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Within-day and between-day variability of transthoracic anatomic M-mode echocardiography in the awake bottlenose dolphin (Tursiops truncatus)
Abstract
The use of transthoracic echocardiography in dolphins has been limited so far owing to technical and anatomical specificities. Anatomic M-mode (AMM) is a postprocessing echocardiographic technique generating M-mode studies from two-dimensional (2D) cineloops independently of the ultrasound beam orientation. The aim of the present study was to determine the within-day (repeatability) and between-day (reproducibility) variability of AMM echocardiography in awake healthy bottlenose dolphins (BN, Tursiops truncatus). Four adult BN trained to lie in left recumbency at the water surface were involved in the protocol. A total of 96 echocardiographic examinations were performed on 4 different days by a trained observer examining each BN 6 times per day. Video clips of 2D left parasternal long-axis views showing the left ventricle (LV) ventrally and the aortic root dorsally were recorded at each examination and analyzed for AMM measurements in a random order. A general linear model was used to determine the within-day and between-day coefficients of variation (CV). All examinations were interpretable allowing calculation of 10 AMM variables (i.e., end-diastolic and end-systolic ventral and dorsal LV myocardial wall thicknesses as well as LV and aortic diameters, mean aortic diameter, and LV shortening fraction). Most within- and between-day CV values (18/20) were <15%, the lowest being observed for the end-diastolic LV diameter (1.6%). In conclusion, AMM provides a simple non-invasive evaluation of heart morphology and function in the awake BN with good repeatability and reproducibility of the measurements. Further studies are required to determine the corresponding reference intervals.
... Unfortunately, similar standardized guidelines do not exist for marine mammals. As such, there is a paucity of Doppler echocardiographic data on cardiac structure and function, in marine mammals, which is restricted to only a few studies (Chetboul et al. 2012;Miedler et al. 2015;Gerlach et al. 2015;Fahlman et al. 2019;Storlund et al. 2021;Linnehan et Aquarium Mar del Plata, Av. De los Trabajadores 5600, Mar del Plata, Provincia de Bs As, Argentina 2021). ...
... The echocardiographic measurements were made adapting the guidelines provided for terrestrial animals (Thomas et al. 1993;Boon 2011) and the few echocardiographic investigations carried out in marine mammals (Chetboul et al. 2012;Miedler et al. 2015;Gerlach et al. 2015;Castro et al. 2018;Fahlman et al. 2019;Storlund et al. 2021;Linnehan et al. 2021). To avoid inter-observer variation echocardiography was performed by a single researcher with certified skills in duplex scan diagnostic procedures, using an M6 Mindray® ultrasound system (Shenzhen, China) with a phased array probe (P7-3s). ...
... Also the systolic function parameters (e.g. LVEF) are comparable with those reported for dolphins (Chetboul et al. 2012) and manatees (Gerlach et al. 2015). However, with comparable ventricular dimensions, those Steller sea lions under anesthesia showed lower values of systolic function parameters (LVEF, FAC, SV, and CO) likely resulting from the negative inotropic effects of isoflurane anesthesia, which commonly reduces fractional shortening in healthy mammals (Shimada et al. 1989). ...
Echocardiography is commonly used in veterinary clinical practice for many species but is not as easily applied in non-sedated marine mammals. Additionally, Doppler echocardiography provides further information on hemodynamics, estimation of cardiac output, characterization of flow patterns, and diastolic function. However, its applications in marine mammals have not been as widely explored either. The present report aimed to characterize the left ventricle structure and function of the Southern Sea Lion (Otaria flavescens) through a Doppler echocardiography study. Data were obtained from a healthy animal trained for different veterinary examination routines. This communication constitutes the first report of Doppler echocardiography with stroke volume, and cardiac output measurement in pinnipeds, and the first report of measurement of myocardial performance index in aquatic mammals. This data brought initial information about in vivo structure and function of the heart of the Southern Sea Lion that can be safely and effectively evaluated with the use of transthoracic Doppler echocardiography. Our data have both clinical and research implications for future studies evaluating diseases of the cardiopulmonary system in pinnipeds, and cardiovascular physiology investigation in diving mammals.
... Recent work has broadened this focus to include other cetacean species (Elmegaard et al., 2016;Bickett et al., 2019;Goldbogen et al., 2019), (Aoki et al., 2021;Blawas et al., 2021b). At present there are few methods available to study cardiac contractility in cetaceans, but recent work has characterized bottlenose dolphin cardiac output, stroke volume, and cardiac anatomy using echocardiography (Sklansky et al., 2006;Chetboul et al., 2012;Miedler et al., 2015;Fahlman et al., 2019;Linnehan et al., 2020). In many of these studies, physiology measurements required the animals to remain tethered to land-based data collection systems. ...
... While a great deal of cetacean cardiovascular research has been conducted since the seminal studies by Scholander and Irving (e.g., Irving & Scholander, 1941a), the majority of efforts have focused on characterizing variation in cardiac cycle frequency during eupnea and apnea in bottlenose dolphins (Tursiops truncatus) using electrocardiography (ECG) (Elsner et al., 1966;Hamlin et al., 1970;Williams et al., 1993;Noren et al., 2004b;Harms et al., 2013;Yaw et al., 2018;Fahlman et al., 2020;Blawas et al., 2021a;Bickett et al., 2019;Goldbogen et al., 2019;Blawas et al., 2021b). Several studies have also used echocardiography to investigate cardiac output, stroke volume, and cardiac anatomy of bottlenose dolphins (Sklansky et al., 2006;Chetboul et al., 2012;Miedler et al., 2015;Fahlman et al., 2019;Linnehan et al., 2020). At least one study has used phonocardiography to record heart sounds in captive bottlenose dolphins and belugas (Miksis et al., 2001). ...
Vertebrate animals undergo a constellation of physiological responses when they experience submersion. These responses, collectively known as the dive response, include apnea (breath-hold), bradycardia (a reduction in heart rate), and peripheral vasoconstriction (the restriction of oxygenated blood to organs critical to life). Cetaceans, the order of mammals that includes whales, dolphins, and porpoise, are obligate air-breathing mammals and one of the few mammalian taxa to become fully aquatic. Given this evolutionary trajectory, cetaceans are an excellent model for investigating the physiological extremity of the dive response.
One limiting factor in dive response research involving cetaceans is the relative lack of non-invasive physio-logging devices that can be attached in free-swimming animal contexts. To address this gap, my collaborators and I invented a new multi-sensor, suction cup-attached device called the FaunaTag. The FaunaTag was custom-built to enable non-invasive collection of cardiovascular physiological data in cetacean species. Equipped with a novel contact sensor column that interfaces with the body surface of the tagged animal, the FaunaTag's near-infrared spatially-resolved diffuse reflectance bio-optical sensor and its accelerometer and gyroscope sensors were used to investigate aspects of the dive response in bottlenose dolphins (Tursiops truncatus), the most accessible and well-studied member of the cetacean order.
In the first set of experimental trials, I used the FaunaTag and a new methodological approach to investigate the extent to which dolphin cardiac heart rate changes during alternating bouts of stationary surface free-breathing and submerged apnea. In these trials, the FaunaTag and its unique contact sensor measured the vibrations associated with the cardiac cycle at the dolphin's chest wall. These vibrations were used to compute instantaneous heart rate and instantaneous kinetic energy associated with cardiac contractility. During these trials, we also tested the efficacy of the FaunaTag's near-infrared bio-optical sensor to measure dolphin heart rate before, during, and after apnea, with the FaunaTag placed at a variety of body locations, and the extent to which optically-computed heart rate estimates matched the cardiac frequency estimates calculated from cardiac vibrations.
I found that instantaneous heart rate estimates measured in this study were consistent with the heart rates computed using electrocardiography in previous studies involving these same animals. I also observed expected patterns of bradycardia during extended apneas, respiratory sinus arrhythmias following respiration events, and a return to a baseline heart rate shortly after respiration. I also found that instantaneous kinetic energy of cardiac contraction varies between free-breathing and breath-holding trial phases, with a decline to a stable apneic baseline during submerged breath-holds, followed by a steep rise following cessation of apnea and an eventual return to a variable but reduced post-apnea baseline. The FaunaTag's near-infrared spectroscopy performed poorly at dorsal body locations, detected 60% of the matched heartbeats while attached to the cardiac window of the bottlenose dolphin, and achieved a match rate exceeding 90% in the best trial. Future efforts involving the FaunaTag will feature an improved bio-optical sensing module which may resolve poor optical cardiography at the dorsal surfaces of the dolphin body and other cetacean species.
... Miedler et al. (48) described a transthoracic echocardiographic technique and presented measured indices in managed dolphins. Chetboul et al. (49) provided quantitative data on left ventricular morphology and function using anatomic M-mode in four managed dolphins. Miedler et al. (50) used echocardiography to estimate systolic left ventricular function during rest and following exercise in 13 trained dolphins, and provided baseline data for normal stroke volume and cardiac output in awake, managed dolphins. ...
Cardiac auscultation is an important, albeit underutilized tool in aquatic animal medicine due to the many challenges associated with in-water examinations. The aims of this prospective study were to (1) establish an efficient and repeatable in-water cardiac auscultation technique in bottlenose dolphins (Tursiops truncatus), (2) describe the presence and characterization of heart murmurs detected in free-ranging and managed dolphins, and (3) characterize heart murmur etiology through echocardiography in free-ranging dolphins. For technique development, 65 dolphins cared for by the Navy Marine Mammal Program (Navy) were auscultated. The techniques were then applied to two free-ranging dolphin populations during capture-release health assessments: Sarasota Bay, Florida (SB), a reference population, and Barataria Bay, LA (BB), a well-studied population of dolphins impacted by the Deepwater Horizon oil spill. Systolic heart murmurs were detected at a frequent and similar prevalence in all dolphin populations examined (Navy 92%, SB 89%, and BB 88%), and characterized as fixed or dynamic. In all three populations, sternal cranial and left cranial were the most common locations for murmur point of maximal intensity (PMI). An in-water transthoracic echocardiogram technique was refined on a subset of Navy dolphins, and full echocardiographic exams were performed on 17 SB dolphins and 29 BB dolphins, of which, 40 had murmurs. Spectral Doppler was used to measure flow velocities across the outflow tracts, and almost all dolphins with audible murmurs had peak outflow velocities ≥1.6 m/s (95%, 38/40); three dolphins also had medium mitral regurgitation which could be the source of their murmurs. The presence of audible murmurs in most of the free-ranging dolphins (88%) was attributed to high velocity blood flow as seen on echocardiography, similar Linnehan et al. Dolphin Auscultation and Heart Murmurs to a phenomenon described in other athletic species. These innocent murmurs were generally characterized as Grade I-III systolic murmurs with PMI in the left or sternal cranial region. This study is the first to describe an efficient technique for in-water dolphin cardiac auscultation, and to present evidence that heart murmurs are common in bottlenose dolphins.
... R Foundation for Statistical Computing, Vienna, Austria). Briefly, and as already described in our previous ultrasound imaging validation studies performed on wild or exotic animals [13,14], the following linear model was used to analyze the within-day and between-day variability of the TTE variables: ...
Introduction
Cardiovascular diseases have been identified as a major cause of mortality and morbidity in Borneo orangutans ( Pongo pygmaeus pygmaeus ). Transthoracic echocardiography is usually performed under anesthesia in great apes, which may be stressful and increase risks of peri-anesthetic complications in case of cardiac alteration. The aim of the present pilot study was hence to develop a quick and non-stressful echocardiographic method (i.e., the COOLEST method) in awake Borneo orangutans (CardiOvascular examination in awake Orangutans: Low-stress Echocardiography including Speckle Tracking imaging) and assess the variability of corresponding variables.
Materials and methods
Four adult Borneo orangutans trained to present their chest to the trainers were involved. A total of 96 TTE examinations were performed on 4 different days by a trained observer examining each orangutan 6 times per day. Each examination included four two-dimensional views, with offline assessment of 28 variables (i.e., two-dimensional (n = 12), M-mode and anatomic M-mode (n = 6), Doppler (n = 7), and speckle tracking imaging (n = 3)), representing a total of 2,688 measurements. A general linear model was used to determine the within-day and between-day coefficients of variation.
Results
Mean±SD (minimum-maximum) images acquisition duration was 3.8±1.6 minutes (1.3–6.3). All within-day and between-day coefficients of variation but one (n = 55/56, 98%) were <15%, and most (51/56, 91%) were <10% including those of speckle tracking systolic strain variables (2.7% to 5.4%).
Discussion
Heart morphology as well as global and regional myocardial function can be assessed in awake orangutans with good to excellent repeatability and reproducibility.
Conclusions
This non-stressful method may be used for longitudinal cardiac follow-up in awake orangutans.
... Further support that anesthesia had an effect on fractional shortening in Steller sea lions comes from the finding that reduced fractional shortening was not observed in bottlenose dolphins and southern sea lions that were examined without anesthesia. 4,5 Measurements of fractional shortening in conscious Steller sea lions are needed for confirmation. ...
Pinniped hearts have been well described via dissection, but in vivo measurements of cardiac structure, function, and electrophysiology are lacking. Electrocardiograms (ECGs) were recorded under anesthesia from eight Steller sea lions (Eumetopias jubatus), five northern fur seals (Callorhinus ursinus), and one walrus (Odobenus rosmarus) to investigate cardiac electrophysiology in pinnipeds. In addition, echocardiograms were performed on all eight anesthetized Steller sea lions to evaluate in vivo cardiac structure and function. Measured and calculated ECG parameters included P-wave, PQ, QRS, and QT interval durations, P-, R-, and T-wave amplitudes, P- and T-wave polarities, and the mean electrical axis (MEA). Measured and calculated echocardiographic parameters included left ventricular internal diameter, interventricular septum thickness, and left ventricular posterior wall thickness in systole and diastole (using M-mode), left atrium and aortic root dimensions (using 2D), and maximum aortic and pulmonary flow velocities (using pulsed-wave spectral Doppler). ECG measurements were similar to those reported for other pinniped species, but there was considerable variation in the MEAs of Steller sea lions and northern fur seals. Echocardiographic measurements were similar to those reported for southern sea lions (Otaria flavenscens), including five out of eight Steller sea lions having a left atrial to aortic root ratio <1, which may indicate that they have an enlarged aortic root compared to awake terrestrial mammals. Isoflurane anesthesia likely affected some of the measurements as evidenced by the reduced fractional shortening found in Steller sea lions compared to awake terrestrial mammals. The values reported are useful reference points for assessing cardiac health in pinnipeds under human care.
... Miedler et al. (48) described a transthoracic echocardiographic technique and presented measured indices in managed dolphins. Chetboul et al. (49) provided quantitative data on left ventricular morphology and function using anatomic M-mode in four managed dolphins. Miedler et al. (50) used echocardiography to estimate systolic left ventricular function during rest and following exercise in 13 trained dolphins, and provided baseline data for normal stroke volume and cardiac output in awake, managed dolphins. ...
Cardiac auscultation is an important, albeit underutilized tool in aquatic animal medicine due to the many challenges associated with in-water examinations. The aims of this prospective study were to (1) establish an efficient and repeatable in-water cardiac auscultation technique in bottlenose dolphins (Tursiops truncatus), (2) describe the presence and characterization of heart murmurs detected in free-ranging and managed dolphins, and (3) characterize heart murmur etiology through echocardiography in free-ranging dolphins. For technique development, 65 dolphins cared for by the Navy Marine Mammal Program (Navy) were auscultated. The techniques were then applied to two free-ranging dolphin populations during capture-release health assessments: Sarasota Bay, Florida (SB), a reference population, and Barataria Bay, LA (BB), a well-studied population of dolphins impacted by the Deepwater Horizon oil spill. Systolic heart murmurs were detected at a frequent and similar prevalence in all dolphin populations examined (Navy 92%, SB 89%, and BB 88%), and characterized as fixed or dynamic. In all three populations, sternal cranial and left cranial were the most common locations for murmur point of maximal intensity (PMI). An in-water transthoracic echocardiogram technique was refined on a subset of Navy dolphins, and full echocardiographic exams were performed on 17 SB dolphins and 29 BB dolphins, of which, 40 had murmurs. Spectral Doppler was used to measure flow velocities across the outflow tracts, and almost all dolphins with audible murmurs had peak outflow velocities ≥1.6 m/s (95%, 38/40); three dolphins also had medium mitral regurgitation which could be the source of their murmurs. The presence of audible murmurs in most of the free-ranging dolphins (88%) was attributed to high velocity blood flow as seen on echocardiography, similar to a phenomenon described in other athletic species. These innocent murmurs were generally characterized as Grade I-III systolic murmurs with PMI in the left or sternal cranial region. This study is the first to describe an efficient technique for in-water dolphin cardiac auscultation, and to present evidence that heart murmurs are common in bottlenose dolphins.
... In preceding works we were able to shed light on a strong association of Hif1a expression with survival in pancreatic cancer and soft tissue sarcomas [4,5]. However, this association was not significant in the examined study group of patients with cholangiocarcinoma which is in concordance with discoveries from other groups examining Hif1a in CCC [18] who were also not able to show a correlation of Hif1a expression to survival. VEGFR 2/3 expression was tested in several studies so far [19]. ...
Background
Cholangiocarcinoma remains to be a tumor with very few treatment choices and limited prognosis. In this study, we sought to determine the prognostic role of fms-related tyrosine kinase 1/vascular endothelial growth factor receptor 1 (FLT1/VEGFR1), heparanase (HPSE) and epidermal growth factor receptor (EGFR) gene expression in patients with resected CCC.
Methods
47 formalin-fixed paraffin embedded FFPE tumor samples from patients with resected CCC were analyzed. FFPE tissues were dissected using laser-captured microdissection and analyzed for FLT1, FLT4, HPSE, Hif1a, VEGFA/C, HB-EGF, PDGFA, PDGF-RA and EGFR mRNA expression using a quantitative real-time RT-PCR method. Gene expression values (relative mRNA levels) are expressed as ratios between the target gene and internal reference genes (beta-actin, b2mg, rplp2, sdha).
Results
EGFR, FLT1 and HPSE expression levels were significantly associated with overall survival (OS). FLT1 showed the strongest significant independent association with overall survival in a multivariate cox regression analysis when compared to the other genes and clinicopathological factors with a nearly 5 times higher relative risk (4.74) of dying earlier when expressed in low levels (p = 0.04). ROC Curve Analysis revealed that measuring EGFR potentially identifies patients at risk of a worsened outcome with a sensitivity of 80% and a specificity of 75% (p = 0.01).
Conclusions
EGFR and FLT1 seem to be potential markers to identify those patients at high risk of dying from cholangiocarcinoma. Therefore these markers may help to identify patient subgroups in need for a more aggressive approach in a disease that is in desperate need for new approaches.
The Deepwater Horizon (DWH) oil spill profoundly impacted the health of bottlenose dolphins ( Tursiops truncatus ) in Barataria Bay, LA (BB). To comprehensively assess the cardiac health of dolphins living within the DWH oil spill footprint, techniques for in-water cardiac evaluation were refined with dolphins cared for by the U.S. Navy Marine Mammal Program in 2018 and applied to free-ranging bottlenose dolphins in BB ( n = 34) and Sarasota Bay, Florida (SB) ( n = 19), a non-oiled reference population. Cardiac auscultation detected systolic murmurs in the majority of dolphins from both sites (88% BB, 89% SB) and echocardiography showed most of the murmurs were innocent flow murmurs attributed to elevated blood flow velocity [1]. Telemetric six-lead electrocardiography detected arrhythmias in BB dolphins (43%) and SB dolphins (31%), all of which were considered low to moderate risk for adverse cardiac events. Echocardiography showed BB dolphins had thinner left ventricular walls, with significant differences in intraventricular septum thickness at the end of diastole ( p = 0.002), and left ventricular posterior wall thickness at the end of diastole ( p = 0.033). BB dolphins also had smaller left atrial size ( p = 0.004), higher prevalence of tricuspid valve prolapse ( p = 0.003), higher prevalence of tricuspid valve thickening ( p = 0.033), and higher prevalence of aortic valve thickening ( p = 0.008). Two dolphins in BB were diagnosed with pulmonary arterial hypertension based on Doppler echocardiography-derived estimates and supporting echocardiographic findings. Histopathology of dolphins who stranded within the DWH oil spill footprint showed a significantly higher prevalence of myocardial fibrosis ( p = 0.003), regardless of age, compared to dolphins outside the oil spill footprint. In conclusion, there were substantial cardiac abnormalities identified in BB dolphins which may be related to DWH oil exposure, however, future work is needed to rule out other hypotheses and further elucidate the connection between oil exposure, pulmonary disease, and the observed cardiac abnormalities.
Information about antemortem cardiac evaluation in sea otters (Enhydra lutris) is limited, despite well-established clinical care and rehabilitation procedures and a reported elevated risk of cardiac disease for this species. Serum cardiac troponin I (cTnI) concentration and echocardiographic assessment are two ways of screening for and diagnosing cardiac disease. However, no baseline data or reference intervals for either evaluation are published for sea otters. The objectives of this prospective study were to establish serum cTnI concentrations and echocardiographic technique and quantitative measurements in anesthetized healthy female southern sea otters (Enhydra lutris nereis) (n=15). Serum cTnI values were assessed by a high-sensitivity assay. Serum cTnI concentration ranged from <0.006 to 0.038 ng/ml. A complete echocardiogram, including two-dimensional and M-mode modalities, was performed. Echocardiographic measurements for left atrial size, aorta size, left ventricular structure, and left ventricular function were reported. The median left atrial size to aorta ratio was 1.22 (range 0.80-1.59) in short-axis and 1.70 (range 1.39-2.15) in long-axis. The median left ventricular internal dimension was 3.53 cm (range 2.87-4.92 cm) when assessed in two dimensions and 3.58 cm (range 2.80-4.48 cm) by M-mode. Serum concentrations of cTnI and transthoracic echocardiography may represent valuable tools for the antemortem diagnosis of cardiac disease in sea otters.
The use of ultrasound has been applied to marine mammals because of the limitations of radiography that require transport and risk. This chapter discusses the uses and limitations of point‐of‐care ultrasound (POCUS) examination of marine mammals and describes the equipment, probe type, and settings for performing POCUS examination of marine mammals. It summarizes the five abdomen focused assessment with sonography for trauma (AFAST) acoustic windows that have been modified from small animals: diaphragmatico‐hepatic view, spleno‐renal view, hepato‐renal view, cysto‐colic reproductive view, and umbilical view. The AFAST‐applied abdominal fluid scoring system may be calculated for peritoneal effusion, providing an objective number for recording in medical records and for tracking static, resolving or worsening ascites. POCUS can be used for venous puncture of the superficial caudal peduncular vein. This provides access for blood letting or phlebotomies, especially in cases of hemosiderosis.
Objectives
To describe characteristics of echocardiography and cranial coelomic radiography in a cohort of iguanas.
Animals
Twenty apparently healthy adult green iguanas (Iguana iguana) from a reptile sanctuary.
Methods
Physical examination, radiography, two-dimensional and color Doppler echocardiography were performed to assess cardiac structures and function, and any related normal or abnormal findings were recorded.
Results
Echocardiographic examination was possible without sedation and allowed visualization of the great vessels, atria, and ventricle. Some structures could not be evaluated in a minority of the iguanas due to individual differences in bony conformation and imaging quality. Suspected abnormal echocardiographic findings in 3 iguanas included pericardial effusion (n=1) and enlarged caudal vena cava and/or sinus venosus (n=2). Objective measurements were repeatable as assessed by within-subject coefficient of variation, and reliable as assessed by intra-observer intraclass correlation coefficient. Left atrial and ventricular measurements were significantly correlated with body weight. Valve regurgitation was common, with atrioventricular valve regurgitation present in 53% (9/17) and aortic or pulmonic valve regurgitation in 71% (12/17) of otherwise normal iguanas. A heart murmur was not appreciated during examination of any of the iguanas. Heart size cannot be measured radiographically due to superimposition and silhouetting of other coelomic structures. Echocardiographic or radiographic findings consistent with mineralization of the great vessels were present in 76% of iguanas (13/17).
Conclusions
Echocardiography in iguanas is well tolerated without sedation and allowed both subjective evaluation and structural measurements. Valve regurgitation and great vessel mineralization were commonly observed in this cohort of apparently healthy adult iguanas.
In humans, fetal echocardiography represents the most important tool for the assessment of the cardiovascular well-being of the fetus. However, because of logistic, anatomic, and behavioral challenges, detailed fetal echocardiographic evaluation of marine mammals has not been previously described. Because the application of fetal echocardiography to cetaceans could have both clinical and academic importance, an approach to evaluating the fetal dolphin's cardiovascular status was developed with conventional, fetal echocardiographic techniques developed in humans. Eight singleton fetal bottlenose dolphins (Tursiops truncatus) were evaluated, each between 6 and 11 mo gestation; six fetuses underwent two fetal echocardiographic evaluations each, four at 3-mo intervals, and two at 0.5-mo intervals. Evaluations were performed without sedation, using conventional, portable ultrasound systems. Multiple transducers, probes, and maternal dolphin positions were used to optimize image quality. Fetal echocardiography included two-dimensional imaging and color flow mapping of the heart and great arteries, as well as pulsed Doppler evaluation of the umbilical artery and vein. Thorough evaluations of the fetal dolphins' cardiovascular status were performed, with the greatest resolution between 8 and 9 mo gestation. With the use of published human fetal echocardiographic findings for comparison, fetal echocardiography demonstrated normal structure and function of the heart and great arteries, including the pulmonary veins, inferior vena cava, right and left atria, foramen ovale, tricuspid and mitral valves, right and left ventricles, ventricular septum, pulmonary and aortic valves, main pulmonary artery and ascending aorta, and ductus arteriosus. Pulsed Doppler techniques demonstrated normal umbilical arterial and venous waveforms, and color flow mapping demonstrated absence of significant valvar regurgitation. Fetal echocardiography, particularly between 8 and 9 mo gestation, can provide a safe and detailed assessment of the cardiovascular status of the fetal bottlenose dolphin.
A free-living adult female Atlantic bottlenose dolphin (Tursiops truncatus) found dead near Panama City, Florida (USA), had necrotizing and ulcerative tracheitis, suppurative and hemorrhagic pneumonia, and necrotizing myocarditis; fungal hyphae were present in these lesions. Additionally, lungs had multifocal proliferative interstitial pneumonia with occasional syncytial cells. Some syncytial cells and type II pneumocytes contained eosinophilic intranuclear or intracytoplasmic inclusion bodies, or both. Based on an immunoperoxidase technique, there was morbilliviral antigen within cytoplasm and nuclei of type II pneumocytes and syncytial cells: antigen also occurred in trachea, skin, liver, stomach, intestine, and uterus. Based on pathologic and immunocytochemical findings, the dolphin had morbillivirus-induced disease. This is the first report of morbilliviral disease in a marine mammal from the Gulf of Mexico.
The M-mode (motion mode) technique has improved the diagnostic possibilities of echocardiography due to its high temporal resolution. The use of a two-dimensional (2D) image as a basis for M-mode analysis at a defined line, independent of the transducer orientation, namely the anatomic M-mode (AMM), has been proposed from the beginning of 2D echocardiography. For several years, however, this could not be accomplished due to several reasons including the limited digital memory, the relatively rough pixels of 2D images, and the low temporal resolution of the screen. The AMM has been improved by the "fully digital" machines. These are able to provide a series of digital data (direction, position, and timing) relative to any single echo received from any point of the tissue. AMM analysis, thus, can be performed in any direction, as a "normal" monodimensional echocardiogram. With respect to traditional M-mode, AMM permits a more detailed analysis of cardiac chambers diameters obtained by linear measurements, regional wall motion of the left ventricle (both at rest and during stress), and location of accessory pathways. In particular, the assessment of left ventricular regional wall motion represents the most important goal of this new technique, which results in marked reduction or even elimination of the limitations due to the subjective character of wall motion evaluation with 2D echocardiography.
Bradycardia is an important component of the dive response, yet little is known about this response in immature marine mammals. To determine if diving bradycardia improves with age, cardiac patterns from trained immature and mature bottlenose dolphins ( Tursiops truncatus) were recorded during three conditions (stationary respiration, voluntary breath-hold, and shallow diving). Maximum (mean: 117+/-1 beats.min(-1)) and resting (mean: 101+/-5 beats.min(-1)) heart rate (HR) at the water surface were similar regardless of age. All dolphins lowered HR in response to apnea; mean steady state breath-hold HR was not correlated with age. However, the ability to reduce HR while diving improved with age. Minimum and mean steady state HR during diving were highest for calves. For example, 1.5-3.5-year-old calves had significantly higher mean steady state diving HR (51+/-1 beats.min(-1)) than 3.5-5.5-year-old juveniles (44+/-1 beats.min(-1)). As a result, older dolphins demonstrated greater overall reductions in HR during diving. Longitudinal studies concur; the ability to reduce HR improved as individual calves matured. Thus, although newly weaned calves as young as 1.7 years exhibit elements of cardiac control, the capacity to reduce HR while diving improves with maturation up to 3.5 years postpartum. Limited ability for bradycardia may partially explain the short dive durations observed for immature marine mammals.
To determine the within-day and between-day variability of regurgitant fraction (RF) assessed by use of the proximal isovelocity surface area (PISA) method in awake dogs with degenerative mitral valve disease (MVD), measure RF in dogs with MVD, and assess the correlation between RF and several clinical and Doppler echocardiographic variables.
Prospective study.
6 MVD-affected dogs with no clinical signs and 67 dogs with MVD of differing severity (International Small Animal Cardiac Health Council [ISACHC] classification).
The 6 dogs were used to determine the repeatability and reproducibility of the PISA method, and RF was then assessed in 67 dogs of various ISACHC classes. Mitral valve regurgitation was also assessed from the maximum area of regurgitant jet signal-to-left atrium area (ARJ/LAA) ratio determined via color Doppler echocardiographic mapping.
Within- and between-day coefficients of variation of RF were 8% and 11%, respectively. Regurgitation fraction was significantly correlated with ISACHC classification and heart murmur grade and was higher in ISACHC class III dogs (mean +/- SD, 72.8 +/- 9.5%) than class II (57.9 +/- 20.1%) or I (40.7 +/- 19.2%) dogs. Regurgitation fraction and left atriumto-aorta ratio, fractional shortening, systolic pulmonary arterial pressure, and ARJ/LAA ratio were significantly correlated.
Results suggested that RF is a repeatable and reproducible variable for noninvasive quantitative evaluation of mitral valve regurgitation in awake dogs. Regurgitation fraction also correlated well with disease severity. It appears that this Doppler echocardiographic index may be useful in longitudinal studies of MVD in dogs.
To study the applicability of anatomical M-mode (AMM) for assessment of left ventricular (LV) size and function in horses, evaluate agreement with conventional M-mode (CMM), determine reliability, and establish reference intervals for AMM measurements.
98 horses; 13.1 +/- 5.6 years; 538 +/- 78 kg.
Two-dimensional and M-mode recordings were analyzed retrospectively. Standard LV dimensions and indices of LV function, including time intervals, were measured in CMM and compared with AMM studies in long-axis (lx) and short-axis (sx) views.
The percentages of measureable cycles were 99%, 97%, and 90% for routine LV studies in CMM(sx), AMM(sx), and AMM(lx) mode. For time intervals, >or= 93% of cycles could be measured using AMM compared to a maximum of 77% using CMM. AMM(sx) measurements agreed well with CMM(sx) measurements for LV studies; the agreement of AMM(lx) with CMM(sx) was markedly lower. The LV ejection time and the duration of electromechanical systole, but not the LV pre-ejection period and the index of myocardial performance, showed fair agreement between methods. Intraobserver and interobserver measurement variabilities were low for most variables.
AMM can replace CMM for assessment of LV dimensions in horses, but is not recommended for measurement of time intervals.
Seventeen striped dolphins (Stenella coeruleoalba) displaying swimming disorders compatible with neurological syndromes were investigated for Brucella infection. Sixteen dolphins had meningoencephalomyelitis. Serum antibody against Brucella antigen was detected in all 14 animals tested and Brucella ceti was isolated from eight out of nine animals. Brucella antigen was detected in the brain by immunofluorescence, but not by immunohistochemical labelling. By contrast, Brucella antigen was demonstrated by immunohistochemistry in the trophoblast of animals with severe placentitis and in the mitral valve of animals with myocarditis. The microscopical lesions observed in the tissues of the infected dolphins were similar to those of chronic brucellosis in man. The severity of brucellosis in S. coeruleoalba indicates that this dolphin species is highly susceptible to infection by B. ceti.
IT has been known for nearly a century that bradycardia is a common feature of the circulatory response to the asphyxia of diving. Heart slowing has been observed in a wide variety of both aquatic and terrestrial animals during submersion1-3. Several lines of evidence indicate that this bradycardia is associated with a decreased cardiac output and widespread arterial vasoconstriction with vastly diminished blood flow in viscera and skeletal muscle2,4,5. Flow in the cerebral and coronary circulations appears to be sustained.
To overcome the limitations of conventional M-mode echocardiography, a new technique referred to as anatomic M-mode has been recently developed. This technique is based on postprocessing of digitally acquired two-dimensional (2D) cineloops, and allows the operator to position one or multiple independent M-mode cursors freely on the 2D images. Initial clinical data show that anatomic M-mode can increase the reproducibility and accuracy of standard M-mode measurements of the left ventricle. Also, this quantitative technique has the potential to improve assessment of left ventricular wall motion and thickening, and could be particularly useful in providing objective measures during stress echocardiography.
To evaluate the accuracy of anatomic M-mode echocardiography (AMM).
Eight phantoms were rotated on a device at different insonation depths (IDs) in a water beaker. They were insonated with different transducer frequencies in fundamental imaging (FI) and second harmonic imaging (SHI), and the diameters were assessed with conventional M-mode echocardiography (CMM) and AMM with the applied angle correction (AC) after rotation. In addition, left ventricular wall dimensions were measured with CMM and AMM in FI and SHI in 10 volunteers.
AC had the greatest effect on the measurement error in AMM followed by ID (AC: R2 = 0. 295, ID: R2 = 0.268; P <.0001). SHI improved the accuracy, and a difference no longer existed between CMM and AMM with an AC up to 60 degrees. In vivo the limit of agreement between AMM and CMM was -1.7 to +1.8 mm in SHI.
Within its limitations (AC < 60 degrees; ID < 20 cm), AMM could be a robust tool in clinical practice.
The objective of this study was to determine intra- and inter-observer variability of echocardiographic measurements in awake cats. Four observers with different levels of experience in echocardiography performed 96 echocardiographic examinations in four cats on four different days over a 3-week period. The examinations were randomized and blinded. The maximum within-day and between-day CV values were 17.4 and 18.5% for inter-ventricular septal thickness in diastole, 18.7 and 22.6% for left ventricular free-wall thickness in diastole, 9.8 and 14.9% for left ventricular end-diastolic diameter, 20.8 and 15.2% for left ventricular end-systolic diameter, and 21.2 and 18.4% for left ventricular shortening fraction. The maximum within-day CV values were most often associated with the least competent observer (i.e. the graduate student) and, the minimum CV values with the most competent observer (i.e. the associate professor in cardiology). A significant interaction between cat and observer was also evidenced. Thus, the most competent observer could not be replaced by any of the other observers.
To determine left ventricular free wall (LVFW) motions and assess their intra- and interday variability via tissue Doppler imaging (TDI) in healthy awake and anesthetized dogs.
6 healthy adult Beagles.
n the first part of the study, 72 TDI examinations (36 radial and 36 longitudinal) were performed by the same observer on 4 days during a 2-week period in all dogs. In the second part, 3 dogs were anesthetized with isoflurane and vecuronium. Two measurements of each TDI parameter were made on 2 consecutive cardiac cycles when ventilation was transiently stopped. The TDI parameters included maximal systolic, early, and late diastolic LVFW velocities.
The LVFW velocities were significantly higher in the endocardial than in the epicardial layers and also significantly higher in the basal than in the mid-segments in systole, late diastole, and early diastole. The intraday coefficients of variation (CVs) for systole were 16.4% and 22%, and the interday CV values were 11.2% and 16.4% in the endocardial and epicardial layers, respectively. Isoflurane anesthesia significantly improved the intraday CV but induced a decrease in LVFW velocities, except late diastolic in endocardial layers and early diastolic in epicardial layers.
Left ventricular motion can be adequately quantified in dogs and can provide new noninvasive indices of myocardial function. General anesthesia improved repeatability of the procedure but cannot be recommended because it induces a decrease in myocardial velocities.
We sought to compare anatomic M-mode (AMM), a new echocardiographic postprocessing option, and conventional M-mode (CMM) using fundamental imaging and tissue harmonic imaging.
Transthoracic echocardiography was performed in 15 selected patients to analyze the reproducibility of AMM and in 47 patients to assess its clinical value versus CMM. Acquisitions were performed successively: CMM fundamental imaging; CMM tissue harmonic imaging; tissue harmonic imaging cineloops for AMM; and fundamental imaging cineloops for AMM. Quantitative analysis was performed offline. The angle alpha between the CMM line and the septal endocardial interface was calculated and the expected percentage of error in measuring left ventricular diameter was derived.
AMM analysis was reproducible. Optimal AMM full echocardiographic definition was obtainable in 77% of the population, whereas CMM was optimal for 49% because of scan line misalignment, causing a measurement overestimation exceeding 5%.
The ability with AMM to reduce the alpha angle to 0 degrees and, thus, avoid overestimation of left ventricular dimensions might improve follow-up in several pathologic conditions.
Anatomic M-mode (AMM) is an echocardiographic technique that is capable of generating M-mode studies from two-dimensional (2D) cine loops. Unlike conventional M-mode (CMM) whose scan line must lie along the axis of the ultrasound signal, AMM produces M-mode studies independent of the orientation of the ultrasound beam. We sought to determine the ability of AMM to measure cardiac dimensions in normal dogs and to assess the accuracy and variability of AMM and CMM vs. 2D measurements. Thirty-eight healthy dogs underwent physical exam and 2D, CMM, and AMM echocardiographic studies. The end-diastolic and end-systolic dimension of the left ventricle and the diameter of the left atrium (LAD) and aortic root were evaluated from the right parasternal short- and long-axis views. Results of the AMM and CMM study were compared with the 2D study via linear regression and calculation of a coefficient of correlation. AMM increased the level of correlation with both the left ventricular dimensions and LAD. Bland-Altman analysis revealed that AMM increased the level of agreement with 2D measurements and CMM greatly underestimated LAD vs. AMM. In healthy dogs, cardiac AMM measurements are associated with greater accuracy and less variability than CMM. AMM has the potential to improve quantification of cardiac dimensions.
Right ventricular myocardial (RVM) motion is poorly documented. The objective of this study was to determine the variability of RVM velocities by tissue Doppler imaging (TDI) in healthy dogs (study 1), to analyze RVM motion in a large healthy canine population (study 2), and to compare the results with those obtained for the left ventricular free wall. Six healthy Beagle Dogs were monitored in study 1, and 64 healthy dogs of 14 different breeds were monitored in study 2. Velocities were recorded in 2 segments (basal and apical) of the right and left myocardial walls. In study 1, 36 TDI examinations were performed for 4 days, whereas a single TDI examination was performed on each dog in study 2. All velocity profiles included 1 positive systolic wave and 2 negative diastolic waves. The lowest intraday and interday coefficient of variation values of the right TDI variables were observed at the base (3.5-16.1%). The variability of the right apical velocities was much higher, with most coefficient of variation values > 15%. RVM velocities were higher in the basal than in the apical segments (P < .001) and were higher than the left velocities of the corresponding segment (P < .01). Body weight and breed had an effect on only a few right and left TDI variables. TDI provides a repeatable and reproducible method for evaluating basal RV function in the dog. These data also demonstrate the heterogeneity of the myocardial velocities between the left and the right ventricles and between the base and the apex.
Strain (St) and strain rate (SR) imaging are new ultrasound modalities based on tissue Doppler imaging (TDI) that allow quantitative assessment of segmental myocardial contraction or stretching and rate of deformation, respectively.
Regional peak systolic St and SR could allow repeatable and reproducible assessment of systolic function of the right (RVW) and left (LVFW) myocardial walls in dogs.
Six healthy Beagle dogs were used to determine the repeatability and reproducibility of regional peak systolic St and SR in the RVW and LVFW (Study 1). These variables were also assessed in 30 healthy dogs of several breeds (Study 2).
Longitudinal peak systolic St and SR were recorded in 2 segments (basal and apical) of the RVW and LVFW. Radial peak systolic St and SR of the LVFW were also assessed.
All within- and most (7/10) between-day coefficients of variation were <15%. Absolute values of the longitudinal deformation indices were significantly higher (P < .001) in the RVW (St = -39.5 +/- 5.5% and SR = -5.2 +/- 0.8 s(-1) at the base; St = -36.3 +/- 4.3% and SR = -4.7 +/- 1.1 s(-1) at the apex) than in the LVFW. Absolute values were also higher for the radial (St = 62.9 +/- 10.4% and SR = 5.8 +/- 1.1 s(-1), P < .001) than for the longitudinal LFVW motions.
St and SR imaging is a repeatable and reproducible method for assessing systolic myocardial function. The combination of these indices with conventional echocardiographic variables may be useful for screening canine myocardial diseases.
The objective of this study was to determine the intra- and inter-observer variability of echocardiographic measurements in dogs. Four observers with different levels of experience in echocardiography performed 192 echocardiographic examinations of six dogs on four different days. The lowest within- and between-day coefficients of variation (CV) (%) were 13.8 and 5.2 for the right ventricle in diastole, 8.9 and 4.5 for the interventricular septal thickness in diastole (6.3 and 7.0 in systole), 7.7 and 9.4 for the left ventricular free-wall thickness in diastole (8.1 and 5.2 in systole), 3.1 and 5.0 for the left ventricular end-diastolic diameter (6.2 and 7.0 for end-systolic diameter), 10.2 and 10.8 for the left ventricular shortening fraction, and 8.2 and 9.8 for the left atrium/aorta ratio, respectively. Most of these lowest CVs were observed by the two most experienced observers. Conversely, all maximum values were obtained with the two less experienced observers. These differences in observer-dependent variability may considerably influence the minimum number of animals required to detect a treatment-associated change in echocardiographic variables.
Safe and effective echocardiography would represent a valuable tool for marine mammal veterinarians and physiologists evaluating the dolphin heart. Unfortunately, conventional ultrasound technology (transthoracic echocardiography) has been limited by logistic, anatomic, and behavioral challenges. Five mature male Atlantic bottlenose dolphins (Tursiops truncatus) were trained for echocardiographic imaging (four for both transthoracic and transesophageal imaging, and one for only transthoracic imaging). It was noted that transesophageal image quality transiently improved when the dolphins spontaneously exhaled. Subsequently, dolphins were conditioned to hold their breath following forced exhalation, and imaging proceeded during such behavioral breath holds. Over 25 transthoracic and 100 transesophageal echocardiographic studies were performed, including both two-dimensional imaging and color flow mapping. Transthoracic imaging yielded poor-quality images of only small portions of the heart. In contrast, transesophageal imaging, which improved dramatically with behavioral breath holding following exhalation, yielded consistently high-quality images of the entire heart (mitral, tricuspid, aortic, and pulmonary valves, atrial and ventricular septa, left and right atria, left and right ventricles, and ascending aorta and main pulmonary artery). Color flow mapping demonstrated mild tricuspid regurgitation in all dolphins, and mild aortic regurgitation in one dolphin found to have a pedunculated mass arising from the sinutubular junction just above the aortic valve. There were no complications in nonsedated dolphins. The heart of the bottlenose dolphin can be safely, effectively, and reproducibly evaluated with the use of transesophageal echocardiography in conjunction with behavioral breath holding following forced exhalation. This approach, and the normative echocardiographic data generated from this work, lays the foundation for future echocardiographic studies of cetaceans.
Two-dimensional (2D) speckle tracking echocardiography (STE) is a new angle-independent ultrasound technique based on tracking of speckles within the myocardium on 2D grayscale images. The aims of this prospective study were as follows: (1) to assess the variability of left ventricular peak systolic radial strain (St) and strain rate (SR) in awake dogs using STE (Protocol 1); and (2) to quantify these variables in a healthy canine population and compare them with tissue Doppler imaging (TDI)-based St and SR values (Protocol 2).
St and SR may be assessed using TDI, which is limited by angle dependency.
Thirty-six STE examinations were performed on 6 healthy dogs for Protocol 1 and 37 healthy dogs were recruited for Protocol 2. In both studies, STE measurements were obtained offline from the right parasternal short-axis view by the same trained observer using automatic frame-to-frame tracking of grayscale speckle patterns.
All within- and between-day coefficients of variation were <10% (Protocol 1). In Protocol 2, St (46.7+/-12.2%) and SR (2.7+/-0.6s(-1)) measured by STE were correlated with heart rate (p<0.01), but not with the ratio of early mitral inflow velocity to early mitral annular velocity. There was a good correlation between STE and TDI for both St and SR values (p<0.001).
STE is a repeatable and reproducible non-Doppler method for assessing radial St and SR. The combination of these indices with conventional echo-Doppler variables could provide a new approach for accurately quantifying canine systolic function.
Left ventricular (LV) torsional deformation plays an important role in myocardial function. However, it has never been assessed in the awake dog, because magnetic resonance imaging and sonomicrometry have been the only methods available so far.
Two dimensional speckle tracking echocardiography (STE), a new ultrasound imaging technique, provides a repeatable and reproducible noninvasive assessment of systolic LV wringing motion in the awake dog.
Six healthy dogs were used to determine the repeatability and reproducibility of STE variables (study 1). These variables also were prospectively assessed in a population of 35 healthy dogs (study 2).
Peak LV basal and apical systolic rotations were measured by STE from right parasternal short-axis views using automatic frame-to-frame tracking of gray-scale speckle patterns. Systolic LV torsion (LVtor, degrees ) was defined as apical rotation relative to the base.
All within-day and between-day coefficients of variation were <20% (6.8-18.0%). Amplitude of apical systolic rotation was significantly higher (P < .001) than the basal value (5.4 +/- 3.2 degrees and -3.1 +/- 1.3 degrees , respectively). Global LVtor was significantly correlated with systolic longitudinal LV myocardial velocity gradient assessed by tissue Doppler (P < .05), but not with either systolic radial LV myocardial velocity gradient or the ratio of early mitral inflow velocity to early mitral annular velocity (Em/Ea).
Speckle tracking echocardiography is a repeatable and reproducible method for assessing systolic LV torsional deformation. The combination of these new STE indices with tissue Doppler variables could provide a new approach for quantifying canine LV systolic function.
End-systolic volume index (ESVI) is a marker of systolic function, which can be assessed by the geometric (GM, based on Teichholz formula) or 2 planimetric methods (PM, Simpson's derived and length area methods).
Systolic dysfunction (SyD) may be observed in dogs with mitral valve disease (MVD) and is better assessed by PM than GM, which does not take into account the longitudinal left ventricular systolic shortening.
Six healthy dogs were used to determine the variability of the tested variables (Study 1). These variables were then prospectively assessed (Study 2) in 101 small breed dogs: 77 dogs with MVD and 24 healthy controls (CD).
ESVI was measured by GM and PM in awake dogs.
All within- and between-day coefficients of variation were <11% (Study 1). For Study 2, a nonlinear overestimation of ESVI was observed by GM compared with PM. PM-derived ESVI was significantly increased in ISACHC class 3 dogs compared with ISACHC class 1 dogs and exerted a significant influence on cardiac events at 5 months in dogs with MVD from ISACHC classes 2 and 3 (P < .05).
ESVI can be calculated by GM and PM with good repeatability and reproducibility. However, GM overestimates ESVI in a nonlinear way. Therefore, PM-derived ESVI should be preferred for the detection of SyD that is present at the late stages of the disease.
In: Dierauf LA, Gulland FMD, editors. CRC handbook of marine mammal medicine
- Van W Bonn
- E Jensen
- Ultrasonography
Brook F, Van Bonn W, Jensen E. Ultrasonography. In: Dierauf LA, Gulland FMD, editors. CRC handbook of marine mammal medicine. 2nd ed. Boca Raton: CRC Press; 2001. p. 596e597.
This study was presented at the 40th Annual Symposium of the European Association of Aquatic Mammals (EAAM, March 10th 2012) and received the " Outstanding Oral Communication by a student Award
- Asté
Asté dolphinarium. This study was presented at the 40th Annual Symposium of the European Association of Aquatic Mammals (EAAM, March 10th 2012) and received the " Outstanding Oral Communication by a student Award " (JL). The authors would like to thank the EAAM committee. References
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Gulland FMD, Lowenstine LJ, Spraker TR. Non infectious diseases. In: Dierauf LA, Gulland FMD, editors. CRC handbook of marine mammal medicine. 2nd ed. Boca Raton: CRC Press; 2001. p. 535e536.
CRC handbook of marine mammal medicine
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Gulland FMD, Lowenstine LJ, Spraker TR. Non infectious
diseases. In: Dierauf LA, Gulland FMD, editors. CRC
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CRC handbook of marine mammal medicine
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Brook F, Van Bonn W, Jensen E. Ultrasonography. In:
Dierauf LA, Gulland FMD, editors. CRC handbook of marine
mammal medicine. 2nd ed. Boca Raton: CRC Press; 2001. p.
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Non infectious diseases
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Quantitative assessment of regional right ventricular myocardial velocities in awake dogs by Doppler tissue imaging: repeatability, reproducibility, effect of body weight and breed, and comparison with left ventricular myocardial velocities
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