Development of diets for red sea bream Pagrus major employing lesser amounts of fish meal

Red Sea bream Pagrus major is an important marine fish cultured in Japan. The success of its culture primarily depends on the availability of reasonably priced high quality commercial feed. In the recent years farmers have had to put up with the high feed costs related to the vagaries in supply of the basic ingredient-fish meal, and inferior quality related to improper feed formulations in the market. Efforts are on in different parts of the world to develop fish feeds that are less dependent on fish meal (FM) as a protein source. This research was motivated by the urgent need to address this issue in the case of red sea bream. The primary aim was to identify and suggest ways of improving the utilization of alternative protein sources. It was also intended to examine the mechanism related to the green-liver syndrome that occurs in red sea bream and yellowtail upon feeding low fish meal diets. Further certain vitamins and/or immunostimulants with health promoting effects in red sea bream were examined as inclusions in the low FM diet. The ingredients examined as possible replacers of FM were soybean meal (SBM), corn gluten meal (CGM), and poultry by-product meal (PBM). The low FM diets were offered to juveniles as well as yearlings of red sea bream. On the basis of the feed performances it was found that SBM could replace up to 40% of FM in diets for juveniles, whereas it was up to 50% in the case of yearlings. In the case of CGM the replacement levels achieved were 30% and 70% of FM for juveniles and yearlings respectively. PBM could replace FM even at greater levels, 70% in juvenile diets and the entire amount in yearling diets. The maximum levels of replacement achieved with the ingredients examined in this study indicate that further substitution might be difficult considering that these alternate proteins are inferior in essential amino acids compared to FM, and contain indigestible carbohydrates and antinutrient factors such as trypsin inhibitor, antigen etc. Comparing the alternate ingredients in this study, SBM has been ranked as the best among the three based on its nutritive value, commercial availability, and price factor. Further experiments focused on improving the suitability of SBM as a protein source in red sea bream diet through refinement and amino acid supplementation. Soy protein concentrate (SPC), the purified form of SBM is higher in crude protein content and lower in antinutrient factors compared to the original form, besides matching it in amino acid balance. It was found that by using SPC up to 50% of FM could be replaced in the diets of juveniles, whereas in the diets of yearlings up to 90% of FM could be substituted. These results indicate that the refined ingredient (SPC) was more efficient than SBM in replacing FM. Further, the quality of the SPC in diets for juveniles was improved by supplementation of amino acids - methionine alone or in combination with lysine. Similar benefits were not evident in the case of SPC dietsfor yearlings, however a combination of the amino acids improved feed conversion. Though the results favour amino acid supplementation to SPC diets, this does not seem to be the right choice considering feed costs. The alternate option to achieve greater balancing of amino acids similar to FM would be to combine several suitable protein sources. This aspect was considered in yet another series of experiments combining SBM, CGM, and PB M in diets for juvenile and yearling red sea bream. SBM in combination with CGM or PB M could replace up to 50% FM in juveniles diets, whereas up to 70% FM could be replaced without amino acid supplementation when the three ingredients were combined. An almost similar combination of ingredients could successfully be used in place of 90% of FM in yearling diets. Subsequently a long term practical study confirmed that the growth performance of alternative protein sources fed diet groups was at par with commercial diet group, except for slight reduction during lower water temperature periods due to the decrease in feed intake. However, hematological and hemochemical characteristics along with the proximate composition of whole body were similar for all groups, indicating the suitability of combining alternative protein sources for practical formulations, both nutritionally and economically. Under practical fish farming, all has been not well with fish feeds containing high levels of alternate protein sources. In yellowtail it has been associated to inferior growth performance, feed conversion, and higher incidence of green liver and anemia. Identical observations have been made in red sea bream in the present studies too. It has been established that the green pigment in hepatopancreas originates from biliverdin, a heme metabolic product and one of the bile pigments. In several fish species, bile pigments conjugate with taurine and are excreted from the hepatopancreas to the bile. Contrary to FM, the alternative protein sources are poor in taurine. It seemed possible that the green liver in fish fed the low or non-FM diets is caused by the abnormal excretion of bile pigments due to dietary taurine deficiency. To tackle this critical issue related to alternate protein ingredients, we evaluated the mechanisms leading to the appearance of the green liver and tested the efficacy of supplemental taurine in overcoming the syndrome in red sea bream and in yellowtail as well, for comparative purposes. In both species, fish fed low or non-FM diets without taurine supplementation resulted in decreased hepatopancreatic content of the amino acid, and higher incidence of green liver and anemia, besides having inferior growth performance and feed conversion. On the other hand dietary taurine supplementation to low or non-FM diets drastically improved the situation. The physiological mechanism causing the green liver in fish fed low or non-FM diet is suggested to be as follows: (i) Biliverdin content rises in hepatopancreas of fish fed these diets without taurine supplementation due to an increase in hemolysis. (ii) Bilirubin is not excreted into the bile as it cannot conjugate with taurine. (iii) This results in biliverdin accumulation in hepatopancreas causing it to appear green. Therefore green liver in fish is caused by hemolytic biliverdin overproduction, and the decrease in excretion of bile pigment due to dietary taurine deficiency. This hypothesis was confirmed when taurine supplementation to low FM diet markedly prevented the occurrence of green liver, apart from improving feed performance and hematological characteristics. Judging from growth performances, it seems that dietary taurine requirement is 10 and 30 mg/g diet for red sea bream and yellowtail respectively. In the final stage of the diet development for red sea bream it was decided to examinethe health benefits of fortifying the low FM diets with established immunostimulants. In the screening test, ascorbic acid (ASP), a-tocopherol (TOC), astaxanthin (AST), and peptidoglycan (PG) were individually included in diets to evaluate the activation of non-specific immune systems and the improvement of resistance to iridovirus infections. All substances tested improved the phagocytic activities of macrophage like cells. Furthermore, the resistance to iridovirus infection was the most effective upon administration of ASP. A dose response study employing ASP followed and it revealed that the nonspecific immunostimulation was linear up to 300mg ASP/100g diet, but the resistance to iridovirus was greater at 70-150mg ASP/100g than at 300mg. These results demonstrate the potential of improving health of red sea bream through dietary manipulation. In conclusion, SBM, CGM and PBM have been identified as useful replacements of FM in red sea bream practical diet formulation and their combinations are more effective rather than as individual substitutes. Refinement of ingredients (as in the case of SPC) and amino acid supplementation are possible means of enhancing the suitability of alternate ingredients. Green liver syndrome of fish fed low or non-FM diets is caused by dietary taurine deficiency. A hypothesis explaining the mechanism of appearance of green liver has been given. ASP has been identified as an immunoenhancer in low FM diets and provided protection for red sea bream from iridovirus infection. This study has succeeded in developing a suitable low FM diet for red sea bream. Minor modifications will allow its commercial application.