Validation of a field-friendly faeces drying and storage method for quantifying faecal glucocorticoid metabolites in African elephants (Loxodonta africana) opens up new perspectives for conservationists

Faecal glucocorticoid metabolites (fGCMs) provide relevant information on wildlife welfare but remain underapplied in conservation due to sample preservation issues. We validated a field-friendly drying and storage method for preserving African elephant faeces, addressing logistical challenges of fGCM quantification, while anticipating the growing interest in considering welfare in conservation management.


Introduction
One of the major coping mechanisms to environmental or anthropogenic perturbations (e.g. lack of food, movement restraints, social disruption, human disturbance, translocation) is the stress response (Wingfield et al., 1998), being defined as a suite of behavioural, physiological and neuroendocrine responses whose aim to neutralize the effect of stressors (Cannon, 1929). These days, markers of physiological stress as an indicator of wildlife disturbance and welfare are among the most popular tools in conservation biology (Busch and Hayward, 2009;Sheriff et al., 2011). Among them, the quantification of faecal glucocorticoid metabolites (fGCMs) has received considerable attention because it has great potential as a non-invasive diagnostic of endocrine functions and as a decision aid for conservationists (Ganswindt et al., 2012;Dantzer et al., 2014;Palme, 2019).
However, a barrier to the popularization and widespread use of fGCM monitoring among conservationists lies in logistic limitations to achieve restrictive sample collection and storage protocols in remote areas (Madliger et al., 2018). To avoid potential alteration of fGCM composition postdefecation, the widely used standard procedure consists of collecting the faeces sample in a quick and defined period after deposition and keep it frozen at −20 • C until steroid extraction and analysis (Sheriff et al., 2011). The strict maintenance of a cold chain between sample collection and analysis in the laboratory is particularly important (Möstl et al., 2005). The composition of fGCMs cannot only be altered by freezing and thawing cycles (Pappano et al., 2010;Gholib et al., 2017), but also by keeping samples at low above-zero temperature (Carbillet et al., 2023), which poses serious logistical challenges in remote areas and hot climates. This has stimulated research into faeces preservation treatments other than freezing for field application. To be widely applicable in the field, the sample collection, pre-treatment and storage protocols must be easy to implement and low energy consuming, while ensuring biologically meaningful fGCMs values and that the signal is stable and repeatable. For example, the use of organic solvents to preserve faeces in the field has proved effective in some species (e.g. in black rhinos (Edwards et al., 2014) and in primates (Khan et al., 2002;Pappano et al., 2010;Shutt et al., 2012;Gholib et al., 2014Gholib et al., , 2018Nugraha et al., 2017)), but with different preservation duration among studies (from a few hours to several weeks). More recently, dehydration of faeces with silica beads has proved effective in preserving fGCMs in horses (Krueger et al., 2019) and African lions (Fowler and Santymire, 2022). However, faeces preservation protocols need to be properly validated for each new species (Palme, 2019), so there is no evidence that any of these protocols are suitable for elephants.
In African elephants, alternatives to freezing protocols for faecal steroid metabolite preservation have been tested on a single captive elephant, including drying in an oven, or with silica gel, or immersing the samples in 90% ethanol, but the fGCM concentrations differed greatly in a time-dependent manner from those in samples preserved at −20 • C (Hunt and Wasser, 2003). Simple drying protocols consisting of passive exposure of elephant dung to sunlight or shade also resulted in a drastic loss of fGCM concentration (Webber et al., 2018), which may be attributed to the long drying time that allows for the alteration of the immunoreactive steroid metabolite composition in faeces presumably by bacterial enzymes (Bokkenheuser and Winter, 1980;Möstl et al., 1999).
Very recently, a dehydration technique using a low-power supplied food dehydrator has been tested on African wild dog faeces and has shown encouraging results on the integrity of fGCMs (Postiglione et al., 2022). Compared with other alternative drying processes, such as exposure to sunlight and the solar oven, this protocol fastens the removal of moisture from the faeces by combining heating with the circulation of a constant air flow. Therefore, the combined use of a food dehydrator and silica beads as a desiccant to trap water in the air inside the food dehydrator would further accelerate the drying process and prevent degradation of fGCMs by bacterial enzymes present in the faeces (Palme, 2019). Whether this protocol is relevant for African elephant dung remains an open question. The question also arises whether fGCMs in field-dehydrated samples are stable when preserved at ambient temperature. Hence, the aim of our study was i) to validate the method for dehydrating African elephant faeces using a food dehydrator and desiccant for subsequent measurement of fGCM concentrations and ii) to test the stability of field-dehydrated samples at ambient temperature over time.

Material and Methods
All the procedures in this study received approval from the Nelson Mandela University Animal Research Ethic Committee (ethical clearance number A21-SCI-NRM-002).

Samples collection
We collected faecal samples from 40 free-ranging African elephants in seven reserves of the North West, Limpopo and Kwa-Zulu Natal Provinces of South Africa from mid-August to late September 2021. All elephants were adults and included 15 males, 5 females and 20 unidentified. Elephants were tracked from a distance and all samples were collected within 1 hour after defecation. For each individual, 50 g of faecal material was sampled from the middle of the bolus to avoid cross-contamination with urine or debris (Ganswindt et al., 2002). Wearing disposable gloves, the samples were kneaded thoroughly to ensure uniform distribution of hormones, and then split in two. One sub-sample was sealed in a plastic bag for subsequent freezing at −20 • C and served as control. The second sub-sample was placed in a folded paper bag for subsequent dehydration protocol ( Figure 2). In the field, samples were placed on ice packs in a cooler box before returning to the camp no more than 8 hours after collection. The samples contained in paper bags were carefully deposited in the cooler box so as not to come into direct contact with the ice packs, which would have wetted them.

Dehydration and storage of faecal samples in the field
Dehydration of faecal samples was performed in a food dehydrator (model BK002 Mellerware, Johannesburg, South Africa) for 24 h (Figure 1). The dehydration process was ensured by heating with a B22 incandescent bulb (60 W) and a constant air flow. We added 20 g of desiccant silica crystals in a small plastic container and we deposited the container open in the bottom of the dehydrator to hasten humidity absorption and optimize the drying process. Each paper bag containing the faecal sample of a single individual was widely opened and hooked with wire to hang in the dehydrator. The dehydrator contained from one to six samples at a time. After Figure 1: Diagram of the dehydrator assembly. The dehydration process was ensured by the heat released from the B22 incandescent bulb (60 W) and the constant air flow produced by the fan. We added 20 g of desiccant silica crystals in a small plastic container and we deposited the container open in the bottom of the dehydrator to hasten humidity absorption and optimize the drying process. Each paper bag containing the faecal sample of a single individual was widely opened and hooked onto a support bar with wire, hanging in the dehydrator.
24 h of drying, the samples were transferred, sealed in plastic containers and maintained at ambient temperature (15-25 • C) until assayed. The average time from dehydration to hormone assay was 34 ± 12 SD days and ranged from 15 to 52 days.
The faecal samples that were frozen in the field were maintained at −20 • C until reaching the Endocrine Research Laboratory, University of Pretoria, South Africa. They were then lyophilized using a freeze-dryer (model ALPHA 1-2 LD plus, Christ, Osterode, Germany) for 5 days at −56 • C.

Steroid extraction and analysis
All samples were pulverized and passed through a mesh sieve to remove undigested material (Ganswindt et al., 2005). For each individual sample and each drying method, we performed three independent steroid extractions using 0.050-0.055 g of faecal powder in 3 ml of 80% ethanol-water ( Figure 2). After vortexing for 15 min, the mixture was centrifuged at 1500g for 10 min and the supernatant was transferred into sealed microcentrifuge tubes for storage at −20 • C until hormone analysis (Ganswindt et al., 2010).
Steroid extracts were analysed using an established 11oxoaetiocholanolone Enzyme Immunoassay (EIA) detecting fGCMs with a 5β-3α-ol-11-one structure (Möstl et al., 2002;Ganswindt et al., 2003). The sensitivity of the assay was 1.5 nanograms per gram of faecal dry weight (ng/g DW). Intra-and inter-assay coefficients of variation, determined by repeated measurements of low-and high-concentration quality controls were 4.9 and 6.8% as well as 9.8 and 10.4%, respectively. The average standard deviation of triplicates was 33 ng/g DW for the freeze-dried samples and 31 ng/g DW for the dehydrated ones. In addition, the average coefficients of variation of triplicates were 10.0 and 10.8% for the freeze- dried and the dehydrated samples, respectively. Assay procedures followed published protocols (Ganswindt et al., 2002) and were conducted in the Endocrine Research Laboratory, University of Pretoria, South Africa.

Statistical analysis
We determined whether the individual sample mean fGCM concentrations differed between the two drying and storage procedures using a paired t test. We performed a linear regression to estimate the equation line describing the relationship between fGCM concentrations from the two sample drying methods. The fGCM values were normally distributed, so we then performed a Bland-Altman analysis to evaluate the mean difference in fGCM concentration between the two methods and estimate an agreement interval (Bland and Altman, 1986). Finally, we performed a linear regression to investigate the effect of the storage time on the differences in fGCM concentrations between the two drying methods. All statistical analyses were carried out using R version 4.1.2 (R Core Team, 2021).

Results
The mean concentration of fGCM measured in the 40 elephants was 332 ± 132 SD ng/g DW when the faeces were stored at −20 • C and freeze-dried before analyses, whereas it was 311 ± 127 SD ng/g DW when dehydrated in the field and then maintained at ambient temperature. The loss of fGCM was 6%; however, this difference was statistically significant (t = 2.9506, P = 0.0053) (Figure 3).  There was a strong linear relationship between fGCM concentrations measured using the two protocols; the slope of the relationship was close to 1 (0.90) and the coefficient of determination was R 2 = 0.88, P < 2.2e-16 (Figure 4). . The x-axis represents the average measures of the two protocols, whereas the y-axis represents the difference between the two protocols. The solid line represents the mean difference, also called 'the bias', and the dashed lines represent the limits of agreement, defined as the mean difference ±1.96 times the standard deviation of the differences. In this study, the mean difference between the two protocols was 21 ng/g DW, with the upper and lower limits of agreement at 110 and −67 ng/g DW, respectively. Most of the data points fall within the limits of agreement, indicating good agreement between the two methods.
The Bland-Altmann plot indicated a mean difference, also called 'the bias', between freeze-dried and dehydrated values of 21 ng/g DW, or 6% of difference, and the limits of agreement were between −67 and 110 ng/g DW, within which 95% of the differences between the two protocols fell ( Figure 5).
Storage time at ambient temperature of faecal samples, ranging from 15 to 52 days, had no effect on the difference in fGCM concentrations between the two protocols (F-statistic = 1.82; P = 0.18) (Figure 6).

Discussion and Conclusion
Our results demonstrate that the protocol combining food dehydrator and desiccant is suitable for dehydrating African elephant faeces without compromising the fGCM integrity and thus provides a field-friendly alternative to the often logistically challenging process of freezing samples on-site and storing them frozen until analysis. Although the paired t test indicates a statistically significant difference in fGCM concentrations between the two protocols (t = 2.9506, P = 0.0053), the very predictive regression (R 2 = 0.88, slope = 0.9, Figure 4) indicates a robust relationship, although with a slight underestimation of fGCM with the food dehydrator protocol. This underestimation is mostly noticeable at relatively higher fGCM levels, i.e. >400 ng/g DW, and this does not contradict the fact that we can clearly generate biologically meaningful results with the food dehydrator approach. The Bland-Altman analysis provided a simple and accurate way to quantify the limits of agreement between two variables (Dogan, 2018), within which 95% of the difference of fGCM values following the field dehydration protocol, compared with the gold standard freezing procedure, fell. This additional analysis enabled us to compare further our method against the gold standard. The small mean difference of 21 ng/g DW, or the 'bias', between the two protocols indicates that, on average, the fGCM concentration is slightly underestimated when measured via the food dehydrator (of ∼6% on average). With a mean standard deviation of triplicates of ∼30 ng/g DW for both protocols, such bias, although statistically significant, is therefore less than the random technical noise associated with the fGCM measurement. This small bias value and the reasonable limits of agreement ranging from −67 to 110 ng/g DW again indicate that the use of dehydration protocol in the field should not affect our interpretation of revealed fGCM concentrations and is unlikely to influence elephant management decisions differently. Nevertheless, the slight overall underestimation of fGCMs with our protocol should be taken into consideration, particularly if comparisons are made with data obtained using another sample conservation protocol. We also checked that the dehydration protocol in the field did not lead to repeatability issues; the mean standard deviation of the triplicates was very similar with both methods, i.e. 31 ng/g DW for the field dehydrated samples versus 33 ng/g DW with the standard freezing procedure, as well as the mean coefficient of variation, i.e. 10.0 and 10.8%, respectively, showing a similar dispersion around the mean.
Finally, we ensured that the fGCMs were stable when the faeces dehydrated in the field were stored at ambient temperature, a necessary condition to secure storage and transport of the samples while minimizing logistical constraints. We found no significant effect of storage time at ambient temperature on the differences between the two protocols up to 52 days (Fstatistic = 1.82; P = 0.18). Although there was no observable trend of increasing or decreasing fGCMs over time, it cannot be ruled out that analyses conducted on a larger sample size would not have slightly altered our conclusions. We also cannot exclude that the differences in fGCM concentrations may be affected over a longer storage time, as observed with other faeces storage methods (Hunt and Wasser, 2003;Pappano et al., 2010). However, the long-term stability of fGCMs should be secured provided that the bacterial activity is kept suppressed (Bokkenheuser and Winter, 1980;Möstl et al., 1999;Lexen et al., 2008), i.e. as long as the samples are kept free of moisture (Möstl et al., 2005). It is thus reasonable to expect that storage of faeces at ambient temperature should be possible for a longer period provided the samples are kept dry (i.e. stored in sealed tubes or maintained in a dry atmosphere). Further experiments are needed to confirm whether there is a limit to the storage time of faeces dehydrated following our protocol.
The use of a food dehydrator and desiccant provides an easy and cost-effective alternative to keeping the samples frozen, preferable when a limited power supply is available or in a remote study area, which may involve complicated transport of samples. Compared with other methods where samples are also dried directly in the field (e.g. sunlight exposure), this alternative method also suppresses any reliance on weather conditions and protects the samples from insects and birds. The drying method using silica beads in contact with faeces has not proved effective in elephants for preserving samples at ambient temperature (Hunt and Wasser, 2003), and therefore does not make it possible to dispense with a freezer in the field. However, we deduced that the addition of silica crystals as a desiccant in the food dehydrator would indeed hasten and optimize the drying process. These aspects indicate that the use of a food dehydrator with desiccant could be an excellent tool to improve and develop the monitoring of African elephant physiological stress in areas where such monitoring programs were not implemented before for logistical reasons.
In a context of growing interest in African elephant welfare in South Africa (South African Department of Forestry, Fisheries and the Environment (DFFE), 2020), the need to consider such physiological metrics in conservation management decision-making is becoming crucial. In addition, the application of a clinical analytical method, such as the Bland-Altman analysis, adds a stone to the edifice of a transdisciplinary approach to conservation and provides an easy tool to researchers to test new field-friendly methods of interest for conservationists. Future research should thus focus on the efficacy of this alternative to freeze-drying for other species and different steroid classes.

Funding
This work was supported by the French doctoral school Evolution, Ecosystèmes, Microbiologie, Modélisation (E2M2), Claude Bernard Lyon 1 University and the French National Centre for Scientific Research (CNRS) through allocations to the International Research Laboratory REHABS.