Fecal glucocorticoid metabolite levels in captive Indian leopards (Panthera pardus fusca) housed under three different enrichment regimes.

Environmental enrichment improves the health and wellbeing of zoo animals. To test this hypothesis, we used Indian leopards (Panthera pardus fusca), one of the popular zoo animals, as a model organism to understand effects of active and passive enrichment elements on stress hormone levels of captive individuals. We included three enrichment categories, category 'A' (having both active: cage size of 1204 m3 with raised platforms and earthen flooring, and passive: controlled temperature, playback of forest sounds and sound proof glass to filter visitors' noise, enrichment elements), category 'B' (active enrichment type I, cage size of 264 m3 with air coolers), and category C (active enrichment type II, cage size of 517 m3 without air coolers) for leopards (n = 14) housed in two Indian zoos. We used a group-specific enzyme immunoassay to measure fecal glucocorticoid metabolites (fGCM) in captive leopards. For comparison, we analysed samples from free-ranging leopards, as well. fGCM levels (Mean±SEM) were 10.45±2.01 and 0.95±0.003 μg/g dry feces in captive and free-ranging leopards, respectively. Our results revealed that fGCM levels of leopards in categories B and C were significantly (P<0.05) different from each other, thus, indicating cage size (an active enrichment element) as an important factor in influencing the physiology of the sampled animals. Overall, the findings of the study will contribute towards informed policies for management of captive Indian leopards.

Introduction

Conservation action requires sustainable management of captive and zoo-housed individuals of a species. Globally, there are approximately 10,000 zoos and captive breeding centers, and India alone has more than 145 zoological gardens [1]. Most of the natural populations of animals are declining due to deforestation and urbanization; thus, zoo or captive populations provide an alternative source of genetic materials for vulnerable or threatened species [2]. Besides conservation research, zoos are also a source of recreation, entertainment and education for the general public [3, 4]. Thus, as a part of the ex-situ conservation action plan, monitoring the health and wellbeing of zoo animals becomes a priority for ecologists and conservation managers, as well. However, being away from their natural habitat, zoo animals often suffer (mentally and physically) within the artificial, unfamiliar, captive environment [5, 6]. Thus, to maintain the wellbeing of the zoo animals, several types of physical and virtual stimuli need to be added to the captive environment with an attempt to supplement the captivity with enriched habitat conditions.

Enrichment is the alteration made to the environment of captive animals for their physiological and psychological wellbeing. Studies have shown that enrichment can be divided into two categories, active and passive. Active enrichment can be defined as enrichment that requires animals to perform some sort of physical activity or the organisms are in direct contact with a physical object [7]. For example, Carlstead and Shepherdson [8] showed that providing structural enrichment that mimicked housing conditions similar to the natural setup, increased success rate of reproduction in animals like rats (Rattus rattus), ferrets (Mustela putorius), great apes and ungulates. Similarly, stereotypic behaviour was reduced in captive bears (Ursus americanus) when food presentation was varied [5]. For example, when food was provided in a log filled with honey and was hidden throughout the exhibit, bears reduced stereotypic pacing from 125min/day to 20min/day, and increased the rate of their exploratory behaviours [5]. On the other hand, passive enrichment can be defined as a regime that does not include any interaction with a physical structure or any kind of direct contact with a living being [7]. Passive enrichment includes visual, olfactory, and auditory enrichment, and mostly consists of modifications that enrich the ambient environment without necessarily involving any physical interaction. Television, video and computer games, mirror and colors are known to be effective as visual, passive enrichment for rhesus macaques (Macaca mulatta), horses (Equus ferus) and chimpanzees (Pan troglodytes) [9]. In the case of auditory enrichment, sounds specific to animal’s natural environment, and even classical music, radio broadcasts and instrumental music acted as enrichment to female African leopards (Panthera pardus pardus), Asian elephants (Elephas maximus), gorillas (Gorilla gorilla), Guinea pigs (Cavia porcellus) and rats [9]. Management of zoo animals is challenging due to vast differences between the natural and the captive environment, and thus, captive conditions often trigger physiological stress reactions, which may eventually impact an animal’s wellbeing [10, 11].

Stress can be defined as loss of homeostasis, and a stressor is an event or force, which causes this disruption [12]. A series of physiological events take place inside the body to restore homeostasis, a way in which the body responds towards the stressor. Stress can be caused by physical factors, for example injuries, conflict [13], environmental factors such as temperature, humidity, sunlight, as well as internal factors such as anoxia, hypoglycemia [14]. Stressful conditions may also impact animal behavior, for example, causing increased aggression or sign of submissive behavior under such conditions [14]. Most kinds of stressors trigger the release of glucocorticoids (GCs) from adrenal glands [14]. GCs are known to increase blood glucose levels, suppress the immune system to help maintain homeostasis, and support metabolism of fats, proteins and carbohydrates [15, 16] to mobilize energy under stressful situations. The GC spike in the bloodstream can be measured by routine hormone assays, and several of GC’s metabolites can be measured in other biological samples, for example, feces, urine, mucus [17]. Studies have shown that captive conditions are highly stressful for most animals and often such artificial environments may lead towards poor reproductive performance and/or higher prevalence of diseases among the zoo populations [18, 19].

Confinement, artificial environment, visitors [20, 21] and isolation of social animals [22] are the key factors known to cause high levels of stress in captive animals. Thus, to improvise ex-situ conservation efforts, enriched habitats are provided to captive animals and their physiological response towards the enrichment can be assessed through measurements of stress hormones, mostly by monitoring levels of GCs [9]. For example, a study on small felids (Leopardus tigrinus and Leopardus wiedii) showed that females when provided with bigger enclosures, an example of active enrichment, had reduced stress hormone levels, and resumed reproductive cyclicity when compared to females maintained under non-enriched, stressful conditions (small and barren enclosures) [23]. Another study on Clouded leopards (Neofelis nebulosa) showed that certain unusual behaviours, for example, fur plucking and nail biting, were relatively higher in captive animals, thus indicating stressful conditions [24]. To reduce such behaviours, different types of enrichment (active and passive) were provided to captive Clouded leopards [24], of which increased time spent with the keeper (a type of active enrichment element) was found to be effective in reducing high fecal glucocorticoid metabolite (fGCM) levels in Clouded leopards. Globally, a large population of different species is present in captivity, and with an aim to provide improvised husbandry practices; studies need to be conducted to understand the effect of diverse enrichment elements (active or passive or a combination of both) on the physiological wellbeing of captive populations.

India is home to a rich biodiversity, but hosts a large population of captive animals, as well. Monitoring wellbeing of the captive animals is one of the priorities for the managers and the veterinarians of the Indian zoos. One such popular animal housed in a large number of Indian zoos is the Indian Leopard (Panthera pardus fusca), categorized as endangered as per the IUCN Red List (2016). Out of 145 zoos in India, leopards are present in 76 different zoos across the country [25], and each zoo having approximately 8–10 leopards. Studies have shown that captivity is very stressful for leopards and induces all sorts of unusual behaviours. For example, Indian leopards kept in zoos in 3 different states of India (Maharashtra, Kerala and Delhi) showed high levels of stereotypic behaviours (repetitive walks, chewing paws and snapping) and had higher fGCM levels under conditions with no enrichment [26]. However, fGCM levels and intensity of stereotypic behaviours decreased when animals were provided with different types of structural enrichment, for example, den or pool within an enclosure, and more time spent with a keeper providing care. Another study showed that captive Indian leopards preferred to spend more time in enriched zones having structural elements, for example, trees, barrels, sleeping platform and logs, and had decreased rates of stereotypic pacing under such conditions [27]. However, both studies on the captive Indian leopards included only components of active enrichment. To the best of our knowledge, so far no study has been conducted to assess the effects of passive enrichment components (auditory, visual or olfactory stimuli) on the physiological wellbeing of captive Indian leopards, and thus, warrants future investigation.

In this study, we monitored the impact of three different enrichment conditions (referred to as categories ‘A’, ‘B’ and ‘C’) on the stress physiology of the zoo leopards. The first category (category ‘A’) had both active and passive elements, whereas active enrichment type-I (category ‘B’) and -II (category ‘C’) had active enrichment elements only. This is the first study to include both active and passive enrichment elements, and assess their effects on the physiological stress levels of the Indian leopards. To measure physiological responses towards a particular enrichment regime, we utilized an enzyme immunoassay to measure fGCM levels in captive Indian leopards. For comparison using the same assay, fGCM concentrations were also measured in scat samples collected from free-ranging leopards. The overarching goal of our study is to develop an improved protocol for better management practices for the target species.

Materials and methods

Study site and animals for captive sampling

Sampling was carried out in 14 leopards (8 males and 6 females) (Table 1). Four male leopards and four females were from Kankaria zoo, Ahmedabad, located in the southeastern part of the city, while remaining four males and two females were from Sayajibaug zoo, Baroda (all in the state of Gujarat, India; Fig 1). Kankaria/Kamala Nehru zoo in Ahmedabad city, Gujarat had two different housing conditions for mammals (indoor and outdoor, Table 1). Sayajibaug zoo (referred to as Baroda zoo from here onwards) in Baroda city, Gujarat, had only one type of housing condition, the outdoor (Table 1). Both zoos are only 72 miles apart from each other, and experience a similar weather pattern in terms of temperature and rainfall. Leopards in both the zoos were adults (Table 1) and were maintained under a similar diet regime. From now onwards, we will refer to the housing conditions in two zoos as category ‘A’ for Kankaria indoor, category ‘B’ for Kankaria outdoor, and category ‘C’ for Baroda outdoor. Table 1 gives details on active and passive enrichment elements that were provided to the leopards within each of these categories, and the number of leopards maintained under these conditions. In all three categories, leopards were housed in individual cages. Since leopards use the height of a cage for climbing and jumping activities, we calculated cage size as a product of length, breadth and height of the cage. Category ‘A’ had the highest enrichment, having the largest (1204 m3) cage size when compared to the other two categories, and had several active (earthen flooring and raised platforms) and passive (sound proof glass to filter visitors’ noise, controlled temperature and playback of natural, forest sounds) elements for enrichment (Table 1). Category ‘A’ was the only regime that had passive enrichment elements for the leopards. Out of all the three categories, category ‘B’ had the smallest cage size (264 m3) and was provided with a few active, structural enrichment elements, for example, air coolers during summer, earthen flooring and raised platforms for climbing. Category ‘C’ had medium size cages (517 m3) but had no coolers, and was provided with similar enrichment elements as category B, having earthen floors and raised platforms (Table 1). The sampled leopards were either captive-born or captured from the wild, and time since captivity varied from 3 to 18 years (Table 2). The detailed information on each of the sampled leopards under each housing category is provided in Table 2.

Table 1

Details on enrichment regimes for leopards in Kankaria and Baroda zoos, Gujarat.

Zoo name	Enrichment category	Number of animals; Sex	Enrichment details	Type of food provided, feeding frequency	Feeding time
Kankaria	Category ‘A’, indoor facility with both active and passive enrichment elements	N = 4; Females	• Artificial light condition	3.6–4 kg buffalo meat/individual. Daily	9:00 A.M.
			• Playback of natural, forest sounds
			• Reverse day and night cycle
			• Sound proof glasses to filter visitors’ noise
			• Raised platforms with stairs
			• Earthen flooring with controlled temperature at 25°C
			• Cage size of 1204 m3
	Category ‘B’, outdoor facility with active enrichment type I	N = 4; Males	• Cages with cemented and earthen flooring	3.6–4 kg buffalo meat/individual. Daily except on Fridays	9:30–10:00 A.M.
			• Raised platforms with stairs
			• Air coolers during summer
			• Cage size of 264 m3
Baroda	Category ‘C’, outdoor facility with active enrichment type II	N = 6; 4 males and 2 females	• Cages with cemented and earthen flooring	3.6–4 kg buffalo meat / individual. Daily	9:30–10:00 A.M.
			• Raised platforms with stairs
			• Cages maintained at ambient temperature across all seasons
			• Cage size of 517 m3

Fig 1

Map showing the study sites for captive and free-ranging populations of the Indian leopards.

Inset panel shows the entire map for the state of Gujarat, India, with approximate locations of Kankaria and Baroda zoos, the study sites for captive leopard populations. Main panel shows the scat collection sites for free-ranging leopards in South Gujarat.

Table 2

Detailed information on sampled individuals at both Kankaria and Baroda zoos, Gujarat.

Animal Name (Zoo name, housing condition)	Place of Birth and date of arrival to respective zoos	Sex	Age at rescue (years)	Approx. age at sampling time (years)	Remarks
Mili (Category ‘A’)	Rescued from Dang, Valsad district, Gujarat	Female	7	19	Died on 08/09/2020 due to old age
	Date: 05/03/2008
Jaya (Category ‘A’)	Rescued from Borivali, Maharashtra	Female	5	19	Died on 24/12/2020 due to old age
	Date:29/09/2005
Vanshankri (Category ‘A’)	Born at Shimoga zoo, Karnataka	Female	NA	5.5	Born on 30/11/2015
	Date:09/07/2018
Tunga (Category ‘A’)	Born at Shimoga zoo, Karnataka	Female	NA	5	Born on 30/01/2016
	Date:09/07/2018
Sunny (Category ‘B’)	Rescued from Borivali, Maharashtra	Male	2.5	18	NA
	Date:29/09/2005
Pradip (Category ‘B’)	Rescued from Shimoga zoo, Karnataka	Male	6	10	NA
	Date:09/07/2018
Pravin (Category ‘B’)	Rescued from Shimoga zoo, Karnataka	Male	5	9	NA
	Date:09/07/2018
Sandip (Category ‘B’)	Born at Shimoga zoo, Karnataka	Male	NA	8	Born on 23/01/2014
	Date:09/07/18
Vishnu (Category ‘C’)	Captured from wild, Dhanpur Forest, Bariya Forest Division, Gujarat.	Male	3	15	NA
	Date:23/03/2009
Naaraj (Category ‘C’)	Born at Baroda zoo on 04/08/2007	Male	NA	15	NA
Neel (Category ‘C’)	Captured from the wild from Tejpur Forest, Assam.	Male	3	16	NA
	Date:05/03/2003
Nayan (Category ‘C’)	Captured from wild, Prankad Village, Jhagadiya Taluka, Gujarat	Male	NA	8	NA
	Date:01/01/2012
Naina (Category ‘C’)	Captured from wild, Prankad Village, Jhagadiya Taluka, Gujarat	Female	NA	8	NA
	Date:01/01/2012
Netra (Category ‘C’)	Captured from wild, Prankad Village, Jhagadiya Taluka, Gujarat Date:01/01/2012	Female	NA	8	NA

For all the three categories, summer sampling (with ambient temperature ranging from 39–42°C) [28] was conducted from June to July 2019 and winter sampling (with ambient temperature ranging from 28–31°C) [29] was carried out from December to February 2020. In total, 119 samples were collected from 14 leopards (n = 68 samples for males and n = 52 samples for females) across the three categories. On an average, we collected 3–7 scats per individual per season. Category ‘A’ had a total of 12 and 21 samples collected during summer and winter seasons, respectively. Accordingly, category ‘B’ had a total of 14 and 13 samples, and category ‘C’ had 36 and 31 total scats, collected during summer and winter seasons, respectively. Due to defecation in water or defecating at an inaccessible spot within the cage, we were not able to collect samples from a few individuals during certain weeks. Samples (~5 g) were collected using ice-cream sticks and zip lock bags. After collection, samples were stored at -20°C until analysis. All necessary permits and ethics approval were obtained from the concerned authorities.

Study site for sampling free-ranging leopards

Four districts, namely, Surat, Bharuch, Valsad and Dang within the southern part of Gujarat state, India, were chosen as the field sites for sampling scats from free-ranging leopards. The annual rainfall in south Gujarat is 900–2800 mm, and the average maximum temperature in summer goes up to 40°C in May, which is the hottest month whereas minimum temperature lies at 26°C. December is the coldest month of the year, and the maximum temperature averages to 25°C and the minimum is 16°C [30]. Overall, south Gujarat has the highest forest cover with dense canopy; wherein Dang district has a forested area of about 1,368 sq. km, Surat has an area of 496.72 sq.km, Bharuch has an area of 1142 sq.km and Valsad having a forest cover of 274.69 sq.km. [30]. According to the census in 2016, the estimated leopard population in Gujarat was about 1395 [31]. Out of which, the population count was 43 in Surat, 18 in Valsad and 43 in Dang districts of Gujarat. However, the census data for Bharuch is not available. The total population across the four districts accounts for 7.4% of the total leopard population for the state of Gujarat [31].

Scat samples were collected from Surat, Bharuch and Dang districts of Gujarat during January-March 2020, and during the month of March 2021. Though we conducted a field survey in Valsad during the month of March 2021, fresh scats were not obtained from the district. A total of 10 field hours were spent daily in tracking and collecting samples of the leopards. Only fresh scat samples were collected in a zip lock bag. Parameters like moisture content, smell and evidence of recent activity, for example fresh tracks and pugmarks, were used as a criterion to determine freshness of the scat [32]. Scats were kept in an icebox at the field site and were transferred to -20°C within 4–6 hours after collection. A total of 12 samples were collected randomly from fairly distant areas (80 to 140 km) across three districts of Gujarat. This was done to avoid pseudo replication by collecting repeated samples from the same individual. However, due to random sampling, the life history or the sex of the sampled leopards could not be determined. Fig 1 shows the sampling sites for free-ranging leopards within the state of Gujarat.

Extraction and analysis of fGCMs from scat samples of both captive and free-ranging leopards

Samples were dried using a hot air oven for 24–36 hrs, until completely dry. After drying, the samples were pulverized and sieved, and the powdered sample was stored in glass bottles at room temperature until further analysis. For sample extraction, 3 ml of 80% methanol was added to 0.1 g of fecal powder. The mixture was vortexed for 3 minutes and then centrifuged at 1500 rpm for 10 minutes [33]. Supernatants were collected (1.5–2 ml) in tubes and stored at -20°C until steroid analysis. Fecal GCMs were quantified using a 5α-pregnane-3β,11β,21-triol-20-one enzyme immunoassay (EIA) kit purchased from Rupert Palme (Department of Biomedical Sciences, University of Veterinary Medicine, Vienna). Details of the EIA, including cross-reactions are given elsewhere [34-36]. The EIA was already validated (with an ACTH challenge test) for African leopards (Panthera pardus pardus,) [13]. The EIA measures fGCMs with a 5α-3β,11β-diol structure [37]. To reduce non-specific binding, the assay was performed on anti-rabbit-IgG-coated (R2004, Sigma-Aldrich) microtiter plates. The assay sensitivity was 2.4 ng per gram DW. The Intra- and inter-assay coefficients of variation were 9.71% and 14.20%, respectively for an internal control sample, whose value was close to the center of the standard curve. Fecal extracts from a sample from a captive and one from a wild animal center were serially diluted and assayed to check for parallelism with the standard curve.

Statistical analyses

To assess the effects of enrichment and season on fGCM values of captive leopards, we used linear mixed effects (LME) model [38] with maximum likelihood method. For the fixed effects, season (with two levels, summer and winter) and enrichment (with three levels, categories ‘A’, ‘B’ and ‘C’) were included as independent variables, and fGCM concentration was included as a dependent variable. Since the housing (category ‘A’) with both active and passive enrichment had only females and active enrichment type I (category ‘B’) had only males, sex was not included in the mixed effect analyses. Log-transformed fGCM values were included in the LME model to meet normality assumption. Each individual leopard was repeatedly sampled over time for both seasons (summer and winter), and thus, individual identity was included as a random effect within the mixed effects model. Model comparisons were conducted to arrive at the best-fit model for the given data set and post-hoc comparisons were done to explore significant interactions within the model. Out of 14 leopards in both the zoos, only four were born in captivity under zoo conditions and the other 10 were captured from the wild (Table 2). Due to the disproportionate sample size between captive and wild born leopards, we did not include the history of individuals as an independent variable in the model. Time since captivity was also not included in the model as all the individuals spent more than 5 years in captivity. Further, all the sampled individuals were adults (Table 2), thus, age was also not included as an independent variable for the mixed effects model. Adult age criterion for the leopards was followed as outlined by [39].

All analyses were conducted on R version 3.2.3 using ‘nlme’, ‘multicomp’ and ‘ggplot 2’ packages [38]. Post hoc multiple comparisons using Tukey’s HSD were done using the R package ‘emmeans’. Parallel displacement between the standard curve and serial dilutions of the fecal extract was used to biochemically validate the fGCM assay for the Indian leopards. The values within the linear range of the curve were subjected to linear regression analysis (PRISM software, version 9) using log molar concentration vs. percent antibody binding of the standard and the sample dilution curves separately. The slopes of the regression lines were compared using Student’s t-test.

All the data are reported as Mean±SEM, and expressed as μg/g dry feces. Significance level was kept at P<0.05 for all the analyses.

Results

Fecal GCM profiles of captive leopards

Overall, fGCM levels of captive leopards were 10.45±2.01 μg/g dry feces, with male and female leopards having an average value of 10.68±2.96 and 10.13±2.71, respectively. During summer, the overall fGCM level was 5.90 ± 2.11 with leopards in ‘A’, ‘B’ and ‘C’ categories had values of 2.42 ± 0.6, 22.62 ± 9.12 and 1.36 ± 0.11 μg/g, respectively (Fig 2). During the winter season, the fGCM level, pooled across all the categories, was 14.92±3.32, with individuals maintained in category ‘A’ having a value of 25.11±6.57, those in category ‘B’ showed a value of 27.83±9.22 and in category ‘C’, the fGCM level was 1.15±0.11 (Fig 2). Fig 3 represents median values and quartile ranges for each enrichment regime for both winter and summer seasons. Fig 4 represents median fGCM values for each individual leopard during summer and winter months across three categories, category ‘A’ (Fig 4A), category ‘B’ (Fig 4B), and category ‘C’ (Fig 4C).

Fig 2

fGCM levels (mean plot) of captive Indian leopards maintained under three different enrichment categories.

fGCM levels (Mean±SEM) in captive Indian leopards maintained under three different enrichment categories, ‘A (active and passive enrichment)’, ‘B (active enrichment type I)’ and ‘C (active enrichment type II)’, during summer and winter seasons.

Fig 3

fGCM levels (median plot) of captive Indian leopards maintained under three different enrichment categories.

fGCM levels (median levels with upper and lower quartile) in captive Indian leopards maintained under three different enrichment categories, ‘A (active and passive enrichment)’, ‘B (active enrichment type I)’ and ‘C (active enrichment type II)’ during summer and winter seasons.

Fig 4

fGCM levels (median plot) of individual captive Indian leopards maintained under three different enrichment categories.

fGCM levels (median values) in individual captive Indian leopards maintained under three different enrichment categories, A (active and passive enrichment)’, ‘B (active enrichment type I)’ and ‘C (active enrichment type II)’ during summer and winter seasons. Panel A represents enrichment category ‘A’, panel B represents enrichment category ‘B’, and panel C represents enrichment category ‘C’. A single color represents an individual indicated by an alphabet.

fGCM levels in captive leopards under different enrichment regimes during summer and winter seasons

A parent LME model was constructed using logfGCM~Enrichment*Season. Further comparison of LME models, with (logfGCM~Enrichment*Season) and without an interaction (logfGCM~Enrichment+Season) between enrichment and season, showed significant difference (P<0.001) (Table 3), and thus, interaction term was retained in the final LME model (Table 4).

Table 3

Comparison of linear mixed effects model with and without an interaction term.

	df	AIC	BIC	logLik	Test	L.Ratio	p-value
Model1a	8	341.07	363.30	-162.5358
Model2b	6	366.79	383.46	-177.3966	1 vs 2	29.72157	<0.0001

aModel1:lme(logfGCM~Enrichment*Season,random = ~1|Ind.ID)

bModel2: lme(logfGCM~Enrichment+Season,random = ~1|Ind.ID)

Table 4

Results from linear mixed effects model showing effects of enrichment categories, seasons and their interaction on fGCM levels in captive leopards.

Linear mixed-effects model fit by maximum likelihood
Random effects:
Formula: ~1 | Ind.ID
(Intercept) Residual
StdDev: 0.1876867 0.3979013
Fixed effects: logfGCM ~ Enrichment*Season
	Value	Std.Error	F	t-value	p-value
(Intercept)	0.2024	0.1339	00	1.5112	0.0149
ERBa	0.7000	0.1893	00	3.6970	0.0068
ERCb	-0.1561	0.1590	00	-0.9814	0.4322
SEASONWNc	0.9611	0.1511	00	6.3590	<0.0001
ERB:SEASONWN	-0.7404	0.2272	00	3.2584	0.0013
ERC:SEASONWN	-1.0395	0.1846	00	-5.6295	<0.0001

aERB = Enrichment category ‘B’

bERC = Enrichment category ‘C’

cSEASONWN = Winter season

Post-hoc Tukey’s test across different enrichment categories showed significant differences in overall fGCM (pooled across seasons) concentrations between categories ‘A’ and ‘C’ (P<0.001) and between ‘B’ and ‘C’ (P<0.001), as well. No significant differences (P>0.05) were obtained between categories ‘A’ and ‘B’. Further, there was a significant difference in overall fGCM levels (pooled across enrichment categories, ‘A’, ‘B’ and ‘C’) between summer and winter (P<0.01). Within group comparisons (grouped by enrichment category) of pairwise interactions showed significant differences in fGCM levels (Tukey post-hoc test, P<0.01) between summer and winter seasons for leopards in category ‘A’, and no significant differences (P>0.05) in fGCM levels between summer and winter seasons for both categories ‘B’ and ‘C’. When grouped by season, Tukey’s post-hoc test showed significant differences in fGCM levels between categories ‘A’ and ‘B’ (P<0.001), and categories ‘B’ and ‘C’ (P<0.001) during summer season, but no significant difference (P>0.05) was obtained between category ‘A’ and ‘C’ during the summer. In contrast, during winter season, significant differences (Tukey post-hoc test, P<0.001) in fGCM levels existed between categories ‘A’ and ‘C’. Significant difference (Tukey post-hoc test, P<0.001) was also obtained between ‘B’ and ‘C’ category-leopards during the winter season; however, there was no significant difference (Tukey post-hoc test, P>0.05) between ‘A’ and ‘B’ category-leopards for the winter season.

fGCM levels of free-ranging leopards

A total of 12 samples was collected from three districts of Gujarat. Overall, fGCM concentrations of free-ranging leopards were 0.95±0.003 μg/g dry feces. The highest and the lowest fGCM values were 0.20 and 1.93, respectively.

Parallelism curves

The slope of the regression lines for standard curve and the serially diluted samples (both captive and wild) (Fig 5) were not significantly different (P>0.05) and thus, the assay can be used to measure fGCM concentrations in both captive and wild Indian leopards.

Fig 5

Parallelism plots of fecal extracts with standard curve.

Parallelism plot showing serial dilution curves of extracted samples collected from free ranging and captive leopards, and the standard curve.

Discussion

Our study presents fGCM levels in Indian leopards housed in two Indian zoos and maintained under three different types of enrichment regimes, defined as active and passive enrichment category (category ‘A’), active enrichment type-I (category ‘B’) and active enrichment type-II (category ‘C’). Overall fGCM levels measured in our study were higher than those measured by Vaz et al. [26] in the scats of captive Indian leopards. However, Vaz et al. [26] utilized a specific corticosterone assay rather than a group-specific assay, which targets metabolites. A native hormone (glucocorticoid) is present only in trace amounts, if at all, in the feces [34]. Thus, using a native hormone assay for non-invasive samples, for example, feces, urine, is not appropriate and may lead to questionable results. Vaz et al. [26] measured corticosterone whereas cortisol is the predominant glucocorticoid released in mammals [35, 40, 41]. This explains the higher fGCM levels in our study when compared to Vaz et al. [26], and further, the assay used in their study [26] was not validated for leopards.

In our study, changes in adrenocortical activity in captive Indian leopards were detected by measuring a group of cortisol metabolites (with a 5α- 3β, 11β-diol structure), and the same assay has been validated and used for African leopards [13]. Thus, we directly compared fGCM levels in captive individuals between the African (pardus subspecies) and the Indian subspecies (fusca subspecies), and found the mean fGCM value to be 36 times higher in Indian leopards than their African counterparts [13]. Such a huge difference in fGCM values between the Indian and the African subspecies can be due to multiple factors, ranging from differences in local environment to physiological differences at a subspecies level. For example, a study on white crowned sparrows (Zonotrichia leucophrys pugetnesis, Zonotrichia leucophrys gambelli and Zonotrichia leucophrys nuttalli) demonstrated that individuals breeding at higher altitude had increased stress hormone levels when compared to the subspecies, which bred at lower altitude [42]. In contrast, Gonzalez et al. [43] demonstrated subspecies-level differences in plasma cortisol concentrations for squirrel monkeys (Saimiri sciureus sciureus and Saimiri sciureus albigena) that were reared under identical laboratory conditions. In contrast, significant differences in fGCM levels were not observed between free-ranging Indian and African leopard subspecies. The fGCM values of free-ranging Indian (the current study) and African leopard subspecies [13] are quite overlapping. However, the sample size for free-ranging Indian leopards was significantly lower in our study. The scat samples collected from free-ranging leopards were for the purpose of validating the fGCM assay through generating parallelism curves, and any further insights on the fGCM patterns of free-ranging leopards will warrant future investigations.

Interestingly, there were no significant differences in fGCM levels between captive male and female leopards for both the African [13] and the Indian subspecies (the current study) (S1 Table). Sex-specific differences in fGCM levels were not so evident in other carnivores [44, 45], as well, except during the gestation phase. Jepsen et al. [45] showed that pregnant female tigers had significantly higher fGCM levels than males and non-pregnant females. The effect of sex on fGCM levels can be further influenced by several physical and social factors, for example, social rank, age, life history, and reproductive status [46]. In our study, the sampled leopards were all adults with no breeding history and were held in social isolation (one leopard per cage). Such lack of differences in physical and social make-up may possibly explain the similar fGCM profiles for both sexes in our study population.

The results from our study indicate a statistically significant effect of enrichment on the fGCM values of captive leopards. The leopards in active enrichment type I (category ‘B’) had the highest mean fGCM values (for both summer and winter seasons, and overall mean including both the seasons, as well) when compared to the other two categories. In contrast, leopards in active enrichment type II (category ‘C’) had the lowest mean (for both summer and winter seasons, and overall mean including both the seasons, as well) value of fGCM. Active enrichment type-I and -II (categories ‘B’ and ‘C’) were similar in quality having only elements of active enrichment (earthen floor, raised platforms). The only difference was that active enrichment type I (category ‘B’) had the smallest cage size and was provided with air coolers during summer. In contrast, active enrichment type II (category ‘C’) had medium size cages (48.94% larger than ‘B’) with no air coolers during summers, and the category having both active and passive enrichment (category ‘A’) had the largest cage size (78.07% larger than ‘B’). Thus, the overall high fGCM levels of leopards in active enrichment type I (category ‘B’), when compared to the other two conditions, indicated that cage size could be an important enrichment element for the given population. Similar results have been documented in other species, as well, where one of the elements had a stronger physiological effect than the rest within a given enrichment regime. Lapinski et al. [47] showed that cage size had no effect on salivary cortisol levels in foxes (Vulpes vulpes). However, gnawing sticks, another type of active enrichment, lowered the hormone levels of captive foxes, from 4.65±0.98 to 3.70±1.01 ng/ml. In this study, we did not measure other variables, for example, keeper’s attitude towards the leopards, occurrence of diseases in captivity and the frequency of visitors, across different enrichment categories, which could possibly impact the fGCM levels and thus, needs further systematic analyses.

Studies have documented that the physiological effects of enrichment elements is quite diverse and usually varies from species to species, and even across different individuals of the same species. For example, a study on Asian elephants [48] showed differences in fGCM levels across individuals, though all were provided with similar kinds of structural enrichment. This pattern is also evident in our study, where conditions with both active and passive enrichment (category ‘A) and active enrichment type I (category ‘B’) had large individual variations in fGCM levels. This was more evident in active enrichment type I (category ‘B’), where individual variations were more prominent irrespective of the differences across seasons. Interestingly, active enrichment type I (category ‘B’) also had the highest mean fGCM values compared to the other two categories. Several studies have documented that individual variations in stress hormone levels are more prevalent when hormone levels increase under stressful conditions [49]. For example, a study in barn swallows [50] showed that with rise in predation pressure, GC levels increased, and this was also accompanied with a large degree of individual variations. Thus, to understand the physiological wellbeing of a target species, systematic monitoring at an individual level needs to be conducted.

Our study is the first of its kind to include passive enrichment elements in understanding the wellbeing of the captive leopards. Category ‘A’ was the only regime where passive enrichment elements, for example, natural sounds, sound proof glass to filter visitors’ noise and artificially maintained lower ambient temperature, were provided along with active enrichment components. However, there were no significant differences in fGCM values between categories ‘A’ and ‘C’ during the summer, where ‘C’ is the regime with active enrichment elements only. Thus, at a physiological level, the combination of active and passive elements in category ‘A’ had a similar effect on fGCM levels when compared to active enrichment regime in category ‘C’. To the best of our knowledge, no study has measured fGCM levels in leopards housed under conditions having both active and passive enrichment elements, and further, has compared the levels with conditions having active enrichment elements only.

We did not find any significant effect of seasons on fGCM levels, except for the leopards in conditions with both active and passive enrichment elements (category ‘A’), where fGCM levels were higher during winter. However, we speculate that the higher fGCM levels in winter was not a ‘season effect’, but could be attributed to the presence of two old (>19 years) individuals in category ‘A’. Both individuals showed high values during winter when compared to summer, and eventually died after a span of 8–10 months. Several studies have found that old age is associated with relatively higher fGCM levels when compared to the younger age classes [51-53]. Studies [51-53] have demonstrated that association between fGCM levels and age of the individual animal is very context-specific, and depends on several environmental and biological factors [51-53]. Thus, in our study, we speculate that the high fGCM levels in the two >19 years old individuals, when compared to other individuals under the same conditions with both active and passive enrichment elements (category ‘A’), could be due to their older age, However, this is only a preliminary finding, which needs to be corroborated with long-term, longitudinal fGCM profiles of captive leopards across different life-history stages.

In conclusion, our study showed that enrichment, in particular size of the cage, influenced fGCM levels of captive Indian leopards within our sampling population. However, our study also revealed that physiological responses were quite diverse, showing huge variations in fGCM levels across individuals, and even for the same individual between summer and winter seasons. To accurately monitor physiological responses, zoo management programmes should focus on collecting several samples from multiple individuals, including equal numbers of males and females. Further, various covariates, including both individual- and environment-specific factors, should be included in the analyses to understand physiological wellbeing of the target species within a captive environment. Individual factors may include, sex ratios, age, life-history stages, duration spent under captive conditions, and social rank, health and disease status of the animal. Environment-specific factors may include types of enrichment element, diet regime, presence of conspecific individuals, seasons, visitors’ frequency, and keeper’s attitude. Our study applied the same fGCM assay method for Indian leopards that has previously been validated for the African ones. Utilizing the same assay for different sub-species in geographically different regions allows for direct comparisons of endocrine profiles. In terms of management practices, the study provides a validated fGCM assay to monitor the wellbeing of Indian leopards, both under captive and free ranging conditions, and underlines the overall importance of cage size as an enrichment element for the sampled leopard population. The findings will contribute towards management and conservation of the Indian leopards.

Comparative overview of fecal glucocorticoid metabolite (fGCM) levels (Mean±SEM), and value range; μg/g dry feces in Indian leopards from our data and in Indian and African leopards from published data.

(DOCX)

Click here for additional data file.

8 Feb 2022

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Reviewer #1: The authors have provided a robust study that provides valuable information related to the housing and welfare of a cryptic and solitary carnivore in captivity. The manuscript meets the requirements for acceptance however I would encourage the authors to obtain assistance from a native English speaker to assist with grammar and sentence structure. Although the information is important it could be more clearly communicated. I therefore recommend acceptance of the manuscript for publication after major revision

Abstract

Line 12 – improves the

Line 13 – first mention of species in the Abstract should have (scientific name)

Line 14 – clarify interacting and non-interacting – are zoo keepers interacting with animals or are animals interacting or not interacting with certain aspects of their environment/enrichment mechanisms – perhaps use the term active and passive

Lines 16-20 – categories should be more specific – the difference between large, medium and small cage size provides no distinction in cage size for the reader

Line 28-29 – The authors need to clarify what policies this information will inform – do you refer to the management of captive, free-ranging leopards or both??

Introduction

I found the introduction difficult to get through, its also very long – with a bit of restructuring, the authors could build a clear and solid foundation that introduces the importance of their work. I would suggest restructuring the introduction and possibly involving an English speaker to assist with sentence structure, grammar and word order.

First introduce the importance of zoos for maintaining genetic integrity of populations and behavioural studies - Focus on India and your species of interest – move lines 118-139 up to the beginning of the intro

Then deal with the difficulties related to the management of zoo animals of different species – which can result in stereotypical behaviour and other negative effects - Then discuss stereotypical behaviours and results of confinement – introduce the concept of enrichment through different mechanisms – food, size of enclosure and define and reference active (Line 58-59) or passive (Line 67-69) enrichments.

Introduce the concept of stress and go into some detail related to stress physiology, which can reduce physiological health, cause reproductive issues or welfare issues in captive settings

Then highlight why it is important to be able to monitor stressors in the most effective, non-invasive and standardized way in this species to ensure optimal health of captive individuals and point out the focus of your study and how it bridges the gaps in our knowledge related to your specific species and their management in zoos in terms of monitoring stress

Line 50 – topmost is not a word

Line 57 – the authors state that studies have shown – but don’t reference these studies also no references are given for definitions of active and passive enrichment within the study setting

Line 60 – I do not understand the term “a study in literature” – are the authors referring to a literature review or are they referring to the fact that they did a literature search

Line 61 refers to ‘proper housing’ - for readers outside of a captive zoo setting, this provides no useful information – be specific in your description of what constitutes proper housing

Line 62 refers to studies conducted on various species but no references have been given for these studies

Line 62 and 75 no scientific names of species mentioned are included in the introduction

Line 63-67 is repetitive – “Bears had higher 
locomotor and exploratory behaviours when food was provided in different ways, such as in a log filled with honey and by hiding food throughout the exhibit. Multiple ways of food 
presentation helped bears to reduce stereotypic pacing from 125min/day to 20min/day, and 
increased the rate of their exploratory behaviours [6].”

Reword to When food presentation was varied, such as in a log filled with honey and by hiding food throughout the exhibit, bears reduced stereotypic pacing from 125min/day to 20min/day, and increased the rate of their exploratory behaviours [6].

Line 81 “Long-term physiological stress is highly detrimental to zoo animals” it is also detrimental to free-ranging animals – I would focus the attention to zoo animals but not restrict your comments to captive individuals only. You also have no references for this and below statements related to the negative physiological effects of stress

Line 84 – definition of stress should be referenced

Distinctions between the stressor and the stress response should be more clearly defined that include physiological and psychological aspects

Line 85 – “A series of physiological events take place to restore this disruption is defined as the stress response” This does not make sense, the authors are saying that disruption (i.e. stress) is restored by physiological events – I think they mean that homeostasis is restored through the stress response –

Line 86-91 – this could be more clearly worded to describe the differences in extrinsic and intrinsic stressors. Utilize terminology suitable for animals – aggression not anger or refer to in humans or in captive animals for these stressors

Lines 91-95 – “Under stressful conditions, stress hormones are released as a biological response 
 helping to cope with “fight or flee” situations. Most kinds of stressors trigger the release of 
 glucocorticoid (GC) hormones [10], and GCs are known to increase blood glucose levels, 
suppress immune system to help maintain homeostasis, and support metabolism of fats, 
 proteins and carbohydrates [11,12] to fight against the stressful situations. 
” I do not think this is an adequate description of the stress response – the authors do not clearly describe the physiological processes of the stress response, where GC come from, why they can provide a measure of stress or how they can be measured or that faeces is used in this study because it meets all of the criteria for non-invasive assessment.

Materials and Methods

This section is quite complicated to read and should be revised to more clearly outline the differences between the sites as well as the categories and number of animals sampled at each site.

I would suggest splitting the section into two section that deal with Captive and Free ranging or into three sections that deal with Captive and Free-ranging and then Sample collection – Either provide a summary of each site in words then add info (number of categories, number of animals, number of samples, etc.) in a table or provide details in text – there is no need to repeat information or leave information out.

If you provide details in the text, make it easier for the reader to go back and check number of categories at a site, or number of samples from an animal

I would start the captive section by describing the overall environmental conditions of the area (as zoos are quite close together) this will help to put any environmental differences between free-ranging and captive animals in context for the reader. Give the same information for each facility – currently you provide size of Kankaria zoo but not number of animals housed but for Sayajibaug zoo you give number of animals and species – Be consistent and be specific.

Then you provide information on each site specifically – I would keep things simple here

e.g. Leopards housed in both facilities are adults. In Kankaria/Kamala Nehru zoo, Ahmedabad, Gujarat indoor and outdoor housing is provided while at the Sayajibaug zoo only outdoor housing is offered. Provide further details in a comparative way regarding cage size, enrichments etc.

Then you describe sample collection methods within each setting – Currently (line 196) you provide total number of samples but do not state from which facilities these samples came from. I would revise Supplementary Table 1 and include it in your methods – or include this information in the text to make it easier for the reader to follow.

The authors state that only fresh faecal sample collection from animals in the wild occurred and that determination of freshness was based on moisture content, smell and state of decomposition. If you do not observe the animal defecating, how did you determine state of decomposition? The moisture content, smell and state of decomposition are all determined by the last meal – if the meal consisted of an old carcass, sinew and bone – moisture content, smell and visual content of the faeces will differ markedly to faeces resulting from a blood rich or flesh meal. The authors should consider rewording this to show that samples were considered fresh if “list your criteria” i.e. warm to the touch, fresh tracks visible, <12 hours etc.

I do not see any mention of why you use wet weight for some samples and dry weight for others – perhaps you can provide some info on this in your methods??

Statistical analysis

You do not include seasonal aspects as one of your stated aims of the project in your introduction or in your data collection sections but you mention it in the statistical analysis and results section – I would suggest making a clear link between possible influences of season on animal welfare in captive individuals and stress levels if this is something you are going to address in your results

You could also add a single sentence in your statistical analysis section indicating when fGCM are reported as micrograms /gram dry weight (DW) or wet weight (WW) instead of repeating this in your reporting of results for each variable.

Results

fGCM profile of captive leopards

If you add a sentence to your statistical analysis for reporting WW and DW – you do not need to state it after reporting each concentration

Your sample size is quite small, how do you account for this limitation in your statistical approach?

Discussion

Throughout the discussion you refer back to the different categories, it may be easier for the reader to follow if you outline the criteria from that category and then add the category in brackets after i.e. active and passive enrichment (Category A)

Lines 382 – 392 – the statement that sex-specific differences in cortisol are not prevalent in other carnivores is a bit misleading as you then state in the next sentence that reproductive state in females influences cortisol concentrations in tigers – the same was observed in the study on African leopards – I suggest rephrasing this to more clearly highlight higher cortisol levels in reproductive females

Line 387 – The effect of sex type is not correct – change to the effect of sex/gender

Line 395 – what do the authors mean by season-wise are you referring to per season?

Be careful of using the phrase “in the current study (Lines 351, 360, 374, 384, 389, 394, 411, 429, 440, 453, 463, 468, 470) and or /our study (349, 357, 378, 393, 419, 449, 455” too often – use only when comparing directly to another study

You sometimes refer to stress hormone metabolites (lines 397, 405) and then other times to fGCM concentrations/values (lines 395, 403) – remember your work might be of use to people outside the field of endocrinology so keeping your terminology consistent will help readers follow your story better

The use of “high” does not convey any quantitative information nor does it contextualise the level – please go through the discussion and reword to be more specific where applicable

Line 403 – you state high fGCM values of leopards in category B – high in comparison to what? Other categories, free ranging animals - you need to be specific

Line 420 high variation – in comparison to what?

Line 422 high levels in comparison to what?

Line 425

Line 426

Line 447

Line 450

Line 415 “Studies have documented that physiological effect “ should read Studies have documented that the physiological effect or physiological effects

Line 431 – should read “sound proof glass” not glasses and sentence should be in active voice “

i.e. Category ‘A’ was the only regime where passive-enrichment elements, for example, natural sounds, sound proof glass to
filter visitor noise ….“

Lines 457 – 463 very long sentence, try breaking up into two

Reviewer #2: The paper submitted by Panchal et al to PLoS ONE reports fecal glucocorticoid metabolite (fGCM) levels in an exploratory study in captive Indian leopards. Animals were kept under three different housing conditions (categorized A-C), providing different types and degrees of enrichments. The authors utilized the same enzyme immunoassay, which has been previously fully validated for African leopards. Overall fGCM levels were much higher in captive animals compared to wild ones, from which a few samples were also included for comparison. Huge individual variations in fGCM levels in captive animals were found, but also differences between seasons, whereas sex did not affect levels. In addition, cage size influenced levels significantly.

Given the setting of the study (for obvious reasons no experiments could be performed), I think it is not possible to speak about “effects” of enrichment. In addition, in categories “A” and “B” only one sex was present, thus the influence of sex and enrichment in those two settings cannot be disentangled. I found the categories, “active” and “passive” enrichment interesting. However, I wonder, whether cage size is really an active enrichment (e.g. line 404)?

Below please find my specific comments. I thought it is easier for all involved to make my suggestions related to style or wording directly into the word file (track mode for changes).

Further detailed comments (ordered by appearance in the ms):

Line 1/2: I suggest rewording: “Fecal glucocorticoid metabolites in captive Indian leopards (Panthera pardus fusca) housed under different enrichment regimes”

Line 14: A (zoo) animal is not a “system”

Line 21 (and elsewhere): You did not “standardize” a non-invasive method. There is no standard available, all immunoassays give relative concentrations when applied to fecal samples (because there is a mixture of different metabolites present there). However, you need to validate such methods (biochemically, but more important physiologically/biologically).

Lines 63-67: I doubt that those details are necessary to describe here. Please delete.

Line 92: The classical “fight or flight” reaction is linked to the sympathetic nervous systems, and includes the release of catecholamines.

Line 103: I suggest citing a review here, at least instead of the primate paper (or why is this cited, and not other ones?)

Lines 127/128: Add the “metabolites”. Even when the original paper has it wrong, there are no glucocorticoids in the feces, only their metabolites.

Table 1: I think it would be better to move it to the supplement. Besides, the column “Breeding history does not give any additional information and should be deleted (also the “Remarks”). “Gender” should be replaced by “Sex” – it is about the biological term here.

Table 2: You give the cage side in m3, but if no details about “length*breadth*height” are given, the latter can be deleted.

Lines 204 and 218: You state that four districts were chosen, but then samples were only collected from three?

Line 215: What accounts for 7.4%, unclear to me?

Lines 237-252: This section needs drastic revision. I have made a suggestion directly in the docx-file. You need to cite the original paper, where the EIA was first described (Touma et al., 2003). 5α-pregnane-3β,11β,21-triol-20-one is the standard of this EIA. However, as this is a corticosterone metabolite, but cortisol is the dominant GC in carnivores, it is most likely not present in the feces. Still, because the EIA picks up metabolites with a 5α-3β,11β-diol structure, which can also be derived from cortisol (respective cross-reactions have only recently been described in Santamaria et al., 2021), this EIA has been proven suited in African leopards [9].

Line 259: Well, category and sex were not independent, because category A had only females and “B” only males.

Results: Overall, I think it is more adequate to present levels as median with range (min-max), and show them in respective figure 2 as boxplot graphs. The variation was so huge (sometimes the SE is greater than the mean) and thus data not normally distributed, or?

Line 287: mean±SE?

Line 287 onwards: “μg/g dry wt of feces” – I think you can state somewhere that fGCM levels are expressed as μg/g per dry weight of the feces and then reduce that to “μg/g feces” throughout.

Line 296 – Figure 2: As the SE is huge (sometimes more than the mean), I think it’s better to give boxplot graphs of the values than presenting mean±SE.

Line 300: For reason outlined above (“effects”), I suggest to delete this heading (it should not have the same level as the one above (line 286)

Table 4: a p value of 0.0000 is impossible, needs to be <0.0001.

Lines 344-347: Move this to the supplements. It is only a small technical details and not of general interest (or importance).

Lines 349-359: Vaz et al (2017) utilized a corticosterone immunoassay. However, this does not mean that they measured corticosterone in the feces (see e.g. Palme, 2019 for a more detailed explanation). They did not characterize the immunoreactive compounds in the fecal samples of Indian leopards. Because glucocorticoids are heavily metabolized prior to excretion and also (as you correctly stated) cortisol and not corticosterone is the predominant GC, it is very likely that their assay picks up (cortisol) metabolites. As cross-reactivity with those metabolites is expected to be low, this perfectly explains the lower fGCM levels reported in [21]. I started to rewrite this paragraph, but I think more needs to be done here. In addition, the assay of Vaz et al. was not validated for leopards, which also warrants mentioning somewhere.

Line 355: I suggest to delete [32, 33], the first does not seem suited at all, and the second is included in the review [34].

Line 361: Actually, 5α-pregnane-3β,11β,21-triol-20-one is a corticosterone metabolite (the 17α-OH, the only difference between cortisol and corticosterone, is missing). However, because the immunogen for the antibody, and the label of this EIA have been coupled at position C20, the assay cross-reacts with both, cortisol and corticosterone metabolites. This was recently proven (see Santamaria et al., 2021).

Line 387: “very variable”: Are there studies available, which report sex differences in a carnivore species? Otherwise this sentence does not make sense.

Lines 423/424: This sentence sounds odd. Please reword.

Lines 438-439: I would be very cautiously in drawing conclusions here. You did not perform experiments, just compared different housing situations. To answer such questions I think it would be necessary to plan longitudinal experiments with the same animals and provide different enrichments.

Line 445: So the two old animals were no longer present in summer?

Lines 457-463: Attention! Suggesting to include a huge number of covariates will decrease statistical power, which will result in more negative findings (no effect found), when sample numbers are not increased at the same time. Thus, I think the first demand should be to increase the number of individuals studied and the number of samples collected!

There is one further study published, which investigated fGCM levels in a single captive Afghan and black leopard each, before, during, and after the period of exhibit construction (Chosy et al., 2014). The authors used a corticosterone and a cortisol EIA, respectively, and found higher levels during construction (up to ~1 µg/g feces). It may be worth including this paper in the discussion.

References: The references need careful revision (e.g. capitalized names in 5; first names spelled out in 41) – why are internet links (doi) given for some papers, but not others? Latin names should be in italics, etc…

Figure 1: The resolution of my copy was rather low (but I hope the original one is better), thus it was hard to get the details.

Figure 2: I suggest presenting the levels as boxplot graphs (because variation was so huge; and data not normally distributed, or?). Comparing levels here, with data given in the “Results” sections (e.g. line 291 for the winter in “A”: 25.11±29.39 µg/g) make me wonder, why they do not match (the error bar should exceed the box)? Y-axes legend: “fGCMs (µg/g dry feces)”

Figure 3: It would be easier to orient oneself, when different colours and also open symbols are used. Also may be better to give median levels, instead of mean. The y-axes should then read: “median fGCMs (µg/g feces)”

Figure 4: As outlined above, I suggest moving it to the supplementary material. And it’s not the sample that is “wild” or “captive” �  .

Supplemental Information (for review purpose only): I encourage the authors to make that information available in a supplement (especially the number of samples is interesting to know). Also, it may be worth noting (remarks) that Vaz et al did not validate their EIA for use in Indian leopards.

Above cited references:

Chosy, J., Wilson, M., Santymire, R. (2014): Behavioral and physiological responses in felids to exhibit construction. Zoo Biol. 33, 267-274. https://doi.org/10.1002/zoo.21142

Palme, R. (2019): Non-invasive measurement of glucocorticoids: advances and problems. Physiol. Behav. 199, 229-243. https://doi.org/10.1016/j.physbeh.2018.11.021

Santamaria, F., Barlow, CK., Schlagloth, R., Schittenhelm, RB., Palme, R., Henning, J. (2021): Identification of koala (Phascolarctos cinereus) faecal cortisol metabolites using liquid chromatography-mass spectrometry and enzyme immunoassays. Metabolites 11, 393. https://doi.org/10.3390/metabo11060393

Touma, C., Sachser, N., Möstl, E., Palme, R. (2003): Effect of sex and time of day on metabolism and excretion of corticosterone in urine and feces of mice. Gen. Comp. Endocrinol. 130, 267-278. https://doi.org/10.1016/s0016-6480(02)00620-2

Reviewer #3: The manuscript is scientifically well-conceived and experimental design strategy to examine the environmental enrichment measures was good. Measures of fGCM as a read out to assess the physiological well-being is interesting. The study outcomes provide improvising opportunities in the zoo management practices and findings are important in the context of strategic development of captive management of big cats, Indian leopards, with implications in their conservation.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Submitted filename: Leopard Draft_Final-rev comments.docx

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Submitted filename: PlosRev(RG)21stJan22.docx

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12 Apr 2022

Dear editor and the reviewers,

Thank you for considering our manuscript. We have addressed all the concerns raised by the editors and the reviewers in the revised version of our manuscript. Please refer to the response to reviewers document for further details.

Sincerely,

Ratna Ghosal

Submitted filename: Response to reviewers.docx

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2 Jun 2022

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Govindhaswamy Umapathy, PhD

Academic Editor

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: Dear Authors,

The revised manuscript addresses many of the comments and suggestions set out by the reviewers, however one of the fundamental issues you have failed to address is the standard of writing. Your findings have merit and would be of value to other conservation entities and practitioners for the care and welfare of captive leopards, however you have not devoted the same time and effort into accurately communicating your results in grammatically correct English. This oversight detracts substantially from your study. Even your title is grammatically incorrect and I therefore encourage you once again, as I did in my first review, to seek the assistance of a native English speaker or to make use of the services of a professional copyeditor.

Reviewer #2: Thanks to the authors for substantially modifying their manuscript. It is much improved now, and I’m happy with it (also their responses to my suggestions). There are only a few, marginal things left, which I kindly ask the authors to correct/modify (see below):

General: Although you describe that levels are expressed “μg/g feces” somewhere in the methods section, it is still necessary to have the dimension when you report values somewhere (for the ease of the reader, but also correctness). You may avoid repetition in a sentence where several concentrations are given, but would need it at least once.

Line 4: delete “levels” it’s obvious and included in “measurement” – otherwise it should read: “fecal glucocorticoid metabolite levels”.

Line 96: [11] – unsure, but I think that this article only marginally deals with sample materials. What about replacing it with Sheriff et al., 2011 – there pros and cons of the different sample matrices are discussed in detail.

Line 280: µg!

Line 319: <.0001 add the 0 --> <0.0001

Line 347: A total of 12 samples was collected.

Line 365: [39] Where do they mention this? I suggest citing [33] here instead.

Line 372: “were detected”

Line 428: effects

Line 472: I suggested rewording, but it’s still here: “demonstrate” is too strong, especially as you did not perform any experiments to prove this – your study is only observational.

Sheriff, MJ., Dantzer, B., Delehanty, B., Palme, R., Boonstra, R. (2011a): Measuring stress in wildlife: techniques for quantifying glucocorticoids. Oecologia 166, 869-887. https://doi.org/10.1007/s00442-011-1943-y

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Reviewer #1: No

Reviewer #2: No

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28 Jun 2022

Uploaded a seperate "Response to reviewers' file

Submitted filename: Response to reviewers.docx

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16 Aug 2022

20 Aug 2022

We have submitted a separate 'Response to Reviewers' document.

Submitted filename: Response to reviewers.docx

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24 Aug 2022

Fecal glucocorticoid metabolite levels in captive Indian leopards (Panthera pardus fusca) housed under three different enrichment regimes

PONE-D-21-38460R3

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30 Aug 2022

PONE-D-21-38460R3

Fecal glucocorticoid metabolite levels in captive Indian leopards (Panthera pardus fusca) housed under three different enrichment regimes

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