Exploring the effect of auditory stimuli on activity levels, milk yield and faecal glucocorticoid metabolite concentrations in Holstein cows.

Health and welfare are inextricably linked within efficient and sustainable dairy production, and several potential risk factors may affect the well-being of dairy cows, including chronic stress. Although auditory stimuli could be used as a tool to decrease the potential stress that cows might experience, it is seldom applied to livestock production systems due to the perception that enrichment is an unnecessary expense. This study aimed to explore the effect of auditory stimuli as a form of enrichment in a Holstein herd by monitoring fecal glucocorticoid metabolite (fGCM) concentrations (a non-invasive, stress-associated biomarker). Cow activity level and milk yield were also measured. Nine cows in their second and third lactation were divided into 3 groups, using a Latin Square experimental design, exposing each cow group to each of the 3 treatments, namely constant exposure (CE), limited exposure (LE), and no exposure (NE) to classical music. FGCMs were quantified using a group-specific enzyme immunoassay detecting 11,17-dioxoandrostanes. Compared to LE and NE animals, cows exposed to constant music had significantly lower fGCM concentrations (P = 0.012), as well as higher milk yields (P < 0.0001) and lowered activity levels during the morning (P = 0.005) and the evening activity period (P = 0.048). These findings indicate that auditory stimuli in the form of classical music may have a positive effect on the welfare of cows as well as milk yield, which hold economic benefits for the producer and potentially reduces the number of cows needed for profitable production.

The endocrine control of reproductive suppression in an aseasonally breeding social subterranean rodent, the Mahali mole-rat (Cryptomys hottentotus mahali).

Cooperative behaviour, sociality and reproductive suppression in African mole-rats have been extensively studied. Nevertheless, endocrine correlates of some species of social mole-rats have been neglected, and these species may hold the key to understanding the behavioural and physiological complexity that allows the maintenance of social groups in African mole-rats. In this study, we investigated endocrine correlates implicated in the suppression of reproduction and cooperative behaviours, namely glucocorticoids (a stress-related indicator) through faecal glucocorticoid metabolites (fGCMs), plasma testosterone (an indicator of aggression) and plasma prolactin in the Mahali mole-rat (Cryptomys hottentotus mahali) across reproductive classes (breeding females and males, non-breeding females and males) and season (wet and dry). Breeders possessed higher levels of testosterone than non-breeders. In reproductively suppressed non-breeding females, fGCMs were significantly higher than in breeders. Furthermore, an adrenocorticotropic hormone stimulation test (ACTH challenge test) on both male and female non-breeders revealed that female non-breeders show a more significant response to the ACTH challenge than males. At the same time, plasma prolactin levels were equally elevated to similar levels in breeding and non-breeding females. Chronically high levels of prolactin and fGCM are reported to cause reproductive suppression and promote cooperative behaviours in non-breeding animals. Furthermore, there was a negative relationship between plasma prolactin and progesterone in non-breeding females. However, during the wet season, a relaxation of suppression occurs through reduced prolactin which corresponds with elevated levels of plasma progesterone in non-breeding females. Therefore, prolactin is hypothesised to be the primary hormone controlling reproductive suppression and cooperative behaviours in non-breeding females. This study provides new endocrine findings for the maintenance of social suppression in the genus Cryptomys.

A non-invasive assessment of essential trace element utilization at different trophic levels in African wildlife.

The complex relationships that exist between terrestrial mammals and their habitats make African ecosystems highly interactive environments. Anthropogenic activities including climate change have altered geochemical cycles, which influence nutrient availability and deficiency at local, regional and global scales. As synergistic and antagonistic interactions occur between essential elements at both deficiency and excess concentrations, the differences in feeding strategy between trophically distinct groups of terrestrial vertebrates are likely to influence the degree to which overall nutrient needs are met or may be deficient. The overall aim of this study was to investigate and compare quantitative differences of nine essential elements in terrestrial vertebrates occupying different trophic levels within two protected areas; Tswalu Kalahari Reserve (TKR) and Manyeleti Nature Reserve (MNR) South Africa, using faeces as an analytical matrix. Results from linear mixed effects models highlight that concentrations varied widely between individuals. Overall, measured concentrations above their respective means were evident for B and Mn in herbivores, Fe in omnivores and Cu, Co, Fe, Se and Zn in carnivores. Measured concentrations of Mo and Ni did not differ significantly between trophic groups. Although site-specific differences were evident for specific elements, measured mean concentrations of B, Co, Cu, Fe, Mo, Ni, Se and Zn were significantly higher overall at the MNR study site compared to the TKR site. This is the first study to non-invasively assess essential element concentrations across trophic levels in free ranging African wildlife species within protected areas of the savannah biome. Combined with the assessment of environmental matrices, this approach can be used as an effective diagnostic tool for the assessment of animal welfare and the management of protected areas globally.

Non-invasive hormone monitoring as a robust method for determining adrenocortical activity in injured, emaciated and oil-contaminated African penguins undergoing rehabilitation.

Anthropogenic activity is a major driver of seabird injury and mortality in the 21st century. Although most seabirds perish within the natural environment as a result of human activities, some are rescued and admitted to rehabilitation centres. Despite the considerable number of admissions, little is known regarding the physiological response seabirds have to specific admission reasons and the rehabilitation process. In this study, we aimed to determine the effect of injury, emaciation, oiling, individual removal from the natural environment and the rehabilitation process on the physiological stress response of the African penguin (Spheniscus demersus). Urofaecal samples were collected from African penguins throughout a three-stage rehabilitation process and quantified for glucocorticoid metabolites (ufGCM). The three stages included an initial ICU crate stage (Stage 1), an ICU pen stage (Stage 2) and a rehabilitation stage (Stage 3). Data were analysed using a generalised linear model in order to determine the effect of admission reason, age and rehabilitation stage (Stage 1, 2 and 3) on ufGCM levels. Although the model indicated that only Stage 1 was a significant driver of adrenocortical activity in the study population, ufGCM levels of injured and emaciated animals within Stage 1 were considerably higher than those of birds with any other admission reason across all stages. This is the first study examining the causes and effect of rehabilitation on the physiological stress response in African penguins. Enhanced care and attention should be given to rescued individuals, especially during the first stage of rehabilitation, to reduce perception of additional stressors and thus increase the chance of full recovery.

Monitoring glucocorticoid metabolite concentrations as a proxy of environmental stress across important life-history stages in captive African penguins.

Due to considerable global decline in wildlife population numbers and species diversity, because of various anthropogenic activities, conservationists increasingly rely on captive and managed populations as important reservoirs to ensure the survival of endangered and vulnerable species. However, very few of these facilities implement robust, non-invasive monitoring techniques to confirm the effectiveness of their management practices to address animal welfare challenges. This study assessed adrenocortical activity as an indication of environmental stress by investigating the effects of both natural (climate, life-history stages) and anthropogenic (visitor presence) factors on captive-housed African penguins. Seven male-female African penguin breeding pairs were housed in a large, naturalistic outside enclosure at the National Zoological Garden (NZG), South Africa. Weekly urofaecal samples were collected from all individuals over one-year to measure urofaecal glucocorticoid metabolite (ufGCM) concentrations. General linear mixed model analysis determined that visitor presence (for males) and rainfall (for females) were the two factors which best explained the variation in ufGCM concentrations of the study population; however, none of the environmental and anthropogenic factors monitored were found to be significant. A posthoc graphical analysis showed considerable individual variation in terms of ufGCM concentrations within and between sexes when comparing life-history stages. This study confirms that non-invasive steroid monitoring can be an effective tool set for defining and assessing environmental stressors for African penguins and potentially other captive seabirds. However, conservationists and wildlife managers should also consider that individual-, sex-, and population-specific differences in the response to environmental stressors can exist. As such, a generalized management protocol for a specific species may not be sufficient and should be customized according to the specific captive population and/or individual.

Evidence for contrasting roles for prolactin in eusocial naked mole-rats, Heterocephalus glaber and Damaraland mole-rats, Fukomys damarensis.

Elevated prolactin (PRL) has been associated with the expression of social and cooperative behaviours in a number of vertebrate species, as well as suppression of reproduction. As social mole-rats exhibit both of these traits, PRL is a prime candidate in mediating their social phenotype. While naked and Damaraland mole-rats (NMRs and DMRs) have evolved eusociality independently within their family, both species exhibit an extreme skew in lifetime reproductive success, with breeding restricted to a single female and one or two males. Non-breeding NMRs of both sexes are physiologically inhibited from reproducing, while in DMRs only the non-breeding females are physiologically suppressed. Newly emerging work has implicated the dopamine system and PRL as a component in socially induced reproductive suppression and eusociality in NMR, but the DMR remains unstudied in this context. To investigate evolutionary convergence in the role of PRL in shaping African mole-rat eusociality, we determined plasma PRL concentrations in breeders and non-breeders of both sexes, comparing DMRs with NMRs. Among samples from non-breeding NMRs 80% had detectable plasma PRL concentrations. As a benchmark, these often (37%) exceeding those considered clinically hyperprolactinaemic (25 ng ml-1) in humans: mean ± s.e.m.: 34.81 ± 5.87 ngml-1; range 0.00-330.30 ng ml-1 Conversely, 85% of non-breeding DMR samples had undetectable values and none had concentrations above 25 ng ml-1: 0.71 ± 0.38 ng ml-1; 0.00-23.87 ngml-1 Breeders in both species had the expected variance in plasma PRL concentrations as part of normal reproductive function, with lactating queens having significantly higher values. These results suggest that while elevated PRL in non-breeders is implicated in NMR eusociality, this may not be the case in DMRs, and suggests a lack of evolutionary convergence in the proximate control of the social phenotype in these mole-rats.

Plains zebra (Equus quagga) adrenocortical activity increases during times of large aggregations in the Serengeti ecosystem.

Adverse environmental stimuli (stressors) activate the hypothalamic-pituitary-adrenal axis and contribute to allostatic load. This study investigates the contribution of environmental stressors and life history stage to allostatic load in a migratory population of plains zebras (Equus quagga) in the Serengeti ecosystem, in Tanzania, which experiences large local variations in aggregation. We expected higher fGCM response to the environmental stressors of feeding competition, predation pressure and unpredictable social relationships in larger than in smaller aggregations, and in animals at energetically costly life history stages. As the study was conducted during the 2016 El Niño, we did not expect food quality of forage or a lack of water to strongly affect fGCM responses in the dry season. We measured fecal glucocorticoid metabolite (fGCM) concentrations using an enzyme immunoassay (EIA) targeting 11ß-hydroxyetiocholanolone and validated its reliability in captive plains zebras. Our results revealed significantly higher fGCM concentrations 1) in large aggregations than in smaller groupings, and 2) in band stallions than in bachelor males. Concentrations of fGCM were not significantly higher in females at the energetically costly life stage of late pregnancy/lactation. The higher allostatic load of stallions associated with females, than bachelor males is likely caused by social stressors. In conclusion, migratory zebras have elevated allostatic loads in large aggregations that probably result from their combined responses to increased feeding competition, predation pressure and various social stressors. Further research is required to disentangle the contribution of these stressors to allostatic load in migratory populations.

Age and socially related changes in fecal androgen metabolite concentrations in free-ranging male giraffes.

In many mammal species, androgen levels in males are elevated during periods of mating activity, often to facilitate aggressive behavior between males over access to fertile females. However, this pattern might be less obvious in species with a rather low male-male aggression rate, or in those that are not strictly seasonal breeders. A complex social structure, as well as additional social and environmental factors, might add more to the complexity. Here, we applied a non-invasive method to monitor fecal androgen metabolite (fAM) levels in free-ranging giraffe bulls over a period of months to examine longitudinal patterns of androgen metabolite concentrations in relation to observed male sexual behavior in different age classes. Giraffes are non-seasonal breeders, living in a fission-fusion social system and males show a roaming strategy to search for fertile females. Our results show that season has an impact on fAM levels in free-ranging giraffes, with respective steroid concentrations being higher in summer. In the presence of females, fAM levels of bulls are significantly higher compared to when found in all-male groups, with old adult bulls showing the highest fAM levels. In contrast, young adult bulls have overall slightly higher fAM levels compared to old adult bulls when residing in all male groups. Sexual behavior increases fAM levels only in old adult bulls.

The impact of age-class and social context on fecal glucocorticoid metabolite levels in free-ranging male giraffes.

One of the primary sources of perceived stress is the social environment of an animal and the interactions with conspecifics. An essential component of the response to a stressor is the activation of the hypothalamic-pituitary-adrenocortical axis, which results amongst others in a temporal increase in circulating glucocorticoid (GC) levels. Giraffes occur in a highly flexible fission-fusion social system and group compositions can change on a daily basis, with bulls establishing an age-related dominance hierarchy and showing a roaming strategy in the search for fertile females. The aim of this study was to non-invasively monitor the influence of different group compositions (mixed sex groups vs. all-male groups) on GC concentrations in free ranging giraffe bulls of different age classes. We collected fecal samples from free-ranging giraffe bulls for 12months in a South African Private Game Reserve to examine age- and social context-related patterns of fecal GC metabolite (fGCM) concentrations. We found that fGCM levels in giraffe bulls are age-class dependent, as well asassociated with changes in the social environment. Independently of the social setting, bulls of the youngest age class exhibited the highest fGCM levels compared to bulls of the other two older age-classes, with differences most pronounced when the bulls are associated in all-male groups. In contrast, an almost reversed picture appears when looking at the fGCM levels of sexually active individuals in mixed sex groups, where highest levels were found for the bulls in the oldest age-class, and the lowest for the bulls in the youngest age-class. The study stresses the importance to taking factors such asage-related status and social settings into account, when interpreting fGCM levels in free ranging giraffes.

A Bayesian latent class model to estimate the accuracy of pregnancy diagnosis by transrectal ultrasonography and laboratory detection of pregnancy-associated glycoproteins in dairy cows.

Accurate diagnosis of pregnancy is an essential component of an effective reproductive management plan for dairy cattle. Indirect methods of pregnancy detection can be performed soon after breeding and offer an advantage over traditional direct methods in not requiring an experienced veterinarian and having potential for automation. The objective of this study was to estimate the sensitivity and specificity of pregnancy-associated glycoprotein (PAG) detection ELISA and transrectal ultrasound (TRUS) in dairy cows of South Africa using a Bayesian latent class approach. Commercial dairy cattle from the five important dairy regions in South Africa were enrolled in a short-term prospective cohort study. Cattle were examined at 28-35days after artificial insemination (AI) and then followed up 14days later. At both sampling times, TRUS was performed to detect pregnancy and commercially available PAG detection ELISAs were performed on collected serum and milk. A total of 1236 cows were sampled and 1006 had complete test information for use in the Bayesian latent class model. The estimated sensitivity (95% probability interval) and specificity for PAG detection serum ELISA were 99.4% (98.5, 99.9) and 97.4% (94.7, 99.2), respectively. The estimated sensitivity and specificity for PAG detection milk ELISA were 99.2% (98.2, 99.8) and 93.4% (89.7, 96.1), respectively. Sensitivity of veterinarian performed TRUS at 28-35days post-AI varied between 77.8% and 90.5% and specificity varied between 94.7% and 99.8%. In summary, indirect detection of pregnancy using PAG ELISA is an accurate method for use in dairy cattle. The method is descriptively more sensitive than veterinarian-performed TRUS and therefore could be an economically viable addition to a reproductive management plan.

Capture of farmed Nile crocodiles (Crocodylus niloticus): comparison of physiological parameters after manual capture and after capture with electrical stunning.

The electric stunner (e-stunner) is commonly used to handle Nile crocodiles (Crocodylus niloticus) on commercial farms in South Africa, but while it seems to improve handling and safety for the keepers, no information regarding physiological reactions to e-stunning is currently available. The aim of this study was therefore to compare various physiological parameters in farmed C niloticus captured either manually (noosing) or by using an e-stunner. A total of 45 crocodiles were captured at a South African farm by either e-stunning or noosing, and blood samples were taken immediately as well as four hours after capture. Parameters monitored were serum corticosterone, lactate, glucose, as well as alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase and creatine kinase. Lactate concentrations were significantly higher in noosed compared with e-stunned animals (P<0.001). No other blood parameter differed significantly between the two methods of capture. In addition, recorded capture time confirmed that noosing takes significantly longer time compared with e-stunning (P<0.001), overall indicating that e-stunning seems to be the better option for restraint of especially large numbers of crocodiles in a commercial setup because it is quicker, safer and did not cause a significant increase in any of the parameters measured.

Characterisation and validation of an enzyme-immunoassay for the non-invasive assessment of faecal glucocorticoid metabolites in cheetahs (Acinonyx jubatus).

The non-invasive measurement of adrenocortical function in cheetahs is an important tool to assess stress in captive and free-ranging individuals, because stress has been suggested to be one of the causes of poor reproductive performance of captive cheetahs. We tested four enzyme immunoassays (EIA) in two captive cheetahs in Germany using adrenocorticotropic hormone (ACTH) challenges and identified the corticosterone-3-CMO EIA to be most sensitive to the increase in faecal glucocorticoid metabolite (fGCM) concentrations after the ACTH challenge. This EIA performed also well in five captive cheetahs in South Africa. The fGCM concentrations across all seven cheetahs increased within 24h by 681% compared to the baseline levels prior to ACTH. Storage of faecal samples at 0-4°C did not strongly affect fGCM concentrations within 24h, simplifying sample collection when immediate storage at -20°C is not feasible. The two cheetahs in Germany also received an injection of [(3)H]cortisol to characterise fGCMs in faecal extracts using high-performance liquid chromatography (HPLC) immunograms. HPLC fractions were measured for their radioactivity and immunoreactive fGCM concentrations with the corticosterone-3-CMO EIA, respectively. The results revealed a polar peak of radiolabelled cortisol metabolites co-eluting with the major peak of immunoreactive fGCMs. Thus, our EIA measured substantial amounts of fGCMs corresponding to the radioactive peaks. The peaks were of higher polarity than native cortisol and corticosterone, suggesting that the metabolites were conjugated, which was confirmed by solvolysis of the HPLC fractions. Our results show that the corticosterone-3-CMO EIA is a reliable tool to assess fGCMs in cheetahs.

Fecal 20-oxo-pregnane concentrations in free-ranging African elephants (Loxodonta africana) treated with porcine zona pellucida vaccine.

Because of overpopulation of African elephants in South Africa and the consequent threat to biodiversity, the need for a method of population control has become evident. In this regard, the potential use of the porcine zona pellucida (pZP) vaccine as an effective means for population control is explored. While potential effects of pZP treatment on social behavior of African elephants have been investigated, no examination of the influence of pZP vaccination on the endocrine correlates in treated females has been undertaken. In this study, ovarian activity of free-ranging, pZP-treated African elephant females was monitored noninvasively for 1 yr at Thornybush Private Nature Reserve, South Africa, by measuring fecal 5α-pregnan-3ß-ol-20-on concentrations via enzyme immunoassay. A total of 719 fecal samples from 19 individuals were collected over the study period, averaging 38 samples collected per individual (minimum, maximum: 16, 52). Simultaneously, behavioral observations were made to record the occurrence of estrous behavior for comparison. Each elephant under study showed 5α-pregnan-3ß-ol-20-on concentrations rising above baseline at some period during the study indicating luteal activity. Average 5α-pregnan-3ß-ol-20-on concentrations were 1.61 ± 0.46 µg/g (mean ± SD). Within sampled females, 42.9% exhibited estrous cycles within the range reported for captive African elephants, 14.3% had irregular cycles, and 42.9% did not appear to be cycling. Average estrous cycle duration was 14.72 ± 0.85 wk. Estrous behavior coincided with the onset of the luteal phase and a subsequent rise in 5α-pregnan-3ß-ol-20-on concentrations. Average 5α-pregnan-3ß-ol-20-on levels positively correlated with rainfall. No association between average individual 5α-pregnan-3ß-ol-20-on concentrations or cyclicity status with age or parity were detected. Earlier determination of efficacy was established via fecal hormone analysis with no pregnancies determined 22 mo post-treatment and onward. Results indicate the presence of ovarian activity amongst pZP-treated female African elephants in 2 yr after initial immunization. Further study should now be aimed toward investigating the long-term effects of pZP vaccination on the reproductive function of female African elephants.

Non-invasive monitoring of glucocorticoid metabolites in brown hyaena (Hyaena brunnea) feces.

The brown hyaena (Hyaena brunnea) is the least known of the large predators of southern Africa. The current IUCN status of the brown hyaena is "Near Threatened", and there are conservation concerns related to a general lack of biological knowledge of the species. For instance, a better knowledge of the responses to environmental and social stressors would improve our abilities to sustainably manage brown hyaena populations in both captive and free-ranging environments. We conducted adrenocorticotrophic hormone (ACTH) challenges in one female and one male adult brown hyaena at Lion Park Zoo, South Africa, to validate measurements of glucocorticoid metabolites (GCM) in brown hyaena feces via an enzyme immunoassay (EIA). We also measured gastrointestinal transit times (GIT times) and the GCM degradation in feces left in ambient temperature for up to 32 hr to more reliably assess the use of this assay as a tool for non-invasive glucocorticoid measurements. Intramuscular injections of synthetic ACTH yielded GCM levels of 388% (female) and 2,682% (male) above baseline with peak increases occurring 25- to 40-hr after injection. The time delay of fecal GCM excretion approximately corresponded with food transit time in the brown hyaenas. Fecal GCM levels declined significantly over time since defecation. Our results provided a good validation that fecal GCMs accurately reflect circulating glucocorticoid stress hormones in brown hyaenas, but we highlight that samples have to be frozen immediately after defecation to avoid bias in the measurements as a result of bacterial degredation.

Comparison of different enzyme-immunoassays for assessment of adrenocortical activity in primates based on fecal analysis.

Most studies published to date that used fecal glucocorticoid measurements to assess adrenocortical activity in primate (and many nonprimate) species applied a specific cortisol or corticosterone assay. However, since these native glucocorticoids are virtually absent in the feces of most vertebrates, including primates, the validity of this approach has recently been questioned. Therefore, the overall aim of the present study was to assess the validity of four enzyme-immunoassays (EIAs) using antibodies raised against cortisol, corticosterone, and reduced cortisol metabolites (two group-specific antibodies) for assessing adrenocortical activity using fecal glucocorticoid metabolite (GCM) measurements in selected primate species (marmoset, long-tailed macaque, Barbary macaque, chimpanzee, and gorilla). Using physiological stimulation of the hypothalamo-pituitary-adrenocortical (HPA) axis by administering exogenous ACTH or anesthesia, we demonstrated that at least two assays detected the predicted increase in fecal GCM levels in response to treatment in each species. However, the magnitude of response varied between assays and species, and no one assay was applicable to all species. While the corticosterone assay generally was of only limited suitability for assessing glucocorticoid output, the specific cortisol assay was valuable for those species that (according to high-performance liquid chromatography (HPLC) analysis data) excreted clearly detectable amounts of authentic cortisol into the feces. In contrast, in species in which cortisol was virtually absent in the feces, group-specific assays provided a much stronger signal, and these assays also performed well in the other primate species tested (except the marmoset). Collectively, the data suggest that the reliability of a given fecal glucocorticoid assay in reflecting activity of the HPA axis in primates clearly depends on the species in question. Although to date there is no single assay system that can be used successfully across species, our data suggest that group-specific assays have a high potential for cross-species application. Nevertheless, regardless of which GC antibody is chosen, our study clearly reinforces the necessity of appropriately validating the respective assay system before it is used.

Non-invasive assessment of adrenocortical function in the male African elephant (Loxodonta africana) and its relation to musth.

Adult male elephants periodically show the phenomenon of musth, a condition associated with increased aggressiveness, restlessness, significant weight reduction and markedly elevated androgen levels. It has been suggested that musth-related behaviours are costly and that therefore musth may represent a form of physiological stress. In order to provide data on this largely unanswered question, the first aim of this study was to evaluate different assays for non-invasive assessment of adrenocortical function in the male African elephant by (i) characterizing the metabolism and excretion of [3H]cortisol (3H-C) and [14C]testosterone (14C-T) and (ii) using this information to evaluate the specificity of four antibodies for determination of excreted cortisol metabolites, particularly with respect to possible cross-reactions with androgen metabolites, and to assess their biological validity using an ACTH challenge test. Based on the methodology established, the second objective was to provide data on fecal cortisol metabolite concentrations in bulls during the musth and non-musth condition. 3H-C (1 mCi) and 14C-T (100 microCi) were injected simultaneously into a 16 year old male and all urine and feces collected for 30 and 86 h, respectively. The majority (82%) of cortisol metabolites was excreted into the urine, whereas testosterone metabolites were mainly (57%) excreted into the feces. Almost all radioactive metabolites recovered from urine were conjugated (86% 3H-C and 97% 14C-T). In contrast, 86% and >99% of the 3H-C and 14C-T metabolites recovered from feces consisted of unconjugated forms. HPLC separations indicated the presence of various metabolites of cortisol in both urine and feces, with cortisol being abundant in hydrolysed urine, but virtually absent in feces. Although all antibodies measured substantial amounts of immunoreactivity after HPLC separation of peak radioactive samples and detected an increase in glucocorticoid output following the ACTH challenge, only two (in feces against 3alpha,11-oxo-cortisol metabolites, measured by an 11-oxo-etiocholanolone-EIA and in urine against cortisol, measured by a cortisol-EIA) did not show substantial cross-reactivity with excreted 14C-T metabolites and could provide an acceptable degree of specificity for reliable assessment of glucocorticoid output from urine and feces. Based on these findings, concentrations of immunoreactive 3alpha,11-oxo-cortisol metabolites were determined in weekly fecal samples collected from four adult bulls over periods of 11-20 months to examine whether musth is associated with increased adrenal activity. Results showed that in each male levels of these cortisol metabolites were not elevated during periods of musth, suggesting that in the African elephant musth is generally not associated with marked elevations in glucocorticoid output. Given the complex nature of musth and the variety of factors that are likely to influence its manifestation, it is clear, however, that further studies, particularly on free-ranging animals, are needed before a possible relationship between musth and adrenal function can be resolved. This study also clearly illustrates the potential problems associated with cross-reacting metabolites of gonadal steroids in EIAs measuring glucocorticoid metabolites. This has to be taken into account when selecting assays and interpreting results of glucocorticoid metabolite analysis, not only for studies in the elephant but also in other species.