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Reference Values and Repeatability of B-mode and M-mode Echocardiographic Parameters in Healthy Donkey (Equus asinus) - The Guide to Assess Healthy Heart
Journal Pre-proof Reference Values and Repeatability of B-mode and M-mode Echocardiographic Parameters in Healthy Donkey (Equus asinus) - The Guide to Assess Healthy Heart Alshimaa M.M. Farag, Hussam M.M. Ibrahim PII:
S0737-0806(20)30020-4
DOI:
https://doi.org/10.1016/j.jevs.2020.102929
Reference:
YJEVS 102929
To appear in:
Journal of Equine Veterinary Science
Received Date: 1 October 2019 Revised Date:
19 December 2019
Accepted Date: 7 January 2020
Please cite this article as: Farag AMM, Ibrahim HMM, Reference Values and Repeatability of Bmode and M-mode Echocardiographic Parameters in Healthy Donkey (Equus asinus) - The Guide to Assess Healthy Heart, Journal of Equine Veterinary Science (2020), doi: https://doi.org/10.1016/ j.jevs.2020.102929. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2020 Elsevier Inc. All rights reserved.
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Reference Values and Repeatability of B-mode and M-mode Echocardiographic Parameters in
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Healthy Donkey (Equus asinus) - The Guide to Assess Healthy Heart
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Alshimaa M.M. Farag; Hussam M.M. Ibrahim*
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Department of Internal Medicine and Infectious Diseases, Faculty of Veterinary Medicine,
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Mansoura University, Mansoura 35516, Egypt
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* Corresponding author:
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Tel.: +2-050-2214233; Mobile: +2-01005290592
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E-mail: [email protected]
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Abstract
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Donkey (Equus asinus), the cheapest form of agricultural power in Egypt, is used principally as
26
draught or pack animals and may also be ridden, or used for threshing, raising water, milling and
27
other work. The aim of the present study was to provide B-mode and M-mode echocardiographic
28
reference ranges from a sample of population of donkeys (Equus asinus) based on the probability
29
theory and statistics. In the current study, B-mode and 2 dimensional guided M-mode
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echocardiography were performed on 44 clinically healthy donkeys (Equus asinus) (22 males and 22
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females), without any neither clinical nor echocardiographic evidences of cardiovascular disease,
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aged 2 – 25 years old, and weighed 150 – 350 kg. The echocardiographic dimensions; including
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interventricular septum thickness at end diastole, interventricular septum thickness at end systole,
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left ventricular internal diameter at end diastole, left ventricular internal diameter at end systole, left
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ventricular posterior wall thickness at end diastole, and left ventricular posterior wall thickness at
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end systole were assessed. The cardiac function indices; including end diastolic volume, end systolic
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volume, stroke volume, ejection fraction, and fractional shortening were also measured. B-mode and
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guided M-mode echocardiographic examination of healthy donkeys revealed that both the
39
interventricular septum thickness and the left ventricular posterior free wall were greater during
40
systole than diastole. However, the left ventricular dimension during diastole was greater than
41
systole. Furthermore, the recorded data of cardiac functional indices; including end diastolic volume,
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end systolic volume, stroke volume, ejection fraction, and fractional shortening using B-mode
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echocardiography were closer to those recorded by guided M-mode echocardiography. To our
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knowledge, this is the first Egyptian study to report the echocardiographic reference ranges of
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donkeys (Equus asinus) based on the probability theory and statistics, the coefficient of variation.
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The results of the current study can be used as a standard and reference value for further
2
47
echocardiographic studies in donkeys and represents a step in the identification, interpretation, and
48
evaluation of cardiovascular disorders in such animals.
49 50
Key
words:
Donkeys
(Equus
asinus),
B-mode
echocardiography,
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echocardiography, Cardiac dimensions, Cardiac function indices.
Guided
M-mode
52 53
1. Introduction
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The quantification of cardiac dimensions and cardiac function indices is the backbone of cardiac
55
imaging, with echocardiography being a safe, non-invasive, powerful standard diagnostic imaging
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tool in equine cardiology. Equine echocardiography uses ultrasound technology to display images of
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a horse’s heart, providing a substantial amount of information about cardiac function, structure, flow
58
and indexes important for their athletic performance [1]. Beside qualitative estimation, measurement
59
of the cardiac dimensions and estimation of the cardiac function indices are inevitable to determine
60
the cardiac performance of healthy animals and to identify injuries, disorders or structural
61
abnormalities of the heart that may impair performance [2]. It is widely used in cardiology clinics
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worldwide to assess heart chambers size, how well the left side of the heart is functioning, intra-
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cardiac and extra-cardiac masses, systolic and diastolic function, valvular structure and function, and
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fluid collections in the pericardial sac around the heart [3,4].
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Echocardiography is the cornerstone for diagnosis of congenital cardiac malformations, valvular
66
diseases, myocardial diseases, acquired cardiac defects, cardiac arrhythmias, and cardiac murmurs.
67
Thus, it is recommended in horses with loud systolic and diastolic murmurs, muffling heart sound,
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pericardial friction rubs, and exercise intolerance. It is also essential to exactly identify the cause of
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the cardiac murmurs and characterize the severity of the underlying cardiac abnormality as well as
3
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assessing its potential impact on the horse performance and life expectancy [5]. Three valuable types
71
of echocardiography, Two-dimension (B-mode), Motion (M-mode) and Doppler are well known
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providing together complementary information about the nature of cardiac abnormalities as well as
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the hemodynamic status.
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B-Mode echocardiography, backbone of cardiac ultrasound, allows imaging a plane of tissue
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(both depth and width) in real time. The images that cut the heart into slices, which may follow the
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length (longitudinal plane) and the width of the heart (transverse plane) represent an
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echocardiography plane [6]. Thus, the anatomical relations between different structures are easier to
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appreciate. There are an infinite number of cardiac imaging planes; however, the standard views are
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essential for evaluation of the intra- and extra-cardiac structures [7,8]. B-Mode echocardiography
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permits imaging the internal cardiac structures, including the heart chambers, the structure and
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thickness of the chamber walls, the structure and function of the valves, myocardium, pericardium
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and great vessels [9,10].
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M-mode echocardiography is produced by transmission and reception of an ultrasound signal
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along only one line, giving a high sensitivity, greater than B-mode, to record the moving structures
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(e.g. valve opening and closing or ventricular wall movement) [11]. M-mode echocardiography
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produces a one-dimension view of the moving cardiac structures over time. A more accurate
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positioning of the M-mode cursor within the heart is performed using two-dimension real-time
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image as a guide. M-mode echocardiogram uses a high sampling rate and can yield cleaner images
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of cardiac borders, allowing the clinician to obtain more accurate measurements of cardiac
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dimensions and more critically evaluate the motion of cardiac structures. Therefore, M-mode
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echocardiography is superior to B-mode echocardiography for timing of events and is used to obtain
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precise cardiac measurements [12]. In equine cardiology, echocardiography has been introduced
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allowing measurement of cardiac dimensions since 1977, but the standardization of the imaging
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techniques and obtaining reference values persisted until recent years. Accurate measurement of
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cardiac dimensions requires knowledge of reference values of normal cardiac anatomy,
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echocardiographic dimensions and cardiac function indices that represents the basis for diagnosing
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cardiac diseases. Thus, the goal of the present investigation was to determine the echocardiographic
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dimensions and cardiac function indices in clinically healthy donkeys (Equus asinus) which will be
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helpful in diagnosing donkeys with cardiac diseases. To our knowledge, this is the first Egyptian
100
study to report on echocardiographic reference ranges from donkeys (Equus asinus) based on the
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probability theory and statistics, the coefficient of variation (CV) which is a standardized measure of
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dispersion of a probability distribution or frequency distribution to express the precision and
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repeatability of an assay.
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2. Materials and Methods
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2.1. Animals
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The present study was conducted on 44 clinically healthy donkeys (Equus asinus) from which
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22 males, all gelding, and 22 females. The donkeys were randomly selected from a wide range of
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ages, to give a fairly even distribution within the age groups < 5 years, 5–15 years and >15 years old
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and weighed 150 to 350 kg. The body condition score (BCS) for the selected donkeys were 3 in 34
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donkeys, 3.5 in 5 donkeys, and 4 in 5 donkeys. All donkeys are selected to be clinically healthy and
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free from any cardiovascular illness, based on clinical examination as well as complete cardiac
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examination, including assessment of the heart rate, regularity of the heart beats, the strength of the
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heart sounds, abnormal heart sounds, and electrocardiography [13]. Therefore, any systemic
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diseases, tachycardia, cardiac arrhythmias, cardiac murmurs, detectable cardiac defects, and those
5
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donkeys non-compliant with handling were considered the exclusion criteria. Donkeys were housed
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indoor in the animal house at the Veterinary Teaching Hospital, Faculty of Veterinary Medicine,
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Mansoura University, Egypt on April, 2018 and received no exercise on the day of each
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examination. The nutrition regimen for all donkeys under investigation was maintenance balanced
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mixed ration containing chopped wheat straw ad libitum, 075 – 3.5 kg of bran and 0.75 – 3.5 kg of
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crushed corn and supplemented by trace elements and minerals mixture according to age of the
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donkey. During the entire experiment, all donkeys were kept under the same management and
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feeding practices and close veterinary supervision. All the institutional and national guidelines for
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animals use and care were followed as outlined by the Medical Research Ethics Committee,
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Mansoura University, Mansoura, Egypt, code No. R/10.
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2.2. Echocardiographic examination
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All the examined donkeys were at rest, calm, physically restrained only by halter, and didn't
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receive any sedative or tranquilizing agents during the examination time. The cardiac examination
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area the right side of the chest; area between the 3rd and the 5th intercostal space just caudal to the
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triceps muscle mass, and 3 – 5 cm below and 5 – 10 cm above the olecranon process, was
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thoroughly clipped, shaved and cleaned with alcohol. Acoustic Coupling gel was applied at the
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examination area to enhance the contact with the probe. B-mode and 2 dimensional (2D) guided M-
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mode echocardiography was performed according to standard method [14] and the recommendations
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of the American Society of Echocardiography [15]. For echocardiographic examination, a 2.5 MHz
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phased array transducer (CHISON D3P64L) (Chison iVis 60 EXPERT Color Doppler Ultrasound,
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Chison Medical Imaging Co, China) was used. In the fourth or fifth intercostal space at the level of
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olecranon process, the transducer was positioned, adjusted, clockwise rotated, and slightly directed
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cranially and / or dorsally to get the required standard image. The right parasternal long axis four-
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chamber apical view was used to obtain the images of B-mode echocardiography. However, the
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guided M-mode echocardiography was obtained using the right parasternal short axis view. Once the
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standard imaging plane was reached, Cube method was used to evaluate the echocardiographic
143
dimensions list and Teichholz method was used to assess the cardiac function indices. For each
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donkey, the assessed echocardiographic dimensions and cardiac function indices were conducted 3
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times in a single batch on the same day of examination, which is necessary to calculate the intra-
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assay coefficient of variation. These measurements were conducted for each donkey on 3 different
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days, in separate batches, which is required to calculate the inter-assay coefficient of variation. Each
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time of echocardiography, three non-consecutive cardiac cycles were measured and the average
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value for each variable was obtained. All echocardiographic examinations were done after at 9:00
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AM and were carried out by one trained examiner and revised again by two experts to avoid any
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individual variations.
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2.3. Echocardiographic views and measurement of cardiac dimensions and cardiac function indices
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Routine transthoracic B-mode and 2D-guided M-mode echocardiography were performed to
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measure the echocardiographic dimensions list as well as the cardiac function indices.
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Echocardiographic examinations began using B-mode echocardiography, with the creation of
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sectional planes, evaluating the morphology and movement of the structures of the heart in a global
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way. The B-mode echocardiographic image was obtained using the right parasternal long axis four-
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chamber view (Figure 1) according to Reef et al. [5]; Youssef et al. [14]; Reef [16]. After that, by
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rotating the transducer through 90 0 angle in a clockwise direction with a slight cranial and/or dorsal
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angulation, the right parasternal short-axis view at the chordal level was obtained observing the
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symmetry of the papillary muscles as well as the shape of the left ventricle. In this view, M-mode
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was then selected to obtain the 2D-guided M-mode of the short-axis view of the left ventricle at the
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level of chordae tendinae when the M-mode cursor bisected the left ventricle, and intersected both
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the interventricular septum and left ventricular free wall in a 90 0 angle, positioning the beam over
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this line (Figure 2) according to the method described by Vajhi et al. [4]; Reef et al. [5]; Youssef et
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al. [14]. Both B-mode and guided M-mode echocardiography were performed for measurement of
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the interventricular septum thickness at end-diastole (IVSd), the interventricular septum thickness at
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end-systole (IVSs), the left ventricle internal diameter at end diastole (LVIDd), the left ventricle
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internal diameter at end systole (LVIDs), left ventricular posterior wall thickness at end-systole
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(LVPWs) and left ventricular posterior wall thickness at end-diastole (LVPWd) by using the Cube
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method [17-22]. However, the use of Teichholz method in both B-mode and guided M-mode
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echocardiography enables calculation of the left ventricular volume at end-diastole (EDV) and at
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end-systole (ESV) [23], stroke volume (SV) and fractional shortening (FS) [24], and ejection
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fraction (EF) [24].
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2.4. Statistical analysis
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All statistical procedures were performed with commercially available software (GraphPad
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Prism for Windows version 5.0; GraphPad Software Inc., CA, USA). To assess the normality of
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distribution of the B-mode and guided M-mode echocardiographic data, the Kolmogorov–Smirnov
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normality test was conducted, and these data were normally distributed. The frequency distribution
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and the summary statistics (mean ± SD, 95% CI, median (range), and 10th, 25th, 75th, and 90th
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percentiles) of the B-mode and guided M-mode echocardiographic measurements of the selected
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donkeys were calculated. The repeatability of the B-mode and guided M-mode echocardiographic
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measurements was estimated by calculating the intra-assay CV. For each of the 3 days, the data were
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collected and the inter-assay CV was calculated by dividing the SD of the measurement by the mean
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of the same measurement. CV < 15% was classified as low variability, CV 15–25% as moderate
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variability and CV > 25% as high variability [25]. Linear regression was performed to explore the
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association between sets of variables, including age, body weight and body condition scores (BCSs),
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on B-mode and guided M-mode echocardiographic data. P < .05 was accepted as representing
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statistical significance for such test.
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3. Results
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All the selected donkeys were considered to be healthy on the basis of complete cardiac check. The
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recorded heart rate during echocardiographic examination was within the normal range values 29 –
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50 beats / minute (mean ± SD 38.75 ± 5.51). The documented respiratory rate was also within
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normal range values 14 – 20 respiratory cycles / minute (mean ± SD 17.34 ± 1.66). There was no
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statistically significant difference between the selected geldings and the selected females in the age
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(P = .132), bodyweight (P = .104), heart rate (P = .118), and respiratory rate (P = -.586).
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The obtained echocardiographic image from the right parasternal long axis four-chamber view,
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showed the right ventricle, aorta, left atrium, left ventricle and left ventricular outflow tract (Figure
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1). Meanwhile, the right parasternal short-axis view at the chordal level displayed the right ventricle,
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left ventricle, interventricular septum, left ventricular free wall, anterior and posterior papillary
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muscles, and the chordae tendinae, but and the leaflets of mitral valve were not visible. In this
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recorded view, the interventricular septum, the right ventricle, the left ventricle, and the free wall of
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the left ventricle were bisected at right angle and the left ventricle appeared to be circular. The 2D-
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guided M-mode echocardiographic image, obtained from right parasternal short axis view, was a
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true short-axis view of the left ventricle, and its lumen was large without the inclusion of the mitral
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valve (Figure 2).
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Regarding the frequency distribution of B-mode echocardiography in clinically healthy
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donkeys, the highest frequency of B-mode echocardiographic dimensions was ≥ 16 – 18 mm for
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IVSTd, ≥ 20 – 22 mm for IVSTs, ≥ 44 – 46 mm for LVIDd, ≥ 28 – 30 mm for LVIDs, ≥ 20 – 22 mm
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for LVPWd, ≥ 22 – 24 mm for LVPWs. Meanwhile, highest frequency of the cardiac function
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indices was ≥ 200 – 205 ml for EDV, ≥ 60 – 70 ml for ESV, ≥ 40 – 50 ml for SV, ≥ 70 – 75 % for
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EF, and ≥ 32 – 39 % for FS (Tables 1).
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Concerning the results of B-mode echocardiographic parameters in the examined donkeys, the
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mean ± SD of the cardiac dimensions was as follows: IVSTd (mm) 18.5 ± 3.6, IVSTs (mm) 23.5 ±
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3.8, LVIDd (mm) 42.7 ± 6.6, LVIDs (mm) 25.5 ± 6.3, LVPWd (mm) 24.2 ± 4.6, LVPWs (mm) 22.5
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± 4.5. However, the mean ± SD of cardiac function indices was as follows: EDV (ml) 202 ± 21.17,
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ESV (ml) 68.6 ± 6.5, SV (ml) 46.3 ± 8.7, EF (%) 76.1 ± 10.9, FS (%) 39.8 ± 11.4 (Table 2).
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The intra-assay (Table 3) and inter-assay (Table 4) CV of B-mode echocardiographic
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dimensions and cardiac function indices in the studied donkeys, the IVSTs, LVIDd, LVIDs, LVPWs,
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EDV, ESV and EF showed low (CV < 15%) variability. Meanwhile, the IVSTd, LVPWd, SV, and
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FS showed moderate (CV 15–25%) variability.
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With regard to the frequency distributions of 2D-guided M-mode echocardiography in clinically
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healthy donkeys, the highest frequency for echocardiographic parameters were ≥ 1.6 – 1.8 mm for
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IVSTd, ≥ 2.8 – 3 mm for IVSTs, ≥ 4.5 – 5 mm for LVIDd, ≥ 3 – 3.5 mm for LVIDs, ≥ 2.4 – 2.6 mm
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for LVPWd, ≥ 2.8 – 3 mm for LVPWs. However, the mean ± SD of cardiac function indices was as
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follows: ≥ 190 – 195 ml for EDV, ≥ 58 – 65 ml for ESV, ≥ 50 – 53 ml for SV, ≥ 74 – 76 % for EF,
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and ≥ 64 – 48 % for FS (Tables 5).
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Concerning the results of the 2D-guided M-mode echocardiographic parameters in the studied
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donkeys, the mean ± SD of the cardiac dimensions was as follows: IVSTd (mm) 1.4 ± 0.4, IVSTs
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(mm) 2.8 ± 0.7, LVIDd (mm) 6.1 ± 1.3, LVIDs (mm) 3.5 ± 0.9, LVPWd (mm) 2.8 ± 0.8, LVPWs
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(mm) 3.2 ± 0.7. Meanwhile, the mean ± SD of cardiac function indices was as follows: EDV (ml)
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200 ± 16.4, ESV (ml) 63.8 ± 9.32, SV (ml) 75.7 ± 13.9, EF (%) 79.9 ± 6.8, FS (%) 42.3 ± 6.9 (Table
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6).
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Concerning intra-assay (Table 7) and inter-assay (Table 8) CV of 2D-guided M-mode
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echocardiographic measurements in healthy donkeys, the IVSTs, LVIDd, LVIDs, LVPWs, EDV,
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ESV, SV, EF and FS showed low (CV < 15%) variability. Meanwhile, the IVSTd, and LVPWd
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showed moderate (CV 15–25%) variability.
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Linear regression analysis of the data demonstrated that there was non-significant association
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between B-mode and 2D-guided M-mode echocardiographic variables and sex, age, body weight,
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and BCS of healthy donkeys (Tables 9,10).
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4. Discussion
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Since echocardiography is a non-invasive procedure, it can be used efficiently for diagnosis of
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diseases of the cardiovascular system as well as investigation of the cardiac structures, cardiac
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dimensions, and cardiac function indices. In ruminants, horses, and pets, the measurement of cardiac
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chamber dimensions as well as cardiac function indices is a vital tool necessary for assessment of the
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severity and prognosis of cardiac diseases [26]. Thus, the basic clinical reference values are required
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to improve identification and assessment of such cardiac diseases allowing an earlier diagnosis and
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more rapid intervention when facing abnormal echocardiographic findings. In the existing study, the
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morphologic features and function indices of the heart were investigated using B-mode and 2D-
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guided M-mode echocardiography.
255
In the present study, for both b-mode and guided M-mode echocardiography, the heart was
256
examined from the right parasternal location, using right parasternal long axis four-chamber view
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and right parasternal short axis view, respectively. Both B-mode and guided M-mode
258
echocardiography were easy to perform and well tolerated in all donkeys, without the need for any
259
sedation or analgesia. Both echocardiographic modes provided valuable information regarding
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cardiac chamber size and dimension, cardiac wall thickness, systolic function, and valvular
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structures. Furthermore, high temporal resolution is a characteristic feature of M-mode
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echocardiography, which makes it a useful tool for estimating the chamber volume as well as left
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ventricular mass when these structures are geometrically uniform and useful for timing valve motion
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[27]. The obtained echocardiographic images were in agreement with those reported by Reef et al.
265
[5]; Patteson et al. [17]; Boon [18]; Bakos et al. [19]; Vöros et al. [28].
266
In the examined donkeys, the recorded mean ± SD of both B-mode and guided M-mode
267
echocardiographic dimensions, including IVSTd, IVSTs, LVIDd, LVIDs, LVPWd, and LVPWs and
268
the mean ± SD of cardiac function indices, including EDV, ESV, SV, EF, and FS were almost
269
similar to those recorded [14,29,30]. The intra-assay and inter-assay CV of B-mode and guided M-
270
mode echocardiographic dimensions and cardiac function indices in the studied donkeys, the IVSTs,
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LVIDd, LVIDs, LVPWs, EDV, ESV and EF showed low (CV < 15%) variability confirming the
272
reliability and uniformity in the values of the recorded data set, while the IVSTd, LVPWd showed
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moderate (CV 15–25%) variability. However, SV and FS in the guided M-mode echocardiography
274
showed low (CV < 15%) variability compared with those of B-mode echocardiography in which
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275
they showed moderate variability (CV 15-25%). Thus, the results of SV and FS in guided M-mode
276
echocardiography were reliable and uniform compared to those in B-mode echocardiography.
277
The overall impressions of heart size and function, obtained using B-mode and guided M-mode
278
echocardiography from the obtained views, were similar to those obtained in sedentary donkeys [29-
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31]. However, it showed some notable differences compared with data generated in another study
280
conducted in Standardbred trotters [19], Endurance horses [9], Quarter horses [8], warm blood
281
horses [2], dogs [32], and human [33,34], which indicate significant differences between species as
282
the length and weight of the heart significantly correlates with weight, height, and athletic
283
performance of individual. In the existing study, B-mode and guided M-mode echocardiographic
284
examination of healthy donkeys (Equus asinus) revealed that both the interventricular septum
285
thickness and the left ventricular posterior free wall were greater during systole than diastole.
286
Meanwhile, the dimension of the left ventricle during diastole was greater than systole. Furthermore,
287
the recorded data of cardiac function indices; including EDV, ESV, SV, EF, and FS using B-mode
288
echocardiography were closer to those recorded by guided M-mode echocardiography. These
289
findings were in agreement with those reported in donkeys [29,30], horses [2,9], dogs [32], human
290
[33,34].
291
The interventricular septum thickness provides a signal about the left ventricular mass that is
292
calculated from the left ventricular free wall thickness, interventricular septum thickness and left
293
ventricular internal diameter. Thus, it is required for evaluation of the left and right ventricular
294
hypertrophy in both human [35] and race horses [36]. The LVIDd and LVIDd are essential to
295
characterize the eccentric hypertrophies in sportsmen [37] and to detect left ventricular overload in
296
cases of valvular insufficiency, regurgitation of mitral and aortic valves or myocardial diseases [38].
297
They are usually increased in exercising horses either as a result of exercise [39] or other
13
298
pathological conditions as aortic valve insufficiency [40]. The LVPWd and LVPWs are essential for
299
investigation of the effects of the training related adaptation, and for detection of left ventricular
300
overload, aortic valve regurgitation, patent ductus arteriosus, and atrial and ventricular septal defect
301
[38]. Cardiac function indices are used to evaluate pumping functions that focus on the ejected blood
302
volume; SV (the difference between the EDV and ESV), the efficiency of ejection; EF, and index to
303
evaluate the strength of myocardial contraction; FS. Thus, EF and FS are indicators of left
304
ventricular systolic function, ventricular compliance and contractibility as well as the cardiac
305
function [36,38]. In heart failure with reduced EF, also called systolic failure, the left ventricle loses
306
its ability to contract normally. Thus, the heart can't pump with enough force to push enough blood
307
into circulation. Meanwhile, heart failure with preserved EF, also called diastolic failure or
308
dysfunction, the left ventricle muscle has become stiff and loses its ability to relax normally. Thus,
309
the heart can't properly fill with blood during the resting period between each beat. In both cases, the
310
cardiac function indices are significantly affected as well as the echocardiographic dimensions.
311
As the donkeys age increases, they gain body weight and both the circumference and the length
312
of the body reach a moderate degree, which does not increase any more with the increasing age of
313
these donkeys. Unlike donkeys, the older the horses are, the greater the body weight and the greater
314
the circumference and length of the body compared to the donkeys of the same age. Fully grown
315
donkey will be around 36 to 54 inches (90- 137 cm) from the withers to the hoof, while horses, on
316
the other hand, are around 56 to 64 inches on average (142 to 163 cm). Thus, the size and
317
dimensions of the heart greatly increase in horses relative to the size and weight of their large body
318
compared to donkeys. This is our suggestion that may explain the non-significant association
319
between B-mode and guided M-mode echocardiographic variables and age, body weight, and BCS
320
of healthy donkeys. Thus, they are not taken into consideration in donkeys when comparing patient
14
321
data with such reference values. These findings were in agreement with those reported by Roberts
322
and Dukes-McEwan [30]. In contrast to data from equine studies, body weight have a significant
323
effect on all echocardiographic dimensions, and also breed have a significant effect on most of them,
324
but not by sex. Furthermore, the age significantly affects only the internal diameter of the aortic and
325
the pulmonary arteries [41]. In human studies, sex, body weight, and age significantly affect the
326
normal heart size. With advancing age, the cardiac chambers seemed to be smaller with thicker walls
327
[42,43]. However, body mass index has no statistical significant influence on echocardiographic
328
variables [44].
329
There are several limitations of this study. First, all studied donkeys were examined in standing
330
position. Diseased donkeys may be in a recumbent position and unable to rise for complete
331
echocardiographic examination. Alternate posture of the donkeys was not investigated in the existing
332
study. Second, the existing study evaluated only standard B-mode and guided M-mode
333
measurements; the authors did not generate reference ranges for spectral Doppler, which may also be
334
important in investigating the effects of diseases. Finally, the used echocardiographic equipment and
335
technical settings in this study were identical for all examinations and the use of other different
336
equipment may increase variability due to differences in resolution and measurement accuracy.
337
In conclusion, echocardiography has been shown to be feasible, repeatable and accurate, and has
338
been demonstrated to be a powerful tool for assessment of the dimensions and function indices of the
339
heart. This study provided new useful reference values of cardiac dimensions and cardiac function
340
indices in healthy donkeys (Equus asinus), considering them normal limits of echocardiographic
341
dimensions and cardiac function indices. Age, body weight, and BCS of healthy donkeys have no
342
effect on the B-mode and guided M-mode echocardiographic variables. Knowing the normal
15
343
parameters of echocardiography facilitates the identification and early diagnosis of cardiac diseases
344
in donkeys, which is considered the first step in managing such problems.
345 346
Conflict of Interest statement: The authors declare that they have no conflict of interest.
347 348
Ethical approval: All applicable international, national, and/or institutional guidelines for the care
349
and use of animals were followed.
350 351
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461
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462
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463
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464
normal heart size. Circ Cardiovasc Imaging 2013; 6:1073 – 9.
465 466
Figure legend
467
Figure 1. Standard echocardiographic view for assessment of left ventricular size and function.
468
Right parasternal long axis four-chamber view centered on the left ventricle. The transducer is
469
positioned in the right fourth intercostal space at the level of or slightly dorsal to olecranon process,
470
angled caudally, and rotated in clockwise direction to the 1 o’clock position. RV, right ventricle,
471
IVS, interventricular septum, LV; left ventricle; LA; left atrium; Ao, aorta.
472
Figure 2. Standard echocardiographic view for assessment of left ventricular size and function. 2D
473
guided M-mode image of the left ventricle obtained from a right-parasternal short-axis view at the
474
chordal level.
475
21
Table 1. Frequency distribution (% of study population) of B-mode echocardiographic dimensions and cardiac function indices in clinically healthy donkeys (Equus asinus) (n = 44) Highest frequency Variables
Lowest frequency
Value
No (%)
Value
No (%)
IVSTd(mm)
≥ 16 – 18
38 (42.2 %)
≥ 26 – 28
4 (4.4 %)
IVSTs (mm)
≥ 20 – 22
36 (40 %)
≥ 32 – 34
2 (2.2 %)
LVIDd (mm)
≥ 44 – 46
22 (24.4 %)
≥ 58 – 60
4 (4.4 %)
LVIDs (mm)
≥ 28 – 30
24 (26.7 %)
≥ 40 – 42
2 (2.2 %)
LVPWd (mm)
≥ 20 – 22
32 (35.6 %)
≥ 32 – 34
6 (6.7 %)
LVPWs (mm)
≥ 22 – 24
36 (40 %)
≥ 32 – 34
6 (6.7 %)
EDV (ml)
≥ 200 – 205
32 (35.6 %)
≥ 166 – 171
10 (11.1 %)
ESV (ml)
≥ 60 – 70
38 (42.2 %)
≥ 35 – 41
8 (8.88 %)
SV (ml)
≥ 40 – 50
46 (51.1 %)
≥ 60 – 70
4 (4.4 %)
EF (%)
≥ 70 – 75
42 (46.7 %)
≥ 95 – 100
8 (8.9 %)
FS (%)
≥ 32 – 39
46 (51.1 %)
≥ 55 – 57
2 (2.2 %)
Table 2. Summary statistics of B-mode echocardiographic dimensions and cardiac function indices in clinically healthy donkeys (Equus asinus) (n = 44) Percentile Median (range)
Mean ± SD
95% CI
Median (range) 10%
25%
75%
90%
IVSTd (mm)
18.5 ± 3.6
17.7 – 19.2
18.1 (12.4 – 26.6)
13.8
15.5
21.2
23.3
IVSTs (mm)
23.5 ± 3.8
22.7 – 24.3
23.2 (16.6 – 32.6)
19.1
20.2
26.9
28.4
LVIDd (mm)
42.7 ± 6.6
41.3 – 44.1
42.7 (30.9 – 58.2)
33.6
38.2
46.7
51.2
LVIDs (mm)
28.5 ± 3.9
24.2 – 26.8
25.3 (11.7 – 40.7)
19.2
21.4
29.6
32.7
LVPWd (mm)
22.5 ± 3.9
21.5 – 23.4
22.2 (14.6 – 32.9)
15.7
19.7
24.9
29.3
LVPWs (mm)
25.2 ± 3.6
23.2 – 25.1
23.9 (15.9 – 33.1)
17.8
21.3
27.7
30.2
EDV (ml)
202 ± 21.17
193.3– 210.8
205 (166– 237)
170.6
180
221.5
227.6
ESV (ml)
68.6 ± 6.5
56.19 – 67.12
66.5 (35 – 78)
38
55.25
71.25
73.6
SV (ml)
46.3 ± 8.7
44.5 – 48.2
46.7 (30.2 – 65.7)
32.8
41.5
52.2
56.8
EF (%)
76.1 ± 10.9
73.8 – 78.4
75 (42 – 96)
64
68.8
84
92
FS (%)
39.8 ± 8.4
37.5 – 42.2
37 (16 – 50)
29
32
46
50
Table 3. Intra-assay coefficient of variation of B-mode echocardiographic dimensions and cardiac function indices in clinically healthy donkeys (Equus asinus) (n = 44) Percentile Median (range)
Mean ± SD
95% CI
Median (range) 10%
25%
75%
90%
IVSTd (mm)
18.8 ± 7.8
15.9 – 21.7
19.9 (7.5 - 33.2)
9.8
11.5
24.8
31.9
IVSTs (mm)
14.8 ± 9.5
11.3 – 18.3
14.4 (1.1 - 37.3)
4.2
6.4
20.8
26.7
LVIDd (mm)
11.5 ± 6.1
9.2 – 13.7
11.7 (3.2 - 27.1)
3.9
6.1
13.7
18.4
LVIDs (mm)
12.9 ± 7.2
10.3 – 15.6
12.4 (4.4 - 30.8)
4.4
6.9
17.9
22.5
LVPWd (mm)
16.7 ± 11.1
12.6 – 20.9
12.5 (2.9 - 35.2)
4.6
7.2
24.9
34.5
LVPWs (mm)
12.8 ± 9.1
9.4 – 16.1
11.4 (2.2 - 34.9)
3.1
5.2
19.1
24.4
EDV (ml)
11.7 ± 6.4
9.3 – 14.1
10.4 (4.4 – 25.5)
4.5
6.4
17.9
21.2
ESV (ml)
11.1 ± 8.8
13.8 – 18.3
18.9 (2.0 – 27.5)
7.3
8.6
21.6
26.5
SV (ml)
16.1 ± 9.7
12.5 – 19.8
18.4 (4.0 – 30.5)
4.0
5.8
25.6
26.8
EF (%)
10.5 ± 8.3
7.4 – 13.6
8.6 (0.6 - 36.6)
3.4
5.6
14.5
15.5
FS (%)
18.3 ± 12.8
13.6 – 23.1
16.4 (3.1 - 57)
5.7
9.4
25.4
27.1
Table 4. Inter-assay coefficient of variation of B-mode echocardiographic dimensions and cardiac function indices in clinically healthy donkeys (Equus asinus) (n = 44) Percentile Median (range)
Mean ± SD
95% CI
Median (range) 10%
25%
75%
90%
IVSTd (mm)
15.8 ± 7.4
12.9 – 18.5
15.6 (1.7 - 28.2)
2.1
12.2
22.4
24.6
IVSTs (mm)
13.6 ± 6.2
11.3 – 15.9
14.5 (3.1 - 23.9)
4.6
9.5
17.9
23.1
LVIDd (mm)
15.1 ± 5.1
13.2 – 17.1
14.7 (7.1 - 23.4)
8.7
11.1
19.4
22.4
LVIDs (mm)
13.7 ± 8.4
19.8 – 28.4
23.7 (1.4 - 38.9)
3.3
21.2
32
36.3
LVPWd (mm)
17.3 ± 5.7
17.9 – 24.4
22.2 (8.9 - 36.1)
11.6
12.6
24.4
34.1
LVPWs (mm)
14.2 ± 8.1
14.2 – 21.1
16.4 (5.4 - 34.8)
6.6
9.1
25.9
31.6
EDV (ml)
13.1 ± 6.2
10.8 – 15.4
11.9 (5.3 - 26.1)
5.6
7.2
20
20.6
ESV (ml)
13.3 ± 7.9
13.3 – 19.2
20.1 (2.6 – 27.9)
4.2
8.0
22.2
23.1
SV (ml)
16.4 ± 9.1
12.9 – 19.8
16.7 (4.8 – 36.5)
5.3
8.3
24.9
27.9
EF (%)
14.4 ± 6.9
11.8 – 16.9
12.5 (4.8 - 33.1)
6.6
10.1
18.3
20.3
FS (%)
21.1 ± 9.1
22.3 – 32
30.5 (8.7 - 46.6)
10.4
15.9
46.6
44.3
Table 5. Frequency distribution (% of study population) of 2 dimensional guided Mmode echocardiographic dimensions and cardiac function indices in clinically healthy donkeys (Equus asinus) (n = 44) Highest frequency variables
Lowest frequency
Value
No (%)
Value
No (%)
IVSTd(mm)
≥ 1.6 – 1.8
44 (48.9 %)
≥ 3 – 3.2
2 (2.2 %)
IVSTs (mm)
≥ 2.8 – 3
26 (28.9 %)
≥ 5.8 – 6
2 (2.2 %)
LVIDd (mm)
≥ 4.5 – 5
24 (26.7 %)
≥ 9 – 9.5
2 (2.2 %)
LVIDs (mm)
≥ 2.6 – 2.9
36 (40 %)
≥ 6.5 – 7
2 (2.2 %)
LVPWd (mm)
≥ 2.4 – 2.6
34 (37.8 %)
≥ 5.8 – 6
2 (2.2 %)
LVPWs (mm)
≥ 2.8 – 3
24 (26.7 %)
≥ 5 – 5.2
2 (2.2 %)
EDV (ml)
≥ 190 – 195
26 (28.8 %)
≥ 173 – 178
6 (6.7 %)
ESV (ml)
≥ 58 – 65
28 (31.1 %)
≥ 39 – 42
4 (4.4 %)
SV (ml)
≥ 50 – 53
66 (28.9 %)
≥ 46 – 48
2 (2.2 %)
EF (%)
≥ 74 – 76
18 (20 %)
≥ 80 – 82
8 (8.9 %)
FS (%)
≥ 41 – 43
16 (17.8 %)
≥ 56 – 58
2 (2.2 %)
Table 6. Summary statistics of 2 dimensional guided M-mode echocardiographic dimensions and cardiac function indices in clinically healthy donkeys (Equus asinus) (n = 44) Percentile Median (range)
Mean ± SD
95% CI
Median (range) 10%
25%
75%
90%
IVSTd (mm)
1.7 ± 0.4
1.6 – 1.9
1.7 (1.1 – 3.1)
1.2
1.5
1.9
2.2
IVSTs (mm)
2.9 ± 0.4
2.7 – 2.9
2.8 (1.9 – 5.8)
2.1
2.4
3.1
3.5
LVIDd (mm)
6.1 ± 0.8
5.8 – 6.3
6.1 (3.8 – 9.1)
4.4
4.9
7.1
7.7
LVIDs (mm)
3.7 ± 0.5
3.3 – 3.7
3.3 (2.1 – 6.5)
2.4
2.6
3.9
4.8
LVPWd (mm)
2.8 ± 0.5
2.6 – 2.9
2.7 (1.5 – 5.9)
1.9
2.4
3.1
3.7
LVPWs (mm)
3.6 ± 0.4
3.1 – 3.3
3.1 (1.9 – 5.1)
2.2
2.8
3.7
3.9
EDV (ml)
200 ± 16.4
193 – 207
198 (173 – 235)
179
189
210
226
ESV (ml)
63.8 ± 9.32
54.9 – 66.7
64 (39 – 78)
42
48
70
76
SV (ml)
54.7 ± 3.1
52.5 – 56.9
54.9 (50.8 – 58.6)
50.84
51.50
57.80
85.52
EF (%)
79.9 ± 6.8
78.5 – 81.4
82 (67 – 91)
70
74
85.3
89
FS (%)
42.3 ± 5.5
40.9 – 43.8
43 (31 – 56)
33
36
47.3
52
Table 7. Intra-assay coefficient of variation of 2 dimensional guided M-mode echocardiographic dimensions and cardiac function indices in clinically healthy donkeys (Equus asinus) (n = 44) Percentile Median (range)
Mean ± SD
95% CI
Median (range) 10%
25%
75%
90%
IVSTd (mm)
17.8 ± 8.3
14.7 – 20.9
16.7 (8.6 – 39.8)
8.6
10.7
21.3
29.6
IVSTs (mm)
14.7 ± 11.9
10.2 – 19.2
10.1 ( 4.1 – 50.5)
5.1
8.6
23.4
27.7
LVIDd (mm)
12.9 ± 6.7
10.1 – 15.1
11.9 ( 3 – 26.3)
6.9
7.3
16.2
25.4
LVIDs (mm)
13.9 ± 8.4
10.8 – 17.1
12.7 (3.7 – 31.6)
4.5
6.3
21.1
21.9
LVPWd (mm)
18.4 ± 8.6
18.2 – 24.6
20.9 (7.4 – 38.9)
11.4
14.5
25.8
42.9
LVPWs (mm)
14.9 ± 9.8
11.2 – 18.6
12.8 (3.4 – 37.3)
4.9
6.8
16.8
35.6
EDV (ml)
10.8 ± 5.6
8.7 – 12.9
11.0 (0.6 – 20.9)
3.1
6.9
15.5
18.5
ESV (ml)
12.2 ± 9.9
13.5 – 18.9
14.2 (5.5 – 35.4)
8.01
11.5
18.6
25.7
SV (ml)
7.13 ± 2.1
12.8 – 18.1
13.8 (7.5 – 35.3)
8.3
9.6
19.7
22.9
EF (%)
6.2 ± 2.9
5.1 – 7.3
7.6 (2.3 – 10.3)
2.3
2.9
8.8
10.3
FS (%)
11.7 ± 5.2
9.8 – 13.6
12.6 (5.1 – 20.7)
5.1
6
15.8
17.9
Table 8. Inter-assay coefficient of variation of 2 dimensional guided M-mode echocardiographic dimensions and cardiac function indices in clinically healthy donkeys (Equus asinus) (n = 44) Percentile Median (range)
Mean ± SD
95% CI
Median (range) 10%
25%
75%
90%
IVSTd (mm)
19 ± 9.3
15.5 – 22.5
17.9 (4.7 – 35.1)
5.3
11.9
22.9
34.5
IVSTs (mm)
13.7 ± 5.5
14.9 – 20.6
15.8 (1.6 – 43.8)
10.5
14.1
20.6
32.0
LVIDd (mm)
13.7 ± 8.7
16.3 – 22.7
17.8 (5.1 – 33.8)
12.8
20.1
27.1
30.3
LVIDs (mm)
13.5 ± 6.1
18.4 – 22.9
17.2 (15.6 – 34.9)
15.8
19.9
31.4
34.2
LVPWd (mm)
17.9 ± 8.2
17.6 – 25.9
17.1 (9.1 – 43.4)
11.7
14.3
31.9
42.9
LVPWs (mm)
11.5 ± 5.6
14.9 – 20.6
14.3 (9.8 – 30.5)
9.9
13.3
25.5
28.6
EDV (ml)
11.9 ± 6.9
9.4 – 14.5
13.6 (0.7 – 24.7)
0.7
6.7
16.4
21.2
ESV (ml)
12.5 ± 8.1
12.4 – 16.5
15.6 (3.1 – 23.8)
4.1
7.9
22.7
24.3
SV (ml)
7.13 ± 2.3
13.8 – 17.8
16.4 (7.9 – 27.2)
10.2
10.7
20.6
21.4
EF (%)
8.8 ± 3.5
7.4 – 10.1
8.4 (3.2 – 15.3)
3.6
6.4
11.9
13.2
FS (%)
13.1 ± 3.4
13.5 – 18.2
14.2 (5.9 – 25.5)
8.3
12.1
20.5
24.3
Table 9. Association between B-mode echocardiographic parameters and sex, age, body weight and body condition score in clinically healthy donkeys (Equus asinus) (n = 44) Sex
Age
BWt
BCS
P-value
P-value
P-value
P-value
0.396 0.734 0.371 0.287 0.154 0.269 0.533 0.430 0.351 0.266 0.621
0.904 0.363 0.465 0.716 0.629 0.724 0.246 0.398 0.304 0.920 0.913
0.725 0.193 0.530 0.687 0.696 0.623 0.360 0.478 0.274 0.556 0.846
0.659 0.831 0.181 0.681 0.961 0.652 0.546 0.144 0.862 0.499 0.145
B-mode variables IVSTd (mm) IVSTs (mm) LVIDd (mm) LVIDs (mm) LVPWd (mm) LVPWs (mm) EDV (ml) ESV (ml) SV (ml) EF (%) FS (%)
P value < 0.05 indicates a significant correlation. BWt, Body weight; BCS, Body condition score.
Table 10. Association between 2 dimensional guided M-mode echocardiographic variable and sex, age, body weight and body condition score in clinically healthy donkeys (Equus asinus) (n = 44) Sex
Age
BWt
BCS
P-value
P-value
P-value
P-value
0.432 0.521 0.211 0.152 0.221 0.311 0.401 0.316 0.241 0.125 0.415
0.298 0.131 0.320 0.203 0.194 0936 0.639 0.402 0.751 0.451 0.364
0.319 0.158 0.400 0.328 0.245 0.946 0.717 0.484 0.922 0.637 0.548
0.130 0.997 0.994 0.154 0.673 0.942 0.904 0.422 0.185 0.823 0.298
M-mode variables IVSTd (mm) IVSTs (mm) LVIDd (mm) LVIDs (mm) LVPWd (mm) LVPWs (mm) EDV (ml) ESV (ml) SV (ml) EF (%) FS (%)
P value < 0.05 indicates a significant correlation. BWt, Body weight; BCS, Body condition score.
Highlights •
•
• • •
B-mode and 2D guided M-mode echocardiography was performed for 44 healthy donkeys (Equus asinus) to assess the cardiac dimensions and cardiac function indices. This study reported the reference ranges of the echocardiographic dimensions and cardiac function indices in donkeys (Equus asinus) based on the probability theory and statistics. The interventricular septum thickness and the left ventricular posterior free wall were greater during systole than diastole. The left ventricular dimension during diastole was greater than systole. The assessed cardiac dimensions and cardiac function indices using B-mode echocardiography were closer to those recorded by 2D guided M-mode echocardiography.
Author statement
We affirm that the submission represents original work that has not been published previously and is not currently being considered by another journal. All other Authors have read the manuscript and have agreed to submit it in its current form for consideration for publication. We confirm that the legal and ethical requirements have been met with regards to the humane treatment of animals described in the study This manuscript is the first report the reference values and repeatability of B-mode and M-mode echocardiography in healthy donkey (equus asinus) - the guide to assess healthy heart Thank you for your cooperation and waiting for your response Kind regards Corresponding author Hussam M. M. Ibrahim
Author’s contributions: To qualify as an author one should 1) have made substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; 2) have been involved in drafting the manuscript or revising it critically for important intellectual content; 3) have given final approval of the version to be published; and 4) agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. We suggest the following kind of format (please use initials to refer to each author's contribution): AB carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. JY carried out the immunoassays. MT participated in the sequence alignment. ES participated in the design of the study and performed the statistical analysis. FG conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. SE: Conceived and designed the experiments, analyzed the data, final revision and submission. MH: Conceived and designed the experiments, and helped to draft the manuscript HI: Performed the experiments, revised the data KA: Performed the experiments RE: drafting the paper Name and signature:
Conflict of Interest statement: The authors declare that they have no conflict of interest.
S0737-0806(20)30020-4
DOI:
https://doi.org/10.1016/j.jevs.2020.102929
Reference:
YJEVS 102929
To appear in:
Journal of Equine Veterinary Science
Received Date: 1 October 2019 Revised Date:
19 December 2019
Accepted Date: 7 January 2020
Please cite this article as: Farag AMM, Ibrahim HMM, Reference Values and Repeatability of Bmode and M-mode Echocardiographic Parameters in Healthy Donkey (Equus asinus) - The Guide to Assess Healthy Heart, Journal of Equine Veterinary Science (2020), doi: https://doi.org/10.1016/ j.jevs.2020.102929. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2020 Elsevier Inc. All rights reserved.
1
Reference Values and Repeatability of B-mode and M-mode Echocardiographic Parameters in
2
Healthy Donkey (Equus asinus) - The Guide to Assess Healthy Heart
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Alshimaa M.M. Farag; Hussam M.M. Ibrahim*
5
Department of Internal Medicine and Infectious Diseases, Faculty of Veterinary Medicine,
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Mansoura University, Mansoura 35516, Egypt
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* Corresponding author:
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Tel.: +2-050-2214233; Mobile: +2-01005290592
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E-mail: [email protected]
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24
Abstract
25
Donkey (Equus asinus), the cheapest form of agricultural power in Egypt, is used principally as
26
draught or pack animals and may also be ridden, or used for threshing, raising water, milling and
27
other work. The aim of the present study was to provide B-mode and M-mode echocardiographic
28
reference ranges from a sample of population of donkeys (Equus asinus) based on the probability
29
theory and statistics. In the current study, B-mode and 2 dimensional guided M-mode
30
echocardiography were performed on 44 clinically healthy donkeys (Equus asinus) (22 males and 22
31
females), without any neither clinical nor echocardiographic evidences of cardiovascular disease,
32
aged 2 – 25 years old, and weighed 150 – 350 kg. The echocardiographic dimensions; including
33
interventricular septum thickness at end diastole, interventricular septum thickness at end systole,
34
left ventricular internal diameter at end diastole, left ventricular internal diameter at end systole, left
35
ventricular posterior wall thickness at end diastole, and left ventricular posterior wall thickness at
36
end systole were assessed. The cardiac function indices; including end diastolic volume, end systolic
37
volume, stroke volume, ejection fraction, and fractional shortening were also measured. B-mode and
38
guided M-mode echocardiographic examination of healthy donkeys revealed that both the
39
interventricular septum thickness and the left ventricular posterior free wall were greater during
40
systole than diastole. However, the left ventricular dimension during diastole was greater than
41
systole. Furthermore, the recorded data of cardiac functional indices; including end diastolic volume,
42
end systolic volume, stroke volume, ejection fraction, and fractional shortening using B-mode
43
echocardiography were closer to those recorded by guided M-mode echocardiography. To our
44
knowledge, this is the first Egyptian study to report the echocardiographic reference ranges of
45
donkeys (Equus asinus) based on the probability theory and statistics, the coefficient of variation.
46
The results of the current study can be used as a standard and reference value for further
2
47
echocardiographic studies in donkeys and represents a step in the identification, interpretation, and
48
evaluation of cardiovascular disorders in such animals.
49 50
Key
words:
Donkeys
(Equus
asinus),
B-mode
echocardiography,
51
echocardiography, Cardiac dimensions, Cardiac function indices.
Guided
M-mode
52 53
1. Introduction
54
The quantification of cardiac dimensions and cardiac function indices is the backbone of cardiac
55
imaging, with echocardiography being a safe, non-invasive, powerful standard diagnostic imaging
56
tool in equine cardiology. Equine echocardiography uses ultrasound technology to display images of
57
a horse’s heart, providing a substantial amount of information about cardiac function, structure, flow
58
and indexes important for their athletic performance [1]. Beside qualitative estimation, measurement
59
of the cardiac dimensions and estimation of the cardiac function indices are inevitable to determine
60
the cardiac performance of healthy animals and to identify injuries, disorders or structural
61
abnormalities of the heart that may impair performance [2]. It is widely used in cardiology clinics
62
worldwide to assess heart chambers size, how well the left side of the heart is functioning, intra-
63
cardiac and extra-cardiac masses, systolic and diastolic function, valvular structure and function, and
64
fluid collections in the pericardial sac around the heart [3,4].
65
Echocardiography is the cornerstone for diagnosis of congenital cardiac malformations, valvular
66
diseases, myocardial diseases, acquired cardiac defects, cardiac arrhythmias, and cardiac murmurs.
67
Thus, it is recommended in horses with loud systolic and diastolic murmurs, muffling heart sound,
68
pericardial friction rubs, and exercise intolerance. It is also essential to exactly identify the cause of
69
the cardiac murmurs and characterize the severity of the underlying cardiac abnormality as well as
3
70
assessing its potential impact on the horse performance and life expectancy [5]. Three valuable types
71
of echocardiography, Two-dimension (B-mode), Motion (M-mode) and Doppler are well known
72
providing together complementary information about the nature of cardiac abnormalities as well as
73
the hemodynamic status.
74
B-Mode echocardiography, backbone of cardiac ultrasound, allows imaging a plane of tissue
75
(both depth and width) in real time. The images that cut the heart into slices, which may follow the
76
length (longitudinal plane) and the width of the heart (transverse plane) represent an
77
echocardiography plane [6]. Thus, the anatomical relations between different structures are easier to
78
appreciate. There are an infinite number of cardiac imaging planes; however, the standard views are
79
essential for evaluation of the intra- and extra-cardiac structures [7,8]. B-Mode echocardiography
80
permits imaging the internal cardiac structures, including the heart chambers, the structure and
81
thickness of the chamber walls, the structure and function of the valves, myocardium, pericardium
82
and great vessels [9,10].
83
M-mode echocardiography is produced by transmission and reception of an ultrasound signal
84
along only one line, giving a high sensitivity, greater than B-mode, to record the moving structures
85
(e.g. valve opening and closing or ventricular wall movement) [11]. M-mode echocardiography
86
produces a one-dimension view of the moving cardiac structures over time. A more accurate
87
positioning of the M-mode cursor within the heart is performed using two-dimension real-time
88
image as a guide. M-mode echocardiogram uses a high sampling rate and can yield cleaner images
89
of cardiac borders, allowing the clinician to obtain more accurate measurements of cardiac
90
dimensions and more critically evaluate the motion of cardiac structures. Therefore, M-mode
91
echocardiography is superior to B-mode echocardiography for timing of events and is used to obtain
92
precise cardiac measurements [12]. In equine cardiology, echocardiography has been introduced
4
93
allowing measurement of cardiac dimensions since 1977, but the standardization of the imaging
94
techniques and obtaining reference values persisted until recent years. Accurate measurement of
95
cardiac dimensions requires knowledge of reference values of normal cardiac anatomy,
96
echocardiographic dimensions and cardiac function indices that represents the basis for diagnosing
97
cardiac diseases. Thus, the goal of the present investigation was to determine the echocardiographic
98
dimensions and cardiac function indices in clinically healthy donkeys (Equus asinus) which will be
99
helpful in diagnosing donkeys with cardiac diseases. To our knowledge, this is the first Egyptian
100
study to report on echocardiographic reference ranges from donkeys (Equus asinus) based on the
101
probability theory and statistics, the coefficient of variation (CV) which is a standardized measure of
102
dispersion of a probability distribution or frequency distribution to express the precision and
103
repeatability of an assay.
104 105
2. Materials and Methods
106
2.1. Animals
107
The present study was conducted on 44 clinically healthy donkeys (Equus asinus) from which
108
22 males, all gelding, and 22 females. The donkeys were randomly selected from a wide range of
109
ages, to give a fairly even distribution within the age groups < 5 years, 5–15 years and >15 years old
110
and weighed 150 to 350 kg. The body condition score (BCS) for the selected donkeys were 3 in 34
111
donkeys, 3.5 in 5 donkeys, and 4 in 5 donkeys. All donkeys are selected to be clinically healthy and
112
free from any cardiovascular illness, based on clinical examination as well as complete cardiac
113
examination, including assessment of the heart rate, regularity of the heart beats, the strength of the
114
heart sounds, abnormal heart sounds, and electrocardiography [13]. Therefore, any systemic
115
diseases, tachycardia, cardiac arrhythmias, cardiac murmurs, detectable cardiac defects, and those
5
116
donkeys non-compliant with handling were considered the exclusion criteria. Donkeys were housed
117
indoor in the animal house at the Veterinary Teaching Hospital, Faculty of Veterinary Medicine,
118
Mansoura University, Egypt on April, 2018 and received no exercise on the day of each
119
examination. The nutrition regimen for all donkeys under investigation was maintenance balanced
120
mixed ration containing chopped wheat straw ad libitum, 075 – 3.5 kg of bran and 0.75 – 3.5 kg of
121
crushed corn and supplemented by trace elements and minerals mixture according to age of the
122
donkey. During the entire experiment, all donkeys were kept under the same management and
123
feeding practices and close veterinary supervision. All the institutional and national guidelines for
124
animals use and care were followed as outlined by the Medical Research Ethics Committee,
125
Mansoura University, Mansoura, Egypt, code No. R/10.
126 127
2.2. Echocardiographic examination
128
All the examined donkeys were at rest, calm, physically restrained only by halter, and didn't
129
receive any sedative or tranquilizing agents during the examination time. The cardiac examination
130
area the right side of the chest; area between the 3rd and the 5th intercostal space just caudal to the
131
triceps muscle mass, and 3 – 5 cm below and 5 – 10 cm above the olecranon process, was
132
thoroughly clipped, shaved and cleaned with alcohol. Acoustic Coupling gel was applied at the
133
examination area to enhance the contact with the probe. B-mode and 2 dimensional (2D) guided M-
134
mode echocardiography was performed according to standard method [14] and the recommendations
135
of the American Society of Echocardiography [15]. For echocardiographic examination, a 2.5 MHz
136
phased array transducer (CHISON D3P64L) (Chison iVis 60 EXPERT Color Doppler Ultrasound,
137
Chison Medical Imaging Co, China) was used. In the fourth or fifth intercostal space at the level of
138
olecranon process, the transducer was positioned, adjusted, clockwise rotated, and slightly directed
6
139
cranially and / or dorsally to get the required standard image. The right parasternal long axis four-
140
chamber apical view was used to obtain the images of B-mode echocardiography. However, the
141
guided M-mode echocardiography was obtained using the right parasternal short axis view. Once the
142
standard imaging plane was reached, Cube method was used to evaluate the echocardiographic
143
dimensions list and Teichholz method was used to assess the cardiac function indices. For each
144
donkey, the assessed echocardiographic dimensions and cardiac function indices were conducted 3
145
times in a single batch on the same day of examination, which is necessary to calculate the intra-
146
assay coefficient of variation. These measurements were conducted for each donkey on 3 different
147
days, in separate batches, which is required to calculate the inter-assay coefficient of variation. Each
148
time of echocardiography, three non-consecutive cardiac cycles were measured and the average
149
value for each variable was obtained. All echocardiographic examinations were done after at 9:00
150
AM and were carried out by one trained examiner and revised again by two experts to avoid any
151
individual variations.
152 153
2.3. Echocardiographic views and measurement of cardiac dimensions and cardiac function indices
154
Routine transthoracic B-mode and 2D-guided M-mode echocardiography were performed to
155
measure the echocardiographic dimensions list as well as the cardiac function indices.
156
Echocardiographic examinations began using B-mode echocardiography, with the creation of
157
sectional planes, evaluating the morphology and movement of the structures of the heart in a global
158
way. The B-mode echocardiographic image was obtained using the right parasternal long axis four-
159
chamber view (Figure 1) according to Reef et al. [5]; Youssef et al. [14]; Reef [16]. After that, by
160
rotating the transducer through 90 0 angle in a clockwise direction with a slight cranial and/or dorsal
161
angulation, the right parasternal short-axis view at the chordal level was obtained observing the
7
162
symmetry of the papillary muscles as well as the shape of the left ventricle. In this view, M-mode
163
was then selected to obtain the 2D-guided M-mode of the short-axis view of the left ventricle at the
164
level of chordae tendinae when the M-mode cursor bisected the left ventricle, and intersected both
165
the interventricular septum and left ventricular free wall in a 90 0 angle, positioning the beam over
166
this line (Figure 2) according to the method described by Vajhi et al. [4]; Reef et al. [5]; Youssef et
167
al. [14]. Both B-mode and guided M-mode echocardiography were performed for measurement of
168
the interventricular septum thickness at end-diastole (IVSd), the interventricular septum thickness at
169
end-systole (IVSs), the left ventricle internal diameter at end diastole (LVIDd), the left ventricle
170
internal diameter at end systole (LVIDs), left ventricular posterior wall thickness at end-systole
171
(LVPWs) and left ventricular posterior wall thickness at end-diastole (LVPWd) by using the Cube
172
method [17-22]. However, the use of Teichholz method in both B-mode and guided M-mode
173
echocardiography enables calculation of the left ventricular volume at end-diastole (EDV) and at
174
end-systole (ESV) [23], stroke volume (SV) and fractional shortening (FS) [24], and ejection
175
fraction (EF) [24].
176 177
2.4. Statistical analysis
178
All statistical procedures were performed with commercially available software (GraphPad
179
Prism for Windows version 5.0; GraphPad Software Inc., CA, USA). To assess the normality of
180
distribution of the B-mode and guided M-mode echocardiographic data, the Kolmogorov–Smirnov
181
normality test was conducted, and these data were normally distributed. The frequency distribution
182
and the summary statistics (mean ± SD, 95% CI, median (range), and 10th, 25th, 75th, and 90th
183
percentiles) of the B-mode and guided M-mode echocardiographic measurements of the selected
184
donkeys were calculated. The repeatability of the B-mode and guided M-mode echocardiographic
8
185
measurements was estimated by calculating the intra-assay CV. For each of the 3 days, the data were
186
collected and the inter-assay CV was calculated by dividing the SD of the measurement by the mean
187
of the same measurement. CV < 15% was classified as low variability, CV 15–25% as moderate
188
variability and CV > 25% as high variability [25]. Linear regression was performed to explore the
189
association between sets of variables, including age, body weight and body condition scores (BCSs),
190
on B-mode and guided M-mode echocardiographic data. P < .05 was accepted as representing
191
statistical significance for such test.
192 193
3. Results
194
All the selected donkeys were considered to be healthy on the basis of complete cardiac check. The
195
recorded heart rate during echocardiographic examination was within the normal range values 29 –
196
50 beats / minute (mean ± SD 38.75 ± 5.51). The documented respiratory rate was also within
197
normal range values 14 – 20 respiratory cycles / minute (mean ± SD 17.34 ± 1.66). There was no
198
statistically significant difference between the selected geldings and the selected females in the age
199
(P = .132), bodyweight (P = .104), heart rate (P = .118), and respiratory rate (P = -.586).
200
The obtained echocardiographic image from the right parasternal long axis four-chamber view,
201
showed the right ventricle, aorta, left atrium, left ventricle and left ventricular outflow tract (Figure
202
1). Meanwhile, the right parasternal short-axis view at the chordal level displayed the right ventricle,
203
left ventricle, interventricular septum, left ventricular free wall, anterior and posterior papillary
204
muscles, and the chordae tendinae, but and the leaflets of mitral valve were not visible. In this
205
recorded view, the interventricular septum, the right ventricle, the left ventricle, and the free wall of
206
the left ventricle were bisected at right angle and the left ventricle appeared to be circular. The 2D-
207
guided M-mode echocardiographic image, obtained from right parasternal short axis view, was a
9
208
true short-axis view of the left ventricle, and its lumen was large without the inclusion of the mitral
209
valve (Figure 2).
210
Regarding the frequency distribution of B-mode echocardiography in clinically healthy
211
donkeys, the highest frequency of B-mode echocardiographic dimensions was ≥ 16 – 18 mm for
212
IVSTd, ≥ 20 – 22 mm for IVSTs, ≥ 44 – 46 mm for LVIDd, ≥ 28 – 30 mm for LVIDs, ≥ 20 – 22 mm
213
for LVPWd, ≥ 22 – 24 mm for LVPWs. Meanwhile, highest frequency of the cardiac function
214
indices was ≥ 200 – 205 ml for EDV, ≥ 60 – 70 ml for ESV, ≥ 40 – 50 ml for SV, ≥ 70 – 75 % for
215
EF, and ≥ 32 – 39 % for FS (Tables 1).
216
Concerning the results of B-mode echocardiographic parameters in the examined donkeys, the
217
mean ± SD of the cardiac dimensions was as follows: IVSTd (mm) 18.5 ± 3.6, IVSTs (mm) 23.5 ±
218
3.8, LVIDd (mm) 42.7 ± 6.6, LVIDs (mm) 25.5 ± 6.3, LVPWd (mm) 24.2 ± 4.6, LVPWs (mm) 22.5
219
± 4.5. However, the mean ± SD of cardiac function indices was as follows: EDV (ml) 202 ± 21.17,
220
ESV (ml) 68.6 ± 6.5, SV (ml) 46.3 ± 8.7, EF (%) 76.1 ± 10.9, FS (%) 39.8 ± 11.4 (Table 2).
221
The intra-assay (Table 3) and inter-assay (Table 4) CV of B-mode echocardiographic
222
dimensions and cardiac function indices in the studied donkeys, the IVSTs, LVIDd, LVIDs, LVPWs,
223
EDV, ESV and EF showed low (CV < 15%) variability. Meanwhile, the IVSTd, LVPWd, SV, and
224
FS showed moderate (CV 15–25%) variability.
225
With regard to the frequency distributions of 2D-guided M-mode echocardiography in clinically
226
healthy donkeys, the highest frequency for echocardiographic parameters were ≥ 1.6 – 1.8 mm for
227
IVSTd, ≥ 2.8 – 3 mm for IVSTs, ≥ 4.5 – 5 mm for LVIDd, ≥ 3 – 3.5 mm for LVIDs, ≥ 2.4 – 2.6 mm
228
for LVPWd, ≥ 2.8 – 3 mm for LVPWs. However, the mean ± SD of cardiac function indices was as
229
follows: ≥ 190 – 195 ml for EDV, ≥ 58 – 65 ml for ESV, ≥ 50 – 53 ml for SV, ≥ 74 – 76 % for EF,
230
and ≥ 64 – 48 % for FS (Tables 5).
10
231
Concerning the results of the 2D-guided M-mode echocardiographic parameters in the studied
232
donkeys, the mean ± SD of the cardiac dimensions was as follows: IVSTd (mm) 1.4 ± 0.4, IVSTs
233
(mm) 2.8 ± 0.7, LVIDd (mm) 6.1 ± 1.3, LVIDs (mm) 3.5 ± 0.9, LVPWd (mm) 2.8 ± 0.8, LVPWs
234
(mm) 3.2 ± 0.7. Meanwhile, the mean ± SD of cardiac function indices was as follows: EDV (ml)
235
200 ± 16.4, ESV (ml) 63.8 ± 9.32, SV (ml) 75.7 ± 13.9, EF (%) 79.9 ± 6.8, FS (%) 42.3 ± 6.9 (Table
236
6).
237
Concerning intra-assay (Table 7) and inter-assay (Table 8) CV of 2D-guided M-mode
238
echocardiographic measurements in healthy donkeys, the IVSTs, LVIDd, LVIDs, LVPWs, EDV,
239
ESV, SV, EF and FS showed low (CV < 15%) variability. Meanwhile, the IVSTd, and LVPWd
240
showed moderate (CV 15–25%) variability.
241
Linear regression analysis of the data demonstrated that there was non-significant association
242
between B-mode and 2D-guided M-mode echocardiographic variables and sex, age, body weight,
243
and BCS of healthy donkeys (Tables 9,10).
244 245
4. Discussion
246
Since echocardiography is a non-invasive procedure, it can be used efficiently for diagnosis of
247
diseases of the cardiovascular system as well as investigation of the cardiac structures, cardiac
248
dimensions, and cardiac function indices. In ruminants, horses, and pets, the measurement of cardiac
249
chamber dimensions as well as cardiac function indices is a vital tool necessary for assessment of the
250
severity and prognosis of cardiac diseases [26]. Thus, the basic clinical reference values are required
251
to improve identification and assessment of such cardiac diseases allowing an earlier diagnosis and
252
more rapid intervention when facing abnormal echocardiographic findings. In the existing study, the
11
253
morphologic features and function indices of the heart were investigated using B-mode and 2D-
254
guided M-mode echocardiography.
255
In the present study, for both b-mode and guided M-mode echocardiography, the heart was
256
examined from the right parasternal location, using right parasternal long axis four-chamber view
257
and right parasternal short axis view, respectively. Both B-mode and guided M-mode
258
echocardiography were easy to perform and well tolerated in all donkeys, without the need for any
259
sedation or analgesia. Both echocardiographic modes provided valuable information regarding
260
cardiac chamber size and dimension, cardiac wall thickness, systolic function, and valvular
261
structures. Furthermore, high temporal resolution is a characteristic feature of M-mode
262
echocardiography, which makes it a useful tool for estimating the chamber volume as well as left
263
ventricular mass when these structures are geometrically uniform and useful for timing valve motion
264
[27]. The obtained echocardiographic images were in agreement with those reported by Reef et al.
265
[5]; Patteson et al. [17]; Boon [18]; Bakos et al. [19]; Vöros et al. [28].
266
In the examined donkeys, the recorded mean ± SD of both B-mode and guided M-mode
267
echocardiographic dimensions, including IVSTd, IVSTs, LVIDd, LVIDs, LVPWd, and LVPWs and
268
the mean ± SD of cardiac function indices, including EDV, ESV, SV, EF, and FS were almost
269
similar to those recorded [14,29,30]. The intra-assay and inter-assay CV of B-mode and guided M-
270
mode echocardiographic dimensions and cardiac function indices in the studied donkeys, the IVSTs,
271
LVIDd, LVIDs, LVPWs, EDV, ESV and EF showed low (CV < 15%) variability confirming the
272
reliability and uniformity in the values of the recorded data set, while the IVSTd, LVPWd showed
273
moderate (CV 15–25%) variability. However, SV and FS in the guided M-mode echocardiography
274
showed low (CV < 15%) variability compared with those of B-mode echocardiography in which
12
275
they showed moderate variability (CV 15-25%). Thus, the results of SV and FS in guided M-mode
276
echocardiography were reliable and uniform compared to those in B-mode echocardiography.
277
The overall impressions of heart size and function, obtained using B-mode and guided M-mode
278
echocardiography from the obtained views, were similar to those obtained in sedentary donkeys [29-
279
31]. However, it showed some notable differences compared with data generated in another study
280
conducted in Standardbred trotters [19], Endurance horses [9], Quarter horses [8], warm blood
281
horses [2], dogs [32], and human [33,34], which indicate significant differences between species as
282
the length and weight of the heart significantly correlates with weight, height, and athletic
283
performance of individual. In the existing study, B-mode and guided M-mode echocardiographic
284
examination of healthy donkeys (Equus asinus) revealed that both the interventricular septum
285
thickness and the left ventricular posterior free wall were greater during systole than diastole.
286
Meanwhile, the dimension of the left ventricle during diastole was greater than systole. Furthermore,
287
the recorded data of cardiac function indices; including EDV, ESV, SV, EF, and FS using B-mode
288
echocardiography were closer to those recorded by guided M-mode echocardiography. These
289
findings were in agreement with those reported in donkeys [29,30], horses [2,9], dogs [32], human
290
[33,34].
291
The interventricular septum thickness provides a signal about the left ventricular mass that is
292
calculated from the left ventricular free wall thickness, interventricular septum thickness and left
293
ventricular internal diameter. Thus, it is required for evaluation of the left and right ventricular
294
hypertrophy in both human [35] and race horses [36]. The LVIDd and LVIDd are essential to
295
characterize the eccentric hypertrophies in sportsmen [37] and to detect left ventricular overload in
296
cases of valvular insufficiency, regurgitation of mitral and aortic valves or myocardial diseases [38].
297
They are usually increased in exercising horses either as a result of exercise [39] or other
13
298
pathological conditions as aortic valve insufficiency [40]. The LVPWd and LVPWs are essential for
299
investigation of the effects of the training related adaptation, and for detection of left ventricular
300
overload, aortic valve regurgitation, patent ductus arteriosus, and atrial and ventricular septal defect
301
[38]. Cardiac function indices are used to evaluate pumping functions that focus on the ejected blood
302
volume; SV (the difference between the EDV and ESV), the efficiency of ejection; EF, and index to
303
evaluate the strength of myocardial contraction; FS. Thus, EF and FS are indicators of left
304
ventricular systolic function, ventricular compliance and contractibility as well as the cardiac
305
function [36,38]. In heart failure with reduced EF, also called systolic failure, the left ventricle loses
306
its ability to contract normally. Thus, the heart can't pump with enough force to push enough blood
307
into circulation. Meanwhile, heart failure with preserved EF, also called diastolic failure or
308
dysfunction, the left ventricle muscle has become stiff and loses its ability to relax normally. Thus,
309
the heart can't properly fill with blood during the resting period between each beat. In both cases, the
310
cardiac function indices are significantly affected as well as the echocardiographic dimensions.
311
As the donkeys age increases, they gain body weight and both the circumference and the length
312
of the body reach a moderate degree, which does not increase any more with the increasing age of
313
these donkeys. Unlike donkeys, the older the horses are, the greater the body weight and the greater
314
the circumference and length of the body compared to the donkeys of the same age. Fully grown
315
donkey will be around 36 to 54 inches (90- 137 cm) from the withers to the hoof, while horses, on
316
the other hand, are around 56 to 64 inches on average (142 to 163 cm). Thus, the size and
317
dimensions of the heart greatly increase in horses relative to the size and weight of their large body
318
compared to donkeys. This is our suggestion that may explain the non-significant association
319
between B-mode and guided M-mode echocardiographic variables and age, body weight, and BCS
320
of healthy donkeys. Thus, they are not taken into consideration in donkeys when comparing patient
14
321
data with such reference values. These findings were in agreement with those reported by Roberts
322
and Dukes-McEwan [30]. In contrast to data from equine studies, body weight have a significant
323
effect on all echocardiographic dimensions, and also breed have a significant effect on most of them,
324
but not by sex. Furthermore, the age significantly affects only the internal diameter of the aortic and
325
the pulmonary arteries [41]. In human studies, sex, body weight, and age significantly affect the
326
normal heart size. With advancing age, the cardiac chambers seemed to be smaller with thicker walls
327
[42,43]. However, body mass index has no statistical significant influence on echocardiographic
328
variables [44].
329
There are several limitations of this study. First, all studied donkeys were examined in standing
330
position. Diseased donkeys may be in a recumbent position and unable to rise for complete
331
echocardiographic examination. Alternate posture of the donkeys was not investigated in the existing
332
study. Second, the existing study evaluated only standard B-mode and guided M-mode
333
measurements; the authors did not generate reference ranges for spectral Doppler, which may also be
334
important in investigating the effects of diseases. Finally, the used echocardiographic equipment and
335
technical settings in this study were identical for all examinations and the use of other different
336
equipment may increase variability due to differences in resolution and measurement accuracy.
337
In conclusion, echocardiography has been shown to be feasible, repeatable and accurate, and has
338
been demonstrated to be a powerful tool for assessment of the dimensions and function indices of the
339
heart. This study provided new useful reference values of cardiac dimensions and cardiac function
340
indices in healthy donkeys (Equus asinus), considering them normal limits of echocardiographic
341
dimensions and cardiac function indices. Age, body weight, and BCS of healthy donkeys have no
342
effect on the B-mode and guided M-mode echocardiographic variables. Knowing the normal
15
343
parameters of echocardiography facilitates the identification and early diagnosis of cardiac diseases
344
in donkeys, which is considered the first step in managing such problems.
345 346
Conflict of Interest statement: The authors declare that they have no conflict of interest.
347 348
Ethical approval: All applicable international, national, and/or institutional guidelines for the care
349
and use of animals were followed.
350 351
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461
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462
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463
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464
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465 466
Figure legend
467
Figure 1. Standard echocardiographic view for assessment of left ventricular size and function.
468
Right parasternal long axis four-chamber view centered on the left ventricle. The transducer is
469
positioned in the right fourth intercostal space at the level of or slightly dorsal to olecranon process,
470
angled caudally, and rotated in clockwise direction to the 1 o’clock position. RV, right ventricle,
471
IVS, interventricular septum, LV; left ventricle; LA; left atrium; Ao, aorta.
472
Figure 2. Standard echocardiographic view for assessment of left ventricular size and function. 2D
473
guided M-mode image of the left ventricle obtained from a right-parasternal short-axis view at the
474
chordal level.
475
21
Table 1. Frequency distribution (% of study population) of B-mode echocardiographic dimensions and cardiac function indices in clinically healthy donkeys (Equus asinus) (n = 44) Highest frequency Variables
Lowest frequency
Value
No (%)
Value
No (%)
IVSTd(mm)
≥ 16 – 18
38 (42.2 %)
≥ 26 – 28
4 (4.4 %)
IVSTs (mm)
≥ 20 – 22
36 (40 %)
≥ 32 – 34
2 (2.2 %)
LVIDd (mm)
≥ 44 – 46
22 (24.4 %)
≥ 58 – 60
4 (4.4 %)
LVIDs (mm)
≥ 28 – 30
24 (26.7 %)
≥ 40 – 42
2 (2.2 %)
LVPWd (mm)
≥ 20 – 22
32 (35.6 %)
≥ 32 – 34
6 (6.7 %)
LVPWs (mm)
≥ 22 – 24
36 (40 %)
≥ 32 – 34
6 (6.7 %)
EDV (ml)
≥ 200 – 205
32 (35.6 %)
≥ 166 – 171
10 (11.1 %)
ESV (ml)
≥ 60 – 70
38 (42.2 %)
≥ 35 – 41
8 (8.88 %)
SV (ml)
≥ 40 – 50
46 (51.1 %)
≥ 60 – 70
4 (4.4 %)
EF (%)
≥ 70 – 75
42 (46.7 %)
≥ 95 – 100
8 (8.9 %)
FS (%)
≥ 32 – 39
46 (51.1 %)
≥ 55 – 57
2 (2.2 %)
Table 2. Summary statistics of B-mode echocardiographic dimensions and cardiac function indices in clinically healthy donkeys (Equus asinus) (n = 44) Percentile Median (range)
Mean ± SD
95% CI
Median (range) 10%
25%
75%
90%
IVSTd (mm)
18.5 ± 3.6
17.7 – 19.2
18.1 (12.4 – 26.6)
13.8
15.5
21.2
23.3
IVSTs (mm)
23.5 ± 3.8
22.7 – 24.3
23.2 (16.6 – 32.6)
19.1
20.2
26.9
28.4
LVIDd (mm)
42.7 ± 6.6
41.3 – 44.1
42.7 (30.9 – 58.2)
33.6
38.2
46.7
51.2
LVIDs (mm)
28.5 ± 3.9
24.2 – 26.8
25.3 (11.7 – 40.7)
19.2
21.4
29.6
32.7
LVPWd (mm)
22.5 ± 3.9
21.5 – 23.4
22.2 (14.6 – 32.9)
15.7
19.7
24.9
29.3
LVPWs (mm)
25.2 ± 3.6
23.2 – 25.1
23.9 (15.9 – 33.1)
17.8
21.3
27.7
30.2
EDV (ml)
202 ± 21.17
193.3– 210.8
205 (166– 237)
170.6
180
221.5
227.6
ESV (ml)
68.6 ± 6.5
56.19 – 67.12
66.5 (35 – 78)
38
55.25
71.25
73.6
SV (ml)
46.3 ± 8.7
44.5 – 48.2
46.7 (30.2 – 65.7)
32.8
41.5
52.2
56.8
EF (%)
76.1 ± 10.9
73.8 – 78.4
75 (42 – 96)
64
68.8
84
92
FS (%)
39.8 ± 8.4
37.5 – 42.2
37 (16 – 50)
29
32
46
50
Table 3. Intra-assay coefficient of variation of B-mode echocardiographic dimensions and cardiac function indices in clinically healthy donkeys (Equus asinus) (n = 44) Percentile Median (range)
Mean ± SD
95% CI
Median (range) 10%
25%
75%
90%
IVSTd (mm)
18.8 ± 7.8
15.9 – 21.7
19.9 (7.5 - 33.2)
9.8
11.5
24.8
31.9
IVSTs (mm)
14.8 ± 9.5
11.3 – 18.3
14.4 (1.1 - 37.3)
4.2
6.4
20.8
26.7
LVIDd (mm)
11.5 ± 6.1
9.2 – 13.7
11.7 (3.2 - 27.1)
3.9
6.1
13.7
18.4
LVIDs (mm)
12.9 ± 7.2
10.3 – 15.6
12.4 (4.4 - 30.8)
4.4
6.9
17.9
22.5
LVPWd (mm)
16.7 ± 11.1
12.6 – 20.9
12.5 (2.9 - 35.2)
4.6
7.2
24.9
34.5
LVPWs (mm)
12.8 ± 9.1
9.4 – 16.1
11.4 (2.2 - 34.9)
3.1
5.2
19.1
24.4
EDV (ml)
11.7 ± 6.4
9.3 – 14.1
10.4 (4.4 – 25.5)
4.5
6.4
17.9
21.2
ESV (ml)
11.1 ± 8.8
13.8 – 18.3
18.9 (2.0 – 27.5)
7.3
8.6
21.6
26.5
SV (ml)
16.1 ± 9.7
12.5 – 19.8
18.4 (4.0 – 30.5)
4.0
5.8
25.6
26.8
EF (%)
10.5 ± 8.3
7.4 – 13.6
8.6 (0.6 - 36.6)
3.4
5.6
14.5
15.5
FS (%)
18.3 ± 12.8
13.6 – 23.1
16.4 (3.1 - 57)
5.7
9.4
25.4
27.1
Table 4. Inter-assay coefficient of variation of B-mode echocardiographic dimensions and cardiac function indices in clinically healthy donkeys (Equus asinus) (n = 44) Percentile Median (range)
Mean ± SD
95% CI
Median (range) 10%
25%
75%
90%
IVSTd (mm)
15.8 ± 7.4
12.9 – 18.5
15.6 (1.7 - 28.2)
2.1
12.2
22.4
24.6
IVSTs (mm)
13.6 ± 6.2
11.3 – 15.9
14.5 (3.1 - 23.9)
4.6
9.5
17.9
23.1
LVIDd (mm)
15.1 ± 5.1
13.2 – 17.1
14.7 (7.1 - 23.4)
8.7
11.1
19.4
22.4
LVIDs (mm)
13.7 ± 8.4
19.8 – 28.4
23.7 (1.4 - 38.9)
3.3
21.2
32
36.3
LVPWd (mm)
17.3 ± 5.7
17.9 – 24.4
22.2 (8.9 - 36.1)
11.6
12.6
24.4
34.1
LVPWs (mm)
14.2 ± 8.1
14.2 – 21.1
16.4 (5.4 - 34.8)
6.6
9.1
25.9
31.6
EDV (ml)
13.1 ± 6.2
10.8 – 15.4
11.9 (5.3 - 26.1)
5.6
7.2
20
20.6
ESV (ml)
13.3 ± 7.9
13.3 – 19.2
20.1 (2.6 – 27.9)
4.2
8.0
22.2
23.1
SV (ml)
16.4 ± 9.1
12.9 – 19.8
16.7 (4.8 – 36.5)
5.3
8.3
24.9
27.9
EF (%)
14.4 ± 6.9
11.8 – 16.9
12.5 (4.8 - 33.1)
6.6
10.1
18.3
20.3
FS (%)
21.1 ± 9.1
22.3 – 32
30.5 (8.7 - 46.6)
10.4
15.9
46.6
44.3
Table 5. Frequency distribution (% of study population) of 2 dimensional guided Mmode echocardiographic dimensions and cardiac function indices in clinically healthy donkeys (Equus asinus) (n = 44) Highest frequency variables
Lowest frequency
Value
No (%)
Value
No (%)
IVSTd(mm)
≥ 1.6 – 1.8
44 (48.9 %)
≥ 3 – 3.2
2 (2.2 %)
IVSTs (mm)
≥ 2.8 – 3
26 (28.9 %)
≥ 5.8 – 6
2 (2.2 %)
LVIDd (mm)
≥ 4.5 – 5
24 (26.7 %)
≥ 9 – 9.5
2 (2.2 %)
LVIDs (mm)
≥ 2.6 – 2.9
36 (40 %)
≥ 6.5 – 7
2 (2.2 %)
LVPWd (mm)
≥ 2.4 – 2.6
34 (37.8 %)
≥ 5.8 – 6
2 (2.2 %)
LVPWs (mm)
≥ 2.8 – 3
24 (26.7 %)
≥ 5 – 5.2
2 (2.2 %)
EDV (ml)
≥ 190 – 195
26 (28.8 %)
≥ 173 – 178
6 (6.7 %)
ESV (ml)
≥ 58 – 65
28 (31.1 %)
≥ 39 – 42
4 (4.4 %)
SV (ml)
≥ 50 – 53
66 (28.9 %)
≥ 46 – 48
2 (2.2 %)
EF (%)
≥ 74 – 76
18 (20 %)
≥ 80 – 82
8 (8.9 %)
FS (%)
≥ 41 – 43
16 (17.8 %)
≥ 56 – 58
2 (2.2 %)
Table 6. Summary statistics of 2 dimensional guided M-mode echocardiographic dimensions and cardiac function indices in clinically healthy donkeys (Equus asinus) (n = 44) Percentile Median (range)
Mean ± SD
95% CI
Median (range) 10%
25%
75%
90%
IVSTd (mm)
1.7 ± 0.4
1.6 – 1.9
1.7 (1.1 – 3.1)
1.2
1.5
1.9
2.2
IVSTs (mm)
2.9 ± 0.4
2.7 – 2.9
2.8 (1.9 – 5.8)
2.1
2.4
3.1
3.5
LVIDd (mm)
6.1 ± 0.8
5.8 – 6.3
6.1 (3.8 – 9.1)
4.4
4.9
7.1
7.7
LVIDs (mm)
3.7 ± 0.5
3.3 – 3.7
3.3 (2.1 – 6.5)
2.4
2.6
3.9
4.8
LVPWd (mm)
2.8 ± 0.5
2.6 – 2.9
2.7 (1.5 – 5.9)
1.9
2.4
3.1
3.7
LVPWs (mm)
3.6 ± 0.4
3.1 – 3.3
3.1 (1.9 – 5.1)
2.2
2.8
3.7
3.9
EDV (ml)
200 ± 16.4
193 – 207
198 (173 – 235)
179
189
210
226
ESV (ml)
63.8 ± 9.32
54.9 – 66.7
64 (39 – 78)
42
48
70
76
SV (ml)
54.7 ± 3.1
52.5 – 56.9
54.9 (50.8 – 58.6)
50.84
51.50
57.80
85.52
EF (%)
79.9 ± 6.8
78.5 – 81.4
82 (67 – 91)
70
74
85.3
89
FS (%)
42.3 ± 5.5
40.9 – 43.8
43 (31 – 56)
33
36
47.3
52
Table 7. Intra-assay coefficient of variation of 2 dimensional guided M-mode echocardiographic dimensions and cardiac function indices in clinically healthy donkeys (Equus asinus) (n = 44) Percentile Median (range)
Mean ± SD
95% CI
Median (range) 10%
25%
75%
90%
IVSTd (mm)
17.8 ± 8.3
14.7 – 20.9
16.7 (8.6 – 39.8)
8.6
10.7
21.3
29.6
IVSTs (mm)
14.7 ± 11.9
10.2 – 19.2
10.1 ( 4.1 – 50.5)
5.1
8.6
23.4
27.7
LVIDd (mm)
12.9 ± 6.7
10.1 – 15.1
11.9 ( 3 – 26.3)
6.9
7.3
16.2
25.4
LVIDs (mm)
13.9 ± 8.4
10.8 – 17.1
12.7 (3.7 – 31.6)
4.5
6.3
21.1
21.9
LVPWd (mm)
18.4 ± 8.6
18.2 – 24.6
20.9 (7.4 – 38.9)
11.4
14.5
25.8
42.9
LVPWs (mm)
14.9 ± 9.8
11.2 – 18.6
12.8 (3.4 – 37.3)
4.9
6.8
16.8
35.6
EDV (ml)
10.8 ± 5.6
8.7 – 12.9
11.0 (0.6 – 20.9)
3.1
6.9
15.5
18.5
ESV (ml)
12.2 ± 9.9
13.5 – 18.9
14.2 (5.5 – 35.4)
8.01
11.5
18.6
25.7
SV (ml)
7.13 ± 2.1
12.8 – 18.1
13.8 (7.5 – 35.3)
8.3
9.6
19.7
22.9
EF (%)
6.2 ± 2.9
5.1 – 7.3
7.6 (2.3 – 10.3)
2.3
2.9
8.8
10.3
FS (%)
11.7 ± 5.2
9.8 – 13.6
12.6 (5.1 – 20.7)
5.1
6
15.8
17.9
Table 8. Inter-assay coefficient of variation of 2 dimensional guided M-mode echocardiographic dimensions and cardiac function indices in clinically healthy donkeys (Equus asinus) (n = 44) Percentile Median (range)
Mean ± SD
95% CI
Median (range) 10%
25%
75%
90%
IVSTd (mm)
19 ± 9.3
15.5 – 22.5
17.9 (4.7 – 35.1)
5.3
11.9
22.9
34.5
IVSTs (mm)
13.7 ± 5.5
14.9 – 20.6
15.8 (1.6 – 43.8)
10.5
14.1
20.6
32.0
LVIDd (mm)
13.7 ± 8.7
16.3 – 22.7
17.8 (5.1 – 33.8)
12.8
20.1
27.1
30.3
LVIDs (mm)
13.5 ± 6.1
18.4 – 22.9
17.2 (15.6 – 34.9)
15.8
19.9
31.4
34.2
LVPWd (mm)
17.9 ± 8.2
17.6 – 25.9
17.1 (9.1 – 43.4)
11.7
14.3
31.9
42.9
LVPWs (mm)
11.5 ± 5.6
14.9 – 20.6
14.3 (9.8 – 30.5)
9.9
13.3
25.5
28.6
EDV (ml)
11.9 ± 6.9
9.4 – 14.5
13.6 (0.7 – 24.7)
0.7
6.7
16.4
21.2
ESV (ml)
12.5 ± 8.1
12.4 – 16.5
15.6 (3.1 – 23.8)
4.1
7.9
22.7
24.3
SV (ml)
7.13 ± 2.3
13.8 – 17.8
16.4 (7.9 – 27.2)
10.2
10.7
20.6
21.4
EF (%)
8.8 ± 3.5
7.4 – 10.1
8.4 (3.2 – 15.3)
3.6
6.4
11.9
13.2
FS (%)
13.1 ± 3.4
13.5 – 18.2
14.2 (5.9 – 25.5)
8.3
12.1
20.5
24.3
Table 9. Association between B-mode echocardiographic parameters and sex, age, body weight and body condition score in clinically healthy donkeys (Equus asinus) (n = 44) Sex
Age
BWt
BCS
P-value
P-value
P-value
P-value
0.396 0.734 0.371 0.287 0.154 0.269 0.533 0.430 0.351 0.266 0.621
0.904 0.363 0.465 0.716 0.629 0.724 0.246 0.398 0.304 0.920 0.913
0.725 0.193 0.530 0.687 0.696 0.623 0.360 0.478 0.274 0.556 0.846
0.659 0.831 0.181 0.681 0.961 0.652 0.546 0.144 0.862 0.499 0.145
B-mode variables IVSTd (mm) IVSTs (mm) LVIDd (mm) LVIDs (mm) LVPWd (mm) LVPWs (mm) EDV (ml) ESV (ml) SV (ml) EF (%) FS (%)
P value < 0.05 indicates a significant correlation. BWt, Body weight; BCS, Body condition score.
Table 10. Association between 2 dimensional guided M-mode echocardiographic variable and sex, age, body weight and body condition score in clinically healthy donkeys (Equus asinus) (n = 44) Sex
Age
BWt
BCS
P-value
P-value
P-value
P-value
0.432 0.521 0.211 0.152 0.221 0.311 0.401 0.316 0.241 0.125 0.415
0.298 0.131 0.320 0.203 0.194 0936 0.639 0.402 0.751 0.451 0.364
0.319 0.158 0.400 0.328 0.245 0.946 0.717 0.484 0.922 0.637 0.548
0.130 0.997 0.994 0.154 0.673 0.942 0.904 0.422 0.185 0.823 0.298
M-mode variables IVSTd (mm) IVSTs (mm) LVIDd (mm) LVIDs (mm) LVPWd (mm) LVPWs (mm) EDV (ml) ESV (ml) SV (ml) EF (%) FS (%)
P value < 0.05 indicates a significant correlation. BWt, Body weight; BCS, Body condition score.
Highlights •
•
• • •
B-mode and 2D guided M-mode echocardiography was performed for 44 healthy donkeys (Equus asinus) to assess the cardiac dimensions and cardiac function indices. This study reported the reference ranges of the echocardiographic dimensions and cardiac function indices in donkeys (Equus asinus) based on the probability theory and statistics. The interventricular septum thickness and the left ventricular posterior free wall were greater during systole than diastole. The left ventricular dimension during diastole was greater than systole. The assessed cardiac dimensions and cardiac function indices using B-mode echocardiography were closer to those recorded by 2D guided M-mode echocardiography.
Author statement
We affirm that the submission represents original work that has not been published previously and is not currently being considered by another journal. All other Authors have read the manuscript and have agreed to submit it in its current form for consideration for publication. We confirm that the legal and ethical requirements have been met with regards to the humane treatment of animals described in the study This manuscript is the first report the reference values and repeatability of B-mode and M-mode echocardiography in healthy donkey (equus asinus) - the guide to assess healthy heart Thank you for your cooperation and waiting for your response Kind regards Corresponding author Hussam M. M. Ibrahim
Author’s contributions: To qualify as an author one should 1) have made substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; 2) have been involved in drafting the manuscript or revising it critically for important intellectual content; 3) have given final approval of the version to be published; and 4) agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. We suggest the following kind of format (please use initials to refer to each author's contribution): AB carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. JY carried out the immunoassays. MT participated in the sequence alignment. ES participated in the design of the study and performed the statistical analysis. FG conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. SE: Conceived and designed the experiments, analyzed the data, final revision and submission. MH: Conceived and designed the experiments, and helped to draft the manuscript HI: Performed the experiments, revised the data KA: Performed the experiments RE: drafting the paper Name and signature:
Conflict of Interest statement: The authors declare that they have no conflict of interest.