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Dietary copper requirement of fingerling channel catfish
Aquaculture, 54 (1986) 277-285 Elsevier Science Publishers B.V., Amsterdam
277 - Printed
in The Netherlands
DIETARY COPPER REQUIREMENT OF FINGERLING CHANNEL CATFISH
DELBERT
M. GATLIN,
III’ and ROBERT
Department of Biochemistry, MS 39762 (U.S.A.)
P. WILSON2
Mississippi State University, Mississippi State,
‘Present address: University of Arkansas at Pine Bluff, Agricultural Station, Pine Bluff, AR 71601 (U.S.A.) ‘To whom requests for reprints should be addressed. (Accepted
6 February
Experiment
1986)
ABSTRACT Gatlin, D.M., III and Wilson, R.P., 1986. nel catfish. Aquaculture, 54: 277-285.
Dietary
copper
requirement
of fingerling
chan-
Purified egg white diets containing incremental levels of copper (as CuSO;B KO) were fed to fingerling channel catfish (Ictalurus punctatus) to determine their dietary copper (Cu) requirement. Catfish in aquaria were fed diets containing 0, 2,4, 6,8 or 10 mg of supplemental copper/kg diet for 13 weeks. Growth and feed efficiency data, as well as hemoglobin, hematocrit and erythrocyte count values were similar for catfish fed the basal diet which contained 0.89 mg Cu/kg diet and all copper-supplemented diets. A diet containing 40 mg of supplemental copper/kg was also fed in this experiment since 16 and 32 mg of supplemental copper/kg had previously been reported to cause suppressed growth of channel catfish. Those fed the 40 mg of supplemental copper/ kg diet had similar growth and feed efficiency values as catfish fed the other diets. At the end of week 13, heart cytochrome c oxidase and liver copper-zinc superoxide dismutase activities were significantly reduced in catfish fed diets containing 0 and 2 mg of supplemental copper/kg as compared to those fed 4 mg/kg or more of supplemental copper. Based on the enzymatic data, the minimum dietary copper requirement of channel catfish was determined to be approximately 5 mg of total copper/kg diet.
INTRODUCTION
The effects of varying levels of dietary copper intake on several fish species have been studied (Ogino and Yang, 1980; Murai et al., 1981; Knox et al., 1982, 1984; Satoh et al., 1983a, b, c). A basal diet containing 0.7 mg Cu/kg diet was reported to suppress growth in carp (Cyprinus carpio) as compared to a control diet containing 3 mg Cu/kg diet (Ogino and Yang, 1980). A differential growth response was not observed in rainbow trout (Salmo gairdneri) fed these same two diets (Ogino and Yang, 1980). In two other studies, similar growth rates and feed conversions were observed
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for rainbow trout fed diets containing 15 and 150 mg of supplemental copper/kg (Knox et al., 1982) and 2.5 and 500 mg of total copper/kg (Knox et al., 1984). In contrast with these studies, suppressed growth and feed efficiency were reported for channel catfish (Ictalurus punctatus) fed diets with 16 and 32 mg of supplemental copper/kg (Murai et al., 1981). The basal diet fed in that study contained 1.5 mg Cu/kg diet and did not produce any observable deficiency signs after 16 weeks. The present study was conducted to characterize copper deficiency in channel catfish and to determine their minimum dietary copper requirement. A diet containing 40 mg of supplemental copper/kg was also fed to reevaluate the toxicity of this level of dietary copper to channel catfish. MATERIALS
AND METHODS
Experimental diets and design The composition of the basal diet is listed in Table 1. Diets were formulated to contain 30% crude protein from egg white and 344 kcal/lOO g diet (Garling and Wilson, 1976). Biotin was included in these diets at 9.3 mg/kg to negate the effect of avidin in egg white. All dietary ingredients were commercially obtained. Minerals used in the premixes and diets were of reagent grade. Experimental diets were prepared by adding incremental levels of a 500 mg/kg copper premix (CuSO,*5 H,O and cellulose) to the basal diet while TABLE 1 Basal diet fed in the copper study Ingredient
% Dry weight
Egg white Dextrin Cellulose’ Corn oil Cod liver oil ADEK mix’ Copper-free mineral mix’ Vitamin mix4 Copper premix’
34.68 28.82 21.00 5.00 4.00 1.00 4.00 1.50 0
‘Celufil (U.S. Biochemical Corp., Cleveland, OH). *Contained (as g/kg): retinyl acetate, 1.80; cholecalciferol, 0.25; menadione, 5.00; dl-a-tocopherol acetate, 6.00; cod liver oil, 986.95. “Contained (as g/kg): CaCO,, 300.00; K$PO,, 319.00; NaHPO;HO, 200.34; MgSO;7 I-JO, 132.00; ZnSO;7 I-JO, 3.00; NaCl, 37.48; CoCA.6 I-JO, 1.00; MnSO;HO, 0.80; FeSO;7 HO, 6.22; KI, 0.15; Na$eO,, 0.011. ‘Same as Gatlin and Wilson (1984) with an additional 0.27 g biotin/kg vitamin mix. Contained 500 pg Cu/g premix.
279
removing corresponding amounts of cellulose. The basal diet contained 0.89 mg Cu/kg diet as determined by atomic absorption spectrophotometry (Association of Official Analytical Chemists, 1980). Supplemental copper concentrations ranged from 0 to 40 mg Cu/kg diet and were confirmed by analyses. Diet preparation and storage procedures were as previously described (Garling and Wilson, 1976). Prior to the initiation of the experiment, catfish underwent a 2-week conditioning period to adjust to a purified diet and standardized environmental conditions (Garling and Wilson, 1976). The experiment was conducted in 110-l flow-through aquaria and all experimental conditions were maintained as previously described (Garling and Wilson, 1976). Rearing water contained 0.11 mg Cu/l as determined by atomic absorption spectrophotometry (United States Environmental Protection Agency, 1979). At the beginning of the experimental period, channel catfish were sorted into groups of 15 fish and each group was placed in an individual aquarium. These individual groups of catfish had initial total weights of 83+3 g which did not differ significantly between groups. Triplicate random groups of catfish were fed diets containing 0, 2, 4, 6, 8 or 10 mg of supplemental copper/kg. Due to a lack of facilities, the diet containing 40 mg of supplemental copper/kg was only fed to duplicate groups of catfish. All diets were fed at a rate equaling 3% of wet body weight per day. This feeding rate is close to the satiation level for the size of fish used in this study. The amount of diet was divided into two equal feedings. Each group of catfish was weighed weekly, and the amount of diet fed was adjusted accordingly. Catfish were fed the test diets for 13 weeks. Sample collection and analytical procedures At the end of week 13, hearts and livers were removed from catfish in each group so that superoxide dismutase (SOD; EC 1.15.1.1) and cytochrome c oxidase (CCO; EC 1.9.3.1) activities could be determined. Enzymatic analyses were conducted on two individual livers per group. Due to the small size of the catfish heart, only one composite sample consisting of three pooled hearts was analyzed per group. Liver and heart samples were homogenized in 5 and 10 volumes of cold Triton X-100 (0.2% vol./vol.), respectively. Half of the homogenate was removed for analysis of CC0 activity while the remainder was centrifuged at 10 000 X g for 30 min. The resulting supematant was assayed for SOD activity by its ability to inhibit the reduction of ferricythochrome c by the xanthine oxidase reaction (McCord and Fridovich, 1969). Total SOD activity was measured in the presence of 0.01 mM KCN to inactivate cytochrome oxidase in the supernatant (Salin and Bridges, 1981). Manganese SOD (Mn SOD) activity was determined in the presence of 1 mM KCN to inhibit any copperzinc SOD (Cu-Zn SOD) (Beau& and Fridovich, 1973). Activity of Cu-Zn SOD was calculated as the difference between total and Mn SOD activities.
280
All rates were corrected for endogenous substrate reduction. One unit of activity was defined as the amount of enzyme that produced a 50% inhibition in the control rate of cytochrome c reduction (McCord and Fridovich, 1969). Cytochrome c oxidase activity was determined in homogenates by measuring the oxidation of ferrocytochrome c at 550 nm in 10 mM potassium phosphate buffer, pH 7.0 (Wharton and Tzagoloff, 1967). The initial concentration of ferrocytochrome c in the reaction mixture was 40 PM. One unit of activity was defined as the oxidation of 1 pmole of ferrocytochrome c/min at 25°C. Blood samples from all remaining fish were taken 15-20 h after the final feeding by severing the caudal peduncle. One composite sample per group was obtained by pooling blood from all fish in each group in heparinized tubes. Hematocrit was determined by the micromethod and the cyanmethemoglobin procedure was used for hemoglobin determination (Brown, 1980). Catfish hemoglobin values were adjusted using the cyanmethemoglobin correction factor determined by Larsen (1964). Erythrocytes were enumerated in a hemacytometer with improved Neubauer ruling (Brown, 1980). Plasma was separated from whole blood by centrifugation and assayed twice for ceruloplasmin (EC 1.16.3.1) activity using o-dianisidine dihydrochloride (Schosinsky et al,, 1974) and p-phenylenediamine (Syed and Coombs, 1982) as substrates. After the remaining fish were exsanguinated, hearts and livers were frozen for subsequent mineral analyses. Five hearts and five livers were randomly selected from each group, pooled and dried at 125°C to a constant weight. These samples were then digested with nitric and perchloric acids and analyzed for copper and iron by atomic absorption spectrophotometry (Association of Official Analytical Chemists, 1980).
Statistical methods All data were subjected to analysis of variance and Duncan’s new multiple-range test (Duncan, 1955) to determine differences in means (P
Catfish weight gain and feed efficiency values (Table 2) were not significantly affected by varying levels of copper intake. A bacterial outbreak occurred early in the experiment and resulted in some mortality (Table 2). Deaths were randomly distributed and not related to dietary treatment. Hematological values of catfish fed the various levels of copper were not significantly different. For all dietary treatments, mean hemoglobin, hematocrit and erythrocyte count values were 6.6+ 0.1 g/dl, 32.0+0.3% and 2.093+0.039 X lo’*, respectively. Varying levels of dietary copper ranging from 0 to 10 mg of supplemental copper/kg did not significantly affect the copper concentrations of catfish
281 TABLE
2
Growth, feed efficiency copper concentrations1
and mortality
of fingerling
channel
Supplemental copper’ (mg/kg)
Weight gain3 (%)
Feed efficiencf
Mortality (%)
0 2 4 6 8 10 40
813* 49 723* 71 800* 41 760*140 720* 12 795+ 34 800* 7
0.93iO.03 0.80*0.07 0.91*0.01 0.83*0.10 0.84iO.04 0.90*0.04 0.91*0.01
6.7 4.4 2.2 11.1 0 2.2 6.6
catfish
fed diets with varying
‘Means of three replicate groups + SEM except for two replicate groups for the 40 mg of supplemental copper/kg treatment. ‘Graded levels of a 500 mg/kg copper premix were added to the basal diet which contained 0.89 mg Cu/kg diet. 3Expressed as the percent increase in initial body weight at the end of week 13. “Wet weight gain/dry weight feed.
liver and heart tissue. However, catfish fed the diet with 40 mg of supplemental copper/kg had a significantly higher liver copper concentration than those fed the diets with the two lowest copper levels. Liver copper concentrations of catfish fed 0 and 40 mg of supplemental copper/kg were 10.92kO.72 and 15.81k1.34 pg/g dry wt, respectively; and heart copper concentrations were 14.63kO.69 and l&59*0.16 ygfg dry wt, respectively. Liver and heart iron concentrations were not affected by dietary copper intake. Liver iron concentrations of catfish fed 0 and 10 mg of supplemental copper/kg were 342.U77.7 and 258.7k4.8 pg/g dry wt, respectively; and heart iron concentrations were 328.3k79.9 and 290.8k34.3 kg/g dry wt. Liver SOD and heart CC0 activities (Table 3) were affected by copper intake. Significantly reduced liver Cu--Zn SOD and heart CC0 activities were found in catfish fed diets containing 0 and 2 mg of supplemental copper/kg as compared to those fed 4 mg/kg or more of supplemental copper. Total SOD activity (Table 3) was also suppressed in catfish fed the two lowest levels of copper due to reduced Cu-Zn SOD activity. A compensatory increase in Mn SOD activity (Table 3) was not observed in those fed the diets with the two lowest copper levels. Catfish fed the diet with 8 mg of supplemental copper/kg had intermediate enzyme activities which were not readily explainable. Suppressed Mn SOD activity was found in catfish fed the diet with 40 mg of supplemental copper/kg; whereas Cu-Zn SOD activity was unaffected. Ceruloplasmin activity was not detected in catfish plasma when odianisidine dihydrochloride was used as a substrate; however, normal activity
282 TABLE 3 Liver superoxide dismutase (SOD) and heart cytochrome c oxidase (CCO) activities of fingerling channel catfish fed diets with varying copper concentrations’** Supplemental coppe? (mg/kg)
Total SOD CuZn SOD (Unit&/g fresh tissue)
Mn SOD
CC0 (~moleslmin~g fresh tissue)
0 2 4 6 8 10 40
488+ 53a 443* 438 660*57b 668+ 46b 575+51ab 677+31b 537+ 9
154i 15abc 115*lP 168+ 9bC 194* 22C 125+ 15ab 201+ 9= 71* 3
19.2i2.48 19.1t1.7a 25.8? l.gb 28.0~0.7~ 24.6i2,gab 28.5i l.gb 26.4~0.5
334*45a 328* 42’ 492i 52b 475+30b 450* 5osb 475*29b 4662 12
‘Means of three replicate groups + SEM except for two replicate groups for the 40 mg of supplemental copper/kg treatment. *Means not sharing a common superscript letter are significantly different (RO.05). The. 40 mg of supplemental copper/kg treatment is included for comparison but was not included in the statistical analysis. ‘Graded levels of a 500 mg/kg copper premix were added to the basal diet which contained 0.89 mg Cu/kg diet. ‘One unit of activity is defined as the amount of enzyme to cause a 50% inhibition of the control rate.
was measured in a control sample of human serum. A low ceruloplasmin activity was measured in catfish plasma as compared to human serum (<30%) when a p-phenylenediamine assay was used. The response of this enzyme to varying levels of dietary copper was generally similar to the Cu-Zn SOD and CC0 activities; however, high variability precluded identification of real differences. Difficulty in reproducibly measuringp-phenylenediamine oxidase activity in plasma from cats has also been reported (Doong et al., 1983). DISCUSSION
Copper is an essential component in several metalloenzymes which catalyze reactions of physiological significance. Certain of these copper-dependent enzymes, such as, ceruloplasmin, Cu-Zn SOD and CCO, have been shown to be excellent indicators of copper nutriture in other animals. Liver Cu-Zn SOD and heart CC0 activities were the most sensitive indicators of copper status in this study. Based on the activities of these enzymes, the dietary copper requirement of channel catfish was estimated to be 4 mg of supplemental copper/kg or approximately 5 mg of total copper/kg. Murai et al. (1981) did not measure the activity of any copperdependent enzymes in channel catfish fed different amounts of copper. In that study, no deficiency signs were observed in catfish fed the basal diet which contained 1.5 mg
283
Cu/kg diet. Rainbow trout fed diets containing 15 and 150 mg of supplemental copper/kg did not have different SOD activities (Knox et al., 1982); whereas, those fed a diet with 500 mg of total copper/kg had lower CU-Zn SOD activity than those fed a diet with 2.5 mg of total copper/kg at similar zinc levels (Knox et al., 1984). The enzyme responses observed in the present study were similar to those reported for mammalian species (Paynter et al., 1979; Doong et al., 1983; L’Abbe’ and Fischer, 1984a, b). Catfish fed 0 and 2 mg of supplemental copper/kg had the lowest heart and liver copper concentrations after 13 weeks, but these reduced tissue levels were not of as great a magnitude as the reduced enzyme activities. The copper requirement determined in the present study is similar to the requirement values of other monogastric animals (National Research Council, 1977, 1978, 1979; Doong et al., 1983). However, the various responses of catfish to graded levels of dietary copper were less pronounced as compared to those of most terrestrial animals (Paynter et al., 1979; Doong et al., 1983; L’Abbe’ and Fischer, 1984a, b). Since fish can absorb certain minerals from the water, copper metabolism of catfish may have been affected by waterborne copper. In the present study, catfish fed 40 mg of supplemental copper/kg responded quite differently than catfish fed similar levels of copper in a previous study (Murai et al., 1981). In that study, catfish fed 16 and 32 mg of supplemental copper/kg for 16 weeks had significantly suppressed growth and feed efficiency values. Reduced growth was detected as early as 4 weeks after the onset of that experiment. Catfish fed a diet containing 40 mg of supplemental copper/kg in the present study had similar weight gain and feed efficiency values as catfish fed lower dietary levels of copper (Table 2). The absence of dietary copper toxicity observed in the present study has also been reported for rainbow trout (Knox et al., 1982,1984). Performance of trout fed diets containing 150 mg of supplemental copper/kg (Knox et al., 1982) and 500 mg of total copper/kg (Knox et al., 1984) was similar to those fed much lower dietary levels of copper. Relatively high levels of dietary copper are also required to produce toxicity in terrestrial animals (National Research Council, 1980). The toxicity of elevated levels of waterborne copper to fish is well known (Lewis and Lewis, 1971); however, this likely did not contribute to the differing responses of catfish fed 40 and 32 mg of supplemental copper/kg in this and the previous study. Flowthrough culture facilities were utilized in both studies. Therefore, the accumulation of excreted dietary copper was prohibited. The rearing water in the previous study also contained less copper than that used in the present study. It has also been reported that catfish fed a diet with 32 mg of supplemental copper/kg had significantly reduced hematocrit and erythrocyte count values as compared to those receiving diets with lower levels of copper; but hemoglobin values were not affected (Murai et al., 1981). There was not a significant reduction in hemoglobin, hematocrit and erythrocyte
284
count values of catfish fed 40 mg of supplemental copper/kg in the present study. Much higher levels of dietary copper also did not reduce hematocrit and hemoglobin values of rainbow trout (Knox et al., 1982,1984). In both catfish studies, the highest level of copper intake affected liver copper concentration in a similar manner. Catfish fed 32 mg of supplemental copper/kg had a significantly higher liver copper concentration than catfish fed the unsupplemented basal diet. However, the effect of lower levels of dietary copper on liver copper concentration was not reported (Murai et al., 1981). Significantly different liver copper concentrations have also been seen in rainbow trout fed diets containing 15 and 150 mg of supplemental copper/kg (Knox et al., 1982) and 2.5 and 500 mg of total copper/kg (Knox et al., 1984). In the present study, the relative distribution of copper in catfish liver and heart tissue was similar to that of the plaice (Pleuronectes plates&z) (Syed and Coombs, 1982). ACKNOWLEDGMENTS
The authors wish to thank William E. Poe, James H. Renfroe and Cathy Shepard for their technical assistance during this investigation. Thanks are also extended to Lynn Pellum, Department of Home Economics, for her assistance in the mineral analyses. The advice of Dr. Marvin L. Salin, Department of Biochemistry, concerning enzymatic analyses is also gratefully acknowledged. Publication number 6067 of the Mississippi Agricultural and Forestry Experiment Station.
REFERENCES Association of Official Analytical Chemists, 1980. Methods of Analysis. AOAC, Washing ton, DC, 1018 pp. Beauchamp, CO. and Fridovich, I., 1973. Isozymes of superoxide dismutase from wheat germ. Biochim. Biophys. Acta, 317: 50-64. Brown, B.A., 1980. Routine hematology procedures. In: Hematology: Principles and Procedures. Lea and Febiger, Philadelphia, pp. 71-112. Doong, G., Keen, C.L., Rogers, Q., Morris, J. and Rucker, R.B., 1983. Selected features of copper metabolism in the cat. J. Nutr., 113: 1963-1971. Duncan, D.B., 1955. Multiple-range and multiple F tests. Biometrics, 11: l-42. Garling, D.L., Jr. and Wilson, R.P., 1976. Optimum dietary protein to energy ratio for channel catfish fingerlings, Zctaluruspunctatus. J. Nutr., 106: 1368-1375. Gatlin, D.M., III and Wilson, R.P., 1984. Zinc supplementation of practical channel catfish diets. Aquaculture, 41: 31-36. Knox, D., Cowey, C.B. and Adron, J.W., 1982. Effects of dietary copper and copper: zinc ratio on rainbow trout Salmo gairdneri. Aquaculture, 27 : 111-119. Knox, D., Cowey, C.B. and Adron, J.W., 1984. Effects of dietary zinc intake upon copper metabolism in rainbow trout (Salmo gairdneri). Aquaculture, 40: 199-207. L’Abbe’, M.R. and Fischer, P.W.F., 1984a. The effects of high dietary zinc and copper deficiency on the activity of copper-requiring metalloenzymes in the growing rat. J. Nutr.. 114: 813-822.
285 L’Abbe’, M.R. and Fischer, P.W.F., 1984b. The effects of dietary zinc on the activity of copper-requiring metalloenzymes in the rat. J. Nutr., 114: 823-828. Larsen, H.N., 1964. Comparison of various methods of hemoglobin determination on catfish blood. Prog. Fish-Cult., 26: 11-15. Lewis, SD. and Lewis, W.M., 1911. The effect of zinc and copper on the osmolality of blood serum of the channel catfish, Zctaluruspunctatus Rafinesque, and golden shiner, Notemigonus crysoleucas Mitchill. Trans. Am. Fish. Sot., 100: 639-643. McCord, J.M. and Fridovich, I., 1969. Superoxide dismutase. An enzymatic function for erythrocuprein (hemocuprein). J. Biol. Chem., 244: 6049-6055. Murai, T., Andrews, J.W. and Smith, R.G., Jr., 1981. Effects of dietary copper on channel catfish. Aquaculture, 22: 353-351. National Research Council, 1911. Nutrient Requirements of Poultry. National Academy of Sciences, Washington, DC, 62 pp. National Research Council, 1918. Nutrient Requirements of Laboratory Animals. National Academy of Sciences, Washington, DC, 96 pp. National Research Council, 1919. Nutrient Requirements of Swine. National Academy of Sciences, Washington, DC, 52 pp. National Research Council, 1980. Mineral Tolerance of Domestic Animals. National Academy of Sciences, Washington, DC, 511 pp. Ogino, C. and Yang, G.-Y., 1980. Requirements of carp and rainbow trout for dietary manganese and copper. Bull. Jpn. Sot. Sci. Fish., 46: 455-458. Paynter, D.I., Moir, R.J. and Underwood, E.J., 1919. Changes in activity of the Cu-Zn superoxide dismutase enzyme in tissues of the rat with changes in dietary copper. J. Nutr., 109: 1510--1516. Salin, M.L. and Bridges, S.M., 1981. Absence of the iron-containing superoxide dismutase in mitochondria from mustard (Brassica campestris). Biochem. J., 195: 229233. Satoh, S., Yamamoto, H., Takeuchi, T. and Watanabe, T., 1983a. Effects on growth and mineral composition of rainbow trout of deletion of trace elements or magnesium from fish meal diet. Bull. Jpn. Sot. Sci. Fish., 49: 425-429. Satoh, S., Yamamoto, H., Takeuchi, T. and Watanabe, T., 1983b. Effects on growth and mineral composition of carp of deletion of trace elements or magnesium from fish meal diet. Bull. Jpn. Sot. Sci. Fish., 49: 431-435. Satoh, S., Takeuchi, T., Narabe, Y. and Watanabe, T., 1983c. Effects of deletion of several trace elements from fish meal diets on growth and mineral composition of rainbow trout fingerlings. Bull. Jpn. Sot. Sci. Fish., 49: 1909-1916. Schosinsky, K.H., Lehmann, H.P. and Beeler, M.F., 1914. Measurement of ceruloplasmin from its oxidase activity in serum by use of o-dianisidine dihydrochloride. Clin. Chem., 20: 1556-1563. Syed, M.A. and Coombs, T.L., 1982. Copper metabolism in the plaice, Pleuronectes plate&w (L.). J. Exp. Mar. Biol. Ecol., 63: 281-296. United States Environmental Protection Agency, 1919. Methods for Chemical Analysis of Water and Wastes. EPA-600 4-19-020, Cincinnati, OH. Wharton, D.C. and Tzagoloff, A., 1961. Cytochrome oxidase from beef heart mitochondria. In: R.W. Estabrook and M.E. Pullman (Editors), Methods in Enzymology, Vol. 10. Academic Press, New York, NY, pp. 245-250.
277 - Printed
in The Netherlands
DIETARY COPPER REQUIREMENT OF FINGERLING CHANNEL CATFISH
DELBERT
M. GATLIN,
III’ and ROBERT
Department of Biochemistry, MS 39762 (U.S.A.)
P. WILSON2
Mississippi State University, Mississippi State,
‘Present address: University of Arkansas at Pine Bluff, Agricultural Station, Pine Bluff, AR 71601 (U.S.A.) ‘To whom requests for reprints should be addressed. (Accepted
6 February
Experiment
1986)
ABSTRACT Gatlin, D.M., III and Wilson, R.P., 1986. nel catfish. Aquaculture, 54: 277-285.
Dietary
copper
requirement
of fingerling
chan-
Purified egg white diets containing incremental levels of copper (as CuSO;B KO) were fed to fingerling channel catfish (Ictalurus punctatus) to determine their dietary copper (Cu) requirement. Catfish in aquaria were fed diets containing 0, 2,4, 6,8 or 10 mg of supplemental copper/kg diet for 13 weeks. Growth and feed efficiency data, as well as hemoglobin, hematocrit and erythrocyte count values were similar for catfish fed the basal diet which contained 0.89 mg Cu/kg diet and all copper-supplemented diets. A diet containing 40 mg of supplemental copper/kg was also fed in this experiment since 16 and 32 mg of supplemental copper/kg had previously been reported to cause suppressed growth of channel catfish. Those fed the 40 mg of supplemental copper/ kg diet had similar growth and feed efficiency values as catfish fed the other diets. At the end of week 13, heart cytochrome c oxidase and liver copper-zinc superoxide dismutase activities were significantly reduced in catfish fed diets containing 0 and 2 mg of supplemental copper/kg as compared to those fed 4 mg/kg or more of supplemental copper. Based on the enzymatic data, the minimum dietary copper requirement of channel catfish was determined to be approximately 5 mg of total copper/kg diet.
INTRODUCTION
The effects of varying levels of dietary copper intake on several fish species have been studied (Ogino and Yang, 1980; Murai et al., 1981; Knox et al., 1982, 1984; Satoh et al., 1983a, b, c). A basal diet containing 0.7 mg Cu/kg diet was reported to suppress growth in carp (Cyprinus carpio) as compared to a control diet containing 3 mg Cu/kg diet (Ogino and Yang, 1980). A differential growth response was not observed in rainbow trout (Salmo gairdneri) fed these same two diets (Ogino and Yang, 1980). In two other studies, similar growth rates and feed conversions were observed
0044-8486/86/$03.50
0 1986 Elsevier
Science
Publishers
B.V.
278
for rainbow trout fed diets containing 15 and 150 mg of supplemental copper/kg (Knox et al., 1982) and 2.5 and 500 mg of total copper/kg (Knox et al., 1984). In contrast with these studies, suppressed growth and feed efficiency were reported for channel catfish (Ictalurus punctatus) fed diets with 16 and 32 mg of supplemental copper/kg (Murai et al., 1981). The basal diet fed in that study contained 1.5 mg Cu/kg diet and did not produce any observable deficiency signs after 16 weeks. The present study was conducted to characterize copper deficiency in channel catfish and to determine their minimum dietary copper requirement. A diet containing 40 mg of supplemental copper/kg was also fed to reevaluate the toxicity of this level of dietary copper to channel catfish. MATERIALS
AND METHODS
Experimental diets and design The composition of the basal diet is listed in Table 1. Diets were formulated to contain 30% crude protein from egg white and 344 kcal/lOO g diet (Garling and Wilson, 1976). Biotin was included in these diets at 9.3 mg/kg to negate the effect of avidin in egg white. All dietary ingredients were commercially obtained. Minerals used in the premixes and diets were of reagent grade. Experimental diets were prepared by adding incremental levels of a 500 mg/kg copper premix (CuSO,*5 H,O and cellulose) to the basal diet while TABLE 1 Basal diet fed in the copper study Ingredient
% Dry weight
Egg white Dextrin Cellulose’ Corn oil Cod liver oil ADEK mix’ Copper-free mineral mix’ Vitamin mix4 Copper premix’
34.68 28.82 21.00 5.00 4.00 1.00 4.00 1.50 0
‘Celufil (U.S. Biochemical Corp., Cleveland, OH). *Contained (as g/kg): retinyl acetate, 1.80; cholecalciferol, 0.25; menadione, 5.00; dl-a-tocopherol acetate, 6.00; cod liver oil, 986.95. “Contained (as g/kg): CaCO,, 300.00; K$PO,, 319.00; NaHPO;HO, 200.34; MgSO;7 I-JO, 132.00; ZnSO;7 I-JO, 3.00; NaCl, 37.48; CoCA.6 I-JO, 1.00; MnSO;HO, 0.80; FeSO;7 HO, 6.22; KI, 0.15; Na$eO,, 0.011. ‘Same as Gatlin and Wilson (1984) with an additional 0.27 g biotin/kg vitamin mix. Contained 500 pg Cu/g premix.
279
removing corresponding amounts of cellulose. The basal diet contained 0.89 mg Cu/kg diet as determined by atomic absorption spectrophotometry (Association of Official Analytical Chemists, 1980). Supplemental copper concentrations ranged from 0 to 40 mg Cu/kg diet and were confirmed by analyses. Diet preparation and storage procedures were as previously described (Garling and Wilson, 1976). Prior to the initiation of the experiment, catfish underwent a 2-week conditioning period to adjust to a purified diet and standardized environmental conditions (Garling and Wilson, 1976). The experiment was conducted in 110-l flow-through aquaria and all experimental conditions were maintained as previously described (Garling and Wilson, 1976). Rearing water contained 0.11 mg Cu/l as determined by atomic absorption spectrophotometry (United States Environmental Protection Agency, 1979). At the beginning of the experimental period, channel catfish were sorted into groups of 15 fish and each group was placed in an individual aquarium. These individual groups of catfish had initial total weights of 83+3 g which did not differ significantly between groups. Triplicate random groups of catfish were fed diets containing 0, 2, 4, 6, 8 or 10 mg of supplemental copper/kg. Due to a lack of facilities, the diet containing 40 mg of supplemental copper/kg was only fed to duplicate groups of catfish. All diets were fed at a rate equaling 3% of wet body weight per day. This feeding rate is close to the satiation level for the size of fish used in this study. The amount of diet was divided into two equal feedings. Each group of catfish was weighed weekly, and the amount of diet fed was adjusted accordingly. Catfish were fed the test diets for 13 weeks. Sample collection and analytical procedures At the end of week 13, hearts and livers were removed from catfish in each group so that superoxide dismutase (SOD; EC 1.15.1.1) and cytochrome c oxidase (CCO; EC 1.9.3.1) activities could be determined. Enzymatic analyses were conducted on two individual livers per group. Due to the small size of the catfish heart, only one composite sample consisting of three pooled hearts was analyzed per group. Liver and heart samples were homogenized in 5 and 10 volumes of cold Triton X-100 (0.2% vol./vol.), respectively. Half of the homogenate was removed for analysis of CC0 activity while the remainder was centrifuged at 10 000 X g for 30 min. The resulting supematant was assayed for SOD activity by its ability to inhibit the reduction of ferricythochrome c by the xanthine oxidase reaction (McCord and Fridovich, 1969). Total SOD activity was measured in the presence of 0.01 mM KCN to inactivate cytochrome oxidase in the supernatant (Salin and Bridges, 1981). Manganese SOD (Mn SOD) activity was determined in the presence of 1 mM KCN to inhibit any copperzinc SOD (Cu-Zn SOD) (Beau& and Fridovich, 1973). Activity of Cu-Zn SOD was calculated as the difference between total and Mn SOD activities.
280
All rates were corrected for endogenous substrate reduction. One unit of activity was defined as the amount of enzyme that produced a 50% inhibition in the control rate of cytochrome c reduction (McCord and Fridovich, 1969). Cytochrome c oxidase activity was determined in homogenates by measuring the oxidation of ferrocytochrome c at 550 nm in 10 mM potassium phosphate buffer, pH 7.0 (Wharton and Tzagoloff, 1967). The initial concentration of ferrocytochrome c in the reaction mixture was 40 PM. One unit of activity was defined as the oxidation of 1 pmole of ferrocytochrome c/min at 25°C. Blood samples from all remaining fish were taken 15-20 h after the final feeding by severing the caudal peduncle. One composite sample per group was obtained by pooling blood from all fish in each group in heparinized tubes. Hematocrit was determined by the micromethod and the cyanmethemoglobin procedure was used for hemoglobin determination (Brown, 1980). Catfish hemoglobin values were adjusted using the cyanmethemoglobin correction factor determined by Larsen (1964). Erythrocytes were enumerated in a hemacytometer with improved Neubauer ruling (Brown, 1980). Plasma was separated from whole blood by centrifugation and assayed twice for ceruloplasmin (EC 1.16.3.1) activity using o-dianisidine dihydrochloride (Schosinsky et al,, 1974) and p-phenylenediamine (Syed and Coombs, 1982) as substrates. After the remaining fish were exsanguinated, hearts and livers were frozen for subsequent mineral analyses. Five hearts and five livers were randomly selected from each group, pooled and dried at 125°C to a constant weight. These samples were then digested with nitric and perchloric acids and analyzed for copper and iron by atomic absorption spectrophotometry (Association of Official Analytical Chemists, 1980).
Statistical methods All data were subjected to analysis of variance and Duncan’s new multiple-range test (Duncan, 1955) to determine differences in means (P
Catfish weight gain and feed efficiency values (Table 2) were not significantly affected by varying levels of copper intake. A bacterial outbreak occurred early in the experiment and resulted in some mortality (Table 2). Deaths were randomly distributed and not related to dietary treatment. Hematological values of catfish fed the various levels of copper were not significantly different. For all dietary treatments, mean hemoglobin, hematocrit and erythrocyte count values were 6.6+ 0.1 g/dl, 32.0+0.3% and 2.093+0.039 X lo’*, respectively. Varying levels of dietary copper ranging from 0 to 10 mg of supplemental copper/kg did not significantly affect the copper concentrations of catfish
281 TABLE
2
Growth, feed efficiency copper concentrations1
and mortality
of fingerling
channel
Supplemental copper’ (mg/kg)
Weight gain3 (%)
Feed efficiencf
Mortality (%)
0 2 4 6 8 10 40
813* 49 723* 71 800* 41 760*140 720* 12 795+ 34 800* 7
0.93iO.03 0.80*0.07 0.91*0.01 0.83*0.10 0.84iO.04 0.90*0.04 0.91*0.01
6.7 4.4 2.2 11.1 0 2.2 6.6
catfish
fed diets with varying
‘Means of three replicate groups + SEM except for two replicate groups for the 40 mg of supplemental copper/kg treatment. ‘Graded levels of a 500 mg/kg copper premix were added to the basal diet which contained 0.89 mg Cu/kg diet. 3Expressed as the percent increase in initial body weight at the end of week 13. “Wet weight gain/dry weight feed.
liver and heart tissue. However, catfish fed the diet with 40 mg of supplemental copper/kg had a significantly higher liver copper concentration than those fed the diets with the two lowest copper levels. Liver copper concentrations of catfish fed 0 and 40 mg of supplemental copper/kg were 10.92kO.72 and 15.81k1.34 pg/g dry wt, respectively; and heart copper concentrations were 14.63kO.69 and l&59*0.16 ygfg dry wt, respectively. Liver and heart iron concentrations were not affected by dietary copper intake. Liver iron concentrations of catfish fed 0 and 10 mg of supplemental copper/kg were 342.U77.7 and 258.7k4.8 pg/g dry wt, respectively; and heart iron concentrations were 328.3k79.9 and 290.8k34.3 kg/g dry wt. Liver SOD and heart CC0 activities (Table 3) were affected by copper intake. Significantly reduced liver Cu--Zn SOD and heart CC0 activities were found in catfish fed diets containing 0 and 2 mg of supplemental copper/kg as compared to those fed 4 mg/kg or more of supplemental copper. Total SOD activity (Table 3) was also suppressed in catfish fed the two lowest levels of copper due to reduced Cu-Zn SOD activity. A compensatory increase in Mn SOD activity (Table 3) was not observed in those fed the diets with the two lowest copper levels. Catfish fed the diet with 8 mg of supplemental copper/kg had intermediate enzyme activities which were not readily explainable. Suppressed Mn SOD activity was found in catfish fed the diet with 40 mg of supplemental copper/kg; whereas Cu-Zn SOD activity was unaffected. Ceruloplasmin activity was not detected in catfish plasma when odianisidine dihydrochloride was used as a substrate; however, normal activity
282 TABLE 3 Liver superoxide dismutase (SOD) and heart cytochrome c oxidase (CCO) activities of fingerling channel catfish fed diets with varying copper concentrations’** Supplemental coppe? (mg/kg)
Total SOD CuZn SOD (Unit&/g fresh tissue)
Mn SOD
CC0 (~moleslmin~g fresh tissue)
0 2 4 6 8 10 40
488+ 53a 443* 438 660*57b 668+ 46b 575+51ab 677+31b 537+ 9
154i 15abc 115*lP 168+ 9bC 194* 22C 125+ 15ab 201+ 9= 71* 3
19.2i2.48 19.1t1.7a 25.8? l.gb 28.0~0.7~ 24.6i2,gab 28.5i l.gb 26.4~0.5
334*45a 328* 42’ 492i 52b 475+30b 450* 5osb 475*29b 4662 12
‘Means of three replicate groups + SEM except for two replicate groups for the 40 mg of supplemental copper/kg treatment. *Means not sharing a common superscript letter are significantly different (RO.05). The. 40 mg of supplemental copper/kg treatment is included for comparison but was not included in the statistical analysis. ‘Graded levels of a 500 mg/kg copper premix were added to the basal diet which contained 0.89 mg Cu/kg diet. ‘One unit of activity is defined as the amount of enzyme to cause a 50% inhibition of the control rate.
was measured in a control sample of human serum. A low ceruloplasmin activity was measured in catfish plasma as compared to human serum (<30%) when a p-phenylenediamine assay was used. The response of this enzyme to varying levels of dietary copper was generally similar to the Cu-Zn SOD and CC0 activities; however, high variability precluded identification of real differences. Difficulty in reproducibly measuringp-phenylenediamine oxidase activity in plasma from cats has also been reported (Doong et al., 1983). DISCUSSION
Copper is an essential component in several metalloenzymes which catalyze reactions of physiological significance. Certain of these copper-dependent enzymes, such as, ceruloplasmin, Cu-Zn SOD and CCO, have been shown to be excellent indicators of copper nutriture in other animals. Liver Cu-Zn SOD and heart CC0 activities were the most sensitive indicators of copper status in this study. Based on the activities of these enzymes, the dietary copper requirement of channel catfish was estimated to be 4 mg of supplemental copper/kg or approximately 5 mg of total copper/kg. Murai et al. (1981) did not measure the activity of any copperdependent enzymes in channel catfish fed different amounts of copper. In that study, no deficiency signs were observed in catfish fed the basal diet which contained 1.5 mg
283
Cu/kg diet. Rainbow trout fed diets containing 15 and 150 mg of supplemental copper/kg did not have different SOD activities (Knox et al., 1982); whereas, those fed a diet with 500 mg of total copper/kg had lower CU-Zn SOD activity than those fed a diet with 2.5 mg of total copper/kg at similar zinc levels (Knox et al., 1984). The enzyme responses observed in the present study were similar to those reported for mammalian species (Paynter et al., 1979; Doong et al., 1983; L’Abbe’ and Fischer, 1984a, b). Catfish fed 0 and 2 mg of supplemental copper/kg had the lowest heart and liver copper concentrations after 13 weeks, but these reduced tissue levels were not of as great a magnitude as the reduced enzyme activities. The copper requirement determined in the present study is similar to the requirement values of other monogastric animals (National Research Council, 1977, 1978, 1979; Doong et al., 1983). However, the various responses of catfish to graded levels of dietary copper were less pronounced as compared to those of most terrestrial animals (Paynter et al., 1979; Doong et al., 1983; L’Abbe’ and Fischer, 1984a, b). Since fish can absorb certain minerals from the water, copper metabolism of catfish may have been affected by waterborne copper. In the present study, catfish fed 40 mg of supplemental copper/kg responded quite differently than catfish fed similar levels of copper in a previous study (Murai et al., 1981). In that study, catfish fed 16 and 32 mg of supplemental copper/kg for 16 weeks had significantly suppressed growth and feed efficiency values. Reduced growth was detected as early as 4 weeks after the onset of that experiment. Catfish fed a diet containing 40 mg of supplemental copper/kg in the present study had similar weight gain and feed efficiency values as catfish fed lower dietary levels of copper (Table 2). The absence of dietary copper toxicity observed in the present study has also been reported for rainbow trout (Knox et al., 1982,1984). Performance of trout fed diets containing 150 mg of supplemental copper/kg (Knox et al., 1982) and 500 mg of total copper/kg (Knox et al., 1984) was similar to those fed much lower dietary levels of copper. Relatively high levels of dietary copper are also required to produce toxicity in terrestrial animals (National Research Council, 1980). The toxicity of elevated levels of waterborne copper to fish is well known (Lewis and Lewis, 1971); however, this likely did not contribute to the differing responses of catfish fed 40 and 32 mg of supplemental copper/kg in this and the previous study. Flowthrough culture facilities were utilized in both studies. Therefore, the accumulation of excreted dietary copper was prohibited. The rearing water in the previous study also contained less copper than that used in the present study. It has also been reported that catfish fed a diet with 32 mg of supplemental copper/kg had significantly reduced hematocrit and erythrocyte count values as compared to those receiving diets with lower levels of copper; but hemoglobin values were not affected (Murai et al., 1981). There was not a significant reduction in hemoglobin, hematocrit and erythrocyte
284
count values of catfish fed 40 mg of supplemental copper/kg in the present study. Much higher levels of dietary copper also did not reduce hematocrit and hemoglobin values of rainbow trout (Knox et al., 1982,1984). In both catfish studies, the highest level of copper intake affected liver copper concentration in a similar manner. Catfish fed 32 mg of supplemental copper/kg had a significantly higher liver copper concentration than catfish fed the unsupplemented basal diet. However, the effect of lower levels of dietary copper on liver copper concentration was not reported (Murai et al., 1981). Significantly different liver copper concentrations have also been seen in rainbow trout fed diets containing 15 and 150 mg of supplemental copper/kg (Knox et al., 1982) and 2.5 and 500 mg of total copper/kg (Knox et al., 1984). In the present study, the relative distribution of copper in catfish liver and heart tissue was similar to that of the plaice (Pleuronectes plates&z) (Syed and Coombs, 1982). ACKNOWLEDGMENTS
The authors wish to thank William E. Poe, James H. Renfroe and Cathy Shepard for their technical assistance during this investigation. Thanks are also extended to Lynn Pellum, Department of Home Economics, for her assistance in the mineral analyses. The advice of Dr. Marvin L. Salin, Department of Biochemistry, concerning enzymatic analyses is also gratefully acknowledged. Publication number 6067 of the Mississippi Agricultural and Forestry Experiment Station.
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