Pregnancy and Fetal Development: Cephalic Presentation and Other

animals

Article Pregnancy and Fetal Development: Cephalic Presentation and Other Descriptive Ultrasonographic Findings from Clinically Healthy Bottlenose Dolphins (Tursiops truncatus) under Human Care

Pietro Saviano 1, Letizia Fiorucci 2,*, Francesco Grande 1, Roberto Macrelli 3 , Alessandro Troisi 4, Angela Polisca 5 and Riccardo Orlandi 6

1 Ambulatorio Veterinario Saviano-Larocca, 41042 Spezzano, Italy; [email protected] (P.S.); [email protected] (F.G.) 2 Facultad de Veterinaria, Universidad de Las Palmas de Gran Canaria, Arucas, 35416 Las Palmas de Gran Canaria, Spain 3 Dipartimento di Scienze Pure e Applicate, Università di Urbino, 61029 Urbino, Italy; [email protected] 4 Scuola di Bioscienze e Medicina Veterinaria, Università di Camerino, 62024 Matelica, Italy; [email protected] 5 Dipartimento di Medicina Veterinaria, Università di Perugia, 06124 Perugia, Italy; [email protected] 6 Tyrus Veterinary Clinic, 05100 Terni, Italy; [email protected] * Correspondence: letiziaﬁ[email protected]

Received: 3 April 2020; Accepted: 22 May 2020; Published: 24 May 2020

Simple Summary: Ultrasound data are vital for monitoring and detecting problems in pregnancies, and although there is a significant amount of data for domestic species, data for marine mammals are scarce. In domestic species, the use of ultrasonography to monitor a pregnancy usually has the following aims: fetal movements, fetal heart rates, measurements of the skull and the thorax for the prediction of the birth date interval, the morphological aspects of the fetal organs, the appearance of the umbilical cord, and the placentation. The purpose of this study is to provide to the clinician additional relevant data on fetal development and well-being during a dolphin pregnancy that may also be useful for wild population monitoring. This study is the result of a retrospective analysis of 192 ultrasound scans over 10 years that, for the first time, describes the sonographic findings of the bottlenose dolphin organogenesis and their correlation with the stage of pregnancy, as well as the calf presentation at birth, according to its position within the uterus, and moreover a complete literature review.

Abstract: Ultrasonography is widely used in veterinary medicine for the diagnosis of pregnancy, and can also be used to monitor abnormal pregnancies, embryonic resorption, or fetal abortion. Ultrasonography plays an important role in modern-day cetacean preventative medicine because it is a non-invasive technique, it is safe for both patient and operator, and it can be performed routinely using trained responses that enable medical procedures. Reproductive success is an important aspect of dolphin population health, as it is an indicator of the future trajectory of the population. The aim of this study is to provide additional relevant data on feto-maternal ultrasonographic monitoring in bottlenose dolphin (Tursiops truncatus) species, for both the clinicians and for in situ population studies. From 2009 to 2019, serial ultrasonographic exams of 11 healthy bottlenose dolphin females kept under human care were evaluated over the course of 16 pregnancies. A total of 192 ultrasound exams were included in the study. For the first time, the sonographic findings of the bottlenose dolphin organogenesis and their correlation with the stage of pregnancy are described. Furthermore, this is the first report that forecasts the cephalic presentation of the calf at birth, according to its position within the uterus.

Animals 2020, 10, 908; doi:10.3390/ani10050908 www.mdpi.com/journal/animals Animals 2020, 10, 908 2 of 16

Keywords: ultrasonography; bottlenose dolphins (Tursiops truncatus); normal appearance; pregnancy; fetal position; fetal development; fetal welfare

1. Introduction A preventative medicine program is one of the key factors in health evaluation for ensuring the welfare of dolphins under human care. Ultrasonography (US) plays an important role in modern-day cetacean preventative medicine because it is a non-invasive technique, it is safe for both patient and operator, and it can be performed routinely using trained responses that enable medical procedures [1,2]. Ultrasound data are vital for monitoring and detecting problems in pregnancies, and while there is a significant amount of data for domestic species [3–11], data for marine mammals are scarce [12–14]. In domestic species, US is often used for the early identification of fetal pathologies and reabsorption, for example, altered/slowed down growth, loss of fetal fluids with decrease in the size of the vesicle and alteration of its shape, absence of heartbeat, blurring of margins and alteration of normal fetal anatomy, and detachment of the placenta from the uterine wall [3–11]. In marine mammal medicine, in the past, US was used only to confirm a pregnancy; however, an increasing number of facilities are now monitoring gestation by US, and further studies are now emerging with additional reference ranges. Reproductive success is an important aspect of dolphin population health, as it is an indicator of the future trajectory of the population [15,16]. Pregnancy determination for wild dolphins, including differentiation of pregnancy stage, is possible during capture–release health assessments through application of diagnostic ultrasound to evaluate fetal development and viability, estimate gestational age, and measure anatomical structures [15,16]. The use of ultrasound for systematic pregnancy determination provides a useful tool for measuring an important component of reproductive success. Application of this approach for conservation of wild populations benefits from the establishment of baseline values, such as the estimates provided herein for the reference population of bottlenose dolphins [15–22]. The brightness (B)-mode technique is based on a process in which focused beams are iteratively sent into the body and the received waves are used to form an image scan-line, covering line-by-line the region of interest. The use of it to monitor bottlenose dolphin pregnancy dates back to the early 1990s, when Williamson et al. (1990) diagnosed pregnancy in a limited number of subjects (n = 4), at approximately the fourth month of gestation, being able to visualize fetal movements and fluids. Periodic monitoring allowed the authors to observe fetal vitality through the observation of cardiac mechanics and to carry out measurements both of the cranial diameter (on the front–occipital axis) and of the thoracic diameter, obtaining linear growth diagrams [23]. The authors showed difficulties in obtaining clear ultrasonographic images, both because the dolphins were uncooperative during the exam due to scarce training, and due to the features of the transducers used at the time. As regards the positioning of the transducer in the first months of gestation, the midline between the genital opening and the navel was used as a landmark, obtaining images in cross section, whereas in late pregnancy the probe was placed longitudinally, at 10–20 cm from the ventral midline [23]. Stone et al. (1999) observed a similar pattern of linear growth in bottlenose dolphins by measuring bi-parietal and thoracic fetal diameters from week 46 up to 1 week after delivery [24]. Lacave in her work developed an easy-to-use computer program to provide better birth prediction for regularly scanned dolphins during their gestation and to predict dolphin delivery dates, even with only one or two ultrasound scans of their animals [25]. Measurement of bi-parietal diameters is only possible when the head is distinguishable from the rest of the body; the head is presented ultrasonographically as a symmetrical ovoid structure and the bi-parietal diameters are measured where they reach their maximum amplitude [25]. For thoracic diameters, Lacave et al. (2004) used, as a reference point, the section where all cardiac chambers, symmetrically surrounded by the lungs, appear in the ultrasound image in the same section and where pectoral fins are also frequently visible. Lacave Animals 2020, 10, 908 3 of 16 showed how the diameters of the skull increase more slowly than the thoracic diameters, and that the thoracic diameters represent the limit of accuracy in late pregnancy [25]. Sklansky et al. (2010) recognize the utility of fetal echocardiography as a safe technique able to evaluate the cardiovascular system in the bottlenose dolphin, especially in the period between the eighth and the ninth month of gestation. As in humans, this technique allows us to identify congenital cardiac anomalies [26–28] and to identify the possible causes of perinatal mortality risk associated with physiological abnormalities and cardiac hemodynamics [29,30]. In most cases, the optimal visualization of the fetal heart is obtained by positioning the pregnant female in lateral decubitus, homolaterally to the uterine horn in which the fetus is housed [26,27]. The optimal window is located near the maternal navel, proximal to the dorsal and caudal–ventral fin compared to the caudal fin. As expected, the cardiac dimensions increased with the approaching birth; passing from 3 to 6 cm of the 9th month up to 8–9 cm of the 10th month [26,27]. Recently, Ivancic et al. (2020) developed a protocol for feto-maternal ultrasonographic monitoring in bottlenose dolphins. In their work, a total of 203 US exams were performed during a 7-year period to monitor 16 pregnancies. The authors reported normal measurements and descriptive findings correlated with a positive outcome—fetal bi-parietal diameter, thoracic width in dorsal and transverse planes, thoracic height in a sagittal plane, aortic diameter, and blubber thickness all demonstrated a high correlation with date of gestation [21]. Umbilical cord accidents were diagnosed in the same dolphin in three consecutive pregnancies in a study by García-Párraga et al. (2014). The trans-abdominal ultrasound evaluation revealed the presence of a wrap of the umbilical cord around the fetal peduncle. All pregnancies ended in in utero death of fetuses and their expulsion [31]. In addition, an omphalocele (an abdominal wall defect at the base of the umbilical cord) in an approximately 16-week-old fetus was detected in the clinical case reported by Smith et al. (2013), thanks to the US prenatal examination. Color Doppler was utilized to study the blood flow within the omphalocele, as well as diagnose an associated anomaly of the umbilical cord, which contained three vessels instead of four [32]. Finally, the case of meconium aspiration syndrome (MAS) in a male neonate of bottlenose dolphin who died immediately after birth was reported by Tanaka et al. In 2014. At necropsy, a knot was found in the umbilical cord [33]. The lungs showed diffuse intra-alveolar edema, hyperemic congestion, and atelectasis due to meconium aspiration with mild inflammatory cell infiltration. Although the exact cause of MAS in this case was unknown, fetal hypoxia due possibly to the umbilical knot might have been associated with MAS, which is the first report in dolphins. MAS due to perinatal asphyxia should be taken into account as a possible cause of neonatal mortality and stillbirth of dolphin calves [33]. Valuable information about the ontogeny of the body systems and their development in cetacean species, the precise time intervals of such developments, and any distinctive growth trajectories are basically unknown, because descriptions are based on occasional recoveries of embryos and fetuses and it very difficult to acquire complete ontogenic series [34–39]. Fetal abnormalities have been observed in cetaceans, as in other species. Brook, in 1994, for the first time, described the ultrasound diagnosis of an anencephaly, a lethal form of cephalic axial skeletal-neuronal disraphism, in a Tursiops aduncus fetus [34]. The fetal skull base appeared disproportionately small, and the cranium could not be identified. Fetal heart motion could be detected throughout the gestation. After a period of 357 days after conception, an uncomplicated, spontaneous delivery produced a stillborn male anencephalic calf. The abnormality was associated with various factors including respiratory tract infection in early gestation and folic acid deficiency. This case illustrates the ability of US to provide assessment of fetal morphology and growth, information not available by other means [34]. Ultrasonography proved to be appropriate to monitor fetal development, placenta and fetal membranes, and also to identify umbilical cord defects, thanks to the monitoring of its position and perfusion. This method has the advantage that it can identify abnormalities at early gestational stages [21,34], and that regular measurements allow monitoring of fetal growth and provide a more accurate prediction of expected delivery [25], so that adequate arrangements can be made in good time. The aim of this retrospective study was Animals 2020, 10, 908 4 of 16 to provide additional relevant descriptive findings on feto-maternal ultrasonographic monitoring protocols in bottlenose dolphin species, thanks to the analysis of 192 ultrasound exams during a 10-year period. The data obtained may be very useful for the future clinical practice for managed population and in situ population studies, as it can be used to improve the understanding of the pathophysiology of reproductive failure.

2. Materials and Methods

2.1. Study Animals This study is the result of a retrospective analysis of 192 ultrasound scans obtained during the routine pregnancy check of bottlenose dolphins and from the marine mammal ultrasound consulting work in 11 different facilities over 10 years. All the examinations were included in the preventative medicine protocol of each facility, and no additional examinations were performed. From 2009 to 2019, serial ultrasonographic exams of 11 healthy bottlenose dolphin females (average age: 18 7 years; ± min–max: 9 to 36 years) kept under human care were evaluated over the course of 16 pregnancies. Three dams were pluriparous. The calves born were 10 males and 6 females; of these, 2 females died 9 days after birth due to a respiratory disease. Neither case was that of cephalic birth. Inclusion criteria involved all pregnancies ended with the birth of alive calves that survived at least 48 h after the delivery. Exclusion criteria included any pre-existing health conditions that could have affected pregnancy, abortion, or any significant health conditions that occurred during pregnancy and required initiation of treatment by the attending clinician. A total of 192 ultrasound exams were included in the study. All examined animals were trained routinely for medical behaviors, including US. Lateral and ventral abdominal scanning was performed using seawater for acoustic coupling. For the study, the urogenital area was explored and the reproductive organs, ovaries, and uterus were evaluated. Uterine fluid echogenicity was assessed as anechoic, hypo-echoic, or hyper-echoic, as well as for the presence of echoic free-floating particles. Umbilical cord vasculature was assessed in cross section. Color Doppler confirmed vascular flow.

2.2. Ultrasonography: Instrumentation and Methodology A portable SonoSite 180 Plus with a 2–5 MHz convex probe, an Esaote Mylab 25 gold with convex probe 2–5 MHz, an Aloka 900 with convex probe 2–5 MHz, and a General Electrics Logiq V2 with a 2–5 MHz curvilinear transducer were used to evaluate the dolphins during pregnancy. During the exam, the machine was covered with a transparent plastic bag to avoid accidental contact of the device with salt water. To avoid direct sunlight, a dark-colored bag was used to cover the instrument. The probe was waterproof. Acoustic gel was unnecessary because water provides an excellent medium through which to conduct ultrasound waves. Still images obtained were stored in DICOM (Digital Imaging and Communications in Medicine) format, whereas videos were also recorded using an external hard-disk.

2.3. Statistical Analyses For each fetus, the time required for the appearance of each organ studied was analyzed. The quantitative variable “organ onset time” was introduced and a descriptive statistic expressed—average organ onset time with relative standard deviation. In addition, left or right ovulation rates and podalic or cephalic birth rates were analyzed. Medcalc, version 11.6.0.0, was used to analyze the data.

3. Results Considering the 16 pregnancies, the percentage of ovulation in the left ovary was 68.75%, whereas the ovulation in the right ovary was 31.25%, and it was of interest that two dams always ovulated in the right ovary. Maximum corpus luteum (CL) longitudinal diameter was 3.63 cm and transversal Animals 2020, 10, 908 5 of 16 diameter was 3.02 cm (Figure1), even though the diameter may vary according to laterality. The results concerningAnimals 2020, 10 the, x gonadal activity correspond to previous studies [18–20]. 5 of 16

Figure 1. Corpus luteum (CL) with measurements.

The embryonic vesicle waswas recognizablerecognizable atat 2929 ± 3 days post-ovulation on the apex of the uterine ± horn as a roundishroundish structurestructure withwith anan averageaverage diameterdiameter ofof 1.211.21 cmcm withwith anan anechoicanechoic content.content. In the following week,week, it it was was possible possible to recognizeto recognize the embryothe embryo inside inside it as an it elongated as an elongated hyperechoic hyperechoic structure (Figurestructure2). (Figure 2).

Figure 2. (a) The embryonic vesicle at 38 2 days post-ovulation appeared as a roundish structure Figure 2. (a) The embryonic vesicle at 38 ± 2 days post-ovulation appeared as a roundish structure withFigure an 2. anechoic (a) The embryonic content and vesicle a hyper-echoic at 38 ± 2 days structure post-ovulation inside (V), appeared under the as CL. a roundish (b) At 52 structure3 days, with an anechoic content and a hyper-echoic structure inside (V), under the CL. (b) At 52 ± 3 days, thewith embryo an anechoic was perfectly content recognizableand a hyper-echo (E). ic structure inside (V), under the CL. (b) At 52 ± 3 days, the embryo was perfectly recognizable (E). The distinction between head and trunk was visible starting from 68 5 days after ovulation. ± FromThe the distinction 216 5 days between of gestation, head measurementsand trunk was startedvisible tostarting be hard from to realize 68 ± 5 withdays accuracy. after ovulation. In fact, ± inFrom the the evaluations 216 ± 5 days after of this gestation, last period, measurements the position star/orientationted to be hard of the to fetus realize and with its sizeaccuracy. meant In that fact, it wasin the not evaluations possible to after take this reliable last period, measurements the position/orientation thereafter. Starting of the from fetus 68 and5 daysits size after meant ovulation, that it ± thewas embryonic not possible cardiac to take mechanisms reliable measurements were displayed ther aseafter. a point Starting of maximum from 68 ﬂuctuation± 5 days after of theovulation, echoes. Thethe embryonic heart rate wascardiac measured mechanisms because were the displayed cardiac mechanics as a point becameof maximum visible fluctuation and remained of the constant echoes. betweenThe heart 155 rate and was 198 measured bpm until because the ninth the monthcardiac of mechanics pregnancy became (Figure visible3). During and remained the last 3 months,constant itbetween stabilized 155 atand 140 198 bpm, bpm to until reach the 85 ninth5 bpmmonth in of the pr lastegnancy 2 weeks (Figure of gestation. 3). DuringThe the last ﬁrst 3 abdominal months, it ± organsstabilized tobe at visualized140 bpm, to were reach the 85 stomach ± 5 bpm and in the the last urinary 2 weeks bladder of gestation. (98 3 and The 110 first 2abdominal days of gestation, organs ± ± to be visualized were the stomach and the urinary bladder (98 ± 3 and 110 ± 2 days of gestation, respectively), which appeared as distinct and anechoic cavities. It was also possible to recognize the eye as an anechoic cavitary structure (Figure 4). Animals 2020, 10, 908 6 of 16

Animals 2020, 10, x 6 of 16 Animalsrespectively), 2020, 10, x which appeared as distinct and anechoic cavities. It was also possible to recognize6 of 16 the eye as an anechoic cavitary structure (Figure4).

Figure 3. ((a)) The distinction between head and trunk was clear betweenbetween 6868 ± 5, up to the 216 ± 55 day Figure 3. (a) The distinction between head and trunk was clear between 68 ± 5,± up to the 216 ± 5 ±day of gestation. It allowed measurement of bi-parietal diameters. The The umbilical umbilical cord cord was already easy of gestation.guessed (as It indicated allowed measurement by yellow arrow). of bi-parietal ( b)) The The fetal fetaldiameters. heart heart rate rateThe (HR) (HR) umbilical was was measured measured cord was once already the easycardiac guessedmechanics (as indicated became by visible. yellow arrow). (b) The fetal heart rate (HR) was measured once the cardiac mechanics became visible.

Figure 4.4. FetalFetal stomach stomach (S) (S) and and fetal fetal urinary urinary bladder bladder (B) were (B) thewere first the abdominal first abdominal organs to organs be visualized, to be Figurevisualized,and appeared4. Fetal and stomach as appeared distinct (S) andasand distinct anechoic fetal andurinary cavities. anechoic bladder The cavities. heart (B) were wasThe recognizableheartthe first was abdominalrecognizable as an anechoic organs as an cavity anechoicto be (H) visualized,cavityand the (H) embryonicand and appeared the embryonic cardiac as distinct mechanics cardiac and wereanechoicmechanics displayed ca vities.were as displayed The a point heart of wasas maximum a recognizablepoint of fluctuation maximum as an of anechoic fluctuation the echoes. cavityTheof the (H) eye echoes. and appeared the The embryonic aseye an appeared anechoic cardiac as cavitary anmechanics anechoic structure were cavitary (E).displayed structure as a(E). point of maximum fluctuation of the echoes. The eye appeared as an anechoic cavitary structure (E). The distinctiondistinction betweenbetween thorax thorax and and abdomen abdomen and, and, thus, thus, the the presence presence of the of diaphragm,the diaphragm, was seenwas seenatThe 92 at distinction 592 days ± 5 days of gestation. between of gestation. Athorax clear A clearand distinction abdomendistinct betweenion and, between thus, lungs lungsthe and presence liver and wasliver of identified was the identifieddiaphragm, at 112 at 112was5 days, ± 5 ± ± seenwhereasdays, at 92 whereas ± the5 days ribs the of were ribsgestation. visible were atvisibleA 153clear at distinct5 153 days. ± ion5 At days. 167between At3 days167 lungs ± of 3 gestation,daysand liver of gestation, was the dorsal identified the fin dorsal was at 112 visible fin ± was5 as ± ± days,visiblea hyper-echoic whereas as a hyper-echoicthe triangular ribs were structuretriangularvisible at in153 structure the ± dorsal5 days. in portion theAt 167dorsal of± the3 daysportion trunk. of gestation,of During the trunk. the the same Duringdorsal period, fin the was itsame was visiblepossibleperiod, as ait to hyper-echoicwas identify possible the totriangular teeth. identify Even thestructure if teeth. the umbilical Evenin the if dorsalthe cord umbilical was portion easily cord of guessed the was trunk. easily previously During guessed (asthe previously shown same in period,(asFigure shown it3 was) from in possible Figure the 119 to3) identifyfrom6 day the of the 119 gestation, teeth. ± 6 day Even it of was ifgestation, the clear umbilical as ait hyper-echoicwas cord clear was as easily cordoniforma hyper-echoic guessed structure, previously cordoniform and it ± (aswasstructure, shown important in and Figure it to was evaluate3) fromimportant the the 119 internal to evaluate± 6 day vascular of the gestation, internal components vascularit was and clear components the as absence a hyper-echoic ofand knots the absence orcordoniform torsions of knots until structure,orthe torsions birth and (Figure untilit was 5the ).important Furthermore, birth (Figure to evaluate it5). was Furthermore, the possible internal to notice,itvascular was possible between components to the notice, 149th and between daythe absence and the of 230th149th knots dayday or andoftorsions gestation, the 230thuntil that theday thebirth of eyegestation, (Figure was open, 5).th atFurthermore, the the lens eye waswas visible,itopen, was possiblethe and lens eyelid wato notice,s movements visible, between and were eyelid the also 149th movements detectable day and(Figurewere the also230th6). detectable day of gestation, (Figure 6). th at the eye was open, the lens was visible, and eyelid movements were also detectable (Figure 6). Animals 2020, 10, x 7 of 16 Animals 2020, 10, x 908 77 of of 16 16

Figure 5. ((aa)) From From day day 119 119 ± 66 of of gestation, gestation, the the umbilical umbilical cord cord was was clear clear as as a a hyper-echoic hyper-echoic cordoniform cordoniform Figure 5. (a) From day 119 ±± 6 of gestation, the umbilical cord was clear as a hyper-echoic cordoniform structure. ( b) The The small small hypo-echoic hypo-echoic central cavity is the urachus, shown by the yellow circle. ( (c)) Color Color structure. (b) The small hypo-echoic central cavity is the urachus, shown by the yellow circle. (c) Color Doppler demonstratingdemonstrating ﬂowflow within within the the umbilical umbilical vasculature: vasculature: (d ()d the) the umbilical umbilical veins veins (in (in blue), blue), and and the Doppler demonstrating flow within the umbilical vasculature: (d) the umbilical veins (in blue), and theumbilical umbilical arteries arteries (in red).(in red). the umbilical arteries (in red).

Figure 6. From day 110 2 of gestation, the eye appeared as anechoic cavitary structures, whereas ± Figurestarting 6. from From the day 149th 110 day,± 2 of the gestation, lens was alsothe eye visible. appeared At 167 as anechoic3 days of cavitary gestation, structures, it was possible whereas to ± Figurestartingidentify 6. from the From teeth. the day 149th 110 day,± 2 ofthe gestation, lens was thealso eye visible. appeared At 167 as ± anechoic 3 days of cavitary gestation, structures, it was possible whereas to startingidentify fromthe teeth. the 149th day, the lens was also visible. At 167 ± 3 days of gestation, it was possible to identifyThe intestine the teeth. was visualized at 189 5 days of gestation. The cardiac chambers were visualized ± 194 The5 days intestine after ovulation,was visualized and afterat 189 about ± 5 days 3 weeks, of gestation. the vascular The cardiac structures chambers (aorta andwere caudal visualized vena ± 194cava) ±The 5 departing days intestine after from ovulation,was them visualized were and visualized afterat 189 about ± 5 days (Figure 3 weeks, of7 gestation.). the vascular The cardiacstructures chambers (aorta andwere caudal visualized vena 194cava) ± 5departing days after from ovulation, them were and visualizedafter about (Figure 3 weeks, 7). the vascular structures (aorta and caudal vena cava) departing from them were visualized (Figure 7). AnimalsAnimals 20202020, 10,, x10 , 908 8 of 816 of 16 Animals 2020, 10, x 8 of 16

FigureFigure 7. Fetal 7. Fetal spinal spinal cord cord (Sp), (Sp), stomach stomach (S), (S),liver liver (Li) (Li),, intestine intestine (I), (I),lungs lungs (Lu), (Lu), and and heart heart (H) (H)were were visualizedFigurevisualized 7.at Fetal194 at 194± spinal5 days5 days cordof gestation. of (Sp), gestation. stomach (S), liver (Li), intestine (I), lungs (Lu), and heart (H) were visualized at 194 ± 5 days of gestation. FromFrom the the230th 230th day day of gestation, of gestation, it was it waspossible possible to observe to observe a ventral a ventral flexion ﬂexion of the of caudal the caudal fin, a ﬁn, hyper-echoica hyper-echoicFrom structure,the 230th structure, dayin contact of in gestation, contact with withthe it wasabdomen. the possible abdomen. From to Fromobserve the 245th the a 245th ventralto the to 288th flexion the 288th day of of daythe gestation, caudal of gestation, fin, it a washyper-echoicit possible was possible to recognizestructure, to recognize thein contact thyroid the thyroid with and the andthymus abdomen. thymus (Figure (Figure From 8). During8the). During245th the to thelast the last 3288th months 3 months day of of gestation, ofgestation, gestation, it it waswasit was possible possible possible to to identify torecognize identify the the kidneys. kidneys.thyroid and thymus (Figure 8). During the last 3 months of gestation, it was possible to identify the kidneys.

Figure 8. (a) At 245 2 to 288 2 days of gestation, it was possible to recognize the thyroid (T) and (b) ± ± Figurethe thymus,8. (a) At respectively.245 ± 2 to 288 ± 2 days of gestation, it was possible to recognize the thyroid (T) and (b) Figurethe thymus, 8. (a) respectively.At 245 ± 2 to 288 ± 2 days of gestation, it was possible to recognize the thyroid (T) and (Inb) addition,the thymus, it respectively. was possible to identify the genitalia and sex the fetus (males have a tri-lobed structure,In addition, whereas it was females possible have to anidentify apricot-shaped the genitalia structure, and sex as shownthe fetus in (m Figureales9 have). a tri-lobed structure,In whereasaddition, females it was possiblehave an apricot-shto identifyaped the structure,genitalia andas shown sex the in fetusFigure (m 9).ales have a tri-lobed structure, whereas females have an apricot-shaped structure, as shown in Figure 9). Animals 2020, 10, x 9 of 16 AnimalsAnimals 20202020, 10, 10, x, 908 9 of9 of16 16

Figure 9. ((aa)) An An ultrasound ultrasound image image of of female female genitals genitals (a (apricot-shaped)pricot-shaped) by by SonoSite SonoSite 180 180 Plus Plus with with a 2– a Figure 9. (a) An ultrasound image of female genitals (apricot-shaped) by SonoSite 180 Plus with a 2– 2–55 MHz MHz convex convex probe; probe; (b ()b the) the same same area area caught caught with with General General Electrics Electrics Logiq Logiq V2, V2, with with a a 2–5 MHz 5 MHz convex probe; (b) the same area caught with General Electrics Logiq V2, with a 2–5 MHz convex probe. convex probe. Starting fromfrom thethe 301th 301th post-ovulation post-ovulation day day until until the the end end of pregnancy, of pregnancy, it is veryit is diveryfficult difficult to obtain to Starting from the 301th post-ovulation day until the end of pregnancy, it is very difficult to imagesobtain images of the caudalof the portionscaudal portions of the fetus, of the due fetus, to the due folded to the position folded itposition assumes it withinassumes the within maternal the obtain images of the caudal portions of the fetus, due to the folded position it assumes within the uterus.maternal To uterus. the authors’ To the knowledge,authors’ knowledge, this is the this first is study the first that reportsstudy that the reports sonographic the sonographic descriptive maternal uterus. To the authors’ knowledge, this is the first study that reports the sonographic findingsdescriptive of thefindings bottlenose of the dolphin bottlen organogenesisose dolphin organogenesis and their correlation and their with correlation the stage with of pregnancy the stage asof descriptive findings of the bottlenose dolphin organogenesis and their correlation with the stage of shownpregnancy in Figure as shown 10). in Figure 10). pregnancy as shown in Figure 10).

Figure 10. BottlenoseBottlenose dolphin dolphin ( (TursiopsTursiops truncatus ) organogenesis timeline table. Figure 10. Bottlenose dolphin (Tursiops truncatus) organogenesis timeline table. Allantoic andand amniotic amniotic fluid fluid are are distinguishable distinguishable in all in examinations all examinations starting starting from the from last trimesterthe last Allantoic and amniotic fluid are distinguishable in all examinations starting from the last oftrimester pregnancy. of pregnancy. Allantoic Allantoic fluid appears fluid appears as an anechoic as an anechoic fluidand fluid the and amniotic the amniotic fluid fluid appears appears as a trimester of pregnancy. Allantoic fluid appears as an anechoic fluid and the amniotic fluid appears hyper-echoicas a hyper-echoic fluid, fluid, with anwith increasing an increasing number number of echoic of particles echoic particles during the during last period the last of pregnancyperiod of as a hyper-echoic fluid, with an increasing number of echoic particles during the last period of (Figurepregnancy 11). (Figure 11). pregnancy (Figure 11). Animals 2020, 10, 908 10 of 16 AnimalsAnimals 20202020,, 1010,, xx 1010 ofof 1616

FigureFigure 11. 11. (((aa))) AllantoicAllantoic (A)(A) andand amnioticamniotic fluidfluid fluid (B (B)(B)) in in early early stage stage of of pregnancy.pregnancy. (( bb)) Allantoic Allantoic fluid fluidfluid appearsappears as as an an anechoic anechoic fluid fluidfluid (A), (A), and and the the amniotic amniotic fluid fluidfluid appears appears as as a a hyper- hyper-echoichyper-echoicechoic fluid, fluid,fluid, with with an an increasingincreasing amount amount of of echoic echoic particles particles duringduduringring the the lastlast periodperiod ofof pregnancypregnancy (B).(B).

Finally,Finally, fetalfetal positionposition waswas was evaluatedevaluated evaluated throughoutthroughout throughout thethe the gestationalgestational gestational period.period. period. ConsideringConsidering Considering thethe the1616 pregnancies,16pregnancies, pregnancies, thethe thepercentagepercentage percentage ofof flukefluke of ﬂuke presentationpresentation presentation waswas was 93.75%,93.75%, 93.75%, whwhereasereas whereas thethe headhead the head presentationpresentation presentation waswas 6.25%.was6.25%. 6.25%. ItIt isis interestinginteresting It is interesting toto notenote to notethatthat thattheythey they werewere were allall successfulsuccessful all successful cephaliccephalic cephalic deliveries.deliveries. deliveries. AccordingAccording According toto thethe to resultstheresults results ofof thethe of thepresentpresent present study,study, study, itit isis it ispossiblepossible possible toto to predictpredict predict thethe the calfcalf calf presentationpresentation presentation atat at birth,birth, birth, consideringconsidering its its positionposition in in the the uterus uterus during during the the la lastlastst trimester. trimester. Using Using the the CL CL asas aa referefe reference,rence,rence, if if thethe fetusfetus skullskull isis locatedlocated closeclose to to the the CL, CL, it it will will have have a a podalic podalic position position atat birthbirth (Figure(Figure 1212).12).).

(a) (b) Figure 12. (a) Using the CL as a reference, if the fetus skull is located close to the CL, it will have a podalicFigure 12.position(a) Using at birth, the CL(b) as shown a reference, in the if image. the fetus skull is located close to the CL, it will have a podalicFigure 12. position (a) Using at birth, the CL (b) as showna reference, in the if image. the fetus skull is located close to the CL, it will have a However,podalic position if the at tail birth, fluke (b )is as located shown closein the toimage. the CL, it will have a cephalic position at birth (Figure 13). However, if the tail ﬂuke is located close to the CL, it will have a cephalic position at birth (FigureToHowever, 13the). authors’ if the tail knowledge, fluke is located the present close to studythe CL, reports it will havethe firsta cephalic bottlenose position dolphin at birth cephalic (Figure presentation13). documented by US. To the authors’ knowledge, the present study reports the first bottlenose dolphin cephalic presentation documented by US. AnimalsAnimals2020 2020, 10, 10, 908, x 1111 ofof 1616

(a) (b)

Figure 13. (a) Using the CL as a reference, if the tail fluke (TF) is located close to the CL, it will have a cephalicFigure 13. position (a) Using at birth, the CL (b )as as a shown reference, in the if the image. tail fluke (TF) is located close to the CL, it will have a cephalic position at birth, (b) as shown in the image. To the authors’ knowledge, the present study reports the first bottlenose dolphin cephalic presentation4. Discussion documented by US. In the present retrospective study, we describe, by US, the genesis of the fetal organs (stomach, 4. Discussion bladder, lungs, eye, intestine) and structures (column, appendices), and note that their appearance can Inbe theused present to estimate retrospective the gestational study, weperiod describe, in the by dolphin, US, the in genesis the absence of the of fetal further organs information, (stomach, bladder,as described lungs, in eye,other intestine) species [40]. and structuresTo the authors’ (column, knowledge, appendices), this is and the notefirst thatstudy their that appearance reports the cansonographic be used to descriptive estimate the findings gestational of bottlenose period in dolphin the dolphin, organogenesis in the absence and their of further correlation information, with the asstage described of pregnancy. in other The species heart, [40 while]. To theappearing authors’ visi knowledge,ble already this in isthe the early first stages study of that gestation, reports was the sonographicdisplayed optimally descriptive between findings the of eighth bottlenose and the dolphin ninth organogenesismonth, allowing and exclusion their correlation of the presence with the of stagedetectable of pregnancy. pathologies. The As heart, reported while by appearing Sedmera et visible al. (2003), already there in is the a scant early amount stages of of gestation,data regarding was displayedheart development optimally in between cetaceans the eighth[41]. In andtheir the stud ninthy, the month, authors allowing examined exclusion samples of the from presence a unique of detectablecollection pathologies.of embryonic As dolphin reported specimens by Sedmera macroscopically et al. (2003), there and is histologically a scant amount to oflearn data more regarding about heartnormal development cardiac development in cetaceans in the [41]. spotted In their dolphin. study, theIt was authors found examined that during samples the spotted from adolphin’s unique collection280 days of of gestation, embryonic the dolphin heart completes specimens septation macroscopically at about 35 and days. histologically However, substantial to learn more trabecular about normalcompaction, cardiac which development normally inoccurs the spotted in terrestrial dolphin. mammals, It wasfound as well that as in during humans the at spotted around dolphin’s the same 280time days period, of gestation, was delayed the heart until completes day 60, when septation corona at aboutry circulation 35 days. However,became established. substantial By trabecular day 80, compaction,however, the which heart normallygained a compacted, occurs in terrestrial characteristic mammals, shape, as with well a as single in humans apex [41]. at around Considering the same the timebottlenose period, dolphin’s was delayed 385 days until dayof gestation, 60, when around coronary 68 circulation± 5 days after became ovulation, established. the results By day of 80,the however,present study the heart show gained how the a compacted, heart was recognizable characteristic by shape, US, and with the a single embryonic apex [cardiac41]. Considering mechanics thewere bottlenose displayed dolphin’s as a point 385 of daysmaximum of gestation, fluctuation around of the 68 echoes.5 days An after accurate ovulation, index the of resultsfetus welfare of the ± presentis the fetal study heart show rate how[26–30]. the The heart heart was rate recognizable was measured by US, once and the the cardiac embryonic mechanics cardiac became mechanics visible, wereand displayedremained asconstant a point between of maximum 155 and fluctuation 198 bpm of un thetil echoes. the ninth An month accurate of index pregnancy. of fetus During welfare the is thelast fetal 3 months, heart rate it stabilized [26–30]. Theat 140 heart bpm, rate to wasreach measured 85 ± 5 bpm once in the the last cardiac 2 weeks mechanics of gestation. became visible, and remainedThe distinction constant between between thorax 155 and and 198 abdomen bpm until and, the ninththus, the month presence of pregnancy. of the diaphragm, During the lastwas 3seen months, between it stabilized 92 ± 5 days at 140 of bpm, gestation, to reach whereas 85 5 bpmthe clear in the distinction last 2 weeks between of gestation. lungs and liver was ± identifiedThe distinction at 112 ± 5 betweendays of gestation. thorax and The abdomen correlation and, between thus, the thepresence dolphin fetus of the organogenesis diaphragm, was and seenthe gestational between 92 period5 days may of gestation,help the clinician whereas to the identify clear distinction the proximity between of the lungs delivery and liver and wasany ± identifiedsuspected at anomaly 112 5 days in fetal of gestation. development. The correlation In huma betweenn literature, the dolphin normal fetus fetal organogenesis lung and lung/liver and the ± gestationalechogenicity period relationships may help facilitate the clinician early to diagnosi identifys theof congenital proximity ofbronchopulmonary the delivery and anyabnormalities, suspected anomalyand a decrease in fetal in development. the fetal lung/liver In human echogenicity literature, has normal been shown fetal lung to predict and lung respiratory/liver echogenicity distress in relationshipsnewborns [21]. facilitate Regarding early the diagnosis two ca oflves congenital who died bronchopulmonary 9 days after birth abnormalities, due to a respiratory and a decrease disease, inthere the fetalare no lung data/liver that echogenicity suggested lung has alteration been shown identified to predict by ultrasound respiratory throughout distress in newbornsthe pregnancy. [21]. RegardingThe two clinical the two cases calves had who fetal died development 9 days after birthcomparable due to awith respiratory the other disease, fetuses there examined. are no data The thatnecropsy suggested of both lung calves alteration revealed identified postpartum by ultrasound pathological throughout process as the cause pregnancy. of death. The two clinical Animals 2020, 10, 908 12 of 16 cases had fetal development comparable with the other fetuses examined. The necropsy of both calves revealed postpartum pathological process as cause of death. The umbilical cord has also been documented in ultrasound images, thus being able to exclude the presence of twisting nodes or echographically detectable defects of the same conditions that proved fatal in the bottlenose dolphins [31–33]. Color Doppler may be utilized to study the blood flow, as well as diagnose an associated anomaly of the umbilical cord, such as in the case of an onphalocele that contained three vessels instead of four [32]. It is worth mentioning that four vessels are needed as normal presentation, as shown in Figure4. As described in other animals such as horses [42], during the last 20 days of pregnancy, amniotic fluid shows an increase of the echogenicity compared with allantoic fluid. Allantoic fluid has to remain completely anechoic during all the stages of pregnancy. Amniotic and allantoic fluids increase their volume during pregnancy, giving protection to the fetus. At the time of birth, allantoic fluid dilates the pathways of birth, whereas amniotic fluid has the function of lubricating the fetus [43]. The allantoic liquid is composed mainly of the urine of the fetus and has an anechoic aspect throughout the pregnancy [43]. The amniotic fluid envelops the fetus, and epithelial cells and meconium are accumulated in it, causing a progressive increase of its echogenicity compared to the allantoic liquid [43]. As confirmed by the present study, the difference in echogenicity between these two liquids reaches the highest level in the last 20 days of pregnancy because of the increase in the number of echogenic particles in the amniotic fluid. The authors hypothesize that these observations could be used to determine the approximate date of birth. Echogenic particles may indicate pathology and fetal stress when associated with infective debris, however, increased fluid echogenicity is not always predictive of pathology [21]. In all pregnancies evaluated in the present study, no changes in the allantoic fluid were observed throughout the gestation period. The results of the present study agree with recently published data by Ivancic et al. (2020) and provide further support for the newly established reference ranges [21]. Fetal position is another aspect to evaluate throughout gestation. In fact, keeping both the CL and the fetus in the same scan, it is possible to evaluate how, during the last three months of pregnancy, the relative topographical position of the head to the CL of the fetus is related to a podalic presentation at birth, which is the normal condition in this species (93.75% of cases). On the contrary, when the tail of the fetus is topographically related to the CL in the same image, it is related to a cephalic birth (6.25%). It is known that a cetacean fetus can change its position in the uterus during pregnancy, but usually moves into a tail-first position by the last months [36,44]. Head presentation of a fetus is reported for several delphinid species, and is thought to be an adaptation to the pregnancy and delivery in the aquatic environment [19,44–53]. It is repeatedly observed under human care—the rate of cephalic delivery was found to be 7% in killer whales (Orcinus orca) and 1.2% in bottlenose dolphins [19]. The labor of eight captive finless porpoises (Neophocaena asiaeorientalis asiaeorientalis and Neophocaena asiaeorientalis sunameri) were described by Deng et al. In 2019. The duration of parturition and the time of particular events in the parturition process were recorded for both podalic and cephalic births. Cephalic births were shorter than podalic births, and the calf position at birth did not seem to have a negative effect on its survival [47]. Successful cephalic delivery in a bottlenose dolphin under human care is described in detail by Essapian (1963)—stage 2 of the parturition lasted 22 min [49]. The result corresponds to that of the present study; in fact, the cephalic birth mentioned here also had a duration of 22 min. Even if it is indicated as a complicating factor, such cases are not referred to as pathologies [49]. In the wild, head presentation of a fetus was observed in a stranded dead white-beaked dolphin (Lagenorhynchus albirostris)[50]; it had ruptured the uterine wall, and thus the head presentation could be a cause of both dystocia and maternal death [50]. Numerous cases of cephalic delivery have been consistently reported for belugas (Delphinapterus leucas) in both nature and under human care; a rate of head-first delivery in captive belugas of 14% has been reported [51–53]. Head presentation seems to be more widespread in belugas than in any cetacean species. The cephalic presentation of a fetus in cetaceans is not a pathology but a natural variation. This idea is also supported Animals 2020, 10, 908 13 of 16 by the documented cases of successful cephalic deliveries and by the occurrence of head presentations both in the wild and under human care. Nonetheless, head presentation of a fetus in a small cetacean can increase the risk of trauma or dystocia. In addition, a change of presentation at an advanced stage can be a pathology, such as that described by Baker and Martin in 1992 [54]. To the authors’ knowledge, the present study reports the first bottlenose dolphin cephalic presentation documented by US. An accurate and early ultrasound diagnosis of the presentation at delivery can help the clinician to adapt the intervention protocol to the needs of the case and, thus, minimize the risk of reproductive failure. The results of the present study reinforce the importance of monitoring health and fetal vitality throughout the duration of pregnancy and at the time of delivery by ultrasound. In spite of the advantages provided by ultrasonographic surveys of dolphins, the procedure has some limitations compared to the same examination carried out in species commonly investigated in clinical practice [1,2]:

(1) it must be conducted by an expert operator; (2) it depends on the features of the device; (3) animals must be trained for the voluntary medical behavior; (4) the animals must remain in water, which may not be safe for the instrumentation; (5) the external environment (and the light level, in particular) negatively aﬀects results.

However, the diﬃculties listed above can be overcome, given the value of the information that can be gained, as the methods have been successfully applied to both under human care and wild dolphins.

5. Conclusions This study adds further findings to the ultrasonographic monitoring protocol of bottlenose dolphin pregnancy. On the basis of the review of the literature, this is the first study that describes the sonographic data of bottlenose dolphin organogenesis and their correlation with the stage of pregnancy. As described in other species [40], these data could be used to estimate the gestational period in the dolphin in the absence of further information (such as measurements that allow derivation of linear growth diagrams, or a known ovulation date). These findings may be useful for investigations of stranding dolphins, providing data on prenatal development. These data are otherwise difficult to establish in wild cetaceans, as the precise time intervals of such developments and any distinctive growth trajectories in most cetacean species are basically unknown [34–39]. Furthermore, this is the first report that describes by ultrasonography the cephalic presentation of the calf at birth, according to its position within the uterus. The results of the present study reinforce the importance of monitoring health and fetal vitality throughout the duration of pregnancy and at the time of delivery by ultrasound. Reproductive success is vital in sustaining cetacean populations, and the systematic use of ultrasound for pregnancy monitoring provides a useful tool for assessing reproductive success, including for free-ranging dolphins. During capture–release health assessments, it is possible through application of diagnostic ultrasound to evaluate fetal development and viability, estimate gestational age, and measure anatomical structures [16]. This wild population conservation approach benefits from the findings of studies of the population of bottlenose dolphins under human care. Deviations from the normal findings during pregnancy could be related to the alteration of the health status and the well-being of the fetus or the mother, and, if detected sufficiently early, could result in timely therapeutic intervention for animals under human care. Thus, findings related to a reproductive failure in the wild may also be elucidated. However, further investigation will be necessary, carried out with a greater number of subjects, in order to validate the results obtained and to apply these diagnostic methods to other cetacean species.

Author Contributions: Conceptualization, P.S., L.F., F.G., A.T., A.P., and R.O.; data curation, R.M.; formal analysis, P.S., F.G., and L.F.; methodology, P.S., F.G., and R.O.; supervision, P.S. and L.F.; writing—original draft, L.F.; writing—review and editing, P.S., L.F., F.G., R.M., A.T., A.P., and R.O. All authors have read and agreed to the published version of the manuscript. Animals 2020, 10, 908 14 of 16

Funding: This research received no external funding. Acknowledgments: We would like to thank the University of Perugia, Faculty of Veterinary Medicine (Dipartimento di Medicina Veterinaria, Università di Perugia, 06124, Perugia, Italy) for their support and for making this study possible. We are especially grateful to all of the staff of veterinarians and trainers who dedicate themselves daily to dolphin welfare. Their dedication and teamwork make medical behavior, investigation, and conservation projects possible. Conflicts of Interest: The authors declare no conflict of interest.

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