Ultrasonographic characteristics of the reproductive tract and serum progesterone and estradiol concentrations in captive female red wolves (Canis rufus) with and without reproductive tract disease

Kadie M. Anderson Point Defiance Zoo and Aquarium, 5400 N Pearl St, Tacoma, WA 98407.

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Mandi W. Schook Cleveland Metroparks Zoo, 4200 Wildlife Way, Cleveland, OH 44109.

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Karen L. Goodrowe Point Defiance Zoo and Aquarium, 5400 N Pearl St, Tacoma, WA 98407.

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William T. Waddell Point Defiance Zoo and Aquarium, 5400 N Pearl St, Tacoma, WA 98407.

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Karen N. Wolf Point Defiance Zoo and Aquarium, 5400 N Pearl St, Tacoma, WA 98407.

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Abstract

OBJECTIVE To describe ultrasonographic characteristics of the reproductive tract and serum progesterone and estradiol concentrations in captive female red wolves (Canis rufus) with and without reproductive tract disease.

DESIGN Prospective study.

ANIMALS 13 adult female red wolves.

PROCEDURES Wolves with varying parity and history of contraceptive treatment were anesthetized to facilitate ultrasonographic examination and measurement of the reproductive tract and blood collection for determination of serum progesterone and estradiol concentrations in December 2011 and June 2012. Additionally, during the December evaluation, fine-needle aspirate samples of the uterus were obtained for cytologic evaluation. Measurements were compared between wolves with and without reproductive tract disease and between wolves that had and had not received a contraceptive.

RESULTS 7 of 13 wolves had or developed reproductive tract disease during the study. Ranges for measurements of reproductive tract structures overlapped between ultrasonographically normal and abnormal tracts, but measurements for abnormal tracts were generally greater than those for normal tracts. The ultrasonographic diagnosis was consistent with the histologic diagnosis for reproductive tracts obtained from wolves that were sterilized, were euthanized, or died during the study. Cytologic results for fine-needle aspirate samples of the uterus and serum progesterone and estradiol concentrations were unable to distinguish wolves with and without reproductive tract disease. Reproductive tract disease was not associated with parity or contraceptive administration.

CONCLUSIONS AND CLINICAL RELEVANCE The ultrasonographic images, reproductive tract measurements, and descriptions of reproductive tract lesions provided in this study can be used as diagnostic guidelines for the treatment and management of red wolves with reproductive tract disease.

Abstract

OBJECTIVE To describe ultrasonographic characteristics of the reproductive tract and serum progesterone and estradiol concentrations in captive female red wolves (Canis rufus) with and without reproductive tract disease.

DESIGN Prospective study.

ANIMALS 13 adult female red wolves.

PROCEDURES Wolves with varying parity and history of contraceptive treatment were anesthetized to facilitate ultrasonographic examination and measurement of the reproductive tract and blood collection for determination of serum progesterone and estradiol concentrations in December 2011 and June 2012. Additionally, during the December evaluation, fine-needle aspirate samples of the uterus were obtained for cytologic evaluation. Measurements were compared between wolves with and without reproductive tract disease and between wolves that had and had not received a contraceptive.

RESULTS 7 of 13 wolves had or developed reproductive tract disease during the study. Ranges for measurements of reproductive tract structures overlapped between ultrasonographically normal and abnormal tracts, but measurements for abnormal tracts were generally greater than those for normal tracts. The ultrasonographic diagnosis was consistent with the histologic diagnosis for reproductive tracts obtained from wolves that were sterilized, were euthanized, or died during the study. Cytologic results for fine-needle aspirate samples of the uterus and serum progesterone and estradiol concentrations were unable to distinguish wolves with and without reproductive tract disease. Reproductive tract disease was not associated with parity or contraceptive administration.

CONCLUSIONS AND CLINICAL RELEVANCE The ultrasonographic images, reproductive tract measurements, and descriptions of reproductive tract lesions provided in this study can be used as diagnostic guidelines for the treatment and management of red wolves with reproductive tract disease.

The red wolf (Canis rufus) is listed as endangered under the Endangered Species Act of the United States and as critically endangered by the International Union for Conservation of Nature and Natural Resources,1 with only 25 wolves estimated to be left in the wild and approximately 190 wolves maintained in zoos and wildlife facilities. A cooperative zoo-based breeding program combined with reintroduction efforts has been responsible for reestablishing the current wild population, which was considered extirpated in the wild in 1980. As with any small population of animals, achieving a self-sustaining, genetically stable population through demographic and genetic management presents unique challenges, which are further compounded by species-specific disease trends that can affect the sustainability of such programs.

The red wolf was recently identified as a species at increased risk for the development of CEH and pyometra,2 and the incidence of pyometra and CEH in red wolves appears to be rising. In a 2009 study,3 endometrial hyperplasia was identified in the reproductive tracts of 15 of 36 (42%) red wolves, whereas in a 2014 retrospective survey2 of records and pathological reports for red wolves that were managed by the Association of Zoos and Aquariums and related facilities, the incidence of CEH or endometrial hyperplasia was 75% and the incidence of pyometra was 11.1%. Results of a 2009 retrospective mortality review4 indicate that 2 of 64 (3.1%) red wolves died of pyometra over a 4-year period, whereas results of a 2016 review5 of mortality records from 1997 to 2012 indicated that 8.8% of female red wolves died of reproductive tract disease and 11.8% of female red wolves had ancillary reproductive lesions such as suppurative endometritis or CEH. Additionally, Veterinary Advisor Reportsa from 2009 to 2013 identified 8 female red wolves that were treated for, died of, or were euthanized because of pyometra. The increasingly frequent identification of CEH and pyometra in red wolves is concerning owing to the critically endangered status of the species and underscores the importance of zoo-based propagation programs to advance population sustainability.

Cystic endometrial hyperplasia is defined as an increase in the size, number, and secretory activity of endometrial glands with excessive proliferation of endometrial cells.6 It is often a precursor to metritis and pyometra in domestic canids (Canis familiaris), with risk factors including nulliparity, increasing age, and exposure to or treatment with exogenous and endogenous hormones, such as estrogen and progestin, at certain stages of the reproductive cycle.6 In domestic dogs, progesterone is believed to have a dominant role in the development of CEH6 by inducing endometrial proliferation and uterine glandular secretions in conjunction with decreased myometrial contractions and leukocyte inhibition that may support bacterial growth.7 Those effects are believed to be cumulative.7 Pyometra can develop secondary to an ascending bacterial infection in an abnormal uterus and lead to potentially life-threatening complications if left untreated.

The risk factors for CEH and pyometra in red wolves are likely similar to those for domestic dogs and may be exacerbated by management methods, which often select for older, more genetically valuable animals to reproduce, which in turn results in younger and more fertile females undergoing several barren reproductive cycles. Because current treatment outcomes and survival rates are poor for canids with pyometra and CEH impairs the reproductive capability of affected animals, identification of this syndrome during its early stages may allow for aggressive management and treatment and potentially improve subsequent reproductive outcomes for affected animals. Addressing CEH and pyometra within the red wolf population has become a top priority for the red wolf species survival plan.

The purpose of the study reported here was to evaluate the reproductive tract health of a subset of female red wolves managed at the PDZA, estimate the prevalence of reproductive tract disease, compare the prevalence of reproductive tract disease between nulliparous wolves and wolves that have received contraceptives, compare serum progesterone and estradiol concentrations between wolves with and without reproductive tract disease, and determine the efficacy of ultrasonography and cytologic evaluation of fine-needle aspirate samples of the uterus collected with ultrasound guidance for detection of the early stages of reproductive tract disease in wolves.

Materials and Methods

Animals

The study was reviewed and approved by PDZA Animal Welfare Committee and the US Fish and Wildlife Service prior to its initiation. Thirteen adult female red wolves with ages ranging from 5 to 10 years (mean, 7 years) were selected for the study (Supplementary Table S1, available at avmajournals.avma.org/doi/suppl/10.2460/javma.252.3.343). Five of the 13 wolves had previously received a 4.7-mg, 6-month deslorelin implantb; 4 were treated once in either 2007 or 2008; and 1 was treated once during both 2007 and 2008. The remaining 8 wolves had never been treated with a contraceptive. Six wolves had been paired for breeding during the previous 5 years, of which 3 wolves successfully produced a litter of pups, whereas the other 3 wolves failed to produce a litter or had reproductive failure (ie, stillborn pups). The remaining 7 wolves were nulliparous. Two of the wolves had been treated for pyometra 6 months and 2 years prior to study initiation. For the wolf treated 6 months before study initiation, the pyometra was confirmed to be resolved by ultrasonographic examination, whereas pyometra was only believed to be resolved for the wolf that was treated 2 years before study initiation.

Study protocol

The breeding season for red wolves is late January through mid-March followed by a 64- to 65-day gestation period, with whelping generally occurring from late March to late May.8–10 Most of the study wolves were evaluated twice, once during late anestrus or early proestrus (the first week of December 2011) and again during early anestrus (the first week of July 2012). Wolves in which pyometra was diagnosed and treated in December 2011 were reevaluated in February 2012 to assess treatment efficacy. Wolves that whelped or were spayed or euthanized following the December evaluation were not assessed during the July evaluation. Additionally, the reproductive tracts of wolves that were spayed or euthanized underwent histologic examination.

Wolves were anesthetized for each evaluation. Anesthesia was induced with a combination of dexmedetomidine (0.02 mg/kg [0.01 mg/lb], IM) and butorphanol (0.4 mg/kg [0.2 mg/lb], IM). Following intubation, anesthesia was maintained with isoflurane delivered in 100% oxygen for the duration of data and sample collection. Each evaluation consisted of ultrasonographic examination and measurement of the reproductive tract and collection of blood samples for determination of serum progesterone and estradiol concentrations. Additionally, during the December 2011 evaluation, ultrasound-guided fine-needle aspirate samples were obtained from either the right or left uterine horn of each wolf for cytologic evaluation. When additional procedures (eg, dental prophylaxis or vaccination) were necessary, they were performed after the ultrasonographic examination. Isoflurane administration was discontinued following completion of the evaluation and any ancillary procedures, and anesthesia was reversed with atipamezole (0.2 mg/kg [0.1 mg/lb], IM) either alone or in combination with naltrexone (0.02 mg/kg, IM). Anesthesia duration ranged from 45 to 60 minutes for the December evaluation and from 20 to 30 minutes for the July evaluation.

Ultrasonographic examination

All ultrasonographic examinations and measurements were performed by 1 experienced ultrasonographer. A 7.5-MHz probe was used to visualize and measure the following structures: uterine body, each uterine horn, and both ovaries. Because ultrasonographic criteria had not been established for ultrasonographically normal reproductive tracts of wolves, all interpretations were based on the ultrasonographer's experience with ultrasonographic examination of the reproductive tracts of domestic dogs. Criteria used to diagnose pyometra included hyperechoic to hypoechoic fluid distention of the uterine lumen with or without a thickened or tortuous endometrial lining and CEH (thickened endometrial lining with hypoechoic cysts within the endometrial lining). Ultrasonographically, mucometra appears similar to pyometra; therefore, the 2 diseases were differentiated on the basis of cytologic and bacterial culture results of a fine-needle aspirate sample collected from the uterine lumen. Mucometra was diagnosed for wolves with aspirate samples that yielded noninflammatory cytologic results and negative bacterial culture results. Uterine aplasia was defined as the presence of ovaries without apparent uterine horns or a uterine body. Metritis was defined as thickening of the endometrial layer without concurrent cystic changes or the presence of intraluminal fluid and cytologic results consistent with inflammation. Ovarian cysts were defined as enlarged (> 0.5 cm) hypoechoic structures on an ovary and were differentiated from follicles on the basis of size.

The following measurements of the reproductive tract were obtained for all wolves evaluated in December and July: the length and width of each ovary, uterine body diameter, and diameter of both the cranial and caudal portions of each uterine horn. The overall measurement for each ovary was calculated by multiplying its length and width. Ovarian cysts, when present, were excluded from measurements to determine ovarian mass because they were not considered to contribute to the physiologic mass of the organ.

Ultrasound-guided fine-needle aspirate of the uterus

During the December evaluation, a fine-needle aspirate sample was collected from either the left or right uterine horn of each wolf by use of a 22-gauge needle attached to a 3-mL syringe. The aspirate sample was ejected onto a microscope slide and submitted to a laboratoryc for cytologic analysis. This procedure was not repeated during the July evaluation because of the high frequency of nonspecific or non-diagnostic results reported for the aspirate samples collected in December.

Determination of serum progesterone and estradiol concentrations

From each wolf, a blood sample (6 to 12 mL) was collected from a jugular or saphenous vein into a sterile serum-separator tube. Blood samples were allowed to clot at room temperature (approx 21° to 24°C) for 10 to 15 minutes and then were centrifuged at 685 × g for 5 minutes. Serum was harvested from each sample and stored at 2.8° to 5°C for 24 to 48 hours until shipped to laboratories for measurement of progesterone and estradiol concentrations.

Serum progesterone concentration was measured by use of a solid-phase,125I competitive chemiluminescent enzyme immunoassayd in accordance with the manufacturer's instructions. This assay was previously validated for use in domestic dogs.11 Assay sensitivity was 0.02 ng of progesterone/mL. Serum progesterone concentrations were interpreted on the basis of the recommended interpretations for domestic dogs. Briefly, a serum progesterone concentration between 0.0 and 0.9 ng/mL was consistent with anestrus or early proestrus, between 1.0 and 1.9 ng/mL was consistent with ovulation in 2 days if vaginal cells were fully cornified on cytologic analysis, between 2.0 and 3.9 ng/mL was consistent with ovulation within 1 day, between 4.0 and 10.0 ng/mL was consistent with ovulation, between 10.1 and 20.0 ng/mL was indicative that ovulation had occurred, and > 20 ng/mL was consistent with diestrus or pregnancy.

Serum estradiol concentration was measured with a commercially available radioimmunoassaye that had been validated for use in red wolves12 and domestic dogs.13 Assay sensitivity was 4 pg of estradiol/mL. Serum estradiol concentrations were again interpreted on the basis of recommendations for domestic dogs. Briefly, a serum estradiol concentration < 20 pg/mL was indicative of male or anestrus, diestrus, or pregnancy, whereas a concentration between 25 and 50 pg/mL was consistent with proestrus or estrus.

Statistical analysis

Reproductive tract disease was not identified during both the December and July evaluations in any wolf. For wolves that were determined to be free of reproductive tract disease during both the December and July evaluations, a Wilcoxon signed-rank test was used to compare measurements between the 2 evaluations. Results confirmed that the measurements did not differ significantly (ie, all values of P ≤ 0.05); thus, for each wolf, the mean was determined for each measurement and used for analysis to avoid pseudoreplication. A Mann-Whitney rank sum test was used to compare ultrasonographic measurements between wolves with and without reproductive tract disease. Although the serum progesterone and estradiol concentration data were normally distributed and homoscedastic, comparisons between wolves with and without reproductive tract disease and wolves that did and did not receive a contraceptive were performed with a Mann-Whitney rank sum test because of the small sample size. All analyses were performed with statistical software,f and values of P ≤ 0.05 were considered significant.

Results

Wolves

Seven of the 13 wolves had or developed some form of reproductive tract disease between December 2011 and July 2012. Of the 5 wolves that had been treated with a deslorelin contraceptive in 2007 or 2008 (n = 4) or both 2007 and 2008 (1), 1 had reproductive tract abnormalities in December and July, 1 had reproductive tract abnormalities in December and April, and 2 had reproductive tract abnormalities in July. Of the 8 wolves that had never received a contraceptive, 1 developed reproductive tract disease in December and February and 2 developed reproductive tract disease in July 2011. Two of the 3 and 5 of the 10 wolves that had and had not produced a litter of pups within the 5 years prior to study initiation, respectively, developed reproductive tract disease between December 2011 and July 2012.

Eight of the 13 study wolves were evaluated during both December 2011 and July 2012. Of the 5 wolves that were not evaluated in July, 2 had successfully whelped and were raising litters and thus excluded from evaluation to avoid interference with pup rearing. One of those wolves was a 7-year-old multiparous female that had been treated with deslorelin in 2007 and 2008; she successfully delivered a litter of 9 pups, all of which survived. The other wolf was an 8-year-old nulliparous female that had never received a contraceptive; she delivered a litter of 8 pups, 2 of which died within 48 hours after birth.

Two of the 3 remaining wolves that were not evaluated in July developed pyometra and were spayed. One of those wolves had pyometra during the December evaluation and was treated orally with prostaglandin F and amoxicillin-clavulanic acid. Results of an ultrasonographic examination in January 2012 suggested that the pyometra had resolved; however, the wolf developed refractory pyometra in April 2012, which necessitated an ovariohysterectomy. Further details regarding the treatment of the 2 wolves that developed pyometra and were spayed is provided elsewhere.14

The final wolf that was not evaluated in July developed severe lymphoplasmacytic enteritis in early 2012. The wolf did not respond to treatment and was euthanized.

A 6-year-old wolf that had received a contraceptive produced a single pup, which was weak with audible respiratory abnormalities and was consumed by the dam within hours after birth. During the July evaluation, that wolf was determined to have CEH as well as a cecal intussusception with severe inflammatory bowel disease. Surgery was performed to repair the intussusception, but the wolf died during the recovery period. Another 6-year-old wolf that had received a contraceptive was suspected to have a closed pyometra in addition to CEH during the July evaluation and subsequently underwent an ovariohysterectomy.

In 2013, 3 of the wolves were paired for breeding. One of those wolves produced a litter of 4 pups despite having ultrasonographic evidence of mild CEH.

Ultrasonographic findings

Representative ultrasonographic images of a uterus without any abnormalities and an inactive and active ovary are provided for reference (Figure 1). All 13 wolves underwent ultrasonographic evaluation during December 2011. Reproductive tract abnormalities identified during the December evaluations included pyometra and CEH (n = 1 wolf), uterine aplasia (1), chronic metritis (1), and ovarian cysts (2). Only 8 of the 13 wolves underwent ultrasonographic evaluation during July 2012. Reproductive tract abnormalities identified during the July evaluations included CEH (n = 3 wolves), suspected closed pyometra (1), mucometra and CEH (1), uterine aplasia (1), and ovarian cysts (1). Cystic endometrial hyperplasia was ultrasonographically detected more frequently in July (n = 3 wolves) than in December (1).

Figure 1—
Figure 1—

Representative ultrasonographic images of the reproductive tracts of red wolves (Canis rufus). A—Ultrasonographic image of an ultrasonographically normal uterine body (arrowheads) of a 7-year-old wolf. B—Ultrasonographic image of an ultrasonographically normal inactive right ovary of an 8-year-old wolf. The boundaries of the ovary are delimited with plus signs. C—Ultrasonographic image of an active right ovary of a 10-year-old wolf. Notice the presence of several small follicles and a potential ovarian cyst (large anechoic structure). The boundaries of both the follicles and probable cyst are delimited with plus signs. D—Ultrasonographic image of the right uterine horn of the wolf in panel C that contains echogenic fluid characteristic of pyometra in addition to CEH and an endometrial cyst. The respective diameters of the uterine horn and endometrial cyst are delimited by plus signs. E—Ultrasonographic image of the right ovary of the wolf in panel C. Notice the 2 large fluid-filled ovarian cysts delimited by plus signs. F—Ultrasonographic image of the right uterine horn of a 10-year-old wolf in which the endometrium is thickened (plus signs) and the lumen has abnormally increased echogenicity, characteristic of metritis. G—Ultrasonographic image of the left uterine horn of an 8-year-old wolf with CEH, endometrial cysts (arrows), and a lumen filled with hypoechoic fluid indistinguishable from that of pyometra. Cytologic analysis of a fine-needle aspirate sample of the uterine fluid indicated that this wolf had mucometra. H—Ultrasonographic image of the left uterine horn of a 6-year-old wolf that was tortuous and filled with both hypoechoic and echogenic fluid consistent with CEH and either pyometra or mucometra. The diameter of the uterus is delimited with plus signs. White lines on the periphery of each panel represent 1-cm increments.

Citation: Journal of the American Veterinary Medical Association 252, 3; 10.2460/javma.252.3.343

A 6-year-old wolf with uterine aplasia had a cyst identified on the left ovary during both the December and July evaluations. The cyst measured 0.48 × 0.63 cm in December and 0.45 × 0.63 cm in July.

The wolf with metritis in December subsequently developed pyometra and CEH and underwent an ovariohysterectomy in April 2012. It also had ovarian cysts. During the December evaluation, that wolf had a primary cyst (2.5 × 1.6 cm) on the left ovary and multiple smaller cysts that ranged in size from 0.6 to 0.9 cm on the right ovary. Ovarian cysts were identified during multiple serial ultrasonographic examinations performed in January and April. At the time of the ovariohysterectomy in April 2012, the wolf had cysts measuring 1.5 × 1.2 cm and 1.1 × 1.0 cm on the right ovary and multiple cysts measuring up to 1.1 cm on the left ovary.

The wolf with mucometra in July 2012 was treated with prostaglandin F and antimicrobials, and the condition was determined to be resolved on the basis of an ultrasonographic examination following completion of the treatment regimen. However, the condition recurred in 2013, and the wolf was euthanized for management purposes. The wolf with uterine aplasia developed lymphoma and was also euthanized in 2013. Gross necropsy confirmed lack of uterine development and small ovaries.

Reproductive tract morphometrics

The median (range) morphometric measurements for ultrasonographically normal and abnormal reproductive tracts were summarized (Table 1). For all reproductive structures evaluated, although there was some overlap in the measurements between ultrasonographically normal and abnormal reproductive tracts, the median for the abnormal tracts was significantly (P ≤ 0.05) greater than that for the normal tracts.

Table 1—

Median (range) morphometric measurements obtained by ultrasonographic examination for ultrasonographically normal (n = 15) and abnormal (6) reproductive tracts of captive red wolves (Canis rufus).

StructureNormal reproductive tracts 
Right ovary (cm)1.05 (0.51–1.73) 
Left ovary (cm)0.84 (0.62–1.96) 
Uterine body (cm)0.58 (0.46–0.81) 
Cranial portion of the right uterine horn (cm)0.37 (0.31–0.54) 
Cranial portion of the left uterine horn (cm)0.45 (0.29–0.68) 
Caudal portion of the right uterine horn (cm)0.42 (0.30–0.69) 
Caudal portion of the left uterine horn (cm)0.45 (0.31–0.71) 

The study population consisted of 13 wolves. All 13 wolves were evaluated in December 2011; only 8 of the 13 wolves were evaluated in July 2012. The 5 wolves that were not evaluated in July included 2 that had whelped and were rearing pups, 2 that developed pyometra and were spayed, and 1 that developed lymphoplasmacytic enteritis and was euthanized. Thus, there were a total of 21 ultrasonographic examinations performed, and each examination is represented in the table above. Measurements for all structures differed significantly (P ≤ 0.05) between ultrasonographically normal and abnormal reproductive tracts.

Value represents the median (range) for only 5 of the 6 abnormal reproductive tracts. The right ovary of 1 abnormal tract was not measured because it contained multiple cysts that precluded accurate measurement; however, measurements for that tract were included in all other medians (ranges) reported.

Cytologic findings

The fine-needle aspirate samples of the uterus obtained from 8 of the 13 wolves during the December ultrasonographic examination were sufficient for cytologic interpretation. Cytologic diagnoses included no cytologic abnormalities (n = 2), mixed inflammation (2), chronic active inflammation (1), septic inflammation (1), neutrophilic inflammation (1), and prior hemorrhage and mild mixed-cell inflammation (1). Cytologic results for wolves with active reproductive tract disease (eg, metritis and pyometra) were consistent with underlying pathology. Of the 2 wolves that successfully produced litters of pups during the observation period, 1 had cytologic evidence of prior uterine hemorrhage and mixed-cell inflammation, and the other had inconclusive cytologic findings. The wolf that produced the single pup had cytologic evidence of minimal mixed-cell inflammation. Cytologic evidence of uterine inflammation in December did not appear to be a precursor for ultrasonographically detectable reproductive tract disease in July.

Histologic findings

The reproductive tracts of all wolves that underwent an ovariohysterectomy (n = 3), died (1), or were euthanized (3) underwent histologic examination. One of the 2 wolves that were spayed in April 2012 had moderate CEH, moderate pyometra and lymphoplasmacytic metritis, and moderate multifocal adenomyosis; the ovaries had multiple large mature corpora lutea with smaller primary and secondary follicles. The other wolf that was spayed in April 2012 had severe CEH with squamous metaplasia, severe pyometra and transmural necrotizing suppurative metritis with serosal mesothelial hyperplasia, a Wolffian duct cyst, and ovaries that contained cystic corpora lutea and subsurface epithelial structures. The wolf with uterine aplasia that was euthanized because of lymphoma had histologic evidence of a cyst on the left ovary. The wolf that developed lymphoplasmacytic enteritis and was euthanized had histologic evidence of active folliculogenesis in both ovaries and no reproductive tract abnormalities. The wolf with recurrent mucometra that was euthanized for management purposes in 2013 had CEH with luminal dilatation, and a granulosa cell tumor in 1 ovary.

Serum progesterone and estradiol concentrations

Serum progesterone and estradiol concentrations were determined for all 13 wolves in December 2011 and for the 8 wolves that underwent ultrasonographic examination in July 2012. Serum hormone concentrations were reported and compared by dividing the study population in 2 ways: wolves that did and did not have ultrasonographically detectable reproductive tract disease and wolves that had and had not received a deslorelin contraceptive in 2007 or 2008 or both 2007 and 2008 (Table 2). During both the December 2011 evaluation and June 2012 evaluation, neither the median serum progesterone concentration nor the median serum estradiol concentration differed significantly between the wolves that did and did not have reproductive tract disease or between wolves that had and had not received a contraceptive.

Table 2—

Median (range) serum progesterone and estradiol concentrations for the 13 captive red wolves in Table 1.

HormoneEvaluation month    
Progesterone (ng/mL)December 2011 July 2012    
Estradiol (pg/mL)December 2011 July 2012    

Serum hormone concentrations were reported and compared by dividing the study population in 2 ways: wolves that did and did not have ultrasonographically detectable reproductive tract disease and wolves that had and had not received a deslorelin contraceptive in 2007 or 2008 or both 2007 and 2008. Numbers in brackets represent the number of wolves that contributed to the median.

Discussion

To the authors' knowledge, the present study was the first to describe the ultrasonographic appearance of clinically normal and abnormal reproductive tracts of female red wolves and evaluate the diagnostic efficacy of cytologic examination of fine-needle aspirate samples of the uterus and serum progesterone and estradiol concentrations for the detection of wolves with reproductive tract abnormalities. Seven of the 13 wolves of the present study developed some form of reproductive tract disease during the observation period (December 2011 to July 2012). Ovarian and uterine measurements obtained during ultrasonographic examinations were generally greater for reproductive tracts with abnormalities, compared with ultrasonographically normal reproductive tracts, but there was some overlap of measurements between ultrasonographically normal and abnormal reproductive tracts. Results of cytologic examination of fine-needle aspirate samples obtained from the uterus and serum progesterone and estradiol concentrations were unable to distinguish wolves with and without reproductive tract disease. The incidence of reproductive tract disease in the wolves of this study did not appear to be associated with parity or exposure to contraceptives, but it may have been confounded by the small sample size. Collectively, the results of the present study provided preliminary information that will be helpful in understanding and diagnosing CEH in red wolves.

Results of retrospective studies2,3,5,14 indicate that red wolves are more likely than other canids to develop CEH and pyometra. Factors that contribute to that increased risk include red wolf management, limited space available for breeding, and possibly a species predilection.2,3,5,14 As the current population of red wolves ages, an increase in the frequency of age-associated diseases such as CEH and pyometra is expected. Results of a study15 involving domestic dogs in Sweden indicate that approximately 23% to 24% of sexually intact female dogs develop pyometra by 10 years of age. Although we anticipated that some of the red wolves of the present study would have or develop CEH during the study, almost half (6/13 [46%]) developed CEH with or without pyometra during the observation period. Two of the 13 (15.4%) red wolves developed pyometra during the present study, which was similar to the incidence rate of pyometra (11.1%) reported in red wolves of another study.2 The frequency of CEH was lower for the red wolves of the present study than that (up to 75%) previously reported,2 which may have been a reflection of the limited ability to detect early CEH lesions by ultrasonographic examination. However, ultrasonography is effective for identification of most forms of reproductive tract disease,16 and the ultrasound equipment used in this study can detect endometrial cysts that are ≥ 1 mm in diameter. Although transcervical endometrial biopsy has been described for the detection of reproductive tract disease in domestic dogs, the high frequency of postbiopsy endometritis and pyometra as well as the difficulty associated with obtaining diagnostic specimens has brought this technique into question.17,18 Moreover, many clinicians have not been trained or do not have experience performing transcervical biopsy, which makes that technique of limited value to most practitioners. Currently, surgical biopsy remains the gold standard for diagnosing CEH during its earliest stages, but ultrasonography might be a viable alternative for detection of patients in the more advanced stages of the disease.

Three of the 4 red wolves of the present study had a recurrence of pyometra or mucometra despite initial medical treatment that appeared to be successful in resolving the condition. The wolf with mucometra also had a granulosa cell tumor that likely contributed to recurrence of the condition. In domestic dogs, ovariohysterectomy is the preferred treatment for pyometra; therefore, reports regarding outcomes following medical management of pyometra are limited. In 1 study,19 the probability of developing pyometra did not differ between bitches that had received previous medical treatment for pyometra and age-matched control bitches that had never had the disease. In another study,20 fertility following medical treatment of pyometra appeared to be promising, with 8 of 15 bitches producing at least 1 litter after treatment and clinical resolution of uterine infections in 42 of 44 bitches, with 38 going on to whelp healthy pups. In yet another study,21 pyometra recurred in 7 of 37 (18.9%) bitches that had been previously treated for pyometra with algepristone (an antigestagen) alone and had concurrent CEH or ovarian cysts (risk factors for pyometra). However, results of other studies22,23 indicate that the recurrence rate of pyometra is up to 77% for bitches that initially received medical treatment for the condition.

The uterine measurements for the red wolves of the present study were comparable to those reported for domestic dogs.24 Although histologic examination of the reproductive tract was performed for only 6 of the 13 wolves of this study, the histologic diagnosis was consistent with the ultrasonographic diagnosis in all but 1 wolf. As anticipated, the uterine measurements for red wolves with CEH or pyometra were greater than those for ultrasonographically normal wolves; however, the ranges of the uterine measurements for wolves with reproductive tract disease overlapped with those for ultrasonographically normal wolves. As with any disease detected by ultrasonography, morphological changes should be considered in conjunction with reproductive tract measurements before a diagnosis (eg, presence of intraluminal fluid or endometrial or ovarian cysts) is made. As pyometra progresses, the uterus becomes distended with purulent fluid that is easily identified during ultrasonographic examination. For the wolf of the present study with mucometra, aspiration of the uterus yielded yellow serous fluid that was negative for bacterial growth and was cytologically consistent with mucometra. Aspiration of the uterus is controversial for animals with suspected pyometra because the procedure can potentially seed the abdomen with bacteria and cause peritonitis. Peritonitis was not identified in any of the wolves of the present study following collection of fine-needle aspirate samples from the reproductive tract. Regardless, although unskilled ultrasonographers may find it difficult to detect minor cystic changes in the uterus of canids, the reproductive tract measurements and ultrasonographic images provided in this study should be useful to clinicians who evaluate red wolves with and without reproductive tract disease.

Interestingly, some wolves of the present study that developed pyometra had large ovarian cysts. Although follicular development could not be ruled out as a differential diagnosis for the ovarian cysts identified during the December evaluation, histologic examination revealed the presence of luteal cysts in 1 of the 2 wolves that underwent an ovariohysterectomy for treatment of pyometra in April 2012 and a cyst in the left ovary of the wolf with uterine aplasia that was euthanized. In domestic dogs, ovarian cysts can be paraovarian, epithelial, or stromal, and most do not secrete hormones. Cysts > 8 mm are often classified as stromal cysts and can secret either progesterone or estrogen and cause uterine pathology.25 Functional luteal cysts frequently secrete progesterone and lead to endometrial proliferation.26 All ovarian cysts identified in the wolves of this study were > 8 mm in diameter, which suggested that they might have been secreting endogenous progesterone or estrogen and could have contributed to the development of CEH.

In the present study, the serum progesterone and estradiol concentrations did not differ significantly between wolves with and without reproductive tract disease regardless of the stage of the estrus cycle. In December 2011, the serum estradiol concentration for all wolves (mean, 31.92 pg/mL) was greater than that anticipated for anestrus bitches, which may have indicated that the wolves were undergoing active folliculogenesis. Results of another study12 involving red wolves indicate that the serum estradiol concentration ranges from 10 to 20 pg/mL during anestrus and peaks at > 30 pg/mL immediately before ovulation. In gray wolves (Canis lupus), serum estradiol concentration also peaks at 30 to 50 pg/mL immediately before ovulation.27 Both estrogen and progesterone are highly conserved across species, so although serum progesterone and estradiol concentrations were not measured by use of the same assays in all studies, the results are believed to be comparable. The duration of proestrus in red wolves is unknown; however, the mean ± SD duration of proestrus is generally 15.7 ± 1.6 days but can last up to 60 days in other wolf species27 and lasts for 2 to 3 months in coyotes.28 Red wolves are seasonally monoestrus, and the onset of estrus generally occurs during the period from late January to March, but on the basis of 1 author's (WTW) experience, the onset of estrus for wolves located in northern climates is often delayed by 2 to 3 weeks. Most wolves at the PDZA exhibited signs of estrus during the period from February to March. The high mean serum estradiol concentration for the wolves of this study at 1 point in December suggested that red wolves may have a prolonged proestrus phase similar to that of coyotes or that the wolves at the PDZA entered estrus earlier than anticipated that year.

The prevalence and incidence rates of reproductive tract disease did not differ significantly between wolves that had and had not received a contraceptive. The only contraceptive received by some of the wolves of the present study was deslorelin, a gonadotropin-releasing hormone agonist commonly used for contraception in carnivores and other zoo species. Gonadotropin-releasing hormone agonists suppress the estrous cycle by downregulation of the pituitary-gonadal axis.29 During the downregulated state, estrogen and progestagen concentrations should be low; thus, deslorelin is a safer alternative than melengestrol acetate, a synthetic progestagen that was commonly used as a contraceptive prior to deslorelin29 and that has been linked to the development of mammary carcinoma in felids30 and endometrial lesions (endometrial hyperplasia, CEH, hydrometra, and adenomyosis) in both felids and canids.3,31 An adverse effect of gonadotropin-releasing hormone agonists is a stimulatory phase,32,33 which was recently associated with the development of reproductive tract disease owing to prolonged progesterone exposure in nondomestic canids.2 In that study,2 wild canids that received deslorelin were just as likely to develop endometrial lesions as those that received melengestrol acetate and wolves that were allowed to undergo multiple nulliparous seasons. Administration of contraceptives to zoo species is complicated at many levels. The Wildlife Contraceptive Advisory Group currently recommends the administration of megestrol acetate 1 week before and after administration of deslorelin for contraception in carnivores.2 Megestrol acetate was not administered before deslorelin implantation in any of the wolves of the present study; thus, it could not be determined whether it affected the outcome. Despite the fact that the present study was unable to find a correlation between reproductive tract disease and contraceptive use, breeding history, or management, the results should be interpreted with caution owing to the small study population.

In the present study, parity did not appear to be protective against the development of reproductive tract disease. Only 1 of the 3 nulliparous wolves successfully delivered a large litter of pups; the remaining 2 wolves developed pyometra or mucometra. Results of other studies2,6 indicate a positive association between nulliparity and the development of reproductive tract disease in canids; but again, owing to the small population, results of this study should be interpreted with caution.

Results of the present study indicated the incidence of reproductive tract disease was high within the red wolf population at the PDZA. Ultrasonography was a useful diagnostic tool for identifying and managing wolves with reproductive tract disease, and the ultrasonographic images, reproductive tract measurements, and descriptions of reproductive tract lesions provided in this study can be used as diagnostic guidelines for other clinicians who treat and manage red wolves. An effective treatment for CEH in red wolves has not been identified, and detection of affected wolves earlier may facilitate management and treatment of this disease. Longitudinal monitoring of serum hormone concentrations, serial ultrasonographic examinations, and histologic evaluation of uterine biopsy specimens may help elucidate the pathogenesis of CEH in red wolves.

Acknowledgments

Supported by the Dr. Holly Reed Wildlife Conservation Fund.

The authors thank Dr. Cindy Nordberg for performing the ultrasonographic examinations; Julie Lemon, Sue Behrns, Jenn Donovan, Natalie Davis, Alicia Pike, Aimee Dahl, and Richard Johnson for technical assistance; and Sheanna Steingass and Jesse Blake for assistance with statistical analysis.

ABBREVIATIONS

CEH

Cystic endometrial hyperplasia

PDZA

Point Defiance Zoo and Aquarium

Footnotes

a.

Reed H, Veterinary Advisor Report, Tacoma, Wash: Unpublished data, 2009–2013.

b.

Suprelorelin, Peptech Animal Health Pty Ltd, Macquarie Park, Australia.

c.

Phoenix Central Laboratories, Mukilteo, Wash.

d.

Immulite 2000, Phoenix Central Laboratories, Mukilteo, Wash.

e.

Coat-a-Count, Siemens Diagnostics, Cornell Animal Health Laboratory, Cornell, NY.

f.

SigmaPlot, version II, Systat Software Inc, San Jose, Calif.

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