Comparing techniques for measuring corticosterone in tadpoles

Abstract

Glucocorticoids play a key role in mediating stress responses in vertebrates. Corticosterone (CORT) is the main glucocorticoid produced in amphibians, birds, and reptiles, and regulates several metabolic functions. The most common methods for quantifying CORT are competitive binding immunoassays: radioimmunoassay (RIA) and enzyme immunoassay (EIA). RIA has been broadly used since the 1980’s but it requires radioactivity. Commercial EIA kits permit quantifying hormone levels without radioactivity although the requirement for a larger sample volume may be a strong limitation for measurements involving larval amphibians. Here we used Xenopus laevis tadpoles to compare the performance of three commonly used procedures for determination of CORT: RIA on a chloroform extract of whole-body homogenate, EIA on plasma, and EIA on supernatant of whole-body homogenate. We treated tadpoles with exogenous CORT at 0, 25, 50, and 100 nM. RIA could distinguish between 0 and 25 nM, and EIA on plasma between 0 and 50 nM, whereas whole-body homogenate EIA only detected significant differences between 0 and 100 nM. Each procedure presents advantages and disadvantages regarding sensitivity, the use of radioactivity, sample size, handling time, and economic cost. RIA is preferred when studying small-bodied animals from which blood samples cannot be obtained. When CORT level differences are intermediate and blood sampling is possible, EIA on plasma is a good non-radioactive alternative. EIA on whole-body homogenates may be useful to assess qualitative changes in CORT levels when considerable differences are expected. Finally, we discuss our findings in the context of previous studies on CORT in amphibians.

Metabolic costs of altered growth trajectories across life transitions in amphibians

“…We determined plasma corticosterone content using a commercial enzymoimmunoassay kit (Arbor Assays, K014‐H5), which has low cross‐reactivity with related molecules such as progesterone (0.24%), cortisol (0.38%) or 11‐deoxycorticosterone (12.3%). This is a reliable method for measuring corticosterone in amphibian larvae (Burraco, Arribas, Kulkarni, Buchholz, & Gomez‐Mestre, ). We used a volume of 5 μl of plasma to perform the assay following the procedure as suggested by suppliers.…”

Non-invasive Methods for Measuring and Monitoring Stress Physiology in Imperiled Amphibians

“…Further, plasma measures of GC only represent a single time point. Alternatively, GCs can be measured from whole-body homogenate, which may be a more integrated measure, but frequently require pooling multiple individuals to collect enough hormones (reviewed by Burraco et al, 2015). Sampling GCs via whole-body homogenate (and sometimes blood sampling) requires euthanizing individuals, which is often not permitted for threatened or endangered species and counterproductive to conservation efforts.…”

Physiological Stress Responses in Amphibian Larvae to Multiple Stressors Reveal Marked Anthropogenic Effects even below Lethal Levels

“…CORT levels were determined from whole-body homogenates by performing enzyme immunoassay (EIA; Burraco et al 2015) using commercial kits (Cayman Chemical). This procedure is a conservative test of CORT differences across treatments, as it has lower sensitivity than radioimmunoassay (RIA) or EIA on plasma samples (Burraco et al 2015).…”

“…This procedure is a conservative test of CORT differences across treatments, as it has lower sensitivity than radioimmunoassay (RIA) or EIA on plasma samples (Burraco et al 2015). However, it does not require the use of radioactive isotopes and allowed us to keep the number of animals used in the study at a minimum given the high combination of factors and levels required (Burraco et al 2015).…”