**A Study of the Head during Prenatal and Perinatal Development of Two Fetuses and One Newborn Striped Dolphin (***Stenella coeruleoalba***, Meyen 1833) Using Dissections, Sectional Anatomy, CT, and MRI: Anatomical and Functional Implications in Cetaceans and Terrestrial Mammals**

**Álvaro García de los Ríos y Loshuertos 1, Alberto Arencibia Espinosa 2, Marta Soler Laguía 3, Francisco Gil Cano 1, Francisco Martínez Gomariz 1, Alfredo López Fernández <sup>4</sup> and Gregorio Ramírez Zarzosa 1,\***


Received: 24 October 2019; Accepted: 9 December 2019; Published: 13 December 2019

**Simple Summary:** The head region of the dolphin has been studied widely to identify its anatomical structures and to compare it with other marine and terrestrial mammals. In this study, specimens stranded off the Spanish coast were used. Our study analyzes four dolphin heads during fetal and perinatal development. All specimens were scanned using modern imaging techniques to study their internal organs and to preserve the specimens, which are difficult to obtain. Only one fetus was transversely cross-sectioned to help us to identify critical organs. The developmental study shows several anatomical structures that are compared with cetaceans and terrestrial mammals. During development of the oral cavity, it was observed that the rostral maxillary and mandible teeth (incisive area) had not completely erupted, in contrast with the rest of teeth, which have done so. Also, the main chewing muscle (masseter) was not observed. In addition, we describe the absence of major salivary glands during these developmental stages. Furthermore, we explain the characteristics of the orbit and its relation to the eyeball. In addition, the fetal dolphin's ear is connected with pharynx in a way similar to that in horses. We conclude that these developmental studies will help cetacean conservation.

**Abstract:** Our objective was to analyze the main anatomical structures of the dolphin head during its developmental stages. Most dolphin studies use only one fetal specimen due to the difficulty in obtaining these materials. Magnetic resonance imaging (MRI) and computed tomography (CT) of two fetuses (younger and older) and a perinatal specimen cadaver of striped dolphins were scanned. Only the older fetus was frozen and then was transversely cross-sectioned. In addition, gross dissections of the head were made on a perinatal and an adult specimen. In the oral cavity, only the mandible and maxilla teeth have started to erupt, while the most rostral teeth have not yet erupted. No salivary glands and masseter muscle were observed. The melon was well identified in CT/MRI images at early stages of development. CT and MRI images allowed observation of the maxillary sinus. The orbit and eyeball were analyzed and the absence of infraorbital rim together with the temporal process of the

zygomatic bone holding periorbit were described. An enlarged auditory tube was identified using anatomical sections, CT, and MRI. We also compare the dolphin head anatomy with some mammals, trying to underline the anatomical and physiological changes and explain them from an ontogenic point of view.

**Keywords:** striped dolphin (*Stenella coeruleoalba*); fetal development; PET/SPECT/CT; MRI; sectional anatomy; head anatomy; ontogenesis

#### **1. Introduction**

Cetaceans are a group of mammals well adapted to their marine environment and whose evolutionary changes are especially marked in the development of the structures of the head. In both suborders of living cetaceans, the skull has been highly modified by changes in feeding apparatus and the elimination or reduction of many structures [1]. The relationship of the bones in the skull to one another is altered due to the caudal migration of the nasal opening, a process known as telescoping [2–4]. In addition, differences occur in the location of the external nasal passages and the structure of the middle and the inner ear.

The study of an extensive collection of embryos and fetuses of these species has produced valuable information about the ontogeny of most of the body systems and about musculoskeletal development. Comparisons with other mammals detected the time lag in ossification, retardation of odontogeny, and the origin and development of the fluke, dorsal fin, and flipper [5].

Nevertheless, the studies performed so far lack information on the prenatal and perinatal development due to the difficulty in establishing the differences in ontogenetic development of cetaceans [6]. The precise time intervals of such development and any distinctive growth trajectories are basically unknown [7].

Even now, studies of cranial anatomy by anatomical sections seem to be scarce, with most studies performed in odontocetes (due to their smaller size) either in adults, for instance in common and striped dolphins [8,9], or in newborn bottlenose dolphins [10,11], pacific spotted dolphins, common dolphins, and narwhals [12], and in the fetal narwhal, common dolphin, Atlantic white-sided dolphin [13–15], and Beaked whale [16]. Fetal studies are least common due to the lack of stranded pregnant females. In the case of mysticetes, the few existent studies are focused almost exclusively on external anatomy: eye, nose, hair, and throat of a neonate gray whale [2]; osteology: skull anatomy in fetal specimens of whales of the genera *Megaptera* and *Balaenoptera* [17]; musculoskeletal: musculoskeletal anatomy of the head of a neonate gray whale [18] or vascular [18]. An exception to this is the work of Schute [19] in which a monograph study of the fetal anatomy of the Sei whale (*Balaenoptera borealis*) was done.

In both orders, outlines of organs are observed during the embryonic period and the organs are almost developed or in a development phase in the fetal period, which is of key importance as this is almost the only different period compared to the adult stage, because, for survival reasons, cetaceans give birth to precocial newborn. During these stages (end of the fetal and all the perinatal period), we can obtain valuable data on the species' ontogeny, so we concur with [7] about the many applications in the fields of biology and animal medicine. One of these applications could be determining an organ's development during the fetal period, helping researchers to calculate approximately the time of fetal development in odontocetes based on anatomical changes during gestation, similar to the Carnegie system designed for the human fetus [20] or for terrestrial mammals [3]. So far, we can only estimate cetacean parameters such as the gestation time by using a mathematical formula in *Stenella longirostris* [21], calculate the time of parturition using ultrasonography in Bottlenose dolphins [22], or estimate the adult's age through dental growing lines in striped dolphins [23].

In the current study, we analyze the head anatomy of two striped dolphins' (*Stenella coreuleoalba*) fetuses and one newborn of the same species. In each case, anatomical sections were correlated with computed tomography (CT) and magnetic resonance imaging (MRI).

Our goal is to accomplish several objectives: (a) to create a cephalic anatomy atlas of images during the fetal period up to the perinatal period, which could have benefits for cetacean conservation; (b) to collaborate with other studies dealing with the chronology of fetal development of these species; (c) to clarify some functional aspects of the anatomical structures of the head during prenatal and perinatal dolphin development; and (d) to accurately describe the structures of the head following the Illustrated Veterinary Anatomical Nomenclature [24].

#### **2. Materials and Methods**

#### *2.1. Animals*

A total of four pre- and perinatal specimens and one adult striped dolphin (*Stenella coeruleoalba*, Meyen 1833) were used in this study (Table 1). The mother of the youngest fetus was stranded on the Spanish Atlantic coast. The mother of the older fetus and two newborn specimens were stranded on the Spanish African coast. The adult specimen was stranded on the Spanish Mediterranean coast. Stranded specimens were found dead and ethics committee clearance was not necessary. Both fetuses and the newborn specimen were transported to the CT and MRI units to perform CT and MRI scans.


**Table 1.** Fetal specimens of striped dolphin used in this study.

*SCOG*: *Stenella coeruleoalba* from Pontevedra, Spain; *SCOCE*: *S. coeruleoalba* from Ceuta, Spain; *SCOMU*: *S. coeruleoalba* from Murcia, Spain; MRI: Magnetic resonance imaging; CT: Computed Tomography, *CEMMA*: Coordinator Center for the study of the marine mammals, Galicia; *CECAM*: Center for the study and conservation of marine animals, Ceuta; *CRFS*: Wildlife rehabilitation Center, Murcia.

#### *2.2. Computed Tomography*

The sco1 was scanned with Positron Emission Tomography (PET), Single Photon Emission Computed Tomography (SPECT)-Computed Tomography (CT) (PET/SPECT/CT AlbiraTM Systems, Valencia, Spain; Centro de Investigación Biomédica, Universidad de Murcia, Spain); single-slice: 1 detector arrays; type of acquisition: helical; thickness: 0.125 mm; image reconstruction interval or index: 0.0125 mm; pitch: 0; tube rotation time: 0.12; mA: 0.4; Kv: 45; FOV 68 cm, Matrix dimensions 2240 × 2360; reconstruction algorithm: FBP filtered back projection; WW: 600/WL: 300). The SPECT-CT images were transferred to a Dicom workstation, while sco2 was scanned with CT (General Electric Medical Systems, Schenectady, NY, USA; Clínica Virgen de Africa, Ceuta, Spain); multislice: 4 detector arrays; type of acquisition: helical; thickness: 5 mm; index: 3.2 mm; pitch: 0.45; tube rotation time: 0.33; mA: 30; kV: 120; FOV 35 cm; matrix dimensions: 512 × 512, reconstruction algorithm: bone; WW: 350/WL: 221; WW 650/WL −34. Finally, sco3 was scanned with a CT (General Electric Medical Systems-HiSpeed dual, Schenectady, NA, USA; Hospital Clínico Veterinario, Universidad de Murcia, Spain); multislice: 2 detector arrays; type of acquisition: helical; thickness: 5 mm; index: 2.5 mm; pitch: 0.35; tube rotation time: 1; mA: 100; Kv: 120; Image field of view FOV 40 cm; matrix dimensions: 512 × 512, reconstruction algorithm: standard; WW: 350/WL: 221; WW 650/WL −34). All dolphin specimens were positioned in ventral recumbency. All CT images were transferred to a DICOM workstation and CT images were analyzed with Radiant DICOM viewer and Osiris 4.0 for Windows. A vascular window setting (WW 600/WL 300) was applied to obtain PET/SPECT/CT images. Mediastinum-vascular window (WW 350/WL 221) and soft-tissue window settings (WW 650/WL −34) were applied to obtain Ceuta and Murcia CT images, respectively.

#### *2.3. Magnetic Resonance Imaging*

In sco1, Magnetic Resonance (MR) images were obtained with a high-field MR apparatus (General Electric Sigma Excite, Schenectady, NA, USA; Centro Veterinario de Diagnóstico por Imagen de Levante, Ciudad Quesada, Alicante, Spain), 1.5 Tesla using a human wrist coil. T1-weighted spin eco (SE) and T2-weighted fast spin scho (FSE) pulse sequences were used. T1-weighted (SE) images were obtained in transverse plane and 2D acquisition, using the following parameters: TE 13 ms, TR 640 ms, TI 0, NEX 1, slices thickness 1 mm, interslice gap 1.3, field of view 75 and matrix dimensions 0\256\192\0. T2-weighted (FSE) images were obtained in transverse plane and 2D acquisition, using the following parameters: TE 84 ms, TR 8100 ms, TI 0, NEX 1, slice thickness 1 mm, interslice gap 1.3, field of view 60, and matrix dimensions 192\0\0\192.

In sco2, MR images were obtained with a high-field apparatus (Philips Medical System Intera, Eindhoven, The Netherlands; Clínica Radiológica, Ceuta, Spain), 1.5 Tesla using a sense-body coil. T1-weighted fast field echo (FFE) and T1-weighted out-of-phase (OOP) gradient echo (GRE) pulse sequences were used. T1-weighted (FFE) images were obtained in transverse plane and 2D acquisition using the following parameters: TE 4.6 ms, TR 183 ms, TI 6, NEX 6, slice thickness 8 mm, interslice gap 9, field of view 68.6, matrix dimensions 0\204\155\0. T1-weighted (OOP) images were obtained in transverse plane and 2D acquisition using the following parameters: TE 2.3 ms, TR 130.3 ms, TI 0, NEX 5, 9 mm slice thickness, interslice gap 10, field of view 69.6, and matrix dimensions 132\0\0\103.

In sco3, MR images were obtained with a high-field apparatus (General Electric Sigma Excite, Schenectady, USA; Centro Veterinario de Diagnóstico por Imagen de Levante, Ciudad Quesada, Alicante, Spain), 1.5 Tesla using a human head coil. T1-weighted spin echo (SE) and T2-weighted fast spin echo (FSE) pulse sequences were used. T1-weighted (SE) images were obtained in transverse plane and 2D acquisition using the following parameters: TE 11 ms, TR 640 ms, TI 0, NEX 1, slice thickness 4 mm, interslice gap 4.5, field of view 75, and matrix dimensions 0\192\192\0. T2-weighted (FSE) images were obtained in transverse plane and 2D acquisition using the following parameters: TE 93.8 ms, TR 6020 ms, TI 0, NEX 1, slice thickness 4 mm, interslice gap 4.5, field of view 75, and matrix dimensions 0\192\192\0. All dolphin specimens were positioned in ventral recumbency. The MR images were transferred to a DICOM workstation. MR images were analyzed with Radiant DICOM viewer and Osiris 4.0 for Windows.

#### *2.4. Anatomic Evaluation*

Sco1, sco3, and sco4 were preserved by immersion in formaldehyde (10%). Sco5 was fixed with embalming solution (formaldehyde, glycerine, isopropyl alcohol, phenol) injecting the right and left carotid arteries and left and right external jugular veins. After 48 h, the carotid arteries and jugular veins were injected with red and blue latex, respectively. These specimens were stored in the Department of Anatomy and Embryology's freezer chamber, Facultad de Veterinaria, Murcia, Spain. Sco3 was preserved frozen (−20 ◦C) in the Department of Anatomy and Embryology's cooling chamber, Facultad de Veterinaria, Murcia, Spain.

Sco2 was frozen at −80 ◦C and then taken out to obtain cross sections cut with a band saw (Anatomical Lab, Department of Anatomy and Embryology, Universidad de Murcia, Murcia, Spain), obtaining 0.7–1 cm thick slices, which were then photographed giving us 57 transverse images used to correlate the sections with CT and MR images. Slices were immersed in acetone for plastination preservation and then stored in a freezer chamber at the Department of Anatomy and Embryology, Facultad de Veterinaria, Universidad de Murcia, Spain.

#### *2.5. Gross Dissections*

A deep head dissection of sco4 showed the melon. At its midpoint, the melon was cut in transverse and horizontal sections, which showed the nucleus and peripheral tissue ring. The nasal plug and nasal cavity were observed after removing the nasal vestibule and spiracle.

The head, face, and adjacent areas of sco5 were superficially dissected showing frontal and facial fat, the melon surface, and the mandible and superficial facial muscles. After carefully removing superficial fat and fibrous tissues, the venous drainage, several depressor mandible muscles, tongue muscles, and the rudiment of external acoustic meatus were exposed.

#### **3. Results**

#### *3.1. Oral Cavity*

The oral cavity of the three studied specimens clearly showed the tongue in anatomical sections, CT, and MRI (Figures 1–5). In sco1, the lateral sublingual recesses were observed only in MRI (Figure 2 Row (from now on R)(R1D-E)) while in sco2 it was identified in CT and anatomical sections Figure 2(R2A–C)). Under the lateral sublingual recess, it was not possible to distinguish sublingual salivary glands (neither polystomatic nor monostomatic). Histological analysis of tissue from this region showed a mixture of adipose and striated muscular tissue.

**Figure 1.** Approximated level sections of fetus dolphin head. Lines represent the location for each transverse anatomical section, CT, and MR images (I–VII).

**Figure 2.** Representative transverse images of the snout made at the level of the rostral portion of the melon and oral cavity. Level I. Images are oriented so that the left side of the head is to the right and dorsal is at the top. Row (from now on R) 1, sco1; R2, sco2; R3, sco3. (**A**) Anatomical section. (**B**) Vascular window PET/SPECT/CT image. (**C**) Soft-tissue window CT image. (**D**) T1-weighted SE sequence. (**E**) T2-weighted fast spin echo (FSE) sequence. (**F**) T1-weighted fast field echo (FFE) sequence. (**G**) T1-weighted out of phase (OOP) gradient echo (GRE) sequence. 1, Mesethmoid cartilage; 2, incisive bone; 3, maxillary bone; 4, vomer bone; 5, supraorbital canal; 6, mandible; 7, canal and mandibular fat; 8, tooth in development; 9, socket of tooth; 10, oral cavity; 11, oral vestibule; 12, tongue; 13, melon; 14, melon rostral muscles; 15, mylohyoid muscle; 16, buccinator and depressor of the lower lip muscles; 17, lateral sublingual recess; 18, epidermis and dermis.

**Figure 3.** Representative transverse images made at the level of root of the snout, caudal portion of the melon, and oral cavity. Level II. Images are oriented so that the left side of the head is to the right and dorsal is at the top. R1, sco1; R2, sco2; R3, sco3. (**A**) Anatomical section. (**B**) Vascular window PET/SPECT/CT image. (**C**) Soft-tissue window CT image. (**D**) T1-weighted SE sequence. (**E**) T2-weighted FSE sequence. (**F**) T1-weighted FFE sequence. (**G**) T1-weighted OOP GRE sequence. 1, Mesethmoid cartilage; 2, incisive bone; 3, maxillary bone; 4, vomer bone; 5, frontal bone; 6, lacrimal bone; 7, zygomatic bone; 8, lacrimal-zygomatic synchondrosis; 9, zygomatic bone: temporal process; 10, maxillary sinus; 11, palatine bone; 12, mandible; 13, canal and mandibular fat; 14, melon; 15, tongue; 16, oral cavity; 17, melon rostral muscles; 18, pterygoid muscles; 19, mylohyoid muscle; 20, digastric muscle; 21, fat and striated muscle; 22, epidermis, dermis and subcutaneous tissue; 23, nostrils.

**Figure 4.** Representative transverse images made at the level of nasal, oral cavities, and orbital craniofacial fossa. Level III. Images are oriented so that the left side of the head is to the right and dorsal is at the top. R1, sco1; R2, sco2; R3, sco3. (**A**) Anatomical section. (**B**) Vascular window PET/SPECT/CT image. (**C**) Soft-tissue window CT image. (**D**) T1-weighted SE sequence. (**E**) T2-weighted FSE sequence. (**F**) T1-weighted FFE sequence. (**G**) T1-weighted OOP GRE sequence. 1, Mesethmoid cartilage; 2, incisive bone; 3, maxillary bone; 4, vomer bone; 5, lacrimal bone; 6, zygomatic bone: temporal process; 7, palatine bone; 8, pterygoid bone; 9, ethmoid bone; 10, mandible; 11, canal and mandibular fat; 12, periorbit and eyeball; 13, tongue; 14, melon external fiber ring; 15, digastric muscle; 16, pterygoid muscle; 17, mylohyoid muscle; 18, oral cavity; 19, melon caudal muscles; 20, frontal bone: orbital recess; 21, pterygopalatine recess; 22, fat and striated muscle; 23, nostrils; 24, nasal diverticulum and nasal plug (arrow); 25, membranous part of nasal septum; 26, nasal cavity; 27, nasal mucosa; 28, melon; 29, nasal vestibule muscles.

**Figure 5.** Representative transverse images made at the level of the rostral part of the cranial cavity, choanas, and eyeball. Level IV. Images are oriented so that the left side of the head is to the right and dorsal is at the top. R1, sco1; R2, sco2; R3, sco3; (**A**) Anatomical section. (**B**) Vascular window PET/SPECT/CT image. (**C**) Soft-tissue window CT image. (**D**) T1-weighted SE sequence. (**E**) T2-weighted FSE sequence. (**F**) T1-weighted FFE sequence. (**G**) T1-weighted OOP GRE sequence. 1, Incisive bone; 2, maxillary bone; 3, frontal bone; 4, vomer bone; 5, presphenoid bone: body and wings; 6, palatine bone; 7, pterygoid bone; 8, basisphenoid bone: pterygoid crest; 9, zygomatic bone: temporal process; 10, ethmoid bone; 11, mandible; 12, eyeball; 13, sclera; 14, lens; 15, eyelids; 16, lateral pterygoid muscle; 17, medial pterygoid muscle; 18, choanae and nasopharyngeal sphincter muscle; 19, tongue; 20, oral cavity; 21, pterygopalatine recess; 22, digastric muscle; 23, subarachnoid space; 24, longitudinal brain fissure; 25, brain: frontal lobe; 26, mandibular canal; 27, mylohyoid muscle; 28, hyoglossus muscle; 29, extraocular muscles; 30, melon caudal muscles; 31, frontal bone: orbital recess; 32, nasal diverticulum; 33, fat and striated muscle.

In sco1, CT showed clearly the dental alveolus dorsal to the mandibular canal but not in the maxillary bone (Figure 2(R1B)), while in sco2, CT and anatomical sections showed the most caudal teeth growing covered by gums in both the mandible and maxillary bones (Figure 2(R2C)). In the CT and MR images of sco3, the mid caudal teeth were forming in both dentary arches. In the three specimens, rostral maxillary and mandible teeth (incisive area) had not completely erupted, whereas the rest of mandible and maxillary teeth have done so in sco3 (Figure 2(R3C)).

Only two of the three pairs of muscles of mastication were identified: temporal and pterygoid (Figure 4). The third, the masseter muscle, originates from the facial crest or maxillary tuber and zygomatic arch which were absent in the studied specimens. Its insertion on the masseter fossa and the caudal and ventral portion of the mandible was not observed. A mixture of adipose tissue and muscle fibers on the caudolateral aspect of the body of the mandible was observed. The cheek area was vestigial and so the buccinator muscle (oral part) and depressor of the lower lip were displaced rostrally under the lower lip (Figures 4–7). The orbicularis oris muscle was absent. Medial to the temporomandibular joint, the pterygoid muscles were easily seen (Figures 3–6). The mandible depressor muscles, digastric and mylohyoid were well developed. The digastric muscle insertion enlarges until the most latero-rostral sections of the mandible body (Figures 3–8). Muscles were easy to differentiate in anatomical sections. CT showed them moderately hypoattenuated and in MR images, slightly hypointense.

**Figure 6.** Representative transverse images made at the level of the pharynx and the caudal part of the orbit. Level V. Images are oriented so that the left side of the head is to the right and dorsal is at the top. R1, sco1; R2, sco2; R3, sco3. (**A**) Anatomical section. (**B**) Vascular window PET/SPECT/CT image. (**C**) Soft-tissue window CT image. (**D**) T1-weighted SE sequence. (**E**) T2-weighted FSE sequence. (**F**) T1-weighted FFE sequence. (**G**) T1-weighted OOP GRE sequence. 1, Nasal bone; 2, maxillary bone; 3, frontal bone; 4, vomer bone; 5, presphenoid bone: body; 6, presphenoid bone: wings; 7, palatine bone; 8, pterygoid bone: hook-like process; 9, pterygoid bone: pterygoid crest; 10, mandible; 11, eyeball; 12, lens; 13, optic nerve; 14, sclera; 15, cornea; 16, eyelids; 17, lateral pterygoid muscle; 18, medial pterygoid muscle; 19, nasopharynx and nasopharyngeal sphincter muscle; 20, pterygopalatine fossa; 21, subarachnoid space; 22, longitudinal brain fissure; 23, brain: temporal lobe; 24, mandibular canal; 25, digastric muscle; 26, sternohyoid muscle; 27, fat and striated muscle; 28, melon caudal muscles; 29, extraocular muscles; 30, temporomandibular joint; 31, auditory tube; 32, pharyngeal opening of the auditory tube; 33, oropharynx; 34, tongue; 35, frontal bone: orbital recess; 36, zygomatic bone: temporal process; 37, brain: lateral ventricle; 38, sagittal dorsal sinus; 39, epidermis, dermis, and subcutaneous tissue.

**Figure 7.** (**A**) Superficial and (**B**) middle head dissection made after removing melon and fat of sco5. 1, Melon; 2, mylohyoid muscle; 3, buccinator and depressor of the lower lip muscles; 4, fat and striated muscle; 5, digastric muscle; 6, geniohyoid muscle; 7, sternohyoid and sternothyroid muscles; 8, orbicularis oculi muscle; 9, external acoustic meatus (cartilaginous); 10, mandible: body; 11, subcutaneous tissue; 12, maxillary vein.

**Figure 8.** Representative transverse images at the level of the cranial vault of the skull involving the temporal lobe of the brain, mesencephalon, middle and inner ear, larynx and hyoid apparatus. Level VI. Images are oriented so that the left side of the head is to the right and dorsal is at the top. R1, sco1; R2, sco2; R3, sco3. (**A**) Anatomical section. (**B**) Vascular window PET/SPECT/CT image. (**C**) Soft-tissue window CT image. (**D**) T1-weighted SE sequence. (**E**) T2-weighted FSE sequence. (**F**) T1-weighted FFE sequence. (**G**) T1-weighted OOP GRE sequence. 1, Occipital bone: basilar part; 2, pterygoid crest; 3, temporal bone: tympanic part; 4, temporal bone: petrous part; 5, auditory ossicles of middle ear (malleus and incus); 6, auditory ossicles of middle ear (stapes); 7, auditory ossicles of middle ear (incus); 8, frontal process of temporal bone; 9, squamous part of temporal bone; 10, parietal bone; 11, interparietal bone; 12, tympanohyoid cartilage; 13, stylohyoid bone; 14, thyrohyoid bone; 15, basihyoid bone; 16, middle ear: tympanic cavity; 17, middle ear: musculotubarius canal; 18, inner ear: cochlea (spiral canal); 19, inner ear: vestibule; 20, arytenoid cartilage; 21, epyglotic cartilage; 22, nasopharynx: intrapharyngeal orifice; 23, laryngopharynx: piriform recess; 24, ramus of the mandible: condylar process; 25, temporal muscle; 26, mesencephalon: tegmentum; 27, mesencephalon: aqueduct; 28, mesencephalon: colliculus; 29, lateral ventricles; 30, corpus callosum; 31, falx cerebri; 32, dorsal sagittal sinus; 33, sinus transversus; 34, brain hemisphere: temporal lobe; 35, cerebellum: cerebellar hemispheres; 36, meninx: cerebellar tentorium; 37, facial and vestibulocochlear nerves and labyrinthic artery; 38, orifice and internal acoustic meatus; 39, sternohyoid muscle; 40, external acoustic meatus: cartilaginous rudiment; 41, peribullar sinus; 42, fontanelles; 43, epidermis, dermis, and subcutaneous tissue.

The mandible was hyperattenuated in CT and hypointense in MRI. The mandibular fat showed some dark color in anatomical sections; it was slightly hyperattenuated in CT sections, slightly hyperintense in T1-weighted, and slightly hypointense in T2-weighted sequences (Figures 3–6). The mandibular canal was observed patent and wide in the three specimens studied, as it is usual in odontocetes.

#### *3.2. Rostrum (Snout)*

Dorsal to the oral cavity and below melon the rostrum is observed. The mesethmoidal cartilage could be seen amongst vomer, maxillary, and incisive bones, being supported only on the groove of the vomer bone. This cartilage acts as an adhesive joining these bones to each other (Figures 2–4). CT showed one of two infraorbital canals inside the incisive bone of sco1 (Figure 2(R1B)). In the anatomical section, under the lateral sublingual recess, only fat and striated muscular tissue was seen, instead of glandular tissue, and this tissue was seen as a slightly hyper/hypointense area depending on the MRI sequence used (Figure 3).

#### *3.3. Melon*

The first two-level sections of the snout showed this particular anatomical structure of cetaceans (Figures 2 and 3). Insco1, the most rostral part of the melon could already be observed (Figure 2(R1E)). The melon was observed hyperintense in T2-weighted FSE sequence but was not detected in PET/SPECT/CT and T1-weighted SE sequence. In sco2, the melon was well appreciated in anatomical sections as well as hypointense structure in MRI sequences (Figure 2(R2A, F)). In sco3, the melon was seen as a large diffuse area in both CT and MRI (Figure 2(R3)).

The caudal part of the melon encloses the nasal cavity. In sco1, it was slightly hyperintense in MRI sequences (Figure 3(R1D, E)) and it was not observed in PET/SPECT/CT (Figure 3(R1B)). In sco2, the anatomical section showed the white nucleus of the melon surrounded by connective tissue and muscles. However, only the nucleus was identified in CT and MRI sequences (Figure 3(R2)). In sco3, CT images showed the central nucleus of the melon as moderately hypoattenuated and its external fibrous ring as slightly hyperattenuated; in a similar way, the nucleus was hyperintense and the external fibrous ring hyper/hypointense depending on the MRI sequence (Figure 3(R3)). Dissection of the melon showed the nucleus and external fibrous ring as in CT and MRI (Figure 9).

#### *3.4. Nasal Cavity and Pasanasal Sinuses*

From the beginning of fetal development until birth, an opening was observed between the melon and the frontal bone. On external examination, the nostrils, also named the spiracle or blowhole, gives the common impression of an access to odd nasal vestibule. But two nostrils closed by a musculomembranous fold were observed. Under the nostrils was the dorsal part of the nasal cavity named the vestibule of the nose, which is divided in two (left and right) by the membranous part of the nasal septum. Between the nasal vestibules and nasal plugs, different diverticula were observed. In sco3, the nasal septum, nasal diverticula (hypoattenuated areas), and plugs were clearly visualized in both CT and MR sequences (Figure 4(R3)). Nasal plugs show a cartilaginous appearance (slightly hyperattenuated in CT and hypointense in MRI), with muscular fibers and mucosa (slightly hypoattenuated in CT, slightly hypointense in T1-weighted SE sequence, and hypointense in T2-weighted FSE sequence) (Figures 4 and 5(R3)).

Under the nasal plugs (Figure 9), two nasal cavities were observed. The rostral boundary is formed by the maxillary bones, medially and caudally the vomer bone and perpendicular lamina of ethmoid bone dominate, respectively, while the lateral boundary is the pterygoid bone. The nasal septum is formed mainly by the vomer bone and by the perpendicular lamina of the ethmoid bone. In sco1, the vomer was observed hypoattenuated in CT and slightly hypointense in MR sequences and the mesethmoidal cartilage was seen hypoattenuated in CT and moderately hyperintense in MR sequences (Figure 4(R1)). In sco2, the vomer together with the ethmoid bone and mesethmoidal cartilage were identified in anatomical section. The vomer was hyperattenuated in CT and hypointense in all MR sequences. The mesethmoidal cartilage was clearly differentiated in all images except in MR sequences (Figure 4(R2)). In sco3, the vomer bone and mesethmoidal cartilage were hyperattenuated in CT, slightly hypointense in T1-weigthed MR SE sequence, and moderately hyperintense in T2-weigthed MR FSE sequence (Figure 4(R3)).

**Figure 9.** Deep head dissection made at the level of the nasal vestibule and melon of sco4. 1, Melon: nucleus; 2, melon: external fiber ring; 3, melon rostral muscles; 4, melon caudal muscles; 5, right nasal cavity (after removing nasal plug); 6, left nasal plug; 7, maxillary bone; 8, ethmoid bone; 9, nasal septum.

In all stages studied and with CT and MRI techniques, the maxillary sinus was observed as a small cavity within the maxillary bone. This sinus was observed in anatomical sections (sco2) filled with a heterogeneous substance observed in the fetal specimens examined in our study (Figure 3(R2A)).

Choanae are openings between the nasal cavity and the nasopharynx, and this space full of air was observed hypoattenuated in CT and hypointense in MRI sequences. In sco1, the mucosa and nasopharyngeal muscle were hypoattenuated in PET/SPECT/CT and slightly hyper/hypointense depending on the MRI sequence (Figure 5(R1)). Anatomical sections of sco2 showed mucosa and nasopharyngeal muscle with a small air space; in CT it was moderately hyperattenuated. MRI sequences showed it moderately hypointense or hypointense (Figure 5(R2)). In sco3, it could be seen as slightly hyperattenuated in CT and moderately hypointense in MRI sequences (Figure 5(R3)).

#### *3.5. Orbit and Eyeball*

CT images showed that the orbit is formed by an incomplete bony rim composed of a supraorbital part formed by the frontal bone; no infraorbital rim was observed. The rostral limit of the supraorbital rim is formed by the zygomatic and lacrimal bones. In sco1, a junction (synchondrosis) between the zygomatic and lacrimal bones was observed in PET/SPECT/CT sections (Figure 3(R1B)). Nevertheless, in sco2 (Figure 3(R2A)), anatomical sections showed both bones undergoing ossification (synostosis). Using CT, it was not possible to differentiate between these bones as the image was very hyperattenuated.

An oblique, thin temporal process of the zygomatic bone crossing under the periorbit and holding it was observed. CT showed the temporal process in sco1 with little bony density (hypoattenuated). Nevertheless, it was observed slightly hyperattenuated in sco2 and sco3 (Figures 3–5).

The eyeball was observed in all MR sequences (Figures 5 and 6) except in PET/SPECT/CT (Figures 5 and 6(R1)). CT showed the lens hyperattenuated and hypointense in MR sequences (Figure 5(R2)). The tapetum lucidum was not appreciated in anatomical sections, but it was clearly seen in the dissection of sco4.

#### *3.6. Central Nervous System*

Brain hemispheres divided by the longitudinal brain fissure (hypointense in MR sequences) were observed in sco1 and sco2 but were not clear in sco3 (Figure 5). The sagittal dorsal sinus at the temporal level was also observed (Figures 6 and 8). In sco1, the lateral ventricle could be distinguished in MR sequences, but it was more difficult to identify in sco2 and sco3, as well as in anatomical sections, CT, and MR sequences. The meninx was observed in both anatomical sections and MR sequences (Figure 8). The mesencephalic aqueduct was clearly seen in all MR sequences. In anatomical sections, the vestibulocochlear and facial nerves as well as the labyrinthine artery passing through the inner auditory meatus were identified (Figure 8(R2)); however, these anatomical structures were not observed in CT and MR sequences.

The cerebellar tentorium appeared hyperattenuated in CT and slightly hyperintense in both MR sequences (Figure 8(R2)). The vermis and cerebellar hemispheres were observed at the level of the cerebellar fossa held by the cerebellar tentorium (Figure 10(R2A)).

#### *3.7. Ear*

The petrous part of the temporal bone was sectioned at the bony labyrinth level, which contains the bony spiral canal, bony vestibule, and spiral canal of the cochlea. Although both petrous and tympanic parts of the temporal bone were seen with both diagnostic imaging techniques (CT and MRI), components such as the auditory ossicles of the middle ear, the spiral canal of the cochlea, and the bony vestibule were visible only in CT and in anatomical sections. The auditory ossicles of the middle ear were observed hypointense in MR sequences and hyperattenuated when using CT. Nevertheless, it was possible to see the malleus, incus, and stapes using anatomical sections and CT (Figure 8). The rudimentary cartilaginous part of the external acoustic meatus was a slightly hyperattenuated small area in sco3 using CT (Figure 8(R3B)) and was also observed in dissections of sco4 and sco5 (Figure 7). CT showed a fatty content slightly hypoattenuated in the tympanic cavity (Figure 8).

Connecting the tympanic cavity with the nasopharynx was an enlargement of the auditory tube which was observed only in sco2 and sco3 (Figure 6). In anatomical sections and in MR sequences, the pharynx was appreciated surrounding aditus laryngis (epiglottic and arytenoid cartilages), and the esophageal vestibule lies dorsally (Figure 8).

#### *3.8. Larynx*

Surrounding the laryngeal cartilages, the laryngopharynx was observed, as well as the hyoid apparatus in their relationship with the ear (Figure 8). In sco1, CT images of the hyoid bones were shown slightly hyperattenuated (Figure 8(R1)). In sco2 (Figure 8(R2)), the hyoid bones were larger, and in sco3 (Figure 8(R3)) were very hyperattenuated; in anatomical sections, the tympanohyoid bone was well appreciated (Figure 8(R2A)). Also observed in this area were the temporomandibular joint and the mandibular canal fat which was very close to the middle ear, making contact with the tympanic wall. The stylohyoid, basihyoid, and thyrohyoid bones were well observed using CT and dissections because they were ossified, while the epihyoid, ceratohyoid, and tympanohyoid bones were not seen because they still remain cartilaginous. The caudal tip of the thyrohyoid bone was not ossified at birth.

#### *3.9. Cranial Cavity*

Fontanelles were wide in sco1 (Figure 8(R1)), closing in sco2 (Figure 8(R2)) and almost closed in sco3. CT in both fetuses showed clearly the fontanelles. Bones of the cranial cavity were analyzed mainly using CT and anatomical sections, since MR sequences showed bones as hypointense in all cases.

The occipital bone has three parts: basilar, lateral, and squamous. The basilar part was not observed in sco1 at this level section (Figure 10(R1)) but fontanelles were clearly seen. The basilar part was observed in sco2 (Figure 10(R2)). In sco3 (Figure 10(R3)) fontanelles are closing, though the bones surrounding the foramen magnum are not totally ossified.

**Figure 10.** Representative transverse images made at the level of the occipital bone, cerebellum, and trunk of encephalon. Level VII. Images are oriented so that the left side of the head is to the right and dorsal is at the top. R1, sco1; R2, sco2; R3, sco3. (**A**) Anatomical section. (**B**) Vascular window PET/SPECT/CT image. (**C**) Soft-tissue window CT image. (**D**) T1-weighted SE sequence. (**E**) T2-weighted FSE sequence. (**F**) T1-weighted FFE sequence. (**G**) T1-weighted OOP GRE sequence. 1, Occipital bone: lateral part; 2, occipital bone: squamous part; 3, occipital bone: basilar part; 4, myelencephalon; 5, cerebellum: vermis; 6, cerebellum: cerebellar hemispheres; 7, subarachnoid space; 8 = esophagus; 9, laryngeal cavity: glottis; 10, vascular and nerve structures of the pharynx and larynx; 11, external jugular vein; 12, longus capitis muscle; 13, scapula; 14, sternohyoid and sternothyroid muscles; 15, cleidocephalic muscle: mastoid part; 16, sternocephalic muscle: mastoid part; 17, longissimus capitis muscle; 18, splenius capitis muscle; 19, semispinalis capitis muscle (digastric); 20, semispinalis capitis muscle (complex); 21, spinalis capitis muscle; 22, epidermis, dermis, and subcutaneous tissue; 23, fontanelles.

#### **4. Discussion**

#### *4.1. Anatomical and Functional Considerations*

#### 4.1.1. Oral Cavity

Comparing our study's anatomical sections and PET/SPECT/CT images, we observed that in the fetus and newborn, the teeth of the rostral alveoli (equivalent to incisive teeth) erupt later than the more caudal alveoli (equivalent to premolars and molars). This would have a functional application in the lactation (perinatal period), where the rostral teeth erupting afterwards would to serve to help to suction milk and at the same time to hold the mother's nipple without harming it, thanks to the fact that the teeth are not yet completely formed (Figures 1–4 and 7). Odontocetes are (eu)homodont with conical teeth without a complete root, polydont, monophyodont [27] and tecodont, isognathous with centric occlusion of both dentary archs and with prognatism even during fetal stages, and are designed to catch prey [28].

We were unable to find the masseter muscle in fetal, newborn, and adult striped dolphin specimens, though it has been described by some authors in the striped dolphin [8,11], in a juvenile common dolphin [9], and in a bottlenose dolphin [29,30]; images from these studies suggest that the papers are describing the buccinator (oral part) and depressor muscles of the lower lip. (Figure 2(R2A) and Figure 9). After performing dissection of the head muscles in an adult striped dolphin, we conclude that the muscle atrophies, finding only remnants of adipose tissue and muscle fibers in its anatomical position (Figure 7). The origin of this muscle (mandible elevator) in domestic mammals extends from the maxillary tuber or facial crest to approximately the middle of the zygomatic arch, the two latter structures being absent in the pre- and perinatal studied specimens. Only a very thin temporal process of the zygomatic bone (jugal bone) is joined to the temporal bone by a symphysis, making it an unsuitable location for the attachment of a strong muscle, which acts to close the mandibles (Figures 3–5). Also, the insertion in domestic animals is the masseteric fossa and medially on the ramus of the mandible [31,32], and the masseteric fossa is absent in odontocetes but not totally in mysticetes. Functionally, the masseter muscle is an extremely powerful muscle of mastication, which varies among species in terms of its topography [32]. Odontocetes have lost this feature throughout evolution, because most of the cetaceans, with the remarkable exception of orcas [33] swallow their prey intact without chewing. Reference [30] described a residual masseter muscle in odontocetes along with a more developed temporal muscle. In addition, mysticetes catch the krill between the whalebone and raise the mandibles full of weight (mostly water) to filter it (Humpback whales [34] and rorquals [35]). The masseter muscle was also described by [19] in the boreal whale fetus and by [36] in the Minke whale.

In this study, salivary glands and lymphatic nodules were not observed in either the fetal or the newborn dolphin heads. Under the lateral sublingual recess and folds (monostomatic and polystomatic), sublingual salivary glands were not observed, only a mass of striated muscle fibers and adipose tissue (Figures 2 and 3). During dissection of the head and neck of an adult striped dolphin, only the superficial cervical lymphatic nodes were identified after histological analysis. This is logical in homodont animals, which swallow whole prey. Reference [30] showed the absence of organized salivary glands in dolphins. These same authors also describe lymphatic nodules of head to be smaller in size (about 1 cm) and therefore difficult to observe. In whales, the lack of salivary glands is described as well as the persistence of its ducts [26]. Strangely enough, an adenocarcinoma affecting the microscopic lingual salivary glands has been described in Beluga whales [37].

#### 4.1.2. Rostrum (Snout)

Telescoping refers to the overlap of the incisive and maxilla bones and the retraction of nasal bones on top of the frontal bone, as well as to the reduction of the temporal fossa and the rostral displacement of some muscles [3,12,28]. However, in the dolphin's snout, there is a link between the vomer, maxilla, and incisive bones. The mesethmoid cartilage serves as an anti-concussive structure. The cartilage appears hypoattenuated in sco1 CT but moderately hyperattenuated in both sco1 and sco2. It does not show more signal intensity in T1-weighted SE sequence as described by [12], except in sco1. In the domestic mammal nose, the nasal septum cartilage extends rostrally to the nasal openings, but in cetaceans the nasal cavity and snout are at different levels due to telescoping. The snout tip has three functions in odontocetes: tactile (protopatic), offensive (together with tip of mandible), and, as a consequence of the mesethmoid cartilage joining these three bones, a shock absorber [8].

#### 4.1.3. Melon

The rostral muscles are inserted in the fibrous external area of the melon in the Tursiops fetus [11] and are called maxilonasolabialis muscles in adult specimens of striped dolphin [8], in the common dolphin [12], and in bottlenose dolphin [38]. These muscles are present in the beluga whale [39]. In our study, topographically, these muscles were located ventrolateral to the melon and belong to the residual group of the facial neuromuscular system [31,32,38] (Figures 2 and 3).

#### 4.1.4. Nasal Cavity and Paranasal Sinuses

After emerging from water, the dolphin exhales air full of CO2 and steam through the nasal openings, nostrils. Surrounding the nostrils are striated muscle fibers opening and closing the nasal openings, acting as sphincter muscles. In our study, we could not clearly identify this group of muscles as they are mixed with melon muscles (Figure 4). In anatomical sections and MRI in a common dolphin, [9] identified the musculature of external sacs system and [40] specially studied them, which concluded that these muscles originate from the residual facial neuromuscular group, for example, orbicularis oris, levator nasolabialis, and the levator of the upper lip.

On dissecting the nasal vestibule in a fetal specimen, we observed that both nostrils have a common area under a musculomembranous sphincter, but ventrocaudally the membranous part of the nasal septum separates the left and right nasal vestibules. Reference [12] stated that the superficial blowhole emerges into an unpaired vestibulum. Ventral to the nostrils, nasal vestibules with several air sacs (diverticula) [41] were observed in the studied specimens (Figures 4 and 5). The nasal vestibule is the rostral part of the nasal cavity, related to the nasal diverticulum (horses) and lined by stratified squamous epithelium [24]. This epithelium is also referred by [30] as "similar to the epidermis of the animal's back" as in the equine vestibule. The nasal diverticulum has been described in horses as a cutaneous blind pouch inside the vestibule. The alar fold is a mucosa supported by the medial accessory cartilage covered dorsally by lateral nasal muscle [24,31,32] in the nasal vestibule in horses. In dolphins, the nasal plug, alar fold, and the nasal vestibule have been modified for sound generation and as a water reservoir. Different authors have named the nasal diverticula as sacs or air sacs or sinuses. References [8,42] described diverticula as vestibular, tubular, and premaxillary sacs. Reference [43] added the nasofrontal, accessory, and connecting air sacs and shows the premaxillary sac under the nasal plug and inside the nasal cavity [8,44]. Reference [12] mentioned three vestibular sacs. Reference [41] added the pterygoid and laryngeal air sacs. Several authors [45–48] explained that the dolphin head shows a very complex structure with unique air sacs and special sound-conducting fats. At the same time, Reference [49] claimed that no paranasal air sinuses form within the skull either prenatally or postnatally. In addition, [8] described the presence of pterygoid and maxillary sinuses with a heterogeneous substance in sections III and IV as we found in sco2 (Figure 3(R2A)). These sinuses may be non-functional. In contrast, references [26,41] describe only the pterygoid sinus (filled with a heterogeneous substance) in CT and anatomical sections in the bottlenose dolphin, whereas the maxillary sinus was the only sinus detected in our study. The paranasal sinuses heat the air and decrease the skull weight in domestic mammals. In cetaceans, moving in a less gravid environment could lead to regression of these cavities. Reference [1] said that there are no paranasal sinuses in *Tursiops truncatus*, and that in the remaining odontocetes, the maxillary sinus is described (Figure 3) (probably without functionality), though it is not well studied in whales. On the other hand, [10] described the ethmoidal sinus and [50] the pterygoid sinuses, which we have not observed in our specimens studied. We have located in striped dolphin some small orifices in the frontal wall of the nasal skull that clearly connect the nasal cavity with the maxillary sinus; however, we do not know if nasal mucosa closes these orifices completely or are vascular nutrition orifices.

The nasal cavities are symmetrical and present a similar diameter compared to those of domestic mammals [31,32]. In odontocetes, there is a skull asymmetry where structures of one side (fluctuating, that means, in one individual a structure on the right side is larger, whereas in another individual from the same species, a structure on the left side is larger) are consistently larger than those on the other [51]. This asymmetry is assumed to be based on biosonar production and reception, but it has been suggested that the asymmetry is directly proportional to the prey size, so through evolution, the larynx and the hyoid apparatus have been pushed to the left in order to extend the pharyngeal canal and allow larger prey to be ingested [52].. The ethmoid bone shows small foramina opening into the

nasal mucosa equivalent to the cribriform plate observed in domestic mammals. Nasal conchae and ethmoturbinates are not observed as are found in other domestic mammals [1]. These same authors state that the ethmoid bone in cetaceans forms the dorsocaudal wall of the nasal cavity. In a striped dolphin newborn skull, we observed an ethmoid bone like the cribriform plate in domestic mammals; therefore, we believe it is possible that olfactory mucosa may be present. We agree with [30] who described the presence of "minute foramina" in very young individuals, reminiscent of the ethmoid bone cribriform plate. Reference [28] also talked about a certain olfactory capacity in cetaceans. On the other hand, [1] claims that, according to [53], in Tursiops and other odontocetes the cribriform plate appears unperforated. We did not observed the vomeronasal organ in our anatomical sections or MR sequences under the nasal mucosa and vomer bone (Figure 4).

Reference [28] described the pterygoid bone as enlarged and much pneumatized at the ventral part of nasal cavities (choanae) in beaked whales, showing medial and lateral bone laminae. According to [54], some cetaceans lost the lateral bone lamina and this lamina is replaced by the tendinous lamina, which holds the palate muscles.

#### 4.1.5. Orbit and Eyeball

Reference [55] described an early stage of development (8–15 cm long.) in the pantropical spotted dolphin fetus, noting the lacrimal bone and later (21–22.5 cm long) the zygomatic (jugal) bone well-ossified and rostrally fused firmly with the lacrimal bone. However, in our study using PET/SPECT/CT, it is possible to observe in sco1, ossification centers which are not fused at this early stage (Figure 3). According to [1,12,30], in odontocetes (except beaked whales), the lacrimal and zygomatic (jugal) bones are fused forming the lacrimozygomatic (lacrimojugal) bone, a fact that we have confirmed in this study. The microtomographies performed on sco1 reveals that these two bones show a slightly hypoattenuated contact line indicating that it is a synchondrosis that will become a synostosis, meaning that they will be fused. As in domestic mammals, the lacrimal bone is placed lateral with respect to the zygomatic bone. The caudal projection of the zygomatic bone, the temporal process (zygomatic arch) [30], holds the periorbit and establishes a symphysis with the orbital surface of the temporal bone. The temporal process of the zygomatic bone in domestic mammals forms the infraorbital border together with the periorbit (fibrous fascia), which holds the eyeball and periorbit. In odontocetes, there is no bony infraorbital border and the only structure running under the ventral eyelid is the facial nerve. In this study, the temporal process of the zygomatic bone holding the periorbit at its middle point was observed in dissections and anatomical sections (Figures 3, 4, 5, 6 and 7B).

#### 4.1.6. Central Nervous System

MR sequences of the brain of sco1 begin to differentiate the diencephalon and telencephalon, though the trunk of the encephalon (medulla oblongata, pons, and mesencephalon) and cerebellum are less defined. In sco2, the different parts of the trunk of the encephalon are better defined, except for the cerebellum. In addition, we have observed the cerebellum and brain hemisphere as described in perinatal dolphins [12]. The lateral ventricles and the mesencephalic aqueduct were appreciated in sco1, sco2, and sco3. We have not observed the fourth ventricle using MR sequences either in sco1 and sco2 or in sco3, even though [15] described it in a sub-adult specimen using sagittal MR sequences (Figures 5–7 and 10). Both developmental fetal stages allow us to differentiate the cerebellar tentorium as hyperintense in MR sequences. Therefore, we have observed that the tentorium cerebelli starts ossification during the fetal stage until the process is complete in adult odontocetes, unlike domestic mammals, where it remains membranous, except in cats [31,32]. We suggest the functional explanation for this relates to both species needing a well-held cerebellum due to their activity during swimming (locomotion), jumping, climbing, etc. Fetal and adult dolphin cerebellar hemispheres are situated laterally and parallel staying almost at the same level of the ventral surface of the encephalon trunk, as it can be observed in the 3D reconstruction of a common dolphin fetal brain [14].

#### 4.1.7. Ear

The external acoustic meatus and the auricular cartilage have their origin from the projection of the first two pharyngeal arches toward the pharyngeal cleft which forms the external ear [56]. The external ear is absent in cetaceans and only remains as a small epidermal depression, barely visible in fetal specimens and newborns. At subcutaneous levels, cartilaginous nests of the external acoustic meatus appear hyperattenuated in CT in sco3. The middle ear or tympanic cavity is formed by the dilatation of the bottom of the first pharyngeal pouch and the remainder of the pouch forms the auditory tube, while the inner ear has an ectodermal origin [57]. We see both middle and inner ears in anatomical sections and tomographies as hyperattenuated (Figure 8), compact bone with thin bony walls in the fetus, with the walls thicker and hyperattenuated at the prenatal stage in agreement with [12]. In our study, MR sequences shows as hypointense the middle and inner ear walls while in our three MR sequences the hyperintense areas correspond with the ossicles and tympanic cavity described by [12] in MR T1-weighted sequence.

At the tympanic cavity, we have observed a semi-open musculotubal canal in sco2 [1], whose opening is the tympanic orifice located in the carotid or rostral wall of middle ear. This continues with the auditory tube to its opening at the pharyngeal orifice in the nasopharynx (Figure 6).

The auditory tube in sco4 could be observed in dissections as a mucosal-bony dilated space close to the perpendicular lamina of the palatine bone, extending to the pharyngeal orifice of auditory tube (eustachian notch) [1] where it opens into the nasopharynx by the pharyngeal orifice of the auditory tube. We agree with [58] about the presence of a maxillary sinus and also about the existence of a connection with the middle ear (auditory tube that we observed dilated), but not about the set of aerial sacs (premaxillary, vestibular, and nasofrontal) [30].

In our fetal study, we have just observed in CT some hypoattenuated areas (air cavities) close to the musculotubal canal of the middle ear, which could be a "pseudo-diverticulum" similar to the horse "guttural pouch" or a pharyngeal diverticulum of the auditory tube [30,31]. That would form a rudimentary osteomucomembranous space or guttural pouch, also placed in the cetaceans under the base of the cranium, connecting the tympanic cavity with the pharyngeal orifice of the auditory tube inside the nasopharynx (Figure 6).

#### 4.1.8. Larynx

During feeding, cetaceans need the hyoid apparatus to expand, be flexible and extensible, and to project the larynx caudally (retraction) and the tongue ventrally (depression) in order to allow food to pass into the esophagus. If the hyoid apparatus was rigid, fractures could occur during feeding. In odontocetes and eschrichtiids (gray whales), increased tongue musculature and enlarged hyoids allow grasping and/or lingual depression to generate intraoral suction for prey ingestion [33]. On the other hand, balaenopterids need the mandible to be open to 90◦ so that the oral cavity holds up to 60 m3 of water, so these specialized mechanisms also affect the anatomical model of the mandible and maxilla [59], as happens, for instance, in the humpback whale [60].

Nevertheless, in fetal specimens, we have observed that the entrance to the larynx (larynx peak) is very circumscribed and is formed by the rostral tips of the epiglottic and arytenoid cartilages. Both cartilages are very enlarged and oriented dorsorostrally toward the choanae. The arytenoid cartilages should not be described as cuneiform cartilages [12,29,30] and perhaps as corniculated tubercles [31]. The cuneiform cartilages are only described in the horse epiglottic cartilage and the dog's arytenoid cartilages. The corniculate tubercules are indeed mucosal eminences formed by the corniculated process of the arytenoid cartilages [24,31,32], a feature not observed in the specimens studied (Figure 8).

In the dolphin anatomical section and dissection, we have observed a cartilaginous tympanohyoid (Figure 8(R2A)) connecting the stylohyoid bone with the tympanic part of the temporal bone, placed externally on the mastoid wall of the middle ear [1,61]. Its function is to hold the tongue root and the larynx to the base of cranium as in the domestic mammals [31] while the thyrohyoid attaches to the paracondylar process of the lateral part of the occipital bone—paraoccipital process of the cranial basal bones according to [30], which is different from domestic mammals, where the thyrohyoid attaches to the rostral horn of thyroid cartilage of the larynx [31]. In addition, in the dolphins studied, the caudal tip of the thyrohyoid does not become ossified at birth, thus remaining in the adult as in gray whale [62].

#### 4.1.9. Cranial Cavity

Three fontanelles were observed in our odontocetes fetus studied: occipital, frontal, and mastoid [1] (the last one less clear) and are confused in *Stenella attenuata* [55]. Studies in mysticetes could not add more information for comparison with dolphins [63].

#### **5. Conclusions**

Fetal anatomical sections have been very important to ensure that certain anatomical structures were correctly identified, but we needed the dissections to confirm the presence of these structures. CT was used to identify the bony and cartilaginous features of both the fetal and newborn specimens. On the other hand, different MRI sequences were used to recognize and differentiate visceral structures, which will help clinicians to diagnose different pathologies in the dolphin's head region.

We have also observed that rostral teeth erupt after lactation and the perinatal period helping suctioning milk and to protect the mother's nipples. Moreover, we have observed the absence or atrophy of the masseter muscle from fetal to adult stage in striped dolphins, mainly due to the presence of adipose tissue mixed with random muscle fibers in its anatomical position and because they swallow their prey.

No major salivary glands and lymphatic nodes were observed during developmental stages in dolphin heads, only a mixed mass of muscle fibers and fat.

A maxillary sinus has been observed filled with a heterogeneous content in our study from fetal to perinatal stage and could be non-functional.

The fusion between the lacrimal and the zygomatic bones was observed in the early fetal specimen. The temporal process of the zygomatic bone holding the periorbit in fetal dolphins has been described.

Finally, we can conclude that a "pseudo-diverticulum" similar to the "guttural pouch" connecting the tympanic cavity (middle ear) with the nasopharynx was observed in fetal anatomical sections.

**Author Contributions:** Conceptualization, A.G.d.l.R.y.L. and G.R.Z.; formal analysis, A.A.E. and F.G.C.; investigation A.G.d.l.R.y.L. and F.G.C.; methodology, A.A.E., M.S.L., F.M.G., and A.L.F.; resources, F.M.G. and A.L.F.; supervision, A.G.d.l.R.y.L., A.A.E., and G.R.Z.; writing—original draft, A.G.d.l.R.y.L. and G.R.Z.; writing—review and editing, A.G.d.l.R.y.L., A.A.E., M.S.L., and G.R.Z.

**Funding:** CT and MRI acquisitions were financed by Departamento de Anatomía y Anatomía Patológica Comparadas. Facultad de Veterinaria. Universidad de Murcia. Spain.

**Acknowledgments:** Many thanks to Nuria García Carrillo and Andrés Parrilla for PET/SPECT/CT image support and scan performed in CEIB, Murcia, Spain. We are grateful to José Mª Gómez-Lama López for the CT and MRI scan performed in la Policlínica Virgen de Africa, Ceuta, Spain. Also, grateful to María Leotte Sánchez, for dolphin head dissections at the Departamento de Anatomía y Anatomía Patológica Comparadas, Facultad de Veterinaria, Murcia, Spain. We are also thankful to image technician Oscar Blázquez Pérez for the MRI scan performed at Centro Veterinario de Diagnostico por Imagen del Levante, Ciudad Quesada, Rojales Alicante, Spain. We give special thanks to Mª José Gens Abujas (Oficina de impulso Socioeconómico del Medio Ambiente, Dirección General de Medio Natural, Consejería de Empleo, Universidades, Empresas y Medio Ambiente, Región de Murcia, Spain). We also thank the CRFS Veterinary Team, in a special way, Fernando Escribano Cánovas, Luisa Lara Rosales y Alicia Gómez de Ramón Ballesta, El Valle, Murcia, Spain, for allowing us to have access to the carcasses stranded in their regional area.

**Conflicts of Interest:** The authors of this manuscript have no conflict of interest to declare.

#### **References**

1. Mead, J.G.; Fordyce, R.E. The therian skull. A lexicon with emphasis on the odontocetes. *Smithson. Contr. Zool.* **2009**, *627*, 1–248. [CrossRef]


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

#### *Article*

## **Comparative Anatomy of the Nasal Cavity in the Common Dolphin** *Delphinus delphis* **L., Striped Dolphin** *Stenella coeruleoalba* **M. and Pilot Whale** *Globicephala melas* **T.: A Developmental Study**

	- 3810-193 Aveiro, Portugal; a.lopez@ua.pt

**Simple Summary:** The developmental anatomy of the dolphin head has been studied mostly in single fetuses and few works have been made using a wide range of specimens. In this study, fetal specimens are the main subjects, but newborn, juvenile and adult specimens were also used. Our study analyzes the external nose and nasal cavities during pre- and postnatal development. The nose and nasal cavities were studied using a high-resolution endoscopy to analyze changes in the mucosa of fetal specimens, newborns and juveniles. Magnetic Resonance Imaging (MRI) was also used in fetuses to locate and identify significant structures. Computed Tomography (CT) allowed us to understand the development of the facial bones and the nasal cavity. The histological samples were compared with a horse, a terrestrial mammal with a complex nasal anatomy. Dissections and anatomical sections in two spatial planes were compared with MRI and CT studies. Endoscopy of the external nose showed interesting morphological changes as only two different diverticula (air sacs) were observed in the vestibular part and one recess in the respiratory and olfactory part. We conclude that nasal cavity development of the striped and common dolphins and the pilot whale is simpler than in the bottlenose dolphin and the melon is part of the nose both anatomically and functionally.

**Abstract:** Our goal was to analyze the main anatomical structures of the dolphin external nose and nasal cavity from fetal developmental stages to adult. Endoscopy was used to study the common development of the external nose and the melon, and nasal mucosa. Magnetic resonance imaging (MRI) and anatomical sections were correlated with anatomical sections. Computed tomography (CT) was used to generate 3D reconstructions of the nasal bones and nasal cavities to study its development. Dissections, histological and pathological studies were carried out on the nasal mucosa to understand its function. These results were compared with the horse. Endoscopy showed an external nose with two lips and the upper lip is divided by a groove due to the nasal septum and an

**Citation:** García de los Ríos y Loshuertos, A.; Soler Laguía, M.; Arencibia Espinosa, A.; López Fernández, A.; Covelo Figueiredo, P.; Martínez Gomariz, F.; Sánchez Collado, C.; García Carrillo, N.; Ramírez Zarzosa, G. Comparative Anatomy of the Nasal Cavity in the Common Dolphin *Delphinus delphis* L., Striped Dolphin *Stenella coeruleoalba* M. and Pilot Whale *Globicephala melas* T.: A Developmental Study. *Animals* **2021**, *11*, 441. https://doi.org/10.3390/ ani11020441

Academic Editor: Matilde Lombardero Received: 16 December 2020 Accepted: 28 January 2021 Published: 8 February 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

obstruction of right nasal cavity was diagnosed in a newborn. Two diverticula (air sacs) were found in the nasal vestibule and an incisive recess (premaxillary sac) in the nasal cavity. These findings were corroborated by 3D reconstructions of the nasal cavities, MRI, anatomical sections and dissections. The presphenoid and ethmoid bones were fused at early stages of fetal development. The ethmoid is the last bone to ossify in the nasal cavity.

**Keywords:** striped dolphin (*Stenella coeruleoalba*); common dolphin (*Delphinus delphis*); pilot whale (*Globocephala melas*); fetal development; nose; nasal cavity; endoscopy; PET/SPECT/CT; MRI; sectional anatomy; dissection; 3D reconstruction; histology; ontogeny

#### **1. Introduction**

Through evolution, mammals that colonized the aquatic environment have undergone numerous adaptations in their anatomical structures, especially those of the cephalic region [1]. These adaptative changes are mainly seen in the asymmetry and telescoping of the skull [2–4] and have direct implications in feeding [5], mechanical protection of cephalic structures, echolocation, breathing and diving. The repositioning of the bones of the dorsal skull affect the position of the nasal opening [6], whose dorsal position enables breathing while the animal is on the surface. Along with the air sacs and sinuses which have respiratory, vocal and structural functions [7–10], these modifications in cephalic anatomy results in the most highly evolved nose of all mammals.

There are not many articles in the scientific literature about the cetacean upper respiratory system, and some of the existing studies differ in their descriptions of structures and the terminology used to identify these structures... Most of those articles are about adult specimens, either odontocetes [11–20] or mysticetes [21–24] and many of them focused on sound production and biosonar [14,25,26]. The few papers on fetal specimens [27–32] do not cover different stages of development and only show one fetus without comparison with other fetal stages, essential to the understanding of cetacean ontogeny and its application in the fields of biology and veterinary medicine [29]. The development of the bones and other nasal structures continues post-partum with opening of the blowhole, expansion of the lungs and independent breathing. In the current study, we analyze the nasal complex of 22 odontocetes belonging to three species: (striped dolphin *Stenella coeruleoalba*, common dolphin *Delphinus delphis* and pilot whale *Globicephala melas*) of all ages (16 fetuses of different stages, three newborn, two juvenile and three adults). We applied several diagnostic techniques: computed tomography (CT), magnetic resonance imaging (MRI), anatomical sections, dissections, 3D reconstructions, histology and histopathology. Endoscopy is commonly used in dolphin medicine especially to view the lower respiratory tract (lungs and bronchus) [33–37] but is not often used to visualize the nasal passages, a region of key importance, not only in live animals, but also during necropsies to confirm any pathology incompatible with a correct air passage and therefore likely to interfere with vital activities such as diving and feeding that will decrease the life expectancy of the animal.

A comparison with terrestrial mammals will serve also to describe the structures of the head following the Illustrated Veterinary Anatomical Nomenclature [38] and to better understand the actual function and position of the different hard and soft tissue structures that form the nasal cavity of small cetaceans.

#### **2. Materials and Methods**

#### *2.1. Animals*

A total of sixteen prenatal, three perinatal specimens, two juvenile and four adult dolphins; one foal fetus and two adult horses were used in this study (Table 1). Additional information is located in Table S1 (Supplementary Materials). The mothers of each fetus were stranded along the Spanish Atlantic coast. The youngest newborn specimen was stranded on the Spanish African coast and the two others on the Mediterranean

coast. The juvenile and adult specimens were stranded on the Spanish Mediterranean coast. All stranded dolphin specimens were found dead, two horse cadaver heads were obtained from the Orihuela abattoir; consequently, ethics committee clearance was not necessary. Endoscopic studies were made between September 2019 and January 2020 in a Veterinary Clinic ("Bonafé"), La Alberca (Murcia). Fourteen fetuses, one newborn and one adult specimen were transported to the image analysis units to perform scans. Six fetuses, one newborn and one adult were transported to the dissection room for silicone injections. The silicone was injected through the blowhole towards the nasal cavity, filling the vestibule first. The amount of silicone went from 1 to 8 cm<sup>3</sup> in the fetuses, 16 cm<sup>3</sup> in the newborn and 20–28 cm<sup>3</sup> in the adult. After that, the blowhole was covered with a tight elastic band to avoid backflow of the silicone. Two adult specimens were used to obtain anatomical sections, one of them for histological analysis, one newborn for pathological study and two adults for dissection. One horse specimen was sectioned and the other dissected.


**Table 1.** Specimens of dolphin used in this study.


**Table 1.** *Cont.*

DDE: *Delphinus delphis* from Galicia, Spain; SCOP: *Stenella coeruleoalba* from Galicia, Spain; SCOCE: *Stenella coeruleoalba* from Ceuta, Spain; SCOMU: *Stenella coeruleoalba* from Murcia, Spain; MRI: Magnetic resonance imaging; CT: Computed Tomography, CEMMA: Coordinator Center for the study of the marine mammals, Galicia; CECAM: Center for the study and conservation of marine animals, Ceuta; CRFS: Wildlife rehabilitation Center, Murcia; ECAL: *Equus caballus* from Alicante, Spain; AMIRA® for FEI systems is a software platform for 3D and 4D data visualization, processing and analysis.

#### *2.2. Endoscopy*

A fixed endoscopy unit (Karl Storz Autocon 200, Tuttlingen, Germany) located at Clínica Veterinaria "Bonafé", La Alberca (Murcia), Spain with a camera processor (Storz image 1 hub, camera head Karl Storz Image 1 H3 HD, a Storz power led 175) was used to obtain endoscopic images. For the external nose we employed a forward telescope (0◦ enlarged view, diameter 3 mm, length 14 cm, with incorporated fiber optic light transmission) and for the nasal cavity we used a forward-oblique telescope 30◦, diameter 2.7 mm, length 18 cm, with incorporated fiber optic light transmission.

The endoscopic procedure was performed on the fetuses and juvenile specimens following standard protocols. Each specimen was placed in ventral recumbency and facing the endoscopist. Initial image acquisition was of the external nose morphology, using an optic of 4 mm diameter and 0◦ vision angle. Following this, the nasal cavity was examined by introducing an optic of 2.7 mm and 30◦ vision angle protected by a 3 mm sheath forming an irrigation channel, visualizing first the vestibule, then turning the optic to obtain a complete exposition of this part of the nasal cavity. After visualizing the nasal plugs, observation and study of the respiratory tract within the nasal cavity was conducted. For this purpose, and with the specimens in a lateral position, the endoscope was introduced through the vestibule towards this respiratory part, which allowed us to observe structures such as the incisive recess and the choanae, while irrigating physiological serum through the endoscopy irrigation channel to clean cavities and to obtain good images. All endoscopic images were stored in external and internal hard disks at the CVB (Bonafé Veterinary Clinic) and at the Department of Anatomy and Embryology, Facultad de Veterinaria, Universidad de Murcia, Spain.

#### *2.3. Magnetic Resonance Imaging*

Magnetic Resonance images were obtained with a high-field MR apparatus (General Electric Sigma Excite, Schenectady, NA, USA; Centro Veterinario de Diagnóstico por Imagen de Levante, Ciudad Quesada, Alicante, Spain), 1.5 Tesla using a human quad knee coil (dde1 to 3, 5, 7, 9 to 12, gma1), wrist coil (scop1, dde1) and head coil (dde13 and 14). All dolphin specimens were positioned in ventral recumbency. The MR images were transferred to a DICOM workstation. MR images were analyzed with Radiant DICOM viewer. MRI parameters used are in Table S2 (Supplementary Materials). All MR images were stored in external and internal hard disks at the CVDIL (Veterinary Center for Imaging Diagnosis), Ciudad Quesada, Alicante, Spain and at the Department of Anatomy and Embryology, Facultad de Veterinaria, Universidad de Murcia, Spain.

#### *2.4. Computed Tomography and 3D Reconstruction of Bony Nasal Cavity*

Four fetuses were scanned with Positron Emission Tomography (PET), Single Photon Emission Computed Tomography (SPECT)—Computed Tomography (CT) (PET/SPECT/CT AlbiraTM Systems, Valencia, Spain; Centro de Investigación Biomédica, Universidad de Murcia, Murcia, Spain). The following parameters were used: single-slice: 1 detector arrays; type of acquisition: helical; thickness: 0.125 mm; image reconstruction interval or index: 0.0125 mm; pitch: 0; tube rotation time: 0.12 s; mA: 0.4; Kv: 45; FOV: 68 cm; Matrix dimensions: 2240 × 2360; reconstruction algorithm: FBP filtered back projection; WW: 600/WL: 300).

Three fetuses (one newborn and one adult) were scanned with a CT (General Electric Medical Systems-HiSpeed dual; Hospital Clínico Veterinario, Universidad de Murcia, Spain). All dolphin specimens were positioned in ventral recumbency. The following parameters were used: multislice: two detector arrays; type of acquisition: helical; thickness: 1 mm; image reconstruction interval or index: 2.5 mm; pitch: 0.35; tube rotation time: 1 s; mA: 45 (dde2), 50 (dde6), 55 (scog1, gma1), 60 (dde9,dde13), 75 (scomu1), 80 (dde14), 150 (scomu5); Kv: 120; image field of view: 40 cm; acquisition matrix: 512 × 512; reconstruction algorithm: standard; WW: 400/WL: 40) (Table 1).

All CT images were transferred to a DICOM workstation and CT images were analyzed with AMIRA for Fei Systems 5.6 (Thermo Fisher Sci and Zuse Institute, Berlin, Germany). Volume rendering was generated to obtain 3D renderings of internal anatomy. All CT images were stored in external and internal hard disks at CEIB (Experimental Center of Biomedical Research) building, SAI (Support Research Facility) building and at the Department of Anatomy and Embryology, Facultad de Veterinaria, Universidad de Murcia, Spain.

#### *2.5. Computed Tomography and 3D Reconstruction of Nasal Cavity Spaces*

The nasal cavities of several fetal, newborn and adult specimens were injected with Silicone Xiameter © RTV-4230-E Base, 2 to 30 mL depending on size of specimen, (Dow Corning Co, Midland, MI, USA; Dissection room, Facultad de Veterinaria, Murcia, Spain) to obtain nasal endocasts and to enhance CT contrast properties. Injected specimens were scanned with CT and DICOM images were used to obtain 3D reconstructions of the nasal cavities. Three-dimensional reconstructions of the nasal cavity were obtained using AMIRA for Fei Systems 5.6. All Dicom images were stored in external and internal hard disks at SAI (Support Research Facility) building and at the Department of Anatomy and Embryology, Facultad de Veterinaria, Universidad de Murcia, Spain.

#### *2.6. Anatomic Evaluation: Sectional Anatomy and Dissection Techniques*

One newborn and one juvenile striped dolphin (*Stenella coeruleoalba*) were frozen at −20 ◦C prior to obtaining coronal and sagittal sections of the head. One adult striped dolphin (*Stenella coeruleoalba*) was frozen at −46 ◦C prior to obtaining sagittal sections. All specimens were cut with a band saw (Anatomical Lab., Department of Anatomy and Embryology, Universidad de Murcia, Murcia, Spain), obtaining 0.5–0.7 cm thick slices. Head sections and slices were immersed in 10% formaldehyde for preservation and then stored in a cooling chamber (5 ◦C) at the Department of Anatomy and Embryology, Facultad de Veterinaria, Universidad de Murcia, Spain.

Fourteen fetuses were preserved by immersion in formaldehyde (10%) and two fetuses were fixed with embalming solution injected into the umbilical arteries and veins. In a striped dolphin (*Stenella coeruleoalba*) (scomu5), the external jugular vein and the left and right atria were injected with embalming solution using an electrical pump. After 48 h, the arteries and veins were injected with red and blue latex, respectively. A deep head dissection of one ill newborn (scoce1) was made to observe abnormal anatomy of the vestibule and nasal cavity and an adult specimen was dissected to observe the normal anatomy of nasal cavity (Table 1). All specimens used were stored in a cooling and

freezer chambers at the Department of Anatomy and Embryology, Facultad de Veterinaria, Universidad de Murcia, Spain.

#### *2.7. Histological Analysis*

The mucosa of the nasal cavity (vestibule and respiratory and olfactory parts) was histologically analyzed in one adult dolphin specimen. Elongated rectangles of nasal mucosa were removed at different levels of the nasal cavity. Samples were oriented perpendicular to the paraffin block base and then processed using a special saw. Paraffin blocs were cut to obtain slices. Routine histological processing was carried out and sections were stained with Haemotoxylin and Eosin. Samples were then photographed with a computed light microscope (Axioskop 40, Zeiss, Jena, Germany) with an incorporated Insight 2 Axiocam 105 color camera. Histological sections were stored at the Department of Anatomy and Embryology, Facultad de Veterinaria, Universidad de Murcia, Spain.

#### **3. Results**

#### *3.1. Endoscopic Study of the External Nose and Nasal Cavity*

The endoscopic figures are described by columns, observing in the left column the external nose closed in its natural position to avoid the entrance of amniotic liquid into the lungs.

#### 3.1.1. External Nose

In the youngest fetus (dde1), a common dolphin *Delphinus delphis*, a very small protuberance was observed between the forehead and the melon. It was seen between a semi-circular line (U-shaped) (rima naris), slightly concave right to angulus naris, closing it hermetically. We observed two lips, the upper (caudal) close to the forehead and the lower (rostral) close to the primordial melon. The lower lip was a little bit prominent compared to the upper (Figure 1A). In the next fetus (dde2), a common dolphin (*Delphinus delphis*), both lips are prominent, the upper due to the fact that it underlies the vestibular fold, muscle fibers and the nasal bones mesenchyme and the lower lip, due to the proximity to the developing melon. The lips line (rima) could be now observed as slightly convex (Figure 1C). In the third common dolphin (*Delphinus delphis*) fetus (dde3) (4.5 months gestation) the epidermis of the external nose differs from the forehead epidermis, with a different colour (light pink) and with a clear separation edge of the upper lip and the forehead skin. This lip, now prominent, was clearly divided vertically by a supranasal groove. The lower lip was undivided and elevated right to the contact with the melon, which has a similar tone to that of the nose. The rima naris was slightly convex (Figure 1E). In the next fetus (scop1), a striped dolphin (*Stenella coeruleoalba*), the external nose maintained its change in colour with respect to adjacent areas. The rima naris shows a slight concavity in both angulus. The upper lip is prominent, compared to the common dolphin (*Delphinus delphis*) fetuses with a light groove dividing them in two while the lower lip descends rostrally, forming a valley, extending rostrally to the melon prominence (Figure 1G).

In the pilot whale (*Globocephala melas*) fetus (gma1), the external nose presented a whitish pigmentation extending to the melon, while the pigmentation of the rest of the head is dark grey. The rima naris is slightly convex and differs from the prominent upper lip divided by the supranasal groove. The rostral lip is visible (Figure 2A). The three next fetuses of the common dolphin (*Delphinus delphis*), more developed than the pilot whale (*Globocephala melas*) fetus, showed the same features as the common dolphin (*Delphinus delphis*) fetus (dde3), with a separation area holding the nose and melon, though the depigmentation can only be observed at the upper lip. The convexity in the rima naris is very slight (Figure 2E,I,M).

**Figure 1.** Endoscopic images of the external nose and nasal cavity at the level of nasal vestibule. Images are oriented so that the left side of the head is to the right of the image and rostral is at the bottom (**arrow**). (**A**,**B**) 1.5 months, dde1; (**C**,**D**) 3.5 months, dde2; (**E**,**F**) 4 months, dde3; (**G**,**H**) 4.5 months, scop1. 1, Rima naris; 2, Upper lip; 3, Lower lip; 4, Angulus naris; 5, Nasal groove; 6, Melon; 7, Forehead; 8, Nasal septum: membranous part; 9, Nasal plugs; 10. Vestibule: right diverticulum (vestibular sac); 11, Vestibule: left diverticulum (vestibular sac); 12, Vestibular folds; 13, Delicate hook; 14, Nasal cavity: respiratory part.

**Figure 2.** Endoscopic images of the external nose and nasal cavity at level of the vestibule. Images are oriented so that the left side of the head is to the right of the image and rostral is at the bottom (**arrow**). Detail of monkey or phonic lips from now on (**K**,**L**,**O**). (**A**–**D**) 5 months, gma1; (**E**–**H**) 5.5 months, dde4; (**I**–**L**) 6 months, dde8; (**M**–**P**) 7 months, dde9. 1, Rima naris; 2, Upper lip; 3, Lower lip; 4, Angulus naris; 5, Nasal groove; 6, Melon; 7, Forehead; 8, Nasal septum: membranous part; 9, Nasal plugs; 10. Vestibule: right diverticulum; 11, Vestibule: left diverticulum; 12, Vestibular folds; 13, Delicate hook; 14, Nasal cavity: respiratory part.

The four next common dolphin (*Delphinus delphis*) fetuses, from 7 to 9 months of gestation, mantain the external features described in the last three, though the midline rima naris forms a triangle with respect to the supranasal groove, and it appears as a horizontal line (Figure 3A,E,I). In the last fetus a light concavity is seen (Figure 3M).

**Figure 3.** Endoscopic images of the external nose and nasal cavity at the level of vestibule. Images are oriented so that the left side of the head is to the right of the image and rostral is at the bottom (**arrow**). (**A**–**D**) 7.5 months, dde10; (**E**–**H**) 8 months, dde11; (**I**–**L**) 8.5 months, dde12; (**M**–**P**) 9 months, dde13 1, Rima naris; 2, Upper lip; 3, Lower lip; 4, Angulus naris; 5, Nasal groove; 6, Melon; 7, Forehead; 8, Nasal septum: membranous part; 9, Nasal plugs; 10. Vestibule: right diverticulum; 11, Vestibule: left diverticulum; 12, Vestibular folds; 13, Delicate hook; 14, Nasal cavity: respiratory part; 15, Diverticulum extension.

In the last common dolphin (*Delphinus delphis*) fetus, the depigmentation of the nose and upper lip remains and misses the delimitation of the nose and melon with respect to the rest of the head. The rima naris keeps the concavity towards the angulus naris, commissures or nose angles (Figure 4A). In the next specimen, a newborn striped dolphin (*Stenella coeruleoalba*), showed a hyperkeratosis between the two lips and the right part of upper lip preventing the access of the endoscope (Figure 4E). In the last specimen studied, a juvenile striped dolphin (*Stenella coeruleoalba*), the nose was tightly closed and uniform pigmentation was seen. The upper lip is divided in two by the supranasal groove. The rima naris was curved towards the commissures or angulus naris and presented many folds sinking to the rima. The lower lip is undivided and prominent remaining close to the melon (Figure 4I). Between both nasal lips, there are striations and a double internal fold hermetically sealing the lips (Figure 4J,K,L).

**Figure 4.** Endoscopic images of the external nose and nasal cavity at the level of vestibule. Images are oriented so that the left side of the head is to the right of the image and rostral is at the bottom (**arrow**). (**A**–**D**) 10 months, dde14; (**E**–**H**) newborn, scoce1; (**I**–**P**) juvenile, scomu4. 1, Rima naris; 2, Upper lip; 3, Lower lip; 4, Angulus naris; 5, Nasal groove; 6, Melon; 7, Forehead; 8, Nasal septum: membranous part; 9, Nasal plugs; 10. Vestibule: right diverticulum; 11, Vestibule: left diverticulum; 12, Vestibular folds; 13, Delicate hook, 14. Hyperkeratosis; 15, Striation marks.

#### 3.1.2. Nasal Cavity: Vestibule

In the second, third and fourth columns the external nose was opened allowing us to see the first part of the nasal cavity, the vestibule (Figures 1–4).

After caudal retraction of the skin of the forehead region of the least developed common dolphin (*Delphinus delphis*) fetus (dde1), the nose was opened to expose the vestibule. Only the membranous part of the nasal septum was seen as were the two nasal plugs. The mucosa is of a similar colour of that of the epidermis, a little clearer at the plugs (Figure 1B). In the next specimen (dde2), we could also see the vestibular folds dorsal to the nasal plugs and laterally the entry to the nasal diverticula (Figure 1D). In the third common dolphin (*Delphinus delphis*) fetus (dde3), the lower lip was pulled rostrally in order to observe the vestibule. The nasal plugs could be seen between the nasal septum. The vestibular folds along with the membranous part of the nasal septum stick to the plugs,

closing the meatus tightly. The nasal diverticula (vestibular sacs) (Figure 1F) of the striped dolphin (*Stenella coeruleoalba*) fetus (scop1), already more developed, showed a mucosa with a whitish tone which was uniform throughout the entire vestibule. Also, the meatus was opened between the 'monkey lips' (both the nasal plugs and the vestibular folds) (Figure 1H). In the pilot whale (*Globocephala melas*) fetus (gma1), the mucosal colour of the vestibule remains the same (Figure 2B,C), though it differs from the greyish tone inside the lips (Figure 2B). The mucosa of the nasal diverticulum remained dark and had folds (Figure 2D). In the three next common dolphin (*Delphinus delphis*) fetuses, the vestibular mucosa goes from pink, to grey to white (Figure 2F,J,N), shown in detail (Figure 2K,L,O). The mucosa of the diverticula has greyish folds arranged in a dorsoventral direction (Figure 2G,H,P). The next group of common dolphin (*Delphinus delphis*) fetuses had a more advanced gestation time. A clamp was used to mark the internal extension of one diverticulum (Figure 3B). We subsequently observed prominent nasal plugs, the nasal septum and well-defined folds and a greyish mucosa, different from the earlier fetal periods (Figure 3C,F,J,N,G,L,O and Figure 4B,C). The mucosa of both left and right diverticula was growing in anfractuosity and the longitudinal folds more abundant (Figure 3D,H,P and Figure 4D).

The newborn (scoce1) was diagnosed with epithelial inflammation with local hyperkeratosis probably caused by a congenital alteration. This obstructed the right nasal cavity (Figure 4E). This specimen had a thickened membranous part of the nasal septum so the nasal plugs are barely visible (Figure 4F). Only the left nasal plug and the dilated mucosa of left diverticulum were visible (Figure 4G,H).

In the juvenile striped dolphin (*Stenella coeruleoalba*), the mucosa of vestibule was wrinkled with striations and was tightly adhered to the plug, septum and vestibular folds (Figure 4M,N). The mucosa of the nasal diverticula presented numerous longitudinal folds (Figure 4O,P).

#### 3.1.3. Nasal Cavity: Respiratory and Olfactory Part

In this part of the nasal cavity, nasal cavities (left and right) were analyzed from superficial to deep regions after passing the nasal plugs. Each row corresponds with each studied specimen, with the first row corresponding to the specimen with the earliest fetal development. This part of the study started analyzing more developed specimens in which the nasal cavity diameter allowed easy passage of the endoscope. In the nasal cavity, we could distinguish four walls: the caudal, once the endoscope passed the nasal plug; the rostral wall, ventral to the nasal plug and containing the incisive recess (premaxillary sac). The lateral wall is the external wall of both cavities and finally, the median wall, built by the vomer bone, separating both choanae.

The study starts in a common dolphin (*Delphinus delphis*) fetus of approximately 5.5 months of gestation. We had observed, caudal to the nasal plug, how the mucosa of longitudinal folds was placed vertically towards the floor of the right nasal cavity (Figure 5A). On turning the endoscope rostrally, we observed the incisive recess (Figure 5B). The mucosa of the deepest part of the recess in the left nasal cavity contained small vesicles. Also, at the bottom of the nasal cavity we could see the choanae and the bony portion of the nasal septum (Figure 5C). Unlike the vestibule, the mucosa of the respiratory and olfactory part shows a pinkish colour in every specimen studied. The presence of vesicles in the mucosa and longitudinal folds was clearly seen (Figure 5D–L). Small mucosal fossae close to the choanae were observed mainly in the common dolphin (*Delphinus delphis*) fetus 8.5 months of gestation (Figure 5L).

**Figure 5.** Endoscopic images of the nasal cavity at the level of respiratory and olfactory parts. Arrows show the entry to the incisive recess (3) and to the nasopharynx (4) (**A**,**B**,**H**) Right nasal cavity, (**C**–**G**,**I**–**L**) Left nasal cavity, (**A**–**C**) 5.5 months, dde4; (**D**–**F**) 6 months, dde7. (**G**–**I**) 7 months, dde9; (**J**–**L**) Detailed images. 8.5 months, dde12. Walls indicate orientation. Vertical view. 1. Nasal plug; 2, Longitudinal folds; 3, Incisive recess (premaxillary sac); 4, Choanae; 5, Nasal septum: bony part; 6, Small vesicles; 7, Small fossae; 8, Caudal wall; 9, Rostral wall; 10, Right lateral wall; 11, Left lateral wall; 12, Medial wall.

In the last two common dolphin (*Delphinus delphis*) fetuses closest to birth, we highlight that the incisive recess is more visible and widened and the mucosa had few folds, a flat aspect and a pinkish colour (Figure 6A–F). In the newborn (scoce1), we could only study the left respiratory part of the nasal cavity since the right one was blocked and was impossible to introduce the endoscope due to hyperkeratosis. The mucosa was greyish in colour and there was inflammation of the nasal mucosa folds. The incisive recess could be observed (Figure 6G–I).

In the last specimen studied with the endoscope, the striped juvenile dolphin, we observed the pinkish mucosa and thick folds, the incisive recess and the choanae (Figure 6J–L). Also, small fossae could be seen close to the choanae (Figure 6L).

**Figure 6.** Endoscopic images of the nasal cavity at the level of respiratory and olfactory parts. Arrows show the entry to the incisive recess (2) and to the nasopharynx (5) 9 months, dde13. (**A**–**C**,**L**) Left nasal cavity. (**D**–**K**) Right nasal cavity. (**A**–**C**) 9 months, dde13; (**D**–**F**) 10 months, dde14; (**G**–**I**) Hypertrophied longitudinal folds. Newborn, scoce1; (**J**–**L**) juvenile, scomu4. Walls indicate orientation. Vertical view. 1, Nasal plug; 2, Incisive recess; 3, Nasal folds; 4, Nasal septum: bony part. 5, Choanae; 6, Caudal wall; 7, Rostral wall; 8, Right lateral wall; 9, Left lateral wall; 10, Median wall.

#### *3.2. MRI Study of the External Nose and Nasal Cavity*

MRI were first used in the sagittal plane in order to have an overall vision of the whole nasal cavity and afterwards in three coronal planes at the level of vestibule, the incisive recess and finally at the choanal region.

The least developed specimen of common dolphin (*Delphinus delphis*) dde1, corresponds to first stage of fetal development. The sagittal MRI allowed us to identify the external nose with two lips and the rima naris. The melon is beginning to develop and was observed hyperintense in both sagittal images. The nasal cavity from the vestibule to the choanae is shown as a mass of mesenchymal tissue slightly hypointense in T1 and hyperintense in T2 (Figure 7A,B). The coronal MRI at the vestibule level allows us to identify the nasal septum slightly hypointense at level of the vestibule (L1) and hyperintense at the respiratory part (L2). Nasal cavities were observed always hypointense (Figure 7C,D).

**Figure 7.** Magnetic Resonance Imaging (MRI) of the external nose and nasal cavity. MR sagittal images (**A**,**B**) are oriented so that the rostral is to the left and the caudal to the top. MR coronal images (**C**,**D**) are oriented so that the rostral is to the bottom and the caudal to the top. (**A**) T1-weighted Spin echo (SE) sagittal plane, (**D**) T2-weighted Fast Recovery Fast Spin Echo (FrFSE) sagittal plane. (**C**,**D**) T1-weighted SE coronal plane. (**C**) Level 1. (**D**) Level 2. 1.5 months, dde1. 1, Rima naris; 2, Upper lip; 3, Lower lip; 4, Nasal cavity: vestibule; 5, Nasal septum: membranous part; 6, Nasal septum: bony part; 7. Nasal cavity: respiratory part.

In the fetus of four months' gestation, dde3, the sagittal sections clearly showed the external nose. The nasal cavity vestibule section does not show the developing cavities, although slightly hypointense (diverticula) were noted. The nasal plug could be seen as slightly hyper/hypointense in T1 and T2, and the respiratory part of the nasal cavity was hypointense in T1 and T2. The main bones which form the walls are more distinct within the mesenchyme especially the presphenoid, but the ethmoid bone was not clearly identified (Figure 2A,B). In the coronal sections we could see the nasal plugs hypointense in

T1 and slightly hyperintense in T2. The nasal septum was observed with different intensity in T1 and T2 as well as the vestibule and respiratory parts of the nasal cavity (Figure 8C,H).

**Figure 8.** MRI of the external nose and nasal cavity. MR sagittal images (**A**,**B**) are oriented so that the rostral is to the left and the dorsal to the top. MR coronal images (**C**,**H**) are oriented so that the rostral is to the bottom and the caudal to the top. (**A**) T1-weighted SE sagittal plane, (**B**) T2-weighted FrFSE sagittal plane. (**C**,**E**,**G**) T1-weighted SE coronal plane (**D**,**F**,**H**) T2-weighted FrFSe coronal plane. (**C**,**D**) Level 1, (**E**,**F**) Level 2, (**G**,**H**) Level 3. 4 months, dde3. 1. Rima naris; 2, Upper lip; 3, Lower lip; 4, Nasal cavity: vestibule (left and right diverticula); 5, Nasal plugs; 6, Nasal septum: membranous part; 7, Nasal septum: bony part; 8, Nasal cavity: respiratory part; 9, Choanae: 10, Nasal bone; 11, Frontal bone; 12, Presphenoid bone; 13, Basisphenoid bone; 14, Pterygoid bone; 15, Incisive bone (premaxilla); 16, Maxillary bone; 17, Vomer bone; 18, Ethmoidal mesenchyme.

In a similar development stage (striped dolphin *Stenella coeruleoalba* fetus, scop1), we could better identify the lumen of the respiratory part of the nasal cavity, but the bones were less defined (Figure 9A–H).

**Figure 9.** Images of the external nose and nasal cavity. MR sagittal images (**A**,**B**) are oriented so that rostral is to the left and dorsal to the top. MRI coronal images (**C**–**H**) are oriented so that the rostral is to the bottom and the caudal to the top. (**A**) T1-weighted SE sagittal plane. (**B**) T2-weighted FrFSE sagittal plane. (**C**,**E**,**G**) T1-weighted SE coronal plane, (**D**,**F**,**H**) T2-weighted FrFSE coronal plane. (**C**,**D**) Level 1. (**E**,**F**) Level 2. (**G**,**H**) Level 3. 4.5 months, scop1. 1, Nasal cavity: vestibule; 2, Nasal plug; 3, Nasal cavity: respiratory part; 4, Nasal septum: membranous part; 5, Nasal septum: bony part; 6, Choanae; 7, Melon; 8, Incisive bone; 9, Maxillary bone; 10, Vomer bone.

In the pilot whale (*Globocephala melas*) fetus, gma1, we found better definition of the nasal plugs, the vestibule and respiratory parts of the nasal cavity in the coronal sections and most of the bony nasal cavity. At this stage, the perpendicular lamina of the ethmoid bone was seen clearly using MRI (Figure 10A–D).

**Figure 10.** Images of the external nose and nasal cavity. MR sagittal images (**A**,**B**) are oriented so that the rostral is to the left and the dorsal to the top. MR coronal images (**C**,**D**) are oriented so that rostral is to the bottom and caudal to the top. (**A**) T1-weighted SE sagittal plane, (**B**) T2-weighted FrFSE sagittal plane. (**C**) T1-weighted SE coronal plane. (**D**) T2-weighted FrFSE coronal plane. (**C**,**D**) Level 1. 5 months, gma1. 1, Rima naris; 2, Upper lip; 3, Lower lip; 4, Melon; 5, Nasal septum: membranous part; 6, Nasal plugs; 7, Nasal cavity: vestibule; 8, Vestibule: right diverticulum; 9, Vestibule: left diverticulum; 10, Nasal cavity: respiratory part; 11, Nasal bone; 12, Frontal bone; 13, Presphenoid bone; 14, Ethmoid bone: perpendicular lamina; 15, Incisive bone; 16, Maxillary bone; 17, Vomer bone, 18, Pterygoid bone; 19, Vestibular folds.

In the common dolphin (*Delphinus delphis*) fetus of six months' gestation, dde7, the melon was observed as more developed and we identified the incisive recess using MRI, as well as the respiratory part of the nasal cavity, as a hypointense cavity in T1 and hyperintense in T2 (Figure 11A,B). In the three anatomical sections we also identified the nasal plugs, the nasal septum (membranous and bony parts) and the mesorostral cartilage (Figure 11C–H).

**Figure 11.** Images of the external nose and nasal cavity. MR sagittal images (**A**,**B**) are oriented so that the rostral is to the left and the dorsal to the top. MR coronal images (**C**,**H**) are oriented so that the rostral is to the bottom and caudal to the top. (**A**) T1-weighted SE sagittal plane, (**B**) T2-weighted FrFSE sagittal plane. (**C**,**E**,**G**) T1-weighted SE coronal plane. (**D**,**F**,**H**) T2-weighted FrFSE coronal plane. (**C**,**D**) Level 1. (**E**,**F**) Level 2. (**G**,**H**) Level 3. 6 months, dde7. 1, Rima naris; 2, Upper lip; 3, Lower lip; 4, Melon; 5, Nasal septum: membranous part; 6, Nasal plugs; 7, Nasal cavity: vestibule; 8, Nasal cavity: respiratory part; 9, Nasal cavity: incisive recess; 10, Choanae; 11, Nasal septum: bony part; 12, Mesorostral cartilage.

In an advanced stage of fetal development all the anatomical structures of the nasal cavity could be seen perfectly defined (Figure 12A–H). We identified the perpendicular lamina of the ethmoid bone in the common dolphin (*Delphinus delphis*) fetus of 7.5 month's

gestation (dde10) as hyperintense in T1 and hypointense in T2. Between the frontal and ethmoid bones mesenchyme still is observed (not bony contact) (Figure 12A,B).

**Figure 12.** Images of the external nose and nasal cavity. MR sagittal images (**A**,**B**) are oriented so that the rostral is to the left and the dorsal to the top. MR coronal images (**C**–**H**) are oriented so that the rostral is to the bottom and caudal to the top. (**A**) T1-weighted SE sagittal plane, (**B**) T2-weighted FrFSE sagittal plane. (**C**,**E**,**G**) T1-weighted SE coronal plane. (**D**,**F**,**H**) T2-weighted FrFSE coronal plane. (**C**,**D**) Level 1. (**E**,**F**) Level 2. (**G**,**H**) Level 3. 7.5 months, dde10. 1, Rima naris; 2, Upper lip; 3, Lower lip; 4, Melon; 5, Nasal cavity: vestibule; 6, Nasal plugs; 7, Nasal septum: membranous part; 8, Nasal cavity: respiratory part; 9, Nasal septum: bony part; 10, Choanae; 11, Incisive bone; 12, Maxillary bone; 13, Presphenoid bone; 14. Vomer; 15, Ethmoid bone: perpendicular lamina; 16, Frontal bone; 17, Nasal bone.

In the common dolphin (*Delphinus delphis*) fetus, ten months old, we could see the lumen of the vestibule and of the nasal (vestibular sacs) and accessory diverticula (nasofrontal sacs), all hypointense in T1 and T2. Also, the nasal plugs, the vestibular folds and their associated muscles are moderately hyperintense in T1 and hypointense in T2 (Figure 13A–D). The respiratory part and incisive recess are hypointense in T1 and T2. The incisive recess is surrounded rostrally by the nasal plug muscles and caudally by the incisive and maxillary bones (Figure 13A–D). This recess could also be observed in the coronal slices

extending to the choanae (Figure 13E–H). The majority of the bones forming the nasal cavity have been identified (Figure 13A,B,E,H).

**Figure 13.** Images of the external nose and nasal cavity. MR sagittal images (**A**,**B**) are oriented so that the rostral is to the left and the dorsal to the top. MR coronal images (**C**,**H**) are oriented so that the rostral is to the bottom and caudal to the top. (**A**) T1-weighted SE sagittal plane, (**B**) T2-weighted FrFSE sagittal plane. (**C**,**E**,**G**) T1-weighted SE coronal plane. (**D**,**F**,**H**) T2-weighted FrFSE coronal plane. (**C**,**D**) Level 1. (**E**,**F**) Level 2. (**G**,**H**) Level 3. 10 months, dde14. 1, Rima naris; 2, Upper lip; 3, Lower lip; 4, Melon; 5, Nasal septum: bony part; 6, Nasal plug; 7, Nasal cavity: vestibule; 8, Vestibule: nasal diverticulum; 9, Vestibule: nasal accessory diverticulum (nasofrontal sac); 10, Caudal vestibular muscles; 11, Rostral vestibular muscles; 12, Nasal plug muscles; 13, Nasal cavity: respiratory part; 14, Nasal cavity: incisive recess; 15, Nasal bone; 16, Frontal bone; 17, Ethmoid bone: perpendicular lamina; 18, Incisive bone; 19, Maxillary bone; 20, Presphenoid bone; 21, Basisphenoid bone; 22, Pterygoid bone; 23, Vomer bone; 24, Mesorostral cartilage; 25, Choanae; 26, Pharyngeal muscles.

#### *3.3. Computed Tomography and 3D Reconstruction of Bony Nasal Cavity*

The 3D reconstruction of the skull bones has been performed in order to show nasal cavity development from early fetal stages where the mesenchymal tissue is abundant and the bones of the skull are being formed by intramembranous and endochondral ossification.

In the less developed second fetus (dde2), we could see the main bones forming the rostral part of the bony nasal cavity such as the incisive and maxillary bones and the vomer groove dividing both cavities (Figure 14A,B). The presphenoid (body) and the ethmoid (body) bones are in close contact from an early developmental stage, and could only be distinguished as separate using MRI (Figure 10). In a dorsal view, we observed the bones of the braincase but not the bones separating the nasal and the cranial cavities (Figure 14C). The choanae were observed formed by the palatine and pterygoid bones and divided ventrally by the vomer crest (Figure 14D).

**Figure 14.** 3D reconstruction images of bony nasal cavity using AMIRA and PET/SPEC/CT. 3.5 months, dde2. (**A**) *Dorsal view.* (**B**) *Caudal view.* (**C**) *Dorsal view.* (**D**) *Ventral view*. 1, Incisive bone; 2, Maxillary bone; 3, Nasal face of maxillary bone; 4, Vomer bone: groove; 5, Vomer bone: ventral crest; 6, Presphenoid bone: body; 7, Presphenoid bone: wings; 8, Basisphenoid bone: body; 9, Basisphenoid bone: wings; 10, Ethmoid bone; 11, Maxillary bone (nasal face): ossification nuclei; 12, Frontal bone: cerebral face; 13, Pterygoid bone; 14 Palatine bone; 15, Choanae; 16, Fontanelles.

A more developed striped dolphin (*Stenella coeruleoalba*) fetus, scop1, showed the frontal bone growing towards the incompletely-ossified ethmoid bone (Figure 15A). The rostral wall of the nasal cavity, mainly formed by the maxillary bone, is closing towards the vomer. The ethmoid bone (ethmoidal fossa) remains unossified and the bones at the base of the skull are quite separated (Figure 15B).

At five and a half months, the fetus (dde5) shows an unossified ethmoidal fossa and bony projections from the frontal bone towards the ethmoid bone were observed (Figure 16A). The choanae are clearly seen, but there are still areas lacking ossification (Figure 16B).

In the next common dolphin (*Delphinus delphis*) fetus of almost 6 months' gestation, dde6, the rostral wall of the nasal cavity is almost closed. The maxilloincisive fissure is almost formed and fontanelles are decreasing in size (Figure 17A,B).

**Figure 15.** 3D reconstruction images of bony nasal cavity using AMIRA and PET/SPEC/CT. 4.5 months, scop1. (**A**) *Rostral view.* (**B**) *Caudal view.* 1, Incisive bone; 2, Incisive bone: nasal process; 3, Maxillary bone: external face; 4, Maxillary bone: nasal face; 5, Frontal bone: external face; 6, Frontal bone: nasal face; 7, Frontal bone: cerebral face; 8, Temporal bone; 9, Nasal bone; 10, Basisphenoid bone: body; 11, Basisphenoid bone: wings; 12, Presphenoid bone: body; 13, Presphenoid bone: wings; 14, Ethmoid bone; 15, Vomer bone: groove; 16, Fontanelles; 17, Maxilloincisive fissure (bony naris opening).

**Figure 16.** 3D reconstruction images of bony nasal cavity using AMIRA and PET/SPEC/CT. 5.5 months, dde5. (**A**) *Caudal view.* (**B**) *Ventrocaudal view*. 1, Maxillary bone: nasal face; 2, Frontal bone: cerebral face; 3, Frontal bone: wall projections; 4, Vomer bone: groove; 5, Vomer: ventral crest; 6, Presphenoid bone: body; 7, Presphenoid bone: wings; 8, Ethmoid bone: crista galli; 9, Palatine bone; 10, Pterygoid bone; 11, Fontanelles; 12, Maxilloincisive fissure (bony naris opening).

**Figure 17.** 3D reconstruction images of bony nasal cavity using AMIRA and PET/SPEC/CT. 5.8 months, dde6. (**A**) *Dorsal view.* (**B**) *Caudal view.* 1, Incisive bone: external face; 2, Maxillary bone: external nasal face; 3, Maxillary bone: nasal face; 4, Nasal bone; 5, Frontal bone: external face; 6, Frontal bone: nasal faces; 7, Frontal bone: cerebral face; 8, Frontal bone: wall projections; 9, Vomer bone: groove; 10, Presphenoid bone: body; 11, Presphenoid bone: wings; 12, Ethmoid bone; 12, Fontanelles; 13, Maxilloincisive fissure (bony naris opening).

In the common dolphin (*Delphinus delphis*) fetus of 9 months' gestation, dde13, the walls of the nasal cavities, near to the choanae, are closing as is the rostral wall, though there are still mesenchymal areas (Figure 18A,B).

**Figure 18.** 3D reconstruction images of bony nasal cavity using AMIRA and CT. 9 months, dde13. (**A**) *Dorsal view.* (**B**) *Caudal view.* 1, Incisive bone; 2, Maxillary bone: external nasal face; 3, Pterygoid bone; 4, Frontal bone: external face; 5, Frontal bone: cerebral face; 6, Frontal bone: wall projections; 7, Vomer bone: groove; 8, Basisphenoid bone; 9, Presphenoid bone; 10, Ethmoid bone: crista galli; 11, Fontanelles; 12, Maxilloincisive fissure (bony naris opening); 13, Nasal cavities.

> In the common dolphin (*Delphinus delphis*) fetus, 10 months of gestation, dde14, the rostral wall of the nasal cavity is totally closed by the maxillary bone, forming the bony walls in both cavities at both sides of the ethmoid bone. The lamina cribosa and the vomer wings are incomplete. The choanae are also seen well delimited (Figure 19A,B).

**Figure 19.** 3D reconstruction images of bony nasal cavity using AMIRA and CT. 10 months, dde14. (**A**) *Caudal view.* (**B**) *Ventrocaudal view.* 1, Maxillary bone: nasal face (rostral nasal wall); 2, Maxillary bone: lateral nasal projections; 3, Ethmoid bone; 4, Ethmoid bone: ossification area (lamina cribosa); 5, Presphenoid bone: body; 6, Presphenoid bone: wings; 7, Basisphenoid bone: body; 8, Basisphenoid bone: wings; 9, Frontal bone: external face; 10, Frontal bone: cerebral face; 11, Vomer bone: groove; 12, Vomer bone: wings; 13, Vomer bone: ventral crest; 14, Interparietal bone; 15, Basisphenoid bone: pterygoid crest; 16, Palatine bone; 17, Pterygoid bone; 18, Choanae; 19, Fontanelles.

> The newborn striped dolphin (*Stenella coeruleoalba*), scomu1, shows the caudal wall of the nasal cavity almost closed, with bony projections from the frontal and maxillary bones towards the ethmoid bone. (Figure 20A,B). The lamina cribosa of the ethmoid bone is closing the wall of right and left nasal cavities (Figure 20C,D).

> In the adult striped dolphin (*Stenella coeruleoalba*), three sections of the nasal cavity have been performed (Figure 21A). We observed the bony nasal septum formed by vomer and perpendicular lamina of ethmoid bone and the closed caudal wall of the nasal cavity (Figure 21B) formed by the ethmoidal and cerebral fossae (Figure 21C). In the last section we could see the completely defined caudal region of the nasal cavities (Figure 21D). Several vestigial perpendicular fissures were observed in the ethmoidal fossae and a small cribriform area in the nasal aspect of the ethmoid bone.

**Figure 20.** 3D reconstruction images of bony nasal cavity using AMIRA and CT. newborn, scomu1. (**A**) *Dorsal view.* (**B**) *Frontal view.* (**C**,**D**) *Caudal view.* 1, Incisive bone; 2, Maxillary bone: external face; 3, Maxillary bone: nasal face; 4, Ethmoid bone: ossification nuclei (lamina cribosa); 5, Frontal bone: external face; 6, Frontal bone: nasal face; 7, Frontal bone: cerebral face; 8, Nasal bone; 9, Interparietal bone; 10, Presphenoid bone: body; 11, Presphenoid bone: wings; 12, Ethmoid bone: crista galli; 13, Ethmoid bone: lamina perpendicular; 14, Fontanelles; 15, Maxilloincisive fissure (bony naris opening); 16, Left nasal cavity; 16, Right nasal cavity.

**Figure 21.** 3D reconstruction images of bony nasal cavity using AMIRA and CT. newborn, scomu1. (**A**) Level sections. *Dorsal view*; (**B**) Level 1 (L1), *Rostral view*; (**C**) Level 2 (L2), *Caudal view*; (**D**) Level 3 (L3), *Ventrocaudal view*. 1, Incisive bone; 2, Maxillary bone; 3, Frontal bone; 4, Ethmoid bone; 5, Ethmoid bone: perpendicular lamina; 6, Ethmoidal fossa: lamina cribosa; 7, Vomer bone: ventral crest; 8, Vomer bone: wings; 9, Presphenoid bone: wings; 10, Palatine bone; 11, Pterygoid bone; 12, Mandibles; 13, Basisphenoid bone: body; 14, Basisphenoid bone: wings; 15, Basisphenoid bone: pterygoid crest.

#### *3.4. Computed Tomography and 3D Reconstruction of Nasal Cavity Spaces*

In order to give a better perspective of the respiratory cavity, 3D casts were observed in both lateral and oblique views along with the 3D reconstructions of the skull bones. In the second less developed common dolphin (*Delphinus delphis*) fetus, dde2, we injected

a small amount of silicone, with similar properties to CT iodinate contrast medium, to enable us to obtain an endocast and dilate the cavities to make the 3D reconstruction. Within the vestibule we could already distinguish the bilateral nasal diverticula. The accessory diverticulum was not clearly seen. We could observe the endocast of respiratory and olfactory part of the nasal cavity (Figure 22A). A more developed striped dolphin (*Stenella coeruleoalba*) fetus, scop1, showed the incisive recesses extending to the snout base overlapping the incisive bone (Figure 22B). In the pilot whale (*Globocephala melas*) fetus of five months' gestation, gma1, we could observe the expansion of the incisive recesses as the fetus is developing (Figure 22C). In common dolphin (*Delphinus delphis*) fetus of 5.5 months' gestation, dde5, the left lateral view allowed us to distinguish the accessory nasal diverticulum, which was very small and oriented rostrally (Figure 22D). In the last studied fetus, dde14, we observed the accessory nasal diverticulum ventral to the left nasal diverticulum (Figure 22E). In the adult striped dolphin (*Stenella coeruleoalba*) specimen, scomu1, we saw well-dilated left and right diverticula, the left accessory diverticulum, left incisive recess and parts of the left respiratory and olfactory parts of the nasal cavity. The right accessory diverticulum and right incisive recess were not dilated by air or the injected silicone, so it could not be reconstructed (Figure 22F).

**Figure 22.** Amira 3D reconstructions of nasal cavity spaces after injecting silicone. Hiperattenuated CT images and air spaces were used to obtain the internal endocast. (**A**) *Right rostral aspect*. 3.5 months, dde2. (**B**) *Right lateral aspect*. 4.5 months, scop1. (**C**) *Left rostral aspect*. 5 months, gma1. (**D**) *Left lateral aspect*. 5.8 months, dde6. (**E**) *Left lateral aspect*. 10 months, dde14. (**F**) *Left rostral aspect*. Adult, scomu5. 1, Nasal cavity: vestibule; 2, Vestibule: left diverticulum; 3, Vestibule: right diverticulum; 4, Vestibule: left accessory diverticulum; 5, Nasal cavity: right respiratory part; 6, Nasal cavity: left respiratory part; 7, Nasal cavity: right incisive recess; 8, Nasal cavity: left incisive recess; 9, Choanae.

#### *3.5. Study of the Nasal Cavity Using Sectional Anatomy and Dissection Techniques* 3.5.1. Sectional Anatomy

The sagittal sections were performed in both a juvenile and an adult striped dolphin (*Stenella coeruleoalba*). The coronal sections were performed in a newborn striped dolphin (*Stenella coeruleoalba*) covering the whole nasal cavity. The two first sections are placed in the vestibule of the nasal cavity while the other four are in the respiratory and olfactory part of the nasal cavity.

At the level of the vestibule we could see many vestibular folds, together with the nasal plugs forming the "monkey lips" involved in sound production. We also observed muscles of the external nose, which enable the opening of its caudal lip. We also could see the accessory diverticulum and the vestibule (Figure 23A,B).

**Figure 23.** (**A**,**B**) Sagittal sections of nasal cavity. Sagittal sections images are oriented so that the rostral is to the left and the caudal to the right. (**A**) Level sections (L1–L6), Fresh section, juvenile, scomu3. (**B**) Fixed section, adult, scomu6. 1, Nasal cavity: vestibule (line) and (arrow) accessory nasal diverticulum; 2, Upper lip and vestibular fold muscles; 3, Vestibular fold; 4, Nasal plug; 5, Nasal plug and lower lip muscles; 6, Nasal cavity: respiratory part; 7, Nasal cavity: incisive recess; 8, Choanae; 9, Aditus laryngis; 10, Pterygopalatine recess: pharyngeal recess of pterygoid and palatine bones; 11, Palatopharyngeal muscles (sectioned); 12, Melon; 13, Nasal bone; 14, Frontal bone; 15, Ethmoid bone; 16, Presphenoid bone; 17, Basisphenoid bone; 18, Incisive bone; 19, Maxillary bone; 20, Mesorostral cartilage; 21, Palatine bone; 22, Pterygoid bone; 23, Vomer bone; 24, Interparietal bone; 25, Hypophysis; 26, Oral cavity; 27, Tongue; 28, "Monkey or phonic lips".

> The coronal sections at the vestibule level showed the left and right diverticulum and nasal plugs close to the vestibular fold (Figure 24A,B).

#### 3.5.2. Dissections

#### Dolphin Specimens

The dissections were performed in a newborn (scoce1) stranded in the Mediterranean coast of Ceuta and two adults (scomu5 and scomu7) stranded in the Mediterranean coast of Murcia.

After removing the external nose in the newborn, we observed dorsally the right nasal diverticulum and ventrally the accessory nasal diverticulum caudal to the right nasal plug (Figure 25A). The right nasal plug was clogged and the left one was normal (Figure 25B). We also observed two functional positions of the left nasal plug, one opened and two closed, and the nasal cavities divided by the nasal septum (Figure 25C,D).

**Figure 24.** Coronal sections of nasal cavity. Coronal sections images are oriented so that the rostral is to the left and the caudal to the right. (**A**,**B**) Level 1 and 2. Nasal cavity: vestibule. (**C**–**F**) Levels 3 to 6. Nasal cavity: respiratory and olfactory parts. Newborn, scomu2. 1, Vestibule: right diverticulum; 2, Vestibule: left diverticulum; 3, Vestibular folds; 4, External nose muscles; 5, Nasal plugs; 6, Nasal plug muscles and connective tissue; 7, Melon; 8, Melon muscles; 9, Nasal cavity: respiratory part (nasal mucosae); 10, Nasal cavity: incisive recess; 11, Choanae; 12, Pharyngeal muscles; 13, Nasal septum: membranous part; 14, Nasal septum: bony part (vomer); 15, Mesorostral cartilage; 16, Frontal bone; 17, Nasal bone; 18, Incisive and maxillary bones; 19, Ethmoid bone.

**Figure 25.** Dissection of nasal cavity. (**A**,**B**) Head is oriented so that the rostral side of the head is to the right up corner and caudal is to the left down corner. (**C**,**D**) Head is oriented so that the rostral to the left and the caudal to the right. scoce1. (**A**) Right nasal cavity: vestibule. Clogged right nasal cavity (hyperkeratosis). (**B**) Nasal cavity: vestibule after removed melon skin. (**C**,**D**) Nasal plug opened and closed. Newborn, scoce1. 1, Nasal cavity: right diverticulum; 2, Nasal cavity: left diverticulum; 3, Nasal cavity: accessory diverticulum opened; 4, Accessory diverticulum opened and mucosa partly removed; 5, Right nasal plug clogged; 6, Left nasal plug normal but hypertrophied; 7, Left nasal plug closed; 8, Left nasal plug opened; 9, Right nasal cavity: respiratory part; 10, Left nasal cavity: respiratory part; 11, Nasal septum: bony part (lamina perpendicular ethmoid bone); 12, Frontal bones.

In a preserved female striped dolphin (*Stenella coeruleoalba*), scomu5, the right nasal diverticulum was dilated to better understand its size and form (Figure 26A). In subsequent images, the rostral wall of diverticulum has been removed to show the dark mucosa and ventrally the closed nasal plug (Figure 26B,C). The final images show in detail the closed nasal plug and the access to the accessory diverticulum, first closed (Figure 26D) and then opened showing a portion of the less pigmented mucosa (Figure 26E).

In a female fresh adult striped dolphin (*Stenella coeruleoalba*), scomu7, we could identify the right and the accessory diverticula. The mucosa of both diverticula is dark in colour. The right nasal plug is tightly closed (Figure 27A). The nasal plug has been displaced rostrally showing the respiratory part of the nasal cavity (Figure 27B). The vestibular fold and nasal plug have been displaced caudally and rostrally, respectively, and inside the respiratory part of the nasal cavity we could observe the dark nasal mucosa (Figure 27C,D). Finally, we examined in detail the closed entrance to the left nasal plug and the respiratory part of the right nasal cavity which was patent after removal of the nasal plug (Figure 27D).

**Figure 26.** Dissection of nasal cavity: vestibule. Detailed images of a nasal diverticulum. Adult, scomu5. (**A**) Nasal cavity: vestibule. Right diverticulum dilated. *Right lateral aspect*. (**B**) Nasal cavity: vestibule. Right diverticulum partially sectioned. *Rostral view*. (**C**) Right diverticulum opened. *Rostral view*. (**D**) Right diverticulum opened. *Caudal view*. (**E**) Right diverticulum border displaced dorsally. *Caudal view*. 1, Nasal cavity: right diverticulum (dilated); 2, Right diverticulum: mucosa; 3, Nasal plug; 4, Accessory diverticulum.

**Figure 27.** Dissection of nasal cavity: vestibule and respiratory part. (**A**,**D**) Head is oriented so that the rostral side of the head is to the top and caudal is at the bottom. Adult, scomu7. (**A**) Right nasal cavity: vestibule open. (**B**) Right nasal cavity: nasal plug displaced rostrally. (**C**) Right nasal cavity: nasal plug displaced rostrally and vestibular fold caudally. (**D**) Right nasal plug has been removed and left is observed closed. 1, Nasal cavity: right diverticulum; 2, Accessory diverticulum opened; 3, Right nasal plug; 4, Vestibular fold; 5, Left nasal cavity: left nasal plug; 6, Right nasal cavity: respiratory part.

Horse Specimens

The dissections were performed in one foal fetus (ecal1) and two adult (ecal2 and 3) horses from the slaughter house (Alicante).

The fetal vestibule was observed in its anatomical position showing the alar fold and nostril (false nostril is the portion of the nostril continuous with the nasal diverticulum) which is the orifice of nasal vestibule (Figure 28A–C). Dissections showed the differences between the mucosa of nasal diverticulum, vestibule, alar fold and respiratory part of the nasal cavity (Figure 28B–D).

**Figure 28.** Nose dissections at right nasal vestibule level in a foal fetus (**A**,**B**) and two adult horses. (**C**,**D**). (**B**) Wing of the nostril has been retracted laterally to observe the nasal diverticulum. (**C**) Forceps are inside the nasal diverticulum. (**D**) The nose has been dissected to observe pigmentation differences between mucosa of the vestibule and the respiratory part of the nasal cavity. (**A**–**D**) *Right lateral view*. 1, Alar fold; 2, Nostril; 3, False nostril; 4, Wing of the nostril; 5, Nasal diverticulum; 6, Forceps; 7, Nasal vestibule: mucosa; 8, Nasolacrimal orifice (plastic tube); 9, Nasal cavity: respiratory part.

#### *3.6. Histological Study of the Nasal Cavity* 3.6.1. Dolphin

The vestibular mucosa, specifically its diverticulae, is a stratified squamous epithelium, both pigmented and keratinized. The papillary layer is wide. The connective tissue base is normal and contains small vessels (Figure 29A). The vestibular folds show a connective tissue base with cartilage and striated muscle (Figure 29B). The nasal plug has a stratified epithelium, narrow and pigmented with a papillary layer wide and flattened. It has stratified musculature in discontinued fascicles. Its connective tissue base is strong with muscular fascicles (Figure 29C). The mucosa in the respiratory part has a pseudostratified arrangement but cilia were not observed (10 layers). A deep and narrow papillary layer is

observed. A dense connective tissue base with a few muscular fibers and blood vessels was seen (white areas enlarged and empty) (Figure 29D). The mucosa of the olfactory part is a pseudostratified epithelium but cilia were not seen and it was more rounded and wider than the respiratory part (15–16 layers). The papillary layer is narrow and vascularized with a connective tissue base (Figure 29E). A stratified squamous epithelium lines the incisive recess. The papillary layer is wide with little vascularization. The connective tissue base containing some nerves was seen (Figure 29F).

**Figure 29.** Histological study of nasal mucosa: vestibule, respiratory and olfactory parts. H-E staining technique. Adult, scomu6. (**A**) Left diverticulum, 10×. (**B**) Vestibular fold, 10×. (**C**) Nasal plug, 10×. (**D**) Respiratory part, 10×. (**E**) Olfactory part, 20×. (**F**) Incisive recess, 20×. 1, Epithelium: stratified squamous keratinized and pigmented; 2, Papillary layer; 3, Connective tissue base; 4, Cartilage; 5, Striated muscular base; 6, Vessels.

#### 3.6.2. Horse

The vestibular mucosa of the nasal cavity, specifically its diverticulae, is a stratified squamous epithelium, both pigmented and keratinized with hair and with associated sebaceous glands. The papillary layer is not well defined. The connective tissue base contains some adipose tissue (Figure 30A). The alar fold has a stratified squamous epithelium which

is not keratinized. The connective tissue base is large and dense. There are groups of glands between the sebaceous tissue (Figure 30B). The respiratory part of the nasal cavity has a pseudostratified epithelium, forming a cylindrical mucosa with cilia and a cartilaginous base (Figure 30C). The mucosa of the olfactory part is a pseudostratified epithelium but olfactory cilia were not clearly seen (Figure 30D).

**Figure 30.** Histological study of nasal mucosa: vestibule, respiratory and olfactory parts. H\_E stain technique. Adult Ecal4 (**A**) Nasal vestibule 10×. (**B**) Alar fold 40×. (**C**) Respiratory part. 40×. (**D**) Olfactory part. 20×. 1, Epithelium: stratatified squamous keratinized and pigmented; 2, Papillary stratum; 3, hair; 4, Fat tissue; 5, Connective tissue base; 6, Cartilage; 7, Respiratory epithelium.

#### **4. Discussion**

#### *4.1. Anatomical, Comparative and Functional Commentaries*

#### 4.1.1. External Nose

The external nose of adult odontocetes and mysticetes is well described [10,11,15,16,19, 21,25,26,41–44] but the information is sometimes confusing, often due to differing terminology. Our study goes from the first development stages of the fetus to the adult stage.

The endoscopy technique to analyze the nasal cavity has allowed us to observe the external nose and nasal cavity morphology caudal to the choanae. In mammals, the external nose is part of the face rostral to the frontal region and dorsal to the infraorbital, buccal and oral region [38]. We have observed the highly characteristic morphology of the cetacean nose with the endoscopy images. The presence of nasal lips (rostral and caudal) and its anatomical position, closed from the beginning of development to avoid the entrance of water and salt into the nasal cavity is one of the first image proofs of the development of the two prominences in the caudal lip and their division by a median groove from the nasal septum to the caudal lip. During early stages of fetal development the external nose and the melon were observed as a common anatomical area delimited by a line and covered by an epidermis paler than the rest of the head. Together with the function of the melon in the projection of sounds produced by "phonic lips" [6,43,44], this supports our idea that the melon is forming part of the external nose both anatomically and functionally, and not merely as a part of the nasal complex.

These changes in morphology will be helpful in determining the stages of fetal development.

The MRI and dissection images allowed us to observe, as well as other authors [19,25,26,41] that both the caudal and the rostral lips present retractor muscles which act to open the nose for breathing when the animal rises above the water. Also, we have observed that the most retracted lip was the caudal one.

We also discovered that closing of the lips becomes more airtight as development progresses to adulthood, whose particular anatomy forces these lips to double their size with an increased number of striations, rendering them more waterproof.

#### 4.1.2. Nasal Cavities

The nasal cavity of odontocetes is similar to that of mysticetes except in the sperm whale, a species with a unique morphology [13,18,42].

The odontocete nasal cavity has only one nostril (naris) and one vestibule with paired diverticula, and two respiratory and olfactory parts divided by a nasal septum and ending caudally at the choanae. The equine nose has two nostril, two vestibules of the nose and a cutaneous blind sac (diverticulum nasi) [38]. Except for the sperm whale, the cetacean nasal cavity has a vertical orientation and the external nose and melon are located dorsally [6] with the choanae ventrally, and as described before, the nasal bones are retracted towards the frontal bones [1–3,15]. Therefore, we suggest that the cetacean nasal cavity should be more properly called the maxillary cavity.

#### Vestibule

The nasal cavity vestibule is a space described in all the terrestrial mammals as the hall of the nasal cavity rostral to therespiratory and olfactory part. Though theoretically it is a simple part of this cavity, most of the studies define it in the bottlenose dolphin as a very complex area [9,15] and in our opinion it is not fully explained in previous publications. The presence of two diverticula of the nasal vestibule is clearly shown by endoscopy, 3D reconstructions, sections and dissection studies. These diverticula present a pigmented mucosa as in other species [18] and also in the dissections, sections and histology of our study. The stratified squamous epithelium is similar to that of the equine nose and corroborates our idea about its function, which is protection from external agents and water [17,28]. The vestibular fold shows a very different mucosa from that of the respiratory region, probably related to is proposed function in sound production. Endoscopy permitted us to observe the morphology of the right and left diverticula as spaces with many folds and an anfractuous arrangement as in other cetacean species [17]. The nasal endocast and the volumetric reconstructions enabled us to study its morphology and to locate another accessory nasal diverticulum that we confirmed by dissections. Though we know the bottlenose dolphin vestibule morphology as very complex [9,15,26], our developmental study shows that the vestibule in common and striped dolphins *Stenella coeruleoalba* and pilot whale *Globocephala melas* is less complex.

Another important aspect of our study is the endoscopic examination of the "monkey or phonic lips" which are responsible for sound production [6,43,44]. According to our study, the dorsal parts of the sound production mechanism were the vestibular folds placed under the upper lip and the ventral parts were the nasal plugs divided by the membranous part of the nasal septum. According to our anatomical idea, the "monkey or phonic lips" are solely in charge of producing sound projected towards the melon. Diverticula, wrongly named as air sacs [7–10] because they are only present in birds in order to decrease the total weight during flight. The bronchi extend outside the lung in a form of thin-walled transparent chambers called air sacs [45] extending through the celomic cavity and bones. Diverticula avoid the entrance of possible water filtrations through the blowhole that could have entered during sound production, diving or under a stressful situation such as vessel crash or an underwater shark attack, since they are very far from the phonic lips. It is interesting to observe that vestibular folds lack an epithelium and present a connective tissue and muscular base explaining its function as sound generator.

#### Respiratory and Olfactory Parts of the Nasal Cavity

This region delivering air towards the lungs is characterized by the absence of nasal conchae or cornettes and paranasal sinuses except for the maxillary sinus [28] which is absent in the adult. The maxiloturbinate, well developed in terrestrial mammals, is vestigial in odontocetes, though there are differences between mysticetes and odontocetes [31]. If the function of heating air at this level of the nasal cavity is the main goal in the domestic mammals due to the presence of cornettes and sinuses. In cetaceans it is not the case. Also, it seems that these structures lack the olfactory function in odontocetes, as our histological analysis did not find olfactory epithelium.

#### Bony Nasal Cavity

The tomographic study of the fetal nasal cavity permitted the analysis of the interior of the cavities Volumetric reconstruction of the nasal cavity allowed us to determine the stages of ossification of the nasal cavity The maxillary bones forming the rostral wall are first to form followed by the frontal bones closing slowly towards the cranium base, but they only slowly ossify and connect with the lamina cribosa of the ethmoid bone. The final bone to ossify is the ethmoid bone, which indicates that it is to allow the passage of the olfactory nerves and form the lamina cribosa, even though the olfactory bulb is vestigial in cetaceans and persists as a remnant of phylogeny [21,30]. The vomer bone is growing rostrally slowly and we can see the groove on which the mesorostral cartilage will be placed. The bones closing the ventral floor of nasal cavity are the pterygoid and the palatine [31]. The ethmoid bones take longer to ossify and close the caudal wall of the nasal cavity which was visible in the volumetric reconstructions. The mold of the respiratory and olfactory parts showed two cavities bending caudally towards the nasopharynx surrounded by the aforementioned bones.

#### Nasal Mucosa

The endoscopic study of the nasal cavity shows at the early developmental stage a smooth mucosa which forms longitudinal folds as development progresses. We have not seen this described by other authors using other techniques, including endoscopy [34,35]. The endoscope allowed us to examine the incisive recess (premaxillary sac according to other authors [8,12,14]) that our study suggests serves to store, along with the vestibular sac [19], water escaping from the nasal vestibule which is expelled when the animal emerges to breathe [28]. The extent of the recesses could be seen in the nasal casts, as was their growth during fetal development, findings supported by the MRI data, anatomical sections and even dissection. As seen in other endoscopy studies [46] small mucosal fossae were detected close to the choanae whose function is uncertain. The nasal folds are very abundant in the adult and the lumen was narrow. Histological analysis of the nasal vestibule produced similar findings to the nasal diverticulum of the horse [7] (Figure 29A and Figure 30A) and the respiratory part had a respiratory mucosa not so similar to that of the horse), but we have found few histological studies at these levels [34,42,46]. The spherical nuclei of the sensorial cells were not observed in the olfactory part confirming the absence of olfactory function (Figure 29E).

#### Pathological Findings

The endoscopic study detected an obstruction in the right nasal cavity vestibule in the newborn from Ceuta stranded with its mother and dying afterwards. Post mortem pathologic analysis confirmed this diagnosis. Many stranded calves with respiratory problems are not fully examined due to the expense of the procedure and insufficient equipment. Using endoscopy, parasites were detected, belonging mainly to two families (*Pseudaliidae* and *Prassicaudidae*) and four genera (*Halocercus*, *Pharurus*, *Pseudalius* and *Stenurus*) [47,48]. This often undiagnosed infestation, along with the obstructions could lead to stranding of neonatal cetaceans. Except for rare cases where it is used during necropsies [46], endoscopy is restricted to live animals to examine of the lower respiratory tract (bronchis) and to diagnose the respiratory pathologies of parasitic, bacterial or fungal kind, either by direct visualization, bronchial flushes, or taking biopsies [36,37]. When necropsies are performed, we think that endoscopy of the nasal cavity should be included in necropsy protocols, despite the difficulty of the technique.

#### **5. Conclusions**

Fetal anatomical endoscopic study allowed us to observe the simultaneous development of the melon and the external nose. Also, we have also seen the form and function of the external nose showing a closing formed by two lips very simple during fetal development and very sophisticated at adult stage. The vestibule showed us the "monkey lips", two diverticula and two incisive recesses. Longitudinal mucosal folds were seen in the respiratory and olfactory parts. 3D reconstructions of nasal spaces and nasal skull gave us a spatial representation of nasal cavity development and confirmed our endoscopic observations. MRI data of sagittal and coronal sections using T1 and T2 MRI sequences were important in confirming that bony anatomical structures were correctly identified such as the presphenoid and ethmoid bone. Also, sectional anatomy and dissections aided our identification of structures seen in endoscopic, CT and MRI studies.

The histological analysis has confirmed the similarity of the dolphin nasal mucosa compared with horse nasal mucosa but cilia were not observed in the respiratory part of the nasal mucosa in odontocetes. Pathological findings which showed hyperkeratosis within the nasal vestibule should be taken into account during necropsies in stranded specimens.

The retraction of the nasal bones, the vertical position of the nasal cavities and the special bony walls leads us to recommend that "nasal cavity" should be referred to as "maxillary cavity" as these bones close the respiratory space in dolphins as nasal bones do in domestic mammals.

The nasal vestibule contains different cavities such as the nasal diverticulum which is described in horses. The cetacean diverticulum functions as a water reservoir and protection against external agents. Vestibular folds are comparable to the modified alar folds described in the horse.

The fusion between the ethmoid and presphenoid bones was seen in the second early fetal specimen.

Finally, we conclude that the lamina cribosa of the ethmoid bone is the last bone to ossify in the nasal cavity in a newborn specimen. It shows a small perpendicular fissure at both sides of the crista galli and a small cribriform area in the nasal aspect of the ethmoid bone. Olfactory cells were not detected during histological analysis of the nasal mucosa.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/2076-261 5/11/2/441/s1, Table S1: Other parameters observed in this study, Table S2: MRI parameters used in this study.

**Author Contributions:** Conceptualization, A.G.d.l.R.yL. and G.R.Z.; Formal analysis, A.A.E. and N.G.C.; Investigation, A.G.d.l.R.yL.; Methodology, A.A.E., M.S.L., F.M.G., A.L.F., P.C.F. and C.S.C.; Resources, F.M.G., A.L.F., P.C.F., N.G.C. and C.S.C.; Supervision, A.G.d.l.R.yL., A.A.E. and G.R.Z.; Writing—original draft, A.G.d.l.R.yL. and G.R.Z.; Writing—review & editing, A.G.d.l.R.yL., A.A.E., M.S.L. and G.R.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** CT and MRI acquisitions were financed by Departamento de Anatomía y Anatomía Patológica Comparadas. Facultad de Veterinaria. Universidad de Murcia. Spain. The Galician stranding network is supported by the regional government Xunta de Galicia—Dirección Xeral de Patrimonio Natural. CESAM/FCT: thanks are due to FCT/MCTES for the financial support to CESAM (UIDP/50017/2020+UIDB/50017/2020), though national funds. Norma transitoria. Alfredo López is funded by national funds (OE), through FCT—Fundaçâo para a Ciência e a Tecnologia, I.P. in the scope of the framenetwork contract foreseen in numbers 4, 5 and 6 of the article 23, of the Decree-Law 57/2016, of 29 August, changed by Law 57/2017, of 19 July.

**Institutional Review Board Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We are grateful to Consejería de Sanidad, Ceuta, Spain. Especial thanks to CESAM and Xunta de Galicia, Spain. We thank CEMMA volunteers that helped with necropsies and sample collection. We are grateful to Miguel Ángel Gómez Sánchez and Serafín Gómez Cabrera for the Histological and Anatomopathological analysis, respectively at the Departamento de Anatomía y Anatomía Patológica Comparadas, Facultad de Veterinaria, Murcia, Spain. We are also thankful to image technician Oscar Blázquez Pérez for the MRI scan performed at Centro Veterinario de Diagnostico por Imagen del Levante, Ciudad Quesada, Rojales Alicante, Spain. We give special thanks to Mª José Gens Abujas (Oficina de impulso Socioeconómico del Medio Ambiente, Dirección General de Medio Natural, Consejería de Empleo, Universidades, Empresas y Medio Ambiente, Región de Murcia, Spain). Thank you very much to the CRFS Veterinary Team, in a special way, Fernando Escribano Cánovas, Luisa Lara Rosales and Alicia Gómez de Ramón Ballesta, El Valle, Murcia, Spain, for allowing us to have access to the carcasses stranded in their regional area.

**Conflicts of Interest:** The authors of this manuscript have no conflict of interest to declare.

#### **References**


#### *Article*

## **Endoscopic Study of the Oral and Pharyngeal Cavities in the Common Dolphin, Striped Dolphin, Risso's Dolphin, Harbour Porpoise and Pilot Whale: Reinforced with Other Diagnostic and Anatomic Techniques**

**Álvaro García de los Ríos y Loshuertos 1,2, Marta Soler Laguía 3, Alberto Arencibia Espinosa 4, Francisco Martínez Gomariz 1, Cayetano Sánchez Collado 1, Alfredo López Fernández 5,6, Francisco Gil Cano 1, Juan Seva Alcaraz <sup>1</sup> and Gregorio Ramírez Zarzosa 1,\*,†**


**Simple Summary:** Developmental studies of the dolphin oral cavity have been scarce and were mostly carried out on adult specimens dealing with teeth and lingual development. Moreover, the adult pharyngeal cavity has been mentioned in cetacean monographic encyclopedias and handbooks. In this work, prenatal and perinatal studies of both the oral and pharyngeal cavities were performed on juvenile and adult specimens to better understand these anatomical structures. Our study analyzes these cavities using high-resolution endoscopy to observe changes in the mucosa and to compare these findings with terrestrial mammals. Even though endoscopy was the main technique used, our study was reinforced with Magnetic Resonance Imaging (MRI), anatomical techniques and fetal histology to locate and identify significant structures. Endoscopy of the oral cavity showed some interesting morphological changes. The incisive papilla, teeth, tongue papillae and lateral sublingual recesses and folds were observed in different development stages. The three different parts of the pharynx (oropharynx, laryngopharynx, and nasopharynx) were examined using endoscopy. The histological study helps us to understand the function of the pharyngeal cavity. The nasopharynx contained important structures such as the orifice of the auditory tube and its expansion, the pharyngeal diverticula of the auditory tubes. This special anatomical area was studied using MRI, serial sections and dissections. Some functional considerations are made about both cavities in the five species of odontocetes studied.

**Abstract:** In this work, the fetal and newborn anatomical structures of the dolphin oropharyngeal cavities were studied. The main technique used was endoscopy, as these cavities are narrow tubular spaces and the oral cavity is difficult to photograph without moving the specimen. The endoscope was used to study the mucosal features of the oral and pharyngeal cavities. Two pharyngeal diverticula of the auditory tubes were discovered on either side of the choanae and larynx. These spaces begin close to the musculotubaric channel of the middle ear, are linked to the pterygopalatine recesses (pterygoid sinus) and they extend to the maxillopalatine fossa. Magnetic Resonance Imaging (MRI), osteological analysis, sectional anatomy, dissections, and histology were also used to better

**Citation:** García de los Ríos y Loshuertos, Á.; Soler Laguía, M.; Arencibia Espinosa, A.; Martínez Gomariz, F.; Sánchez Collado, C.; López Fernández, A.; Gil Cano, F.; Seva Alcaraz, J.; Ramírez Zarzosa, G. Endoscopic Study of the Oral and Pharyngeal Cavities in the Common Dolphin, Striped Dolphin, Risso's Dolphin, Harbour Porpoise and Pilot Whale: Reinforced with Other Diagnostic and Anatomic Techniques. *Animals* **2021**, *11*, 1507. https:// doi.org/10.3390/ani11061507

Academic Editors: Matilde Lombardero and Mar Yllera Fernández

Received: 31 March 2021 Accepted: 19 May 2021 Published: 22 May 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

understand the function of the pharyngeal diverticula of the auditory tubes. These data were then compared with the horse's pharyngeal diverticula of the auditory tubes. The histology revealed that a vascular plexus inside these diverticula could help to expel the air from this space to the nasopharynx. In the oral cavity, teeth remain inside the alveolus and covered by gums. The marginal papillae of the tongue differ in extension depending on the fetal specimen studied. The histology reveals that the incisive papilla is vestigial and contain abundant innervation. No ducts were observed inside lateral sublingual folds in the oral cavity proper and caruncles were not seen in the prefrenular space.

**Keywords:** Striped dolphin (*Stenella coeruleoalba*); Common dolphin (*Delphinus delphis*); Pilot whale (*Globicephala melas*); Risso's dolphin (*Grampus griseus*); Harbour porpoise (*Phocoena phocoena*); fetal development; mouth; buccal; oral; pharyngeal; cavity; endoscopy; sectional anatomy; dissection; histology; ontogeny; MRI

#### **1. Introduction**

The anatomy of the cephalic region of marine mammals has undergone many evolutionary changes [1]. Amongst all those adaptations in cetaceans, the complete independence of the digestive and respiratory system is one of the most important and has direct implications in the colonization of the aquatic environment. Even though the process of elongation of the skull and the migration of certain flat bones mainly affected the nasal and cranial cavities, the oral cavity also underwent a change in comparison with terrestrial animals, and the repositioning of these bones and the vertical placement of the nasal structures meant that the nasal bones and orifice disappeared from the face.

Inside the cavities surrounded by these bones, the muscles, organs and other anatomical structures also adapted to the new aquatic environment. The tongue, which occupies most of the space within the oral cavity, has the most important role (followed by the teeth) in maintaining the thermoregulatory and feeding [2] functions of terrestrial mammals, but also in acquiring the new function of expelling water from the mouth after feeding, a feature especially seen in Mysticetes.

The oral cavity is not the most studied region in the cetacean head, when compared, for example, with the nasal cavity [3–8]. The snout is a sensitive anatomical structure, the first one to have contact with the environment and it performs specific interactions, such as courtship (epicritic tactile sensibility) and defence (protopathic tactile sensibility). There are few articles in the scientific literature about the oral cavity and pharynx, and some of the existing studies may need to be clarified. Most of these articles are about adult specimens, [9–12], and the few papers on development, both in mysticetes [13,14] and odontocetes [15,16], focus mainly on the tongue of neonates, and do not cover different stages of development, being restricted to only one species [17].

Throughout gestation, bones and other oral structures are in the process of development and their formation continues after birth. Endoscopy is commonly used in dolphin medicine, especially to view the lower respiratory tract (lungs and bronchus) [18–23]. It is not often used to visualize the oropharyngeal tract, a region of key importance in stranded dolphins where pathological changes (such as tooth infections or oral obstructions) can be the cause of death.

We include the pharyngeal cavity, as food goes towards the oesophagus and stomach via the laryngopharynx. A comparison with terrestrial mammals will also serve to describe the structures of the head, following the Illustrated Veterinary Anatomical Nomenclature [24] and will aid in our understanding of the function and position of the different structures forming the buccopharyngeal cavity of our small cetaceans.

#### **2. Materials and Methods**

#### *2.1. Animals*

In the current study, we analysed the oral complex of 24 odontocetes belonging to five species (*Stenella coeruleoalba*, *Delphinus delphis*, *Globicephala melas*, *Grampus griseus* and *Phocoena phocoena)* of all ages (17 fetuses of different stages, three newborn, two juveniles and two adults) carrying out several diagnostic techniques on all specimens: namely, endoscopy, magnetic resonance imaging (MRI), dissections and histology (Table 1). Additional information is contained in Table S1 (Supplementary Materials). The mothers of each fetus were stranded along the Spanish Atlantic and Mediterranean coasts. The newborn and adult specimens were stranded along the Spanish Mediterranean coast. All stranded specimens were found dead and ethics committee clearance was not necessary. An endoscopic study was carried out, transporting sixteen fetuses and one juvenile to Veterinary Clinic "Bonafé", La Alberca, Murcia, (Spain) during November 2020. Eight fetuses were transported to the MRI unit to perform scans. One newborn, one juvenile and one adult specimen were used to obtain anatomical sections, the same adult and two fetuses for histological analysis and one newborn for dissection.

**Table 1.** Specimens of dolphin used in this study. dde: *Delphinus delphis* (Linnaeus 1758) from Galicia, Spain; scop: *Stenella coeruleoalba* (Meyen 1833) from Galicia, Spain; gma: *Globicephala melas* (Traill 1809) from Galicia, Spain; scoce: *Stenella coeruleoalba* (Meyen 1833) from Ceuta, Spain; scomu: *Stenella coeruleoalba* (Meyen 1833) from Murcia, Spain; phog: *Phocoena phocoena* (Linnaeus 1758) from Galicia, Spain; grgr: *Grampus griseus* (Cuvier 1912) from Valencia, Spain; MRI: Magnetic resonance imaging.


#### *2.2. Endoscopy*

A fixed endoscopy unit (Karl Storz Autocon 200, Tuttlingen, Germany) located at Clínica Veterinaria "Bonafé", La Alberca (Murcia), Spain with a camera processor (Storz image 1 hub, camera head Karl Storz Image 1 H3 HD, a Storz power led 175) was used to obtain endoscopic images. For the oral, oropharyngeal and laryngopharyngeal cavities, we employed a forward telescope (0◦ enlarged view, diameter 4 mm, length 14 cm, with incorporated fiber optic light transmission) and for the nasopharyngeal cavity, we used a forward-oblique telescope 30◦, diameter 2.7 mm, length 18 cm, with incorporated fiber optic light transmission.

The endoscopic procedure was performed on the fetuses and juvenile specimens following standard protocols. Each specimen was placed in ventral recumbency and facing the endoscopist. Initial image acquisition was of the oral, oropharyngeal and laryngopharyngeal cavities using an optic of 4 mm diameter and 0◦ vision angle. Following this, the pharyngeal cavity was examined, first visualizing the oropharynx, then turning the optic to obtain a complete exposition of this part of the pharyngeal cavity. After visualizing the epiglottic mucosa, the endoscope was introduced through the left pyriform recess towards the oesophageal vestibule to reach the laryngoesophageal limit and the oesophageal mucosa.

The nasopharyngeal cavity was examined by introducing an optic of 2.7 mm and 30◦ vision angle protected by a 3 mm sheath forming an irrigation channel, visualizing first the vestibule, then the nasal plugs to the nasal cavity and finally the nasopharynx. All endoscopic images were stored in external and internal hard disks at the CVB (Bonafé Veterinary Clinic) and at the Department of Anatomy and Embryology, Facultad de Veterinaria, Universidad de Murcia, Spain.

#### *2.3. Magnetic Resonance Imaging*

Magnetic Resonance (MR) images were obtained with a high-field MR apparatus (General Electric Sigma Excite, Schenectady, NA, USA; Centro Veterinario de Diagnóstico por Imagen de Levante, Ciudad Quesada, Alicante, Spain), 1.5 Tesla using a human quadknee coil (dde3, 5, 8, 11,12, gma1) and head coil (dde13-14, grgr1). Images were used to analyze the oral and pharyngeal cavities, and for a special study of the pharyngeal diverticulum of the auditory tube during fetal development. All dolphin specimens were positioned in ventral recumbency. The MR images were transferred to a DICOM workstation. MR images were analyzed with Radiant DICOM viewer. MRI parameters used are in Table S2 (Supplementary Materials). All MR images were stored in external and internal hard disks at the CVDIL (Veterinary Center for Imaging Diagnosis), Ciudad Quesada, Alicante, Spain and the Department of Anatomy and Embryology, Facultad de Veterinaria, Universidad de Murcia, Spain.

#### *2.4. Anatomic Evaluation: Sectional Anatomy and Dissection Techniques*

One newborn, one juvenile and one adult *Stenella coeruleoalba* were frozen at −20 ◦C prior to obtaining coronal and sagittal sections of the head. One adult *Stenella coeruleoalba* was frozen at −46 ◦C prior to obtaining sagittal sections. All specimens were cut with a band saw (Anatomical Lab, Department of Anatomy and Embryology, Universidad de Murcia, Murcia, Spain), obtaining 0.5–0.7 cm thick slices. Head sections and slices were immersed in 10% formaldehyde for preservation and then stored in a cooling chamber (3 ◦C) at the Department of Anatomy and Embryology, Facultad de Veterinaria, Universidad de Murcia, Spain.

Sixteen fetuses were preserved by immersion in formaldehyde (10%). Two fetuses and one adult were fixed with embalming solution injected into the fetal umbilical artery and vein, and the adult external jugular vein and left auricule. In a *Stenella coeruleoalba* (scomu5), the external jugular vein and the auricles were injected with embalming solution using an electrical pump. Head coronal and sagittal sections and a deep head dissection of one newborn were made to observe the pharyngeal diverticulum of the auditory tube. One newborn specimen was used to study the normal anatomy of the oral and nasal cavities and one fetus and one adult specimen were used to inspect the bony anatomy of the oral and pharyngeal cavities (Table 1).

All specimens used were stored in a cooling and freezer chambers at the Department of Anatomy and Embryology, Facultad de Veterinaria, Universidad de Murcia, Spain.

#### *2.5. Histological Analysis*

The mucosa of the oral and pharyngeal cavities was histologically analyzed in two fetuses, one *Delphinus delphis* and one adult *Stenella coeruleoalba*. Elongated rectangles of nasal mucosa were removed at different levels of these cavities. Samples were oriented perpendicular to the paraffin block base and then processed using a special saw. Paraffin blocks were cut to obtain slices. Routine histological processing was carried out and sections were stained with Haematoxylin and Eosin. Samples were then photographed with a computed light microscope (Zeiss Axioskop 40, Jena, Germany) with Camera Insight 2 Axiocam 105 color incorporated. Histological sections were stored at the Department of Anatomy and Embryology, Facultad de Veterinaria, Universidad de Murcia, Spain.

#### **3. Results**

#### *3.1. The Oral Cavity*

The oral region is closed during fetal development. We have opened it to view the different parts, starting with the vestibule placed between the lips and the teeth. Beyond the vestibule is the oral cavity proper, which extends from the rostral part of the palatoglossal arch or folds to the lingual aspect of the incisive teeth. The dorsal limit is the oral cavity's roof, and the ventral limit is defined by the tongue and the proper oral cavity and the prefrenular space (Figure 1).

**Figure 1.** Head of a dolphin fetus showing the oral cavity opened. (**A**) Left aspect; (**B**) Right aspect. 6 months, dde7. 1, Upper lip; 2, Lower lip; 3, Angulus oris; 4, Rima oris; 5, Oral vestibule; 6, Oral cavity roof; 7, Tongue and oral cavity floor: 8, Palatoglossal archs or folds; 9, Incisive teeth (not erupted yet); 10, Eyelids (closed); 11, Oral region.

The bony part of the oral cavity's roof is composed of the palatine processes of the maxillary and incisive bones as seen in the *Phocoena phocoena* (phog1). The bony ventral limit of the oral cavity is represented by the left and right mandibles. In this specimen, close to birth, teeth have a conic shape. The intermandibular symphysis can be seen. Four incisive teeth were observed (Figure 2).

**Figure 2.** Bony anatomical base of the oral and pharyngeal cavities in a skull. (**A**) Ventral view. Right and left mandibles. (**B**) Dorsal view. Dots show the extension and form of the right pharyngeal diverticulum of the auditory tube. Photography Francisco Gil Cano. 9 months, phop1. 1, Palatine bone: perpendicular lamina; 2, Palatine bone: horizontal lamina; 3, Basisphenoid bone: body; 4, Vomer bone; 5, Pterygoid bone: medial lamina; 6, Pterygoid bone: lateral lamina (incomplete); 7, Choana; 8, Maxillary bone: palatine process; 9, Incisive bone: palatine process; 10, Temporal bone: petrous and tympanic part; 11; Left mandible: body; 12, Right mandible: body; 13, Dental alveolus; 14, Incisive teeth; 15, Mandibular channel; 16, Mandible: condyle; 17, Bony area of pharyngeal diverticulum of the auditory tube.

#### 3.1.1. Endoscopic Study

In the least developed fetus a *Delphinus delphis* (dde1), the lips are proportionally large and immobile. The oral rim, like the blowhole, is tightly closed. A frenulum of the superior and inferior lips is absent. The buccal vestibule is not well-defined at this stage of development. The gums of the mandibles are wider than those of the maxilla. A hard palate is forming. An incisive papilla was not observed. The tongue is short, wide and without papillae (Figure 3).

**Figure 3.** Endoscopic images of the oral cavity. The arrows point to the tip of the mouth. **L** (Left) **R** (Right). (**A**) Oral cavity open. (**B**) Oral cavity proper. 1,5 months, dde1. 1, Upper lip; 2, Lower lip; 3, Gums; 4, Hard palate; 5, Tongue: tip; 6, Tongue: border; 7, Tongue: dorsum; 8, Tongue: ventral part; 9, Tongue: root; 10, Angulus oris.

In the next fetuses (dde2, dd3), *Delphinus delphis*, a labial vestibule between the gums and lips was observed (not a buccal vestibule). The hard palate shows a middle palatine raphe in all species studied, and only in *Delphinus delphis* are both lateral palatine grooves parallel to the middle hard palate. No transverse ridges of the mucosa of hard palate were observed. We observed the incisive papilla at the tip of hard palate. Marginal papillae extend from the edges to the middle of the tongue. The dorsum of the tongue is smooth with an indistinguishable central groove. In the oral cavity proper, there are two lateral sublingual recesses, with a lateral sublingual fold which thickened rostrally (Figure 4).

**Figure 4.** Endoscopic images of the oral cavity. The arrows show the tip of the mouth. **L** (Left) **R** (Right). (**A**,**B**) Oral cavity open. (**C**) Tongue: lateral part. (**D**) Oral cavity proper. 3,5 months, dde2. 1, Upper lip; 2, Lower lip; 3, Incisive papilla; 4, Labial vestibule; 5, Gums; 6, Hard palate; 7, Palatine raphe; 8, Greater palatine groove; 9, Tongue: tip; 10, Tongue: border; 11, Marginal papilla; 12, Tongue: dorsum; 13, Tongue: ventral part; 14, Lingual frenulum; 15, Lateral sublingual recesses; 16, Lateral sublingual folds; 17, Tongue: root; 18, Angulus oris.

In dde3, the lateral sublingual folds are very thin (Figure 5).

**Figure 5.** Endoscopic images of the oral cavity. In the different images, the arrows point to the tip of the mouth. **L** (Left) **R** (Right). (**A**,**B**) Hard palate. (**C**) Tongue1. (**D**–**G**) Oral cavity proper. 4 months, dde3. 1, Upper lip; 2, Lower lip; 3, Incisive papilla; 4, Labial vestibule; 5, Gums; 6, Hard palate; 7, Palatine raphe; 8, Greater palatine groove; 9, Tip of the tongue; 10, Tongue: border; 11, Marginal papilla; 12, Tongue: dorsum; 13, Tongue: ventral part; 14, Tongue: longitudinal prominence; 15, Lingual frenulum; 16, Lateral sublingual recess; 17, Lateral sublingual folds; 18, Tongue: root; 19, Angulus oris.

The ventral part of the tongue shows a simple lingual frenulum. In the pre-frenular space no sublingual caruncles were observed. Gums are long and smooth (Figures 4 and 5). In the *Stenella coeruleoalba* (scop1), one month older than *Delphinus delphis* (dde3), the hard palate shows a palatine raphe and the incisive papilla is a little larger than in *Delphinus delphis* (dde2). The lateral sublingual folds are thin (Figure 6).

**Figure 6.** Endoscopic images of the oral cavity. The arrows show the tip of the mouth. **L** (Left) **R** (Right). (**A**) Hard palate. (**B**,**C**) Tongue. (**D**) Oral cavity proper. 4,5 months, scop1. 1, Upper lip; 2, Lower lip; 3, Incisive papilla; 4, Labial vestibule; 5, Gums; 6, Hard palate; 7, Palatine raphe; 8, Tongue: tip; 9, Tongue: border; 10, Marginal papilla; 11, Tongue: dorsum; 12, Tongue: ventral part; 13, Lingual frenulum; 14, Lateral sublingual recess; 15, Lateral sublingual folds; 16, Angulus oris; 17, Tongue: root.

In the *Globicephala melas* (gma1), a little older than *Stenella coeruleoalba* (scop1), the hard palate has a deep palatine raphe and the incisive papilla is larger than in other fetuses. The marginal papillae extend from the tip of the tongue almost to the root. The lingual frenulum is thinner than in other small fetuses. The gums are covering the teeth in both superior and inferior arcades. Teeth are well differentiated in this *Globicephala melas* at this stage (5 months). Also, the sublingual lateral folds are thin but their extremities are starting to thicken (Figure 7).

**Figure 7.** Endoscopic images of the oral cavity. In the different images, the arrow points to the tip of the mouth. **L** (Left) **R** (Right). (**A**,**B**) Oral cavity open. (**C**,**D**) Tongue: lateral part. (**E**,**F**) Oral cavity proper. 5 months, gma1. 1, Upper lip; 2, Lower lip; 3, Incisive papilla; 4, Oral vestibule; 5, Gums covering teeth; 6, Hard palate; 7, Palatine raphe; 8, Tongue: tip; 9, Tongue: border; 10, Marginal papilla; 11, Dorsum of the tongue; 12, Tongue: ventral part; 13, Lingual frenulum; 14, Lateral sublingual recess; 15, Lateral sublingual folds; 16, Angulus oris.

> In a more developed fetus (dde8) *Delphinus delphis,* it was possible to observe marginal papillae extending from the tip to the middle of the tongue, and the lateral sublingual folds were thickening throughout their length (Figure 8).

**Figure 8.** *Cont*.

**Figure 8.** Endoscopic images of the oral cavity. In the different images, the arrow points to the tip of the mouth. **L** (Left) **R** (Right). (**A**) Hard palate. (**B**) Tongue. (**C**,**D**) Oral cavity proper. 6 months, dde8. 1, Upper lip; 2, Lower lip; 3, Incisive papilla; 4, Labial vestibule; 5, Gums; 6, Hard palate; 7, Palatine raphe; 8, Greater palatine groove; 9, Tip of the tongue; 10, Tongue: border; 11, Marginal papilla; 12, Tongue: dorsum; 13, Tongue: ventral part; 14, Tongue: longitudinal prominence; 15, Lingual frenulum; 16, lateral sublingual recesses; 17, Lateral sublingual folds; 18, Angulus oris; 19, Tongue: root.

In a *Delphinus delphis* (dde13) close to birth, the teeth of the superior arcade are well developed, unlike those of the inferior arcade. Additionally, the lateral sublingual folds are well dilated (Figure 9).

**Figure 9.** *Cont*.

**Figure 9.** Endoscopic images of the oral cavity. In the different images, the arrow points to the tip of the mouth. **L** (Left) **R** (Right). (**A**) Hard palate. (**B**) Detail of gums. (**C**) Tip of the tongue and prefrenular space. (**D**) Right mandible and lateral part of the tongue. (**E**) Tongue. (**F**) Detail of left lateral sublingual recess. (**G**) Oral cavity proper: prefenular space. 9 months, dde13. 1, Upper lip; 2, Lower lip; 3, Incisive papilla; 4, Labial vestibule; 5, Gums; 6, Hard palate; 7, Palatine raphe; 8, Greater palatine groove; 9, Tongue: tip; 10, Tongue: marginal papilla; 11, Tongue: border; 12, Tongue: dorsum; 13, Tongue: ventral part; 14, Lingual frenulum; 15, lateral sublingual recesses; 16, Lateral sublingual folds; 17, Tongue: root; 18, Angulus oris.

In the youngest newborn *Stenella coeruleoalba* (scoce1), all teeth are covered by gums. Teeth eruption can be seen in the two older newborn specimens (scomu1 and scomu2) where the caudal teeth start to erupt, but the rostral teeth are covered by gums and the incisive teeth are not well developed. Also, the marginal papillae are well developed and decrease progressively towards the root. (Figure 10).

#### 3.1.2. MRI Study

The T2 MRI image of the oral cavity in this *Delphinus delphis* (dde11) shows that the maxillary bone is medium hyperintense with respect to the hypointense hard palate. The superficial mucosa of tongue is slightly hyperintense with respect to the hypointense depressor, protractor and retractor muscles of the tongue. A hyperintense stratum under the tongue muscles is probably due to the high rate of irrigation of these muscles. We observed that non-erupted teeth can be seen under the gums (Figure 11).

**Figure 10.** Endoscopic images of the oral cavity. In the different images, the arrow points to the tip of the mouth. **L** (Left) **R** (Right). (**A**) Hard palate. (**B**) Tongue. (**C**–**F**) Tongue, oral cavity proper and prefenular space. newborn, scumu2. (**G**) Deep dissection of the newborn dolphin head after removing skin and partial section of the right mandible. Observe that rostral teeth during lactation period do not erupt to protect mother's nipple. newborn, scoce1. (**H**) Detailed image of the marginal papilla and teeth without gum covering the clinic crown. A palatal raphe in hard palate is present. newborn, scomu1. 1, Upper lip; 2, Lower lip; 3, Incisive papilla; 4, Labial vestibule; 5, Gums; 6, Hard palate; 7, Palatine raphe; 8, Tongue: tip; 9, Tongue: border; 10, Tongue: marginal papilla; 11, Tongue: dorsum (middle tongue groove); 12, Tongue: ventral part; 13, Lingual frenulum; 14, lateral sublingual recess; 15, Lateral sublingual folds; 16, Tongue: root; 17, Teeth roots; 18, Angulus oris.

#### 3.1.3. Histological Study

Vestigial incisive papillae appear with a keratinized pseudostratified epithelium. Conduits arriving at papilla were not seen, nor was the vomeronasal organ *Delphinus delphis* fetus (dde14). Abundant lymphatic vessels were observed in the dermis. Developing teeth were observed covered by gum tissue. The histological structure of teeth shows the inner dentin, covered by enamel and all parts (crown, neck, and root) covered by cementum. The sublingual lateral folds have a mucosa with abundant vessels and mucous glands while neither the sublingual caruncule nor the orobasal organ were observed (Figures 12 and 13).

**Figure 11.** Images of the oral cavity. MR sagittal images is oriented so that the rostral is to the right. (**A**) T2 FrFSE sagittal plane. 6 months, dde8. Image of the oral cavity. (**B**) T2 FrFSE sagittal plane. Quadknee coil. 8 months, dde11. 1, Teeth (under gum); 2, Tongue: body; 3, Maxillary bone; 4, Hard palate; 5, Soft palate; 6, Tongue: apex; 7, Tongue: root; 8, Mandibles; 9, Pterygoid and palatine bones.

**Figure 12.** (**A**,**B**) Histological study of the oral cavity. (**A**–**C**) Hard palate: incisive papilla. (**D**) Tooth. (**E**) Oral cavity proper: sublingual lateral fold. (**F**) Tongue: root. 10 months, dde14. 1, Epidermis; 2, Papillary stratum; 3, Nervous tissue; 4, Connective tissue; 5, Lymphatic vessels; 6, Bony tissue; 7, Dental structure initial development transverse sectioned; 8, Dental structure development sagittal sectioned; 9, Dental papilla; 10, Dentin; 11, Enamel; 12, Cementum; 13, Venous vessels; 14, Striated muscle; 15, Mucous glands.

**Figure 13.** (**A**,**B**) Histological study of the oral cavity. (**A**) Sublingual lateral fold: pigmented epithelium in basal stratum (**\***). (**B**) Tooth. (**C**,**D**) Tongue: root. 7.5 months, dde10. 1, Epidermis; 2, Papillary stratum; 3, Mucous glands; 4, Secretor duct; 5, Striated muscle: proper muscle tongue; 6, Dental papilla 7, Dentin; 8, Enamel; 9, Cementum.

The sublingual lateral fold showed a pigmented epithelium at its basal stratum. The tongue shows a striated muscle base and abundant sub-epithelial mucous glands are visible, along with their secretory ducts (Figure 13).

In an adult *Stenella coeruleoalba* (scomu6), the incisive papilla showed a well-developed papillary stratum. The dermis contains abundant fat tissue and nests of epithelial ducts in regression (Figure 14A,B). The lateral sublingual recess has a keratinized epithelium with mucous glands and a well-developed papillary stratum (Figure 14).

**Figure 14.** (**A**,**B**) Histological study of the oral cavity. (**A**,**B**) Hard palate: incisive papilla. (**C**,**D**) Oral cavity proper: sublingual lateral fold. Adult, scomu6. 1, Epidermis; 2, Papillary stratum; 3, Corneum stratum; 4, Fat tissue; 5, Connective tissue; 6, Remains of epithelial duct; 7, Lymphatic vessels.

#### *3.2. The Pharyngeal Cavity*

The pharynx is a musculo-membranous cavity divided into three parts: the oropharynx linking the oral cavity with the oesophagus, the nasopharynx connecting the nasal cavity with the larynx, and the laryngopharynx, which is an intermediate cavity caudal to the oropharynx and caudoventral to the nasopharynx. The laryngopharynx connects the oral cavity to the stomach and allows the aditus laryngis enter to the nasopharyngeal cavity crossing, only in cetaceans (Figure 15), the intrapharyngeal orifice.

**Figure 15.** Head of a dolphin fetus, showing the pharyngeal cavity. (**A**) Oropharynx; (**B**) Laryngopharynx; (**C**) Nasopharynx. MRI sagittal T1 SE sequence. 10 months, dde14. 1, Isthmus of the fauces; 2, Fauces; 3, Epiglottic vallecula; 4, Piriform recess; 5, Oesophageal vestibule; 6, Intrapharyngeal orifice: 7, Oesophagus mucosa.

The bony roof of oropharynx is composed of the palatine bone and the pterygoid bone laminae (lateral and medial). Additionally, the nasopharynx is delimited dorsally by the vomer wings and the medial lamina of the pterygoid bone (Figure 2). Ventrally, the hyoid apparatus attaches to the root of the tongue and the larynx to the base of the cranium.

3.2.1. Study of Oropharynx, Nasopharynx and Laryngopharynx

The oropharynx begins at the isthmus of the fauces, continues with a conduit (fauces) and finishes at the lingual aspect of the epiglottic cartilage (Figure 15).

#### Endoscopic Study

The endoscopic study began at the **oropharynx**, showed a tightly closed isthmus of the fauces in a young fetus, a *Delphinus delphis* (dde2). The endoscope could not cross this gate (Figure 16).

The endoscope was passed into the fauces in an older fetus, a *Delphinus delphis* (dde3) showing a bright mucosa. No lymphoreticular tissue in the floor (tongue), walls (palatoglossus archs or folds) or roof (soft palate) of the fauces was observed. The soft palate is inserted into the ventral crest formed between the lateral and medial lamina of the pterygoid bones. The palatoglossal archs or folds connect to the soft palate through the tongue root. At the end of fauces a soft vallecula continues dorsally with the lingual aspect of the mucosa of epiglottic cartilage. At this level we dorsally observed the intrapharyngeal orifice to allow entry of the larynx into the **nasopharynx**. Additionally, the **laryngopharynx** begins with a piriform recess on either side of the larynx cartilages. The left piriform recess

is wider than the right one. The dilated oesophageal vestibule is caudal to the recesses whose mucosa is arranged in longitudinal folds changing to small quadrangular folds where the oesophageal mucosa begins (Figure 17).

**Figure 16.** Endoscopic image of the pharyngeal cavity: oropharynx. **L** (Left) **R** (Right). Fauces: isthmus. 3.5 months, dde2. 1, Hard palate; 2, Isthmus of the fauces; 3, Arcus palatoglossus or palatoglossus folds; 4, Soft palate; 5, Tongue: root.

Both the *Stenella coeruleoalba* (scop1) and *Globicephala melas* (gma1) fetuses had a welldeveloped mucosa at the isthmus and only a narrow passage to the fauces which had a pale lingual mucosa and a grey/brown colour in its walls and roof (Figures 18 and 19).

**Figure 17.** *Cont*.

**Figure 17.** Endoscopic image of the pharyngeal cavity. **L** (Left) **R** (Right). (**A**–**C**) Oropharynx, (**A**) Fauces. (**B**–**D**) Laryngopharynx. 4 months, dde3. 1, Arcus palatoglossus or palatoglossus folds; 2, Soft palate; 3, Tongue: root; 4, Epiglottic vallecula; 5, Epiglottis: lingual surface (mucosa); 6, Piriform recess; 7, Intrapharyngeal orifice (nasopharynx); 8, Oesophageal vestibule; 9, Pharyngoesophageal limit; 10, Oesophageal mucosa.

**Figure 18.** Endoscopic image of the pharyngeal cavity: oropharynx. The arrows show where is the tip of the mouth. **L** (Left) **R** (Right). (**A**) Fauces: isthmus. (**B**) Fauces: inside. 4.5 months, scop1. 1, Hard palate; 2, Isthmus of the fauces (closed); 3, Arcus palatoglossus or palatoglossus folds; 4, Soft palate; 5, Tongue: root.

A well-defined fauces was observed in an older *Delphinus delphis* (dde8) and also a broad left piriform recess, with longitudinal folds finishing at the oesophageal vestibule (Figure 20).

In this well-developed fetus (*Delphinus delphis*) (dde9), the endoscope could pass into the choanae to see the nasopharynx and the pharyngeal orifice of the auditory tube; alsothe longitudinal folds changing to small quadrangular folds where the oesophageal mucosa begins (Figure 21). The oropharyngeal mucosa is thickening, the longitudinal folds in the piriform recesses of the laryngopharynx are thin, and a clear difference between the mucosa of the oesophageal vestibule and oesophagus was seen.

**Figure 19.** Endoscopic image of the pharyngeal cavity: oropharynx. The arrows show where is the tip of the mouth. **L** (Left) **R** (Right). (**A**) Fauces: isthmus. (**B**) Fauces: inside. 5 months, gma1. 1, Hard palate; 2, Isthmus of the fauces (closed); 3, Arcus palatoglossus or palatoglossus folds; 4, Soft palate; 5, Tongue: root.

**Figure 20.** Endoscopic images of the pharyngeal cavity. **L** (Left) **R** (Right). (**A**–**C**) oropharynx. (**B**) Fauces. (**C**–**F**) Laryngopharynx. 6 months, dde8. 1, Isthmus of the fauces; 2, Arcus palatoglossus; 3, Tongue: root; 4, Soft palate; 5, Epiglottic vallecula; 6, Piriform recess; 7, Epiglottis: mucosa. 8, Oesophageal vestibule; 9, Pharyngoesophageal limit; 10, Oesophageal mucosa.

**Figure 21.** Endoscopic images of the pharyngeal cavity. **L** (Left) **R** (Right). (**A**,**B**) oropharynx: fauces. (**B**–**F**) Laryngopharynx. (**G**) Left nasopharynx. 7 months, dde9. 1, Arcus palatoglossus; 2, Tongue: root; 3, Soft palate; 4, Piriform recesses (laryngopharynx); 5, Epiglottis: mucosa. 6, Intrapharyngeal orifice (entrance to nasopharynx); 7, Oesophageal vestibule; 8, Pharyngoesophageal limit; 9, Oesophageal mucosa; 10, Pharyngeal orifice of the auditory tube; 11, Choanae; 12, Nasopharyngeal mucosa: longitudinal or striated folds; 13, Nasal septum: vomer bone.

The mucosa of the fauces continues to thicken and has a bright aspect in a *Delphinus delphis* fetus (dde11). Additionally, in the nasopharynx, the mucosa shows longitudinal folds and small openings surrounding the pharyngeal orifice of the auditory tube (Figure 22).

**Figure 22.** Endoscopic images of the pharyngeal cavity. **L** (Left) **R** (Right). (**A**) Oropharynx: fauces. (**B**) Left nasopharynx. (**C**) Right nasopharynx. 8 months, dde11. 1, Arcus palatoglossus or palatopharyngeal folds; 2, Tongue: root; 3, Soft palate; 4, Pharyngeal orifice of the auditory tube; 5, Nasal septum: vomer bone; 6, Nasopharyngeal mucosa: longitudinal or striated folds and small openings.

> In a juvenile dolphin, we could observe the pinkish mucosa of the nasopharynx with longitudinal folds, but the small holes had less border definition (Figure 23).

#### Histological Study

The histological results show that the epidermis of the oropharynx at the soft palate level has a tightly papillary stratum with deep mucous glands and abundant mucous glands in its submucosa. Additionally, at the level of the isthmus of the fauces, histology shows a connective tissue stratum deep to the epidermis, containing many deep mucous glands (Figure 24A,B). The nasopharynx shows a respiratory mucosa with an anfractuous papillary stratum, below which is a wide connective stratum, and finally a deep serous gland close to striated muscle. Additionally, we have located Vater–Paccini corpuscles near the auditory duct between striated muscles (Figure 24C–E). No lymphoreticular tissue was observed.

**Figure 23.** Endoscopic images of the pharyngeal cavity. **L** (Left) **R** (Right). (**A**,**B**) Left nasopharynx. (**C**,**D**) Right nasopharynx. Juvenile, scomu4. 1, Pharyngeal orifice of the auditory tube; 2, Nasal septum: vomer bone; 3, Nasopharyngeal mucosa: longitudinal or striated folds with small holes.

**Figure 24.** (**A**,**B**) Histological study of the pharyngeal cavity. (**A**) Fauces: soft palate. (**B**) Tongue: root. (**C**) Pharynx: mucosa (**D**,**E**). Detail of pharyngeal mucosa. Adult, scomu6. 1, Epidermis; 2, Papillary stratum; 3, Secretor ducts; 4, Connective tissue; 5, Deep mucous glands; 6, Deep serosa glands; 7, Vater-Paccini corpuscle.

#### MRI Study

The MRI sagittal images show a pharyngeal cavity in a *Globicephala melas* fetus (gma1) and we could appreciate the oropharynx (fauces), the **nasopharynx** and the oesophageal vestibule hypointense in both T1 and T2 sequences(Figure 25A,B). Coronal T1 and T2 sequences show the piriform recess alongside the larynx (Figure 25C,D).

**Figure 25.** Images of the oral and pharyngeal cavity. MR sagittal and coronal images are oriented so that the rostral is to the right. (**A**) T1 SE sagittal, (**B**) T2 FrFSE sagittal, (**C**) T1 SE coronal and (**D**) T2 FrFSE coronal planes. 5 months, gma1. 1, Hard palate; 2, Tongue; 3, Oral cavity (closed); 4, Oropharynx: fauces; 5, Oropharynx: soft palate; 6, Laryngopharynx: left piriform recess; 7, Laryngopharynx: oesophageal vestibule; 8, Epiglottis cartilage; 9, Epiglottic vallecula; 10, Arytenoid cartilages; 11, Nasopharynx; 12, Larynx.

3.2.2. Special Study of Nasopharynx and Pharyngeal Diverticulum of the Auditory Tube (PDAT)

(a) MRI study

In MRI, we can appreciate, in early fetal stages, a bilateral structure within the laryngopharyngeal cavity, each named as a *pharyngeal diverticulum of the auditory tube* (PDAT). These are connected through the musculotubaric channel with the middle ear (temporal bone: petrous and tympanic part). In a young *Delphinus delphis* fetus (dde3), it appears in sagittal sections as a hyper/hypointense area seen caudal and rostrally, respectively (Figure 26A,B), and also in coronal sections (Figure 26C,D).

**Figure 26.** Images of the pharyngeal cavity. MR sagittal and coronal images are oriented so that the rostral is to the right. (**A**) T1 SE sagittal, (**B**) T2 FrFSE sagittal, (**C**) T1 SE coronal and (**D**) T2 FrFSE coronal planes. 4 months, dde3. 1, Inner and middle ear; 2, Pharyngeal diverticulum of the auditory tube.

In older *Delphinus delphis* fetuses (dde5, dde8, dde11) this double space at both sides of the laryngopharynx is more evident and shows the same intensity, but now we can distinguish the vascular area (hyperintense) and the air-filled area (hypointense) (Figures 27–31).

**Figure 27.** *Cont*.

**Figure 27.** Images of the pharyngeal cavity. MR coronal and sagittal images are oriented so that the rostral is to the right. (**A**,**B**) T2 FrFSE sagittal, (**C**) T1 SE and (**D**) T2 FrFSE coronal planes. 5.5 months, dde5. 1, Inner ear; 2, Pharyngeal diverticulum of the auditory tube: moderate hyperintense area (vascular); 3, Pharyngeal diverticulum of the auditory tube: hypointense area (air).

**Figure 28.** Images of the pharyngeal cavity. MR sagittal and coronal images are oriented so that the rostral is to the right. (**A**) T1 SE sagittal, (**B**) T2 FrFSE sagittal, (**C**) T1 SE coronal and (**D**) T2 FrFSE coronal planes. 6 months, dde8. 1, Inner ear; 2, Pharyngeal diverticulum of the auditory tube: moderate hyperintense area (vascular); 3, Pharyngeal diverticulum of the auditory tube: moderate hypointense area; 4, Pharyngeal diverticulum of the auditory tube: hypointense area (air); 5, Pharyngeal diverticulum of the auditory tube: hyperintense area (vascular).

**Figure 29.** Images of the pharyngeal cavity. (**A**,**B**) MR coronal and sagittal images are oriented so that the rostral is to the right. (**A**,**B**) T2 FrFSE coronal and sagittal planes. 8 months, dde11. 1, Oropharynx: fauces; 2, Nasopharynx; 3, Laringopharynx: oesophageal vestibule; 4, Nasopharynx: pharyngeal diverticulum of the auditory tube; 5, Pharyngeal diverticulum of the auditory tube: hyperintense area (vascular); 6, Pharyngeal diverticulum of the auditory tube: hypointense area (air); 7, Larynx; 8, Middle and inner ear.

**Figure 30.** *Cont*.

**Figure 30.** Images of the pharyngeal cavity. MR sagittal and coronal images are oriented so that the rostral is to the right. (**A**) T1 SE sagittal, (**B**) T2 FrFSE sagittal, (**C**,**E**) T1 SE coronal and (**D**,**F**) T2 FrFSE coronal planes. 4 months, dde14. 1, Inner and middle ear; 2, Pharyngeal diverticulum of the auditory tube (vascular); 3, Pharyngeal diverticulum of the auditory tube (air); 4, Auditory tube; 5, Nasopharinx; 6, Intrapharyngeal orifice.

**Figure 31.** Images of the pharyngeal cavity. MR sagittal and coronal images are oriented so that the rostral is to the right. (**A**) T1 SE sagittal, (**B**) T2 FrFSE sagittal, (**C**) T1 SE coronal and (**D**) T2 FrFSE coronal planes. 9 months, grgr1. 1, Inner and middle ear; 2, Pharyngeal diverticulum of the auditory tube: vascular; 3, Pharyngeal diverticulum of the auditory tube: air; 4, Mandibles.

> In more advanced fetal development, it is possible to observe air (hypointense) and vascular (moderate hyperintense) areas, and even the auditory tube (slightly hypointense) (Figure 30).

> PDAT were clearly seen in sagittal and coronal sections in a *Grampus griseus* fetus (grgr1). The T2 sequences are clearer than T1 because they differentiate two areas: slightly hypointense (vascular) and hyperintense (air) (Figure 31).

(b) Histological study

The histological analysis of the PDAT shows two well-defined areas inside: a pharyngeal vascular plexus (Figure 32A) with abundant and dilated vascular endothelium and the respiratory epithelium area in contact with air (Figure 32 E). A detailed image reveals the luminal vessels filled with blood (Figure 32B,C). The wall of PDTA is filled with air, which is in contact with respiratory epithelium (Figure 32D,E).

**Figure 32.** Histological study of the pharyngeal cavity: pharyngeal diverticulum of the auditory tube. (**A**) Pharyngeal vascular plexus wide. (**B**,**C**) Detail of plexus with blood in lumen. (**D**,**E**) Detail of plexus wall. Adult, scomu6. 1, Vascular lumen; 2, Respiratory lumen; 3, Blood vessel lumen; 4, Vascular endothelium; 5, Respiratory epithelium.


The PDAT is a well delimited area, even in the early stages of fetal development (Figure 2). This area is medially extended to the bony choanae and extends dorsally to the maxillopalatine fossa, medially to the pterygopalatine recess (pterygoid sinus) and rostrally to the petrous and tympanic parts of the temporal bone (cochlea) (Figure 33).

(c2) Sectional anatomy

The three coronal sections, in a newborn *Stenella coeruleoalba* (scomu2), extend from the floor (Figure 34A) to the roof of the oral and pharyngeal cavities (Figure 34B,C). In these images it was possible to observe the proximity to the mandible channel tissue and the pharyngeal orifices of the auditory tubes crossing the pharyngeal muscles. It is easy to medially differentiate the air area (near the auditory tube and nasopharynx and laterally to the cribriform area) (Figure 34).

**Figure 33.** Common dolphin skull. Dots show the extension and form of the right pharyngeal diverticulum of the auditory tube. Photography Francisco Gil Cano. Courtesy from Ángel Tórtola. Spanish naturalist. Oblique view. dde15. 1, Greater palatine groove; 2, Palatine bone: perpendicular lamina; 3, Palatine bone: horizontal lamina; 4, Vomer bone; 5, Pterygoid bone: medial lamina; 6, Pterygoid bone: lateral lamina; 7, Pterygoid bone: crest; 8, Lacrimal and zygomatic bone; 9, Temporal process of the zygomatic bone; 10, Frontal bone; 11, Presphenoid bone: wings; 12; Basisphenoid bone: wings; 13, Temporal bone: squamous part; 14, Temporal bone: petrous and tympanic parts; 15, Occipital bone: basilar part; 16, PDAT area; 17, Maxilopalatine fossa (pterygopalatine fossa in mammals); 18, Pterygopalatyne recess (pterygoid sinus); 19, Maxillary bone: palatine process.

**Figure 34.** *Cont*.

**Figure 34.** (**A**–**C**) Coronal sections of head at level of eyes, ear, pharyngeal and oral cavity. These three sections show the extension and connection between the pterygopalatine recess (pterygoid sinus) and the PDAT and between the nasopharynx and PDAT. (**A**,**B**) Dorsal view (**C**) Ventral view. scomu2. 1, Middle and inner ear; 2, Pharyngeal orifices of the auditory tube; 3, Pharyngeal diverticulum of the auditory tube: air area; 4, Pharyngeal diverticulum of the auditory tube: vascular area; 5, Vomer and choanas; 6, Pharyngeal muscles; 7, Piriform recess; 8, Laryngeal cartilages: aditus laryngis; 9, Hard palate (maxillary bones); 10, Tongue (sectioned) 11, Mandibles; 12, Labial vestibule; 13, Oral cavity.

The two sagittal sections in a juvenile *Stenella coeruleoalba* (scomu3) were made parasagittally at the level of the ear. It shows that this area (PDAT) extends rostrally to the inner and middle ear crossing below the basal bones of the cranium to arrive to the pterygopalatine recess (pterygoid sinus) and finish dorsally at the maxillopalatine fossa (Figure 35).

In the adult *Stenella coeruleoalba* (scomu6), the pharyngeal orifice of the auditory tube is canalized by a trocar (Figure 36A) and the PDAT area is located ventrally. The enlarged image shows, after removing the pharyngeal muscles, the trajectory of the auditory tube towards the pharyngeal diverticulum (Figure 36B).

**Figure 35.** (**A**,**B**) Detailed serial sagittal sections at level of the pharyngeal diverticulum of the auditory tube with an anfractuous mucosa filled with a heterogeneous content. It extends up to the maxillopalatine fossa rostral to the eyeball. scomu3. 1, Middle and inner ear; 2, Pharyngeal diverticulum of the auditory tube; 3, Occipital bone: basilar part; 4, Basisphenoid bone; 5, Presphenoid and ethmoid bones; 6, Pterygoid bone; 7, Palatine bone; 8, Maxilopalatine fossa (pterygopalatine fossa in domestic mammals); 9, Pterygopalatine recess (pterygoid sinus).

**Figure 36.** *Cont*.

**Figure 36.** (**A**) Sagittal section of head at level of nasal, pharyngeal and oral cavity. (**B**) Detail of the trajectory of the trocar towards the pharyngeal diverticulum after removing the pharyngeal muscles around the auditory tube. Adult, scomu6. 1, Nasal cavity: vestibule; 2, External nares muscles; 3, Phonic lips; 4, Nasal plug; 5, Nasal plug muscles; 6, Nasal cavity: respiratory part; 7, Nasal cavity: incisive recess; 8, Choanae; 9, Melon; 10, Pharyngeal muscles; 11, Nasal bone; 12, Frontal bone; 13, Ethmoid bone; 14, Presphenoid bone; 15, Basisphenoid bone; 16, Incisive bone; 17, Maxillary bone; 18, Pterygoid bone; 19, Mesethmoid cartilage; 20, Pharyngeal diverticulum of the auditory tube (rostral part is pterygoid sinus); 21, Pterygopalatine recess (pterigoyd sinus); 22, Hypophysis; 23, Connection orifice; 24, Tongue: proper lingual muscle; 25, Hyoglossus muscle; 26, Geniohyoid muscle; 27, Mylohyoid muscle; 28, Digastricus muscle; 29, Musculotubaric channel; 30, Middle ear (petrotympanic bone); 31, Trocar inserted in the pharyngeal orifice of the auditory tube; 32, Trocar (showing duct trajectory).

#### (c3) Dissection

A deep dissection in a newborn *Stenella coeruleoalba* (scoce1) shows the extent of the PDAT area (Figure 37).

**Figure 37.** Deep dissection of the dolphin head after removing petrous and tympanic part of the temporal bone. Discontinuous line shows the extension and form of the right pharyngeal diverticulum of the auditory tube. Newborn, scoce1. 1, Pterygopharyngeal recess (pterygoid sinus); 2, Maxilopalatine fossa (pterygopalatine fossa in domestic mammals).

#### **4. Discussion**

*4.1. Oral Cavity*

4.1.1. Vestibule

The oral vestibule in terrestrial mammals could be sub-divided in oral and labial vestibules, due to the presence of a space between lips and teeth (labial vestibule), and between cheeks and teeth (buccal vestibule). In cetaceans, which lack cheeks and, therefore, masseter muscles during development [3], only the labial one is present.

Additionally, in terrestrial mammals, there are superior and inferior labial frenula to capture air and grab food. In cetaceans, the lips are tightly sealed, so the mouth is hermetically closed as it happens with the blowhole. Therefore its lips lack the mobility of the terrestrial mammal. Therefore, the labial vestibule is unique in cetaceans because it lacks a buccal vestibule and it is different from terrestrial mammals.

#### 4.1.2. Oral Cavity Proper

(a) Roof

The rostral part of hard palate has only a vestigial incisive papilla. Since the ontogeny reflects the phylogeny, the ducts are more formed in early stages of development and degenerate as gestation progresses. The innervation observed in this incisive papilla is probably related to the tactile sensitivity to test the mucosa of some prey. This sensation is collected by the major palatine nerve from the maxillary branch of trigeminal nerve.

(b) Tongue

The lingual papillae in the *Globicephala melas*, a teuthophagous (consuming cephalopods) suction feeder [2], may enable its specific feeding strategy, especially when combined with the modifications to its hyoid apparatus, and often to the skull and jaws [28]. On the other hand, *Stenella coeruleoalba* and *Delphinus delphis* dolphin's lingual papillae do not extend caudally, and these disappear as the animal matures (as in terrestrial mammals).

The regressing papillae may be due to the adult type of feeding and with the prolonged lactation period, 19–20 months in *Stenella coeruleoalba* and *Delphinus delphis* and 24 months or even longer in *Globicephala melas* [26]. Newborn carnivores and suids also have marginal papillae [24].

In birds, there are blade-like lamellae in the inner and outer margin of the bill. For instance, anatids generally have filtering papilla, while ducks have a double row of overlapping bristles in the tongue that interdigitate with a double row of lamellae on the bill [29].

#### 4.1.3. Histological Considerations

Refs. [10,26] show the lingual seromucous and mucous glands in *Delphinus delphis*. We also confirm the presence of mucous glands in an adult *Stenella coeruleoalba* (scomu6) at these levels as well as in the oesophagus, confirmed by the endoscopic images. We also observed mucous glands in *Delphinus delphis* fetus (dde10). The large number of mucous glands has a double function, first to serve as mechanical protection against the abrasive diet, i.e., the fins and scales of fishes and second, to favour lubrication to enable a proper food movement during swallowing. The same conclusions apply to the oropharynx. In addition, the absence of salivary glands explains the abundant mucous glands as a substitute.

In birds, there is a similar compensation process [29], which is of great utility in the digestive process of certain avian species such as galliforms, which ingest seeds, grains and even stones and mud to help trituration.

As for the immune system, the large number of lymphatic vessels (Figure 13) explains the absence of nodular formations (tonsils and nodules) which, in cetaceans, could jeopardize deglutition. The absence of lymphoreticular tissue does not indicate lack of immune function.

The frenulum in most terrestrial animals is wider and more mobile. In dogs and horses, it is sharper and more angled like *Delphinus delphis*, *Stenella coeruleoalba* and *Globicephala melas*. In cetaceans the tongue has limited mobility, serving first during lactation and then during the rest of life, as it is useful to grab food and expel extra water.

The small grooves may, in fact, be the location of the hitherto unveiled taste receptors [10]. We did not locate receptors for taste or olfaction in the oral or nasal cavity [3].

#### (c) Floor of oral cavity

The sublingual recess and labial vestibule are quite shallow, perhaps due to the absence of mastication, which nullifies the need for a functional space outside the vestibule (outer) surface of the teeth [26].

We see this narrowness of the labial vestibules and sublingual recess during fetal development. Within the lateral sublingual folds, we did not observe the presence of any duct transporting saliva from the major salivary glands, as would happen in terrestrial mammals. Neither did we observe sublingual papillae, as in domestic mammal species.

The monostomatic salivary glands are absent in cetaceans [3]. Stomas opened along the lateral sublingual folds (polystomatic gland) and major sublingual and mandibular ducts, with the latter atrophied in cetaceans, as we have observed in histology (Figure 14).

Even though the mouth is a tight opening, when the dolphin feeds under the water, the entrance of some salt water is unavoidable, but this will be ejected later with the help of the tongue. In a humid environment, the saliva would be unnecessary.

(d) Teeth

Tooth formation occurs below the gums [3]. The final eruption takes place during the perinatal period after lactation (this, along with the few development of the incisive teeth, protects the mother's nipple), probably by the scratching of the gums against the rough surfaces of food.

In *Delphinus delphis,* teeth are developed just in the molar region; the mandibular teeth develop later. *Grampus griseus* (Figure 31) and *Globicephala melas* (Figure 7) have fewer teeth due to their shorter and rounder jaw. In this last species, the caudal teeth do not develop, since the mandible grows and the teeth remain rostral in position. In *Phocoena phocoena* (phog1) the conic teeth (Figure 2) with which they are born will flatten and round with age by the process of wear.

For the first time, we discovered that the teeth, during development, are covered by cementum under the gums. We can state that odontocete teeth are phylogenetically closer to the hypsodont dentition of ruminants and equids, in which teeth are composed from the crown to root as follows: a core of dentine covered internally and externally by enamel which is, in turn, covered by cementum (Figures 12d and 13d).

Cetacean teeth are designed to grab and swallow but not to chew, so molars are not necessary.

#### *4.2. Pharyngeal Cavity*

During fetal development, we observed that the soft palate of odontocetes stays attached caudally along the crest of the pterygoid bone (Figure 13), while in terrestrial mammals, it remains attached to the horizontal lamina of the palatine bone (palatine aponeurosis).

The isthmus of the fauces is tightly closed, as happens with the blowhole [3], because it is formed by musculomembranous walls, like those of the oesophagus.

The tightly closed oropharynx (Figure 12) can be opened (Figure 15) during gestation to allow the entrance of amniotic fluid into the fetal alimentary canal to stimulate the glands producing gastric enzymes [30,31]. Once born, neither the oropharynx nor the oesophagus open until food is swallowed.

The endoscope overcomes the resistance of the isthmus of the fauces by exerting pressure similar to the effect produced by the ingestion of prey.

(a) Histological considerations

The lateral asymmetry in dolphins exists to the right externally, and to the left internally. [28] This defends a trophic explanation since the internal asymmetry allows the ingestion of larger prey, though we believe the asymmetry described is the opposite, since it is not the right piriform recess but the left one which is the larger (Figure 21). The voluntary dislocation of the larynx to ingest larger prey has already been described [32].

Nasopharynx

The pharyngeal orifice of the auditory tube is an open membranous duct (Figure 36a) close to the pharyngeal muscles. The PDAT connects to the musculotubaric channel and finishes in the tympanic orifice of the auditory tube.

The pterygopalatine recess (pterygoid sinus) is not isolated but connected to the pharyngeal diverticulum of the auditory tube at the level of the auditory tube notch [33]). We do not consider the pterygopalatine recess as an isolated space of the auditory tube diverticulum, but instead as part of the structure.

In the horse, the auditory tube diverticulum extends from the skull base to the atlas dorsally and ventrally to the pharyngeal cavity roof [34,35].

The fact that the auditory tube is not a closed duct but dilating and folding according to pressure, has its application in decompression. The auditory tube protects from threats (i.e., strong sounds which are potentially dangerous to the small bones) forcing the dolphin to emerge quickly releasing high-pressure air (or liquid) through the orifice. This theory can be reinforced by the fact that the Vater–Paccini corpuscles (Figure 24) are pressure receptors observed in the nasopharyngeal mucosa close to the pharyngeal orifice of the auditory tube (Figure 36).

This would also prevent the collapse of the tracheal and respiratory walls while simultaneously ensuring that the air at great depths is distributed to essential areas.

The blood present can have a double function of refrigeration and a reservoir of extra oxygen, available for exchange with blood vessels in part of the epithelium and within the sinuses. It could be very interesting to further examine this process in Mysticetes. A similar process happens in other parts of the lower respiratory system [36].

#### (b) Comparative Anatomy

In the horse, the function of these diverticula is to cool the blood going to the brain via the internal carotid and the external carotid artery. In equids, the diverticula are divided by a membranous septum extending to the ventral face of the atlas. In odontocetes, it extends only to the pterygopalatine recess (pterygoid sinus) and medially to the pterygoid bone. The opposite happens in terrestrial mammals (where the diverticulum of the auditory tube is divided by the basilar portion ridge, the basihyoid, and the medial lamina of the pterygoid bone [34] and [35]. Both diverticula are divided by bone, so the entrance to the larynx (aditus laryngis) is not interrupted at this level.

(c) Histological considerations

The deep serosal glands observed in the nasopharynx secrete towards the small openings observed in the deepest area of the nasal cavity [3] and to the small holes observed in the nasopharynx area close to the pharyngeal orifice of the auditory tube (Figures 21–23). Close to these orifices, a pressure corpuscle (Figure 24) was observed. This possibly indicates that, when the vascular plexus observed in the pharyngeal diverticulum of the auditory tube is filled with blood (Figures 27–32 and 34–36), this process promotes the expulsion of the air stored to the auditory tube into the nasopharynx (Figure 36). The air pressure within the nasopharynx is detected by the Vater–Paccini corpuscles (Figure 24).

#### **5. Conclusions**

Inside the oral cavity proper, incisive papilla are present in all specimens to test food texture and hardness and, during lactation, this is also reinforced by the lack of incisive teeth to prevent nipple harming. Comparatively, we can state that *Delphinus delphis* palate is morphologically different from the other species due to an additional groove.

We have also seen the form and function of the tongue showing the marginal papillae disappearing gradually after lactation, lasting more in some species like *Globicephala melas*. The teeth in formation present three layers under the gum, developed during fetal life but erupting through the gums after lactation.

We found mucous glands in the oral and pharyngeal cavities, with a double function of lubrication and mechanical protection. In the nasopharynx, we found serous glands to humidify the area as well as pressure corpuscles.

In the laryngopharynx, we realized that the left piriform recess was larger than the right one, probably to allow the ingestion of larger prey.

In the nasopharynx, one of the achievements of this study was finding and visualizing, for the first time, the diverticula of the auditory tube from early stages of the development, using MRI and the dissection of fetus skulls. This space extends rostrodorsally from the ear to the nasopharynx and contains air and a vascular plexus, similar to those mammals which possess such a structure.

Buccopharyngeal cavity endoscopies would also be useful to highlight alterations in the anatomical structures at these levels, creating situations incompatible with life.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/ani11061507/s1, Table S1: Other parameters observed in this study. Table S2: MRI parameters used in this study.

**Author Contributions:** Conceptualization, Á.G.d.l.R.y.L. and G.R.Z.; Formal analysis, A.A.E. and F.G.C.; Investigation, Á.G.d.l.R.y.L.; Methodology, A.A.E., M.S.L., F.M.G., A.L.F., J.S.A. and C.S.C.; Resources, F.M.G., A.L.F., F.G.C., J.S.A. and C.S.C.; Supervision, Á.G.d.l.R.y.L., A.A.E. and G.R.Z.; Writing—Original draft, Á.G.d.l.R.y.L. and G.R.Z.; Writing—Review and editing, Á.G.d.l.R.y.L., A.A.E., M.S.L. and G.R.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** MRI acquisitions were financed by Departamento de Anatomía y Anatomía Patológica Comparadas. Facultad de Veterinaria. Universidad de Murcia. Spain. The Galician stranding network is supported by the regional government Xunta de Galicia—Dirección Xeral de Patrimonio Natural. CESAM/FCT: thanks are due to FCT/MCTES for the financial support to CESAM (UIDP/50017/2020+UIDB/50017/2020), through national funds. Norma transitoria. Alfredo López is funded by national funds (OE), through FCT—Fundaçâo para a Ciência e a Tecnologia, I.P. in the scope of the framenetwork contract foreseen in numbers 4, 5 and 6 of the article 23, of the Decree-Law 57/2016, of August 29, changed by Law 57/2017, of July 19.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We are grateful to Consejería de Sanidad, Ceuta, Spain. Many thanks to Mariano Orenes Hernández for bony preparation. Spanish technician. Anatomía Veterinaria. Murcia. Thanks to Ángel Tórtola, a Spanish Naturalist, for the adult dolphin skull picture. Special thanks to Xunta de Galicia and CESAM. We thank all the CEMMA volunteers that helped with necropsies and sample collection. Thanks are due to the Valencia Oceanographic and the University of Valencia for allowing us to perform image diagnostic analysis on the Risso's dolphin fetus, and especially to their Veterinarian Daniel García-Párraga and José Luis Crespo and the biologist Patricia Gozalbes. We are also thankful to image technician Oscar Blázquez Pérez for the MRI scan performed at Centro Veterinario de Diagnostico por Imagen del Levante, Ciudad Quesada, Rojales Alicante, Spain. We give special thanks to M. José Gens Abujas (Oficina de impulso Socioeconómico del Medio Ambiente, Dirección General de Medio Natural, Consejería de Empleo, Universidades, Empresas y Medio Ambiente, Región de Murcia, Spain). Thank you very much to the CRFS Veterinary Team, in a special way, Fernando Escribano Cánovas, Luisa Lara Rosales and Alicia Gómez de Ramón Ballesta, El Valle, Murcia, Spain, for allowing us to have access to the carcasses stranded in their regional area.

**Conflicts of Interest:** The authors of this manuscript have no conflict of interest to declare.

#### **References**


## *Article* **Cranial Structure of** *Varanus komodoensis* **as Revealed by Computed-Tomographic Imaging**

**Sara Pérez 1, Mario Encinoso 2, Juan Alberto Corbera 1, Manuel Morales 1, Alberto Arencibia 3, Eligia González-Rodríguez 1, Soraya Déniz 2, Carlos Melián 2, Alejandro Suárez-Bonnet <sup>3</sup> and José Raduan Jaber 3,\***


**Simple Summary:** We investigated the head of Komodo dragons using CT imaging. Cross-sections show that all cranial bones can be delineated, while soft tissue structures are evident but not clearly identifiable without an anatomical atlas. Additional three-dimensional reconstructed and maximum intensity projection images of the head were presented to depict bony structures. The anatomical structures identified on the CT images could help further assess the head of the Komodo dragon.

**Abstract:** This study aimed to describe the anatomic features of the normal head of the Komodo dragon (*Varanus komodoensis*) identified by computed tomography. CT images were obtained in two dragons using a helical CT scanner. All sections were displayed with a bone and soft tissue windows setting. Head reconstructed, and maximum intensity projection images were obtained to enhance bony structures. After CT imaging, the images were compared with other studies and reptile anatomy textbooks to facilitate the interpretation of the CT images. Anatomic details of the head of the Komodo dragon were identified according to the CT density characteristics of the different organic tissues. This information is intended to be a useful initial anatomic reference in interpreting clinical CT imaging studies of the head and associated structures in live Komodo dragons.

**Keywords:** computed tomography; head; Komodo dragon

#### **1. Introduction**

The introduction of imaging diagnostic techniques has revolutionized the knowledge in reptile medicine. The radiographic evaluation has been traditionally used by clinicians [1]. Nonetheless, the progressive increase in modern imaging modalities such as computed tomography (CT) and magnetic resonance imaging has improved diagnostic abilities in reptile medicine and research [2]. Therefore, these techniques represent an enormous resource that allows for fast, non-invasive anatomy visualizations of internal structures that are challenging to interpret [2].

In recent years, the contributions of zoo veterinarians, researchers, and specialized technicians (anatomists, radiologists, and wildlife and exotic specialists) working with captive and free-ranging animals to prevent and treat diseases that threaten the survival of species in wildlife conservation have increased [3]. Since 1996, the Komodo dragon (*Varanus komodoensis*) is listed as vulnerable by the Red List of the World Conservation Union [4]. To our knowledge, the anatomy of different species of reptiles has already been

**Citation:** Pérez, S.; Encinoso, M.; Corbera, J.A.; Morales, M.; Arencibia, A.; González-Rodríguez, E.; Déniz, S.; Melián, C.; Suárez-Bonnet, A.; Jaber, J.R. Cranial Structure of *Varanus komodoensis* as Revealed by Computed-Tomographic Imaging. *Animals* **2021**, *11*, 1078. https:// doi.org/10.3390/ani11041078

Academic Editors: Matilde Lombardero and Mar Yllera Fernández

Received: 1 March 2021 Accepted: 7 April 2021 Published: 9 April 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

thoroughly described by radiology and CT [1,5–10], but only sparse numbers of these studies reported comprehensive descriptions of computed tomographic features of the head [1,5,6,9–12]. To date, not one of these reports investigates to what extent structures of the varanid head could be visualized and identified in low-resolution clinical CT-image data. In the Komodo dragon and other reptiles, the head conforms to a complex structure, which is challenging to interpret. The purpose of this study was to describe the normal anatomy of the head of the *Varanus komodoensis* by computed tomography, and threedimensional head reconstructed images to assist in the understanding of the head and its associated structures.

#### **2. Materials and Methods**

#### *2.1. Animals*

Two 17-year-old female specimens born in captivity at Reptilandia Park (Las Palmas, Spain) were imaged at the Veterinary Clinic Hospital of Las Palmas de Gran Canaria University. One female had a length of 225 cm (snout-vent length) and weighed 36 kg, whereas the other had a length of 190 cm and weighed 24 kg. No physical abnormalities were detected before the study. The Ethical Committee of Las Palmas de Gran Canaria University, College of Veterinary Medicine Section authorized the research protocol (MV-2019/04). The owner of the animals was informed of the study and signed consent for participation in the study.

#### *2.2. CT Technique*

Sequential transverse CT slices were obtained using a 16-slice helical CT scanner (Toshiba Astelion, Toshiba Medical System, Madrid, Spain). The animals were positioned symmetrically in ventral recumbency on the CT couch, and a standard clinical protocol (120 kVp, 80 mA, 512 × 512 acquisition matrix, 1809 × 858 field of view, a spiral pitch factor of 0.94, and a gantry rotation of 1.5 s) was used to acquire sequential transverse CT images of 1 mm thickness slice. The original transverse data were stored and transferred to the CT workstation. No CT density or anatomic variations were detected in the head of the dragons used in the study. In the CT technique, tissue density can be assessed directly on the image using the Hounsfield Unit scale, The range of values assumes 2000 shades of gray (between −1000 and +1000 HU). However, as the human eye cannot distinguish more than 30 shades, representing the entire range of values in an image implies not being able to visualize a large amount of information. Therefore, only a partial sector of the TC values previously selected by the operator (window selection) is represented by grayscale. Ultimately, the use of windows allows extracting the information that the computer has, showing only a part of it, which is of interest in each anatomical region. Therefore, bone window, soft tissue windows, brain window, and pulmonary window can be applied, delivering alternate streams of information. Thus, two CT windows were applied by adjusting the window widths (WW) and window levels (WL): a bone window setting (WW = 1500; WL = 300), and a soft tissue window setting (WW = 350; WL = 40). The original data were used to generate head volume-rendered reconstructed images after manual editing of the transverse CT images to remove soft tissues using a standard dicom 3D format (OsiriX MD, Geneva, Switzerland). In addition, maximum intensity projection (MIP) images were obtained to better display the outlines between bones and other lowerattenuation structures using an image viewer (OsiriX MD, Apple, Cupertino, CA, USA). MIP is a specific type of rendering in which the brightest voxel is projected into the 3D image. There tends to be much less variability in MIP image reconstruction than in volume rendering because fewer parameters are factored into the MIP algorithm [13].

#### **3. Results**

#### *3.1. Transverse Computed Tomography Images*

Transverse sections are provided that demonstrate critical anatomical features of the varanid cranium (Figures 1–6). Figure 1 consists of three images: (C) Sagittal image of the head, where each line and number (I–VI) represents the approximate level of the following transverse CT images, (A) CT bone window, (B) CT soft tissue window. Figures 2–6 represent transverse CT images where (A) CT bone window, and (B) CT soft tissue window. The CT images are presented in a cranial to caudal progression from the septomaxilla level (Figure 1) to the brain stem level (Figure 6). The comparison between available literature and CT images enabled us to identify most of the clinically relevant anatomic structures of the head. These features were identified according to location and the degree of attenuation of the different tissues.

With regards to hard tissues, the CT images acquired using the bone window setting (Figures 1–6A) provided good differentiation between the bones and the soft tissues of the head. Thus, the bones of the cranium (prefrontal, frontal, postorbital, parietal, squamosal, quadrate, jugal, pterygoid, basioccipital, parabasisphenoid, and maxilla), the mandible (dentary, angular, surangular, and articular bones) and hyoid bones were easily recognizable because of the high CT density in cortical bone and the low CT density in their medullary cavities. Most of these structures were also visualized with the soft tissue window setting (Figures 1–6B).

Air-filled structures, such as the nasal cavity, larynx, trachea, and the oral cavity gave negligible CT-tissue density and appeared black with both window settings.

Soft-tissue structures—such as the jaw muscles, the labial and nasal glands, the eyes, and the Harderian glands—gave an intermediate CT density and appeared grey. The nervous structures (brain, cerebellum, lateral ventricles, brain stem, and spinal cord) were appreciated in both CT window modalities (Figures 3–6).

**Figure 1.** Sagittal image of the head of *Varanus komodoensis*. The lines and numbers (I–VI) represent the approximate level of the following transverse CT images (**C**). Transverse CT image of the head at the level of the nasal cavity corresponding to line I. (**A**) CT bone window. (**B**) CT soft tissue window. These images are displayed so that the right side of the head is to the viewer's left and the dorsal view is at the top. N: Nasal bone. Sm: Septomaxilla. Cnd: Dorsal nasal conchae. Cnv: Ventral nasal conchae. Prf: Prefrontal bone. 6. Maxillary bone. T: Tooth. T': Tooth. Co: Cavum oris. Tg: Tongue. Ms: Musculus intermandibularis + Musculus geniohyoideus + Musculus genioglossus. D: Dentary bone. Ig: Infralabial glands.

**Figure 2.** Transverse CT image of the head of *Varanus komodoensis* at the level of the nasal bone corresponding to line II. (**A**) CT bone window. (**B**) CT soft tissue window. These images are displayed so that the right side of the head is to the viewer's left and the dorsal view is at the top. N: Nasal bone. Prf: Prefrontal bone. Ns: Nasal septum. Ng: Nasal glands. St: Stammteil, V: Vomer. Sr: Subconchal recess. Cht: Choanal tube. Nld: Nasolacrimal duct. Mx: Maxillary bone. T: Tooth. Co: Cavum oris. Tg: Tongue. Ms: Musculus intermandibularis + Musculus geniohyoideus + Musculus pterygoideus + Musculus hyoglossus. D: Dentary bone. Mk.fs: Meckelian fossa. Ig: Infralabial glands. Fr: Frontal bone.

**Figure 3.** Transverse CT image of the head of *Varanus komodoensis* at the level of the eyes corresponding to line III. (**A**) CT bone window. (**B**) CT soft tissue window. These images are displayed so that the right side of the head is to the viewer's left and the dorsal view is at the top. Frs: Frontal sinus. Br: Brain. Sco: Scleral ossicles. Ls: Lens. Vh: Vitreous humor. Hg: Harderian gland. Sc: Sclera. J: Jugal bone. Ec: Ectopterygoid bone. Pt: Pterygoid bone. D: Dentary bone. Mk.fs: Meckelian fossa. mPt: Musculus pterygoideus. Lxc: Laryngeal cavity. Lxm: Laryngeal muscles. Ms: Musculus intermandibularis + Musculus geniohyoideus + Musculus genioglossus + Musculus hyoglossus. Crc: Cricoid cartilage. Arc: Arytenoid cartilage. Co: Cavum oris. Sr: Sublingual recess.

**Figure 4.** Transverse CT image of the head of *Varanus komodoensis* at the level of the parietal bone corresponding to line IV. (**A**) CT bone window. (**B**) CT soft tissue window. These images are displayed so that the right side of the head is to the viewer's left and the dorsal view is at the top. P: Parietal bone (frontoparietal suture). Pfr-Po: Postfrontal + Postorbital bone. Br: Brain. Sp: Sphenoid bone. Pt: Pterygoid bone. San: Surangular bone. Co: Coronoid bone. Co: Cavum oris. Tr: Trachea. Hs: Hyobranchial skeleton. Ms: Musculus intermandibularis + Musculus geniohyoideus + Musculus hyoglossus. J: Jugal bone. Ig: Infralabial glands. mAEM: Musculus adductor mandibulae externus. mPt-mAPM: Musculus pterygoideus + Musculus adductor mandibularis posterior.

**Figure 5.** Transverse CT image of the head of *Varanus komodoensis* at the level of the postorbital + postfrontal bone corresponding to line V. (**A**) CT bone window. (**B**) CT soft tissue window. These images are displayed so that the right side of the head is to the viewer's left and the dorsal view is at the top. P: Parietal bone. Lv: Lateral ventricle. Br: Brain. Ep: Epipterygoid bone. Pt: Pterygoid bone. Psp: Parabasisphenoid bone. Pfr-Po: postfrontal + postorbital bone. San: Surangular bone. Co: Cavum oris. Tr: Trachea. Hs: Hyobranchial skeleton. Ms: Musculus intermandibularis + Musculus geniohyoideus + Musculus hyoglossus. mAEM-mPSTs: Musculus adductor mandibulae externus + Musculus pseudotemporalis superficialis. mLPt: Musculus levator pterygoideus. mPt: Musculus pterygoideus.

**Figure 6.** Transverse CT image of the head of *Varanus komodoensis* at the level of the squamosal bone corrsponding to line VI. (**A**) CT bone window. (**B**) CT soft tissue window. These images are displayed so that the right side of the head is to the viewer's left and the dorsal view is at the top. Sq: Squamosal bone. P: Parietal bone. Cr: Cerebellum (vermis). Bs: Brain stem. Psp: Parabasisphenoid bone. Pt: Pterygoid bone. San: Surangular bone. Co: Cavum oris. Tr: Trachea. Hs: Hyobranchial skeleton. Ms: Musculus intermandibularis + Musculus geniohyoideus + Musculus hyoglossus. mAEM: Musculus adductor externus mandibularis. mPSTs-p: Musculus pseudotemporalis superficialis and profundus. mPt: Musculus pterygoideus.

#### *3.2. Head Volume-Rendered Reconstructed Images*

We provide images of the three-dimensional structure of the Varanus cranium in dorsal and ventral view (Figures 7 and 8, respectively) and the left lateral view (Figure 9). Volume-rendered reconstructed CT images provided good visualization of the different bones that compose the skull. Thus, the orbital border was circumscribed by the lacrimal, the prefrontal, and the jugal bones (Figures 7 and 9). Moreover, the jugal bone was distinguishable from the ectopterygoid (Figure 9). At the posterodorsal border of the orbit, the fusion of postorbital and postfrontal bones could be seen in the lateral and dorsal reconstructed CT images (Figures 7 and 9). In ventral view, the following bones of the neurocranium were clearly delineated: the parabasisphenoid, the basioccipital, and the prootic (Figure 8). The junction between premaxilla and maxilla with the tooth arranged in a curved row was identified in the lateral and ventral view (Figures 8 and 9). In the lateral view, this tooth row curved with the margin of the mandible and maxillary. Besides, the primary curvature of the maxilla was convex, whereas that of the mandible was concave. In addition, the coronoid process was quite prominent, and the surangular and articular bones were observed extending caudally (Figure 9).

**Figure 7.** Three-dimensional volume-rendered reconstruction image of the cranium of *Varanus komodoensis*. Dorsal aspect. Pmx: Premaxillary bone. Mx: Maxillary bone. Sm: Septomaxilla. N: Nasal bone. Prf: Prefrontal bone. Fr: Frontal bone. L: Lacrimal bone. J: Jugal bone. Q: Quadrate bone. Sq: Squamosal. Pfr-Po: Postfrontal + postorbital. P: Parietal. Pro: Prootic. San: Surangular bone. Cv1: First cervical vertebra. Cv2: Second cervical vertebra.

**Figure 8.** Three-dimensional volume-rendered reconstruction image of the cranium of *Varanus komodoensis*. Ventral aspect. Mx: Maxillary bone. D: Dentary bone. San: Surangular bone. Art: Articular bone. Q: Quadrate bone. Bo. Basioccipital bone. Psp: Parabasisphenoid bone. Pro: Prootic bone. Pt: Pterygoid bone. Pa: Palatine bone. V: Vomer. Sm: Septomaxilla. N: Nasal bone. Fr: Frontal bone. P: Parietal bone. Ha: Hyoid apparatus. Cv2: Second cervical vertebra.

**Figure 9.** Three-dimensional volume-rendered reconstruction image of the cranium of *Varanus komodoensis*. Lateral aspect. Pmx: Premaxillary bone. Mx: Maxillary bone. Prf: Prefrontal bone. N: Nasal bone. Fr: Frontal bone. L: Lacrimal bone. J: Jugal bone. Ec: Ectopterygoid bone. Pt: Pterygoid bone. Ep: Epipterygoid. Q: Quadrate bone. Sq: Squamosal. Pfr-Po: Postfrontal + postorbital. Pro: Prootic. D: Dentary bone. T: Tooth. San: Surangular bone. Art: Articular bone. Co: Coronoid bone. Ha: Hyoid apparatus.

#### *3.3. Maximum Intensity Projection (MIP) Images*

Two MIP images corresponding to dorsal (Figure 10) and ventral (Figure 11) views of the varanid skull were selected. These images were able to resolve the relation between the bones that comprise the head. The dorsal MIP image showed the junction between the premaxilla and the maxilla. We were also able to show how the laminar disposition of the vomer supports the septomaxilla (Figure 10). This last finding could be better distinguished in the ventral MIP image (Figure 11). The relation between the lacrimal, the prefrontal, and the frontal bones was seen in the dorsal view (Figure 10). At the posterodorsal border of the orbit, the fusion of postorbital and postfrontal bones could be easily seen. In addition to these observations, the junction of the frontal and the parietal bones were identified in dorsal (Figure 10) and ventral (Figure 11) MIP images. The ventral MIP image showed excellent visualization of the pterygoid, a flat, Y-shaped bone. This bone provides a rounded process that contacts the caudal border of the palatine. Moreover, this view displayed the junction between the parabasisphenoid, the prootic, and the basioccipital bones. This last bone forms the ventral portion of the occipital condyle.

**Figure 10.** Dorsal MIP image of the cranium of *Varanus komodoensis*. Px: Premaxillary bone. Mx: Maxillary bone. Sm: Septomaxilla. V: Vomer. N: Nasal bone. Prf: Prefrontal bone. Fr: Frontal bone. L: Lacrimal bone. Sco: Scleral ossicles. J: Jugal bone. Pfr-Po: Postfrontal + postorbital. P: Parietal. So: Supraoccipital. Sq: Squamosal. Ost: Osteoderms. Cv1: First cervical vertebra. Cv2: Second cervical vertebra.

**Figure 11.** Ventral MIP image of the cranium of *Varanus komodoensis*. D: Dentary bone. Mx: Maxillary bone. San: Surangular bone. Art: Articular bone. Bo. Basioccipital bone. Psp: Parabasisphenoid bone. Oc: Occipital condyle. Pro: Prootic bone. Sm: Septomaxilla. V: Vomer. Pa: Palatine bone. Pt: Pterygoid bone. Cv1: First cervical vertebra. Cv2: Second cervical vertebra.

#### **4. Discussion**

In recent years, the contribution of imaging techniques to reptile medicine has increased the knowledge in veterinary practice and research [4–6,12]. Traditionally, radiography and ultrasonography have been used to obtain information on the bony and the main soft-tissue structures of different reptile regions [14,15]. More advanced imaging techniques, such as computed tomography, have become increasingly common in veterinary clinical practice [15]. Third and fourth generation CT scanners give considerable advantages over traditional radiography: body sections from different tomographic planes, fair anatomic resolution without superimposition of the tissues, and a higher differentiation of tissue densities allow better detection of several diseases [6,15,16]. Nonetheless, its use in reptile medicine is still limited because of the cost of the equipment, availability, and logistic problems of acquiring CT images in these animals [6,15].

The cranium of the genus *Varanus* is a complex structure that has received some attention in morphofunctional studies [12,17], perhaps due to the enormous disparity in the form that evolved among varanid lizards [18]. The head of this iconic varanid represents a complex structure, composed of various tissues with varying degrees of attenuation in radiographic images, making it a challenging object to assess. The two window settings used in our CT study facilitated the identification of the main head structures such as the bones of the skull, mandible, muscles, air-filled structures of the respiratory and digestive system, the nervousranium and other associated structures. Visualizing images through use of the "bone window" provided good resolution for skeletal structures, whereas the "soft tissue window" allowed us to distinguish the eyes and the nervous structures from the remaining soft tissues. Similar results were described in other studies conducted in reptiles [1,5,6,15]. Several causes have been reported to explain the low resolution of soft tissue structures showed in our study [1,15], such as the small volume of these species, the impossibility of reducing the field of view of the CT scanner, and the presence of bones embedded within the skin. These bones, called cephalic osteoderms, vary in shape and complexity and serve primarily as a defensive anatomical system to protect individuals during aggressive confrontations with other specimens [11]. To avoid this low resolution, some investigations reported the use of micro-CT scanners [17], although this equipment is not usually available in veterinary clinics [15].

Employing computed tomography, we were able to fully visualize the cranium in virtual reconstructions and MIP images. Thus, the reconstructed images showed a broad, dorsoventrally compressed cranium. The mandible was curved, and teeth were laterally compressed. This morphology contrasts with most other varanids, which feature a relatively narrow rostrum, a dorsoventrally tall cranium, and a straight ventral margin of the maxilla [18]. Additionally, an enormous variation of the orbit was observed, especially along the posterior margin of the orbit, which is closed in lizards, or semiclosed in these varanids. This fact is determined by variation in the shape, size, and presence of the jugal bone and variations in the postorbital and postfrontal bones [19]. MIP images proved a helpful tool in delineating bones in volume-rendered images.

In conclusion, the CT images obtained in this study provided an adequate anatomical interpretation of the head of *Varanus komodoensis*. This information could be used to diagnose disorders involving the head of lizards, such as abscesses, metabolic bone diseases, fractures, and neoplasia.

**Author Contributions:** Conceptualization, A.A., S.P. and J.R.J.; Methodology, S.P., J.A.C., E.G.-R., A.S.-B. and M.E.; Formal analysis, J.R.J., M.E. and A.A.; Investigation, S.D. and M.M.; Writing original draft, A.A. and J.R.J.; Writing—review and editing, C.M.; Supervision, J.R.J. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding and the University of Las Palmas de Gran Canaria funded the study.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethical Committee of Las Palmas de Gran Canaria University, College of Veterinary Medicine Section (protocol code MV-2019/04).

**Data Availability Statement:** Not applicable.

**Acknowledgments:** In loving memory of Alvaro Domingo Rodriguez Garcia. We also thank Marisa Mohamad for her support and constructive comments.

**Conflicts of Interest:** The authors declare no conflict of interest.

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