Margaret V. Root Kustritz, DVM, PhD, DACT
Assistant Clinical Specialist, Small Animal Reproduction
University of Minnesota College of Veterinary Medicine
St. Paul, MN 55108

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Bitches undergo puberty and achieve reproductive maturity when they go through their first heat. The average age of onset of puberty is 10 to 12 months, but anywhere from 6 to 24 months is normal. Small breed dogs come into heat at a younger age than do bitches of the large or giant breeds.


* Ovaries - The two ovaries are within the abdomen, just behind the kidneys. The ovaries contain the developing follicles, which contain the eggs and produce estrogen. The eggs are released from the follicles at the time of ovulation during standing heat. The ruptured follicle then becomes the corpus luteum, which produces progesterone.
* Uterus - The bitch has a bicornuate uterus, with long, torturous horns extending from each ovary back to the small common uterine body. The uterus is a sterile environment.
* Cervix - The cervix joins the uterus to the vagina. It is within the abdomen, and has a vertical orientation, making it difficult to visualize and manipulate. The cervix usually is inaccessible in the bitch.
* Vagina - The vagina of the bitch is very long. It is not a sterile environment.
* Vestibule / Vulva - The vestibule is the outermost portion of the reproductive canal. It contains the urethral papilla, which is the external opening of the urinary tract, and the clitoral fossa. The vulvar lips are the external portion of the genitourinary tract.


The estrous cycle consists of four stages. These are proestrus, estrus, diestrus, and anestrus. The stages of the estrous cycle are defined by the bitch's behavior, type of vaginal discharge present, if any, changes in concentrations of the hormones estrogen and progesterone, and vaginal cytology. Assessment of vaginal cytology specimens allows veterinarians to predict what is happening in the heat cycle by assessing the cells present. There are four types of vaginal lining (epithelial) cells; parabasal and intermediate cells (non-cornified cells), and superficial and anuclear squame cells (cornified cells).


Proestrus is the first stage of heat. The bitch shows a variable amount of vulvar swelling and vaginal discharge. The discharge is most characteristically bloody and serum-like (serosanguinous) but may range in color from milky to frankly bloody. Male dogs are attracted to the bitch but she will not stand for breeding, and often tucks her tail under tightly, sits down, or turns on the male dog. This stage lasts an average of 9 days with a range from 0 to 17 days.

Vaginal cytology specimens contain a population of epithelial cells that undergo a gradual transition from a predominance of non-cornified cells to complete cornification. Red blood cells (RBCs), white blood cells (WBCs), and bacteria are present early and disappear as the bitch nears estrus. Veterinarians can only make estimates from these swabs as to when a dog may come into standing heat.

Progesterone is low until near the end of proestrus, when it may begin to rise slightly. Estrogen is the primary hormone being produced, and is the hormone responsible for the physical and behavioral changes in the bitch, and the changes in the vaginal epithelial cells seen by vaginal cytology. Under the influence of estrogen, the vaginal wall becomes very thick and more cornified cells are collected on the vaginal swab.


Estrus is the technical name for standing heat. In this stage, the bitch allows mounting and breeding by the male dog. Her vulva is still swollen but may be softer than in proestrus. Vaginal discharge is still present, and classically attains a straw color at this point, although in many dogs it remains serosanguinous. This stage lasts an average of 9 days with a range from 3 to 21 days.

A bitch is in estrus by cytology when she has 100% cornification with >50% anuclear squames. There are virtually no RBCs, WBCs, or debris present. Veterinarians cannot predict ovulation time prospectively by vaginal cytology alone.

Progesterone rises abruptly early in estrus, and estrogen falls off somewhat. The most important hormonal event taking place at this time is the release of a surge of luteinizing hormone (LH). The significance of this is described later. Ovulation occurs during estrus. Until a bitch ovulates, she cannot conceive. Ovulation is the most important event occurring in the heat cycle.


The bitch enters diestrus when she no longer stands to be bred. This is not an all-or-none phenomenon; some dogs are classified as being in diestrus by vaginal cytology and will still stand to be bred. The vulva will gradually decline in size through this stage. Some dogs continue to exhibit vaginal discharge, which usually is mucoid. Most dogs have no vaginal discharge during this stage. Diestrus lasts an average of 60 days and occurs in every dog, whether they were bred or not, and whether or not they conceive.

As the bitch enters diestrus, there is an abrupt shift back to complete non-cornification and WBCs appear on vaginal cytology specimens. This occurs consistently six days after ovulation.

Progesterone remains high throughout this period. All other hormones are fairly low until the time of whelping.


This is the interval between periods of estrous activity. This is a time of reproductive quiescence, when there is minimal hormonal activity and the dog shows no outward physical changes or unusual behaviors. Anestrus lasts an average of 4.5 to 5 months. At this stage, vaginal cytology specimens contain only a scant number cells, all of which are non-cornified.


Measurement of progesterone allows veterinarians to predict when a bitch will ovulate. It is not the progesterone itself that makes her ovulate. The hormone that induces ovulation in the bitch is luteinizing hormone (LH), which is difficult to assay accurately and quickly. An in-house LH assay is commercially available. Daily blood samples must be drawn at the same time of day to allow identification of the LH surge, because LH is secreted as a surge lasting at most 24 hours in duration, and samples drawn before and after the surge are indistinguishable. The manufacturer recommends confirmation of LH surge and ovulation with progesterone assays.

Progesterone happens to begin its normal rise at the time of the LH surge that stimulates ovulation. Serum progesterone concentrations are about 2 ng/ml on the day of the LH surge, and 4 to 10 ng/ml on ovulation day. Knowing this, veterinarians can measure serum progesterone concentrations in a dog serially, and watch for the time of its rise to predict ovulation. Once progesterone begins to rise, reaching a concentration of about 2 ng/ml, ovulation will occur 2 days later. The egg is not ready to be fertilized immediately upon ovulation; optimal breeding day is 2 days post-ovulation. Therefore, once progesterone begins to rise, optimal breeding day for that dog is about 4 days later. There will be exceptions to this rule, depending on the quality and type of semen to be used and the type of breeding to be performed. In general, remember the 2-2-2 rule; a serum progesterone concentration of 2 ng/ml means ovulation will occur in 2 days and optimal breeding day is 2 days after that.

Two types of progesterone assay are available. In-house assays identify ranges of progesterone, and must be run daily to counter the inherent inaccuracy of the method. Turn-around time is very good; many hospitals can provide results within 30 to 60 minutes of drawing blood. Assays at commercial laboratories usually are either radioimmunoassays (RIA) which use a radioactive tracer to quantify progesterone, or chemiluminescence assays. These are much more accurate, providing a number instead of a range for progesterone concentration, and samples need only be submitted every 3 to 4 days. Turn-around time is longer than for in-house assays and is dependent on the laboratory used.

Serum progesterone (ng/ml)
Event occurring
< 1.0
Anestrus or proestrus
1.0 - 1.9
Ovulation minus 3 days - Recommend recheck
2.0 - 2.9
Ovulation minus 2 days
3.0 - 3.9
Ovulation minus 1 day
4.0 - 10.0
Ovulation day
Optimal breeding day is 2 days after ovulation
> 10.0 with cornified vaginal cytology
Ovulation plus 1 to 5 days
> 10.0 with non-cornified vaginal cytology
Diestrus - Too late to breed this season


Prospective methods include progesterone assay, as described above, and examination of breeding history. Many bitches ovulate on or about the same day of their cycle from one heat to the next. Retrospective methods include vaginal cytology and examination of whelping history. The abrupt transition from the complete cornification seen on cytology during estrus to the complete non-cornification seen on the first day of diestrus happens six days after ovulation. Whelping occurs 62 to 64 days after ovulation. Good record-keeping of dates from first discharge noted, breeding dates, and whelping dates allows you to count back and figure out on which day of the bitch's cycle she ovulated.


In dogs with no history of infertility or reproductive tract disease that are to be bred by natural service, there is nothing wrong with presenting the bitch to the stud dog when she has been in heat about seven days, and breeding the dogs every other day while the female will stand to be bred and the male is interested. The technologies described above are important for (1) bitches that do not stand to be bred after having been in heat 7-10 days, (2) lack of interest of an experienced male when the bitch will allow mounting, and (3) dogs that have been bred as described above with no success.

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