**1.3. Recurrent estrus (shortened interestrous intervals or polyestrous)**

The mean duration between estruses is 7 months (4–13 months) in bitches, and the long part of the cycle (2–10 months) comprises a mandatory anestrus phase following diestrus. In the anestrous phase, the uterus enters the involution process and the endometrium is regenerated. Anestrus shorter than 2 months naturally results in repetition of estrus at 4-month or shorter intervals, which is defined as "recurrent estrous." It should be kept in mind that the estrous period is shorter in breeds such as the *German Shepherd Dog*, *Rottweiler*, *Basset Hound*, *Cocker Spaniel*, and *Labrador Retriever*. Fertility decreases in bitches with recurrent estrus [29] from two major causes: (1) overstimulation of the ovaries due to the formation of follicular cysts and granulose cell tumors and (2) a premature decrease of progesterone during diestrus [21].

The period between estruses may be prolonged up to 6 months by using a weak androgen, mibolerone (Cheque Drops), recommended at a dose of 30–180 μg per day to suppress estruses [1, 30].

Furthermore, if infertility resulted from short estrous period, bitches are reported to return to normal fertility in the following cycles when estrus is suppressed with synthetic progestin administration. For this purpose, 2 mg/kg per day megestrol acetate or 0.5 mg/kg per day of chlormadinone acetate may be administered for 8 days, so administration should begin within a maximum of 3 days following the beginning of proestrus [29].

## **1.4. Prolonged interestrous interval**

Prolonged interestrous interval is defined as an interestrous period longer than 12 months. While estrus repeats 12–13 months after the previous estrus (prolonged interestrous interval) in some adult bitches, some are not observed to experience estrus again for a long time (secondary anestrus). It should be kept in mind that the interestrous period is longer in breeds such as the *Basenji* and *Tibetan Mastiff* compared to other breeds. Secondary anestrus may result from hypothyroidism, administrations of hyperadrenocortisolism, hyperprolactinemia, progesterone-secreting ovarian cysts, progestogen, androgenic or anabolic steroid substances, systemic diseases, poor nutrition, or housing in an inappropriate environment. Thyroid function should be evaluated first in bitches with prolonged interestrous intervals. Cycles typically return to normal when the underlying cause is treated [5].

## **1.5. Prolonged estrus (persistent estrus)**

successful when implanted subcutaneously or submucosally. Deslorelin-containing implants

Hypophyseal (FSH and LH) and chorionic (PMSG and hCG) gonadotropins are also used for inducing estruses. It has been found that chorionic gonadotropins are more successful than hypophyseal gonadotropins in bitches. Although various protocols have been attempted, successful results have been reported with 20 IU/kg/d PMSG applied subcutaneously for 5 days, with 500 IU intramuscular hCG on day 5. PG600, a preparation containing PMSG and hCG first produced for pigs (80 IU PMSG and 40 IU hCG/mL), is quite effective for inducing

The mean duration between estruses is 7 months (4–13 months) in bitches, and the long part of the cycle (2–10 months) comprises a mandatory anestrus phase following diestrus. In the anestrous phase, the uterus enters the involution process and the endometrium is regenerated. Anestrus shorter than 2 months naturally results in repetition of estrus at 4-month or shorter intervals, which is defined as "recurrent estrous." It should be kept in mind that the estrous period is shorter in breeds such as the *German Shepherd Dog*, *Rottweiler*, *Basset Hound*, *Cocker Spaniel*, and *Labrador Retriever*. Fertility decreases in bitches with recurrent estrus [29] from two major causes: (1) overstimulation of the ovaries due to the formation of follicular cysts and granulose cell tumors and (2) a premature decrease of progesterone during diestrus [21].

The period between estruses may be prolonged up to 6 months by using a weak androgen, mibolerone (Cheque Drops), recommended at a dose of 30–180 μg per day to suppress es-

Furthermore, if infertility resulted from short estrous period, bitches are reported to return to normal fertility in the following cycles when estrus is suppressed with synthetic progestin administration. For this purpose, 2 mg/kg per day megestrol acetate or 0.5 mg/kg per day of chlormadinone acetate may be administered for 8 days, so administration should begin within

Prolonged interestrous interval is defined as an interestrous period longer than 12 months. While estrus repeats 12–13 months after the previous estrus (prolonged interestrous interval) in some adult bitches, some are not observed to experience estrus again for a long time (secondary anestrus). It should be kept in mind that the interestrous period is longer in breeds such as the *Basenji* and *Tibetan Mastiff* compared to other breeds. Secondary anestrus may result from hypothyroidism, administrations of hyperadrenocortisolism, hyperprolactinemia, progesterone-secreting ovarian cysts, progestogen, androgenic or anabolic steroid substances, systemic diseases, poor nutrition, or housing in an inappropriate environment. Thyroid function should be evaluated first in bitches with prolonged interestrous intervals. Cycles

(Suprelorin®, 4.7 mg deslorelin) are used most frequently for this purpose [23–27].

**1.3. Recurrent estrus (shortened interestrous intervals or polyestrous)**

a maximum of 3 days following the beginning of proestrus [29].

typically return to normal when the underlying cause is treated [5].

**1.4. Prolonged interestrous interval**

estruses [8, 28].

132 Canine Medicine - Recent Topics and Advanced Research

truses [1, 30].

The mean duration of estrus is 9 days in an adult dog, which may sometimes be prolonged up to 3 weeks. Estrus of longer than 21 days with the absence of ovulation at the end of this long period is defined as prolonged estrus. Prolonged estrus is related to persistent and elevated estrogen levels, which remain continuously high during the estrous phase of the cycle. This disorder is encountered frequently in younger bitches, especially during the second cycle [1].

The most important clinical signs of the continuation of estrus include cornification in vaginal epithelial cells, continuation of the desire for copulation, vulvar edema and swelling, and hyperemia in vaginal mucosa for longer than 21 days. The serum progesterone level is low, while estrogen level is high.

Persistent estruses are usually related to an estrogen-releasing source, which may be an anovulatory follicle, follicular cysts, or functional ovarian tumors (granulose cell tumors). The follicles that develop in bitches receiving exogenous gonadotropins in order to experience estrus may sometimes lead to prolonged estruses. Exogenous estrogen administration for the treatment of urinary incontinence or vaginitis, hormone replacement therapy, and prevention of undesired pregnancy may also cause persistent estruses. Persistent estrus may also develop alongside tumors of the hypophysis or the hypothalamus or in a hepatic disease defined as portosystemic shunting in which an abnormal vascular junction is formed between the hepatic portal vein and the systemic circulation. In these cases, metabolism of estrogen in the liver is impaired.

Determination that 90% or more of the cells in a vaginal smear specimen are permanently cornified on cytological examination and nondetection of the normal increase in serum progesterone levels (remaining within the preovulatory range <2 ng/mL) indicate prolonged estrus. Detection of serum estrogen level is not a reliable method for diagnosis [1].

The first step in treatment should include a determination of the source of estrogen causing prolonged estrus. For this purpose, the ovaries should be examined ultrasonographically for the presence of abnormal structures (e.g., ovarian cyst, granulose cell tumor); if ovarian structures cannot be identified by ultrasonographic examination, exploratory laparotomy should be performed, followed by biopsy, if required.

Follicles or follicular cysts causing prolonged estrus may heal spontaneously. If estrus is determined to last longer than 3 weeks, interventions are recommended in order to prevent bone marrow hypoplasia and/or pyometra. Treatment options should match the pet owner's expectations regarding having a puppy; if a puppy is not expected, ovariohysterectomy is the best option.

Ovulation or luteinization may be obtained by GnRH or hCG injections into follicles if the pet owner wants a puppy. hCG administration at a dose of 22 IU/kg via the intramuscular route for 3 days and GnRH (gonadorelin) administration at a dose of 10 μg/kg via the intramuscular route for 3 days are recommended. Copulation is not recommended, as the target of these applications is not the induction of ovulation but rather the termination of the signs of prolonged estrus [1]. Ovariohysterectomy is inevitable in cases in which no response is obtained from medical applications and in prolonged estrus cases due to ovarian tumors [5].

Megestrol acetate (Ovaban, Ovarid) may be applied to reduce the signs of prolonged estrus. Low doses of megestrol acetate are recommended via the peroral route for 2 weeks. A dose of 0.1 mg/kg is proper for the first week, and a dose of 0.05 mg/kg is proper for the second week. Although progesterone treatment with megestrol acetate is effective in bitches with persistent estrus, there is the potential to trigger the development of cystic endometrial hyperplasia. Therefore, the treatment is contraindicated for bitches to be later considered for copulation. In general, ovariohysterectomy is performed within 3 weeks after treatment with progesterone in bitches with persistent estrus.

Bone-marrow suppression related to long-term estrogen toxicity may develop in bitches with persistent estrus. Nonregenerative anemia and thrombocytopenia are observed in such bitches [31]. Therefore, erythropoiesis-stimulating agents, such as synthetic erythropoietin, darbepoetin, granulocyte-colony stimulating factor, and granulocyte-macrophage colonystimulating factor, may be used beside proper antibiotic and blood products in bitches that have anemia due to bone-marrow suppression. In addition, lithium [30, 32], synthetic anabolic steroids such as nandrolone decanoate (Deca-Durabolin), or a dihydrotestosterone derivative such as stanozolol (Winstrol) can be quite useful [33, 34].

#### **1.6. Prolonged proestrus (persistent proestrus)**

Prolonged proestrus is defined as a proestrous phase that is not followed by an estrous phase and that lasts 3 weeks or longer. In bitches with prolonged proestrus, estrus and ovulation do not occur, as the estrogen level insufficiently increases during the proestrous phase.

Prolonged hemorrhagic vaginal discharge, cornified cells higher than 50–90% on examination of vaginal smear, and serum progesterone level remaining below 2 ng/mL indicate prolonged proestrus. Treatment principles are similar to those for prolonged estrus.

## **1.7. Split estrus**

Split estrus is a disorder in which no or quite short estrous signs develop despite the presence of proestrous signs. In this situation, pregnancy usually does not develop even if copulation occurs; the bitch is observed to enter proestrus again within 3–4 weeks. In these bitches, the next cycle is usually a normal ovulatory cycle.

Split estrus is usually seen in young bitches that have shown first estrus. However, continuous or frequent split estruses should suggest chronic premature luteolysis or hypothyroidism. Split estrus may be confused with recurrent estrus (short interestrous interval). Ovulation will not develop in dogs showing split estrus but without the typical progesterone elevation. The condition usually recovers spontaneously [1, 7].

## **1.8. Anovulatory cycle**

A serum progesterone level not exceeding 2 ng/mL despite cytological estrous signs is defined as anovulation. Although the cell type in vaginal cytology is noncornified, diestrous-specific progesterone elevation does not occur, and the bitch enters anestrus.

The most typical signs are low serum progesterone levels and the absence of ovulation during the days after copulation in a bitch showing proestrous and estrous signs. Its incidence is about 1%. The following ovulatory cycles were observed to be normal in 45% of bitches that had an anovulatory cycle [1, 21, 35].

Thus, treatment is usually not required in bitches with an anovulatory cycle. hCG or GnRH may be administered, if desired; however, their application carries the potential to trigger pyometra [1, 21].
