Long-term storage effects in steroid metabolite extracts from baboon (Papio sp.) faeces – a comparison of three commonly applied storage methods

1. The measurement of steroid hormone metabolites from faeces in wild animal populations is a powerful, non-invasive tool in behavioural endocrinology of all major vertebrate taxa. However, because such research is often done in remote areas with limited infrastructure, storage of samples for hormone analysis over long periods at high temperature is a critical issue in field endocrinology. Previous studies have indicated that storage of alcoholic faecal extracts is more reliable than storage of unprocessed faeces if no freezer is available, but a standard method has not been established yet. 2. We tested the validity of three commonly applied storage conditions - liquid extracts, dried extracts, and extracts placed on solid phase extraction (SPE)-cartridges - to preserve concentrations of glucocorticoid and androgen metabolites from faecal extracts of olive baboons (Papio anubis) at high temperature over one year. 3. Temporal variation in concentrations was detected for all metabolites and all storage conditions, including values measured from the control condition, i.e. extracts stored at 20°C. This suggested that most variation was due to inter-assay variability, corroborated by comparisons of variation in ‘quality controls’ and samples. 4. Compared to frozen control samples, liquid extracts were stable for up to 24 weeks, extracts on SPE-cartridges were stable for up to 50 weeks, while steroid metabolite concentrations in dried extracts decreased slightly over time. 5. If steroid samples have to be stored at ambient temperature, we suggest storage of liquid extracts for up to 24 weeks in a dark and cool place. For longer periods, SPE-cartridges should be applied as evaporation, a potential confound arising with long-term storage of liquid extracts at higher temperatures, is not a problem in this storage condition. Storage of dried extracts is more cost-effective, but may result in small time-dependent changes in steroid concentrations.

1. 对野生动物种群粪便中的类固醇激素代谢物进行检测，是针对所有主要脊椎动物类群（vertebrate taxa）的行为内分泌学（behavioural endocrinology）研究中一种高效且非侵入性的研究手段。然而，此类研究常开展于基础设施有限的偏远地区，因此在高温环境下长期储存样本用于激素分析，是野外内分泌学（field endocrinology）领域的关键难题。既往研究表明，在无法使用冷冻设备的情况下，酒精浸泡的粪便提取物的储存方式比未处理的粪便样本更可靠，但目前尚未形成标准化的储存方法。
2. 本研究针对三种常用储存条件——液体提取物、干燥提取物以及置于固相萃取（solid phase extraction, SPE）小柱上的提取物——进行了有效性验证，以探究它们在高温环境下保存橄榄狒狒（olive baboons, Papio anubis）粪便提取物中糖皮质激素（glucocorticoid）与雄激素代谢物（androgen metabolites）浓度长达一年的稳定性。
3. 所有代谢物与所有储存条件下均检测到浓度随时间的变化，包括对照条件（即储存于-20℃的提取物）的检测结果。这提示绝大多数变异源于批间变异（inter-assay variability），该结论通过对比"质量控制（quality controls）"样本与实际样本的变异程度得到了验证。
4. 与冷冻对照样本相比，液体提取物的稳定性可达24周，置于SPE小柱上的提取物稳定性可达50周，而干燥提取物中的类固醇激素代谢物浓度则随时间出现小幅下降。
5. 若类固醇样本需在环境温度（ambient temperature）下储存，我们建议将液体提取物置于避光阴凉处储存，最长保存期限为24周。若需长期储存，则应采用SPE小柱储存方式：高温环境下长期储存液体提取物可能引发蒸发问题，而该储存方式则不存在此隐患。干燥提取物的储存方式成本更具经济性，但可能导致类固醇浓度随时间出现小幅变化。