Adrenocorticotropic Hormone Challenge Research Articles

Non-invasive assessment of fecal glucocorticoid and androgen metabolites in the pygmy hippopotamus (Choeropsis liberiensis)

The pygmy hippopotamus (Choeropsis liberiensis) is an endangered species endemic to the Upper Guinea Forest ecosystem in West Africa. We have limited information concerning the species’ reproduction and well-being under managed care. We therefore developed non-invasive methods for characterizing gonadal androgen and adrenal hormone profiles in pygmy hippos using fecal samples collected from 12 males and 12 females housed in North American zoological institutions. We aimed to: 1) identify and validate enzyme immunoassays (EIAs) for measuring metabolites of corticosteroids and testosterone in feces; and 2) test whether gonadal activity is correlated with previous breeding history, season or type of housing. For glucocorticoids, several EIAs for measuring metabolites were investigated. A group-specific EIA exhibiting cross-reactivity with 11,17-dioxoandrostane (DOA) metabolites of cortisol most clearly reflected adrenocortical activity in response to pharmocological challenge with adrenocorticotropic hormone (ACTH) in both males and females. However, day-to-day concentrations of this metabolite in the feces of pygmy hippos that did not undergo ACTH challenge were near the detection limits of the assay, making this EIA impractical for assessing glucocorticoid activity in this species. Another group-specific EIA, exhibiting cross-reactivity with 5α-pregnane-3β,11β,21-triol-20-one, produced biologically relevant data and evidence of an appropriate response to pharmacological challenge with exogenous ACTH. The testosterone metabolite assay C196 (Arbor Assays, Ann Arbor, Michigan, USA) also produced biologically coherent data: adult males exhibited the highest mean androgen metabolite concentrations (477 ng/g), followed by adult females (259 ng/g) and juvenile males (160 ng/g). Proven breeding males had higher, but not significantly different, mean concentrations (472 ng/g) to unproven males (352 ng/g; P = 0.400). Similarly, adult males housed outdoors year-round in subtropical climates exhibited higher, but not statistically different mean concentrations (554 ng/g) to males in temperate climates that were housed indoors at least part of the year (412 ng/g; P = 0.208). There were, however, significant differences in mean concentrations among seasons for adult males, with higher values in spring (546 ng/g) and summer (542 ng/g) than in autumn (426 ng/g) and winter (388 ng/g, P = 0.003). In conclusion, we identified EIAs for the measurement of fecal metabolites of androgens and glucocorticoids that can be used for further studies to monitor gonadal activity in male pygmy hippos and adrenocortical activity in both sexes. We also identified a seasonal trend in male gonadal activity in this species under managed care in North America. Finally, our findings highlight an important consideration when using non-invasive methods for evaluating fecal cortisol metabolites: ACTH used for pharmacological validation of an EIA does not necessarily equate to biological relevance.

Validation of Enzyme Immunoassays via an Adrenocorticotrophic Stimulation Test for the Non-Invasive Quantification of Stress-Related Hormone Metabolites in Naked Mole-Rats.

Small size in mammals usually restricts long-term, frequent monitoring of endocrine function using plasma as a matrix. Thus, the non-invasive monitoring of hormone metabolite concentrations in excreta may provide an invaluable approach. The aim of the current study was to examine the suitability of enzyme immunoassays (EIAs) for monitoring responses to stressors in the naked mole-rat (Heterocephalus glaber, NMR) using urine and feces as hormone matrices. A saline control administration, and a high- and low-dose adrenocorticotropic hormone (ACTH) challenge were performed on six male and six female disperser morph NMRs. The results revealed that a 5α-pregnane-3β,11β,21-triol-20-one EIA detecting glucocorticoid metabolites (GCMs) with a 5α-3β-11β-diol structure is the most suitable assay for measuring concentrations in male urine samples, whereas an 11-oxoaetiocholanolone EIA detecting GCMs with a 5β-3α-ol-11-one structure appears the most suitable EIA for quantifying GCMs in female urine. An 11-oxoaetiocholanolone EIA detecting 11,17 dioxoandrostanes was the most suitable EIA for quantifying GCMs in the feces of both sexes. There were sex-related differences in response to the high- and low-dose ACTH challenge. We recommend using feces as a more suitable matrix for non-invasive GCM monitoring for NMRs which can be valuable when investigating housing conditions and other welfare aspects.

Environmental stress during the last trimester of gestation in pregnant cows and its effect on offspring growth performance and response to glucose and adrenocorticotropic hormone.

Winter and spring precipitation are predicted to increase in the Midwest region of the United States, causing muddy conditions. In a previous experiment, Angus cows (8 per treatment) were paired based on initial body weight (BW) and one cow from each pair was randomly allocated to either the mud or control treatment. Though cows consumed the same amount of dry matter, cows in the mud treatment weighed 37.4 kg less than cows in the control treatment by day 269 of gestation. The objective of this experiment was to evaluate developmental programming effects of steers born to cows in the mud treatment (MUD; n = 7) or the control treatment (CON; n = 6). Steers were weighed at birth and then weekly from approximately 56 d of age until weaning and were subjected to a glucose tolerance test (GTT) and adrenocorticotropic hormone (ACTH) challenge after weaning. Steers were then placed in the feedlot for an 84-d growing phase and were weighed weekly and 12th rib back fat (BF) and ribeye area (REA) were imaged every 28 d using ultrasonography. Data were analyzed as a randomized complete block design with repeated measurements when appropriate (SAS 9.4). Although there was a 37.4 kg decrease in BW of cows by the end of gestation, there was no evidence of a pen treatment effect on calf birth weight (P = 0.60) or weaning weight (P = 0.99). Additionally, there was no evidence of a pen treatment × day effect for steer BW from birth to weaning (P = 0.67) or growing phase BW (P = 0.60). There was evidence of a treatment × day of growing phase effect (P = 0.02) for BF, such that CON steers had greater BF on day 28 of the growing phase; however, there was no evidence of a treatment × day effect for REA (P = 0.20). Furthermore, there was no evidence of a pen treatment effect for the growing phase average daily gain (P = 0.74), dry matter intake (P = 0.65), gain:feed (P = 0.48), plasma glucose concentration (P = 0.67) or plasma insulin concentration (P = 0.61) in response to the GTT, or plasma cortisol concentration in response to the ACTH challenge (P = 0.51). These results indicate that while mud increased net energy requirements for cows in the MUD treatment, there were no subsequent effects observed for steer BW, gain:feed, or response to glucose and ACTH during the growing phase.

S-16-4: PHARMACOKINETICS, PHARMACODYNAMICS, AND SAFETY RESULTS FROM A PHASE 1 MULTIPLE ASCENDING DOSE STUDY OF THE SELECTIVE ALDOSTERONE SYNTHASE INHIBITOR CIN-107

Objective: To evaluate the safety, pharmacokinetics, and pharmacodynamics of multiple ascending doses of the highly potent, selective small-molecule inhibitor of aldosterone synthase (CIN-107) in a randomized, double-blind, placebo-controlled phase 1 study in healthy volunteers. Design and methods: Subjects were randomized into 5 cohorts to receive CIN-107 or placebo once daily for 10 days. Cohorts 1 and 2 were placed on a low salt diet to stimulate aldosterone production and were administered 2.5 or 5.0 mg oral CIN-107, respectively. They also underwent an adrenocorticotropic hormone challenge to increase aldosterone and cortisol levels to evaluate the specificity of CIN-107 for aldosterone synthase. Cohorts 3, 4, and 5 were placed on a normal salt diet and were administered 1.5, 2.5, or 0.5 mg oral CIN-107, respectively. Blood samples were collected prior to and after dosing on days 1 and 10 for measurement of plasma CIN-107 concentrations to characterize single-dose and steady-state pharmacokinetics. Pharmacodynamic measurements included aldosterone, cortisol, and electrolytes. Safety assessments included physical examination, electrocardiograms, orthostatic vital signs, and clinical laboratory evaluations, including renal function. Results: Fifty-four subjects completed the study. There were no deaths, serious adverse events, or discontinuations due to treatment-emergent adverse events (TEAEs). All TEAEs in subjects receiving CIN-107 were mild in severity. Plasma concentrations of CIN-107 increased proportionally with ascending doses. CIN-107 was rapidly absorbed, with peak concentrations observed within 4 hours after dosing. Mean half-life was 26–31 hours. A dose-dependent reduction of plasma aldosterone occurred with CIN-107 doses ≧1.5 mg, regardless of normal or low salt diet. Decreases in plasma aldosterone started on Day 1 and were sustained, with levels reduced by approximately 51–73% on Day 10 (Figure 1). CIN-107 had no meaningful impact on plasma cortisol levels. The urine sodium:potassium ratio increased on Day 1 and diminished by Day 10, an effect mediated by greater elimination of sodium on Day 1 compared to Day 10. A mild increase in the blood urea nitrogen:creatinine ratio and a mild reduction in the glomerular filtration rate (<15%) suggest that CIN-107 produced a mild diuretic effect. Conclusions: CIN-107 was safe and well tolerated and resulted in dose-dependent increases in plasma CIN-107 with a half-life that supports once-daily dosing. The dose-dependent decrease in plasma aldosterone, transient increase in sodium excretion, and mild diuretic effect support continued study in ongoing phase 2 clinical trials evaluating the efficacy and safety of CIN-107 for treatment-resistant or uncontrolled hypertension and primary aldosteronism.

Validation of a non-invasive assessment technique for quantifying faecal glucocorticoid metabolite concentrations in the short-beaked echidna (Tachyglossus aculeatus)

The monotreme adrenocortical response to stress may not rely as heavily on the hypothalamic–pituitaryadrenal (HPA) axis compared to other mammals. This study aimed to validate a technique in which glucocorticoid metabolites could be quantified non-invasively in short-beaked echidna faeces by examining the secretion of glucocorticoids (GC) using an adrenocorticotrophic hormone (ACTH) challenge on sexually mature captive echidnas. Echidnas were housed individually for 15 days, with the ACTH challenge occurring on day five. Blood samples were collected on day five during the challenge and faecal samples were collected each morning for the 15 days. Both sample types were analysed for glucocorticoids (GC) or its metabolites. Plasma corticosterone concentrations increased significantly after 30 min and 60 min relative to time 0, whilst plasma cortisol concentrations increased significantly after 60 min. The ACTH challenge also resulted in an increase in glucocorticoid metabolite concentration in faecal samples from four of the six echidnas detected one to two days post ACTH injection, thereby validating a non-invasive method to assess adrenal response in the echidna. These results confirm that echidnas respond to a synthetic ACTH challenge in a similar manner to that of eutherian species indicating that echidnas appear to use the HPA axis in their stress response.

Patterns of faecal glucocorticoid metabolite levels in captive roan antelope (Hippotragus equinus) in relation to reproductive status and season

Populations of roan antelope (Hippotragus equinus) in southern Africa have experienced a drastic decline over the past few decades and this situation has led to the development of intensive breeding programmes to support conservation efforts. However, little is known about related welfare aspects, including stress-related physiological biomarkers. The present study set out to establish a non-invasive method to monitor faecal glucocorticoid metabolite (fGCM) concentrations as a measure of stress and determine fGCM concentrations in relation to male reproductive activity and female reproductive status in the roan antelope. An adrenocorticotrophic hormone challenge was performed using two adult roan antelope (one male and one female) at Lapalala Wilderness Nature Reserve, South Africa, to determine the suitability of five enzyme immunoassays (EIA) for monitoring adrenocortical function in roan antelope. An 11-oxoaetiocholanolone I EIA detecting 11,17 dioxoandrostanes performed best showing 17–20 folds increases in fGCM concentrations after 12 h-17 h post-injection. The identified EIA was then used to monitor fGCM concentrations during active and non-active reproductive periods in males (n = 3), and during periods of cyclicity, gestation, and postpartum in females (n = 10). Males showed an overall 80% increase in fGCM concentrations when reproductively active and females showed a progressively significant increase in fGCM levels throughout pregnancy, with overall fGCM concentrations being 1.5 to 2.6-fold higher than the respective fGCM concentrations during periods of postpartum and cyclicity, respectively. Furthermore, fGCM concentrations remained above baseline for up to 21 days post-partum. A correlation between ecological parameters (rainfall and temperature) and fGCM concentrations revealed elevated fGCM concentrations during the dry season for males, but not females. The non-invasive method validated in this study provides a valuable tool to quantify stress-related biomarkers in roan antelope, and findings can be used to support management decisions in conservation breeding facilities.

Measuring Faecal Glucocorticoid Metabolite Concentrations as an Indicator of Stress in Blue Wildebeest (Connochaetes taurinus)

In South Africa, blue wildebeest (Connochaetes taurinus) are routinely captured for relocation purposes. To monitor the stress caused by this practise, a non-invasive method assessing adrenocortical function as a measure of stress would minimize disturbance during sample collection. In our study, an adrenocorticotropic hormone challenge, and a mass-capture event were used to examine the suitability of five enzyme immunoassays (EIAs) for monitoring stress-related physiological responses using faeces as a sample matrix. The tested 11-oxoaetiocholanolone I EIA performed best, showing a 2126% increase above baseline after 22 h for a male, and a 474% increase for a female 23 h post-ACTH injection. Baseline faecal glucocorticoid metabolite (fGCM) concentrations did not differ between animals captured on either day 1 or day 2 of the capture event, indicating that the frequent presence of a helicopter during the two days did not influence fGCM concentrations. However, during capture-related restraint for up to 10 h, an overall 1.5-fold elevation in (fGCM) concentrations was found. Storage of faeces at ambient temperature post-defecation indicated a fair stability of fGCMs for up to 8 h. The ability to reliably assess adrenocortical function provides a solid basis to examine endocrine responses to putative stressful circumstances in blue wildebeest.

Salivary cortisol concentrations changes in horses during daily routine

Article Details: Received: 2020-10-20 | Accepted: 2020-11-27 | Available online: 2021-01-31 https://doi.org/10.15414/afz.2021.24.mi-prap.1-4 Abstract The aim of the study was to observe the salivary concentrations of cortisol in horses during daily routine over the course of four weeks lasting experiment in three different stages of day to monitor the changing values of cortisol in horse saliva during the day. Saliva was obtained from 12 Slovak warmblood horses – 1 stallion, 6 mares and 5 geldings. In the experiment we focused purely on changes of salivary cortisol concentrations over the course of the daily routine.The results shown us multiple significant changes between individual sample collections and thus, we can state that according to our results the highest concentration of cortisol in horse saliva is in the morning and it decreases throughout the day with lowest measured concentrations being in the final sample collection of the day (at 22:00). These changes had no visible effect on horses´ organism throughout entirety of the experiment and were caused due to horses daily rhythm. Keywords: horse, cortisol, saliva, rest, daily routine References Bohak, Zs, Szabo, F, Beckers, J-F, Melo de Sousa, N, Kutasi, O., Nagy, K, Szenci, O. 2013. Monitoring the circadian rhythm of serum and salivary cortisol concentrations in the horse . Domest. Anim. Endocrinol. 45:38-42. https://doi.org/10.1016/j.domaniend.2013.04.001 Contreras-Aguilar, MD, Lamy, E, Escribano, D, Ceron, JJ, Tecles, F, Quiles, AJ, Hevia, ML. 2020. Changes in salivary analytes of horses due to circadian rhythm and season: A Pilot Study. Animals. 10(9):1486. https://doi.org/10.3390/ani10091486 Halo, M, Hollý, A, Mlynekova, E, Polyakova, L, Horný, M, Kovalcik, E. 2009. Influence feeding and training on the metabolic profil sport horses . J Cent. Eur. Agric. 10(4):411-416. Halo, M, Strapak, P, Mlynekova, E, Kovalcik, E, Horný, M. 2008. Influence stres on the training process of the horses. J. Cent. Eur. Agric. 9(1):217-224. Ille, N, von Lewinski, M, Erber, M, Wulf, M, Aurich, J, Mostl, E, Aurich, C. 2013. Effects of the level of experience of horses and their riders on cortisol release, heart rate and heart rate variability during a jumping course. Anim. Welf. 22:457-465. 10.7120/09627286.22.4.457 Irvine, CHG, Alexander, SL. 1994. Factors affecting the circadian rhythm in plasma cortisol concentrations in the horse. Domest. Anim. Endocrinol. 11(2):227-238. https://doi.org/10.1016/0739-7240(94)90030-2 Kang, O-D, Lee W-S. 2016. Changes in salivary cortisol concentration in horses during different types of exercise . Asian-Australas J Anim Sci. 29(5):747-752. 10.5713/ajas.16.0009 Kang, O-D, Yun Y-M. 2016. Influence of Horse and Rider on Stress during Horse-riding Lesson Program. Asian Australas J Anim Sci. 29(6):895-900. 10.5713/ajas.15.1068 Leal, BB, Alves, GES, Douglas, RH, Bringel, B, Young, RJ, Haddad, JPA, Viana, WS, Faleiros, RR. 2011. Cortisol circadian rhythm ratio: A simple method to detect stressed horses at higher risk of colic? Equine Vet. J. 31:188-190. https://doi.org/10.1016/j.jevs.2011.02.005 Massanyi, P, Stawarz, R, Halo, M, Formicki, G, Lukac, N, Cupka, P, Schwarcz, P, Kovacik, A, Tusimova, E, Kovacik, J. 2014. Blood concentration of copper, cadmium, zinc and lead in the horses and its relation to hematological and biochemical parameters. J. Environ. Sci. Health A. 49:973-979. 10.1080/10934529.2014.894322 Peeters, M, Sulon J, Beckers J-F, Ledoux D, Vandenheede M. 2011. Comparison between blood serum and salivary cortisol concentrations in horses using an adrenocorticotropic hormone challenge . Equine Vet J. 43(4):487-493. 10.1111/j.2042-3306.2010.00294.x Schmidt, A, Mostl, E, Wehnert, Ch, Aurich, J, Muller, J, Aurich, Ch. 2010. Corisol release and heart rate variability in horses during road transport . Hormones Behaviour. 57: 209-215. 10.1016/j.yhbeh.2009.11.003 Strzelec, K, Kankofer, M, Piertzak, S. 2011. Cortisol concentration in the saliva of horses subjected to different kinds of exercise. Act Vet. Brno. 80:101-105. 10.2754/avb201180010101 Stull, CL, Morrow, J, Aldridge, BA, Stott, JL, McGlone, JJ. 2008. Immunophysiological responses of horses to 12-hour rest during 24 hours of road transport. Vet. Rec. 162:609-614. 10.1136/vr.162.19.609 Van der Kolk, JH, Nachreiner, RF, Schott HC, Refsal KR, Zanella AJ. 2001. Salivary and plasma concentration of cortisol in normal horses and horses with Cushing´s disease. Equine Vet J. 33(2):211-213. 10.1111/j.2042-3306.2001.tb00604.x

Trace mineral metabolism and nutrient digestibility in lambs supplemented with zinc sulfate during an adrenocorticotropic hormone challenge

The objective was to evaluate the effects of Zn supplementation and adrenocorticotropic hormone (ACTH) injections on nutrient digestibility, trace mineral metabolism and status. It was hypothesized that ACTH would decrease nutrient digestibility and Zn retention, but Zn supplementation would improve Zn retention, regardless of ACTH administration. Twenty-two Polypay cross weathers previously enrolled in a trial, consumed a moderately Zn deplete diet (22 mg Zn/kg DM) for 52 d, followed by being housed in metabolism crates for 15 d while receiving one of two diets: CON (Zn deplete diet) or ZINC (CON plus 40 mg Zn/d from ZnSO4). Lambs rested for one day outside of crates then returned to crates for this 5 d study. Lambs continued to receive dietary Zn treatments and were randomly assigned to receive injectable treatments, once daily on d 1–4 at feeding: SALINE (2 mL saline I.M.), STRESS (80 I.U. ACTH I.M.); lamb was experimental unit (n = 5–6/treatment) in a 2 × 2 factorial design. Total fecal and urine collection was conducted on d 3–5, and blood samples were collected on d 0, 1, and 4, 3 h post feeding. Day 4 plasma Zn concentrations, covariate adjusted with d 0 concentrations, displayed a diet × injection tendency (P = 0.06) where ACTH given to ZINC lambs depressed plasma Zn to values similar to CON lambs. Day 1 and 4 cortisol concentrations determined 3 h after injection administration were increased in STRESS vs. SALINE (P < 0.01). Day 4 plasma Cu concentrations, covariate adjusted with d 0 concentrations were lesser in STRESS vs. SALINE (P = 0.04). Day 4 plasma Fe concentrations were unaffected by diet or injection treatment (P ≥ 0.18). Apparent absorption of Zn was increased (P < 0.01) in CON vs. ZINC, but were similar in STRESS vs. SALINE (P = 0.13). Zinc retention, and apparent absorption of Mn and Cu was similar across treatments (P ≥ 0.19). Organic matter (OM) digestibility tended to be decreased in ZINC vs. SALINE (P = 0.07); neutral detergent fiber (NDF) and acid detergent fiber (ADF) digestibility were decreased (P ≤ 0.04) in ZINC vs. SALINE. Injection treatment had no effect on macronutrient digestibility (P ≥ 0.37). The CON lambs were marginally Zn deficient based on plasma Zn concentrations and rate of apparent absorption, while fiber digestion was impaired by Zn supplementation. Plasma cortisol concentrations indicate lambs receiving ACTH experienced physiological stress, however this had limited effects on macro or micronutrient metabolism.

Cortisol and Dehydroepiandrosterone Response to Adrenocorticotropic Hormone and Frailty in Older Women.

The response to adrenocorticotropic hormone (ACTH) is poorly characterized in old-old adults and may provide insight into the physiologic response to stress. We performed a standard 250 µg ACTH stimulation test in a home-based substudy of 51 women aged 85-96 years enrolled in the Women's Health and Aging Study II who were not taking corticosteroids. We examined the cortisol and dehydroepiandrosterone (DHEA) responses at 0, 30, 60, and 120 minutes, overall and by frailty status. The peak cortisol response to ACTH could not be determined, with the highest levels at the 120-minute time point. Pre- and post-ACTH stimulated cortisol levels did not differ by frailty status over this time frame, with no difference in the characteristics of the dose-response curves. Pre- and post-ACTH stimulated DHEA levels also did not differ by frailty status, though the dose-response curves suggested divergence after stimulation, with a more rapid DHEA response with increasing frailty. Our data demonstrate a robust cortisol response to ACTH challenge testing, but inadequate negative feedback in old-old women, resulting in prolonged exposure to cortisol. Future studies should examine dynamic cortisol and DHEA responses in this age group, using a less potent ACTH stimulus and longer collection period.

Sex and dose-dependent responses of urinary and faecal glucocorticoid metabolite concentrations following an ACTH challenge in eastern rock sengis (Elephantulus myurus)

Non-invasive methods for measuring glucocorticoids and their metabolites are frequently used in ecological, behavioural and physiological studies of mammals. Using faeces, urine and other matrices for such a measurement has considerable advantages in comparison to more traditional methods, but also requires thorough validation of the methods used. Eastern rock sengis (Elephantulus myurus) are fascinating African mammals and the non-invasive monitoring of the adrenocortical activity opens up new opportunities to study their biology. We were able to validate two assays for measuring urinary (uGCM) and faecal glucocorticoid metabolite (fGCM) concentrations in this species using a dose-dependent challenge with adrenocorticotropic hormone (ACTH). A higher concentration of ACTH elicited higher uGCM and fGCM concentrations in both males and females. Interestingly, uGCM and fGCM concentrations and the responses to ACTH were higher in females than in males and small changes in faecal glucocorticoid metabolites could not be reliably detected in males. In contrast to ACTH, a saline injection did not result in an increase in uGCM or fGCM concentrations. The study also provided insight into when responses to a stressor are likely to be detected in the urine and faeces of sengis and opens up new opportunities to study the stress physiology of this and other sengi species. It further emphasises the importance of thoroughly validating non-invasive methods for measuring hormones in both sexes of a species and for incorporating dose-dependent approaches.

Longitudinal fecal hormone monitoring of adrenocortical function in zoo housed fishing cats (Prionailurus viverrinus) during institutional transfers and breeding introductions.

The ex situ population of fishing cats (Prionailurus viverrinus) has become increasingly important for the conservation of this species. Unfortunately, captivity-induced stress is a concern and potential factor for lack of breeding success in this small felid, resulting in an unsustainable population. The objectives of this study were to: 1) validate an enzyme immunoassay for monitoring of fecal glucocorticoid metabolite (FGM) concentrations in the fishing cat; 2) identify potential exogenous stressors in the captive environment; 3) pinpoint management techniques that may lower FGM concentrations; and 4) determine if FGM concentrations are related to breeding success. Through a successful adrenocorticotrophic hormone challenge and additional laboratory methods, a cortisol enzyme immunoassay was validated as an effective tool for detecting FGM in this species. Between 2010 and 2013, longitudinal FGM monitoring was conducted in 26 fishing cats in the North American Species Survival Plan®. Exogenous stressors that elevated FGM concentrations included: chemical immobilizations; permanent transfers between facilities; construction; facility events; and fights/aggression among breeding pairs. Management factors that lowered FGM concentrations included: increased animal-keeper interaction through formal training; and providing indoor, off-exhibit, holding areas. In addition, social housing of individuals (either established breeding pairs or same sex pairs) decreased FGM concentrations. Individuals with breeding success (defined as observed copulations during the study period) also had lower FGM concentrations than unsuccessful individuals. Findings indicate that management factors play a role in lowering glucocorticoid (stress) levels in fishing cats, which may ultimately affect breeding success in the ex situ population.

Validation of a noninvasive technique to quantify stress in northern bobwhite (Colinus virginianus).

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.

279 Effects of pre-mortem stress on heat shock protein expression in the longissimus lumborum following harvest

Abstract Despite similar production practices, meat products from beef cattle exhibit undesirable variation in tenderness and stability of flavor and color. The objective of this study was to determine whether pre-mortem stress impacts expression of heat shock proteins (HSP) in the skeletal muscle following harvest. In this study, 40 Holstein steers underwent a 6 h adrenocorticotropic hormone (ACTH) challenge designed to emulate pre-mortem stress prior to harvest. Skeletal muscle biopsies from the longissimus lumborum were taken prior to the ACTH challenge. During the challenge, complete blood counts (CBC) were collected every 2 h and serum cortisol every 0.5 h from -2 to 6 h of the ACTH challenge. Following the 6 h ACTH challenge, skeletal muscle and blood samples were collected from 10 animals each harvested at 2, 12, 24 and 48 h post-challenge. Immediately following harvest, samples were collected from the longissimus lumborum and again after 14 d of aging. Protein expression of HSPβ1 was analyzed in skeletal muscle samples taken prior to the ACTH challenge, at harvest, and after 14 d of aging. Expression of HSPβ1 was different (P &amp;lt; 0.05) at harvest between animals that were harvested at different points following induction of stress where steers that were harvested at 12 h had decreased expression when compared to those harvested at 48 h. In addition, time of harvest had an effect (P &amp;lt; 0.01) on cortisol concentrations, hemoglobin, hematocrit, white blood cells, neutrophils, monocytes, and eosinophils. Number of platelets, lymphocytes, and basophils did not differ (P &amp;gt; 0.05) between animals harvested at different time points. These data demonstrate that HSPβ1 protein expression in the longissimus lumborumafter harvest may be related to pre-mortem stress.

Diagnosis and Management of Adrenal Insufficiency in Hospitalized Patients.

Plasma cortisol is commonly obtained in hospitalized hypotensive patients, and adrenocorticotropic hormone (ACTH) challenge is typically conducted to further workup hypocortisolemia. It is important to recognize that relative adrenal insufficiency (AI) is the most common cause of low cortisol levels and failed ACTH challenge in ill patients. Both cortisol and synthetic ACTH challenge assays are unreliable in critically ill patients. In clinical practice, corticosteroid therapy in septic shock patients results in immediate hemodynamic benefits with less vasopressor and ventilator dependence. There is no consensus about the diagnostic criteria of relative AI, appropriate cortisol level, and the dose used for synthetic ACTH challenge in patients with septic shock. There is controversy about the mortality benefits of supplemental steroid therapy and about the use of adjunctive fludrocortisone. PubMed search of randomized control trials and meta-analyses. Despite all the controversies, hospital physicians frequently use steroids in patients with septic shock with hypocortisolemia. Hydrocortisone should be the choice of steroid for most relative AI patients, and fludrocortisone can be added on a case-by-case basis in refractory shock. Most of the adverse effects induced by a short course of steroids are easily managed in the inpatient setting.

Non-invasive stress monitoring and temperament of chestnut-bellied seed-finch (Passeriformes, Thraupidae)

Songbirds are kept as pets around the world, and bird-singing contests occur in several countries. However, there is a lack of scientific support for songbird welfare and stress monitoring. Thus, we aimed to validate the measurement of glucocorticoid metabolites (GCM) concentrations in bird droppings as a way to assess endogenous adrenal activity in the chestnut-bellied seed-finch (Sporophila angolensis) by using an adrenocorticotropic hormone (ACTH) challenge. We also aimed to describe behaviors potentially indicative of stress and to evaluate the role of temperament in the expression of stress in this species. For the ACTH challenge, 12 seed-finch adult males were randomly submitted to one of the three treatments: S – 100 μL saline solution (control); D1 – 0.5 μg of ACTH; and D2 – 1.0 μg of ACTH. Total bird droppings were collected during pre-challenge and challenge days at intervals of four and two hours, respectively. We also quantified the total time birds spent on the following behavioral states: alertness, freezing, comfort, flying and foraging during the pre-challenge, challenge and post-challenge days. The birds’ temperament was assessed during three challenge tests involving novelty. The mean baseline GCM concentration, determined on the pre-challenge day, was 68.7 ± 20.2 ng/g of dry bird droppings. Birds that received treatments D1 and D2 showed an increase of 2.2 times in the GCM concentration 12 h after treatment injections. The chestnut-bellied seed-finch showed individual behavioral distinctiveness, which was correlated with the time they spent on foraging in response to acute stress. Our study showed that GCM concentration reflects endogenous adrenal activity in the chestnut-bellied seed-finch. However, this measure alone is not enough as an indicator of songbirds’ welfare. Therefore, the valences (positive or negative) of freezing, alertness, flying, and foraging behaviors can be used together with the physiological measure for noninvasive stress monitoring in S. angolensis.

Validation of water-borne cortisol and corticosterone in tadpoles: Recovery rate from an acute stressor, repeatability, and evaluating rearing methods

Amphibian populations are declining globally, so understanding how individuals respond to anthropogenic and environmental stressors may aid conservation efforts. Using a non-invasive water-borne hormone assay, we measured the release rates of two glucocorticoid hormones, corticosterone and cortisol, in Rio Grande Leopard frog, Rana berlandieri, tadpoles. We validated this method pharmacologically and biologically using an adrenocorticotropic hormone (ACTH) challenge, exposure to exogenous corticosterone, and an agitation test. We calculated the repeatability of hormone release rates, the recovery time from an acute stressor, and explored rearing methods for tadpoles. Tadpole corticosterone release rates increased following an ACTH challenge, exposure to exogenous corticosterone, and agitation, validating the use of water-borne hormone methods in this species. After exposure to an acute stressor via agitation, corticosterone release rates began to decline after 2 h and were lowest after 6 h, suggesting a relatively rapid recovery from an acute stressor. Tadpoles reared in groups had higher corticosterone release rates than tadpoles reared individually, and lost mass by Day 7, while tadpoles reared individually did not show a stress response, therefore either rearing method is viable, but have differing physiological costs for tadpoles. Repeatability of corticosterone release rates was moderate to high in R. berlandieri tadpoles, indicating that this species can show a response to selection and potentially respond to rapid environmental change. Our results show that the water-borne hormone assay is a viable way to measure glucocorticoids in this species and is useful in the field of conservation physiology for rare and endangered species.

The ACTH challenge and its repeatability in fattening bulls—influences of physiological state, challenge time standardization, and horn status

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 < 0.05) in ST bulls, and no effects were observed for AUCG between C1 and C2 in ST or AT bulls, nor for any AUC between C2 and C3 (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 < 0.01, ICC = 0.41) and lacked in AT bulls (AUCG: r = 0.07, 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 < 0.001; ICC = 0.53; AUCI: r = 0.58, P < 0.001, ICC = 0.52). Assignment to high and low responder groups based on peak cortisol concentration in C1 did not persist over time. H+ bulls showed higher AUCI in C2 and C3 (P < 0.1 and P < 0.05, respectively) than H− bulls. The M group differed from the H− group only in C3 (P < 0.05). Thus, the effect of horn status on ACTH challenges needs further investigation. In conclusion, our results report poor repeatability of the cortisol response to ACTH challenges for challenges performed in different physiological states and moderate repeatability for challenges performed within the same physiological state. Moreover, they point out the importance of standardization of TOD when performing repeated ACTH challenge.

Responsiveness of swine divergently selected for feed efficiency to exogenous adrenocorticotropic hormone and glucose challenges

Increasing the feed efficiency of lean tissue gains is an important goal for improving sustainable pork production and profitability for swine producers. To study feed efficiency, genetic selection based on residual feed intake (RFI) was used to create two divergent lines. Low-RFI pigs consume less feed for equal weight gain compared with their less-efficient, high-RFI counterparts. As cortisol and insulin are important energy control and growth regulators, our objective was to evaluate the role of the adrenocorticotropic hormone (ACTH)-cortisol and the glucose-insulin axes in pigs divergently selected for RFI. Adrenocorticotropic hormone (0.2 IU/kg BW)–stimulated cortisol and non-esterified fatty acids (NEFA) concentrations and intravenous glucose tolerance test (IVGTT; 0.25 g/kg BW)–stimulated glucose, insulin, and NEFA concentrations were assessed in six low-RFI and six high-RFI gilts (68 ± 5.2 kg). Before the ACTH challenge, low-RFI gilts tended to have less baseline plasma cortisol (P = 0.08) but no difference in NEFA concentrations (P = 0.63) compared with high-RFI gilts. After the ACTH challenge, low-RFI gilts had less cortisol (P = 0.04) and NEFA concentrations (P = 0.05) compared with high-RFI gilts. Glucose, insulin, and NEFA concentrations did not differ between genetic lines before the IVGTT. After glucose infusion, low-RFI gilts had greater insulin concentrations (P = 0.003) but did not differ in glucose or NEFA concentrations compared with high-RFI gilts. These results indicate that genetic selection for reduced RFI (improved feed efficiency) resulted in less stress responsiveness and an increase in insulin after glucose infusion. These data have implications for identifying and selecting more feed efficient pigs and for understanding the physiological mechanisms underlying feed efficiency.

55 Apparent absorption, zinc retention and nutrient digestibility in lambs during an adrenocorticotropic hormone challenge.

This study sought to determine if Zn supplementation would increase Zn retention and nutrient digestibility in lambs exposed to adrenocorticotropic hormone. Twenty-two crossbred poly-pay wethers (n = 5–6/treatment) enrolled in a previous trial had been stagger started on a common diet (22 mg Zn/kg DM) for 52-d, and were placed in metabolism crates for 15-d of dietary treatments: CON: common diet + no supplemental Zn; ZINC: CON + 40 mg/d ZnSO4 top dressed daily in fine ground corn. On d16 lambs were assigned within diet to injected challenge treatments: Saline: 2 mL saline; ACTH: 80 IU adrenocorticotropic hormone. On d17 lambs were placed in crates to facilitate fecal and urine collection, and received injections at 0700h for d17-20. Plasma cortisol was assessed on d16 3h post-feeding, and 3h after injections 1 (d17) and 4 (d20). Data were analyzed as a 2 × 2 factorial; no interactions were found (P ≥ 0.09). Plasma cortisol was greater in ACTH post-injection 1 and 4 (P ≤ 0.002). Zinc intake was greater in ZINC (P < 0.001). Zinc apparent absorption was greater in CON (16.0) vs. ZINC (7.04%; P = 0.002) and tended (P = 0.12) to be lesser in ACTH (9.57%) vs. Saline (13.5%). Day-20 plasma Zn was lesser in CON (0.67) vs. ZINC (0.78 mg/L; P = 0.03). Digestibility was greater in CON vs. ZINC for NDF (45.8% vs. 41.8%) and ADF (33.3% vs. 26.7%; P ≤ 0.03). Dry matter and OM digestibility tended (P ≤ 0.09) to be greater in CON compared to ZINC. Retained Zn was unaffected (P ≥ 0.24) by diet (CON: 4.09 mg/d; ZINC: 4.29 mg/d) or injection (Saline: 4.79 mg/d; ACTH: 3.59 mg/d) suggesting that Zn requirements in lambs may not increase in response to ACTH. Nutrient digestibility may also be affected by supplementation with ZnSO4.