USE OF A LOW-VOLUME UTERINE FLUSH FOR MICROPIOLOGIC AND

CYTOLOGIC EXAMINATION OF THE MARE'S ENDOMETRIUM

B. A. Ball,' S. J. Shin,* V. H. Patten,'

D. H. Lein2 and G. L. Woods3

'DepartBent of Clinical Sciences and

Diagnostic Laboratory,
New York State College of Veterinary Medicine,
Cornell University, Ithaca, NY 14853

Received for publication: June 19, 1987

Accepted: January 19, 1988

ABSTRACT

A guarded uterine swab and a how-vo~urn~uterine flush were

compared for microbiologic and cytologic examination of the endometrium
of young, normal mares (n = 24) and aged, subfertile mares (n = 27).
Mares from each group had clitoral sinus swabs, guarded endometrial
swabs (X2), uterine flushes and endometrial biopsy samples taken for
microbiologic, cytologic and histologic examination.

The guarded swab technique yielded significantly fewer (P < 0.05)

microbiologic cultures with pathogenic microorganisms than the flush
technique for both normal mares (O/24 vs 6/24) and subfertile mares
(4/27 vs 14127). The swab technique also yielded significantly fewer
(P < 0.05) endometrial smears with cytologic evidence of inflammation
than the flush technique for subfertile mares (12/27 vs 20/27);
however, there was no significant difference (P > 0.1) for the two
techniques for cytologic results in normal mares (3124 vs 7/24).
Urea lasma spp. were isolated from the uterine flushes (3/27) and
-r?l-*
c I ora sinus swabs (6/27) from subfertile mares. Mycoplasma spp.
were isolated from the uterine flush from one subfertile mare and
clitoral sinus swab from another subfertile mare. Significantly fewer
(P < 0.01) normal than subfertile mares had histologic evidence of
inflammation in endometrial tissue sections (5/?4 vs 25/27). Based on
microbiologic and cytologic results, the low-volume uterine flush
appeared superior to the guarded endometrial swab for the diagnosis of
endometritis in mares.

Key words: mare, endometritis, diagnosis, microbiology, cytology

The authors thank Thornbrook Farms and the Harry M. Zweig Memorial
Trust for financial support and Dr. T. French for advice on preparation
and interpretation of cytologic specimens. Technical assistance was
provided by P. Kelley and K. Roneker.
3
Present address: Department of Veterinary Clinical Medicine
and Surgery, WOI Regional Program of Veterinary Medicine,
University of Idaho, Moscow, Idaho 83843

JUNE 1988 VOL. 29 NO. 6 1269

THERIOGENOLOGY

INTRODUCTION

Correct interpretation of microbiologic cultures from the mare's

endometrium requires consideration of possible false positive and false
negative culture results (l-4). False positive cultures have been
defined as endometrial samples with microbiologic but not cytologic
evidence of endometritis (4,5). False positive cultures have been
associated with contamination of the culture instrument from the
environment, the external genitalia and the vagina (1,4). This
contamination is reduced with guarded culture instruments (1,6).
Conversely, false negative endometrial cultures have been defined as
endometrial samples without microbiologic isolates but with cytologic
or histologic evidence of endometritis (2-4,7,8). False negative
cultures may be associated with inadequate sampling of the endometrium
or with the use of aerobic culture techniques which may not recover
anaerobic bacteria, viruses or mycoplasma (7,9-13). The association of
microorganisms such as mycoplasma or ureaplasma with endometritis,
however, has not been well defined (9,11,12).

The problems associated with false positive and false negative

cultures indicate a need for improved methods to diagnose endometritis.
Uterine flushes have been used for microbiologic and cytologic
examination of the mare's endometrium and represent an alternative to
endometrial swabs for examination of the endometrium (14-20). In a
study of 200 barren mares, three separate two-liter uterine flushes
were used for microbiologic and cytologic examination (20). Although
77% of the mares in this study had positive cultures, only about 25% of
the isolates represented microorganisms normally considered endometrial
pathogens (3,6,8,21-23). No differences in cytologic findings were
observed between mares with positive and negative microbiologic
findings, and the authors suggested that most of the microorganisms
isolated represented contaminants (20).

The objectives of the current study were 1) to compare a guarded

endometrial swab and a low-volume uterine flush for the diagnosis of
endometritis, based on quantitative microbiologic and cytologic
results; 2) to determine the frequency of isolation of ureaplasmae and
mycoplasmae from the endometrium and clitoral sinuses of normal and
subfertile mares; and 3) to examine the correlation of endometrial
histopathology with microbiologic and cytologic findings in normal and
subfertile mares.

MATERIALS AND METHODS

The normal mares that were used in the study consisted of 24

nulliparous, Standardbred, Thoroughbred, Quarter Horse or Morgan mares
between 3 and 8 yr of age; the subfertile mares consisted of 27
Standardbred, Thoroughbred or Arabian mares from 10 to 23 yr of age
with a 2-yr history of reproductive failure. All examinations were

1270 JUNE 1988 VOL. 29 NO. 6

 THERIOGENOLOGY

conducted, regardless of the stage of the estrous cycle, during

February and March of 1985,

Each mare was restrained in stocks, and a swab of a c‘litoralsinus

was obtained for microbiologic culture. The vulva and perineum were
then alternately scrubbed with povidone-iodine scrub (Betadine Surgical
Scrub, Purdue Frederick, Co., Norwalk, CT) and rinsed three times with
water. A double-guarded culture instrument (McCullough Swab,
McCullough-~artwright Co., Barrington, IL) was then passed per vaginum
into the uterus by an examiner whose arm was covered by a clean plastic
sleeve and a sterile surgical glove. This swab was kept in contact
with the endometrium for a minimum of 15 set and was placed in Aime's
transport medium with charcoal (Baltimore Biological Laboratories
fBBL), Cockeysville, MD) for subsequent microbiologic culture. A
second endometrial swab was taken, rolled onto a clean glass slide,
air-dried, and stained with a modified Wright's stain (Diff-Quik,
Harleco Division, American Hospital Supply) for cytologic examination.

After the endometrial swabs were taken, the tip of a guarded (l?
20"French Gibbons balloon catheter (Franklin Medical, Bucks) was placed
through the cervix, and the balloon was inflated with 60 ml of air to
ensure retention within the uterus. Sixty milliliters of sterile
phosphate-buffered saline (PBS) was then flushed into the uterus, and
the uterus was manipulated per rectum to distribute the fluid into both
uterine horns. As much of the 60 ml of PBS was recovered as possible,
and the recovered fluid was divided into two 15-ml aliquots. The first
15"ml aliquot was transported to the laboratory immediately for
microbiologic examination. The second 15-ml aliquot was centrifuged at
1000 to 1500 rpm for 5 min; the supernatant was decanted, and the cells
were resuspended in 0.5 ml of fetal calf serum (Cibco, Grand Island,
NV). This suspension was used to prepare smears to be stained with
Diff-Quik for cytologic examination. An endometria~ biopsy was
obtained after the uterine flush for histopathologic examination (7).

Ten milliliters of the first aliquot of PBS recovered from the

uterus was centrifuged at 15000G for 15 min in a refrigerated
centrifuge, the supernatant was decanted, and the pedicle was
resuspended in 1.0 ml of sterile PBS. Both 0.1 ml of this resuspended
volu~ and the initial endo~trial swab were plated onto 5% Columbia
sheep-blood agar, chocolate agar, Columbia sheep-blood colistin-
nalidixic-acid (CNA) agar and eosin-methylene blue (EM62,agar plates
(BBl, Cockeysville, MD). Plates were incubated at 35.5 C in 5% CO
with 100% relative humidity for 48 h, and complete characterizatiog of
each isolate was performed based on biochemical tests (24,25).
Qugntitative plate counts were performed, and microbial growth was
scored as none (0 colonies}, +l (1 to 20 colonies}, +2 (21 to IO0
colonies) or +3 (greater than lO@ colonies). microbiologic cultures
were considered positive for pathogenic microorganisms if greater than
20 colonies (+2 or t3 score) of Candida spp., Cor nebacterium spp
B-hemolytic streptocccci, coagulase-positive s*schekichia
ap y ococcl,
fi, Pseudomonas aeru inosa or Klebsiella pneumoniae were recovered
per plate (4,6,8,2Ii or quantitation of plate counts, plates with

JUNE 1988 VOL. 29 NO. 6 1271

THERIOGENOLOGY

too numerous to count (TNTC) colonies were arbitrarily assigned a value

of 500 or greater colonies.

Ureaplasma and mycoplasma cultures were performed on uterine flush

and clitoral sinus samples from all mares. For culture of ureaplasma,
0.02 ml of the resuspended uterine flush and the clitoral sinus swab
were inoculated into Shepard's agar and broth (26,27) and incubated
under anaerobic conditions at 37 C for 4 to 5 d. Broth cultures were
subcultured at 48 and 96 h. For culture of mycoplasma, 0.02 ml of the
resuspended uterine flush and the clitoral sinus swab were inoculated
into Hayflick's agar with 10% equine or 20% porcine serum and
Hayflick's broth (28) and incubated at 35.5'C in 5% CO2 for 4 to 5 d.
Broth cultures were subcultured at 48 and 96 h.

For cytologic examination, differential cell counts were erformed

on smears prepared from both the endometrial swab and flush (5 .
Cytologic smears were considered indicative of infla~ation (5P if
greater than 2% of the 100 cells counted were neutroph~ls (Pan).
Infla~atory changes on endometrial biopsies were scored as none = 0,
mild = 1, moderate = 2 or severe = 3 by two evaluators (7,29).

Statistical analysis of quantitative data was based on the

appropriate paired or independent samples t-test (30,31). Ranked data
were analyzed by Chi-square ana'lysisor by nonparametric methods via
Fischer's randomization test (30). Correlation analysis (Pearson's
product moment coefficient) between scores was also performed (30,31).

RESULTS

Kfcrobiologic Findings

The mean volumes of PBS recovered from the uterine flushes were
not significantly different between normal or subfertile mares
(34.7 rl:
1.5 vs 35.5 + 1.6 ml). The swab technique yielded
significantly fewer positive endometrial cultures than the flush
technique in normal (P < 0.05) and in subfertile (P < 0.01) mares
(Table I). Normal mares had significantly fewer (P < 0.05) positive
endometrial cultures than subfertile mares based on both the swab and
flush techniques (Table 1).

The overall mean colony counts per plate for pathogenic

microorganisms were significantly lower (P < 0.01) for the swab versus
flush technique in both normal and subfertile mares (Table 2). When
examined by distribution of culture scores, there were significant
differences between the distribution of culture scores for the swab
compared to the flush technique in subfertile mares (P < 0.05) but not
in normal mares (P > 0.25; Table 3).

When examined by the type of pathogenic microorganism (Table Z),

the swab technique yielded a lower mean plate count than the flush
technique for endometrial cultures of C. parapsilosis (P < 0.01) and

1272 JUNE 1988 VOL. 29 NO. 6

Corynebacterium spp. (P < 0.05). There were positive correlations
between the type of pathogenic microorganism from the swab and flush
cultures in both normal mares (rr0.51, P < 0.01) and in subfertile
mares (r=0.89, P < 0.01). Overall, there was a positive correlation
(rr0.64, P < 0.01) between culture scores for pathogenic microorganisms
recovered via the swab and flush techniques. When examined by mare
group, there was a positive correlation between the swab and flush
techniques for culture scores of pathogenic microorganisms from
subfertile mares (r=0.74, P < 0.01) but not from normal mares (r =
0.20, P > 0.05).

Table 1. Microbiologic and cytologic results for normal and subfertile

mares sampled with a guarded endometrial swab and a
low-volume uterine flush

Technique
Item Swab blush

Positive microbiologic culturese

(Wo. mares/no. mares sampled)

Normal mares

Subfertile mares

Cytologic evidence of inflamnationf

(No. mares/no. mares sampled)

Normal mares 3/24c 7/24'

Subfertile mares ~2,27d____*----_20,27d

Values with different superscripts (a,b P < 0.05; c,d P < 0.01) within
columns were different.
Values within rows were different f* P < 0.05; ** P < 0.01).
@Cultures with > 20 colonies per plate (+2 or +3) of a defined
pathogenic microorganism.
fSmears with > ZPMN/lOO cells on differential counts.

The swab technique yielded significantly fewer (P < 0.01)

endometrial cultures with no microorganisms than the flush technique in
both normal and subfertile mares (Table 4). Overall, the swab
technique yielded fewer (P < C.025) nonpathogenic microor anisms than
the flush technique (8/51 vs 19/51, respectively; Table 4B.
Nonpathogenic microorganisms isolated from endometrial cultures from
both normal and subfertile mares included diptheroids, alpha- and

JUNE 1988 VOL. 29 NO. 6 1273

THERIOGENOLOGY

Table 2. Mean (+SEM) colony counts of pathogenic microorganisms

recovered from endometrial swab and flush cultures and
clitoral sinus cultures from normal and subfertile mares

Flush Swab
Clitoral
Organism Culturee Cytologyf Culturee Cytologyf sinus

-C. parapsilosis 402 + 74c 19 _+gd 34 _+ 15

(n = 5) (5) (n = 5) (5) (n = 4)

Corynebacterium 141 f 5oa 24 + 13b 104 a 41

SPP. (n = 12) (10) (n = 4) (4) (n = 12)

E. coli 16 f 6 5*3 28 f 19
(n = 9) (6) (n = 4) (2) (n = 5)

LK pneumoniae 6+2 100

(n = 3) (1) (n f 0) __ (n = 1)

-P. aeruginosa (0)

(n l 1) (0) (n L 1) (G

Beta-streptococci 10 f 8 4?2 29 + 20
(n = 5) (2) (n = 3) (1) (n = 3)

Overall

Normal
mares 29 + 1oa 2 f 0.3b 53 f 13
(n = 16) (n = 2) (n = 12)
Subfertile
mares 182 A 49' 16 f gd 112 f 45
(n = 19) (n = 15) (n = 14)

Total 112 + 3cc 13 + qd 67 * 19

(n = 35) (n = 17) (n = 26)

Values within rows with different superscripts were different

(a,b P < 0.05; c,d P < 0.01).

eMean (+ SEM) colony counts of pathogenic microorganisms where n = number

of isolates.

fNumber of mares with cytologic evidence of inflammation with the

corresponding technique.

1274 JUNE 1988 VOL. 29 NO. 6

 THERIOGENOLOGY

Tab7e 3. Distribution of microbiologic and histo'logicscores for

endometrial samples from normal and subfertile mares

Scorea
Item 0 1 2 3

Norma7 maresb (n = 24)

Swab culture
Flush culture : (2) ! (2)
Riopsy 19 3 1 1

Subfertile maresbyc (n = 27)

Swab culture g (4) 4 (2)
Flush culture :: 1:; 3 (I) 5 (5) 9" (6)
Biopsy 2 12 13 0

aCulture scores of 0 (no growth), +l (1 to 20 colonies), +2 (21 to 100

colonies) or +3 (greater than 100 colonies) of a pathcgenic
microorganism. Biopsy scores of 0 (none), 1 (mild), ? (moderate), or
b3 (severe) inflammation.
Distribution of biopsy scores were different between normal and
subfertile mares (P < 0.01).
'Distribution of culture scores were different between swab and flush
dtechnique for subfertile mares (P < 0.05).
Numbers in parentheses represent the number of mares in each category
with cytologic evidence of inflamnation with the corresponding
technique.

nonhemolytic streptococci, noncoagulase positive staphylococci,

Bacillus spp. and micrococcus.

Clitoral sinus cultures yielded pathogenic microorganisms from 12

of 24 normal mares and 14 of 27 subfertile mares (P > 0.5; Table 2).
Th'erewas a positive correlation between the type of pathogenic isolate
recovered from the clitoral sinus culture and flush culture (r=0.43, P
< O.Ol), but not between the clitoral sinus and endometrial swab
cultures (r-=0.17,P > 0.1).

Ureaplasma spp. were isolated from the clitoral sinus swabs from 6
of 27 subfertire mares; three of these six subferti'lemares also had
spp. isolated from the uterine flush. The isolation rate of
spp. from the clitoral sinus swab of normal mares tended
to be less than that of subfertile mares (l/24 vs 6/27).
spp. were isolated from the clitoral sinus swab from one
mare and from the uterine flush of another subfertile mare.

JUNE 1988 VOL. 29 NO. 6 1275

 Table 4. Number of normal and subfertile mares
. . with* 1 to
_ 20 colonies
. .
of pathogenic ml~roorgan~sms, nonpatnogenic microorganisms or
no microbial growth from endo~trial swab and flush cultures

Item Normal mares Subfertile mares

Pathogenic organism (+1 growth)

Swab 2/24_____*____ g/27a

Flush 6124 3/27b

Nonpathogenic organism
Swab 4/24 4/27

Flush lo/24 9127

No microbial growth
Swab ~~/24c~~~**~~~~~~/27c

Flush 2/24d 1/27d

lalues with different superscripts (a,b P < 0.05; c,d P < 0.01) within
columns were different.
Values within rows were different (* P < 0.05; ** P < 0.01).

Cytologic Findings

The swab technique yielded significantly fewer endometrial smears

with cytologic evidence of inflammation than the flush technique in
subfertile mares (P < 0.05), but not in normal mares (P > 0.1; Table
1). There were significantly fewer (P < 0.01) normal than subfertile
mares with cytoloqic evidence of inflammation for both the swab and
flush techniques (Table I). For differentia? cell counts, the mean
number of P~N/lOO cells on smears from both normal and subfertile mares
was significantly lower (P < 0.01) for the swab compared to the flush
technique (3.7 i: 1.2 vs 18 k 3.5, respectively).

Based on combined cytologic and microbiologic results, the

distributions of positive (t/+), negative f-/-), false negative f+/-)
or false positive (-/+) diagnoses of endo~tritis between the swab and
flush techniques were different in normal mares (P < 0.1) and in
subfertile mares (P < 0.01; Table 5). The swab technique yielded
significantly fewer (P < 0.01) positive diagnoses and significantly
more (P < 0.01) negative diagnoses than the flush technique in
subfertile mares.

1276 JUNE 1988 VOL. 29 NO. 6

 THERIOGENOLOGY

Table 5. Agreement between cytologic and microbiologic results for

swab and flush techniques in normal and subfertile mares

Cytology/Cultures
+/+ _- t- -/t
Mare group POS. Neg. False Neg. False Pos.

Normalb (n = 24)
swab 0 21 3 0

flush 4 15 3 2

Subfertileb (n = 27)
swab 2 13 10 2

flush 13 6 7 1

aDenotes positive (> 2 PMN per 100 cells) or negative cytologic results
and positive (> 20 colonies of a defined pathogenic microorganism per
plate) or negative microbiologic results.

bDistribution of cytologic and microbiologic results were different

between swab and flush techniques for normal (P <O.l) and subfertile
(P < 0.01) mares.

Histologic Findings

Significantly fewer (P < 0.01) normal versus subfertile mares had

mild, moderate or severe inflammatory changes detected on the
endometrial biopsy (5/24 vs 25/27; Table 3), and the mean inflammatory
score on endometrial biopsy was significantly lower (P < 0.01) for
normal versus subfertile mares (0.4 k 0.1 vs 1.3 * 0.1, respectively).
Overall, there was a significant, positive correlation between
endometrial biopsy score and endometrial microbiologic score or
cytologic results for the swab technique and between endometrial biopsy
score and cytologic results for the flush technique; however, these
correlations were nonsignificant when examined for normal and
subfertile mares as separate groups (Table 6).

DISCUSSION

The flush technique was superior to the swab technique for the
diagnosis of endometritis based on quantitative cytologic and
microbiologic findings in both normal and subfertile mares. For
cytologic findings, the flush technique resulted in significantly more
subfertile mares with evidence of inflaannation;however, in normal

JUNE 1988 VOL. 29 NO. 6 1277

THERIOGENOLOGY

Table 6. Correlation coefficients between culture scores and cytologic

results for the swab and flush techniques and endometrial
biopsy scores for normal and subfertile mares

Culture
Swab l-lush Biopsy

Cytology

Swab NS 0.30a 0.30a

Flush NS 0.47b 0.35a

Biopsy 0.3gb NS ___

Coefficients were significant (a, P < 0.05; b, P < 0.01).

NS indicates that coefficients were not significant (P > 0.05).

mares, the flush technique did not significantly increase the frequency
of cytologic diagnosis of endometritis. For microbiologic findings,
the flush technique yielded more samples that were positive for
pathogenic microorganisms and a higher mean colony count of pathogenic
microorganisms in both groups of mares. Although the flush technique
increased the number of positive microbiologic results in normal mares,
four of six normal mares with positive microbiologic results also had
cytologic evidence of inflammation with the flush technique.
Therefore, the increased number of positive microbiologic results with
the flush technique in normal mares did not appear to represent false
positive cultures.

The increased frequencies of positive cytologic and microbiologic

findings with the flush technioue were in agreement because the flush
technique significantly increased the number of positive (t/t)
diagnoses and significantly decreased the number of negative (-/-)
diagnoses of endometritis in the subfertile mares. Concurrently, there
were no significant changes in the numbers of false negative (+/-) or
false positive (-/t) diagnoses of endometritis in subfertile mares
between the swab or flush techniques. Agreement between cytologic and
microbiologic results with the flush technique was further supported by
the significant positive correlation between these findings.

The increased colony counts of pathogenic microorganisms with the

flush compared to the swab technique could have been due to 1) an
increase in the area of endometrium sampled with the uterine flush, 2)
a concentration of microorganisms during centrifugation, 3) an increase

1278 JUNE 1988 VOL. 29 NO. 6

 THERIOGENOLOGY

in contaminants from the external genitalia or vagina recovered with

the flush technique or 4) a combination of these factors. It has been
previously suggested that swabs may be an ineffective method of
sampling the endometrium due to the small surface area normally
contacted by the culture swab (6,3?). When mares were experimentally
infected with Klebsiella pneumoniae, the organism was recovered less
often by direct swabbing of the endometrium than by a method with an
intrauterine tampon that sampled a larger surface area of endometrium
(32). The effect of sampling area would be exacerbated if certain
endometrial pathogens were distributed in a focal rather than a diffuse
pattern.

Contamination from the external genitalia during the uterine flush

must also be considered as a source of the increased number of cultures
with pathogenic microorganisms because such contaminants have been
shown to occur more frequently in semiguarded (i.e., the flush
technique) than in completely guarded (i.e., the swab technique)
endometrial culture techniques (6). This result may be reflected in
the current study because the flush culture results were significantly
correlated with the clitoral sinus culture results, which may indicate
an increased rate of contamination from the external genitalia with the
flush compared to the swab technique. In addition, the order in which
the endometria? samples were taken from each mare (i.e., swab first,
then flush) may have increased the probability of positive
microbiologic findings with the flush technique. The increased number
of positive cultures with the flush technique, however, was associated
with an increased number of cytologic diagnoses of endometritis.

The flush technisue did increase the interpretive difficulty of

microbiologic results, because the flush technique resulted in fewer
cultures with no growth of microorganjsms and more cultures with
nonpathogenic microorganisms. Uhen microbiologic and cytologic results
were combined, the interpretive problems with the flush technique did
not appear to confuse the diagnosis.

The types of bacterial pathogens isolated from the endometrium of

normal and subfertile mares in this study were similar to those
reported in ear?ier studies (4,6,8,21-23). The relative distribution of
these pathogens, however, appeared different in our study compared to
that previously reported (3,4,21,23). Several investigators have
reported that beta-hemolyti~ streptococci were the most colon clinical
isolate recovered from the mare's uterus; however, in our study,
Corynebacterium spp. were the most common pathogenic microorganisms
recovered from the uterus of selected subfertile mares. Although
Cor neba~terium spp. have been reported as potential pathogens, the
?-i&&F- isolation was only 3 to 4% (3,21,23). In our study, 10 of
12 uterine flush cultures that had Corynebacterium spp. recovered had
evidence of infla~ation on cytologic examination and 8 had evidence of
inflammation on histopathologic examination of the endometrial biopsy.
Cytologic preparations from mares with Cor nebacterium spp. revealed
numerous macrophage and lymphocytes in -s.
a This
pyogranulomatous type of infla~atory response has heen reported with

JUNE 1988 VOL. 29 NO. 6 1279

 detect with routine endometrial swabs.

The frequency of isolation of M co lasma spp. from the clitoral

fossa (33.7%), vagina (63%) and cervix
G of mares has been
previously reported; however, the significance of this organism in the
mare's genital tract is not known (9,11,12). The frequency of
isolation of F! co lasma spp. from the uterus of subfertile mares in
this study appeare
-=+-. slmmlar to that reported for isolation from the
mare's cervix (11). Reports on the frequency of isolation of
Ureaplasma spp. from the genital tract of mares were not found. The
frequency of fsolation of Ureaplasma spp. from the clitoral fossa of
subfertile mares tended to be higher than that from normal mares;
however, the importance of this finding requires further testing.

An approxi~tion of the number of pathogens present in the uterus

can be Made based on the dilutions used in recovery of microorganisms
via the flush technique. The colony counts listed in Table 2 for the
flush technique multiplied by 100 give the mean concentration of
microorganisms recovered per milliliter of flush media. On the
assumption that microorganisms in the uterus are uniformly suspended in
the 60 ml of flush media, the concentration (per milliliter) multiplied
by 60 gives an approxi~tion of the total number of bacteria
recoverable from the uterus of these mares. For example, C.
parapsilosis had a mean plate count of 402 colonies or 4.02 x lo4
organisms per milliliter. Therefore the total estimaged number of 2.
parapsilosis recoverable from the uterus was 2.4 x 10 organisms.

The weak positive correlations between microbiologic findings

(swab) or cytologic findings (swab and flush) and endometrial biopsy
score were similar to other studies that indicate only a moderate
relationship between microbiologic and histologic findings in
endometritis (6,7,29,34). One study reported no significant
correlations between culture result and histologic evidence of
inflammation in 77 barren mares, although there was a significant
positive correlation between culture results and the number of
inflammatory cells migrating through the uterine luminal epithelium
(6). Although cytologic results were not reported in this study (6),
the correlation between migrating infla~atory cells and culture
results is in agreement with the correlation between microbiologic and
cytologic results in our study.

In conclusion, the low-volume uterine flush improved the ability

to diagnose endometritis based on quantitative microbiologic and
cytologic findings. The increased frequency of diagnosis of
endometritis with the flush technique was more evident in subfertile
than normal mares; therefore, the flush technique may be best applied
in the clinical evaluation of barren or subfertile mares.

1280 JUNE 1988 VOL. 29 NO. 6

 THERIOGENOLOGY

REFERENCES

1. Blanchard, T.L., Garcia, M.C., Hurtgen, J.P. and Kenney, R.M.

Comparison of two techniques for obtaining endometrial
bacteriologic cultures in the mare. Theriogenology -16:85-93
(1981).

2. Knudsen, 0. Endometrial cytology as a diagnostic aid in mares.

Cornell Vet. -54:415-422 (1964).

3. Digby, N.J.W. and Ricketts, S.W. Results of concurrent

bacteriological and cytological examination of the endometrium of
mares in routine stud farm practice 1978-1981. J. Reprod. Fertil.
(Suppl.) -32:181-185 (1982).

4. Ricketts, S.W. Bacteriological examination of the mare's cervix:

Techniques and interpretation of results. Vet. Rec. -108:46-51
(1981).

5. Digby, N.J.W. The technique and clinical application of

endometrial cytology in mares. Equine Vet. J. -10:167-170 (1978).

6. Blanchard, T.L., Cummings, MR., Garcia, M.C., Hurtgen, J.P. and

Kenney, R.M. Comparison between two techniques for endometrial
swab culture and between biopsy and culture in barren mares.
Theriogenology -16:541-552 (1981).

7. Kenney, R.M. Cyclic and pathologic changes of the mare

endometrium as detected by biopsy, with a note on early embryonic
death. J. Am. Vet. Med. Assoc. -172:241-262 (1978).

8. Bain, A.M. The role of infection in infertility in the

Thoroughbred mare. Vet. Rec. -78:168-173 (1966).

9. Moorthy, A.R.S., Spradbrow, P.B. and Eisler, M.E.D. Isolation of

Mycoplasmas from the genital tract of horses. Aus. Vet. J.
-53: 167-169 (1977).
10. Ogata, M., Watabe, J. and Koshimizu, K. Classification of
Acholeplasmas from horses. Jap. J. Vet. Sci. 36:43-51 (1974).

11. Krabisch, P., Kirchoff, H. and Lepel, J.F. Nachweis von

Mykoplasmen auf genitalschleimhauten von stuten. Dtsch.
Tierarztl. Wschr. -80:493-516 (1973).

12. Bermudez, V., Miller, R., Johnson, W., Roeendal, S. and Ruhnke, L.
The prevalence of Mycoplasma spp. and their relationship to
reproductive performance in equine herds in southern Ontario. J.
Reprod. Fertil. (Suppl.) -35:671-673 (1987).

JUNE 1988 VOL. 29 NO. 6 1281

 13. Ricketts, S.W. and Mackintosh, M.E. Role of anaerobic bacteria in
equine endometritis. J. Reprod. Fertil. (Suppl.) -35:343-351
(1987).

14. Rasbech, N.O. Effect of equine genital infections on

reproduction. Nord. Vet. Med. -17:305-317 (1965).

15. Stremienski, P.J. and Kenney, R.M. Effect of stage of cycle,

sampling frequency and recovery of micro-organisms on total
protein content of mare uterine flushings. J. Reprod. Fertil.
-70:327-332 (1984).

16. Williamson, P., Dunning, A,, O'Conner, J. and Penhale, W.J.

Iannunoglobulinlevels, protein concentrations and alkaline
phosphatase activity in uterine flushings from mares with
endometritis. Theriogenology -19:441-448 (1983).

17. Slusher, S.H., Freeman, t&P. and Roszef, J.F. Eosinophils in

equine uterine cytology and histology specimens. J. Am. Vet. Med.
Assoc. -184:665-669 (1984).

18. Bowman, T. Early response of the equine uterus to induced

streptococcal endometritis. Master's Thesis, University of
Maryland, 1972.

19. Slusher, S.H. The relationship between endometrial biopsy,

cytology and bacterial pathogens in mares. Master's Thesis,
Oklahoma State University, 19&4.

20. El Sanousi, S.M., El Tayeb, A.B. and Shadad, E.Y. Bacteria

isolated from uterine washings from mares in the Sudan. Equine
Vet. J. z:219-222 (1979).

21. Shin, S.J., Lein, D.H., Aronson, A.L. and Nusbaum, S.R. The
bacteriological culture of equine uterine contents, in-vitro
sensitivity of organisms isolated and interpretation. J. Reprod.
Fertil. (Suppl.) -27:307-315 (1979).

22. Allen, W.E. and Newcombe* J.R. Aspects of genital infection and
swabbing techniques in the mare. Vet. Rec. -104:228-231 (1979).

23. Porchester, Lord. Uterine infections in mares. Vet. Rec.

-77:110-111 (1965).

24. Lennete, E.H., Balows, A., Hausler, W.J. and Truant, J.P. Manual
of Clinical Microbiology. American Society Microbiology,
Washjngton, DC, 1985.

25. Finegold, S.M. and Baron, E.J. Bailey and Scott's Diagnostic
Microbiology. C.V. Mosby, St. Louis, 1986.

1282 JUNE 1988 VOL. 29 NO. 6

26. Shepard, M.C. and Lunceford, C.D. Differential agar medium (A-7)
for identification of Urea lasma urea1 ticum (human T-mycoplasmas)
in primary cultures o&m&. Clin. Microbial.
3:613-625 (1976).

27. Shepard, M.C. and Lunceford, C.D. Urease color test medium U-9
for the detection of “T" mycoplasmas In clinical material. Appl.
Microbial. -20:539-543 (1970).

28. Hayflick, L. Tissue cultures and mycoplasmas. Tox. Rep. Biol.

Med. (Suppl,) 1:285-303 (1965).

29. Doig, P.A., McKnight, J.D. and Miller, R.B. The use of
endometrial bi@PSy in the infertile mare. Can. Vet, J. 22~72-76
(1981).

30. Ryan, T.A., Jr., Joiner, B.L. and Ryan, B.F. Minitab Student
Handbook. Duxbury Press, Boston, 1976, pp. 134-148, 222-241.

31. Snedecor, G.W. and Cochran, W.G. Statistical Methods. Iowa State
Univ. Press, Ames, 1980, pp. 83-193.

32. Stratton, L.G., Corstvet, R., Brown, J. and Corley, L. Isolation

of Klebsiella pneumoniae from the urogenital tract of
experimentally infected mares. J. Reprod. Fertil. (Suppl.)
-273317-320 (3979).

33. Hartigan, P.J., Griffin, J.F.T. and Nunn, W.R. Some observations
on Cor nebacterium pyogenes infection of the bovine uterus.
TheA5m974).

34. Witherspoon, D.M., Goldston, R.T. and Adsit, M.E. Uterine culture
and biopsy in the mare. J. Am. Vet. Med. Assoc. -161:1365-1366
(1972) *

JUNE 1988 VOL. 29 NO. 6 1283