Fish shellfish Immunology ELSEVIER Fish Shellfish Immunology 23(2007)154-163 www.elsevier.com/locate/fsi Supra dietary levels of vitamins C and E enhance antibody production and immune memory in juvenile milkfish, Chanos chanos(forsskal) to formalin-killed Vibrio vulnificus I.S. Azad", J Syama Dayal, M. Poornima, SA.All Central Institute of Brackishwater Aquaculture, 75 Santhome High Road, RA. P Chennai 600028. india Received 26 May 2006: revised 16 September 2006: accepted 29 September 2006 Available online 10 October 2006 Abstract Juveniles of milkfish, Chanos chanos(Forsskal), were fed two independent supra dietary levels of vitamins C(500 and 1500 mg kg feed, TI and T2)and E(50 and 150 mg kg, T3 and T4). Milkfish fed diets with supra(in addition to the vitamins present in the control diet) and normal levels(T5 containing 90 and 1.2 mg of vitamins C and E, respectively, kg- of feed)of vitamins were immunized (ip) with formalin-killed Vibrio vulnificus(FKVV). Priming and booster antibody responses to the injected bacterin were significantly(P<0.05)better in the milkfish juveniles fed supra dietary levels. Survival response of the experimental fish fed supra dietary levels of vitamins (Tl, T2 and T3) was significantly (P<0.01) better than that of the control set. Protective response against virulent bacterial challenge of the vaccinated fish fed vitamin-supplemented diets ( t2 and T3)was better than the control (T5)and TI and T4 Memory factor reflecting immunological memory was superior in the fish fed vitamin- supplemented diets. Diets supplemented with either 1500 mg of Vitamin C or 50 mg of Vitamin E kg- produced the best antibody responses, final survival and protective response upon challenge. No conclusive inferences could be drawn on the growth respons from the experiment C 2006 Elsevier Ltd. All rights reserved Keywords: Milkfish; Immune response; V. vulnificus Vitamins C and E: Memory factor 1. ntroduction The biological role played by vitamins C and E is very vital for the sustained growth and health of many living organisms. These vitamins exhibit antioxidant properties that scavenge reactive oxygen species in membranes [1] and biological fluids [2]. Vitamin deficiencies in fish under aquaculture are known to produce biochemical dysfunction leading to tissue and cellular level clinical manifestations. Several morphological and functional abnormalities have been reported in various fish species deprived of vitamins. Properties of disease resistance in fish fed ascorbic acid and Vitamin E have been reported by several researchers [3-7]. Dietary vitamins were reported to have antibod s Corresponding author. Present address: Mariculture and Fisheries Department, Kuwait Institute for Scientific Research, P.O. Box No. 1638 Salmiya22017.Tel:+9655711295;fax:+9655711090. E-mailaddress:azadi@rediffmail.com(L.S.Azad) 1050-4648/.see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/jfsi.2006.09.014
Supra dietary levels of vitamins C and E enhance antibody production and immune memory in juvenile milkfish, Chanos chanos (Forsskal) to formalin-killed Vibrio vulnificus I.S. Azad*, J. Syama Dayal, M. Poornima, S.A. Ali Central Institute of Brackishwater Aquaculture, 75 Santhome High Road, R.A. Puram, Chennai 600028, India Received 26 May 2006; revised 16 September 2006; accepted 29 September 2006 Available online 10 October 2006 Abstract Juveniles of milkfish, Chanos chanos (Forsskal), were fed two independent supra dietary levels of vitamins C (500 and 1500 mg kg1 feed, T1 and T2) and E (50 and 150 mg kg1 , T3 and T4). Milkfish fed diets with supra (in addition to the vitamins present in the control diet) and normal levels (T5 containing 90 and 1.2 mg of vitamins C and E, respectively, kg1 of feed) of vitamins were immunized (ip) with formalin-killed Vibrio vulnificus (FKVV). Priming and booster antibody responses to the injected bacterin were significantly (P < 0.05) better in the milkfish juveniles fed supra dietary levels. Survival response of the experimental fish fed supra dietary levels of vitamins (T1, T2 and T3) was significantly (P < 0.01) better than that of the control set. Protective response against virulent bacterial challenge of the vaccinated fish fed vitamin-supplemented diets (T2 and T3) was better than the control (T5) and T1 and T4. Memory factor reflecting immunological memory was superior in the fish fed vitaminsupplemented diets. Diets supplemented with either 1500 mg of Vitamin C or 50 mg of Vitamin E kg1 produced the best antibody responses, final survival and protective response upon challenge. No conclusive inferences could be drawn on the growth responses from the experiment. 2006 Elsevier Ltd. All rights reserved. Keywords: Milkfish; Immune response; V. vulnificus; Vitamins C and E; Memory factor 1. Introduction The biological role played by vitamins C and E is very vital for the sustained growth and health of many living organisms. These vitamins exhibit antioxidant properties that scavenge reactive oxygen species in membranes [1] and biological fluids [2]. Vitamin deficiencies in fish under aquaculture are known to produce biochemical dysfunction leading to tissue and cellular level clinical manifestations. Several morphological and functional abnormalities have been reported in various fish species deprived of vitamins. Properties of disease resistance in fish fed ascorbic acid and Vitamin E have been reported by several researchers [3e7]. Dietary vitamins were reported to have antibody * Corresponding author. Present address: Mariculture and Fisheries Department, Kuwait Institute for Scientific Research, P.O. Box No. 1638, Salmiya 22017. Tel.: þ965 5711295; fax: þ965 5711090. E-mail address: azadis@rediffmail.com (I.S. Azad). 1050-4648/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2006.09.014 Fish & Shellfish Immunology 23 (2007) 154e163 www.elsevier.com/locate/fsi
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 final
enhancement 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 commercial 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 obtaining 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 conditioned 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. Azad et al. / Fish & Shellfish Immunology 23 (2007) 154e163 155
l-. Azad et al. I Fish shellfish Immunology 23(2007)154-163 Table 1 Ingredient and composition of feed used in milkfish feeding trial Ingredients io composition Vitamins C and E(mg kg diet) Treatments Fish meal Wheat flour 22 01 02 02 Vitamin mixture Moisture 9.65 protein Ether extract Nitrogen free extract Control feed (T5); TI and T2-500 and 1500 mg kg, respectively, of additional stable Vitamin C; T3 and T4-50 and 150 mg kg, respectively, Poly methyl carbonide itamin mixture(mg/100 g): Vitamin A 2.0, Vitamin D0.4. Vitamin E 12.0, Vitamin K6.0, Choline ch 0, Pyridoxine 18.0, Niacin 108.0, Pantothenic acid 72.0, Biotin 0.2, Folic acid 3.0, Vitamin B12 0.015, Inositol 150.0, Vitamin C 900.0. Mineral mixture(g/kg): CaCO3-280, NaHPO4-220, K2SO4-100 MgSO4-125, CusO4-02, FeCl3-05 Mr SO4-1.0, CoSO4 -0.01, Cr2SO4-0.05, Bread fiour-7 14 d nFe= 100-(moisture % Crude protein %+ Crude fibre %+ Ether extract %+Ash %) suspension of the bacterium corresponded to a cell density of 10 CFU ml and the count was confirmed through pread plate enumeration. Formalin inactivation was carried out adjusting the final formalin concentration in the bac terial suspension to 0.5%o(v/v of formalin, for 24 h). Inactivated cells were harvested as explained above and the final suspension(in sterile PBS)of formalin-killed V vulnificus(FKVV) was used for immunization and for estimating the antibody levels Fish of set-Il were conditioned to experimental diet for a week, anaesthetized using 20 ppm crude clove oil (fr stock suspension of FKVV. Set-Il had two subsets of experimental fish one each for priming and booster o) with the local pharmacy)emulsion in filtered sea water, injected intraperitoneally(0. 1 ml corresponding to 10 CF The booster sets were injected with FKVV, as stated above, at 21 days post-priming(dpp). All the treatment groups (Tl, T2, T3, T4 and T5) of set-Il were vaccinated as detailed above and fed respective designated diets for 6 weeks. 2. 4. ELISA for anti-FKVV antibodies Random samples of five fish from different treatments(set-ID)were drawn at 0, 7, 14, 21, 28(7 days post-booster dpb), 35(14 dpb) and 42 (21 dpb)dpp. The fish were anaesthetized as explained above and the blood was drawn using I-ml sterile disposable tuberculin syringe via caudal vein. Blood was allowed to clot at room temperature for I h and held overnight at 4C, centrifuged at 3500 x g for 5 min and the serum was used for the ELISA. Rabbit polyclonal antisera against Fk WV bacterin previously produced in the laboratory were used for quantitative titration of antibodies in the fish serum using standard sandwich ELISA protocols. Briefly, 100 ul of test serum from milkfish in 1: 100 dilutions with sterile carbonate bicarbonate coating buffer( CBC, pH9.6) was loaded in 96 well ELISA plates for overnight incubation at 4C. The plates were flipped off to remove unbound serum from the wells and washed three times with 1% tween-20 in sterile phosphate buffered saline (T-PBS, pH 7.4). The plates were inverted on paper towels to remove excess moisture and unbound sites of the wells in the plates were blocked using O5% BSA in sterile
suspension of the bacterium corresponded to a cell density of 108 CFU ml1 and the count was confirmed through spread plate enumeration. Formalin inactivation was carried out adjusting the final formalin concentration in the bacterial suspension to 0.5% (v/v of formalin, for 24 h). Inactivated cells were harvested as explained above and the final suspension (in sterile PBS) of formalin-killed V. vulnificus (FKVV) was used for immunization and for estimating the antibody levels. Fish of set-II were conditioned to experimental diet for a week, anaesthetized using 20 ppm crude clove oil (from a local pharmacy) emulsion in filtered sea water, injected intraperitoneally (0.1 ml corresponding to 107 CFU) with the stock suspension of FKVV. Set-II had two subsets of experimental fish one each for priming and booster vaccination. The booster sets were injected with FKVV, as stated above, at 21 days post-priming (dpp). All the treatment groups (T1, T2, T3, T4 and T5) of set-II were vaccinated as detailed above and fed respective designated diets for 6 weeks. 2.4. ELISA for anti-FKVV antibodies Random samples of five fish from different treatments (set-II) were drawn at 0, 7, 14, 21, 28 (7 days post-booster, dpb), 35 (14 dpb) and 42 (21 dpb) dpp. The fish were anaesthetized as explained above and the blood was drawn using 1-ml sterile disposable tuberculin syringe via caudal vein. Blood was allowed to clot at room temperature for 1 h and held overnight at 4 C, centrifuged at 3500 g for 5 min and the serum was used for the ELISA. Rabbit polyclonal antisera against FKVV bacterin previously produced in the laboratory were used for quantitative titration of antibodies in the fish serum using standard sandwich ELISA protocols. Briefly, 100 ml of test serum from milkfish in 1:100 dilutions with sterile carbonate bicarbonate coating buffer (CBC, pH 9.6) was loaded in 96 well ELISA plates for overnight incubation at 4 C. The plates were flipped off to remove unbound serum from the wells and washed three times with 1% tween-20 in sterile phosphate buffered saline (TePBS, pH 7.4). The plates were inverted on paper towels to remove excess moisture and unbound sites of the wells in the plates were blocked using 0.5% BSA in sterile Table 1 Ingredient and composition of feed used in milkfish feeding trial Ingredients % composition Vitamins C and E (mg kg1 diet) Treatments C E Fish meal 35 T1 0590 01.20 Squid 05 T2 1590 01.20 Squilla 10 T3 0090 50.00 Acetes 05 T4 0090 150.00 Soya cake 15 T5 0090 01.20 Wheat flour 22 Bindera 01 Fish oil 02 Lecithin 02 Vitamin mixtureb 01 Mineral mixturec 02 Proximate components % composition Moisture 09.65 Crude protein 39.81 Ether extract 06.94 Crude fibre 04.01 Nitrogen free extractd 24.91 Ash 14.68 Control feed (T5); T1 and T2 e 500 and 1500 mg kg1 , respectively, of additional stable Vitamin C; T3 and T4 e 50 and 150 mg kg1 , respectively, of additional Vitamin E. a Poly methyl carbomide. b Vitamin mixture (mg/100 g): Vitamin A 2.0, Vitamin D 0.4, Vitamin E 12.0, Vitamin K 6.0, Choline chloride 600.0, Thiamine 18.0, Riboflavin 24.0, Pyridoxine 18.0, Niacin 108.0, Pantothenic acid 72.0, Biotin 0.2, Folic acid 3.0, Vitamin B12 0.015, Inositol 150.0, Vitamin C 900.0. c Mineral mixture (g/kg): CaCO3 e 28.0, NaHPO4 e 22.0, K2SO4 e 10.0, MgSO4 e 12.5, CuSO4 e 0.2, FeCl3 e 0.5, MnSO4 e 0.5, KI e 0.01, ZnSO4 e 1.0, CoSO4 e 0.01, Cr2SO4 e 0.05, Bread flour e 7.14. d NFE ¼ 100 (moisture % þ Crude protein % þ Crude fibre % þ Ether extract % þ Ash %). 156 I.S. Azad et al. / Fish & Shellfish Immunology 23 (2007) 154e163
L.S. Azad et al. Fish& Shellfish Immunology 23(2007)154-163 PBS for I h. The blocking solution was Hipped off, washed with T-PBS three times and the wells were loaded with 100 ul of FKVV bacterial suspension, incubated for I h and washed as above. The wells were then loaded with 100 ul of rabbit anti-FKVV polyclonal serum(1: 500 dilutions in sterile PBS, pH 7.4)and incubated for 1 h. After the next washing, 100 ul of goat anti-rabbit HRP conjugated antibody(Bangalore Genei, India)was added, incubated for 1 h, washed and 50 ul of the substrate (TMB-H2O2, Bangalore Genei, India) was added. The colour development was stopped after 20 min using 1N HCI and optical density (oD)of colour developed was measured at 450 nm usin an ELISA reader. Negative control consisted of similarly treated wells where un-immunized rabbit serum was used instead of anti-FKVV polyclonal serum. The difference in ODs of treatment sets and that of the negative controls was taken as the anti-FKVV titre of the test serum 25. Challenge and relative percent survival(RPS) A random sample of eight fish from each replicates of primed and boosted groups of set -Il and 8 fish from unva inated control (T5)of set-I were challenged, at the end of the experiment using live bacterial culture of V. vulnificus The bacterium was grown in brain-heart infusion broth(.5% Naci)to get a final cell suspension of 10'CFU ml Challenge was carried out in 100-1 FRP tanks with eight fish from each tank. The fish were maintained for recording mortalities till 10 days post-challenge. Relative percent survival (RPS)was calculated following Amend [13 as tality in vaccinated grou 100 mortality in C-l or C-lI where C-I and C-lI are control fish (T5) from set-I and set-lI 2.6. Memory factor(MF) Evaluation of the efficacy of booster response to quantify the immunological memory [14] was carried out using the eliSa titres measured at different time intervals and the memory factor(MF) was calculated as follows: Tb(x)-T(r Tp(x) where Tb(r): titre of boosted fish at x dpb; T(r): titre of primed fish on the day of booster; and Tp(x): titre of primed fish at x dpb 2.7. Statistical analysis The data on antibody titres was subjected to ANOVA for testing the significance of difference between treatment parameters. Pair-wise multiple comparisons for final growth and survival were made following Dennets test using PEPI-404 statistical software. Tukey's HSD test was used for comparing the ELISa titres due to treatments, due to days post-immunization and protective response of different treatment groups. 3.. Growth and survival Information on the stocking, growth and survival of milkfish juveniles is presented in Table 2a. Growth of milkfish juveniles fed higher levels of Vitamin E(T4)was lower than those of T2 and T3; however, the ANOVA of weight gain data revealed that the parameters did not differ significantly(P>0.05) from one another. It was interesting to note that all the treatments differed significantly(P<0.01)in their survival responses from that of the control (Table 2b).Fish from the control group(T5)showed the lowest survival (80%)followed by those fed higher dietary Vitamin E levels (150mgkg-)
PBS for 1 h. The blocking solution was flipped off, washed with TePBS three times and the wells were loaded with 100 ml of FKVV bacterial suspension, incubated for 1 h and washed as above. The wells were then loaded with 100 ml of rabbit anti-FKVV polyclonal serum (1:500 dilutions in sterile PBS, pH 7.4) and incubated for 1 h. After the next washing, 100 ml of goat anti-rabbit HRP conjugated antibody (Bangalore Genei, India) was added, incubated for 1 h, washed and 50 ml of the substrate (TMBeH2O2, Bangalore Genei, India) was added. The colour development was stopped after 20 min using 1 N HCl and optical density (OD) of colour developed was measured at 450 nm using an ELISA reader. Negative control consisted of similarly treated wells where un-immunized rabbit serum was used instead of anti-FKVV polyclonal serum. The difference in ODs of treatment sets and that of the negative controls was taken as the anti-FKVV titre of the test serum. 2.5. Challenge and relative percent survival (RPS) A random sample of eight fish from each replicates of primed and boosted groups of set-II and 8 fish from unvaccinated control (T5) of set-I were challenged, at the end of the experiment using live bacterial culture of V. vulnificus. The bacterium was grown in braineheart infusion broth (1.5% NaCl) to get a final cell suspension of 107 CFU ml1 . Challenge was carried out in 100-l FRP tanks with eight fish from each tank. The fish were maintained for recording mortalities till 10 days post-challenge. Relative percent survival (RPS) was calculated following Amend [13] as: RPS ¼ 1 % mortality in vaccinated group % mortality in C-I or C-II 100 where C-I and C-II are control fish (T5) from set-I and set-II. 2.6. Memory factor (MF) Evaluation of the efficacy of booster response to quantify the immunological memory [14] was carried out using the ELISA titres measured at different time intervals and the memory factor (MF) was calculated as follows: MF ¼ TbðxÞ TðrÞ TpðxÞ where Tb(x) : titre of boosted fish at x dpb; T(r): titre of primed fish on the day of booster; and Tp(x): titre of primed fish at x dpb. 2.7. Statistical analysis The data on antibody titres was subjected to ANOVA for testing the significance of difference between treatment parameters. Pair-wise multiple comparisons for final growth and survival were made following Dennet’s test using PEPI-404 statistical software. Tukey’s HSD test was used for comparing the ELISA titres due to treatments, due to days post-immunization and protective response of different treatment groups. 3. Results 3.1. Growth and survival Information on the stocking, growth and survival of milkfish juveniles is presented in Table 2a. Growth of milkfish juveniles fed higher levels of Vitamin E (T4) was lower than those of T2 and T3; however, the ANOVA of weight gain data revealed that the parameters did not differ significantly (P > 0.05) from one another. It was interesting to note that all the treatments differed significantly (P < 0.01) in their survival responses from that of the control (Table 2b). Fish from the control group (T5) showed the lowest survival (80%) followed by those fed higher dietary Vitamin E levels (150 mg kg1 ). I.S. Azad et al. / Fish & Shellfish Immunology 23 (2007) 154e163 157
l-. Azad et al. I Fish shellfish Immunology 23(2007)154-163 ble 2a Stocking and growth details of juveniles of Chanos chanos fed vitamin-supplemented diets Treatments wf(g±SE) Growth (g) Survival(%±SE) 0916±0.138 1606±0.256 87.78±1.93 80090) 1082±0.036 1.777±0.118 8889±1.93 1682±0.173 84(90 0.868±0.011 1411±0.l17 9333±3.33 (90) 0952±0.107 1.562士0.217 8000±3.33 3. 2. Antibody resp Antibody titres elicited by the fish fed supra dietary levels of vitamins were significantly(P0.05)difference between the higher level of Vitamin C(T2) and lower level of Vitamin E (T3)in their protective responses 4. Discussion The dietary protein level (39%)used in the present investigation is higher than that suggested by Borlongan and Satoh[15]. They recommended 24% dietary protein for economic grow-out production of milkfish. However, a higher level of protein was used in the present study: keeping in view the commercial shrimp diet used in shrimp production ponds and aquaculture potentials of fish-shrimp mixed farming as done in many of the traditional farms of West Bengal and Kerala in India. Growth responses due to additional supplementation of vitamins C and e, in the preser study, did not result in a statistically significant improvement compared to that of the control. Supra dietary levels of Vitamin C in yellow perch(Perca flavescens) resulted in better growth and feed efficiency [16]. Similar growth promoting properties of sufficient dietary levels of Vitamin C are well documented [17-20]. Short duration of the experiment was probably responsible for the statistically insignificant difference in growth enhancement between the groups, though, there was an apparent improvement in treatments Tl, T2 and T3 compared to that of T5. However, Table 2b Multiple comparisons for survival response(Dennett test) Comparison treatments S.E. of difference Two-tailed P I ys. 5 <0.01 2vs.5 <0.01 15.556 <0.01 13.333 <0.01
3.2. Antibody response Antibody titres elicited by the fish fed supra dietary levels of vitamins were significantly (P 0.05) difference between the higher level of Vitamin C (T2) and lower level of Vitamin E (T3) in their protective responses. 4. Discussion The dietary protein level (39%) used in the present investigation is higher than that suggested by Borlongan and Satoh [15]. They recommended 24% dietary protein for economic grow-out production of milkfish. However, a higher level of protein was used in the present study; keeping in view the commercial shrimp diet used in shrimp production ponds and aquaculture potentials of fish-shrimp mixed farming as done in many of the traditional farms of West Bengal and Kerala in India. Growth responses due to additional supplementation of vitamins C and E, in the present study, did not result in a statistically significant improvement compared to that of the control. Supra dietary levels of Vitamin C in yellow perch (Perca flavescens) resulted in better growth and feed efficiency [16]. Similar growth promoting properties of sufficient dietary levels of Vitamin C are well documented [17e20]. Short duration of the experiment was probably responsible for the statistically insignificant difference in growth enhancement between the groups, though, there was an apparent improvement in treatments T1, T2 and T3 compared to that of T5. However, Table 2a Stocking and growth details of juveniles of Chanos chanos fed vitamin-supplemented diets Treatments Nt (N0) Wi (g SE) Wf (g SE) Growth (g) Survival (% SE) T1 79 (90) 0.916 0.138 1.606 0.256 0.69 87.78 1.93 T2 80 (90) 1.082 0.036 1.777 0.118 0.69 88.89 1.93 T3 86 (90) 0.995 0.117 1.682 0.173 0.69 95.56 1.93 T4 84 (90) 0.868 0.011 1.411 0.117 0.54 93.33 3.33 T5 72 (90) 0.952 0.107 1.562 0.217 0.61 80.00 3.33 Nt e number at termination; N0 e Number at start; Wi e initial average weight; Wf e final average weight. Table 2b Multiple comparisons for survival response (Dennett test) Comparison treatments Difference S.E. of difference Two-tailed P 1 vs. 5 7.778 0.667 <0.01 2 vs. 5 8.889 <0.01 3 vs. 5 15.556 <0.01 4 vs. 5 13.333 <0.01 158 I.S. Azad et al. / Fish & Shellfish Immunology 23 (2007) 154e163
L.S. Azad et al. Fish& Shellfish Immunology 23(2007)154-163 Table ANOVA of ELISA titres in milkfish fed Vitamins( C and E)-supplemented diets Source of variatio df P- value 0.009471 0.002368 83E-41 462613 0.006016 0.001504 9591786 41E33 2.462613 0.003905 0.000244 15.56677 1.88E-20 1745647 0.001568 57E05 Total 0.02096 higher levels of Vitamin E(T4) produced growth retardation. Higher levels of vitamins are required by fish in tropical aquaculture due to increased physiological stress [21, 22]. Enhanced growth and survival responses of hybrid striped bass juveniles fed vitamins C and e was noticed by Sealy and Galtin [23]. It is evident from the present study that the supra dietary levels of both vitamins C and e resulted in a better survival response of milkfish juveniles compared to he fish in control group e, Role of dietary vitamins in the context of disease resistance of farmed fish has been very well established. Ascorbic deficiency in rainbow trout[24], channel catfish [25] and Atlantic salmon [26] was found to increase disease ole comparisons (Tukey's HSD) of mean ELISA titres in different treatments at different time intervals after priming or booster vaccination st-priming/booster keys HSD'q Treatments Difference betwee TI T2 0 dpp 0.00725 0.0004 0.002 0.0008 0.0016 0.0004 0.0012 7 dpp 0.00738 2BTT 0 -0.0052 0.0022 0.0104* 0.0074* 0.0156* 0.018* 14 dpp 0.00511 0.013 0.01 0.0234 345 21 dpp 0.00579 0.006 345 42 dpp 0.01065 0.006 -0.0066 -0.0006 7 dpb 0.01045 -0.0048 0.004 0.0008 0.0152* 0012 0.003462 0.0112* 0.006
higher levels of Vitamin E (T4) produced growth retardation. Higher levels of vitamins are required by fish in tropical aquaculture due to increased physiological stress [21,22]. Enhanced growth and survival responses of hybrid striped bass juveniles fed vitamins C and E was noticed by Sealy and Galtin [23]. It is evident from the present study that the supra dietary levels of both vitamins C and E resulted in a better survival response of milkfish juveniles compared to the fish in control group. Role of dietary vitamins in the context of disease resistance of farmed fish has been very well established. Ascorbic acid deficiency in rainbow trout [24], channel catfish [25] and Atlantic salmon [26] was found to increase disease Table 3a ANOVA of ELISA titres in milkfish fed Vitamins (C and E)-supplemented diets Source of variation SS df MS F P-value F crit Duration 0.009471 4 0.002368 151.002 1.83E-41 2.462613 Treatments 0.006016 4 0.001504 95.91786 2.41E-33 2.462613 Interaction 0.003905 16 0.000244 15.56677 1.88E-20 1.745647 Within 0.001568 100 1.57E-05 Total 0.02096 124 Table 3b Multiple comparisons (Tukey’s HSD) of mean ELISA titres in different treatments at different time intervals after priming or booster vaccination Days post-priming/booster Tukey’s HSD ‘q’ Treatments Difference between mean antibody titres T1 T2 T3 T4 T5 0 dpp 0.00725 T1 0 0.0004 0.002 0.0008 0.0012 T2 0 0.0016 0.0004 0.0008 T3 0 0.0012 0.0008 T4 0.0004 T5 0 7 dpp 0.00738 T1 0 0.0052 0.0022 0.0104* 0.0128* T2 0 0.0074* 0.0156* 0.018* T3 0 0.0082* 0.0106* T4 0 0.0024 T5 0 14 dpp 0.00511 T1 0 0.013* 0.003 0.0076* 0.0104* T2 0 0.01* 0.0206* 0.0234* T3 0 0.0106* 0.0134* T4 0.0028 T5 0 21 dpp 0.00579 T1 0 0.006* 0.0052 0.003 0.0072* T2 0 0.0008 0.009* 0.0132* T3 0 0.0082* 0.0124* T4 0 0.0042 T5 0 42 dpp 0.01065 T1 0 0.006 0.0066 0.0058 0.007 T2 0 0.0006 0.0118* 0.013* T3 0 0.0124* 0.0136* T4 0 0.0012 T5 0 7 dpb 0.01045 T1 0 0.0048 0.004 0.0104 0.00672 T2 0 0.0008 0.0152* 0.01152* T3 0 0.0144 0.01072 T4 0 0.00368 T5 0 21 dpb 0.003462 T1 0 0.0112* 0.006* 0.0203* 0.0238* T2 0 0.0052* 0.0315* 0.035* T3 0 0.0263* 0.0298* T4 0 0.0035* T5 0 *Significant at P ¼ 0.05. I.S. Azad et al. / Fish & Shellfish Immunology 23 (2007) 154e163 159
l-. Azad et al. I Fish shellfish Immunology 23(2007)154-163 0 dpp 008 00 T4 Days post priming dpp/booster(dpb Fig. 1. Immune response(ELISA)of juveniles of Chanos chanos fed different vitamin( C and E)enriched diets to Vibrio vulnificus susceptibility Supra dietary levels of vitamins C and e were found to have direct influence on the immune response of juvenile milkfish as evident from the enhanced anti-FKVV antibody titres(tl, T2 and T3)in the present study Supra dietary levels of Vitamin C probably helped in neutralizing the stress responses [4, 22] of confinement in the present study and thus, resulted in enhanced antibody production. Similar enhancements in antibody production of channel catfish against Edwardsiella ictaluri [25] and in rainbow trout against V. anguillarum [4] have been reported. Working on grouper(Epinehelus awoara), Wei and co-workers [27] reported higher antibody production in the fish fed supra dietary levels of Vitamin C. Protective response of milkfish in the present study following vaccination was significant compared to the unvac cinate fish. High protective response and immunological memory in the present study can be attributed to a combi- nation of enhanced specific antibody production and probable elevation of non-specific immune responses as reported in many fish species receiving varying levels of dietary Vitamin C [23, 28-31. High specific antibody response and enhanced protective responses to bacterial challenge exhibited by milkfish juveniles is supported by the enhanced immunological memory. Similar findings were reported by Wei et al. [27] who tested higher levels of dietary Vitamin C in grouper resulting in enhanced specific antibody response against injected formalin-killed v. vulnificus and protective response against the bacterium, delivered live via injection/bath. 7222 400 4000 3000 Fig. 2. Memory factor(MF) as an index of specific immunity in vaccinated juveniles of Chanos chanos fed vitamin(C and E) enriched diets Treatment plot points with common letter-lable are not significantly(P=0.05) different from one another
susceptibility. Supra dietary levels of vitamins C and E were found to have direct influence on the immune response of juvenile milkfish as evident from the enhanced anti-FKVV antibody titres (T1, T2 and T3) in the present study. Supra dietary levels of Vitamin C probably helped in neutralizing the stress responses [4,22] of confinement in the present study and thus, resulted in enhanced antibody production. Similar enhancements in antibody production of channel catfish against Edwardsiella ictaluri [25] and in rainbow trout against V. anguillarum [4] have been reported. Working on grouper (Epinehelus awoara), Wei and co-workers [27] reported higher antibody production in the fish fed supra dietary levels of Vitamin C. Protective response of milkfish in the present study following vaccination was significant compared to the unvaccinated fish. High protective response and immunological memory in the present study can be attributed to a combination of enhanced specific antibody production and probable elevation of non-specific immune responses as reported in many fish species receiving varying levels of dietary Vitamin C [23,28e31]. High specific antibody response and enhanced protective responses to bacterial challenge exhibited by milkfish juveniles is supported by the enhanced immunological memory. Similar findings were reported by Wei et al. [27] who tested higher levels of dietary Vitamin C in grouper resulting in enhanced specific antibody response against injected formalin-killed V. vulnificus and protective response against the bacterium, delivered live via injection/bath. Fig. 1. Immune response (ELISA) of juveniles of Chanos chanos fed different vitamin (C and E) enriched diets to Vibrio vulnificus. Fig. 2. Memory factor (MF) as an index of specific immunity in vaccinated juveniles of Chanos chanos fed vitamin (C and E) enriched diets. Treatment plot points with common letter-lable are not significantly (P ¼ 0.05) different from one another. 160 I.S. Azad et al. / Fish & Shellfish Immunology 23 (2007) 154e163
l.S. Azad et al. Fish Shellfish Immunology 23(2007)154-163 —21db TI Fig. 3. Protective response of vaccinated juveniles of Chanos chanos fed vitamin(C and E)enriched diets to Vibrio vulnificus(plot points o treatment means sharing common labels are not significantly (P 0.05) different from one another. RPS(C-1): protective response unvaccinated control. RPS( C-ll): protective response relative to the vaccinated control ooster vaccination was rendered highly efficient in milkfish juveniles fed high Vitamin C (T2)and low Vitamin E (T3)indicating a probable interaction between the two vitamins. It is widely accepted that Vitamin C, in the water phase, spares Vitamin E and helps in its regeneration from the radical form [32]. Lower antibody titres and protective responses as reported in rainbow trout from a feeding trial with varying combinations of vitamins C and E(2 O response of milkfish juveniles fed higher Vitamin E (T4)are probably due to the reduced lymphoprolifera Protective response of milkfish juveniles followed closely the results of antibody production with high levels of Vitamin C not differing significantly from that of low levels of Vitamin E fed fish. Rainbow trout fed double deficient or double low vitamin(vitamins C/E)diets recorded high mortalities upon challenge with Yersinia ruckeri [28].Low protective response of T5 is probably due to the negligible levels of vitamins C and E(90 mg kg and 1.2 mg kg of diet, respectively) in the control diet and these levels were not enough to make the fish overcome the vaccination stress.It has been very well shown by previous researchers that sampling and confinement stress can be managed with Vitamin C supplementation [20, 21]. Stress is known to reduce the immune response and disease resistance in fish [33; hence, low levels of vitamins C and E (T5)resulted not only in the reduced antibody production but also in protective response upon challenge with live V. vulnificus. High protective response of T2 and T3 in the present tudy is probably due to both enhanced specific immune response and non-specific immune response. Results of the present study open up new avenues of making milkfish an alternative for mixed crop species in hrimp aquaculture ponds utilizing the high nutrient feed and supplementing additionally to keep the immune system fit to fight diseases Table 3c comparisons(Tukey's HSD)of average percentage of mortality in different treatments following challenge with live Vibrio vuinificr Calculated difference between means 0.00 8.33 .Difference between
Booster vaccination was rendered highly efficient in milkfish juveniles fed high Vitamin C (T2) and low Vitamin E (T3) indicating a probable interaction between the two vitamins. It is widely accepted that Vitamin C, in the water phase, spares Vitamin E and helps in its regeneration from the radical form [32]. Lower antibody titres and protective response of milkfish juveniles fed higher Vitamin E (T4) are probably due to the reduced lymphoproliferation responses as reported in rainbow trout from a feeding trial with varying combinations of vitamins C and E [28]. Protective response of milkfish juveniles followed closely the results of antibody production with high levels of Vitamin C not differing significantly from that of low levels of Vitamin E fed fish. Rainbow trout fed double deficient or double low vitamin (vitamins C/E) diets recorded high mortalities upon challenge with Yersinia ruckeri [28]. Low protective response of T5 is probably due to the negligible levels of vitamins C and E (90 mg kg1 and 1.2 mg kg1 of diet, respectively) in the control diet and these levels were not enough to make the fish overcome the vaccination stress. It has been very well shown by previous researchers that sampling and confinement stress can be managed with Vitamin C supplementation [20,21]. Stress is known to reduce the immune response and disease resistance in fish [33]; hence, low levels of vitamins C and E (T5) resulted not only in the reduced antibody production but also in protective response upon challenge with live V. vulnificus. High protective response of T2 and T3 in the present study is probably due to both enhanced specific immune response and non-specific immune response. Results of the present study open up new avenues of making milkfish an alternative for mixed crop species in shrimp aquaculture ponds utilizing the high nutrient feed and supplementing additionally to keep the immune system fit to fight diseases. Fig. 3. Protective response of vaccinated juveniles of Chanos chanos fed vitamin (C and E) enriched diets to Vibrio vulnificus (plot points of treatment means sharing common labels are not significantly (P ¼ 0.05) different from one another. RPS (C-I): protective response relative to unvaccinated control. RPS (C-II): protective response relative to the vaccinated control. Table 3c Multiple comparisons (Tukey’s HSD) of average percentage of mortality in different treatments following challenge with live Vibrio vulnificus Treatments Calculated difference between means T1 T2 T3 T4 T5 T1 0.00 25.00* 27.78* 5.56 13.89 T2 0.00 2.78 30.56* 38.89* T3 0.00 33.33* 41.67* T4 0.00 8.33 T5 0.00 Tukey’s HSD at a ¼ 0.05 16.48 *Difference between means significant at P < 0.05. I.S. Azad et al. / Fish & Shellfish Immunology 23 (2007) 154e163 161
l-. Azad et al. I Fish shellfish Immunology 23(2007)154-163 Acknowledgements The authors are thankful to the Director, CIBA for his constant support and critical suggestions. Thanks are also due to Joseph Sahay Rajan, Technician of CiBA for the help in laboratory analysis References [1 Burton Gw.J KU. Is vitamin E y lipid-soluble, chain-breaking antioxidant in human blood plasma and erythrocyt membranes? Arch Brophy1983:221:28 [2 Frei B. Stocker R, nd L, Ames BN. Ascorbate: the most effective antioxidant in human blood plasma. Adv Exp Med Biol 990264:55-163. [3] Hardie LJ, Marsden MJ, Fletcher TC, Secombes CJ In witro addition of vitamin C affects rainbow trout lymphocyte responses. Fish Shellfish immunol1983:3:207-19 [4] Navarre O, Halver JE Disease resistance and humoral antibody production in rainbow trout fed high levels of vitamin C. Aquaculture [5] Satyabudhy AMA, Grant BF, Halver JE. Effects of L-ascorbyl phosphates(AsPP) on growth and immunoresistance of rainbow trout Oncorhynchus mykiss)to infectious hematopoietic necrosis(IHN)virus. In: Proceedings from the third international symposium on feeding and nutrition in fish, Toba, Japan: 1989. p. 411-26 [6] Hardie LJ, Fletcher TC, Secombes CJ. The effect of vitamin E on the immune response of the Atlantic salmon(Salmo salar L ) Aquaculture 99187:1-13 [7] Wagba R, Glette J, Nilsen ER, Sandnes K. Dietary vitamin C, immunity and disease resistance in Atlantic salmon (Salmo salar). J Fish hysiol Biochem 1993: 12: 61-73 [8] Lall SP, Olivier G. Role of micronutrients in immune response and disease resistance in fish. In: Kaushik SJ, Luquet P, editors. Fish nutrition in practice. Paris: INRA: 1993. P. 101-18 I Lovell RT, Miyazaki T, Rabegnator S. Requirement for a-tocopherol by channel catfish fed diets low in polyunsaturated triglycerides. J Nutr 984114:894-901 [10] Hamre K, Waagbo R, Berge RK. Lie O. Vitamins C and E interact in juvenile Atlantic salmon Salmo salar L. Free Radic Biol Med 997;22:137-49 [11] Pillay TVR, Bose B. Observations on the culture of brackishwater fishes in paddy fields in West Bengal(India). Proc Indo-Pacific Fish Council1958:7:187-92. [12] AOAC. Official methods of analysis. 15th ed. Washington DC: Association of Official Analytical Chemists Inc USA: 1990. P. 1-30 [13] Amend DE. Potency testing of fish vaccines. In: Anderson DP, Hennessen W, editors. Fish biologics: serodiagnostics and vaccines. Developments in biological standardization, vol. 49. Basel: S. Karger; 1981. P. 447-54. [ 15] Borlongan I, Satoh S Studies on the nutrition and feed development for milkfish( Chanos chaney. Immunology 1965: 9: 333-48 [14] Nossal GJ, Austin CM, Ada GL. Antigens in ty. VIl. Analysis of immunological e-ronpaku/2003/17BORLONGAN. pdf>: 2003. p 1-42 [accessed 28 10.06] [16] Lee K-J, Dabrowski K. Interaction between vitamins C and E affects their tissue concentrations, growth, lipid oxidation, and deficiency symptoms in yellow perch(Perca fiavescens). Br J Nutr 2003: 89: 89-596 [17] Dabrowski K, El-fiky N, Kock G, wieser w. Requirement and utilization of ascorbic acid and ascorbic sulphate in juvenile rainbow trout. Aquaculture 1990: 91: 317 18] Dabrowski K, Moreau R, El-saidy D. Ontogenic sensitivity of channel catfish to ascorbic acid deficiency. J Aquat Anim Health 1996: 8: 22-7. [19] Lee K-J, Kim K-W, Bai SC. Effects of different dietary levels of L-ascorbic acid on growth and tissue vitamin C concentrations in juvenile Korean rockfish, Sebastes schlegeli(Hilgendorf). Aquac Res 1998: 29: 237-44. [20] Shiau S-Y, Hsu T-S. Quantification of C requirement for juvenile hybrid tilapia, Oreochromis niloticus x Oreochromis aureus, with ascorbyl-2-monosulphate-Na and L-ascorbyl-2-monophosphate-Mg. Aquaculture 1999: 17: 317-26. ward w. Dietary vitamin requirements of cultured young fish, with special emphasis on quantitative estimates for salmonids. Aquaculture 1994: 124: 133-68. [22] Montero D, Marrero M, Izquierdo MS, Robaina L, Vergara JM, Tort L Effect of vitamin E and C dietary supplementation on some immune of gilthead seabream (Sparus aurata) juveniles subjected to crowding stress. Aquaculture 1999: 171: 269-78 [23] Sealey WM, Galtin DM. Dietary C and vitamin E interact to influence growth and tissue composition of juvenile hybrid striped bass (Morone chrysops 9 x M. saxatilis 8) but have limited effects on immune response. J Nutr 2002: 132: 748-55 [24 Blazer VS. Nutrition and disease resistance in fish. Ann Rev Fish Dis 1992: 1: 309-23 [25] Li Y, Lovell RT. Elevated levels of dietary ascorbic acid increase the immune responses in channel catfish. J Nutr 1985: 198: 123-31 [26] Hardie L, Fletcher TC, Secombes CJ. The effect of dietary vitamin C on the immune response of Atlantic salmon. Aquaculture 19919:201-14. [27] Wei QQ, He WZ, Pei PJ. Disease resistance and humoral and immunomodulatory effects of vitamin C on grouper, Epinephelus awoara. Chin J Oceano Limnol 2000: 18: 247-52 [28] Verlhac V, Gabaudan J, Obach A, Schuep W, Hole R. Influence of dietary glucan and vitamin C on non-specific and specific immune responses of rainbow trout(Oncorhynchus rykiss). Aquaculture 1996: 143: 123-3
Acknowledgements The authors are thankful to the Director, CIBA for his constant support and critical suggestions. Thanks are also due to Joseph Sahay Rajan, Technician of CIBA for the help in laboratory analysis. References [1] Burton GW, Joyce A, Ingold KU. Is vitamin E the only lipid-soluble, chain-breaking antioxidant in human blood plasma and erythrocyte membranes? Arch Biochem Biophy 1983;221:281e90. [2] Frei B, Stocker R, England L, Ames BN. Ascorbate: the most effective antioxidant in human blood plasma. Adv Exp Med Biol 1990;264:55e163. [3] Hardie LJ, Marsden MJ, Fletcher TC, Secombes CJ. In vitro addition of vitamin C affects rainbow trout lymphocyte responses. Fish Shellfish Immunol 1983;3:207e19. [4] Navarre O, Halver JE. Disease resistance and humoral antibody production in rainbow trout fed high levels of vitamin C. Aquaculture 1989;79:207e21. [5] Satyabudhy AMA, Grant BF, Halver JE. Effects of L-ascorbyl phosphates (AsPP) on growth and immunoresistance of rainbow trout (Oncorhynchus mykiss) to infectious hematopoietic necrosis (IHN) virus. In: Proceedings from the third international symposium on feeding and nutrition in fish, Toba, Japan; 1989. p. 411e26. [6] Hardie LJ, Fletcher TC, Secombes CJ. The effect of vitamin E on the immune response of the Atlantic salmon (Salmo salar L.). Aquaculture 1991;87:1e13. [7] Wagbo¨ R, Glette J, Nilsen ER, Sandnes K. Dietary vitamin C, immunity and disease resistance in Atlantic salmon (Salmo salar). J Fish Physiol Biochem 1993;12:61e73. [8] Lall SP, Olivier G. Role of micronutrients in immune response and disease resistance in fish. In: Kaushik SJ, Luquet P, editors. Fish nutrition in practice. Paris: INRA; 1993. p. 101e18. [9] Lovell RT, Miyazaki T, Rabegnator S. Requirement for a-tocopherol by channel catfish fed diets low in polyunsaturated triglycerides. J Nutr 1984;114:894e901. [10] Hamre K, Waagbo R, Berge RK, Lie O. Vitamins C and E interact in juvenile Atlantic salmon Salmo salar L. Free Radic Biol Med 1997;22:137e49. [11] Pillay TVR, Bose B. Observations on the culture of brackishwater fishes in paddy fields in West Bengal (India). Proc Indo-Pacific Fish Council 1958;7:187e92. [12] AOAC. Official methods of analysis. 15th ed. Washington DC: Association of Official Analytical Chemists Inc USA; 1990. p. 1e30. [13] Amend DF. Potency testing of fish vaccines. In: Anderson DP, Hennessen W, editors. Fish biologics: serodiagnostics and vaccines. Developments in biological standardization, vol. 49. Basel: S. Karger; 1981. p. 447e54. [14] Nossal GJ, Austin CM, Ada GL. Antigens in immunity. VII. Analysis of immunological memory. Immunology 1965;9:333e48. [15] Borlongan I, Satoh S. Studies on the nutrition and feed development for milkfish (Chanos chanos Forsskal), ; 2003. p. 1e42 [accessed 28.10.06]. [16] Lee K-J, Dabrowski K. Interaction between vitamins C and E affects their tissue concentrations, growth, lipid oxidation, and deficiency symptoms in yellow perch (Perca flavescens). Br J Nutr 2003;89:89e596. [17] Dabrowski K, El-fiky N, Kock G, Wieser W. Requirement and utilization of ascorbic acid and ascorbic sulphate in juvenile rainbow trout. Aquaculture 1990;91:317e37. [18] Dabrowski K, Moreau R, El-saidy D. Ontogenic sensitivity of channel catfish to ascorbic acid deficiency. J Aquat Anim Health 1996;8:22e7. [19] Lee K-J, Kim K-W, Bai SC. Effects of different dietary levels of L-ascorbic acid on growth and tissue vitamin C concentrations in juvenile Korean rockfish, Sebastes schlegeli (Hilgendorf). Aquac Res 1998;29:237e44. [20] Shiau S-Y, Hsu T-S. Quantification of vitamin C requirement for juvenile hybrid tilapia, Oreochromis niloticus Oreochromis aureus, with L-ascorbyl-2-monosulphate-Na and L-ascorbyl-2-monophosphate-Mg. Aquaculture 1999;17:317e26. [21] Woodward W. Dietary vitamin requirements of cultured young fish, with special emphasis on quantitative estimates for salmonids. Aquaculture 1994;124:133e68. [22] Montero D, Marrero M, Izquierdo MS, Robaina L, Vergara JM, Tort L. Effect of vitamin E and C dietary supplementation on some immune parameters of gilthead seabream (Sparus aurata) juveniles subjected to crowding stress. Aquaculture 1999;171:269e78. [23] Sealey WM, Galtin DM. Dietary vitamin C and vitamin E interact to influence growth and tissue composition of juvenile hybrid striped bass (Morone chrysops \ M. saxatilis _) but have limited effects on immune response. J Nutr 2002;132:748e55. [24] Blazer VS. Nutrition and disease resistance in fish. Ann Rev Fish Dis 1992;1:309e23. [25] Li Y, Lovell RT. Elevated levels of dietary ascorbic acid increase the immune responses in channel catfish. J Nutr 1985;198:123e31. [26] Hardie LJ, Fletcher TC, Secombes CJ. The effect of dietary vitamin C on the immune response of Atlantic salmon. 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L.S. Azad et al. Fish& Shellfish Immunology 23(2007)154-163 [29] Wahli T, Verlhac V, Gabaudan J, Schuep W, Meier W. Influence of combined vitamin C and E on non-specific immunity and disease of rainbow trout, Oncorhynchus mykiss(Walbaum). J Fish Dis 1998: 21: 127-37. [30] Anbarasu K, Chandran MR. Effect of ascorbic acid on the immune response of the catfish, Mystus gulio(Hamilton), to different bacterins of Aeromonas hydrophila. Fish Shellfish Immunol 2001: 11355 [31] Chen R, Lochmann R, Goodwin A, Praveen K, Dabrowski K, Lee K-J. Effects of dietary vitamins C and E on alternative complement ty, haematology, tissue composition, vitamin concentrations and response to heat stress in juvenile golden shiner (Notemigonus crysoleucas). Aquaculture 2004: 242: 3-569. [32] Tappel AL. will antioxidant nutrients slow aging processes? Geriatrics 1968: 23: 97-105 [33] Li MH, Wise DJ, Robinson VH. Effect of dietary vitamin C on weight gain, tissue ascorbate concentration, stress response and disease resistance of channel catfish Ictalurus punctatus. J World Aquac Soc 1998: 29: 1-8
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