Validation of a field-friendly extraction and storage method to monitor fecal steroid metabolites in wild orangutans

Abstract

Measuring hormone metabolites from feces is the most often used method to assess hormonal status in wildlife. Although immediate freezing of fecal samples collected in the field is the best method to minimize the risk of degradation of hormones over time, this is often not possible in remote field sites. Therefore, alternative storage and preservation methods for fecal samples are required in these conditions. We conducted an experiment to investigate if fecal glucocorticoid (FGCM) and progesterone metabolite (pregnanediol-3-glucuronide; PdG) levels measured from samples that were extracted with a simple, field-friendly methodology correlate with those generated from frozen samples. We also evaluated whether storing fecal samples in alcohol is a suitable alternative to preserve FGCM and PdG concentrations long-term (i.e. over a 9-month period) at locations where fecal extraction is not feasible. Finally, we tested if the hormone concentrations in unpreserved fecal samples of orangutans change over 14 h when stored at ambient conditions, representing the maximum duration between sample collection and return to the camp. FGCM and PdG levels measured from samples that were extracted with the field-friendly method showed strong correlations with those generated from frozen samples, and mean levels did not differ significantly between these methods. FGCM concentrations showed no significant change compared to control samples when fecal samples were stored for up to 6 months in alcohol at ambient temperature and PdG concentrations even remained stable for up to 9 months of storage. FGCM concentrations of fecal samples kept at ambient temperature for up to 14 h post-defecation did not significantly differ compared to control samples frozen immediately after collection. These results provide the basis for the successful monitoring of the physiological status of orangutans living in remote natural settings, like those included in the Indonesian reintroduction programs.

Repeated Freeze-Thaw Cycles but Not Short-Term Storage of Fecal Extracts at Ambient Temperature Influence the Stability of Steroid Metabolite Levels in Crested Macaques

“…Moreover, different species have different diets and gut microflora which may affect potential degradation and the chemical nature of the metabolites in a species-specific manner (Hunt and Wasser, 2003;Goymann, 2012). Different assay antibodies may be more or less prone to detect these changes (Nugraha et al, 2016). From a practical point of view, our results suggest that repeated freeze-thaw of fecal extracts may compromise the stability of fGCM and fEM levels, although the changes in hormone metabolite levels recorded were relatively minor, i.e.…”

“…Our experiment used a group-specific measurement of 5ß-reduced cortisol metabolites using an 11ß-hydroxyetiocholanolone EIA that has been previously validated by Gholib (2011) and used successfully for other primate species (e.g. Girard-Buttoz et al, 2009;Nugraha et al, 2016), while Pappano et al (2010) used a commercial corticosterone radioimmunoassay. Second, our experiment used savelock (Eppendorf ®) tubes for storing fecal extracts, while the previous study used SPE cartridges for storing the fecal extracts.…”

“…However, analyzing hormone metabolites from fecal samples is not as easy as it seems (Heistermann, 2010). There are several aspects concerned with pre-analytical factors that need to be considered prior to analysis, such as time of sample collection (Turriani et al, 2016), handling/processing (Kalbitzer and Heisterman, 2013), preservation/storage (Shutt et al, 2012;Nugraha et al, 2016) and assay validation (Gholib et al, 2016).…”

“…However, this was not always successful as hormone levels still showed alterations after long-term storage. To overcome this problem, other researchers extracted the fecal samples immediately after collection using a "field friendly extraction" method (Shutt et al, 2012;Kalbitzer and Heisterman, 2013;Nugraha et al, 2016). Ziegler and Wittwer (2005) assumed that once the hormone metabolites are extracted from the feces, the levels will remain stable even if the extracts are stored at ambient temperature.…”

A Review of Hormone Analysis on Primate Ecology

“…。しかし，尿は排泄後に時間が経 過すると乾燥して回収できなかったり，排尿場所によっ ては全く回収ができない場合がある。そのため，より 採取が容易な糞を用いた研究が，その後 1990 年頃から 飼育下および野生下で発表されるようになった (Lasley and Kirkpatrick, 1991;Wasser et al, 1988;Ziegler et al, 1989) 。雌の性ホルモン(特に種特異性のない性ステロ イドホルモン)濃度を分析することで，春機発動(繁 殖能力の発現開始) ，発情期(排卵期) ，妊娠の有無， 陰部腫脹との関係などが明らかにされてきた (Carroll et al, 1990;Curtis et al, 2000;Dahl et al, 1987;Heistermann et al, 1996;Kinoshita et al, 2017;Shimizu, 2005;Shimizu et al, 2003) Fourie et al, 2016) Fairbanks et al, 2011;Henley et al, 2013;Vliegenthart et al, 2016;Yamanashi et al, 2013) (Maggioncalda et al, 1999;Marty et al, 2015) ，その間に成長ホルモンの濃度も上昇 することが確認されている (Maggioncalda et al, 2000) 。 ただし，野生オランウータンにおける遺伝解析により， アンフランジ(フランジがない)雄でも子の父親になっ ていることが確認されており (Goossens et al, 2006) (Prudom et al, 2007;Roberts et al, 2001;Soltis et al, 2005;Ziegler and Snowdon, 2000;Ziegler et al, 1996) (Woods, 1975) Pettitt et al, 2007) 。糞中の水分含量が高いと，より一層細菌による 代謝は活発になる (Palme, 2005 (Khan et al, 2002;Palme, 2005;Pettitt et al, 2007;Woods, 1975 (Kinoshita et al, 2016) 。オランウータンやチンパンジーにおいて， 有機溶媒を用いてフィルターペーパーからホルモンを 抽出し分析に成功している研究例もある (Seraphin et al, 2008;Shideler et al, 1995;Thompson and Knott, 2008) 。 このように，調査地の状況に合わせてどのような保存 方法をとるべきかについても，予備実験で確認してお くことが重要である。また，近年は「Field-Friendly 法」 として，保存過程を経ずに野外で直接サンプルからホ ルモンを抽出し実験室に抽出物を持ち帰って分析して い る 例 も あ る (Nugr...…”