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Effect of age and endometrial degenerative changes on uterine blood flow during early gestation in mares
Accepted Manuscript Effect of age and endometrial degenerative changes on uterine blood flow during early gestation in mares J.C. Ferreira, H.S. Canesin, F.S. Ignácio, N.S. Rocha, C.R. Pinto, C. Meira PII:
S0093-691X(15)00318-0
DOI:
10.1016/j.theriogenology.2015.06.013
Reference:
THE 13236
To appear in:
Theriogenology
Received Date: 13 February 2015 Revised Date:
10 June 2015
Accepted Date: 18 June 2015
Please cite this article as: Ferreira JC, Canesin HS, Ignácio FS, Rocha NS, Pinto CR, Meira C, Effect of age and endometrial degenerative changes on uterine blood flow during early gestation in mares, Theriogenology (2015), doi: 10.1016/j.theriogenology.2015.06.013. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Effect of age and endometrial degenerative changes on uterine blood flow during
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early gestation in mares
3 4 J. C. Ferreira1,3*, H. S. Canesin1, F. S. Ignácio1; N.S. Rocha2; C.R. Pinto3; C.
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Meira1
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Univ Estadual Paulista (UNESP), Department of Animal Reproduction and
Veterinary Radiology, SP18618-970, Brazil
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970, Brazil
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USA
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Univ Estadual Paulista (UNESP), Department of Veterinary Clinics, SP18618-
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School of Veterinary Medicine, Louisiana State University, Baton Rouge 70803,
* corresponding author:
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Jair Camargo Ferreira
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phone: 225-733-2384
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e-mail: [email protected]
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address: 5284 Brightside View apt 2, Baton Rouge, LA 70820, USA
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REVISED
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Doppler ultrasonography is a noninvasive pulse wave technique currently
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used for in vivo evaluation of the uterine blood flow of pregnant and non-pregnant
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mares [1]. An uninterrupted embryo-maternal interaction is essential for the
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establishment and maintenance of the gestation [2, 3]. The uterine blood supply
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increases from the second week of pregnancy in mares [4] subsequently to a
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conceptus-induced endometrial vasculogenesis [5]. However, there has been no
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studies reporting on waveforms from arteries of mesometrium attachment during
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the diestrus and first days of pregnancy. Therefore, the correlation between
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objective and subjective evaluation of the equine uterine blood flow during the
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early gestation remains unknown.
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In multiparous-aged mares, poor uterine blood flow [6, 7, 8] and subfertility [9,
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10] have been related with elastosis of endometrial vessels. Reduced uterine
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blood flow associated with poor placental development [11] and compromised
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haematological contact at the fetomaternal interface [12] have been observed in
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pregnant aged mares.
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Disturbances of the uterine blood flow during the early gestation in mares with
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age-related endometrial degenerative changes have not been investigated using
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Doppler ultrasonography. Therefore, the overall purpose of the present study was
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to characterize the uterine blood flow of mares of different ages and endometrial
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histopathological classification during the first 20 days of gestation.
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2. Materials and Methods
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2.1 Animals and experimental groups
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The mares were kept under natural light in an open shelter and outdoor
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paddock at the Reproduction Center of the Department of Animal Reproduction
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and Veterinary Radiology at the São Paulo State University (UNESP), Campus of
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Botucatu. Animals were handled in accordance with the São Paulo State
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University Guide for Care and Use of Agricultural Animals in Research (protocol
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number 13/2012-CEUA). The mares were of mixed breeds weighing 270 to 410
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kg and were 4 to 18 years old. Animals were maintained on grass hay and
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pelleted feed with free access to water and trace-mineralized salt. Score for body
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condition for all mares throughout the experiment was ≥ 7 [13].
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B-mode ultrasonography exam was done once daily for monitoring of follicular
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development and detection of ovulation. Day of ovulation was considered D0. All
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mares were submitted to ovulation inducing treatment with 2500 IU of hCG when
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a dominant follicle reached a diameter ≥ 35mm associated with uterine edema.
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Study 1: Effect of semen infusion and conception on uterine hemodynamics of mares
Twenty-eight mares were retrospectively assigned into three experimental
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groups according to insemination procedure and presence of the embryonic
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vesicle: Non-inseminated group (n=7 mares; non-AI), Inseminated Non-pregnant
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group (n=7 mares; AI-NP) and Inseminated pregnant group (n=14 mares; AI-P).
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The mean ages of non-AI, AI-NP, and AI-P ranged from 7 to 12 years (9.5±0.8,
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10.3±0.9 and 9.7±0.9, respectively).
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Study 2: Effect of age on the uterine blood flow of mares during the early gestation
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Twenty-one pregnant mares were used to evaluate the temporal relationship
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between age and uterine blood flow throughout the first 20 days of gestation. The
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mares were assigned to three age groups (n=7 mares/ group): young (≤6 years,
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5.5±0.2 years), adult (from 8 to 12 years, 10.1±0.5 years) and old (≥15 years,
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17.2±0.6 years). Age of mares was estimated from dental characteristics as
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previously described [14].
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Study 3: Effect of endometrial degenerative changes on the uterine blood flow of mares during the early gestation
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Twenty-one pregnant mares were used to study the influence of endometrial
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degenerative changes in the uterine blood flow during early gestation. Initially,
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endometrial samples from all mares were obtained using transcervical uterine
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biopsy and stained with hematoxilin and eosin. Endometrial degenerative
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changes were classified into four categories (Figure 1) as previously established
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[15]. Therefore, the mares were assigned in three groups (n=6 mares/group)
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based on the degree of endometrial histopathological changes: GI (category I),
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GII (categories IIA and IIB) and GIII (category III).
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2.2 Ultrasonography and Doppler End Points
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Mares were scanned using a triplex B-mode (gray scale) and pulse wave
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color Doppler ultrasound instrument (Sonoace Pico; Medison do Brasil Ltda)
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equipped with a linear-array multifrequency transducer (LV5-9CDn, 5 to 9 MHz)
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with a beam-field width of 60 mm. Pharmacological sedation was not used for
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any examination using ultrasonography. The principles, techniques, and
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interpretations of B-mode examinations of the mare reproductive tract have been
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reviewed [16]. The brightness and contrast controls of the monitor, and the gain
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controls of the scanner, were standardized to constant settings [17]. Uterine blood flow of pregnant and non-pregnant mares was based on the
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pulsatility index (PI) from arteries attached to the mesometrium and uterine
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vascular perfusion. In Study 1, data collection was done once daily during the first
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20 days of pregnancy in AI-P group and between D0 and D12 in AI-NP and non-
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AI groups. In Studies 2 and 3, Doppler end points were evaluated daily from D0
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and D20.
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Vascular perfusion from both uterine horns was subjectively estimated by one
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operator using power-flow Doppler mode. Only color signals that appeared to be
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within the limits of the uterus (endometrium and myometrium) were considered.
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Uterine vascular perfusion was scored from 0 to 4 (minimum to maximum,
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including fractional scores), considering the extension of uterine tissue with color
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signals of blood flow, during real-time cross-sectioning scan of the uterus in a
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slow continuous motion for two minutes.
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Spectral assessment of arterial PI was performed as described previously [2].
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The formula for calculating spectral data has been previously described [18]. To
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produce a spectral waveform, each entire mesometrium attachment was initially
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scanned in a slow, continuous motion using the power-flow Doppler mode.
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Subsequently, in spectral mode, the sample-gate cursor was placed on an artery
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of the mesometrium attachment. Spectral waveform, with a minimum of three
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cardiac cycles, was generated and one of the cycles was used to measure the
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spectral end points. The procedure was repeated twice, and the average was
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used for statistical analyses.
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Mares from AI-NP and AI-P groups and from Part 2 and 3 were artificially
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inseminated 24 hours after induction of ovulation with 2500 UI of hCG,
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administered when a preovulatory follicle reached a diameter ≥ 35 mm
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associated with uterine edema. Semen from one stallion of proven fertility was
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used. After collection, semen was diluted with skim milk extender (BotuSÊMEN®;
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Botupharma) to obtain an insemination dose of 50 x 106/mL progressively motile
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spermatozoa in a final volume of 20 to 30 mL. The insemination dose was
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maintained cooled at 15oC using an insulated container (BotuBOX®; Botupharma)
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until the time of insemination. The semen dose was placed into the body of the
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uterus using a standart AI catheter.
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Pregnancy exam was performed once daily, beginning on D9, using
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conventional B-mode ultrasonography. In AI-NP mares, a final ultrasound exam
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was performed on D15 to confirm the absence of an embryonic vesicle. Early
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pregnancy termination was induced with a single injection of Dinoprost
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tromethamine (10mg; IM) after end point collection ending.
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2.4 Statistical analyses
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The scores for uterine vascular perfusion and spectral end points were
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analyzed by a two-way repeated measure (PROC MIXED, SAS Institute, 2009).
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To determine the main effects and interactions, a repeated statement was used
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to account for autocorrelation between sequential measurements. A Tukey’s test
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was used to identify the differences both within and between horns and between
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groups when significant effects or interactions were identified. A probability of
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P≤0.05 was used to indicate significant difference. Data are presented as
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mean±S.E.M.
152 153 3. Results
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No difference (P>0.05) between uterine horns (ipsilateral vs. contralateral to
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the CL) was found for mesometrial PI and uterine vascular perfusion from D0 to
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D12 in any part of the experiment. Therefore, the average was used for
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subsequent statistical analyses throughout the first 12 days after ovulation. All
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embryonic vesicles were first detected by D9 or D10. The embryo almost always
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remained in the uterine body between days 9 and 11 of gestation in all pregnant
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mares during the ultrasonography evaluation. Consequently, the statistical
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analysis according to the position of the embryonic vesicle (horn with embryo vs.
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opposite horn) was performed only from D12. Embryo fixation occurred in the
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caudal segment of one of the uterine horns in all pregnant mares. No embryo
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loss was detected after the visualization of the embryonic vesicle.
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3.1 Study 1
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Effect of group and day was observed for uterine vascular perfusion (Figure
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2). In all groups, the vascular perfusion of the uterus was constant and low
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(P>0.1) during the first two days after ovulation. A pronounced and transitory
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increase in uterine vascular perfusion was detected (P<0.001) between D3 and
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D5 in non-AI and AI-P mares. AI-P and non-AI groups had greater (P<0.05)
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uterine vascular perfusion than AI-NP group on D4. Uterine vascular perfusion
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was significantly greater (P<0.001) in AI-P group than in non-AI and AI-NP
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groups on D12. The uterine vascular perfusion of AI-NP group did not change
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(P>0.05) throughout the experiment. An effect of day within each group was observed for mesometrial PI during
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the first 12 days post-ovulation (Figure 2). A transitory decrease in PI was
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detected (P<0.01) between D3 to D5 in non-AI and AI-P groups, while mares
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from AI-NP showed decreased PI only in D3. A progressive decrease of PI
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(P<0.001) was observed from D7 only in AI-P mares. After D10, mesometrial PI
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of AI-NP and non-AI mares was constant and similar (P>0.1) to the observed
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during the first two days post-ovulation.
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An effect of pregnancy was detected (P<0.004) on the uterine vascular
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perfusion and mesometrial PI of the uterine horn containing the embryo between
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D12 and D20 in AI-P mares (Figures 3). Progressive increase in uterine vascular
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perfusion was observed (P<0.001) in both uterine horns from D12. However, the
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uterine horn with embryo showed greater (P<0.001) vascular perfusion than the
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opposite horn from D15 onward. Between D12 and D20 of gestation, greater
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values (P<0.05) for mesometrial PI were observed in the uterine horn with
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embryo compared to the opposite horn.
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3.2 Study 2
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There was an effect of day and age on uterine vascular perfusion. Similar to
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the observed in the Study 1, an early and transient increase on uterine
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vascularity was detected in the three age groups. However, the uterine vascular
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changes were more prominent (P<0.05) in young and old mares when compared
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to mares with intermediary age.
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Progressively increased uterine vascular perfusion was detected (P<0.02)
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after D12 in all age groups (Figure 5). In young and adult mares, the vascularity
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of the uterine horn with embryo was often greater than in the opposite one
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(P<0.001). However, both uterine horns of old mares showed similar (P>0.1)
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vascular perfusion from D12 to D20, with exception of D15.
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Effect of horn (P<0.001) and day (P=0.009) for PI from arteries attached to
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the mesometrium were detected only in young mares (Figure 5). In young and old
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pregnant mares, PI of the uterine horn with the embryo was lower when
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compared to the opposite one. Mesometrial PI from both uterine horns of adult
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mares were similar (P>0.2) between D12 and D20.
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During the first 12 days of gestation, a transient increase on uterine vascular
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perfusion was detected (P<0.05) between D3 and D5 in the three groups (GI, GII
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and GIII). Additionally, decreased mesometrial PI was detected between D2-5
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and D3-4 in GI and GIII groups, respectively.
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Effect of embryo location, day and group were observed (P<0.002) for uterine
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vascular perfusion after D12 (Figure 6). A progressive increase in vascular
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perfusion with distinctive intensities were observed (P<0.001) in the uterine horn
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with embryo from all groups. In GI group, the uterine horn with embryo showed
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higher vascular perfusion than the opposite horn from D15 (P<0.001). In GII and
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GIII pregnant mares, significant difference (P<0.05) between uterine horns for
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vascularity were detected only on days 16 and 19 of gestation, respectively.
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When compared to GI and GII groups, both uterine horns of GIII pregnant mares
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had lower vascular perfusion after D12 (P<0.001). Additionally, the vascular
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perfusion of the opposite horn (without embryo) of GIII mares did not increase
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after D12. (P>0.1). Effect of embryo location and day was observed in pregnant mares from GI
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and GII groups for PI from arteries attached to the mesometrium (P<0.002;
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Figure 7). In GIII group, mesometrial PI of the uterine horn with embryo was
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lower (P<0.001) than in the opposite horn and no changes on PI values were
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detected through the experiment (P>0.1).
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232 4. Discussion
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The present study demonstrated the negative effect of age and endometrial
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degeneration on the uterine blood flow of mares during early gestation. These
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findings will be important in determining normal standards for blood flow of the
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reproductive tract and further characterizing vascular dysfunctions that could be
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related to early pregnancy loss, particularly in older mares or in mares with
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significant endometrial degeneration.
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As a major parameter indicator of blood flow, the pulsatility index can be
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related to the hemodynamics of a tissue supplied by an artery [19] and a
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decrease in its value indicates lower arterial resistance and higher blood flow
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[18]. In one report, endometrial vessels generated inadequate signals for spectral
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evaluation during diestrus and the first 12 days of gestation in mares [2].
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However, spectral analysis of mesometrial attachment arteries, that is the area of
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entrance of the arteries into the uterus [20], showed to be efficient for the
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objective exam of the uterine blood flow during the first 20 days of gestation and
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thought the early and mid diestrum in pregnant and non-pregnant mares,
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respectively. The description of an earlier and transient increase in uterine vascular
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perfusion accompanied by decreased PI indices of pregnant and non-
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inseminated mares is a new finding that contrasts with a previous study [2]. The
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early detection of increased uterine blood perfusion in the present study might be
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explained by the use of a more complete approach to evaluate the uterus, such
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as the use of Power-flow Doppler as previously suggested [21, 22]. Power-flow
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Doppler recognizes different intensities of blood flow velocities, showing a greater
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sensitivity to weak blood flow and reduced blooming artifacts when compared to
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the conventional Color-mode imaging [1].
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Apparently, infusion of semen did not affect the uterine hemodynamics during
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early diestrus. The relatively lower uterine blood flow during the first two days
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post-ovulation observed in AI-P and non-AI groups suggests that the vascular
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changes detected between D3 and D5 were not associated with a post-breeding
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inflammatory response. In mares, several studies have shown that introduction of
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semen to the uterine lumen will give rise to an acute endometritis [23,24]
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characterized by increased myometrial activity [25] and local innate immune
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reactions [26, 27]. However, changes on blood flow velocity of the uterine arteries
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have been observed only on the first hour after infusion of raw semen [28].
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Furthermore, our research group has recently described a transient increase in
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uterine vascular perfusion without mesometrial PI changes during the first 8
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hours after artificial insemination [22].
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The low and constant uterine vascular perfusion observed between D1 and
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D12 only in the inseminated non-pregnant (AI-NP) group suggests that vascular
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disturbance during the first days post-mating may be associated with poor
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reproductive efficiency in mares. Infertile women with documented abnormal
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uterine blood flow were unable to conceive following in vitro fertilization [29, 30,
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31]. Also, a higher uterine artery impedance have been observed in women with
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recurrent pregnancy loss [32] and different causes of infertility [33].
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A lower mesometrial PI from D10 onward was associated with a progressive
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increase in uterine vascular perfusion after D12 in young pregnant mares with
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minimal endometrial degeneration. In these mares (AI-P, young and GI groups),
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a greater vascular perfusion was recorded for the uterine horn with embryo gravid
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uterine horn than the opposite one. This finding might reflect the effect of
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vasoactive factors produced by pre-implantation embryos as described in horses
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[2] and cattle [34]. The equine conceptus secretes significant quantities of
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prostaglandins and estradiol during the first days of gestation [35,36] and PGE2
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levels remain high until D32 [37]. In human in vitro assays, estradiol stimulated
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the proliferation of angiogenic factors by endometrial endothelial cells [38] and
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increased the response of endometrial cells to VEGF [39]. A greater abundance
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of VEGF mRNA, VEGFR2 transcript and blood vessel proliferation has been
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found in the uterine tissue of mares during early pregnancy [5].
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Our findings support the hypothesis that poor blood flow of the gravid uterus
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is correlated with advanced age and diffuse endometrial degenerative changes
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during early pregnancy. In contrast to observations in young and adult mares, the
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influence of embryo mobility within the uterus on the local vascular perfusion was
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not evident in mares with advanced age. This apparent inability of the aged
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uterus to respond to embryo-derived vasoactive factors may be associated to
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progressive changes on the architecture of the vascular network of the equine
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uterus observed in older mares [40,41]. In fact, it has been shown that age-
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related endometrial degenerative changes might affect the development of
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placental microcotyledons [12] and their associated blood flow [11]. In the present study, significant difference in vascularity between uterine
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horns (with embryo vs. opposite) were rarely detected in pregnant mares with
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moderate or intense endometrial fibrosis. Moreover, mares with diffuse
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endometrial degeneration showed reduced uterine vascular perfusion when
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compared to pregnant mares with unaltered endometria. It has been
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hypothesized that severe angiosis may reduce the ability of the vessels to adapt
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to the varying demands of uterine circulation [10]. Moreover, endometrial
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pathological changes has been strongly associated with degeneration of uterine
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vessels in mares [7].
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Disturbed uterine blood flow has been recently associated to other uterine
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pathologies such as uterine cysts [21] and endometrial elastosis [8] in subfertile
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mares. However, in the present study, no early embryo loss was observed in
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pregnant mares (studies 2 and 3), despite the fact that older mares and those
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with significant endometrial degeneration (GIII) had reduced uterine blood flow.
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The relevance of vascular perturbations on conception and initial embryo
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development need further studies.
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5. Conclusion
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Under the current experimental conditions, power-Doppler ultrasonography
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associated with Doppler velocimetry generated adequate color signals for
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measuring the Doppler indices of mesometrial attachment arteries during diestrus
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and early pregnancy of mares. Early and transient increase in uterine blood flow
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was detected in pregnant mares independent of age and endometrial alterations,
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while a deficient uterine vascularity was found only in inseminated non-pregnant
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mares. Changes on the uterine vascularity according to the position of the
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embryo during the mobility phase was confirmed in mares under 12 years of age
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and with minimally altered endometrium. However, these changes were not
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consistent in mares of advanced age and in mares with significant endometrial
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degenerative changes.
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6. Acknowledgments
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This project was financially supported by FAPESP.
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7. References
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secretion by day 6 to day 9 equine embryos. Prostaglandins 1992;43:55-59.
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[38] Kayisli UA, LUK J, Guzeloglu-Kayisli O, Seval Y, Demir R, Arici A.
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Regulation of angiogenic activity of human endometrial endothelial cells in culture
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by ovarian steroids. J Clin Endocrinol Metab 2004;89:5794-802.
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[39] Iruela-Arispe ML, Rodriguez-Manzaneque JC, Abu-Jawdeh G. Endometrial
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morphometrical changes of arteries of uterine wall in mares. J Vet Med
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1995;42:383-387.
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[41] Hanada M, Maeda Y, Oikawa, M.A. Histopathological characteristics of
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endometrosis in thoroughbred mares in Japan: results from 50 necropsy cases. J
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Equine Sci 2014;25:45-52.
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Figure 1. Uterine classification in mares according to the intensity of
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degenerative endometrial changes. Adapted from Kenney and Doig, 1986 [15].
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Figure 2. Mean (±SEM) score (0-4) for uterine vascular perfusion (A) and mean
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(±SEM) mesometrial pulsatility index ( B) of inseminated pregnant (●; n=14
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mares), inseminated non-pregnant (▼, n=7 mares) and non-inseminated (□, n=7
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mares) mares during the first 12 days after ovulation. * indicates P<0.05 between
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AI-P and AI-NP groups within day. G = group; D = day; GD = interaction between
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group and day
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Figure 3. Mean (±SEM) score (0-4) for uterine vascular perfusion (A) and mean
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(±SEM) mesometrial pulsatility index (B) of uterine horn with embryo (●) and
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opposite horn (□) from inseminated pregnant mares (n=14 mares) between days
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12 and 20 of gestation. * indicates P<0.05 between horns within day. H = uterine
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horn; D = day; HD = interaction between uterine horn and day.
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embryo (●) and opposite horn (□) from young (A), adult (B) and old (C) pregnant
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mares (n=7 mares/group) between days 12 and 20 of gestation. * indicates
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P≤0.05 between horns within day. H = uterine horn; D = day.
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Figure 5. Mean (±SEM) mesometrial pulsatility index of uterine horn with embryo
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and (●) opposite horn (□) from young (A), adult (B) and old (C) pregnant mares
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(n=7 mares/group) between days 12 and 20 of gestation. * indicates P<0.05
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between uterine horns within day. H = uterine horn; D = day.
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Figure 6. Mean (±SEM) score (0-4) for vascular perfusion of uterine horn with
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embryo (A) and opposite horn (B) between days 12 and 20 of gestation in mares
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divided in three groups (n=7 mares/group) according to the endometrial
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degenerative changes: GI (●), GII (□), and GIII (▼).* indicates P≤0.05 between
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GI and GIII within day. H = uterine horn; D = day.
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Figure 7. Mean (±SEM) mesometrial pulsatility index of uterine horn with embryo
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(●) and opposite horn (□) between days 12 and 20 of gestation in mares divided
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in three groups (n=7 mares/group) according to the endometrial degenerative
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changes: GI (A), GII (B), and GIII (C). H = uterine horn; D = day.
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Figure 1. Uterine classification in mares according to the intensity of
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degenerative endometrial changes. Adapted from Kenney and Doig, 1986 [15].
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Figure 2. Mean (±SEM) score (0-4) for uterine vascular perfusion (A) and mean
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(±SEM) mesometrial pulsatility index ( B) of inseminated pregnant (●; n=14
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mares), inseminated non-pregnant (▼, n=7 mares) and non-inseminated (□, n=7
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mares) mares during the first 12 days after ovulation. * indicates P<0.05 between
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AI-P and AI-NP groups within day. G = group; D = day; GD = interaction between
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Figure 3. Mean (±SEM) score (0-4) for uterine vascular perfusion (A) and mean
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(±SEM) mesometrial pulsatility index (B) of uterine horn with embryo (●) and
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opposite horn (□) from inseminated pregnant mares (n=14 mares) between days
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12 and 20 of gestation. * indicates P<0.05 between horns within day. H = uterine
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horn; D = day; HD = interaction between uterine horn and day.
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Figure 4. Mean (±SEM) score (0-4) for vascular perfusion of uterine horn with
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embryo (●) and opposite horn (□) from young (A), adult (B) and old (C) pregnant
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mares (n=7 mares/group) between days 12 and 20 of gestation. * indicates
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P≤0.05 between horns within day. H = uterine horn; D = day.
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Figure 5. Mean (±SEM) mesometrial pulsatility index of uterine horn with embryo
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and (●) opposite horn (□) from young (A), adult (B) and old (C) pregnant mares
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(n=7 mares/group) between days 12 and 20 of gestation. * indicates P<0.05
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between uterine horns within day. H = uterine horn; D = day.
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Figure 6. Mean (±SEM) score (0-4) for vascular perfusion of uterine horn with
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embryo (A) and opposite horn (B) between days 12 and 20 of gestation in mares
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divided in three groups (n=7 mares/group) according to the endometrial
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degenerative changes: GI (●), GII (□), and GIII (▼).* indicates P≤0.05 between
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GI and GIII within day. H = uterine horn; D = day.
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Figure 7. Mean (±SEM) mesometrial pulsatility index of uterine horn with embryo
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(●) and opposite horn (□) between days 12 and 20 of gestation in mares divided
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in three groups (n=7 mares/group) according to the endometrial degenerative
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changes: GI (A), GII (B), and GIII (C). H = uterine horn; D = day.
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RESEARCH HIGHLIGHTS:
An early and transient increase in uterine vascular perfusion accompanied by decreased mesometrial
pulsatility index was documented in pregnant mares.
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2. Poor blood flow of the gravid uterus during the early gestation is correlated with advanced age in mares.
Pregnant mares with diffuse endometrial degeneration have reduced uterine blood flow during the early
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S0093-691X(15)00318-0
DOI:
10.1016/j.theriogenology.2015.06.013
Reference:
THE 13236
To appear in:
Theriogenology
Received Date: 13 February 2015 Revised Date:
10 June 2015
Accepted Date: 18 June 2015
Please cite this article as: Ferreira JC, Canesin HS, Ignácio FS, Rocha NS, Pinto CR, Meira C, Effect of age and endometrial degenerative changes on uterine blood flow during early gestation in mares, Theriogenology (2015), doi: 10.1016/j.theriogenology.2015.06.013. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Effect of age and endometrial degenerative changes on uterine blood flow during
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early gestation in mares
3 4 J. C. Ferreira1,3*, H. S. Canesin1, F. S. Ignácio1; N.S. Rocha2; C.R. Pinto3; C.
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Meira1
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Univ Estadual Paulista (UNESP), Department of Animal Reproduction and
Veterinary Radiology, SP18618-970, Brazil
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970, Brazil
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USA
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Univ Estadual Paulista (UNESP), Department of Veterinary Clinics, SP18618-
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School of Veterinary Medicine, Louisiana State University, Baton Rouge 70803,
* corresponding author:
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Jair Camargo Ferreira
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phone: 225-733-2384
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e-mail: [email protected]
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address: 5284 Brightside View apt 2, Baton Rouge, LA 70820, USA
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REVISED
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Doppler ultrasonography is a noninvasive pulse wave technique currently
28
used for in vivo evaluation of the uterine blood flow of pregnant and non-pregnant
29
mares [1]. An uninterrupted embryo-maternal interaction is essential for the
30
establishment and maintenance of the gestation [2, 3]. The uterine blood supply
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increases from the second week of pregnancy in mares [4] subsequently to a
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conceptus-induced endometrial vasculogenesis [5]. However, there has been no
33
studies reporting on waveforms from arteries of mesometrium attachment during
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the diestrus and first days of pregnancy. Therefore, the correlation between
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objective and subjective evaluation of the equine uterine blood flow during the
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early gestation remains unknown.
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In multiparous-aged mares, poor uterine blood flow [6, 7, 8] and subfertility [9,
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10] have been related with elastosis of endometrial vessels. Reduced uterine
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blood flow associated with poor placental development [11] and compromised
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haematological contact at the fetomaternal interface [12] have been observed in
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pregnant aged mares.
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Disturbances of the uterine blood flow during the early gestation in mares with
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age-related endometrial degenerative changes have not been investigated using
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Doppler ultrasonography. Therefore, the overall purpose of the present study was
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to characterize the uterine blood flow of mares of different ages and endometrial
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histopathological classification during the first 20 days of gestation.
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2. Materials and Methods
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2.1 Animals and experimental groups
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The mares were kept under natural light in an open shelter and outdoor
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paddock at the Reproduction Center of the Department of Animal Reproduction
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and Veterinary Radiology at the São Paulo State University (UNESP), Campus of
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Botucatu. Animals were handled in accordance with the São Paulo State
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University Guide for Care and Use of Agricultural Animals in Research (protocol
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number 13/2012-CEUA). The mares were of mixed breeds weighing 270 to 410
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kg and were 4 to 18 years old. Animals were maintained on grass hay and
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pelleted feed with free access to water and trace-mineralized salt. Score for body
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condition for all mares throughout the experiment was ≥ 7 [13].
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B-mode ultrasonography exam was done once daily for monitoring of follicular
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development and detection of ovulation. Day of ovulation was considered D0. All
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mares were submitted to ovulation inducing treatment with 2500 IU of hCG when
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a dominant follicle reached a diameter ≥ 35mm associated with uterine edema.
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Study 1: Effect of semen infusion and conception on uterine hemodynamics of mares
Twenty-eight mares were retrospectively assigned into three experimental
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groups according to insemination procedure and presence of the embryonic
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vesicle: Non-inseminated group (n=7 mares; non-AI), Inseminated Non-pregnant
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group (n=7 mares; AI-NP) and Inseminated pregnant group (n=14 mares; AI-P).
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The mean ages of non-AI, AI-NP, and AI-P ranged from 7 to 12 years (9.5±0.8,
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10.3±0.9 and 9.7±0.9, respectively).
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Study 2: Effect of age on the uterine blood flow of mares during the early gestation
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Twenty-one pregnant mares were used to evaluate the temporal relationship
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between age and uterine blood flow throughout the first 20 days of gestation. The
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mares were assigned to three age groups (n=7 mares/ group): young (≤6 years,
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5.5±0.2 years), adult (from 8 to 12 years, 10.1±0.5 years) and old (≥15 years,
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17.2±0.6 years). Age of mares was estimated from dental characteristics as
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previously described [14].
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Study 3: Effect of endometrial degenerative changes on the uterine blood flow of mares during the early gestation
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Twenty-one pregnant mares were used to study the influence of endometrial
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degenerative changes in the uterine blood flow during early gestation. Initially,
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endometrial samples from all mares were obtained using transcervical uterine
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biopsy and stained with hematoxilin and eosin. Endometrial degenerative
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changes were classified into four categories (Figure 1) as previously established
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[15]. Therefore, the mares were assigned in three groups (n=6 mares/group)
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based on the degree of endometrial histopathological changes: GI (category I),
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GII (categories IIA and IIB) and GIII (category III).
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2.2 Ultrasonography and Doppler End Points
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Mares were scanned using a triplex B-mode (gray scale) and pulse wave
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color Doppler ultrasound instrument (Sonoace Pico; Medison do Brasil Ltda)
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equipped with a linear-array multifrequency transducer (LV5-9CDn, 5 to 9 MHz)
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with a beam-field width of 60 mm. Pharmacological sedation was not used for
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any examination using ultrasonography. The principles, techniques, and
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interpretations of B-mode examinations of the mare reproductive tract have been
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reviewed [16]. The brightness and contrast controls of the monitor, and the gain
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controls of the scanner, were standardized to constant settings [17]. Uterine blood flow of pregnant and non-pregnant mares was based on the
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pulsatility index (PI) from arteries attached to the mesometrium and uterine
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vascular perfusion. In Study 1, data collection was done once daily during the first
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20 days of pregnancy in AI-P group and between D0 and D12 in AI-NP and non-
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AI groups. In Studies 2 and 3, Doppler end points were evaluated daily from D0
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and D20.
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Vascular perfusion from both uterine horns was subjectively estimated by one
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operator using power-flow Doppler mode. Only color signals that appeared to be
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within the limits of the uterus (endometrium and myometrium) were considered.
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Uterine vascular perfusion was scored from 0 to 4 (minimum to maximum,
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including fractional scores), considering the extension of uterine tissue with color
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signals of blood flow, during real-time cross-sectioning scan of the uterus in a
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slow continuous motion for two minutes.
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Spectral assessment of arterial PI was performed as described previously [2].
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The formula for calculating spectral data has been previously described [18]. To
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produce a spectral waveform, each entire mesometrium attachment was initially
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scanned in a slow, continuous motion using the power-flow Doppler mode.
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Subsequently, in spectral mode, the sample-gate cursor was placed on an artery
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of the mesometrium attachment. Spectral waveform, with a minimum of three
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cardiac cycles, was generated and one of the cycles was used to measure the
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spectral end points. The procedure was repeated twice, and the average was
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used for statistical analyses.
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Mares from AI-NP and AI-P groups and from Part 2 and 3 were artificially
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inseminated 24 hours after induction of ovulation with 2500 UI of hCG,
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administered when a preovulatory follicle reached a diameter ≥ 35 mm
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associated with uterine edema. Semen from one stallion of proven fertility was
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used. After collection, semen was diluted with skim milk extender (BotuSÊMEN®;
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Botupharma) to obtain an insemination dose of 50 x 106/mL progressively motile
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spermatozoa in a final volume of 20 to 30 mL. The insemination dose was
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maintained cooled at 15oC using an insulated container (BotuBOX®; Botupharma)
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until the time of insemination. The semen dose was placed into the body of the
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uterus using a standart AI catheter.
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Pregnancy exam was performed once daily, beginning on D9, using
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conventional B-mode ultrasonography. In AI-NP mares, a final ultrasound exam
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was performed on D15 to confirm the absence of an embryonic vesicle. Early
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pregnancy termination was induced with a single injection of Dinoprost
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tromethamine (10mg; IM) after end point collection ending.
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2.4 Statistical analyses
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The scores for uterine vascular perfusion and spectral end points were
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analyzed by a two-way repeated measure (PROC MIXED, SAS Institute, 2009).
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To determine the main effects and interactions, a repeated statement was used
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to account for autocorrelation between sequential measurements. A Tukey’s test
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was used to identify the differences both within and between horns and between
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groups when significant effects or interactions were identified. A probability of
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P≤0.05 was used to indicate significant difference. Data are presented as
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mean±S.E.M.
152 153 3. Results
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No difference (P>0.05) between uterine horns (ipsilateral vs. contralateral to
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the CL) was found for mesometrial PI and uterine vascular perfusion from D0 to
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D12 in any part of the experiment. Therefore, the average was used for
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subsequent statistical analyses throughout the first 12 days after ovulation. All
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embryonic vesicles were first detected by D9 or D10. The embryo almost always
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remained in the uterine body between days 9 and 11 of gestation in all pregnant
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mares during the ultrasonography evaluation. Consequently, the statistical
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analysis according to the position of the embryonic vesicle (horn with embryo vs.
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opposite horn) was performed only from D12. Embryo fixation occurred in the
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caudal segment of one of the uterine horns in all pregnant mares. No embryo
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loss was detected after the visualization of the embryonic vesicle.
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3.1 Study 1
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Effect of group and day was observed for uterine vascular perfusion (Figure
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2). In all groups, the vascular perfusion of the uterus was constant and low
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(P>0.1) during the first two days after ovulation. A pronounced and transitory
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increase in uterine vascular perfusion was detected (P<0.001) between D3 and
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D5 in non-AI and AI-P mares. AI-P and non-AI groups had greater (P<0.05)
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uterine vascular perfusion than AI-NP group on D4. Uterine vascular perfusion
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was significantly greater (P<0.001) in AI-P group than in non-AI and AI-NP
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groups on D12. The uterine vascular perfusion of AI-NP group did not change
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(P>0.05) throughout the experiment. An effect of day within each group was observed for mesometrial PI during
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the first 12 days post-ovulation (Figure 2). A transitory decrease in PI was
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detected (P<0.01) between D3 to D5 in non-AI and AI-P groups, while mares
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from AI-NP showed decreased PI only in D3. A progressive decrease of PI
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(P<0.001) was observed from D7 only in AI-P mares. After D10, mesometrial PI
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of AI-NP and non-AI mares was constant and similar (P>0.1) to the observed
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during the first two days post-ovulation.
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An effect of pregnancy was detected (P<0.004) on the uterine vascular
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perfusion and mesometrial PI of the uterine horn containing the embryo between
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D12 and D20 in AI-P mares (Figures 3). Progressive increase in uterine vascular
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perfusion was observed (P<0.001) in both uterine horns from D12. However, the
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uterine horn with embryo showed greater (P<0.001) vascular perfusion than the
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opposite horn from D15 onward. Between D12 and D20 of gestation, greater
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values (P<0.05) for mesometrial PI were observed in the uterine horn with
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embryo compared to the opposite horn.
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3.2 Study 2
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There was an effect of day and age on uterine vascular perfusion. Similar to
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the observed in the Study 1, an early and transient increase on uterine
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vascularity was detected in the three age groups. However, the uterine vascular
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changes were more prominent (P<0.05) in young and old mares when compared
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to mares with intermediary age.
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Progressively increased uterine vascular perfusion was detected (P<0.02)
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after D12 in all age groups (Figure 5). In young and adult mares, the vascularity
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of the uterine horn with embryo was often greater than in the opposite one
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(P<0.001). However, both uterine horns of old mares showed similar (P>0.1)
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vascular perfusion from D12 to D20, with exception of D15.
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Effect of horn (P<0.001) and day (P=0.009) for PI from arteries attached to
205
the mesometrium were detected only in young mares (Figure 5). In young and old
206
pregnant mares, PI of the uterine horn with the embryo was lower when
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compared to the opposite one. Mesometrial PI from both uterine horns of adult
208
mares were similar (P>0.2) between D12 and D20.
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209 3.3 Study 3
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During the first 12 days of gestation, a transient increase on uterine vascular
212
perfusion was detected (P<0.05) between D3 and D5 in the three groups (GI, GII
213
and GIII). Additionally, decreased mesometrial PI was detected between D2-5
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and D3-4 in GI and GIII groups, respectively.
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Effect of embryo location, day and group were observed (P<0.002) for uterine
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vascular perfusion after D12 (Figure 6). A progressive increase in vascular
217
perfusion with distinctive intensities were observed (P<0.001) in the uterine horn
218
with embryo from all groups. In GI group, the uterine horn with embryo showed
219
higher vascular perfusion than the opposite horn from D15 (P<0.001). In GII and
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GIII pregnant mares, significant difference (P<0.05) between uterine horns for
221
vascularity were detected only on days 16 and 19 of gestation, respectively.
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When compared to GI and GII groups, both uterine horns of GIII pregnant mares
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had lower vascular perfusion after D12 (P<0.001). Additionally, the vascular
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perfusion of the opposite horn (without embryo) of GIII mares did not increase
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after D12. (P>0.1). Effect of embryo location and day was observed in pregnant mares from GI
227
and GII groups for PI from arteries attached to the mesometrium (P<0.002;
228
Figure 7). In GIII group, mesometrial PI of the uterine horn with embryo was
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lower (P<0.001) than in the opposite horn and no changes on PI values were
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detected through the experiment (P>0.1).
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232 4. Discussion
234
The present study demonstrated the negative effect of age and endometrial
235
degeneration on the uterine blood flow of mares during early gestation. These
236
findings will be important in determining normal standards for blood flow of the
237
reproductive tract and further characterizing vascular dysfunctions that could be
238
related to early pregnancy loss, particularly in older mares or in mares with
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significant endometrial degeneration.
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As a major parameter indicator of blood flow, the pulsatility index can be
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related to the hemodynamics of a tissue supplied by an artery [19] and a
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decrease in its value indicates lower arterial resistance and higher blood flow
243
[18]. In one report, endometrial vessels generated inadequate signals for spectral
244
evaluation during diestrus and the first 12 days of gestation in mares [2].
245
However, spectral analysis of mesometrial attachment arteries, that is the area of
246
entrance of the arteries into the uterus [20], showed to be efficient for the
247
objective exam of the uterine blood flow during the first 20 days of gestation and
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thought the early and mid diestrum in pregnant and non-pregnant mares,
249
respectively. The description of an earlier and transient increase in uterine vascular
251
perfusion accompanied by decreased PI indices of pregnant and non-
252
inseminated mares is a new finding that contrasts with a previous study [2]. The
253
early detection of increased uterine blood perfusion in the present study might be
254
explained by the use of a more complete approach to evaluate the uterus, such
255
as the use of Power-flow Doppler as previously suggested [21, 22]. Power-flow
256
Doppler recognizes different intensities of blood flow velocities, showing a greater
257
sensitivity to weak blood flow and reduced blooming artifacts when compared to
258
the conventional Color-mode imaging [1].
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Apparently, infusion of semen did not affect the uterine hemodynamics during
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early diestrus. The relatively lower uterine blood flow during the first two days
261
post-ovulation observed in AI-P and non-AI groups suggests that the vascular
262
changes detected between D3 and D5 were not associated with a post-breeding
263
inflammatory response. In mares, several studies have shown that introduction of
264
semen to the uterine lumen will give rise to an acute endometritis [23,24]
265
characterized by increased myometrial activity [25] and local innate immune
266
reactions [26, 27]. However, changes on blood flow velocity of the uterine arteries
267
have been observed only on the first hour after infusion of raw semen [28].
268
Furthermore, our research group has recently described a transient increase in
269
uterine vascular perfusion without mesometrial PI changes during the first 8
270
hours after artificial insemination [22].
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The low and constant uterine vascular perfusion observed between D1 and
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D12 only in the inseminated non-pregnant (AI-NP) group suggests that vascular
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disturbance during the first days post-mating may be associated with poor
274
reproductive efficiency in mares. Infertile women with documented abnormal
275
uterine blood flow were unable to conceive following in vitro fertilization [29, 30,
276
31]. Also, a higher uterine artery impedance have been observed in women with
277
recurrent pregnancy loss [32] and different causes of infertility [33].
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A lower mesometrial PI from D10 onward was associated with a progressive
279
increase in uterine vascular perfusion after D12 in young pregnant mares with
280
minimal endometrial degeneration. In these mares (AI-P, young and GI groups),
281
a greater vascular perfusion was recorded for the uterine horn with embryo gravid
282
uterine horn than the opposite one. This finding might reflect the effect of
283
vasoactive factors produced by pre-implantation embryos as described in horses
284
[2] and cattle [34]. The equine conceptus secretes significant quantities of
285
prostaglandins and estradiol during the first days of gestation [35,36] and PGE2
286
levels remain high until D32 [37]. In human in vitro assays, estradiol stimulated
287
the proliferation of angiogenic factors by endometrial endothelial cells [38] and
288
increased the response of endometrial cells to VEGF [39]. A greater abundance
289
of VEGF mRNA, VEGFR2 transcript and blood vessel proliferation has been
290
found in the uterine tissue of mares during early pregnancy [5].
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Our findings support the hypothesis that poor blood flow of the gravid uterus
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is correlated with advanced age and diffuse endometrial degenerative changes
293
during early pregnancy. In contrast to observations in young and adult mares, the
294
influence of embryo mobility within the uterus on the local vascular perfusion was
295
not evident in mares with advanced age. This apparent inability of the aged
296
uterus to respond to embryo-derived vasoactive factors may be associated to
297
progressive changes on the architecture of the vascular network of the equine
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uterus observed in older mares [40,41]. In fact, it has been shown that age-
299
related endometrial degenerative changes might affect the development of
300
placental microcotyledons [12] and their associated blood flow [11]. In the present study, significant difference in vascularity between uterine
302
horns (with embryo vs. opposite) were rarely detected in pregnant mares with
303
moderate or intense endometrial fibrosis. Moreover, mares with diffuse
304
endometrial degeneration showed reduced uterine vascular perfusion when
305
compared to pregnant mares with unaltered endometria. It has been
306
hypothesized that severe angiosis may reduce the ability of the vessels to adapt
307
to the varying demands of uterine circulation [10]. Moreover, endometrial
308
pathological changes has been strongly associated with degeneration of uterine
309
vessels in mares [7].
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Disturbed uterine blood flow has been recently associated to other uterine
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pathologies such as uterine cysts [21] and endometrial elastosis [8] in subfertile
312
mares. However, in the present study, no early embryo loss was observed in
313
pregnant mares (studies 2 and 3), despite the fact that older mares and those
314
with significant endometrial degeneration (GIII) had reduced uterine blood flow.
315
The relevance of vascular perturbations on conception and initial embryo
316
development need further studies.
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5. Conclusion
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Under the current experimental conditions, power-Doppler ultrasonography
321
associated with Doppler velocimetry generated adequate color signals for
322
measuring the Doppler indices of mesometrial attachment arteries during diestrus
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and early pregnancy of mares. Early and transient increase in uterine blood flow
324
was detected in pregnant mares independent of age and endometrial alterations,
325
while a deficient uterine vascularity was found only in inseminated non-pregnant
326
mares. Changes on the uterine vascularity according to the position of the
327
embryo during the mobility phase was confirmed in mares under 12 years of age
328
and with minimally altered endometrium. However, these changes were not
329
consistent in mares of advanced age and in mares with significant endometrial
330
degenerative changes.
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6. Acknowledgments
334
This project was financially supported by FAPESP.
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337 338
7. References
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[36] Vanderwall DK, Woods GL, Weber JA, Lichtenwalner AB. PGE2 secretion by
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[41] Hanada M, Maeda Y, Oikawa, M.A. Histopathological characteristics of
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Figure 1. Uterine classification in mares according to the intensity of
504
degenerative endometrial changes. Adapted from Kenney and Doig, 1986 [15].
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Figure 2. Mean (±SEM) score (0-4) for uterine vascular perfusion (A) and mean
507
(±SEM) mesometrial pulsatility index ( B) of inseminated pregnant (●; n=14
508
mares), inseminated non-pregnant (▼, n=7 mares) and non-inseminated (□, n=7
509
mares) mares during the first 12 days after ovulation. * indicates P<0.05 between
510
AI-P and AI-NP groups within day. G = group; D = day; GD = interaction between
511
group and day
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Figure 3. Mean (±SEM) score (0-4) for uterine vascular perfusion (A) and mean
514
(±SEM) mesometrial pulsatility index (B) of uterine horn with embryo (●) and
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opposite horn (□) from inseminated pregnant mares (n=14 mares) between days
516
12 and 20 of gestation. * indicates P<0.05 between horns within day. H = uterine
517
horn; D = day; HD = interaction between uterine horn and day.
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520
embryo (●) and opposite horn (□) from young (A), adult (B) and old (C) pregnant
521
mares (n=7 mares/group) between days 12 and 20 of gestation. * indicates
522
P≤0.05 between horns within day. H = uterine horn; D = day.
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Figure 5. Mean (±SEM) mesometrial pulsatility index of uterine horn with embryo
525
and (●) opposite horn (□) from young (A), adult (B) and old (C) pregnant mares
526
(n=7 mares/group) between days 12 and 20 of gestation. * indicates P<0.05
527
between uterine horns within day. H = uterine horn; D = day.
528
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Figure 6. Mean (±SEM) score (0-4) for vascular perfusion of uterine horn with
530
embryo (A) and opposite horn (B) between days 12 and 20 of gestation in mares
531
divided in three groups (n=7 mares/group) according to the endometrial
532
degenerative changes: GI (●), GII (□), and GIII (▼).* indicates P≤0.05 between
533
GI and GIII within day. H = uterine horn; D = day.
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Figure 7. Mean (±SEM) mesometrial pulsatility index of uterine horn with embryo
536
(●) and opposite horn (□) between days 12 and 20 of gestation in mares divided
537
in three groups (n=7 mares/group) according to the endometrial degenerative
538
changes: GI (A), GII (B), and GIII (C). H = uterine horn; D = day.
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Legends
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Figure 1. Uterine classification in mares according to the intensity of
4
degenerative endometrial changes. Adapted from Kenney and Doig, 1986 [15].
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Figure 2. Mean (±SEM) score (0-4) for uterine vascular perfusion (A) and mean
7
(±SEM) mesometrial pulsatility index ( B) of inseminated pregnant (●; n=14
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mares), inseminated non-pregnant (▼, n=7 mares) and non-inseminated (□, n=7
9
mares) mares during the first 12 days after ovulation. * indicates P<0.05 between
10
AI-P and AI-NP groups within day. G = group; D = day; GD = interaction between
11
group and day
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Figure 3. Mean (±SEM) score (0-4) for uterine vascular perfusion (A) and mean
14
(±SEM) mesometrial pulsatility index (B) of uterine horn with embryo (●) and
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opposite horn (□) from inseminated pregnant mares (n=14 mares) between days
16
12 and 20 of gestation. * indicates P<0.05 between horns within day. H = uterine
17
horn; D = day; HD = interaction between uterine horn and day.
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Figure 4. Mean (±SEM) score (0-4) for vascular perfusion of uterine horn with
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embryo (●) and opposite horn (□) from young (A), adult (B) and old (C) pregnant
21
mares (n=7 mares/group) between days 12 and 20 of gestation. * indicates
22
P≤0.05 between horns within day. H = uterine horn; D = day.
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Figure 5. Mean (±SEM) mesometrial pulsatility index of uterine horn with embryo
25
and (●) opposite horn (□) from young (A), adult (B) and old (C) pregnant mares
26
(n=7 mares/group) between days 12 and 20 of gestation. * indicates P<0.05
27
between uterine horns within day. H = uterine horn; D = day.
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Figure 6. Mean (±SEM) score (0-4) for vascular perfusion of uterine horn with
30
embryo (A) and opposite horn (B) between days 12 and 20 of gestation in mares
31
divided in three groups (n=7 mares/group) according to the endometrial
32
degenerative changes: GI (●), GII (□), and GIII (▼).* indicates P≤0.05 between
33
GI and GIII within day. H = uterine horn; D = day.
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Figure 7. Mean (±SEM) mesometrial pulsatility index of uterine horn with embryo
36
(●) and opposite horn (□) between days 12 and 20 of gestation in mares divided
37
in three groups (n=7 mares/group) according to the endometrial degenerative
38
changes: GI (A), GII (B), and GIII (C). H = uterine horn; D = day.
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RESEARCH HIGHLIGHTS:
An early and transient increase in uterine vascular perfusion accompanied by decreased mesometrial
pulsatility index was documented in pregnant mares.
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2. Poor blood flow of the gravid uterus during the early gestation is correlated with advanced age in mares.
Pregnant mares with diffuse endometrial degeneration have reduced uterine blood flow during the early
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gestation