The study, conducted at a protected wildlife rehabilitation centre in Kerman province, examined ten mature long-legged buzzards weighing between 650 and 750 grams. Because the species is monomorphic, meaning males and females cannot be distinguished by external appearance, and because the birds were imaged outside the breeding season with no active gonads visible on radiographs, the sex of each animal remained undetermined. An avian specialist confirmed all ten were mature based on physical characteristics, though precise ages could not be established. Each bird received a physical examination to rule out health complications that might skew the results, and body weights were measured individually on a digital balance so that anaesthetic doses could be calculated with precision.

Imaging required careful sedation with a combination of ketamine at 20 milligrams per kilogram and diazepam at 1 milligram per kilogram, administered intramuscularly to minimise stress and movement. The researchers then captured two standard orthogonal radiographic views of each wing, a medio-lateral projection and a ventro-dorsal projection from the coelomic cavity, using a direct digital radiography system set at 60 kilovoltage peak and 2.5 milliamperage-seconds with a source-to-detector distance of 90 centimetres. The birds were placed in dorsal recumbency with their wings extended, and all images were archived in a picture-archiving and communication system for later analysis.

Computed tomography followed on a 16-slice scanner, with the animals again positioned in dorsal recumbency and their wings and legs secured with microporous surgical tape. The CT protocol used 120 kilovoltage peak and 150 milliamperage-seconds, a rotation time of one second, a slice thickness of just 1 millimetre and a pitch of 1. After scanning, the images were reconstructed into bone planes on a specialised workstation, allowing the team to interrogate the humerus in cross-section in a way no plain radiograph can match. Eight parameters were then measured by a single observer across both modalities: the cranio-caudal diameter of the bone, the diameter of the medullary cavity, cortical thickness, total proximal-to-distal length, soft tissue thickness at three regions along the bone, and bone density expressed both as pixel values on radiographs and Hounsfield units on CT.

The headline finding is a stark demonstration of how much conventional radiography can mislead. On plain films, the medullary cavity of the humerus appeared to average 7.32 millimetres in diameter, while CT revealed the true figure to be just 5.67 millimetres. Cortical thickness told the mirror-image story: radiography suggested a cortex of only about 1.06 millimetres, whereas CT measured it at 1.86 millimetres, nearly 75 percent thicker. The explanation lies in the fundamental physics of projection radiography. Because an X-ray compresses a three-dimensional structure into a two-dimensional image, overlapping anatomical structures blur the boundary between dense cortical bone and the air-filled cavity within, systematically overestimating the medullary space and underestimating the cortex. For a surgeon planning fracture repair, that difference is not academic; it determines implant selection and the feasibility of intramedullary pinning.

Not every measurement diverged. The cranio-caudal diameter of the bone itself, around 9.4 millimetres by CT and 9.46 millimetres by radiography, showed only a negligible difference between the two techniques, and this was the one parameter where the comparison did not reach statistical significance. But for medullary cavity diameter, cortical thickness, total bone length, and soft tissue thickness at the proximal, middle and distal regions, the differences between modalities were all statistically significant, with CT consistently delivering the more precise values. Total humeral length, for instance, measured 140.68 millimetres on radiographs but 132.84 millimetres on CT, a discrepancy the authors attribute to the magnification and superimposition inherent in two-dimensional projection imaging.

Perhaps the most biologically intriguing numbers came from the density measurements. On CT, the cortex registered approximately 822 Hounsfield units, the medullary cavity around negative 805 Hounsfield units, and the surrounding soft tissue about 66 Hounsfield units, with no meaningful difference between the right and left wings for any parameter. The medullary value is the giveaway: a strongly negative Hounsfield reading indicates air, confirming the extensive pneumatisation that defines the avian humerus. In birds, the humerus is not filled with marrow in the mammalian sense but with air-filled cavities connected to the respiratory system, an adaptation that lightens the skeleton for flight and assists in respiration and air humidification. The researchers note that a Hounsfield value of 800 plus or minus 50 in the cortex reflects the degree of this pneumatisation, and the symmetry between wings suggests a tightly regulated developmental programme.

That pneumatisation is a double-edged sword. By hollowing the bone, evolution has made pneumatic bones more prone to shattering and fragmenting on impact, and the distal extremities of the humerus lack substantial soft tissue protection, leaving them vulnerable to open, comminuted fractures that are notoriously difficult to repair. Fractures of pneumatic bones, particularly the femur, can even trigger respiratory complications when the injury is open. This is precisely why an accurate anatomical baseline matters. The CT-derived reference values now published for Buteo rufinus, including cortical thickness, medullary diameter, total length and regional soft tissue measurements, give veterinarians a species-specific yardstick against which traumatic injuries can be assessed and surgical plans drawn.

The findings dovetail with a growing body of comparative work. Previous tomographic studies of common buzzards and peregrine falcons documented significant interspecies variation in bone density, cortical thickness and trabecular architecture, suggesting that structural resilience in raptors is closely tied to anatomical adaptation to the demands of flight. Other researchers have used calibrated CT to quantify the avian centre of mass and inertia tensor for biomechanical flight modelling, while anatomical atlases combining CT with cross-sections have supplied reference values for parrots and other species. Studies of roadside hawks have similarly combined radiography and CT with three-dimensional digital modelling to map long-bone anatomy, an approach that improves diagnostic precision and supports surgical planning and post-injury rehabilitation. Even archaeology has benefited, with multi-resolution CT and deep-learning image segmentation now used to identify bird species in Egyptian animal mummies, a testament to how versatile the technology has become.

For the long-legged buzzard itself, the practical implications are immediate. As apex predators and indicators of local biodiversity, these birds occupy dry and semi-dry steppes, grasslands, desert margins, hills and mountains, and every individual successfully returned to the wild after fracture treatment represents a small conservation victory. The study’s authors conclude that while digital radiography remains a valuable first-line tool yielding useful data on humeral indices, CT scanning is the superior diagnostic instrument, its high-resolution three-dimensional views enabling precise structural assessment that directly informs targeted treatment plans and rehabilitation protocols. In a field where the difference between flight and permanent grounding can hinge on fractions of a millimetre of cortical bone, that precision may prove decisive for the raptors of Kerman and beyond.

Subject of Research: Comparative diagnostic imaging of humerus bone structure in the long-legged buzzard using radiography and computed tomography

Article Title: Diagnostic Imaging Analysis of Humerus Bone Structure in Long‐Legged Buzzard (Buteo rufinus): Radiology and CT‐Scan Insights

Article References: Alinejad, N., Esmailinejad, M. R., Shafiei, H., & Jahedi, M. (2026). Diagnostic Imaging Analysis of Humerus Bone Structure in Long‐Legged Buzzard ( Buteo rufinus ): Radiology and CT‐Scan Insights. Veterinary Medicine and Science, 12(6), Article e71233. https://doi.org/10.1002/vms3.71233

Image Credits: AI Generated

DOI: 10.1002/vms3.71233

Keywords: long-legged buzzard, Buteo rufinus, computed tomography, digital radiography, humerus, avian skeletal pneumatisation, raptor medicine, fracture diagnosis, veterinary imaging, wildlife rehabilitation, Hounsfield units, cortical bone thickness

The research was born of necessity. Temminck’s pangolin (Smutsia temminckii) is classified as Vulnerable by the International Union for Conservation of Nature, and its populations have declined drastically across its range due to poaching for illegal wildlife and traditional medicine trades, habitat loss, electric fences, and overexploitation. Individuals rescued from traffickers often arrive at rehabilitation facilities in poor condition and require extended care before they can be released. Yet the species is notoriously stress-prone, and ethical, logistical, and conservation constraints make it impossible to capture, hold, or conduct experimental manipulations on pangolins purely for research purposes. Traditional physiological validation, which involves administering a compound such as adrenocorticotropic hormone to artificially activate the stress axis, was therefore off the table.

To understand why this matters, it helps to look at how stress hormones work. The physiological stress response involves two main systems: the sympathetic nervous system, which triggers the familiar ‘fight or flight’ release of catecholamines, and the hypothalamic–pituitary–adrenal (HPA) axis, which drives the secretion of glucocorticoid steroid hormones. An acute rise in glucocorticoids is adaptive, mobilising energy, enhancing cardiovascular activity, and shifting behaviour to restore homeostasis. But chronic elevation carries serious costs, including immune and reproductive suppression and a general decline in fitness. Researchers therefore track glucocorticoid concentrations as a proxy for the stress an individual experiences and its likely consequences for survival and reproduction.

Blood sampling, the traditional route for hormone monitoring, is particularly problematic for pangolins. Capturing and restraining an animal to draw blood activates the very stress response researchers want to measure, with blood glucocorticoids rising significantly within three to five minutes of capture. Repeated collections are impractical for small-bodied species with limited blood volumes, and serum or plasma hormone levels fluctuate with pulsatile secretion patterns, circadian rhythms, and feeding state. For threatened species, invasive endocrine monitoring has fallen out of favour across much of the research community.

Non-invasive monitoring sidesteps these problems. Once steroid hormones are secreted into the bloodstream by the HPA axis, the liver metabolises them and excretes them in bile and faeces. Because metabolites pool in the gut, faecal glucocorticoid metabolite (fGCM) concentrations provide a smoother, longer-term signal spanning several hours, rather than a snapshot distorted by episodic hormone pulses. Collecting faeces requires no direct human–animal interaction, eliminates stress-related feedback from handling, and allows repeated longitudinal sampling from small-bodied species. But there is a catch: hormone metabolism and excretion are sex- and species-specific, so any EIA must be validated in each species before its results can be trusted.

The team, led by Juan Scheun of the BioPulse Research Group at Tshwane University of Technology, together with colleagues from the South African National Biodiversity Institute, Tshwane University of Technology, and the Mammal Research Institute at the University of Pretoria, turned to biological validation. They opportunistically collected 28 faecal samples from five Temminck’s pangolins—three females and two males—confiscated from the illegal wildlife trade and admitted to the Johannesburg Wildlife Veterinary Hospital during 2020 and 2021. Samples were gathered before and after known stressors such as confiscation, veterinary handling, and transport, with samples collected during periods of no handling serving as baselines. For security reasons, the animals were housed at an undisclosed off-site location, and the hospital’s welfare policies meant no strict sampling regime could be imposed; staff simply collected fresh faeces whenever animals defecated, sometimes days apart.

In the laboratory, frozen samples were lyophilised, pulverised, and sieved, then extracted with 80% ethanol and analysed at the Endocrine Research Laboratory of the University of Pretoria using five different EIAs: a cortisol assay, a corticosterone assay, two 11-oxoaetiocholanolone assays (designated 72a and 72T), and a 5α-pregnane-3ß,11ß,21-triol-20-one assay (37e). Serial dilutions produced displacement curves parallel to the standard curves in all assays, confirming analytical validity. The researchers then applied a stringent criterion: an assay was considered reliable only if it detected peak fGCM increases exceeding 150% above baseline, a tougher threshold than the 100% often used in comparable studies, to reduce the risk of false positives.

The results were striking. In female 1, the 72a assay recorded a 982% increase in fGCM concentrations following a stressful event, with 72T close behind at 763%, followed by the cortisol assay at 481% and 37e at 317%. Female 2 showed increases above the 150% threshold for 37e (325%), 72T (221%), and 72a (201%), and her secondary fGCM rise coincided with a move to a holding facility ahead of release. Female 3 exceeded the threshold on both 72a (339%) and 72T (251%). Among the males, only 72a detected increases above 150% in both individuals, reaching 168% in male 1 and 695% in male 2, where the cortisol assay also responded strongly at 437%. Notably, in female 2, fGCM levels rose considerably before a decline in health that led to her readmission to the rehabilitation centre, hinting that hormone monitoring might one day flag deteriorating welfare before clinical signs appear.

The standout performer was the 72a assay, the only one to exceed the 150% threshold in every study animal of both sexes, making it the recommended tool for monitoring fGCM levels in Temminck’s pangolin. The 72T assay proved a strong alternative for females, while 37e, which exceeded 100% increases in all females, can serve as a back-up when the primary assays are unavailable. The cortisol assay should be treated as equivocal pending further validation. Interestingly, the finding that cortisol metabolites dominated the faecal profile contrasts with work on the Taiwanese pangolin, where mass spectrometry revealed higher faecal corticosterone concentrations, though that study measured actual hormones rather than immunoreactive metabolites, and the authors suggest mass spectrometry could further clarify active circulating glucocorticoids in Temminck’s pangolin.

Beyond the assay validation itself, the study delivers an uncomfortable but important message: direct human interaction, even in the context of care and rehabilitation, elicits pronounced physiological stress responses in pangolins, with increases exceeding 100% across all study animals. Similar patterns have been documented in rehabilitated African penguins and koalas. The findings argue for refining rehabilitation protocols to minimise human contact and for assessing individuals physiologically before release to improve survival odds. The authors caution that the small sample size limits generalisability, that excretion time lags could not be determined for the species—gut passage times in other pangolins range from roughly 20 to 60 hours—and that individual variation in stress perception likely shapes adrenal responses. Even so, the validated 72a EIA now offers researchers, conservationists, and rehabilitation managers a way to track stress in captive settings and, crucially, in free-ranging populations, where pressures such as climate change are expected to intensify. For an animal that rolls into an armour-plated ball when threatened, the ability to read its stress from a dropping may prove one of the most powerful conservation tools yet devised.

Subject of Research: Biological validation of non-invasive faecal glucocorticoid metabolite enzyme immunoassays for measuring physiological stress in rehabilitated Temminck's pangolins

Article Title: Biological validation of enzyme immunoassays for measuring physiological stress in rehabilitated Temminck’s pangolins

Article References: Scheun, J., Labuschagne, K., Jansen, R., Ganswindt, A., & Long, C. (2026). Biological validation of enzyme immunoassays for measuring physiological stress in rehabilitated Temminck’s pangolins. Discover Conservation, 3(1), Article 20. https://doi.org/10.1007/s44353-026-00090-2

Image Credits: AI Generated

DOI: 10.1007/s44353-026-00090-2

Keywords: Temminck's pangolin, faecal glucocorticoid metabolites, enzyme immunoassay, stress physiology, wildlife rehabilitation, non-invasive hormone monitoring, conservation, HPA axis, animal welfare, illegal wildlife trade, biological validation, South Africa