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Fecal corticosterone metabolite (FCM) concentrations, which can be determined noninvasively, have recently been explored as a stress indicator in birds. In our study, we measured FCM concentrations in penguins under nonmolting or molting conditions, cool or hot season, diseased condition, and incubation period. These measurements were conducted in an aquarium that housed king penguins, gentoo penguins, and African penguins. This study aimed to investigate the validity of fecal matter as a stress indicator. Our findings revealed that FCM concentrations were significantly higher in molting individuals than in nonmolting individuals. Compared with the cool season, FCM concentrations were significantly higher in penguins housed outdoors during the hot season. However, no differences were observed in penguins housed indoors. Diseased individuals and an incubating individual showed notably higher FCM concentrations than healthy individuals. Interestingly, the FCM concentration in king penguin that underwent cataract surgery was extremely high before the surgery. However, 1 month postsurgery, it decreased to a level similar to that of healthy individuals. We observed increased FCM concentrations in penguins considered to be exposed to stressors. Notably, FCM concentration decreased after removing the stress factor. The FCM concentration was found to be consistent with the stress state of penguins, suggesting its usefulness as a stress indicator.

Fecal samples are a non-invasive and relatively accessible matrix for investigating physiological processes in resident killer whale (Orcinus orca) populations. The high lipid content of the diet (primarily salmonids) leads to lower density fecal material and slower dispersion, facilitating sample collection. As fecal discharge is relatively infrequent and the volume of sample is variable, maximizing analytical options is an important consideration. Here we present an extraction methodology to measure hormones and lipid content from the same fecal aliquot. Lipid extractions are commonly conducted using chloroform and methanol from Folch or Bligh and Dyer (B&D), while alcohol is the primary solvent for hormone extraction. We evaluated the possibility of using the methanol layer from lipid extractions to assess fecal steroid hormone levels. Folch and B&D methanol residues were assayed form metabolites of progesterone (PMs) and corticosterone (GCs), and results were compared to aliquots extracted in 70 % ethanol. Hormone concentrations measured in the methanol layer from Folch and B&D extractions were 55 % to 79 % lower than concentrations in 70 % ethanol. We developed mathematical corrections, using linear regression models fitted to Folch or B&D methanol vs 70 % ethanol hormone concentrations (p < 0.01). Fecal concentrations of PMs and GCs from methanol extractions were biologically validated and are significantly higher in confirmed pregnant females compared to non-pregnant individuals (p < 0.05). This study demonstrates that lipid extraction protocols may be used for the analysis of multiple biomarkers, maximizing the use of small-volume samples., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2024. Published by Elsevier Inc. All rights reserved.)

Intrafacility transport of mice is an essential function for both laboratory and husbandry personnel. However, transport may induce a stress response that can alter research findings and negatively impact animal welfare. To determine minimally adverse intrafacility transport methods, in-cage noise and vibration exposure during transport on a variety of transport vehicles (hand carrying, stainless steel rack, flatbed cart, metal teacart, plastic teacart, and a cart with pneumatic wheels) were measured. Under-cage and in-cage padding was tested for its ability to decrease noise and vibration on each vehicle. Behavioral (open field test and elevated plus maze) and corticosterone responses of mice were then measured following transport on the most adverse (metal teacart) and least adverse (pneumatic cart) methods of multicage transport. Behavioral measures showed no difference between transported mice and untransported mice in both single- and group-housed settings. Plasma corticosterone was significantly elevated in mice transported on the metal teacart immediately following transport and continued to have elevated trends in circadian peaks during the 48h of sampling. The cart with pneumatic wheels was most effective at reducing noise and vibration, reflected in posttransport corticosterone readings that remained equivalent to those in untransported mice. This study demonstrates that mitigation of noise and vibration during cart transport may decrease the impact of transport on certain stress parameters in mice.

Identifying reliable bioindicators of population status is a central goal of conservation physiology. Physiological stress measures are often used as metrics of individual health and can assist in managing endangered species if linked to fitness traits. We analysed feather corticosterone, a cumulative physiological stress metric, of individuals from historical, translocated, and source populations of an endangered endemic Hawaiian bird, the Laysan duck ( Anas laysanensis ). We hypothesized that feather corticosterone would reflect the improved reproduction and survival rates observed in populations translocated to Midway and Kure Atolls from Laysan Island. We also predicted less physiological stress in historical Laysan birds collected before ecological conditions deteriorated and the population bottleneck. All hypotheses were supported: we found lower feather corticosterone in the translocated populations and historical samples than in those from recent Laysan samples. This suggests that current Laysan birds are experiencing greater physiological stress than historical Laysan and recently translocated birds. Our initial analysis suggests that feather corticosterone may be an indicator of population status and could be used as a non-invasive physiological monitoring tool for this species with further validation. Furthermore, these preliminary results, combined with published demographic data, suggest that current Laysan conditions may not be optimal for this species.

Avian embryos develop in an egg composition which reflects both maternal condition and the recent environment of their mother. In birds, yolk corticosterone (CORT) influences development by impacting pre- and postnatal growth, as well as nestling stress responses and development. One possible mechanism through which maternal CORT may affect offspring development is via changes to offspring DNA methylation. We sought to investigate this, for the first time in birds, by quantifying the impact of manipulations to maternal CORT on offspring DNA methylation. We non-invasively manipulated plasma CORT concentrations of egg-laying female zebra finches (Taeniopygia castanotis) with an acute dose of CORT administered around the time of ovulation and collected their eggs. We then assessed DNA methylation in the resulting embryonic tissue and in their associated vitelline membrane blood vessels, during early development (5 days after lay), using two established methods - liquid chromatography-mass spectrometry (LC-MS) and methylation-sensitive amplification fragment length polymorphism (MS-AFLP). LC-MS analysis showed that global DNA methylation was lower in embryos from CORT-treated mothers, compared to control embryos. In contrast, blood vessel DNA from eggs from CORT-treated mothers showed global methylation increases, compared to control samples. There was a higher proportion of global DNA methylation in the embryonic DNA of second clutches, compared to first clutches. Locus-specific analyses using MS-AFLP did not reveal a treatment effect. Our results indicate that an acute elevation of maternal CORT around ovulation impacts DNA methylation patterns in their offspring. This could provide a mechanistic understanding of how a mother's experience can affect her offspring's phenotype., (© 2024 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Certain life stressors having enduring physiological and behavioral consequences, in part by eliciting dramatic signaling shifts in monoamine neurotransmitters. High monoamine levels can overwhelm selective transporters like the serotonin transporter. This is when polyspecific transporters like plasma membrane monoamine transporter (PMAT, Slc29a4 ) are hypothesized to contribute most to monoaminergic signaling regulation. Here, we employed two distinct counterbalanced stressors-fear conditioning and swim stress-in mice to systematically determine how reductions in PMAT function affect heterotypic stressor responsivity. We hypothesized that male heterozygotes would exhibit augmented stressor responses relative to female heterozygotes. Decreased PMAT function enhanced context fear expression, an effect unexpectedly obscured by a sham stress condition. Impaired cued fear extinction retention and enhanced context fear expression in males were conversely unmasked by a sham swim condition. Abrogated corticosterone levels in male heterozygotes that underwent swim stress after context fear conditioning did not map onto any measured behaviors. In sum, male heterozygous mouse fear behaviors proved malleable in response to preceding stressor or sham stress exposure. Combined, these data indicate that reduced male PMAT function elicits a form of stress-responsive plasticity. Future studies should assess how PMAT is differentially affected across sexes and identify downstream consequences of the stress-shifted corticosterone dynamics.

Background: As a prey species, rabbits tend to hide their illnesses and injuries. Consequently, pet rabbit owners often do not notice that their pet may be suffering., Methods: Data on the housing and health of 34 dwarf rabbits were collected from a total of 22 rabbit owners. A score from 0 (not stressed) to 3 (highly stressed) was assigned to each rabbit based on the owners' reports. Afterwards, the hair corticosterone concentration (HCC) of the rabbits was analysed as an indicator of long-term stress., Results: The mean HCC of all rabbits was 1.54 ± 0.64 pg/mg. Animals with a stress score of 3 had a higher HCC (2.17 pg/mg) than those with a score of 0 (1.09 pg/mg) or 1 (1.44 pg/mg) (p < 0.05). HCC was not significantly affected by sex or age (p > 0.05)., Limitation: Only a small number of rabbits were included in the study. Therefore, the findings need to be confirmed in a larger cohort of animals., Conclusion: HCC seems to be a promising stress marker in pet rabbits., (© 2023 The Authors. Veterinary Record published by John Wiley & Sons Ltd on behalf of British Veterinary Association.)

Understanding how natural aging impacts rodent performance in translational behavior tests is critical to teasing apart impairments due to age-related decline from neurodegenerative disorder modeling. Reduced neuropilin and tolloid-like 1 (NETO1), an accessory protein of ionotropic glutamate receptors involved in synaptic plasticity, was associated with Alzheimer's disease, yet aging effects on Neto1 remain unclear. For these reasons, our goal was to characterize how Neto1 expression corresponded with social, repetitive, and spatial learning behaviors and stress response across the C57BL/6J mouse lifespan. We measured social preferences in three-chamber tests, and motor stereotypies by marble burying. Cognitive flexibility is typically assessed in the Morris water maze (MWM), wherein C57BL/6J mice exhibit deficits with age. However, fatigue or locomotor impairment may confound interpretation of MWM performance. Therefore, we used a less arduous water T-maze (WTM) to compare spatial learning flexibility in 2, 9-15, and 24-month-old male and female mice to test the hypothesis that deficits would emerge with age. In both sexes, 9-15-month-olds made more chamber entries during social preference tests, while 2-month-olds did less social sniffing than aged mice. No age or sex differences emerged in marble burying or serum corticosterone measurements. In 24-month-olds hippocampal Neto1was increased relative to 2-month-olds, and male cognitive flexibility was strong, while spatial learning and reversal learning of 24-month-old females was impaired in WTM irrespective of Neto1 expression. The WTM is a useful alternative assessment for cognitive flexibility deficits in aged mice, and the role of hippocampal Neto1 in promoting social sniffing is of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The widespread use of phthalates (PAEs) has drawn increasing attention due to their endocrine disruption and reproductive toxicity, while the steroid metabolome is essential for follicular development. However, the mechanism by which PAE exposure affects ovarian reserve through the steroid metabolome remains unclear. This study recruited 264 childbearing-age women in Tianjin (China) from April 2019 to August 2020 in a cross-sectional design. Target metabolome analysis of 16 steroids was performed in follicular fluid (FF) to compare diminished ovarian reserve (DOR) against normal ovarian reserve (NOR) women and differential steroids were identified using binary logistic analyses. Further analysis of eleven PAE metabolites (mPAEs) in FF was conducted, and the retrieved oocyte number (RON) representing ovarian reserve was counted. Multiple linear regression and quantile-based g-computation (qgcomp) models were used to associate individual mPAEs and mPAE mixture with the DOR-related differential steroids in FF. Mediation analysis was used to discuss the mediating effect of DOR-related steroids on the association between mPAEs and RON. Androstenedione (A4), corticosterone (CORT), cortisol (COR) and cortisone were significantly down-regulated in FF from women with DOR. Nine mPAEs with detection frequencies greater than 60% and median concentrations of 0.02-4.86 ng/mL were incorporated into statistical models. Negative associations with COR and CORT were found for mono-ethyl phthalate (mEP), mono-(2-ethyl-5-oxohexyl) phthalate (mEOHP), and mono-2-ethylhexyl phthalate (mEHP). A positive association with cortisone was found for mEOHP, mEHP, monobutyl phthalate (mBP), and mono (2-isobutyl) phthalate (miBP). The qgcomp and mediation analyses revealed that mEP and mEOHP not only significantly contributed to the decline of COR and CORT in the mixed exposure but also indirectly reduced RON through the mediating effects of COR and CORT. In conclusion, PAE exposure may decrease ovarian reserve by downregulating COR and CORT., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Exposure to persistent organic pollutants (POPs), such as organochlorines (OCs) and polybrominated diphenyl ethers (PBDEs), is associated with adverse health effects in wildlife. Many POPs have been banned and consequently their environmental concentrations have declined. To assess both temporal trends of POPs and their detrimental impacts, raptors are extensively used as biomonitors due to their high food web position and high contaminant levels. White-tailed eagles (WTEs; Haliaeetus albicilla) in the Baltic ecosystem represent a sentinel species of environmental pollution, as they have suffered population declines due to reproductive failure caused by severe exposure to dichlorodiphenyltrichloroethane (DDT) and polychlorinated biphenyls (PCB) during the 1960s through 1980s. However, there is a lack of long-term studies that cover a wide range of environmental contaminants and their effects at the individual level. In this study, we used 135 pooled samples of shed body feathers collected in 1968-2012 from breeding WTE pairs in Sweden. Feathers constitute a temporal archive for substances incorporated into the feather during growth, including corticosterone, which is the primary avian glucocorticoid and a stress-associated hormone. Here, we analysed the WTE feather pools to investigate annual variations in feather corticosterone (fCORT), POPs (OCs and PBDEs), and stable carbon and nitrogen isotopes (SIs; dietary proxies). We examined whether the expected fluctuations in POPs affected fCORT (8-94 pg. mm -1 ) in the WTE pairs. Despite clear temporal declining trends in POP concentrations (p < 0.01), we found no significant associations between fCORT and POPs or SIs (p > 0.05 in all cases). Our results do not support fCORT as a relevant biomarker of contaminant-mediated effects in WTEs despite studying a highly contaminated population. However, although not detecting a relationship between fCORT, POP contamination and diet, fCORT represents a non-destructive and retrospective assessment of long-term stress physiology in wild raptors otherwise not readily available., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Early life stress (ELS) is associated with perturbed neural development and augmented vulnerability to mental health disorders, including addiction. How ELS changes the brain to increase addiction risk is poorly understood, and there are no therapies which target this ELS-induced vulnerability. ELS disrupts the oxytocin system, which can modulate addiction susceptibility, suggesting that targeting the oxytocin system may be therapeutic in this ELS-addiction comorbidity. Therefore, we determined whether adolescent oxytocin treatment after ELS could: (1) reduce vulnerability to anxiety, social deficits, and methamphetamine-taking and reinstatement; and (2) restore hypothalamic oxytocin and corticotropin-releasing factor expressing neurons and peripheral oxytocin and corticosterone levels. Long Evans pups underwent maternal separation (MS) for either 15 min or 360 min on postnatal days (PND) 1-21. During adolescence (PNDs 28-42), rats received a daily injection of either oxytocin or saline. In Experiment 1, adult rats were assessed using the elevated plus-maze, social interaction procedure, and methamphetamine self-administration procedure, including extinction, and cue-, methamphetamine- and yohimbine-induced reinstatement. In Experiment 2, plasma for enzyme immunoassays and brain tissue for immunofluorescence were collected from adult rats after acute stress exposure. Adolescent oxytocin treatment ameliorated ELS-induced anxiety and reduced methamphetamine- and yohimbine-induced reinstatement in both sexes, and suppressed methamphetamine intake and facilitated extinction in males only. Additionally, adolescent oxytocin treatment after ELS restored oxytocin-immunoreactive cells and stress-induced oxytocin levels in males, and attenuated stress-induced corticosterone levels in both sexes. Adolescent oxytocin treatment reverses some of the ELS effects on later-life psychopathology and vulnerability to addiction., (© 2022. The Author(s).)

Objectives: To explore the associations of environmental endocrine disruptors on precocious puberty in girls., Methods: This was a case-control study in which 30 girls with precocious puberty and 46 age- and race-matched prepubertal females were enrolled. The concentrations of 10 environment endocrine disruptors (bisphenol A, bisphenol B, butylparaben, propylparaben, ethvlparaben, methylparaben, mono-butyl phthalate, mono-2-ethylhexyl phthalate, monoethyl phthalate, and monomethyl phthalate) in urine and 10 steroid hormones (dihydrotestosterone, corticosterone, hydrocortisone, 11-deoxycortisol, 17α-hydroxy progesterone, 4-androstene-3,17-dione, estrone, deoxycorticosterone, pregnenolone, and dehydroepiandrosterone) in serum were detected with the liquid chromatography-mass spectrometry (LC-MS)., Results: According to the Mann-Whitney U test, urinary levels of bisphenol A, monobutyl phthalate, and monomethyl phthalate were significantly higher in the precocious group than in the prepubertal group, and blood levels of hydrocortisone, 11-deoxycortisol, corticosterone, deoxycorticosterone, and pregnenolone were significantly lower in the precocious group than in the prepubertal group (p<0.05, VIP>1)., Conclusions: Our findings confirm the association between phthalate exposure and the incidence of precocious puberty in girls. Control and reduction of children exposure to phthalate esters should be considered as a health priority., (© 2022 Fang Zhou et al., published by De Gruyter, Berlin/Boston.)

Glucocorticoids (GCs) are critical modulators of the immune system. The hypothalamic-pituitary-adrenal (HPA) axis regulates circulating GC levels and is stimulated by endotoxins. Lymphoid organs also produce GCs; however, it is not known how lymphoid GC levels are regulated in response to endotoxins. We assessed whether an acute challenge of lipopolysaccharide (LPS) increases lymphoid levels of progesterone and GCs, and expression of steroidogenic enzymes and key HPA axis components (eg, corticotropin-releasing hormone [CRH], adrenocorticotropic hormone [ACTH]). We administered LPS (50 µg/kg intraperitoneally) or vehicle control to male and female C57BL/6J neonatal (postnatal day [PND] 5) and adult (PND90) mice and collected blood, bone marrow, thymus, and spleen 4 hours later. We measured progesterone, 11-deoxycorticosterone, corticosterone, and 11-dehydrocorticosterone via liquid chromatography-tandem mass spectrometry. We measured gene expression of key steroidogenic enzymes (Cyp11b1, Hsd11b1, and Hsd11b2) and HPA axis components (Crh, Crhr1, Pomc, and Mc2r) via quantitative polymerase chain reaction. At PND5, LPS induced greater increases in steroid levels in lymphoid organs than in blood. In contrast, at PND90, LPS induced greater increases in steroid levels in blood than in lymphoid organs. Steroidogenic enzyme transcripts were present in all lymphoid organs, and LPS altered steroidogenic enzyme expression predominantly in the spleen. Lastly, we detected transcripts of key HPA axis components in all lymphoid organs, and there was an effect of LPS in the spleen. Taken together, these data suggest that LPS regulates GC production by lymphoid organs, similar to its effects on the adrenal glands, and the effects of LPS might be mediated by local expression of CRH and ACTH., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Complete Freund's adjuvant (CFA)-induced arthritis in rats is a common animal model for studying chronic inflammatory pain. However, modelling of the disease is associated with unnecessary pain and impaired animal wellbeing, particularly in the immediate post-induction phase. Few attempts have been made to counteract these adverse effects with analgesics. The present study investigated the effect of buprenorphine on animal welfare, pain-related behaviour and model-specific parameters during the disease progression in a rat model of CFA-induced monoarthritis. The aim was to reduce or eliminate unnecessary pain in this model, in order to improve animal welfare and to avoid suffering, without compromising the quality of the model. Twenty-four male Sprague Dawley rats were injected with 20 μl of CFA into the left tibio-tarsal joint to induce monoarthritis. Rats were treated with either buprenorphine or carprofen for 15 days during the disease development, and were compared to a saline-treated CFA-injected group or a negative control group. Measurements of welfare, pain-related behaviour and clinical model-specific parameters were collected. The study was terminated after 3 weeks, ending with a histopathologic analysis. Regardless of treatment, CFA-injected rats displayed mechanical hyperalgesia and developed severe histopathological changes associated with arthritis. However, no severe effects on general welfare were found at any time. Buprenorphine treatment reduced facial pain expression scores, improved mobility, stance and lameness scores and it did not supress the CFA-induced ankle swelling, contrary to carprofen. Although buprenorphine failed to demonstrate a robust analgesic effect on the mechanical hyperalgesia in this study, it did not interfere with the development of the intended pathology., Competing Interests: The authors have declared that no competing interests exist.

Steroid hormones such as glucocorticoids and their metabolites are closely related to mental diseases and neuroendocrine diseases. Quantitative analysis of these substances will help in understanding their roles in related research fields. In this study, an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed to detect the concentration of corticosterone (CORT) and its metabolites, progesterone (PROG) and testosterone in rat plasma and prefrontal cortex (PFC), and was applied to investigate the changes in hormones in rats with depression induced by chronic unpredictable mild stress (CUMS). The method was shown to be linear in the quantitation range for all analytes. Intra- and inter-day accuracy and precision were between 80% and 120%. Furthermore, we found that the level of CORT in plasma and PFC increased, whereas that of 11-dehydrocorticosterone (11-DHCORT) as well as the ratio of 11-DHCORT and CORT declined in rats with CUMS-induced depression. The trends of these changes in central PFC and peripheral plasma were consistent. In conclusion, this study successfully established an UPLC-MS/MS method for simultaneous measurement of CORT and its metabolites, PROG and testosterone in rat plasma and PFC, and applied it to rats with depression. The method could be further applied to the research of depression and diseases related to these steroid hormones., (© 2021 John Wiley & Sons, Ltd.)

Objective: The effects of neonatal caffeine therapy in adults born preterm are uncertain. We studied the impact of neonatal caffeine on systemic blood pressure, vessel reactivity, and response to stress in adult mice., Study Design: Mice pups were randomized to caffeine (20 mg/kg/d) or saline by intraperitoneal injection for 10 days after birth. We performed tail-cuff BP (8/12 weeks), urinary 8-hydroxydeoxyguanosine and fecal corticosterone (14 weeks), and vessel reactivity in aortic rings (16 weeks) in adult mice., Results: No differences were noted in systolic, diastolic, and mean blood pressures between the two groups at 8 and 12 weeks of age. However, norepinephrine-induced vasoconstriction was substantially higher in aortic rings in CAF-treated male mice. More significant vasodilator responses to nitric oxide donors in aortic rings in female mice may suggest gender-specific effects of caffeine. Female mice exposed to caffeine had significantly lower body weight over-time. Caffeine-treated male mice had substantially higher fecal corticosterone and urinary 8-hydroxydeoxyguanosine at 14 weeks, suggestive of chronic stress., Conclusion: We conclude sex-specific vulnerability to the heightened vascular tone of the aorta in male mice following neonatal caffeine therapy. Altered vessel reactivity and chronic stress in the presence of other risk factors may predispose to the development of systemic hypertension in adults born preterm., Competing Interests: None declared., (Thieme. All rights reserved.)

Background: Brain development is a prolonged process and it is sensitive to the environment during critical periods. Stress in early life is believed to increase vulnerability to depression, while enriched environment (EE) has beneficial effects on neural plasticity and depression. In this study, we compared the therapeutic effect of EE during different periods on early life stress-induced depression, and investigated the role of brain-derived neurotrophic factor (BDNF) and protein kinase B (AKT) on the effect of EE. Plasma corticosterone level was also detected to evaluate the reactivity of hypothalamic-pituitary-adrenal axis., Methods: C57BL/6 mice were subjected to a 4-h maternal separation (MS) procedure during postnatal days 2-21. After this separation, the mice were assigned to standard environment groups (SE), EE in the early period groups (3-8 weeks, EEE) and EE in the adult groups (8-13 weeks, EEA). Depression and anxiety behavior were evaluated at 14-weeks of age. The plasma corticosterone was quantified utilizing enzyme-linked immunosorbent assay. Hippocampus BDNF and AKT/p-AKT were detected using Western blotting., Results: The results showed that MS increased depression and anxiety level, while EE in both intervention periods alleviated the symptoms of depression and anxiety. The EEE group showed better effects in terms of anhedonia and anxiety than the EEA group. The difference in despair behavior between the EEE and EEA groups was not significant. MS increased plasma corticosterone level, while EE decreased corticosterone level in both intervention periods. EE increased BDNF and p-AKT expression in the hippocampus, with stronger effects in the EEE group., Conclusion: EE during the early development period was more effective in alleviating depression and anxiety induced by early life stress. BDNF and AKT may play a significant role in the effect of EE, and further research is needed to explore the detailed neurobiological mechanisms., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Muskoxen (Ovibos moschatus), a taxonomically unique Arctic species, are increasingly exposed to climate and other anthropogenic changes. It is critical to develop and validate reliable tools to monitor their physiological stress response in order to assess the impacts of these changes. Here, we measured fecal glucocorticoid metabolite (FGM) levels in response to the administration of adrenocorticotropic hormone (ACTH) in the winter (1 IU/kg) and summer (2 IU/kg) using two enzyme immunoassays, one targeting primarily cortisol and the other targeting primarily corticosterone. Fecal cortisol levels varied substantially within and among individuals, and none of the animals in either challenge showed an increase in fecal cortisol following the injection of ACTH. By contrast, two of six (winter) and two of five (summer) muskoxen showed a clear response in fecal corticosterone levels (i.e., maximal percentage increase as compared to time 0 levels > 100%). Increases in fecal corticosterone post-ACTH injection occurred earlier and were of shorter duration in the summer than in the winter and fecal corticosterone levels were, in general, lower during the summer. These seasonal differences in FGM responses may be related to the use of different individuals (i.e., influence of sex, age, social status, etc.) and to seasonal variations in the metabolism and excretion of glucocorticoids, intestinal transit time, voluntary food intake, and fecal output and moisture content. Results from this study support using FGMs as a biomarker of hypothalamic-pituitary-adrenal axis activity in muskoxen, advance our understanding of the physiological adaptations of mammals living in highly seasonal and extreme environments such as the Arctic, and emphasize the importance of considering seasonality in other species when interpreting FGM levels., Competing Interests: The authors have declared that no competing interests exist.

Species that live in degraded habitats often show signs of physiological stress. Glucocorticoid hormones (e.g., corticosterone and cortisol) are often assessed as a proxy of the extent of physiological stress an animal has experienced. Our goal was to quantify glucocorticoids in free-ranging small mammals in fragments of Interior Atlantic Forest. We extracted glucocorticoids from fur samples of 106 small mammals (rodent genera Akodon and Oligoryzomys, and marsupial genera Gracilinanus and Marmosa) from six forest fragments (2-1200 ha) in the Reserva Natural Tapytá, Caazapá Department, Paraguay. To our knowledge, this is the first publication of corticosterone and cortisol levels for three of the four sampled genera (Akodon, Oligoryzomys, and Marmosa) in this forest system. We discovered three notable results. First, as predicted, glucocorticoid levels were higher in individuals living withing small forest fragments. Second, animals captured live using restraint trapping methods (Sherman traps) had higher glucocorticoid levels than those animals captured using kill traps (Victor traps), suggesting that hair glucocorticoid measures can reflect acute stress levels in addition to long-term glucocorticoid incorporation. These acute levels are likely due to urinary steroids diffusing into the hair shaft. This finding raises a concern about the use of certain trapping techniques in association with fur hormone analysis. Finally, as expected, we also detected genus-specific differences in glucocorticoid levels, as well as cortisol/corticosterone ratios.

Back-translating the clinical manifestations of human disease burden into animal models is increasingly recognized as an important facet of preclinical drug discovery. We hypothesized that inbred rat strains possessing stress hyper-reactive-, depressive- or anxiety-like phenotypes may possess more translational value than common outbred strains for modeling neuropathic pain. Rats (inbred: LEW, WKY, F344/ICO and F344/DU, outbred: Crl:SD) were exposed to Spared Nerve Injury (SNI) and evaluated routinely for 6 months on behaviours related to pain (von Frey stimulation and CatWalk-gait analysis), anxiety (elevated plus maze, EPM) and depression (sucrose preference test, SPT). Markers of stress reactivity together with spinal/brain opioid receptor expression were also measured. All strains variously developed mechanical allodynia after SNI with the exception of stress-hyporesponsive LEW rats, despite all strains displaying similar functional gait-deficits after injury. However, affective changes reflective of anxiety- and depressive-like behaviour were only observed for F344/DU in the EPM, and for Crl:SD in SPT. Although differences in stress reactivity and opioid receptor expression occurred, overall they were relatively unaffected by SNI. Thus, anxio-depressive behaviours did not develop in all strains after nerve injury, and correlated only modestly with degree of pain sensitivity or with genetic predisposition to stress and/or affective disturbances.

Pangolins are 'keystone species' driven towards extinction due to a lack of profound awareness and illegal trade. The drivers urge for immediate development in the understanding of demographics and reproductive dynamics of this species. In this study, we developed and validated a quantitative method to measure pangolin fecal extracts using the electrospray (ESI-MS/MS) interface in positive ionization mode. The method aids in the measurement of hormones from the hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-gonadal (HPG) axis, making it a possibly appropriate technique to understand the cross-talk between the axes. The study aims to measure the relative abundance of adrenal and gonadal hormones such as corticosterone, cortisol, estrone, estradiol-17β, progesterone, testosterone, and a number of its metabolites. From the dried fecal extract, the principal metabolite identified from the estrogen family was estradiol-17β, whereas the gestagen family revealed that the pregnane series is predominated in 5α-configuration. On the other hand, epiandrosterone was seen as the dominant form in the male fecal extracts. Additionally, the glucocorticoids are excreted majorly as corticosterone, but traces of cortisol are also present in both the male and female fecal samples. The physiological validation confirmed that the ESI-MS/MS technique is suitable to determine physiologically caused differences in the fecal steroid concentrations. Physiologically, the age structure in pangolin is not responsible for causing differences within gender. However, the results revealed that glucocorticoids might vary between the sexes, i.e., males have a higher relative abundance of glucocorticoids over females. Therefore, our studies show that some of the main adrenal and gonadal metabolites can be predicted by exploiting MS/MS, which can steer research to potentially assess the reproductive status of captive and free-ranging pangolin species., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Although chronic stress is an acknowledged risk factor for the development of somatic and affective disorders, the cellular and molecular mechanisms underlying stress-induced pathologies are not fully understood. Interestingly, rodent studies involving immune cell transfer suggest that CD4 + T cells might be at least in part involved in reactivation of a chemically-induced colitis by stress. However, until now evidence is lacking that these immune cell types are indeed involved in the development of a "stressed phenotype". The aim of the present study was, therefore, to assess the effects of adoptively transferring total mesenteric lymph node cells (mesLNCs) and CD4 + mesLNCs isolated from chronically-stressed mice into healthy recipient mice on various physiological, immunological and behavioral parameters. To induce chronic psychosocial stress in donor mice we employed the chronic subordinate colony housing (CSC) paradigm. Our data indicate that transfer of total or CD4 + mesLNCs from CSC mice, compared with respective cells from single-housed control (SHC) mice, promoted splenomegaly and interferon (IFN)-γ secretion from in vitro anti-CD3-stimulated mesLNCs in naïve recipient mice. This effect was independent of recipient mice additionally being administered with dextran sulfate sodium (DSS) or not. Transfer of CD4 + mesLNCs additionally increased adrenal weight and secretion of IL-6 from in vitro anti-CD3 stimulated mesLNCs in recipients administered with DSS. Importantly, transfer of neither cell type from CSC vs. SHC donor mice affected anxiety-related behavior of recipient mice in the light-dark box. Taken together, our data demonstrate that typical physiological and immunological, but not behavioral, effects of chronic stress can be induced in naïve recipient mice by adoptively transferring mesLNCs, in particular CD4 + mesLNCs, from chronically stressed donor mice., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Density-dependence is an important mechanism in the population regulation of small mammals. Stressors induced by high-density (e.g., crowding and aggression) can cause physiological and neurological disorders, and are hypothesized to be associated with alterations in gut microbiota, which may in turn reduce the fitness of animals by increasing stress- or disease-associated microbes. In this study, we examined the effects of housing density on the hormone levels, immunity, and composition of gut microbiota in male Brandt's voles (Lasiopodomys brandtii) by conducting two specific housing density experiments with or without physical contact between voles. Voles in high density groups exhibited higher serum corticosterone (CORT), serotonin (5-HT), and immunoglobulin G (IgG) levels, as well as higher testosterone (T) levels only in the experiment with physical contact. Meanwhile, high-density treatments induced significant changes in the composition of gut microbiota by increasing disease-associated microbes. The levels of hormones and immunity (i.e., CORT, 5-HT, and IgG) elevated by the high density treatment were significantly correlated with some specific microbes. These results imply that high-density-induced stress may shape the fitness of animals under natural conditions by altering their gut microbiota. Our study provides novel insights into the potential roles of gut microbiota in the density-dependent population regulation of small rodents as well as the potential mechanisms underlying psychological disorders in humans and animals under crowded conditions., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

The measurement of corticosterone in feathers is an appealing tool for assessing glucocorticoids in wild and domestic bird species. Feather corticosterone measurements can be performed noninvasively and can provide a means for comparing glucocorticoid secretion between individual birds; thus, such measurements can be used to assess the welfare of domestic poultry. The focus of this study was to assess the validity of detecting corticosterone in turkey (Meleagris gallopavo) feathers using an ELISA and investigate differences between genetic lines. Primary feather 9 was obtained at a processing plant from 123 individuals from 3 different purebred turkey lines (line A [N = 46], line B [N = 24], and line C [N = 53]). Assay validation tests were performed using a commercially available ELISA kit (Cayman Chemicals, Cedarlane Labs, Canada). Indicators of accuracy, recovery, precision, and sensitivity were sufficient. Significant differences in feather corticosterone concentration between the 3 lines were observed. Line C had significantly higher feather corticosterone than line A (P < 0.0001) and line B (P = 0.036). These results indicate that the quantification of feather corticosterone using an ELISA is a valid method for assessing glucocorticoid levels in turkeys. This is the first report of differences in feather corticosterone between different purebred turkey lines. Differences observed between purebred lines provide an intriguing basis for further investigation into the genetic parameters of glucocorticoid levels in turkeys., (Copyright © 2020. Published by Elsevier Inc.)

Anxiety disorders are the most prevalent mental health disorder worldwide, with a lifetime prevalence of 5-7 % of the human population. Although the etiology of anxiety disorders is incompletely understood, one aspect of host health that affects anxiety disorders is the gut-brain axis. Adolescence is a key developmental window in which stress and anxiety disorders are a major health concern. We used adolescent female mice in a gastrointestinal (GI) colonization model to demonstrate that the commensal fungus Candida albicans affects host health via the gut-brain axis. In mice, bacterial members of the gut microbiota can influence the host gut-brain axis, affecting anxiety-like behavior and the hypothalamus-pituitary-adrenal (HPA) axis which produces the stress hormone corticosterone (CORT). Here we showed that mice colonized with C. albicans demonstrated increased anxiety-like behavior and increased basal production of CORT as well as dysregulation of CORT production following acute stress. The HPA axis and anxiety-like behavior are negatively regulated by the endocannabinoid N-arachidonoylethanolamide (AEA). We demonstrated that C. albicans-colonized mice exhibited changes in the endocannabinoidome. Further, increasing AEA levels using the well-characterized fatty acid amide hydrolase (FAAH) inhibitor URB597 was sufficient to reverse both neuroendocrine phenotypes in C. albicans-colonized mice. Thus, a commensal fungus that is a common colonizer of humans had widespread effects on the physiology of its host. To our knowledge, this is the first report of microbial manipulation of the endocannabinoid (eCB) system that resulted in neuroendocrine changes contributing to anxiety-like behavior., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Glucocorticoids (GCs) circulate in the plasma bound to corticosteroid-binding globulin (CBG). Plasma CBG may limit access of glucocorticoids to tissues (acting as a sponge: the free hormone hypothesis), or may solely serve as a transport molecule, releasing GCs to tissues as the plasma moves through capillaries (the total hormone hypothesis). Both biomedical (focused on human health) and comparative (focused on ecological and evolutionary relevance) studies have worked to incorporate CBG in glucocorticoid physiology, and to understand whether free or total hormone is the biologically active plasma fraction. The biomedical field, however, has been well ahead of the comparative physiologists, and have produced results that can inform comparative research when considering the import of total vs. free plasma hormone. In fact, biomedical studies have made impressive strides regarding the function of CBG in tissues as well as plasma; we, however, focus solely on the plasma functions in this review as this is the primary area of disagreement amongst comparative physiologists. Here we present 5 sets of biomedical studies across genomics, pharmacology, cell culture, whole animal research, and human medicine that strongly support a role for CBG limiting hormone access to tissue. We also discuss three areas of concern across comparative researchers. In contrast to former publications, we are not suggesting that all comparative studies in glucocorticoid physiology must measure CBG, or that only free corticosterone levels are valid. However, we propose that comparative physiologists be aware of biomedical results as they investigate glucocorticoids and interpret how total hormone may or may not impact behavior and physiology of free-living vertebrates., (Published by Elsevier B.V.)

Laboratory male mice are often housed individually due to aggressive behavior or experimental requirements, though social isolation can cause welfare issues. As a strategy to refine housing of male mice, we introduce the separated pair housing system. A perforated transparent wall divides the cage into two compartments and allows olfactory, acoustic, and visual communication between the two mice but prevents fighting and injuries. Long-term effects of separated pair housing on well-being and distress of adult male C57BL/6JRj mice were investigated and compared with both single- and group-housed mice. Behavioral analysis after eight weeks in three different housing systems revealed no differences in burrowing performance, social interaction, anxiety, and stress hormone concentrations. However, pair-housed mice built more complex nests compared to single-housed mice and the nest position suggested that pair-housed mice preferred the close proximity to their cage mates. Moreover, pair-housed mice showed less locomotor activity compared to group- and single-housed mice. Body weight was higher in group-housed mice. All in all, no unambiguous long-term beneficial effects of pair housing on the well-being were found. However, the findings emphasized that effects of the housing systems on behavioral, physical, and biochemical parameters must be considered in the design of animal experimental studies.

The effect of gut microbiota on the activity of the HPA and HPG axes and ECS is a short-term or long-lasting process remains unclear in rodents. However, the extant studies focused only on its short-term effect on the HPA activity because there is lack of reliable biomarkers characterizing short-term activity of the HPG axis and ECS and long-term activities of the three endocrine systems. The endogenous levels of aldosterone (ALD), 11-dehydrocorticosterone (11-DHC), estradiol (E2), estrone (E1), androstenedione (A4), dihydrotestosterone (DHT), corticosterone (CORT), dehydroepiandrosterone (DHEA), testosterone (T), progesterone (P), N-arachidonoyl ethanoamide (AEA) and 1-arachydonoyl glycerol (1-AG) in hair and plasma are the potential long-term and short-term biomarkers of the three systems. This study aimed to develop the sensitive and selective methods for simultaneous quantitation of the twelve compounds in rodent's hair and plasma. Then the methods were used to explore the differences in the hair levels of the twelve compounds between the mice in XZ group possibly having gut microbiota with more diversity and SPF group possibly having gut microbiota with less diversity and the inter-group differences in the plasma levels in the response to 1-h restraint stress. The methods were based on high performance liquid chromatography tandem mass spectrometry with atmospheric pressure chemical ionization in positive mode. The methods were adopted for 20 mg hair and 100 μL plasma, respectively. Hair samples were incubated in methanol at 40 ℃ for 24 h, and were performed by solid phase extraction. Plasma samples were implemented by liquid-liquid extraction with ethyl acetate. The methods showed limit of quantification at 0.06-1.3 pg/mg and 0.03-0.6 ng/mL and recovery ranging between 87.7-115.1 % and 86.1-114.6 % for all compounds in rodent's hair and plasma, and the intra-day and inter-day coefficients of variation less than 15 %, and good freeze/thaw and short-term stability. The present methods also had good reliability as demonstrated by the sex difference in the testosterone levels in hair and plasma. The inter-group comparison revealed that the mice in XZ group showed significantly higher hair levels than those in SPF group for 1-AG and most of hormones except for T and P. The non-stressed female mice in SPF showed significantly higher plasma levels than those in XZ for AEA and most of hormones except for E2, A4, DHT, T and 1-AG, but there were no inter-group differences for the stressed mice except for DHEA and 11-DHC and for the non-stressed male mice. Additionally, the stressed mice showed significantly higher corticosterone level in plasma than controls for male and female mice in XZ and male mice in SPF, but it was not true for female mice in SPF., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The aim of this study was to explore the neurobiological background of individual susceptibility and resistance to the development of posttraumatic stress disorder (PTSD)-like behaviours. Rats were divided into susceptible, PTSD(+), and resistant, PTSD(-), groups based on freezing duration during exposure to aversive context and the time spent in the central area in open field test one week after threefold stress experience (modified single prolonged stress). PTSD(-) rats showed increased concentrations of corticosterone in plasma and changes in GAD67 expression: decreased in the infralimbic cortex (IL) and increased in the lateral amygdala (LA), dentate gyrus (DG), and CA1 area of the hippocampus. Moreover, in this group, we found an increase in the number of CRF-positive nuclei in the parvocellular neurons of the paraventricular hypothalamic nucleus (pPVN). The PTSD(+) group, compared to PTSD(-) rats, had decreased concentrations of corticosterone in plasma and reduced CRF expression in the pPVN, higher CRF expression in the CA1, increased expression of CRF-positive nuclei and GR receptors in the CA3 area of the hippocampus, and increased expression of GR receptors in the DG and the central amygdala (CeA). Biochemical analysis showed higher concentrations of noradrenaline, glutamic acid in the dorsal hippocampus and amygdala and lower levels of dopamine and its metabolites in the amygdala of the PTSD(+) group than in the PTSD(-) group. The study revealed different behavioural and biochemical profiles of PTSD(+) and PTSD(-) rats and suggested that individual differences in hypothalamic-pituitary-adrenal (HPA) axis activity may determine hippocampal- and amygdala-dependent memory and fear processing., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Several neuropharmacological actions of cannabidiol (CBD) due to the modulation of the endocannabinoid system as well as direct serotonergic and gamma-aminobutyric acidergic actions have recently been identified. The current study aimed to reveal the effect of a long-term CBD treatment in the chronic unpredictable mild stress (CUMS) model of depression. Adult male Wistar rats (n = 24) were exposed to various stressors on a daily basis in order to induce anhedonia and anxiety-like behaviors. CBD (10 mg/kg body weight) was administered by daily intraperitoneal injections for 28 days (n = 12). The effects of the treatment were assessed on body weight, sucrose preference, and exploratory and anxiety-related behavior in the open field (OF) and elevated plus maze (EPM) tests. Hair corticosterone was also assayed by liquid chromatography-mass spectrometry. At the end of the experiment, CBD-treated rats showed a higher rate of body weight gain (5.94% vs. 0.67%) and sucrose preference compared to controls. A significant increase in vertical exploration and a trend of increase in distance traveled in the OF test were observed in the CBD-treated group compared to the vehicle-treated group. The EPM test did not reveal any differences between the groups. Hair corticosterone levels increased in the CBD-treated group, while they decreased in controls compared to baseline (+36.01% vs. -45.91%). In conclusion, CBD exerted a prohedonic effect in rats subjected to CUMS, demonstrated by the increased sucrose preference after three weeks of treatment. The reversal of the effect of CUMS on hair corticosterone concentrations might also point toward an anxiolytic or antidepressant-like effect of CBD, but this needs further confirmation.

There is extensive evidence that glucocorticoid hormones enhance memory consolidation, helping to ensure that emotionally significant events are well remembered. Prior findings suggest that the anteroventral region of bed nuclei of the stria terminalis (avBST) regulates glucocorticoid release, suggesting the potential for avBST activity to influence memory consolidation following an emotionally arousing learning event. To investigate this issue, male Sprague-Dawley rats underwent inhibitory avoidance training and repeated measurement of stress hormones, immediately followed by optogenetic manipulations of either the avBST or its projections to downstream regions, and 48 h later were tested for retention. The results indicate that avBST inhibition augmented posttraining pituitary-adrenal output and enhanced the memory for inhibitory avoidance training. Pretreatment with a glucocorticoid synthesis inhibitor blocked the memory enhancement as well as the potentiated corticosterone response, indicating the dependence of the memory enhancement on glucocorticoid release during the immediate posttraining period. In contrast, posttraining avBST stimulation decreased retention yet had no effect on stress hormonal output. Subsequent experiments revealed that inhibition of avBST input to the paraventricular hypothalamus enhanced stress hormonal output and subsequent retention, whereas stimulation did not affect either. Conversely, stimulation-but not inhibition-of avBST input to the ventrolateral periaqueductal gray impaired consolidation, whereas neither manipulation affected glucocorticoid secretion. These findings indicate that divergent pathways from the avBST are responsible for the mnemonic effects of avBST inhibition versus stimulation and do so via glucocorticoid-dependent and -independent mechanisms, respectively., Competing Interests: The authors declare no competing interest.

Artificial light at night (ALAN) can disrupt adaptive patterns of physiology and behavior that promote high fitness, resulting in physiological stress and elevation of steroid glucocorticoids (corticosterone, CORT in birds). Elevated CORT may have particularly profound effects early in life, with the potential for enduring effects that persist into adulthood. Research on the consequences of early-life exposure to ALAN remains limited, especially outside of the laboratory, and whether light exposure affects CORT concentrations in wild nestling birds particularly remains to be elucidated. We used an experimental setup to test the hypothesis that ALAN elevates CORT concentrations in developing free-living birds, by exposing nestling great tits (Parus major) to ALAN inside nest boxes. We measured CORT in feathers grown over the timeframe of the experiment (7 nights), such that CORT concentrations represent an integrative metric of hormone release over the period of nocturnal light exposure, and of development. We also assessed the relationships between feather CORT concentrations, body condition, nestling size rank and fledging success. In addition, we evaluated the relationship between feather CORT concentrations and telomere length. Nestlings exposed to ALAN had higher feather CORT concentrations than control nestlings, and nestlings in poorer body condition and smaller brood members also had higher CORT. On the other hand, telomere length, fledging success, and recruitment rate were not significantly associated with light exposure or feather CORT concentrations. Results indicate that exposure to ALAN elevates CORT concentrations in nestlings, which may reflect physiological stress. In addition, the organizational effects of CORT are known to be substantial. Thus, despite the lack of an effect on telomere length and survivorship, elevated CORT concentrations in nestlings exposed to ALAN may have subsequent impacts on later-life fitness and stress sensitivity., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Yolk hormones are substances which transmit non-genetic factors from the mother to the next generation. The systematic changes of yolk hormone concentrations within asynchronously hatching clutches have been interpreted as a means to adaptively shape the offspring's phenotype. However, in synchronously hatching clutches the role of yolk hormones is less understood. We investigated whether seasonal changes between eggs in the yolk hormones testosterone (Testo), progesterone (Prog) and corticosterone (Cort) also occur in the grey partridge, a synchronously hatching precocial species without direct food competition between siblings. Specifically we asked whether yolk hormone concentrations systematically vary with season and whether they affect the offspring's hatching mass, mass gain, circulating baseline and stress-induced Cort. Additionally, we investigated the effect of genetic background and food availability on yolk hormone concentrations by subjecting grey partridge hens of two strains (wild and domesticated) to two different feeding regimes (predictable vs. unpredictable feeding) during egg laying. We hypothesized that egg hormone concentrations change over the season, but breeding in captivity over many generations and ad libitum food access could have resulted in domestication effects which abolished potential seasonal effects. Results showed that progressing season had a strong positive effect on yolk Prog and yolk Testo, but not on yolk Cort. Feeding regimes and strain had no effect on yolk hormones. Offspring mass and mass gain increased and baseline Cort decreased with progressing season. In addition, yolk Testo correlated positively with offspring mass gain and negatively with baseline Cort, while yolk Prog had a positive correlation with baseline Cort. Strain and feeding regimes of the mother had no effect on offspring traits. In conclusion, grey partridge chicks hatching late in the season might benefit from the increased concentrations of the growth-stimulating yolk Testo and by this catch-up in development. Hence, yolk hormone concentration could adaptively shape the offspring phenotype in a precocial species., (Copyright © 2019 Elsevier Inc. All rights reserved.)

There is growing interest in the use of glucocorticoid (GC) hormones to understand how wild animals respond to environmental challenges. Blood is the best medium for obtaining information about recent GC levels; however, obtaining blood requires restraint and can therefore be stressful and affect GC levels. There is a delay in GCs entering blood, and it is assumed that blood obtained within 3 min of first disturbing an animal reflects a baseline level of GCs, based largely on studies of birds and mammals. Here we present data on the timing of changes in the principle reptile GC, corticosterone (CORT), in four reptile species for which blood was taken within a range of times 11 min or less after first disturbance. Changes in CORT were observed in cottonmouths (Agkistrodon piscivorus; 4 min after first disturbance), rattlesnakes (Crotalus oreganus; 2 min 30 s), and rock iguanas (Cyclura cychlura; 2 min 44 s), but fence lizards (Sceloporus undulatus) did not exhibit a change within their 10-min sampling period. In both snake species, samples taken up to 3-7 min after CORT began to increase still had lower CORT concentrations than after exposure to a standard restraint stressor. The "3-min rule" appears broadly applicable as a guide for avoiding increases in plasma CORT due to handling and sampling in reptiles, but the time period in which to obtain true baseline CORT may need to be shorter in some species (rattlesnakes, rock iguanas), and may be unnecessarily limiting for others (cottonmouths, fence lizards)., (Copyright © 2019. Published by Elsevier Inc.)

Level of corticosterone (CORT), which is a predominant glucocorticoid in birds, has become the main indicator for assessing the stress level of birds in ecological studies. Feather corticosterone (CORT f ) provides information about corticosterone levels during feather growth, however, the underlying causes of individual variation of CORT f between individuals and individual persistency of CORT f are not yet fully understood. Therefore, this study addresses individual consistency in CORT f and the association of variation in CORT f with behaviour that results in damage to tail feathers. We studied relations between CORT f , plasma CORT, and behaviour in wild-caught male greenfinches in captivity. CORT f in wild-grown feathers correlated positively with CORT f in lab-grown feathers. CORT f levels were about 20% lower in lab-grown feathers than in those grown in the wild. Four birds that died in captivity had significantly higher average CORT f levels in wild-grown feathers than the survivors. Plasma CORT levels of two measurements taken in the lab seven days apart correlated positively, however, no correlations between plasma CORT and CORT f were found. In order to study the link between CORT f and behaviour, the extent of tail damage from flapping against cage bar was assessed. Contrary to our prediction, birds with higher CORT f had less tail damage. This study adds to the evidence that CORT f levels can be considered as informative markers of some persistent component of individual phenotypic quality that can predict survival under standardized laboratory conditions., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The endocannabinoid system plays a key role in the control of emotional responses to environmental challenges. CB1 receptors are highly expressed within cortico-limbic brain areas, where they modulate stress effects on memory processes. Glucocorticoid and endocannabinoid release is influenced by circadian rhythm. Here, we investigated how different stress intensities immediately after encoding influence rat short-term memory in an object recognition task, whether the effects depend on circadian rhythm and if exogenous augmentation of anandamide levels could restore any observed impairment. Two separate cohorts of male adult Sprague-Dawley rats were tested at two different times of the day, morning (inactivity phase) or afternoon (before the onset of the activity phase) in an object recognition task. The anandamide hydrolysis inhibitor URB597 was intraperitoneally administered immediately after the training trial. Rats were thereafter subjected to a forced swim stress under low or high stress conditions and tested 1 h after training. Control rats underwent the same experimental procedure except for the forced swim stress (no stress). We further investigated whether URB597 administration might modulate corticosterone release in rats subjected to the different stress conditions, both in the morning or afternoon. The low stressor elevated plasma corticosterone levels and impaired 1 h recognition memory performance when animals were tested in the morning. Exposure to the higher stress condition elevated plasma corticosterone levels and impaired memory performance, independently of the testing time. These findings show that stress impairing effects on short-term recognition memory are dependent on the intensity of stress and circadian rhythm. URB597 (0.3 mg kg -1 ) rescued the altered memory performance and decreased corticosterone levels in all the impaired groups yet leaving memory unaltered in the non-impaired groups., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Minimization and alleviation of stress are generally viewed as desirable aspects of laboratory animal management and use. However, achieving that goal requires an unambiguous and valid measure of stress. Glucocorticoid concentrations are commonly used as a physiologic index of stress. Measurement of glucocorticoids in blood, serum or plasma clearly reflects many types of both acute and chronic stress. However, the rapid rise in concentrations of circulating glucocorticoids that occurs even with relatively simple manipulations such as handling has led to the increased use of fecal glucocorticoid metabolite (FCM) assays, which provide a temporally integrated measure that may allow a more accurate interpretation of chronic stressors. In this review, we consider 3 aspects of glucocorticoids as a measure of stress. First, we discuss the analytic and interpretational pitfalls of using FCM concentrations as an index of stress in mice and rats. Second, we consider evidence that some degree of stress may benefit animals by priming physiologic and behavioral adaptations that render the animals more resilient in the face of stress. Finally, we use 2 situations-social housing and food restriction-to illustrate the concept of hormesis-a biologic phenomenon in which a low dose or intensity of a challenge has a beneficial effect, whereas exposure to high doses or intensities is detrimental.

Repeated exposure to stress produces cognitive impairments that can lead to psychiatric disorders. How and when these cognitive impairments occur during repeated exposure to stress is not well understood. The present study investigates the time course effects of the exposure to intermittent episodes of social stress on cognitive flexibility performance. Male rats were trained to perform a cognitive flexibility (set-shifting) task. Then they were submitted to intermittent social defeat stress, which consisted of exposing animals to social defeat once every three days for ten days (four stress episodes). Set-shifting performance was evaluated before and in between social stress episodes, and up to ten days after the end of the stress protocol. Plasma levels of corticosterone after tail-pinch were also measured after the last set-shifting session. Intermittent exposure to social stress did not impair cognitive flexibility but produced short-and long-term changes in set-shifting performance. After the third social defeat episode, stressed animals required significantly more time to respond to cues (instrumental action) and to poke in the food-trough when no food pellet was delivered. These effects were reversed ten days after stress and suggest a decrease in motivation to pursue rewards. In contrast, stressed animals responded more accurately to light cues that predicted rewards. This effect, which emerged ten days after stress, suggests an increase in the reactivity to salient cues in the long-term. These time-dependent behavioral changes might help to understand the transition from social stress to stress-related disorders including drug abuse., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Cold stress can induce neuroinflammation in the hippocampal dentate gyrus (DG), but the mechanism underlying neuronal apoptosis induced by cold stress is not well-understood. To address this issue, male and female C57BL/6 mice were exposed to a temperature of 4 °C for 3 h per day for 1 week, and glial cell activation, neuronal apoptosis, and neuroinflammation were evaluated by western blotting, immunofluorescence, terminal deoxynucleotidyl transferase 2'-deoxyuridine 5'-triphosphate (dUTP) nick end labeling, Nissl staining, and immunohistochemistry. Additionally, BV2 cells were treated with different concentrations of cortisol (CORT) for 3 h to mimic stress and molecular changes were assessed by western blotting, immunofluorescence, and co-immunoprecipitation. We found that excess CORT activated glial cells and increased neuroinflammation in the DG of mice exposed to cold temperatures, which was associated with increased acetylation and nuclear factor-κB signaling. These effects were mediated by the acetylation of lysine 9 of histone 3 and lysine 310 of p65, which resulted in increased mitogen-activated protein kinase phosphorylation, nuclear translocation of p65, microglia activation, and acetylation of high-mobility group box 1. Neuroinflammation was more severe in male compared to female mice. These findings provide new insight into the mechanisms of the cold stress response, which can inform the development of new strategies to combat the effects of hypothermia.

Mild traumatic brain injury is a common outcome of blast exposure, and current literature indicates high rates of comorbid posttraumatic stress disorder (PTSD) in military personnel. Blast-exposed rats display PTSD-like behavior, suggesting relationships may exist between PTSD and blast exposure. Other studies demonstrate the roles of stathmin and corticosterone associated with fear- and anxiety-like behaviors in rodent models. Furthermore, studies have observed ranges of responses to both physical and psychological trauma in animal populations (Elder 2012, Ritov 2016). This study exposed rodents to repeated blast overpressure (BOP) and analyzed behavioral responses and molecular variables at 3 weeks and 6 months after exposure. We applied a modified version of a previously reported behavioral profiling approach that separates "affected" and "unaffected" rats based on the presence of anxiety-like behaviors (Ritov, 2016). We report that "affected" 3 week animals showed higher plasma corticosterone and amygdalar stathmin levels, while "affected" 6 month animals had lower prefrontal cortex stathmin. Higher corticosterone also paralleled anxiety behavior in "affected" 3 week animals, which was not observed in 6 month animals, indicating possible negative feedback loop mechanisms. Elevated levels of amygdalar stathmin correlated with anxiety behaviors in "affected" 3 week and 6 month animals, indicating sustained molecular changes. We conclude that this unique analysis may provide more information about response to blast. This type of analysis should also be considered when treating clinical populations, since individual differences may affect behavioral and long-term outcomes. Future studies should elucidate relationships of stress and fear responses in the context of BOP., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Maternal corticosterone in avian eggs may modify offspring phenotype in order to increase survival in poor environments. In the Greater Rhea (Rhea americana), we previously found that yolk immunoreactive corticosterone is influenced by the quality of the maternal environment: eggs laid by females of the intensive rearing system (IRS), living in poor captive conditions, had higher yolk immunoreactive corticosterone than those produced by females of the semi-extensive rearing system (SRS), living in better conditions. Here, we evaluate if these different hormone levels are associated with the production of different phenotypes. We collected eggs from the IRS and SRS for hormonal quantification and artificial incubation. Then, half of the chicks selected from each environment were exposed to a capture and restraint protocol, and the rest remained undisturbed and were used as controls. In the IRS, we found that higher yolk immunoreactive corticosterone was associated with the production of chicks that had reduced hatchability, lower hatchling mass and higher baseline fecal glucocorticoid metabolites (FGM) than those produced by SRS females. Moreover, after capture and restraint, IRS chicks did not modify their FGM nor their behaviors compared to their controls, while SRS chicks increased their FGM and spent more time ambulating and less time pecking, compared to their controls. These results indicate that yolk immunoreactive corticosterone could modify offspring phenotype. Although future studies are needed to elucidate their implications for fitness, our results suggest that yolk corticosterone could be mediating an adaptive maternal effect that allows individuals to better cope with poor conditions., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Research into the molecular basis of stress resilience is a novel strategy to identify potential therapeutic strategies to treat stress-induced psychopathologies such as anxiety and depression. Stress resilience is a phenomenon which is not solely driven by effects within the central nervous system (CNS) but involves multiple systems, central and peripheral, which interact with and influence each other. Accordingly, we used the chronic social defeat stress paradigm and investigated specific CNS, endocrine and immune responses to identify signatures of stress-resilience and stress susceptibility in mice. Our results showed that mice behaviourally susceptible to stress (indexed by a reduction in social interaction behaviour) had higher plasma corticosterone levels and adrenal hypertrophy. An increase in inflammatory circulating monocytes was another hallmark of stress susceptibility. Furthermore, prefrontal cortex mRNA expression of corticotrophin-releasing factor (Crf) was increased in susceptible mice relative to resilient mice. We also report differences in hippocampal synaptic plasticity between resilient and susceptible mice. Ongoing studies will interpret the functional relevance of these signatures which could potentially inform the development of novel psychotherapeutic strategies., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Environmental contaminants, such as the trace element selenium (Se), are a continuing concern to species worldwide due to their potential pathophysiological effects, including their influence on the stress response mediated through glucocorticoids (GCs; stress hormones). Environmental concentrations of Se are increasing due to anthropogenic activities, including the incomplete combustion of coal and subsequent disposal of coal combustion wastes. However, most studies examining how Se affects GCs have been focused on lower trophic organisms. The objectives of this study were to investigate the effects of long-term Se exposure on traditionally used stress parameters and to identify which of these parameters best indicate Se accumulation in liver and kidney of the American alligator (Alligator mississippiensis), a top trophic carnivore found in the southeastern United States and known to inhabit Se-containing areas. Alligators were divided into three dietary treatments and fed prey spiked with 1000 or 2000 ppm of selenomethionine (SeMet) or deionized water (control treatment) for 7 weeks. Following the 7-week treatment protocol, blood and tissue samples were obtained to measure plasma corticosterone (CORT; the main crocodilian GC), tail scute CORT, the ratio of peripheral blood heterophils (H) to lymphocytes (L) as H/L ratio, and body condition. To evaluate which parameter best indicated Se accumulation in the liver and kidney, principal component and discriminant analyses were performed. The only parameter significantly correlated with liver and kidney Se concentrations was scute CORT. Our results suggest that measurement of CORT in tail scutes compared with plasma CORT, H/L ratios, and body condition is the best indicator of Se-exposure and accumulation in crocodilians.

The host microbial community is thought to have an important role in the host endocrine system and behavioral phenotype. We investigated chronological changes of levels of gonadal hormones and corticosterone in the feces of 4- to 8-week-old female germ-free (GF) mice, and conducted odor preference test at 8 weeks of age. We further evaluated the developmental impact of the microbial community by analyzing 4-week-old GF mice orally administered the fecal microbiota of specific pathogen-free (SPF) mice or guinea pigs (GF-SPF mice or GF-Guinea pig mice). The fecal estradiol, progesterone, and corticosterone levels of GF mice were lower than those of SPF mice. Furthermore, the increased levels in GF mice were suggested to be caused by colonization of microbiota of SPF mice or guinea pigs. However, the degree of recovery of progesterone and corticosterone by microbiota of guinea pigs was lower than that by SPF mice. In odor preference tests, interestingly, female GF mice preferred female odors to male odors, although this preference was not seen in other mice. These findings suggested that the microbial community plays an important role in the development of the host endocrine system for gonadal hormones and corticosterone, and odor preference in mice., (© 2019 Wiley Periodicals, Inc.)

Human activities have introduced a variety of chemicals, including pesticides, fertilizers, and salt, into the environment, which may have deleterious effects on the organisms inhabiting these areas. Amphibians are especially susceptible to absorption of chemical pollutants. To determine the possible combined effects of these chemicals on amphibian development and stress levels, Southern leopard frog (Lithobates sphenocephala) larvae were exposed to one of eight individual or combined treatments of atrazine, ammonium nitrate fertilizer, and sodium chloride salt. Stress levels, indicated by release of the stress hormone corticosterone, were measured premetamorphosis at week 8 of development. Water hormone samples were processed to analyze corticosterone levels. Changes in tadpole growth were determined by surface area measurements taken from biweekly photographs. The combined chemical treatment of atrazine, salt, and fertilizer had a significant interactive effect by increasing stress levels before metamorphosis (p = 0.003). After a month of larval development, tadpoles exposed to ammonium nitrate had larger surface area (p = 0.035). Tadpoles exposed to atrazine had a lower growth rate throughout larval development (p = 0.025) and the lowest number of individuals reaching metamorphosis at 33%. However, the frogs in the atrazine treatment that did successfully metamorphose did so in fewer days (p = 0.002). Because amphibians are exposed to multiple chemicals simultaneously in the environment, assessing the effects of a combination of contaminants is necessary to improve application strategies and ecosystem health.

Availability of food resources affects animal survival and reproduction. Thus, coping with changes in food availability is one of the most crucial behavioural and physiological processes in wildlife. Food intake is a key concept in animal ecology that is directly conditioned by food quality and abundance or diet choice, but may also vary according to individual-related factors (e.g. foraging behaviours, social rank or energy-demanding periods) and the influence of the endocrine system on energy metabolism. Here, we studied food intake in relation to individual characteristics (sex, breeding condition and age) and whether steroid hormones (testosterone and corticosterone metabolites) mediate food intake in wild wood mouse (Apodemus sylvaticus). Field work was carried out in February-March 2014 in Monte de Valdelatas (Madrid, Spain). Wood mice were live-trapped for 10 consecutive days in four independent plots. Traps were baited with 4 g of toasted corn and food intake was calculated by subtracting the remaining bait found inside traps. Fresh faecal samples from 130 different wood mice were collected and faecal testosterone and corticosterone metabolites (FTM and FCM, respectively) were analysed by enzyme immunoassays. Food intake was higher in females than males, probably due to greater energy requirements. Non-breeders and young individuals also showed a higher food intake. These individuals usually hold a lower social rank which is associated to limited food resources because of dominants; thus, increased food intake may be a result of freely exploit food bait inside traps while avoiding risky competition. In addition, food intake negatively correlated with FTM levels and positively with FCM levels indicating that both hormones have an active role mediating food intake in the wood mouse. Our data suggest that food intake is a function of both individual traits and the endocrine system that accordingly respond throughout different energy-demanding periods.

Emerging evidence indicates that ingestion of specific probiotics, known as "psychobiotics", confer beneficial effects on mental health. This study investigated antidepressant-like effects and possible underlying mechanisms of Lactobacillus paracasei PS23 (PS23), live or heat-killed, in a mouse model of corticosterone-induced depression using fluoxetine as standard drug. PS23 were orally gavaged to mice from day 1 to 41 or fluoxetine from day 17 to 41 and injected with corticosterone from day 17 to 37. After the last corticosterone treatment, anxiety- and depression-like behaviors were tested within 4 days. On day 42, serum and brain tissue were collected 24 min after forced swim stress. Abnormal behavioral changes induced by corticosterone were ameliorated by treatment with live PS23 in open field and sucrose preference tests, with heat-killed PS23 in open field, forced swim and sucrose preference tests, and with fluoxetine in open field and forced swim tests. Furthermore, both live and heat-killed PS23 and fluoxetine reversed corticosterone-reduced protein levels of brain-derived neurotropic factor, mineralocorticoid, and glucocorticoid receptors in the hippocampus. In addition, live PS23 also reverses corticosterone-reduced serotonin levels in hippocampus, prefrontal cortex and striatum; whereas heat-killed PS23 reverses corticosterone-reduced dopamine levels in hippocampus and prefrontal cortex. And fluoxetine normalized reduced corticosterone level in serum. These studies showed that both live and heat-killed PS23 can reverse chronic corticosterone-induced anxiety- and depression-like behaviors and that may provide insights into the mechanism and a potential psychobiotic for depression management., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Wild animals typically suffer from stress when brought into captivity. This stress is characterized by elevated circulating glucocorticoid levels and weight loss. We here describe for the first time a case where a wild animal, the long-distance migrating northern wheatear, does not show signs of stress when caged. We captured these birds on a stopover site during their spring migration and caged them individually with ad libitum access to food and water. The birds were divided into four groups and were blood-sampled immediately in the field, a few hours after caging, one day after caging, or three days after caging, respectively. From these blood-samples we determined circulating corticosterone level. Food intake and body mass were also monitored. We found that, with very few exceptions, corticosterone levels were low and did not differ among the groups. Accordingly, almost all birds consumed huge quantities of food and substantially increased their body mass. Together these results clearly show that caging does not result in indications of stress in wild migrating northern wheatears. Confinement-specific conditions such as restricted movement normally stress animals. We suggest migratory birds may not perceive such conditions as stressors due to their hyperphagic state, a notion that requires further testing., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Feathers gradually accumulate hormones and reflect long-term average plasma steroid levels during their growth. Feather hormone levels thus provide for the measurement of plasma hormones concentrations integrated over a period of several days or weeks. In this study, we focused on the development of a method to determine testosterone (TEST) and corticosterone (CORT) levels in extracts from feathers of small bodied birds with a limited amount of feathers available per individual. For this purpose, the method had to be verified for a small weight of samples. The present study describes the effect of the conditions of sample preparation and keto-derivatisation on the sensitivity of the LC-ESI-MS/MS analysis of TEST and CORT. Generally, chemical derivatization improves the sensitivity and selectivity of LC-MS/MS analysis. It can be used particularly in situations when the total amount of collected sample is limited (such as in our studies). Both the conditions of feather sample preparation (the selection of the extraction solvent, the time of extraction, and the conditions of solid phase extraction) and the reaction conditions affecting the formation of keto-derivatives (such as reaction temperature and reaction time) were tested. Methanol as the extraction solvent, 8 h as the extraction time, 50 °C as the reaction temperature of derivatization, and 90 min as the reaction time of derivatization are the most suitable conditions in terms of achieving a high sensitivity of analyses. Calibration curves are linear, at least in the range 25-2500 pg mL -1 , which is usually found in feather extracts. The limit of detection (LOD) for TEST and CORT was 1.0 and 0.3 pg per mL, respectively. The limit of quantification (LOQ) for TEST and CORT was 3.3 and 1.0 pg per mL, respectively. The optimized procedure was successfully applied for the analysis of TEST and CORT in real feather samples. The method could be used in a variety of research direction including wildlife, agricultural or veterinary studies., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Recent studies have shown that mild traumatic brain injury (mTBI) is associated with higher risk for anxiety-related disorders. Dysregulation in the hypothalamus-pituitary-adrenal (HPA) axis following mTBI has been proposed to be involved in the development of neurobehavioral abnormalities; however, the underlying mechanisms are largely unknown. The aim of this study was to determine whether the corticotropin-releasing-factor-1 (CRF-1) receptor is involved in the regulation of anxiety-related symptoms in a mouse model of mTBI. Animals with or without mTBI received intracerebroventricular injections of a CRF-1 receptor agonist (CRF; 0.01 nmol/mouse) or antagonist (antalarmin; 1 µg/mouse) for 5 days, and then the animals were subjected to anxiety tests (light-dark box and zero maze). The levels of adrenocorticotropic hormone and corticosterone, the most important markers of HPA axis, were also measured after behavioral tests. Our results indicated that mTBI-induced anxiety-related symptoms in mice through increased levels of adrenocorticotropic hormone and corticosterone, showing HPA axis hyperactivity. Interestingly, activation of CRF receptor by a subthreshold dose of CRF resulted in significant increases in anxiety-like behaviors and HPA axis response to stress, whereas blockade of CRF receptors by a subthreshold dose of antalarmin decreased anxiety-related symptoms and HPA axis response to stress in mTBI-induced mice. Collectively, these findings suggest that the CRF-1 receptor plays an important role in the regulation of anxiety-related behaviors following mTBI induction in mice and support the hypothesis that blockade of the CRF-1 receptor may be a promising therapeutic target for anxiety-related disorders in patients with TBI.