Faecal Cortisol Metabolites Research Articles

Endocrine Correlates of Musth in Free-Ranging Asian Elephants (Elephas maximus) Determined by Non-Invasive Faecal Steroid Hormone Metabolite Measurements

The occurrence of musth, a period of elevated levels of androgens and heightened sexual activity, has been well documented for the male Asian elephant (Elephas maximus). However, the relationship between androgen-dependent musth and adrenocortical function in this species is unclear. The current study is the first assessment of testicular and adrenocortical function in free-ranging male Asian elephants by measuring levels of testosterone (androgen) and cortisol (glucocorticoid – a physiological indicator of stress) metabolites in faeces. During musth, males expectedly showed significant elevation in faecal testosterone metabolite levels. Interestingly, glucocorticoid metabolite concentrations remained unchanged between musth and non-musth periods. This observation is contrary to that observed with wild and captive African elephant bulls and captive Asian bull elephants. Our results show that musth may not necessarily represent a stressful condition in free-ranging male Asian elephants.

Assessing the impact of live-capture, confinement, and translocation on stress and fate in eastern gray squirrels

Live-capture and translocation are methods to deal with nuisance eastern gray squirrels in North America, but it is unknown how these methods affect squirrel physiology or survival. In this study we validated an enzyme immunoassay (EIA) to measure fecal cortisol metabolites (FCMs) in gray squirrels; assessed their stress response relative to the positioning of the live trap (sun, shade, or control); and assessed the impact of translocation on their long-term stress, movement patterns, and survival using FCM levels, body mass changes, and radiotelemetry. We found that a 5α-pregnane-3β,11β,21-triol-20-one EIA reliably detected acute stress in gray squirrel feces 12–24 h after the stressor; live traps positioned in the sun resulted in higher peak FCM levels compared with traps positioned in the shade; translocated squirrels experienced a 10% mortality rate, compared with no mortality in the controls, although overall fates were the same; translocated squirrels initially explored more and dispersed farther than controls, but after 2 weeks made similar movements; and after controlling for the effect of season, translocation did not affect long-term FCM levels or body mass; this conclusion must be tempered by the low number of recaptures. Our study demonstrates the utility of the FCM assay; that gray squirrels are extremely sensitive to capture, handling, and confinement; and that live-capture must be done in a way that minimizes exposure to additive environmental stressors.

Variation in the hindgut microbial communities of the Florida manatee,Trichechus manatus latirostrisover winter in Crystal River, Florida

The Florida manatee, Trichechus manatus latirostris, is a hindgut-fermenting herbivore. In winter, manatees migrate to warm water overwintering sites where they undergo dietary shifts and may suffer from cold-induced stress. Given these seasonally induced changes in diet, the present study aimed to examine variation in the hindgut bacterial communities of wild manatees overwintering at Crystal River, west Florida. Faeces were sampled from 36 manatees of known sex and body size in early winter when manatees were newly arrived and then in mid-winter and late winter when diet had probably changed and environmental stress may have increased. Concentrations of faecal cortisol metabolite, an indicator of a stress response, were measured by enzyme immunoassay. Using 454-pyrosequencing, 2027 bacterial operational taxonomic units were identified in manatee faeces following amplicon pyrosequencing of the 16S rRNA gene V3/V4 region. Classified sequences were assigned to eight previously described bacterial phyla; only 0.36% of sequences could not be classified to phylum level. Five core phyla were identified in all samples. The majority (96.8%) of sequences were classified as Firmicutes (77.3 ± 11.1% of total sequences) or Bacteroidetes (19.5 ± 10.6%). Alpha-diversity measures trended towards higher diversity of hindgut microbiota in manatees in mid-winter compared to early and late winter. Beta-diversity measures, analysed through PERMANOVA, also indicated significant differences in bacterial communities based on the season.

Preliminary study of faecal cortisol and corticosterone as an index of acute cortisol secretion in the koala (Phascolarctos cinereus).

Stress can play a role in disease incidence in all species via immunosuppression and has been implicated as a contributing factor in significant infectious diseases of koalas. Faecal cortisol measurement may represent a non-invasive methodology for quantifying stress in koalas. We used an ACTH (adrenocorticotropic hormone) stimulation test (10 IU) to induce sustained secretion of cortisol, which was measured in serum samples from four koalas and subsequently it was attempted to locate a corresponding elevation in either cortisol or corticosterone measurements within the faeces. Although ACTH administration resulted in an elevation of serum cortisol for at least 4 h post injection, it was not possible to identify a corresponding peak in corticosterone or cortisol concentrations in extracts from the faeces, consistent with the known gut transit time of the koala. Faecal cortisol and corticosterone metabolites may not be reliable indices of acute changes in cortisol secretion in the koala and studies that attempt to use faecal cortisol as an index of stress will need to be interpreted with caution.

Physiological stress in koala populations near the arid edge of their distribution.

Recent research has shown that the ecology of stress has hitherto been neglected, but it is in fact an important influence on the distribution and numbers of wild vertebrates. Environmental changes have the potential to cause physiological stress that can affect population dynamics. Detailed information on the influence of environmental variables on glucocorticoid levels (a measure of stress) at the trailing edge of a species’ distribution can highlight stressors that potentially threaten species and thereby help explain how environmental challenges, such as climate change, will affect the survival of these populations. Rainfall determines leaf moisture and/or nutritional content, which in turn impacts on cortisol concentrations. We show that higher faecal cortisol metabolite (FCM) levels in koala populations at the trailing arid edge of their range in southwestern Queensland are associated with lower rainfall levels (especially rainfall from the previous two months), indicating an increase in physiological stress when moisture levels are low. These results show that koalas at the semi-arid, inland edge of their geographic range, will fail to cope with increasing aridity from climate change. The results demonstrate the importance of integrating physiological assessments into ecological studies to identify stressors that have the potential to compromise the long-term survival of threatened species. This finding points to the need for research to link these stressors to demographic decline to ensure a more comprehensive understanding of species’ responses to climate change.

Assessment of adrenocortical activity and behavior of the collared anteater (Tamandua tetradactyla) in response to food‐based environmental enrichment

One of the current standard approaches to the study of animal welfare is measuring hypothalamic-pituitary-adrenal activity, frequently in association with behavioral assessment. We studied the effects of food-based environmental enrichment on adrenocortical activity and behavior in zoo-housed collared anteaters (Tamandua tetradactyla; n = 5). We successfully validated measurements of fecal cortisol metabolites (FCMs) using an 11-oxoetiocholanolone enzyme immunoassay by stimulating (ACTH injection) and suppressing (dexamethasone administration) adrenocortical activity. Three months later, we subjected animals to an ABA-type experiment (three 6-week periods): pre-enrichment (routine diet: A), enrichment (modified diet: B), and post-enrichment (routine diet: A) periods. We assessed adrenocortical activity by collecting individual feces three times a week (total number of samples: 228), and evaluated behavior by performing 3 days of behavioral observations per period (with a total of 3,600 behavioral data points for the individuals studied). Statistical analysis revealed changes in FCM concentrations (µg/g) over the periods (3.04 ± 0.68, 2.98 ± 0.66, and 4.04 ± 0.90, respectively). Additionally, it showed that the number of FCM peaks was highly reduced during enrichment; meanwhile active natural behaviors were significantly increased. We consider that these changes in response to food-based environmental enrichment improved the welfare of individual zoo-housed collared anteaters. This research might contribute to in situ and ex situ studies on the physiology and behavior of this endemic South American species.

Faecal cortisol metabolites in Bengal (Panthera tigris tigris) and Sumatran tigers (Panthera tigris sumatrae)

The tiger (Panthera tigris) faces a great risk of extinction as its wild numbers have plummeted due to poaching and habitat destruction so ex-situ conservation programs are becoming ever more necessary. Reliable non-invasive biomarkers of the stress hormone (cortisol) are necessary for assessing the health and welfare of tigers in captivity. To our knowledge, non-invasive stress endocrinology methods have not been tested as widely in tigers. The first aim of this study was to describe and validate a faecal cortisol metabolite enzyme-immmunoassay (FCM EIA) for two tiger sub-species, the Bengal tiger (Panthera tigris tigris) and the Sumatran tiger (Panthera tigris sumatrae). Individual tigers (n=22) were studied in two large Zoos in Queensland, Australia (Dreamworld Theme Park and Australia Zoo). Fresh faecal samples (<12h old) were collected each morning from both Zoos over a study period of 21days. Biological validation was conducted separately by collecting feces 5days before and 5days after blood was taken from four male and five female tigers. Results showed that mean FCM levels increased by 138% and 285% in the male and female tigers within 1day after bloods were taken, returning to baseline in 5days. Laboratory validations of the FCM EIA were done using an extraction efficiency test and parallelism. Results showed >89% recovery of the cortisol standard that was added to tiger faecal extract. We also obtained parallel displacement of the serially diluted cortisol standard against serially diluted tiger faecal extract. Our second aim was to determine whether the FCM levels were significantly different between tiger sub-species and sex. Results showed no significant difference in mean FCM levels between the Bengal and Sumatran tiger sub-species. Mean levels of FCMs were significantly higher in females than in male tigers. Those male and female tigers with reported health issues during the study period expressed higher FCM levels than the reportedly healthy tigers. Interestingly, those tigers that took part in some activity (such as walks, photos, presentations and guest feeds) expressed moderately higher FCM levels at Dreamworld and lower FCM levels at Australia Zoo in comparison to those tigers that did not take part in such activities. These results indicate potential habituation in some tigers for routine activity through specialized training and pre-conditioning. In conclusion, the FCM EIA described in this study provides a reliable non-invasive method for evaluating the stress status of tigers in Zoos.

Additional foraging elements reduce abnormal behaviour – fur-chewing and stereotypic behaviour – in farmed mink (Neovison vison)

We investigated whether provision of additional appetitive and consummatory elements of foraging reduces baseline stress and abnormal behaviour – in terms of fur-chewing and stereotypic behaviour – in farmed mink. We studied 200 juveniles (n=100 females and 100 males) during the 5-month growth period with plenty of feed, and subsequently the females as adults during the 2-month feed restriction period before mating. The mink were distributed in four equally sized groups: (i) FARM, conventional finely ground feed (<8mm) without additional foraging elements; (ii) ROPE, access to biting ropes; (iii) CONS, chunky feed (parts up to 42mm), replacing conventional feed; (iv) BOTH, access to both biting ropes and chunky feed. In growing mink, biting ropes reduced fur-chewing (P=0.044) and chunky feed reduced stereotypic behaviour (P=0.038) and fur-chewing in female mink (P=0.019). During the season of feed restriction, the wear/tear of biting ropes increased. Females on the chunky diet had a higher concentration of faecal cortisol metabolites (P=0.033), probably due to a more severe slimming resulting in a 6.2% lower body weight (P=0.006) than the mink on the finely ground diet; still the chunky diet reduced time spent in pre-feeding stereotypies (P=0.001). In the restrictively fed females, fur-chewing was reduced both by access to biting ropes (P=0.005) and chunky feed (P=0.007). Consequently, 54% of group FARM mink displayed fur-chewing compared to 21% in group BOTH. In conclusion, stereotypic behaviour was reduced by provision of chunky feed, increasing the consummatory element in daily foraging. Fur-chewing was reduced upon access to either biting ropes or chunky feed in female mink throughout the study. Our findings support frustrated foraging, mainly consummatory, behind abnormal behaviour.

Age‐dependent baseline values of faecal cortisol metabolites in the American mink (Neovison vison) under semi‐natural housing conditions

The welfare of an animal is ensured if it is able to fully satisfy its essential species-typical needs in all functional aspects of behaviour. In mink, stereotypies and apathy, internal and/or external injuries as well as increased susceptibility to disease have been known to occur as a result of chronic stress. The non-invasive method of analysing faecal cortisol metabolites (FCM) allows conclusions to be drawn about the stress level in the respective housing system. The objective of this study is to find out how the cortisol metabolites content in the faecal changes with increasing age of the mink under semi-natural housing conditions. Thus, 40 American mink (Neovison vison) were housed in two outdoor enclosures imitating natural conditions. Throughout the entire study (13th to 32nd week of life), faecal samples were collected to measure cortisol metabolites. No differences in FCM concentrations between the two outdoor enclosures were found. In the young mink lower, less fluctuating FCM levels were found than in older animals. After the first faecal collection in the 13th/14th week of life, the level of metabolites decreased slightly (p = 0.032; 17th/18th week). From the 22nd/23rd week onwards until the 30th/31st week, shortly before the animals were pelted, continuously increasing concentrations were then measured. Increasing FCM levels with advancing age of the animals are probably attributable to the onset of sexual maturity and/or the respective season. This has to be taken into account in future studies using this method for assessing welfare and when comparing different mink housing systems.

The effect of ACTH upon faecal glucocorticoid excretion in the koala

Environmental changes result in physiological responses of organisms, which can adversely affect population dynamics and reduce resistance to disease. These changes are expressed in chronic levels of stress. The measurement of glucocorticoid (GC) concentrations in faeces is a non-invasive method for monitoring stress in wildlife. The metabolism and excretion of steroids differ significantly between species and, as a consequence, non-invasive methods must be physiologically validated for each species. Koalas (Phascolarctos cinereus) are declining in numbers through much of their range. The role of chronic stress in koala populations has not been identified. Prior to the assessment of faecal GC concentrations in wild koala populations, the excretion timing and concentrations of GCs need to be determined. In this study, we assessed a method for identifying and measuring the concentrations of GC metabolites in faecal pellets of captive koalas following ACTH treatment. The results show that an elevation of plasma cortisol concentrations, using sustained release of ACTH, results in elevated concentrations of faecal cortisol/cortisol metabolites. Taking into account the excretion time lag, an increase in faecal cortisol metabolite concentrations corresponds to the release of GCs from the adrenal cortex as early as 36 h before faecal pellet collection. The calculations of steroid partitioning of plasma cortisol showed that the ACTH-stimulated values were significantly different from the control values for the concentrations of free, corticosteroid-binding globulin-bound and albumin-bound cortisol. This study validates the use of faecal cortisol analysis to assess the activity of the hypothalamo-pituitary-adrenocortical axis in freshly collected koala faecal pellets and indicates that the method should be suitable to assess the adrenocortical status of koalas in wild populations.

Providing ‘get-away bunks’ and other enrichments to primiparous adult female mink improves their reproductive productivity

This study investigated whether simple, cheap enrichments – ‘get-away bunks’ (a wire mesh semi-cylinder attached to the cage ceiling) and small manipulable objects (balls and suspended chewing items) – could improve welfare and productivity in nursing mink dams (Neovison vison) in commercial farm conditions in southern Ontario (Canada). Experiment 1 replicated a study conducted the previous whelping season on the same farm. It evaluated whether providing bunks to multiparous dams (n=164) could decrease their morbidity and mortality or boost kit weaning weight, and assessed dams’ overnight use of these structures to compare day and night utilization. Overnight bunk-use proved to be no different from daytime use, and night- and day-use co-varied; bunk use recorded in daylight is thus a good proxy for overall use. Bunk use did not, however, influence kit weights at weaning, nor reduce dam deaths from nursing sickness (replicating the previous year's findings), nor significantly improve subjectively scored teat health (unlike the previous year's findings). Experiment 2 reassessed bunk effects using larger sample sizes, and investigated their interaction with enrichment objects. Focusing on primiparous dams (n=318), it evaluated whether providing balls and items to chew, along with bunks in a cross-factored design, could decrease stereotypic behaviour, glucocorticoid output (assessed via faecal cortisol metabolites: FCM), kit losses, and, again dam mortality. Objects were provided c. 10 months earlier (since the previous July) for approximately 60% of the object-enriched dams, and c. 5 months earlier (since January) for the remaining 40%. Analyses showed that effects of bunks and enrichment-objects did not significantly interact for any variable. Bunks significantly reduced kit mortality: kit losses/litter were reduced by c. 0.3 infants, resulting in negligible levels of mortality, and bunks tended to reduce dam stereotypy levels by about half (from approximately 12.5% to 6% of time spent active). However, bunks had no significant effects on FCM or dam mortality rates. Bunk-use also significantly co-varied with litter size, being greatest in dams with bigger litters. Enrichment objects tended to increase weaning litter size, an effect caused by dams provided with these objects for 5 months weaning 0.9 more kits per litter than females without these items. However, this type of enrichment again had no significant effects on FCM or dam mortality. Instead, which farm animals lived on appeared to be the major determinant of FCM and dam mortality: both significantly varied between farms, with one farm showing notably higher levels of both. Also, even controlling for farm, females who died tended to have had elevated glucocorticoids when sampled 2–3 weeks prior to death. In conclusion, bunks and manipulable enrichment objects seem ineffective against nursing sickness, but had independent, additive effects on the productivity of young adult females, possibly acting by improving primiparous dams’ welfare.

Novelty exploration, baseline cortisol level and fur-chewing in farm mink with different intensities of stereotypic behaviour

The present study aimed to examine the extent to which abnormal behaviours, stereotypic behaviour and fur-chewing, commonly used indicators of reduced welfare, are interrelated and linked to other welfare indicators in mink. Three groups were used based on behavioural observations, mink with no stereotypic behaviour (NST), mink performing stereotypic behaviour with only few repetitions (up to 4; ST1) and mink performing stereotypic behaviour with few (up to 4) and more repetetions (5 and above), in their repertoire ST2. Indicators of welfare included stereotypic behaviour observations, fur-chewing evaluation, baseline cortisol (faecal cortisol metabolites, FCM), and approach/avoidance behaviour in a novel object test and a stick test. There was no significant relation between the performance of stereotypic behaviour and fur-chewing. We found a higher level of FCM in the ST1 group with no tail-chewing behaviour and in tail-chewing mink in general, indicative of stress. However, unexpectedly tail-chewing mink were more explorative towards novel objects. The results supports that stereotypic behaviour and fur-chewing in mink are elicited by different environmental factors, suggested to be related to foraging and under-stimulation respectively.

Effect of the duration of road transport on the physiology and meat quality of lambs

To assess the effect of transport duration on animal welfare and meat quality of lambs, two trials were performed: Forty Ile-de-France × Merino lambs were used in Trial 1 and 40 Comisana lambs in Trial 2. In both trials, the lambs, aged between 14 and 16 weeks, were divided into two groups of 20 animals. One group was subjected to a 1-h period of transportation (T1) and the other to a 24-h period of transportation (T24), both in the same truck and arriving to the same slaughterhouse at the same time. The effect of transport on serum biochemistry variables (cortisol, aspartate transaminase, lactate dehydrogenase, blood urea nitrogen, creatine kinase, creatinine and total proteins), salivary cortisol, metabolites of cortisol in faeces, intra-ruminal temperature and meat quality (pH, conductivity, expressible juice, colour and shear force) was assessed. In both studies, the duration of transport did not affect serum and salivary cortisol concentration (P &gt; 0.05). However, in Trial 2, lambs exposed to 24-h transport had a higher concentration of faecal cortisol metabolites than did those transported for 1 h (P &lt; 0.05). Blood variables were not affected by transport in either trial (P &gt; 0.05), with the exception of blood urea nitrogen which was higher in Trial 1 for the T24 group (P &lt; 0.05) than it was to T1 group. Although no signs of dehydration were found, intra-ruminal loggers showed that animals did not drink during the transportation in the way they did before transport. In Trial 1, no effect of transport duration was found on meat-quality traits (P &gt; 0.05). Nevertheless, in Trial 2, lambs exposed to 24-h transport had higher values of colour attribute of a* (red trend) and less tenderness or higher values of shear force (P &lt; 0.05). The present study showed that although there is little effect on meat quality, signs of stress are detectable in lambs transported for 24 h. Therefore, in the case of lambs, the effect of long transportation periods must be considered more in terms of animal welfare than in terms of product quality.

Use of infrared thermography to assess the influence of high environmental temperature on rabbits

The aim of this work was to ascertain if infrared thermography (IRT) can be used on rabbits to assess differences in surface body temperature when they are subjected to two different environmental temperatures outside the comfort zone. Rabbits housed in room A were maintained at a temperature of below 30°C and rabbits in room B at a temperature of above 32°C for a year. Faeces were collected six times during the year to assess stress by means of faecal cortisol metabolites (FCM). The assessment of IRT was carried out to assess maximum and minimum temperatures on the eyes, nose and ears. FCM concentration was higher in room B than A, to confirm that stress conditions were higher in room B. Significant differences in IRT were found between the animals housed in both rooms. It was observed that it was more difficult for animals from room B to maintain a regular heat loss. Although all the body zones used to assess temperature with IRT gave statistical differences, the correlations found between the eyes, nose and ears were moderate, suggesting that they were giving different information. In addition, differences up to 3.36°C were found in the eye temperature of rabbits housed in the same room, with a clear effect of their position in relation to extractors and heating equipments. Therefore, IRT could be a good tool to assess heat stress in animals housed on typical rabbit farm buildings, giving a measure of how the animal is perceiving a combination of humidity, temperature and ventilation. Some face areas were better for analysing images. Minimum temperature on eyes and temperatures on nose are suggested to assess heat losses and critical areas of the farm for heat stress in rabbits.

On the relationships between glucocorticoids and feed efficiency in beef cattle

Improvement in the utilization of feed in livestock is an important target of breeding and nutritional programs. Recent evidence indicates a potential association between feed efficiency and fecal cortisol metabolites, which could eventually be used as an indirect assessment of this trait. This evidence is more comprehensively evaluated in here with samples for plasma cortisol (PC; ng/ml) and fecal cortisol metabolites (FCM; ng/ml) collected more often during the entire finishing phase in beef steers. Individual daily feed intake of 112 steers fed a high-moisture corn-based and haylage diet was measured over 168d. Body weight, blood and fecal samples were collected every 14d and ultrasound measures of backfat thickness and longissimus muscle area were taken every 28d. Four productive performance traits were calculated: daily dry matter intake (DMI), average daily gain (ADG), feed to gain ratio (F:G) and residual feed intake (RFI). At the end of the feedlot phase, steers were ranked according to RFI and samples were analyzed for PC and FCM from the 32 steers with greatest and 32 steers with lowest feed efficiency. In addition, a sub-group of 12 steers from each of these two groups with divergent feed efficiency were subjected to hourly blood sampling for 24h. Less efficient steers had greater DMI, F:G and consumed 1.5kg/d more DMI (P<0.05) than steers with improved feed efficiency. No differences (P>0.10) in PC over the 12 biweekly sampling periods between steers with divergent feed efficiency were observed. However, a trend toward significance between 19:00 and 02:00h over the hourly sampling evaluation was noticed, with the sub-group of more feed efficient steers presenting higher levels of PC in this period of the day (P=0.08). On the other hand, FCM levels displayed a distinct pattern between RFI groups over the biweekly sampling period, with more efficient cattle presenting greater levels of these metabolites (P<0.05). This study reinforces the positive association between improved feed efficiency and FCM levels over the finishing phase; and the lack of association between feed efficiency and PC when single samples are collected every two weeks through a single jugular venipuncture performed after handling the cattle for sampling. Further studies to develop sampling protocols for assessing FCM as an indicator trait for feed efficiency are warranted, as well as, studies to understand the role of endogenous glucocorticoids in the performance of the bovine.

Behavioural and physiological reactions of goats confronted with an unfamiliar group either when alone or with two peers

When introduced into a new herd, goats are confronted with unfamiliar animals. Their behavioural and physiological reactions during this confrontation are likely to differ depending on the presence or absence of familiar conspecifics (peers). To assess these reactions, we confronted 12 goats both alone and with two peers (confrontees) with established groups (n=4 groups) consisting of goats unfamiliar to the confrontee (unfamiliar goats) (12 goats×2 confrontations=24 confrontations in total). Each confrontation lasted for one hour. Agonistic interactions, sniffing behaviour and level of activity were recorded throughout the confrontations. In addition, concentrations of cortisol metabolites were measured in faecal samples taken in the evening before the confrontation and three successive samples after the confrontation. Before the start of the experiment, we evaluated the dominance relationships of the involved goats within their respective housing groups by direct observations made during the main feeding times. Data were analysed using generalised linear mixed-effects models with the fixed effects presence of peers (yes, no), rank category (high, medium, low) and repeated confrontation (numeric variable). For the analysis of activity level and concentrations of faecal cortisol metabolites, period (minutes 0–15, 16–30, 31–45, 46–60) and sample (control, 13, 14, 15h after the confrontation), respectively, were included as additional fixed effects. Unfamiliar goats directed fewer agonistic interactions towards confrontees when the latter were accompanied by peers compared to when they were alone (without peers: 57; with peers: 20 interactions per animal and confrontation). The same was true for the proportion of agonistic interactions involving physical contact (without peers: 69; with peers: 53%) and the number of sniffing behaviours (without peers: 16; with peers: 9 interactions per animal and confrontation). On the other hand, confrontees with peers were more likely to direct agonistic and sniffing behaviour towards unfamiliar goats than those on their own. Confrontees with peers had lower concentrations of faecal cortisol metabolites after confrontations (without peers: 273; with peers: 198ng/g). For confrontees (with and without peers), activity level was highest during the first 15min of the confrontation and decreased over its course. For the unfamiliar goats, the activity pattern was similar but was modulated by rank, with higher values for low-ranking goats than for medium- and high-ranking ones. In conclusion, our results indicate that the presence of peers is advantageous for goats being introduced into groups of unfamiliar goats.

Non-invasive evaluation of physiological stress in an iconic Australian marsupial: The Koala (Phascolarctos cinereus)

Koalas (Phascolarctos cinereus) are the only extant representatives of Australia’s unique marsupial family Phascolarctidae and were listed as nationally Vulnerable in 2012. Causes of mortality are diverse, although the disease chlamydiosis, dog attacks, collisions with cars, and loss of habitat represent the principal reasons for the continued species decline. Koala breeding facilities in Queensland and New South Wales, Australia have been established for conservation and tourism. Non-invasive monitoring of physiological stress is important for determining the sub-lethal effects of environmental stressors on the well-being, reproduction and survival of Koalas in Zoos and also in the wild. In this study, we developed a faecal cortisol metabolite (FCM) enzyme-immunoassay (EIA) for monitoring physiological stress in Koalas from two established Zoos in Australia and also within a free-living sub-population from Queensland. Biological validation of the FCM EIA was done using an adrenocorticotropic hormone (ACTH) challenge. We discovered excretory lag-times of FCM of 24h in females (n=2) and 48h in male (n=2) Koalas in response to the ACTH challenge. FCM levels showed an episodic and delayed peak response lasting up to 9days post ACTH challenge. This finding should be taken into consideration when designing future experiments to study the impacts of short-term (acute) and chronic stressors on the Koalas. Laboratory validations were done using parallelism and recovery checks (extraction efficiency) of the cortisol standard against pooled Koala faecal extracts. Greater than 99% recovery of the cortisol standard was obtained as well as a parallel displacement curve against Koala faecal extracts. FCM levels of the captive Koalas (n=10 males and 13 females) significantly differed by sex, reproductive condition (lactating versus non-lactating Koalas) and the handling groups. Handled male Koalas had 200% higher FCM levels than their non-handled counterparts, while females were not affected by handling as long they were not undergoing lactation. There was no significant difference in FCM levels between the captive and wild Koalas (n=9 males and 7 females). Overall, these results provide foundation knowledge on non-invasive FCM analysis in this iconic Australian marsupial. Non-invasive stress endocrinology opens up opportunities for evaluating the sub-lethal physiological effects of management activities (including caging, translocation) on the nutritional status, reproductive behaviors and disease status of captive and managed in situ Koala populations.

Fecal cortisol levels predict breeding but not survival of females in the short-lived rodent, Octodon degus

The cort-adaptation hypothesis indicates that an association between glucocorticoid (cort) levels and fitness may vary with the extent to which reproduction or breeding effort is a major determinant of cort levels. Support for a context dependent association between cort and fitness comes mostly from relatively long-lived, bird species. We tested the hypothesis that there are gender and context (life-history) specific cort-fitness relationships in degus, a short-lived and generally semelparous social rodent. In particular, we used demographical records on a natural population to estimate adult survival through seasons and years and linked that to records of baseline cort (based on fecal cortisol metabolites). We found no evidence for a direct relationship between baseline cort and adult survival across seasons, and this lack of association was recorded irrespective of sex and life history stage. Yet, cort levels during early lactation predicted the probability that females produce a second litter during the same breeding season, supporting a connection between baseline cort levels and breeding effort. Overall, the differential effects of cort on survival and breeding supported that the extent of cort-fitness relationships depends on the fitness component examined.

Assessment of adrenocortical activity by non-invasive measurement of faecal cortisol metabolites in dromedary camels (Camelus dromedarius)

The aim of this study was to determine whether glucocorticoid production could be monitored non-invasively in dromedary camels by measuring faecal cortisol metabolites (FCMs). Five Sudanese dromedaries, two males and three females, were injected with a synthetic adrenocorticotropic hormone (ACTH) analogue. Blood samples were collected pre- and post-ACTH injection. Faeces were sampled after spontaneous defecation for five consecutive days (2 days before and 3 days after ACTH injection). Baseline plasma cortisol values ranged from 0.6 to 10.8 ng/ml in males and from 1.1 to 16.6 ng/ml in females, while peak values after ACTH injection were 10.9-41.9 in males and 10-42.2 ng/ml in females. Peak blood cortisol values were reached between 1.5 and 2.0 h after ACTH injection. The concentration of FCMs increased after ACTH injection in the faeces of both sexes, although steroid levels peaked earlier in males [24 h; (286.7-2,559.7 ng/g faeces)] than in females [36-48 h; (1,182.6-5,169.1 ng/g faeces)], reflecting increases of 3.1-8.3- and 4.3-8-fold above baseline levels. To detect chromatographic patterns of immunoreactive FCMs, faecal samples with high FCM concentrations from both sexes were pooled and subjected to reverse phase high performance liquid chromatography (RP-HPLC). RP-HPLC analysis revealed sex differences in the polarity of FCMs, with females showing more polar FCMs than males. We concluded that stimulation of adrenocortical activity by ACTH injection resulted in a measurable increase in blood cortisol that was reliably paralleled by increases in FCM levels. Thus, measurement of FCMs is a powerful tool for monitoring the adrenocortical responses of dromedaries to stressors in field conditions.

Sleeping tight or hiding in fright? The welfare implications of different subtypes of inactivity in mink

Effects of sub-optimal housing on inactivity vary across species and experiments, probably because inactivity is heterogeneous, reflecting both positive states (e.g. relaxation) and negative ones (e.g. fear). We therefore aimed to identify specific subtypes of inactivity that could indicate poor welfare in mink, by comparing their behaviour in enriched and non-enriched conditions (the former having been previously demonstrated to be highly preferred by mink and to enhance their welfare). We assessed this in three groups of subjects, as well as after housing conditions were reversed for the last group. During live scans, inactive animals were scored for posture, location, and whether awake or apparently asleep. Data on temperament and physiological stress indicators were also collected for one group; these confirmed that non-enriched housing increased faecal cortisol metabolites (FCM; P=0.040). Non-enriched housing also increased locomotor stereotypy in females (sex×housing: P=0.004). Inactivity in the nest-box (vs. in the open cage) was higher among females in non-enriched housing (housing×sex: P<0.001), and increased by 20% of observations after enrichment removal (P=0.018) for both sexes. Furthermore, males with fearful temperaments spent the most time inactive in the nest-box (sex×temperament: P=0.054), while females whose FCM decreased most when given enrichment also showed the largest decreases in this behaviour (sex×FCM change: P=0.019). Together, this suggests that inactivity in the nest-box may reflect anxiety-induced hiding. Lying awake (i.e. prone with eyes open) was also higher in non-enriched housing (3.1% of observations vs. 1.7%; P=0.002); furthermore, this subtype of inactivity increased after enrichment removal (by 1.0% of observations; P=0.021), and decreased when non-enriched mink were given enrichment (by 2.4% of observations; P=0.004). This behaviour did not co-vary with fearfulness, however, nor with FCM (both P>0.05). This suggests that lying awake is not fear-related (e.g. not reflecting enhanced vigilance) but instead reflects some other negative state. Effects on inactivity subtypes as defined by posture were less consistent. For example, time spent lying belly down tended to decrease in mink moved from non-enriched to enriched cages (P=0.054), but enriched mink spent significantly less time belly down (in one of the three groups; P=0.002). Overall, two subtypes of inactivity, lying in the nest-box and lying awake seem likely to be valid indicators of housing-induced poor welfare in this species, being consistently increased by non-enriched cages. Lying in the nest-box may indicate fear or anxiety, and lying awake, a boredom-like state.