submitted with permission by Marj Brooks
with thanks to Kevin & Donna Frizzell of DeSaix St. Bernards for generously allowing us to use many of their superb array of articles

Proestrus

Proestrus is the phase of the estrus cycle when ovarian follicles develop and produce oestrogen. The effects of increased serum oestrogen concentration in the bitch include enlargement of the vulva, attraction of male dogs, and thickening and hemorrhage of the endometrium in preparation for pregnancy. Uterine hemorrhage is seen outwardly as vaginal discharge and its onset marks the first day of proestrus. Proestrus typically has a nine-day duration, but may range from 2 to 15 days. The vaginal mucosa also thickens during proestrus, and the additional cell layers protect the vaginal lining during intromission and contribute to changes observed in vaginal cytology.

The predominant cells observed in a vaginal cytological specimen from a bitch in proestrus include Para basal cells, which are small epithelial cells with round borders and large round nuclei that stain dark blue with Wright’s stain. Para basal cells originate near the germinal cell layer (deepest layer) of the mucosa. As more layers of cells are added to the vaginal mucosa, the cells on the superficial layer become far removed from the underlying blood supply and therefore degenerate. These are superficial cells, which are the largest epithelial cells; they have angular borders and are a nucleate or have very small, faint-staining nuclei. Intermediate cells are those that range between the Para basal and superficial cells in regard to origin and appearance.

Early in proestrus, Para basal cells predominate the vaginal cytology. Red blood cells (RBCs) are also seen and they signal that proestrus has begun. As proestrus continues, the relative percentage of Para basal cells decreases, and the percentages of intermediate and superficial cells increase. White blood cells (WBCs) and bacteria may be seen on the smear and are normal findings. Late in proestrus WBCs will no longer be observed, and the number of RCSs may decrease.

Estrus

Oestrus is defined as the period of sexual receptivity. Oestrus duration is typically from seven to nine days, but may range from 3 to 21 days. The bloody vaginal discharge apparent in proestrus usually diminishes and the vulva tends to soften in tone during oestrus. When a male dog attempts to mount the oestrous bitch, the bitch will “flag” (shift her tail to the side), and stand for copulation. Serum oestrogen concentration during oestrus rapidly decreases and the progesterone (also produced by the ovaries) concentration increases. Lutenizing hormone (LH) serum concentration increases approximately one to two days after the onset of oestrus and this increase initiates ovulation within 24 to 48 hours and the formation of the corpora lutea. Ovulation occurs over a 24 to 96 hour period during which immature ova (primary oocytes) are released. Primary oocytes require an additional 24 to 48 hours to mature (to secondary oocytes) and are then viable for fertilization for an additional 24 hours.

Vaginal cytology during estrus is characterized by a predominance of superficial cells, no WBCs, rare RBCs, and often large amounts of bacteria in the background.

Diestrus

Diestrus is the phase in which the bitch is under the influence of progesterone. The serum concentrations of progesterone (produced by corpora lutea in the ovary) are increased over basal levels for approximately 60 days. Diestrus ends when luteolysis occurs (corpora lutea degenerate) and the serum progesterone concentration falls to basal levels. At this time parturition occurs in the pregnant female or signs of pseudo pregnancy (i.e., mammary gland enlargement, lactation, nesting) may occur in the non-pregnant bitch. These signs represent a bitch’s response to a normal hormonal event (declining progesterone) and should not be interpreted as an abnormality; some bitches respond more intensely to these hormonal changes than do others.

The onset of diestrus is defined behaviourally as the first day of refusal of the male for breeding, and cytologically as the first day of an apparent change from a predominance of the superficial cells of estrus to as little as 20% superficial cells, with a concomitant increase in Para basal cells. WBCs may also become apparent. These cellular changes represent a loss of the superficial vaginal mucosal layers. The onset of both behavioral and cytological diestrus may not occur on the same day, and it is important to know when each occurred in retrospectively determining the bitch’s fertile period as described below.

Anestrus

Anestrus is the phase between the end of diestrus and the onset of the next proestrus. Although anestrus was once thought to be a hormonally quiescent period, it is now known not to be. It is still not fully understood, however, what triggers the onset of the next proestrus period. The normal length of anestrus may range from two to 10 months but is commonly four to five months. The normal interestrous interval ranges from 4.5 months in German Shepherds to 12 months in Basenji, and although the usual interval is seven months in other breeds, individuals within a breed may vary.