Irregularities of the Estrous Cycle and Ovulation in Mares (Including Seasonal Transition)

Irregularities of the Estrous Cycle and Ovulation in Mares (Including Seasonal Transition)

144 35. 36. 37. 38. 39. 40. 41. 42. 43. CHAPTER 18 sis of livestock abortion, 3rd ed. Ames, Iowa: Iowa State University Press, 1990, p 207. G...

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sis of livestock abortion, 3rd ed. Ames, Iowa: Iowa State University Press, 1990, p 207. Golnik W: Viruses isolated from aborted foetuses and stillborn foals. Equine infectious diseases VI: Proceedings of the Sixth International Conference, Newmarket, UK, 1992, p 314. Murray MJ, Del Piero F, Jeffrey SC, et al: Neonatal herpesvirus type 1 infection on a Thoroughbred breeding farm. J Vet Intern Med 1998;12:36–41. Perkins G, Ainsworth DM, Erb HN, et al: Clinical, haematological and biochemical findings in foals with neonatal equine herpesvirus-1 infection compared with septic and premature foals. Equine Vet J 1999;31:422–426. von Nowotny N, Burki F: Drei durch Virusisolierung aus Pferdefeten abgesicherte Equine Arteritis Virus (EAV) Aborte aus Gestuten mit unterschiedlichen Zuchtrassen. Berl Munch Tierarztl Wschr 1992;105:181. Vaala W, Hamir A, Dubovi E, et al: Fatal congenitally acquired infection with equine arteritis virus in a neonatal Thoroughbred. Equine Vet J 1992;24:155–158. Del Piero F: Equine viral arteritis. Vet Path 2000;37:287– 296. Swerczek T, Hong C: Campylobacter fetus abortion in the mare. In Kirkbride CA (ed): Laboratory diagnosis of livestock abortion, 3rd ed. Ames, Iowa: Iowa State University Press, 1990, p 237. Welsh R: Equine abortion caused by Listeria monocytogenes serotype 4. J Am Vet Med Assoc 1983;182:291. Burgess E, Gendron-Fitzpatrick A, Mattison M: Foal mortality associated with natural infection of pregnant mares with

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Borrelia burgdorfferi. Equine infectious diseases V: Proceedings of the Fifth International Conference, Newmarket, UK, 1991, pp 217–220. Eisner R, Meirs D, Meirs D, et al: Lack of correlation between exposure to Lyme disease (Borrelia burgdorfferi) and pregnancy loss in mares. J Equine Vet Sci 1994;14:102–105. Long M, Goetz T, Kakoma I, et al: Evidence for fetal infectivity and abortion caused by Ehrlichia risticii. Proc Am Assoc Equine Pract 1992;571–577. Dawson J, Ristic M, Holland C, et al: Isolation of Ehrlichia risticii from the fetus of a mare with Potomac horse fever. Equine infectious diseases V: Proceedings of the Fifth International Conference, Newmarket, UK, 1991, pp 214–216. Cline J, Schlafer D, Callihan D, et al: Abortion and granulomatous colitis due to Mycobacterium avium complex infection in a horse. Vet Path 1991;28:89–91. Dwyer R: Fitting the pieces of the puzzle: mare reproductive loss syndrome update. Equine Vet Educ 2003;56–58. Cohen N, Carey V, Donahue J, et al: Case-control study of late-term abortions associated with mare reproductive loss syndrome in central Kentucky. J Am Vet Med Assn 2003;222:199–209. Tobin T, Harkins JD, Roberts JF, et al: The mare reproductive loss syndrome and the eastern tent caterpillar II: a toxicokinetic/clinical evaluation and a proposed pathogenesis: septic penetrating setae. Intern J Appl Res Vet Med 2004;2:142–158. Turner B, Savva D: Evidence of Toxoplasma gondii in an equine placenta. Vet Rec 1990;127:96. Dubey J, Porterfield M: Neospora caninum (Apicomplexa) in an aborted equine fetus. J Parasit 1990;76:732–734.

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Irregularities of the Estrous Cycle and Ovulation in Mares (Including Seasonal Transition) KATRIN HINRICHS

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rregularities of the estrous cycle may be associated with pathology of the ovary (granulosa cell tumor, gonadal dysgenesis) or uterus (shortened luteal activity due to endometritis) or, rarely, may be due to apparent functional abnormalities of the ovarian/hypophysial axis (anovulation with hematoma formation). However, wide variations in cycle length and ovarian characteristics are associated with normal fertility. Abnormalities of endocrinology, cyclicity, and ovulation are uncommon in mares with normal reproductive tracts, and thus other causes of infertility should be ruled out before implicating abnormal cyclicity as a cause of infertility.

Apparent estrous cycle irregularities that are not associated with pathology include prolonged estrus without ovulation during the seasonal transition period, “silent heat” (apparent failure to show estrous behavior), prolonged luteal activity, and anestrus or constant estrus during pregnancy. Additionally, aging of mares is associated with changes in cyclicity. Abnormal behavior may be related to ovarian pathology, most commonly granulosa cell tumors, in mares. Because this link is known, many other behavioral changes may be attributed to the ovaries by mare owners when in fact they have no association with reproduction.

Irregularities of the Estrous Cycle and Ovulation in Mares For this reason, mares with abnormal behavior may be presented for examination of the reproductive tract, and it becomes a diagnostic challenge on the part of the theriogenologist to determine the cause of the change in behavior. Diagnosis of the cause of estrous cycle irregularities in mares is based on history, teasing records, findings on palpation and ultrasonography per rectum, and determination of hormone concentrations, when indicated.

ASSESSMENT OF THE ESTROUS CYCLE AND OVULATION The Estrous Cycle A complete review of mare endocrinology is given in Chapter 7. An important factor to remember when evaluating apparent cycle abnormalities is the normal variation in length of estrus. Estrus length in normal cycling mares can vary at least from 2 to 12 days. Estrus length is generally repeatable within mares, and is longer at the beginning and end of the breeding season (the peak of the breeding season being the first day of summer, June 21 in the Northern Hemisphere). In some mares, estrus becomes so short in midseason that it may be missed if teasing is only performed sporadically. Owners may report that their mare was showing good heat in April and May but that they haven’t seen her in heat in June. Teasing is a major determinant of apparent cyclicity. Individual teasing of mares with an active stallion is the best method of heat detection. To adequately detect cyclical changes in behavior, teasing should be performed thoroughly at least three times weekly, and the mare’s behavior scored by a knowledgeable individual. “Silent heat” may occur in mares that have normal ovarian function. The meaning of this term is relative, indicating only that the observer did not recognize estrous behavior. Knowledge of the estrous and diestrous reactions of each individual mare is crucial in heat detection; one mare in heat may be less demonstrative than another mare in diestrus. Some mares may show signs of estrus immediately on contact with the stallion; others—especially young or timid mares—may require teasing for 3 to 4 minutes before they respond. A mare that shows no change in behavior at all during her cycle, as assessed by an experienced observer using good heat detection methods, may present a challenge. Such a mare may need to have the reproductive tract examined regularly by transrectal palpation and ultrasonography to detect estrus (see later discussion). However, assessing the willingness of the mare to allow mounting by a stallion may reveal that she is receptive to him, even though teasing evoked no signs of estrus. Mares that do not show good signs of estrus to a stallion may begin to show estrous behavior if they are bred by natural cover, during estrus, on a few occasions. Estrous behavior in mares does not necessarily indicate follicular activity. Estrous behavior can be seen in mares in seasonal anestrus, in mares that have been ovariectomized, and in mares with gonadal dysgenesis. This is because sexual behavior in the mare is largely regulated

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by progesterone; that is, estrous behavior is suppressed by progesterone. In the absence of progesterone, even the small amount of estrogen produced by the adrenals is sufficient to cause the mare to show some signs of estrus. Further stimulation with estrogen does, however, increase the intensity of estrous behavior. This is reflected in the finding that mares in true estrus (during the follicular phase of the cycle) have fewer negative reactions to mounting or intromission than do seasonally anestrous or ovariectomized mares.1 The length of diestrus is more repeatable among mares than is the length of estrus, at 15 ± 2 days.1 When evaluating heat detection records, repeated diestrus intervals of normal length, interspersed with estrous intervals, are a good indication that corpora lutea are forming and being lysed normally. The formation of corpora lutea in turn suggests that normal follicle growth and ovulation are occurring. Hormone analysis can also be helpful in defining the pattern of cyclicity in a mare; progesterone concentrations are of most value. Reflecting teasing records, a pattern of high progesterone (increasing to >4 ng/ml) for approximately 14 days followed by low progesterone (<1 ng/ml) for 3 or more days is strongly indicative of normal cyclicity.

Ovarian and Uterine Characteristics of Normal Cyclicity In assessing the normality of follicle growth and ovulation, the large variation in follicle size at ovulation should be recognized. The size of the follicle the day before ovulation is detected is commonly 35 to 45 mm in diameter, but mares can ovulate much smaller follicles (less than 30 mm), or larger follicles, with normal fertility. Follicle size at ovulation is often repeatable for a given mare. Small follicle size at ovulation may be a cause of apparent infertility because, unless this tendency is known, the mare may not be bred at the appropriate time. When double ovulations occur, the follicles ovulate at a smaller size than for single ovulations. Follicle size at ovulation also decreases toward the middle of the breeding season (July and August). Numerous large follicles and corpora lutea are normally present on the ovaries of pregnant mares, especially between 30 and 120 days. These are sometimes mistaken for ovarian pathology. Transrectal palpation and ultrasonography are indispensable tools in the evaluation of the estrous cycle. Familiarity with the normal appearance of the ovary and uterus on ultrasonographic examination throughout the cycle is a great help when assessing possible abnormalities.2 Multiple follicles may be present on the ovaries at any stage of the cycle. During estrus, the dominant preovulatory follicle is usually identifiable at approximately 25 mm diameter, when it is about 5 mm larger than other follicles on the ovary. In some mares, however, multiple follicles appear to grow to a large size (>30 mm) from which one or two ovulate; this is associated with normal fertility. On ultrasonographic examination, follicles usually are round and should be echolucent (black) in appearance. In the day or so before ovulation, infrequent small echodensities may be seen within the follicle, and

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the follicle may become less round, reflecting a palpable loss of tone. Immediately before ovulation, follicles may appear pointed on ultrasound examination, as they apparently protrude toward the ovulation fossa. On the first day after ovulation, the collapsed follicle may be difficult to define ultrasonographically. The only finding may be that the follicle seen previously is gone; however, a thick echodense line or mass is usually visible within the ovary. At this time, a depression may be palpable on the ovary, and the ovary may be sensitive to palpation. The normally developing corpus luteum (CL) has two major forms on ultrasonographic examination.2 Over the first few days after ovulation, the CL may become more and more apparent as an echodense structure within the ovary; then, after about 7 days, it may become less dense and less easily delineated. Alternatively, the ovulated follicle may fill with clotted blood over 1 to 2 days. This corpus hemorrhagicum has a typical ultrasonographic appearance of a round structure with an echodense rim and an echolucent interior, crossed with a lattice of echodense fibers. The echolucent area decreases in size over the next few days as the echodense rim thickens; after 5 or more days this structure is indistinguishable from the CL that did not have an interior clot. A normal corpus hemorrhagicum is typically no larger than was the original follicle; a larger structure with similar appearance on ultrasonography may be a hematoma (see later discussion). Uterine changes detectable on ultrasonographic examination are also helpful in staging the cycle in mares with questionable estrous behavior. The uterus in estrus becomes edematous. This is characterized on palpation as a heaviness and lack of tone, and ultrasonographically by presence of echolucent areas of fluid within the uterine folds, giving a “wagon wheel” or “orange slice” effect to the cross-sectional uterine image. In diestrus, the uterus is homogenous in appearance on ultrasonographic examination and tends to be more tightly circular in cross section, reflecting increased tone. Changes in palpable cervical tone reflect those of the uterus but may be more influenced by individual variation between mares; cyclical ultrasonographic changes in the cervix are not obvious.

PHYSIOLOGIC ESTROUS IRREGULARITY— THE TRANSITIONAL PERIOD The mare is a seasonal, long-day breeder, and the majority of mares enter anestrus in the winter. Ten to 25% of mares may cycle through the winter, this percentage being higher in mares having good nutrition and housing. In anestrus, gonadotropin-releasing hormone (GnRH) and gonadotropin secretion is low; follicles reach a maximum diameter of 10 to 15 mm and then regress. The period between anestrus and the first ovulation of the year is termed the transitional period. During this time, pituitary gonadotropin output is sufficient to cause follicular growth, but insufficient to stimulate normal follicle maturation and ovulation. The follicles produced at this time are indistinguishable from normal follicles on palpation and ultrasonographic examination; however,

they may be structurally and hormonally abnormal. For this reason, transition follicles will not ovulate in response to one-time administration of gonadotropins or GnRH analogues. During the transitional period, mares may be presented for examination because of constant or irregular estrus, as they respond to rising and falling estrogen from waves of nonovulatory follicles. Owners may become frustrated at this time with breeding mares over periods of weeks as the mares remain in heat. When estrous behavior does subside, it may recur within days; there is no normal diestrus interval. Unless the mare is bred immediately before the first ovulation of the year, the breedings will not result in pregnancy. On palpation and ultrasonographic examination, the ovaries have multiple small (in early transition) to large (in late transition) follicles. Extreme cases may have many (5–10) follicles greater than 30 mm in diameter on each ovary. No corpora lutea are present on the ovaries; however, corpus hemorrhagicum–like structures may be present, as these anovulatory follicles sometimes become filled with blood. The structures do not luteinize and so do not progress over time to the normal CL appearance on ultrasonography as described previously. The lack of luteal progesterone (<1 ng/ml in peripheral blood throughout this period) is reflected in poor uterine tone. Some practitioners feel that the presence of uterine edema signals the presence of a functional preovulatory follicle; however, recent studies have not supported this relationship. As gonadotropin secretion reaches functional levels, one of the follicles on the ovary will grow and ovulate; this signals the end of transition and the mare will cycle normally through the season from this point. Irregular estrous behavior and multiple anovulatory follicles are a normal aspect of the transition period and do not represent an abnormality. Diagnosis of seasonal transition is based on the season of the year (usually February through April in the Northern Hemisphere), lack of evidence of ovulation (no diestrous periods; no corpora lutea; poor uterine tone; low progesterone on assay), and multiple small to large follicles present on both ovaries.

MANAGEMENT OF SEASONAL TRANSITION Because of the January 1 birthdate imposed by many breed registries, foals born in a given year compete with one another. This has resulted in a demand for foals to be born early in the year. To have a foal born in January, the mare must conceive the previous February, during the period of seasonal transition. Many management methods have been developed to shorten the transitional period or move it to an earlier time of year (December to January). Currently no method is both commercially feasible on a large scale and highly predictable; however, it is likely that in the future this may be achieved using some combination of the treatments currently under investigation. The most widely used method for management of seasonal transition is increasing perceived day length. This

Irregularities of the Estrous Cycle and Ovulation in Mares may be done by housing mares under artificial light (100 lux, or equivalent of a 200-watt bulb 12 ft from the ground in a 12 ft by 12 ft stall) starting approximately December 15. Mares should be exposed to 14.5 to 16 hours of light per day; there is no benefit to increasing the apparent day length slowly, and lengths of light greater than 20 hours may actually reduce effectiveness. Alternatively, it has been shown that a short (1- to 2-hour) burst of light timed to occur at 9.5 to 10.5 hours after dusk is equally effective as having light on constantly for 16 hours. Although this technique saves electricity and may allow large groups of horses to be treated, application is more difficult because the time of the light burst must be correlated with changing day length. Recent studies have shown that reduced lux and reduced treatment periods may be equally as effective as is the traditional regimen.3 Mares typically ovulate and resume normal cyclicity 6 to 10 weeks after the onset of increased day length. Use of GnRH administration to induce cyclicity in anestrous or seasonal transition mares has been studied extensively.4 Infusion of pulses of GnRH (25 to 250 μg/ hour) at hourly intervals results in ovulation in essentially all anestrous mares within 11 days. This ovulation may be fertile, but mares may not continue to cycle if not pregnant. Although native GnRH is commercially available, this application requires the attachment and use of a pump. Constant infusion of GnRH or its analogues may also be effective, but responsiveness appears to be greater when the mare has entered transition. Constant infusion of GnRH using an osmotic minipump (an implant that releases drug as it accumulates fluid osmotically), delivering 100 to 200 ng GnRH per kg body weight per hour, induced ovulation in 60 to 70% of mares within 30 days. Injection of long-acting GnRH analogues (e.g., 40 μg buserelin twice daily) induced ovulation in 50% of anestrous mares in 10 to 25 days; however, this compound is not available commercially in the United States. Use of the commercially available implant containing 2.2 mg of the GnRH analogue, deslorelin, administered every other day has been shown to induce ovulation in about 50% of transitional mares. However, recent reports that deslorelin implants suppress pituitary responsiveness to native GnRH after administration suggest that repeated use of the implant may not be advisable. Indeed, when deslorelin was used to induce cyclicity, ovarian suppression was noted in the treated mares that did not ovulate. Injection of equine pituitary extract (once daily for 14–20 days; dosage is dependent on strength of the preparation) is effective for induction of follicular growth and ovulation in anestrous mares, but this compound is not currently commercially available. Progesterone and its analogues (e.g., altrenogest) have been used in an attempt to induce early cyclicity with the thought of suppressing luteinizing hormone (LH) release from the pituitary and then having a “rebound” when the progesterone is withdrawn. Progestins are effective in suppressing estrous behavior in transition mares and for this reason may be used simply to reduce the client’s concern about whether to breed the mare. The progestin is typically given for 10 to 15 days. Mares return to estrus

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approximately 3 days after the progestin is withdrawn, because the suppressive action of progesterone on estrous behavior is lost. Administration of progestin is not effective in hastening ovulation if given to mares in anestrus or early transition. In late transition, the first ovulation of the year in mares treated with progestin (e.g., 0.44 mg/kg altrenogest for 12 days) may be hastened by about 10 days. Other methods for inducing cyclicity during seasonal transition are currently under study. Dopamine antagonists, such as sulpiride (0.5 mg/kg, IM) or domperidone (1.1 ng/kg PO) daily for 10 to 60 days have been shown to induce cyclic activity in seasonally anestrous and transition mares.3 However, a repeatable method for use of these compounds has not yet been developed, and the response seems to depend on mare, environmental conditions, and stage of transition at the time of treatment. The variability in effect may be due to the mechanism of action of dopamine antagonists in the mare. In contrast to its action in ewes, dopamine antagonists do not appear to directly increase gonadotropin secretion in mares. Dopamine antagonists may work via increasing prolactin concentrations, which sensitize the ovary to circulating gonadotropins;3 therefore, there must be some native gonadotropin activity for ovarian response to occur.

CYCLE IRREGULARITIES ASSOCIATED WITH AGING Mares cycle less efficiently after about 20 years of age. There is a delayed entry into the ovulatory season, longer follicular phase (estrus), and fewer ovulations per year.5 Mares over 25 years of age may cease cycling altogether. These mares may be presented for examination because of erratic or constant heat during the breeding season, or because multiple breedings have not resulted in pregnancy. They may also be apparently anestrous during the breeding season. Diagnosis is based on the mare’s age; these changes are not usually seen until the mare is near 20 years of age. The delay of the first ovulation of the year is associated with a prolonged transitional period in the spring, which extends into the normal breeding season. Breedings during this time will not result in pregnancy because the mare is not ovulating. Findings on palpation and ultrasonographic examination will be similar to those described earlier for the transitional period. Once the mare does ovulate, however, the corpus luteum appears to have normal function and cycles may be regular. Old mares that have ceased cycling will appear to be anestrous (have no follicular activity, no uterine tone, and low progesterone) throughout the breeding season. Management of old mares that are still intended for breeding would include monitoring the ovaries by palpation and ultrasonographic examination over time to detect follicular growth leading to ovulation. In the case of an old mare that is anestrous, treatment with pulsatile GnRH may be effective in inducing follicular growth and ovulation. It must be remembered, however, that even when cycling normally, old mares have low fertility,

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associated with decreased oocyte viability and uterine changes.

Prolonged Luteal Activity A prolonged luteal phase, that is, greater than 17 days, is a common occurrence in mares. It is estimated that failure of normal return to estrus may occur in 4% to 18% of cycles, and in these cases the luteal phase may be prolonged to over 60 days.1 The first work in this area was done before ultrasonography, and it was presumed that the primary corpus luteum (from ovulation during the previous estrus) was somehow maintained after the normal time of luteolysis. Work with ultrasonography has shown that a prolonged luteal phase is seen when a diestrous follicle ovulates after day 10 of diestrus. This new corpus luteum is too immature to respond to the endogenous prostaglandin release around day 14. Persistent luteal activity can also be associated with an apparently luteinized hematoma. It is not known whether idiopathic maintenance of the primary corpus luteum can also be a cause of prolonged diestrus. Another possible cause of prolonged luteal activity is severe damage to the endometrium, as seen in overt pyometra. If the damage is severe enough that prostaglandin production is impaired, retention of the primary corpus luteum results. Inflammation of the endometrium, as seen in endometritis, may result in release of prostaglandin and thus shortening of the luteal phase (see later discussion). Mares with prolonged luteal activity may be presented because of failure to return to estrus. The other major differential diagnoses for failure to return to estrus are pregnancy, which can happen even if no breeding is recorded; silent heat (the mare that is cycling but does not show behavioral estrus); and poor estrus detection, or short heats that are missed, especially near the middle of the breeding season. Diagnosis of prolonged luteal activity is based on finding a normal-appearing nonpregnant diestrous tract with failure to show estrus or have estrustype examination findings for more than 17 days after ovulation. Progesterone concentrations will be high (usually >4 ng/ml) for more than 2 weeks. Treatment of prolonged luteal activity is simple and effective: administration of prostaglandin F2α (10 mg IM). To assure a response, the prostaglandin should be given at least 5 days after the most recent ovulation. The mare should return to heat in about 3 days. It should be verified that the mare is not pregnant before treatment, even if the mare has had a previous negative pregnancy check.

Shortened Luteal Phase A decrease in the length of diestrus (less than 13 days) may be indicative of premature luteolysis. Primary defects of the corpus luteum have not been confirmed in the mare.1 The most common factor associated with premature luteolysis is endometritis; prostaglandin production associated with uterine inflammation or bacterial endotoxin production is the causative factor. Some interval of diestrus is seen even if an active endometritis is present,

as the corpus luteum is not completely responsive to prostaglandin for about the first 5 days after ovulation. After this, the relatively low but constant production of prostaglandin is sufficient to cause lysis of the corpus luteum over a period of days, typically resulting in a 7to 11- day diestrus. If a shortened luteal phase is detected, an endometrial culture and biopsy should be obtained to determine if endometritis is present, and, if so, which organism may be responsible. Resolution of the endometritis should result in return of normal diestrous intervals.

CYCLE IRREGULARITIES ASSOCIATED WITH OVARIAN PATHOLOGY Gonadal Dysgenesis Gonadal dysgenesis refers to the congenital lack of development of the ovaries. Only a “streak” (small, almost cylindrical) gonad is present, with no follicular activity. The remainder of the tract is intact, as the female tract will form in the absence of gonads, but the tract is juvenile because no ovarian steroids are present to stimulate growth. This abnormality is most commonly associated with defects of the X chromosome, including XO (Turner’s syndrome) and XXX; however, it may be seen in XY mares as well as in mares with apparently normal XX karyotypes. Mares with gonadal dysgenesis may be presented for examination because of anestrus, erratic estrus, or constant estrus. Exhibition of estrous behavior is due to the lack of progesterone. In cases of erratic or constant estrus, the mares may have been bred repeatedly without conceiving. Mares with XO karyotypes may be unthrifty, be shorter than anticipated, or have increased angulation of the hind legs. Diagnosis of gonadal dysgenesis is based on history, karyotype, and repeated palpation and ultrasonography or progesterone determination. Mares with gonadal dysgenesis have never foaled and have never been pregnant. Palpation and ultrasonography reveal very small or apparently absent ovaries; if ovaries are present, no follicular activity is seen. It should be noted that some normal mares, especially young mares of smaller breeds, can have very small ovaries that function normally (raise follicles and ovulate with normal fertility). For this reason, serial examinations or progesterone determinations at least once weekly for 5 or more weeks during the breeding season may be needed to definitively state that the ovaries are nonfunctional. Presence of nonfunctional ovaries does not necessarily mean that the mare has gonadal dysgenesis: small, inactive ovaries during seasonal anestrus are normal; very old mares (>24 years) may have inactive ovaries in the breeding season because they have stopped cycling altogether; and mares having a severe energy imbalance due to starvation or disease (or, theoretically, mares in extremely hard work) may have inactive ovaries. A chromosomal abnormality on karyotype supports the diagnosis of gonadal dysgenesis. No treatment is possible for mares with gonadal dysgenesis.

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Granulosa Cell or Other Functional Tumor Granulosa cell tumors (GCTs) are by far the most common ovarian tumors in mares. These neoplasms of the sex-cord stroma may also contain thecal cells (granulosa cell–theca cell tumors). They may be found in mares of any age, and are almost invariably benign. Behavioral changes associated with GCTs include aggressive or stallion-like behavior, constant or erratic estrus, or anestrus, with about equal frequency. These changes are attributed to steroid production by the neoplasm. Theoretically, production of testosterone or high concentrations of estrogen may be associated with aggressive or stallion-like behavior; estrogen with constant estrus; and progesterone with constant diestrous-like behavior; however, progesterone is typically low in cases of GCT. Anestrous behavior (including erratic signs of heat) may be associated with neoplasms that do not produce steroids. The contralateral ovary is typically inactive and small. This is attributed to high concentrations of inhibin or steroids from the tumor, causing suppression of follicle-stimulating hormone (FSH) release. In rare cases, apparently no pituitary suppression is present and mares with GCT cycle normally, ovulating from the contralateral ovary. Diagnosis of GCT is based largely on the history (abnormal behavior or cyclicity) and findings on palpation and ultrasonography per rectum. The affected ovary is typically large, smooth walled, and spherical. The ovulation fossa is usually not present. The contralateral ovary is usually small and inactive, resembling an anestrous ovary. Ultrasonography is useful for differentiating normal follicular activity from pathologic changes in the ovarian stroma and for confirming the lack of activity of the contralateral ovary. There is no one typical appearance of GCT on ultrasonography, however. The most common ultrasonographic appearance is that of multiple cystic structures (“honeycomb” appearance); however, GCT may also appear as unilocular cysts or may be solid throughout.6 The honeycomb appearance of a GCT may be confused with that of a clotting hematoma (see following discussion). A hematoma may be differentiated from a GCT by several features: with a hematoma, the activity of the contralateral ovary is normal and the mare has normal cycles; the ovulation fossa is present on the ovary, as the hematoma causes enlargement of only one pole; and the hematoma appears abruptly and regresses over time (usually within 30 days). Serum testosterone concentrations are elevated in 50% to 90% of GCT, and inhibin concentrations are elevated in over 85% of mares with GCT; tests for these hormones may be used as a diagnostic aid.7 Management is by surgical removal of the affected ovary; approximately 75% of mares resume normal cyclicity from the remaining ovary within 2 years of removal of a GCT. Teratomas are relatively rare in comparison with GCTs; they may be difficult to diagnose as the ovarian enlargement may not be pronounced, and ultrasonographic findings may be unremarkable. Although not commonly considered to be functional tumors, teratomas may be associated with disruption of cyclicity. Serous cystadenomas of mare ovaries have also been reported; these are

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associated with multiple large cystic structures resembling normal follicles on palpation and ultrasonography. Serous cystadenomas may be associated with elevated serum testosterone concentrations, but do not appear to affect cyclicity.

INTERSEXUALITY Conditions in which testicular tissue is present in the gonad such as true or pseudo-hermaphroditism affect cyclicity but usually are suspected early in life because of intersexuality of the external genitalia. However, conditions may occur in which testicles are present but the external genitalia is that of a normal female (some XY sex reversals, including testicular feminization syndrome). These are associated with presence of internal testicles instead of ovaries, absence of cyclicity, stallion-like behavior, and usually failure of formation of the internal female tract, resulting in the vagina ending in a blind pouch. These conditions may be diagnosed on the basis of high serum testosterone levels, abnormal size, shape, consistency, or ultrasonographic appearance of the gonads, and abnormalities of the internal genitalia.

ABNORMALITIES OF OVULATION Failure of normal ovulation of the dominant preovulatory follicle may occur in mares. This is identified on repeated ultrasonographic examination. Two abnormalities of ovulation have been noted clinically: anovulation with hematoma formation, and apparent failure of the follicle to completely empty at ovulation. The former abnormality has been documented but the latter has only been reported anecdotally among clinicians. No information on the pathogenesis of either abnormality has been reported. In anovulation with hematoma formation (persistent anovulatory follicles), the preovulatory follicle appears to grow normally, but instead of ovulating, starts to rapidly enlarge. Whereas normal follicles typically grow about 3 mm per day, anovulatory follicles may suddenly increase 10 mm or more in diameter per day, becoming much larger than a normal preovulatory follicle. Echogenic particles become visible within the follicle, and may increase in number over a few days until the follicle may appear to be diffusely echogenic. At this time the contents of the follicle are fluid, and can be seen to swirl on ultrasonography when the follicle is balloted. Aspiration of the follicular contents at this time yields bloody fluid, and so these follicles are considered to be hemorrhagic. Finally, in the majority of anovulatory follicles, the contents of the follicle appear to clot (nonechogenic spaces form, delineated by strands of echogenic material).6 Increasing echogenicity over time and apparent clotting, resulting in formation of a hematoma, is not pathologic in follicles that would not normally be destined to ovulate (e.g., diestrus follicles, secondary follicles during estrus, dominant estrus follicles during fall transition [autumn follicles]). Anovulation of the dominant preovulatory follicle during the breeding season is less common and is associated with infertility. A 4.7% incidence of hematoma formation was found in one study,

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but it was unclear how many of these were seen in mares that successfully ovulated another follicle at the same time.1 In a second study, 8% of cycles in mares of varied reproductive history resulted in anovulation. This proportion was age dependent, being 4% in mares 6 to 10 years of age and 13% in mares 16 to 20 years of age.8 When anovulation occurs, more than 84% of mares progress into diestrus with progesterone concentrations greater than 1 ng/ml.8 In these mares, the wall of the follicle surrounding the hematoma appears to thicken, suggesting that it is luteinizing. In one study all such structures responded to prostaglandin administration, but the day of administration was not given.8 Clinically these luteal structures may appear not to respond to prostaglandin as readily as do normal corpora lutea, and repeated doses of prostaglandin may be needed to cause regression of the structure and return of the mare to estrus. Formation of an anovulatory follicle is associated with failure of conception in inseminated mares.8 Mares that experience anovulation and hematoma formation of the dominant follicle tend to do this over repeated cycles.8 The author observed one Warmblood mare that formed a hematoma instead of ovulating at each estrus, throughout a breeding season. Treatment with human chorionic gonadotropin (hCG), suppression of follicular activity with progesterone and estrogen with subsequent release, and short-cycling with prostaglandin had no effect on hematoma formation. The mare was bred on each estrus but did not get pregnant. The next year, she was bred on an early estrus, ovulated normally, and became pregnant. Mares experiencing anovulation/hematoma formation of the dominant preovulatory follicle may in fact appear to cycle normally, due to luteinization of the follicle wall. Some cases may be associated with prolonged diestrus; however, the presenting complaint may be simply infertility. Diagnosis entails monitoring follicle growth during estrus daily by ultrasound examination. Findings are those for normal follicle growth up to about ovulatory size (35–40 mm); then these follicles continue to grow, often rapidly, sometimes to a large size (60–90 mm). Echogenic particles usually are seen at the time of follicle enlargement. The echogenicity increases, then apparent clotting occurs. No specific causes or treatment for this syndrome are currently known. Apparent incomplete emptying of the follicle at ovulation is much less well documented, but has been noted by practitioners. The follicle grows normally and then is seen to be partially collapsed, suggesting that the examination caught the follicle in the process of ovulation (this in fact would be a rare occurrence, as normal ovulation results in follicle emptying within about 5 minutes). Examination the following day shows the follicle to have an unchanged appearance, (i.e., to still be “ovulating”), irregular in outline and containing 15 to 25 mm of fluid within it. This appearance is maintained over the following days, then the fluid regresses slowly. The follicle wall appears to luteinize, and progesterone levels during the ensuing diestrus may be normal. While no hard data is available, the clinical impression is that the pregnancy rate for mares having this follicular appearance at ovula-

tion is low. Again, no specific causes or treatment for this syndrome are known.

FOLLICLE GROWTH AND REGRESSION WITHOUT OVULATION Follicle growth followed by regression, without ovulation, during the breeding season is extremely rare, but may occur. It is difficult to find documentation of this phenomenon, but it is recognized clinically. The author has observed this syndrome in two 14-hand, 5- and 10year-old ponies, both pintos. The ponies were obtained from a dealer without knowledge of previous breeding history. Ovarian activity was followed throughout the breeding season by ultrasonography at least three times weekly. The ponies had apparently normal reproductive tracts on palpation and ultrasonography, and the ovaries were of normal size. Multiple follicles smaller than 10 mm were present, and periodically, a follicle would grow to 18 to 20 mm, only to gradually regress. Treatment with prostaglandin to lyse any luteal tissue present, and administration of hCG at peak follicle growth had no effect. No karyotype was done on these mares; however, because of the normal ovarian size and follicle growth, they could not be classified as having gonadal dysgenesis.

BEHAVIORAL PROBLEMS ATTRIBUTED TO THE OVARIES Persistent Estrus “Persistent estrus” is a fairly common complaint in working and racing mares. The major factor in the complaint is that the mare is constantly showing a behavior, interpreted as estrus, and it is interfering with the mare’s function. Upon investigation, this complaint is usually not associated with any actual abnormality in cyclicity, and it is seldom identified in mares in a teasing program where true estrous behavior (response to a stallion) can be assessed. When evaluating histories of persistent estrus, the normal variation of estrous length, from 2 to over 10 days, should be considered. The pattern of estrous cyclicity noted by the owner, and exactly what behavior the owner is seeing, should be ascertained. A major confusing issue in these mares is what is considered to be estrous behavior. Such mares may be characterized by their owner as being irritable, kicking when their sides are touched, leaning on the handler, striking, urinating, or wringing their tails. These signs are interpreted as indicating that the mare is in heat; however, they may be simply signs of agitation, discomfort, or submission. To diagnose the cause of the apparent constant estrous behavior, findings on palpation and ultrasonography of the tract are evaluated with the history, and the mare should be teased with an active stallion. Response of the mare to the advances of the stallion with signs of estrus, such as urinating, raising the tail, winking the vulva, and hesitating to move away from him, accompanied by estrus-type signs on palpation and ultrasonography as outlined earlier, indicate that the mare is in estrus at the

Irregularities of the Estrous Cycle and Ovulation in Mares time of the examination. Mares with large follicles (>33 mm) may respond to administration of hCG (2500 IU IV) by ovulating approximately 2 days later and going out of heat in another 1 or 2 days. If monitored, mares in estrus with small follicles will usually show normal follicle growth, ovulation, and entrance into diestrus. The reason for the history of prolonged heat in these mares before the examination is not clear. Constant or erratic true estrous behavior (response to a stallion) may be seen during seasonal transition (see previous discussion). This behavior may also be related to ovarian pathology such as gonadal dysgenesis or granulosa cell and related tumors, and these possibilities should be investigated as outlined earlier. In addition, pregnant mares may show constant or irregular estrus in response to teasing by a stallion. In a nontransitional mare with a normal nonpregnant tract showing persistent behavior interpreted as heat, any ovarian basis for the behavior may be determined by establishing whether there is a correlation between behavior and cyclicity. The reproductive tract should be examined two to three times weekly for a month or more while the owner notes the mare’s behavior. If the mare cycles normally, and no correlation of cyclicity with behavior is present, the mare’s behavior is probably not associated with her ovaries. Many nonreproductive behaviors may be interpreted as signs of “heat.” Mares that have constant agitated behavior, rather than true estrous behavior, may be in any stage of the cycle at the time of examination. Agitated, estrous-type behavior is often seen (or at least complained about) in mares in fairly intense training. This behavior has been termed “submissive, cowering behavior” and has been equated to the urinating behavior of a scared puppy.9 When mares with this behavior are evaluated critically, although some signs associated with estrus may be present (raising the tail, urinating, leaning), the behavior of the agitated mare is anxious, or guarding.9 She is resentful of the approach of a stallion when teased, and may lean away from the stallion or appear fearful and attempt to escape. This behavior is in contrast to that shown by the mare in true estrus, which during teasing is typically calm and interested in maintaining contact with the stallion. Mares with cowering behavior may improve given changes in their environment and gentle handling, with the goal of increasing the mare’s confidence and decreasing intimidating stimuli while reacclimating her to her training schedule. Long-acting tranquilizers have also been felt to aid in management of these mares.10 Signs interpreted as persistent estrus may be attributable to vaginal inflammation due to a foreign body, or to aspiration of air into the vagina when the mare is working. The latter seems to be most common in racingfit mares (mares with little body fat) in training. These mares lack perineal fat, which affects the tone of the vulvar lips, the angle of the vulva, and the weight of the perineal body, which closes the entrance to the vagina. The affected mare has signs of vaginitis such as frequent urination, hunching the back, dragging the hind feet, and wringing the tail. Racing mares with this complaint commonly have a history of “stopping” part way through

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training periods; this may be due to pneumovagina incurred during work. Examination of the tract shows the mare in any stage of the cycle; bright echogenic particles representing air may be seen in the uterus on ultrasonographic examination; the vagina may be ballooned with air. Speculum examination of the vagina may reveal an inflamed vagina and the mare may show extreme signs of irritation (hunching the back, attempting to urinate) after the speculum is inserted or removed. This problem may occur even after a Caslick’s operation has been performed. A modified Caslick’s procedure (perineal reconstruction) may help to prevent pneumovagina by narrowing the vestibule and increasing the bulk of the perineal body. Abnormalities of the bladder or urethra, such as cystitis or urethral masses, may result in frequent urination that is interpreted as persistent estrus. One mare, presented for examination for “nymphomania,” exhibited agitated, pacing behavior, and frequent urination. The cause was found to be neurogenic bladder atony with associated cystitis. The distended bladder constantly ejected urine as the mare moved. When the bladder was kept empty by catheterization, the mare’s constant tail raising, posturing, and agitated behavior all ceased; the agitation may have been due to pain from bladder distention. The mare’s reproductive tract and cyclicity were normal. Sometimes stallion-like behavior is interpreted to be estrous behavior by the owner. Assessment of stallion-like behavior may be done by turning the mare out with other mares and observing whether she exhibits courting or mounting behavior, by placing her in a stallion’s paddock to observe how she responds to fecal piles (stallion-like behavior includes smelling fecal piles, defecating or urinating on the same pile, and smelling the pile again), and by bringing mares up to the fenceline to see if the mare under study prances up, vocalizes or otherwise behaves in a stallion-like manner. Stallion-like behavior is almost always related to exposure to androgens, possibly from an ovarian tumor or presence of testicular tissue (see previous discussion) or from administration of exogenous anabolic steroids. The effects of exogenous androgens may last for months after the last administration.

Cyclic or Sporadic Behavioral Problems Some mares do become harder to manage, perform irregularly, or even appear lame when in heat; this behavior is intermittent and corresponds to specific stages of the estrous cycle.9 Such problems may be due simply to exaggerated exhibition of normal estrous behavior at times that are deemed inappropriate by the owner (e.g., while under saddle). However, there may also be behavioral and physical effects of estrogen, such as increased joint laxity, that affect performance. Some part of this behavior may also be related to pain from the growing follicle stretching the ovarian tunic, or from rupture of the follicle at ovulation. Most mares exhibit signs of pain when the ovary is palpated immediately after ovulation. Altrenogest treatment (at 0.044 to 0.132 mg/kg orally daily) is commonly used to suppress problem estrous behavior in mares, and is usually effective. Although

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progestin-containing implants produced for estrus synchronization in cattle have been used for this purpose, and anecdotally have had some effect, studies have shown that most synthetic progestins do not suppress estrus or interact with equine progesterone receptors. Sometimes owners request ovariectomies for mares that are sporadic or constant “bad actors.” This should be reserved only for cases in which the mare has cyclic behavioral problems corresponding to a specific part of the estrous cycle when evaluated as described earlier; this behavior should seem to improve over the winter (during anestrus). Mares rarely fall into this category, but when they do, ovariectomy can be an effective cure for the behavior. The mare’s tendency to react violently should be considered when making the decision on whether to do the ovariectomy standing (via colpotomy) or under general anesthesia. As with “persistent estrus,” however, owners may attribute objectionable behavior in a mare to ovarian problems when in fact there is no connection. Any type of sporadic behavior (even refusing jumps when under saddle) may be interpreted by the owner as a sign that the mare is in heat. Owners may assign a behavior to 21day cycles because they are associating it with estrus, and they are aware of the length of the estrus cycle; however, questioning may reveal that the actual duration of the behavior and the period between behavioral episodes is unknown. These cases should be approached with the presumption that the behavior is not related to cyclicity until the association is proved. Examples of this include a mare that was presented for examination with a history that she was in pain during estrus, showing signs of colic and inappetence. The owners had not kept a written record but stated that the episodes occurred about every 21 days. A work-up for recurrent colic was performed, and on gastroscopy the mare was found to have stomach ulcers. Treatment of the ulcers with cimetidine resulted in cessation of the colicky episodes. In another case, a mare was periodically resentful of being handled, to the point of shaking and attempting to lie down; complete examination, including exploratory laparotomy, revealed an abscess in the stomach wall.

Aggressive or Abnormal Behavior Constant aggressive, quasi-stallion-like or otherwise abnormal behavior may be related to presence of a granulosa cell tumor or testicular tissue (see previous discussion). Interestingly, it appears that some mares may show changes in behavior for a year or more before the detec-

tion of ovarian changes indicative of GCT.10 Therefore, if the reproductive tract is normal on initial examination, but no other cause of abnormal behavior is documented, a repeat examination of the reproductive tract should be performed every 6 months or so. A warning should be made regarding mares presented for assessment of ovarian causes of abnormal behavior. Occasionally a mare is presented that has vicious outbursts of aggressive behavior—in one case that the author evaluated, the mare broke through a fence to attack a child. These mares may be presented for reproductive examination on the possibility that violent or vicious behavior is related to an ovarian abnormality. Such mares should be approached with caution. Little information is available on the causes of this behavior but it is unlikely to be related to the reproductive tract. In two cases of such behavior seen by the author, pituitary abnormalities were suggested. One mare had preneoplastic changes in the pituitary on histopathologic examination at necropsy, and one mare had adrenocorticotropic hormone (ACTH) concentrations and dexamethasone suppression test results midway between those expected for normal and for Cushing’s disease patients.

References 1. Ginther OJ: Reproductive biology of the mare: basic and applied aspects, 2nd ed. Cross Plains, WI: Equiservices, 1992. 2. Ginther OJ: Ovaries and uterus. In Ultrasonic imaging and reproductive events in the mare. Cross Plains, WI: Equiservices, 1996, pp 115–194. 3. Daels P: Seasonal anestrus in mares: physiology, diagnosis and treatment. Proc Soc Theriogenol 2000; pp 189–196. 4. Hyland J: Uses of gonadotropin releasing hormone (GnRH) and its analogues for advancing the breeding season in the mare. Anim Reprod Sci 1993;33:195–207. 5. Vanderwall DK, Woods GL, Freeman DA, et al: Ovarian follicles, ovulations, and progesterone concentrations in aged versus young mares. Theriogenology 1993;40:21–32. 6. Hinrichs K, Hunt PR: Ultrasound as an aid to diagnosis of granulosa cell tumor in the mare. Equine Vet J 1990; 22:99–103. 7. McCue PM: Equine granulosa cell tumors. Proc 38th Ann Mtg AAEP 1992;587–593. 8. McCue PM, Squires EL: Persistent anovulatory follicles in the mare. Theriogenology 2002;58:541–543. 9. McDonnell SM: Evaluation and modification of mare behavior problems. Proc Soc Theriogenol 1993; pp 185–189. 10. McDonnell SM, University of Pennsylvania, personal communication, 2001.