Nutrient Interactions and Toxicity

Transcription

1 Nutrient Interactions and Toxicity 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 Responses 1,2 Wendy M. Sealey 3 and Delbert M. Gatlin III 4 Department of Wildlife and Fisheries Sciences and Faculty of Nutrition, Texas A&M University System, College Station, TX ABSTRACT Juvenile hybrid striped bass (initially 12.0 g) were fed diets containing deficient, adequate or excessive amounts of vitamin C and/or vitamin E in a factorial arrangement to investigate potential nutritional interaction and effects on immune responses. Nine semipurified diets were supplemented with 0, 25 or 2500 mg vitamin C/kg and 0, 30 or 300 mg vitamin E/kg and fed to fish in triplicate aquaria for 10 wk. Weight gain, feed efficiency, mortality and tissue vitamin levels were significantly (P 0.05) affected by dietary vitamin levels. In addition, a significant interaction between vitamin C and vitamin E was observed. At inclusion levels of 25 and 2500 mg/kg, dietary vitamin C improved feed efficiency and protected fish fed vitamin E deficient diets from growth depression and mortality. At inclusion levels of 30 and 300 mg/kg, vitamin E prevented mortality in fish fed vitamin C deficient diets; however, 300 mg vitamin E/kg was necessary to prevent growth depression in vitamin C deficient fish but was unable to improve feed efficiency. Lysozyme, bacterial killing ability, as well as plasma protein and total immunoglobulin levels of fish were not affected by dietary vitamin levels, whereas respiratory burst activity increased with vitamin E supplementation. Thus, interactions between vitamin C and vitamin E were observed in hybrid striped bass. These interactions may be due to the ability of vitamin C to regenerate vitamin E to its functional form but also suggest an ability of vitamin E to spare vitamin C. J. Nutr. 132: , KEY WORDS: hybrid striped bass vitamin C vitamin E sparing immune responses The hypothesis that vitamin C spares vitamin E by regenerating it from the radical form was first proposed by Tappel (1) and proven by in vitro studies of Packer et al. (2). An alternative explanation of the interaction in vivo between these vitamins suggests that vitamin C may not recycle vitamin E but serve instead to spare it by quenching radicals that would otherwise consume -tocopherol (3). However, the nature of these interactions and the extent to which they actually occur in vivo remain poorly understood and often debated (4). In cultured aquatic species such as the channel catfish (Ictalurus punctatus), the presence of a vitamin C/E sparing mechanism has been suggested as an explanation for the large 1 Presented in part at World Aquaculture 99, 26 April 2 May, 1999, Sydney, Australia [Sealey, W. M. & Gatlin, D. M., III (1999) Interactions between vitamin C and vitamin E in hybrid striped bass (Morone chrysops M. saxatilis). World Aquaculture 99 Book of Abstracts, p. 683]. The work was derived from the Ph.D. dissertation [Sealey,W. M. (2000) Examination of interactions between vitamin C and vitamin E and effects on immune responses and disease resistance of hybrid striped bass, Morone chrysops M. saxatilis. Doctoral thesis, Texas A&M University, College Station. TX. 2 Funded in part by the Texas Agricultural Experiment Station under project H-6556 and the Texas Advanced Technology Program under project Funding for W.M.S. was provided in part by a Tom Slick Fellowship awarded by the Texas A&M University College of Agriculture and Life Sciences. 3 Present address: Department of Animal Ecology, Iowa State University, Ames, IA To whom correspondence should be addressed. d-gatlin@tamu.edu. variability observed in sensitivity to vitamin E deficiency (5). The possibility that a sparing mechanism was responsible was investigated by Gatlin et al. (6) who examined the hypothesis that marginal vitamin C status would increase sensitivity of channel catfish to vitamin E deficiency. In that study, channel catfish fed diets without vitamin C had reduced weight gain and feed efficiency regardless of vitamin E supplementation. Gross signs of vitamin E deficiency were not observed in fish fed the vitamin E deficient diet with supplemental vitamin C, although elevated lipid peroxidation was observed. The authors interpreted these results and those of Wilson et al. (7) and Gatlin et al. (8), in which vitamin E deficient diets supplemented with high or adequate levels of vitamin C still caused elevated lipid peroxidation, as the lack of a vitamin C sparing effect on vitamin E nutrition of channel catfish. Results of research investigating the interactions between vitamin C and vitamin E in cold-water salmonid species (9,10) contrast somewhat with those obtained from studies of the warm-water channel catfish. Frischknecht et al. (9) demonstrated the ability of dietary vitamin C to protect rainbow trout (Oncorhynchus mykiss) from anemia and mortality due to dietary deficiency of vitamin E. More recently, research with Atlantic salmon (Salmo salar) has indicated that vitamin C may protect fish against vitamin E deficiency in a dose-dependent manner (10). Previous experiments in this laboratory established the minimum dietary requirements of juvenile hybrid striped bass /02 $ American Society for Nutritional Sciences. Manuscript received 5 September Initial review completed 30 October Revision accepted 2 January

2 INTERACTIONS OF VITAMINS C AND E IN HYBRID STRIPED BASS 749 (Morone chrysops M. saxatilis) for vitamin E and vitamin C to be 30 and 25 mg/kg, respectively (11,12). Hybrid striped bass have been identified as fish with considerable potential for aquaculture throughout the United States (13) as well as in other countries such as Israel and Taiwan. However, some constraints that limit advancements in the intensive production of these fish remain, including the lack of optimized prepared diets. Interactions between vitamin C and vitamin E have not been addressed in hybrid striped bass and if present, could affect recommended dietary vitamin levels. Therefore, the first phase of the present study was to investigate the presence of these interactions in hybrid striped bass juveniles by feeding diets containing deficient, adequate or excessive amounts of vitamin C and/or vitamin E in a factorial arrangement. Additionally, although some previous research findings indicate that dietary supplementation with immunomodulatory vitamins such as vitamins C and E can improve immune response and disease resistance of a variety of fish species (14 20), other studies have failed to show positive responses with overfortification of such vitamins (21 24). Recently, Ortuno et al. (25) suggested that an intermediate level rather than a megadose level of vitamin E was necessary to enhance nonspecific immune response of gilthead seabream (Sparus auratus) due to imbalances of vitamin E with other antioxidant vitamins, such as vitamin C. Therefore, the purpose of the second phase of this study was to investigate the effects of dietary vitamin C and vitamin E and any potential interactions of these vitamins on nonspecific immunity and antibody responses of hybrid striped bass. MATERIALS AND METHODS Experimental diets and design. A3 3 factorial design was employed to evaluate deficient, requirement and megadose levels of dietary vitamin C and vitamin E and their interaction on growth and immune responses of hybrid striped bass. The basal diet in this experiment was similar to the 40% crude protein diets used by Craig and Gatlin (26), which contained hexane-extracted menhaden fish meal as the protein source to limit intrinsic levels of vitamins (Table 1). This diet was formulated to meet all other known nutrient requirements of hybrid striped bass or the established requirements of other warm-water fishes except for vitamins C and E (27). Dietary lipid consisted of tocopherol-stripped corn oil at 4.4% and menhaden oil at 5.0% of the diet to satisfy the essential fatty acid requirements of hybrid striped bass (28). Nine experimental diets consisted of the basal diet supplemented with vitamin C (as ascorbate polyphosphate) at 0 (deficient), 25 (requirement) or 2500 (megadose) mg/kg and vitamin E (as 2-ambo- -tocopherol) at 0 (deficient), 30 (requirement) or 300 (megadose) mg/kg. Procedures for diet preparation were as previously described (29). The basal diet was analyzed to contain 4.4 mg ascorbic acid/kg and 6.1 mg -tocopherol/kg; supplemental levels were confirmed by analysis to be within 5% of targeted levels. Diets were stored at 18 C before feeding. Experimental fish and feeding trial. Before initiation of the feeding trial, age-0 reciprocal cross hybrid striped bass (Morone chrysops M. saxatilis ) obtained from a commercial producer were subjected to a 2-wk conditioning period for acclimation to standardized regimens and the basal diet in a water-recirculating culture system consisting of 110-L aquaria. Freshwater with hardness of 150 mg/l as CaCO 3 was supplied to each aquarium at a flow rate of 0.5 L/min. Optimal water quality was maintained by biological and mechanical filtration as well as supplemental aeration. Water temperature was maintained at 25 2 C. A diurnal 12-h light:dark cycle was provided by fluorescent lights controlled by timers with lights on at 0700 h. After conditioning, juvenile hybrids (average initial weight 12.0 g) were stocked in the aquaria as groups of 20 fish ( g) and fed one of the nine experimental diets. Each diet was fed Ingredient TABLE 1 Formulation of the basal diet1 g/100 g Menhaden fish meal Dextrin 12.5 Menhaden oil 5.0 Corn oil3 4.4 Mineral premix4 4.0 Vitamin premix5 3.0 Carboxymethyl cellulose 1.0 Cellulose Analyzed to contain 4.4 mg ascorbic acid/kg and 6.1 mg tocopherol/kg. 2 Solvent extracted; provided by Omega Protein, Reedville, VA. 3 -Tocopherol stripped. 4 Contains (as g/kg): Ca(C 6 H 10 O 6 ) 5H 2 O, ; Ca(H 2 PO 4 ) 2 H 2 O, 136.0; FeSO 4 7H 2 O, 5.0; MgSO 4 7H 2 O, 132.0; K 2 HPO 4, 240.0; NaH 2 PO 4 H 2 O, 88.0; NaCl, 45.0; AlCl 3 6H 2 O, 0.15; KI, 0.15; CuSO 4 5H 2 O, 0.5; MnSO 4 H 2 O, 0.7; CoCl 2 6H 2 O, 1.0; ZnSO 4 7H 2 O, 3.0; Na 2 SeO 3, Contains (as g/kg): DL-calcium pantothenate, 5.0; choline bitartrate, 100.0; inositol, 5.0; menadione, 2.0; niacin, 5.0; pyridoxine HCl 1.0; riboflavin, 3.0; thiamine mononitrate, 0.5; retinyl acetate (20,000 IU/g), 5.0; biotin, 0.05; cholecalciferol (1 g 40 IU), 0.002; folic acid, 0.18; vitamin B-12, 0.002; cellulose, to fish in three replicate aquaria for 10 wk. All groups of fish were fed their respective diets to apparent satiation twice daily (morning and evening). Growth and feed efficiency were monitored by weighing each group of fish weekly. At the end of wk 10, fish were sampled (described below) to examine the effects of diet on tissue vitamin levels, oxidative status and nonspecific immune responses. After sampling, eight fish from each of three replicate aquaria per diet were pooled, immunized and redistributed into two replicate aquariums at a density of 12 fish per aquarium and monitored for an additional 4 wk to evaluate the effects of dietary vitamin C and E on antibody production. Procedures used in this study were approved by the Texas A&M University Laboratory Animal Care Committee and ensured humane treatment of the fish. Specific immune function sampling. Fish were immunized with formalin-killed Streptococcus iniae cells/l in PBS by intraperitoneal (IP) injection. Fish were given a booster IP injection of formalin-killed S. iniae cells/l in PBS 3 wk postimmunization (wk 13). Fish continued to be fed their respective diets twice daily to approximate satiation throughout the antibody segment and were weighed and counted biweekly. Serum antibody responses were determined from three fish in each replicate aquarium at immunization (wk 10 of feeding) and at 14 wk. Blood was collected from the caudal vasculature with a nonheparinized, 27-gauge needle. Blood was allowed to clot at room temperature for 2 h and serum was separated by centrifugation at 2000 g. Serum agglutination antibody titers were determined by microtitration agglutination as described by Eldar et al. (30). A positive reaction was described by the presence of a button with fuzzy edges, whereas a negative reaction consisted of a round precipitate with clearly defined circular margins. Antibody titer was expressed as the reciprocal log base 10 of the highest dilution demonstrating a positive reaction as suggested by Klesius et al. (31). Nonspecific immune function sampling. To examine the effects of dietary vitamins C and E on nonspecific immune responses, four fish were randomly sampled from each aquarium (12 fish/dietary treatment) after the 10-wk feeding trial. Fish were anesthetized with tricaine methane sulfonate and 0.5 ml of blood was collected from the caudal vasculature using a heparin-treated syringe and 27-gauge needle. The blood was centrifuged and plasma treated as described previously for serum. Total plasma protein and immunoglobulin were measured using a colorimetric assay (32). After blood collection, fish were killed by pithing, and head kidneys were removed. Kidneys from four fish per aquarium were

3 750 SEALEY AND GATLIN pooled and single cell suspensions were collected according to the methods described by Stave et al. (33). Phagocytic leukocytes were then separated on a Percoll gradient (33) and enumerated as described by Ellsaesser et al. (34). Respiratory burst activity as measured by nitroblue tetrazolium reduction, and ability of phagocytic leukocytes to kill S. iniae were determined in vitro as described by Secombes (35) and Anderson and Siwicki (32), respectively, with minor modifications. Lysozyme levels of fish at the end of the 10 wk were determined in sera from three fish per aquarium, which also were sampled for determination of liver microsomal lipid peroxidation. Fish were bled as previously described and a portion of blood was immediately used for determination of hematocrit by microcentrifugation. The remaining blood was allowed to clot for 1 h before centrifugation; then sera was separated and frozen at 80 C until lysozyme determination. Lysozyme activity was determined by turbidimetric assay as described by Parry et al. (36). A lysozyme activity unit was defined as the amount of enzyme producing a decrease in absorbance of 0.001/min. Nutritional analyses. At the end of 10 wk (feeding trial segment) and 14 wk (antibody segment), three fish from each aquarium were sampled and condition indices including intraperitoneal fat ratio, hepatosomatic index and muscle ratio were determined (28). Blood ( 0.5 ml) was collected from the caudal vasculature using a heparin-treated syringe and 27-gauge needle, and plasma was separated following centrifugation at 3000 g. After blood sampling, liver and head kidney samples were obtained, immediately frozen and stored at 80 C until determination of tissue vitamin levels. All samples were kept separate and analyzed independently. Total ascorbic acid concentrations in liver and plasma after the feeding trial segment and in head kidney and plasma after the antibody segment were determined using the dinitrophenylhydrazine method with correction for interfering substances (37). Concentrations of -tocopherol in these tissues were determined by reverse-phase HPLC (38). The CV for the total ascorbate and -tocopherol assays were 5.3 and 4.0%, respectively. Three additional fish per aquarium were sampled at the end of 10 wk for determination of liver microsomal lipid peroxidation. Thiobarbituric acid reactive substances (TBARS) in liver were determined as described by Draper et al. (39) with modifications in sample homogenization and ascorbate challenge. Briefly, 0.5 g of liver tissue was diluted 1:10 with 33 mmol/l phosphate buffer and homogenized. After homogenization, samples were centrifuged at 20,800 g for 30 min. A 0.25-mL aliquot of supernatant was then added to a 2-mL iron ascorbate solution as described by Cowey et al. (40), consisting of 33 mmol/l phosphate, ph 7.5, 100 mmol/l KCl, mmol/l FeCl 3 and 1.0 mmol/l ascorbic acid. Samples were then incubated at 37 C for 0, 50, or 100 min as suggested by Baker and Davies (41). After ascorbate challenge, samples were processed as described by Draper et al. (39) and TBARS were quantified by determination of malondialdehyde by reverse-phase HPLC. The CV for the TBARS assay was 6.6%. Statistical analysis. All data were analyzed by factorial ANOVA using SAS (42). A significant (P 0.05) main effect and no interaction allowed data to be pooled before mean separation by Duncan s multiple range test. An individual aquarium (three replicates per diet) served as the basic statistical unit in all analyses except for antibody responses. For antibody data, nested ANOVA was conducted to detect possible effects of aquaria within diet treatments. The effect of aquaria was not significant, which allowed all data from fish in both replicate aquaria to be pooled for ANOVA per diet (n 6). Differences were considered significant at P 0.05 for all analyses. Values in the text are means SD. RESULTS Production characteristics in the feeding trial. Weight gain, feed efficiency and survival were significantly affected by dietary vitamin levels and significant interactions between vitamin C and vitamin E also were observed (Table 2). Mortality was first observed in fish fed the basal diet without supplemental vitamin C or vitamin E by wk 6 and survival of fish fed that diet was significantly lower than that of fish fed TABLE 2 Effect of dietary vitamin C and vitamin E on growth, feed efficiency and survival of juvenile hybrid striped bass fed deficient, requirement or megadose levels of vitamin C and/or vitamin E for 10 wk1 Supplemental Vitamin C Vitamin E Weight gain Feed efficiency Survival mg/kg diet % increase g gain/g feed % Pooled SEM ANOVA, Pr F2 Vitamin C Vitamin E Vitamin C Vitamin E Means of three replicate groups. 2 Significance probability associated with the F statistic. supplemented diets by wk 9. Dietary vitamin C at 25 and 2500 mg/kg significantly improved feed efficiency and protected fish fed vitamin E deficient diets from growth depression and mortality. Dietary vitamin E at 30 and 300 mg/kg significantly improved survival and at 300 mg/kg prevented significant growth depression in fish fed vitamin C deficient diets. Body condition indices, including the intraperitoneal fat ratio ( ), relative liver weight ( ) and muscle ratio ( ), were not significantly affected by dietary vitamin level. Tissue vitamin levels after the feeding trial. Tissue total ascorbate and -tocopherol levels increased in response to dietary supplementation of vitamin C or vitamin E, respectively (Table 3). A significant interaction between vitamins C and E was observed for plasma ascorbate level in that fish fed 2500 mg vitamin C/kg and 300 mg vitamin E/kg had significantly lower plasma total ascorbate than fish fed 2500 mg vitamin C/kg and 0 or 30 mg vitamin E/kg. Liver ascorbate levels were increased significantly in fish fed diets with 25 and 2500 mg vitamin C/kg compared with fish fed the vitamin C deficient diets. No significant dietary vitamin E effect on liver ascorbate was observed. Plasma -tocopherol increased significantly in a graded response to incremental dietary vitamin E levels. Liver -tocopherol was significantly lower in fish fed the vitamin E deficient diet than in fish fed diets with 30 and 300 mg vitamin E/kg. In addition, vitamin C at 25 and 2500 mg/kg significantly lowered liver -tocopherol levels without a significant interaction between the vitamins. TBARS after the feeding trial. A significant interaction between vitamin C and vitamin E was observed in the formation of liver TBARS at 0 and 100 min after oxidation (Table 4). Dietary vitamin E at 30 and 300 mg/kg reduced TBARS formation in fish fed 0 and 25 mg vitamin C/kg. Dietary vitamin C at 25 and 2500 mg/kg reduced TBARS formation in fish fed 0 and 30 mg vitamin E/kg. Dietary supplementation with vitamin C or vitamin E had no effect on TBARS forma-

4 INTERACTIONS OF VITAMINS C AND E IN HYBRID STRIPED BASS 751 TABLE 3 Effect of dietary vitamin C and vitamin E on tissue total ascorbic acid and -tocopherol levels of juvenile hybrid striped bass fed deficient, requirement or megadose levels of vitamin C and/or vitamin E for 10 wk1 Supplemental Total ascorbate -tocopherol Vitamin C Vitamin E Plasma Liver Plasma Liver mg/kg diet mol/l nmol/g mol/l nmol/g Pooled SEM ANOVA, Pr F2 Vitamin C ( )3 ( ) Vitamin E ( ) ( ) Vitamin C Vitamin E Means of three individual fish from each of three replicate groups. After initial conditioning, five fish were sampled and mean tissue vitamin levels were as follows: plasma ascorbate 23 mol/l; liver ascorbate 68 nmol/g; plasma -tocopherol 16 mol/l; liver -tocopherol: 21 nmol/g. 2 Significance probability associated with the F statistic. 3 A significant (P 0.05) main effect and no interaction allowed data to be pooled before mean separation by Duncan s multiple range test. Parentheses indicate that significant differences among main effect means for dietary vitamin C (0, 25 or 2500 mg/kg diet) or vitamin E (0, 30 or 300 mg/kg diet) were detected. tion when fish were fed 300 mg vitamin E/kg or 2500 mg vitamin C/kg, respectively. Hematocrits after the feeding trial. A significant dietary effect of vitamin C and a significant interaction between vitamin C and vitamin E was observed for hematocrit (Table 5). Fish fed 0 or 2500 mg vitamin C/kg had significantly lower hematocrits than those fed 25 mg vitamin C/kg. Fish fed diets supplemented with 30 and 300 mg vitamin E/kg had significantly higher hematocrits than fish fed the vitamin E deficient diet, when vitamin C was supplemented to the diet at 0 or 25 mg/kg. Fish fed vitamin E deficient diets supplemented with 25 and 2500 mg vitamin C/kg had hematocrits that were comparable to fish fed diets with the requirement level of both vitamins. Nonspecific immune responses. Serum lysozyme levels were highly variable, with no significant effects of dietary vitamin levels or interactions observed (Table 5). Plasma protein was not significantly affected by dietary vitamin level and no significant interaction between vitamins C and E was observed (Table 5). Total plasma immunoglobulin was similarly unaffected by dietary vitamin levels (Table 5). Dietary vitamin E at 300 mg/kg significantly increased the oxidative burst of phagocytic head kidney cells isolated from fish fed 25 or 2500 mg vitamin C/kg (Fig. 1). Killing of S. iniae by phagocytic head kidney cells was low and highly variable. Bacterial killing ranged from 12.4% for head kidney cells from fish fed 2500 mg vitamin C/kg and 300 mg vitamin E/kg to 45.5% for fish fed 0 mg vitamin C/kg and 30 mg vitamin E/kg with no significant effect of diet (data not shown). Production characteristics in antibody trial. Significant effects of diet on weight gain, feed efficiency and survival that were observed at the end of 10 wk were further intensified after the additional 4 wk of feeding during the antibody segment of the study (data not shown). TABLE 4 Effect of dietary vitamin C and vitamin E on liver thiobarbituric acid reactive substances (TBARS) of juvenile hybrid striped bass fed deficient, requirement or megadose levels of vitamin C and/or vitamin E for 10 wk1 Supplemental Time, min2 Vitamin C Vitamin E mg/kg diet nmol/g Pooled SEM ANOVA, Pr F3 Vitamin C ( )4 Vitamin E ( ) Vitamin C Vitamin E Means of three individual fish from each of three replicate groups. 2 Liver TBARS were determined after 0, 50 and 100 min incubation in iron ascorbate solution. 3 Significance probability associated with the F statistic. 4 A significant (P 0.05) main effect and no interaction allowed data to be pooled before mean separation by Duncan s multiple range test. Parentheses indicate that significant differences among main effect means for dietary vitamin C (0, 25 or 2500 mg/kg diet) or vitamin E (0, 30 or 300 mg/kg diet) were detected.

5 752 SEALEY AND GATLIN TABLE 5 Hematocrit, serum lysozyme, plasma protein and total immunoglobulin levels of juvenile hybrid striped bass fed deficient, requirement or megadose levels of vitamin C and/or vitamin E for 10 wk Supplemental Vitamin C Vitamin E Hematocrit1 Lysozyme1 Plasma protein2 Total immunoglobulin2 mg/kg diet 103 units/l g/l g/l Pooled SEM ANOVA, Pr F3 Vitamin C Vitamin E Vitamin C Vitamin E Means of three individual fish from each of three replicate groups. 2 Means of four individual fish from each of three replicate groups. 3 Significance probability associated with the F statistic. Tissue vitamin levels in antibody trial. Tissue total ascorbate and -tocopherol levels at 14 wk (end of the antibody trial) significantly increased in a graded response to dietary supplementation of vitamin C or vitamin E, respectively (Table 6). A significant interaction between vitamins C and E was observed for plasma ascorbate level in that fish supplemented with vitamin E at 30 or 300 mg/kg had higher levels of plasma ascorbate than those fed diets deficient in vitamin E. No significant dietary effect of vitamin E on kidney ascorbate was observed. FIGURE 1 Intracellular O 2 production of hybrid striped bass head kidney cells from fish fed various levels of vitamin C and/or vitamin E as measured by nitroblue tetrazolium (NBT) reduction. Data are expressed as means SD (n 4 replicate wells for each composite sample obtained from four fish in each of three replicate groups). Superscripts in each column group indicate significant (P 0.05) effects of dietary vitamin E. Agglutinating antibody titers. No detectable levels of antibody were observed at wk 10 before immunization. At the conclusion of the antibody trial (wk 14), antibodies were detected in fish from all treatments but no significant effect of diet was observed (Table 6). DISCUSSION Previous experiments in this laboratory have established the minimum dietary requirements of hybrid striped bass for vitamins E and C to be 30 and 25 mg/kg, respectively, based on growth and survival (11,12). Data from the present study support these values in that dietary supplementation with additional levels of either vitamin above the minimum requirement resulted in no significant improvements in weight gain, feed efficiency or survival when the other vitamin was provided at the previously determined requirement level. This was not the case, however, when either vitamin was lacking in the diet. Dietary vitamin C at 25 and 2500 mg/kg significantly improved feed efficiency and protected hybrid striped bass fed vitamin E deficient diets from growth depression and mortality. These results are in agreement with those of Frischknecht et al. (9) who found that dietary vitamin C protected rainbow trout from anemia and mortality associated with a dietary deficiency of vitamin E. Hamre et al. (10) also observed similar responses in Atlantic salmon. In addition, dietary vitamin E at 30 and 300 mg/kg significantly improved survival of hybrid striped bass and at 300 mg/kg prevented significant growth depression in fish fed vitamin C deficient diets. These data contrast with those of Gatlin et al. (6) who found that channel catfish fed diets without vitamin C had reduced weight and feed efficiency regardless of vitamin E supplementation. Interestingly, mortality of hybrid striped bass fed the diet without vitamin C but supplemented with 30 mg vitamin E/kg was not observed until wk 8 and may indicate an ability for vitamin E to delay onset of mortality due to vitamin C deficiency. Hamre et al. (10) fed Atlantic salmon vitamin C deficient diets supplemented with 300 mg vitamin E/kg and found an

6 INTERACTIONS OF VITAMINS C AND E IN HYBRID STRIPED BASS 753 TABLE 6 Tissue total ascorbate and -tocopherol concentrations, and agglutinating antibody titers of juvenile hybrid striped bass fed deficient, requirement or megadose levels of vitamin C and/or vitamin E at the conclusion of the antibody segment of the experiment (14 wk)1,2 Supplemental Total ascorbate -Tocopherol Vitamin C Vitamin E Plasma Kidney Plasma Kidney Agglutinating antibody titers mg/kg diet mol/l nmol/g mol/l nmol/g log base NT3 NT NT NT NT NT NT NT Pooled SEM ANOVA, Pr F4 Vitamin C ( )5 Vitamin E ( ) Vitamin C Vitamin E Means of three individual fish from each of two replicate groups. 2 Individual fish within a dietary treatment were used to calculate means after nested ANOVA (n 6). 3 NT, not tested due to lack of sufficient sample. 4 Significance probability associated with the F statistic. 5 A significant (P 0.05) main effect and no interaction allowed data to be pooled prior to mean separation by Duncan s multiple range test. Parentheses indicate that significant differences among main effect means for dietary vitamin C (0, 25 or 2500 mg/kg diet) or vitamin E (0, 30 or 300 mg/kg diet) were detected. earlier onset of vitamin C deficiency, which was attributed to prooxidant effects of high -tocopherol levels when tissue vitamin C was marginal. Hamre et al. (10) also observed decreased levels of liver ascorbate in fish fed a diet with 300 mg vitamin E/kg compared with those fed a diet with 150 mg vitamin E/kg. In the current study, plasma total ascorbate data at the end of the 10-wk feeding trial lend additional support for a prooxidant effect of high dietary levels of vitamin E. A significant interaction between vitamins C and E was observed for plasma total ascorbate in that fish fed 2500 mg vitamin C/kg and 300 mg vitamin E/kg had lower plasma ascorbate than fish fed 2500 mg vitamin C/kg and 30 mg vitamin E/kg. However, after an additional 4 wk of feeding during the antibody segment of the study, increased plasma ascorbate was observed in fish fed diets with vitamin E supplemented at 30 or 300 mg/kg. These contrasting results may indicate a difference in the effect of length of feeding on tissue oxidative status or possibly effects of stress because fish in the antibody segment were responding to an antigentic stimulus and were subjected to repeated sampling. Tissue -tocopherol levels of hybrid striped bass also indicated significant interactions between dietary vitamins C and E. After 10 wk of feeding, plasma -tocopherol increased with dietary vitamin E supplementation and there was a tendency (P ) for plasma -tocopherol to increase with vitamin C supplementation. Chen et al. (43) observed that rats given supplemental vitamin C had higher plasma -tocopherol concentrations than those ingesting the same diet without supplemental vitamin C. In inherently scorbutic rats, Tanaka et al. (44) observed that vitamin C deficiency decreased plasma -tocopherol. Liver -tocopherol similarly increased with dietary vitamin E supplementation. However, in contrast to plasma -tocopherol data, vitamin C at 25 and 2500 mg/kg significantly lowered liver -tocopherol levels without a significant interaction between the vitamins. These data contrast with those of Bendich et al. (45) who found that guinea pigs fed high levels of vitamin C had increased vitamin E in lung tissue. In contrast, Chen (46) found that excessive vitamin C intake lowered tissue antioxidant potential with marginal vitamin E intake, suggesting an increased vitamin E requirement with high vitamin C intake. However, some tissue vitamin data from salmonids do not support that hypothesis. White et al. (47) found that dietary vitamin C levels between 50 and 2750 mg/kg had no effect on liver -tocopherol levels in Atlantic salmon. Liver peroxidation and hematocrit data from the present study further support the presence of significant interactions between vitamin C and vitamin E in juvenile hybrid striped bass. Similar to the growth data, dietary vitamin E at 30 and 300 mg/kg reduced formation of TBARS in fish fed 0 and 25 mg vitamin C/kg, and dietary vitamin C at 25 and 2500 mg/kg reduced formation of TBARS in fish fed 0 and 30 mg vitamin E/kg. Additionally, while no prooxidant effects of dietary vitamin C or vitamin E at their highest level were observed for formation of TBARS, dietary vitamin C at 2500 mg/kg did decrease hematocrits, possibly indicating cellular damage and suggesting a need to carefully consider megadose supplementation of vitamin C. In contrast to growth, tissue vitamin and oxidation data, immune responses of hybrid striped bass were only slightly affected by dietary supplementation of either vitamin C or vitamin E or both. In channel catfish, megadose levels of vitamin C added to the diet have been shown to improve antibody response, complement activity and survival after

7 754 SEALEY AND GATLIN infection with Edwardsiella tarda and E. ictaluri (17,48). Similar improvements in immune responses of rainbow trout after dietary supplementation with vitamin C (16,20) or vitamin E (15,16,49) also have been observed. However, other studies with fish have not shown positive responses due to overfortification of vitamin C (23,24) or E (21). Lysozyme, plasma protein, total immunoglobulin levels, bacterial killing ability as well as antibody response of hybrid striped bass in the current study were not significantly affected by dietary vitamin level, whereas respiratory burst activity did respond slightly. In summary, significant interactions between vitamin C and vitamin E were observed for growth, feed efficiency, survival, tissue vitamin levels and oxidation status in the present study. Taken together, these results clearly indicate the presence of an in vivo vitamin C sparing of vitamin E as well as a vitamin E sparing of vitamin C. The mechanism(s) behind these interactions remain unclear but lend support to the theory of sparing through the quenching of radicals by one vitamin that would otherwise consume the other antioxidant vitamin. This contrasts with the in vitro explanation of vitamin E radical regeneration by vitamin C due to the fact that both vitamins demonstrated sparing ability. The presence of sparing mechanisms noted in the current studies also may explain some of the previously observed discrepancies regarding the immunostimulatory effects of these vitamins because very limited research has addressed both vitamins simultaneously or made any attempts to control for interactions between them. Future studies should therefore be designed with the knowledge that vitamin C and E interactions do exist and adjustments in experimental design should be made accordingly to limit interactions. Additionally, reevaluation of results from previous studies that did not control for these interactions may be warranted. To fully address the question of nutritional immunostimulation by vitamins C and E, additional study using standardized protocols that closely mimic the natural environment is warranted to address questions of fish and pathogen species variability, vitamin dosages necessary to elicit protection, and required duration of treatments. ACKNOWLEDGMENTS The authors thank Mike Freeze (Keo Fish Farms, Keo, AR) for providing the fish for this study. We also wish to acknowledge Omega Protein Corporation for the kind donations of the menhaden fish meal (Houma, LA) and the menhaden fish oil (Reedville, VA). The authors also wish to acknowledge Stewart Anderson at Hoffmann-La Roche (Nutley, NJ) for donation of the Rovimix Stay-C. LITERATURE CITED 1. Tappel, A. L. (1962) Vitamin E as the biological lipid antioxidant. Vitam. Horm. 20: Packer, J. E., Slater, T. F., & Willson, R. 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