In the following sections, we describe current standard methods for physical assessment, laboratory examinations, and diagnostic imaging techniques to evaluate mammary glands. In humans, regular check of these organs is vital for prevention, early detection and treatment of pathologies such as breast cancer [37]. For veterinarians, maintaining udder health in dairy animals is vital for optimal sustained milk production (galactopoiesis), as mammary gland inflammation (mastitis) can affect not only the overall health of the animal, but also milk yield, resulting in economic losses [38]. In contrast, clinical examination methods are limited in marine mammal medicine due to the animals’ aquatic environment and transportation difficulties, with a consequent lack of standardized approaches in these species. Moreover, mammary tumorigenesis in marine mammals is extremely rare, with only a few cases reported in beluga whales (Delphinapterus leucas) [39], suggesting a low incidence or underestimation of such neoplasms compared to humans and domestic mammals [40]. As a result, for species under human care (e.g. in zoos and aquaria), mammary glands are typically ignored in clinical examinations, only receiving focused evaluation when they are visibly swollen in association with pregnancy or pathology (pers obs., Sánchez-Contreras).

Mammary glands are under hormonal control for their development and growth, especially during important ontogenetic stages like lactation. Although multiple hormones regulate aspects of milk production (e.g. oxytocin, progesterone, estrogens), prolactin is the most important [41]. Prolactin can promote mammary gland growth, initiate the production of milk (lactogenesis), and sustain lactation (galactopoiesis) with the help of other metabolic, local, and reproductive processes [42]. Baseline concentrations of prolactin fluctuate depending on the reproductive stage of species such as humans, rodents, and ruminants [43]. In a diversity of mammals (from humans [44] to bottlenose dolphins [6]), prolactin concentrations rise with the progression of pregnancy and reach their highest concentrations in the last month before parturition. In marine mammals, prolactin is known to be of the utmost importance for the development of mammary gland secretory cells, for example, increasing in otariids (eared seals) usually one or two days before parturition and showing the highest levels ~ 0–3 days postpartum [45]. Measuring this hormone is therefore extremely valuable for monitoring the development of important reproductive events such as pregnancy and lactation, which can help differentiate healthy and unhealthy pregnancies and predict successful lactation, which is vital for offspring survival.

Blood is the most common biological sample taken for measuring prolactin levels in humans and other animals [46, 47]. Prolactin levels are affected by age, sex, stress, circadian rhythm, and even drug ingestion, and therefore need to be considered when measuring hormone concentrations [48]. Medical and veterinary researchers have sought less invasive methods for measuring prolactin, in urine [49, 50] and saliva from humans, dogs, and rhesus macaques (Macaca mulatta) [51,52,53]. To our knowledge, only urinary analysis has been used in marine mammals as a non-invasive method to determine prolactin levels, but only in bottlenose dolphins [6] and belugas [54] under human care. In fact, whereas other reproductive hormones (e.g. progesterone, estrogen) are regularly monitored in managed animals during pregnancy [54,55,56], prolactin is not commonly measured in marine mammals, in part due to the lack of specific essays for these species, limiting understanding of normal ranges of this hormone and its role in reproduction. Veterinarians and researchers would therefore benefit greatly from better knowledge of baseline ranges of prolactin in healthy female cetaceans of various ages and reproductive stages, to predict optimal mammary gland development for lactation and calf survival.

Other factors surely regulate mammary gland development (e.g. the milk protein α-lactalbumin, mentioned above; [20]) and therefore could prove useful for regular diagnostics, but are yet to be thoroughly explored for cetaceans. Placental lactogens, for example, are peptide hormones produced and secreted by the placenta [57, 58], identified in both primates and non-primates, including rodents [58, 59] and ruminants (cows, goats, and sheep; [60, 61]. These hormones serve key regulatory roles throughout pregnancy, promoting fetal and placental growth [60, 62] and facilitating development and functionality of the mammary gland during and after gestation. Even though growth of mammary ducts is primarily regulated by ovarian steroids, complete lobuloalveolar growth is only fully achieved with lactogenic hormones, like placental lactogens [61]. Placental lactogen concentrations fluctuate throughout pregnancy [61], with concentrations positively correlating with placental mass in humans [59], the stage of gestation in cows and sheep, and fetal number/milk production in sheep [60]. In humans, after the placenta is delivered, this hormone is quickly cleared from the maternal bloodstream [62]. In cetaceans, placental lactogens were identified immunohistochemically in three rorqual whales: Bryde’s (Balaenoptera brydei), sei (B. borealis), and common minke (B. acutorostrata) [63]. Localization of the hormone to placental trophoblast cells in these species suggest a primary secretion in the maternal bloodstream, yet the precise effects of placental lactogens on either mother or fetus remain uncertain.

The most common way to evaluate mammary gland health in humans and other mammals is physical examination. In humans, as part of an integrative assessment, a clinical breast exam (CBE) provides vital information for diagnosis of breast diseases and abnormalities [64] and can aid in early detection of breast cancer as a complement to mammography [37]. The basic components of a CBE include visual inspection and palpation of the breasts and nipples, characterizing various parameters such as color, symmetry, shape, temperature, texture, venous patterns, and size and allowing detection of lesions or masses [37, 65, 66]. Although CBE can successfully detect breast cancer without additional tests, multiple factors relating to the patient and examiner can impact examination sensitivity and specificity [37]; as a result, CBE has been increasingly conducted alongside modern diagnostic imaging tools (see below), particularly for diagnosing breast malignancies [67, 68].

In veterinary medicine, physical assessment of the mammary gland has been performed mainly in farm animals such as cattle and to a lesser extent for small ruminants [69,70,71]. As in human CBE, physical examination of the udder and teats in veterinary medicine includes visual inspection followed by palpation, allowing inexpensive and quick early detection of pathologies such as mastitis or intramammary infections [69]. Examinations describe a diverse range of features that speak to the health of mammary glands: the texture, volume, symmetry, and thickness of the teat and nipple orifice; udder shape and edema; mastitis, knotty tissue; as well as presence of wounds, inflammation or swelling of the teat (thelitis) and inflammation of the teat cistern (cisternitis). Microbiological tests and milk somatic cell counts (MSCCs) are often used in follow-up to verify the presence of infections [69, 70]. These evaluations are not only useful for determining herd health with regard to milk production [72], but can also aid in identifying inadequate use of milking equipment that could damage mammary tissues (e.g. in dairy goat farms) [73].

In marine mammals, only a few descriptions of physical evaluation of mammary glands exist and only for species under human care, such as polar bears (under anesthesia) [74]. The limited adoption of this approach is due in part to challenges faced by the habitats and anatomy of species. In captive dolphins, for example, the blubber and the internal positioning of the glands makes palpation largely ineffective as a diagnostic technique. As a result, physical evaluation of mammary glands is performed only when the animal’s external visual appearance indicates the presence of infection. In these cases, when animals are lactating, milk samples might also be taken to verify infection [75, 76]. For these reasons, imaging techniques such as ultrasound (see below), are particularly important complements for laboratory analyses in marine mammals.

Across species and medical contexts (human or veterinary), the most common imaging techniques used for mammary gland assessment include radiography (mammography), magnetic resonance imaging (MRI), positron emission tomography (PET), computed tomography (CT) scanning, and ultrasonography [11]. Because of its comparative portability and ability to render soft tissues in real-time, ultrasonography is particularly apt for mammary gland imaging and so we devote more time to discussing its different modalities and future applications.

Each method described below is apt for imaging particular tissues, creating visual contrast based on characteristics of tissue composition and structure, whether healthy or pathological [11, 77]. As a result, the properties of tissues also affect their imaging: in humans, age, hormones and the use of prescription drugs can cause tissue changes (e.g. increased fibrosis) that limit imaging and hamper the diagnosis of breast pathologies. Imaging techniques, therefore, are usually considered complementary to visual and physical assessments as diagnostic tools for detecting abnormalities or monitoring pathology progression in mammary glands [78]. Depending on the findings, laboratory tests might still be necessary companions to imaging to reach a definitive diagnosis [79,80,81,82].

Mammography is one of the most common imaging techniques used in human medicine to assess women’s breast tissues. This radiographic method creates an X-ray shadowgram by irradiating the breast, compressed onto an underlying platform, above an X-ray sensitive film or receptor, where the X-ray tube can rotate to create specific radiographic projections from diverse angles [77, 83]. To detect abnormalities (e.g. micro-calcifications, masses), high resolution and low contrast are needed with a low dose of radiation to minimize patient exposure risk [84]. Compression of the breast during the examination is important to lower the dose of radiation, enhance contrast, avoid movement artifacts, and minimize breast tissue overlap (superimposition), which can hamper assessment and pathology localization [77]. Based on image grayscale variation (a function of radiodensity), mammography can detect pathological soft tissue alterations, calcifications of different shapes and sizes, as well as disruptions in breast architecture [77]. Moreover, this imaging technique is efficient for early cancer detection, reducing mortality and helping improve treatment outcomes [85, 86]. Risks associated with this technique, such as radiation exposure, have dropped significantly due to the development of full-field digital mammography [84], where an electronic detector is used to absorb the radiation applied to breast tissues [83, 87]. In contrast to traditional film mammography, digital processing methods can be applied after image acquisition to modify brightness and contrast without the need to expose the patient multiple times [87]. In veterinary research and practice, mammography is applied mainly in preclinical settings (e.g. animal subjects used for human medical research) and not as a diagnostic tool in a veterinary clinical setting. For example, studies have examined the mammary glands of mice in-vivo [86] or udders from slaughtered sheep [88] to improve mammography in human patients.

In marine mammal medicine, mammography is not feasible, due to the internal and ventral abdominal location of the glands (Fig. 1), which prevents isolation of the mammary glands within the scanning field. However, with the development of portable and wireless X-ray machines, radiographic studies have been successfully performed in live marine mammals for skeletal, soft tissue and intraoral examinations (e.g. Figure 4) [89, 90]. The same risks and limitations associated with radiological exams exist for marine mammals, but in some cases these species present even greater challenges: given their reproductive anatomies and aquatic habitats, patient movement, correct positioning of structures of interest, and equipment damage (e.g. from salt water) are all relevant obstacles [89].

Field-based plane X-ray procedure for a male Atlantic bottlenose dolphin to evaluate the spine. a The dolphin is positioned for imaging, with two trainers stabilizing it while two veterinarians conduct the procedure. The veterinarian in the red shirt holds the X-ray source while another veterinarian holds a digital detector on the contralateral side of the animal. b The veterinarian analyzes the digital X-ray image displayed on a laptop. Photo credit: The Dolphin Company

Like mammography/plane radiography, computed tomography measures the differential attenuation of X-ray beams as they pass through various tissues. A CT scanner involves a circular rotating structure (gantry), which holds the X-ray source and associated radiation detectors on opposite sides of the patient, as well as a moving table that passes through the gantry where the patient is lying [89]. In this way, a CT scanner creates many (often thousands of) radiographic projections taken from multiple positions around the subject to reconstruct tissue arrangements in 3D, enabling cross-sectional and volume renderings of anatomical features and differentiation among diverse tissue types based on their radiodensity [91]. CT is widely utilized in human medicine to evaluate both normal and pathologically altered organs, particularly to image tissues with high attenuation (e.g. bones, teeth) or soft tissues injected with contrast media (e.g. mammary gland vasculature) [92]. Compared to MRI (see below), the achievable resolution is similar, but CT is considerably faster [91, 93]. The application of CT in breast tissue evaluation in women has been limited, primarily due to concerns of high radiation exposure (e.g. to organs in addition to the mammary glands); as a result, mammography has remained the preferred screening method [92, 94, 95]. However, several studies have focused on developing CT technology specifically for breast imaging (e.g [96,97,98]). , which can be particularly effective for elements with high attenuation, such as microcalcifications and neoplastic soft tissue elements, where CT imaging can deliver excellent anatomical detail and volumetric imaging [94].

The increasing accessibility of CT scanning, its lower cost relative to other advanced imaging techniques, and the availability of free viewers for DICOM (Digital Imaging and Communications in Medicine) files, have facilitated the adoption of this modality in veterinary medicine [91, 99]. CT has been used to examine a diverse range of species, encompassing companion animals, wildlife, and even aquatic species, and to evaluate various anatomical elements, including the thoracic and abdominal cavities, central nervous and musculoskeletal systems [91, 100]. The broadened interest in this imaging modality and in digital imaging approaches has also supported a rise in online imaging databases (e.g. Morphosource.org, including the openVertebrate project: https://www.floridamuseum.ufl.edu/overt/), often geared toward skeletal research, where comparative anatomical data can be flexibly shared and explored. However, the same attention has not been given to mammary glands, with existing research primarily focusing on dairy species such as goats and cattle. In goats, CT has allowed the visualization of udder substructures and surrounding fascia and muscles. Moreover, visualization of the mammary vessels after the injection of contrast medium was also possible, allowing different areas of the lactiferous sinus to be identified and their volumes calculated non-invasively [92]. Additionally, CT imaging helped distinguish between tissue types in dairy heifers undergoing somatotropin treatment [101], successfully differentiating between parenchymal and extraparenchymal tissues [101, 102]. This enabled non-destructive cross-sectional and volumetric measurements of the mammary gland, which are far more precise and less labor-intensive than previous estimates derived from manual dissections [103].

In marine mammal medicine, CT has already been employed in various experimental and anatomical studies of live animals: for example, to investigate body composition (blubber, skeletal muscle) in grey seal pups (Halichoerus grypus) [104] and measure splenic volume in harbor seals (Phoca vitulina) and California sea lions (Zalophus californianus), either restrained, sedated, or anesthetized for the procedure [105] (Fig. 5). Although plane radiography remains a more standard approach, CT scanning has shown great utility in marine mammal medicine by offering superior imaging detail and the ability to visualize complex structures in 3D (i.e. without the superimposition of 2D projections), improving the evaluation of disease severity and surgical approach planning [106]. In a clinical context, CT scans of marine mammal patients have revealed various conditions that are undetectable through common diagnostic methods or palpation [