Nutrient Requirements

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1 Nutrient Requirements Choline Is Required by Tilapia when Methionine Is Not in Excess 1 Craig S. Kasper, M. Randall White* and Paul B. Brown 2 Department of Forestry and Natural Resources, and *Department of Veterinary Pathobiology and Animal Disease Diagnostic Laboratory, Purdue University, West Lafayette, IN ABSTRACT Choline is essential in diets fed to most young vertebrates, but previous studies did not confirm the essentiality of choline in diets fed to tilapia. Two experiments were conducted to evaluate the essentiality of dietary choline in such diets. The basal diet used in both experiments contained 32 g crude protein/100 g diet (10.1 g crude protein from casein and gelatin, and 21.9 g from a crystalline L-amino acid mixture). The total sulfur amino acid (TSAA) concentration of the basal diet was 0.28 g/100 g diet, Met:Cys 89:11. In Experiment 1, a 4 2 design was used in which crystalline L-methionine was added to the basal diet resulting in four levels of TSAA (0.28, 0.50, 0.75 or 1.0 g/100 g diet, Met:Cys 89:11, 94:6, 96:4, or 97:3, respectively). At each level of TSAA, diets also contained either 0 or 1 g choline/kg diet supplied as choline chloride. Weight gain, feed efficiency (FE) and serum methionine concentrations were significantly affected by dietary TSAA concentration, but not by dietary choline concentration or the interaction between TSAA and choline. Weight gain, feed efficiency and serum methionine concentrations indicated that the TSAA requirement was 0.5 g/100 g diet. In the second experiment, diets were formulated to contain either 0.28 or 0.5 g TSAA/100 g diet, Met:Cys 89:11 or 94:6, respectively, and graded levels of choline ranging from 1 to 4 g/kg diet in gradations of 1 g/kg. Dietary methionine significantly affected weight gain and FE, whereas dietary choline significantly affected weight gain, FE and survival, and the interaction of methionine and choline significantly affected weight gain. Fish fed diets containing 0.5 g TSAA/100 g diet and 3 g choline chloride/kg diet exhibited the highest weight gain, feed efficiency and survival. On the basis of these data, it seems clear that juvenile tilapia require choline in certain dietary formulations. J. Nutr. 130: , KEY WORDS: tilapia methionine sulfur amino acid requirements choline Choline is an essential nutrient in diets fed to a wide range of vertebrates including humans (Zeisel 1990), cats (Anderson et al. 1979, Caravalho da Silva et al. 1959), pigs (Lovett et al. 1986, Russett et al. 1979), rats (Kroening and Pond 1967), guinea pigs (Casselman and Williams 1954), rabbits (Hove et al. 1954), dogs (Dutra and McKibbon 1945), poultry (Jukes 1941) and at least nine species of fish (Craig and Gatlin 1996, Griffin et al. 1994b, Hung 1989, NRC 1993). However, Roem et al. (1990a and 1990b) did not establish the essentiality of choline in diets fed to blue tilapia (Oreochromis aureus). Choline is an important intermediate in the catabolic pathway that begins with methionine, thus there are several potential interactions of choline with other dietary constituents (Byington et al. 1972, Miles et al. 1983a and 1983b, Vemury et al. 1980). Those interactions have not been evaluated in fish and may have confounded previous studies with tilapia. Further, there are ontogenetic differences in choline essentiality in vertebrates. As adults, some vertebrates are able to synthesize sufficient choline to meet metabolic needs (Anderson et al. 1979, Casselman and Williams 1954, Dutra and McKibbon 1945, Kroening and Pond 1967, Russett et al. 1979). However, juveniles of several species lack phosphatidylethanolamine-n-methyl 1 Supported by the Purdue Agricultural Research Programs (IND ) contribution number To whom correspondence should be addressed. transferase activity and cannot synthesize sufficient choline, as phosphatidylcholine. Further, synthesis of choline from other sources is insufficient to meet the animals needs (Combs 1992, Hove et al. 1954, Jukes 1941 and 1945, Molitoris and Baker 1976). Thus, dietary choline is essential for maximum growth and health regardless of the level of dietary methionine or other methyl donors. In the previous studies with tilapia, methionine concentrations were 1.0 and 1.13 g/100 g diet, amounts that are well in excess of the requirement of 0.75 g/100g for Nile tilapia (O. niloticus) with an initial weight of 62 mg (Santiago and Lovell 1988). The excessive methionine concentration may have flooded the sulfur amino acid catabolic pathway with sufficient metabolic precursors for synthesis of choline, thereby masking any effect from dietary supplementation of choline. Most diets for tilapia will not contain excessive levels of methionine because this essential amino acid tends to be one of the first limiting in diets fed to fish. The most common clinical signs and lesions associated with choline deficiency in fish are decreased weight gain and feed efficiency, hepatic lipidosis and renal hemorrhage (Chan 1991, Griffin et al. 1994b, Halver 1957, Ketola 1976, McLaren et al. 1947, Ogino et al. 1970, Wilson and Poe 1988). However, these findings vary across species. Graded additions of choline in diets fed to red drum (Sciaenops ocellatus) resulted in an accumulation of liver lipid (Craig and Gatlin 1991). Addition of choline to purified diets fed to rainbow trout (Oncorhynchus mykiss; Rumsey 1991) and yellow perch (Perca flavescens, un /00 $ American Society for Nutritional Sciences. Manuscript received 11 May Initial review completed 18 June Revision accepted 18 October

2 ESSENTIALITY OF CHOLINE IN THE DIET OF TILAPIA 239 TABLE 1 Composition of the basal diet fed to juvenile tilapia in both experiments Ingredient1 Amount g/kg dry diet Casein 90.0 Gelatin 18.0 Amino acid premix L-Methionine 0.0 L-Glutamic acid 7.2 Dextrin Mineral premix Vitamin premix3 3.3 Ascorbic acid 2.0 Carboxymethylcellulose 20.0 Menhaden oil 70.0 Cellulose Casein, gelatin, dextrin, cellulose, carboxymethylcellulose, crystalline L-amino acids and vitamins were obtained from U.S. Biochemical (Cleveland, OH). Reagent-grade minerals were obtained from Sigma Chemical (St. Louis, MO), and menhaden oil was obtained from Zapata Proteins, (Reedville, VA). 2 Composition shown in Table 2. 3 Mineral and vitamin premixes were the same as those reported by Griffin et al. (1992). published data from our laboratory) did not have a significant effect on liver lipid concentrations. Choline requirements of fish range from 0.05 g/kg diet for juvenile rainbow trout (Poston 1991) to 3.4 g/kg diet for sturgeon, Acipenser transmontanus, (Hung 1989). An inverse relationship exists between the dietary choline requirement and size of rainbow trout. Requirements were 3.0, 0.813, and between 0.05 and 0.1 g/kg diet for trout weighing 0.12, 1.4, 3.2 and 3.5 g, respectively (McLaren et al. 1947, Poston 1991, Rumsey 1991). Thus, the ontogenetic differences observed in some terrestrial vertebrates might be present in trout. The purposes of the following two studies were to reexamine the essentiality of choline in diets fed to tilapia and investigate the potential interaction between methionine and choline. MATERIALS AND METHODS Fish and animal husbandry. Juvenile Nile tilapia were acquired from a private producer in Ladoga, IN, and transported to the Purdue University Aquaculture Research Facility. Fish were acclimated to laboratory conditions for 2 wk before initiation of the experiment. Experimental protocols used in these studies were approved by the Purdue Animal Care and Use Committee (PACUC No , Nutritional Studies with Aquatic Animals, Principal Investigator Qualifications No. BRO-249.) The recirculating experimental culture system that was used in both experiments was the same as that previously described by Griffin et al. (1992). The diurnal light:dark cycle was 16 h light:8 h dark, and temperature was maintained at 28 C. Fish (n 15) were randomly selected and stocked into each aquarium, and dietary treatments were randomly assigned to triplicate aquaria in both experiments. Fish were acclimated to their new environment and to a crystalline amino acid diet for 2 wk before initiation of the experiments. Initial fish weights ranged from 3.1 to 3.4 g in Experiment 1 and 2.6 to 3.0 g in Experiment 2. Fish were fed 5 g feed/(100 g body weight d) divided into two equal meals. Fish were weighed every 14 d to adjust food allotments. Each experiment lasted 8 wk. Diets. The basal diets used in both experiments were identical and were formulated to contain 32 g crude protein/100 g diet (Table 1). Casein and gelatin provided 10.1 g crude protein/100 g diet, and the methionine- and cyst(e)ine-free crystalline L-amino acid mixture (Table 2) provided 21.9 g crude protein/100 g diet. Total sulfur amino acid (TSAA) concentration provided from the casein and gelatin was 0.28 g/100 g diet (0.25 g methionine and 0.03 g cyst(e)ine/100 g diet). The basal diet contained 7 g menhaden oil and 30 g dextrin/100 g, resulting in an energy/protein ratio of 63 kj/g protein. A choline-free, but otherwise complete vitamin premix and a nutritionally complete mineral premix were added to diets in both experiments (Griffin et al. 1992). These formulations are similar to those used to quantify the essential amino acid requirements of tilapia (Santiago and Lovell 1988). Diets used in Experiment 1 contained graded additions of L- methionine, resulting in TSAA concentrations of 0.28, 0.50, 0.75 or 1.0 g/100 g of the dry diet, methionine/cyst(e)ine (Met/Cys) 89:11, 94:6, 96:4 or 97:3, respectively, and either 0 or 1 g/kg choline provided as choline chloride. In Experiment 2, the eight experimental diets contained either 0.28 or 0.50 g TSAA/100 g dry diet, Met/Cys, 89:11 or 94:6, respectively, and graded concentrations of choline (1, 2, 3 or 4 g/kg dry diet). L-Methionine was added to the diets as an isonitrogenous replacement for glutamic acid. Choline chloride was added at the expense of cellulose. Dry ingredients, except for choline chloride, were mixed in a V-mixer (Patterson-Kelly, East Stroudsburg, PA), transferred to a Hobart mixer (Hobart, Troy OH) and mixed with lipid and water. Before pelleting, diets were adjusted to ph (Wilson et al. 1977) with saturated sodium hydroxide. Choline was then added in a water carrier (Halver 1989) and mixed into the diets. All diets were then pelleted, dried at 60 C in a forced-air oven and stored under air-tight conditions at 20 C until required for feeding (Griffin et al. 1992). Serum and liver collection. At the conclusion of each experiment, all fish were weighed 24 h after the final feeding and then killed with tricane methanesulfonate (Argent Chemical, Redmond, WA). Livers from three randomly chosen fish in each replicate were removed and pooled for determination of liver lipid content. Liver lipid was quantified by chloroform/methanol extraction (Folch et al. 1957) in a Soxhlet extractor. Livers from three randomly selected fish from each treatment group were removed and preserved in 100 ml/l neutral buffered formalin solution for subsequent histologic evaluation. Tissue sections were prepared using routine techniques (Sheehan and Hrapchak 1980). Paraffin-embedded tissues were sectioned TABLE 2 Amino acid composition of basal diet fed to juvenile tilapia in both experiments1 Amino acid Supplied by casein and gelatin Supplied by amino acid mixture g/kg diet Total L-Methionine L-Cysteine L-Arginine L-Histidine L-Isoleucine L-Leucine L-Lysine L-Phenylalanine L-Tyrosine L-Threonine L-Tryptophan L-Valine Amino acid mixture provided 126 g/kg each of L-aspartic acid, L-glutamic acid, L-serine and L-proline. 2 Arginine and lysine were provided as L-arginine HCl and L-lysine HCl, respectively.

3 240 KASPER ET AL. TABLE 3 Initial weight, weight gain, feed efficiency, survival, liver lipid concentration and serum methionine concentration of juvenile tilapia fed graded levels of total sulfur amino acids and 0 or 1 g/kg choline chloride for 8 wk (Experiment 1)1 Total sulfur amino acids Choline Initial weight Weight gain2 Feed efficiency3 Survival Liver lipid4 Serum methionine g/100 diet g/kg diet g g/8 wk g gain/g feed % g/100 g mol/l b 0.28b b b 0.27b b a 0.50a b ab 0.41a b a 0.47a a a 0.51a a ab 0.36ab a ab 0.38ab a Pooled SEM Probability Methionine Choline Methionine choline Values are means of three replications. 2 Means in the same column with different superscripts were significantly different. 3 Feed efficiency was calculated as g wet gain/g dry feed. 4 Liver lipid was calculated on a dry matter basis. 5 Probability of treatment differences as determined by ANOVA. at 5 6 m, stained with hematoxylin and eosin, and subjected to microscopic evaluation. At the end of each experiment, blood was collected from a minimum of three anesthetized fish in each aquarium and pooled by dietary replicate for determination of serum methionine concentrations. Serum was obtained from pooled blood samples by centrifuging at 3000 g for 15 min and deproteinized with HPLC-grade acetonitrile. Serum amino acids were then subjected to precolumn derivitization with phenylisothiocyanate, and separated and quantified using a Waters Pico Tag system (Waters Chromatography Division, Millipore, Milford, MA) according to the methods of Sarwar and Botting (1990). Statistical analyses. Data were analyzed with the use of two-way ANOVA, using each aquarium as the experimental unit. Accepted level of significance was P All statistical analyses were performed using software from the Statistical Analysis System (SAS 1996). If differences in treatment means were detected by ANOVA, then a Student-Neuman-Keuls test was used to separate means. RESULTS In Experiment 1, graded additions of L-methionine significantly affected weight gain, feed efficiency (FE) and serum methionine concentrations of juvenile tilapia, whereas survival and liver lipid concentrations were not significantly affected (Table 3). Weight gain and FE of fish increased as dietary concentrations of TSAA increased from 0.28 to 0.50 g/100 g dry diet. Fish fed dietary TSAA concentrations 0.50 g/100 g dry diet did not exhibit significant improvements in either variable. Serum methionine concentrations were not significantly different between fish fed the two lowest concentrations of TSAA, but were significantly higher in fish fed dietary TSAA concentrations 0.50 g/100 g diet. None of the variables measured were significantly affected by dietary choline or the interaction between choline and methionine. In Experiment 2, weight gain and FE of tilapia was significantly affected by dietary methionine (Table 4). Values for fish fed 0.28 g/100 g and any level of dietary choline were significantly lower than those of fish fed 0.50 g/100 g and 1 3 g/kg choline. Weight gain and FE of fish fed 0.50 g/100 g TSAA and 4 g/kg choline were significantly lower than those in any other dietary treatment. Dietary choline significantly affected weight gain, FE and survival of tilapia. Regardless of the dietary TSAA concentration, fish fed 1 and 2 g/kg choline exhibited significantly lower weight gain and FE than fish fed 3 g/kg. Fish fed 4 g/kg gained significantly less weight and exhibited significantly lower FE than fish fed 3 g/kg. Fish fed 0.28 g TSAA/100 g and 3 g choline/kg, and 0.50 g TSAA/100 g and 4 g choline/kg exhibited significantly lower survival than those in other dietary treatments. The interaction between methionine and choline significantly affected weight gain of tilapia, but not other variables. Fish fed diets containing 0.50 g TSAA/100 g diet and 4 g choline chloride/kg diet exhibited significantly lower FE. Hepatic lipid or serum methionine concentrations of fish were not significantly affected by dietary methionine, choline or their interaction. In both experiments, histopathologic evaluation of livers revealed variable amounts of both macrovesicular and microvesicular degeneration of fish from all groups. However, no differences were discernible among treatment groups or between studies. DISCUSSION On the bases of weight gain, FE and serum methionine concentrations, the quantitative requirement for dietary total sulfur amino acids is 0.5 g/100 g diet or 1.6 g/100 g protein for tilapia with an initial weight of 3 g. This is lower than the requirement reported by Santiago and Lovell (1988) of 1.0 g/100 g diet. However, the fish used by Santiago and Lovell (1988) weighed 62 mg at the beginning of their experiment. Decreasing nutritional requirements are relatively common in vertebrates; thus, differences in initial weight may explain the differences in values. On the basis of the results observed in Experiment 1, it was unclear whether dietary choline was essential. In Experiment 2, when juvenile fish were fed diets that contained minimal sulfur amino acid concentrations and

4 ESSENTIALITY OF CHOLINE IN THE DIET OF TILAPIA 241 TABLE 4 Initial weight, weight gain, feed efficiency, survival, liver lipid and serum methionine concentrations of juvenile tilapia fed either 0.28 or 0.50 g/100 g total sulfur amino acids and graded levels of choline for 8 wk (Experiment 2)1 Total sulfur amino acids Choline Initial weight Weight gain2 Feed efficiency3 Survival Liver lipid4 Serum methionine g/100 diet g/kg diet g g/8 wk g gain/g feed % g/100 g mol/l by 0.21by 89x by 0.21by 94x bx 0.22bx 81x bz 0.22bz 94y ay 0.29ay 92x ay 0.36ay 87x ax 0.41ax 100x az 0.09az 66y Pooled SEM Probability Methionine Choline Methionine choline Values are means of three replications. 2 Means in the same column with different superscripts were significantly different. Differences denoted with a or b refer to total sulfur amino acids; differences denoted by x, y or z refer to choline. 3 Feed efficiency was calculated as g wet gain/g dry feed. 4 Liver lipid was calculated on a dry matter basis. 5 Probability of treatment differences as determined by ANOVA. higher concentrations of choline, it was clear that dietary choline was required. Within sulfur amino acid levels, weight gain increased as choline concentration increased from 1 to 3 g/kg diet, then decreased in fish fed 4 g/kg diet. On the bases of weight gain and feed efficiency in fish fed 0.5 g/100 g TSAA, the requirement for choline in juvenile tilapia is 3 g/kg diet. Hepatic lipid concentrations were not responsive to dietary treatments in these studies, although they have been responsive to dietary choline concentrations in some other species of fish. The exceptions have been red drum (Craig and Gatlin 1991), rainbow trout (Rumsey 1991) and yellow perch (unpublished data from our laboratory). Despite the reduction in total liver lipids observed in Experiment 2, histopathologic evaluation indicated the presence of macrovesicular and microvesicular degeneration in fish from all treatments, indicative of intracellular lipid and glycogen accumulations, respectively. This has been a relatively common finding in nutritional studies with fish (Brown et al. 1993). However, association between microscopic lesions and hepatic function is lacking in fish. The dietary TSAA requirement determined in the first experiment is lower than most other TSAA requirements for fish, which range from 1.9 to 4.0 g/100 g dietary protein. However, the requirement estimate based on weight gain and FE was confirmed with serum methionine values. Our ability to confirm dietary essential amino acid requirements of fish by serum amino acid concentrations has not been universally successful (Cowey et al. 1992); however, it is considered an important response variable (Griffin et al. 1994a, Harding et al. 1977). More importantly, the requirement estimate determined in these studies could be an overestimate. If tilapia can use absorbed methionine as a source of choline, the diets used in the first study were deficient in choline, and a higher proportion of the methionine would have been used to supply choline. Further, the TSAA requirement for vertebrates can be lower when a maximal proportion of cyst(e)ine is supplied in the diet (Chung and Baker 1992). Although the TSAA requirement for tilapia determined in these studies was on the lower end of existing values for fish, the dietary choline requirement was comparatively high. Most choline requirements for fish range from 0.05 to 3.4 g/kg diet (McLaren et al. 1947, NRC 1993, Poston 1991, Rumsey 1991); thus the value for tilapia is on the high end of that range. In our second study, dietary TSAA concentration was maintained at the minimal requirement, which diminishes the amount that could be used for choline synthesis. Thus, our requirement estimate could be viewed as maximal, appropriate for dietary formulations that contain a minimal amount of potential precursors for choline synthesis. Sulfur amino acids in formulations for fish are often first limiting (Brown et al. 1997), and excessive levels are rare in most formulations. The dietary choline requirement might be lower if excess sulfur amino acids or other compounds are provided in the diet. However, it seems clear that a source of choline is required in certain diets fed to tilapia. Further, it appears that choline is toxic when fed at 4 g/kg diet regardless of the level of TSAA. Weight gain, FE and survival were clearly lower in fish fed 4 g choline/kg diet compared with fish fed other levels in either study. Byington (1978) reported that the 50% lethal dose (LD 50 ) for rats was g choline chloride/kg diet. Zeisel and Blusztajn (1994) reported that excess dietary choline chloride depressed growth and increased mortality in rats, and caused diarrhea in humans. The toxicity was attributed to an ionic imbalance caused by the chloride component. Choline toxicity was reported by Griffin et al.(1994a) when hybrid striped bass where fed 8 g/kg of choline bitartrate. Fish in that experiment showed no adverse affects to lower concentrations of the bitartrate form or to the chloride form at dietary concentrations of 0.25 to 8 g/kg. In the previous studies with tilapia, initial weight of fish was g and dietary choline concentrations were 0 2 g/kg diet (Roem et al. 1990a and 1990b). Initial fish size was similar in those studies compared with this study; thus, age or size of fish does not seem to have contributed to the previous attempts to establish the essentiality of choline. Dietary TSAA

5 242 KASPER ET AL. concentrations were well above either of the established requirements for juvenile tilapia and may have provided sufficient precursor to mask any effects of dietary choline supplementation. However, it is also clear that dietary choline concentrations evaluated in the previous studies were below those that resulted in maximal response in our studies. Roem et al. (1990b) evaluated graded levels of choline that included 1 and 2 g/kg diet. In agreement with our results, fish fed those diets did not exhibit significantly different weight gain or FE. Thus, the inability to establish the essentiality of dietary choline in tilapia appears to have been the result of excessive sulfur amino acid precursors, insufficient dietary levels of choline or a combination of the two factors. LITERATURE CITED Anderson, P. A., Baker, D. H., Sherry, P. A. & Corbin, J. E. (1979) Cholinemethionine interrelationships in feline nutrition. J. Anim. Sci. 49: Brown, P. B., Griffin, M. 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