Effects of dietary protein and energy levels on growth and lipid composition of juvenile snail (Semisulcospira gottschei).
KEY WORDS: dietary protein and energy, snail, Semisulcospira gottschei
Protein is one of the most important nutrient affecting animal growth and the feed cost. Protein requirements have been studied in aquaculture species with the aim of determining the minimum amount required to produce maximum growth and not be used for energy. The protein requirement of fish varies with fish species, fish size, dietary protein quality, and environmental conditions (NRC 1993). The non protein energy levels may also influence the dietary protein requirement of fish. When insufficient non-protein energy is available in feeds, dietary protein is deaminated in the body to supply energy for metabolism rather than being used for tissue growth, and excreted ammonia can reduce water quality (Phillips 1972, Shyong et al. 1998). Because fish consume food to satisfy their energy requirement, excess dietary energy may limit intake of essential nutrients like protein and amino acids. Thus, excesses of energy can lead to growth reduction and increase fat deposition in fish (Daniels & Robinson 1986).
The snail (Semisulcospira gottschei) is becoming a candidate shellfish for aquaculture, because this species has high consumer's demand as the health food in Korea. However, limited study has been performed on nutritional requirements of this species except for essential fatty acids requirement and carbohydrate use (Lee et al. 2002, Lim et al. 2003). Therefore, this study investigates the effects of dietary protein and energy levels on growth and lipid composition of snails.
MATERIALS AND METHODS
A 5 x 2 factorial design using three replications was used. Ten experimental diets were formulated to contain 5 protein levels (12%, 22%, 32%, 42%, and 52%) and 2 energy levels (3.3 and 3.9 kcal/g diet). Gross energy levels of diets were calculated based on 4 kcal/g protein, 9 kcal/g lipid and 4 kcal/g N-free extract (Garling & Wilson 1976). Ingredients and nutrients contents of the experimental diets, and fatty acid compositions of dietary lipid sources are presented in Table 1 and Table 2, respectively. Casein increased mainly at the expense dextrin to increase the protein level in diets. The mixture of squid liver oil, linseed oil, and corn oil was added 4.5% and 12% for low and high-energy diets, respectively, in each protein level. Procedures for feed preparation were adapted from the method of Mat et al. (1995a) who studied for abalone feed. Experimental diets were dried at room temperature and stored at -25[degrees]C until used. Crude lipid contents in some experimental diets were somewhat reduced during the process of rolling and dipping into the solution of Ca[Cl.sub.2] to make a flake type of feed.
Juvenile snails (Semisulcospira gottschei) were obtained from a private hatchery (Pyungchang, Korea). They were acclimated to a recirculating system in our laboratory for 1 wk by feeding a commercial abalone diet containing 30% protein and 5% lipid. They were then randomly redistributed into 25-L tanks (20 L of water each) at a density of 100 snails (37 mg/snail) per tank. Three replicate groups of snails were fed once in 2 days for 12 wk. Before feeding, uneaten diets in each tank were cleaned by siphoning and 20% of water volume in system was replaced with fresh water every 2 days. Fresh water was supplied at a flow rate of approximately 0.3 L/min in the recirculating system. Photoperiod was left at the natural condition, and water temperature ranged from 15[degrees]C to 22[degrees]C during the feeding trial. Snails in each tank were collectively weighed on the day of initiation and on the day of termination of the feeding trial, after being fasted for 24 h.
Sample Collection and Chemical Analysis
Three hundred snail samples at the beginning and all snails at the end of the feeding trial were sacrificed and stored at -70[degrees]C for chemical analysis. Proximate composition of experimental diets and snails were analyzed as follows. Crude protein was determined by Kjeldahl method using Auto Kjeldahl System (Buchi B-324/ 435/412, Switzerland). Crude lipid was determined with ether extraction in a soxhlet extractor, moisture was determined by dry oven (105[degrees]C for 12 h), and ash was determined by a muffle furnace (550[degrees]C for 4 h). Lipid for fatty acid analyses was extracted by the method of Folch et al. (1957), and fatty acid methyl esters were prepared by transesterification with 14% B[F.sub.3]-MeOH (Sigma, Chemical Co., USA). Fatty acid methyl esters were analyzed by using a gas chromatography (HP 5890, Hewlett-Packard, USA) with flame ionization detector, equipped with HP-INNOWax capillary column (30 m x 0.32 mm i.d., film thickness 0.5 [micro]m, Hewlett-Packard, USA). Injector and detector temperatures were 250[degrees]C and 270[degrees]C, respectively. The column temperature was programmed from 170[degrees]C to 225[degrees]C at a rate of 1[degrees]C/min. Helium was used as the carrier gas. Fatty acids were identified by comparison with retention times of the standard fatty acid methyl esters (Sigma, USA).
The data of result were subjected to 1- and 2-way ANOVA to test the effects of dietary protein and energy levels. Where significant (P < 0.05) differences were found in the 1-way ANOVA test, Duncan's multiple range test (Duncan 1955) was used to rank the groups. All statistical analyses were carried out using the SPSS Version 10.0 (SPSS, Michigan Avenue, Chicago, IL).
RESULTS AND DISCUSSION
Survival and growth of snails fed the diets containing various protein and energy levels for 12 wk are presented in Table 3. Survival of each group was all >80% and no significant difference among treatments. Mean weight gain was significantly (P < 0.001) affected by only dietary protein level. No significant interactions were observed between dietary protein and energy levels (P > 0.05) regarding growth and survival responses. Mean weight gain of snails was improved with increasing dietary protein level up to 22% and 32% at low and high energy levels (P < 0.05), respectively and reached a plateau above these levels (P > 0.05). This trend, which is in agreement with other studies, showed that growth of fish linearly increased up to the minimum required protein level, and then growth was similar without difference above these levels of protein (Mai et al. 1995b, Shyong et al. 1998). In this study, mean weight gain of snails fed the diets containing 22% protein with low energy level was not significantly (P > 0.05) different from that of snails fed the diets containing 32% to 52% protein with both energy levels. Considering this growth response, optimum dietary protein and energy levels are about 22% and 3.3 kcal/g diet for growth of snails. The optimum dietary protein level for snails determined in this study is comparable with that reported for abalone, Haliotis discus hannai (Uki et al. 1986, Mai et al. 1995b).
In addition, the nonprotein energy sources may also influence the dietary protein requirement of fish. Adequate levels of nonprotein energy sources in diets can minimize use of protein as a source of energy (NRC 1993), and the protein sparing effect by fat and carbohydrate in diets has been reported in other fish (Cho & Kaushik 1990, De Silva et 'al. 1991). However, no significant difference on weight gain of snails was observed between low and high energy levels at same protein level in this study. This indicates that protein sparing by dietary lipid is not expecting for snail growth.
Lipid content and fatty acid composition of snails fed the diets containing various protein and energy levels for 12 wk are presented in Table 4. Lipid content was significantly (P < 0.01) affected by only dietary energy level, and lipid content of snails fed the high energy diets showed tendency toward higher values than that of the low energy diets at all protein levels in this study. The effect of dietary energy on body fat in this study was in agreement with reports for other studies that showed that high dietary energy level increased lipid content of fish (Lee et al. 2000, Lee et al. 2003).
It has been reported that dietary lipid level influence fatty acid compositions of fish lipid (Silver et al. 1993), and similar results were observed in this study. In this study, a good agreement between the fatty acid compositions of snail lipid and dietary lipid was observed as reported from the previous snail study (Lee et al. 2002). The 16:0, 18:1n-9, 18:2n-6, and 18:3n-3 were the most abundant saturated, monoenoic, n-6 and n-3 poly unsaturated fatty acids in snails fed the experimental diets, respectively, in this study. Snails fed the high-energy diets showed a tendency toward higher in 18:1n-9, 18:2n-6, 18:3n-3, and 22:6n-3 and lower in 20:2n-6, 20:4n-6, and 22:1n-9 than those of snails fed the low energy diets. Relative high levels (5.0% to 7.6%) of 20:4n-6 were observed in snails fed the experimental diets though 20:4n-6 content was about 0.2% in dietary lipid, in this study. Lee et al. (2002) reported that considerable content of 20:4n-6 was found in snails fed the diets containing no 20:4n-6. These results indicate that this snail may have capacity to elongate or desaturate 20:4n-6 from shorter chain polyunsaturated fatty acids. It has been known that eicosanoids derived from 20:4n-6 are physiologically active in reproductive processes of fish (Tocher & Sargent 1984) and immune function (Kinsella & Lokesh 1990). The 20:4n-6 is also one of the main components of phosphatidylinositol in fish tissues (Bell & Dick 1990, Sargent et al. 1999). Furthermore, Castell et al. (1994) reported that dietary 20:4n-6 is essential for the normal growth and development of juvenile turbot. Whereas, Lee et al. (2002) suggested that snail require n-3 highly unsaturated fatty acids as essential fatty acids in diets for normal growth, and plant oil could be used as an energy source when n-3 highly unsaturated fatty acids requirement is satisfied. However, limited information on the biologic function of 20:4n-6 in snails is available. Therefore, more detailed studies on biologic function of 20:4n-6 in snails are needed.
The results of this study indicate that a diet containing 22% protein and 3.3 kcal/g diet with P/E ratio of 69 mg protein/kcal was recommended for snail growth.
TABLE 1. Ingredients and nutrients content of the experiment diets. 12LE 12HE 22LE 22HE 32LE Ingredients (%) Casein 10.3 10.3 20.7 20.7 31.0 Lipids (1) 4.5 12.0 4.5 12.0 4.5 Dextrin 50.0 50.0 40.0 40.0 30.0 Vitamin premix (2) 2.0 2.0 2.0 2.0 2.0 Mineral premix (3) 3.0 3.0 3.0 3.0 3.0 Na alginate 18.0 18.0 18.0 18.0 18.0 [alpha]-cellulose 11.7 4.2 11.3 3.8 11.0 Choline salt 0.5 0.5 0.5 0.5 0.5 Nutrients content (dry matter basis) Crude protein (%) 11.7 12.4 22.8 23.0 33.0 Crude lipid (%) 2.4 7.2 3.6 8.7 3.6 Ash (%) 8.4 9.3 8.5 9.2 8.9 Crude fiber (%) 13.7 5.7 13.3 5.2 13.0 N-free extract (%) (4) 77.5 72.1 65.1 59.1 54.5 Energy (kcal/g diet) (5) 3.24 3.80 3.31 3.86 3.31 P/E ratio (mg/kcal) 36 33 69 60 100 32HE 42LE 42HE 52LE 52HE Ingredients (%) Casein 31.0 41.3 41.3 51.7 51.7 Lipids (1) 12.0 4.5 12.0 4.5 12.0 Dextrin 30.0 20.0 20.0 10.0 10.0 Vitamin premix (2) 2.0 2.0 2.0 2.0 2.0 Mineral premix (3) 3.0 3.0 3.0 3.0 3.0 Na alginate 18.0 18.0 18.0 18.0 18.0 [alpha]-cellulose 3.5 10.7 3.2 10.3 2.8 Choline salt 0.5 0.5 0.5 0.5 0.5 Nutrients content (dry matter basis) Crude protein (%) 32.4 42.1 43.1 52.2 51.3 Crude lipid (%) 10.3 4.4 10.8 5.0 11.5 Ash (%) 9.9 9.5 9.9 10.2 9.7 Crude fiber (%) 4.9 12.6 4.6 12.2 4.2 N-free extract (%) (4) 47.4 44 36.2 32.6 27.5 Energy (kcal/g diet) (5) 3.92 3.33 3.96 3.35 4.02 P/E ratio (mg/kcal) 83 126 109 156 127 (1) The mixture of squid liver oil, linseed oil and corn oil (1:1:1). (2) Vitamin mix contained the following amount which were diluted in cellulose (g/kg mix): L-ascorbic acid, 200; DL-[alpha]-tocopheryl acetate, 20; thiamin hydrochloride, 5; riboflavin, 8; pyridoxie hydrochloride, 2; niacin, 40; Ca-D-pantothenate, 12; myo-inositol, 200; D-biotin, 0.4; folic acid, 1.5; p-amino benzoic acid, 20; menadione, 4; retinyl acetate, 1.5; cholecalciferol, 0.003; cyanocobalamin, 0.003. (3) Mineral mix contained the following ingredients (g/kg mix): NaCl, 10; MgS[O.sub.4] * 7[H.sub.2]O, 150; Na[H.sub.2]P[O.sub.4] * 2[H.sub.2]O, 250; K[H.sub.2]P[O.sub.4], 320; Ca[H.sub.4][(P[O.sub.4]).sub.2] * [H.sub.2]O, 200; Ferric citrate, 25; ZnS[O.sub.4] * 7[H.sub.2]O, 4; Ca-lactate, 38.5; CuCl, 0.3; Al[Cl.sub.3] * 6[H.sub.2]O, 0.15; KI[O.sub.3], 0.03; [Na.sub.2][Se.sub.2][O.sub.3], 0.01; MnS[O.sub.4] * [H.sub.2]O, 2; Co[Cl.sub.2] * 6[H.sub.2]O, 0.1. (4) Calculated by the difference (= 100-crude protein-crude lipid-ash-crude fiber). (5) Calculated based on 4 kcal/g protein, 9 kcal/g lipid and 4 kcal/g N-free extract (Garling & Wilson, 1976). TABLE 2. Fatty acid composition (% of total fatty acids) of dietary lipid sources. Dietary Lipid Squid Liver Oil Linseed Oil Corn Oil Fatty acids 12:0 3.6 1.9 0.3 14:0 6.5 0.9 0.3 16:0 13.3 5.7 11.6 16:1n-7 9.1 18:0 1.7 2.7 3.3 18:1n-9 12.8 16.1 19.1 18:2n-6 1.4 15.6 55.3 18:3n-3 1.0 53.6 6.7 18:4n-3 3.6 20:1 n-9 8.5 20:4n-6 0.7 20:4n-3 0.8 20:5n-3 15.2 22:1n-9 4.7 22:5n-3 1.0 22:6n-3 13.7 n-3HUFA (1) 30.7 (1) Highly unsaturated fatty acids (C [greater than or equal to] 20). TABLE 3. Survival and weight gain of snails fed the diets containing various protein and energy levels for 12 weeks. (1) Initial Mean Survival Mean Weight Gain Diets Weight (mg) (%) (mg/snail) 12LE 38 [+ or -] 4.0 82.3 [+ or 15.4 [+ or -] -] 4.98 0.40 (ab) 12HE 41 [+ or -] 3.8 81.7 [+ or 13.9 [+ or -] -] 2.33 2.21 (a) 22LE 37 [+ or -] 7.6 83.0 [+ or 29.3 [+ or -] -] 3.21 5.22 (cd) 22HE 34 [+ or -] 3.7 80.0 [+ or 24.7 [+ or -] -] 2.08 1.27 (bc) 32LE 38 [+ or -] 5.7 81.7 [+ or 29.4 [+ or -] -] 2.73 0.44 (cd) 32HE 38 [+ or -] 2.2 80.3 [+ or 34.6 [+ or -] -] 5.04 7.10 (cd) 42LE 37 [+ or -] 2.2 85.0 [+ or 34.4 [+ or -] -] 1.00 4.78 (cd) 42HE 38 [+ or -] 4.9 87.3 [+ or 38.9 [+ or -] -] 3.67 2.58 (d) 52LE 34 [+ or -] 1.8 84.3 [+ or 29.7 [+ or -] -] 2.19 1.85 (cd) 52HE 34 [+ or -] 1.2 87.3 [+ or 38.0 [+ or -] -] 2.67 0.77 (d) Two-way ANOVA Dietary protein P < 0.4 P < 0.001 Dietary energy P < 0.9 P < 0.3 Interaction P < 0.9 P < 0.4 (1) Values (mean [+ or -] SE of three replications) in the same column not having a common superscript are significantly different (P < 0.05).
The authors thank Mr. Y.-J. Kim for donation of snail samples. This research was supported by a grant from the Pyongchang Agricultural Development and Technology Center, Gangwondo, Korea.
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SANG-MIN LEE * AND TAE-JUN LIM
Faculty of Marine Bioscience and Technology, Kangnung National University, Gangneung 210-702, Korea
* Corresponding author. E-mail: firstname.lastname@example.org