Reproductive success is vital in sustaining free‐ranging and managed bottlenose dolphin ( Tursiops truncatus ) populations. Ultrasonography is an invaluable, non‐invasive tool in assessing the fetomaternal unit in humans and animals, including dolphins and horses. The purpose of this prospective longitudinal cohort study was to develop a protocol for fetomaternal ultrasonographic monitoring in dolphins and to report normal measurements and descriptive findings correlated with a positive outcome. From 2010 to 2017, serial ultrasonographic evaluations of 12 healthy dolphins were performed over the course of 16 pregnancies. A total of 203 ultrasound examinations were included in the study. Several metrics were accurate in predicting fetal age. Fetal biparietal diameter (BPD), thoracic width in dorsal and transverse planes, thoracic height in a sagittal plane, aortic diameter, and blubber thickness all demonstrated high correlation with gestational age ( r > 0.94, P < .00001). Regional uteroplacental thickness significantly increased with each trimester (range 0.22–0.40 cm; P < .00011 cranial uterus, P < .00057 mid, and P < .000011 caudal). Lung:liver mean pixel intensity was 2.57 ± 0.46 (95% confidence interval 2.47‐2.67). Ultrasonographic characteristics of normal pregnancy in dolphins are described and an equation for prediction of parturition date in Tursiops is reported: days to parturition = 348.16 − (26.03 × BPD(cm)) ( R 2 = 0.99). Future applications of these normal data will help identify in utero abnormalities indicative of fetal morbidity, and improve understanding of reproductive failure in wild and managed populations.

Abbreviations

BPD biparietal diameter CL corpus luteum MPI mean pixel intensity

1 INTRODUCTION Ultrasonographic evaluation of pregnancy is a valuable non‐invasive diagnostic tool, well‐established in human and veterinary medicine. Advances in human perinatology and maternal‐fetal medicine have been shown to reduce morbidity and mortality,1 and fetomaternal ultrasound is vital in monitoring pregnancy and maximizing neonatal survival.2 Ultrasound is also a critical component of bottlenose dolphin (Tursiops truncatus) medicine and has been used to evaluate pulmonary, cardiac, reproductive, hepatic, urinary, gastrointestinal, and lymphatic systems.3-9 Improved understanding of Tursiops reproduction through routine imaging in managed care has led to advancements in hormone analysis, gonadal evaluation, early fetal monitoring, parturition prediction, and artificial insemination.10-13 This knowledge has successfully been applied to longitudinal investigations of wild dolphin health.14 A number of congenital lesions have been reported in Tursiops fetuses and perinates, including umbilical cord abnormalities,15, 16 vascular pathologies,17 and cranial malformations.18 Following the “Deepwater Horizon” oil spill in 2010, wild dolphins living in oil‐impacted bays had increased prevalence of perinatal mortality, fetal distress, fetal pneumonia,19 and maternal pulmonary disease and reproductive failure.20-22 The most likely cause of these abnormalities was exposure to petroleum products.23, 24 To better understand these findings, promptly diagnose congenital anomalies, and enhance early detection of pregnancy complications in wild and managed dolphins, there is a need for more sophisticated assessments of fetoplacental health. Standard ultrasonographic methodology to assess fetal growth and well‐being has been established in humans and horses.25-28 Such evaluations help predict positive versus negative outcomes and aid in understanding of pregnancy failure pathophysiology.29-31 As dolphins and horses have a similar diffuse epitheliochorial placenta, equine reproductive ultrasound provides a reasonable species model.32, 33 The purpose of this study was to expand upon early work in gestational ultrasound of bottlenose dolphins34-36 and develop a reproducible protocol for ultrasonographic monitoring of fetomaternal health in the bottlenose dolphin, reporting normal measurements and descriptive findings correlating with a positive outcome.

3 RESULTS A total of 203 ultrasound examinations were included in the study. Every dam had a single fetus. Up to 70 ultrasonographic parameters were assessed in each scan (see Supporting Information 1 for a sample datasheet). Nine dams had a left ovarian CL and seven had a right ovarian CL. Maximum CL diameter was 3.7 cm and did not vary based on laterality (right: 2.5‐3.7 cm, left: 2.1‐3.6 cm). A central anechoic cavity was seen in five of 16 corpora lutea; others were solid. Static cyst‐like structures (0.9‐3.5 cm diameter) were noted on the gravid‐side ovary in four pregnancies of four animals. Cyst laterality was split evenly in these pregnancies. One animal had two cyst‐like structures. Other cysts were single. Transient follicles (>3) were detected in the non‐CL ovary in three examinations of two dams; both occurred during the first half of gestation. The uteroplacental unit had a thin layered wall. There was a significant increase in thickness over the course of gestation in all three locations (Table 1). Maximum was 5.6 mm, noted in the caudal region on day 357 of one pregnancy. No abnormalities suggestive of uteroplacental disruption were found. In four dams, echogenic internal roughening was noted, with/without frond‐like projections that extended ≤1 cm into the lumen (Figure 4B). Table 1. Regional thickness of the uteroplacental unit (UPU) based on trimester UPU (cm) First trimester Second trimester Third Trimester P‐value Cranial 0.22−0.31 (8) 0.26−0.29 (37) 0.30−0.32 (50) <.00011* Mid ND 0.29−0.33 (22) 0.33 −0.37 (38) <.00057* Caudal 0.27−0.31 (7) 0.30−0.33 (33) 0.36 − 0.40 (45) <.000011* Uterine fluid assessment was possible in 148 of 203 examinations. Allantoic and amniotic fluid were distinguishable in 100 of 148 examinations. In all 100, the allantoic fluid was anechoic (Figure 3B). Amniotic fluid was identified in 102 examinations. It was diffusely hyperechoic in 24% (49/102). Echogenic FFPs were only seen in amniotic fluid (25% or 26/102) in the second half of gestation (Figure 4A). Maximum recorded amniotic depth (10.4 ± 2.67 cm) exceeded allantoic (7.8 ± 2.67 cm). Fluid depths did not correlate with gestation. The umbilical cord was examined in 64% (n = 130). Four umbilical vessels (two arteries and two veins)40 were identified in all examinations where the umbilical vasculature was seen in cross section (48%; Figure 5A,B). In this same subset of examinations, the urachus was identified in 46 studies. Cord diameter near the fetal insertion was measured in 77 of 130 cord examinations (Figure 5C). Minimum and maximum cord diameter were 1.1 cm (day 95) and 3.9 cm (day 364). Cord diameter was positively correlated with gestational age (R2 = 0.7). In three exams of two animals, a loose, ball‐shaped coil was identified in the cord (Figure 5D) that resolved by the following evaluation. Fetal activity was characterizable in 122 exams. It was frequent in 62%, rare in 32%, and continuous in 6%. One cursory exam performed on gestational day 377 of 380 did not record fetal movement. Fetal BPD, thoracic width, thoracic height, aortic diameter, and blubber thickness all demonstrated very high correlation with gestational age (R2 > 0.94) (Figure 11). Table 2 serves as a clinical reference for normal growth progression of fetal structures. The strongest linear correlation (P = .00001) with actual fetal age was BPD. Days until parturition = 348.16 – (26.03 × BPD) is a highly accurate equation of prospective fetal age determination (BPD in cm) (R2 = 0.99). The earliest and latest possible gestational age for six fetal measurements is outlined in Table 3. Fetal eye dimensions were less tightly correlated to gestational age than other parameters (R2 = 0.77, n = 48). Figure 11 Open in figure viewer PowerPoint Six ultrasonographic parameters of fetal growth based on day of gestation and trimester [Color figure can be viewed at wileyonlinelibrary.com Table 2. Mean fetal ultrasonographic measurements based on month and trimester Fetal Measurements First trimester (days 0–126) Second trimester (days 127–253) Third trimester (days 254–380) Pre‐birth Month → n 2 3 4 5 6 7 8 9 10 11 12 13 Aortic diameter (cm) 72 0.02−0.13 0.13−0.23 0.23−0.34 0.34−0.45 0.45−0.56 0.56−0.67 0.67−0.77 0.77−0.88 0.88−0.99 0.99−1.10 1.10−1.21 1.21−1.31 Biparietal diameter (cm) 117 0.01−1.13 1.13−2.27 2.27−3.42 3.42−4.56 4.56−5.70 5.70−6.84 6.84−7.99 7.99−9.13 9.13−10.27 10.27−11.42 11.42−12.56 12.56−13.70 Thorax width dorsal (cm) 45 0.0−1.43 1.43−2.99 2.99−4.55 4.55−6.11 6.11−7.67 7.67−9.23 9.23−10.79 10.79−12.35 12.35−13.91 13.91−15.47 15.47−17.03 17.03−18.59 Thorax height (cm) 84 0.97−2.42 2.42−3.97 3.97−5.32 5.32−6.77 6.77−8.22 8.22−9.67 9.67−11.12 11.12−12.56 12.56−14.01 14.01−15.46 15.46−16.91 16.91−18.36 Blubber thickness (cm) 102 0.17−0.20 0.20−0.24 0.24−0.29 0.29−0.34 0.34−0.40 0.40−0.48 0.48−0.57 0.57−0.67 0.67−0.80 0.80−0.95 0.95−1.13 1.13−1.34 Table 3. Determination of earliest and latest possible gestational day based on fetal measurements Fetal Measurement (x) Earliest Possible Gestational Day (y) Latest Possible Gestational Day (y) Aortic Diameter y = 278.95 x − 24.74 y = 281.82 x + 63.00 Biparietal diameter y = 23.92 x + 31.21 y = 26.82 x + 41.26 Thorax width (dorsal) y = 16.09 x + 35.64 y = 19.51 x + 61.31 Thorax width (trans) y = 15.35 x + 43.93 y = 14.53 x + 137.08 Thorax height y = 15.82 x + 28.38 y = 20.95 x + 39.07 Blubber thickness y = 211.21 x + 26.28 y = 236.11 x + 140.50 Fetal cardiac motion was recorded in 86% of examinations. Heart rate was normally distributed and had a strong negative correlation with gestation, decreasing during pregnancy. Maximum and minimum rates were 188 bpm (day 120) and 76 bpm (day 379). Rate varied with gestational age as follows: 1st trimester, difficult to assess; 2nd trimester, 118‐173 bpm (n = 34); 3rd trimester, 76‐154 bpm (n = 95); last 2 weeks of gestation, 76‐125 bpm (n = 40). The earliest cardiac movement was recorded opportunistically on day 70 during a monthly examination. Cardiac flicker was seen in two first‐trimester examinations. Correlations were not found between fetal heart rate and activity, nor between activity and gestational age. Fluid was seen in the fetal forestomach in 42% of exams and the urinary bladder in 41%. The genitalia became conspicuous in 12 of 16 pregnancies; these 12 fetuses were all correctly sexed ultrasonographically (six male, six female). Fetal lung was discernible from liver as early as day 112, and in all fetuses by the end of the first trimester. Fetal lung was subjectively and objectively hyperechoic to fetal liver (lung MPI = 87.9 ± 14.5 [95% confidence interval 84.7‐91.1], liver MPI = 35.7 ± 7.1 [95% confidence interval 34.1‐37.2]). Mean pixel intensity did not correlate to gestational age. Lung:liver MPI ratio was 2.57 ± 0.46 (95% confidence interval 2.47‐2.67), denoting lung was roughly twice as echogenic as liver. The lung was nearly always homogeneous in echotexture (92%). No anomalies or focal lesions of the fetal calvarium, eyes, blubber, heart, aorta, lungs, pleural space, peritoneum, liver, forestomach, kidneys, urinary bladder, or genitalia were noted.

4 DISCUSSION This study describes a standardized ultrasound method for obtaining in utero measurements in dolphins. The reference values detailed in these 16 successful pregnancies are reproducible, critical to ultrasonographic assessment of pregnant dolphins, and may facilitate early diagnosis of abnormalities. The highly accurate updated13 equation presented in this publication for determining gestational age based on biparietal diameter (days to parturition = 348.16 − (26.03 × BPD)) aids in timing of clinical decisions as parturition approaches. We also demonstrate that several other fetal measurements (thorax, aorta, blubber) are practical indices to obtain and are highly accurate in predicting gestational age (Figure 11). New equations define the earliest and latest possible day of gestation for each of six fetal measurements (Table 3). In addition, the medical illustrations of dolphin fetomaternal anatomy (Figure 10) presented in this study correlate to ultrasound and further understanding of spatial relationships. Based on our review of the literature, this study is the first report of the normal ultrasonographic appearance of the Tursiops uteroplacental unit. We report normal reference ranges and demonstrate that regional uteroplacental thickness significantly increases with each trimester. Disruption of uteroplacental contact, a sign of placentitis and abnormal separation of the allantochorion from the endometrium,41 was not seen in any of the successful pregnancies in this study. Significant deviations from the values reported here may aid in diagnosis of placentitis or other placental pathology. A thorough ultrasonographic evaluation of the reproductive tract should routinely be performed in sexually mature female dolphins. Identification and monitoring of ovarian corpora, developing follicles, and cyst‐like lesions establishes baseline "normal" for each animal, detects reproductive cycle changes, and facilitates diagnosis of pregnancy and pathology. Echogenic particles in amniotic fluid of human fetuses can indicate the presence of vernix (shedding of fetal skin), and are also seen in the allantoic fluid of healthy pregnant mares within 10 days of foaling.29 Echogenic particles or FFPs may indicate pathology and fetal stress when associated with infective debris, blood, or meconium;42 however, increased fluid echogenicity is not always predictive of pathology.43 No allantoic FFPs were seen in our study, but they were noted in amniotic fluid. In light of the successful pregnancy outcomes for these animals and lack of findings indicative of fetal stress, we suspect the presence of amniotic echogenic particles may be normal in Tursiops. The finding of a ball‐shaped umbilical coil in three of 203 exams in our study was surprising, given the focally convoluted appearance of the cord and the clinical concern warranted for antenatal diagnosis of true umbilical knots in humans39 and neonatal dolphins.44 A convoluted or tangled cord, sometimes found in monoamniotic twin pregnancies in women, was not anticipated in this species (twins are exceedingly rare in dolphins). Given that both coils resolved and no clinical sequela resulted, we suggest this may be transiently seen in a normal pregnancy. Some limitations in our study are worth noting. The sample size (16 pregnancies) is relatively low, but this should be weighed against the complexity involved in gaining longitudinal access to long‐lived, legislatively protected animals such as dolphins and the value of acquiring such data.45 Behavioral cooperation of the dam and position of the fetus during any given exam dictated the quality/quantity of data acquired. In addition, the retrospective nature of image analysis despite prospective image acquisition necessarily resulted in dataset truncation. Not all measurements could be derived from each set of captured cine and still images. With regards to fetal fluid depths, for example, measurements obtained may not have represented the deepest fluid pocket present but rather the greatest depth captured in the exam. The lack of correlation between fetal fluid depth and gestational age in dolphins has also been found in horses.31 Clinically, it may be of most value to note the fluid characteristics reported and maximum fluid depths seen in these successful pregnancies. Retrospective assessment of fetal movement was also limiting. The inability to evaluate activity in 40% of exams was likely due to inadequate recording of fetal motion during abbreviated scans. Further, the compression of the fetus by the fusiform, hydrodynamic abdomen of a cetacean, also limits its movement near term. Anecdotally, fetal movement was always seen in first or second trimester evaluations. In general, a multifactorial approach is essential to pregnancy evaluation, as a single abnormal parameter may not be representative of the overall clinical state. Lack of fetal movement during a given exam may not indicate lack of fetal viability, however if coupled with undetectable cardiac motion, abnormalities in organ conspicuity or echogenicity, or abnormal fetometrics, this likely represent fetal loss.46 The simplified use of MPI as a proxy for echogenicity47 rather than gray level histogram width assessment48 or gray‐scale histogram49 is also a limitation. However, the importance of establishing the normal ultrasonographic appearance of fetal dolphin lung and its relationship to liver in light of recent reports of fetal pneumonia and perinatal mortality in wild dolphins necessitated straightforward, reproducible methodology. Dolphin fetal lung in our study, as expected, was largely homogeneous and hyperechoic to liver due to a high density of fluid‐tissue interfaces.2 Lung lesions in human fetuses are generally echogenic.50, 51 None were seen in this study. Normal fetal lung and lung/liver echogenicity relationships facilitate early diagnosis of congenital bronchopulmonary abnormalities in human fetuses, and a decrease in the fetal lung:liver echogenicity has been shown to predict respiratory distress in newborns.49 As such, comparison of fetal dolphin lung:liver MPI to values published here could enable future identification of respiratory pathology in perinatal dolphins. In conclusion, this study provides reference information on the qualitative and quantitative fetomaternal ultrasonographic findings associated with successful pregnancy in bottlenose dolphins. This information may be used as the basis for identifying gestational abnormalities in managed populations and their wild counterparts. Lesions affecting morbidity or mortality of the fetus or dam detected early could result in timely therapeutic intervention for animals in human care, and findings indicating reproductive failure in the wild may be elucidated. Additional studies are needed to corroborate our findings, expand the dataset, and apply these diagnostic imaging methods to other cetacean species of interest.

LIST OF AUTHOR CONTRIBUTIONS Category 1 (a) Conception and Design: Ivančić, Smith, Gomez, Meegan

Conception and Design: Ivančić, Smith, Gomez, Meegan (b) Acquisition of Data: Ivančić, Smith, Gomez, Meegan, Musser, Cárdenas Llerenas, Waitt

Acquisition of Data: Ivančić, Smith, Gomez, Meegan, Musser, Cárdenas Llerenas, Waitt (c) Analysis and Interpretation of Data: Ivančić, Smith, Gomez, Meegan, Musser, Barratclough, Jensen Category 2 (a) Drafting the Article: Ivančić, Musser, Barratclough

Drafting the Article: Ivančić, Musser, Barratclough (b) Revising Article for Intellectual Content: Ivančić, Smith, Gomez, Meegan, Musser, Barratclough, Jensen, Cárdenas Llerenas, Waitt Category 3 (a) Final Approval of the Completed Article: Ivančić, Smith, Gomez, Meegan, Musser, Barratclough, Jensen, Cárdenas Llerenas, Waitt

ACKNOWLEDGMENTS The authors thank Veronica Cendejas for her technical expertise and dedication in creating the anatomical drawings in this text. This research was made possible by a grant from The Gulf of Mexico Research Initiative (GoMRI), Award SA 16–17. Data are publicly available through the Gulf of Mexico Research Initiative Information & Data Cooperative (GRIIDC) at https://data.gulfresearchinitiative.org (https://doi.org/10.7266/n7-eydg-cy02).

CONFLICT OF INTEREST The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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