Impact of Indigestible Materials on the Efficiency of Fecal Corticosterone Immunoassay Testing in Pituophis Species

SIMPLE SUMMARY: Evaluating stress in animals is important for improving animal welfare and husbandry. However, it has been challenging to establish reliable and noninvasive methods for quantifying stress. Steroid hormones released during prolonged periods of stress are metabolized by the liver and excreted in feces. In snakes, corticosterone is the primary hormone of stress and is often measured in fecal samples collected from these animals. Assays can be used to measure the metabolites of corticosterone from feces, but are limited by compounding factors (season, reproductive status, diet, etc.) that influence the ability to confidently conclude the results. Our overall objective is to standardize these methods by first improving the extraction methodology. We found that 75% of the fecal sample contained indigestible materials (hair, teeth, bone, etc.) and, therefore, interferes with the extraction process. After removing the indigestible material, we found that we had a 95% improvement in overall yield. These findings alleviate one limitation to using fecal samples to measure stress in animals. ABSTRACT: Measuring fecal glucocorticoid metabolites (FGM) has recently become a sought-after method for assessing stress in animals. While there are many benefits to this methodology, there are also recognized limitations, including the apprehensive interpretation of results. While many factors can influence FGM levels, we aimed to standardize and improve these methods in snakes. Fecal samples were collected from Pituophis species and FGMs were extracted by two different sample collection methods: (1) fecal sample containing undigested materials and (2) fecal samples with undigested materials removed. These extracts were then used to quantify FGM concentrations using a corticosterone EIA kit. The results indicated that the samples with the undigestible materials removed had a 95% increase in overall yield (p < 0.01). Since the collected fecal samples contain 75% undigestible materials by weight, these results support our hypothesis that removing these materials will improve extraction methods for a more reliable measurement of corticosterone. This is the first step towards standardizing the methods for assessing stress by measuring fecal glucocorticoid metabolites in snakes.