Effect of Dietary Protein and Lipid Levels on Growth Performance, Carcass Composition, and Digestive Enzyme of the Juvenile Spotted Babylon, Babylonia areolata Link 1807 Shu Y. Chi Laboratory of Aquatic Economic Animal Nutrition and Feed, College of Fisheries, Guangdong Ocean University, Zhanjiang 524025, China
Qi C. Zhou1 Laboratory of Fish Nutrition, College of Life Science and Biotechnology, Ningbo University, Ningbo 315211, China
Bei P. Tan, Xiao H. Dong, Qi H. Yang, and Jian B. Zhou Laboratory of Aquatic Economic Animal Nutrition and Feed, College of Fisheries, Guangdong Ocean University, Zhanjiang 524025, China
Abstract This study was undertaken to evaluate the effects of dietary protein and lipid levels on growth performance, feed utilization, carcass composition, and digestive enzyme activity of the juvenile spotted babylon, Babylonia areolata. Six experimental diets were formulated to contain three protein levels (25, 35, and 45%) at two lipid levels (8 and 12%). Triplicate groups of 40 animals (average weight 5.05 ± 0.08 g) were stocked in 120-L tanks and fed to apparent satiation twice daily for 8 wk. Growth performance and feed utilization were significantly affected by dietary protein and lipid levels (P < 0.05). Protein efficiency ratio (PER), specific growth rate (SGR), and weight gain (WG) were the best at 45%/8% treatment (P < 0.05). There was no significant interaction between different levels of dietary protein and lipid on survival rate and the soft body to shell ratio (SB/SR). There was an interaction effect between dietary treatments on PER, SGR, and WG, in which shellfish fed with 45% protein at 8% lipid had the highest interaction (P < 0.05). There was an interaction effect between dietary protein and lipid levels on pepsin, tryptase, and lipase activities in soft body. Tryptase enzyme activity of 45%/8% treatment was the lowest and the highest was found in 25%/8% treatment which also had the highest activity of lipase. Results indicated that the juvenile spotted babylon would obtain better growth performance when fed with diets containing 45% dietary protein at 8% dietary lipid.
Members of the genus Babylonia are distributed in the Indo-Pacific region, of which spotted babylon, Babylonia areolata, a large marine gastropod (adult size 50–60 mm) extends from Sri Lanka and the Nicobar Islands through the Gulf of Siam, along the Vietnamese and Chinese coast to Taiwan (Altena and Gittenberger 1981). Juvenile spotted babylon is one of the most extensively cultured marine mollusks in the Southeast Asian countries, and 1
it is the second most economically important marine gastropods for human consumption in Thailand (Kritsanapuntu et al. 2009). It is a carnivore inhabiting the muddy/sandy subtidal zone at depths of 4–20 m (summer) and 40–60 m (winter) (Cai et al. 1995). It was previously abundant, but declined in number because of overfishing since the late 1980s. In recent years, there has been a rapid increase in market demand for this species in Thailand and other Asian countries. As a result, this
species has attracted a great interest of shellfish
farmers because of its resistance to handling, rapid growth, delicious meat, and high market price (Zhou et al. 2007a). However, a main constraint to spotted babylon culture development is the limited supply of trash fish or crabs that are presently the main feed source for growout. The use of formulated feeds for growing to marketable size would be practical and efficient in terms of labor cost compared with the present practice of using trash fish as the rearing diet. The requirement level for dietary nutrients is the basis for their inclusion levels in the feed formula. Limited studies have been reported on the nutrient requirements of spotted babylon (Ke et al. 1997; Xu 2006; Zhou et al. 2007a, 2007b; Zhang et al. 2009). Protein is one of the most important nutrient categories for growth and the most expensive macrocomponent of fish feed because of its bulk in the feed formula (National Research Council 1993). Protein requirements are always studied in aquaculture species with the aim of determining the minimum amount requirement to produce maximum growth. The relevant studies for shellfish mainly focused on scallop, abalone, and spotted babylon. Uriarte and Farías (1999) reported that the postlarvae of Chilean scallop, Argopecten purpuratus, showed significantly better growth and survival when fed with the higher protein diet. For abalone, some researchers reported that protein requirements ranged from 20 to 44% (Uki et al. 1985; Mai et al. 1995; Coote et al. 2000; G´omez-Montes et al. 2003; Thongrod et al. 2003). As for spotted babylon, the protein requirements ranged from 25 to 48% (Ke et al. 1997; Xu 2006; Zhou et al. 2007a). Lipid is one of the important nutrients to provide energy for mollusk, especially at larval and juvenile stages. Lipid provides a source of energy, essential fatty acids and other lipid classes such as phospholipids, sterols, and fatsoluble vitamins (Watanabe 1982). The optimal dietary lipid level had been demonstrated for mollusk species, such as Haliotis discus hannai (Uki et al. 1985; Mai et al. 1995) and Haliotis tuberculata (L.) (Mai et al. 1995). Zhou et al. (2007b) reported that the optimal dietary lipid requirement for maximum mean
protein gain of juvenile spotted babylon was about 6.54% of dry diet with 43% crude protein. Britz and Hecht (1997) reported that the combination of 34% protein and 2–6% lipid was optimum for the growth of abalone Haliotis midae. Therefore, this study was undertaken to determine the optimal levels of dietary protein and lipid to support optimum growth response, body composition, and digestive enzyme of the juvenile spotted babylon. Materials and Methods Diet Preparation Six diets were formulated to contain three protein levels (25, 35, and 45%) at two lipid levels (8 and 12%) for each protein (Table 1). Fish meal and soybean meal were used as protein sources. Fish oil/soybean oil (1:1) and wheat meal were used as lipid and carbohydrate sources, respectively. Diet ingredients were ground through an 80-mesh sieve. Lipid and distilled water (40% w/w) were added to the premixed dry ingredients and thoroughly mixed until homogenous in a Hobart-type mixer. The 1-mm diameter pellets were wet extruded using a pelletizer (Institute of Chemical Engineering, South China University of Technology, Guangzhou, China), air-dried, and then sealed in plastic bags and stored at −20 C before use. Animal Rearing Juvenile spotted babylon was obtained from a local shellfish farm (Dongding breeding farm, Zhanjiang, China). Management was as described in our previous study (Zhou et al. 2007a). Prior to the start of the trial, juvenile spotted babylon was acclimatized to a commercial diet (containing 42% crude protein and 6% crude lipid) and was fed twice daily to apparent satiation for 2 wk. A 2 × 3 factorial experiment in a completely randomized design was used. Each experimental diet was randomly assigned to three tanks. The acclimated spotted babylon (initial mean weight, 5.05 ± 0.08 g) was sorted into 18 120-L cylindrical fiberglass tanks at a stocking density of 40 spotted babylon per tank. Juvenile spotted babylon was fed to visual satiety twice daily at a rate of 4% wet
EFFECT OF DIETARY PROTEIN AND LIPID LEVELS
Table 1. Ingredients and composition of experimental diets (g/kg dry matter). Diets (protein, %/lipid, %)
Ingredients Fish meala Soybean mealb Wheat mealb Fish oil/soybean oil (1:1)b Celluloseb Mineral mixturec Vitamin premixd Squid meale Lecithine Calcium dihydrogen phosphatef Choline chlorideg Ascorbyl-2-monophosphateh Sodium alginateg Composition Moisture Gross energy (kJ/g) Crude protein Crude lipid Crude ash Calcium Available phosphors
from New Zealand (g/kg dry matter: protein, 670.5; lipid, 92.0). from Yuhai Feed Company Ltd., Zhanjiang, China (g/kg dry matter: protein, 430.0; lipid, 19.0). c Mineral premix was based on Zhou et al. (2007a). mg/kg: MnSO4 ·H2 O, 10; CuSO4 ·5H2 O, 1; FeSO4 ·H2 O, 4; ZnSO4 ·7H2 O, 8; Na2 SeO3 , 0.05; KI, 0.2; and CoCl2 ·6H2 O, 0.05. d Vitamin premix was based on Zhou et al. (2007a). mg/kg or IU: vitamin A, 2400; vitamin D, 400; vitamin E, 30; vitamin K (menadione sodium bisulfate), 14; thiamin, 10; riboflavin, 9; pyridoxine, 14; vitamin B12 , 0.008; niacin, 40; d-calcium pantothenate, 30; folic acid, 2.4; biotin, 0.2; and inositol, 60. e Purchased from Yuehai Feed Company Ltd., Zhanjiang, China. f Sichuan Lomon Corporation Phosphorous Co., Sichuan, China. g China National Medicines Corporation Ltd., Shanghai, China. h 35% ascorbic acid activity, Hoffmann-La Roche Ltd., Basel, Switzerland. b Purchased
body weight for 8 wk, 30% of the ration was fed at 0900 h, and 70% at 1800 h, which was the start of the dark phase during which most feeding activity occurs (Liu and Xiao 1998). Feed consumption was recorded for each tank and animals were bulk weighed and counted every 2 wk to adjust the quantity of feed. Uneaten feed were removed daily before the next feeding, dried, and weighed to calculate the feed intake. They were provided with sandfiltered seawater (2 L/min) with continuous aeration. The bottom of each tank was covered with about 4 cm clean sea sand, which simulated the natural environment that they normally inhabit. Tanks were thoroughly cleaned and the sea sand was changed biweekly. Water quality
parameters were monitored daily between 0900 and 1800 h. During the feeding trial, water temperature ranged from 28.5 to 30.5 C, salinity from 27 to 32 ppt, and pH from 7.8 to 8.0. Ammonia nitrogen was maintained from 0.02 to 0.03 mg/L and dissolved oxygen was from 6.0 to 6.5 mg/L. Sample Collection and Chemical Analyses At the end of the growth trial, spotted babylon was starved 24 h and weighed. A sample of 150 animals at the initiation of the feeding trial and 20–25 animals per tank at termination were used for carcass proximate analysis, and then shell and soft body tissues were individually weighed for the calculation of soft body to
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shell ratio (SB/SR) (Mai et al. 1995). Soft body of spotted babylon was sampled, sealed in plastic bags, and stored frozen (−20 C) for the analysis of nutrient composition. Crude protein, crude lipid, moisture, and ash in diets and soft body were determined by standard methods (AOAC 1995). Moisture was determined by oven-drying at 105 C for 24 h. Crude protein (N × 6.25) was determined by the Kjeldahl method after acid digestion using an Auto Kjeldahl System (1030-Auto-analyzer, Tecator, H¨ogan¨as, Sweden). Crude lipid was determined by ether extraction using a Soxtec System HT6 (Tecator). Ash was determined by muffle furnace at 550 C for 24 h. Digestible Enzyme Analyses The soft body of 15 spotted babylon from each tank was homogenized in 10 volumes (w/v) of ice-cold double distilled water by an electric homogenizer (IKA, T-25, Staufen, Germany). Homogenates were centrifuged at 10,000 g for 30 min at 4 C to analyze protease activity and 1660 g for 20 min at 4 C to analyze lipase activity, respectively. After centrifugation, the supernatants were collected and stored frozen at −70 C until analyzed. The assays for pepsin and tryptase activity were measured using the casein hydrolysis method of Liu et al. (1991) and Pan et al. (2005). The substrate was 0.5% casein (Sigma, St. Louis, MO, USA) in citric acid (China National Medicines Corporation Ltd., Shanghai, China) buffer (pH 3.0) for pepsin and in borax–sodium hydroxide (China National Medicines Corporation Ltd.) buffer (pH 9.8) for tryptase. The reaction proceeded at 37 C for 15 min and was stopped with trichloroacetic acid (China National Medicines Corporation Ltd.). Percolate was filtered and mixed with 0.5 mol/L Na2 CO3 . Color was allowed to develop for 20 min after adding forint-hydroxybenzene. At 20 min, the enzyme activity was calculated from the light absorption at 680 nm. One unit of protease activity was defined as 1-μg tyrosine liberated by hydrolyzing casein in 1 min at 37 C. Lipase activity was determined by the method of Pan and Wang (1997). Homogenates
were incubated with 2% polyvinyl alcohol (Sigma, N81384) in 25-mM phosphate buffer, pH 7.5, containing 25% olive oil (China National Medicines Corporation Ltd.) as an emulsifying agent at 40 C for 15 min, and then 15-mL 95% ethanol was added to terminate the reaction. Two to three drops of phenolphthalein were added to the solution and a titration action with 0.05 mol/L sodium hydroxide was performed. Consumed volume of sodium hydroxide was measured when the solution showed light red. One unit of lipase activity was defined as the amount of enzyme that catalyzed the release of 1 μmol of fatty acids in 1 min at pH 7.5 and 40 C. Specific activities were expressed as enzyme activity per milligram protein. The protein concentration in homogenates was determined by Bradford (1976) and bovine serum albumin (China National Medicines Corporation Ltd.) as the standard. Calculations and Statistical Analysis The parameters were calculated as follows: Specific growth rate (SGR, %) = (ln Wt − ln Wi ) × 100/t. Percent weight gain (WG, %) = 100 × (Wt − Wi )/Wi . Protein efficiency ratio (PER) = (Wt − Wi )/ protein intake (g). Soft body to shell ratio (SB/SR) = Ws /shell weight (g). where Wt (g) is final body weight, Wi (g) the initial body weight, Ws (g) the soft body weight, and t the experimental duration in day. Results are presented as mean ± SE of the three replicates. All data were analyzed using two-way ANOVA and Tukey’s multiple range test (Puri and Mullen 1980). All statistical analyses were performed by SPSS version 13.0 (SPSS, Chicago, IL, USA). Results Growth performance and feed utilization of the juvenile spotted babylon fed with different dietary protein and lipid levels are shown in Table 2. There was no significant interaction
EFFECT OF DIETARY PROTEIN AND LIPID LEVELS
Figure 1. Effect of dietary protein levels on the protein efficiency ratio (PER) and specific growth rate (SGR) of juvenile spotted babylon. Regardless of lipid levels, Fig. 1 showed significant differences among protein levels on PER and SGR. The highest PER and SGR were found in 45% protein treatments (P < 0 .05 ).
a b c a
Pepsin Tryptase Lipase
20 18 16 14 12 10 8 6 4 2
Figure 2. Effect of dietary protein levels on the weight gain (WG) of juvenile spotted babylon. Regardless of lipid levels, Fig. 2 showed significant differences among protein levels on WG, which was found the highest in 45% protein treatments (P < 0 .05 ).
Figure 3. Effect of dietary protein levels on the digestive enzyme activities of juvenile spotted babylon. Regardless of lipid levels, significant differences of pepsin activities were found in three protein treatments (P < 0 .05 ). Tryptase and lipase activities of 25% protein treatments were higher than those of 45% protein treatments (P < 0 .05 ).
Digestible enzymes activity (U/mg pro.)
extract content than those fed with the other diets (P < 0.05) (Table 3). Pepsin, tryptase, and lipase activities in soft body were significantly affected by the dietary protein and lipid levels (P < 0.05) (Figs. 3, 4 and Table 4). The highest pepsin activity was found in animals fed with the 35%/8% diet (P < 0.05). The tryptase activity was lowest in spotted babylon fed with the 45%/8% diet; however, animals fed with the 25%/8% diet had a significantly higher tryptase activity than those fed with the other diets (P < 0.05). The lowest lipase activities were found in animals fed with the 35% protein at 8 and 12% lipid diets. Digestible enzymes activity (U/mg pro.)
between different levels of dietary protein and lipid on survival rate and SB/SR. Regardless of protein levels, the SB/SR of shellfish fed with 8% lipid (0.41 ± 0.02) was significantly higher than 12% lipid (0.39 ± 0.04) (P < 0.05). However, there was an interaction on PER, SGR, and WG among the dietary treatments, in which shellfish fed with 45% protein at 8% lipid had the highest interaction (P < 0.05). Regardless of lipid levels, PER, SGR, and WG of shellfish fed with 45% protein were the highest (P < 0.05) (Figs. 1, 2). Moisture, crude protein, and ash in soft body were not significantly affected by the dietary protein and lipid levels (P > 0.05) (Table 3). Juvenile spotted babylon fed with 45% protein at 12% lipid diet had a significantly higher ether
14 12 10
Pepsin Tryptase Lipase
4 2 0
12 Lipid levels%
Figure 4. Effect of dietary lipid levels on the digestive enzyme activities of juvenile spotted babylon. Regardless of protein levels, significantly higher digestible enzyme activities were found at 8% lipid treatments (P < 0 .05 ).
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Table 2. Effect of dietary protein/lipid ratio on growth performance, survival, and feed utilization of juvenile spotted babylon. Diets (protein, %/ lipid, %) 25/8 25/12 35/8 35/12 45/8 45/12
PER 1.23 1.63 1.69 2.97 5.11 4.29
± ± ± ± ± ±
0.90 0.73 1.11 1.84 2.55 2.17
0.34d 0.07d 0.21c 0.35a 0.26b
± ± ± ± ± ±
65.44 51.63 86.00 180.22 318.81 237.73
0.12e 0.04d 0.08c 0.10a 0.07b
± ± ± ± ± ±
95.00 95.83 96.7 100 99.2 100
10.46d 4.32d 11.80c 24.55a 13.66b
± ± ± ± ± ±
2.50 2.20 3.33 0.00 0.83 0.00
0.40 0.38 0.41 0.37 0.43 0.44
± ± ± ± ± ±
0.01 0.01 0.01 0.00 0.01 0.01
PER = protein efficiency ratio; SGR = specific growth rate; WG = weight gain; and SB/SR = soft body to shell ratio. Data represent mean ± SE (n = 3). Values in the same column sharing different superscript letters are significantly different (P < 0.05). Table 3. Effect of dietary protein/lipid ratio on soft body composition of juvenile spotted babylon. Diets (protein, %/ lipid, %) 25/8 25/12 35/8 35/12 45/8 45/12
± ± ± ± ± ±
± ± ± ± ± ±
± ± ± ± ± ±
73.81 73.35 72.78 71.87 72.41 71.85
0.35 0.51 0.42 0.37 0.57 0.35
50.01 52.18 54.05 46.76 53.83 50.51
1.00 1.20 4.45 2.34 1.17 4.03
22.13 21.56 19.84 23.42 23.21 30.84
18.38 16.89 20.36 17.41 16.24 15.10
1.38b 3.12b 0.72b 0.22b 1.69a
± ± ± ± ± ±
0.98 0.52 1.64 1.49 0.70 0.46
Data represent mean ± SE (n = 3). Values in the same column sharing different superscript letters are significantly different (P < 0.05). Table 4. Effect of dietary protein/lipid ratio on the digestive enzyme activities of juvenile spotted babylon. Diets (protein, %/ lipid, %)
Data represent mean ± SE (n = 3). Values in the same column sharing different superscript letters are significantly different (P < 0.05).
Discussion In this study, PER, SGR, and WG of the shellfish were significantly affected by dietary protein and lipid levels. Similar results were observed in abalone fed with diets containing three protein levels at 24, 34, and 44%, each with three lipids levels at 2, 6, and 10%, respectively (Britz and Hecht 1997). Juvenile green abalone, Haliotis rufescens, fed 40.5 and 44.1%
protein diets showed significantly better growth performance than those fed the other diets (26, 31 and 35% protein with the same energy content at about 4.1 kcal/g) (G´omez-Montes et al. 2003). Xu (2006) reported that the optimal protein and lipid requirement of juvenile spotted babylon (initial weight 2.16 ± 0.05 g) should be 36.5–43.1% and 7.8–10.7%, respectively; growth performance would be restrained when the dietary lipid level was under 7.8%. In this
EFFECT OF DIETARY PROTEIN AND LIPID LEVELS
study, the maximum growth performance of spotted babylon was observed in diet containing 45% protein at 8% lipid. However, Liu et al. (2006) indicated that Babylonia formosae (initial weight 1.60 ± 0.11 g) fed with different dietary protein levels (crude protein from 25 to 48%) have no significant differences in growth performance, which was different from our results. Dietary protein is not enough to meet the growth requirement; lower growth rates would be observed (Smith 1989). If the dietary energy level is insufficient in the diets, protein will be used as energy for maintenance (National Research Council 1983). The estimation of protein requirements is affected by some factors such as rearing conditions, stage of growth, and sources of protein, but a more significant factor may be the dietary energy content in quantitative determination (Wilson 1989). Lee et al. (2002) reported that a positive correlation was found between the levels of dietary digestible protein/digestible energy ratio and growth performance at the same lipid levels. The results of juvenile spotted babylon fed with 25 and 35% protein indicated a sparing effect of the lipid for protein on growth performance. Juvenile spotted babylon fed with diet containing 12% lipid had higher PER than that fed with 8% lipid diet. The trend indicated that spotted babylon can effectively utilize dietary lipid as an energy source and dietary protein will be used for growth. The theory behind a protein sparing effect is that, when protein provides essential amino acids to meet growth requirements, extra dietary protein will be used for energy purposes. Increases in the nonprotein energy component of diets (at a specific protein concentration) have been reported to improve growth and reduce the protein requirement through protein sparing in the American lobster (Capuzzo and Lancaster 1979). When spotted babylon was fed with a diet containing excess energy, WG may be decreased because of the reduced feed consumption. However, when spotted babylon was fed with a diet deficient in energy, dietary protein will be used as an energy source and this elevates the
production cost. In this study, there was no significant sparing effect when the dietary protein increased to 45%. Mai et al. (1995) found that SB/SR of abalone did not differ significantly when fed with diets containing 20–50% protein. In this study, although the protein and lipid had significant influences on SB/SR, the interaction between protein and lipid was not significant. The main difference in protein and lipid utilization may be because of the carnivorous feeding activity of spotted babylon, whereas abalone is a herbivorous mollusk. Protein level in diet would affect the body protein and lipid contents of scallop spat, but there were no effects on protein deposition with the growth change (Uriarte and Farías 1999). However, the increase of dietary lipid levels should be carefully considered as it may affect carcass quality, mainly because of an increase of lipid deposition (Cowey 1993; Hillestad and Johnsen 1994). Zhou et al. (2007a) reported that lipid content in soft body (initial weight = 93.50 ± 1.70 mg) decreased with increasing dietary protein levels from 27 to 49% with lipid levels from 15 to 3%. In this study, by comparison with the spotted babylon fed with the different protein and lipid levels, ether extracts of soft body of juvenile spotted babylon were significantly affected by the dietary protein and lipid levels; however, there was no significant difference in the protein content of soft body. Increasing dietary protein level did not influence the protein content in soft body. However, our previous studies reported that crude protein, moisture, and ash content in soft body significantly decreased when the dietary lipid level increased from 1.83 to 11.73% at 43% dietary protein, but the lipid content was reversed (Zhou et al. 2007b). It is speculated that excretive nitrogen level would increase with increasing dietary protein level (Hawkins and Bayne 1991). In this study, the digestive enzyme activities in soft body were significantly affected by the dietary protein and lipid levels. Protease activity in the digestive gland is a key determinant enzyme of the digestibility and assimilation efficiency of ingested proteins. The results
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showed that spotted babylon fed with a diet with 35% protein and 8% lipid had the highest pepsin activities of those fed the diets. With the dietary protein level increasing at the same lipid level, pepsin activity showed a downtrend, except 35%/8%. On the contrary, Pan et al. (2005) and Zhou et al. (2007a) reported that the activities of pepsin and tryptase in soft body were elevated with an increase in the dietary protein. The main reason may be because of different species or different dietary lipid level and/or development stage. At the low lipid level, tryptase activity significantly declined with the protein increasing. However, at the high lipid level, the trend was adverse precisely without difference. Lipase activities of lipid level at 12% were lower than those at 8% with dietary protein level at 25 and 35%. The lipase activities were improved at 45% protein level. To the juvenile spotted babylon, lower protein level would limit the utilization of higher lipid. The juvenile spotted babylon could digest lipid and utilize the dietary lipid as an energy source at higher protein levels. Conclusion In summary, this study provides some insight into the nutrition of juvenile spotted babylon. The levels of protein and lipid at 45 and 8% were recommended for the best growth of juvenile spotted babylon (initial mean weight = 5.05 ± 0.08 g). Acknowledgments This work was supported by Zhanjiang Science and Technology Research Program (grant number 200401). The authors are grateful for J. C. Zhang and S. L. Zeng for their skilled technical assistance. Literature Cited Altena, C. O., Van Regteren, and E. Gittenberger. 1981. Zoologische Verhandelingen. Leiden E-Journal of Brill 188:1–57. AOAC (Association of Official Analytical Chemists). 1995. Official method of analysis, 16th edition. Association of Official Analytical Chemists, Arlington, Virginia, USA.
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