus.

Progesterone starts to rise during estrus and the initial rise usually coincides with the LH peak.  The preovulatory rise in progesterone is caused by estrual luteinization of follicles and a "backup" in progesterone from the estrogen sythesis.  The progesterone rise that coincides with the LH peak can be measured every other day, and can be used to predict the fertile period.  Since progesterone starts to rise coincidentally with the LH peak, when estrus ends, the progesterone is already greatly elevated over baseline.  Diestrus is then the time of progesterone dominance.

Diestrus in the non-pregnant bitch could be called covert pseudopregnancy, since progesterone remains elevated, but there are no signs of pregnancy.  During diestrus the bitch refuses male's advances.  Bitches may remain receptive after the fertile period.  At the beginning of diestrus there is an abrupt change from the 100% to less than 50% corninfication, marking the first day of diestrus.  Neutrophils return to clean up all the sloughed cells and debris.  Intermediate cells return as well as "metestrum" cells and foam cells.  Progesterone is secreted by the corpora lutea, the only source of progesterone in the bitch.  Prolactin in diestrus is higher in pregnant bitches than in nonn-pregnant bitches.  It rises with the decline in progesterone in both pregnant and non-pregnant bitches.  Prolactin causes mammary development.  The cessation of diestrus is not known to be associated with PGF secretion from the uterus in non-pregnant bitches.

Bitches presented for failure to permit breeding or failing to conceive after a forced breeding, or artificial insemination, during the perceived fertile period need to be evaluated first for kennel management errors.  The timing of receptive and fertile periods during estrus varies significantly among normal bitches during different estrus cycles.  These periods may not correlate with the handler's choice of predetermined breeding dates, typically between days 10-14 after the onset of vaginal bleeding.  Ovulation timing protocols utilizing serial vaginal cytology, repeated vaginoscopy and serum progesterone and LH concentrations are useful in identifying the actual fertile period when breeding should occur, allowing evaluation of breeding management decisisons.

Breeder clients should be advised to notify the clinic when they first notice that the bitch under evaluation is in season, based on vaginal discharge or vulval swelling/attraction to males.  Even the most astute owner can fail to notice the true onset of proestrus for a few days.  Early proestrus should be documented by vaginal cytology (<50% cornification/superficial cells).  A baseline progesterone level might be informative if the true onset of the cycle is unknown (usually 0-1 ng/ml in proestrus).  Vaginal cytology should be performed every 2-4 days until a significant progression in cornification is seen, usually>70% superficial cells.  At that point, serial hormonal assaying should begin.  For routine breedings, progesterone testing may be done every other day, until a rise in progesterone>2 ng/ml is identified.  This day of the initial rise in progesterone is identified as day "zero".  Breedings are advised on days 2, 4 and 6 (1, 5, 7).

When increased accuracy of ovulation timing is necessary (e.g. frozen or chilled semen breedings, bitch infertility cases, breedings with subfertile stud dogs) daily LH testing is recommended if available.  Once the LH surge is identified, breeding days may be planned based on the basis that this is called day "zero".  As with progesterone testing, vaginal cytology dictates when to begin LH testing (>70% superficial cells).  Vaginal cytology may be continued until the diestral shift is identified, which gives a retrospective evaluation of the breeding just completed (LH peak likely 7-9 days previously).  In addition, at least one progesterone assay should be performed after the LH surge or when indicative initial rise in progesterone is identified.  The purpose is to document that levels continue to rise above 5 ng/ml, confirming ovulation and luteinization.  Extended chilled breedings should occur on days 4 and 6, or 3 and 5 after day "zero".  Which two days are chosen can depend upon overnight shipping possibilities and the involved clients' schedules.  Frozen semen breedings should occur day 5 or 6 after day "zero". 

If client economics dictate minimal testing, serum can be batched on a daily basis and quantitive progesterone tests perfomred as advised above.   When the initial rise in progesterone is identified, the batched serum can be specifically evaluated for the day of the LH surge, confirming identification of day "zero".

Vaginoscopy may be performed throughout the cycle as an adjunct to vaginal cytology and hormonal assays, especially when evaluating an unusual cycle.  Behavior and other observations should also be made at each examination, but less weight should be put on these parameters.  The clinician should keep in mind that the most accurate ovulating timing occurs when information from several tessts is pooled (vaginal cytologies, vaginoscopy, and progesterone or LH tests).

Ultrasonography may be used to help identify ovulation in the bitch.  Early attempts were discouraging; the small size of the ovaries and their similarity to close structures made them difficult to visualize.  However, recent reports have identified ovulation as occuring when a detectable decrease in the number of hypoechoic follicles is seen during serial imaging (3 times daily).  The data has shown a close correlation to the ovulation time established by LH and progesterone levels.

Behavioral or physical problems can interfere with a bitch's acceptance of a male for breeding.  dominant bitches expose to an inexperienced male may not allow breeding even during the appropriate time.  Vulval or vaginal abnormalities such as strictures and septate bands, and vaginal hyperplasia may make natural breeding painful and result in a bitch refusing to permit copulation even when in estrus.  The pre-breeding veterinary examination permits early detection of such anatomic problems, enabling their correction or adjustment in breeding plans  (artificial insemination versus natural) before the onset of proestrus.

Male Problems:
Fertility of the male must be confirmed before initiating an infertility work up on the bitch failing to become pregnant.  Although physical findings and semen evaluation including sperm count, mmorphology and motillity are helpful, there is not method for evaluating sperm function other than a history of conception with other bitches close to the time a stud dog is bred to the bitch with infertility concerns.

True Infertility
Abnormal estrus cycles
Abnormal estrus cycles can be categorized and simplified into several patterns reflecting either a prolongation or abbreviation of a phase of the cycle, or an alteration in the normal sequence of events.  An owner's interpretation of a bitch's behavior and physical characteristics may not equate with the actual physiologic events, necessitating prospective documentation of the cycle through vaginal cytology, vaginoscopy, behavioral analysis and serum progersterone and LH levels when the bitch is in season.

Prolonged proestrus or estrus occurs when a bitch displays vaginal bleeding for more than 21-28 consecutive days, accompanied by attractiveness to males.  Greater than 80-90% superficial cells are found on vaginal cytology.  Such bitches may or may not be receptive to breeding.  Prolonged proestrus and/or estrus most likely results from persistent secretion of estrogens, with or without small elevations in progesterone secretion.  If secreted, progesterone enhances the presence of sexual receptivity.

Endogenous sources of prolonged estrogen exposure in the bitch, with or without progesterone, include ovarian follicular cysts and secretory ovarian neoplasias, and theoretically, adrenal gland origin.  Secretory, anovulatory follicular ovarian cyts tend to be solitary, lined with granulosa cells, and exceed normal preovulatory follicles in size, ranging from 1 to 5 cm in diameter.  Bilateral follicular cysts can indicate a problem with the hypothalamic pituitary ovarian axis.  Follicular cysts tend to occur in bitches <3 years of age.  Ovarian neoplasias capable of producing estrogen occur and include tumors of epithelial origin (cystadenomas and adenocarcinomas) as well as tumors of gonadal-stromal origin (granulosa-theca cell tumors).  Ovarian neoplasia tends to occur in bitches >5 years of age.  Ovarian tumors can occur unilaterally or, less commonly, bilaterally.  Function ovarian neoplasia and cystic ovarian pathology can occur simultaneously.  Cysts found in the contralateral ovary and endometrial hyperplasia accompanying a functional tumor occur most frequently with gonadal-stromal origin tummors.  Adrenal disorders causing hyperestrogenism are rare.

There are a few differential diagnoses for prolonged vaginal bleeding:
Vaginal bleeding secondary to infection, inflammation or neoplasia of the genitourinary tract
Vaginal foreign bodies
Coagulopathy
These differentials should be differentiated from prolonged proestrus or estrus due to ovarian disease.  Excessive exogenous (iatrogenic) administration of estrogen may be encountered when a bitch is treated for urethral sphincter incompetence with diethlstilbestrol (DES), or from attempts to prevent unwanted pregnancy using DES or estradiol cypionate. 

After confirmation of naturally occurring hyperestrogenism is obtained through vaginal cytologies (they can be confirmed with serum estrogen level measurement), abdominal ultrasonography is recommended in the attempt to identify an ovarian follicular cyst or functional neoplasia.  Normal preovulatory follicles usually measure 4 to 9 mm in diameter, smaller than most follicular cysts and most functional neoplasias.  Analysis of the estrogen and progesterone levels in fluid from abnormal cystic ovarian structures can confirm the diagnosis.  These analyses are obtained via ultrasound guidance and histologic analysis of tissues obtained surgically.  Estradiol levels from cysts are usually much higher than serum levels.

Because follicular cysts may spontaneously undergo atresia or luteinization, not all bitches experiencing prolonged proestrus or estrus require treatment.  Progression of the follicular cyst to an atretic follicle or a copora lutea can be monitored ultrasonographically, via vaginal cytologies, by serum estrogen and progesterone levels.
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