2007 Annual Report 1a.Objectives (from AD-416) Objective 1 - Examine the effect of alternative protein sources and dietary nutrients on stress response, immune response, and disease resistance of fish. Objective 2 - Evaluate the effect of immunostimulants and probiotics on fish stress resistance, immune response and disease resistance. Objective 3 - a) Determine the optimum dietary level of marine fish oil and feeding duration necessary to optimize n-3 highly unsaturated fatty acids (n-3 HUFA) content in fillets of channel catfish and b) Determine the relationship between n-3 HUFA content in diets and the catfish vitamin E requirement with regard to stress, the immune response, and disease resistance. 1b.Approach (from AD-416) Fish farmers in the U.S. reported that disease related mortality represents the major economic loss to the aquaculture industry. Historically, antibiotics and chemicals have been used to treat diseases in aquatic animals. However, given the fact that diseased fish eat poorly, only a limited number of FDA approved and efficacious drugs/chemicals are available for treatment, and the increasing problem of emerging drug-resistant pathogens and the resultant food and environmental contamination, disease prevention is a better means of controlling infectious diseases. Nutrition has proved to be a key factor in maintaining fish health. All essential nutrients are required in diets in adequate quantity to promote growth, sustain health, and maintain the ability of fish to withstand stress and resist disease-causing agents. Dietary modulation of stress and immune responses has been accomplished in numerious terrestrial animals as well as in some fish species using various approaches, including use of additives and supplementation of certain essential nutrients at levels above the minimum requirements for growth. In this proposed project, we will investigate the effect of alternative protein sources, selected dietary nutrients, immunostimulants and their interactions, and probiotics on fish health. This project will lead to discovery of compounds, nutrients, non-nutrient dietary additives, probiotic microorganisms, and their concentrations and interactions that are effective in increasing stress resistance, immune responses, and resistance to infectious diseases of channel catfish and Nile tilapia. Research will also be conducted to define dietary levels of fish oil and feeding duration to optimize highly unsaturated fatty acid content in catfish fillets. The outcome of this project will lead to increased demand for alternative protein sources and development of least-cost diets to improve growth and fish health and value added catfish products to improve human health will be available for consumers. 4.Accomplishments 1. Alternative protein sources and agricultural byproducts. As a result of the recent expansion and increase in ethanol production for fuels due to the shortage and rising cost of petroleum-based fuel, the distiller’s dried grains with solubles (DDGS) production in the U.S. has been estimated to greatly increase in the years to come. Thus, increased and new uses are needed. A feeding study indicated that, with lysine supplementation, at least 40% DDGS can be included in catfish feeds as replacement of a combination of Soybean Meal (SBM) and corn meal on an equal protein basis without affecting their growth performance. Incorporating 20 to 40% DDGS with lysine also improved some immune parameters and the resistance of catfish to E. ictaluri. For tilapia, 30% DDGS can be used as a substitute for soybean and corn meal without requiring the addition of lysine. With lysine supplementation, DDGS can be included at 60% level as a total replacement of soybean meal without affecting fish growth and feed efficiency. Dietary levels of DDGS, however, had no effect on immune response and resistance of tilapia to S. iniae. With lysine supplementation, 60% DDGS or 30% DDGS without addition of lysine can be used in tilapia diets as a replacement of soybean meal and corn meal. For catfish, at least 40% DDGS plus lysine can be used to replace a mixture of soy meal and corn meal. Inclusion of 20% or more DDGS also improved catfish resistance to E. ictaluri. NP 106 (Action Plan components VIb- Nutrient use and feed evaluation and VIc- Sustainable source of nutrient). 2. Dietary nutrients and their interaction on fish growth and health. Dietary levels of antioxidant vitamin A and E, and dietary lipids have been shown to have profound effect on immunity and disease resistance in terrestrial animals and some fish species. Studies on the effect of dietary levels of vitamin A using practical-type basal diets containing cod liver oil (for tilapia) and menhaden fish oil (for channel catfish) indicated that supplementation of graded levels vitamin A (up to 8000 IU/kg diet)had no effect on fish growth performance, feed utilization efficiency, immune response and resistance to fish disease challenge.The practical basal diets supplemented with fish oil probably contain vitamin A at levels sufficient for maintaining normal physiological and immunological functions of both tilapia and catfish. NP 106 (Action Plan components IVc- Influence of nutrition on immunity and disease resistance). The study on the interaction between dietary levels of lipid (6, 10 and 14% of 1:1 mixture of corn and menhaden fish oil) and vitamin E (50, 100 and 200 mg/kg diet) showed that dietary lipid and vitamin E levels or their interaction had no effect on growth performance and hematological parameters of tilapia. Serum protein was not affected by vitamin E levels but increased in fish fed the 14% lipid diet. Lysozyme activity was not affected by dietary lipid levels but increased in fish fed the 200 mg vitamin E diet. Serum complement activity decreased in fish fed 10 or 14% lipid diets but increased when dietary vitamin E levels increased to 100 or 200 mg. However, lipid and vitamin E levels or their interaction had no effect on the resistance of tilapia to S. iniae challenge. Dietary levels of lipid at 6% and vitamin E at 100 mg/kg diet were sufficient for good growth and sustain good health of tilapia. NP 106 (Action Plan components IVc- Influence of nutrition on immunity and disease resistance). 3. Dietary immunostimulants on stress and disease resistance. Reducing fish stress and increasing their resistance to disease by nutritional means or through proper use of nutritional additives, such as immunostimulants, may offer increased protection during times of stress and can benefit farmers by reducing fish losses. Research to evaluate the effect of dietary levels of bovine lactoferrin showed that in both species, tilapia and catfish, dietary levels of these additives had no effect on growth performance and feed utilization efficiency. However, 800 mg/kg of lactoferrin in diet appears to be optimal for increasing disease resistance. Although bovine lactoferrin had no effect on growth performance, supplementation of 800 mg/kg of this compound improved the resistance of fish to bacterial infection. NP 106 (Action Plan components IVc- Influence of nutrition on immunity and disease resistance). Studies on the effects of commercial yeast or yeast subcomponents (YYS; beta-glucan or mannan oligosaccharide) showed limited positive effects on fish health. Although dietary YYS shows some promise in increasing stress resistance in channel catfish, we did not find any therapeutic benefit of the commercial glucan sources that we tested on disease resistance or immunity of channel catfish or Nile tilapia. The information provided by this research will prevent the unnecessary expense of using yeast or its subcomponents in diets of channel catfish or tilapia. NP 106 (Action Plan components IVc- Influence of nutrition on immunity and disease resistance). 4. Dietary probiotics on disease resistance. Thus far, we have tested several commercially available probiotic bacteria in both Nile tilapia and channel catfish, but none affected growth, immune function, or disease resistance in either species. Because none of the tested probiotics had any significant effect on improving fish health, no further research related to commercial probiotics was conducted. However, we have isolated and identified a number of bacteria and yeasts which inhabit the intestine of healthy catfish and tested for their potential probiotic activity. So far, none of the isolated microorganisms have shown any beneficial effect in improving catfish health. Isolation and identification of bacteria and yeasts from catfish and tilapia will continue in an effort to identify potential probiotics. NP 106 (Action Plan components IVc- Influence of nutrition on immunity and disease resistance). 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of non-peer reviewed presentations and proceedings 15 Review Publications Shoemaker, C.A., Lim, C.E., Aksoy, M., Welker, T.L., Klesius, P.H. 2006. Growth response and acquired resistance of Nile tilapia, Oreochromis niloticus (L.) that survived Streptococcus iniae infection. Aquaculture Research. 37: 1238-1245. Welker, T.L., Lim, C.E., Aksoy, M., Klesius, P.H. 2007. Growth, immune function, and disease and stress resistance of juvenile Nile tilapia (Oreochromis niloticus) fed graded levels of bovine lactoferrin. Aquaculture.262(2007):156-162. Aksoy, M., Lim, C.E., Davis, A.D., Shelby, R.A., Klesius, P.H. 2007. Influence of Dietary Lipid Sources on the Growth Performance, Immune Response and Resistance of Nile Tilapia, Oreochromis niloticus, to Streptoccus iniae Challenge. Journal of Applied Aquaculture. Vol. 19(2) 2007. pgs. 29-49. Shelby, R.A., Lim, C.E., Aksoy, M., Klesius, P.H. 2007. Effects of probiotic bacteria as dietary supplements on growth and disease resistance in young channel catfish, Ictalurus punctatus (Rafinesque). Journal of Applied Aquaculture. 19(1) pgs. 81-91. Lim, C.E., Garcia, J.C., Aksoy, M., Klesius, P.H., Shoemaker, C.A., Evans, J.J. 2007. Growth Responses and Resistance to Streptococccus iniae of Nile Tilapia, Oreochromis niloticus Fed Diets Containing Distiller's Dried Grains with Solubles. Journal of the World Aquaculture Society. Vol. 38, No. 2. p. 231-237. Welker, T.L., Lim, C.E., Aksoy, M., Shelby, R.A., Klesius, P.H. 2007. Immune response and resistance to stress and Edwardsiella ictaluri challenge in channel catfish, Ictalurus punctatus, fed diets containing commercial whole-cell yeast or yeast subcomponents. Journal of the World Aquaculture Society. 38(1):24-35. Welker, T.L., Lim, C.E., Aksoy, M., Klesius, P.H. 2007. Effect of buffered and unbuffered tricaine methanesulfonate (ms-222) at different concentrations on the stress responses of channel catfish (Ictalurus punctatus Rafinesque). Journal of Applied Aquaculture. 19(3)pgs. 1-18. Welker, T.L., Mcnulty, S.T., Klesius, P.H. 2007. Effect of sub-lethal hypoxia on immune response and susceptibility of channel catfish,Ictalurus punctatus,to enteric septicemia. Journal of the World Aquaculture Society. 38(1):12-23. Lim, C.E., Webster, C.D. editors. 2006. Tilapia: Biology, Culture, and Nutrition. Complete Book. Binghamton, NY: Haworth Press. 703 p. Lim, C.E., Webster, C. 2006. Nutrient requirements. In: Lim, C.E. and Webster C.D. editors. Tilapia Biology. Binghamton, NY: Haworth Press. p. 469-501. Lim, C.E., Webster, C.D., Li, M.H. 2006. Feeding practices. In: Lim, C. and Webster, C. D. editors. Tilapia: Biology, Culture and Nutrition. Binghamton, NY: Haworth Press. p. 547-559. Li, M., Lim, C.E., Webster, C.D. 2006. Feed Formulation and Manufacture. In: Tilapia, Biology, Culture, and Nutrition, C. Lim and C.D. Webster, Eds. The Haworth Press, Inc., Binghamton, New York, New York, pp. 517-543. Hughes, S.G., Lim, C.E., Webster, C.D. 2006. Nonnutrient Components of Fish Diets. In: Tilapia: Biology, Culture, and Nutrition, C. Lim and C.D. Webster, Eds. The Haworth Press, Inc., Binghamton, New York, New York, pp. 503-516. Lim, C.E., Aksoy, M., Welker, T.L., Veverica, K. 2006. Effect of feeding duration of sodium chloride containing diets on growth performance and some osmoregulatory parameters of nile tilapia (Oreochromis niloticus) after transfer to water of different salinities. Journal of Applied Aquaculture 18: 1-17.