Non-invasive assessment of adrenocortical function in captive Nile crocodiles (Crocodylus niloticus)

The occurrence of stress-inducing factors in captive crocodilians is a concern, since chronic stress can negatively affect animal health and reproduction, and hence production. Monitoring stress in wild crocodiles could also be beneficial for assessing the state of health in populations which are potentially threatened by environmental pollution. In both cases, a non-invasive approach to assess adrenocortical function as a measure of stress would be preferable, as animals are not disturbed during sample collection, and therefore sampling is feedback-free. So far, however, such a non-invasive method has not been established for any crocodilian species. As an initial step,we therefore examined the suitability of two enzyme-immunoassays, detecting faecal glucocorticoid metabolites (FGMs) with a 11β,21-diol-20-one and 5β-3α-ol-11-one structure, respectively, for monitoring stress-related physiological responses in captive Nile crocodiles (Crocodylus niloticus). An adrenocorticotropic hormone (ACTH) challenge was performed on 10 sub-adult crocodiles, resulting in an overall increase in serum corticosterone levels of 272% above the pre-injection levels 5 h post-injection. Saline-treated control animals (n=8) showed an overall increase of 156% in serum corticosterone levels 5 h post-administration. Faecal samples pre- and post-injection could be obtained from three of the six individually housed crocodiles, resulting in FGMconcentrations 136–380% above pre-injection levels, always detected in the first sample collected post-treatment (7–15 days post-injection). FGM concentrations seem comparatively stable at ambient temperatures for up to 72 h post-defaecation. In conclusion, non-invasive hormone monitoring can be used for assessing adrenocortical function in captive Nile crocodiles based on FGM analysis.University of Pretoria.The IUCN-SSC Crocodile Specialist Group (SRAS Project 10/12) and Novartis/SAVF Wildlife Research Fundhttp://www.elsevier.com/locate/cbpahb201

Non-invasive monitoring of glucocorticoid metabolite concentrations in urine and faeces of the Sungazer (Smaug giganteus)

Developing non-invasive techniques for monitoring physiological stress responses has been conducted in a number of mammal and bird species, revolutionizing field-based endocrinology and conservation practices. However, studies validating and monitoring glucocorticoid concentrations in reptiles are still limited. The aim of the study was to validate a method for monitoring glucocorticoid metabolite concentrations in urine (uGCM) and faeces (fGCM) of the cordylid lizard, the Sungazer (Smaug giganteus). An adrenocorticotropic hormone (ACTH) challenge was conducted on one male and two females with both urine and faecal material being collected during baseline and post-injection periods. Steroid extracts were analysed with four enzyme immunoassays (EIAs)namely: 11-oxoaetiocholanolone, 5α-pregnane-3β-11β-21-triol-20-one, tetrahydrocorticosterone, and corticosterone. A considerable response in fGCM and uGCM concentrations following ACTH administration was observed in all subjects, with the 5α-pregnane-3β-11β-21-triol-20-one and tetrahydrocorticosterone EIAs appearing to be the most suited for monitoring alterations in glucocorticoid metabolite concentrations in S. giganteus using faeces or urine as hormone matrix. Both EIAs showed a significantly higher concentration of glucocorticoid metabolites in faeces compared to urine for both sexes. Collectively, the findings of this study confirmed that both urine and faeces can be used to non-invasively assess adrenocortical function in S. giganteus

Non-invasive assessment of adrenocortical function as a measure of stress in the endangered golden langur

The golden langur (Trachypithecus geei) is an endangered primate endemic to northern India and Bhutan. The main stressors to the species are habitat degradation and fragmentation. Non‐invasive fecal glucocorticoid metabolite (fGCM) analysis is a powerful tool for assessing stress associated with environmental disturbances in wildlife. However, interspecific differences in glucocorticoid metabolism require careful selection of the antibody used in their quantification. The goals of this study were to: 1) validate an enzyme immunoassay (EIA) to determine fecal GC metabolite (fGCM) concentrations in the golden langur and 2) compare fGCM concentrations between golden langurs living under different environmental conditions. We compared five enzyme immunoassays for determining stress related physiological responses in golden langurs by performing an adrenocorticotropic stimulation test in both sexes. Our validations identified an 11‐oxoaetiocholanolone EIA detecting 11, 17 dioxoandrostanes as the most suitable assay for monitoring adrenocortical activity in the species. FGCM concentrations from semi‐captive temple langurs were significantly higher than respective stress steroid concentrations in free‐ranging or pet/zoo langurs. This study presents a validated practical method for non‐invasive monitoring of adrenocortical function in this rare and declining species.http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1098-236http://wileyonlinelibrary.com/journal/zoo.2018-07-13hj2018Anatomy and Physiolog

Determining the adrenocortical activity as a measure of stress in domestic pigs (Sus scrofa domesticus) based on salivary and faecal analysis

Domestic pigs are subjected to farm management procedures, some of which might be perceived as causing stress and therefore may have welfare concerns. When confronted with a stressor, animals display a response that consists of a suite of physiological and behavioural alterations to restore homeostasis. Physiologically, the response is usually determined using glucocorticoid concentrations (GC) albeit invasively, with the disadvantage of a possible handling-induced stress response. To date, no non-invasive method exists for determining stress-related responses in pigs. This study aimed to examine the suitability of enzyme-immunoassays (EIAs) for determining GC concentrations in saliva and faeces of domestic pigs by performing an ACTH challenge. A total of 6 animals were studied with 4 receiving 10µg/kg of Synacthen® (Novartis, South Africa Pty Ltd) and the remaining 2 receiving 0.5 ml physiologic saline. Baseline salivary glucocorticoid (sGC) concentrations of 2.38±1.83ng/ml (mean±SEM) increased by six-fold (14.03±6.83 ng/ml) within 40-90 minutes after administration of ACTH (P < 0.0001) and the elevated salivary cortisol levels were maintained for up to nine hours. Administration of saline caused no significant differences in sGC concentrations (P = 0.82). Similarly, baseline faecal glucocorticoid metabolite (fGCM) level of 235±46μg/g rose significantly up to 393±164μg/g within 36 hours post ACTH administration (P < 0.0001). No significant difference was found between baseline and post saline administration (P = 0.57). Significantly higher sGC concentrations were found in samples collected in the morning compared to those collected in the afternoon, indicating a circadian rhythm. In terms of stability of fGCMs, post-defecation levels only changed by 4% over the course of 50h (P = 0.76). In conclusion, EIAs can be used to determine sGC and fGCM concentrations to assess adrenocortical function in pigs. FGCM levels are relatively stable for at least two days post-defecation, which facilitates field sample submissions.Poster presented at the University of Pretoria, Faculty of Veterinary Science Faculty Day, August 25, 2016, Pretoria, South Africa.ab201

Degradation of Temminck's pangolin (Smutsia temminckii) scales with a keratinase for extraction of reproductive steroid hormones

Hormone monitoring in keratinous tissues has become increasingly popular. The insoluble keratin materials are generally pulverised before hormone extraction; however, this is difficult for thicker keratin structures like baleen plates or hooves. A new method, involving the use of keratinase, allows enzymatic digestion of keratin and hormone analysis in the resulting suspension. Pangolins are unique mammals covered in keratinous scales, which are one of the reasons these animals are extensively trafficked. This study aimed to investigate the suitability of Temminck's pangolin scales as hormone matrix for quantifying reproductive steroids. A protocol was developed to digest scales with a keratinase before measuring hormone concentrations. This method can be used to investigate the reproductive endocrinology of Temminck's pangolins but may also be extended to the other extant pangolin species. • Keratinase digests Temminck's pangolin scales and reproductive steroid metabolite concentrations are measurable in the resulting suspension. • Isopropanol is an ideal washing solvent for scales to remove surface contaminants and scale sample mass should be standardised to allow comparisons. • Any section of a scale and scales from any pangolin body region can be used as samples for hormone quantification.The Rufford Foundation (UK) and the Pangolin Consortium (US).http://www.elsevier.com/locate/mexhj2021Mammal Research InstituteZoology and Entomolog

Non-invasive monitoring of adrenocortical activity in free-ranging Namaqua rock mice Micaelamys namaquensis from South Africa in response to anthropogenic land use and season

Stress in animals has been linked to behavioural and physiological changes in response to environmental, social and anthropogenic stimuli. Hence, stress-related responses in animals, especially in rodents, have been used as biological indicators of ecosystem health. This study aimed to establish an enzyme immunoassay (EIA) for monitoring adrenocortical activity in free-ranging Namaqua rock mice (Micaelamys namaquensis) (Rodentia: Muridae) using faeces as a prerequisite for assessing the effects of anthropogenic land use and season on faecal glucocorticoid metabolite (fGCM) concentration. Rodents were live-trapped seasonally across four land use types: an agricultural crop farm, an agricultural livestock farm, a human-populated site, and a nature reserve; all situated in Limpopo Province, South Africa. Determined fGCM concentrations from capture and recapture events were used for biologically validating an EIA detecting steroids with a 5α-3β-11β-diol structure. Recapturing resulted in a significant overall 40% elevation of individual fGCM concentrations demonstrating the effectiveness of the chosen EIA to reliably detect glucocorticoid output in the study species. Neither land use type nor season affected fGCM concentrations in the species, suggesting that land use and season-related environmental changes do not necessarily act as stressors for M. namaquensis, presumably due to their adaptive and resilient nature. Such species can be used to identify ecosystems affected by human-mediated disturbances and allow insights into the management and restoration of these threatened ecosystems and their associated species.Centre of Excellence for Invasion Biology (CIB)http://www.wildlifebiology.orgpm2020Zoology and Entomolog

Validation of enzyme immunoassays via an adrenocorticotrophic stimulation test for the non-invasive quantification of stress-related hormone metabolites in naked mole-rats

DATA AVAILABILITY: Data supporting the reported results will be sent by the corresponding author upon request.SUPPLEMENTARY MATERIALS : TABLE S1: Summary of saline volume and synthetic adrenocorticotropic hormone (Synacthen® depot, Novartis, South Africa (Pty) Ltd.) administered to each individual during the ACTH challenge; TABLE S2: Summary of EIA sensitivities and intra-assay and inter-assay coefficients of variation in high- and low-value quality controls for all EIAs applied to measure immunoreactive urine glucocorticoid metabolite (uGCM) and fecal glucocorticoid metabolite in naked mole-rats.Small size in mammals usually restricts long-term, frequent monitoring of endocrine function using plasma as a matrix. Thus, the non-invasive monitoring of hormone metabolite concentrations in excreta may provide an invaluable approach. The aim of the current study was to examine the suitability of enzyme immunoassays (EIAs) for monitoring responses to stressors in the naked mole-rat (Heterocephalus glaber, NMR) using urine and feces as hormone matrices. A saline control administration, and a high- and low-dose adrenocorticotropic hormone (ACTH) challenge were performed on six male and six female disperser morph NMRs. The results revealed that a 5α-pregnane-3β,11β,21-triol-20-one EIA detecting glucocorticoid metabolites (GCMs) with a 5α-3β-11β-diol structure is the most suitable assay for measuring concentrations in male urine samples, whereas an 11-oxoaetiocholanolone EIA detecting GCMs with a 5β-3α-ol-11-one structure appears the most suitable EIA for quantifying GCMs in female urine. An 11-oxoaetiocholanolone EIA detecting 11,17 dioxoandrostanes was the most suitable EIA for quantifying GCMs in the feces of both sexes. There were sex-related differences in response to the high- and low-dose ACTH challenge. We recommend using feces as a more suitable matrix for non-invasive GCM monitoring for NMRs which can be valuable when investigating housing conditions and other welfare aspects.SARChI Chair of Mammalian Behavioral Ecology and Physiology from the DST–NRF South Africa and the National Research Foundation.https://www.mdpi.com/journal/animalsMammal Research InstituteZoology and Entomolog

From killer to carer : steroid hormones and paternal behaviour

Mammalian parental investment (i.e., care of descendant offspring) is largely biased towards maternal contributions due to the specific feeding needs of mammalian offspring; however, varying degrees of paternal investment have been reported in about 10% of all mammalian species. Within the order Carnivora, paternal contribution to rearing offspring is particularly high: an estimated 32% of all studied carnivore species exhibit direct paternal care. Despite the prominence of paternal investment in carnivores, the endocrine basis of this behaviour is not well understood. This review examines the current – highly constrained -- state of knowledge about the endocrine basis of carnivore paternal investment. We attempt to link changes in androgen and glucocorticoid levels with variation in direct and indirect paternal care behaviour making specific predictions regarding the way forward. Well studied species such as bat-eared foxes (Otocyon megalotis), dwarf mongoose (Helogale parvula) and meerkats (Suricata suricatta), where social dynamics are relatively well understood, can act as ideal model systems through which we may further investigate the endocrine basis of paternal investment in carnivores.P.R. de Bruin would like to thank the South African National Research Foundation (NRF) and the University of the Free State for their financial support while writing this manuscript.http://www.nisc.co.za/products/59/journals/african-zoology2017-12-31hb2017Anatomy and Physiolog

Faecal glucocorticoid metabolite concentrations and their alteration post-defaecation in African wild dogs Lycaon pictus from South Africa

Non-invasive techniques for the monitoring of animal well-being, such as faecal hormone analysis, are increasingly becoming some of the most desirable methods for addressing practical conservation questions. Despite the widespread use of faecal hormone measurements for monitoring responses to stressors and its known applicability to African wild dogs Lycaon pictus, the potential influence of time of collection post-defaecation on stress-steroid concentrations in faecal matter has not yet been investigated. In the present study, we determined the rate at which African wild dog faecal glucocorticoid metabolite (fGCM) concentrations change over a 16-day period post-defaecation, in order to provide recommendations for best sampling practice. No significant changes in fGCM concentrations were found for the first 48 h post-defaecation. However, an approximately 30% increase in fGCM concentrations were already noted between day 1 and day 2, followed by a significant 150% increase at 96 h post-defaecation. We therefore suggest that respective faecal material should be collected within the first 24 h post-defaecation to ensure the reliability of fGCM analysis. In addition, we collected baseline data denoting the fGCM concentrations of captive African wild dogs sampled across three South African captive sites. Determined baseline fGCM concentrations differed between African wild dogs at the sites sampled. These data could be used in future studies aimed at identifying the key stressor complexes perceived by captive African wild dogs in order to improve management strategies.This study was funded in part by the National Research Foundation (NRF).am2019Anatomy and PhysiologyZoology and Entomolog

First record of the marine turtle leech (Ozobranchus margoi) on hawksbill turtles (Eretmochelys imbricata) in the inner granitic Seychelles

Ozobranchus spp. are leeches that feed solely on turtle blood. They are common ectoparasites found on a range of marine turtle species, with some species of the leech being implicated as vectors of fibropapilloma-associated turtle herpesvirus (FPTHV). Green (Chelonia mydas) and hawksbill (Eretmochelys imbricata) turtles are the two commonly occurring species in the inner granitic islands of the Seychelles. Routine monitoring of nesting turtles on Cousine Island, Seychelles, allowed for opportunistic sightings of leeches on two hawksbill females. In both cases infestation was low, with three leeches collected off one female turtle and five off the other. No obvious signs of papillomas secondary to infection of FPTHV were seen. All of the turtle leeches collected were determined to be Ozobranchus margoi as they had five pairs of lateral digiform branchiae. The specimens were deposited in the Seychelles Natural History Museum on Mahé. To the best of our knowledge this is the first record of Ozobranchus margoi recorded in the inner granitic Seychelles on hawksbill turtles