Does sex matter in the cheetah? Insights into the skeletal muscle of the fastest land animal.

The cheetah is considered the fastest land animal, but studies on their skeletal muscle properties are scarce. Vastus lateralis biopsies, obtained from male and female cheetahs as well as humans, were analysed and compared for fibre type and size, and metabolism. Overall, cheetah muscle had predominantly type IIX fibres, which was confirmed by the myosin heavy chain isoform content (type I: 17±8%, type IIA: 21±6%, type IIX: 62±12%), whereas humans contained predominantly type I and IIA fibres (type I: 49±14%, type IIA: 43±8%, type IIX: 7±7%). Cheetahs had smaller fibres than humans, with larger fibres in the males compared to their female counterparts. Citrate synthase (16±6 vs. 28±7 µmol/min/g prot, P<0.05) and 3-hydroxyacetyl co-enzyme a dehydrogenase (30±11 vs. 47±15 µmol/min/g prot, P<0.05) activities were lower in cheetahs, whereas lactate dehydrogenase activity was 6 times higher in cheetahs (2159±827 vs. 382±161 µmol/min/g prot, P<0.001). The activities of creatine kinase (4765±1828 vs. 6485±1298, P<0.05 µmol/min/g prot) and phosphorylase (111±29 vs. 216±92 µmol/min/g prot) were higher in humans, irrespective of higher type IIX fibres in cheetahs. Superoxide dismutase and catalase, markers of antioxidant capacity, were higher in humans, but overall antioxidant capacity was higher in cheetahs. To conclude, fibre type, fibre size and metabolism differ between cheetahs and humans, with limited differences between the sexes.

A cross-over dietary intervention in captive cheetahs (Acinonyx jubatus): Investigating the effects of glycine supplementation on blood parameters.

Captive cheetahs are prone to unusual diseases which may be attributed to their high muscle meat, collagen deficient captive diet. Glycine is a simple amino acid that is abundant in collagen rich tissues and has many physiological functions, specifically in collagen synthesis and in the conjugation of detrimental by-products produced during gut bacterial fermentation. Therefore, the aim of this study was to investigate the effect of a 4 week glycine supplementation on the body measurements, haematology and serum blood parameters of 10 captive cheetahs using a randomised controlled cross-over design. This approach has not yet been used to investigate the effect of diet in captive cheetahs. Cheetahs were randomly assigned to a control diet (horse meat only) or a glycine diet (30 g glycine per 1 kg meat) for 4 weeks before being crossed over. Blood was collected at baseline and after each intervention. The glycine diet resulted in a decreased serum albumin, alkaline phosphatase and total calcium concentration and increases in eosinophils and basophils counts compared to the control diet. Body weight also decreased on the glycine diet which may be due to increased ß-oxidation and fat loss. This was the first study to investigate the effect of glycine supplementation, which resulted in slight body and blood changes, in captive cheetahs using a cross-over design and this approach should be utilised for future dietary studies.

Agreement of high-definition oscillometry at two cuff locations with invasively measured arterial blood pressures in anaesthetised cheetahs.

OBJECTIVES: To evaluate the agreement between high-definition oscillometry (HDO) used on the metatarsus or tail base with invasive arterial blood pressures measured in the dorsal pedal artery in anaesthetised cheetahs. STUDY DESIGN: Prospective clinical study. ANIMALS: A group of 13 captive adult cheetahs. METHODS: Cheetahs were immobilised with medetomidine (32-45 µg kg-1) and tiletamine/zolazepam (0.93-1.39 mg kg-1) administered intramuscularly, and anaesthesia was maintained with either isoflurane in oxygen or continuous propofol infusion. Invasive blood pressure was measured via a 20 gauge intra-arterial catheter in the dorsal pedal artery in the metatarsus and used as a reference method for pressures simultaneously estimated using HDO on the contralateral metatarsus and tail base. Bland-Altman plots (for repeated measurements) and criteria defined by the American College of Veterinary Internal Medicine (ACVIM) were used to compare agreement according to the anatomical location of the cuff, the anaesthetic maintenance agent and magnitude of the blood pressure. RESULTS: A total of 147 paired measurements were obtained with HDO on the metatarsus and 135 on the tail. Agreement with invasive pressures was better when HDO was used on the tail (rather than on the metatarsus) with all ACVIM criteria being met. Mean bias (a positive bias meaning that HDO overestimated the invasively measured pressures) ± standard deviation of differences for systolic, diastolic and mean arterial pressures were -7.0 ± 13.9, 4.2 ±12.1 and 4.6 ±11.2 mmHg, respectively, for HDO on the tail, and -11.9 ±15.1, 2.8 ±16.5 and 2.1 ±13.2 mmHg, respectively, for HDO on the metatarsus. Agreement was better during isoflurane anaesthesia than propofol, and at lower blood pressures than at higher. CONCLUSIONS AND CLINICAL RELEVANCE: When used on the tail base of anaesthetised cheetahs, HDO met the ACVIM validation criteria for a noninvasive device, as compared to invasively measured pressures in the dorsal pedal artery.

Sublingual papillomas of cheetahs in southern Africa.

Nine distinct papillomaviruses (Lambdapapillomavirus) have been described in domestic and nondomestic cats, but not in cheetahs. These viruses have been associated with cutaneous papillomas or plaques, bowenoid in situ carcinomas, feline cutaneous squamous cell carcinomas (SCC), feline sarcoids, and oral (often sublingual) papillomas. Fourteen cheetahs from the AfriCat foundation (Namibia) and one from the Ann van Dyk Cheetah center (South Africa) presented with sublingual lesions reminiscent of sublingual papillomas. Two animals were biopsied and the histopathology revealed benign proliferative epithelial lesions with prominent thickening of the overlying squamous epithelium. Throughout the squamous epithelial layers were cells with nuclear enlargement, irregularity of the nuclear membranes and cell contours, focal hyperchromasia of the nuclei, and perinuclear halos, reminiscent of a virus-associated process as seen in papillomavirus infections. Thirteen more cheetahs were sampled and the tissue snap frozen for molecular characterization. Amplification and sequencing of the papillomavirus L1, E6, E7, and E1 gene regions was achieved with modified primers. Maximum likelihood phylogenetic analyses revealed all 15 cheetah papilloma samples were 99.99% genetically similar and closely related to, but genetically distinct from any known felinepapillomaviruses. All cheetahs were FIV and FeLV negative. The results suggest the samples identified in this study can be considered a previously undescribed or novel feline papillomavirus and the authors propose "Acinonyx jubatus papillomavirus type 1" (AjPV-1), within the Lambdapapillomavirus 1 genus (Family: Papillomaviridae).

Validating a non-invasive technique for monitoring physiological stress in the samango monkey.

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.

EVALUATION OF TWO DOSES OF BUTORPHANOL-MEDETOMIDINE-MIDAZOLAM FOR THE IMMOBILIZATION OF WILD VERSUS CAPTIVE BLACK-FOOTED CATS (FELIS NIGRIPES).

The efficacy, safety, physiologic effects, and reversibility of butorphanol-medetomidine-midazolam (BMM) immobilization were evaluated in black-footed cats (Felis nigripes) and compared between captive and wild animals. Nine captive and 14 wild black-footed cats were hand injected into an accessible hind limb muscle group with the BMM combination. The captive cats (captive group) received a lower dose of the combination (butorphanol, 0.25 ± 0.03 mg/kg; medetomidine, 0.06 ± 0.01 mg/kg; midazolam, 0.13 ± 0.02 mg/kg), whereas the wild cats received a higher dose (butorphanol, 0.53 ± 0.11 mg/kg, medetomidine, 0.13 ± 0.03 mg/kg, midazolam, 0.27 ± 0.05 mg/kg). Two capture methods were required to restrain the wild cats; previously collared cats were tracked and excavated out of their burrows during daylight hours (excavated group), whereas uncollared cats were randomly located using spotlights and pursued by a vehicle at night (pursued group). Inductions were rapid and no spontaneous arousals occurred. Mean arterial blood pressure in all cats was within normal limits for domestic cats. Initial rectal temperatures varied greatly among the groups, but decreased in all groups as the immobilization progressed. In the pursued animals, heart rates and respiratory rates were initially elevated. All cats had moderate hypoxemia, hypocapnia, and metabolic acidosis. Intramuscular administration of naltrexone, atipamezole, and flumazenil resulted in rapid, uncomplicated recoveries. BMM is thus a safe, effective immobilizing drug combination for both captive and wild black-footed cats, but higher doses are required in wild animals. The capture methods exerted a greater influence on the physiology of the immobilized animals than did the doses of the drugs used. Although this drug combination can be used safely to immobilize black-footed cats, supplemental oxygen should always be available for use, especially in pursued animals due to hypoxia.

A veterinary survey of factors associated with capture-related mortalities in cheetahs (Acinonyx jubatus).

The objective of this study was to gain better insight into factors associated with the capture-related mortality rate in cheetahs. A link to an online questionnaire was sent to zoo and wildlife veterinarians through the Species Survival Plan Programme and European Endangered Species Programme coordinators and via the 'Wildlife VetNet' Google group forum. The questionnaire consisted of 50 questions relating to the veterinarians' country of residence and experience, the medicine combinations used, standard monitoring procedures, capture-related complications and mortalities experienced in this species under different capture conditions. In addition, necropsy data from the national wildlife disease database of the National Zoological Gardens of South Africa were examined for cases where anaesthetic death was listed as the cause of death in cheetahs. A total of 75 veterinarians completed the survey, with 38 from African countries and a combined total of 37 from Europe, the United States (US) and Asia. Of these, 24% (n = 18/75) had experienced at least one capture-associated cheetah mortality, with almost all of the fatalities (29/30) reported by veterinarians working in Africa. A lack of anaesthetic monitoring and the absence of supplemental oxygen were shown to be significant risk factors for mortality. Hyperthermia, likely to be associated with capture stress, was the most common reported complication (35%). The results suggest that free-ranging rather than habituated captive cheetahs are particularly at risk of dying during immobilisation and transport. The capture-related fatalities in this species do not appear to be associated with either the veterinarian's level of clinical experience or the immobilisation agents used.

INTRAOCULAR PRESSURE, TEAR PRODUCTION, AND OCULAR BIOMETRY IN HEALTHY ADULT CHEETAHS (ACINONYX JUBATUS).

Schirmer tear test (STT), intraocular pressure (IOP), and ocular biometry were measured in 58 healthy cheetahs (Acinonyx jubatus) between the ages of 2 and 14 yr in two captive groups. During routine health examination under immobilization, each animal underwent ophthalmic examination including STT, IOP, slit-lamp biomicroscopy, indirect ophthalmoscopy, and ocular ultrasonography. Mean ± SD values for STT and IOP were 13.58 ± 5.29 mm/min and 31.40 ± 5.01 mm Hg, respectively. The time between immobilization and examination significantly influenced both STT and IOP measurements. For IOP measurements, a significant group effect was also demonstrated, possibly due to the different anesthetic drug combinations used. Mean ± SD values for biometry were as follows: globe length (32.73 ± 1.74 mm), lens thickness (7.25 ± 1.09 mm), anterior chamber (8.54 ± 1.21 mm), and posterior segment (16.31 ± 2.06 mm). Surprisingly, all of the biometry measurements differed significantly between the two groups. Establishing STT, IOP, and biometric reference values is important for evaluating the ocular health status of the species. These reference values will assist veterinarians working with cheetahs to perform a full and informed ophthalmic examination.

Assessment of a portable lactate meter for field use in the white rhinoceros (Ceratotherium simum).

Blood lactate is a predictor of mortality in critically ill humans and animals. Handheld lactate meters have the potential to be used in the field to evaluate the condition of severely injured rhinoceroses but have not been compared with laboratory-based methods. Agreement between a handheld lactate meter and a laboratory method was assessed, as was the stability of rhino blood lactate in the anticoagulant sodium fluoride/potassium oxalate (fluoride/oxalate). Blood samples were obtained from 53 white rhinos that had been immobilised for management reasons. Lactate was measured by means of a handheld meter using whole blood in heparin (WBHEP), whole blood in fluoride/oxalate (WBFO) and fluoride/oxalate plasma (PFO). Results were recorded in both blood (BL) and plasma (PL) modes and compared to an established laboratory method for measuring plasma lactate. To assess the stability of lactate over time, blood lactate in fluoride/oxalate was measured on the handheld meter at intervals for up to 91 h. Agreement was best using WBFO in PL mode, with small bias (-0.16), tight 95% limits of agreement (LOA) (-1.46, 1.14) and a Pc (95% CI) of 0.97 (0.92, 0.99). The agreement was improved for all sample types when using the PL mode compared to the blood lactate (BL) mode. Blood lactate was stable in fluoride/oxalate for 91 h, with a mean change from baseline of 0.15 (-0.178, 0.478) mmol/L (mean, 95% CI). The handheld meter was found to be suitable for field use in white rhinos but provided more reliable results with the device in PL mode. Furthermore, rhino blood lactate was found to be stable in fluoride/oxalate for as long as 3 days.

Ultrasonographically determined renal values and comparisons to serum biochemistry renal variables in aged semi-captive cheetahs (Acinonyx jubatus).

BACKGROUND: Cheetahs in captivity have a high prevalence of chronic renal diseases. We ultrasonographically evaluated the renal volumes, a variety of renal dimensions, interarcuate artery resistive indices (RI) as well as aortic diameters and the length of the ventral aspect of the 6th lumbar vertebrae in 27 aged semi-captive anesthetized cheetahs. Renal size, dimensions and ratios were compared to urine specific gravity, serum creatinine and urea values. RESULTS: There were minimal differences for all values between left and right kidneys. Mean kidney length was 65.1 mm (range 55.2-76.9) with left kidney length ratios to L6 length being 1.60 (range 1.27-2.06) and to the aortic diameter 7.69 (range 4.54-10.72). Significant correlations between left renal length as well as length:L6 ratio to creatinine values were found ((r - 0.66) and (r - 0.60) respectively). The mean RI values of the different sedation/anesthetic protocols ranged from 0.46-0.55. CONCLUSIONS: Left renal length and L6 ventral vertebral body length as well as left kidney RI values should be routinely measured in all cheetah abdominal ultrasound examinations. These measurements, together with serum creatinine, urea and urine specific gravity values may be relatively sensitive indicators of early renal pathology in the absence of gross ultrasonographic changes.

Comparative Serum Fatty Acid Profiles of Captive and Free-Ranging Cheetahs (Acinonyx jubatus) in Namibia.

Cheetahs (Acinonyx jubatus) are highly specialised large felids, currently listed as vulnerable on the IUCN red data list. In captivity, they are known to suffer from a range of chronic non-infectious diseases. Although low heterozygosity and the stress of captivity have been suggested as possible causal factors, recent studies have started to focus on the contribution of potential dietary factors in the pathogenesis of these diseases. Fatty acids are an important component of the diet, not only providing a source of metabolisable energy, but serving other important functions in hormone production, cellular signalling as well as providing structural components in biological membranes. To develop a better understanding of lipid metabolism in cheetahs, we compared the total serum fatty acid profiles of 35 captive cheetahs to those of 43 free-ranging individuals in Namibia using gas chromatography-mass spectrometry. The unsaturated fatty acid concentrations differed most remarkably between the groups, with all of the polyunsaturated and monounsaturated fatty acids, except arachidonic acid and hypogeic acid, detected at significantly lower concentrations in the serum of the free-ranging animals. The influence of age and sex on the individual fatty acid concentrations was less notable. This study represents the first evaluation of the serum fatty acids of free-ranging cheetahs, providing critical information on the normal fatty acid profiles of free-living, healthy individuals of this species. The results raise several important questions about the potential impact of dietary fatty acid composition on the health of cheetahs in captivity.

Non-invasive assessment of adrenocortical activity as a measure of stress in giraffe (Giraffa camelopardalis).

BACKGROUND: Numbers of giraffes are declining rapidly in their native habitat. As giraffe research and conservation efforts increase, the demand for more complete measures of the impact of conservation interventions and the effects of captive environments on animal health and welfare have risen. We compared the ability of six different enzyme immunoassays to quantify changes in fecal glucocorticoid metabolites (FGM) resulting from three sources: adrenocorticotropic hormone stimulation test, transport, and time of day that samples were collected. RESULTS: Two male giraffes underwent ACTH injections; all six assays detected FGM increases following injection for Giraffe 1, while only three assays detected FGM increases following injection for Giraffe 2. Consistent with other ruminant species, the two 11-oxoetiocholanolone assays (one for 11,17-dioxoandrostanes and the other for 3α,11-oxo metabolites) measured the most pronounced and prolonged elevation of FGM, while an assay for 3ß,11ß-diol detected peaks of smaller magnitude and duration. Both of the 11-oxoetiocholanolone assays detected significant FGM increases after transport in Giraffes 3-7, and preliminary data suggest FGM detected by the assay for 11,17-dioxoandrostanes may differ across time of day. CONCLUSIONS: We conclude the assay for 11,17-dioxoandrostanes is the most sensitive assay tested for FGM in giraffes and the assay for FGM with a 5ß-3α-ol-11-one structure is also effective. 11-oxoetiocholanolone enzyme immunoassays have now been demonstrated to be successful in a wide variety of ruminant species, providing indirect evidence that 5ß-reduction may be a common metabolic pathway for glucocorticoids in ruminants. As FGM peaks were detected in at least some giraffes using all assays tested, giraffes appear to excrete a wide variety of different FGM. The assays validated here will provide a valuable tool for research on the health, welfare, and conservation of giraffes.

Correction: Measuring Faecal Epi-Androsterone as an Indicator of Gonadal Activity in Spotted Hyenas (Crocuta crocuta).

[This corrects the article DOI: 10.1371/journal.pone.0128706.].

Ultrasonographic adrenal gland findings in healthy semi-captive cheetahs (Acinonyx jubatus).

Cheetahs in captivity are believed to suffer from stress predisposing them to poor health. To date fecal glucocorticoids have been used as a non-invasive indicator of chronic stress. This study examines, the feasibility of transabdominal adrenal gland ultrasonography in cheetahs and determined normal adrenal measurements that can potentially be used as a more reliable indicator of chronic stress and/or adrenal function. Thirty-three adult cheetahs, aged between 2 and 13 years, accommodated in large off-display camps were examined over 9 days under general anesthesia. The adrenals were readily identified, with the right adrenal being more difficult to find and measure than the left, and were smaller than those expected in similar sized dogs. The left adrenal was shorter and slightly more oval shaped than the right with a length and cranial pole width at a 95% prediction interval of 16.3-22.4 and 4.1-8.7 mm. The same measurements for the right adrenal were 16.8-26.2 and 3.4-10.8 mm, respectively. Corticomedullary ratios were larger for the left adrenal. When corrected for body size, females had significantly longer and greater left adrenal corticomedullary ratios than males. Adrenal measurements did not correlate with left renal length, body size measurements, or enclosure size. Measurements that increased with age included the cortical and total adrenal widths. Adrenal ultrasonography offers potential benefits in assessment of individual cheetah adrenal pathology or the evaluation of stress induced adrenomegally especially in combination with other evaluations such as non-invasive fecal glucocorticoid analyses. Zoo Biol. 35:260-268, 2016. © 2016 Wiley Periodicals, Inc.

Reference intervals for selected serum biochemistry analytes in cheetahs Acinonyx jubatus.

Published haematologic and serum biochemistry reference intervals are very scarce for captive cheetahs and even more for free-ranging cheetahs. The current study was performed to establish reference intervals for selected serum biochemistry analytes in cheetahs. Baseline serum biochemistry analytes were analysed from 66 healthy Namibian cheetahs. Samples were collected from 30 captive cheetahs at the AfriCat Foundation and 36 free-ranging cheetahs from central Namibia. The effects of captivity-status, age, sex and haemolysis score on the tested serum analytes were investigated. The biochemistry analytes that were measured were sodium, potassium, magnesium, chloride, urea and creatinine. The 90% confidence interval of the reference limits was obtained using the non-parametric bootstrap method. Reference intervals were preferentially determined by the non-parametric method and were as follows: sodium (128 mmol/L - 166 mmol/L), potassium (3.9 mmol/L - 5.2 mmol/L), magnesium (0.8 mmol/L - 1.2 mmol/L), chloride (97 mmol/L - 130 mmol/L), urea (8.2 mmol/L - 25.1 mmol/L) and creatinine (88 µmol/L - 288 µmol/L). Reference intervals from the current study were compared with International Species Information System values for cheetahs and found to be narrower. Moreover, age, sex and haemolysis score had no significant effect on the serum analytes in this study. Separate reference intervals for captive and free-ranging cheetahs were also determined. Captive cheetahs had higher urea values, most likely due to dietary factors. This study is the first to establish reference intervals for serum biochemistry analytes in cheetahs according to international guidelines. These results can be used for future health and disease assessments in both captive and free-ranging cheetahs.

COMPARISON OF HIGH-DEFINITION OSCILLOMETRIC AND DIRECT ARTERIAL BLOOD PRESSURE MEASUREMENT IN ANESTHETIZED CHEETAHS (ACINONYX JUBATUS).

Blood pressure measurement reveals important insights into the health of conscious and anesthetized individuals. This is of particular interest in cheetahs (Acinonyx jubatus), which in captivity are known to suffer from chronic diseases that may be associated with hypertension and which often require immobilization for transport or veterinary treatment. Invasive testing methods are considered the gold standard but are not practical in many settings. Consequently, it is important to evaluate the use of noninvasive methods in this species. Measurements for systolic, diastolic, and mean arterial pressure obtained using high-definition oscillometry (HDO) at the coccygeal artery were compared to simultaneous direct measurements obtained via catheterization of the femoral or dorsal pedal artery in eight anesthetized captive cheetahs during nine anesthetic events. Overall, HDO and direct measurements agreed most closely for mean arterial pressure, and the poorest agreement was observed for systolic pressure. There was a tendency for low diastolic pressures to be underestimated and for high diastolic pressures to be overestimated. Across all three parameters, HDO measurements from the tail overestimated directly measured pressures in the femoral artery and underestimated those in the dorsal pedal artery. HDO agreed most closely with directly measured dorsal pedal pressures. Mean arterial pressure showed the greatest precision (standard deviation of 10.2 mm Hg) and lowest bias (-1.2 mm Hg), with 75.9% of readings within 10 mm Hg of the direct dorsal pedal pressure. Agreement with systolic pressure was hindered by a high bias (-10.4 mm Hg), but if a correction factor of +10 mm Hg was applied to all systolic measurements, agreement was improved and 65.7% of readings were within 10 mm Hg of the direct pressure. When compared to criteria defined by the American College of Veterinary Internal Medicine for validation of blood pressure devices, results were favorable, but a limited sample size prevented formal validation.

Measuring Faecal Epi-Androsterone as an Indicator of Gonadal Activity in Spotted Hyenas (Crocuta crocuta).

Enzyme immunoassays (EIA) that measure faecal testosterone metabolites (fTM) are useful tools to monitor gonadal activity. The aim of this study was to validate an "in-house" epiandrosterone EIA to monitor fTM in spotted hyenas. FTM were characterised in a male and a female hyena that each received an injection of 3H-testosterone. High-performance liquid chromatography (HPLC) analyses revealed a cluster of highly polar enzyme-hydrolysable hormone metabolite conjugates. We performed hydrolysis using ß-glucuronidase to deconjugate metabolites and improve sensitivity of the assay. Because ß-glucuronidase from Helix pomatia has been reported to bias testosterone measurements in some species, we compared the enzymatic activity of the commonly used ß-glucuronidase extracted from H. pomatia with the same enzyme from Escherichia coli. Our results showed that ß-glucuronidases from both sources produced similar results from spotted hyena faeces. We therefore hydrolysed samples with H. pomatia enzymes. HPLC analyses also demonstrated that following hydrolysis the epiandrosterone EIA measured significant amounts of immunoreactive metabolites corresponding to radiolabelled metabolites in both sexes. Additionally, HPLC and GC-MS analyses confirmed the presence of epiandrosterone in faeces of spotted hyenas. The biological relevance of the epiandrosterone EIA was validated by demonstrating (1) a significant increase in fTM levels in response to a testosterone injection within 16 h, (2) no biological responsiveness to an adrenocorticotropic hormone (ACTH) injection and (3) significant differences in fTM levels between juvenile males and adult immigrant males in a free-ranging wild population. Our results clearly demonstrate that the epiandrosterone EIA is a reliable non-invasive method to monitor gonadal activity in spotted hyenas.