Early-life seasonal, weather and social effects on telomere length in a wild mammal.

Early-life environmental conditions can provide a source of individual variation in life-history strategies and senescence patterns. Conditions experienced in early life can be quantified by measuring telomere length, which can act as a biomarker of survival probability in some species. Here, we investigate whether seasonal changes, weather conditions and group size are associated with early-life and/or early-adulthood telomere length in a wild population of European badgers (Meles meles). We found substantial intra-annual changes in telomere length during the first 3 years of life, where within-individual effects showed shorter telomere lengths in the winter following the first spring and a trend for longer telomere lengths in the second spring compared to the first winter. In terms of weather conditions, cubs born in warmer, wetter springs with low rainfall variability had longer early-life (3-12 months old) telomeres. Additionally, cubs born in groups with more cubs had marginally longer early-life telomeres, providing no evidence of resource constraint from cub competition. We also found that the positive association between early-life telomere length and cub survival probability remained when social and weather variables were included. Finally, after sexual maturity, in early adulthood (i.e., 12-36 months) we found no significant association between same-sex adult group size and telomere length (i.e., no effect of intrasexual competition). Overall, we show that controlling for seasonal effects, which are linked to food availability, is important in telomere length analyses, and that variation in telomere length in badgers reflects early-life conditions and also predicts first year cub survival.

Seed dispersers shape the pulp nutrients of fleshy-fruited plants.

The dispersal-syndrome hypothesis posits that fruit traits are a product of selection by frugivores. Although criticized as adaptationist, recent studies have suggested that traits such as fruit or seed size, colour and odour exhibit signatures that imply selection by animal mutualists. These traits imply nutritional rewards (e.g. lipid, carbohydrate), attracting frugivores; however, this remains incompletely resolved. Here, we investigated whether fruit nutrients (lipid, sugar, protein, vitamin C, water content) moderate the co-adaptation of key disperser-group mutualisms. Multivariate techniques revealed that fruit nutrients assembled non-randomly and grouped according to key dispersal modes. Bird-dispersed fruits were richer in lipids than mammal-dispersed fruits. Mixed-dispersed fruits had significantly higher vitamin C than did mammal- or bird-dispersed fruits separately. Sugar and water content were consistently high irrespective of dispersal modes, suggesting that these traits appeal to both avian and mammalian frugivores to match high-energy requirements. Similarly, protein content was low irrespective of dispersal modes, corroborating that birds and mammals avoid protein-rich fruits, which are often associated with toxic levels of nitrogenous secondary compounds. Our results provide substantial over-arching evidence that seed disperser assemblages co-exert fundamental selection pressures on fruit nutrient trait adaptation, with broad implications for structuring fruit-frugivore mutualism and maintaining fruit trait diversity.

Estimation of environmental, genetic and parental age at conception effects on telomere length in a wild mammal.

Understanding individual variation in fitness-related traits requires separating the environmental and genetic determinants. Telomeres are protective caps at the ends of chromosomes that are thought to be a biomarker of senescence as their length predicts mortality risk and reflect the physiological consequences of environmental conditions. The relative contribution of genetic and environmental factors to individual variation in telomere length is, however, unclear, yet important for understanding its evolutionary dynamics. In particular, the evidence for transgenerational effects, in terms of parental age at conception, on telomere length is mixed. Here, we investigate the heritability of telomere length, using the 'animal model', and parental age at conception effects on offspring telomere length in a wild population of European badgers (Meles meles). Although we found no heritability of telomere length and low evolvability (<0.001), our power to detect heritability was low and a repeatability of 2% across individual lifetimes provides a low upper limit to ordinary narrow-sense heritability. However, year (32%) and cohort (3%) explained greater proportions of the phenotypic variance in telomere length, excluding qPCR plate and row variances. There was no support for cross-sectional or within-individual parental age at conception effects on offspring telomere length. Our results indicate a lack of transgenerational effects through parental age at conception and a low potential for evolutionary change in telomere length in this population. Instead, we provide evidence that individual variation in telomere length is largely driven by environmental variation in this wild mammal.

Alternative reproductive strategies provide a flexible mechanism for assuring mating success in the European badgers (Meles meles): An investigation from hormonal measures.

Selection-pressures differ with population density, but few studies investigate how this can affect reproductive physiology. European badger (Meles meles) density varies from solitary to group-living across their range, with reported mating periods throughout the entire year to specific seasonal periods. Badger reproduction is evolutionarily distinct, interrupting the direct progression from conception to gestation with delayed implantation (DI), allowing for superfecundation (SF). To establish the tactical mating flexibility afforded by DI*SF, we used cross-sectional population-level seasonal variation of circulating sex-steroids for 97 females from a high-density population. Oestradiol was highest in spring among non-parous females, then lower in summer, and remained low during following seasons, suggesting that the mating period was restricted to just spring. Oestrone was consistently higher than oestradiol; it was elevated in spring, lowest during summer, peaked in autumn, and remained elevated for pregnant females in winter. This suggests that oestrone sustains pre-implanted blastocysts throughout DI. Progesterone was low throughout, except during winter pregnancy, associated with implantation and luteal development. In contrast to multiple mating periods reported by lower-density studies, our oestradiol data suggest that, at high-density, females exhibit only one mating period (congruent with testosterone patterns in males studied previously in this same population). While additional mating periods during DI enhance fertility assurance at low-density, at high-density, we propose that when coitus is frequent, fertilisation is assured, precluding the need for further cycles and associated mating risks. This endocrinologically flexible DI*SF mating strategy likely represents a form of balancing selection, allowing badgers to succeed at a range of regional densities.

A non-invasive method to assess the reproductive status of the European badger (Meles meles) from urinary sex-steroid metabolites.

Due to their unique reproductive physiology and behaviour, European badgers (Meles meles) are often used as a model to study mammalian reproduction. For reproductive endocrinology, circulating hormone levels are conventionally measured directly from blood samples. However, routine blood sampling is often not practical for wild animals and may induce stress affecting measurement accuracy. Non-invasive alternatives are thus of interest. Circulating hormones are metabolized through different routes, either by the kidneys, to be excreted through urine, or by the liver, to be excreted through faeces. These metabolites can thus be used as a proxy of hormone measurements, provided the species-specific metabolic characteristics are known. Here we tested the suitability of measuring urinary metabolites of circulating plasma sex-steroid hormones (testosterone in males and oestrogen in females) with enzyme immunoassays to assess the reproductive status of the European badger (Meles meles). Biological validation evidenced that urinary testosterone metabolite (UTM) and urinary total oestrogen metabolite (UEM) excretion patterns both corresponded with seasonal badger reproductive patterns on a population level, signaling correlation over a broad time frame. On an individual level, concurrent sampling of urine and plasma showed that male plasma testosterone and UTM levels correlated significantly across seasons, but no short term correlation was evident for total oestrogen and UEM in females. Thus, in badgers, urinary sex-steroid metabolites can be used reliably in the short term to assess male reproductive status at the individual level, but only at the broader population level for females.

What lies beneath? Population dynamics conceal pace-of-life and sex ratio variation, with implications for resilience to environmental change.

Life-history and pace-of-life syndrome theory predict that populations are comprised of individuals exhibiting different reproductive schedules and associated behavioural and physiological traits, optimized to prevailing social and environmental factors. Changing weather and social conditions provide in situ cues altering this life-history optimality; nevertheless, few studies have considered how tactical, sex-specific plasticity over an individual's lifespan varies in wild populations and influences population resilience. We examined the drivers of individual life-history schedules using 31 years of trapping data and 28 years of pedigree for the European badger (Meles meles L.), a long-lived, iteroparous, polygynandrous mammal that exhibits heterochrony in the timing of endocrinological puberty in male cubs. Our top model for the effects of environmental (social and weather) conditions during a badger's first year on pace-of-life explained <10% of variance in the ratio of fertility to age at first reproduction (F/α) and lifetime reproductive success. Conversely, sex ratio (SR) and sex-specific density explained 52.8% (males) and 91.0% (females) of variance in adult F/α ratios relative to the long-term population median F/α. Weather primarily affected the sexes at different life-history stages, with energy constraints limiting the onset of male reproduction but playing a large role in female strategic energy allocation, particularly in relation to ongoing mean temperature increases. Furthermore, the effects of social factors on age of first reproduction and year-to-year reproductive success covaried differently with sex, likely due to sex-specific responses to potential mate availability. For females, low same-sex densities favoured early primiparity; for males, instead, up to 10% of yearlings successfully mated at high same-sex densities. We observed substantial SR dynamism relating to differential mortality of life-history strategists within the population, and propose that shifting ratios of 'fast' and 'slow' life-history strategists contribute substantially to population dynamics and resilience to changing conditions.

Social effects on age-related and sex-specific immune cell profiles in a wild mammal.

Evidence for age-related changes in innate and adaptive immune responses is increasing in wild populations. Such changes have been linked to fitness, and knowledge of the factors driving immune response variation is important for understanding the evolution of immunity. Age-related changes in immune profiles may be owing to factors such as immune system development, sex-specific behaviour and responses to environmental conditions. Social environments may also contribute to variation in immunological responses, for example, through transmission of pathogens and stress arising from resource and mate competition. Yet, the impact of the social environment on age-related changes in immune cell profiles is currently understudied in the wild. Here, we tested the relationship between leukocyte cell composition (proportion of neutrophils and lymphocytes [innate and adaptive immunity, respectively] that were lymphocytes) and age, sex and group size in a wild population of European badgers (Meles meles). We found that the proportion of lymphocytes in early life was greater in males in smaller groups compared to larger groups, but with a faster age-related decline in smaller groups. By contrast, the proportion of lymphocytes in females was not significantly related to age or group size. Our results provide evidence of sex-specific age-related changes in immune cell profiles in a wild mammal, which are influenced by the social environment.

Normalizing Gas-Chromatography-Mass Spectrometry Data: Method Choice can Alter Biological Inference.

We demonstrate how different normalization techniques in GC-MS analysis impart unique properties to the data, influencing any biological inference. Using simulations, and empirical data, we compare the most commonly used techniques (Total Sum Normalization 'TSN'; Median Normalization 'MN'; Probabilistic Quotient Normalization 'PQN'; Internal Standard Normalization 'ISN'; External Standard Normalization 'ESN'; and a compositional data approach 'CODA'). When differences between biological classes are pronounced, ESN and ISN provides good results, but are less reliable for more subtly differentiated groups. MN, TSN, and CODA approaches produced variable results dependent on the structure of the data, and are prone to false positive biomarker identification. In contrast, PQN exhibits the lowest false positive rate, though with occasionally poor model performance. Because ESN requires extensive pre-planning, and offers only mixed reliability, and ISN, TSN, MN, and CODA approaches are prone to introducing artefactual differences, we recommend the use of PQN in GC-MS research.

Functional adaptation rather than ecogeographical rules determine body-size metrics along a thermal cline with elevation in the Chinese pygmy dormouse (Typhlomys cinereus).

Phenotypic plasticity is crucial for how organisms respond to variation in their environment, affecting their diversity and distribution, especially in the light of rapid environmental change. Ecogeographical rules predict an association between specific adaptive morphological and physiological traits with cooler conditions due to higher latitude, elevation, or climate change. Such ecogeographical effects are often most evident in ancient species due to continuous selective adaptation occurring over long periods of time. Here, we use the suitably ancient Chinese pygmy dormouse (Typhlomys cinereus) to test whether body-size, appendage length and heart size vary in accordance with Bergmann's, Allen's and Hesse's rule, respectively. Based on a sample of 67 adult individuals (female, n = 29; male n = 38) trapped at 37 sites transcending an elevational range from 414 to 1757 m, we tested for trait concordance with Bergmann's rule (body mass, length and SMI), Allen's rule (length of tail, foot, ear, snout), and Hesse's rule (wet and dry heart mass). Effects of elevation (and thus temperature lapse rate; calculated as 0.61 °C per 100 m) on body size, appendage length and heart size, were tested by fitting Standardized Major Axis (SMA) models. We observed substantial heterogeneity in morphometric traits allowing for the detection of ecogeographical clines. However, none conformed with Bergmann's, Allen's (except ear size), or Hesse's rule. However, our results indicate some support for Geist's rule of net primary productivity. We conclude that pervasive functional life-history adaptations in this blind, arboreal, echolocating ancient species exceeded selection for morphological energy efficiency constraints, with the notable exception of reduced ear pinnae size at colder, elevated sites. This is an important consideration for predicting how species, and populations in general, may adapt to human induced rapid environmental change, contrary to expectations of warming driving selection for smaller body-size.

Individual variation in early-life telomere length and survival in a wild mammal.

Individual variation in survival probability due to differential responses to early-life environmental conditions is important in the evolution of life histories and senescence. A biomarker allowing quantification of such individual variation, and which links early-life environmental conditions with survival by providing a measure of conditions experienced, is telomere length. Here, we examined telomere dynamics among 24 cohorts of European badgers (Meles meles). We found a complex cross-sectional relationship between telomere length and age, with no apparent loss over the first 29 months, but with both decreases and increases in telomere length at older ages. Overall, we found low within-individual consistency in telomere length across individual lifetimes. Importantly, we also observed increases in telomere length within individuals, which could not be explained by measurement error alone. We found no significant sex differences in telomere length, and provide evidence that early-life telomere length predicts lifespan. However, while early-life telomere length predicted survival to adulthood (≥1 year old), early-life telomere length did not predict adult survival probability. Furthermore, adult telomere length did not predict survival to the subsequent year. These results show that the relationship between early-life telomere length and lifespan was driven by conditions in early-life, where early-life telomere length varied strongly among cohorts. Our data provide evidence for associations between early-life telomere length and individual life history, and highlight the dynamics of telomere length across individual lifetimes due to individuals experiencing different early-life environments.

Knowing Me, Knowing You: Anal Gland Secretion of European Badgers (Meles meles) Codes for Individuality, Sex and Social Group Membership.

European badgers, Meles meles, are group-living in the UK, and demarcate their ranges with shared latrines. As carnivores, badgers possess paired anal glands, but olfactory information on the content of badger anal gland secretion (AGS) is largely uninvestigated. Here, we examined the volatile organic compounds (VOCs) of AGS samples from 57 free-living badgers using solid-phase microextraction (SPME) and gas chromatography-mass spectrometry. AGS was rich in alkanes (C7-C15, 14.3% of identified compounds), aldehydes (C5-C14, 9.7%), phenols (C6-C15, 9.5%), alcohols (C5-C10, 7.3%), aromatic hydrocarbons (C6-C13, 6.8%), ketones (C6-C13, 6.3%) and carboxylic acids (C3-C12, 5.6%) and contained a variety of esters, sulfurous and nitrogenous compounds, and ethers. The number of VOCs per profile ranged from 20 to 111 (mean = 65.4; ± 22.7 SD), but no compound was unique for any of the biological categories. After normalization of the raw data using Probabilistic Quotient Normalization, we produced a resemblance matrix by calculating the Euclidian distances between all sample pairs. PERMANOVA revealed that AGS composition differs between social groups, and concentration and complexity in terms of number of measurable VOCs varies between seasons and years. AGS VOC profiles encode individual identity, sex and vary with female reproductive state, indicating an important function in intraspecific communication. Because AGS is excreted together with fecal deposits, we conclude that chemical complexity of AGS enables particularly latrine-using species, such as badgers, to advertise more complex individual-specific information than in feces alone.

Badger setts provide thermal refugia, buffering changeable surface weather conditions.

Den use can be crucial in buffering environmental conditions and especially to provide an insulated environment for raising altricial young. Through Sept-Dec 2016 we monitored temperature and humidity at 11 badger setts (burrow systems), using thermal probes inserted over 4-13 sett entrances to a depth of ca. 2 m, supplemented by continuous daily logging at one entrance per sett. Setts were cooler than exterior conditions Sept-Oct, and warmer than exterior conditions Nov-Dec. Setts cooled down when badgers left them to forage by night, and warmed up when badgers occupied them by day. Soil type and aspect also influenced sett temperature. Sett temperature did not affect the weight or body-condition of either adults or maturing cubs in autumn. However, cubs born into setts that were relatively warmer through the preceding autumn-winter were heavier in the following spring than contemporaries born in cooler setts (badgers exhibit delayed implantation), and so warmer setts might benefit early cub growth. We posit that sett quality may be important in providing badgers with a stable thermal refuge from variable weather conditions. More broadly, den use may buffer climate change effects for many fossorial carnivore species.

Avoiding verisimilitude when modelling ecological responses to climate change: the influence of weather conditions on trapping efficiency in European badgers (Meles meles).

The signal for climate change effects can be abstruse; consequently, interpretations of evidence must avoid verisimilitude, or else misattribution of causality could compromise policy decisions. Examining climatic effects on wild animal population dynamics requires ability to trap, observe or photograph and to recapture study individuals consistently. In this regard, we use 19 years of data (1994-2012), detailing the life histories on 1179 individual European badgers over 3288 (re-) trapping events, to test whether trapping efficiency was associated with season, weather variables (both contemporaneous and time lagged), body-condition index (BCI) and trapping efficiency (TE). PCA factor loadings demonstrated that TE was affected significantly by temperature and precipitation, as well as time lags in these variables. From multi-model inference, BCI was the principal driver of TE, where badgers in good condition were less likely to be trapped. Our analyses exposed that this was enacted mechanistically via weather variables driving BCI, affecting TE. Notably, the very conditions that militated for poor trapping success have been associated with actual survival and population abundance benefits in badgers. Using these findings to parameterize simulations, projecting best-/worst-case scenario weather conditions and BCI resulted in 8.6% ± 4.9 SD difference in seasonal TE, leading to a potential 55.0% population abundance under-estimation under the worst-case scenario; 38.6% over-estimation under the best case. Interestingly, simulations revealed that while any single trapping session might prove misrepresentative of the true population abundance, due to weather effects, prolonging capture-mark-recapture studies under sub-optimal conditions decreased the accuracy of population estimates significantly. We also use these projection scenarios to explore how weather could impact government-led trapping of badgers in the UK, in relation to TB management. We conclude that population monitoring must be calibrated against the likelihood that weather conditions could be altering trap success directly, and therefore biasing model design.

Giant panda (Ailuropoda melanoleuca) neonates use broadband calls to communicate with their mothers.

Infant call structure should have evolved to elicit maximum maternal attention and investment. Neonates of giant pandas produce three types of vocalizations reported to be vitally important in the context of mother-infant communications. However, how cubs, 0-15 days old, communicate with their mothers to elicit maternal care remains unknown. We analyzed 12 different call parameters of 3475 squawks, 1355 squalls, and 491 croaks from 11 captive giant panda (Ailuropoda melanoleuca) neonates from age 0 to 15 days. In playback experiments, we also tested whether mothers could detect ultrasound. Our results show that neonates use broadband calls with ultrasonic frequencies up to 65 kHz to convey information about their physiological needs and to attract maternal care. In playback experiments, we tested if mothers reacted differently to broadband calls (BBC) than to artificially altered calls that included only frequencies <20 kHz (AUDC) or calls that included only frequencies >20 kHz (USC). Playback confirmed that, although adult females responded significantly less often to USC, BBC than to or AUDC, they could detect USC, BBC and generally made appropriate behavioral responses, indicating a potential benefit for neonates to utilize ultrasonic and broadband frequencies. Our findings provide a new insight into mother-infant communication in giant pandas and will be helpful for reducing the mortality of cubs, younger than 1 month old, in captivity.

Friend or foe? Using eye-tracking technology to investigate the visual discrimination ability of giant pandas.

The role that visual discriminative ability plays among giant pandas in social communication and individual discrimination has received less attention than olfactory and auditory modalities. Here, we used an eye-tracker technology to investigate pupil fixation patterns for 8 captive male giant pandas Ailuropoda melanoleuca. We paired images (N = 26) of conspecifics against: 1) sympatric predators (gray wolves and tigers), and non-threatening sympatric species (golden pheasant, golden snub-nosed monkey, takin, and red panda), 2) conspecifics with atypical fur coloration (albino and brown), and 3) zookeepers/non-zookeepers wearing either work uniform or plain clothing. For each session, we tracked the panda's pupil movements and measured pupil first fixation point (FFP), fixation latency, total fixation count (TFC), and duration (TFD) of attention to each image. Overall, pandas exhibited similar attention (FFPs and TFCs) to images of predators and non-threatening sympatric species. Images of golden pheasant, snub-nosed monkey, and tiger received less attention (TFD) than images of conspecifics, whereas images of takin and red panda received more attention, suggesting a greater alertness to habitat or food competitors than to potential predators. Pandas' TFCs were greater for images of black-white conspecifics than for albino or brown phenotypes, implying that familiar color elicited more interest. Pandas reacted differently to images of men versus women. For images of women only, pandas gave more attention (TFC) to familiar combinations (uniformed zookeepers and plain-clothed non-zookeepers), consistent with the familiarity hypothesis. That pandas can use visual perception to discriminate intra-specifically and inter-specifically, including details of human appearance, has applications for panda conservation and captive husbandry.

Links between energy budgets, somatic condition, and life history reveal heterogeneous energy management tactics in a group-living mesocarnivore.

BACKGROUND: Optimal management of voluntary energy expenditure is crucial to the survival and reproductive success of wild animals. Nevertheless, a growing appreciation of inter-individual variation in the internal state driving movement suggests that individuals may follow different, yet equally optimal tactics under the same environmental conditions. However, few studies in wild populations have investigated the occurrence and demographic context of different contemporaneous energetic expenditure tactics. Here, we explore this neglected aspect of energy budgeting in order to determine the effect of life-history traits such as age and reproductive status on the co-occurrence of different energy-budgeting tactics in wild populations. METHODS: We investigated inter-individual heterogeneity in energy expenditure within a wild population of European badgers (Meles meles) by quantifying individual overall dynamic body acceleration (ODBA, from tri-axial accelerometry collars) and total daily energy expenditure (DEE, from doubly-labelled water) during 6-9 day deployments and dosing periods over six different seasons (spring, summer, and autumn) in 2018-2019. We obtained ODBA values for 41 deployments (24 unique badgers) and DEE measurements for 41 dosings (22 unique badgers). We then evaluated correlations between these energetic metrics and computed individual ratios of ODBA/DEE as a proxy for the proportion of total energy spent on activity. We measured the impact of alternative ODBA/DEE ratios on body condition, and use survival models constructed using 29 years of demographic data from the same population to situate body-condition changes in the context of age and reproductive status. RESULTS: Both ODBA and DEE were highly variable between individuals and exhibited season-specific relationships with individual body condition and life-history factors. DEE scaled allometrically with body weight, but only in summer and autumn; post-reproductive female badgers were lighter than other badgers during the spring but expended on average 350 kJ/day more than predicted from allometric scaling. Older badgers expended significantly less energy on movement during the summer than did younger adults. The ratio of ODBA to DEE (OD) provides a measure of proportional investment into movement. This ratio correlated more significantly with next-season body condition than either energetic metric did independently. However, the majority of individuals with high OD ratios were either younger badgers or reproductive females, for which lower body condition typically presented less of a mortality risk in previous analyses of this population. CONCLUSIONS: Within a single population under the same environmental conditions, we found wide inter-individual variation in both mechanical and total energy expenditure. The adoption of different tactics aligns with relationships between life-history parameters and mortality risk previously studied within the population. Crucially, younger badgers and reproductive females appeared able to tolerate energy expenditure tactics that depleted their body condition more than other badgers. These findings provide a mechanism by which differences in individual energetic context set by life history can maintain heterogeneity in wild populations, providing a wide range of potential energetic tactics under changing environmental conditions.

Assessment of stress levels and reproductive condition in giant pandas: insights from hair, faecal and saliva samples.

Concerted conservation efforts have brought the giant panda (Ailuropoda melanoleuca) back from the brink of extinction, but pandas continue to face anthropogenic threats in the wild and breeding success in captivity remains low. Because stress can have detrimental impacts on reproduction, monitoring stress- and sex-steroid levels would help assess the effectiveness of conservation mitigation measures in panda populations as well as monitor the welfare and reproductive health of captive animals. In this proof-of-concept study, we used faecal sex steroid and cortisol concentrations (n = 867 samples collected from five males and five females at Beijing Zoo every 4 days over the course of 12 months) as a reference to investigate if testosterone, estradiol, progesterone and cortisol can be meaningfully measured in panda hair (n = 10) using radio-immuno-assays. Additionally, we calculated the ratio of testosterone to cortisol (T:C ratio) for each male, which can provide a biomarker of stress and physical performance. Our findings revealed distinct monthly variations in faecal sex-steroid and cortisol concentrations, reflecting reproductive seasonality and visitor-related stress among individual pandas. Notably, the oldest male had a significantly lower T:C ratio than other males. Our results confirm that the level of sex steroids and cortisol can be assayed by panda hair, and the hair cortisol concentrations correlate significantly with that in faeces with one month lag behind (r = 0.68, P = 0.03). However, the concentrations of hormones detected in saliva are lower than those in faeces by two orders of magnitude, making it difficult to ensure accuracy. By assessing the applicability of hair, faecal and salivary sampling, we can infer their utility in monitoring the reproductive status and acute and chronic stress levels of giant pandas, thereby providing a means to gauge the success of ongoing habitat restoration efforts and to discuss the feasibility of sample collection from wild populations.

Assessing chronic stress in wild mammals using claw-derived cortisol: a validation using European badgers (Meles meles).

Measuring stress experienced by wild mammals is increasingly important in the context of human-induced rapid environmental change and initiatives to mitigate human-wildlife conflicts. Glucocorticoids (GC), such as cortisol, mediate responses by promoting physiological adjustments during environmental perturbations. Measuring cortisol is a popular technique; however, this often reveals only recent short-term stress such as that incurred by restraining the animal to sample blood, corrupting the veracity of this approach. Here we present a protocol using claw cortisol, compared with hair cortisol, as a long-term stress bio-indicator, which circumvents this constraint, where claw tissue archives the individual's GC concentration over preceding weeks. We then correlate our findings against detailed knowledge of European badger life history stressors. Based on a solid-phase extraction method, we assessed how claw cortisol concentrations related to season and badger sex, age and body-condition using a combination of generalized linear mixed models (GLMM) (n = 668 samples from 273 unique individuals) followed by finer scale mixed models for repeated measures (MMRM) (n = 152 re-captured individuals). Claw and hair cortisol assays achieved high accuracy, precision and repeatability, with similar sensitivity. The top GLMM model for claw cortisol included age, sex, season and the sex*season interaction. Overall, claw cortisol levels were significantly higher among males than females, but strongly influenced by season, where females had higher levels than males in autumn. The top fine scale MMRM model included sex, age and body condition, with claw cortisol significantly higher in males, older and thinner individuals. Hair cortisol was more variable than claw; nevertheless, there was a positive correlation after removing 34 outliers. We discuss strong support for these stress-related claw cortisol patterns from previous studies of badger biology. Given the potential of this technique, we conclude that it has broad application in conservation biology.