L.S. Azad et al. Fish& Shellfish Immunology 23(2007)154-163 enhancement effects in salmon [4, 7]. Disease resistance and humoral antibody production in rainbow trout wa directly and positively related to the levels of Vitamin C in the trout diet [8]. Interaction between these vitamins is also known to influence the beneficial effects they induce in cultured fish. Vitamin C/E sparing action in channel catfish was studied to explain the variability observed in its sensitivity to Vitamin E deficiency [9]. A dose dependant rotection of dietary Vitamin C against dietary deficiency of Vitamin E was demonstrated in Atlantic salmon [10] With the persistent losses due to diseases in shrimp aquaculture, coastal aquaculture farmers in India are constantly on the look-out for sustainable aquaculture and mixed farming of fish with shrimp. Milkfish is one such fish species that is traditionally harvested from extensive paddy-fish culture systems [11]. Information generated on nutrition and disease management will not only help enhance productivity from milkfish aquaculture but make it possible to tackle the disease problems that are increasingly becoming a part of aquaculture. Private shrimp farms in India use commer- cial feeds with vitamins C and E supplementations. The present investigation was carried out with an aim of obtaining information on the immune response of milkfish to supra dietary vitamins C and E. This study also is aimed at obtain ing the information on the protective response and ological memory 2. Materials and methods 2/. Fish Fingerlings of milkfish(0.87+0.01 to 1.08+0.04 g)collected from the coastal waters off north Chennai, India, sere stocked in 10-tonne cement tanks supplied with filtered aerated seawater( Salinity -32-34 ppt: DO-62 4 ppm) for acclimatisation The experiments were conducted in two sets of rearing systems. Set-I was used for evaluating the effect of supra dietary levels of vitamins C and e on the growth and survival after 6 weeks of feeding. Set-ll was used to immunize (priming and booster)and evaluate the efficacy of supra dietary vitamins on the antibody production and protective response Set-1: Fish were stocked (30 per tank) in fibre glass reinforced plastic(FRP)tanks of 0.5 tonne capacity itioned to experimental environment and control diet for a week. Five treatments were randomly laid out each with three replicates Set-ll: Fish were stocked in 200-1 FRP tanks. Duplicates of primed and booster sets(5 x 2 X 2)containing 12 fish in each tank were immunized and fed as stated above. All the tanks were supplied with filtered aerated seawater with more than 80% daily replenishment. 2. 2. Feed preparation 4 Vitamin incorporated feed was prepared using locally available feed ingredients (Table 1). The ingredients such as e dry fish(Anchovy sp ) squid (Loligo sp ) mantis shrimp(Oratosquilla nepa), Acetes and soya cake were ground in a micropulveriser, passed through a 300-um mesh screen and mixed with binder( Aquastab)in an electric blender Fish oil was added into the blender and thoroughly homogenized. Feed ingredients were mixed with additional levels of stable Vitamin C(SD Fine Chemicals, India, TI and T2 with 500 and 1500 mg kg feed, respectively)and Vitamin E (Merck, India, T3 and T4 with 50 and 150 mg kg feed, respectively) for the supra dietary supplementation. Control feed, as per the ingredients(Table 1), contained 90 mg of Vitamin C and 1. 2 mg of Vitamin e kg in the prepared diet. The ingredients were kneaded into a dough(to an approximate moisture level of 30%0), steamed at atmospheric ressure for 5 min and pelletized (2 mm diameter)in a bench top pelletizer. The pellet was dried in a hot air oven at 0C for 2-3 days to a uniform moisture level of%. Proximate composition of the feed was analysed as per the AOAC [12] methods 23. Immunization V. vulnificus, isolated from diseased wild collections of gray mullet collected from the backwaters of Muttukadu, south of Chennai, India was grown in brain-heart infusion broth(Hi Media, India) with a final salt concentration of 1.5%o at 32C for 34 h. The bacterium was harvested by spinning the suspension at 13,000 x g for 10 min; the process was repeated three times with sterile phosphate buffered saline(PBs, pH 7.2)as the resuspension medium. The finalenhancement effects in salmon [4,7]. Disease resistance and humoral antibody production in rainbow trout was directly and positively related to the levels of Vitamin C in the trout diet [8]. Interaction between these vitamins is also known to influence the beneficial effects they induce in cultured fish. Vitamin C/E sparing action in channel catfish was studied to explain the variability observed in its sensitivity to Vitamin E deficiency [9]. A dose dependant protection of dietary Vitamin C against dietary deficiency of Vitamin E was demonstrated in Atlantic salmon [10]. With the persistent losses due to diseases in shrimp aquaculture, coastal aquaculture farmers in India are constantly on the look-out for sustainable aquaculture and mixed farming of fish with shrimp. Milkfish is one such fish species that is traditionally harvested from extensive paddyefish culture systems [11]. Information generated on nutrition and disease management will not only help enhance productivity from milkfish aquaculture but make it possible to tackle the disease problems that are increasingly becoming a part of aquaculture. Private shrimp farms in India use commer￾cial feeds with vitamins C and E supplementations. The present investigation was carried out with an aim of obtaining information on the immune response of milkfish to supra dietary vitamins C and E. This study also is aimed at obtain￾ing the information on the protective response and immunological memory. 2. Materials and methods 2.1. Fish Fingerlings of milkfish (0.87 0.01 to 1.08 0.04 g) collected from the coastal waters off north Chennai, India, were stocked in 10-tonne cement tanks supplied with filtered aerated seawater (Salinity e 32e34 ppt; DO e 6.2e 7.4 ppm) for acclimatisation. The experiments were conducted in two sets of rearing systems. Set-I was used for evaluating the effect of supra dietary levels of vitamins C and E on the growth and survival after 6 weeks of feeding. Set-II was used to immunize (priming and booster) and evaluate the efficacy of supra dietary vitamins on the antibody production and protective response. Set-I: Fish were stocked (30 per tank) in fibre glass reinforced plastic (FRP) tanks of 0.5 tonne capacity and con￾ditioned to experimental environment and control diet for a week. Five treatments were randomly laid out each with three replicates. Set-II: Fish were stocked in 200-l FRP tanks. Duplicates of primed and booster sets (5  2  2) containing 12 fish in each tank were immunized and fed as stated above. All the tanks were supplied with filtered aerated seawater with more than 80% daily replenishment. 2.2. Feed preparation Vitamin incorporated feed was prepared using locally available feed ingredients (Table 1). The ingredients such as the dry fish (Anchovy sp.), squid (Loligo sp.), mantis shrimp (Oratosquilla nepa), Acetes and soya cake were ground in a micropulveriser, passed through a 300-mm mesh screen and mixed with binder (Aquastab) in an electric blender. Fish oil was added into the blender and thoroughly homogenized. Feed ingredients were mixed with additional levels of stable Vitamin C (SD Fine Chemicals, India, T1 and T2 with 500 and 1500 mg kg1 feed, respectively) and Vitamin E (Merck, India, T3 and T4 with 50 and 150 mg kg1 feed, respectively) for the supra dietary supplementation. Control feed, as per the ingredients (Table 1), contained 90 mg of Vitamin C and 1.2 mg of Vitamin E kg1 in the prepared diet. The ingredients were kneaded into a dough (to an approximate moisture level of 30%), steamed at atmospheric pressure for 5 min and pelletized (2 mm diameter) in a bench top pelletizer. The pellet was dried in a hot air oven at 40 C for 2e3 days to a uniform moisture level of 9e10%. Proximate composition of the feed was analysed as per the AOAC [12] methods. 2.3. Immunization V. vulnificus, isolated from diseased wild collections of gray mullet collected from the backwaters of Muttukadu, south of Chennai, India was grown in braineheart infusion broth (Hi Media, India) with a final salt concentration of 1.5% at 32 C for 34 h. The bacterium was harvested by spinning the suspension at 13,000  g for 10 min; the process was repeated three times with sterile phosphate buffered saline (PBS, pH 7.2) as the resuspension medium. The final I.S. 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