Comparison of Sample Extraction and Storage Methods across Time for Fecal Progesterone Metabolite Analysis in Female Verreaux’s Sifaka

AbstractResearchers commonly collect fecal samples from wild animals to monitor steroid metabolites and associate them with ecology and behavior. This method requires that we develop and validate species-specific methods that are appropriate for various field conditions and compatible with laboratory resources. We compared multiple extraction and storage methods for analyzing fecal progesterone metabolites (fPms) in wild female Verreaux's sifaka (<i>Propithecus verreauxi</i>). We created a subset of samples from a single homogenized fecal pool. We preserved samples by freezing, desiccation, and suspension on four different brands of solid-phase extraction (SPE) cartridges and extracted or eluted samples under each condition at 0-, 90-, 180-, and 270-d intervals for subsequent enzyme immunoassay. Mean progesterone metabolite levels varied across conditions (<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mrow><mml:mn>655.41</mml:mn><mml:mo>±</mml:mo><mml:mn>247.46</mml:mn></mml:mrow></mml:math> to <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mrow><mml:mn>846.09</mml:mn><mml:mo>±</mml:mo><mml:mn>217.44</mml:mn></mml:mrow></mml:math> ng/g). Frozen samples generally yielded the highest fPm levels; post hoc comparisons, with Bonferroni correction, indicated that samples that were desiccated and samples that were suspended on Alltech Maxi-Clean SPE cartridges were the most similar to frozen samples overall (<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mrow><mml:mi>P</mml:mi><mml:mo>></mml:mo><mml:mn>0.05</mml:mn></mml:mrow></mml:math>). Alltech SPE cartridges produced the most consistent results within and across time points. We were unable to suspend or elute sample extracts using SPE cartridges from two brands that were not developed for long-term storage. Based on samples from the remaining four conditions, fPm levels did not vary from time point 1, except for an increase in time point 4 (<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mrow><mml:mi>β</mml:mi><mml:mo>=</mml:mo><mml:mn>0.050</mml:mn></mml:mrow></mml:math>, <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mrow><mml:mi>SE</mml:mi><mml:mo>=</mml:mo><mml:mn>0.025</mml:mn></mml:mrow></mml:math>, <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mrow><mml:mi>t</mml:mi><mml:mo>=</mml:mo><mml:mn>2.002</mml:mn></mml:mrow></mml:math>, <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mrow><mml:mi>P</mml:mi><mml:mo>=</mml:mo><mml:mn>0.046</mml:mn></mml:mrow></mml:math>), suggesting long-term stability to at least 180 d. However, no consistent trend across time was apparent within any condition. Overall, we found that freezing, desiccation, and field extraction using Alltech SPE cartridges are reliable methods for measuring fPms in <i>Propithecus edwardsi</i>. We provide guidance for choosing methods under certain field and laboratory conditions, as well as detailed protocols that can help guide protocol development for analyzing fecal steroid metabolites in other species.