Killer whale fecal samples: How to get the most out of a single extraction

Fecal samples are a non-invasive and relatively accessible matrix for investigating physiological processes in resident killer whale (Orcinus orca) populations. The high lipid content of the diet (primarily salmonids) leads to lower density fecal material and slower dispersion, facilitating sample collection. As fecal discharge is relatively infrequent and the volume of sample is variable, maximizing analytical options is an important consideration. Here we present an extraction methodology to measure hormones and lipid content from the same fecal aliquot. Lipid extractions are commonly conducted using chloroform and methanol from Folch or Bligh and Dyer (B&D), while alcohol is the primary solvent for hormone extraction. We evaluated the possibility of using the methanol layer from lipid extractions to assess fecal steroid hormone levels. Folch and B&D methanol residues were assayed form metabolites of progesterone (PMs) and corticosterone (GCs), and results were compared to aliquots extracted in 70 % ethanol. Hormone concentrations measured in the methanol layer from Folch and B&D extractions were 55 % to 79 % lower than concentrations in 70 % ethanol. We developed mathematical corrections, using linear regression models fitted to Folch or B&D methanol vs 70 % ethanol hormone concentrations (p < 0.01). Fecal concentrations of PMs and GCs from methanol extractions were biologically validated and are significantly higher in confirmed pregnant females compared to non-pregnant individuals (p < 0.05). This study demonstrates that lipid extraction protocols may be used for the analysis of multiple biomarkers, maximizing the use of small-volume samples.