Validation,use, and disadvantages of enzyme-linked immunosorbent assay kits for detection of cortisol in channel catfish,largemouth bass,red pacu,and golden shiners

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.     

Development of a whole-body cortisol extraction procedure for determination of stress in golden shiners, <Emphasis Type="Italic">Notemigonus crysoleucas</Emphasis>

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; inter-assay %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.     

Effects of Amylopectin/Amylose Starch Ratio on Growth, Body Composition and Glycemic Response of Sunshine Bass Morone chrysops × M. saxatilis

Manipulation of the ratio of amylopectin (α‐[1,4] and α‐[1,6] linked glucose) to amylose (α‐[1,41 linked glucose) starches in the carbohydrate fraction of the diet has been used to improve carbohydrate and lipid metabolism in mammalian models. A 10‐wk feeding trial was conducted to determine the effect of dietary amylopectin/amylose ratio on growth and composition of growth of advanced sunshine bass (Morone chrysops × M. saxatilis) fingerlings (60 g, initial weight). Fish were fed cold‐pelleted, semipurified, isonitrogenous (35% crude protein), isocaloric (3.6 kcaVg protein), isolipidic (5%) diets containing 25% carbohydrate. The carbohydrate fraction of the diets was composed of either glucose, dextrin, 100% amylopectin/0% amylose, 70% amylopectin/30% amylose, or 30% amylopectin/70% amylose. Diets differing in ratios of amylopectin/amylose were achieved by adjusting the proportion of high‐amylopectin (100% amylopectin) to high‐amylose (70% amylose) corn starch. Diets were fed to fish in quadruplicate 76‐L tanks (seven fish/tank) connected to a brackish water (5‐7%v) recirculating culture system with biofiltration. Weight gain ranged from 195 to 236% of initial weight (60 g) and was significantly greater (P < 0.1) for fish fed diets containing 25% carbohydrate as dextrin or as 70% amylose and significantly lower in fish fed diets in which carbohydrate was composed of 30% amylose, 100% amylopectin, or glucose. Feed efficiency ranged from 0.52 to 0.61 and was higher in fish fed the diet containing the highest concentration of amylose and lower in fish fed the diet containing glucose. Hepatosomatic index was highest (2.71) in fish fed the diet containing glucose and lowest (1.401.45) in fish fed diets containing high‐amylose cornstarch. Intraperitoneal fat ratio was distinctly lower in fish fed diets containing some amylose as compared to those fed diets without amylose. Liver lipid was significantly lower (4.8%) in fish fed the diet containing glucose and almost twice as high (7.3‐8.9%) in fish fed the diets containing any starch. Glycogen content of the liver decreased from approximately 12% in fish fed the diet containing glucose to 5% in fish fed the diets containing amylose. Muscle proximate composition and ratio were unaffected by the dietary treatments. Fasting levels (15 h) of blood glucose in fish reared for 10 wk on the diet containing glucose were significantly elevated (5.5 mmol/L) when compared to fasting levels of those that had been reared on diets containing starch (3.4‐1.1 mmol/L). Fish fed the diet containing glucose exhibited maximum blood concentrations (14.6 mmoVL) 4 h postprandial then rapidly declined to nearly fasting levels within 8 h postprandial. In contrast, maximum plasma glucose concentrations in fish fed diets containing starch were roughly half (6.8‐8.1 mmol/L) those of fish fed the diet containing glucose. Blood glucose in fish fed diets containing dextrin or predominantly amylopectin starch remained elevated longer than that of fish fed diets containing glucose or predominantly amylose starch. Glycemic response appeared to decrease with increasing dietary amylose content. These data suggest that feeding diets in which a greater portion of the starch is amylose may be a useful strategy for improving carbohydrate use in sunshine bass.     

Massive Hepatic Necrosis and Nodular Regeneration in Largemouth Bass Fed Diets High in Available Carbohydrate

Largemouth bass Micropterus salmoides are piscivorous fish raised on farms then sold live in Asian fish markets on the east and west coasts of the United States. In the winter of 1998, a major producer of feed‐trained bass suffered a significant increase in fish mortality both during shipping and while the fish were still in ponds. No bacterial, viral, or significant parasitic pathogens were found at necropsy. Livers of affected fish were pale and translucent with 3–10 mm pink nodules on their surface and deeper in the parenchyma. Histological examination of these livers showed that the translucent regions of the liver contained few hepatocytes and were composed of tissue consistent with a chronic inflammatory response. Also present were eosinophils, islands of pancreatic and biliary cells, and granulomas that did not stain positively for mycobacteria. The pink nodules were areas of multifocal regeneration of normal hepatocytes. Sequential studies of bass in ponds revealed that the bass were progressively accumulating glycogen in their hepatocytes to an extent sufficient to explain the massive necrosis of that organ. In order to determine the effect of diets varying in available carbohydrate on fish growth, survival, and liver glycogen content, a 12‐wk feeding trial was conducted in aquaria with juvenile largemouth bass. Nitrogen‐free extract values indicated that an extruded trout diet, a steelhead trout diet, and a diet designed to contain 45% protein and 25% fat, contained 35, 27, and 21% carbohydrate, respectively. Weight gain was lowest in fish fed the extruded trout diet, while liver glycogen was significantly higher in fish fed diets with >27% carbohydrate than in fish fed the diet with 21% carbohydrate. The farmer switched to a diet similar to the 45–25 diet used in our trial. Subsequently, 16‐mo‐old fish examined in October 2000 had no hepatic nodules or necrosis, were significantly larger and less variable in size than previous crops, and the farmer experienced no significant losses in ponds or during shipping.     

Responses of Juvenile Golden Shiner Notemigonus crysoleucas Fed Semipurified or Practical Diets With or Without Supplemental Ascorbic Acid

A 12-wk feeding trial was conducted in a flow-through system to determine whether juvenile golden shiner Notemigonus crysoleucas have a dietary requirement for ascorbic acid. Triplicate groups of 30 fish each weighing 0.44 g initially were fed semi-purified (casein) diets supplemented with 0 or 250 mg ascorbic acid/kg, or practical (fish meal) diets supplemented with 0 or 250 mg ascorbic acid/kg. The form of ascorbic acid used was L-ascorbyl-2-polyphosphate. Survival was higher in fish fed the semi-purified diet with 250 mg ascorbic acid/kg than in those fed the unsupplemented semi-purified diet, while weight gain did not differ between treatments. Weight gain was higher in fish fed the practical (fish meal) diet supplemented with 250 mg ascorbic acid/kg than in those fed the unsupplemented practical diet, while survival did not differ between treatments. No other signs of ascorbic acid deficiency were observed, but fish fed the casein diets had skin and fin erosion indicative of a possible bacterial infection. Whole-body concentrations of ascorbic acid (total, reduced, and percent of reduced ascorbic acid) were higher in fish fed semi-purified or practical diets supplemented with 250 mg ascorbic acid/kg than in fish fed unsupplemented diets. Golden shiners appear to require a dietary source of ascorbic acid for growth or survival, depending on experimental conditions. The two main differences in the study were in diet composition (semi-purified versus practical diets) and apparent health status of fish fed the different diet types. Additional studies are needed to define requirements further.     

Evaluation from a food regulatory aspect of tuberculous slaughtered cattle at the end of the 18th century--together with a study of the position of the veterinarian in public service at that time]

A decree of the Hannoverian sovereign GEORGE III passed in 1787 regarding the treatment of slaughtering cattle affected with tuberculous changes of the pleura is presented. Special emphasis is placed on the practice of food hygiene at that time and on the fact that the veterinarian was regarded an equal expert compared to the physician.     

Effects of Carbohydrate‐Rich Alternative Feedstuffs on Growth,Survival, Body Composition,Hematology, and Nonspecific Immune Response of Black Pacu,Colossoma macropomum,and Red Pacu,Piaractus brachypomus

To facilitate economical culture of black pacu, Colossoma macropomum, and red pacu, Piaractus brachypomus, in the Amazon region of South America, we assessed locally available alternative energy sources for practical diets. We tested the effects of control diets (containing wheat products) versus diets with different Amazonian feedstuffs (yucca, Manihot sculenta, plantain, Musa paradisiaca, or pijuayo, Bactris gasipaes) on the performance of the pacus in three feeding trials. Black pacu (22.5 ± 0.03 g; Trial 1) or red pacu (2.56 ± 0.01 g; Trial 2) were fed diets containing 30% wheat bran (control) or cooked or uncooked yucca, plantain, or pijuayo for 12 wk. In Trial 3, larger black pacu (86.9 ± 6.4 g) were grown to market size in 24 wk on similar diets. Weight gain, feed conversion, survival, alternative complement activity, and lysozyme were similar among diets. Hepatosomatic index, liver glycogen, and dry matter were affected by diet in Trials 1 and 2, but effects were not consistent among trials. In Trial 3, protein efficiency ratio was lower in fish fed the diet containing wheat middlings. However, relative to wheat bran or wheat middlings, all feedstuffs tested were effective energy sources for juvenile black pacu and red pacu.     

Performance of Alternative Diets Containing Solvent‐Extracted Distillers Dried Grains with Solubles Compared to Traditional Diets for Pond‐Raised Channel Catfish,Ictalurus punctatus,and Hybrid Catfish,Ictalurus punctatus × Ictalurus furcatus

Two concurrent pond studies were initiated in April 2011 to evaluate feeds containing solvent‐extracted distillers dried grains with solubles (SE‐DDGS) in both 28 and 32% protein catfish feeds. Channel catfish, Ictalurus punctatus, were stocked in multiple‐batch production ponds at the University of Arkansas at Pine Bluff (UAPB) and hybrid catfish, ♀ Ictalurus punctatus × ♂ Ictalurus furcatus, in single‐batch production ponds at the National Warmwater Aquaculture Center (NWAC), Mississippi State University, Stoneville, Mississippi, USA. The experimental design was a 2 × 2 factorial with two protein levels (32 and 28% protein), with and without 20% SE‐DDGS. Fish were fed once daily to apparent satiation, and managed similarly to a commercial farm for 186 d at UAPB and 172 d at NWAC. For channel catfish, two‐way ANOVA showed significant effects of both protein level (28% being better than 32%) and protein source (20% SE‐DDGS diets being better than traditional diets) on yield, survival, total diet fed, and feed conversion ratio (FCR), due primarily to effects on the understocked fingerlings. Partial budget analysis suggested that the 28% protein diet with the 20% SE‐DDGS was preferred economically for the prevailing feed costs. For hybrid catfish, two‐way ANOVA did not show significant differences in total diet fed, yield, survival, and processing yields, but use of SE‐DDGS resulted in a lower FCR. Results showed that both channel and hybrid catfish can utilize 20% SE‐DDGS in the diet without adversely affecting production or processing characteristics.