We collected the pituitary glands from four orcas (Orcinus orca) that died between 2021 and 2025 and had been born under human care at Loro Parque (Canary Islands, Spain). The sample included three females (two adults and one juvenile) and one adult male. Full post-mortem examinations were conducted within 12 hours post-mortem, following the standardized protocol described by Kuiken and García-Hartmann (1991), incorporating specific adaptations implemented by the pathology team at the Institute for Animal Health and Food Safety, Universidad de Las Palmas de Gran Canaria (IUSA, ULPGC) (Arbelo et al., 2013; Díaz-Delgado et al., 2018). These were performed by the veterinary pathology team (IUSA) at the Faculty of Veterinary Medicine, ULPGC. At the time of necropsy, all individuals were classified as either “very fresh” or “fresh,” according to the categories established by Kuiken and García-Hartmann (1991). The individual characteristics of the animals, including sex, age class, and cause of death, are summarized in Table 1.

Anatomical extraction of the pituitary gland was performed following the protocol established by Alonso-Almorox et al. (2025). The glands were subsequently processed for histological, ultrastructural, and immunohistochemical analyses using optical microscopy, transmission electron microscopy (TEM), and immunostaining techniques. Additionally, a computed tomography (CT) scan was performed on the head of the juvenile female prior to dissection to assess the precise anatomical location and structural characteristics of the pituitary gland.

The head of a juvenile female orca was dissected and scanned using a 16-slice helical CT scanner (Toshiba Astelion, Canon Medical System, Tokyo, Japan) at the Veterinary Hospital HCV (Hospital Clínico Veterinario) of the Universidad de Las Palmas de Gran Canaria (ULPGC), acquiring transverse CT sequential images. For fitting reasons, only the head was placed inside the scanner. The head was positioned in dorsoventral position during the scan. The acquired images had a slice thickness of 1 mm wish a slice increment of 0.80 mm. We used a variety of window settings by adjusting the window widths (WWs), and window levels (WLs): a bone window setting (WW = 2,700; WL = 550), a soft tissue window setting (WW = 500; WL = 50). The scan was conducted at 135 kilovolt peak (kVp) and an exposure of 75 mAs. The gantry tilt was set to 0°. The analysis was performed with OsiriX MD© software (Pixmeo, Geneva, Switzerland).

For histological evaluation, pituitary samples were embedded in paraffin and sectioned into 5 μm slices. Tissue sections were then stained using hematoxylin and eosin (HE), periodic acid-Schiff–orange G (PAS-OG), Masson’s trichrome (MT), Masson’s trichrome–orange G (MT-OG) and Luxol fast blue stain (LFB). All staining reagents were sourced from Panreac Applichem (Barcelona, Spain) or VWR Chemicals (Radnor, PA, USA). Microscopic imaging was carried out either using an Olympus BX51 microscope, equipped with a DP21 camera and a 0.5X (U-TV0.5XC-3) adapter (Olympus Corp., Tokyo, Japan); or with a Motic Easy Scan One slide scanner (Motic, Hong Kong, China) and the Motic DSAssistant© Software (Motic, Hong Kong, China).

Formalin-fixed, paraffin-embedded (FFPE) pituitary sections (3 μm thick) were placed on Vectabond^®-treated slides (Vector Laboratories, Newark, CA, USA) before undergoing immunohistochemical processing. The following primary antibodies were used: polyclonal anti-ACTH antibody (206A-74) at a 1:75 dilution (Merck, Darmstadt, Germany), polyclonal anti-alpha-MSH antibody (M0939-0.2ml) at a 1:500 dilution (Merck, Darmstadt, Germany), and polyclonal anti-TSH antibody (211A-14) at a 1:100 dilution (Merck, Darmstadt, Germany). Standardized protocols were followed to ensure accuracy.

To evaluate the suitability of commercially available antibodies for use in orcas, we performed in silico validation using BLASTp analysis (NCBI Protein-Protein BLAST) to compare the amino acid sequences of the immunogenic regions targeted in the host species (Homo sapiens) with the corresponding O. orca protein sequences. For each antibody, we retrieved the target protein sequence from the UniProt or NCBI database, identified the epitope or immunogenic region when available, and aligned it with the orthologous orca sequence using BLASTp. Sequence similarity was assessed based on percent identity and query coverage. All three primary antibodies used (anti-ACTH, anti-MSH, and anti-TSH) showed high similarity with their respective orca targets, with 100% identity for POMC-based epitopes (ACTH and MSH) and 89.13% identity for TSH. These results suggest strong to likely cross-reactivity with orca tissue (Supplementary Table 1), supporting their application in this study.

The antibodies and immunohistochemical conditions used in this study are detailed in Table 2. Positive controls consisted of common bottlenose dolphin pituitary tissue, while negative controls were prepared by replacing the primary antibody with non-immune homologous 10% serum in phosphate-buffered saline (PBS) or by using tissues that did not express the target antigen.

Immunostaining was performed using 3,3’-diaminobenzidine (DAB) as the chromogen. Tissue sections were first deparaffinized in xylene and progressively rehydrated in ethanol solutions. To inhibit endogenous peroxidase activity, sections were treated with 3% hydrogen peroxide in methanol for 30 min at room temperature. Antigen retrieval was carried out using either heat-induced epitope retrieval in citrate buffer (pH 6.0) at 96°C for 15 min or enzymatic digestion with 0.1% protease from Streptomyces (P5147) (Merck, Darmstadt, Germany) in PBS. Samples were incubated with primary antibodies at 4°C overnight, followed by a 30-min incubation with a biotinylated secondary antibody at room temperature. Detection was achieved using the VECTASTAIN^® Elite ABC-Peroxidase Kit (PK-6100) (Vector Laboratories, Newark, CA, USA), with DAB development lasting between 2 and 7 min. Mayer’s hematoxylin was applied for counterstaining (5–10 min), and slides were mounted using Faramount Aqueous Medium (S3025) (Agilent, Santa Clara, CA, USA).

For ultrastructural examination, rectangular sections of the adenohypophysis, measuring approximately 2 × 5 mm with a thickness of less than 1 mm, were carefully excised from a sagittal plane using a precision blade. The study did not include samples from the neurohypophysis, as the analysis focused on the endocrine components of the pituitary gland.

Tissue samples were rinsed in PBS for 10 min, followed by overnight fixation at 4°C in a 2.5% glutaraldehyde solution prepared in 0.1 M phosphate buffer (pH 7.4). Post-fixation was carried out in 1% osmium tetroxide (Merck, Darmstadt, Germany) in the same phosphate buffer for 30 min. After dehydration through a graded ethanol series, samples were embedded in Araldite resin M (10951) (Merck, Darmstadt, Germany). Semi-thin sections were prepared using an LKB ultramicrotome and stained with toluidine blue, while ultrathin sections underwent staining with uranyl acetate and lead citrate. Transmission electron microscopy (TEM) imaging was performed with a JEM 1400 (JEOL, Ltd.) at the Central Microscopy Research Facilities, Universidad de Córdoba (Spain).

The results below summarize the structural and cellular features of the orca pituitary gland as revealed by our multimodal investigation.

Due to the limited soft tissue contrast of computed tomography, direct visualization of the pituitary gland was not possible. However, by orienting the scan to the midbrain region—where the hypophysis is anatomically expected—we were able to approximate its location. At this level, within the central base of the skull, a hypodense area was observed, consistent with the vascularized tissue that typically surrounds the gland (Figures 1A, B).

Although no definitive margins of the hypophysis could be discerned, this low-density region appeared embedded within a subtle concavity of the sphenoid bone (Figure 1A), likely representing the shallow depression of the sphenoid bone where the pituitary gland sits. This indirect evidence, along with anatomical positioning, provided the best estimate of the gland’s location in the CT images.

In all four orcas examined, the pituitary gland presented as a flat, bean-shaped structure, broad and elongated, yet notably thin in its dorsoventral profile. Its length consistently exceeded its height by approximately a factor of two. As the glands were extracted in their anatomical relation, still attached to the ventral aspect of the brain, the adenohypophysis was the predominant portion visible on gross examination, appearing considerably larger than the neurohypophysis (Figure 2A).

The gland was embedded within a dense, fibrous pad that was remarkably thick and highly vascularized, enveloping the entire pituitary region. This pad contained a rich and intricate network of arteries and veins, arranged in a branching and interlacing pattern (Figure 2A). This vascular plexus followed the organization pattern and morphology of the rete mirabile. On gross examination, the vascular meshwork extended dorsally and laterally around the gland, forming a prominent connective and vascular interface between the hypophysis and the surrounding cranial structures.

Upon performing a mid-sagittal section through the gland, the neurohypophysis became apparent, located dorsally and slightly caudally relative to the central region of the adenohypophysis. It was approximately three times smaller in volume than the adenohypophysis and was clearly delineated by a fibrous band of tissue, which then continued to form a partial capsule around the larger adenohypophyseal lobe (Figure 2B).

In terms of coloration, the neurohypophysis exhibited a pale, whitish to yellowish hue, consistent with its neural composition. In contrast, the adenohypophysis displayed a deeper brownish-wine coloration, which intensified around the area corresponding to the pars tuberalis and infundibulum, likely due to the increased vascularization of this region.

Histological examination revealed a well-preserved gland with clearly distinguishable adenohypophyseal and neurohypophyseal regions. The entire structure was enveloped by a dense connective tissue capsule containing a complex and highly vascularized retiform architecture. Numerous thin-walled blood vessels were observed running in parallel and perpendicular orientations, branching and converging within the fibrous tissue surrounding the gland (Figures 3A, B). This vascular mesh extended into the glandular parenchyma, particularly at the dorsal and lateral boundaries of the adenohypophysis, and was especially prominent at the interface between the two hypophyseal lobes. At this junction, the rete-like structure formed a thick dural recess, acting as a distinct anatomical boundary between the lobes (Figure 4C).

The adenohypophysis was organized as epithelial hormone-secreting cells arranged in cords, clusters, and anastomosing ribbons, supported by a prominent capillary network and interstitial stroma (Figure 3C). Within the pars distalis, cells were densely packed and showed differential staining affinities in hematoxylin and eosin (H&E), corresponding to chromophilic (acidophilic and basophilic) and chromophobic types. Histochemical staining further differentiated cell populations: PAS–Orange G (PAS-OG) highlighted glycoprotein-rich basophilic cells, particularly those in the central and rostral pars distalis; trichrome Masson (TCM) and TCM–OG staining accentuated the connective tissue framework, capillary distribution, and interstitial stroma, aiding in the delineation of endocrine cell cords and vascular associations (Figures 4D, F). The pars tuberalis was composed of elongated cords of endocrine cells in close association with blood vessels. The infundibular stalk appeared particularly long and slender in this species, owing to the extended pars distalis, and was flanked by connective tissue that maintained the gland’s integrity up to its junction with the hypothalamus (Figures 3A, B, 4A, B).

The neurohypophysis was composed of loosely arranged fibrous tissue, with low cellular density. It contained unmyelinated axons interspersed with glial-like pituicytes and a dense network of capillaries (Supplementary Figure 1 and Figure 3D). PAS-positive eosinophilic accumulations, consistent with neurosecretory material (Herring bodies), were observed in one of the female adult samples (Figure 4E). At the junction with the infundibulum, a distinct but intimate interface was observed, where nervous and endocrine tissues were tightly interwoven in a digitiform manner—finger-like projections of each tissue type interlacing with the other, yet remaining histologically distinct (Figure 4B). This arrangement suggested a structurally complex but clearly demarcated neuroendocrine interface in the orca.

Immunolabelling of the adenohypophysis in orcas revealed specific distribution patterns for ACTH, MSH, and TSH, consistent with a structurally and functionally organized gland.

Transmission electron microscopy revealed that the adenohypophysis of orcas is composed of a heterogeneous population of endocrine cells, distinguishable by differences in granule morphology, cytoplasmic organization, and intracellular components. Although preservation was not optimal in all samples, six hormone-secreting cell types were identifiable based on general ultrastructural features (Figure 7) and one non-granular cell type was also identified (Figure 8). To complement the morphological classification, quantitative comparisons of cell sizes across age and sex groups (Figure 9A) and secretory granule diameters among the granular cell types (Figure 9B) were performed. These measurements further supported the distinction between cell populations and suggested potential variations in cell structure related to biological variables such as age and sex.

The overall architecture of the orca hypophysis adheres to the classical mammalian organization (Gorbman, 1941; Betchaku and Douglas, 1981; Lechan and Toni, 2000; Tziaferi and Dattani, 2018), with a clearly delineated adenohypophysis and neurohypophysis. However, it also exhibits particularities shared only with other cetacean species, and species-specific traits reflecting the unique anatomical and physiological adaptations of orcas. Extraction of the gland in continuity with the brain, following previously described protocols (Alonso-Almorox et al., 2025), ensured the preservation of both the whole gland and its associated structures. Notably, the sphenoid bone in orcas was exceptionally thick, necessitating the use of appropriate dissection tools to facilitate ventral access via the basisphenoid, as outlined in the protocol.

Gross anatomical examination revealed a distinctly flattened and elongated gland, embedded within a thick, fibrous, and highly vascularized connective tissue pad. This flattened morphology and robust vascular bed contrast with findings in smaller delphinids, which typically exhibit a more globular gland and a proportionally thinner vascular cushion (Pilleri, 1969; Yablokov et al., 1972; Cozzi et al., 2016). This morphology is aligned with the previous the findings observed through magnetic resonance imaging (MRI) in a 12 year-old male orca, where the adenohypophysis appeared to be independent and trifolded the neurohypophysis in size, and showed a very characteristic flat and elongated shape (Wright et al., 2017). These morphological differences with other cetaceans may reflect cranial constraints in large delphinids or represent functional adaptations for enhanced vascular integration (Cowan et al., 2008; Vuković et al., 2011; Panin et al., 2013; Alonso-Almorox et al., 2025).

The thick, highly vascularized fibrous pad observed surrounding the gland in orcas closely resembles the rete cushion previously described in dolphins, both in gross appearance and histological architecture (Lillie et al., 2022; Alonso-Almorox et al., 2025). Although this structure has not been fully characterized functionally in cetaceans, its gross configuration is consistent with the definition of a rete mirabile, where a single artery branches into a dense network of smaller vessels before converging again (Cozzi et al., 2016). In terrestrial artiodactyls, such retia are often involved in thermoregulatory processes and countercurrent heat exchange, playing a role in protecting sensitive neural tissues from thermal fluctuations (Mitchell and Lust, 2008). The dense vascular network observed in our study may similarly contribute to regulating the temperature and perfusion of the pituitary gland, which is critical for its endocrine function. While direct physiological measurements are lacking, the morphological prominence of this rete-like structure in orcas—especially in comparison to smaller odontocetes—may suggest a possible adaptive role in supporting the metabolic demands of larger brains or in mitigating pressure-related stress during deep dives. Further studies integrating anatomical, physiological, and imaging data are needed to clarify the function and evolutionary significance of this vascular specialization.

In situ, the ventral aspect of the adenohypophysis constituted the most visible portion of the gland, while the neurohypophysis was positioned dorsally and slightly caudally, obscured by a thick connective tissue septum. Moreover, both the darker coloration in all orca adenohypophysis examined, in contrast to the white to yellow appearance in dolphin species; and the elongated infundibular stalk, resulting from the extended pars distalis reaching toward the hypothalamus, contrast with the shorter stalks observed in smaller odontocetes such as the bottlenose and common dolphins, appear to be unique to orcas and may reflect a vascular and morphological adaptation in these species (Alonso-Almorox et al., 2025).

These anatomical distinctions, while requiring validation with larger sample sizes and more diverse age and sex classes, may reflect ecological, or physiological divergence within the Delphinidae, and emphasize the necessity of developing species-specific reference data for large odontocetes, as their endocrine architecture may diverge from that of smaller cetaceans due to variations in skull morphology, brain positioning, and vascular organization.

Histological analysis of the orca pituitary revealed a well-organized, highly vascularized gland, enclosed within a robust and similarly vascularized dural capsule. A prominent dural recess—composed of dense connective tissue—clearly demarcated the adenohypophysis from the neurohypophysis, as is characteristic in other studied cetaceans (Wislocki and Geiling, 1936; Vuković et al., 2011).

Within the adenohypophysis, hormone-producing cells were arranged in classical cords, clusters, and ribbons, embedded in a dense capillary network. The pars distalis exhibited high cellularity, with clear histochemical distinction between chromophilic and chromophobic cell types, indicative of functional diversity; and the neurohypophysis or pars nervosa also showed the expected sparse cellularity, consisting primarily of unmyelinated axons, pituicytes, and a dense capillary plexus, both as previously noted in dolphin species (Fair et al., 2011; Suzuki et al., 2018; Alonso-Almorox et al., 2025). In contrast, the pars tuberalis appeared more developed in orcas, composed of elongated cords of endocrine cells often closely associated with vasculature. This region featured a striking neuroendocrine interface not yet reported in other studies, with digitiform histologically distinct interdigitations between neural (infundibular) and endocrine tissues, suggesting enhanced neurovascular integration and efficient hormonal exchange.

Immunohistochemical analysis of the orca pituitary revealed distinct regional expression patterns for ACTH, MSH, and TSH, supporting functional compartmentalization within the adenohypophysis. These hormones were selected for their involvement in major neuroendocrine axes and their relevance to critical physiological functions in marine mammals. ACTH is a central effector of the hypothalamic-pituitary-adrenal (HPA) axis and a well-established marker of stress-related activity (Kurusomi and Kobayashi, 1966; Hauger and Dautzenberg, 2000). MSH, derived from the same POMC precursor as ACTH, plays roles in energy balance, appetite regulation, and environmental adaptation, and its cellular localization may shed light on the debated presence or absence of the pars intermedia in cetaceans (Panin et al., 2013). TSH was included due to its essential function within the hypothalamic-pituitary-thyroid (HPT) axis, regulating thyroid hormone production (T3 and T4), which are vital for metabolism, thermoregulation, and growth. Moreover, thyroid disruption has emerged as a critical area of investigation in orcas and other marine mammals due to the bioaccumulation of endocrine-disrupting pollutants—such as persistent organic pollutants (POPs)—that interfere with thyroid hormone homeostasis and have been linked to metabolic, developmental, and reproductive impairments (Tanabe, 2002; Bennett et al., 2021; Bjørneset et al., 2023). The spatial distribution of immunoreactive cells aligned with the classical subdivisions of the pars distalis and pars tuberalis, as seen in other mammals and odontocetes (Cowan and Gatalica, 2002; Cowan et al., 2008; Alonso-Almorox et al., 2025), indicating a conserved endocrine topography across species.

ACTH-positive cells were the most abundant and showed robust staining across all specimens. Their prominent presence in both the pars distalis and tuberalis—especially near vascular sinusoids—highlights their key role in the hypothalamic-pituitary-adrenal (HPA) axis (Yoshitomi et al., 2016). The adult male specimen exhibited a higher density of ACTH-immunoreactive cells, potentially indicating sex-related differences in corticotroph activity or stress-related hormonal demands. Sexual dimorphism in glucocorticoid physiology and corticotroph responsiveness has been reported in other mammals, including mice and humans (Kroon et al., 2020; Moisan, 2021; Duncan et al., 2023). This individual was also the oldest among the studied (29 years old), and studies in humans have demonstrated that the volume density of ACTH-producing cells tends to increase with aging (Pavlović et al., 2021). Additionally, animal models have shown similar increases in corticotroph labeling under chronic stress conditions (Kapitonova et al., 2010). Given that these orcas were captive individuals of varying ages and medical histories—including some with documented chronic pathologies—these factors may have contributed to interindividual variability in ACTH expression. While our sample size limits definitive conclusions, the combined effects of age, stress, and health status should be considered when interpreting corticotroph distribution.

MSH-expressing cells were widely distributed, primarily within the pars distalis, where they formed small clusters or linear arrangements consistent with patterns reported in other delphinids (Panin et al., 2013; Alonso-Almorox et al., 2025). Notably, these cells were more frequently observed in the dorsal region of the pars distalis, adjacent to the dural recess that separates the two hypophyseal lobes, while fewer were detected in the pars tuberalis. This distribution pattern is of particular interest because in many mammalian species, MSH-producing cells (melanotrophs) are primarily localized in the pars intermedia, a distinct zone juxtaposed between the adenohypophysis and neurohypophysis (Naik, 1973; Takeuchi, 2001). However, the presence of a true pars intermedia in cetaceans has long been debated. Most studies suggest that cetaceans lack a distinct pars intermedia, with melanotrophs instead dispersed throughout the adenohypophysis, and the dural recess clearly separates the anterior and posterior hypophyseal lobes (Wislocki and Geiling, 1936; Slijper, 1962; Harrison, 1969; Arvy, 1971; Panin et al., 2013; Alonso-Almorox et al., 2025). A similar condition has been reported in other species like manatees, which also appear to lack this intermediate zone (Quiring and Harlan, 1953). However, some authors have described the presence of a so-called “dorsal shoulder” in dolphin species, a distinct region located dorsally to the pars distalis and adjacent to the dural septum, characterized by a different cellular organization and arrangement of cell cords. This area has been interpreted as a possible remnant of the pars intermedia (Cowan et al., 2008), raising questions about the evolutionary and functional significance of this anatomical variation in cetaceans. The predominance of MSH-expressing cells in the dorsal pars distalis of orcas, adjacent to the dural recess, reinforces the hypothesis of a functionally distinct dorsal region in cetaceans and underscores the need for further investigation into the evolutionary and physiological implications of this potential pars intermedia remnant.

TSH-positive cells were the least numerous, localized mainly in the pars distalis. Their compact, polygonal shape and discrete staining suggest a tightly controlled secretory function, reflecting the thyroid axis’s sensitivity to metabolic feedback (Hoermann et al., 2015). Despite their low abundance, their proximity to vascular channels likely facilitates effective hormone release. In cetaceans, the hypothalamic-pituitary-thyroid (HPT) axis plays a pivotal role in maintaining metabolic homeostasis amidst the physiological challenges of an aquatic environment, such as thermoregulation or energy balance (Fair et al., 2011; Suzuki et al., 2018). Given these unique adaptations, the structure and function of the cetacean HPT axis deserves further investigation to better understand its role in marine mammal endocrinology and environmental resilience.

Beyond the ACTH-, MSH-, and TSH-immunoreactive cells analyzed, the adenohypophysis comprises additional hormone-producing cell populations that were not targeted in this study but are known to play key physiological roles in mammals- Cells producing growth hormone (GH) contribute to somatic growth, protein synthesis, and metabolic regulation; GH may be particularly relevant for supporting blubber deposition, tissue maintenance, fasting and diving or other metabolic adaptations in cetaceans (Weber et al., 1978; Hemming et al., 1986). Prolactin (PRL)-producing cells are involved in lactation, but also play roles in osmoregulation, immune function, and behavioral modulation, with potential relevance in highly complex social species like orcas. Cells that secrete follicle-stimulating hormone (FSH) and luteinizing hormone (LH) regulate reproductive function and are influenced by both internal and environmental cues, including stress, nutritional status, and contaminant exposure (Hart et al., 1984; Ottinger et al., 2009; Delbes et al., 2022). While the present study focused on a selected set of hormones to provide a first morphological and immunohistochemical framework for orca adenohypophyseal organization, further work is needed to identify and characterize the full range of hormone-secreting cell types in this species. Future studies employing broader immunolabelling approaches will be essential to develop a more complete understanding of the endocrine architecture of the cetacean pituitary and its functional significance in the context of marine mammal physiology.

Together, these findings confirm a structurally and functionally specialized adenohypophysis in orcas, with distinct endocrine cell populations contributing to systemic homeostasis. The immunohistochemical profiles reported here, documented for the first time in this species, generally mirror those observed in other odontocetes, reinforcing the conserved nature of pituitary architecture across body sizes and ecological contexts (Cowan et al., 2008; Alonso-Almorox et al., 2025). While also revealing unique, species-specific features that may reflect the particular physiological demands of this apex marine predator.

Transmission electron microscopy revealed multiple endocrine cell types in the adenohypophysis of orcas, distinguishable by differences in size, granule morphology, and cytoplasmic organization. Although ultrastructural preservation was suboptimal in some specimens, limiting detailed resolution and requiring broader morphological descriptions compared to prior odontocete studies (Young and Harrison, 1970, 1977; Alonso-Almorox et al., 2025), the overall cell types and architecture aligned with those described in mammals, allowing clear cellular identification (Moriarty, 1973; Uei and Kanzaki, 1976; García-Ayala et al., 1997). The small sample size, a consequence of limited postmortem access to orcas, also constrained statistical comparisons, especially for sex- or age-related variation. Quantitative estimation of the relative abundance of different endocrine cell types was not feasible due to both the limited number of samples and inconsistent tissue preservation. Moreover, our study focused on the endocrine aspects of this gland, leaving the neurohypophysis ultrastructurally unexplored. Future research should include the pars nervosa as well, to obtain a fuller perspective of the neuroendocrine nature of this gland. Nonetheless, these findings offer a valuable reference for future neuroendocrine and welfare studies in this species and enrich broader knowledge on cetacean pituitary complexity.

The spatial distribution and organization of endocrine cells were comparable between orcas and smaller odontocetes like common dolphins (Young and Harrison, 1970, 1977; Alonso-Almorox et al., 2025). Both showed marked cellular pleomorphism, with secretory cells arranged singly or in clusters, often near fenestrated capillaries—facilitating hormone release, as expected in vascularized glands. The parenchymal structure, including a delicate reticulin network, and the identification of well-developed rough endoplasmic reticulum and Golgi complex in the endocrine cells were also similar across species, underscoring conserved roles in protein synthesis and granule formation, though ultrastructural assessment in orcas was hindered by preservation quality.

Lactotrophs showed pleomorphic morphology, with heterogeneous granules and prominent Golgi zones, consistent with their lactogenic function as seen in other mammals (Hemming et al., 1986; Christian et al., 2015). In orcas, in opposition to what was observed in common dolphins, lactotrophs were the largest endocrine cells, while somatotrophs, on the other hand, were relatively small cells, featuring the largest secretion granules of the adenohypophysis, supporting their role in growth hormone production (Alonso-Almorox et al., 2025). Among non-granular cells previously described in dolphins, only follicular cells were observed. These lacked granules and displayed elongated projections forming intercellular scaffolds and cavities, with proportions comparable to dolphins. Capsular cells were not identified, possibly due to tissue degradation, but further studies are needed to confirm their presence.

Corticotrophs were identifiable by their small, spherical, electron-dense granules adjacent to plasma membranes and capillaries. Their cytoplasmic profile suggested active ACTH secretion, consistent with the observed strong immunohistochemical labeling. A greater abundance of secretory granules was qualitatively noted in the adult individuals compared to the juvenile, with the adult male exhibiting the highest proportion of granules per cell, indicative of heightened ACTH synthesis or release. These findings align with the results in common dolphins (Alonso-Almorox et al., 2025), and likely reflect differing hormonal demands for stress and energy regulation (Naik, 1973; Uei and Kanzaki, 1976; Duncan et al., 2023). The pronounced granule density in the adult male may reflect multiple contributing factors. As the oldest individual (29 years), age-related changes in corticotroph function are likely, as aging has been associated with increased ACTH cell activity (Pavlović et al., 2021). His documented chronic pathology may have further stimulated the HPA axis, given that prolonged stress and illness are known to affect corticotroph dynamics (Kapitonova et al., 2010; Herman et al., 2016). While such factors are common in captive settings, similar health-related biases also affect studies of stranded or free-ranging individuals. Additionally, the higher granule content in the male may relate to sex-linked differences in corticotroph function and glucocorticoid metabolism, as reported in other mammals (Kroon et al., 2020; Moisan, 2021; Duncan et al., 2023). These findings, together with immunohistochemical data, highlight a multifactorial modulation of corticotroph morphology and physiology. Understanding this complexity is essential for accurately evaluating the welfare and physiological state of cetaceans, especially given the central role of the HPA axis in mediating responses to environmental and internal challenges.

This study provides the first integrative anatomical, histological, and ultrastructural description of the orca pituitary gland, establishing a robust baseline for future neuroendocrinological research. By revealing both conserved and species-specific features, our findings deepen the understanding of cetacean neuroendocrinology and highlight the importance of species-specific reference data. The pituitary emerges as a sensitive indicator of endocrine function, stress physiology, and overall health status. As anthropogenic pressures continue to mount, interdisciplinary efforts such as this one are essential for informing welfare assessments and guiding conservation strategies in large, long-lived marine mammals like orcas. This foundational work opens critical pathways for future studies linking endocrine architecture to environmental, physiological, and behavioral variables in both managed and wild cetacean populations.