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FSN Research Center, University of Rhode Island, West Kingston, RI 02892;
* School of Agriculture, Fisheries, and Human Sciences, University of Arkansas at Pine Bluff, Pine Bluff, AR 71601;
College of Veterinary Medicine, University of Georgia, Athens, GA 30602;
** School of Natural Resources, The Ohio State University, Columbus, OH 43210; and
School of Applied Marine Science, Cheju National University, Jeju, Korea
2To whom correspondence should be addressed. E-mail: rlochmann{at}uaex.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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10 times the amount of AA required to prevent deficiency signs.
KEY WORDS: • ascorbic acid • vitamin deficiency • golden shiner • alternative complement • heat stress
The golden shiner (Notemigonus crysoleucas) is a small cyprinid fish cultured primarily as bait for recreational fishing in the United States. Commercial diets used to culture this fish are based on nutrient requirements for channel catfish (1), which are similar to those that are established for golden shiners (2,3). However, the micronutrient requirements of golden shiners have not been established. Golden shiners in ponds fed practical diets with or without a vitamin and mineral supplement had similar weight gain and net yield (4). Golden shiners fed semipurified or practical diets in aquariums required a dietary source of ascorbic acid (AA) for optimal growth or survival, depending on diet composition (5). However, no quantitative requirements of golden shiners for AA for growth or survival have been determined, and no definitive deficiency signs have been observed in fish fed AA-free diets for 12 wk. Ascorbic acid is an essential nutrient for normal growth and reproduction in teleosts (6), and it enhances immune function during stress and pathogen invasion (7–9). Numerous factors such as species, age, environmental conditions, stress levels, and initial tissue levels of AA affect the quantitative dietary requirement for the vitamin (10). Signs of vitamin C deficiency have been well characterized in warm water omnivorous fish including channel catfish (11–13), common carp (14), Indian major carp (15), Nile tilapia (16), blue tilapia (17) and Mexican native cichlid Cichlasoma urophthalmus (18). The signs of vitamin C deficiency described in these warm water fish include structural deformities in vertebrae, fins, gill opercula and support cartilage, and blood vessels. Hematological abnormalities in channel catfish fed diets without vitamin C have been reported (19). Deficient fish had reduced hematocrit and erythrocyte and leukocyte counts and had abnormally large erythrocytes and spectacle cells. Fish fed a diet with a marginal level of AA (20 mg/kg) also had greater numbers of other abnormalities such as binuclear erythrocytes and condensation of the nucleus.
The AA requirement for immunostimulation in fish is much higher than the level required for prevention of deficiency signs. Channel catfish fed a diet with 0 mg of AA/kg had significantly lower levels of serum hemolytic activity on sensitized sheep RBC than those fed AA-supplemented diets (7). Catfish fed diets with megadoses of AA (3000 mg/kg) had almost twice the hemolytic activity of fish fed diets with 30 or 300 mg of AA/kg. Hardie et al. (8) and Verlhac et al. (20) also showed that complement activity was compromised by AA deficiency, and that high AA intake increased complement levels in rainbow trout and Atlantic salmon.
Stress generally increases the AA requirement of fishes (21,22), and increased oxidative stress in livers of fishes exposed to high temperatures has been documented in several species. Lipid peroxidation in liver of carp exposed to a temperature of 35°C was lower in those fed diets with a vitamin C supplement than in those fed an unsupplemented diet (23). Similarly, lipid peroxidation in liver of thornfish (Terapon jarbua) fed a diet without vitamin C was higher at 36°C than that of fish fed a diet with vitamin C (24). The quantitative dietary vitamin C requirement of thornfish increased from 80 to 400 mg/kg diet with an increase in water temperature from 32 to 36°C (25). Parihar et al. (26) also reported increased oxidative stress and depletion of AA in livers of freshwater catfish (Heteroneustes fossilis) as temperature increased from 25 to 37°C.
The dietary requirement for ascorbic acid for growth and other functions decreases with age in fishes as the rate of metabolism and tissue synthesis slows and the capacity for tissue storage of AA increases (27). However, differences in experimental conditions among studies contribute to discrepancies in estimates of dietary AA requirements within and between species. Therefore, the dietary AA requirement must be determined for a specific set of experimental conditions using a defined set of criteria (6).
Ascorbic acid deficiencies are relatively common in intensively cultured fish due to the vitamin’s instability during processing and storage, and its rapid depletion during stress (6). Golden shiners are subjected to frequent stress from capture, handling, and transportation during production and marketing processes (28), and elevated dietary levels of ascorbic acid might be beneficial.
The purpose of this study was to determine the effects of different dietary levels of ascorbic acid on the growth and health of golden shiners in aquariums. The health of the fish was assessed by examination for gross deformities, hematology, determination of alternative complement activity and survival of fish that were challenged by exposure to elevated temperature.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The formula and analyzed proximate composition of the basal diet are shown in Table 1. The diets were formulated to contain 13.4 kJ of digestible energy/g diet, on the basis of previous work with golden shiners (2). Vitamin-free casein (ICN Biomedicals, Aurora, OH) was the main protein source. The gelatin, dextrin, cellulose and carboxymethylcellulose were extracted for 1 h with boiling ethanol to minimize their vitamin content before inclusion in diets (29). Diet ingredients were mixed and pelleted following routine procedures (30) and stored at -18°C until needed. The diets were formulated to contain 0, 25, 50, 100 or 250 mg of ascorbic acid equivalent/kg dry diet, supplemented as L-ascorbyl-2-monophosphate (Stay C-35, F. Hoffmann-La Roche, Basel, Switzerland). The AA content was analyzed by Hoffmann-La Roche, and the proximate composition of the diets was determined at the University of Arkansas at Pine Bluff (UAPB), Pine Bluff, AR (Table 1).
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Juvenile golden shiners produced on a commercial fish farm (Anderson’s Minnow Farm, Lonoke, AR) were fed a vitamin C-free diet for 2 wk while acclimating to experimental conditions at UAPB. Pretrial fish (n = 50) were frozen at -70°C for subsequent analysis of proximate composition and ascorbic acid content. The fish had no clinical signs of diseases before stocking. Uniform golden shiners (n = 30) were randomly selected and stocked into each of three 110-L aquariums, which in turn were assigned to each treatment. Individual initial weight of golden shiners was 1.04 ± 0.002 g (mean ± SEM). Aquariums were configured in a flow-through system using dechlorinated municipal water with a hardness of 10–30 mg/L as calcium carbonate. Water quality was maintained by continuous aeration and a flow rate of 1 L/min per aquarium. Dissolved oxygen and ammonia levels were maintained at acceptable levels for golden shiners (28), and water temperature was 25 ± 2°C (mean ± SD). A diurnal 12-h light:dark cycle was maintained during the feeding trial. Fish were fed to apparent satiation, which was 4–5% of their body weight daily, divided into two equal feedings (0900 and 1600 h). Fish from each tank were weighed collectively after stocking to monitor growth and adjust feed rations. Mortality, general health, and specific vitamin C deficiency signs were recorded. During the feeding trial, fish were counted and weighed by tank every 2 wk for the first 10 wk. Mean individual weight gain, the feed conversion efficiency and the protein efficiency ratio (wet weight gain/dry weight of protein intake) were calculated at 10 wk. Fish were returned to their tanks and fed their respective diets for another 6 wk. During this time, fish were not weighed to minimize the influence of handling stress on results of the alternative complement assay and hematological tests. At 16 wk, 7–10 fish/tank (depending on the number of live fish remaining in each tank) were collected and used for hematology and the alternative complement assays. One tank (from the AA-free treatment) contained no additional live fish, but six fish from each of the other 14 tanks were used for a high temperature stress test. Due to the limited number of fish available in some tanks at the end of the study, fish bled for hematological and hemolytic tests were also frozen at -70°C until analyzed for protein [Kjeldahl method; (31)], total lipid (32), dry matter, ash and ascorbic acid concentration. All procedures for handling fish complied with the guidelines specified by the Institutional Animal Care and Use Committee at UAPB.
Observation of fish skeletal components.
After 10 wk, 8 fish from the vitamin C-free treatment and two from vitamin C-supplemented treatments were rendered transparent and the bones were stained by alizarin red according to a modified method described by Cailliet et al. (33) to observe skeletal features.
Blood sample collection and analysis.
After 16 wk, golden shiners were not fed for 24 h, then anesthetized with tricaine methanesulfonate (MS-222). The caudal peduncle was severed with a scalpel and blood was collected using heparinized microhematocrit tubes. Analyses of hematocrit (34), hemoglobin (Hb) content [Hb cyanide method (34)], and differential count were performed on 5–10 fish/tank. The hematological variables were analyzed using blood from individual fish so that mean corpuscular Hb concentration (MCHC) could be calculated on an individual basis. The MCHC was calculated by the formula: MCHC = Hb concentration/hematocrit fraction. After centrifugation (3500 x g for 10 min) of each blood sample, 25 µL of fish serum was collected for a hemolytic assay of alternative complement activity. When serum volume from some fish was insufficient, fish samples from the same tank were pooled to obtain 25 µL of serum for the assay.
Hemolytic assay (alternative complement pathway).
Hemolytic activity driven by the alternative complement pathway was measured using rabbit RBC in EGTA, magnesium, gelatin buffer (GVB) as described by Tort et al. (35). Golden shiner serum (25 µL) was diluted in 175 µL of GVB and serial doubling dilutions made down a 96-well plate. The optical density of the diluted serum solution was measured at 414 nm by a Kinetic microplate reader (Molecular Devices, Sunnyvate, CA). After reading, 25 µL of rabbit RBC washed in GVB was added to each well. The plate was incubated at 20°C for 90 min with manual shaking. After incubation, each sample was transferred to a 48-well plate with 1 mL of cold 20 mmol/L EDTA-GVB buffer to stop the hemolytic reaction. The 48-well plates were centrifuged at 600 x g for 5 min. The upper supernatant (200 µL) from each well was transferred to a new 96-well plate. The extent of hemolysis was determined by measuring the optical density of the supernatant at 414 nm. Complete (100%) and no (0%) hemolysis were determined by adding 25 µL of the washed rabbit RBC suspension to 100 µL of distilled water, and 25 µL of the washed rabbit RBC suspension to 100 µL of GVB buffer, respectively. The alternative complement pathway hemolytic activity (ACH) was reported as the reciprocal of the serum dilution causing 50% lysis of rabbit RBC (ACH50) (36). Mathematica for Students (Wolfram Research, Champaign, IL) was used to predict ACH50 (kU/L of fish serum) based on the value of Y/1 - Y against the reciprocal of serum dilutions. The value of Y was defined as the ratio of adjusted serum hemolysis to complete hemolysis.
Relative differential count of blood leukocytes.
Smears were prepared from heparinized blood and stained (Hema 3 Stain, Biochemical Sciences, St. Louis, MO). Visual observation for the differential blood count was done using a X100 oil immersion objective on a compound microscope, and the different cells (lymphocytes, thrombocytes, neutrophils, monocytes and eosinophils) were identified according to Grizzle and Rogers (37). The differentiated cell types were counted and calculated as the number of cells of a specific type (e.g., lymphocyte, thrombocyte) per 100 total leukocytes counted (n = 200 leukocytes).
Ascorbic acid analysis.
After fish were bled for hematological and hemolytic assays, viscera from the same fish (4 per tank) were analyzed for total ascorbic acid (TAA) and dehydroascorbic acid (DHAA) using the dinitrophenylhydrazine (DNPH) spectrophotometric method described by Terada et al. (38) and modified by Dabrowski and Hinterleitner (39). Modifications included the background level determination of interfering substances that form color complexes with DNPH.
Stress test: exposure to high water temperature.
The live fish reserved for this test were maintained in a recirculating system and fed the experimental diets to satiation from wk 10–16 after final weights were obtained in the feeding trial. At the end of wk 15, the water temperature was 17°C. Fish were gradually acclimated to 26°C for an additional week with all fish surviving. Then temperature was raised gradually (1–2°C/d) to 34°C when the first deaths were observed. This was considered the beginning of the stress test. The temperature increased to 35.3 ± 0.3°C where it remained for the rest of the stress test. Mortalities (number of deaths per unit time, and cumulative deaths) were recorded in each tank for 96 h. The exposure temperatures in this stress test were comparable to temperatures observed in the late summer in ponds in Arkansas where most golden shiners are cultured (28).
Statistical analysis.
Each of the five dietary treatments was assigned to three aquariums in a completely randomized design. Weight gain, survival (postfeeding trial), TAA, DHAA, ratio of reduced AA to TAA, hematocrit, leukocyte percentage, Hb, MCHC and ACH50 level were tested by ANOVA. Mortality during the heat stress test was analyzed with a repeated-measures ANOVA. All statistical analyses were conducted using a StatView program (SAS Institute, Cary, NC) (40). When significant differences among treatment means were found (P ≤ 0.05), treatment means were compared using Fisher’s least significant difference test (41).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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There were no differences in the mean individual weight gain, survival, feed conversion efficiency and protein efficiency ratio of golden shiners fed diets with 0, 19.5, 40.3, 81.2 or 218.5 mg AA/kg at 10 wk.
Vitamin C deficiency signs.
Fish fed the AA-free diet began to show gross signs of vitamin C deficiency after 9 wk (excluding the 2-wk conditioning period). Externally visible signs included scoliosis, lordosis and exophthalmia. Skin hemorrhaging on the abdomen, dorsum, fins, head and eye rim were also evident in some fish. The extent of the damage to bones in the spine, ribs, fin rays and cranium was especially evident in transparent fish.
Hematology.
At 16 wk there were no differences in hematocrit, Hb concentration or MCHC among the groups. Lymphocyte percentage increased from 38.3 to 50.2% with increasing AA levels from 19.5 to 218.5 mg/kg (Table 2). Fish fed the AA-free diet or the diet with 218.5 mg of AA/kg had a significantly higher percentage of lymphocytes than those fed the diet with 19.5 mg of AA/kg (Table 2). The percentage of thrombocytes and other leukocytes (dominated by neutrophils) were not affected by dietary AA concentration (Table 2).
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The ACH50 differed significantly between fish fed the diet with the highest ascorbic acid concentration (218.5 mg/kg) and those fed diets with less AA (Table 3).
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The TAA, DHAA and ratio of reduced ascorbic acid (RAA) to TAA in viscera of fish fed the diet with 218.5 mg of AA/kg were significantly higher than those of fish fed diets with less AA (Table 4). The DHAA was also higher in viscera of fish fed the diet with 81.2 mg of AA/kg compared with fish fed diets with less AA. There were moderately high concentrations of TAA and DHAA in initial fish, even after 2 wk of consuming the AA-free conditioning diet. There were no differences in crude protein, total lipid, dry matter and ash content of golden shiners fed diets with different levels of ascorbic acid for 16 wk.
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The mean cumulative mortality of golden shiners fed diets with 0 or 19.5 mg of AA/kg diet was higher than that of fish fed diets with 40.3 or 81.3 mg of AA/kg after exposure to high water temperatures (34–35.5°C) (Fig. 1). The rate of mortality was also higher in fish fed diets with 0 mg of AA/kg diet than in those fed supplemented diets.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Mean survival of golden shiners during the feeding trial was not affected by dietary AA at 10 wk, but variability in survival of fish fed the AA-free diet was high compared with those fed the AA-supplemented diets. High mortality occurred in the vitamin C-deficient fish with the development of advanced deficiency signs. The most obvious signs of vitamin-C deficiency in golden shiners were structural deformities in the vertebral elements, similar to those described for other warm water omnivorous fish (11–18). No more than 19.5 mg of AA/kg of diet was required for normal growth, survival, and prevention of AA-deficiency signs in golden shiners. However, body weight increased only 2.5-fold during the 10-wk trial. The AA stored initially in the bodies of golden shiners appeared to be depleted slowly because growth was not depressed and deficiency signs took ≥9 wk to appear (excluding the 2-wk conditioning period).
Hematological abnormalities did not appear in golden shiners fed the AA-free diet for 16 wk. However, the percentage of lymphocytes was higher in fish fed 40.3–218.5 mg AA/kg than in fish fed a diet with 19.5 mg AA/kg. The percentage of lymphocytes in fish fed the AA-free diet was not different from that of fish fed ≥40.3 mg AA. This apparent discrepancy is probably due to the lower number of live fish left for sampling in the AA-free treatment at 16 wk. Possibly, fish with fewer lymphocytes died before 16 wk. Elevated levels of vitamin C can stimulate lymphocyte activity and increase survival of lymphocytes by protecting their cell membranes and extending their life span (44–46). Therefore, supplementation of the diet with high levels of AA might improve the ability of golden shiners to fight infectious diseases.
The alternative complement pathway is an important humoral defense mechanism especially against gram-negative bacteria due to its functions in the absence of specific antibodies (35). The alternative complement activity increased in golden shiners only at the highest dietary AA level tested (218.5 mg/kg). Like other fish, the AA requirement for immunostimulation in golden shiners is much higher than the level required for prevention of deficiency signs. The mechanism for the changes in hemolytic activity is not known. Li and Lovell (7) speculated that AA affects the collagen-like regions of the C1 complex of complement through its role in proline hydroxylation.
The visceral tissues of initial fish contained 10 µg of TAA/g after they had consumed an AA-free conditioning diet for 2 wk. The total ascorbic acid concentration was comprised mainly of DHAA. At 16 wk, the TAA and reduced AA concentration increased in viscera of golden shiners with increasing dietary AA in a dose-dependent manner. The significant increase in the reduced AA (RAA/TAA, Table 4) indicated considerably improved tissue antioxidant properties. However, viscera were not saturated with AA within the range of dietary levels tested, as indicated by the lack of a plateau. Maximum tissue storage of AA is a useful response criterion for determining dietary AA requirements, but the response is dependent on growth rate (47–50). Golden shiners in this study showed modest growth and additional data are needed to determine the dietary AA concentration required for tissue saturation.
The lethal temperature is near 40°C for golden shiners (51). In this study, the high mortality of AA-deficient or marginally AA-deficient golden shiners occurred within 96 h of exposure to sublethal water temperatures of 34–35.5°C. Fish exposed to high temperatures have an increased AA requirement due to increased oxidative stress in the liver (23–26). Lipid peroxidation and enzyme activities were not measured in golden shiners exposed to heat stress in this study, but it is likely that similar mechanisms explained the higher mortality of heat-exposed golden shiners fed diets with <40 mg ascorbic acid/kg diet. This result has practical importance for baitfish culture because the water temperature in ponds in Arkansas commonly reaches 36°C in the summer.
In summary, a dietary AA level of 19.5 mg/kg was sufficient to prevent deficiency signs and optimize survival of golden shiners. No dietary AA requirement for growth was demonstrated, possibly due to the small increase in body weight during the experiment and the high level of AA present in tissues at the initiation of the study. The dietary AA requirement of golden shiners for maximal survival upon exposure to heat stress increased to 40.3 mg/kg. The highest quantitative AA requirements (≥218.5 mg/kg) were demonstrated for elevation of alternative complement activity, i.e., improved disease resistance, and highest concentration of AA in visceral tissues.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: AA, ascorbic acid; ACH50, alternative complement pathway hemolytic activity; DHAA, dehydroascorbic acid; DNPH, dinitrophenylhydrazine; GVB, gelatin buffer; Hb, hemoglobin; MCHC, mean corpuscular hemoglobin concentration; RAA, reduced ascorbic acid; TAA, total ascorbic acid; UAPB, University of Arkansas at Pine Bluff.
Manuscript received 6 February 2003. Initial review completed 19 February 2003. Revision accepted 6 May 2003.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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