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There is currently a poor understanding of the impact of chronic stress on the behaviour and physiology of beef cattle. In monogastrics, hormones released in response to stress can have deleterious effects on the balance of the microbiota present in the gut, which can last long after stress hormone levels have returned to normal. If comparable changes occur in the rumen microbiota, these could have important consequences for ruminal fermentation and digestibility, leading to suboptimal use of nutrients and increased methane emissions. However, there is little information regarding the effects of stress on the rumen microbiome. The overall aim of this thesis was to understand how commercially relevant stressors affect ruminal microbial populations, as well as concurrent effects on behaviour, feed efficiency and methane emissions in beef cattle. The first objective was to assess the direct contribution of glucocorticoids such as cortisol in mediating the effect of stress on the microflora and feed efficiency. Dexamethasone, a potent synthetic cortisol analogue, was used to evaluate the effect of an exogenous glucocorticoid on feed efficiency and the rumen microbial populations. This treatment was applied to 516 ± 50-day old Limousin cross steers selected based on extremes of feed efficiency, to contrast the effects on ruminal microbial communities of more efficient and less efficient animals. Animals in the treatment group (n=24) were injected with 0.05 mg/kg dexamethasone intramuscularly for 3 consecutive days, while matching extremes of feed efficiency controls (n=16) were treated with the equivalent volume of saline solution (Control group). The effect of dexamethasone was assessed on faecal cortisol metabolites, feeding behaviour, locomotor activity, as well as metagenomic information obtained from 16S rRNA gene sequencing of rumen fluid samples. Treatment with exogenous glucocorticoid dexamethasone induced transient changes in activity and faecal cortisol. Nonetheless, this glucocorticoid did not cause any significant changes in the rumen archaea population or microbial diversity, an indication that ruminal microbial populations might be resilient to the direct effects of exogenous glucocorticoids. The second objective was to quantify the behavioural and physiological responses to a putative composite chronic stressor treatment, by applying a series of commercially relevant stressors and assessing any changes in behaviour and HPA axis responsiveness. The commercially relevant stressors used were reduced space allowance, in addition to being subjected every week to regrouping, transport and a short period of isolation. Limousin (n=32) and Angus (n=32) crossbred beef steers, 400 ±13 days old at the beginning of the trial, were assigned in a balanced way to a composite stressor (S) or control (C) treatment, each treatment with four replicate groups. Blood samples and faecal samples were collected to measure cortisol levels. An ACTH challenge was performed using 0.5ug/kg of Synacthen Depot® with blood samples taken just before, 30 min post and 60 min post ACTH challenge to assess changes in HPA axis responsiveness on a subsample of animals (S=22; C=19). Behaviour was assessed from activity monitors, feed intake, an attention bias test, video observations of agonistic behaviour at the feeders and affiliative behaviour (rubbing and licking) in the home pen. Results showed differences between treatments in some parameters of activity and an attention bias test. However, there were no effects of treatment on agonistic or affiliative behaviour. Cortisol responses, although different between groups, could not be specifically attributed to the composite stressor treatment and the ACTH challenge employed did not detect any significant differences in adrenal sensitivity between treatments. Making use of metagenomic information obtained from 16S rRNA gene sequencing of rumen fluid samples taken during the composite stressor experiment, the last objective of this project assessed changes in the rumen microbiota in response to the applied stressors. Although there was a particular interest in the methanogenic populations, analyses also looked at effects on productivity and methane emissions. To this end a small cohort of 12 animals (six from each C and S treatments) was used to assess any effects of the composite stressor treatment on methane emissions. Although changes were detected in some microbial genera throughout the experiment, there were no major changes in the rumen archaea population or microbial diversity directly associated with the composite stressor treatment. Additionally, by the end of the experiment, there was no effect of stress on growth performance or methane emissions. In conclusion, circulating glucocorticoids do not appear to affect the rumen microbiota balance directly, although it is possible that they may affect microbial communities in other sites of the gastrointestinal tract. Some differences in behaviour but not cortisol were found in response to the composite stressor treatment, suggesting that beef cattle might be resilient to repeated but predictable stressors. The stressor regime applied did not cause substantial changes in rumen microbial diversity or methanogenic archaea populations. This thesis was a first step towards enhancing our understanding of the dynamics of the rumen microbiome in response to stress. Further research needs to examine more closely the links between biological changes in response to severe chronic stress and microbiota resilience in the rumen and other sites of the gastrointestinal tract.

The study of the endocrine status can be useful to understand wildlife responses to the changing environment. Here, we validated an enzyme immunoassay (EIA) to non-invasively monitor adrenocortical activity by measuring fecal corticosterone metabolites (FCM) in three sympatric gerbil species (Gerbillus andersoni, G. gerbillus and G. pyramidum) from the Northwestern Negev Desert&rsquo, s sands (Israel). Animals included into treatment groups were injected with adrenocorticotropic hormone (ACTH) to stimulate adrenocortical activity, while control groups received a saline solution. Feces were collected at different intervals and FCM were quantified by an EIA. Basal FCM levels were similar in the three species. The ACTH effect was evidenced, but the time of FCM peak concentrations appearance differed between the species (6&ndash, 24 h post-injection). Furthermore, FCM peak values were observed sooner in G. andersoni females than in males (6 h and 18 h post-injection, respectively). G. andersoni and G. gerbillus males in control groups also increased FCM levels (18 h and 48 h post-injection, respectively). Despite the small sample sizes, our results confirmed the EIA suitability for analyzing FCM in these species as a reliable indicator of the adrenocortical activity. This study also revealed that close species, and individuals within a species, can respond differently to the same stressor.

Examination of the endocrine system through non-invasive fecal sampling may improve population management more than using demographic indicators alone. By addressing the physiological mechanisms that are influencing fitness, management actions can be proactively developed to alleviate stressors. Proactive determination of vulnerable populations is critical for species of concern, such as the Northern Bobwhite (Colinus virginianus), which have suffered decades of population decline. We validated an assay to noninvasively measure the adrenocortical response of captive reared bobwhite through fecal corticosterone metabolites (FCM). All individuals received three sequential 48-hour treatments in which samples were collected every 4 hours, including a reference period, an adrenocorticotropic hormone (ACTH) challenge and a biological stressor (exposure to a hunting dog). Reference FCM values had a mean concentration of 16.75 pg/mg (95% CrI: 13.68, 19.91) with adrenocortical activity increasing by 73% for the duration of the ACTH challenge (29.00 pg/mg; CrI: 25.01, 33.78). FCM concentrations remained similar to that of the reference levels during the biological stressor (16.56 pg/mg; CrI: 13.33, 19.92). Our study validates the use of feces to detect changes in FCM levels in our subject species but also demonstrates the complexity of FCM and the importance of both physiological and biological validation prior to field implementation.

The non-invasive monitoring of physiological stress can provide conservation and wildlife managers with an invaluable tool for assessing animal welfare and psychological health of captive and free-ranging populations. A significant decrease in free-ranging primate populations globally and an increase in captive-housed primates have led to a need to monitor the stress and general welfare of these animals. We examined the suitability of three enzyme immunoassays (EIAs) for monitoring stress-related physiological responses in the samango monkey, Cercopithecus albogularis erythrarchus. We conducted an adrenocorticotropic hormone (ACTH) challenge on a male and female at the National Zoological Garden, Pretoria, South Africa. Individual faecal samples were collected 8 days pre- and post-ACTH administration and subsequently analysed for faecal glucocorticoid metabolite (fGCM) concentrations. During the study, biological stressors occurred for both the male and female. Two of the three EIAs tested (11-oxoetiocholanolone I and II) were able to reliably monitor fGCM alterations throughout the study period in both sexes. The 11-oxoetiocholanolone I EIA, however, had the lowest mean deviation from the calculated baseline value and was thus chosen as the preferred assay. Both the physiological activation of the stress response and the biological response to a stressor could be monitored with the chosen assay. The successful establishment of a reliable, non-invasive method for monitoring adrenocortical activity in C. albogularis erythrarchus will now allow conservationists, scientific researchers and wildlife managers to evaluate the level of stress experienced, and general welfare, by animals in captivity as well as free-ranging populations.

Reliable methods to measure stress-related glucocorticoid responses in free-ranging animals are important for wildlife management and conservation. Such methods are also paramount for our ability to improve our knowledge of the ecological consequences of physiological processes. The brown bear (Ursus arctos) is a large carnivore of ecological and cultural importance and is important for management. Here, we provide a physiological validation for an enzyme immunoassay (EIA) to quantify glucocorticoid metabolites in brown bear feces. We also provide an evaluation of the effects of sample exposure to ambient temperature on measured fecal glucocorticoid metabolite (fGCM) concentrations. We evaluated three EIA systems: a cortisol assay, an 11-oxoetiocholanolone assay, and an 11β-hydroxyetiocholanolone assay. Of these, the cortisol assay provided the best discrimination between peak fGCM concentrations detected 1–4 d after injections of synthetic adrenocorticotrophic hormone and preinjection baseline concentrations in four individual brown bears. The time of exposure to ambient temperature had substantial but variable effects on measured fGCM concentrations, including variation both between samples from the same individual and among samples from different bears. We propose that the validated EIA system for measuring fGCM concentrations in the brown bear could be a useful noninvasive method to monitor stress in this species. However, we highlight that this method requires that fecal samples be frozen immediately after defecation, which could be a limitation in many field situations., This research project has been partially funded by FOA, Fundación Biodiversidad, and the Servicio de Medio Natural, Consejería de Desarrollo Rural, Agroganadería y Pesca del Principado de Asturias to M.M.D. In addition, F.D. was financially supported by a Spanish Ramon y Cajal grant funded by the Spanish Ministry of Economy and Competitiveness (RYC-2013-16263); V.P. was supported by a Grupos de Investigación (GRUPIN) research grant from the Regional Government of Asturias (IDI/2018/000151); andV.P., A.O., and R.G.G. were financially supported by an Excellence Project financed by the Spanish Ministry of Science, Innovation and Universities, the Agencia Estatal de Investigación, and the Fondo Europeo de Desarrollo Regional (EU, CGL2017- 82782-P).

Greyhounds are routinely bred and managed for racing and are usually housed in individual kennels. To address the question of whether greyhounds would benefit from an increase in the minimum kennel size specified in the Victorian Code of Practice, the effects of kennel size and routine exercise on dog behaviour, stress physiology and injuries were examined. A total of 36 healthy greyhounds (aged 17 months (replicate 1 and 2) and 12 months (replicate 3)) were studied in three time replicates in a 2 × 2factorial experiment (kennel size of 3 m2 and 10 m2 of floor space, and two levels of exercise (‘no exercise’ and ‘exercise’(consisting of 2 × 10 min per day and 3 times per week chasing a lure)) over 6 weeks. During the 1st and 5th week of treatment, dog behaviours were recorded for 2 days in their kennels using time-lapse video. During the 6th week, six saliva samples were collected to measure basal cortisol concentrations, an ACTH challenge test was performed and injuries were assessed. Intensity of exercise had no significant (p ≫> 0.05) effect on behaviour, stress physiology and injuries. In the 1st week the adult dogs in large kennels spent more time in the front of the kennels (p ≪ 0.05), less time in the back of the kennels (p ≪ 0.01), less time lying (p ≪ 0.05) and more time standing (p ≪ 0.01) than those adult dogs in the small kennels. In week 5, the younger dogs in the large kennels spent more time at the front of the kennel (p ≪ 0.05), less time in the back (p ≪ 0.01) and spent more of their lying down time at the front and middle of the kennel (p ≪ 0.001) than dogs in the small kennels. No significant differences in any of the measurements were found in the adult dogs in week 5. No abnormal or stereotypic behaviour was observed in Week 1 or 5. No significant differences in injuries, saliva cortisol concentrations and maximum cortisol concentrations in response to ACTH were found. The results of this study do not provide evidence that the smaller kennels and limited exercise increase the risks to dog welfare based on behaviour, stress physiology and injuries. Nevertheless, the observation that dogs tended to spend more time in the front of the large kennels where they were in view of other dogs could indicate that larger kennels may enable better welfare by providing additional social contact.

Amphibians are experiencing drastic population declines. New semi-invasive conservation methods are required to protect threatened species. The collection of mucus, from dorsal and ventral body regions, is an ideal matrix for monitoring stress hormones, and thus physiological stress, in African amphibians. This technique will assist in amphibian conservation in Africa., The complex interaction between factors leading to amphibian declines is responsible for the inability to develop robust, standardized conservation action plans. Monitoring physiological stress responses in amphibians may provide an ideal tool to assist conservationists in this regard. This study aimed to validate dermal secretions as a robust matrix for monitoring glucocorticoid alterations in an African amphibian, the edible bullfrog (Pyxicephalus edulis). Both a biological (handling) and physiological (ACTH challenge) validation were conducted to determine which of five available enzyme immunoassays (EIAs) tested is most suited for monitoring alterations in dermal glucocorticoid (dGC) concentrations. Additionally, the most optimal body region for monitoring dGC concentrations in P. edulis was identified. To confirm the activation of the hypothalamic–pituitary–interrenal (HPI) axis following ACTH administration, urinary glucocorticoid metabolites (uGCM) were also quantified. The tested corticosterone EIA was the only assay able to monitor alterations in dGC concentrations following the handling event in P. edulis. Further validation during the ACTH challenge supported the corticosterone EIA in this regard. Dermal secretions collected from both the ventral and dorsal regions were suitable for monitoring dGC concentrations in both sexes. A considerable increase in uGCM concentrations following ACTH administration was found, suggesting that the dGC concentrations observed were derived from the HPI axis. This study offers further support for the use of dermal secretions as an important matrix for monitoring physiological stress, and thus general health, in amphibian species.

We studied the adrenocortical and vagal tone responses to a single ACTH challenge in lame (n = 9) vs nonlame (n = 9) dairy cows. Cows were paired according to parity, days in milk, and milk yield. Plasma cortisol and dehydroepiandrosterone concentrations and cardiac vagal tone response (high-frequency component of heart rate variability) were compared after intravenous ACTH administration. Baseline, minimum or maximum, amplitude of the response and area under the response curve were compared. No difference was detected between groups in the cortisol response. Dehydroepiandrosterone was irresponsive to ACTH treatment, and concentrations did not differ between lame and nonlame cows. Vagal tone decreased in response to the ACTH treatment. High frequency component of heart rate variability was lower in the lame group at all sampling times. Lameness was associated with delayed return to baseline. We concluded that the adrenal response capacity is not influenced by lameness, which supports the concept of lameness being a chronic intermittent rather than a chronically persistent stressor. Dehydroepiandrosterone concentrations were not proven to be useful indicators of hypothalamus–pituitary axis dysfunction in cattle. A decreased vagal contribution to heart rate variability—possibly coupled with increased sympathetic modulation—was observed in lame cows, which suggests that lameness affects the mechanisms underlying the action of ACTH on cardiovascular activity.

Using faecal matter to monitor stress levels in animals non-invasively is a powerful technique for elucidating the effects of biotic and abiotic stressors on free-living animals. To validate the use of droppings for measuring stress in southern pied babblers (Turdoides bicolor) we performed an ACTH challenge on captive individuals and determined the effect of temporary separation from their social group on their faecal glucocorticoid metabolite (fGCM) concentration. Additionally, we compared fGCM concentrations of captive babblers to those of wild conspecifics and examined the effects of dominance rank on fGCM concentration. We found droppings to be a suitable matrix for measuring physiological stress in babblers and that individual separation from the group caused an increase in fGCM levels. In addition, babblers temporarily held in captivity had substantially higher fGCM concentrations than wild individuals, indicating that babblers kept in captivity experience high levels of stress. In wild, free-living individuals, dominant males showed the highest levels of stress, suggesting that being the dominant male of a highly territorial social group is stressful. Non-invasive sampling allows field-based researchers to reduce disturbance related to monitoring adrenocortical function, thereby avoiding artificially increasing circulating corticosterone concentration as it is not necessary to physically restrain study animals.

Adrenocorticotropic hormone (ACTH) challenges are frequently performed in repetition when evaluating stress or welfare in animals. To our knowledge, the repeatability of ACTH challenges in cattle, although fundamental to further studies of this type, has not yet been the subject of research. Therefore, the objective of this study was to evaluate the repeatability of ACTH challenges in fattening bulls of different horn status. Eight-one bulls were subjected to 3 consecutive ACTH challenges. The first challenge (C1) was performed in calves aged 1.5 mo. Subsequently, animals were characterized as high or low cortisol responders and either disbudded or left with horns. They were then assigned to 1 of 3 rearing groups: a horned group (H+), a disbudded group (H−), and a mixed group (M; 50% horned and 50% disbudded), with each group containing an equal number of high and low responders. The second ACTH challenge (C2) was performed at the age of 11 mo. Time of day (TOD) of challenge was either fixed (ST = same TOD) or alternated (AT = alternate TOD) between C1 and C2. The third ACTH challenge (C3) was performed 7 d after and at the same TOD as C2. Saliva samples were taken 60 and 30 min before and 30, 60, 90, 120, and 150 min after each intravenous ACTH injection. The area under the curve (AUC) was calculated with respect to both ground (AUCG) and to increase (AUCI). The AUCI increased markedly between C1 and C2 (P 0.1). The overall repeatability of AUCG and AUCI between C1 and C2, reflecting the repeatability between 2 different physiological states, was poor and moderate, respectively, for ST bulls (AUCG: r = 0.24, P > 0.1, intraclass correlation coefficient (ICC) = 0.21; AUCI: r = 0.48, P 0.1; ICC = 0.03; AUCI: r = 0.08, P > 0.1, ICC = 0.06). The repeatability of AUCG and AUCI between C2 and C3, reflecting the repeatability within the same physiological state, was moderate (AUCG: r = 0.59, P

Changes in ACTH challenge test characteristics in dairy cows changing their physiological status at different lactational stages and different feeding levels were not investigated in terms of repeatability yet. In 23 multiparous Holstein cows (10 cows fed a sole fresh herbage diet without concentrate, 13 cows fed with concentrate), three ACTH challenge tests were performed: once during pregnancy shortly prior to drying off (T1), and in week 3 (T2) and 8 (T3) after parturition. Test characteristics were correlated to performance and metabolic parameters: DMI, BW, energy balance (EB), plasma concentrations of free fatty acids (NEFA) and beta-hydroxybutyrate (BHB). Basal plasma cortisol concentrations were higher at T1 compared with T2 and T3 (p < .05). The adrenal cortex sensitivity (expressed as total AUC (AUCt ) of cortisol response after ACTH application) was lowest at T2 compared with T1 and T3 (p < .05). Ranking of the individual animals' responses was not repeatable between time points of the ACTH tests. Enhancing the energy deficiency during early lactation by omission of concentrate did not affect baseline cortisol concentrations in plasma, but decreased peak height at T2 (p < .05). Baseline plasma cortisol concentrations were positively correlated with cortisol peak values after ACTH application, previous lactation performance, milk yield and BW (p < .05). The AUCt was positively correlated with baseline cortisol concentrations, EB and DMI. Cortisol release after ACTH injection was lower in animals with high plasma concentrations of NEFA, BHB and with higher contents of fat and free fatty acids in milk (p < .05). Cortisol peak height after ACTH administration was higher in cows with a more positive EB, higher DMI and lower plasma concentrations of NEFA and BHB. In summary, cortisol responses to ACTH challenges in this study were not repeatable in dairy cows changing their physiological status.

Supplementary data to this article can be found online at https://doi.org/10.1016/j.physbeh.2018.07.019; Entire male pigs display more aggressive and sexual behaviour. This might cause a condition of chronic stress and impair their welfare. In order to assess chronic stress in entire and castrated male pigs, as well as effects of providing grass silage as occupational and feed material on behaviour and health, we carried out a 2 × 2 × 2-factorial experiment with 147 growing-finishing pigs. Factors investigated were castration (entire/castrated), chronic intermittent social stress exposure (yes/no) and access to grass silage (yes/no), as well as their interactions. The stress exposure treatment consisted of repeated short-term confrontations and separations. We recorded different behavioural variables, circadian rhythm of salivary cortisol, response to an ACTH (adrenocorticotropic hormone) challenge test, pathological changes in the gastric mucosa and morphology of the intestinal epithelium. Stress exposure caused a decrease in posture changes and head knocks/bites in the home pen. Reference indicators affected by stress exposure did not differ between entire and castrated male pigs, indicating that there is no permanently increased baseline level of stress in entire male pigs. However, entire males responded more pronouncedly to the stress exposure compared to castrated males in terms of posture changes and play behaviour. Pigs provided with grass silage showed more play behaviour and less manipulative behaviours than pigs not receiving grass silage. Stress treated pigs had more hyperkeratosis in the gastric mucosa and gastric ulcers, while offering grass silage reduced such changes. In conclusion, our results indicate that the increased behavioural stress response of entire male pigs might require some adaptations in housing and management of entire male pigs. Gastric ulceration scoring turned out to be a potential post mortem indicator for chronic stress. Finally, providing roughages like grass silage could be a means to positively affect behaviour and gastric health in pigs.

Cortisol levels reflect hypothalamic-pituitary-adrenocortical (HPA) axis activity. While most plasma cortisol is supposed to be bound to the soluble corticosteroid-binding globulin (CBG), only free cortisol (FC) actively regulates metabolic and immunological processes. We aimed to establish a multispecies suitable method to assess FC in cows and horses which in combination with total cortisol (TC) allows interpreting proportional changes of cortisol in saliva as well as in blood in response to a standardized HPA axis activation via ACTH. We further investigated if the ratios of cortisol fractions as obtained at basal levels in healthy horses (herbivorous and monogastric) and dairy cows (herbivorous and ruminant) change during HPA axis activation, and to which extent saliva cortisol (SC) is representative for alterations in plasma FC and adrenal cortex reactivity. However, it was not the objective of the present study to directly compare the two species. Dosages of ACTH applied in cows and horses were based on published data. Synthetic ACTH was intravenously administered to eight dairy cows (0.16 µg/kg BW) and five horses (1 µg/kg BW). Blood and saliva were collected every 30 min for 3 h from a jugular vein catheter, and analyzed for TC and SC, the ratio of free cortisol (rFC), and the concentration of FC (cFC) in plasma. During the entire sampling period of the ACTH test, plasma TC was paralleled by blood cFC, rFC, and SC in both cows and horses. All cortisol fractions increased within 30 min of ACTH administration compared to basal values (0 min, P < 0.05). Peak TC concentration reached 63.2 ± 9.6 ng/mL and 73.2 ± 11.8 ng/mL in bovine and equine plasma, respectively. Peak values of rFC averaged 17.9 ± 4.5% in cows and 19.2 ± 7.8% in horses. The ratio of SC to cFC in horses remained similar during the ACTH challenge suggesting that SC is recruited from plasma FC. However, SC increased less compared to plasma TC and FC during HPA axis activation in cows. In conclusion, the short-term activation of the HPA axis caused not only an elevation of TC, but also a similar increase of rFC in both species. SC closely reflected changes of FC in horses, but less accurately in cows. The concomitant evaluation of changes among cortisol fractions might give further indications on adaptation mechanisms in glucocorticoid regulation as well as differentiate cortisol-related health disorders.

The bank vole is a commonly used model species in behavioral and ecophysiological studies. Thus, presenting a validated method for noninvasive monitoring of corticosterone and testosterone secretion is of high relevance. Here, we evaluated the effect of time of day and an ACTH challenge test on measured fecal corticosterone (FCM) and testosterone (FTM) metabolites in both sexes. Furthermore, we performed radiometabolism experiments for both steroids and sexes to study metabolism and excretion of 3H-corticosterone and 3H-testosterone. FCM and FTM were analysed with a 5α-pregnane-3β,11β,21-triol-20-one enzyme immunoassay (EIA) and a testosterone (measuring 17β-hydroxyandrostanes) EIA, respectively. Males had significantly higher FCM levels than females and their main excretion route was via the feces (∼72%), whereas females excreted nearly equal portions in both feces and urine. For testosterone the main excretion route was via the feces in both sexes (∼80%). The time course of excretion was similar in both sexes, but for the first time a significant difference between injected steroids was found: Corticosterone was excreted faster than testosterone, both in urine (median of peak levels: 4 h vs 6 h) and feces (6 h vs 8 h). Several metabolites were present in the feces and the tested EIAs reacted with some of them. Time of day had a significant effect on measured fecal steroid metabolites. As expected, males had significantly higher FTM levels than females. ACTH administration significantly increased FCM values; peaks were observed 4–8 h after injection. In conclusion, both tested EIAs proved suited for a noninvasive measurement of glucocorticoids and androgens in bank voles. peerReviewed

Stress hormone levels are important indicator of an animal’s well-being, as stress has harmful effects on reproduction, growth and immune function. The development of enzyme immunoassays (EIA) to monitor faecal glucocorticoid metabolites (fGM) contributes a powerful tool to assess an animal’s adrenal status non-invasively. We aimed to identify a suitable EIA for monitoring fGM by assessing the suitability of six different EIAs for detecting quantitative changes in fGM concentrations in response to an ACTH challenge test in Eurasian lynx. FGM were characterised in a male Eurasian lynx that received an injection of 3H-cortisol. Using HPLC analyses radiolabeled metabolites were compared with immunoreactive metabolites. The second aim was to biologically validate the established EIA for monitoring adrenocortical activity of captive Iberian lynxes after a translocation to new enclosures in relation to behaviour. Additionally faecal samples of ten pregnant Iberian lynxes from the peripartal period were analysed. The ACTH challenge revealed an 11β-hydroxyetiocholanolone EIA as the most sensitive assay to reflect acute fGM elevations in the Eurasian lynx. HPLC immunograms demonstrated that the 11β-hydroxyetiocholanolone EIA measured significant amounts of immunoreactivities corresponding to radiolabeled metabolites with strong similarities across both lynx species. Additionally, HPLC and GC–MS analyses confirmed the presence of 11β-hydroxyetiocholanolone in faeces of both, the Eurasian and the Iberian lynx. Longitudinal fGM profiles of Iberian lynx revealed increases in concentrations associated with management events. During the peripartal period, however, fGM concentrations were not significantly elevated. Our results show that the 11β-hydroxyetiocholanolone EIA is a reliable tool to assess fGM in both lynx species.

Enzyme-immunoassays (EIA) that detect fecal testosterone metabolites (fTM) are powerful tools to monitor gonadal activity non-invasively. However, a challenge with testosterone EIAs might be their potential for cross-reactivities with structurally similar glucocorticoid metabolites. Therefore, we aimed to verify the capability of four different testosterone EIAs to monitor fTM without reflecting changes in adrenocortical activity in spotted hyenas by analyzing fecal samples following testosterone and ACTH challenge tests. We demonstrated that none of the testosterone EIAs is appropriate to measure fTM as all of them showed substantial cross-reactivities to unknown metabolites. Our study underlines the importance of validating androgen EIAs.

Up to the present no studies have been conducted either on baseline concentrations of adrenal hormones or on hormonal responses to stress in Greater rhea (Rhea americana) and most ratite species. The aims of this work were to assess the presence of corticosterone in plasma of Greater rhea, to validate a corticosterone 125I-radioimmunoassay for determining corticosterone levels in plasma samples and to study the activation of the adrenal gland after an adrenocorticotrophic hormone (ACTH) challenge. Six captive Greater rhea juveniles of 10 months of age received an intravenous ACTH injection. Blood samples were taken at 0 min (baseline pre-ACTH levels), and post-injection at 15, 30, 60 min and at 24 and 48 h. The high pressure liquid chromatography (HPLC) analysis of pooled plasma showed that corticosterone is the glucocorticoid found in the plasma of Greater rhea. Biochemical assays of standard validation (e.g., parallelism, exogenous corticosterone recovery) showed that measurements of corticosterone present in the plasma of the Greater rhea provided by commercial corticosterone 125I-radioimmunoassay were accurate and precise. ACTH challenge induced a more than 40-fold increase in plasma corticosterone at 60 min post-ACTH (from 4.0 to 166.5 ng/ml, on average). The corticosterone response to ACTH in Greater rhea was higher than is usual in birds, an apparently typical characteristic of ratites.

Hypothalamic-pituitary-adrenal (HPA) axis function, in Brahman heifers of differing temperament, was evaluated using separate challenges with CRH and ACTH. Exit velocity (EV) measurement was used to classify heifer temperament as calm [C; consisted of 6 slowest heifers (EV=1.05+/-0.05 m/s)] or temperamental [T; 6 fastest heifers (EV=3.14+/-0.22 m/s)]. During the 6 h prior to CRH challenge, areas under the ACTH (P=0.025) and cortisol (P

In this review, we address the function of the hypothalamus-pituitary-adrenocortical (HPA) axis with special emphasis on the comparison between the bovine and equine species. The pars intermedia of the pituitary gland is particularly well developed in horses and cattle. However, its function is not well appreciated in cattle yet. The Wulzen's cone of the adenohypophysis is a special feature of ruminants. Total basal cortisol concentration is much higher in horses than that in cows with similar free cortisol fractions. Corticotropin-releasing factor (CRF) concentrations in equine pituitary venous blood are lower compared with other species, whereas plasma ACTH concentrations in cows are higher than those in horses. A CRF challenge test induced a more pronounced cortisol response in horses compared with cattle, whereas regarding ACTH challenge testing, the opposite seems true. Based on data from literature, the bovine species is characterized by relatively high basal blood CRF and ACTH and low cortisol and glucose concentrations. Obviously, further lowering of blood cortisol in cattle is easily prevented by the high sensitivity to ACTH, and as a consequence, subsequent increased gluconeogenesis prevents imminent hypoglycemia. Hypoglycemia is less likely in horses given their high muscle glycogen content and their relatively high cortisol concentration. When assessing HPA axis reactivity, response patterns to exogenous ACTH or CRH might be used as a reliable indicator of animal welfare status in cows and horses, respectively, although it is emphasized that considerable caution should be exercised in using measures of HPA activity solely to assess animal welfare.

Our research group, under PI Thea Bechshft, has recently shown that it is possible to extract and measure cortisol in cetacean skin (Bechshoft et al. Submitted). The goal of the current project is to validate this novel method by measuring baseline cortisol levels in cetacean skin and demonstrating the correlation between an acute stress exposure and the ensuing skin cortisol response. At the conclusion of the project period, we expect to have provided a greater understanding of a) the relationship in cetaceans between blood cortisol levels and skin cortisol levels , b) the period of time it takes before an acute stress response is measurable in the cetacean skin matrix, and c) cetacean baseline cortisol levels in skin, as well as inter-and intra-individual fluctuations.

Stereotypies are repetitive and relatively invariant patterns of behavior, which are observed in a wide range of species in captivity. Stereotypic behavior occurs when environmental demands produce a physiological response that, if sustained for an extended period, exceeds the natural physiological regulatory capacity of the organism, particularly in situations that include unpredictability and uncontrollability. One hypothesis is that stereotypic behavior functions to cope with stressful environments, but the existing evidence is contradictory. To address the coping hypothesis of stereotypies, we triggered physiological reactions in 22 horses affected by stereotypic behavior (crib-biters) and 21 non-crib-biters (controls), using an ACTH challenge test. Following administration of an ACTH injection, we measured saliva cortisol every 30 min and heart rate (HR) continuously for a period of 3 h. We did not find any differences in HR or HR variability between the two groups, but crib-biters had significantly higher cortisol responses than controls (mean ± SD: CB, 5.84 ± 2.62 ng/ml, C, 4.76 ± 3.04 ng/ml). Moreover, crib-biters that did not perform the stereotypic behavior during the 3- h test period (Group B) had significantly higher cortisol levels than controls, which was not the case of crib-biters showing stereotypic behavior (Group A) (A, 5.58 ± 2.69 ng/ml; B, 6.44 ± 2.38 ng/ml). Our results suggest that crib-biting is a coping strategy that helps stereotypic individuals to reduce cortisol levels caused by stressful situations. We conclude that preventing stereotypic horses from crib-biting could be an inappropriate strategy to control this abnormal behavior, as it prevents individuals from coping with situations that they perceive as stressful. Postprint

In this study, we analysed the effects of social and human contact on calves ‘ behaviour and stress responses. We also measured the effect of this contact on calves ‘ reactions to novel conspecifics and novel humans. Sixty-four calves were housed either alone or in pairs and received either minimal human contact or ‘additional’ human contact (stroking and talking). At six, 10 and 14 weeks of age, the behaviour of the calves was recorded in their home pens. Calves were then tested in an unfamiliar arena either alone, with an unfamiliar calf, or with an unfamiliar man, and in a Y-maze with one arm leading to a calf and the other to a man. An adrenocorticotrophic hormone (ACTH) challenge was performed in order to assess chronic stress responses. Compared with individually housed calves, pair-housed calves were more active and made fewer contacts with their neighbours when in their home pens; they were also less active in the arena, spent more time near the calf in the Y-maze, and had lower Cortisol responses to ACTH. Calves that had received additional human contact interacted more with the man in the arena and had lower mean heart rates than those that had received minimal contact. This study confirmed that calves feel a need for social contacts and that pair-housing can lower the stress felt by calves separated from their conspecifics. Additional contact from stockpersons increases calves ‘ likelihood of approaching humans but cannot compensate for their lack of social partners. Hence, when calves are separated, the duration of the separation should be limited, and visual and physical contact with other calves should be provided.

Attempts have been made to establish relationship between the response to ACTH challenge in female calves, growth and first lactation performance. A total of 19 Holstein calves weighing 100 kg i. v. were given 0.50 IU of ACTH/kg BW .75 (EXACTHIN inj., Richter G., Budapest) at 60 days of age. Serial blood samples were taken at times 0, 0.5, 1, 2, 3, 4 and 5 hours and analyzed for cortisol, glucose insulin and FFA levels. From challenge series the area under the curve from time of administration and the following 5 h were calculated. Negative, and mostly loose relationship between response to ACTH challenge for cortisol, insulin, or FFA and ADWG during growth have been established (p>0.05) with positive one for glucose. Bivariate coefficients of correlation varied within the range from -0.35 to 0.15. Estimations reveal negative correlation between the length of first lactation and cortisol or insulin (r=-0.80, p

Animals in captivity and in the wild face numerous challenges, including the risk of enduring acute or chronic stress. In captivity, facilities attempt to alleviate the risk of chronic stress by providing environmental enrichment, shown to minimize behavioral disorders and stress in several species. One potential form of enrichment in zoos is training animals to provide rides for guests, however, the effect of this activity on the welfare of individual animals has never been examined. We validated the use of saliva for assessing stress in dromedary camels ( Camelus dromedarius ), an animal commonly used for rides. We then measured variation in salivary cortisol in four male camels while providing rides of differing frequency for guests at the Toronto Zoo. The camels were sampled during the ride season (June to September) using four treatments: (1) in their pasture, (2) at the ride area when not performing rides, (3) while providing a low number of rides ( n = 50/day) and (4) while providing a high number of rides ( n = 150/day). Furthermore, samples were taken before and after the ride season for comparison. There was a significant difference between the post-ride season treatment and the three treatments involving guest presence during the ride season (ride area, low rides, high rides). In general, cortisol concentrations were lower during the ride season and higher during the non-ride season. Based on the metrics we used, performing rides is not a stressful experience for these dromedary camels and suggests that rides may be a form of enrichment.

1. In order to reduce injurious pecking, the influence of environmental enrichment on pecking and perching behaviour was studied in young male and female turkeys. 2. Two different types of enrichment and a control treatment (TO) were tested: T1, metal objects and straw; T2, similar to T1 + wood perches. Birds were housed in 36 m2 pens at a light intensity of 5 lux with 4 replicates per treatment in a 2x3 factorial design. 3. Pecking at objects and perching behaviour were observed weekly. Behaviour was video recorded at weeks 5 and 10. Birds were examined daily for the occurrence of injuries. At the end of the rearing period, an ACTH challenge was performed and H/L ratio was measured. 4. Objects were regularly pecked at. Perching was more common in females, peaked at week 5 (10% to 13% of birds perched) and declined to 0% by week 10. Aggressive pecking was more frequently observed in males in T0 than in T1 or T2 at week 10. Wing (in males and females), tail and head (in males) injuries were more common in T0 than in T1 or T2. T1 and T2 were similar Response to ACTH challenge and H/L ratio were not consistently influenced by treatments. 5. It is concluded that metal objects and straw reduced injurious pecking in young female and male turkeys by redirecting, pecking activity.

The effects of chronic toxic stress on the hypothalamo-pituitary-interrenal (HPI) axis were investigated in yellow perch (Perca flavescens) captured at a reference site (Lake Memphremagog) and two sites contaminated with heavy metals and organic contaminants (Ile Perrot and Iles de la Paix, Lake St. Louis) in spring, summer, and fall. Cortisol secretion of the fish was stimulated in situ by an acute capture stress or by an i.p. injection of 4 IU/100 g body mass of porcine corticotropin (ACTH1-39). The response to both these challenges was lower in perch from the highly contaminated site than in perch from the reference site in the spring but not in summer. In fall, fish from the highly contaminated site had, as in spring, a lower response to ACTH than fish from the reference site. The reduced ability of perch to respond to capture stress or to ACTH indicates that the interrenal tissue in fish from contaminated sites is functionally impaired. Cortisol-impaired fish also had abnormal carbohydrate metabolism. The reduced ability of wild fish from contaminated sites to respond to a standardized ACTH challenge may be used as an early indicator of contamination-induced chronic stress.

A trial was designed to determine the effect of season and feed restriction on LH and prolactin secretion, adrenal response, insulin and FFA in the early pregnant gilt. Groups of cross bred gilts (n = 24) were mated and allocated to two feeding levels; a non-restricted group received close to ad libitum feeding of 3.6 kg whereas, the restricted group received 1.8 kg as recommended by the NRC. The trial was carried out in winter-spring and repeated in summer-autumn to investigate the effects of season. The feeding regimen were fed to the group housed animals for the first two weeks of pregnancy. A 12 h period of blood sampling every 15 min thereafter revealed higher amplitude LH pulses with larger area under the curve in winter compared with summer (1.17 +/- 0.03 vs. 0.69 +/- 0.03 ng ml(-1) and 65.09 +/- 1.46 vs. 33.60 +/- 1.25, P0.05). Overall, feed restriction reduced LH pulse frequency (2.5 +/- 0.1 and 1.6 +/- 0.1 pulses/12 h for high and low feeding levels, P0.05), but the difference was large in winter and no difference was detected in summer. An ACTH challenge test carried out the day after the frequent sampling revealed greater response to the ACTH challenge in winter in comparison with summer. Plasma prolactin values were generally very low and ranged from 1 to 4.5 ng/ml with highest values detected in the feed restricted group in summer. Plasma FFA and insulin concentrations showed greater pre- versus post-prandial variation in the feed restricted groups. It was concluded, that feed restriction and season affected LH secretion and those effects appeared to be related to the metabolic changes in the early pregnant group housed gilt.

A remote infusion and blood collection device (Dracpac) was used on free-ranging red deer stags to deliver i.v. a range of ACTH1–24 doses and collect blood for cortisol determination. In Experiment 1, conducted in September, saline and 1, 4, and 16 IU ACTH/100 kg bodyweight were infused (n = 5–7 per treatment). In Experiment 2 (November), 16 and 64 IU ACTH/100 kg were infused (n = 5 per treatment); and the response of animals to restraint was also assessed (n = 6). Pre-infusion concentrations of plasma cortisol were low (mean 6.5 ± 1.0 ng/ml), and unaffected by infusion of saline. ACTH significantly elevated plasma cortisol concentrations, with mean peak concentrations occurring 20–40 min postinfusion. Duration of the response was dose dependent, ranging from 80–160 min. With increasing doses of ACTH, maximal peak heights plateaued at approximately 40 and 60 ng/ml in September and November, respectively; whereas the areas under the curves tended to increase. The minimum dose of ACTH that resulted in a maximal peak cortisol response was 4 IU. The maximum peak height of the cortisol response to 16 IU tended to be higher (P < 0.10) in November compared with September, suggesting that adrenal responsiveness may change over this period. The adrenal response to a 16-IU ACTH challenge in Experiment 2 resulted in similar plasma cortisol concentrations to an acute stress event (restraint). Four IU/100 kg ACTH i.v. is recommended as an appropriate dose for ACTH infusion studies in red deer stags. When used in conjunction with the Dracpac technique, it becomes a stress-free, repeatable procedure for assessing aspects of adrenal cortex physiology in free-ranging deer.

ACTH challenge tests for the assessment of stress on the first and third week postpartum and bacteriologic examination of uterine swabs on the second and fourth week, were conducted in dairy cows following clinically normal (n=12) and abnormal (n=21) puerperium. Postpartum ovarian activity and uterine involution were also investigated. The basal plasma cortisol concentrations (mean ± S.E.) during the first and third week postpartum were 3.1 ± 0.9 ng/ml and 3.4 ± 1.1 ng/ml in normal cows, and 6.5 ± 1.9 ng/ml and 4.9 ± 1.2 ng/ml in abnormal cows, respectively. The mean basal plasma cortisol values of abnormal cows were elevated and significantly higher (P

Summary Birth timing, a relative measure of the timing of births within a season, has been shown to be related to the ways mothers and infant interact as well as to infant behavior and physiology. Although effects of birth timing on the hypothalamic–pituitary–adrenal (HPA) axis have previously been associated with variation in social relationships, these effects could also be related to seasonal variation in climate conditions when the birth season is long. The current study examines the effects of birth timing and ambient temperature on the activity and regulation of the HPA axis in 3–4 month old rhesus monkeys ( N = 338). Subjects were part of a BioBehavioral Assessment in which infants were separated from their mothers and relocated to a novel testing environment for a period of 25 h. Four blood samples were collected and assayed for cortisol concentrations and reflected HPA response to (1) 2 h maternal separation and relocation, (2) 7 h maternal separation and relocation (sustained challenge), (3) dexamethasone suppression, and (4) ACTH challenge. Nonlinear mixed modeling was used to examine the independent effects of birth timing and temperature on HPA axis activity and regulation over the study period. Results indicated that birth timing and ambient temperature both had significant, but opposing effects on the cortisol response to sustained challenge. Chronic exposure to low ambient temperatures was associated with higher cortisol levels. After controlling for the effect of ambient temperature, birth timing was positively associated with cortisol such that late-born infants exhibited higher cortisol concentrations than did early-born infants. These results highlight the fact that climate conditions, even mild, subtropical conditions, can have potentially important influences on the activity and development of the HPA axis.

Land use changes are a significant factor influencing the decline of felid populations. However, additional research is needed to better understand how these factors influence populations in the wild. Hormone analysis can provide valuable information on the basic physiology and overall health of an animal, and enzyme immunoassays (EIA) are generally used for hair hormone analysis but must first be validated for the substrate of choice and species of interest. To date, hormone assays from hair have not been validated for Felidae, despite that the method holds considerable promise for non-invasive sampling of free-ranging animals. We sought to: (1) evaluate whether increased adrenocorticotrophin hormone (ACTH) during the period of hair growth results in elevated hair cortisol; (2) validate the enzyme immunoassay used; and (3) identify any variations in hair cortisol between age, sex and body regions, using Canada lynx. We quantified hair cortisol concentrations in captive animals through an ACTH challenge and collected samples from legally harvested lynx to compare variability between body regions. An EIA was validated for the analysis of hair cortisol. Lynx (n = 3) had a qualitative increase in hair cortisol concentration following an ACTH challenge in captive animals (20 IU/kg of body weight weekly for 5 weeks), thereby supporting the use of an EIA to quantify cortisol values in hair. Based on our analysis of sampled lynx pelts, we found that hair cortisol did not vary between age and sex, but varied within the foot/leg region to a greater extent than between individuals. We recommend that future studies identify a standardized location for hair cortisol sampling.

Analysis of fecal glucocorticoid (GC) metabolites has recently become the standard method to monitor adrenocortical activity in primates noninvasively. However, given variation in the production, metabolism, and excretion of GCs across species and even between sexes, there are no standard methods that are universally applicable. In particular, it is important to validate assays intended to measure GC production, test extraction and storage procedures, and consider the time course of GC metabolite excretion relative to the production and circulation of the native hormones. This study examines these four methodological aspects of fecal GC metabolite analysis in tufted capuchins (Cebus apella). Specifically, we conducted an adrenocorticotrophic hormone (ACTH) challenge on one male and one female capuchin to test the validity of four GC enzyme immunoassays (EIAs) and document the time course characterizing GC metabolite excretion in this species. In addition, we compare a common field-friendly technique for extracting fecal GC metabolites to an established laboratory extraction methodology and test for effects of storing “field extracts” for up to 1 yr. Results suggest that a corticosterone EIA is most sensitive to changes in GC production, provides reliable measures when extracted according to the field method, and measures GC metabolites which remain highly stable after even 12 mo of storage. Further, the time course of GC metabolite excretion is shorter than that described yet for any primate taxa. These results provide guidelines for studies of GCs in tufted capuchins, and underscore the importance of validating methods for fecal hormone analysis for each species of interest.

Blood CORT is commonly used to assess stress in rodents, but sampling can trigger a rapid stress response. This study aims to identify whether faecal CORT metabolites (FCM) can reflect changes in CORT induced by 7-day food restriction (FR) and an ACTH challenge. Blood and 24hr faecal samples were collected at baseline and Day 7 for control (n=8) and FR (n=10) rats. On Day 8, after a baseline blood sample, an ACTH injection was administered and followed by blood and fecal sampling. Results showed increased serum CORT and FCM in response to FR. Increased adrenal sensitivity with FR was illustrated by a greater increase in serum CORT compared to control in response to ACTH. Lastly, although it appeared that ACTH induced an increase in FCM in FR and control, only the latter reached statistical significance. Thus FCM might be better suited for quantifying chronic rather than acute changes in CORT.

Analysis of fecal glucocorticoid (GC) metabolites has recently become the standard method to monitor adrenocortical activity in primates noninvasively. However, given variation in the production, metabolism, and excretion of GCs across species and even between sexes, there are no standard methods that are universally applicable. In particular, it is important to validate assays intended to measure GC production, test extraction and storage procedures, and consider the time course of GC metabolite excretion relative to the production and circulation of the native hormones. This study examines these four methodological aspects of fecal GC metabolite analysis in tufted capuchins (Cebus apella). Specifically, we conducted an adrenocorticotrophic hormone (ACTH) challenge on one male and one female capuchin to test the validity of four GC enzyme immunoassays (EIAs) and document the time course characterizing GC metabolite excretion in this species. In addition, we compare a common field-friendly technique for extracting fecal GC metabolites to an established laboratory extraction methodology and test for effects of storing “field extracts” for up to 1 yr. Results suggest that a corticosterone EIA is most sensitive to changes in GC production, provides reliable measures when extracted according to the field method, and measures GC metabolites which remain highly stable after even 12 mo of storage. Further, the time course of GC metabolite excretion is shorter than that described yet for any primate taxa. These results provide guidelines for studies of GCs in tufted capuchins, and underscore the importance of validating methods for fecal hormone analysis for each species of interest. peerReviewed

The relationship between stress and feed efficiency in finisher swine was investigated through an adrenocorticotropin hormone (ACTH) challenge. The objective of this study was to evaluate the role of the ACTH-cortisol axis and the stress response in feed efficiency using pigs divergently selected for RFI. Gilts divergently selected for residual feed intake (RFI) were used as a model for feed efficiency and were challenged with 0.2 IU/kg BW of exogenous porcine ACTH. Serial blood samples were collected and plasma cortisol concentrations were determined. These results indicate that high-RFI pigs(lower feed efficiency) are more responsive to an ACTH challenge, but cope equally as well as pigs selected for low-RFI (higher feed efficiency).

Four yearling Hampshire ewes were bilaterally adrenalectomized by a technique simplified by the exclusive use of ligaclips on the blood vessels of the adrenal glands and by the employment of a specially modified forceps to facilitate excision of the adrenal glands. One ewe developed severe symptoms of cortical insufficiency, while the other three remained in good condition. In two of the latter three animals, an accessory adrenal gland, consisting of cortex and medulla, was found on the left side, while in the third ewe a small mass of cortical cells was found on the right side. Since there are considerable difficulties in locating accessory adrenals at necropsy, it is concluded that a judgment on completeness of removal of cortical tissue must be based on testing the living animal (development of adrenocortical insufficiency or ACTH challenge test).

The objective of this study was to evaluate how behavioral and physiological parameters are affected based on a cow's level of success at displacing others at an overstocked feed bunk. Forty Holstein nonlactating, late-gestation dairy cattle were housed in an overstocked pen [5 stalls/10 cows and 0.34 m of linear feed bunk (FB) space/cow] in groups of 10 (4 heifers and 6 multiparous cows) for 14 d. Plasma nonesterified fatty acids, glucose, and fecal cortisol metabolites (11,17-dioxoandrostanes) were measured in blood and feces sampled every 2d. A glucose tolerance test and an ACTH challenge were conducted on all cows on d 13 and 14, respectively to further explore the effects of competitive success on energy metabolism and stress physiology. Feeding behavior and displacements at the FB were recorded between d 7 to 10 of the observation period. A competition index (CInd) was calculated for each cow by dividing the number of times the cow displaced another at the FB by the total number of displacements the cow was involved in, either as an actor or reactor. Cows were then divided into 3 subgroups based on their CInd: high success (HS: CInd ≥0.6), medium success (0.4 ≤ CInd0.6), and low success (LS: CInd0.4). Heifers accounted for 7, 36, and 79% of the total number of animals in the HS (n=15), medium success (n=11), and LS (n=14) groups, respectively. No differences were observed in daily feeding time, total number of displacements, and time to approach the FB following fresh feed delivery between the 3 CInd groups; however, cows in the LS group had greater daily nonesterified fatty acid and 11,17-dioxoandrostane concentrations relative to cows in the HS group. No differences existed in cortisol response to an ACTH stimulation test between CInd categories. During the glucose tolerance test, glucose response curves were the same between all 3 CInd categories; however, the peak insulin response of LS cows was 130 μIU/mL greater than the peak HS response, indicating that LS cows may have decreased tissue responses to insulin or increased pancreatic responses to glucose. In an overstocked environment, dairy cattle physiology is associated with a cow's level of success at displacing other individuals at the feed bunk.

Climate change and industrial development are contributing to synchronous declines in Rangifer populations across the Arctic. Chronic stress has been implicated as a proximate factor associated with decline in free-ranging populations, but its role in Rangifer is unspecified. Analysis of glucocorticosteroid (GC) concentration in feces, and more recently in hair, is a non-invasive method for monitoring stress in wildlife. Adrenocorticotropic hormone (ACTH) released from the pituitary gland stimulates GC release from the adrenals and can be administered to reflect adrenal activation. In this study, we assessed concentrations of GC metabolites in feces and cortisol in hair of Alaskan caribou (Rangifer tarandus granti) and reindeer (R. t. tarandus) following ACTH treatment. We predicted that ACTH challenge would increase concentrations of fecal GCs, but not hair cortisol because steroid deposited into the hair shaft occurs over an extended period of time (months) and is likely insensitive to acute adrenal stimulation. Adult caribou (n=10; mean age, 6.5 years old) exhibited a peak increase in fecal GCs 8h following a 2 IU/kg dose of ACTH compared to pre-injection concentrations. In contrast, sub-adult reindeer (n=10, 0.8 years old) elicited a diminished response to the same dose. Quadrupling the dose (8 IU/kg) prolonged the fecal GC response in female reindeer, but male reindeer were unresponsive. Hair cortisol was unaffected by a single ACTH challenge. Further investigation is required to ascertain whether subspecific differences in adrenal sensitivity are attributed to age or sex differences, or historical selective pressures from semi-domestication and/or sedentary life cycle in reindeer.

Plasma concentrations of growth hormone (GH) were studied in 40 Friesian cows in a balanced block design. The cows were assigned to four treatments: (1) control, tie stalls; (2) deprivation of lying from 09:00 to 16:00 and from 22:00 to 05:00, tie stalls; (3) social isolation, tie stalls; (4) slatted floor, crowding in box. Plasma GH concentrations were measured after 23 d of treatment during an 8-h serial blood sampling as well as prior to and after a short test. On day 24, GH response was measured in a serial blood sampling for 0.5 h prior to and for 5 h following intravenous administration of adrenocorticotrophic hormone (ACTH). Deprivation of lying down reduced the level of GH in all sampling series, while no difference was found between treatments 1, 3, and 4. Plasma concentrations of GH prior to and after the test were not different. While concentration of plasma GH of deprived cows did not respond to ACTH challenge, an immediate and transient increase in plasma GH was found in the cows on the other three treatments. Mean plasma concentrations of GH were found to be repeatable between successive days. The results demonstrate that alterations in GH levels, among others, may be a valuable indicator of the biological cost of different environmental challenges. Key words: Dairy cows, stress, ACTH, GH release

Glucocorticoids or their metabolites can be measured in several body fluids or excreta, including plasma, saliva, urine and faeces. In recent years the measurement of glucocorticoid metabolites (GCMs) in faeces has gained increasing attention, because of its suitability for wild populations. In horses, however, the group-specific enzyme immunoassay described so far has a limited practicability due to its complex extraction procedure. Therefore, we tested the applicability of other enzyme immunoassays for glucocorticoid metabolites. The present study clearly proved that an enzyme immunoassay (EIA) for 11-oxoaetiocholanolone using 11-oxoaetiocholanolone-17-CMO: BSA (3alpha,11-oxo-A EIA) as antigen showed high amounts of immunoreactive substances. Therefore it was possible to use just a small amount of the supernatant of a methanolic suspension of faeces. The results correlated well with the already described method for measuring GCMs in horse faeces, i.e. analysing the samples with an EIA after a two step clean up procedure of the samples (Merl et al. 2000). In addition, the 3alpha,11-oxo-A EIA has the advantage of providing a bigger difference between baseline values and peak values after ACTH stimulation. The new assay increased the accuracy of the test, lowered the expenses per sample, and storing samples at room temperature after collection was less critical than with other assays investigated in our study. This is a big advantage both in the field of wildlife management of equids and in the field of equestrian sports and it shows the importance of choosing an assay which is in good accordance with the metabolites excreted in a given species.

In our previous studies we had demonstrated that, in children affected with isolated GH deficiency (IGHD), a short-term recombinant growth hormone (rGH) therapy increases the 11-deoxycortisol (S) secretion and induces an IGF-I responsiveness to the ACTH challenge. The aim of the present study was to further investigate the mechanisms by which IGF-I is secreted after ACTH challenge in children affected with IGHD by correlating IGF-I versus cortisol (F) time courses after ACTH administration. Ten children affected with IGHD were subjected to rGH therapy (4 IU/day subcutaneously) for 10 days. The responsiveness of IGF-I, F and S to the ACTH 1-17 test were evaluated before and at the end of the therapy. No IGF-I response to the ACTH test was recorded in the patients before the rGH treatment, whereas after rGH administration ACTH induced a significant IGF-I release (p < 0.001) which started at the 1st hour, reached a peak value between the 5th and 6th hours and disappeared at the 10th hour. In conclusion, our study confirms that a short-term rGH therapy induces an IGF-I responsiveness to ACTH and helps to better define the kinetics and the mechanism of this IGF-I response to ACTH.

We verified the relevance of measuring fecal glucocorticoid metabolites (FGM) to assess the stress response of the Syrian hamster. Male and female hamsters (n = 10 each) were submitted to an adrenocorticotropic hormone (ACTH) challenge test, whereas animals in the control group received 0.5 mL of sterile isotonic saline solution. All feces voided by each animal were collected at 4 h intervals from 24 h before (baseline) until 48 h after injections. FGM were quantified using an 11-oxoetiocholanolone enzyme immunoassay (EIA). Basal concentrations of FGM were almost four times higher in males than in females. Following ACTH administration, FGM levels started rising from 8 h onwards, reaching peak concentrations 20 or 28 h post injection in males and females, respectively. Despite the much higher absolute concentrations present in males, the relative increase (500%) in response to the ACTH stimulation was similar in both sexes. Sex differences in FGM levels are in accordance with results reported by others regarding the hamster adrenal physiology. The comparison of the adrenocortical response of males and females to an ACTH challenge provided new information about the amplitude and the timing of such a response and the excretion of glucocorticoids in both sexes. We demonstrated for the first time in the Syrian hamster that adrenocortical activity can be monitored in fecal samples in a noninvasive way. Our study provides a humane, practical, and noninvasive alternative to blood removal and therefore a powerful tool for stress-related studies in a species frequently used as an animal model in medical research.

Determination of fecal steroid metabolites is a noninvasive technique that characterizes the physiological state of organisms without the physiological and psychological stress of handling. Although this technique has many applications in the study of wildlife and/or captive animals without the necessity of capturing individuals, it requires a species-specific validation before use. A complete validation includes an analytical and a physiological one. In the latter changes in fecal hormone metabolites are induced by previous manipulations of the respective plasma hormones. Here we validated a method for measuring fecal cortisol metabolites (FCM) in the hystricomorph rodent Octodon degus. We extracted feces with 80% ethanol and quantified steroids using a commercial available cortisol radioimmunoassay. We first compared baseline levels of blood cortisol and FCM, and then performed a challenge test with adrenocorticotropic hormone (ACTH) to demonstrate that FCM accurately reflect adrenocortical activity. We found a significantly positive relationship between concentrations of blood cortisol and its fecal metabolites. During the ACTH challenge test, blood cortisol levels peaked 30 min after injection, and FCM mirrored this peak with a delay of about 6 hr. Our successfully validated noninvasive method provides new opportunities for studies assessing the influence of social and ecological factors on degus under natural conditions.

The ocelot (Leopardus pardalis) is included in list of wild felid species protected by CITES and is part of conservation strategies that necessarily involve the use of assisted reproduction techniques, which requires practical and minimally invasive techniques of high reproducibility that permit the study of animal reproductive physiology. The objective of this study was to compare and validate two commercial assays: ImmuChem Double Antibody Corticosterone 125I RIA from ICN Biomedicals, Costa Mesa, CA, USA; and Coat-a-Count Cortisol 125I RIA from DPC, Los Angeles, CA, USA, for assessment of fecal glucocorticoid metabolites in ocelots submitted to ACTH (adrenocorticotropic hormone) challenge. Fecal samples were collected from five ocelots kept at the Brazilian Center of Neotropical Felines, Associação Mata Ciliar, São Paulo, Brazil, and one of the animals was chosen as a negative control. The experiment was conducted over a period of 9 days. On day 0, a total dose of 100 IU ACTH was administered intramuscularly. Immediately after collection the samples were stored at 20C in labeled plastic bags. The hormone metabolites were subsequently extracted and assayed using the two commercial kits. Previously it was performed a trial with the DPC kit to check the best extraction method for hormones metabolites. Data were analyzed with the SAS program for Windows V8 and reported as means ± SEM. The Schwarzenberger extraction method was slightly better when compared with the Wasser extraction method (103,334.56 ± 19,010.37ng/g of wet feces and 59,223.61 ± 12,725.36ng/g of wet feces respectively; P=0,0657). The ICN kit detected an increase in glucocorticoid metabolite concentrations in a more reliable manner. Metabolite concentrations (ng/g wet feces) on day 0 and day 1 were 66,956.28 ± 36,786.93 and 92,991.19 ± 28,555.63 for the DPC kit, and 205,483.32 ± 83,811.32 and 814,578.75 ± 292,150.47 for the ICN kit, respectively. The limit of detection for the ICN kit was 7.7 ng/mL for 100% B/Bo (25ng/mL for 88%B/Bo) and for the DPC kit it was 0.2ug/dL for 90.95% B/Bo (1ug/dL for 81.27% B/Bo). In conclusion it was confirmed that the Schwarzenberger extraction method and the ICN kit are superior for extracting and measuring fecal glucocorticoid metabolites in ocelot fecal samples. A jaguatirica (Leopardus pardalis) é uma das espécies de felídeos selvagens listadas no CITES e integrantes de planos de conservação que necessariamente envolvem o emprego de técnicas de reprodução assistida. Para isso, é fundamental o desenvolvimento de técnicas práticas, de alta repetibilidade e pouco invasivas, que permitam o estudo da fisiologia reprodutiva animal. Objetivou-se neste experimento comparar e validar os ensaios realizados com os conjuntos comerciais ImmuChem Double Antibody Corticosterone 125I RIE da ICN Biomedicals, Costa Mesa, CA, USA e Coat-a-count Cortisol 125I RIE da DPC, Los Angeles, CA, USA, na mensuração dos metabólitos de glicocorticóides das fezes de jaguatiricas por meio do desafio com ACTH. Para este estudo foram utilizadas amostras fecais de cinco indivíduos que se encontravam no Centro Brasileiro de Felinos Neotropicais, Associação Mata Ciliar, Pedreira, SP, Brasil, sendo que um destes animais foi escolhido como controle negativo. O experimento foi conduzido em um período de nove dias. No dia 0 foi administrada uma dose total de 100 UI IM de ACTH (hormônio adrenocorticotrópico). Imediatamente após a colheita, as amostras de fezes foram conservadas a -20ºC em sacos plásticos identificados e posteriormente os metabólitos hormonais foram extraídos e mensurados utilizando-se os dois conjuntos comerciais. Previamente foi realizado com o conjunto comercial da DPC um teste para comparar dois métodos de extração. Os dados foram analisados utilizando o programa The SAS System for Windows V8. A média ± erro padrão dos valores obtidos pela técnica de Schwarzenberger foi levemente superior à média dos valores obtidos pela técnica de Wasser (103.334,56 ± 19.010,37ng/g de fezes úmidas e 59.223,61 ± 12.725,36ng/g de fezes úmidas, respectivamente; P=0.0657). O conjunto comercial da ICN detectou com maior clareza o aumento das concentrações dos metabólitos de glicocorticóides. Os valores do dia 0 e do dia 1 para os conjuntos comerciais DPC e ICN foram respectivamente 66.956,28 ± 36.786,93ng/g de fezes úmidas e 92.991,19 ± 28.555,63ng/g de fezes úmidas; 205.483,32 ± 83.811,32ng/g de fezes úmidas e 814.578,75 ± 292.150,47ng/g fezes úmidas. Para o conjunto comercial da ICN a sensibilidade foi de 7,7ng/mL para 100% B/ Bo (25 ng/mL para 88%B/Bo) e para o conjunto comercial da DPC foi de 0,2ug/dL para 90,95% B/Bo (1ug/dL para 81,27% B/Bo). Confirmou-se, portanto, que a melhor técnica de extração foi a descrita por Schwarzenberger e que o conjunto comercial da ICN Biomedicals teve um melhor desempenho para a quantificação de metabólitos de glicocorticóides em fezes de jaguatiricas.

Non-invasive methods for determining the stress responses through the faecal cortisol metabolite levels have been adopted for various animals, including several deer species like Roosevelt elk, white-tailed deer, roe deer and red deer. Despite the close relationship between these species, faecal glucocorticoid assay must be validated for each species prior to its application. Using adrenocorticotropic challenge and comparing the results obtained with negative control we validated the physiological relevance of 11, 17 – dioxoandrostanes (DOA) analysis in fallow deer faeces. During the experiment three fallow deer yearlings received an ACTH injection and faecal samples were collected after 6, 12, 18 and 22 hours, respecting the influence of time interval between defecation and freezing on metabolite levels. The ACTH administration resulted in gradual increase of 11, 17 – DOA concentrations over the observed period to up to 4.3-fold increase when compared to initial concentrations. These concentrations were significantly higher than those in control animal. The results obtained indicate that faecal glucocorticoid analysis could be used to evaluate stress responses in fallow deer.

This paper analyses the consequences of repeated regrouping of dairy heifers according to their behaviour, stress physiology and production. Thirty-two Holstein heifers were housed in pairs. Between 11 and 13 months of age, half were subjected to 16 pen relocations each time with a new peer (regrouped heifers), while the other half remained in the same pen with the same peer (controls). The heifers were observed in standardised behavioural tests comprising sudden (opening of an umbrella), novel (an unfamiliar arena) or predator-related (dog) stimuli. All behavioural tests were conducted on three sessions between the 13th and the 16th regroupings. The functioning of the HPA axis was assessed through blockade by dexamethasone and stimulation by ACTH after the 14th regrouping and stimulation by CRF after the 15th regrouping. Weight gain was assessed during the regrouping period and reproduction (No. of inseminations before successful insemination) thereafter. Regrouped heifers reacted less than the controls to the umbrella ( = 8.23, P < 0.05). They started eating more quickly in the arena (F = 10.8, P < 0.01). In the presence of the dog, they were less active (F = 6.26, P < 0.05) and tended to look at the dog less often (F = 3.63, P < 0.10). The reduction in behavioural responses from one session to the next one was more pronounced in regrouped heifers (e.g. number of eating bouts: F (session treatment) = 4.23, P < 0.05). Regrouped heifers had lower cortisol responses to CRF. In conclusion, repeatedly regrouped heifers appear less disturbed by unusual situations and habituate to those more quickly. According to the behaviour of heifers and as suggested by their lower cortisol responses, diversity rather than stability of the social environment appears more beneficial to them., Cet article rapporte les conséquences de mélanges répétés d'animaux sur le comportement, le stress et la production de génisses de race laitière. Trente deux génisses Holstein ont été élevées par paires. Au sein de huit paires, les animaux ont changé de partenaires une à deux fois par semaine entre 11 et 13 mois d'âge, pour un total de 16 mélanges (génisses " mélangées "). Ces mélanges s'accompagnaient également de changements de parc. Les huit autres paires sont restées inchangées (génisses " témoins "). La réactivité comportementale a été mesurée en exposant les génisses à un événement soudain (ouverture d'un parapluie), à un environnement nouveau (alimentation dans un nouvel enclos) et à un prédateur (chien). Chaque test était répété trois fois, entre le 13 et le 16 mélange. Le fonctionnement de l'axe corticotrope a été évalué à l'aide d'un test de freination par la dexamethasone puis stimulation par l'ACTH après le 14 mélange et par un test de stimulation au CRF après le 15 mélange. Le gain de poids a été évalué par pesée hebdomadaire au cours des 16 mélanges. Par la suite, le nombre d'inséminations avant insémination fécondante a été relevé. Les génisses mélangées ont réagi à l'ouverture du parapluie moins fortement que les témoins ( = 8,23, P < 0,05). Elles ont commencé à manger plus rapidement dans l'enclos nouveau (F = 10,8, P < 0,01). En présence du chien, elles ont été moins actives (F = 6,26, P < 0,05) et tendaient à le regarder moins souvent (F = 3,63, P < 0,10). En outre, la diminution des réponses comportementales d'une séance sur l'autre était plus marquée chez les génisses mélangées (par exemple, nombre de prises alimentaires : F (séance traitement) = 4,23, P < 0,05). Par ailleurs, l'élévation du taux plasmatique de cortisol consécutive à l'injection de CRF a été moins prononcée chez les génisses mélangées. En conclusion, les génisses préalablement exposées à des réallotements répétés sont moins perturbées par des situations non familières et s'y habituent plus facilement par comparaison aux génisses maintenues avec le même partenaire. Dans ces conditions, il semblerait que ce soit la diversité de l'environnement social plutôt que sa stabilité qui ait permis d'améliorer la réactivité émotionnelle des génisses.

Shortened and weakened estrous expressions could be one of the causes of poor heat detection rate. Non-specific acute stresses are assumed to depress expression of estrus by an increase of plasma progesterone which may originate from the adrenal cortex. The objective of the present study was to examine whether the adrenal cortex can secrete significant amounts of progesterone in response to exogenous adrenocorticotropic hormone (ACTH) in lactating cows. Four cows had estrus synchronized and were ovariectomized in the luteal phase. The cows were given 25 IU ACTH through an indwelling catheter 5 h after catheterization. Blood samples were collected at an interval of 30 min. In 3 of the 4 cows, plasma progesterone concentrations increased significantly 0.5-1.5 h after the first ACTH challenge with a mean peak value of 4.2 +/- 0.4 (S.D.) ng/ml. A similar response was also observed after the second ACTH challenge. Peak plasma progesterone concentrations in the 3 cows after first ACTH challenge were comparable with the progesterone values in the luteal phase of each cow. The results suggest that lactating cows have the capability to secrete a significant amount of progesterone from the adrenal cortex.

Cortisol is a hormone released when animals experience stress. We validated the measurement of cortisol from hair for use in wildlife using wild chipmunks, and tested the use of hair cortisol by measuring cortisol from chipmunks captured in natural and logged sites., Stress levels of individuals are documented using glucocorticoid concentrations (including cortisol) in blood, saliva, urine or faeces, which provide information about stress hormones during a short period of time (minutes to days). In mammals, use of hair cortisol analysis allows for the assessment of prolonged stress over weeks and months and provides information on chronic stress levels without bias associated with handling. Here, we validate hair cortisol analysis in wild rodents using exogenous adrenocorticotrophic hormone (ACTH challenge) and apply the technique to evaluate stress in eastern chipmunks inhabiting logged and natural sites. Chipmunks were subjected to a mark–recapture study and injected weekly with ACTH (Synacthen Depot) or saline, with hair being collected at the conclusion of the challenge. Subsequently, faecal and hair samples were collected from chipmunks occupying logged and natural sites to assess the utility of hair cortisol in comparison with faecal cortisol metabolites. Following extraction, cortisol concentrations were quantified in hair and faecal extracts by enzyme immunoassay. Hair cortisol concentrations were significantly elevated in samples from ACTH-injected chipmunks compared with saline-injected control animals (five times higher). Chipmunks inhabiting logged sites had increased faecal cortisol metabolite concentrations compared with those in natural sites, but no differences were observed in hair cortisol concentrations. Faecal cortisol metabolite levels were positively correlated with hair cortisol levels in chipmunks. Hair cortisol levels reflect changes in circulating cortisol levels and can be used to evaluate the adrenal stress response, and thus stress, in natural populations. Nonetheless, because of the differences in the temporal scale of stress that hair and faeces represent, we caution the use of hair cortisol for detecting differences in physiological stress when comparing individuals within populations and suggest that it is best suited to examining population-level differences.

Angora goats do not cope well with stress compared with goats of other breeds. Our hypothesis that this involves subclinical primary hypoadrenocorticism associated with low cortisol release in response to ACTH stimulation was tested by measuring adrenocortical response (plasma cortisol) in six Spanish (37 +/- 2 kg BW) and six Angora wethers (39 +/- 3 kg BW) under simulated acute and chronic ACTH challenges. In Exp. 1 (acute ACTH challenge), wethers were dosed i.v. with high (2.5 IU/kg BW) or low (.4 IU/kg BW) quantities of ACTH. In Exp. 2 (chronic ACTH challenge), ACTH at the rate of .015 IU/(kg BW x min) or saline (.15 M NaCl) was infused i.v. at 15 mL/h for 6 h. The mean baseline plasma cortisol concentration before ACTH stimulation was similar (P > .05) between Angora and Spanish goats in Exp. 1 (averaged over days) and in Exp. 2. The cortisol concentration response area (ng/ (mL x min) x 10(-3)) above the baseline was similar (P > .05) between Angora and Spanish goats during low (7.6 +/- .5 and 9.0 +/- 1.7, respectively) and high (12.8 +/- 1.0 and 16.0 +/- 1.8, respectively) levels of acute ACTH challenge (Exp. 1) and during chronic ACTH challenge (45.1 +/- 5.9 and 41.8 +/- 7.3, respectively; Exp. 2). In conclusion, these data indicate that, under the conditions of this study, adrenocortical responsiveness to ACTH stimulation is not different between Angora and Spanish goat wethers and, thus, may not contribute to stress susceptibility in Angora goats.