Nutrient Requirements

Transcription

1 Nutrient Requirements Dietary Choline Requirement of Juvenile Yellow Perch (Perca flavescens) 1 Ronald G. Twibell and Paul B. Brown 2 Purdue University, Department of Forestry and Natural Resources, West Lafayette, IN ABSTRACT We conducted an 11-wk feeding trial to determine the dietary choline requirement of juvenile yellow perch (Perca flavescens) and to investigate whether dietary phosphatidylcholine (PC) could meet this requirement. Six dietary treatments contained choline concentrations of 0.11, 0.23, 0.34, 0.75, 1.22 or 3.37 g/kg diet. Two additional diets contained 31 g of lecithin/kg diet, with or without supplemental choline chloride (4.0 g choline/kg diet). The total sulfur amino acid concentration was maintained at 1.0 g/100 g diet (methionine/cyst(e)ine, 49:51). Diets were fed to satiation twice daily to triplicate groups of yellow perch initially weighing 16.0 g/fish. Weight gain, feed intake and carcass proximate composition were significantly affected by dietary choline. Weight gains and feed intakes increased as dietary choline increased from 0 to 0.75 g/kg. Both values tended to plateau in fish fed dietary choline levels above 0.75 g/kg. Broken-line analyses of weight gain and feed intake data indicated the dietary choline requirement was and g/kg diet, respectively. Hepatic lipid concentrations and feed efficiency values were not significantly different. Whole-body fat concentrations increased significantly, whereas ash levels decreased significantly in fish fed increasing levels of dietary choline. Weight gain and feed intake of fish fed diets containing PC were not significantly different from fish fed 0.75 g/kg of dietary choline. However, hepatic lipid concentrations were significantly higher in fish fed the diet containing PC and no choline chloride. Thus, yellow perch require a maximum of g of choline/kg diet for maximum growth and this requirement may potentially be met with 31 g of lecithin/kg diet. J. Nutr. 130: 95 99, KEY WORDS: yellow perch phosphatidylcholine choline fish nutrition Choline is considered a vitamin in diets fed to most young vertebrates (Canty and Zeisel 1994, NRC 1993). However, the study of choline nutrition is confounded by other dietary components, including amino acids, lipids and other vitamins. The most commonly studied interaction has been with amino acids. Kroening and Pond (1967) reported that dietary methionine concentrations only slightly in excess of those required for maximum growth eliminated the need for dietary choline in pigs and rats. Excess methionine was also shown to eliminate the need for choline in diets fed to young cats (Anderson et al. 1979). However, excess methionine replaced only a small portion of the dietary choline requirement of young chicks, and excess dietary choline had only a minimal capacity to reduce the chick s requirement for dietary methionine (Baker et al. 1982). Fewer studies have been conducted with fish. We recently identified a significant interaction between methionine and choline in diets fed to tilapia (unpublished data). Rumsey (1991) reported that a dietary source of betaine spared 50% of the choline requirement of rainbow trout (Oncorhynchus mykiss). Excess methionine was not able to supply the choline requirement in trout. The other potentially confounding issue in fish has been phosphatidylcholine (PC) 3 or lecithin. In contrast to most terrestrial vertebrates (Overland et al. 1993a, 1993b and 1994), dietary PC improved growth rates and feed efficiency in several species of fish (Craig and Gatlin 1997, Hung 1989, Hung and Lutes 1988, Poston 1990, Poston 1991a and 1991b). However, lecithin contains choline, fatty acids and a glycerol backbone. Responses to lecithin could, therefore, be responses to one or more of the constituent components (Canty and Zeisel 1994, Hung and Lutes 1988) particularly in species for which few nutritional requirements have been quantified. Further, sources of PC used in previous studies were typically food grade and contained numerous other components. At this time, probably only the studies with rainbow trout and red drum provide meaningful indications of the benefits of lecithin in diets fed to fish as most of the potentially confounding nutritional requirements were quantified prior to evaluation. Brown et al. (1996) identified purified diets that could be used in nutritional research with yellow perch. They reported that a purified diet containing crystalline amino acids as the primary source of crude protein supported weight gain that was not significantly different from that of fish fed the best commercial diet. This dietary formulation has been used in subsequent research in our laboratory to estimate the dietary arginine (Twibell and Brown 1997), lysine (unpublished data) and 1 Supported by the Purdue Agricultural Research Programs contribution number This publication is a result of work sponsored by the North Central Regional Aquaculture Center Program under grant number from the U.S. Department of Agriculture. The U.S. Government and the North Central Regional Aquaculture Center are authorized to produce and distribute reprints for governmental purposes notwithstanding any copyright notation appearing hereon. 2 To whom correspondence should be addressed. 3 Abbreviations used: PC, phosphatidylcholine; /00 $ American Society for Nutritional Sciences. Manuscript received 2 April Initial review completed 28 May Revision accepted 7 October

2 96 TWIBELL AND BROWN TABLE 1 Composition of basal diet fed to juvenile yellow perch Ingredient1 Amount g/kg dry mixture Casein 90.0 Gelatin 18.0 Amino acid mixture Dextrin Mineral premix Vitamin premix4 3.3 STAY-C Carboxymethylcellulose 20.0 Cellulose Menhaden oil Vitamins, casein, gelatin, dextrin, carboxymethylcellulose, crystalline L-amino acids and cellulose were acquired from U.S. Biochemical (Cleveland, OH). Purified soybean lecithin, containing 90 g/100 g of phosphatidylcholine and 3 g/100 g of lysophosphatidylcholine, was provided by the American Lecithin Company (Oxford, CT). Ascorbic acid, as L-ascorbyl 2-polyphosphate, was obtained from Roche Inc. (Nutley, NJ). Menhaden oil and reagent-grade minerals were obtained from commercial suppliers (Zapata-Protein, Reedville, VA, and Sigma Chemical, St. Louis, MO, respectively). 2 Composition of the amino acid mixture is presented in Table 2. 3 Mineral premix consisted of (g/kg of premix): Na 2 SeO 3, 0.4; CaCO 3, 350; NaH 2 PO 4 HOH, 200; KH 2 PO 4, 200; MgSO 4 7HOH, 10; MnSO 4 HOH, 2; CuCl 2 2HOH, 1; ZnSO 4 7HOH, 2; FeSO 4 7HOH, 2; NaCl, 12; KI, 0.1; CoCl 2 6HOH, 0.1; Na 2 MoO 4 2HOH, 0.5; AlCl 3 6HOH, 1; and KF, 1. 4 Vitamin premix supplied the diets with (mg/kg dry diet): retinyl acetate, 40; cholicalciferol, 0.1; DL- -tocopheryl acetate, 80; menadione, 15; niacin, 168; riboflavin, 22; pyridoxine HCl, 40; thiamin mononitrate, 45; D-Ca pantothenate, 102; biotin, 0.4; folic acid, 10; cyanocobalamin 0.04; and, inositol Contained 25 g/100 g of ascorbic acid equivalents, as L-ascorbyl- 2-polyphosphate. total sulfur amino acid requirements (unpublished data) of yellow perch. The same basic dietary formulation was used in the present study to estimate the dietary choline requirement of yellow perch and the ability of PC to meet the choline requirement. MATERIALS AND METHODS Fish and animal husbandry. All-female yellow perch were obtained from a commercial producer (Coolwater Farms, Cambridge, WI) and transported to the Purdue University Aquaculture Research Facility. All fish were acclimated to laboratory conditions for 6 mo prior to initiation of the experiment. Procedures used during transport, quarantine and experimental period were approved by the Purdue Animal Care and Use Committee (PACUC No , Nutritional Studies with Aquatic Animals, Principal Investigator Qualification No. BRO-249). The closed recirculating system used in this experiment contained 24 individual 38-L aquariums. The experimental system was equipped with two submerged filtration tanks for solid material removal and denitrification of the water. Water was pumped through a sand filter to each aquarium at a rate of 1 L/min and was maintained at 20 1 C throughout the experiment. The diurnal light/dark cycle of the aquaculture facility remained at 16 h light/8 h dark throughout the study. Groups of 20 randomly chosen fish were stocked into each aquarium. Fish were acclimated to the experimental system and their respective diets for 3 wk prior to the experiment. During the first 2 wk of the acclimation period, all fish were fed a commercial diet (Bioproducts, Warrenton, OR). During wk 3, all fish were offered their respective experimental diets, which had been randomly assigned to triplicate aquariums. Following the acclimation period, the number of fish in each tank was reduced to 13 so that the total weight of fish in each tank was g. Fish were fed to satiation twice daily during the 11-wk experiment. Water quality was monitored daily and was within acceptable limits throughout the experiment. Dissolved oxygen concentrations were not below 6.0 mg/l, ammonia-nitrogen concentrations were not 0.22 mg/l, and nitrite-nitrogen did not exceed 0.07 mg/l at any point during the study. Diets. The basal diet was formulated to provide 33.3 g of crude protein/100 g diet (Table 1). Casein and gelatin served as intact protein sources and provided a total of 10.1 g of crude protein/100 g diet. An L-amino acid mixture (Table 2) supplied the remaining 23.2 g of crude protein/100 g diet. The L-amino acid mixture was formulated so the basal diet contained 1.41 g of arginine/100 g diet (Twibell and Brown 1997) and 1.10 g of lysine/100 g diet (unpublished data from our laboratory), thus meeting the dietary requirement of yellow perch for these amino acids. The L-amino acid mixture was also formulated to provide 1.0 g of TSAA/100 g diet as a ratio of 49:51 met/cys, which was determined to be near the requirement for this species (unpublished data). The remaining dietary essential amino acid concentrations met or exceeded the highest known requirements for fish (NRC 1993). The basal diet contained 6.0 g of lipid (menhaden oil)/100 g diet and 33.5 g of carbohydrate (dextrin)/ 100 g diet (Cartwright 1998). Six dietary treatments were formulated to contain choline concentrations of 0, 0.25, 0.50, 1.00, 2.00 or 4.00 g/kg diet as choline chloride. The analyzed dietary choline concentrations were 0.11, 0.23, 0.34, 0.75, 1.22 and 3.37 g/kg diet, as determined by standard methods (AOAC 1964). Two additional diets were supplemented with 31 g of lecithin/kg diet. One of those diets was supplemented with 4.00 g of choline/kg diet; the other was not supplemented with choline. A level of 31 g of lecithin/kg diet provided the equivalent of 4.00 g of choline/kg diet from the choline moieties of PC and lysophosphatidylcholine, based on the guaranteed analysis provided by the supplier. The analyzed choline concentration of the diet containing 31 g of lecithin/kg diet and no choline chloride was 4.65 g of choline/kg diet. The analyzed choline concentration of the diet containing 31 g of lecithin/kg diet and 4.00 g of choline/kg diet was g of choline/kg diet. Choline chloride was added to the basal diet at the expense of cellulose. Lecithin was added at the expense of menhaden oil. Dry ingredients were thoroughly mixed in a twin-shell V-mixer Amino acid TABLE 2 Amino acid composition of basal diet fed to juvenile yellow perch1 Supplied by casein and gelatin g/kg dry diet Supplied by amino acid mixture Total L-Arginine L-Histidine L-Isoleucine L-Leucine L-Lysine L-Methionine L-Phenylalanine L-Threonine L-Tryptophan L-Valine L-Cysteine L-Tyrosine Amino acid mixture also provided 25.9 g/kg of L-aspartic acid, -glutamic acid, -serine and -proline. 2 Arginine and lysine were provided as L-arginine-HCl and L-lysine- HCl, respectively, but values shown represent only the contribution of the amino acid fraction.

3 CHOLINE IN PERCH 97 TABLE 3 Initial weight, relative weight gain, feed efficiency, feed intake and total liver lipid concentration of juvenile yellow perch fed various levels of choline or lecithin1,2 Choline Dietary treatment Lecithin3 Initial weight Weight gain Feed efficiency Feed intake Total liver lipids g/kg g/fish g/100 g initial weight g gain/g dry feed g/fish g/100 g dry liver d e 30.1b c,d d,e 30.4b b,c c,d 29.9b a,b b,c 33.3b a,b a,b 29.8b a a 34.3b 4.00 (10.12) a,b a,b 38.1b 0.00 (4.65) a,b a,b 49.4a Pooled SEM Probability Means in the same column with different superscripts were significantly different as determined by ANOVA and Student-Newman-Keuls test. 2 Values are means of three replications. 3 Lecithin contained 90 g/100 g of phosphatidylcholine and 3 g/100 g of lysophosphatidylcholine. 4 Concentrations of choline and lecithin in these diets were expressed as a function of the supplemental concentrations added to the respective diets. Values in parentheses are analyzed choline concentrations. (Patterson-Kelly, East Stroudsburg, PA). Diets were then transferred to a Hobart mixer (Hobart Corporation, Troy, OH) where choline chloride was added to the diet in a water carrier. Lipid and additional water were then added to the dry ingredients and mixed. For the diets containing lecithin, the material was heated to a liquid and added to the dry ingredients prior to mixing. Diets were adjusted to ph with saturated sodium hydroxide (Wilson et al. 1977) and pelleted. The diets were air-dried for 48 h then stored under air-tight conditions at 20 C until needed. Sample collection and analysis. All fish were anesthetized(tricaine methanesulfonate; Argent Chemical, Redmond, WA) and weighed 24 h after the final feeding. Three randomly chosen fish were collected from each dietary replicate and frozen at 20 C for subsequent proximate analysis. Whole-body moisture was determined by drying fish for 24 h in a forced-air oven at 100 C. Crude protein was estimated from whole-body nitrogen values, which were determined in an elemental nitrogen analyzer (LECO Corporation, St. Joseph, MI). Ash content was determined by incinerating samples at 600 C for 24 h in a muffle furnace. Whole-body lipid concentration was determined as described by Folch et al. (1957). Livers from three randomly chosen fish in each dietary replicate were removed and weighed for calculation of relative liver weight (liver weight 100/body weight). The livers were then frozen at 20 C for subsequent determination of total lipids using the method of Folch et al. (1957). Visceral fat was removed from an additional group of three randomly chosen fish in each dietary replicate for calculation of intraperitoneal fat (IPF) ratio (IPF 100/body weight). Statistical analyses. Data were analyzed as a completely randomized design using each aquarium as an experimental unit and dietary treatment as the independent variable. The data were analyzed as two distinct sets: one to establish the quantitative dietary choline requirement and one to compare the dietary treatments containing lecithin to those containing choline. In the first statistical analysis, only data from fish fed graded levels of choline were included in the data set and broken-line analyses of weight gain and feed intake data were used to estimate the dietary choline requirement (Robbins et al. 1979). In the second analysis, all data were included and Student- Neuman-Keuls test separated mean values when significant differences were detected by ANOVA. Both data sets were analyzed with the Statistical Analysis System (SAS 1990). Accepted level of significance was RESULTS There were no mortalities during the experiment. Additions of choline chloride to the basal diet significantly increased weight gain and feed intake of juvenile yellow perch (Table 3). Broken-line analysis of weight gain data indicated the dietary choline requirement of yellow perch was g/kg diet. Broken-line analysis of feed intake data indicated that the dietary choline requirement was g/kg diet. Weight gains of fish fed the two diets containing PC were not significantly different from each other, but were significantly higher than those of fish fed the basal diet or the diet containing 0.23 g of choline/kg diet (Table 3). Weight gain of fish fed either diet containing lecithin was not significantly different from fish fed 0.34 g of choline/kg diet or higher concentrations. Feed intake of fish fed diets with lecithin was significantly higher than that of fish fed 0.34 g of choline/kg diet and lower concentrations, but not significantly different from fish fed 0.75 g of choline/kg and higher concentrations. There were no significant differences in feed efficiency among fish fed any level of choline or PC. Choline or PC did not significantly affect relative liver weight, and values ranged from 1.2 to 4.4 (data not shown). There were no significant differences in total liver lipid concentrations among fish fed any level of dietary choline (Table 3). However, fish fed the diet with lecithin and deficient in choline exhibited significantly higher liver lipid concentrations compared to fish fed any of the other diets, including those fed lecithin with 4.00 g of choline/kg diet. Dietary choline and PC significantly affected carcass composition of yellow perch (Table 4). While ANOVA indicated significant differences in IPF, Student-Neuman Keuls test did not differentiate among means. Choline or PC did not significantly affect moisture or protein content of the fish. Wholebody fat concentrations were significantly different and generally increased in fish fed increasing concentrations of choline. Fish fed diets containing lecithin, as well as fish fed the diet containing 1.22 g of choline/kg diet, had significantly higher whole-body fat concentrations compared to fish fed the

4 98 TWIBELL AND BROWN TABLE 4 Carcass composition of juvenile yellow perch fed various levels of choline or lecithin1,2 Choline Dietary treatment Lecithin3 Intraperitoneal fat ratio Moisture Crude protein Fat Ash g/kg g/100 g g/100 g dry carcass wet body c 19.5a b,c 19.4a,b a,b,c 18.8a,b,c b,c 18.5a,b,c a,b 17.9b,c a,b,c 17.4c 4.00 (10.12) a 17.6c 0.00 (4.65) a,b 18.2a,b,c Pooled SEM Probability Means in the same column with different superscripts were significantly different as determined by ANOVA and Student-Newman-Keuls test. 2 Values are means of three replications. 3 Lecithin contained 90 g/100 g of phosphatidylcholine and 3 g/100 g of lysophosphatidylcholine. 4 Concentrations of choline and lecithin in these diets were expressed as a function of the supplemental concentrations added to the respective diets. Values in parentheses are analyzed choline concentrations. basal diet. Whole-body ash levels were significantly different among dietary treatments and decreased as dietary choline concentration increased. Fish fed the diets containing lecithin and 4.00 g of choline/kg had significantly lower whole-body ash concentrations compared to fish fed the basal diet. DISCUSSION This study indicates that a dietary source of choline is essential for juvenile yellow perch when sulfur amino acid concentrations are not in excess of the requirement. The quantitative requirement for 16 g yellow perch is to g of choline/kg diet supplied as choline chloride. Because the dietary TSAA concentration was maintained near the required level (1.0 g/100 g diet), these estimates should represent the maximum dietary choline requirement for yellow perch weighing 16 g. Dietary choline requirements of rainbow trout range from to 4.00 g of choline/kg diet and are apparently related to initial weight of the fish (McLaren et al. 1947, Poston 1991c, Rumsey 1991). Dietary choline requirements of other fish are 1.00 g/kg for 5.0 g lake trout (Salvelinus namaycush) (Ketola 1976), g/kg for 34.1 g white sturgeon (Hung 1989), 0.59 g/kg for 5.5 g red drum (Craig and Gatlin 1996), 0.50 g/kg for 10.5 g hybrid striped bass (Morone saxatilis x M. chrysops) (Griffin et al. 1994) and 0.40 g/kg for 5.6 g channel catfish (Ictalurus punctatus) (Wilson and Poe 1988). Although comparisons of dietary choline requirements among species are difficult due to differences in initial weight of fish, dietary composition and experimental conditions, apparently, the estimates obtained in the present study are within the range of dietary choline requirements for fish, which are 0.05 to 4.00 g/kg diet. Weight gain of yellow perch fed the diets containing lecithin, with or without choline, was not different from that of fish fed at least 0.34 g of choline/kg diet. This indicates that yellow perch can use lecithin as a source of choline for growth. Further, perch likely have some form of phospholipase. Apparently, there are no reports of phospholipase activity in the gastrointestinal tract of fish, but there are reports of cellular forms in muscle (Jonas and Bilinski 1967), liver (Neas and Hazel 1984) and whole larval fish (Evans et al. 1998). However, since there was no difference in weight gain between fish fed the diets containing lecithin, it appears that lecithin provides no additional benefit when diets contain choline at approximately six times the dietary requirement. Furthermore, liver lipid concentration was significantly higher in fish fed lecithin and deficient in choline. Further studies should be conducted to evaluate potential interactions of dietary choline and PC. Liver lipid concentration has been used as an indicator of choline status in a variety of animals. It is thought that accumulation of liver lipids in choline-deficient animals is a result of impaired hepatic lipoprotein secretion and subsequent accumulation of triglycerides (Chan 1991). However, liver lipid concentration has not been a consistent indicator of choline status in fish. Dietary choline supplied at or above the requirement resulted in significantly lower liver lipid concentrations in channel catfish (Wilson and Poe 1988), lake trout (Ketola 1976) and hybrid striped bass (Griffin et al. 1994), but higher liver lipid levels in red drum (Craig and Gatlin 1996). Liver lipid levels were not responsive to dietary choline in rainbow trout (Rumsey 1991) or yellow perch in the present study. However, carcass composition of yellow perch was responsive to increasing dietary choline. A reduction in protein and increase in fat in fish fed increasing concentrations of choline were observed. Similar results were reported for Atlantic salmon (Poston 1990) and rainbow trout (Poston 1991c). When sulfur amino acid concentrations are provided at the requirement, choline is clearly an essential vitamin in diets fed to juvenile yellow perch with an initial weight of 16 g. This is similar to studies with other species of fish and terrestrial vertebrates, yet a complete understanding of choline nutrition in fishes has not been developed. Specifically, there have not been studies designed to understand ontogenetic differences in choline nutrition in fishes, nor is there a complete understanding of the numerous interactions that can occur in the sulfur amino acid catabolic pathway.

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