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.

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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.

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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.

Columnaris, caused by Flavobacterium columnare, is a major bacterial disease of the golden shiner Notemigonus crysoleucas, especially when fish become stressed during handling and transport. Feed additives, such as a dairy‐yeast prebiotic, can decrease disease susceptibility in some fish species. Previous studies have indicated that diets with higher fat concentrations have improved the growth and survival of golden shiners. We conducted a study to determine whether a high‐fat diet alone or supplemented with a dairy‐yeast prebiotic could decrease mortality rates of golden shiners subjected to columnaris challenge. Golden shiners were assigned one of three diets with four replicate aquaria per diet and fed to apparent satiation twice daily for 16 weeks before the challenge. Diets (30.3 ± 0.31% crude protein; mean ± SE) were similar to a commercial formula and contained (1) 4% poultry fat (control), (2) 10% poultry fat, or (3) 10% poultry fat and 2% dairy‐yeast prebiotic. After the 16‐week feeding period, 15 golden shiners (2.2 ± 0.03 g) from each aquarium were stocked into different aquaria (experimental replicates were maintained) and exposed to 20 mL of columnaris bacteria in Sheih broth (optical density, 0.395 Å; at 560 nm; Sheih broth blank) for 18 h. Kidney cultures taken from moribund fish were streaked on Sheih agar to confirm the presence of columnaris and an active infection. Mortality in the 4% poultry fat (41.7 ± 12.9%) and 10% poultry fat (40.0 ± 6.1%) diets was high and not significantly different. Mortality for the dairy‐yeast prebiotic (6.7 ± 2.7%) diet was much lower and significantly different from that for the 4% and 10% poultry fat diets. Thus, the dairy‐yeast prebiotic effectively reduced mortality rates in golden shiners exposed to columnaris, but a high‐fat diet alone provided no protection relative to the lower‐lipid control diet.

Anesthetics are widely used for surgical, field sampling, and experimental procedures in fisheries sciences. Given the high cost and the U.S. Food and Drug Administration's (FDA) mandatory withdrawal time of the only FDA-approved fisheries anesthetic, tricaine methanesulfonate (MS-222), clove oil has emerged as an alternative anesthetic that is generally regarded as safe. However, studies regarding the effectiveness of clove oil in retarding the stress response of fish are contradictory. This study evaluated the effectiveness of MS-222 (60 mg/L), clove oil (30 mg/L) emulsified in ethanol, and clove oil (30 mg/L) mechanically emulsified in water in reducing the stress response of rainbow trout Oncorhynchus mykiss during a 15-min confinement. Clove oil emulsified in ethanol (mean, 56.3 ng cortisol/mL) was not as effective in reducing the cortisol response as MS-222 (mean, 33.4 ng/mL). Furthermore, clove oil emulsified in ethanol and clove oil mechanically emulsified in water (means, 49.0 and 56.2 ng/mL, respectively) produced a greater cortisol response in undisturbed fish. Our results suggest that clove oil is less effective than MS-222 in reducing cortisol responses in rainbow trout subjected to handling and confinement. Our results also indicate that clove oil can induce stress and possibly lead to greater release mortality of fieldhandled fish.

A feeding trial was conducted to determine the effects of soy lecithin supplementation on production performance of juvenile channel catfish, Ictalurus punctatus (mean ± SE; 5.8 ± 0 g). The basal diet consisted of a practical dietary formulation for channel catfish, containing 4.3% endogenous phospholipids (PL) from dietary ingredients, to which supplemental PL from soybean lecithin were added. The study diets were 1 control and 2 experimental diets to which 0, 2, or 4% supplemental lecithin was added, respectively. Soy lecithin inclusion did not affect survival, growth, feed consumption, whole‐body total lipid, innate immune response, plasma cholesterol or triglyceride concentrations, or hepatosomatic index. Feed conversion (gain/intake) improved in fish fed 4% supplemental lecithin compared with 0% lecithin. Whole‐body crude protein was greater in fish fed 2% supplemental lecithin compared with 0% lecithin, while 4% supplemental lecithin was intermediate. Phosphatidylcholine (PC) content was greater in fish fed 2 or 4% lecithin than 0% lecithin. Plasma concentrations of PC were inversely proportional to dietary concentrations. Liver glycogen was greater in fish fed 0% lecithin compared with 2 or 4% lecithin. Liver lipid and phospholipid were lower in fish fed 0% lecithin than 2 or 4% lecithin. The dietary phospholipid requirement, if any, of juvenile channel catfish for growth and survival is less than or equal to 4.3% (1.5% PC) of the diet. Feed conversion is improved in channel catfish fed diets supplemented with 4% soy lecithin (7.2% phospholipid; 5.1% PC), which might offset additional costs due to phospholipid supplementation. Dietary soy lecithin inclusion altered plasma and liver lipid composition, but it is unknown whether these effects can alter the ability of juvenile catfish to survive and grow under various conditions.