2006 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Under intensive production systems, fish are exposed to various stressful conditions. Stress is a major problem in fish production, leading to growth reduction, immuno-suppression and susceptibility to infectious diseases, which can result in major economic loss to fish farmers. Fish producers in the U.S. indicate that disease prevention by dietary means, rather than treatment of sick fish should be a top research priority. The potential for reducing stress and enhancement of immunity and disease resistance by manipulation of nutritional factors and use of feed additives (such as immunostimulants, bacterins and probiotics) has been demonstrated in warm-blooded animals. However, very little work in this area has been conducted in fish. Thus, the effects of dietary nutrients and their interactions, anti-nutritional factors, additives, feed and feeding strategy need to be assessed to develop economically viable feeds and feeding practices to optimize growth, improve stress resistance, immune response and disease resistance, and improve product quality of aquaculture species. 2.List by year the currently approved milestones (indicators of research progress) FY 2006 (1) Evaluation of dietary anti-oxidant nutrients and nutritional values of distiller's grains with solubles with and without lysine supplementation on growth performance and health of tilapia and catfish. (2) Determine if dietary supplementation of sodium chloride affects growth performance, immune response and disease resistance in catfish. The effect of dietary NaCl levels and feeding duration on nitrite toxicity were also conducted. (3) Identification of optimum concentration of bovine lactoferrin in tilapia diets and catfish based on immune function and resistance to stress and disease. (4) Evaluation of commercially available probiotics for tilapia and catfish based on immunity and resistance to diseases. FY 2007: (1) Define the essentiality of antioxidant nutrients such as vitamin A in catfish and the interactions of vitamin E and C on tilapia immunity, stress, and disease resistance. (2) Determine if supplementation of lysine to diets containing dry distiller's grains with solubles improve growth, immune response, and resistance of tilapia to S. iniae. (3) Determine, under pond conditions, optimum feeding duration and fish oil levels necessary to increased highly unsaturated fatty acids (HUFA) content in catfish fillets. (4) Define interactions between dietary lipid and vitamin E on tilapia stress and disease resistance. (5) Assess the effect of cottonseed meal immunostimulants (flavonoids) on growth and disease resistance in fish (tilapia and catfish). (6) Establish the optimum level of bovine lactoferrin, and feeding duration and concentrations of the best-performing B-glucan on immune function and susceptibility to stress and disease. (7) Conduct isolation and identification (in vitro) of bacteria from tilapia and catfish digestive tracts with probiotic activities. FY 2008: (1) Define the effects of vitamin A on tilapia and vitamin E and selenium interactions on tilapia and catfish growth, stress, immunity and disease resistance. (2) Identify and determine the optimum concentration of the best performing E. ictaluri bacterin and interaction between B-glucan and vitamin C on immune response and disease resistance. (3) Compare the efficacy of flavonoids from cottonseed and soybean on stress, immune response and disease resistance in catfish and tilapia. (4) Evaluate the effect of probiotic isolates (in vivo) on immune response and disease resistance in catfish and tilapia. FY 2009: (1) Determine established concentrations of cottonseed and soybean flavonoids necessary to improve the immune response and disease resistance in catfish and tilapia. (2) Establish interactive effects of B-glucan and E. ictaluri bacterin and the interaction between bacterin and vitamin C on fish performance and health. (3) Assess defining optimum concentrations of probiotics and feeding duration to improve the resistance of tilapia and catfish to diseases. 4a.List the single most significant research accomplishment during FY 2006. Highly unsaturated fatty acids (HUFA) in catfish fillets increased by feeding diets supplemented with fish oil. There is evidence that HUFA in marine fish oil reduces the incidence of heart disease in human by lowering cholesterol and triglyceride levels and increasing blood clotting time. Juvenile channel catfish were fed a commercial diet sprayed with menhaden fish oil at levels of 0, 3, 6 and 9% for 15 weeks. Fish were sampled at 0 6, 9, 12 and 15 weeks for whole body and fillet proximate composition and determination of fatty acid profile. Levels of dietary fish oil did not significantly influenced final weight gain, feed intake, feed efficiency ratio or survival of catfish. Whole body and fillet lipid contents were directly correlated to the dietary lipid levels, whereas protein content of both whole body and fillet were significantly lower in fish fed 6% or 9% fish oil diet compared to control. As feeding duration increased, fillet moisture content progressively decreased, but the lipid content increased. Whole-body and fillet fatty acid composition reflected dietary fatty acid composition. Progressive increases in saturated, total n-3 fatty acids, n-3 HUFA, and decreases in monoenoic and total n-6 fatty acids were observed in whole body and fillet as supplementation of fish oil increased. Fish fed the 9% fish oil diet had significantly higher percentages of total n-3 and n-3 HUFA, and the values were similar for fish fed from 9 and 15 weeks. When expressed in mg/g of fillet, n-3 HUFA content increased with increasing levels of dietary fish oil and feeding period. 4b.List other significant research accomplishment(s), if any. 1. Dietary supplementation of bovine lactoferrin significantly reduced susceptibility of Nile tilapia to Streptococcus iniae. 2. Supplementation of fish oil to the diets at 3 to 6% improved some immune responses, but had no effect on the resistance of catfish to E. ictaluri infection. 3. Growth performance and immune response of catfish were not affected by inclusion of up to 40% dry distiller's grains with solubles (DDGS) (supplemented with lysine) as partial replacement of soybean meal, but a level of 10% DDGS appears to improve the resistance of catfish to E. ictaluri infection. 4. Supplementation of NaCl up to 4% in diets had no effect on growth and hematological parameters of channel catfish, but increased their resistance to nitrite toxicity. 5. Three commercially available probiotics and a yeast added to commercial feeds at manufacturer's recommended doses had no effects on improving disease resistance in catfish or tilapia, but showed some promise in increasing resistance to stress in catfish. 4c.List significant activities that support special target populations. Increasing the n-3 HUFA in fish fillets by feeding diets supplemented with fish oil will create a value added product for fish producers and improve the health (by reducing the risk of cardio-vascular disease) of consumers. Results of studies on the use of DDGS, interactions between dietary nutrients and feed additives (B-glucan and lactoferrin) will lead to an increased use of agricultural byproducts, a decrease in feed cost, and an improvement in fish health. This information is beneficial to the fish feed industry and fish farmers. It will also benefit the rapidly expanding distiller industry and row-crop farmers through increased demand of agricultural products/byproducts for use in aquaculture diets. Scientists involved in nutrition and fish health will benefit and will use the new information gained from this research. 4d.Progress report. None. 5.Describe the major accomplishments to date and their predicted or actual impact. A study on the interaction between vitamins E and C showed that vitamin E present in a soybean-based diet is sufficient for normal growth of catfish, but supplementation of vitamin C is needed. Grow-out diets should be supplemented with 100 mg/kg of each of these vitamins for good growth, increased liver storage of vitamins and improvement of immune responses. Increasing dietary fish oil levels beyond 6% and vitamin E beyond 50 mg/kg diet had no effect on growth, feed utilization efficiency, or hematological parameters. The study on dietary fish oil levels and vitamin E requirement indicated that increasing dietary fish oil levels had no effect on vitamin E requirement of catfish. Addition of 10% fish oil had positive effect on some immune parameters, but fish oil and vitamin E levels had no effect on the resistance of catfish to ESC. Dry distiller's grains with solubles (DDGS), a by-product of alcohol distilleries can be incorporated in Nile tilapia diets at a level of 20% as a replacement of soybean meal without affecting growth, hematological values, the immune response, and resistance to S. iniae. For channel catfish, growth performance and immune responses were not affected by inclusion of up to 40% DDGS (supplemented with lysine) as partial replacement of soybean meal, but a level of 10% DDGS appeared to improve the resistance of catfish to E. ictaluri infection. Feeding Nile tilapia diets supplemented with 6% NaCl improved growth, but had no effect on some osmoregulatory parameters. Nile tilapia can be directly transferred from fresh water to 20 ppt salt water without mortality. Increased osmoregulatory parameters were detected at 6 to 12 hours after exposure, but returned to normal levels after 48 hours. Dietary NaCl at levels up to 4% had no effect on growth of catfish, but increased their resistance to nitrite toxicity. Severe growth reduction was observed within 2 weeks in catfish fed the 8% NaCl-diet. Nile tilapia fed the diet containing beef tallow, which is deficient in both n-6 and n-3 fatty acids, had the poorest growth and lowest immune responses compared to those fed diets containing oil rich in n-3, n-6, or both. Addition of fish oil (high in n-3 HUFA) as the sole dietary oil source, provided good growth, but caused some hematological abnormalities. The dietary lipid sources tested, however, had no effect on the resistance of fish to S. iniae challenge. In catfish, addition of 3, 6 or 9% fish oil to a commercial diet had no effect on growth and feed efficiency, but affected body lipid content. Nine-week feeding with the diet supplemented with 9% fish oil was sufficient to obtain a high percentage of n-3 HUFA in fillets. However, the fillet concentration in mg/kg of n-3 HUFA was highest in fish fed the highest level (9%) of added oil for 15 weeks. The effects of added oil levels on immune parameters were not consistent, and the level of added fish oil had no effect on the resistance of catfish to E. ictaluri infection. The study on the stress response of catfish exposed to various concentrations of MS-222 with and without buffering showed that 90 mg/L of MS-222 was optimum for anesthetizing juvenile catfish with minimal stress to fish. We also found that water with neutral to slightly alkaline pH can be used without requiring buffering. Dietary supplementation of bovine lactoferrin is effective in reducing susceptibility of Nile tilapia to Streptococcus iniae infection, and 800 mg/kg of lactoferrin in diet appears to be optimal. The benefit of dietary lactoferrin to disease resistance appears to be from a reduction in iron and an increase in iron-binding capacity in plasma. Although dietary B-glucan shows some promise in increasing stress resistance in channel catfish, no therapeutic benefit was found of the commercial glucan sources that we tested on disease resistance of channel catfish or Nile tilapia. Seven separate studies (4 with tilapia and 3 with catfish) were performed to determine the efficacy of commercially available probiotic bacteria and yeast to improve immune responses and disease resistance of both catfish and tilapia. Probiotics/yeast were incorporated in commercial diets at concentrations recommended by the manufacturers. To date, no positive response was detected of the various microorganisms tested on immune parameters or disease resistance, which is in contrast with results of several studies in the published literature that have reported positive effects of probiotics/yeast on immunity and disease resistance in several aquaculture species. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Most of the information generated has been transferred to farmers, extension specialists, feed formulators and scientists through presentations at farmer meetings, scientific conferences, publications in scientific journals and personal contacts. Review Publications Pasnik, D.J., Evans, J.J., Aksoy, M., Lim, C.E., Klesius, P.H. 2006. Natural content and processing of alternative protein sources: histologic effects in fingerling channel catfish (ictalurus punctatus). 31st Annual Eastern Fish Health Workshop. Charleston, SC. March 28-30, 2006. page 54. Welker, T.L., Klesius, P.H., Arias, C.R., Small, B.C. 2005. Effect of hypoxia on stress and heat shock protein expression in channel catfish (ictalurus punctatus rafinesque). Aquaculture America Conference. Welker, T.L., Congleton, J. 2004. Effect of a low-water stressor on oxidative stress in sub-yearling chinook salmon (Oncorhynchus tshawytscha). Aquaculture Research, 35: 881-887. Welker, T.L., Lim, C.E., Aksoy, M., Klesius, P.H. 2005. Effect of MS-222 concentration with and without buffering on the stress response of channel catfish. Annual Meeting of the American Fisheries Society. Anchorage, Alaska. p. 403. Aksoy, M., Lim, C.E., Li, M.H., Klesius, P.H. 2006. Interaction between dietary levels of vitamin C and E on growth and immune responses in channel catfish Ictalurus punctatus. Aquaculture America Conference. Las Vegas, NV. Shoemaker, C.A., Lim, C.E., Aksoy, M., Welker, T.L., Klesius, P.H. 2006. Growth response and acquired resistance of Streptococcus iniae-recovered Nile tilapia, Oreochromis niloticus. 31st Annual Eastern Fish Health Workshop. Charleston, SC. p. 55. Welker, T.L., Mcnulty, S.T., Klesius, P.H. 2006. Effect of hypoxia stress on blood glucose, plasma cortisol, and susceptibility to Edwardsiella ictaluri in channel catfish (Ictalurus punctatus Rafinesque). Aquaculture America Conference. Las Vegas. NV. p. 166. Welker, T.L., Congleton, J.L. 2004. Relationship between dietary lipid source, oxidative stress, and the physiological response to stress in sub-yearling chinook salmon (Oncorhynchus tshawytscha). Fish Physiology and Biochemistry. 29: 225-235. Welker, T.L., Congleton, J.L. 2005. Oxidative stress in migrating spring chinook salmon (Oncorhynchus tshawytscha) smolts of hatchery origin: changes in vitamin E and lipid peroxidation. American Fisheries Society Transaction. 134(6): 1499-1508. Lim, C.E., Aksoy, M., Shelby, R.A., Li, M.H., Klesius, P.H. 2006. Influence of dietary levels of fish oil and vitamin E on growth and resistance of channel catfish to Edwarsiella ictaluri challenge. Aquaculture America Conference. Las Vegas. NV. Shelby, R.A., Lim, C.E., Aksoy, M., Delaney, M.A. 2006. Effects of probiotic feed supplements on disease resistance and immune response of young Nile tilapia (Oreochromis niloticus). Journal of Applied Aquaculture 2006, 18(2) 22-34. Welker, T.L., Lim, C.E., Aksoy, M., Klesius, P.H. 2006. Effect of dietary bovine lactoferrin on disease and stress resistance of Nile tilapia (Oreochromis niloticus). Symposium of the Comparative Nutrition Society. August 4-9, 2006. Pages 208-212.