FETAL ECHOCARDIOGRAPHIC EVALUATION OF THE BOTTLENOSE DOLPHIN (TURSIOPS TRUNCATUS)

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1 Journal of Zoo and Wildlife Medicine 41(1): 35 43, 2010 Copyright 2010 by American Association of Zoo Veterinarians FETAL ECHOCARDIOGRAPHIC EVALUATION OF THE BOTTLENOSE DOLPHIN (TURSIOPS TRUNCATUS) Mark Sklansky, M.D., Michael Renner, D.V.M., Patricia Clough, Gregg Levine, D.V.M., Michelle Campbell, Rae Stone, D.V.M., Todd Schmitt, D.V.M., Ruey-Kang Chang, M.D., and Jayne Shannon-Rodriguez Abstract: 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. Key words: Bottlenose dolphin, fetal echocardiography, fetal heart, fetal ultrasound, Tursiops truncatus. INTRODUCTION Sonographic evaluation of the fetal heart, or fetal echocardiography, represents an exquisitely sensitive tool for the evaluation of the structural and functional integrity of the human cardiovascular system. The technique can evaluate for structural congenital heart disease 11,14 and provide a detailed assessment of overall fetal cardiovascular health and well-being, 1,4,9,18 identifying fetuses at risk for perinatal mortality secondary to fetal cardiovascular physiologic derangements 4,18 or placental pathology. 1 In From the Department of Pediatrics, Pediatric Cardiology, Mail Stop #34, Childrens Hospital Los Angeles, 4650 Sunset Boulevard, Los Angeles, California 90027, USA (Sklansky); Dolphin Research Center, Grassy Key, Florida 33050, USA (Clough, Renner, Shannon- Rodriguez); Dolphin Quest, Kona, Hawaii 96738, USA (Levine, Campbell); Dolphin Quest, Middleburg, Virginia 20118, USA (Stone); SeaWorld San Diego, San Diego, California 92109, USA (Schmitt); Department of Pediatrics, Harbor UCLA Medical Center, Torrance, California 90502, USA (Chang). Correspondence should be directed to Dr. Sklansky (msklansky@chla. usc.edu). contrast, echocardiographic evaluation of the fetal cetacean, limited by logistic, anatomic, and behavioral challenges, has not previously been described. Moreover, although ultrasound has been used to evaluate much of adult marine mammal extracardiac anatomy, only recently has the echocardiographic evaluation of the bottlenose dolphin (Tursiops truncatus) been described. 15 As a result, understanding of the fetal cetacean heart relies largely on examination of postmortem specimens, 3,13 in conjunction with limited invasive catheterization studies in adult dolphins. 17 Although, during adult life, the right (pulmonary) side of the four-chambered cetacean heart appears to be slightly larger 15 and at lower pressure 17 than the left (systemic) side, remarkably little data are available on the cardiovascular physiology of the fetal dolphin. It was hypothesized that application of fetal echocardiography to the bottlenose dolphin might dramatically improve the ability to evaluate the cardiovascular health of fetal cetaceans and that comparison with human fetal echocardiography might provide insights into the cetacean s specialized cardiovascular development. This report describes, for the first time, the safe 35

2 36 JOURNAL OF ZOO AND WILDLIFE MEDICINE Table 1. Subject site, gestational age, and outcome. Subject Site a Gestational age at evaluation (mo) Outcome 1 DRC 6/9 healthy calf 2 DRC 6.5/9.5 healthy calf 3 DRC 6.5/9.5 healthy calf 4 DRC 7/10 neonatal demise (inadequate lactation) 5 DRC 11 neonatal demise (birth during hurricane) 6 DQ 6 intrapartum demise (fetal size/lie) 7 SW 7.5/8 healthy calf 8 SW 7.5/8 healthy calf a DQ, Dolphin Quest, Kona, Hawaii; DRC, Dolphin Research Center, Grassy Key, Florida; SW, SeaWorld, San Diego, California. and effective application of fetal echocardiography to the bottlenose dolphin. MATERIALS AND METHODS Subjects Fetal echocardiographic evaluations were performed on eight singleton fetal dolphins between 6 and 11 mo gestation, corresponding to the second and third trimesters (average gestation, 12 mo). Six fetuses underwent two fetal echocardiographic evaluations each, four at 3-mo intervals and two at 0.5-mo intervals (Table 1). Five fetuses were evaluated at the Dolphin Research Center (Florida Keys, Florida), one fetus at Dolphin Quest (Hawaii), and two fetuses at SeaWorld (San Diego, California). All dolphins at the Dolphin Research Center and Dolphin Quest were Atlantic bottlenose dolphins (Tursiops truncatus truncatus). At Sea World, one fetus (subject 7) was seven-eighths Atlantic and one-eighth Pacific bottlenose dolphin (Truncatus truncatus gilli), and the other fetus (subject 8) was three-quarters Atlantic and one quarter Pacific bottlenose dolphin. Approval for this work was obtained from each center s research committee. Scanning logistics All imaging was performed without sedation, with the pregnant dolphins in the water, and using standard behavioral reinforcement techniques. Because of behavioral issues, one pregnant dolphin (subject 6) was scanned with the use of passive restraint. The other seven fetal dolphins each underwent two to three voluntary scanning sessions, lasting between 10 and 45 min each, at every gestational age examined. Scanning was performed dockside/poolside by a single, experienced fetal echocardiographer (MS) using conventional portable ultrasound systems (Cypress, Siemens Medical Systems, Mountain View, California 94039, USA, and GE Logic, GE Healthcare, Milwaukee, Wisconsin 53219, USA). A minority of fetal and maternal positions required scanning with the transducer submerged under water. Voluntary dolphin behaviors were trained through operant conditioning, rewarding desired behaviors with positive reinforcement. One or more animal trainers, in addition to a marine mammal veterinarian, monitored each pregnant dolphin s well-being throughout each imaging session, stabilizing the dolphin s fluke, pectoral fin, or both. Multiple transducers (Siemens, 3V2c and 4C1; GE, 4C-RS 2 6 MHz), probe positions, and maternal dolphin positions were used to optimize image quality. Use of the 4C1 curvilinear probe consistently generated problematic behavioral issues in the pregnant dolphins. The 3V2C and 4C-RS probes, in contrast, were consistently well tolerated. Optimal positioning of the pregnant dolphins, as expected, varied with fetal lie. In most cases, optimal visualization of the fetal heart could be obtained with the pregnant dolphin floating in either left or right lateral recumbency, ipsilateral to the occupied uterine horn. In general, the optimal window typically approximated the maternal umbilicus, on the lateral abdominal wall, somewhat anterior to the dorsal fin and inferior/caudal to the pectoral fin. In contrast, prone positioning placed the optimal window in an inaccessible position under the water. Supine positioning, with the pregnant dolphin apneic, did not improve visualization over lateral decubitus positioning. Scanning protocol and data analysis Fetal echocardiographic modalities used included two-dimensional imaging of the heart and great arteries, pulsed wave Doppler evaluation of the umbilical artery and vein, and color flow

3 SKLANSKY ET AL. FETAL ECHO OF BOTTLENOSE DOLPHIN 37 mapping of the heart, great arteries, and ductus arteriosus. Short (2 3 sec) video clips were digitally acquired, stored, and subsequently evaluated offline. The maximum annulus dimensions of the mitral, tricuspid, aortic, and pulmonary valves were measured multiple times, and the average measurements were recorded. To evaluate the relationships between valve annulus dimensions and gestational age, scatter diagrams were plotted with gestational age (Xaxis) and valve dimension (Y-axis). Paired Student s t-tests were performed to compare mitral and aortic valve dimensions (at each point in gestation) with the respective tricuspid and pulmonary valve dimensions. RESULTS Scanning limitations Although no complications occurred with any of the dolphins, a wide variety of imaging limitations was encountered. Most dolphins voluntarily allowed up to 3 5 min of uninterrupted scanning. The dolphin that, for behavioral reasons, required passive restraint did not hold still well enough to allow high-quality imaging. On the other hand, in the other seven dolphins, several other imaging limitations were partially overcome by changing the pregnant dolphin s position, eliciting various swimming/jumping activities meant to effect a change in fetal lie, or both. Early gestation fetuses (6 7 mo) were occasionally too deep ( cm) for adequate ultrasound penetration, a limitation frequently overcome with a change in fetal lie/position. Throughout gestation, but particularly earlier in gestation, the fetal pectoral fin (Fig. 1A), spine (Fig. 1B), or uterine horn membrane (Fig. 1C) occasionally interfered with effective imaging. Early scanning faced general resolution limitations, as well. Beyond 9 10 mo gestation, imaging of the heart became increasingly limited by diminishing amniotic fluid, decreasing fetal motion, and increasing density of fetal bony structures. Echocardiographic findings Fetal echocardiography consistently demonstrated absence of findings suggestive of pathology. The optimal window for evaluating the fetal heart was found to be between 8 and 9 mo gestation. At this gestational age, a complete fetal echocardiographic evaluation required min or longer, depending upon the fetal lie and the degree of maternal and fetal cooperation. The size of the heart between 6 and 7 mo gestation Figure 1. A. Sonographic image of thorax and pectoral fins of fetal dolphin at 6 mo gestation. B. Cross-sectional image of thorax of fetal dolphin at 6 mo gestation demonstrating anatomic barriers to effective imaging. C. Sagittal image of thorax of fetal dolphin at 6 mo gestation. Abbreviations: LV, left ventricle; RV, right ventricle; Membrane, dividing membrane between two uterine horns; PecFins, pectoral fins. limited the ability to evaluate the cardiac structure consistently and to make reproducible measurements of valve dimensions. As expected, fetal heart size increased with increasing gesta-

4 38 JOURNAL OF ZOO AND WILDLIFE MEDICINE Figure 2. Echocardiographic image of four-chamber view in fetal dolphin at 10 mo gestation. Abbreviations: LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle. tion. Between 6 and 9 mo gestation, the maximum fetal heart dimension varied from 3 to 6 cm, roughly equal to the number of months minus three; thus, the heart at 7 mo gestation measured 4 cm. At or beyond 10 mo gestation, the maximum fetal heart dimension varied between 8 and 9 cm, roughly equal to the number of months minus two. Every fetus was found to have a relatively symmetric four-chamber view during diastole (Fig. 2) and early systole (Fig. 3), with the left ventricle appearing more smooth-walled and globular than the relatively hypertrophied right ventricle. Serving as useful landmarks, the descending aorta and pulmonary veins could be Figure 3. Echocardiographic image of foramen ovale in fetal dolphin at 11 mo gestation. Note the presence of two pulmonary veins immediately posterior to the left atrium and the larger descending aorta further posteriorly. Abbreviations: as in Figure 1. Figure 4. Fetal echocardiographic image of flow across the foramen ovale (red flow from right atrium to left atrium) in fetal dolphin at 9 mo gestation. Abbreviations: LA, left atrium; LV, left ventricle; PFO, patent foramen ovale; PV, pulmonary vein; RA, right atrium; RV, right ventricle. seen consistently in cross-section behind the left atrium (Fig. 3). Right-to-left shunting across the foramen ovale (Fig. 4) could be seen readily from the four-chamber view with color flow Doppler. The mitral and tricuspid valves were consistently well seen in every fetus. The mitral valve was statistically smaller than the tricuspid valve throughout the gestational period studied (P, 0.01). Both tricuspid and mitral valve annulus dimensions demonstrated linear growth with gestational age (Fig. 5A, B). Interestingly, the fetus with one-quarter Pacific bottlenose genotype tended to have larger valve dimensions than the fetus with one-eighth Pacific bottlenose genotype. At later gestations, at least one pulmonary vein (Fig. 4), as well as the inferior vena cava, could be visualized. In three fetuses (subjects 1, 3, and 5, all at or beyond 9 mo gestation), color flow mapping demonstrated a trace amount of tricuspid regurgitation (Fig. 6). Evaluation of the outflow tracts required additional image manipulation. The aortic valve could be visualized from either the long axis (Fig. 7A) or short axis (Fig. 7B) views, and the pulmonary valve could be seen best from the short axis view (Fig. 7B). Average maximum measurements of the aortic and pulmonary valves (Fig. 8A, B) demonstrated linear growth, with the pulmonary valve dimensions measuring statistically larger than the respective aortic valve dimensions (P, 0.01). Limited visualization of the ductus arteriosus could be performed, and only with optimal windows.

5 SKLANSKY ET AL. FETAL ECHO OF BOTTLENOSE DOLPHIN 39 Figure 5. A. Scatter plot demonstrating relationship between tricuspid valve diameter and gestational age in the fetal dolphin. B. Scatter plot demonstrating relationship between mitral valve diameter and gestational age in the fetal dolphin. Pulsed Doppler evaluation of the free-floating umbilical artery demonstrated pulsatile systolic flow with continuous forward flow throughout diastole (Fig. 9A); evaluation of the free-floating umbilical vein demonstrated nonpulsatile continuous flow (Fig. 9B). DISCUSSION Cetacean fetal echocardiography might be expected to have both clinical and academic importance. From a clinical perspective, fetal echocardiography of bottlenose dolphins could substantially improve the power of routine surveillance ultrasounds to evaluate the overall well-being of routine pregnancies, as well as pregnancies considered to be at high risk. Whereas normative quantitative data for human fetal hearts have been well described, 12 this report represents the first such in vivo data for fetal cetaceans. In low-risk dolphin pregnancies, fetal echocardiography could be performed electively between 8 and 9 mo gestation. Extrapolating

6 40 JOURNAL OF ZOO AND WILDLIFE MEDICINE Figure 6. Echocardiographic image of trace tricuspid regurgitation (orange jet) in fetal dolphin at 9 mo gestation. Abbreviations: LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle; TR, tricuspid regurgitation. from data from human fetuses, ill or at-risk cetacean fetuses might be expected to have diminished ventricular systolic function, 4,11,14,18 pericardial effusion, 4,11,14,18 moderate or severe tricuspid regurgitation, 4,5,9,14 pulsatility in the umbilical vein, 4,18 or a combination of conditions. Moreover, whereas holodiastolic flow within the umbilical artery reflects low placental resistance and generally suggests absence of significant placental pathology, absent end-diastolic flow might suggest an increased risk for intrauterine growth retardation and fetal demise. 1 An additional clinical benefit to the application of fetal echocardiography to cetacean populations would be to increase the ability to detect forms of congenital heart disease currently going undiagnosed. The incidence of congenital heart disease in cetaceans remains unknown, likely in part because many affected fetuses might not come to term. Only two cases of congenital heart disease in bottlenose dolphins have been published. 2,8 In humans, congenital heart disease occurs in approximately eight of 1,000 live births. 14 Only routine fetal echocardiography could determine whether a similar incidence occurs in cetaceans. From an academic perspective, fetal echocardiographic evaluation of bottlenose dolphins and other cetaceans could provide important insights into comparative cardiac anatomy, physiology and morphogenesis. In humans, fetal echocardiography has not only demonstrated right heart dominance throughout gestation, 6,12,14 but has also allowed the study of fetal pulmonary/ systemic blood flows and resistance, gestational Figure 7. A. Echocardiographic image of left ventricular outflow tract in fetal dolphin at 11 mo gestation. B. Echocardiographic image of right ventricular outflow tract in fetal dolphin at 10 mo gestation. Abbreviations: AoV, aortic valve; LA, left atrium; LV, left ventricle; MPA, main pulmonary artery; PulmV, pulmonary valve; RV, right ventricle. age-dependent flow across the foramen ovale, and umbilical arterial/venous flow patterns. 6,18 Previous work in adult dolphins has demonstrated pressure 17 and size 15 ratios between right and left hearts similar to those seen in humans. Application of fetal echocardiography to cetaceans could lead to a greater understanding of how the cetacean s cardiovascular development contributes to the specialized cardiovascular adaptations found among adult marine mammals. For instance, although the fetal bottlenose dolphin shares the right-heart dominance seen in human fetuses, the fetal dolphin s right ventricle appears more hypertrophied than the right ventricle in the human fetus. This relative hypertrophy of the cetacean right ventricle might represent one adaptation common to marine mammals, conceivably in preparation for a subpulmonary

7 SKLANSKY ET AL. FETAL ECHO OF BOTTLENOSE DOLPHIN 41 Figure 8. A. Scatter plot demonstrating relationship between pulmonary valve diameter and gestational age in the fetal dolphin. B. Scatter plot demonstrating relationship between aortic valve diameter and gestational age in the fetal dolphin. ventricle with greater immediate postnatal demands than required by the human right ventricle. As another example, the pulmonary veins visualized in cross-section in the dolphin s four-chamber view appear longitudinally in the human fetal four-chamber view. This difference in pulmonary vein orientation might reflect important morphologic differences in pulmonary architecture between cetaceans and humans. Finally, the application of fetal echocardiography to cetaceans could deepen our understanding of the relationship between cardiac development and morphogenesis (ontogeny) and evolutionary differences among species in cardiac anatomy/physiology (phylogeny). Although postnatal echocardiography of cetaceans 15 and reptiles 16 has offered some insights into whether, in some ways, ontogeny might recapitulate phylogeny, fetal echocardiography has profound potential to illuminate the connection between cardiac development and comparative anatomy. In humans, for instance, a four-vessel umbilical cord (a normal finding in cetaceans) represents a highly unusual

8 42 JOURNAL OF ZOO AND WILDLIFE MEDICINE uterus. 10 Finally, this work did not include quantitative assessment of chamber size or ventricular systolic/diastolic function. Such data, although not critical to the routine fetal echocardiographic evaluation, 14 might be of potential clinical and academic interest and could be pursued in future studies. Despite these limitations, in this paper, we demonstrate the feasibility of safe and effective fetal echocardiography in the bottlenose dolphin and describe the correlation of fetal cardiac valve size with gestational age. Figure 9. A. Spectral Doppler display of umbilical artery flow within umbilical cord of fetal dolphin at 9 mo gestation. B. Spectral Doppler display of umbilical vein flow within umbilical cord of fetal dolphin at 8 mo gestation. Abbreviations: D, diastole; S, systole; Umb- Vein, umbilical vein. finding, frequently associated with fetal pathology. 7 Fetal echocardiography offers a unique window to study umbilical arterial and venous blood flow patterns which might, ultimately, shed light on why cetaceans have four umbilical vessels and humans only three. This preliminary work with cetacean fetal echocardiography has several limitations. First, the numbers are too small to generate complete and robust normative curves relating valve size to gestational age. Further studies will be needed to describe, with greater statistical power, morphologic differences between the human and cetacean fetal echocardiographic evaluation. Second, whereas human umbilical waveforms have been previously studied in detail, interpretation of cetacean umbilical waveforms could be complicated by anatomic and morphologic differences between the human and cetacean placenta and CONCLUSIONS Fetal echocardiography by commercially available equipment can safely and effectively evaluate the heart of the bottlenose dolphin, without the use of sedation. The combination of two-dimensional, color flow, and pulsed Doppler modalities enables a comprehensive evaluation of fetal cardiovascular structure and function, particularly between 8 and 9 mo gestation. Various behavioral and logistic maneuvers can overcome many imaging limitations. Other than the cetacean heart s larger size, relative hypertrophy of the right ventricle, and pulmonary vein orientation, no major difference was found between the cetacean fetal heart and published descriptions of the human fetal heart. Applied to cetaceans, fetal echocardiography has the potential to become an important clinical tool for the routine surveillance of cetacean pregnancies, evaluation of fetal cetaceans felt to be at risk for fetal distress or demise, or both. Further experience with this technique could increase our understanding of normal fetal cetacean cardiovascular development and physiology, provide additional insights into the specialized cardiovascular adaptations of diving mammals, and contribute to the field of comparative anatomy and physiology. Acknowledgments: The authors thank the Dolphin Research Center, Dolphin Quest Hawaii, Sea World San Diego, and Siemens Medical Systems for their support of this work, including Linda Erb, Cheryl Sullivan, Debbie Rose, Denise Cabrisas, and Lynne Albert (DRC), Carla Buczyna (DQ), and Valerie Maples, Robert Guttierez, and Hugo Amaya (SMS). LITERATURE CITED 1. Brar, H., and L. Platt Reverse end diastolic flow velocity on umbilical artery veclocimetry in high risk pregnancies: an ominous finding with adverse pregnancy outcome. Am. J. Obstet. Gynecol. 159:

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