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In utero development of the fetal intestine: Sonographic evaluation and correlation with gestational age and fetal maturity in dogs
Accepted Manuscript In-utero development of the foetal intestine – sonographic evaluation and correlation with gestational age and foetal maturity in dogs Elaine M.U. Gil, Daniela A.A. Garcia, Tilde R. Froes PII:
S0093-691X(15)00231-9
DOI:
10.1016/j.theriogenology.2015.04.030
Reference:
THE 13181
To appear in:
Theriogenology
Received Date: 26 February 2015 Revised Date:
28 April 2015
Accepted Date: 29 April 2015
Please cite this article as: Gil EMU, Garcia DAA, Froes TR, In-utero development of the foetal intestine – sonographic evaluation and correlation with gestational age and foetal maturity in dogs, Theriogenology (2015), doi: 10.1016/j.theriogenology.2015.04.030. 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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In-utero development of the foetal intestine – sonographic evaluation and correlation
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with gestational age and foetal maturity in dogs
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Elaine M. U. Gil1*, Daniela A. A. Garcia1, Tilde R. Froes1
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of Paraná, Brazil
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* Corresponding author:
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Elaine M. U. Gil
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[email protected]
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Department of Veterinary Medicine, School of Veterinary Medicine, Federal University
Rua dos Funcionários 1540. Juvevê. Zip Code 80035-050
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Curitiba City, Paraná State, Brazil
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Phone: 55 (41) 3350-5767, Fax: 55 (41) 3350-5725
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Abstract
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Modern high-resolution ultrasound images enable earlier assessment of measures of foetal
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development, including identification of bowel. The aim of this study was to describe the
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ultrasonographic development of foetal bowel and correlate this with gestational age;
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define whether ultrasonographic visualization of foetal intestinal peristalsis in utero is
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associated with foetal maturation and determine whether there is a difference in foetal
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intestinal peristalsis detection time between foetuses delivered by normal delivery and
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caesarean. A cohort study was conducted in pregnant bitches presented to a veterinary
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hospital, to assess foetal bowel development. Statistical analysis was used to establish the
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correlation of the stage of foetal bowel development, as recorded by ultrasound, with
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outcomes of normal delivery and caesarean section. The study was broken down into three
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stages: the first stage was a descriptive analysis of foetal bowel development by
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ultrasound; the second stage compared time (in days) of bowel development between
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groups (normal delivery versus caesarean); and the third stage was correlated survival
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probability for foetuses born on any day after detection of intestinal peristalsis with foetal
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maturity. All statistical analyses were significant. It is possible to monitor pregnancy
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progression using ultrasonographic evaluation of bowel development and this can reliably
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identify the end of foetal organogenesis. However, ultrasonographic detection of bowel
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segments with visualization of wall layers and associated peristalsis should not be used as
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the sole indicator for caesarean section planning because it is not possible to determine
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ultrasonographically whether the bowel is functional (mature).
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Keywords: Canine, pregnancy, gestational ultrasound, foetal bowel ultrasonography,
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maturation
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1. Introduction Ultrasonography is the preferred method for early diagnosis of pregnancy, evaluation
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of foetal viability, and estimation of gestational age [1]. Early and accurate determination
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of gestational age is useful for predicting time and management of deliver, including
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planning caesarean section (C-section) [2]. In some breeds of dogs in which C-section is
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considered necessary, assessment of foetal maturity and appropriate timing of C-section
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can reduce neonatal mortality [3].
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As in human medicine, there are a variety of methods for ultrasonographic estimation
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of foetal age in pregnant bitches, these include calculation from crown-rump length in
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early pregnancy, biparietal diameter and assessment of foetal organogenesis by serial
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examinations [2,3,4,5,6,7]. The assessment of foetal organogenesis is an interesting model
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for determination of foetal age in dogs. The bowel is the last organ to be identified by
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ultrasonography as foetal dogs develop [5,7].
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According to some authors visualization of bowel and identification of peristalsis by
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ultrasound determines the end of foetal organogenesis, indirectly indicating that the foetus
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is full term. This happens around 57 to 63 days of gestation [5,8,9]. Several authors
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[7,8,9,10] describe a range of gestational ages for detection of peristalsis by
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ultrasonography from 57 days after the peak of luteinizing hormone.
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However, modern high-resolution ultrasound images enable earlier assessment of
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measures of foetal development, including identification of bowel. There are no previous
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studies describing the ultrasonographic assessment of bowel development in canine
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foetuses. Our hypothesis is that ultrasonographic visualization of foetal bowel is possible at
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earlier foetal ages than previously described. The purpose of this study was threefold: 1. To
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describe the ultrasonographic development of foetal bowel and correlate this with
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gestational age; 2. To define whether ultrasonographic visualization of foetal intestinal
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peristalsis in utero is associated with foetal maturation; 3. To determine whether there is a
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difference in foetal intestinal peristalsis detection time (days before delivery), between
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foetuses born by normal delivery and caesarean.
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2. Materials and Methods 2.1 Patient selection
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A cohort study was conducted in pregnant bitches to assess foetal bowel
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development. The sample population consisted of bitches with confirmed pregnancy
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presented to a veterinary hospital between May 2013 and April 2014. All procedures were
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carried out in accordance with Animal Use Committee guidelines. All owners provided
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informed, written consent to be included in the study. Exclusion criteria were bitches with
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positive pregnancy unavailable for serial examinations, late pregnancy (more than 30 days
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gestation), at time of first presentation, date of delivery not reported by owners and bitches
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presented with concomitant disease.
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2.2 Ultrasound imaging and equipment
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Ultrasound scanning was performed with a high-resolution linear multifrequency
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transducer (7.5 to 12 MHz), model MyLab 30 [Esaote, Genova, Italy]. The gain, focus and
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other adjustments were made during examination to obtain the best image for each foetus
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evaluated. All pregnant bitches were prepared with hair clipped prior to the examination,
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positioned in dorsal recumbency and ultrasound gel was used to optimize image
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acquisition.
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The pregnant bitches were followed by ultrasonographic examination from the 14th
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day after the first mating and/or insemination. Ultrasonography examinations were
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performed every four days until intense peristalsis (movement every three seconds) of
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foetal bowel was visible. From that examination onwards daily assessments were made
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until delivery. Gestational age was confirmed after delivery, in days to delivery, by counting
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backwards (delivery as day zero), normal gestational length was considered to be 57 to 63
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days, due to the variable pro-estrus and estrus periods in dogs [11]. During ultrasound
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examination gestational age was estimated in days of gestation, using the descriptions of
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Yeager et al.[5].
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Each sonographer scanned all foetuses in each pregnant bitch. Each bitch was
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scanned independently by two experienced sonographer (one of whom is a member of the
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Brazilian College of Veterinary Radiology) at each examination. However, both
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sonographers were in the room during the examinations. The ultrasound interpretation was
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achieved by consensus of the two sonographers. The examination method followed a
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protocol to examine the whole abdomen by scanning clockwise, starting with foetuses in
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the left uterine horn (cranial to caudal) followed by the right horn (caudal to cranial). In
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bitches with large numbers of foetuses, at least four foetuses were examined at all
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examinations, and in bitches with four or fewer foetuses all foetal abdomens were
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examined.
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Intrauterine foetal abdomens were assessed in the longitudinal and dorsal planes of
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each foetus. All foetal organogenesis was monitored; however, the priority was
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examination of the bowel.
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After data collection pregnant females were classified according to delivery method
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(C-section or natural) and divided in two groups. The decision to perform a C-section was
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based on ultrasound indications of foetal distress [12]. Foetuses were considered to be
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distressed if foetal heart rate remained low (between 160 and 190 beats per minut), and
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without peaks of acceleration and deceleration as described by Gil et al. [13]. Foetal heart
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rate was measured using M-mode for five minutes per foetus in all pregnant bitches up to
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maximum four foetuses.
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2.3 Experimental Design
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Statistical analysis was used to establish the correlation of the stage of foetal bowel
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development, as recorded by ultrasound, with outcomes of normal delivery and C-section.
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The study was broken down into three stages.
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The first stage was a descriptive analysis of foetal bowel development by ultrasound.
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In the second and third stages a rating from one to four, was used for various phases of
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foetal bowel development. Phase 1 was defined as the first time bowel could be visualized
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and phase 4 was the last phase, when intestinal peristalsis (movement every three seconds)
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was identified. Once phase 4, had been identified this was used for statistical analysis of
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the second and third stages of the experiment. For each phase (1 to 4) of intestinal
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development respective foetal age ranges were calculated.
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The aim of the second stage was to compare time (in days) of bowel development
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between groups (normal delivery versus caesarean). For this, statistical testing was used: 1.
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Student t-Test which compared all phases (1/1, 2/2, 3/3 and 4/4) between the two groups;
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2. The semi-parametric regression Cox model [14] was used to specifically assess Phase 4
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of bowel development, testing whether there was a difference in time (days) of ultrasound
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visualisation of bowel between the two groups. In the Cox model the group of bitches with
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normal delivery were used as the control.
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The aim of third stage of the study was to correlate survival probability for foetuses
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born on any day in Phase 4, at which time foetuses were assumed to be mature, and to this
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end a survival curve was generated. The survival curve was constructed using the Kaplan-
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Meier method [15] and statistical comparison between groups was performed by the Log-
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Rank test, and then identifying whether there were differences between the survival
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probabilities of puppies born by normal delivery or caesarean. In all analyzes, a P-value of
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α = 0.05 was considered significant and the significance was analyzed relative to the value
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set.
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All statistical tests were selected and performed by one author (E.G) and one
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statistical professional (E.C). Data collation and analysis were performed using Microsoft
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Office Excel (Microsoft ® Office 2007 for Windows, Redmond, WA, USA) and R for
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Windows (version 3.1.1 Ri386, R Foundation for Statistical Computing, Vienna, Austria,
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2014) through packages "Survival", "KMsurv" and "Coin".
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3. Results
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Eighteen pregnant bitches were included in this study. Several breeds were
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represented and ages ranged from 1 to 6 years. Seventy-five foetuses were evaluated and
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all were born healthy, including those delivered by caesarean. The litter size of each bitch
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was between 2 and 9 pups (Table 1).
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After ultrasound monitoring of foetal organogenesis and evaluation of development
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of foetal intrauterine bowel in all females, four different phases of development were
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defined. All foetuses displayed the same ultrasonographic patterns of bowel development.
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The four phases were:
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• Phase 1. One uniform echogenic area caudal to the foetal liver in the topographic region of bowel (Figure 1A);
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• Phase 2. In the same anterior region, some portions of bowel segments were better
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defined allowing visualization of the intestinal wall ie anechoic areas intermixed
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with multifocal normal intestinal wall. Peristalsis was absent (Figure 1B); • Phase 3. Bowel segments had clearly defined intestinal walls and anechoic areas
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were fewer when compared to Phase 2; defined segments had mucosal intraluminal
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content, with better definition of mucosal surface versus intestinal wall. Peristalsis
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in some intestinal portions (Figure 1C);
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• Phase 4. Identification of complete intestinal wall, visual distinction between
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mucosal surface and intestinal wall, ultrasonographic determination of wall layers,
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surface mucosa - hyperechoic; mucosa, submucosa, muscle - hypoechoic; serous -
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hyperechoic. Segmental dilatation of the bowel by intraluminal mucous and fluid
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content. Peristalsis in all segments of the bowel (movement every 3 seconds -
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Figure 1D).
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Table 2 shows gestational ages (days to delivery and days of estimated gestation),
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mean, standard deviation and median of four phases of intrauterine foetal bowel
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development by ultrasound according to type of delivery.
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Table 3 presents results of Student t-test for the four phases of intrauterine foetal
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bowel development comparing the type of delivery (normal or caesarean), showing a
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significant (α = 0.05) difference between groups only in phase 4 of bowel development.
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Semiparametric COX model, coefficient value of reference group (normal delivery
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bitches) was -1.316, resulting in exp (-1.316) = 0.268. This confirms that the time of
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ultrasound visualization of bowel in Phase 4 (in which there is peristalsis) is different in
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foetuses born by C-section from those delivered naturally, in other words, the duration of
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phase 4 is earlier in foetuses delivered by C-section.
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The probability of survival of foetuses born on any day during phase 4 was different
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when comparing the puppies born by normal delivery with those delivered by caesarean,
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resulting in a Kaplan-Meier curve with a stairway shape (Figure 2). This difference in
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survival probability in phase 4 was tested by Log-Rank test indicating that the duration of
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Phase 4 was different between groups.
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4. Discussion
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In view of the continuous technical improvements in ultrasound equipment it is
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essential to reconfirm data previously published in the veterinary literature. In this study
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ultrasonographic description of the development of foetal bowel was studied because
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bowel is the organ whose development is reported to terminate foetal organogenesis,
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indicating a foetal age of 57 days of gestation [5,8,9].
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Accurate determination of foetal age is important to reduce the risk of premature
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birth and consequent foetal deaths by informed decisions on timing of caesarean, if this is
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indicated. Accurate foetal ageing is particularly important in brachicephalic breeds, in
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which surgical intervention is often necessary for delivery [16,17,18,19,20,21].
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In this study the first ultrasonographic identification of foetal bowel occurred around
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39 to 44 days of gestation (Table 2) period. At that time (Phase 1) foetal bowel has
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ultrasonographic features not described in previous studies. In Phase 1, ultrasound
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characteristics correspond to primitive intestine development, originating from the dorsal
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mesenchymal layer (dorsal mesentery) and is interposed between vascular supply,
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lymphatics and neurones. Phase 2 is accompanied by the formation of bowel walls, which
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are derived from endoderm (bowel inner layer) and splanchnic mesoderm (outer layer) that
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will develop into the connective muscle of bowel wall and will subsequently organize to
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form four layers of intestinal wall and contribute to increased intestinal length [22]. The ability to see defined layers of foetal intestinal wall and lumen interface,
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associated with the first visualisation of intestinal peristalsis, heralds the beginning of
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Phase 3, which occurs at approximately 48 to 54 days of gestation (Table 2). In this phase
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peristalsis is not constant, and some regions of bowel exhibit no peristaltic movement
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(Figure 1C). It is this intermittent peristalsis that defines that the foetus is still in phase 3 of
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development and has not completed organogenesis. Errors in interpretation of peristaltic
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motion may result in inaccurate determination of foetal age and consequently inappropriate
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planning of surgical intervention (C-section), whereas in fact in phase 3 there are still
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approximately eight to fifteen days before full term (Table 2).
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Phase 4 coincides with an approximate foetal age of 57 to 62 days of gestation (Table
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2) and probably corresponds with the descriptions of foetal intestine as reported in the
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literature [5,8,9,10]. This is the last stage of foetal organogenesis. At this time, it is
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possible to recognize the bowel wall and distinguish the surface mucosa versus the wall
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(Figure 1D). With high-resolution ultrasound equipment and appropriate adjustments to
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examine the foetus, three layers of intestinal wall (as in adult dogs) [23] can be identified.
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However, ultrasonographic visualization of five wall layers (as in adult intestine) is not
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possible, presumably because of the size of structures when compared to adult patients.
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In Phase 4 intraluminal contents are well defined, we suggest that this finding is
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related to increased meconium in the gastrointestinal tract, which in man correlates to
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progression of pregnancy [24,25]. In human foetuses, it is known that propulsion of
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meconium along the intestine causes gut distension and sufficient peristalsis to move the
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luminal contents [26], a finding also observed in our study.
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Identification of anechoic intraluminal fluid is a characteristic of intestinal
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maturation of human foetuses, and corresponds to meconium. Meconium accumulates in
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the intestine partially distending the bowel and facilitating identification of peristalsis. In
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human foetuses this finding, together with visualization of stomach and bladder are some
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of ultrasonographic features that assist in determining progression of foetal organogenesis
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[27].
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Care should be taken during ultrasound examination when differentiating phases 3
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and 4 of intestinal development as this distinction is subtle. Assessment of peristalsis is the
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key point - in Phase 4 intestinal peristalsis is visible immediately, whereas in Phase 3
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identification of peristalsis is only possible after one minute’s observation. Another
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important point for distinction between phase 3 and 4 is that intraluminal fluid is more
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evident in Phase 4. It is essential that decisions with regard to planning caesarean are not
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rushed as this could result in death of premature foetuses.
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Phases 1, 2 and 3 of foetal bowel development occurred, on average, at the same
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foetal ages (as determined by ultrasonography), in both groups of bitches (normal delivery
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and C-section) (Table 2); demonstrating that development of organs is equivalent in both
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groups. However, in this study we show that there were, on average, twice as many days
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until delivery from onset of phase 4 in bitches which subsequently had caesarean deliveries
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compared to those bitches which achieved normal delivery. The difference in duration of
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phase 4 (in days) was confirmed statistically by the Student t-test. It is likely that those
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bitches which subsequently had caesarean deliveries went beyond full term whilst
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investigators waited for puppies to be born by normal delivery. Caesareans were only
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requested once normal delivery had failed to occur, and foetuses became distressed. The
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Cox model of proportional hazards resulted in a significant coefficient demonstrating the
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differences in duration of Phase 4 development stage between foetuses of bitches born by
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caesarean deliveries compared to foetuses of bitches which had a normal delivery. The probability of survival for foetuses born on a particular day is illustrated by the
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Kaplan-Meier curve (Figure 2) and is correlated to the degree of foetal maturation on each
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day of Phase 4 in both groups of dogs ie once intestinal peristalsis is consistently
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demonstrated ultrasonographically. Foetuses born at this time had a 10% chance of
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survival if delivered naturally and a 20% chance of survival if delivered by C-section . This
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suggests that foetuses from both groups were probably insufficiently mature to survive
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extra-utero. This demonstrates that ultrasound detection of foetal intestinal peristalsis on
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the first day of phase 4 is insufficient alone to indicate foetal maturity. Natural delivery
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occurred four days after first detection of peristalsis and after six days in bitches submitted
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to C-section. When intestinal peristalsis is demonstrated on a single ultrasound
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examination foetal maturation should be confirmed by additional evaluation of other
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organs.
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The Kaplan-Meier curve (Figure 2) shows a difference in the probability of survival
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between foetuses of the two groups in stage 4. This difference between groups was tested
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by Log-Rang test which proved to be statistically significant (P=0.05). The difference in
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probability of survival between the groups is because there are more days of development
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required for foetuses in the group of bitches undergoing caesarean deliveries. Once again,
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these extra days are probably due to delays in delivery - normal delivery was expected in
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the caesarean delivery group until such time as the foetus became distressed [12,13], and
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surgery was indicated.
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The limitations of this study are that all foetuses were born healthy, despite
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indications for caesarean section, foetal distress was based on ultrasound detection of
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variations in foetal heart rate [13]. Another factor that must be considered is that there was
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a wider than expected age at which bitches were presented for examination, because these
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were owned pets and not experimental dogs. Other limitations of this study were breed
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variations, the small number of animals included and the absence of luteinizing hormone
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measurements.
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Intestinal peristalsis is an important finding that provides evidence of completion of
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canine foetal organogenesis. However, it should be noted that, even after complete
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development of bowel, ultrasonography cannot be relied upon to determine functionality
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(maturation) of this organ. This was demonstrated in this study because even after
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ultrasonographic confirmation of foetal intestinal development, up to a further four days
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elapsed before natural delivery and up to six days before signs of foetal distress were
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evident.
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Ultrasonographic evidence of completion of bowel organogenesis should not be used
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as the sole parameter for scheduling caesarean section. We suggest daily foetal monitoring
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by ultrasound from Phase 4 of intestinal development since oscillations of foetal heart rate
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[13] begins at this time and this may be a more reliable predictor of day of parturition.
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5. Conclusions
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Evaluation of the development of a pregnancy is possible using ultrasonographic
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monitoring of intestinal development and this can reliably identify the end of foetal
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organogenesis. Ultrasonographic detection of intestinal segments with visualization of wall
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layers and associated peristalsis should not however be used as the sole indicator for C-
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section planning because it is not possible to determine ultrasonographically whether the
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bowel is functional (mature).
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statistical help. Our main thanks go to the owners who agreed to cooperate with this study.
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We thank CAPES for the scholarship granted and Eliane de Fátima Coimbra for
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6. Acknowledgements
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Figure legends
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Figure 1. Ultrasound image showing (A) Phase 1: presence of echogenic and homogeneous
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areas (arrows) in caudal region to the foetal liver. (B) Phase 2: beginning of differentiation
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of wall layers in the foetal bowel (blue arrow), with intermixed anechoic areas (red arrow).
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No intestinal peristalsis. (C) Phase 3: identification of intestinal wall segment intermixed
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with fewer anechoic areas (red arrow) when compared to Phase 2. The defined segments
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showed intraluminal mucosal content (blue arrow) and there is a better definition of the
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mucosal surface versus intestine wall. Intestinal peristalsis identified in some bowel
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segments. (D) Phase 4: well defined intestinal wall, mucosa versus intestinal wall clearly
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defined, ultrasonographic definition of bowel wall layers into mucosal surface -
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hyperechoic; mucosa, submucosa, muscle - hypoechoic; serosa- hyperechoic; segmental
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dilatation of the bowel by intraluminal mucous (arrow) [23]. Intestinal peristalsis in all
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bowel segments.
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Figure 2. Kaplan-Maier curve showing probability of survival for foetuses born on any day
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during Phase 4.
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15. Kaplan EL, Meier PJ. Nonparametric estimation from incomplete observations. J Amer Statist Assoc 1958; 53(282): 457–60.
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16. Johnston SD, Root Kustritz MV, Olson PNS. Canine pregnancy. In: Johnston SD,
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17. Jackson PGG. Dystocia in dog and cat. In: Jackson PGG, editor. Handbook of
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2004 p. 141-166. 18. Linde-Forsberg C. Abnormalities in pregnancy, parturition and the periparturient
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19. Bergstrom A, Nodtvedt A, Lagerstedt AS, Egenvall A. Incidence and breed
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20. Evans KM, Adams VJ. Proportion of litters of purebred dogs born by Caesarean section. J Small Anim Pract 2010; 51(2): 113–8.
21. Wydooghe E, Berghmans E, Rijsselaere T, Van Soom A. International breeder inquiry into the reproduction of the English bulldog. In Pract 2013; 82: 38–43.
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23. Penninck DG, Nyland TG, Fisher PE, Kerr LY. Normal ultrasonography of the canine gastrointestinal tract. Vet Radiol Ultrasound 1989; 30(6): 272-76.
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24. Becker RF, Marth EE, Schulz MD. Fetal swallowing, gastro-intestinal activity and defecation in amnio. Surg Gynecol Obstet 1940; 70: 603-14.
25. McLain CRJr. Amniography studies of the gastrointestinal motility of the human fetus. Am J Obstet Gynecol 1963; 15(86): 1079-87. 26. Crelin, ES. Functional Anatomy of the newborn. New Haven, Yale University, 1973, p. 56-58.
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Gestational age and Fetal Maturity. Obstet Gynecol 1983; 62(5): 569-73.
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Table 1 - Description of breeds, age and litter size of bitches classified according to the type of delivery May/ 2013 to April/ 2014. Type of Age Litter Breed Delivery (years) Size 2 Dog Chinese Crested 2 5 French Bulldog 3 5 Miniature Schnauzer 3 1 Siberian Husky 7 Normal 2 Pug 7 Delivery 2 Pekingese 5 1 Maltese 3 2 Chihuahua 3 6 Miniature Schnauzer 4 2 Dog Chinese Crested 3 5 Yorkshire Terrier 4 3 American Staffordshire Terrier 3 2 English Bulldog 9 Caesarean 2 English Bulldog 3 1 English Bulldog 5 2 Pug 3 6 Miniature Schnauzer 4 3 Miniature Schnauzer 4
ACCEPTED MANUSCRIPT Table 2 - Descriptive statistics of four phases of ultrasound visualization of foetal bowel development intrauterine correlated foetal age determined counting backward considering delivery day zero, categorized according to type of delivery (normal delivery or caesarean), May/ 2013 to April/ 2014.
19 to 23 15 to 19 9 to 13 1 to 4
21 16 10 2
20 to 24 14 to 19 8 to 15 2 to 6
22 16 11.5 4
20.86 16.67 10.67 2.22
1.35 1.22 1.41 1.09
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Normal delivery Phase 1 Phase 2 Phase 3 Phase 4 Caesarean Phase 1 Phase 2 Phase 3 Phase 4
21.44 16.56 11.75 4.11
Estimated gestacional age (Days of gestation)
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Gestacional age Standard Median Average (Days from delivery) deviation
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1.51 1.81 2.25 1.36
40 to 44 44 to 48 50 to 54 59 to 62 39 to 43 44 to 49 48 to 55 57 to 61
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Abbreviations: US, ultrasonographic; Phase 1: viewing a echogenic area in topographic region of bowel; Phase 2: beginning visualization of definition intestinal wall without peristalsis; Phase 3: peristalsis visualization in some segments of bowel; Phase 4: peristalsis visualization in all segments (movement every 3 seconds).
ACCEPTED MANUSCRIPT Table 3 - Values obtained in Student t-test in which time (in days) of intestinal development visualization is analyzed in four phases of development, comparing the bitches who had normal delivery versus caesarean section.
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US phases development of Confidence interval 95% foetal bowel Lower limit Higher limit D. F. p- value Phase 1 -2.1248 0.9502 14 0.4257 Phase 2 -1.451 1.6732 14 0.881 Phase 3 -3.1085 0.9419 12 0.2653 Phase 4 -3.1289 -0.6489 15 0.0054**
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Abbreviations: US, ultrasonographic; Phase 1: viewing a echogenic area in topographic region of bowel; Phase 2: beginning visualization of definition intestinal wall without peristalsis; Phase 3: peristalsis visualization in some segments of bowel; Phase 4: peristalsis visualization in all segments (movement every 3 seconds). ** Significant at 5%.
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ACCEPTED MANUSCRIPT Highlights Describes the ultrasonographic development of foetal bowel and correlates this with gestational age. Defines as ultrasonographic visualization of foetal intestinal peristalsis in utero is not
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associated with foetal maturation.
Determines that there is a difference in foetal intestinal peristalsis detection time (days
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before delivery), between foetuses born by normal delivery and caesarean.
S0093-691X(15)00231-9
DOI:
10.1016/j.theriogenology.2015.04.030
Reference:
THE 13181
To appear in:
Theriogenology
Received Date: 26 February 2015 Revised Date:
28 April 2015
Accepted Date: 29 April 2015
Please cite this article as: Gil EMU, Garcia DAA, Froes TR, In-utero development of the foetal intestine – sonographic evaluation and correlation with gestational age and foetal maturity in dogs, Theriogenology (2015), doi: 10.1016/j.theriogenology.2015.04.030. 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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In-utero development of the foetal intestine – sonographic evaluation and correlation
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with gestational age and foetal maturity in dogs
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Elaine M. U. Gil1*, Daniela A. A. Garcia1, Tilde R. Froes1
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of Paraná, Brazil
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* Corresponding author:
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Elaine M. U. Gil
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[email protected]
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Department of Veterinary Medicine, School of Veterinary Medicine, Federal University
Rua dos Funcionários 1540. Juvevê. Zip Code 80035-050
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Curitiba City, Paraná State, Brazil
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Phone: 55 (41) 3350-5767, Fax: 55 (41) 3350-5725
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Abstract
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Modern high-resolution ultrasound images enable earlier assessment of measures of foetal
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development, including identification of bowel. The aim of this study was to describe the
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ultrasonographic development of foetal bowel and correlate this with gestational age;
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define whether ultrasonographic visualization of foetal intestinal peristalsis in utero is
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associated with foetal maturation and determine whether there is a difference in foetal
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intestinal peristalsis detection time between foetuses delivered by normal delivery and
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caesarean. A cohort study was conducted in pregnant bitches presented to a veterinary
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hospital, to assess foetal bowel development. Statistical analysis was used to establish the
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correlation of the stage of foetal bowel development, as recorded by ultrasound, with
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outcomes of normal delivery and caesarean section. The study was broken down into three
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stages: the first stage was a descriptive analysis of foetal bowel development by
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ultrasound; the second stage compared time (in days) of bowel development between
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groups (normal delivery versus caesarean); and the third stage was correlated survival
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probability for foetuses born on any day after detection of intestinal peristalsis with foetal
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maturity. All statistical analyses were significant. It is possible to monitor pregnancy
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progression using ultrasonographic evaluation of bowel development and this can reliably
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identify the end of foetal organogenesis. However, ultrasonographic detection of bowel
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segments with visualization of wall layers and associated peristalsis should not be used as
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the sole indicator for caesarean section planning because it is not possible to determine
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ultrasonographically whether the bowel is functional (mature).
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Keywords: Canine, pregnancy, gestational ultrasound, foetal bowel ultrasonography,
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maturation
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1. Introduction Ultrasonography is the preferred method for early diagnosis of pregnancy, evaluation
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of foetal viability, and estimation of gestational age [1]. Early and accurate determination
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of gestational age is useful for predicting time and management of deliver, including
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planning caesarean section (C-section) [2]. In some breeds of dogs in which C-section is
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considered necessary, assessment of foetal maturity and appropriate timing of C-section
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can reduce neonatal mortality [3].
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As in human medicine, there are a variety of methods for ultrasonographic estimation
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of foetal age in pregnant bitches, these include calculation from crown-rump length in
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early pregnancy, biparietal diameter and assessment of foetal organogenesis by serial
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examinations [2,3,4,5,6,7]. The assessment of foetal organogenesis is an interesting model
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for determination of foetal age in dogs. The bowel is the last organ to be identified by
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ultrasonography as foetal dogs develop [5,7].
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According to some authors visualization of bowel and identification of peristalsis by
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ultrasound determines the end of foetal organogenesis, indirectly indicating that the foetus
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is full term. This happens around 57 to 63 days of gestation [5,8,9]. Several authors
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[7,8,9,10] describe a range of gestational ages for detection of peristalsis by
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ultrasonography from 57 days after the peak of luteinizing hormone.
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However, modern high-resolution ultrasound images enable earlier assessment of
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measures of foetal development, including identification of bowel. There are no previous
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studies describing the ultrasonographic assessment of bowel development in canine
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foetuses. Our hypothesis is that ultrasonographic visualization of foetal bowel is possible at
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earlier foetal ages than previously described. The purpose of this study was threefold: 1. To
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describe the ultrasonographic development of foetal bowel and correlate this with
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gestational age; 2. To define whether ultrasonographic visualization of foetal intestinal
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peristalsis in utero is associated with foetal maturation; 3. To determine whether there is a
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difference in foetal intestinal peristalsis detection time (days before delivery), between
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foetuses born by normal delivery and caesarean.
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2. Materials and Methods 2.1 Patient selection
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A cohort study was conducted in pregnant bitches to assess foetal bowel
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development. The sample population consisted of bitches with confirmed pregnancy
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presented to a veterinary hospital between May 2013 and April 2014. All procedures were
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carried out in accordance with Animal Use Committee guidelines. All owners provided
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informed, written consent to be included in the study. Exclusion criteria were bitches with
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positive pregnancy unavailable for serial examinations, late pregnancy (more than 30 days
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gestation), at time of first presentation, date of delivery not reported by owners and bitches
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presented with concomitant disease.
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2.2 Ultrasound imaging and equipment
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Ultrasound scanning was performed with a high-resolution linear multifrequency
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transducer (7.5 to 12 MHz), model MyLab 30 [Esaote, Genova, Italy]. The gain, focus and
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other adjustments were made during examination to obtain the best image for each foetus
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evaluated. All pregnant bitches were prepared with hair clipped prior to the examination,
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positioned in dorsal recumbency and ultrasound gel was used to optimize image
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acquisition.
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The pregnant bitches were followed by ultrasonographic examination from the 14th
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day after the first mating and/or insemination. Ultrasonography examinations were
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performed every four days until intense peristalsis (movement every three seconds) of
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foetal bowel was visible. From that examination onwards daily assessments were made
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until delivery. Gestational age was confirmed after delivery, in days to delivery, by counting
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backwards (delivery as day zero), normal gestational length was considered to be 57 to 63
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days, due to the variable pro-estrus and estrus periods in dogs [11]. During ultrasound
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examination gestational age was estimated in days of gestation, using the descriptions of
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Yeager et al.[5].
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Each sonographer scanned all foetuses in each pregnant bitch. Each bitch was
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scanned independently by two experienced sonographer (one of whom is a member of the
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Brazilian College of Veterinary Radiology) at each examination. However, both
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sonographers were in the room during the examinations. The ultrasound interpretation was
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achieved by consensus of the two sonographers. The examination method followed a
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protocol to examine the whole abdomen by scanning clockwise, starting with foetuses in
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the left uterine horn (cranial to caudal) followed by the right horn (caudal to cranial). In
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bitches with large numbers of foetuses, at least four foetuses were examined at all
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examinations, and in bitches with four or fewer foetuses all foetal abdomens were
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examined.
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Intrauterine foetal abdomens were assessed in the longitudinal and dorsal planes of
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each foetus. All foetal organogenesis was monitored; however, the priority was
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examination of the bowel.
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After data collection pregnant females were classified according to delivery method
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(C-section or natural) and divided in two groups. The decision to perform a C-section was
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based on ultrasound indications of foetal distress [12]. Foetuses were considered to be
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distressed if foetal heart rate remained low (between 160 and 190 beats per minut), and
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without peaks of acceleration and deceleration as described by Gil et al. [13]. Foetal heart
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rate was measured using M-mode for five minutes per foetus in all pregnant bitches up to
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maximum four foetuses.
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2.3 Experimental Design
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Statistical analysis was used to establish the correlation of the stage of foetal bowel
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development, as recorded by ultrasound, with outcomes of normal delivery and C-section.
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The study was broken down into three stages.
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The first stage was a descriptive analysis of foetal bowel development by ultrasound.
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In the second and third stages a rating from one to four, was used for various phases of
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foetal bowel development. Phase 1 was defined as the first time bowel could be visualized
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and phase 4 was the last phase, when intestinal peristalsis (movement every three seconds)
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was identified. Once phase 4, had been identified this was used for statistical analysis of
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the second and third stages of the experiment. For each phase (1 to 4) of intestinal
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development respective foetal age ranges were calculated.
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The aim of the second stage was to compare time (in days) of bowel development
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between groups (normal delivery versus caesarean). For this, statistical testing was used: 1.
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Student t-Test which compared all phases (1/1, 2/2, 3/3 and 4/4) between the two groups;
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2. The semi-parametric regression Cox model [14] was used to specifically assess Phase 4
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of bowel development, testing whether there was a difference in time (days) of ultrasound
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visualisation of bowel between the two groups. In the Cox model the group of bitches with
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normal delivery were used as the control.
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The aim of third stage of the study was to correlate survival probability for foetuses
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born on any day in Phase 4, at which time foetuses were assumed to be mature, and to this
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end a survival curve was generated. The survival curve was constructed using the Kaplan-
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Meier method [15] and statistical comparison between groups was performed by the Log-
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Rank test, and then identifying whether there were differences between the survival
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probabilities of puppies born by normal delivery or caesarean. In all analyzes, a P-value of
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α = 0.05 was considered significant and the significance was analyzed relative to the value
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set.
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All statistical tests were selected and performed by one author (E.G) and one
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statistical professional (E.C). Data collation and analysis were performed using Microsoft
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Office Excel (Microsoft ® Office 2007 for Windows, Redmond, WA, USA) and R for
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Windows (version 3.1.1 Ri386, R Foundation for Statistical Computing, Vienna, Austria,
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2014) through packages "Survival", "KMsurv" and "Coin".
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3. Results
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Eighteen pregnant bitches were included in this study. Several breeds were
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represented and ages ranged from 1 to 6 years. Seventy-five foetuses were evaluated and
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all were born healthy, including those delivered by caesarean. The litter size of each bitch
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was between 2 and 9 pups (Table 1).
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After ultrasound monitoring of foetal organogenesis and evaluation of development
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of foetal intrauterine bowel in all females, four different phases of development were
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defined. All foetuses displayed the same ultrasonographic patterns of bowel development.
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The four phases were:
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• Phase 1. One uniform echogenic area caudal to the foetal liver in the topographic region of bowel (Figure 1A);
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• Phase 2. In the same anterior region, some portions of bowel segments were better
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defined allowing visualization of the intestinal wall ie anechoic areas intermixed
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with multifocal normal intestinal wall. Peristalsis was absent (Figure 1B); • Phase 3. Bowel segments had clearly defined intestinal walls and anechoic areas
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were fewer when compared to Phase 2; defined segments had mucosal intraluminal
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content, with better definition of mucosal surface versus intestinal wall. Peristalsis
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in some intestinal portions (Figure 1C);
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• Phase 4. Identification of complete intestinal wall, visual distinction between
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mucosal surface and intestinal wall, ultrasonographic determination of wall layers,
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surface mucosa - hyperechoic; mucosa, submucosa, muscle - hypoechoic; serous -
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hyperechoic. Segmental dilatation of the bowel by intraluminal mucous and fluid
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content. Peristalsis in all segments of the bowel (movement every 3 seconds -
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Figure 1D).
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Table 2 shows gestational ages (days to delivery and days of estimated gestation),
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mean, standard deviation and median of four phases of intrauterine foetal bowel
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development by ultrasound according to type of delivery.
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Table 3 presents results of Student t-test for the four phases of intrauterine foetal
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bowel development comparing the type of delivery (normal or caesarean), showing a
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significant (α = 0.05) difference between groups only in phase 4 of bowel development.
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Semiparametric COX model, coefficient value of reference group (normal delivery
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bitches) was -1.316, resulting in exp (-1.316) = 0.268. This confirms that the time of
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ultrasound visualization of bowel in Phase 4 (in which there is peristalsis) is different in
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foetuses born by C-section from those delivered naturally, in other words, the duration of
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phase 4 is earlier in foetuses delivered by C-section.
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The probability of survival of foetuses born on any day during phase 4 was different
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when comparing the puppies born by normal delivery with those delivered by caesarean,
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resulting in a Kaplan-Meier curve with a stairway shape (Figure 2). This difference in
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survival probability in phase 4 was tested by Log-Rank test indicating that the duration of
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Phase 4 was different between groups.
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4. Discussion
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In view of the continuous technical improvements in ultrasound equipment it is
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essential to reconfirm data previously published in the veterinary literature. In this study
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ultrasonographic description of the development of foetal bowel was studied because
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bowel is the organ whose development is reported to terminate foetal organogenesis,
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indicating a foetal age of 57 days of gestation [5,8,9].
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Accurate determination of foetal age is important to reduce the risk of premature
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birth and consequent foetal deaths by informed decisions on timing of caesarean, if this is
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indicated. Accurate foetal ageing is particularly important in brachicephalic breeds, in
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which surgical intervention is often necessary for delivery [16,17,18,19,20,21].
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In this study the first ultrasonographic identification of foetal bowel occurred around
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39 to 44 days of gestation (Table 2) period. At that time (Phase 1) foetal bowel has
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ultrasonographic features not described in previous studies. In Phase 1, ultrasound
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characteristics correspond to primitive intestine development, originating from the dorsal
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mesenchymal layer (dorsal mesentery) and is interposed between vascular supply,
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lymphatics and neurones. Phase 2 is accompanied by the formation of bowel walls, which
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are derived from endoderm (bowel inner layer) and splanchnic mesoderm (outer layer) that
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will develop into the connective muscle of bowel wall and will subsequently organize to
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form four layers of intestinal wall and contribute to increased intestinal length [22]. The ability to see defined layers of foetal intestinal wall and lumen interface,
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associated with the first visualisation of intestinal peristalsis, heralds the beginning of
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Phase 3, which occurs at approximately 48 to 54 days of gestation (Table 2). In this phase
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peristalsis is not constant, and some regions of bowel exhibit no peristaltic movement
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(Figure 1C). It is this intermittent peristalsis that defines that the foetus is still in phase 3 of
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development and has not completed organogenesis. Errors in interpretation of peristaltic
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motion may result in inaccurate determination of foetal age and consequently inappropriate
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planning of surgical intervention (C-section), whereas in fact in phase 3 there are still
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approximately eight to fifteen days before full term (Table 2).
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Phase 4 coincides with an approximate foetal age of 57 to 62 days of gestation (Table
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2) and probably corresponds with the descriptions of foetal intestine as reported in the
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literature [5,8,9,10]. This is the last stage of foetal organogenesis. At this time, it is
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possible to recognize the bowel wall and distinguish the surface mucosa versus the wall
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(Figure 1D). With high-resolution ultrasound equipment and appropriate adjustments to
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examine the foetus, three layers of intestinal wall (as in adult dogs) [23] can be identified.
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However, ultrasonographic visualization of five wall layers (as in adult intestine) is not
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possible, presumably because of the size of structures when compared to adult patients.
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In Phase 4 intraluminal contents are well defined, we suggest that this finding is
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related to increased meconium in the gastrointestinal tract, which in man correlates to
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progression of pregnancy [24,25]. In human foetuses, it is known that propulsion of
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meconium along the intestine causes gut distension and sufficient peristalsis to move the
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luminal contents [26], a finding also observed in our study.
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Identification of anechoic intraluminal fluid is a characteristic of intestinal
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maturation of human foetuses, and corresponds to meconium. Meconium accumulates in
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the intestine partially distending the bowel and facilitating identification of peristalsis. In
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human foetuses this finding, together with visualization of stomach and bladder are some
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of ultrasonographic features that assist in determining progression of foetal organogenesis
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[27].
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Care should be taken during ultrasound examination when differentiating phases 3
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and 4 of intestinal development as this distinction is subtle. Assessment of peristalsis is the
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key point - in Phase 4 intestinal peristalsis is visible immediately, whereas in Phase 3
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identification of peristalsis is only possible after one minute’s observation. Another
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important point for distinction between phase 3 and 4 is that intraluminal fluid is more
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evident in Phase 4. It is essential that decisions with regard to planning caesarean are not
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rushed as this could result in death of premature foetuses.
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Phases 1, 2 and 3 of foetal bowel development occurred, on average, at the same
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foetal ages (as determined by ultrasonography), in both groups of bitches (normal delivery
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and C-section) (Table 2); demonstrating that development of organs is equivalent in both
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groups. However, in this study we show that there were, on average, twice as many days
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until delivery from onset of phase 4 in bitches which subsequently had caesarean deliveries
244
compared to those bitches which achieved normal delivery. The difference in duration of
245
phase 4 (in days) was confirmed statistically by the Student t-test. It is likely that those
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bitches which subsequently had caesarean deliveries went beyond full term whilst
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investigators waited for puppies to be born by normal delivery. Caesareans were only
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requested once normal delivery had failed to occur, and foetuses became distressed. The
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Cox model of proportional hazards resulted in a significant coefficient demonstrating the
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differences in duration of Phase 4 development stage between foetuses of bitches born by
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caesarean deliveries compared to foetuses of bitches which had a normal delivery. The probability of survival for foetuses born on a particular day is illustrated by the
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Kaplan-Meier curve (Figure 2) and is correlated to the degree of foetal maturation on each
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day of Phase 4 in both groups of dogs ie once intestinal peristalsis is consistently
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demonstrated ultrasonographically. Foetuses born at this time had a 10% chance of
256
survival if delivered naturally and a 20% chance of survival if delivered by C-section . This
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suggests that foetuses from both groups were probably insufficiently mature to survive
258
extra-utero. This demonstrates that ultrasound detection of foetal intestinal peristalsis on
259
the first day of phase 4 is insufficient alone to indicate foetal maturity. Natural delivery
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occurred four days after first detection of peristalsis and after six days in bitches submitted
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to C-section. When intestinal peristalsis is demonstrated on a single ultrasound
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examination foetal maturation should be confirmed by additional evaluation of other
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organs.
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The Kaplan-Meier curve (Figure 2) shows a difference in the probability of survival
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between foetuses of the two groups in stage 4. This difference between groups was tested
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by Log-Rang test which proved to be statistically significant (P=0.05). The difference in
267
probability of survival between the groups is because there are more days of development
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required for foetuses in the group of bitches undergoing caesarean deliveries. Once again,
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these extra days are probably due to delays in delivery - normal delivery was expected in
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the caesarean delivery group until such time as the foetus became distressed [12,13], and
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surgery was indicated.
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The limitations of this study are that all foetuses were born healthy, despite
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indications for caesarean section, foetal distress was based on ultrasound detection of
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variations in foetal heart rate [13]. Another factor that must be considered is that there was
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a wider than expected age at which bitches were presented for examination, because these
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were owned pets and not experimental dogs. Other limitations of this study were breed
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variations, the small number of animals included and the absence of luteinizing hormone
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measurements.
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Intestinal peristalsis is an important finding that provides evidence of completion of
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canine foetal organogenesis. However, it should be noted that, even after complete
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development of bowel, ultrasonography cannot be relied upon to determine functionality
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(maturation) of this organ. This was demonstrated in this study because even after
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ultrasonographic confirmation of foetal intestinal development, up to a further four days
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elapsed before natural delivery and up to six days before signs of foetal distress were
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evident.
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Ultrasonographic evidence of completion of bowel organogenesis should not be used
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as the sole parameter for scheduling caesarean section. We suggest daily foetal monitoring
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by ultrasound from Phase 4 of intestinal development since oscillations of foetal heart rate
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[13] begins at this time and this may be a more reliable predictor of day of parturition.
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5. Conclusions
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Evaluation of the development of a pregnancy is possible using ultrasonographic
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monitoring of intestinal development and this can reliably identify the end of foetal
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organogenesis. Ultrasonographic detection of intestinal segments with visualization of wall
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layers and associated peristalsis should not however be used as the sole indicator for C-
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section planning because it is not possible to determine ultrasonographically whether the
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bowel is functional (mature).
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statistical help. Our main thanks go to the owners who agreed to cooperate with this study.
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We thank CAPES for the scholarship granted and Eliane de Fátima Coimbra for
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6. Acknowledgements
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Figure legends
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Figure 1. Ultrasound image showing (A) Phase 1: presence of echogenic and homogeneous
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areas (arrows) in caudal region to the foetal liver. (B) Phase 2: beginning of differentiation
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of wall layers in the foetal bowel (blue arrow), with intermixed anechoic areas (red arrow).
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No intestinal peristalsis. (C) Phase 3: identification of intestinal wall segment intermixed
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with fewer anechoic areas (red arrow) when compared to Phase 2. The defined segments
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showed intraluminal mucosal content (blue arrow) and there is a better definition of the
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mucosal surface versus intestine wall. Intestinal peristalsis identified in some bowel
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segments. (D) Phase 4: well defined intestinal wall, mucosa versus intestinal wall clearly
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defined, ultrasonographic definition of bowel wall layers into mucosal surface -
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hyperechoic; mucosa, submucosa, muscle - hypoechoic; serosa- hyperechoic; segmental
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dilatation of the bowel by intraluminal mucous (arrow) [23]. Intestinal peristalsis in all
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bowel segments.
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Figure 2. Kaplan-Maier curve showing probability of survival for foetuses born on any day
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during Phase 4.
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Table 1 - Description of breeds, age and litter size of bitches classified according to the type of delivery May/ 2013 to April/ 2014. Type of Age Litter Breed Delivery (years) Size 2 Dog Chinese Crested 2 5 French Bulldog 3 5 Miniature Schnauzer 3 1 Siberian Husky 7 Normal 2 Pug 7 Delivery 2 Pekingese 5 1 Maltese 3 2 Chihuahua 3 6 Miniature Schnauzer 4 2 Dog Chinese Crested 3 5 Yorkshire Terrier 4 3 American Staffordshire Terrier 3 2 English Bulldog 9 Caesarean 2 English Bulldog 3 1 English Bulldog 5 2 Pug 3 6 Miniature Schnauzer 4 3 Miniature Schnauzer 4
ACCEPTED MANUSCRIPT Table 2 - Descriptive statistics of four phases of ultrasound visualization of foetal bowel development intrauterine correlated foetal age determined counting backward considering delivery day zero, categorized according to type of delivery (normal delivery or caesarean), May/ 2013 to April/ 2014.
19 to 23 15 to 19 9 to 13 1 to 4
21 16 10 2
20 to 24 14 to 19 8 to 15 2 to 6
22 16 11.5 4
20.86 16.67 10.67 2.22
1.35 1.22 1.41 1.09
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Normal delivery Phase 1 Phase 2 Phase 3 Phase 4 Caesarean Phase 1 Phase 2 Phase 3 Phase 4
21.44 16.56 11.75 4.11
Estimated gestacional age (Days of gestation)
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Gestacional age Standard Median Average (Days from delivery) deviation
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1.51 1.81 2.25 1.36
40 to 44 44 to 48 50 to 54 59 to 62 39 to 43 44 to 49 48 to 55 57 to 61
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Abbreviations: US, ultrasonographic; Phase 1: viewing a echogenic area in topographic region of bowel; Phase 2: beginning visualization of definition intestinal wall without peristalsis; Phase 3: peristalsis visualization in some segments of bowel; Phase 4: peristalsis visualization in all segments (movement every 3 seconds).
ACCEPTED MANUSCRIPT Table 3 - Values obtained in Student t-test in which time (in days) of intestinal development visualization is analyzed in four phases of development, comparing the bitches who had normal delivery versus caesarean section.
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US phases development of Confidence interval 95% foetal bowel Lower limit Higher limit D. F. p- value Phase 1 -2.1248 0.9502 14 0.4257 Phase 2 -1.451 1.6732 14 0.881 Phase 3 -3.1085 0.9419 12 0.2653 Phase 4 -3.1289 -0.6489 15 0.0054**
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Abbreviations: US, ultrasonographic; Phase 1: viewing a echogenic area in topographic region of bowel; Phase 2: beginning visualization of definition intestinal wall without peristalsis; Phase 3: peristalsis visualization in some segments of bowel; Phase 4: peristalsis visualization in all segments (movement every 3 seconds). ** Significant at 5%.
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ACCEPTED MANUSCRIPT Highlights Describes the ultrasonographic development of foetal bowel and correlates this with gestational age. Defines as ultrasonographic visualization of foetal intestinal peristalsis in utero is not
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associated with foetal maturation.
Determines that there is a difference in foetal intestinal peristalsis detection time (days
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before delivery), between foetuses born by normal delivery and caesarean.