Development of a whole-body cortisol extraction procedure for determination of stress in golden shiners, Notemigonus crysoleucas

Abstract

Baitfish such as golden shiners are subjected to stress during harvesting, grading, and transport. Their small size makes it difficult to measure the stress response with the biological indicator cortisol using conventional assay methods for plasma. This paper examines the development and validation of methods for whole-body cortisol extraction from individual baitfish. Three types of extracts were tested: (1) an ethyl ether unaltered extract (UA); (2) an extract reconstituted in phosphate buffered saline (PBS); (3) an extract that had been increased in volume by the addition of food-grade vegetable oil (VO). These extracts were evaluated using validation tests with radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA). The UA extract produced inadequate volumes of extract for multiple assays and could not be used for the determination of cortisol in a single fish. The PBS reconstitution method failed the precision recovery of serial dilutions (62.3%), linearity (R 2 : 0.7864), and parallelism validation tests. The VO volume-boosting method passed all validation tests [intra-assay coefficent of variation (%CV): 16.3 for ELISA and 5.9 for RIA; interassay %CV: 10.3; spiked recovery: 102.0%; dilution recovery: 93.0%; linearity R 2 : 0.9435; log of serial dilutions was parallel] and provided enough extract for multiple assays from an individual baitfish. Based on these results, we conclude that the VO volume-boosting method presents a means for determining cortisol from individual baitfish using either RIA or ELISA assays.

Comparing the efficacy of dietary α-tocopherol with that of dl-α-tocopheryl acetate, both either alone or in combination with ascorbic acid, on growth and stress resistance of angelfish, Pterophylum scalare, juveniles

“…To evaluate whole-body cortisol levels as a stress-response indicator, frozen juveniles were sectioned with a scalpel and then were used for cortisol extraction using the method described by Sink et al (2007). Whole-body cortisol was then measured using the commercially available EIAgen cortisol test (BioChem ImmunoSystems) according to Barcellos et al (2007).…”

Validation of a whole-body cortisol extraction procedure for channel catfish (Ictalurus punctatus) fry

“…We hypothesized zero calibrator A would be the most appropriate diluent to reconstitute the lipid extracts with since this is the same buffer used in the cortisol standards. In the PBS method, lipid extract was reconstituted with 1 ml of PBS (PBS method) to increase volume of the sample, as described by Sink et al (2007) and Ramsay et al (2006). In the VO method, 100 ll of vegetable oil per gram of body weight was added to increase the volume of the extract before adding the homogenized sample and ethyl ether, as described in Sink et al (2007).…”

“…The zero calibrator A buffer was the same buffer used to make the standard curve. In the second method, the lipid extract was reconstituted with 1 ml of BupH TM Modified Dulbecco's Phosphate Buffered Saline (Pierce, Rockford, IL) (PBS method) to increase volume of the sample as described by Ramsay et al (2006) and Sink et al (2007). In the third method, 100 ll of food-grade vegetable oil (Enova TM oil, Kao Health and Nutrition, Itasca, IL) (VO method) per gram of body weight was added to increase the volume of the extract before adding the homogenized sample and ethyl ether, as described in Sink et al (2007).…”

“…Cortisol extraction was performed using the method described by Sink et al (2007), with some modifications. Briefly, the samples, still in the storage tube, were homogenized in 1 ml of PBS, using a Qiagen Tissue Lyser for 15 min.…”

“…Whole-body cortisol assays of larval rainbow trout (Oncorhynchus mykiss) and yellow perch (Perca flavescens) (Barry et al 1995;Jentoft et al 2003), juvenile summer flounder (Paralichthys dentatus) and winter flounder (Pseudopleuronectes americanus) (Hinkle and Specker 2003;Breves and Specker 2002), and zebra fish (Danio rerio) and golden shiners (Notemigonus crysoleucas) (Ramsay et al 2006;Sink et al 2007) have been published. The methodology of the extraction procedures varies in these papers, the description of the methods used is sometimes incomplete, and the validation of the cortisol assay is not always complete.…”