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

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Abstract

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 24 h 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 24 h, simplifying sample collection when immediate storage at −20 °C is not feasible. The two cheetahs in Germany also received an injection of [3H]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.

Graphical abstract

We developed an enzyme immunoassay (EIA) based on an antibody directed against corticosterone-3-CMO which is a reliable tool to assess faecal lucocorticoid metabolites (fGCM) in cheetahs. Faecal GCM concentrations measured with the corticosterone-3-CMO assay before and after ACTH administration in three female and four male cheetahs. Concentrations are expressed as % (mean ± SEM) from the pre-challenge baseline.

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Highlights

► We tested four enzyme immunoassays (EIA) in two captive cheetahs to monitor adrenocortical activity. ► The assay comparison was carried out based on two ACTH challenges. ► The corticosterone-3-CMO EIA can be used to reliably assess faecal glucocorticoid (fGM) concentrations in cheetahs. ► A radiometabolism study used to identify fGM revealed a polar peak of radiolabelled cortisol metabolites. ► We suggest that these metabolites were conjugated, because solvolysis released free, unpolar metabolites.

Introduction

The number of cheetahs (Acinonyx jubatus) in the wild are diminishing rapidly which is mainly a consequence of habitat loss and a declining prey base [17]. Thus, captive breeding of cheetahs might become increasingly important as an insurance measure, facilitating re-introductions in the wild once conditions for cheetahs have improved. However, whereas reproductive performance of free-ranging cheetahs is high [16], [29], cheetahs in captivity reproduce rather poorly [18], [32].

Numerous studies in zoos and in free-ranging populations investigated possible reasons for this phenomenon [5], [14], [19], [20], [27]. Recently, Wachter et al. [29] tested the predictions of four hypotheses and demonstrated that the “asymmetric reproductive aging” hypothesis was the most likely explanation. This hypothesis predicts rapid aging of the inner reproductive organs associated with the early development of pathologies in older, nulliparous captive females compared to younger nulliparous captive females or young and old multiparous free-ranging females [11], [12]. In captivity, females often start to reproduce later than in the wild which diminishes their reproductive performance, as also found in other species [11], [12].

Previously published findings on the possible impact of stressors that may impair reproductive performance in female cheetahs are inconsistent. Wachter et al. [29] used ultrasonography to assess the potential extent of chronic stress in free-ranging and captive cheetahs in Namibia and demonstrated similar sizes of the adrenal glands in both study groups, with little indication that they were enlarged, suggesting that – if anything – levels of stress were at best moderate and similar for both conditions. They described substantial pathologies in the inner reproductive organs in the captive individuals but found little evidence of such anomalies in free-ranging animals. On the other hand, female cheetahs in North American zoos had significantly higher stress levels than free-ranging cheetahs in Namibia or cheetahs kept in large enclosures in their natural Namibian environment, as measured by the size of their adrenal cortices and faecal corticoid metabolite concentrations [26], [27]. Without reliable measurements of stress levels in captive cheetah females on a regular basis and a large scale the conclusion that in zoos stress might complement asymmetric reproductive aging in depressing reproductive performance of cheetah females should remain tentative.

The development of immunoassays to measure faecal steroid metabolites in domestic and non-domestic felids provides a useful approach to non-invasively assess an individual’s endocrine status. Steroid metabolism studies in domestic cats demonstrated that the majority of steroid excretion occurs mainly via faeces rather than urine [4], [9], [23]. Steroid metabolites in faeces represent a pooled value of steroids excreted by the adrenal glands during the previous 12–24 h (depending on defaecation rates), thus are less affected by episodic fluctuations or the pulsatility of hormone secretion and are therefore useful to evaluate the adrenal activity in felids in an integrated manner.

The development of immunoassays to measure faecal steroid metabolites in domestic and non-domestic felids provides a useful approach to non-invasively assess an individual’s endocrine status but need to be validated for each species and each hormone, respectively, because they are often based on antibodies generated against the native steroid but with unknown cross-reactivities with the excreted steroid metabolites. Steroid metabolism by the liver as well as microbial impact during the intestinal passage, and re-absorption into the enterohepatic circulation generate a vast number of faecal steroid metabolites, different in even closely related species [10], [21]. In addition, metabolism of stress hormones such as cortisol, and gonadal hormones such as testosterone may lead to faecal metabolites with similar structures because the native hormones have similar structures. Thus, immunoassays designed to assess stress by measuring the concentration of faecal glucocorticoid metabolites (fGCM) may cross-react with other steroid metabolites, distorting the results. To ensure that a proposed immunoassay reliably measures an animal’s endocrine status, it should be experimentally demonstrated that the concentrations of fGCMs increase with defined stressors experienced by the animal. The most widely used approach is to stimulate the adrenocortical activity with an adrenocorticotropic hormone (ACTH) challenge test. If the assay works well, there should be a sharp increase of fGCM concentrations with a lag time proportional to the defaecation rate after ACTH application. Furthermore, to characterise the species-specific predominance of fGCMs and to identify the affinity of the antibody to these fGCMs, an injection of 3H labelled cortisol and high-performance liquid chromatography (HPLC) immunograms are a useful approach (e.g. [2]). Comparing the pattern of excreted radiolabelled metabolites with the metabolites produced after an ACTH challenge reveals the major fGCMs for a particular species and the fit of the respective immunoassay.

For cheetahs several RIAs and one EIA validated by ACTH challenges are available [14], [25], [30], [33], but no comprehensive characterisation of the fGCMs and the exact affinity of the immunoassays were conducted, or the performances of the different EIAs compared. Our first aim was therefore to test four available EIAs and to identify the most reliable antibody for accurately assessing adrenal status with a non-radioactive immunoassay in the cheetah by conducting ACTH challenges. Our second aim was to characterise the fGCMs and measure their relative abundance in the best assay by performing a radiometabolism study and subsequent HPLC analyses. Owing to logistical reasons, especially in the field, faecal samples can be often only kept cooled in an ice box rather than be deep frozen immediately after defaecation. Our third aim was therefore to examine the stability of fGCM concentrations when faeces are stored for variable amounts periods of time at temperatures of 0–4 °C.

Section snippets

Study animals and housing conditions

Four adult male cheetahs and three adult female cheetahs were involved in this study. One male and one female were kept in German zoos in Münster and Wuppertal, respectively, whereas the other cheetahs were kept at the Ann van Dyk cheetah centre, North West Province, South Africa. The cheetahs in Germany were fed daily with whole rabbit or beef meat, the cheetahs in South Africa received daily a mixture of cat food (Iams®), horse-, and chicken mince. Water was available ad libitum. As two males

Comparison of different antibodies

In general, higher quantities were measured with corticosterone than with cortisol EIA’s. In the male (Fig. 1A), the cortisol-21-HS and the corticosterone-21-HS assays showed a 1.7 and a 2.3-fold increase in fGCM levels, respectively, after ACTH administration. In contrast, the corticosterone-3-CMO assay showed an 18-fold increase in fGCM concentration (+1700%) over the baseline (286.0 ± 76.3 ng/g) 23 h after ACTH injection and thus was considered the be the most sensitive EIA.

The results for the

Discussion

We established a new corticosterone EIA for cheetahs that has a high affinity to immunoreactive fGCMs as demonstrated with the injection of 3H labelled cortisol in two cheetahs and ACTH challenges of seven animals. Our EIA is based on a polyclonal antiserum raised in rabbits against corticosterone-3-CMO, similar to an antibody used in a commercially available corticosterone RIA that is frequently used to identify changes in adrenal activity in cheetahs and other wild felid species [26], [27],

Acknowledgments

We thank the Allwetterzoo Münster and the Zoo Wuppertal in Germany for their permission to conduct the study and Torsten Bohm and Stephanie Wandelmaier for the help in collecting the faecal samples. We further thank the De Wildt Cheetah centre, especially Ann van Dyk, for allowing us to perform the research, Cézanne Valk and Kirsten in ’t Ven for their help in collecting faecal samples, and Stefanie Ganswindt for expert help in laboratory techniques. We also thank Marlies Rohleder and Katrin

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