2008 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. 3.Progress Report A study evaluating the nutritional values of cottonseed meal (CSM) in tilapia diets showed that this species utilizes CSM poorly relative to catfish. Supplementation of lysine alone did not improve the nutritional value of CSM-containing diets, but supplementation of lysine + methionine + isoleucine improved the performance of the 16.9%-CSM diet. CSM levels and amino acid supplementation had no effect on the resistance of tilapia to S. iniae. Study on the interaction between Se and vitamin E suggested that the practical diets used in this study probably contains selenium at a level sufficient for maintaining normal physiological and immunological functions of tilapia. However, supplementation of 50 mg vitamin E/kg diet is needed for good Ig level and maintaining normal erythrocyte peroxidase activity. We also found that Nile tilapia are sensitive to thiamin (vitamin B1) deficiency and a dietary supplementation of 2 mg thiamin/kg was sufficient to prevent classical deficiency signs. Higher than this level had no effect on tilapia performance. Fish fed a thiamin-deficient diet had reduced growth and increased susceptibility to bacterial infection. A pond study to increase n-3 high unsaturated fatty acid (HUFA) (beneficial fatty acids) content in catfish showed that, in terms of mg/g fillet, levels of these fatty acids progressively increased with increasing dietary fish oil levels and feeding duration. In five separate studies (two with tilapia and three with channel catfish) using practical diets supplemented with commercial oligosaccharides at levels recommended by manufacturers, we found no beneficial effects of these products on fish immunity and disease resistance. Results of another study showed that bovine lactoferrin had no effect on growth performance; however, dietary supplementation 1,136 mg/kg and 493 mg/kg diet is effective in reducing susceptibility in catfish and tilapia to E. ictaluri and S. iniae infections, respectively. Concerning the use of probiotics as feed additives, thus far, we tested five and seven different bacteria in Nile tilapia and channel catfish, respectively, for use as potential probiotics, but none affected growth, immune function, or disease resistance in either species. NP 106, Components: 5 and 6, Problem Statement: 5d, 6b and h. 4.Accomplishments 1. Cottonseed meal (CSM) in tilapia diets. Feed cost is the largest expense in intensive and semi-intensive aquaculture production. Reducing feed costs by using CSM would benefit fish producers. We evaluated the nutritional value of various levels of CSM with the addition of lysine, methionine, and isoleucine as replacement of soybean meal (SBM) in diets. We discovered that CSM is deficient in essential amino acids (EAA) other than lysine. Supplementation of lysine, methionine, and isoleucine improved the performance of tilapia fed 16.9% but not higher levels of CSM. CSM may contain antinutritional factors other than gossypol that are toxic to Nile tilapia. On an equal protein basis, with the addition of lysine, methionine and isoleucine, CSM can be used to replace up to 1/3 SBM in Nile tilapia diets. Inclusion of CSM in tilapia diets would decrease feed cost. Identification of antinutritional factors and deficient nutrients would increase the use of CSM in tilapia diets and further reduce feed cost. NP 106, Components: 5, Problem Statement: d. 2. Interaction of selenium and vitamin E in Nile tilapia. Selenium (Se) acts along with vitamin E as biological antioxidants to protect polyunsaturated phospholipids from oxidative damage. Selenium deficiency in animals decreases antibody responses, especially if associated with vitamin E deficiency. We evaluated the interaction between dietary levels of Se and vitamin E using practical diets on tilapia performance and health. Dietary selenium and vitamin E levels or their interaction had no effect on growth performance, hematology, and glutathione peroxidase activity. Supplementation of vitamin E appeared to improve some immune parameters. However, dietary selenium and vitamin E levels or their interaction had no effect on the resistance of tilapia to S. iniae infection and antibody production against that bacterium. Commercial diets probably contain a sufficient level of Se for maintaining normal physiological and immunological functions of tilapia. However, supplementation of 50 mg vitamin E/kg diet is recommended to maintain proper fish health, and supplementation of vitamin E higher than this level is uneconomical. NP 106, Components: 6, Problem Statement: h. 3. Vitamin B1 (thiamin) in diet of Nile tilapia. The water-soluble vitamin B1 (thiamin) is essential in the diet of fish. Severe thiamin deficiency is often associated with neurological disturbances and impaired carbohydrate metabolism. We conducted a study to evaluate the effect of graded levels of thiamin on growth performance and health of tilapia. Nile tilapia show signs of thiamin deficiency within two weeks (poor growth, reduced feed intake, poor survival, decreased hematocrit, and increased serum pyruvate). Nile tilapia require dietary thiamin supplementation at a level of 2 mg/kg; higher than this level had no effect on tilapia performance. Fish fed a thiamin-deficient diet had increased susceptibility to bacterial infection. Thiamin is dietary essential for tilapia. A level of 2 mg/kg in diet is sufficient to prevent deficiency signs and improve the resistance to S. iniae infection, and addition at higher than this level would unnecessarily increase feed cost. NP 106, Components: 6, Problem Statement: h. 4. Addition of dietary fish oil to increase n-3 highly unsaturated fatty acid (n-3 HUFA) in catfish. The public is aware that eating fish rich in n-3 HUFA is beneficial to health, mainly due to reduced incidence of cardio-vascular diseases. Therefore, increasing these fatty acids in catfish would benefit the consumer and increase the demand for catfish. Channel catfish were fed in ponds for 5 months with a commercial feed supplemented with 0, 3 and 6% menhaden fish oil. Adding marine fish oil to the diets of farm-raised channel catfish is an efficient means to increase fillet n-3 HUFA content. Fillet n-6 fatty acids and ratio of n-3/n-6, however, were unaffected by feeding duration. When expressed in terms of mg/g fillet, total n-3 and n-3 HUFA progressively increased with increasing dietary levels of fish oil and feeding duration. n-3 HUFA content in catfish fillets can be increased by feeding commercial diets supplemented with fish oil. Increasing n-3 HUFA content of catfish would benefit consumer health and expand the catfish market. NP 106, Components: 6, Problem Statement: b. 5. Immunological and disease resistance effects of commercial oligosaccharides. Although there have been reports that the cell wall components of yeast consisting mainly of oligosaccharides may have immunostimulatory effect in fish diets, our earlier studies have been generally unsuccessful in showing any effect on measured immunological parameters from these supplements, and in particular no effect on disease resistance in channel catfish or Nile tilapia. We conducted five separate experiments, two with tilapia and three with channel catfish, using practical diets supplemented with commercial oligosaccharides at levels recommended by manufacturers. Immune response parameters (immunoglobulin (Ig), lysozyme and alternative complement) and disease resistance were not affected by oligosaccharide supplementation in the diets of Nile tilapia. However, feeding oligosaccharides to channel catfish for 1 week appeared to provide protection against enteric septicemia of catfish (ESC). This same effect was not seen in fish fed for 2 or 4 weeks. Based on our research, we would not recommend the routine use of oligosaccharides as dietary supplements to improve the disease resistance in commercial aquaculture, which would potentially save producers unnecessary expenses. NP 106, Components: 6, Problem Statement: h. 6. Immunological and disease resistance effect of dietary lactoferrin. Lactoferrin (Lf) is an iron-transporting protein with important immunological properties. Supplementation of Lf has been shown to improve health and disease resistance of some fish species. We investigated the effect of supplementation of graded levels of bovine Lf in diets of channel catfish and Nile Tilapia. Bovine Lf had no effect on growth performance; however, dietary supplementation is effective in reducing susceptibility of Nile tilapia to S. iniae and channel catfish to E. ictaluri infections. The minimum dietary concentration which reduced mortality in channel catfish from E. ictaluri infection was 1136 mg/kg and in Nile tilapia from S. iniae infection was 493 mg/kg diet. Dietary Lf supplementation of 1136 and 493 mg/kg diet improved the resistance of catfish and tilapia to E. ictaluri and S. iniae, respectively. However, due to the high cost of this product, supplementation of bovine Lf at these levels is currently not economically feasible. NP 106, Components: 6, Problem Statement: h. 7. Bacteria as potential probiotics in Nile tilapia and channel catfish. Increasing resistance to disease by nutritional means or through proper use of nutritional additives, such as probiotics, are beneficial to farmers by reducing fish losses. Thus far, we tested five and seven different bacteria in Nile tilapia and channel catfish, respectively, for use as potential probiotics. Addition of probiotics to diets of tilapia or catfish had no effect on growth, immune function, or disease resistance in either species. Based on our findings, we would not recommend the use of probiotics as dietary supplements to improve growth or disease resistance in commercial aquaculture. The information provided by this research will prevent the unnecessary expense of probiotic bacteria in diets of channel catfish or tilapia. NP 106, Components: 6, Problem Statement: h. 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of New Commercial Licenses Executed 5 Review Publications Aksoy, M., Shelby, R.A., Lim, C.E., Klesius, P.H. 2007. Growth performance, and proximate and fatty acid composition of channel catfish, Ictalurus punctatus, fed for different duration with a commercial diet supplemented with various levels of menhaden fish oil. Journal of the World Aquaculture Society. 38(4): 461-474. Venero, J.A., Davis, D., Lim, C.E. 2008. Use of plant protein sources in crustacean diets. In: Lim. C.E., Webster, C.D., and Lee C.S., editors. Alternative Protein Sources in Aquaculture Diets. New York: NY. Haworth Press. p. 163-203. Lim, C.E., Li, M.H., Robinson, E., Aksoy, M. 2008. Cottonseed meal in fish diets. In: Lim, C.E., Webster, C.D., and Lee, C.S., editors. Alternative Protein Sources in Aquaculture. New York, NY: Haworth Press. p. 313-342. Peres, H., Lim, C.E. 2008. Utilization of soybean products in non-salmonid marine fish diets. In: Lim, C.E., Webster, C.D.; and Lee, C.S.; editors. Alternative Protein Sources in Aquaculture Diets. New York, NY: Haworth Press. p. 281-312. Lim, C.E., Webster, C.D., Lee, C.S. 2008. Alternative Protein Sources in Aquaculture Diets. New York:Haworth Press. 571 p. Welker, T.L., Congleton, J.L. 2008. Effect of dietary alpha-tocopherol + ascorbic acid, selenium, and iron on oxidative stress in sub-yearling Chinook Salmon (Oncorhynchus tshawytscha Walbaum). Journal of Animal Physiology and Animal Nutrition. 92: 414-425. Li, M. H., Robinson, E., Lim, C.E. 2008. Use of meatpacking by-products in fish feeds. In: Lim, C.E., Webster, C.D., and Lee, C.S., editors. Alternative Protein Sources in Aquaculture. New York, NY: Haworth Press. p. 95-116.