Journal of Equine Veterinary Science 119 (2022) 104147

Contents lists available at ScienceDirect

Journal of Equine Veterinary Science

journal homepage: www.j-evs.com

Original Research

Comparison Between Gynecological Examination Methods and Sample

Collection Techniques for the Diagnosis of Endometritis in Subfertile
Mares
Carlos Mattos Teixeira-Soares a,∗, Arabela Guedes de Azevedo Viana a,
Iara Magalhães Ribeiro a, Kamilla Dias Paes Silva a, Yamê Fabres Robaina Sancler-Silva b,
Mariana Machado-Neves a,c
a
Programa de Pós-graduação em Medicina Veterinária, Departamento de Medicina Veterinária, Universidade Federal de Viçosa, Viçosa, Brazil
b
Departamento de Zootecnia Universidade Federal de Viçosa, Viçosa, Brazil
c
Departamento de Biologia Geral, Universidade Federal de Viçosa, Viçosa, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Endometritis is a relevant cause of subfertility in mares. However, the accurate diagnosis, essential for
Received 29 June 2022 effective treatment, can be difficult due to the variability of results and interpretations resulting from dif-
Received in revised form 12 September
ferent examination methods and sample collection techniques. The present work compared gynecological
2022
evaluation methods and sample collection techniques to diagnose endometritis in subfertile mares. Forty
Accepted 20 October 2022
Available online 23 October 2022 animals with a history of subfertility were selected for gynecological evaluation using clinical methodolo-
gies, such as perineal conformation, transrectal palpation and ultrasonography, vaginoscopy, and digital
Keywords: examination of the cervix. In addition, we performed laboratory analyses, including uterine microbio-
Gynecological exam logical culture and endometrial cytology and histology, of which the latter is the gold standard for the
Endometrial cytology diagnosis of endometritis. Samples were collected for microbiological culture and endometrial cytological
Endometrial histology evaluations using three different techniques: a commercial cytobrush/swab collector, low-volume uterine
Uterine microbiological culture
flush, and a new tested technique, by flush the fragment resulting from the endometrial biopsy. Transrec-
tal palpation and ultrasound showed the best results among clinical examinations. However, they were
less efficient in laboratory tests of endometrial cytology and uterine microbiological culture, in which the
latter showed the highest sensitivity and specificity for endometritis compared with endometrial histol-
ogy. The use of multiple results from different methods has also proved to be an effective alternative for
diagnosis. Among the techniques used to collect endometrial material for cytology and microbiological
culture, the most effective and practical in this study was the commercial cytobrush/swab collector.
© 2022 Elsevier Inc. All rights reserved.

1. Introduction of the endometrium with a multifactorial etiology, have a hostile

Endometritis is the main cause of reduced fertility in mares
[1,2], contributing to substantial economic losses. Mares with en-
dometritis, characterized by the persistent inflammatory process
Conflict of interest statement: The authors declare that they have no conflicts of
interest to disclose.
Animal welfare/ethical statement: Animal Use Ethics Committee of the Universi-
dade Federal de Viçosa, Brazil (CEUA, protocol number 16/2016)
∗
Corresponding author at: Carlos Mattos Teixeira Soares, Student in Programa
de Pós-graduação em Medicina Veterinária, Departamento de Medicina Veterinária,
Universidade Federal de Viçosa, Av. Peter Henry Rolfs s/n, Campus Universitário,
CEP: 36570-900. Viçosa, Minas Gerais, Brazil.
E-mail address: carlosmattos1991@gmail.com (C.M. Teixeira-Soares).
uterine environment for the survival and implantation of the em-
bryo [3,4]. Accurate diagnosis of endometritis must be based on a
complete gynecological assessment, using multiple clinical findings
associated with laboratory tests [5,6]. Although there are several
methods of gynecological evaluation, many are used inappropri-
ately and generate incorrect results interpretations of the results
[7,8]. Consequently, endometritis diagnosis becomes problematic,
where speed and safety are required [9,10].
Transcervical uterine sampling for laboratory evaluations can be
performed using a cotton swab or cytological brush collectors, low-
volume uterine flush, or endometrial biopsy [2]. These methods al-
low the safe collection of uterine material [11], but present high
discrepancy sensitivity and specificity between results [8]. Regard-
less, each technique has advantages and disadvantages in practical-

https://doi.org/10.1016/j.jevs.2022.104147
0737-0806/© 2022 Elsevier Inc. All rights reserved.
C.M. Teixeira-Soares, A.G.d.A. Viana, I.M. Ribeiro et al.
ity, costs, sampling locations of the uterus, quantity, and quality of
the collected sample.
In this context, there is still no single method that comprehends
ease in execution, low cost, and safe results for an accurate diag-
nosis [12]. Several studies have tested exam methods and sam-
ple collection techniques [1,8,9,13–18], however, there is still no
standardization of methodologies used for diagnosis. This makes
it difficult to compare the results and leads to the search for new
techniques and the use of multiple methods [5,17]. Therefore, this
study aimed to evaluate clinical and laboratory methods used to
identify endometritis in mares compared to the histopathological
analysis of the endometrium, a current gold standard for diagnosis.
Furthermore, three approaches for uterine sample collection used
for uterine microbiological culture and endometrial cytology exams
were compared for sensitivity and specificity: a commercial cyto-
brush/swab collector, low-volume flush, and biopsy fragment flush
which is a new sampling method tested to unify sample collection
for laboratory tests.
2. Material and Methods
All procedures performed on animals in this study were ap-
proved by the Animal Use Ethics Committee of the Universidade
Federal de Viçosa, Brazil (CEUA, protocol number 16/2016). The
study was conducted in the 2018/2019 breeding season on seven
different farms in the Zona da Mata region of Minas Gerais, Brazil.
2.1. Animals
Forty Mangalarga Marchador mares, 5–20 years old (mean 13.2
± 3.80 years old) were selected for the study. Seven mares were
younger than 10 years old, 15 mares were between 10 and 14 years
old, and 18 were considered elderly between 15 and 20 years old.
All selected mares were classified as subfertile due to repro-
ductive history of early embryonic losses (eight mares) or sugges-
tive findings of endometritis (32 mares), such as, intrauterine fluid
or excessive endometrial edema during a routine transrectal ultra-
sound examination.
The gynecological examination was performed during the
diestrus stage of reproductive cycle, when the corpus luteum was
detected in screening examination of the mares by palpation and
transrectal ultrasound. Posteriorly, the clinical methods of gyneco-
logical examination and the collection of samples for the labora-
tory methods were performed by an experienced veterinarian.
2.2. Clinical Methods of Gynecological Evaluation
2.2.1. Transrectal Palpation and Ultrasonography
Individual stocks were used for proper restraint and the tail was
bandaged. With a lubricated palpation glove and ultrasound with
a 5-MHz linear transrectal transducer (Sonoscape A5 Vet, Shen-
zhen, China), the internal reproductive organs were evaluated to
determine the stage of the estrous cycle, ovarian structures, uter-
ine edema, cysts and intrauterine fluid, if present. The uterine and
cervical tone was determined by rectal palpation and graded in
1 = ideal tone typical of diestrus; 2 = intermediate or softening;
3 = flaccid as an indication of estrus and compared with the ul-
trasound findings to determine the phase of the cycle [19]. Uterine
edema was categorized in 0 = none, 1 = little, 2 = mild, 3 = mod-
erate, 4 = significant and 5 = excessive, and mares were consid-
ered positive for endometrial inflammation when they presented
excessive edema or not compatible with the stage of cycle [20].
The presence of intrauterine fluid was also considered an indica-
tion of endometritis, either in any amount or appearance.
2
Journal of Equine Veterinary Science 119 (2022) 104147
2.2.2. Perineal Conformation
Perineal conformation was evaluated through visual analysis,
palpation, and measured with a vulvometer [21]. Mares were clas-
sified as positive in this evaluation method with compromised
physical protection barriers when the vulva did not promote ad-
equate closure, and there was an accumulation of air or urine in
the vagina, total length of the vulvar commissure greater than 15
cm, or Caslick Index greater than 150 [22].
2.2.3. Vaginoscopy
The perineum was washed with water and gentle soap. Vagina
was accessed with a sterile Polansky vaginal speculum lubricated
with a sterile solution. With the aid of a flashlight to facilitate in-
ner vision, the vagina and the caudal ostium of the cervix were
evaluated. Mares were classified as positive in this examination
method when they showed alterations indicative of inflammation
or breakdown in natural defense, presenting a tortuous opening of
the cervix, secretion during the evaluation, pale or hyperemic mu-
cosa coloration, or abnormalities such as signs of trauma and lac-
erations in the vagina or cervix [23].
2.2.4. Examination of the Cervix
Using a sterile palpation sleeve, the vagina was accessed. A fin-
ger was inserted into the cervix and a digital examination was
performed, evaluating its integrity and detecting possible changes,
such as lacerations, fibrosis, adhesions, diverticulums, and/or tor-
tuosity. Mare with one or more of these alterations were classified
as positive, presenting risk factors for contamination and efficient
clearance uterine [24].
2.3. Sample Collection Techniques
2.3.1. Commercial Cytobrush/Swab Collector
A double-guarded cytobrush collector (Provar Commercial Ltda,
São Paulo, Brazil) was introduced manually through the vulva,
vagina, and cervix and guided into the uterus. After being in con-
tact with the uterine wall for at least 1 minute, with alternating
smooth rotational movements, it was removed to avoid contami-
nation [5]. The double-guarded swab collector (Provar Commercial
Ltda, São Paulo, Brazil) was then introduced following the same
procedure performed with the cytobrush.
2.3.2. Low-volume Uterine Flush
A volume of 60 mL of 0.9% sodium chloride (Fresenius Kabi,
Bad Homburg, Germany) was transcervical infused into the uterus
using a disposable sterile catheter without a cuff (Embramed, São
Paulo, Brazil), to reduce costs and make the technique more prac-
tical. After 1 minute of transrectal uterine massage for body and
horns perfusion, fluid was recovered into sterile 15 mL plastic
tubes (Corning Incorporated Life Science, Tewksbury, England). The
recovered fluid was evaluated, and those samples that presented
non-translucent and high cellularity aspects were classified as pos-
itive, indicative of endometritis in this method of examination. Af-
ter this evaluation, the tube was centrifuged at 400 G for 10 min-
utes, and most of the supernatant was discarded. The main sedi-
ment was recovered by cytological brush for cytological evaluation
and by swab for microbiological evaluation [5,15].
2.3.3. Biopsy Fragment Flush
A new sampling technique for cytology and microbiological cul-
ture was performed from the biopsy fragment, called "biopsy frag-
ment flush," to improve cell preservation and avoid contamination.
The technique aims to make sample collection more practical, as
only uterine biopsy would be required to perform uterine culture,
cytology, and histology.
C.M. Teixeira-Soares, A.G.d.A. Viana, I.M. Ribeiro et al. Journal of Equine Veterinary Science 119 (2022) 104147

A sterilized biopsy forceps (Botupharma, Botucatu, Brazil) was
used to perform the uterine biopsy transcervical collecting the
uterine fragment. The fragment was apprehended with sterile
anatomical forceps and washed in a sterile 15 mL plastic tube con-
taining 0.9% sodium chloride solution (Fresenius Kabi, Bad Hom-
burg, Germany). Then, the tube was centrifuged at 400 G for 10
minutes. Most supernatant was discarded, leaving 2 mL at the bot-
tom of the tube. The sediment was resuspended and used for sam-
pling with a sterile cytology brush and swab (Labor Import, Osasco,
Brazil). Both were immersed in the sediment for 1 minute, alter-
nating gentle rotational movements. The collected samples were
used for cytological and microbiological evaluation, respectively, as
well as in other collection techniques.
2.4. Laboratory Methods of Gynecological Evaluation
2.4.1. Endometrial Cytology
The cytology brushes from the three sampling techniques were
slightly slid in a rotational motion by the microscopic slide, per-
forming the smear. The slides were air-dried and stained with
Diff-quick (Laborclin, Pinhais, Brazil). Then, samples were evaluated
to ascertain the proportion of polymorphonucleate to endometrial
cells, 300 cells per sample were counted, in 400x magnification by
light microscopy (P207/B Coleman, São Paulo, Brazil) [8]. The in-
flammation was classified as positive when the percentage of poly-
morphonucleate was greater than 2% [1].
2.4.2. Uterine Microbiological Culture
Swab samples from the three collection techniques were sown
on 5% sheep blood agar plates and incubated at 37°C. Microorgan-
isms growth was checked after 48 hours and, when present, iden-
tified in a specialized laboratory (Microvet, Viçosa, Brazil). Mares
with at least one microorganism growth were classified as positive
and considered to have uterine contamination and, consequently,
endometritis [9]. The susceptibility test to antimicrobials was per-
formed employing the Kirby Bauer method using the standard con-
centrations of ampicillin, penicillin, gentamicin, tetracycline, ceftio-
fur, sulfamethoxazole/trimethoprim, enrofloxacin, florfenicol, and
neomycin. The results were interpreted according to the CLSI rec-
ommendations [25].
positive diagnoses by the evaluated methodologies on the prob-
ability of positive histological diagnosis of endometritis was an-
alyzed by univariate logistic regression (Logistic Procedure). The
level of significance adopted was P = .05.
3. Results
Transrectal palpation and ultrasonography showed that 52.5%
of the mares presented changes such as intrauterine fluid or ex-
cess edema, which are consistent with uterine inflammation. These
mares were classified as positive for endometritis in this evalua-
tion method. Perineum conformation, vaginoscopy and cervical ex-
amination, were diagnosed, respectively, in 32.5%, 25%, and 37.5%
of the mares (Table 1). These findings indicate breaks in uterine
physical barriers, known endometritis risk factors.
The low-volume flush using a catheter without a cuff recovered
in average 30 mL of efflux and, in a total of 40 animals evaluated,
17.5% had abnormal appearance and cellularity, being classified as
positive in this suggestive exam of endometritis (Table 1).
In laboratory evaluations, endometrial cytology detected 52.5%
of positive mares for endometritis with more than 2% polymor-
phonucleates, while uterine microbiological evaluation detected
62.5% of positive mares for this pathology, presenting at least one
microorganism in the culture (Table 1). Among positive animals in
the microbiological exam, 56% showed only one species of microor-
ganism, while 44% showed mixed growth, meaning two or more
species. The isolated microorganisms were Staphylococcus spp., Es-
cherichia coli, Enterobacter spp., and Citrobacter spp. All microorgan-
isms found were sensitive to sulfa/trimethoprim, enrofloxacin, and
florfenicol, according to the in vitro sensitivity test to antimicro-
bials (Table 2).
According to the histopathological evaluation, the gold standard
for endometritis diagnosis, 65% of the 40 mares evaluated pre-
sented endometrium inflammation (Fig. 1). Regarding the meth-
ods of clinical reproductive evaluation compared in the study with
Table 1
Diagnostic methods used during gynecological evaluation in mares (n = 40)
checked for endometritis.
Methods Positive Diagnosed Changes (n)
Animals

2.4.3. Endometrial Histopatology Palpation and ultrassonogaphy 21 Intrauterine fluid (15)

Biopsies fragments collected were fixed in 10% formalin solu- Abnormal edema (10)
Perineal conformation 13 Inefficient vulvar closure (11)
tion and embedded in paraffin. Histological slides were processed
Pneumovagina (10)
using hematoxylin and eosin staining. The slides were examined Caslick Index > 150 (9)
under optical microscope (P207/B Coleman, São Paulo, Brazil) in Urovagina (2)
400x magnification, and the classification system of Kenney & Doig Vaginoscopy 10 Intravaginal secretion (6)
[26] was used, modified by Schoon et al. [27] for inflammatory Tortuous cervical ostium (5)
Traumas (4)
severity according to the number of inflammatory cells; periglan- Pale mucous(3)
dular fibrosis; glandular distribution; and lymphatic lacunae. The Cervical examination 15 Tortuosity (12)
mares were classified as grade 1 if there were no polymorphonu- Diverticuluns (6)
clear cells in the compact stratum or signs of inflammation or de- Fibrosis (5)
Adherence (4)
generation, being considered negative for endometritis. Those clas-
Laceration (1)
sified as grades 2A (mild inflammation), 2B (moderate inflamma- Effluent evaluation of 7 Abnormal appearance (7)
tion), and 3 (severe inflammation) were considered positive for en- low-volume uterine flush
dometrial histological evaluation and diagnosed with endometritis Endometrial citology 21 Polymorphonucleates > 2%
in this study. (21)
Uterine microbiological culture 25 Staphylococcus spp. (15)
Escherichia coli (10)
2.5. Statistical Analysis Enterobacter spp. (7)
Citrobacter spp. (5)
Endometrial histopalogy 26 Grade 2A – Mild endometritis
For data analysis, the Statistical Analysis System [28] was ap-
(15)
plied. Histological evaluation was considered the gold standard for Grade 2B – Moderate
the diagnosis of endometritis (100% correct). The agreement be- endometritis (7)
tween each methodology used and the histological evaluation was Grade 3 – Severe endometritis
(4)
assessed using the Kappa Statistic (Freq Procedure). The number of

3
C.M. Teixeira-Soares, A.G.d.A. Viana, I.M. Ribeiro et al. Journal of Equine Veterinary Science 119 (2022) 104147

Fig. 1. Histological images of mare’s endometrial biopsies (Hematoxylin and Eosin stain X 100). (A) Grade 1 - Normal endometrium, negative for endometritis. (B) Grade
2A–Mild endometritis. (C) Grade 2B - Moderate endometritis. Arrow: Endometrial glands form nests and build up secretion in the lumen. (D) Grade 3 - Severe endometritis.

Table 2 Table 3
In vitro susceptibility to antimicrobials of isolated microorganisms harvested from Comparison between the different methods of diagnosis of endometritis in mares
the uterine fluid of mares. used in gynecological evaluation in contrast to the histopathological evaluation of
the endometrium.
Antimicrobials
Microorganism Methods Kappa Agreement Sensitivity Specificity
Neo Flo Pen Gen Tet Cef Str Enr Amp
Coefficient Strengtha
Staphylococcus spp. R S R R S S S S R
Palpation and 0.6447 Substantial 0.76 0.92
Escherichia coli R S R S R R S S R
ultrassonogaphy
Enterobacter spp. S S R S S S S S R
Perineal conformation 0.2308 Mild 0.42 0.85
Citrobacter spp. R S R S S S S S R
Vaginoscopy 0.1304 Poor 0.30 0.85
Abbreviations: Amp, ampicillin; Cef, ceftiofur; Enr, enrofloxacin; Flo, florfenicol; Examination of the cervix 0.3023 Mild 0.50 0.85
Gen, gentamicin; Neo, neomycin; Pen, penicillin; R, resistant to antimicrobial; S, Microbiological culture CC 0.7938 Substantial 0.84 1.00
sensitive to antimicrobial; Str, sulfa/trimethoprim; Tet, tetracycline. Microbiological culture LVF 0.6907 Substantial 0.80 0.92
Microbiological culture BFF 0.2373 Mild 0.30 1.00
Endometrial citology CC 0.7000 Substantial 0.76 1.00
Endometrial citology LVF 0.7000 Substantial 0.76 1.00
the histological evaluation, palpation and ultrasound showed the Endometrial citology BFF 0.2373 Mild 0.30 1.00
Effluent evaluation of LVF 0.2050 Mild 0.26 1.00
best results for sensitivity and specificity, followed by the examina-
tion of the cervix, conformation of the perineum, and vaginoscopy. Abbreviations: BFF, biopsy fragment flush; CC, commercial collector; LVF, low-
Uterine cytology and uterine microbiological culture, which are volume flushing.
a
Agreement strength: Kappa coefficient 0.00–0.19 (poor); 0.20–0.39 (Mild); 0.40–
laboratory evaluation methods, showed higher values than clinical
0.59 (moderate); 0.60–0.79 (substantial); 0.80–1.00 (almost perfect).
methods (Table 3).
Comparing the three sample collection techniques for uterine
cytological evaluation, the double-guarded commercial cytobrush
collector and the low volume flush presented similar sensitivity a low volume flush and a biopsy fragment flush, which, as in the
and specificity results. The biopsy fragment flush technique, in cytological evaluation, obtained a lower sensitivity value (Table 3).
turn, obtained a lower sensitivity result, showing a high index When comparing the seven evaluation methods used (pal-
false-negative result. For the evaluation of uterine microbiological pation and transrectal ultrasonography, perineum conformation,
culture, the commercial device had the best results, followed by vaginoscopy, cervical examination, low volume flush evaluation,

4
C.M. Teixeira-Soares, A.G.d.A. Viana, I.M. Ribeiro et al.
Fig. 2. Probability of obtaining a positive diagnosis for endometritis in mares by
histological examination in relation to the number of positive evaluations.
uterine cytology, and uterine microbiological culture) with the
golden standard histopathological exam, it was observed that the
number of positive methods influenced the probability of obtain-
ing a diagnosis for endometritis. Thus, the greater the number of
positive methods, the greater the probability of the correct diagno-
sis of endometritis (Fig. 2).
4. Discussion
Endometritis is the main reproductive concern in mares and
generates negative impacts in equine rearing due to subfertility in
mares. In our study the prevalence of positive mares, using his-
tological evaluation as the gold standard for diagnosis, was 65%,
a value similar to that of other authors [8,13]. This pathology is
the main reproductive concern in mares and generates significant
negative impacts on equine rearing due to its high prevalence, em-
phasizing the importance and challenge of correct diagnosis, for
consequent efficient treatment and success of pregnancy rates in
mares.
During the endometritis diagnostic gynecological exam, the
clinical evaluation methods are simpler and more practical to be
performed, being more commonly used than routine laboratory
methods by veterinarians. However, when comparing the sensitiv-
ity and specificity of clinical and laboratory methods, the values
were lower, demonstrating the need to combine the practicality
of clinical methods with the effectiveness of laboratory methods.
An alternative would be to use several clinical assessment meth-
ods combined since when more exams were performed, the more
accurate was the diagnosis of endometritis in our study. Further-
more, clinical methods can be used as screening for further labo-
ratory exams.
Among the clinical evaluation methods, palpation and transrec-
tal ultrasound showed the best sensitivity and specificity values.
This finding reinforces the judicious use of this method, not only
for routine monitoring of the estrous cycle but also for the ob-
servation of signs suggestive of endometritis, such as abnormal
endometrial edema and intrauterine fluid, which were correlated
with the results of the microbiological evaluation. In addition, the
professional’s experience during thorough examination combined
with complete mare history can improve the accuracy of transrec-
tal palpation and ultrasound diagnosis.
The uterine microbiological culture showed that 62.5% of the
mares were positive. Despite the different sampling techniques and
interpretations, this result is similar to other studies that range
between 61.9% and 73.3% [19,29–32]. All bacteria found in this
study are commonly isolated from mares’ uterus and fungal infec-
tion was not detected. However, the most frequent bacteria found
in this study (Staphylococcus spp.) differed from Streptococcus spp.
5
Journal of Equine Veterinary Science 119 (2022) 104147
[9,10,13] and Escherichia coli [19,30,31] reported by previous stud-
ies. Exposure to antimicrobials and geographic location may be a
hypothesis that explains this difference in the frequency of isolated
microorganisms [32].
The cytological examination is practical and simple to perform
and presents rapid results compared to other laboratory meth-
ods. Although the results may be affected by the chronicity of en-
dometritis and the type of bacteria isolated, the sensitivity and
specificity found in the study were satisfactory for the diagnosis.
The percentage of positive animals in the present study was com-
parable to the percentage range (20%–56%) described in the litera-
ture [1,9,15,16]. However, the use of different sampling techniques
and analysis of results demonstrates the need for standardization
of methodologies.
When comparing sample collection techniques, they have ad-
vantages and disadvantages themselves. Commercial collectors pre-
sented good results and greater practicality of execution. The sam-
pling technique with the cytobrush for cytology has been shown to
recover more cells for analysis. This observation is similar to that
found by Kozdrowski et al. [8] and can be explained by the fact
that the brush fibers enter the epithelium and allow the collection
of a higher number of cells, which, consequently, allows a faster
evaluation of the smear [1]. For microbiological evaluation, swabs
proved to be effective and safe to avoid sample contamination.
The low-volume flush technique was developed to allow sam-
pling of a larger area of the uterus since the solution goes through
the whole uterine surface and allows the evaluation of the efflux
[15]. Nevertheless, in the present study, the sensitivity and speci-
ficity values of the low-volume flow did not exceed those of the
commercial collector, which allows a punctual uterine sampling.
For cytological evaluation, the low uterine volume flow diluted the
sample, making interpretation and reading of the slide more dif-
ficult, even after centrifugation. Regarding microbiological evalua-
tion, even with satisfactory sensitivity and specificity values, cen-
trifugation increases the risk of environmental contamination of
the sample.
Low-volume flushing was performed efficiently with catheters
without cuffs. On average, 30 mL of efflux were recovered. Coc-
chia et al. [11] and Leblanc et al. [15], infusing the same volume
of solution with a cuffed catheter, recovered an average of 30 mL
and 34 mL of efflux, respectively. Thus, average values of fluid re-
covered were similar to those of the present study, showing the
use of a catheter without a cuff is feasible, with the advantage of
being less expensive and more practical. Efflux evaluation can be
an efficient auxiliary tool for diagnosis when associated with other
methods, especially uterine microbiological culture. Leblanc et al.
[15] correlated abnormalities in the efflux with the presence of mi-
croorganisms, which was also observed in the present study since
all positive samples in the low-volume flush were also positive in
the microbiological culture.
The endometrial biopsy fragment flushing technique was devel-
oped to reduce the number of procedures and uterine manipula-
tion when the biopsy is performed in the gynecological exam. In
addition, the technique is an alternative to printing on the cytolog-
ical slide and the culture plate to decrease contamination and in-
crease the quality of the collected sample. However, concerning the
other collection methods, it is not efficient for cytology and uter-
ine culture, with lower values of sensitivity and specificity. Thus,
the technique of flushing the biopsy fragment must be improved
to obtain more relevant results.
5. Conclusion
The methods of laboratory evaluation showed higher sensi-
tivity and specificity than the clinical evaluation. However, mul-
tiple findings made the diagnosis more accurate, showing the
C.M. Teixeira-Soares, A.G.d.A. Viana, I.M. Ribeiro et al.
importance of carrying out the gynecological examination with the
largest possible number of evaluation methods. The technique of
double-guarded commercial cytobrush/swab collector, either the
brush type for endometrial cytology or swab for a microbiology
culture, was quick to perform and demonstrated the best values
of sensitivity and specificity. Endometrial biopsy fragment flush,
although presenting lower sensitivity results compared to other
techniques, has the advantage of allowing the histological exami-
nation to be performed later, which may make this technique pos-
sible when a biopsy is performed.
Acknowledgments
This work was supported by Fundação de Amparo à Pesquisa
de Minas Gerais (FAPEMIG) [doctoral fellowship to CMT Soares -
12170], Coordenação de Aperfeiçoamento de Pessoal de Nível Supe-
rior (CAPES), and Conselho Nacional de Desenvolvimento Científico
e Tecnológico (CNPq).
References
[1] Overbeck W, Witte TS, Heuwieser W. Comparison of three diagnostic methods
to identify subclinical endometritis in mares. Theriogenology 2011;75:1311–18.
[2] Canisso IF, Segabinazzi LGTM, Fedorka CE. Review persistent breeding-induced
endometritis in mares - a multifaceted challenge: from clinical aspects to im-
munopathogenesis and pathobiology. Int J Mol Sci 2020;21:1432.
[3] Davies Morel MCG. Equine reproductive physiology, breeding and stud man-
agement. CAB Int 2003;3rd ed:1–428.
[4] Canisso IF, Stewart J, Silva MAC. Endometritis – managing persistent
post-breeding endometritis. Elsevier Inc 2016;16:0739–49.
[5] M Diel de Amorim, Gartley CJ, Hill A, Scholtz EL, Chenier TS, Signs Foster
RAComparison of Clinical. Endometrial culture, endometrial cytology, uterine
low-volume lavage, and uterine biopsy and combinations in the diagnosis of
equine endometritis. J Equine Vet Sci 2016;44:54–61.
[6] Katila T. Evaluation of diagnostic methods in equine endometritis. Reprod Biol
2016;16:189–96.
[7] McCue PM. The problem mare: management philosophy, diagnostic proce-
dures, and therapeutic options. J Equine Vet Sci 2008;28:619–26.
[8] Kozdrowski R, Sikora M, Buczkowska J, Nowak M, Raś A, Dziecioł M. Effects of
cycle stage and sampling procedure on interpretation of endometrial cytology
in mares. Anim Reprod Sci 2015;154:56–62.
[9] Nielsen JM. Endometritis in the mare: a diagnostic study comparing cultures
from swab and biopsy. Theriogenology 2005;64:510–18.
[10] Riddle WT, Leblanc MM, Stromberg AJ. Relationships between uterine cul-
ture, cytology and pregnancy rates in a thoroughbred practice. Theriogenology
2007;68:395–402.
[11] Cocchia N, Paciello O, Auletta L. Comparison of the cytobrush, cottonswab,
and low-volume uterine flush techniques to evaluate endometrial cytology
for diagnosing endometritis in chronically infertile mares. Theriogenology
2012;77:89–98.
[12] Ferris RA. Endometritis - diagnostic tools for infectious endometritis. Equine
Pract 2016;32:481–98.
Journal of Equine Veterinary Science 119 (2022) 104147
[13] Buczkowska J, Kozdrowski R, Nowak M, Ras A, Staroniewicz Z, Siemieniuch MJ.
Comparison of the biopsy and cytobrush techniques for diagnosis of subclinical
endometritis in mares. Reprod Biol Endocrinol 2014;12:27.
[14] Christoffersen M, MajaSöderlind M, Rudefalk SR, Pedersen HG, Allen J,
Krekeler N. Risk factors associated with uterine fluid after breeding caused by
Streptococcus zooepidemicus. Theriogenology 2015;84:1283–90.
[15] Leblanc MM, Magsig J, Stromberg AJ. Use of a low-volume uterine flush
for diagnosing endometritis in chronically infertile mares. Theriogenology
2007;68:403–12.
[16] Rasmussen CD, Morten PR, Bojesen AM. Equine infectious endometritis clinical
and subclinical cases. J Equine Vet Sci 2015;35:95–104.
[17] Silva Rua MA, Quirino CR, Ribeiro RB, Queiroz Carvalho EC, de Lourdes Amaral
Bernadino M, Junior AB, Cipagalta LF, MarcusAntô Pessanha Barreto. Diagnostic
methods to detect uterus illnesses in mares. Theriogenology 2018;114:285–92.
[18] Mathew A, Patel D, Sensitivity Hadiya K. Specificity and predictive values
of cytological and microbiological findings of endometrial biopsy, cytobrush
and low volume uterine lavage in relation to endometrial histology in barren
mares. Indian J Vet Sci Biotechnol 2020;15(04):75–9.
[19] Brogan PT, Henning H, Stout TAE, de Ruijter-Villani M. Relationship between
color flow Doppler sonographic assessment of corpus luteum activity and pro-
gesterone concentrations in mares after embryo transfer. Anim Reprod Sci
2016;166:22–7.
[20] Samper JC. A review of a practitioner’s perspective on endometrial edema.
Pferdeheilkunde 2010;26:14–18.
[21] Pascoe RR. Observations on the length and angle of declination of the vulva
and its relation to fertility in the mare. J Reprod Fertil 1979;27:299–305.
[22] Caslick EA. The vulva and the vulvo-vaginal orifice and its relation to genital
health of the thoroughbred mare. Cornell Vet 1937;27:178.
[23] Zent WW, Steiner JV. Vaginal examination. Equine Reproduction. McKin-
non AO, Squires EL, Vaala WE, Varner DV, editors. Second Edition editors. U.K:
Wiley-Blackwell; 2011.
[24] Sitters S. Digital examination of the vagina/cervix. Equine Reproductive Proce-
dures. Dascanio JJ, McCue PM, editors editors. Jersey: John Wiley & Sons, Inc;
2014.
[25] Clinical and Laboratory Standards Institute. Clinical and Laboratory Standards
Institute (CLSI). Performance standards for antimicrobial susceptibility testing-
CLSI Supplement M100-S25. 30th ed. Wayne, PA.: CLSI; 2020.
[26] Kenney RM, Doig PA. Equine endometrial biopsy. In: Current Therapy in Theri-
ogenology. Newberg: W.B. Saunders Company; 1986. p. 723–9.
[27] Schoon HA, Schoon D, Klug E. Uterus biopsienals Hilfsmittelfür diagnose und
prognose von Fertilitätsstörungen der Stute. Pferdeheilkunde 1992;8:355–62.
[28] Institute Inc SAS. 2002: SAS/STAT® 9.0 User’s guide. Cary, NC: SAS Institute
Inc; 2002.
[29] Jacob JCF, VLT Jesus, Barbosa HP, Zimmerman MF, Silva AG, Melo CM. Antimi-
crobic susceptibility of uterine and clitoral swabs of mares with endometritis.
Rev. Univ. Rural, Sér. Ciên. da Vida 2002;22:109–14.
[30] Oliveira IB, Peixoto RM, Silva DR, Pinheiro Junior JW, Oliveira AAF, Mota RA.
Etiologia e perfil de sensibilidade de bactérias aeróbias isoladas de éguas sus-
peitas de endometrite procedentes do Estado de Pernambuco. MedicinaVeter-
inária 2007;1:19–25.
[31] Carvalho CFPM, Borba HS, Ribas JAS, Barros RR. Diagnóstico bacteri-
ológico, citológico e histopatológico da endometrite equina. R. bras. Ci. Vet.
2011;18:19–22.
[32] Cabrera T, Pastorello M, Alvarenga MA. Prevalência e perfil de sensibilidade
bacteriana em éguas com Endometrite. Enciclopédia Biosfera, Centro Científico
Conhecer 2016;13:1013.