2005 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? What does it matter? Under intensive production system, 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 resulting in major economic loss to fish farmers. The potential for reducing stress and enhancing immunity and disease resistance by nutritional factors/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 the milestones (indicators of progress) from your Project Plan. a. Nutritional value, and effect of alternative protein sources and agricultural byproducts on fish health. b. Dietary nutrients and their interactions on fish stress, immune system functions and disease resistance. c. Dietary immunostimulants and orally-administered bacterin and stress and fish health. d. Probiotics and disease resistance. 4a.What was the single most significant accomplishment this past year? Vitamins C and E are dietary essential for growth and maintenance of normal immune responses. Channel catfish juveniles were fed practical diets supplemented with graded levels of both vitamins for 12 weeks. Vitamin E presence in soybean-based diets is sufficient to promote good growth but supplementation of 100-500 mg/kg diet is needed for maintaining normal blood parameters, improving white blood cell superoxide anion production and maintaining high liver storage levels. Vitamin C supplementation (100 mg/kg diet) is required for good growth, normal bone formation, improving immune responses and increasing liver storage of both vitamins C and E. Although supplementation of vitamin E to practical diets is not required for normal growth, it is recommended that grow-out diets for catfish should be supplemented with vitamins E and C, each at 100 mg/kg diet for optimum growth, improving immunity and increasing liver storage levels of these vitamins. Since minimal feed was offered for fish held through winter months, much higher levels of these vitamins may be required for winter feeding to maintain high levels of these vitamins in early spring. 4b.List other significant accomplishments, if any. 1. Feeding duration of sodium chloride containing diets on salinity adaptation of Nile tilapia: Feeding Nile tilapia 6% sodium chloride-containing diets for 2 to 6 weeks consistently improved fish growth, although not statistically different. Nile tilapia can tolerate up to 20 ppt salinity, but not 30 ppt. Feeding tilapia a diet containing 6% sodium cloride does not influence some osmoregulatory parameters such as blood glucose, hematocrit, and serum osmolality and cortisol levels. Nile tilapia can survive direct transfer from fresh water to salt water up to 20 ppt salinity. This transfer associated with temproray elevation of blood glucose, serum osmolality and serum cortisol levels but hematocrit decreases with increasing water salinity. The duration of salt water exposure significantly effected blood glucose, hematocrit and serum cortisol levels. Highest blood glucose and serum cortisol levels were observerd at 12 h and 6 h of salt water exposure, respectively. At 48 h of salt water exposure, serum cortisol returned to pre-transfer level, but blood glucose level and hematocrit did not recover. Feeding tilapia salt-containing diets to improve fish growth should be explored. 2. Substitution of soybean meal with distiller’s dried grains with soluble (DDGS) in Nile tilapia diets: Nile tilapia were fed diets containing 0, 10, 20 and 40% DDGS with and without added lysine as replacements of soybean meal protein for 12 weeks to evaluate their effect on growth, immunity, and resistance to Streptococcus iniae challenge. DDGS at a level of 20% can be included in diets of juvenile tilapia as a replacement of soybean meal protein without affecting weight gain, nutritional value of the diets, hematological and immunological parameters. Increasing the level of DDGS to 40% significantly decreased the growth and diet feeding value, but these can be improved by adding lysine. The improvement, however, was still inferior to those of the diets with 20% or lower DDGS. The resistance of tilapia to S. iniae challenge was not affected by inclusion level of DDGS. DDGS at a dietary level of 20% can be included in tilapia diets to reduce feed cost. 3. Effect of dietary lipid levels on vitamin E requirement and their influence on growth and resistance of channel catfish to E. ictaluri challenge: Purified diets containing 6, 10 and 14% menhaden fish oil were each supplemented with 50, 100 and 200 mg vitamin E/kg and were fed to catfish for 12 weeks to assess their effect on growth, tissue fatty acid content and resistance to E. ictaluri challenge. Dietary lipid or vitamin E levels had no effect on weight gain, feed consumption and feed efficiency ratio. High dietary fish oil levels (14%), regardless of vitamin E content, increased natural mortality. Tissue n-3 highly unsaturated fatty acids increased whereas n-9 fatty acids decreased with increasing dietary fish oil level. Liver vitamin E increased with increasing dietary level of vitamin E. Neither fish oil nor vitamin E levels affected hematological parameter, serum protein, total immunoglobulin, lysozyme activity and antibody titer against E. ictaluri. Fish oil at 14% in diets increased natural hemolytic complement activity. High level (200 mg/kg) of vitamin E decreased natural hemolytic complement activity but increased superoxide anion production of leukocytes. However, fish oil at 10% level was sufficient to stimulate macrophage chemotaxis and phagocytosis. Neither lipid nor vitamin E levels affected the resistance of catfish to E. ictaluri challenge. 4. Effect of MS-222 concentration with and without buffering on the stress response of channel catfish: Based on the cortisol response to handling stress, 90 mg/L of MS-222 was the optimal concentration for anesthetizing channel catfish with the water conditions present at our research facility. We also discovered that buffering of the MS-222 was unnecessary, even though MS-222 treated water had significantly lower pH than untreated water. The results suggest that water with neutral to slightly alkaline pH may not require buffering when MS-222 is used to anesthetize juvenile catfish. However, the results may not be applicable for water possessing different chemical properties than those at our research facility, because the pH and osmotic characteristics may affect the efficacy of the anesthetic and response of fish to handling. Researchers and fish farmers should determine the best conditions for anesthetization of fish at their own research facilities to reduce stress to fish from handling. 5. Effect of dietary bovine lactoferrin on susceptibility of Nile tilapia to disease and stress: Identification of the optimum dietary concentration of bovine lactoferrin in tilapia and catfish based on immune function and susceptibility to stress and disease was substantially met. The first half of the lactoferrin project, the effect of lactoferrin in tilapia has been completed and laboratory analyses are ongoing. The same project using catfish will be conducted in the latter half of FY 2005. 6. Effect of probiotic microorganisms on disease resistance of channel catfish and Nile tilapia: To date, five separate experiments utilizing commercially available probiotic microorganisms (bacteria and yeasts) have been conducted. Three experiments involved Nile tilapia and two involved channel catfish. Commercially available probiotics were incorporated in commercial diets at concentrations recommended by manufacturers and stored under refrigerated conditions to maintain microorganisms’ viability. Fish were fed probiotic diets to apparent satiation twice daily. After 8 weeks fish were tested for serum protein, immunoglobulin, lysozyme, complement and antibody titer. Thereafter, they were challenged with S. iniae for tilapia and E. ictaluri for catfish. No significant immune responses and disease resistance were observed as a result of any probiotic treatment thus far. Since all commercially available probiotics are developed for terrestrial vertebrates, they appear to have limited effectiveness for aquatic animals. Other dietary supplements effective for aquatic animals should be identified and refined. 4c.List any significant activities that support special target populations. Studies on the use of DDGS, interaction between dietary nutrients and feed additives lead increasing use of agricultural byproducts, decreasing feed cost and improving fish health which are beneficial to fish feed industry and fish farmers. It will also benefit agriculture 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. Channel catfish were fed commercial catfish feed (6% fat) supplemented with 0, 3, 6 and 9% fish oil for 15 weeks and fatty acid content of fillets measured at week 6, 9, 12 and 15 to determine optimum dietary lipid level and feeding duration necessary to maximize fillet content of the n-3 highly unsaturated fatty acids. The feeding portion of the study has been completed and total lipid and fatty acid content of fillets are being determined. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. This research project was started this fiscal year. Thus, the accomplishments described above represent those over the life of the project. 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, publication in scientific journals and personal contacts. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). 1. Lim, C., Yildirim-Aksoy, M., Wan, Peter, 2005. Gossypol in purified and practical diets: Effect on growth performance, immune responses and resistance of channel catfish, Ictalurus punctatus to Edwardsiella ictaluri infection. [Abstract]. Aquaculture America. 17-20 January 2005. New Orleans, LA. p 242. 2. Pasnik, D., Evans, J., Lim, C., Klesius, P.H., Yildirim, M., Barros, Margarida, Peres, H. 2005. Plant-based diets containing anti-nutritional factors can cause tissue changes in channel catfish, Ictalurus punctatus. [Abstract]. Aquaculture America. 17-20 January 2005. New Orleans, LA. p. 315. 3. Klesius, P.H., Evans, J., Shoemaker, C.A., Pasnik, D., Lim, C. 2005. Dietary strategy to improve vaccination efficacy. [Abstract]. Aquaculture America. 17-20 January 2005, New Orleans, LA., p. 216. 4. Lim, C. 2004. Aquaculture industry and fish nutrition research in the United States. Abstract, the Fifth Symposium of World Chinese Scientists on nutrition and Feeding of Finfish and Shellfish. 5-9 September 2004. Zhuhai, Guangdong, China. P. I-11. Review Publications Evans, J.J., Pasnik, D.J., Peres, H., Lim, C.E., Klesius, P.H. 2005. No apparent differences in intestinal histology of channel catfish (Ictalurus punctatus) fed heat-treated and non-heat-treated raw soybean meal. Aquaculture Nutrition. 11;123-129.