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Collection of shed skin samples from a Komodo dragon. Samples were collected under protected contact using tweezers.
Source publication
The analysis of corticosterone (CORT), the main glucocorticoid in reptiles, via blood or faeces provides an index of hormone
concentrations over a relatively short time period. Unlike these conventional matrices, snake shed skin is supposed to
incorporate circulating CORT over the period of skin growth, thus reflecting long-term retrospective level...
Contexts in source publication
Context 1
... skin samples were collected between March and October 2014 from five Komodo dragons; two adult males, one adult female and two juvenile females, housed at Barcelona Zoo (Spain). Over the period of the study, 39 shed skins were collected with an interval time of 10.56 ± 16.29 days (mean ± SD) between sheds and stored at room temperature. Shed skin was collected with tweezers during training sessions under protected contact ( Fig. 1). This technique allowed the collection and identification of shed skin samples originated from three different body regions (head, body and limbs) of each individual (Fig. ...
Context 2
... skin samples were collected between March and October 2014 from five Komodo dragons; two adult males, one adult female and two juvenile females, housed at Barcelona Zoo (Spain). Over the period of the study, 39 shed skins were collected with an interval time of 10.56 ± 16.29 days (mean ± SD) between sheds and stored at room temperature. Shed skin was collected with tweezers during training sessions under protected contact ( Fig. 1). This technique allowed the collection and identification of shed skin samples originated from three different body regions (head, body and limbs) of each individual (Fig. ...
Citations
... Studies of corticosterone in keratinized tissues of reptiles are limited in number compared to hair and feather glucocorticoid studies in avian and mammalian species, but warrant continued research, as they can be obtained in a minimally invasive manner and may reflect more chronic glucocorticoid levels than can be measured in plasma or other tissues (Berkvens et al., 2013;Baxter-Gilbert et al., 2014;Matas et al., 2016;Carbajal et al., 2018;Hamilton et al., 2018;Gormally and Romero, 2020;Dillon et al., 2021). The chronic presence of a stressor can lead to a persistent physiologic stress response and continuous activation of the hypothalamic-pituitary-adrenal axis due to reduced efficacy of negative feedback mechanisms (Sapolsky, 1992;Sheriff et al., 2009). ...
... In reptiles, corticosterone has been quantified from turtle and chameleon claws (Baxter-Gilbert et al., 2014;Matas et al., 2016), as well as the shed skin of lizards (Carbajal et al., 2018;Zena et al., 2022) and snakes (Berkvens et al., 2013). No effect of a chronic stressor was reflected in shedskin corticosterone in snakes in the study by Berkvens et al. (2013). ...
... No effect of a chronic stressor was reflected in shedskin corticosterone in snakes in the study by Berkvens et al. (2013). Studies on lizard have demonstrated seasonal variations in shed-skin corticosterone (Carbajal et al., 2018;Zena et al., 2022). Further research is needed regarding the ability of shed-skin corticosterone to reflect chronic stress and its relationship to circulating corticosterone levels in reptiles. ...
Renal disease is often identified as a cause of morbidity and mortality in avian patients. However, currently, early antemortem detection of renal disease in avian patients is difficult. Anatomical and physiological differences between mammals and birds mean the use of commonly employed diagnostic testing (ie, measurement of blood urea nitrogen [BUN] and serum creatinine, urinalysis, and ultrasonography) are either nondiagnostic or difficult to achieve. Symmetric dimethylarginine (SDMA) is considered a more sensitive marker for renal disease in humans, dogs, and cats. However, SDMA has not yet been assessed for diagnostic use in any psittacine species. In this study, we establish reference ranges for SDMA in both Hispaniolan Amazon parrots (Amazona ventralis, HAP) and Quaker parrots (Myiopsitta monachus, QP). Blood was collected from 23 Amazon parrots and 32 Quaker parrots maintained in research facilities. Measurement of SDMA through a commercially available immunoassay (IA-SDMA) as well as creatinine, BUN, uric acid, phosphorus, calcium, sodium, potassium, and chloride were determined through IDEXX Laboratories. Plasma SDMA concentrations ranged from 6 to 15 µg/dL and 3 to 15 µg/dL for the HAP and QP, respectively. Sex was a confounding factor for the QP population, but sex did not have a significant effect on SDMA for the HAP population. No significant correlations were identified between SDMA concentrations and other parameters in either psittacine species. Our results show proof of concept for the IA-SDMA and provide reference intervals for SDMA in HAP and QP. Further investigation is required to determine the validity of this assay and the predictive power of SDMA in the detection of renal impairment for parrots and other common companion birds.
... The hypothesis is that circulating hormones are deposited in the new epidermal layer while it is being formed, and potentially, during the time it is in place until the upcoming skin shedding process begins (Berkvens et al., 2013;Zena et al., 2022). In light of this, levels of CORT have been measured in crocodile tail scutes (Hamilton et al., 2018) and in the shed skin of various snake and lizard species (Berkvens et al., 2013;Carbajal et al., 2018;Carbajal, Fern andez-Bellon, et al., 2014;Zena et al., 2022). Increases of CORT levels in these keratinous tissues are thought to reflect prolonged or repeated stress exposures, as described in American alligator (Alligator mississippiensis) subjected to long-term environmental contamination (Finger et al., 2019). ...
... Despite that, the authors revealed a positive association in CORT levels between shed skin and faeces providing, albeit modest, grounds for hope for the use of this alternative non-invasive sample. Further proof that this tool shows promise was given by the biological variation identified in shed skin CORT levels of Komodo dragons (Carbajal et al., 2018). Hormonal patterns in Komodo' shed skin were related to sex, age and season, revealing that there are potential variables that should be considered to overcome confounding influences, as also supported by Zena et al. (2022). ...
Recent advancements in stress physiology, driven by the relevance of the stress response in animal welfare and conservation, have focused on alternative techniques beyond blood sampling for measuring glucocorticoids (GC). While blood samples have been traditionally used, practical and ethical concerns have spurred exploration into minimally invasive media like saliva, feces, milk, hair, and feathers. This review addresses the dearth of research on reptile endocrinology, offering insights into measuring GC or their metabolites in reptiles through various biological tissues. It underscores the importance of considering temporal dynamics in stress response evaluation and advocates for further exploration of alternative tools to enhance our understanding of reptilian stress responses.
... Understanding and identifying, for example, when, how or why reptiles are stressed are important elements for welfare practice, and different authors highlighted the need to further explore non-invasive measures [10,11]. Samples, such as fecal, urinary, salivary or skin, have been already used in some species, e.g., [12][13][14], instead of traditional invasive methods such as blood testing, representing a promising area of research to measure glucocorticoid metabolite levels. Along with this, other authors stated the importance of assessing the welfare of reptiles at an individual level within species, since differences has been found already in responses to novel objects and environments [15]. ...
... However, a recent meta-analysis study found no evidence for widespread sex differences in variability in non-human animal personality [69], which is in line with what we saw. Something similar was obtained for the stress response with no significant differences between FCM levels of males and females, in contrast to what we previously thought, since in other reptiles males show higher corticosterone levels than females [12,40,70] or the opposite with females showing higher levels [41]. What we detected were differences according to the species, with crested geckos showing significantly greater FCM levels than leopard geckos. ...
Many reptiles are maintained in captivity and heavily traded, although welfare measures for many species are not well established and are under-researched compared to other animals. In this study, we focused on two of these species: crested geckos (Correlophus ciliatus) and leopard geckos (Eublepharis macularius). To better interpret their behavior in captivity, the individual reaction to novel objects and the fecal glucocorticoid metabolite levels were measured in an attempt to identify the potential correlation between them. Also, we explored if some characteristic of the objects (e.g., color, shape, or smell) resulted in being more attractive to some species and/or individuals. Equivalent responses to different objects were not obtained for all the geckos, the behavioral response being highly individual and context-dependent, although modulated by the species. Individuals which manipulated earlier and interacted longer with novel objects showed lower basal fecal corticosterone metabolite (FCM) levels. Differences according to the species suggested that crested geckos have significantly greater and more variable FCM levels than leopard geckos. Our results can help to understand the reaction of geckos to novelty and have the potential to serve in their welfare assessment, although more studies are needed to proper establish welfare protocols.
... Thus, reptilian sheds could be useful as a sample type for retrospective evaluation of physiological status, since its collection is minimally or noninvasive, avoids capture stress, and might be useful for species from which blood sampling is not advisable or permitted. However, because few studies have used sheds as a potential matrix for assessments of a reptile's endocrine history (Berkvens et al. 2013;Carbajal et al. 2018), it remains unclear whether sheds accumulate and retain hormones at physiologically meaningful levels. Thus, the value of sheds as a sample type for determining longitudinal profiles of reproduction, nutritional status, exposure to stressors, etc., is not known. ...
... Previous studies have investigated the potential effect of body location on hormone deposition in sheds of reptiles, more specifically corticosterone. One study performed in Komodo dragons (Varanus komodoensis) showed no effect of body region on corticosterone concentration (Carbajal et al. 2018). In another study performed in African House Snake (Lamprophis fuliginosus), no difference in shed skin corticosterone along the length of the snake's body was observed, except for the tail section, which showed higher levels than the other parts of the snake's body (Berkvens et al. 2013). ...
... So far, only the present study and the study by Berkvens and coworkers (2013) have quantified hormone levels of keratinized tissues and tested for correspondence with sample types that reflect relatively short time periods of incorporation, like blood (immediate) and fecal samples (hours to days). Other studies on epidermal tissues (sheds, claws) have not assessed potential correlations with plasma hormones (Baxter-Gilbert et al. 2014;Carbajal et al. 2018). ...
Sampling blood for endocrine analysis from some species may not be practical or ethical. Quantification of hormones extracted from nontypical sample types, such as keratinized tissues, offers a less invasive alternative to the traditional collection and analysis of blood. Here, we aimed to validate assays by using parallelism and accuracy tests for quantification of testosterone, corticosterone, progesterone, and triiodothyronine (T3) in shed skins of tegu lizards. We assessed whether hormone content of sheds varied across one year similar to what was previously detected in plasma samples. In addition, we aimed to identify the phase relationship between hormone levels of shed skin and plasma levels obtained from the same animals. High frequency of shedding occurred during the active season for tegus (spring/summer), while shedding ceased during hibernation (winter). All hormones measured in shed skins exhibited seasonal changes in concentration. Levels of testosterone in shed skins of male tegus correlated positively with plasma testosterone levels, while corticosterone in both males and females exhibited an inverse relationship between sample types for the same month of collection. An inverse relationship was found when accounting for a lag time of 3 and 4 months between sheds and plasma testosterone. These results indicate that endocrine content of sheds may be confounded by factors (i.e., seasons, environmental temperature, thermoregulatory behavior, among others) that affect frequency of molting, skin blood perfusion, and therefore hormone transfer from the bloodstream and deposition in sheds of squamates.
... Traditionally, circulating Corticosterone (Cort), the main glucocorticoid in reptiles, has been used as a biomarker of stress in sea turtles (Gregory et al., 1996;Milton & Lutz, 2003;Tokarz & Summers, 2011). Circulating Cort concentration increases in reptiles that face acute stressful conditions (Carbajal et al., 2018;Cockrem, 2013). Changes in circulating Cort of wild sea turtles have been studied under different field conditions. ...
Frequently, stranded sea turtles require rehabilitation under controlled conditions. Currently, few publications have described the conditions under which rehabilitation is to take place, particularly with respect to the hatchling life stage. To address this paucity of data, we conducted some experiments to assist rehabilitating facilities assess their handling of hatchlings. While in captivity, hatchlings are routinely handled, for example, for data collection and cleaning. Standardization of handling and housing protocols is necessary to define the most adequate rearing conditions to maintain hatchling welfare. Accordingly, the aim of this study was to assess plasma circulating corticosterone (Cort) concentration and growth, as a biomarker for the stress of hatchling loggerhead sea turtles (Caretta caretta) under controlled conditions. We performed two experiments to analyze handling frequency and stocking density. In both, Cort was measured and correlated with variations in animal weight and length. In handling experiments, Cort exhibited no significant increase when hatchlings were handled once a week, whereas Cort was significantly elevated when hatchlings were handled once every 2 weeks, suggesting that hatchlings have the ability to acclimate to frequent handling. However, hatchlings exhibited similar growth and mortality, regardless of handling regime. In stocking density experiments, hatchling isolation induced a significant elevation of Cort, in comparison with hatchlings placed with conspecifics at increasing densities. Growth increased in singly housed hatchlings, while mortality increased in tanks with three or more hatchlings. The results obtained suggest that Cort, growth, and mortality should be measured to assess hatchling welfare when kept under controlled conditions.
... Non-invasive sampling techniques that have been established so far include the collection of both keratinized (e.g., hair, skin, feathers) and non-keratinized (e.g., urine, feces, saliva) tissues (Sheriff et al., 2011). Although previous studies implementing alternative tissue types as biomarkers for stress have been largely conducted across several taxa, not many have focused on reptiles (e.g., Berkvens et al., 2013;Baxter-Gilbert et al., 2014;Carbajal et al., 2018;Hamilton et al., 2018). The efficacy of measuring glucocorticoid metabolites in feces, however, is gaining recognition as corroborated by this study and those similarly conducted on other reptile species (e.g., Kalliokoski et al., 2012;Umapathy et al., 2015;Scheun et al., 2018). ...
Agonistic encounters necessary for territory establishment and maintenance can be stressful for those involved. Stress responsiveness associated with territorial behavior can occur on both acute and chronic temporal scales contingent upon social status. Social interactions that recur for territory maintenance pose periodic stressors that incur variable physiological costs across social ranks. Adult males of the Green Anole, Anolis carolinensis, experience stressful social encounters during territorial disputes as individuals contest status within a dominance hierarchy. Dominant males in stable territories are known to exhibit greener body coloration and lower levels of stress hormone, corticosterone, relative to their subordinate counterparts. Periodic interactions with novel competitors, however, may induce comparable levels of cumulative glucocorticoid secretion regardless of social status. Glucocorticoid metabolites excreted in feces can be quantified to assess the chronic hypothalamic–pituitary–adrenal (HPA) axis response to periodic social stressors. Fecal glucocorticoid metabolite (FGM) levels in male A. carolinensis were hypothesized to increase in response to novel social encounters that simulated territory establishment and maintenance. Adrenocortical response to recurring episodes of territoriality was predicted to generate similar longitudinal FGM levels across social ranks. FGM analysis was combined with behavioral assessment of body coloration to further contextualize measured stress levels of dominant and subordinate anoles. Prolonged social interaction led to similarly increased levels of fecal glucocorticoid metabolites in both dominant and subordinate anoles relative to those that were solitary. This study provides an alternative perspective on the activity of the HPA axis in dominant-subordinate relationships of A. carolinensis over prolonged periods of territoriality.
... Corticosterone is typically measured invasively via a blood sample. However, less invasive sampling methods are available, such as fecal, urinary, salivary, tear, skin shed, and hair samples [22,61]. Due to the lack of baseline reference ranges in reptiles, interpretation is problematic [5]. ...
... In this study, biological variation in corticosterone, as demonstrated by other researchers, was present. For example, corticosterone values were higher in males, juveniles and after spring skin shedding [61]. Further characterization of non-invasive measurement methods is an area ripe for future research, as these alternative methods are being more extensively used in mammalian species. ...
Reptiles are held at wildlife parks and zoos for display and conservation breeding programs and are increasingly being kept as pets. Reliable indicators of welfare for reptiles need to be identified. Current guidelines for the captive management of reptiles utilize resource-based, rather than animal-based indicators; the latter being a more direct reflection of affective state. In this paper we review the literature on welfare assessment methods in reptiles with a focus on animal-based measures. We conclude that, whilst a number of physiological and behavioral indicators of welfare have been applied in reptiles, there is need for further validation of these methods across the diversity of species within the Class. Methods of positive welfare state assessment are comparatively understudied and need elucidation. Finally, we examine some widely-used welfare assessment tools in mammals and explore the application of the Welfare Quality® Protocol to the endangered pygmy blue-tongue skink, Tiliqua adelaidensis. We propose that this framework can form the basis for the development of taxon-specific tools with consideration of species-specific biology.
Measurement of corticosterone in various tissues has been used to investigate the stress response in reptile and amphibian species for decades. The tissue source from which corticosterone is measured reflects different periods of time and chronicity of stress levels in the subject, and different tissue collection methods differ in degree of invasiveness. Studies of corticosterone in keratinized tissues of reptiles, such as shed skin, are limited in number compared to hair and feather glucocorticoid studies in avian and mammalian species, but warrant continued research as they may reflect more different periods of time and chronicity of corticosterone levels than plasma or other tissues, and can be obtained in a minimally invasive manner. In this study, we measured corticosterone concentrations in both plasma and shed skin of Louisiana pine snakes (Pituophis ruthveni) that were all previously diagnosed with subclinical Cryptosporidium serpentis infection. We also tracked stressors experienced by different individuals to identify potential relationships between periods of increased stress and corticosterone levels in plasma and shed skin. There were no significant correlations between individual plasma and shed skin corticosterone levels, or between corticosterone levels in either tissue type and stressors experienced. This is the first study where corticosterone levels were measured in plasma and shed skin of P. ruthveni, and is the first known evaluation of plasma and shed skin corticosterone levels in a snake population previously testing positive for Cryptosporidium serpentis.
Objective:
Measurement of steroid hormones in skin appendages such as mammalian hair or claws and in avian feathers represents a recognized non-invasive method for the determination of these parameters. The aim of this pilot study in the Gila Monster was to investigate whether the measurement of sex steroids in shed skin may be employed for the monitoring of endocrine gonadal function or sex determination in reptiles.
Material and methods:
Shed skins were available from 11 female and 7 male adult and sexually mature animals. Large pieces of skin were initially cut into smaller pieces with scissors. The resultant dermal fragments were finely ground under liquid nitrogen and finally extracted with organic solvents. The following parameters were determined radioimmunologically in the dried and re-dissolved extracts: progesterone (P4), estradiol-17β (E2), testosterone (T), free total estrogens (fGÖ) and free plus conjugated total estrogens (fkGÖ).
Results:
For P4 (p = 0.0052) and E2 (p = 0.0079) significant sex differences were found with higher concentrations in females compared to males. Unexpectedly, the measured values for T were also significantly higher in females (p = 0.0232) than in males, with the concentrations overall only slightly above the detection limit. Compared to fGÖ, the concentrations of fkGÖ were only slightly higher, with no significant differences between both sexes.
Conclusion and clinical relevance:
In this pilot study, the methods employed did not allow for reliable sex determination in individual animals, neither alone nor in combination, due to an overlap between the sexes. In principle, however, the measurement of sex steroids in shed skins could represent a useful method for non-invasive sex determination or endocrine gonadal function assessment in certain reptile species.
