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1 Assessment of the bovine uterus with endometritis using Doppler ultrasound

3 Bruno Leonardo Mendonça Ribeiroa; Enoch Brandão de Souza Meira Júniora; Mario Augusto

4 Aleman Reyesa; Eduardo Carvalho Marquesa; Alessandra Figueiredo de Castro Nassarb; Lilian

5 Gregorya.

7 a Departamento de Clínica Médica, Faculdade de Medicina Veterinária e Zootecnia, Universidade de

8 São Paulo. Av. Prof. Orlando Marques de Paiva, 87, Cidade Universitária, São Paulo, Brazil, CEP
9 05508-270. lgregory@usp.br
10 b Laboratório de Bacteriologia Geral. Instituto Biológico. Av. Conselheiro Rodrigues Alves, 1252,
11 Vila Mariana, São Paulo, Brazil, CEP: 04014-002.
12

13 ABSTRACT

14 Postpartum uterine diseases such as metritis and endometritis are highly prevalent in dairy

15 cows. These diseases negatively affect the reproductive performance and consequently economic

16 activity. Cows in the puerperal period presenting endometritis may have alterations in the

17 hemodynamics of the uterine tissue and the uterine arteries, which differ them from healthy cows.

18 Therefore, this study aimed to use the Doppler ultrasonography to describe the hemodynamic changes

19 in the uterus of cows showing endometritis diagnosed between 25 - 35 days postpartum. Eighty-nine

20 Holstein Friesian females with 25 to 35 days postpartum were studied. Cows were assigned to two

21 experimental groups, infected or not infected, according to the results of the endometrial cytology.

22 Clinical examination, vaginoscopy, Doppler ultrasound and sample collection (saline solution was

23 injected and recovered by endoscopie method aiming cytological and microbiological evaluation of

24 the uterus) were also performed. Cows with endometritis had the cervix (P = 0.040) and the left horn

25 (P = 0.020) increased compared to healthy cows. 78.6% of the endometritic cows showed abnormal

26 uterine discharge, while 57.6% of healthy cows had this same condition (P = 0.0005). The spectral

27 Doppler evaluation of the uterine arteries revealed no differences between groups. Bacillus spp.,
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28 Trueperella pyogenes, Escherichia coli and Staphylococcus intermedius were the most isolated

29 bacteria among samples. Higher score or increase of uterus vascularization of the endometrial

30 Doppler was correlated with Trueperella pyogenes (P = 0.0003) and intrauterine heterogeneous

31 content (P = 0.0047). Finally, mesometrial Doppler was correlated with endometrial Doppler (P <

32 0.001), uterine bacteria (P = 0.001) and intrauterine heterogeneous content (P = 0.049). Regarding

33 the evolution of uterine alterations, Doppler ultrasonography provides fast results and is a lesser

34 invasive technique such as uterus biopsie and endometrial citology and gives answers about fertility

35 and uterus health.

36 Keywords: Endometritis, clinical reproduction, infertility

37

38 1.INTRODUCTION

39 High milk production causes high negative energy balance and could cause changes in

40 hormone levels, embryonic losses and a higher incidence of reproductive disorders in dairy cows

41 [1,2]. Unsatisfactory reproductive performance negatively affects productivity [3], decreasing milk

42 production and number of calves [4].

43 The uterine infection, like metritis and endometritis is the most important cause of infertility

44 in dairy cows. Therefore causing decrease in reproductive performance with economic injuries [5,6].

45 Metritis is defined as a severe alteration of all layers of the uterus. In the clinical uterine examination

46 the infected has reddish-brown fetid uterine discharge and systemic clinical signs such as fever and

47 reduced milk production, normally within 21 days postpartum [7]. In contrast endometritis occurs

48 after 21 days postpartum and is the inflammation of the endometrium. Animals show no systemic

49 clinical signs [8]. Cytological endometritis may also occur, with absence of purulent discharge but

50 high number of neutrophils on the endometrial cytological slide[8].

51 By showing the presence, direction and type of blood flow the Doppler ultrasonography

52 technique determines hemodynamics changes wich are important to understand the

53 morphophysiological aspects of the female reproductive tract [9]. This concept is used in women
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54 ginecology to tell a part fertile [10] and infertile women [11]. Doppler ultrasound shows up the

55 changes in the uterine circulation and it is used widely in veterinary reproduction [12,13].

56 It is hypothesized that alterations in the hemodynamics of the uterus tissue and arteries may

57 happened in cows presenting endometritis in the puerperal. Thus, the use of more than one diagnostic

58 technique may help in the identification of uterine diseases. Therefore, this study aimed to use the

59 Doppler ultrasonography to describe the hemodynamic changes in the uterus of cows showing

60 endometritis diagnosed between 25 - 35 days postpartum.

61

62 2.MATERIALS AND METHODS

63

64 2.1 Area characterization and case definition

65 This study was carried out in dairy farms from the State of São Paulo (N = 2) and from the

66 State of Minas Gerais (N=1), Brazil, under the strict regulations of CONCEA (Brazilian commetee

67 for Animal use in experiments, protocol number 5135030214 and approved 27/08/2014 at CEUA,

68 commetee for animal use in experiments at the Veterinary School in the University of São Paulo),

69 equivalent to EU Directive 2010/63/EU for animal experiments. All farms have freestall breeding

70 system and assisted calving. Eighty-nine Holstein Friesian females with 25 to 35 days postpartum

71 were studied. Cows were assigned to two experimental groups according to the results of the

72 endometrial cytology [14] as follows:

73 a) Control group (C): healthy cows (neutrophil polymorphonuclear count < 10%).

74 b) Endometritis group (E): cows diagnosed by cytology (neutrophil polymorphonuclear

75 count ≥10%).

76 Clinical examination, vaginoscopy, Doppler ultrasound and sample collection were also

77 performed, all in the same day.

78
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79 2.2 Clinical examination

80 Female genital tract was evaluated by rectal palpation as proposed by Grunert, Birgel and Vale

81 (2005) [15]. Briefly, ovaries were evaluated searching for follicles and corpus luteum (CL). In the

82 cervix, morphological alterations such as volume was evaluated. In the uterus, location, size,

83 symmetry of the horns and consistency were also investigated. Changes were described according to

84 Grunert, Birgel and Vale (2005) [15] using the classification: from I (not very thick) to VI (very

85 bulky). The horns were classified as symmetrical (s) and asymmetrical (As). According to consistency

86 (C), the uterus was classified as flaccid (CI), reactive (CII), and with vigorous and prolonged

87 contraction (CIII).

88

89 2.3 Vaginoscopy

90 Vaginoscopy was performed after rectal palpation. The speculum was introduced in the vagina

91 to observe the vaginal mucosa, cervix and the presence of discharge according to Grunert, Birgel and

92 Vale (2005) [15].

93
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94 2.4 Ultrasonography and Doppler mode

95 The ultrasound was performed with a m-turbo, Fuji film Sonosite (Bothel, WA, USA) with a

96 micro convex probe (8-5 mhz), as proposed by Meira Jr et al. (2012) [16] and Heppelmann, Krüger

97 and Leidl (2013)[17]. Diameters of the cervix and horns, ovarian structures and the presence of

98 uterine discharge were evaluated. Cervical and uterine diameters were classified as small or negative

99 (< 3.5 cm), medium or suspect (between 3.5 and 5 cm) and large or positive (> 5 cm) (Figure 1).

100 Uterine dimensions were evaluated according to Meira Jr et al. 2012 [16] (Figure 1). The presence of

101 heterogeneous intrauterine contents (hic) and the uterine wall with hyperechoic characteristics were

102 associated with purulent discharge according to Descoteaux, Gnemmi and Colloton (2010) [18]

103 (Figure 2).

104 After the conventional ultrasonographic evaluation, hemodynamic patterns of the left and

105 right uterine arteries were analyzed using the spectral Doppler mode [12]. Maximum flow velocity,

106 pulsatility index and systole/diastole ratio were determined as described by Heppelmann, Krüger and

107 Leidl (2013) [17] (Figure 3).

108 Color Doppler mode was used to evaluate the hemodynamic pattern of the uterine tissue in

109 the intercornual ligament. The colorimetric scale for reproductive patterns [19] was used to establish

110 a parallel with the inflammatory process. The procedures involved a subjective evaluation of the

111 endometrial vascularization (0 - without vascularization to 2 - very vascularized) and the

112 mesometrium (0 - without vascularization to 4 - extremely vascularized), as described by Ginther

113 (2007) [19] (Figures 4 to 8).

114 The ovaries were evaluated for their size, consistency and structures (CL, follicles and cysts).

115 Morphological characteristics of the ovaries were assessed to verify the presence of abnormalities

116 such as cysts or tumors, to determine the existence of cyclic luteal ovarian activity and to estimate

117 the probable phase of the cycle, factors that may affect animal's fertility [20]. Particularly, the

118 vascularization of the CL was assessed by colorimetric Doppler mode, and it was classified as

119 containing or not containing vascularization (Figure 9).

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120
121
122
123
124
125
126
127
128
129 Figure 1: Ultrasound image in transverse section of the cervix (left) and uterine horns (right),
130 using micro probe convex 8.5 Mhz. Measurement in centimeters of the diameter of cervix and uterine
131 horns in bovine.
132

133
134 Source: (RIBEIRO, B.L.M. 2016) [21]
135
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136 Figure 2 : Ultrasound imaging in transverse section of the uterus, using a microconvex 8.5

137 Mhz probe, characterizing intrauterine contents in abnormal proportions; intrauterine-fluid (left) and

138 hyperechoic intrauterine content.

139

140
141 Source: (RIBEIRO, B.L.M. 2016) [21]
142
143
144
145
146
147
148
149
150
151 Figure 3: Ultrasound image in cross section of uterus, using 8.5 Mhz micro probe. Spectral
152 Doppler of right left uterine arteries.
153

154
155 Source: (RIBEIRO, B.L.M. 2016) [21]
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156
157 Figure 4: Characterization of 0 score for the subjective evaluation of the vascularization
158 pattern of mesometrium (M) and 0 score for endometrium (e) by colorimetric evaluation technique
159 in Doppler color mode.

160
161 Source: (RIBEIRO, B.L.M. 2016) [21]
162
163
164
165
166
167
168 Figure 5: Characterization of a score 1 for the subjective evaluation of the vascularization
169 pattern of mesometrium 0 by colorimetric evaluation technique in Doppler color mode.

170
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171 Source: (RIBEIRO, B.L.M. 2016) [21]

172
173 Figure 6: Characterization of score 2 for the subjective evaluation of the pattern of
174 vascularization of the mesometrium and score 0 for endometrium by colorimetric evaluation in
175 Doppler color mode.

176
177 Source: (RIBEIRO, B.L.M. 2016) [21]
178
179 Figure 7: Characterization of score 3 for the subjective evaluation of the vascularization
180 pattern of the mesometrium (m) and score 1 for endometrium (e) by colorimetric evaluation technique
181 in color Doppler mode.

182
183 Source: (RIBEIRO, B.L.M. 2016) [21]
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10

184
185 Figure 8: Characterization of the score 4 for the subjective evaluation of the vascularization
186 pattern of the mesometrium (m) and score 2 for the endometrium ( e ) by colorimetric evaluation in
187 Doppler color mode.

188
189 Source: (RIBEIRO, B.L.M. 2016) [21]
190
191
192
193 Figure 9: Ovarian evaluation with Doppler color mode: ovary with CL without vascularization
194 (left); ovary with CL vascularized (right).

195
196 Source: (RIBEIRO, B.L.M. 2016) [21]
197
198
199 2.5 Hysteroscopy
200
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201 The hysteroscopic examination was performed using a rigid endoscope (430 X 6 mm) with 2
202 channels: one for the passage of 0.9% sterile saline for uterine lumen distension and another for
203 cytological and microbiological samples collection. The optic part of the endoscope was introduced
204 into the vagina, guided by transrectal palpation. Once inside the uterus, the sterile saline was infused
205 to explore this anatomic site. The presence of mucopurulent or purulent inflammatory lesions in the
206 mucosa were considered as signs of endometritis [22]. The images were classified as healthy or
207 unhealthy (presence of pus, fibrin and hyperemia) mucosa. After exploration, a small portion of saline
208 was recovered and used for macroscopic, cytological and microbiological evaluation of the uterus
209 content. After each examination, the optic cable was washed with water only. The work channels
210 were brushed with a flexible 75cm X 0.20mm brush immersed in enzymatic detergent (Hs-enzyme®,
211 Strattner®) and, then, disinfected using paracetic acid (Peroxylife®), according to the manufacturer's
212 instructions.
213 2.6 Uterine cytology

214 One hundred and fifty microliters of the recovered saline were transferred to a cytocentrifuge

215 chamber and samples were fixed and centrifuged on a glass slide for microscopy at approximately

216 550 rpm for 6 minutes [6]. The slides were stained using the Panotipo Rapido® kit, and cytology was

217 performed by counting 100 cells, under magnification of 400x using an optical microscope. The

218 percentage of neutrophil polymorphonuclear cells (% pmn) (Figure 12) was determined. Cows with

219 %Ne > 10% were considered positive to the cytology test [23].

220
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221 2.7 Microbiological

222 Five hundred microliters of the recovered saline were added to thioglycolate broth (Difco®)

223 and kept at 2°-10 °C until processing at the Laboratory of General Bacteriology, Biological Institute,

224 São Paulo. Ten microliters were plated in 5% sheep blood agar and incubated for 72 hours at 37 °C

225 under microaerophilic conditions. Morphological characteristics of each colony and Gram staining

226 were performed. Bacteria species were determined using biochemical tests according to Winn et al.

227 (2008).

228 2.8 Statistical analysis

229 Error normality and homogeneity of variance were analyzed by Shapiro-Wilk Test and

230 Bartlett Test, respectively. Non-normal data were analyzed using a non-parametric test (Kruskal-

231 Wallis test) (proc npar1way). Differences between the groups (control and endometritis) were

232 evaluated by ANOVA, reaffirmed by a t-test (JMP 12.0 - SAS). Same conditions (proc glimmix from

233 SAS) were used for binary data ("subjective evaluation score of endometrial perfusion” and

234 “subjective evaluation score of mesometrium perfusion"). Correlation between variables were

235 performed using Spearmann correlation test for variables that did not present normal distribution

236 (proc corr from SAS). A significance level of 5% was used for all tests. All tests were performed in

237 SAS.

238
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13

239 3. Results
240

241 One hundred cows were initially examined, but due to problems with the ultrasound device,

242 eleven samples were lost. So, 89 cows were enrolled in this study, and they were divided accordingly

243 to citology results into two groups: control group (E) (N= 33) and endometritis group (N = 56).

244 3.1 Ultrasonographic exam

245 Cows with endometritis had the cervix (P = 0.040) and the left horn (P = 0.020) increased

246 compared to healthy cows (Table 1). In the endometritis group, 78.6% of the cows showed abnormal

247 uterine discharge, while in health group only 57.6% of the animals showed this same condition (P =

248 0.0005) (Table 2). Besides, intrauterine heterogeneous content were increased in sick cows (66.0%)

249 compared to healthy cows (30.3%) (P = 0.0011) (Table 3). Evaluation of endometrial and

250 mesometrium vascularization by the colorimetric method revealed differences in the vascularization

251 score (vc) of the endometrium between healthy (C) and endometritis group (C) (P < 0.05) (Table 4

252 and 5). During ovarian examination, presence or absence of CL were not diferent between group (C)

253 and group (E) (Table 6). In addition, the absence of vascularizated corpus luteum in the left ovary

254 was increased in the endometritis group (P = 0.029) (Table 7). The spectral Doppler evaluation of

255 the uterine arteries revealed no differences between groups (Table 8).

256
257 Table 1 - Average diameter between measurements of linear ultrasonography
258 of the cervix and uterine horns with Endometritis
Uterine structures Control Endometritis P value
Cervical (cm) 3.55(±0,13) 3.89(±0,01) 0.040
Right horn (cm) 2.56(±0,11) 2.77(±0,08) 0.100
Left horn (cm) 2.48(±0,13) 2.89(±0,10) 0.020
259 Source: (RIBEIRO, B.L.M., 2016).
260
261
262 Table 2- Presence of intrauterine fluid (IUF) in cows with and without Endometritis
Control Endometritis
P value
n (%) n (%)
Presence de IUF (14/33) 42.42% (44/56) 78.57%
0,0005
Absence de IUF (19/33) 57.58% (12/56) 21.43%
263 Source: (RIBEIRO, B.L.M., 2016).
264
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14

265
266 Table 3 - Animals that presented intrauterine heterogeneous content (IUHC) in abnormal
267 amounts throughout the experiment
Control Endometritis
P value
n (%) n (%)
Presence de IUHC (10/33) 30.30% (37/56) 66.07%
0,0011
Absence de IUHC (23/33) 69.70% (19/56) 33.93%
268 Source: (RIBEIRO, B.L.M., 2016).
269
270 Table 4 - Evaluation of vascular pattern of endometrium
Control Endometritis
EV- Doppler Endometrium P value
n (%) n (%)
0 (31/33) 93.94% (42/56) 75.00%
0,0246
1+2 (2/33) 6.06% (14/56) 25.00%
271 Source: (RIBEIRO, B.L.M., 2016).
272 Legend: 0 = non-vascularized; 1 = poorly vascularized; 2 = very vascularized
273
274 Table 5- Frequency of evaluation of the vascular pattern of Mesometrium
Control Endometritis
EV- Doppler Mesometrium P value
n (%) n (%)
0 (10/33) 30.30% (8/56) 14.29%
1 (11/33) 33.33% (9/56) 16.07%
2 (5/33) 15.15% (14/56) 25.00% 0,0344
3 (5/33) 15.15% (12/56) 21.43%
4 (2/33) 6.06% (13/56) 23.21%
275 Source: (RIBEIRO, B.L.M., 2016).
276 Legend: 0 = non-vascularized; 1 = poorly vascularized; 2 = vascularized; 3 = very vascularized;
277 4 = extremely vascularized.
278
279
280 Table 6 - Frequency of Presence of CL as a function of Endometritis
Control Endometritis
P value
n (%) n (%)
Presence de CL Right (15/33) 45.45% (24/56) 42.86%
0.811
Absence de CL Right (18/33) 54.55% (32/56) 57.14%
Presence de CL Left (16/33) 48.48% (18/56) 32.14%
0.125
Absence de CL Left (17/33) 51.52% (38/56) 67.86%
281 Source: (RIBEIRO, B.L.M., 2016).
282
283 Table 7 - Presence of CLv as a function of Endometritis
Control Endometritis
P value
n (%) n (%)
Presence de CLv Right (12/33) 36.36% (19/56) 33.93%
0.815
Absence de CLv Right (21/33) 63.64% (37/56) 66.07%
Presence de CLv Left (15/33) 45.45% (13/56) 23.21%
0.029
Absence de CLv Left (18/33) 54.55% (43/56) 76.79%
284 Source: (RIBEIRO, B.L.M., 2016).
285
286
287 Table 8 - Spectral Doppler average in Uterine Arteries
Doppler spectral Control Endometritis P value
Vmax-Right 88.47 (±5.33) 93.84 (±4.14) 0.428
IR- Right 0.85 (±0.35) 0.84 (±0.02) 0.766
IP-Right 2.57 (±0.20) 2.66 (±0.16) 0.744
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15

S/D Right 22.16 (±6.39) 12.63 (±4.96) 0.242

MTM-Right 20.01 (±2.35) 21.84 (±1.83) 0.541
Flow- Right 0.94(0.11) 1.02 (0.086) 0.540
Vmax-Left 90.19 (±4.47) 85.77 (±3.38) 0.433
IR- Left 0.80 (±0.03) 0.85 (±0.03) 0.217
IP-Left 2.62 (±0.27) 2.70 (±0.20) 0.809
S/D- Left 19.47 (±5.67) 12.19 (±4.30) 0.309
MTM-Left 21.50 (±2.05) 18.93 (±1.55) 0.321
Flow- Left 1.01(0.09) 0.89(0.07) 0.321
288 LEGEND: VMAX- MAXIMUM SPEED; IR- INDEX RESISTANCE; IP-INDEX PULSATILITY; S / D-
289 SYSTOLE AND DIATST; MTM- AVERAGE TIME
290
291
292
293
294

295 3.3 Microbiological findings

296 Bacillus spp., Trueperella pyogenes, Escherichia coli and Staphylococcus intermedius were

297 the most isolated bacteria among samples. Yeast was also detected in 25% of samples (Table 9).

298

299 Table 9 Identification of the microorganisms in samples

Endometritis
n (%)
Bacterium+Yeast (6/85) 7.06%
Yeast (21/85) 24.71%
Bacterium (38/85) 44.71%
T. pyogenes (10/85) 11.76%
S. intermedius (7/85) 8.24%
S.auerus (1/85) 1.18%
Streptococcussp. (1/85) 1.18%
Bacillussp.; (13/85) 15.29%
E. coli (7/85) 8.24%
Proteussp. (2/85) 3.53%
300 Source: (RIBEIRO, B.L.M., 2016).
301

302

303 3.4 Correlations with variables

304 A positive correlation between mixed culture (bacteria + yeasts) and endometritis was

305 observed (P = 0.0003). In addition, weak correlations were observed between intrauterine

306 heterogeneous content (r = 0.34), intrauterine fluid (r = 0.37) and endometritis. The disease was also
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16

307 correlated with Doppler ultrasonography (P = 0.0027) and endometrial Doppler (P = 0.0383). A weak

308 and negative correlation with the presence of corpus luteum in the vascularized left ovary (P = 0.0359)

309 and endometritis was also detected (Table 10).

310 Table 10 - Correlation between Endometritis and other

311 variables
Endometritis
P value
-1<r>1
Bacterium+ Yeast 0.38 0.0003
IUHC 0.34 0.0012
IUF 0.37 0.0005
Doppler Mesometrium 0.32 0.0027
Eclv -0.23 0.0359
Doppler Endometrium 0.22 0.0383
312 Source: (RIBEIRO, B.L.M., 2016).
313 Legend: IUHC = intrauterine heterogeneous content;
314 IUF = intrauterine fluid; eclv = left vascularized
315 corpus luteum.

316

317 Intrauterine fluid (IUF) was correlated with Trueperella pyogenes (r = 0.27), yeast (r = 0.25),

318 Staphylococcus intermedius (r = 0.23), and Escherichia coli (r = 0.23) (P < 0.05) (Table 13). Strong

319 correlations between intrauterine heterogeneous content (IUHC), and IUF (P = 0.0001) and the

320 presence of bacteria (P = 0.0001) were detected. IUHC was also correlated with Trueperella pyogenes

321 (r = 0.36), Escherichia coli (r = 0.30), Bacillus spp. (r = 0.25), Staphylococcus intermedius (r = 0.22)

322 and yeast (P < 0.05) (Table 14). Endometrial Doppler was correlated with Trueperella pyogenes (P

323 = 0.0003) and IUHC (P = 0.0047) (Table 15). Finally, mesometrial Doppler was correlated with

324 endometrial Doppler (P < 0.001), uterine bacteria (P = 0.001) and IUHC (P = 0.049). A negative

325 correlation with the presence of yeast (P = 0.022) was also observed (Table 16).

326

327

328

329

330

331
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332 Table 11 - Correlation between Presence of Intrauterine Fluid (IUF)

333 compared to Presence of microorganisms
IUF
P value
-1<r>1
Bacterium 0.52 0.0001
T.pyogenes 0.27 0.0109
Yeast 0.25 0.0215
S.intermedius 0.23 0.0344
E. coli 0.23 0.0345
334 Source: (RIBEIRO, B.L.M., 2016).
335 Legend: T. pyogenes= Trueperella pyogenes; S. intermedius=
336 Staphylococcus intermedius E. coli = Escherichia coli
337
338
339
340 Table 12 - Correlation of animals with heterogeneous content compared to other variables
341
342 IUHC
P value
-1<r>1
IUF 0.72 0.0001
Bacterium 0.64 0.0001
T. pyogenes 0.36 0.0007
E. coli 0.3 0.0052
Yeast 0.27 0.0135
Bacillussp. 0.25 0.0223
S. intermedius 0.22 0.0434
343 Source: (RIBEIRO, B.L.M., 2016).
344 Legend: IUF = Intrauterine fluid; T. pyogenes = Trueperella pyogenes; S. intermedius=
345 Staphylococcusintermedius;E.coli = Escherichia coli.
346
347
348 Table 13 -Endometrium color Doppler correlation compared to other variables
Doppler Endometrium
P value
-1<r>1
T. PYOGENES 0.38 0.0003
Bacterium 0.38 0.0003
IUHC 0.36 0.0047
349 Source: (RIBEIRO, B.L.M., 2016).
350 Legend: IUHC = intrauterine heterogeneous content;T. pyogenes = Trueperella pyogenes.
351
352
353 Table 14 - Doppler color correlation of Mesometrium compared with other
354 variables
Doppler
P value
Mesometrium
Doppler
0.52 0.000
Endometrium
Bacterium 0.35 0.001
Yeast -0.25 0.022
IUHC 0.21 0.049
355 Source: (RIBEIRO, B.L.M., 2016).
356 Legend: IUHC = intrauterine heterogeneous content.
357

358
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359 4. Discussion
360
361 Endometritis is a uterine disease and is related to negative reproductive performance [8].

362 Besides physical examination, new diagnostic tools are important to improve the precocity in the

363 diagnosis of this disease [24].

364 Our results showed differences between the size of the cervix and a larger uterus in cows with

365 endometritis. After delivery, the uterus is greatly enlarged (approximately 8 to 10 kg). In general, the

366 uterine macroscopic involution occurs between 3 to 5 weeks postpartum, when the uterus should

367 weight approximately 0.9 kg and the diameter of the pre-gravid uterine horn should be less than 5

368 cm, total involution of the cervix occurs between 4 to 6 weeks postpartum [25]. A new pregnancy

369 depends on the anatomical and functional return of the genital tract to a state similar to that before

370 pregnancy [26]. In the present research, the increased left horn in animals with endometritis was

371 observed. This information is in agreement with other researches that proved that a uterine infection

372 provides a delayed involution [27, 26]. In this study, cows with endometritis presented larger cervix

373 diameter compared to healthy animals. LeBlanc et al. (2002) [5] highlighted that cows with clinical

374 endometritis showed cervical measurement ≥7.5 cm in diameter after 20 days postpartum and they

375 presented mucopurulent or purulent discharge detected by vaginoscopy after 26 days postpartum. Our

376 study highlights that endometritis was increased in cows showing abnormal intrauterine fluid

377 accumulation, and it was in agreement with several studies [28, 29, 16]. Intrauterine fluid was

378 correlated with Trueperella pyogenes, yeast, Staphylococcus intermedius, Escherichia coli, which

379 suggests that these agents cause inflammation of the uterine mucosa and proliferation of the

380 endometrial glands, leading to a greater accumulation of intrauterine fluid.

381 Endometritis was highly detected in cows showing intrauterine heterogeneous content (IHC).

382 Meira Jr. et al. (2012) [16] also described high correlation between IHC and the diagnosis of

383 endometritis. IHC was also associated with Trueperella pyogenes, Escherichia coli, Bacillus spp. and

384 Staphylococcus intermedius, and yeast, demonstrating that these microorganisms may cause genital

385 discharge [16].

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386 The presence of vascularization in the endometrium (degrees 1 and 2) was increased in cows

387 with endometritis, suggesting differences between healthy and unhealthy cows. The color Doppler

388 mode provides colored images of the blood fow, allowing estimation of the tissue vascularization

389 [34]. Ginther (2007) [19] proposed a model to evaluate the uterine hemodynamic associated with

390 endometritis. This model was modified in the present research, and our results showed a few animals

391 with alterations in the subjective evaluation score of endometrial vascularization, and a positive

392 correlation with Trueperella pyogenes.

393 Ultrasonographic evaluation of the endometrium using color Doppler mode showed a positive

394 correlation with endometritis. This information emphasizes the possible combination of this new

395 noninvasive technique with the standard diagnostic methods to predict uterine disease. No difference

396 was detected at data analisys of spectral Doppler mode. Herzog and Bollwein (2013) [2] reported

397 increased pulsatility at 24 hours postpartum with a peak on the 28th day, decreasing progressively

398 until 90th day. An inverse proportional relation was observed in blood flow, which already starts high

399 in the postpartum and decreases until around the 28th day maintaining basal levels up to 90th day.

400 Clinical and histologic uterine involution ranges from 21 to 50 postpartum, and this may differs from

401 the recovery of hemodynamic patterns of the uterus that requires more time [30].

402 Advancing the understanding of uterine perfusion, Bollwein et al. (2002) [13] when evaluating

403 pregnant animals, concluded that throughout pregnancy there is a strong increase of blood flow in the

404 uterine arteries. However, they observed that the resistivity index decreased in the first 8 months of

405 gestation and remained at a relatively constant level until the calf was born. It is known that during

406 the diestro the blood flow velocity remains constant and are correlated to the plasma concentration

407 estrogen and progesterone, indicating a moderate positive correlation Bollwein et al. (2016) [30].

408 These results indicate that there are other factors involved in the regulation of blood flow [31].

409 In healthy cows the uterine blood flow decreased after the complete uterine involution.

410 However, cows with uterine disease presents slow uterine involution, occurring between 45 and 65

411 days postpartum. These results indicate an association between incomplete uterine involution and
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412 regeneration of the uterine vascular layer in cows with puerperal disease. Changes in uterine perfusion

413 are greatly pronounced during the first 4 days after delivery. Uterine puerperal diseases such as

414 retention fetal attachments and metritis have a negative impact on uterine involution [17]. Thus,

415 uterine blood flow is affected by puerperal uterine diseases. Cows with retained placenta have higher

416 resistivity index than healthy cows. On the 8th day postpartum, cows with metritis had higher blood

417 flow and decreased pulsatility in the arteries compared to healthy cows [17].

418 Evaluation of the uterine and ovarian vascularization helps to determine the best moment to

419 perform artificial insemination [31]. The vascular perfusion of corpus luteum and uterus is greater

420 during the first follicular wave compared to the second follicular wave of the estrous cycle [31]. The

421 color Doppler mode identifies follicles in normal development and predicts the next ovulate. In this

422 research, the ovarian evaluation showed that some cows with endometritis had no corpus luteum in

423 both the right and left ovary. However, animals with right and left CL presented 61.54% and 52.94%

424 vascularized, respectively. Color Doppler mode showed that cows with no vascularized left and right

425 corpus luteum had endometritis, demonstrating that there is a interference in reproduction

426 performance even in endometritic cows exhibiting normal estrous cycle.

427 Microbiological results showed the presence of Bacillus spp., Trueperella pyogenes,

428 Staphylococcus intermedius, Escherichia coli, Streptococcus spp., Proteus spp., Staphylococcus

429 aureus, Enterobacteria and Serratia spp. besides fungus and yeast. Santos, Gilbert and Bicalho

430 (2011) [32] reported that Escherichia coli, Streptococcus spp., Trueperella pyogenes and

431 Fusobacterium necrophorum are the main bacteria that commonly contaminate the uterine lumen

432 after childbirth. These microorganisms were also associated with some uterine diseases. Trueperella

433 pyogenes acts in synergisms with Fusobacterium necrophorum, Bacteroides spp., and Prevotella spp.

434 [33]. Potter et al. (2010) [34] reported that Trueperella pyogenes is recognized as an important

435 pathogen associated with endometritis due to its persistence in the contaminated uterus. Recently,

436 Boer et al. (2015) [35] found that cows with any bacterial growth at 21 days postpartum, regardless

437 of bacterial species, had a lower conception rate. Using the 16S rRNA sequencing, Machado et al.
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438 (2012) [36] detected Trueperella pyogenes increased in cows with endometritis. Yeasts have been

439 associated with chronic endometritis in women [40]. In the present research, yeasts were detected in

440 22.5% of cows. The onset of Trueperella pyogenes was correlated with uterine inflammation seen by

441 the colorimetric ultrasonographic method.

442 Although endometritis has a spontaneously healing condition, Dubuc et al. (2011) [7] found

443 63% of spontaneous cure, while LeBlanc et al. (2002a) [5] described 77% self-healing. It still causes

444 many economic and reproductive damages in the dairy industry. In order to reduce this problem, it is

445 important to use new tools to improve the precision and the precocity in the diagnostic, upgrading the

446 reproductive indices.

447 5. Conclusions

448 It is detected the relationship between the diameter of the cervix and the uterine horns with

449 endometritis. This relationship was notorious when the presence of intrauterine fluid and intrauterine

450 heterogeneous content was verified. Color Doppler showed the association between the

451 vascularization of inflamed tissue and endometritis. However, performing spectral Doppler

452 ultrasound showed no differences between healthy and unhealthy animals. The microbiological

453 examination showed that Trueperella pyogenesis and Escherichia coli have an important role in the

454 development of endometritis, corroborating other researches. Less invasive techniques with fast

455 results such as Doppler ultrasonography can provide satisfactory answers regarding to the evolution

456 of uterine alterations, improving reproductive rates.

457

458 Acknowledgements and Funding: Authors thank São Paulo Research Foundation (Process

459 number 2014/02676-4)

460 Declarations of Interest: None

461

462

463
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464 REFERENCES

465

466 [1] Radostits OM; Gay CC; Hinchcliff KW; Constable PD. Veterinary medicine: a textbookof disease
467 of cattle, sheep, pigs, goats and horses. 9. Ed. Philadelphia: WB. Sauders, 2007.
468 [2] Bollwein H; Luttgenau J; Herzog K. Bovine luteal blood flow: Basic mechanism and clinical
469 relevance. Reprod, Feril, Dev. V.25, n.1, p.71-79, 2013.
470 [3] Grohn YT; Rajala-Schults PJ. Epidemiology of reproductive performance in dairy cows. Anim
471 Reprod Sci, 60-61:605-614, 2000.
472 [4] Leblanc S. Economics if improving reproductive performance in dairy herds. WCDS advances in
473 Dairy technology. 19:201-214, 2007.
474 [5] Leblanc SJ; Duffield TF; Leslie KE; Bateman KG; Keefe GP; Walton JS; Johnson WH. Defining
475 and diagnosing postpartum clinical endometritis and its impact on reproductive performance in dairy
476 cows. J. Dairy Sci, 85(9):2223-2236, 2002.
477 [6] Gilbert RO; Shin ST; Guard CL; Erb HN; Frajblat M. Prevalence of endometritis and its effects
478 on reproductive performance of dairy cows. Theriogenology. 64(9):1879-1888, 2005.
479 [7] Dubuc J; Duffield TF; Leslie KE; Walton JS; Leblanc SJ. Effects of postpartum uterine diseases
480 on milk production and culling in dairy cows. J. dairy Sci. 94(3):1339-1346, 2011.
481 [8] Sheldon IM; Lewis GS; Leblanc S; Gilber RO. Defining postpartum uterine disease in cattle.
482 Theriogenology. 65(8):1516-1530, 2006.
483 [9] Carvalho CF; Cjammas MC; Cerri GG. Princípios físicos do Doppler em ultra-sonografia. Cienc
484 Rural. 38(3):872-879, 2008.
485 [10] Steer CV; Campbell S; Pampiglione JS; Kingsland CR; Mason BA; Collins WP. Transvaginal
486 colour flow imaging of the uterine arteries during the ovarian and menstrual cycles. Hum Reprod.
487 5(4):391-395, 1990.
488 [11] Steer CV; RTan SL; Mason BA; Campbell S. Midluteal-phase vaginal color Doppler assessment
489 of uterine artery impedance in subfertile population. Fertil Steril. 61(1):53-58, 1994.
490 [12] Bollwein H; Mayer HH; Maierl J; Weber F; Baumgartner U; Stolla R. Transrectal color
491 Dopplersonography of the A uterine in cyclic mares. Theriogenology. 49:1483-1488, 2000.
492 [13] Bollwein H; Baumgartner U; Stolla R. Transrectal Doppler sonography of uterine blood flow in
493 cows during pregnancy. Theriogenology. 57(8):2053-2061, 2002.
494 [14] Kasimanickam R; Duffield TF; Foster RA; Gartley CJ; Leslie KE; Walton JS; Johnson WH. The
495 effect of a single administration of cephapirin or cloprostenol on the reproductive performance of
496 dairy cows with subclinical endometritis. Theriogenology. 63(3):818-830, 2005.
 bioRxiv preprint doi: https://doi.org/10.1101/826065; this version posted October 31, 2019. The copyright holder for this preprint (which
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under aCC-BY 4.0 International license.

23

497 [15] Grunert E; Birgel EH; Vale WG. Patologia e clínica da reprodução dos animais mamíferos
498 domésticos: ginecologia. São Paulo: Varela,2005. 551p.
499 [16] Meira JR EBS; Henriques LCS; Sá LRM; Gregory L. Comparison of ultrasonography and
500 histopathology for diagnosis of endometritis in Holstein-Friesian cows. J Dairy Sci. 95(2):6969-6973,
501 2012.
502 [17] Heppelmann M; Kruger L; Leidl HB. Science transrectal Doppler sonography of uterine blood
503 flow during the first two weeks after parturition in Simmenthal heifers. J Vet Sci 14(3):323-327,
504 2013.
505 [18] Descoteaux L; Gnemmi G; Colloton J. Ruminant and camelid reproductive ultrasonography.
506 Ames:Blackwell Publishing, 2010.
507 [19]Ginter OJ. Ultrasonic imaging and animal reproduction: color-Doppler ultrasonography. Cross
508 Plains, WI: equiservices publishing, 2007, v.4, 258p.
509 [20] Mello RC. Perdas reprodutivas em femeas bovinas. Agropecuária científica no semiárido.
510 10(4):07-23, 2014.
511 [21] Ribeiro,BLM. Avaliação do útero bovino com endometrite utilizando a técnica de
512 ultrassonografia Doppler. Dissertação de Mestrado, Universidade de São Paulo, Faculdade de
513 Medicina Veterinária e Zootecnia 72 f., 2016.
514 [22] Madoz LV; Giuliodori MJ; Migliorisi AL; Jaureguiberry M; De la Sota RL. Endometrial
515 cytology, biopsy and bacteriology for the diagnosis of subclinical endometritis in grazing dairy cows.
516 J Dairy Sci. 97(1):195-201, 2014.
517 [23] Kasimanickam R; Duffield TF; Foster RA; Gartley CJ; Leslie KE; Walton JS; Johnson WH.
518 Endometrial cytology and ultrasonography for the detection of subclinical endometritis in postpartum
519 dairy cows. Theriogenology. 62(1-2): 09-23, 2004.
520 [24] Dubuc J; Duffield TF; Leslie KE; Walton JS; Leblanc SJ. Definitions and diagnosis of
521 postpartum endometritis in dairy cows. J Dairy Sci. 93(11):5225-5233, 2010.
522 [25] Morrow DA; Roberts SJ; Mcentee K; Gray HG. Postpartum ovarian activity and uterine
523 involution in dairy cattle. J Am Vet Med Assoc. 149:1596-1609, 1966.
524 [26] Mateus, L; da Costa LL; Bernardo F; Silva JR. Influence of puerperal uterine infection on uterine
525 involution and postpartum ovarian activity in dairy cows. Reprod Domest Anim. 37(1):31-35, 2002.
526 [27] Slama H.; Vaillancourt D; Goff AK. Pathophysiology of puerperal period: relationship between
527 prostaglandin E2 (PGE@) and uterine involution in the cow. Theriogenology. 36(6):1071-1090,
528 1991.
529 [28] Barlund, C S; Carruthers, T D; Waldner, C L; Palmer, CWA. Comparison of diagnostic
530 techniques for postpartum endometritis in dairy cattle. Theriogenology, v. 69, n. 6, p. 714-723, 2008.
531 [29] Oral , H; Sozmen, M.; Serin, G.; Kaya, S. Comparison of the cytobrush technique, vaginoscopy
532 and transrectal ultrasonography methods for the diagnosis of postpartum endometritis in cows. J
 bioRxiv preprint doi: https://doi.org/10.1101/826065; this version posted October 31, 2019. The copyright holder for this preprint (which
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
available under aCC-BY 4.0 International license.

24

533 Anim Vet Adv, v. 8, n. 7, p. 1252-1255, 2009.

534 [30] Bollwein, H; Heppelmann, M; Johannes, L. Ultrasonographic Doppler use for female
535 reproduction management. Vet Clin North Am Small Anim Pract, v. 32, n. 1, p. 149-164, 2016.
536 [31] Honnens, A; Voss, C; Herzog, K; Niemann, H; Rath, D; Bollwein, H. Uterine blood flow during
537 the first 3 weeks of pregnancy in dairy cows. Theriogenology, v. 70, p. 1048–56, 2008.
538 [32] Santos TM, Gilbert RO, Bicalho RC. Metagenomic analysis of the uterine bacterial microbiota
539 in healthy and metritic postpartum dairy cows. J Dairy Sci 94(1): 291–302, 2011.
540 [33] Bicalho ML, Machado VS, Oikonomou G, Gilbert RO, Bicalho RC. Association between
541 virulence factors of Escherichia coli, Fusobacterium necrophorum, and Arcanobacterium
542 pyogenes and uterine diseases of dairy cows. Vet Microbiol 57(1-2):125-31, 2011.
543 [34] Potter, TJ; Guitian, J; Fishwick, J; Gordon, PJ; Sheldon, IM. Risk factors for clinical endometritis
544 in postpartum dairy cattle.Theriogenology, v.74, p.127–134, 2010.
545 [35] Boer, M; Buddle, BM; Heuer, C; Hussein, H; Zheng, T; Leblanc, SJ; McDougall, S. Associations
546 between intrauterine bacterial infection, reproductive tract inflammation, and reproductive
547 performance in pasture-based dairy cows. Theriogenology, v. 83, p. 1514-1515, 2015.
548 [36] Machado, VS; Oikonomou, G; Bicalho, MLS; Knauer, WA; Gilbert, R; Bicalho, R C
549 Investigation of postpartum dairy cows’ uterine microbial diversity using
550 metagenomicpyrosequencing of the 16S rRNA gene. Vet Microbiol, v. 159, n. 3, p. 460-469, 2012.
551 [37] Cicinelli, E; De Ziegler, D; Nicoletti, R; Colafiglio, G; Saliani, N; Resta, L; Rizzi, D; De Vito,
552 D. Chronic endometritis: correlation among hysteroscopic, histologic, and bacteriologic findings in a
553 prospective trial with 2190 consecutive office hysteroscopies. Fertil Steril, v. 89, n. 3, p. 677-684,
554 2008.