Development of a field‐friendly technique for fecal steroid extraction and storage using the African wild dog (Lycaon pictus)

Abstract

Hormonal analysis provides information about wildlife populations, but is difficult to conduct in the field. Our goal was to develop a rapid and effective field method for fecal steroid analysis by comparing: (1) three extraction methods (laboratory (LAB), homogenize (HO) and handshake (HS)) and (2) two storage methods (solid-phase extraction (SPE) tubes vs. plastic tubes (PT)). Samples (n=23) from captive African wild dogs (Lycaon pictus) were thoroughly mixed, three aliquots of each were weighed ( approximately 0.5 g) and 5 ml of 90% ethanol was added. For LAB, samples were agitated (mixer setting 60; 30 min), centrifuged (1,500 rpm; 20 min) and poured into glass tubes. Or aliquots were HO (1 min) or HS (1 min) and poured through filter paper into glass tubes. Samples were split, analyzed for corticosterone (C) and testosterone (T) metabolites using enzyme immunoassays or stored in SPE or PT. Samples were stored (room temperature) for 30, 60 or 180 days, reconstituted in buffer and analyzed. Mean C and T recoveries of HO were greater (P=0.03) than HS compared with LAB, which was similar to HO (P>0.05). After 30 days <21% of C and T was recovered from SPE, but approximately 100% of each was recovered from HO-PT and HS-PT. Similarly, after 60 and 180 days, approximately 100% of C and T was recovered from HO-PT and HS-PT. Results demonstrated that, for C and T, HO was more comparable (P<0.001) to LAB than HS and PT storage was more efficient than SPE (P<0.001).

Non-invasive monitoring of physiological stress in the Western lowland gorilla (Gorilla gorilla gorilla): Validation of a fecal glucocorticoid assay and methods for practical application in the field

“…The strong correlation between field-and laboratory-extracted FGCM values in our study supports this contention indirectly, and we recommend ethanol for extraction of feces in the field as it is often readily available in primate habitat countries. Our data also suggest that simple hand-shaking of samples for a constant amount of time, as suggested by Ziegler and Wittwer (2005), is sufficient to obtain reliable results, although use of a battery-powered homogenizer might increase hormone extraction efficiency slightly (Santymire & Armstrong 2010) especially if feces are hard in texture.…”

“…Having a validated option to extract feces in the field is increasingly important as it removes the risk of unknown alterations in hormone concentrations when feces are stored in alcohol for prolonged periods of time (Khan et al 2002;Hunt & Wasser 2003;Daspre et al 2009), and also allows the researcher to collect fecal samples quickly whilst following wild animals without the need for lengthy or complicated treatments. The best solvent for extracting hormones from feces may be species-and hormone-specific (Palme & Möstl 1997;Pappano, Roberts & Beehner 2010), but several studies have reported high extraction efficiency for steroids using ethanol at 80-100% (Palme & Möstl 1997;Mateo & Cavigelli 2005;Freeman et al 2010;Santymire & Armstrong 2010). The strong correlation between field-and laboratory-extracted FGCM values in our study supports this contention indirectly, and we recommend ethanol for extraction of feces in the field as it is often readily available in primate habitat countries.…”

“…Various extraction/storage techniques have been developed, but the efficiency of these techniques in extracting the hormones to be measured, and the reliability of the storage methods used to stabilize the FGCMs long-term varies with the species and context (Beehner & Whitten 2004;Wielebnowski & Watters 2007;Freeman et al 2010;Pappano, Roberts & Beehner 2010;Santymire & Armstrong 2010). For example, while storing fecal extracts in solid-phase-extraction cartridges is highly efficient for preserving gelada (Theropithecus gelada) FGCM levels in for up to three weeks (Pappano, Roberts & Beehner 2010), it results in a decline of FGCM levels of about 80% within 30 days of storage in feces from African wild dogs (Lycaon pictus pictus) (Santymire & Armstrong 2010).…”

“…For example, while storing fecal extracts in solid-phase-extraction cartridges is highly efficient for preserving gelada (Theropithecus gelada) FGCM levels in for up to three weeks (Pappano, Roberts & Beehner 2010), it results in a decline of FGCM levels of about 80% within 30 days of storage in feces from African wild dogs (Lycaon pictus pictus) (Santymire & Armstrong 2010). Field-friendly extraction and storage methods should therefore also be validated for each species and context.…”

Monitoring stress in captive and free-ranging African wild dogs (Lycaon pictus) using faecal glucocorticoid metabolites

“…We focused on the metabolites that have previously been measured in African wild dogs (de Villiers et al, 1995;de Villiers et al, 1997;Monfort et al, 1998;Santymire and Armstrong, 2009) and that are commonly used to asses stress (Touma and Palme, 2005). We hypothesised that faecal concentrations of GC metabolites in wild dogs would a) be higher in captive than in free-ranging animals, as found in previous studies, b) vary with conditions of captivity; and c) be affected by age and reproductive status, in both captive and free-ranging animals.…”