Measurement of cortisol response is an important tool to asses stress in fisheries research. Radioimmunoassay (RIA) is a common method for the measure of cortisol in fish. Use of enzyme-linked immunosorbent assays (ELISA) to detect cortisol would eliminate health hazards, costs of handling radioisotopes, and the short stability time associated with RIA. Enzyme-linked immunosorbent assays have been used for the determination of cortisol in several fish species. However, the ELISA method of cortisol determination in fish lacks proper validation testing. We conducted validation procedures for multiple commercial cortisol ELISA kits and compared the results to RIA. The assays were tested for four species: (1) channel catfish Ictalurus punctatus, (2) largemouth bass Micropterus salmoides, (3) red pacu Piaractus brachypomus, and (4) golden shiners Notemigonus crysoleucas. We evaluated the ELISA methods against RIA, and determined that at least one kit is suitable (accuracy: mean recovery of spiked samples, 102.8%; reproducibility: interassay coefficient of variation < 10.5% for all species; precision: intra-assay coefficient of variation < 16.8% for all species; linearity: R (2) > 0.96 for all species) for the measurement of cortisol response in fish and comparative determination of stress. All of the ELISA assay results varied by more than 10% from the cortisol concentrations detected by the RIA. The high variability of the kit results indicates that commercial ELISA kits could be utilized for qualitative determination of cortisol in fish, but should be fully validated in each laboratory for each species before being used for research.
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.
We conducted preliminary studies to evaluate the effects of including dairy-yeast prebiotics in the diets of golden shiners Notemigonus crysoleucas with access to natural foods on their resistance to challenge with Flavobacterium columnare. In trial 1, fish were fed either a control diet or a 2% dairy-yeast prebiotic diet for 10 weeks in outdoor pools before challenge. In trial 2, fish fed the experimental diets were either subjected to confinement stress or left unmolested before challenge. Mortality (mean 6 SE) was not significantly different in the control diet (23.4 6 3.4%), the prebiotic diet (10.0 6 3.3%), and the prebiotic diet with stress (16.7 6 3.4%) treatments. However, mortality was significantly greater in the control diet with stress treatment (50.0 6 3.3%) than in the other treatments. This preliminary investigation suggests that prebiotic supplementation in golden shiner feeds before a stressful event would significantly reduce the mortality from F. columnare.
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