Leopards (Panthera pardus) are listed in the IUCN Red list as vulnerable, primarily due to habitat loss, natural prey base depletion and exploitation caused by various anthropogenic activities. Although protected areas are important for leopard conservation, the majority of suitable leopard habitat lies beyond protected area boundaries exposing individuals to different environmental, physiological and psychosocial stressors. This study aimed to examine the suitability of five different enzyme immunoassays (EIAs) for monitoring adrenocortical function in the leopard based on faecal glucocorticoid metabolite (fGCM) analysis. After performing an adrenocorticotrophic hormone (ACTH) stimulation test and investigating the stability of fGCM post-defaecation, faeces from free-ranging leopards in a peri-urban and a conservation area was collected to investigate the potential impact of habitat variability on glucocorticoid output. An EIA measuring fGCM with a 5α-3β-11β-diol structure performed best, demonstrating a ~200-330% increase in fGCM concentrations approximately 40 h post-ACTH administration. Concentrations of fGCM remained quite stable for up to six-days post defaecation and showed a maximum increase of 8% and a maximum decrease of 9%. Although not significantly different (MWRS T = 86, n = 16, p = 0.920), overall median fGCM concentrations were 68% higher in individuals utilising the peri-urban area compared to leopards utilising the conservation area. The ranges of fGCM concentrations between sites however were similar. Individual median fGCM concentrations differed distinctly between free-ranging males and females, possibly linked to female reproductive status. The established method can now assist in addressing some of the issues facing local wildlife managers, conservationists and researchers tackling 1 various aspects related to leopard conservation and management under different land use practices.

1. Animals concentrate key nutrients in their bodies. In fenced wildlife reserves where nutrient input and/or retention is low, the off-site removal of animals may constitute a significant loss of nutrients for the ecosystem.2. Here we add wildlife capture and removal into the phosphorus (P) and calcium (Ca) budget for a 121,700 ha fenced game reserve located in the southern Kalahari.We then use faecal P concentrations from 11 mammal herbivores >10 kg as an indicator of potential nutrient stress in this system to investigate whether the implications of nutrient loss via off-site wildlife removal may be cause for concern.Finally, we assess the role of natural predation as a mechanism to minimise the need for wildlife removal and concomitant nutrient loss.3. During the period 2009-2018, mean loss of P and Ca via wildlife removal was 2.9 and 6.2 kg km −2 year −1 , respectively. This compares to 1.0 and 2.1 kg km −2 year −1 of P and Ca added via the provision of mineral licks. If it is assumed that natural fluxes of these elements are in steady state, then anthropogenic activities have resulted in a net deficit of 18.5 kg/km 2 of P and 40.6 kg/km 2 of Ca over the decade. 4. We found that dry season herbivore faecal P concentrations are close to or below a widely cited minimum threshold of 2,000 mg/kg, below which most vertebrates begin suffering growth and reproductive issues. Large animals were more likely to be under this threshold. Prolonged continuation of off-site wildlife removal may result in nutrient losses that can lead to long-term ecological degradation. Natural predation levels were, however, found sufficient to mitigate the need for wildlife removal and present a management strategy whereby herbivore populations can be regulated without a loss of nutrients. Synthesis and applications.We find that the capture and permanent removal of large-bodied animals from wildlife reserves can be a significant cause of nutrient loss. Over time, in sites where nutrient input and/or retention is low, this may | 1361

In nutrient-poor wildlife reserves it has become common-place to provide supplemental mineral resources for wildlife. Yet, the impacts of anthropogenic mineral supplementation on community-wide wildlife nutrition, behaviour and subsequent impact on ecosystem processes remain poorly understood. Here, we examine the contribution of anthropogenic mineral lick provision to wildlife nutrient intake across a community of large mammals (>10kg) in the southern Kalahari Desert. Based on predicted daily nutrient requirements and a faecal nutrient assessment, large herbivores appear deficient in phosphorus (P), sodium (Na) and zinc (Zn). For these nutrients, anthropogenic salt and mineral licks constitute an important (>10%) source of nutrient intake helping to reduce or overcome requirement deficits. Larger-bodied species disproportionately consumed licks (p<0.01), acquiring more nutritional benefits. A comprehensive assessment of animal body condition indicated that in general large herbivores display good health. However, bulk grazers, hindgut fermenters and females were more likely to display signs of malnourishment. We discuss how provisioning of anthropogenic mineral licks may be inflating large herbivore populations beyond the long-term carrying capacity of the reserve, with subsequent impacts for ecosystem integrity and herbivore population instability. Based on results presented here, it is clear that anthropogenic provision of mineral licks should be considered carefully by wildlife managers aiming to conserve or restore natural processes in conservation and rewilding landscapes.

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