Canine estrous cycle and ovulation (Proceedings)

Bitches generally attain puberty two to three months after reaching adult body size. The average is six to 12 months with a range of four to twenty-four months. Smaller breeds reach adult size earlier and therefore reach puberty sooner than larger breeds. The first estrus can be abnormal in that it can be long or split. It generally will occur sooner if young bitches are housed with mature cycling bitches. The Basenji, Dingo and wolf cycle is usually once a year. Cycles begin at all times of the year but there is a small, yet significant increase in the occurrence of estrus in the late winter and early spring months.

The estrous cycle interval averages approximately six months with a range of 4 to 24.

Clinical evaluations to determine the stage of the estrous cycle are based on: behavior, vulvar appearance, vaginal appearance, vaginal cytology, uterine tone, progesterone concentrations and luteinizing hormone concentrations.

Proestrus

Proestrus lasts approximately 9 days with a range of 3 to 17 days. It has been reported that several weeks prior to the onset of proestrus there may be an improvement in appetite and appearance and somewhat more tolerant of the presence of males. Immediately prior to proestrus the bitch may become listless, become inappetent, and demonstrate nervous signs. The beginning of proestrus is designated by the commencement of a serosanguinous vulvar discharge. During early proestrus the bitch will be aggressive toward males. As the bitch approaches late proestrus she no longer attacks the male but still may be unreceptive as evidenced by sitting down. During proestrus estrogen from the developing follicles, which began to increase 3 to 4 weeks prior to the onset of proestrus, continues to increase due to the effects of sustained follicle stimulating hormone (FSH) and luteinizing hormone (LH) release. The estrogen creates a positive feedback on the release of LH and possibly FSH.

The increasing estrogen causes edema of the vulvar lips. The swelling increases until the vulva is very firm and very enlarged. The source of the bloody vulvar discharge is the uterus. The blood vessels within the endometrium increase in size and length and vasodilatation occurs thereby cause a loss of blood into the uterine lumen by diapedesis. This discharge, although bloody, should not contain clots. The appearance of clots could be an indication of cystic ovaries or other abnormalities of the reproductive tract. The increasing estrogen also causes a growth of the endometrial crypts and differentiation of the glandular epithelial cells into well-developed secreting cells.

Vaginal cytology

One of the most commonly employed clinical aids used to determine the reproductive stage of the bitch and whether it is normal is vaginal cytology. A vaginal smear is obtained by moistening a sterile cotton swab with sterile saline and inserting it into the anterior vagina. The swab is gently rolled against the vaginal wall and removed. The swab is then rolled onto a microscopic slide and permitted to air dry. It is fixed with alcohol and stained. The slide is permitted to remain in the alcohol for 15 to 30 seconds, rinsed with tap water, and placed into eosin for 10 seconds. The slide is again rinsed with water, placed into new methylene blue for 15 seconds, and rinsed. The stains most commonly but not solely utilized are eosin and new methylene blue. The slide is rinsed a final time with tap water and air dried, patted dry with a towel or dried with a warm air drier. The slide can be examined at 100X, 200X or 400X magnification.

The order of slide examination that I have found most beneficial is to first note cellularity. It does not matter the type of cells at this time just whether or not there is an increase in number. The importance of this will be explained later. The amount of mucus and debris is next noted. This is followed by determining if there is an increase in the number of WBC's in the sample. Next the presence of RBC's is noted. The last part of the examination is to determine number and types of epithelial cells in the sample.

During proestrus the sample should appear as follows: high number of cells throughout proestrus; increased debris and mucus early decreasing late; few WBC's (1 to 3 per field) early and becoming absent late; numerous RBC's early and disappearing late in proestrus; parabasal epithelial cells will disappear and superficial cells will predominate late in proestrus. The noncornified cells are called parabasal cells. These cells have a rounded uniform cytoplasm with large healthy nucleus. These cells are close to the basement membrane and have a rich blood supply. The cornified cells are divided into intermediate cells and superficial cells. The intermediate cells have a cytoplasmic border that is roughened or irregular. The cytoplasm demonstrates signs of degeneration as evidenced by density changes. The nucleus is still apparent and normal or near normal in appearance. The superficial cell has a cytoplasmic border that is irregular and degenerating with a nucleus that is degenerating. This is apparent by the decreased density and the increase in size. It may also become pyknotic, very dark and small. An anuclear cell is the most cornified superficial epithelial cell with a cytoplasm that is degenerated and the nucleus is absent.

Since each bitch is unique, it is necessary to do several smears in order to know the rate of progression and what is normal for her.

Estrus

Estrus lasts approximately 9 days with a range of 3 to 18 days. It is suggested that the total time of proestrus and estrus should not exceed 21 days to be considered normal. The signs of estrus include a willingness to accept the male characterized by standing with the rear legs firmly placed and the tail lifted over her back or held to the side; known as flagging. It is important to note that not all bitches will stand for all males making it sometimes difficult to detect estrus by signs of receptiveness. The normal bitch may stand for the male as early as 11 days before or as late as 8 days following ovulation. The vulvar edema decreases rapidly such that wrinkles appear in the vulva and thus the vulva appears smaller than during proestrus. The vulvar discharge generally becomes serous or slightly brownish in color. In some bitches the discharge may remain red but examination of vaginal cytology will not reveal intact red blood cells indicating that the cells have previously lysed. The continued release of FSH and the increase in release of LH causes ovulation to occur to within 24 to 72 hours of the luteinizing hormone peak. The LH surge or peak occurs within 24 hours of the beginning of estrus. Estrogen concentrations are dropping and progesterone concentrations begin to rise more rapidly then during proestrus. Younger bitches tend to ovulate within 24 hours of the increase and older bitches ovulate slightly later. The bitch may remain in estrus for 7 or 8 days following ovulation even though a functional corpus luteum (CL) is present. Estrogen concentrations have decreased several days prior to the refusal of the bitch to stand. The increasing concentrations of progesterone and decreasing estrogen reduce the attractiveness of the bitch to the males.

The ova in the bitch are ovulated prior to the shedding of the polar body; therefore, the ova are not fertilizable at the time of ovulation. Another 48 hours are necessary in order to permit maturation of the ovum prior to the capability of fertilization. The release of ova are generally thought to be nearly simultaneous as evidenced by reports suggesting that over 93% of follicles released ova within 96 hours of the LH peak.

The vaginal cytology characterizing early estrus is: high cellularity; little or no mucus and debris; no WBC's; few or no RBC's; and no parabasal cells with superficial cells predominating. Epithelial cell cornification is nearly complete approximately 2 days prior to the estrogen peak or about 4 days prior to the onset of estrus. As the bitch approaches late estrus (metestrus), the parabasal cells and leukocytes may begin to reappear. Temperature fluctuations are not reliable enough to be used for the prediction or determination of ovulation.

Metestrus

Metestrus I lasts approximately 4 to 6 days. This is the period during the cycle when the corpus luteum is forming. In the bitch this actually occurs during estrus and is not a separate specific period of time from that of estrus. The vaginal cytology is characterized by the reappearance of leukocytes and the increase parabasal cells. Metestrum cells, which are noncornified epithelial cells with vacuoles present in the cytoplasm, are found during metestrus. The glandular epithelium during this period undergoes hypertrophy and hyperplasia.

Diestrus

Diestrus (Metestrus II) lasts approximately 60 days. The vaginal cytology of the bitch in diestrus is characterized by fewer cells present on the slide and reappearance of debris. The cells present are parabasal and intermediate cells and few leukocytes. Late diestrus and anestrus vaginal cytology appear identical. Unless the bitch enters into a pseudopregnancy, her clinical appearance will be indistinguishable from anestrus. Another unique characteristic of the bitch is that the CL is functional for approximately the same length of time in the nonpregnant and the pregnant bitch. Progesterone concentrations are highest at approximately 25 days following ovulation. This is followed by a transient plateau and a prolonged decline until progesterone is less than 1 ng/ml. The only modification in progesterone concentration between pregnant and nonpregnant bitches is that there may be an increased progesterone concentration at day 15 post-ovulation in pregnant bitches above that observed in nonpregnant bitches. Diestrus ends when a pregnant bitch whelps or when a pseudopregnant bitch's progesterone concentration drops below 1 ng/ml.

Pseudopregnancy is a condition that occurs in the bitch and is characterized by deposition of abdominal fat during the diestrus. Other characteristics include increased mammary development with lactation and mothering instincts occurring at approximately 60 days post ovulation. There does not appear to be any difference in progesterone concentrations between the bitch, which demonstrates pseudopregnancy, and the one, which does not. The specific reasons for the occurrence of pseudopregnancy are unknown. Some authors refer to all nonpregnant bitches during the latter stages of diestrus as being pseudopregnant. This is probably clinically inaccurate and inappropriate. Only those bitches demonstrating signs of pseudopregnancy should be classified as pseudopregnant. Prostaglandin F2 alpha does not appear to be important as a natural controller of CL regression in the bitch. Parturition occurs approximately 24 hours after the CL is no longer functional.

Uterine involution following whelping or in the nonpregnant bitch is not complete until approximately 120 days following ovulation. This delayed involution especially in nonpregnant bitches is possibly due to the prolonged luteal phase following ovulation and due to an apparent genetic sensitivity to progesterone. These periods of prolonged endometrial stimulation predisposes the uterus to glandular hyperplasia and cyst formation. This may result in endometritis and pyometra.

Anestrus

This is the period of least reproductive activity. There is some activity occurring as the uterus is regenerating from the previous estrus and diestrus or pregnancy. There is no ovarian activity until near the end of anestrus. Anestrus lasts approximately 120 days with a range of 60 to 200 days. The occurrence of a pregnancy prior to the anestrus does not affect the length of anestrus. Estrogens during this stage of the cycle may be elevated but not as high as during proestrus. Progesterone remains below 0.5 ng/ml. The vaginal cytology is characterized by the presence of a few cornified epithelial cells and a variable number of noncornified cells. There should be no erythrocytes and few leukocytes present. The total number of cells will be considerably less then during the other stages of the cycle. There will be some debris and possibly some mucus.