Stages of the canine estrous cycle can be defined by sexual behavior, physical signs (vulvar swelling, vaginal bleeding) or by vaginal cytology. The period of receptivity to a male varies considerably among bitches; some bitches are receptive well before and after the period of potential fertility. Similarly, signs such as "proestrus bleeding" are often unreliable indicators; some bitches bleed very little and other show bleeding through estrus and into diestrus.
Since cytologic changes reflect the underlying endocrine events of the cycle, they are almost always a better predictor of the "fertile time" and gestation length than are behavioral or physical signs.
Cytologic changes through the canine estrous cycle reflect changes in blood concentrations of estrogen. As depicted, estrogen levels rise prior to and during proestrus and fall in conjunction with the preovulatory surge of luteinizing hormone. Rising levels of estrogen induce the "cornification" that is characteristic of smears examined during estrus. Ovulation occurs two days after the LH surge.
The sections below describe the cytologic picture typical of different stages of the canine estrous cycle. Examination of a single smear can sometimes provide useful information, but can also be quite misleading. For example, it is often difficult to differentiate proestrus and diestrus from an isolated smear. It is therefore highly recommended that multiple smears be evaluated.
Intermediate and parabasal cells predominate in smears taken during anestrus. Superficial cells are absent or found in very small numbers. Neutrophils may also be present or absent.
Serum concentrations of estrogen rise during proestrus, leading to capillary breakage and leakage of red blood cells through uterine epithelium, as well as proliferation of the vaginal epithelium.
Examination of vaginal smears from early to late proestrus will reveal a gradual shift from intermediate and parabasal cells to superficial cells. Typically, red blood cells are present in large numbers and neutrophils are commonly observed. Large numbers of bacteria are also often present.
The defining characteristic of cytologic estrus is the predominance of superficial cells. Most, but not all, bitches will undergo full cornification, and the smear will reveal a monotonous pattern composed almost exclusively of anucleate superficial cells.
If the bitch has been bred within a day of preparing a vaginal smear, it is quite likely that sperm will be observed among the epithelial cells. Indeed, careful examination for sperm in a smear taken within a few hours of an alleged breeding is a fairly reliable means of confirming or denying such an incident. In the image below, an intact sperm (left panel) and a sperm head (right panel) are present next to superficial cells.
The onset of diestrus is marked by a precipitous decline in the number of superficial cells and reappearance of intermediate and parabasal cells. Most commonly, the cellular profile changes within a single day from essentially 100% superficial cells to less than 20% superficial cells. However, it is best to confirm the onset of diestrus by examining a smear prepared on diestrus day 2.
Dogs ovulate 5-7 days prior to the onset of diestrus (7-9 days after the preovulatory LH surge), and hence, gestation length is usually 57 + 1 day from the onset of diestrus day 1. The period of behavioral estrus is variable, and often extends up to several days before and/or after cytologic estrus. Gestation lengths calculated from the onset or cessation of receptivity are correspondingly inaccurate. The onset of diestrus also correlates well with loss of fertility, and breedings after the diestrus shift are rarely fertile.