Aquaculture, 68 (1988) 65-71 Elsevier Science Publishers B.V., Amsterdam -
65 Printed in The Netherlands
Choline Nutrition of Fingerling
ROBERT P. WILSON and WILLIAM E. POE Department of Biochemistry, Mississippi State University, Mississippi State, MS 39762 (U.S.A.) (Accepted 27 July 1987)
ABSTRACT Wilson, R.P.. and Poe, W.E., 1988. Choline nutrition of fingerling channel catfish. Aquaculture, 68: 65-71. Channel catfish can apparently utilize dietary methionine to spare, at least in part, their need for dietary choline. In order to determine a choline requirement for channel catfish, it was necessary to use a diet limiting in methionine to induce an increase in liver lipid content indicative of a choline deficiency. A dietary methionine reduction was accomplished by using soybean protein as the protein source in the test diet. An optimal level of 400 mg choline/kg of diet based on liver lipid content was determined for catfish under the dietary conditions used. This value would appear to represent a maximum requirement level because it was determined at the lowest level of dietary methionine that could be used while still meeting the total sulfur amino acid requirement of the catfish. The observations made in this study are consistent with those reported for other animals indicating that fish can synthesize choline from appropriate dietary precursors. Thus, it would appear that many of the differences in requirement values of choline for various fishes reported by previous workers may be explained by species differences in enzyme activities associated with choline synthesis and degradation, as well as, various dietary factors known to directly affect the rate of choline synthesis.
Choline is generally classified as an essential vitamin by most comparative nutritionists. Choline is an important component of the phospholipid lecithin and certain other complex lipids. It serves as a source of labile methyl groups for the synthesis of various methylated metabolites and as a precursor of acetylcholine. Most animals can synthesize choline if adequate methyl donors such as methionine are present in the diet; however, the rate of choline synthesis has been shown to be insufficient to meet the metabolic and physiologic needs in some young animals. Several factors have been shown to affect the need for dietary choline in animals. Species differences in enzyme activities associated with
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choline synthesis and degradation have been detected. Dietary factors such as the amounts of dietary methionine or betaine and the presence of folacin and vitamin B-12 will directly affect rates of choline synthesis. For these reasons a quantitative dietary requirement for choline cannot be stated for most animals ( Appel and Briggs, 1973 ) . Dietary essentiality of choline and deficiency signs have been reported for rainbow trout (McLaren et al., 1947; Kitamura et al., 1967)) chinook salmon (Halver, 1957)) coho salmon (Coats and Halver, 1958)) channel catfish (Dupree, 1966)) common carp (Ogino et al., 1970)) Japanese eel (Arai et al., 1972)) red sea bream (Yone, 1975) and lake trout (Ketola, 1976). Deficiency signs have been reported to include poor growth and feed efficiency, anorexia, fatty livers, and hemorrhagic areas in kidneys, liver and intestine. Only limited information is available on the requirement of choline for fish. McLaren et al. (1947) suggested that 50-100 mg of choline/kg of diet was adequate for rainbow trout. The choline requirement of common carp appears to be no more than 1500 mg choline/kg of diet (Ogino et al., 1970). Halver (1972) reported that chinook and coho salmon require 600-800 mg of choline/kg of diet based on maximum liver storage data. Ketola (1976) concluded that the choline requirement of lake trout was no greater than 1000 mg/kg of diet based on growth data. In a preliminary experiment, fingerling channel catfish were fed our standard casein-gelatin diet (Gatlin and Wilson, 1984) that was supplemented with graded levels of choline. After 12 weeks, catfish fed these diets did not have significantly different growth rates, feed efficiency values or liver lipid content. The casein-gelatin diet contained 0.81% methionine and 0.10% cystine which exceeds the total sulfur amino acid requirement of fingerling channel catfish (0.69% for a 30% crude protein diet) as reported by Harding et al., (1977). Therefore, this study was designed to evaluate the choline requirement of fingerling channel catfish using a diet limiting in dietary methionine.
MATERIALS AND METHODS
The basal diet was formulated to contain a minimum amount of methionine with adequate cystine to meet the total sulfur amino acid requirement of the catfish. This was accomplished by supplying 30% crude protein as soybean protein (Table 1) . This basal diet contained 0.39% methionine and 0.38% cystine. Six diets containing 0 ,200,400,800,1200 and 1600 mg choline/kg of diet were used to evaluate the choline requirement. Three additional diets with supplemental L-methionine and no added choline were fed to determine if catfish could meet their choline requirement from added methionine. The diets
67 TABLE 1 Composition of basal diet Ingredient
% Dry weight
Soybean protein (70% CP) Dextrin Cellulose’ Corn oil Cod liver oil Mineral premix2 Choline-free vitamin premix3 Carboxymethyl cellulose Calcium carbonate Choline premix4 Methionine premix5
43.07 22.50 12.43 5.00 5.00 4.00 3.00 4.00 1.00 0 0
‘Celufil (United States Biochemical Corp., Cleveland, OH). ‘Contained (as g/kg) : Ca (H,PO,) 2*Hz0, 135.49; Ca (O,CCHOHCH,) 2, 327; FeCsH507*Hz0, 29.7; MgS04e7H,0, 132; K,HP0,,239.8; NaH,PO,*H,O, 87.2; NaCl, 43.5; AlC&, 0.15; KI, 0.15; CuCl, 0.2; MnSO,*H,O, 0.8; CoCl,*GH,O, 1; ZnSO,*7H,O, 3; Na,SeO,, 0.011. 3Contained (as g/kg) : ascorbic acid, 45; inositol, 5; niacin, 4.5; calcium d-pantothenate, 3; riboflavin, 1; pyridoxine HCl, 1; thiamin*HCl, 1; biotin, 0.02; folic acid, 0.09; vitamin B-12,0.00135; retinyl acetate, 0.6; cholecalciferol, 0.083; menadione, 1.67; DL-a-tocopheryl acetate (powder 250 U/g), 8; cellulose, 929.04. 4Contained 20 mg choline as choline chloride/g in a cellulose premix. 5Contained 0.2 g L-methionine/g in a cellulose premix.
contained an estimated energy level of 3.44 kcal/g (Garling and Wilson, 1976). Procedures for diet preparation and storage were as previously described ( Garling and Wilson, 1976). Prior to initiation of the experiment, catfish underwent a 2-week conditioning period during which they readily adjusted to the soybean protein based diet and standardized environmental conditions. The feeding trial was conducted in 110-l flow-through aquaria with flow rates of approximately 900 ml/min. Water temperature was maintained at 26.7 & 1.1’ C and a diurnal 1ight:dark cycle was regulated at 14:lO h. At the start of the experiment, groups of 16 catfish weighing 902 5 g per group were stocked into individual aquaria. Each diet was fed to triplicate random groups of fish for 15 weeks. Diets were fed at a rate equaling 3% of wet body weight per day in two equal feedings. Each group was weighed weekly, and the amount of diet fed was adjusted accordingly. At the termination of the experiment, the fish were killed by an overexposure to MS-222. The fish from each aquarium were divided into two groups. Total body weights and liver weights were recorded for calculation of liver-weightto-body-weight ratios. The livers from each group were pooled, frozen and stored for lipid analysis. Each pooled liver sample was finely minced into a composite homogeneous sample. Total liver lipid was determined by the method of Folch
et al. (1957). Moisture was determined on a portion of the composite liver sample by drying for 3 h at 125 ‘C. The average of the results for the two groups of pooled samples represented one observation per aquarium. All data were subjected to analysis of variance and Duncan’s Multiple Range Test (Duncan, 1955) to determine differences in means (P-c 0.05). RESULTS
Catfish weight gain was not significantly affected by either added dietary choline or methionine (Table 2). Feed efficiency values were not affected by dietary choline; however, a significant decrease in feed efficiency was observed at the highest level of supplemental methionine. Mean liver-weight-to-bodyweight ratios ranged from 1.79 to 1.96 and did not differ among treatments. Only one death and no gross deficiency signs were observed during the 15-week period. Liver lipid content was significantly affected by both dietary choline and methionine (Table 2). Catfish fed increasing levels of dietary choline exhibited a linear ( r2 = 0.87) decrease in liver lipid content from 0 to 400 mg choline/kg of diet. Liver lipid content did not differ for fish fed diets containing 400-1600 mg choline/kg of diet, thus indicating the optimal level of choline to be about 400 mg/kg of diet for the dietary conditions used in this study. Methionine supplementation in the absence of dietary choline also significantly TABLE 2 Growth, feed efficiency and liver lipid content of fingerling channel catfish fed diets with varying choline and methionine content for 15 weeks; means of three replicates + SEMI Dietary level Choline (mg/kg)
200 400 800 1200 1600 0 0 0
0.39 0.39 0.39 0.39 0.39 0.59 0.79 0.99
weight gain as % of initial weight
Feed efficiency ( g gain/g fed )
Liver lipid (% dry weight)
1022 + 36 1100+48 1111+18 1028k21 1092 + 62 1096 t 43 1112i35 1041 f 39 1002 ?I 21
0.88 + 0.01”s 0.88 +_0.02”b 0.88 f O.Ol”b 0.87 + 0.02”b 0.88 f 0.02”s 0.88 + O.Olab 0.89 + 0.02” 0.86 + 0.03”b 0.82 f 0.02b
15.24 + 0.25” 14.01 z!I0.58b 12.51* 0.43” 12.31 kO.50” 11.96?0.28” 11.91 kO.21” 14.45 +o.ls”s 13.93+0.16b 14.08 + 0.40s
‘Means within each column not sharing a common superscript letter are significantly different (PcO.05). 2Each diet also contained 0.38% cystine, thus meeting or exceeding the total sulfur amino acid requirement.
reduced liver lipid content when the methionine content was increased from 0.39% to 0.79% of the diet. These data indicate that the catfish can utilize dietary methionine to spare a portion of their choline requirement; however, the conversion was apparently not adequate to result in a lowering of the liver lipid content comparable to that resulting from feeding 400 mg or more choline/kg of diet. DISCUSSION
The results of this study indicate that the channel catfish can apparently utilize methionine to spare a portion of their need for dietary choline. This observation may explain why we were unable to induce a choline deficiency in a 12-week preliminary study using our standard casein-gelatin diet which contained excess methionine. We were, however, able to induce a choline deficiency with the soybean protein diet because the amount of methionine was reduced to a level which just met the dietary need for methionine; i.e., about 50% of the total sulfur amino acid requirement of the catfish can be provided by methionine and 50% by cystine (Harding et al., 1977). This observation is also consistent with the results of the choline essentiality study conducted by Dupree (1966). He did not observe any significant difference in growth rates of channel catfish fed casein-gelatin diets with and without choline until week 24 of the study. The difference in growth rates was more pronounced at 36 weeks. Perhaps, we would have observed a growth difference in the current study if it had been continued for a much longer period. It appears that certain other dietary factors may affect the ability of the catfish to utilize dietary methionine to spare their need for dietary choline. For example, the addition of excess methionine to the soybean protein diet did not result in a reduction of the liver lipid content to a level comparable to that resulting from feeding 400 mg or more choline/kg of diet (Table 2) ; however, comparable low liver lipids were observed in the preliminary study with the casein-gelatin diet with no added choline. Based on the above observations, it should be emphasized that the choline requirement value for channel catfish will vary depending on the diet fed, and the value of 400 mg choline/kg of diet would only apply to the dietary conditions used in this study. It seems reasonable to suggest, however, that this value of 400 mg choline/kg of diet represents a maximum requirement level because it was determined at the lowest level of dietary methionine that could be used while still meeting the total sulfur amino acid requirement of the catfish. Ogino et al. (1970) also suggested that the methionine content of casein might spare the need for dietary choline. These workers found it necessary to reduce the casein content of their test diet from 54% to 30% in order to induce a choline deficiency in carp. Ketola (1976) demonstrated that methyl donors such as methylaminoethanol and dimethylaminoethanol could be used to re-
place dietary choline in lake trout. Ketola (1976) also summarized several of the differences in growth responses and deficiency signs which have been reported for various fishes fed choline deficient diets. Based on the observations made in the current study, many of the differences in requirement values of choline for various fishes reported by previous workers may be explained by species differences in enzyme activities associated with choline synthesis and degradation, as well as, various dietary factors known to directly affect the rate of choline synthesis. ACKNOWLEDGMENTS
The authors wish to thank Mr. William Tierce, Jr. for his technical assistance during this investigation. Publication number 6656 of the Mississippi Agricultural and Forestry Experiment Station.
REFERENCES Appel, J.A. and Briggs, G.M., 1973. Choline. In: R.S. Goodhart and M.E. Shils (Editors), Modern Nutrition in Health and Disease, 6th edition. Lea and Febiger, Philadelphia, PA, pp. 282-286. Arai, S., Nose, T. and Hashimoto, Y., 1972. Qualitative requirements of young eels Anguilla japonica for water-soluble vitamins and their deficiency symptoms. Bull. Freshwater Fish. Res. Lab., Tokyo, 22: 69-83. Coats, J.A. and Halver, J.E., 1958. Water-soluble vitamin requirements of silver salmon. Special Scientific Report, Fisheries No. 281, U.S. Dept. of Interior, Fish and Wildlife Service, Washington, D.C., 9 pp. Duncan, D.B., 1955. Multiple-range and multiple F tests. Biometrics, 11: l-42. Dupree, H.K., 1966. Vitamins essential for growth of channel catfish, Ictuluruspunctatus. Technical Paper No. 7, Bureau of Sport Fisheries and Wildlife, Washington, D.C., 12 pp. Folch, J., Lees, M. and Stanley, G.H.S., 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem., 226: 497-509. Garling, D.L., Jr. and Wilson, R.P., 1976. Optimum dietary protein to energy ratio for channel catfish fingerlings, lctulu~us punctatus. J. Nutr., 106: 1368-1375. Gatlin, D.M., III and Wilson, R.P., 1984. Dietary selenium requirement of fingerling channel catfish. J. Nutr., 114: 627-633. Halver, J.E., 1957. Nutrition of salmonoid fishes. III. Water-soluble vitamin requirement of chinook salmon. J. Nutr., 62: 225-243. Halver, J.E., 1972. The vitamins. In: J.E. Halver (Editor), Fish Nutrition. Academic Press, New York, NY, pp. 29-103. Harding, D.E., Allen, O.W., Jr. and Wilson, R.P., 1977. Sulfur amino acid requirement of channel catfish: L-methionine and L-cystine. J. Nutr., 107: 2031-2035. Ketola, H.G., 1976. Choline metabolism and nutritional requirement of lake trout (Saloelinus namaycush) . J. Anim. Sci., 43: 474-477. Kitamura, S., Suwa, T., Ohara, S. and Nakagawa, K., 1967. Studies on vitamin requirements of _rainbow trout. II. The deficiency symptoms of fourteen kinds of vitamins. Bull. Jpn. Sot. Sci. Fish., 33: 1120-1125. McLaren, B.A. Keller, E., O’Donnell, D.J. and Elvehjem, C.A., 1947. The nutrition of rainbow trout. I. Studies of vitamin requirements. Arch. Biochem. Biophys., 15: 169-178.
71 Ogino, C., Uki, N., Watanabe, T., Iida, Z. and Ando, K., 1970. B vitamin requirements of carp. IV. Requirements for choline. Bull. Jpn. Sot. Sci. Fish., 36: 1140-1146. Yone, Y., 1975. Nutritional studies of red sea bream. In: K.S. Prince, W.N. Shaw and K.S. Danbert (Editors), Proceedings of the First International Conference on Aquaculture Nutrition, Lewes/Rehoboth, University of Delaware, pp. 34-39.