Aquaculture 295 (2009) 238–242 Contents lists available at ScienceDirect Aquaculture j o u r n a l h o m e p a g e : w w w e l s ev i e r c o m / l o c a t e / a q u a - o n l i n e Effect of dietary carbohydrate level on growth performance of juvenile spotted Babylon (Babylonia areolata Link 1807) Li-Li Zhang, Qi-Cun Zhou ⁎, Yi-Qiu Cheng Laboratory of Aquatic Economic Animal Nutrition and Feed, College of Fisheries, Guangdong Ocean University, Zhanjiang 524025, People's Republic of China a r t i c l e i n f o Article history: Received 26 April 2009 Received in revised form 25 June 2009 Accepted 29 June 2009 Keywords: Babylonia areolata Carbohydrate utilization Growth Enzyme activity a b s t r a c t A growth trial was conducted to determine the effects of dietary carbohydrate level on growth performance, feed utilization and metabolism of juvenile spotted babylon Six isonitrogenous and isoenergetic experimental diets (48% crude protein and 15 MJ kg− diet) using wheat starch as the carbohydrate source, were formulated to contain six carbohydrate levels Triplicate groups of 45 animals (initial average weight, 168.39 ± 0.69 mg) were stocked in 120-l tanks and fed to apparent satiation twice daily for 10 weeks Growth performance and feed utilization were significantly affected by dietary carbohydrate level Maximum weight gain and specific growth rate occurred at 20% dietary starch inclusion, survival and soft body to shell ratios were not significantly different among diets There were significant differences in protein, lipid, moisture and glycogen content in soft body Glycogen content in soft body was positively correlated with dietary starch level The activities of glucose-6-phosphate dehydrogenase and fructose-1,6-bisphosphatase were significantly affected by dietary starch level, with both peaking in the 20% treatment; however, there were no significant differences in 6-phosphofructokinase activity in any treatment Quadratic regression analysis of weight gain against dietary starch level indicated that the optimal dietary carbohydrate level for maximum weight gain of juvenile spotted babylon is 27.1% of dry diet © 2009 Elsevier B.V All rights reserved Introduction The known geographical distribution of spotted babylon (Babylonia areolata) extends from Sri Lanka and the Nicobar Islands through the Gulf of Siam, along the Vietnamese and Chinese coast to Taiwan (Altena et al., 1981) B areolata is nowadays one of the most extensively cultured marine mollusks in the Southeast Asian countries, and it is the second most economically important marine gastropods for human consumption in Thailand (Kritsanapuntu et al., 2009) It had many biological attributes and market characteristics necessary for profitable aquaculture, and it is considered a promising new candidate for aquaculture in China (Zhou et al., 2007a) Traditional culture of this spotted babylon mainly depends on minced small fish or crabs However, the limited supply of trash fish or crabs as the main feed sources for grow-out could be the main constraint to culture of spotted babylon in China, because of difficulty in storage, variable nutritional quality and low feed conversion rate Therefore it is necessary to conduct nutritional research and to develop nutritionally balanced feeds for the spotted babylon Limited research has been conducted on the nutrient requirements of spotted babylon (Ke et al., 1997, 2007; Xu et al., 2006; Zhou et al., 2007a,b) Based on the above mentioned research, optimum protein and lipid requirements of spotted babylon ranged from 37% to 45% and ⁎ Corresponding author Tel.: +86 759 2362270; fax: +86 759 2362290 E-mail addresses: zhouqc@gdou.edu.cn, qicunzhou@21cn.com (Q.-C Zhou) 0044-8486/$ – see front matter © 2009 Elsevier B.V All rights reserved doi:10.1016/j.aquaculture.2009.06.045 from 6.54% to 10.74%, respectively Information on nutritional requirements of major dietary components such as protein and energy is a prerequisite for the formulation of an inexpensive and balanced diet for aquatic species Carbohydrate has been given priority in nutritional studies for its protein-sparing effect because it is one of the principal energetic components which has lower relative cost than protein and lipid (Shiau and Lin, 2001; Keshavanath et al., 2002; Stone et al., 2003) Information on carbohydrate utilization in mollusks has mainly been focused on abalone (Thongrod et al., 2003) and scallops (Enomoto et al., 2000) However, to our knowledge, no information has been published to evaluate carbohydrate utilization of spotted babylon Therefore, the present study was designed to investigate the effects of dietary carbohydrate level on growth performance, feed utilization, carcass composition and key enzyme activities in glycolysis and gluconeogenesis of juvenile spotted babylon Materials and methods 2.1 Diet preparation Six isonitrogenous (48% crude protein) and isoenergetic (ca 15 MJ gross energy kg− 1) semi-purified diets were formulated to contain graded levels of wheat starch (uncooked) from to 30% (Table 1) Fish meal, casein and gelatin were used as protein sources, and pollock liver oil was used as the lipid source Isoenergetic diets were made by adjusting the lipid and cellulose content Diet ingredients were L.-L Zhang et al / Aquaculture 295 (2009) 238–242 Table Composition and proximate analysis of the experiment diets (% dry weight) Ingredient Fish meal Casein Gelatin Wheat starch Pollock oil Lecithin Choline chloride Monocalcium phosphate Ascorbyl-2-polyphosphate Vitamin mixture a Mineral mixture a Cellulose Sodium alginate Proximate composition (% dry Crude protein Crude lipid Ash Fiber Digestible carbohydrate b Gross energy c (MJ kg− 1) Dietary carbohydrate levels (%) 10 15 20 25 30 30.00 21.84 5.46 5.00 10.90 1.50 0.20 1.50 0.03 0.50 0.50 20.57 2.00 30.00 21.84 5.46 10.00 8.73 1.50 0.20 1.50 0.03 0.50 0.50 17.74 2.00 30.00 21.84 5.46 15.00 6.56 1.50 0.20 1.50 0.03 0.50 0.50 14.91 2.00 30.00 21.84 5.46 20.00 4.39 1.50 0.20 1.50 0.03 0.50 0.50 12.08 2.00 30.00 21.84 5.46 25.00 2.22 1.50 0.20 1.50 0.03 0.50 0.50 9.25 2.00 30.00 21.84 5.46 30.00 0.05 1.50 0.20 1.50 0.03 0.50 0.50 6.42 2.00 matter) 47.51 13.70 7.94 25.76 5.09 14.55 47.61 12.03 7.95 22.30 10.11 14.71 48.98 11.23 8.25 16.52 15.02 15.39 47.97 9.84 8.48 13.86 19.85 15.413 48.92 7.76 8.11 10.32 24.89 15.59 48.58 5.79 8.94 7.03 29.66 15.52 a Vitamin and mineral mixture was based on Zhou et al (2007a) Digestibility carbohydrate = 100 − protein − lipid − ash – fiber Gross energy were calculated using energy equivalents 18.81, 35.57, and 14.59 kJ g− for protein, lipid and digestible carbohydrate, respectively b c ground through an 80-mesh screen Vitamins and minerals were mixed by the progressive enlargement method (Zhou et al., 2007a) 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, and then airdried, sealed in plastic bags and stored frozen at − 20 °C until used 2.2 Animal rearing and experimental procedures Juvenile spotted babylon (B areolata) were obtained from a local farm Prior to the start of the trial, animals were acclimated to a commercial diet (containing 42% crude protein and 6% crude lipid) for weeks and were fed twice daily to apparent satiation At the beginning of the feeding trial, juvenile spotted babylon were starved for 24 h, weighed, and then they were randomly distributed into 18, 120-l cylindrical fiberglass tanks at 45 shells in each tank The bottom of each tank was covered with about cm clean sea sand, which simulated the natural environment that they normally inhabit Animals were provided with a continuous flow of sand-filtered seawater (2 l min− 1) with continuous aeration Water quality parameters were monitored daily During the feeding trial, water temperature ranged from 27.5 to 32.5 °C, salinity from 25 to 27 psu, pH from 7.6 to 8.0 Ammonia nitrogen was maintained lower than 0.03 mg l− and dissolved oxygen was not less than 6.0 mg l− Each experimental diet was randomly assigned to three tanks Juvenile spotted babylon were fed twice daily at a rate of to 4% wet body weight for 10 weeks, 30% of the ration was fed at 08:00 h and 70% at 19:00 h at the start of the dark phase when most feeding activity occurs (Liu and Xiao, 1998) Feed consumption was recorded for each tank every day Animals were bulk weighed and counted every weeks to adjust the feeding rate Tanks were thoroughly cleaned and the sea sand was changed biweekly 239 body tissue were weighed for calculation of soft body to shell ratio Soft-body tissues of spotted babylon were pooled, sealed in plastic bags and stored frozen at −20 °C until analysis Also, 10 to 15 spotted babylon animals in each tank were immediately frozen in liquid nitrogen and then stored at −80 °C until analyzed for glycogen content and enzymatic activities Chemical composition of diets and soft body of B areolata were determined by standard methods (Association of Official Analytical Chemists, AOAC, 1995) Moisture was determined by oven-drying at 105 °C for 24 h Crude protein content (N × 6.25) was determined according to the Kjeldahl method after acid digestion using an Auto Kjeldahl System (1030-Auto-analyzer, Tecator, Hoganos, Sweden) Crude lipid was determined by ether-extraction using a Soxtec extraction System HT (Soxtec System HT6, Tecator, Sweden) Ash was determined by muffle furnace at 550 °C for 24 h Glycogen of soft body was determined spectrophotometrically at 620 nm using the anthrone reaction method as previously described by Garcia de Frutos et al (1990) 2.4 Enzyme activity analysis 2.4.1 Fructose-1,6-bisphosphatase activities To measure the activity of fructose-1.6-bisphosphatase (FBPase; EC 3.1.3.11), a frozen sample of soft body was homogenized (dilution 1/10) in ice-cold buffer (85 mM imidazole-HCl, pH 7.7, mM MgCl2, 0.5 mM NADP, 12 mM 2-mercaptoethanol, 0.05 mM fructose-1,6-bisphosphate, 2.5 U ml− phosphate glucose isomerase, 0.48 U ml− G6PDH) The homogenate was centrifuged at 20,000 ×g for 30 at °C (Metón et al.,1999, 2003) Enzyme assay was performed as previously described (Foste and Moon, 1985; Bonamusa et al., 1992) using a Boi-Tek µ-Quart Microplate Spectrophotometer 2.4.2 Glucose-6-phosphate dehydrogenase activities To analyze the glucose-6-phosphate dehydrogenase activity (G6PD; EC 1.1.1.49), a frozen sample of soft body was homogenized (dilution 1/10) in ice-cold buffer (8 mM imidazole-HCl, pH 7.7, mM MgCl2, mM NADP and mM glucose-6-phosphate) The homogenate was centrifuged at 20,000 ×g for 30 at °C (Metón et al., 1999, 2003) The assay was performed as previously described (Foste and Moon, 1985; Bonamusa et al., 1992) using a Boi-Tek µ-Quart Microplate Spectrophotometer 2.4.3 6-phosphofructokinase activities For measurement of 6-phosphofrutokinase (PFK; EC 2.7.1.11) activity, a frozen sample of soft body was homogenized (dilution 1/10) in icecold buffer (100 mM Tris–HCl, pH 8.25, mM MgCl2, 50 mM KCl, 0.15 mM ammonium sulfate, mM 2-mercaptoethanol, 10 mM fructose-6-phosphate, 30 mM glucose-6-phosphate, 0.675 U ml− aldolase, U ml− triose phosphate isomerase, U ml− glycerol 3-phosphate dehydrogenase) The homogenate was centrifuged at 20,000 ×g for 30 at °C with the assay performed as previously described (Foste and Moon, 1985; Bonamusa et al., 1992) using a Boi-Tek µ-Quart Microplate Spectrophotometer All enzyme activities were expressed per mg of total protein (specific activity) The total protein content in crude extracts was determined at 30 °C using bovine serum albumin as a standard based on the method of Bradford (1976) One unit of enzyme activity was defined as the amount of NADH or NADPH generated by per mg protein per minute at 30 °C 2.5 Calculations and statistical analysis 2.3 Samples collection and chemical analyses The parameters were calculated as follows: At the end of the growth trial, spotted babylon were starved for 24 h and weighed A sample of 135 spotted babylon (B areolata) at the initiation of the feeding trial and 25 to 30 spotted babylon per tank at termination were used for carcass proximate analysis Shell and soft- Specific growth rate (SGR) =(Ln Wt − Ln Wi) × 100 / t Percent weight gain (WG, %) =Wt (g) × 100 / Wi (g) Feed conversion ratio (FCR) =feed consumed (g, DW)/weight gain (g) 240 L.-L Zhang et al / Aquaculture 295 (2009) 238–242 Protein efficiency ratio (PER) =weight gain (g) / protein intake (g) Soft body to shell ratio (SB/SR) =soft-body weight (g)/shell weight (g) Mean protein gain (MPG) =SBt · (1 − Mt)·Pt − SBi·(1 − Mi)·Pi where Wt is final body weight, Wi is initial body weight, t is experimental times in days, SBt is final soft-body weight (mg), SBi is initial soft-body weight (mg), Mt is final moisture level in soft body (%), Mi is initial moisture level in soft body (%), Pt is final protein level in soft body (%), and Pi is initial protein level in soft body (%) (Mai et al., 1995) Results are presented as mean ± sd All data were subjected to oneway ANOVA When there were significant differences, the group means were further compared with Duncan's multiple-range test A quadratic regression analysis method (Snedecor and Cochran, 1978) was used to analyze the correlation between weight gain and dietary wheat starch level of juvenile spotted babylon All statistical analyses were performed using the SPSS 15.0 (SPSS, IL USA) Fig Relationship between weight gain and dietary carbohydrate levels of juvenile spotted babylon (B areolata) fed the experimental diets Results Growth performance and feed utilization of juvenile spotted babylon fed different dietary carbohydrate levels are shown in Table Survival in all treatments was 100% Weight gain (WG) and specific growth rate (SGR) were significantly affected by the dietary carbohydrate levels, with the highest WG and SGR occurring at the 20% dietary starch level WG and SGR significantly increased with dietary starch level from 5% to 20% However, WG and SGR slightly decreased at dietary starch levels of 20% to 30% The secondary curve equation between weight gain and dietary starch level was y=−0.6562x2 +35.541x+50.881 (R2 =0.9372) (Fig 1) The optimal dietary starch level was determined to be 27.1% for maximum weight gain Feed conversion ratio of spotted babylon fed dietary starch levels from to 10% was significantly lower than that of animals fed 15% starch and greater Protein efficiency ratio significantly increased with dietary starch level, increasing from to 20%, with no significant differences among the treatments with over 20% starch Soft body to shell ratio was not significantly affected by the dietary starch levels Mean protein gain significantly increased with increasing dietary starch levels from to 20%; there were no significant differences at dietary starch levels over 20% Soft body composition of spotted babylon was significantly affected by the dietary starch levels (Table 3) Moisture and protein content in soft body significantly increased with increasing dietary starch level However, lipid content in soft body significantly decreased with increasing dietary starch level Glycogen content in soft body significantly increased with dietary starch level from to 25%; however, glycogen content in soft body significantly decreased when the dietary starch level increased from 25 to 30% PFK activities in soft body did not differ among all treatments G6PD and FBPase activities were significantly affected by dietary starch levels (Table 4) The highest G6PD and FBPase activities were found in animals fed 20% starch There were no differences in G6PD among treatments, except for animals fed the 20% starch diet which had higher activities than those fed the other diets The FBPase activity was lowest in spotted babylon fed the 5% dietary starch level, which was significantly lower than that of animals fed the 20% and 25% dietary starch diets Discussion The present study showed that weight gain of juvenile spotted babylon increased with increasing wheat starch level from to 20%, and slightly decreased thereafter with further increase in dietary wheat starch A secondary curve equation according to regression analysis of weight gain against dietary starch level indicated that optimal dietary starch level for maximum weight gain was 27.1% These results are lower than those reported for Haliotis asinine at 47.81% (Thongrod et al., 2003) The main difference in carbohydrate utilization between spotted babylon and abalone may be due to the carnivorous feeding activity of spotted babylon, while abalone is a herbivorous mollusk However, the carbohydrate level is higher than the values reported for shrimp (Alava and Pascual, 1987; Rosas et al., 2000; Guo et al., 2006) and some fish (Catacutan and Coloso, 1997; Enes et al., 2006, 2008) The ability of different species to utilize carbohydrate depends on their ability to oxidize the glucose from the digestion of carbohydrate, and to store the excess glucose as glycogen or fat (Guo et al., 2006) Meanwhile, the ability to utilize dietary carbohydrate as an energy source depends on digestibility, endogenous metabolic enzymes, and assimilation of different dietary carbohydrates (Stone et al., 2003) The incorporation of appropriate carbohydrate levels in the diet has been reported to improve growth performance in some fish and shrimp species (Anderson et al., 1984; Alava and Pascual, 1987; Hemre et al., 1995; Peragón et al., 1999; Hung et al., 2003) Similar results were observed in the present study Both carbohydrate and lipid in the diet are important energy sources for mollusk species (Mai et al., 1995) Generally, herbivorous and omnivorous species, such as certain fish and mollusks, can use higher carbohydrate levels for optimal growth, and have the ability to utilize carbohydrate for energy However, no growth improvement was observed due to dietary starch incorporation in other species (Hemre et al., 2000; Enes et al., 2006, 2008) In the present study, to keep energy invariable in all treatments, lipid content decreased when dietary starch level increased The growth performance results indicated that spotted Table Growth performance, feed utilization, SB/S ratio and mean protein gain of juvenile spotted babylon (B areolata) fed on the experimental diets Dietary carbohydrate levels (%) Initial weight (mg) Final weight (mg) Weight gain (%) SGR FCR PER SB/S ratio MPG (mg/shell) 10 15 20 25 30 167.87 ± 0.81 168.40 ± 1.00 169.07 ± 0.46 168.40 ± 0.69 167.93 ± 0.46 168.67 ± 0.12 546.67 ± 9.59a 720.13 ± 25.15b 846.87 ± 28.01c 1046.09 ± 77.50d 1042.66 ± 32.02d 1045.95 ± 44.78d 225.68 ± 7.26a 327.60 ± 13.02b 400.91 ± 16.68c 548.89 ± 8.10d 520.91 ± 20.73d 520.12 ± 26.25d 1.69 ± 0.03a 2.06 ± 0.05b 2.28 ± 0.02c 2.63 ± 0.01d 2.57 ± 0.03d 2.59 ± 0.09d 1.10 ± 0.04c 0.83 ± 0.04b 0.73 ± 0.02a 0.69 ± 0.01a 0.70 ± 0.02a 0.72 ± 0.05a 1.92 ± 0.06a 2.53 ± 0.13b 2.79 ± 0.06c 3.00 ± 0.01d 2.91 ± 0.07cd 2.86 ± 0.19cd 0.70 ± 0.03 0.71 ± 0.04 0.71 ± 0.06 0.78 ± 0.04 0.74 ± 0.06 0.76 ± 0.05 21.81 ± 2.48a 27.26 ± 1.54ab 32.10 ± 3.83b 46.64 ± 4.34c 44.59 ± 1.85c 48.86 ± 4.28c Values are means + sem (n = 3) Values in the same column followed by the same letter are not significantly different L.-L Zhang et al / Aquaculture 295 (2009) 238–242 Table Composition and glycogen content in soft body of juvenile spotted babylon (B areolata) fed on the experimental diets Dietary carbohydrate levels (%) Moisture (%) Protein (%)⁎ Lipid (%)⁎ Glycogen (mg/g) 10 15 20 25 30 70.37 ± 1.06a 71.42 ± 0.91ab 70.84 ± 0.45ab 72.39 ± 1.04bc 73.04 ± 1.05c 73.17 ± 0.34c 53.51 ± 1.14a 54.28 ± 0.49ab 53.94 ± 0.50ab 54.77 ± 0.48b 57.41 ± 0.03c 59.12 ± 0.49d 17.59 ± 0.60c 17.51 ± 0.92c 16.62 ± 0.20c 16.22 ± 2.40c 10.43 ± 0.03b 7.88 ± 0.19a 26.65 ± 0.04a 29.80 ± 1.02b 29.47 ± 0.74b 31.86 ± 0.94c 44.34 ± 1.97d 29.30 ± 0.39b 241 spotted babylon was not depressed by increasing the dietary starch level The activities of the lipogenic enzyme glucose-6-phosphate dehydrogenase (G6PD) increased in animals fed the high-carbohydrate diets This is in agreement with the results reported in some fish (Lin and Shiau, 1995; Enes et al., 2008) In conclusion, this study provides some insight into the carbohydrate nutrition of juvenile spotted babylon and indicates that the optimal carbohydrate (wheat starch) level for juvenile spotted babylon for maximum weight gain was 27.1% Dietary starch enhanced glycolytic and lipogenic pathways in soft body of spotted babylon Values are means + sem (n = 3) Values in the same column followed by the same letter are not significantly different ⁎On dry weight basis Acknowledgements babylon have less ability to utilize higher dietary lipid; similar results also have been reported in our previous study (Zhou et al., 2007b) In the present study, glycogen content in soft body significantly increased when the dietary starch levels increased from to 25% However, glycogen content in soft body significantly decreased with the dietary starch levels increasing from 25 to 30% Similar results were observed in gilthead sea bream (Enes et al., 2008) Nevertheless, our data showed the negative correlation between lipid content in soft body and dietary starch levels These results may indicate that when the dietary lipid was supplied in excess (dietary lipid content was 13.70%), a proportion of dietary lipid was deposited as lipid not glycogen in soft body Protein content in soft body significantly increased with increase of the dietary starch level, it indicated dietary carbohydrate improved protein utilization This is in agreement with the results reported for some species (Thongrod et al., 2003; Enes et al., 2008) Several studies have showed that high starch digestibility was observed in European sea bass (Enes et al., 2006) and gilthead sea bream (Enes et al., 2008) It indicated that some aquatic fish could decompose starch into glucose and utilize it well Such inducible enzymatic response and digestibility of starch may also contribute to explain the reason why higher glycogen levels obtained in spotted babylon than those fed higher starch levels (diets and 5) It appears that carnivorous species make more efficient use of carbohydrates than herbivorous (Furuichi and Yone, 1982), however, the result that B areolata fed on highcarbohydrate diets showed stimulation of key visceral enzymes for glycolysis and the pentose phosphate pathway suggests its ability to utilize high-carbohydrate diets in this study The activities of phosphofructokinase (PFK) and fructose-1,6-biphosphatase (FBPase) in soft body of spotted babylon fed the diet containing 20% starch had higher activities than those fed the other diets This is in agreement with the results reported for some species (Borrebaek and Christophersen, 2000; Enes et al., 2008) Higher activity of the glycolytic and gluconeogenesis enzymes may suggest that spotted babylon have metabolic ability to adapt to high-carbohydrate levels (about 20% to 25% carbohydrate) Moreover, our data indicated a promotive effect on FBPase activity with carbohydrate level increasing These results are consistent with other results in fish (Metón et al., 1999; Fernández et al., 2007; Enes et al., 2008) It suggests that endogenous gluconeogenesis in Table PFK, G6PD and FBPase activities in soft body of juvenile spotted babylon (B areolata) fed the experimental diets Dietary carbohydrate levels (%) PFK (U/mg protein) G6PD (U/mg protein) FBPase (U/mg protein) 10 15 20 25 30 0.96 ± 0.08 0.97 ± 0.03 0.97 ± 0.05 1.06 ± 0.07 1.05 ± 0.07 0.96 ± 0.10 0.97 ± 0.01a 0.97 ± 0.02a 0.99 ± 0.04a 1.10 ± 0.12b 1.00 ± 0.02ab 0.98 ± 0.09a 0.83 ± 0.04a 0.91 ± 0.09ab 0.91 ± 0.09ab 1.03 ± 0.05b 0.99 ± 0.05b 0.96 ± 0.12ab Values are means + sem (n = 3) Values in the same column followed by the same letter are not significantly different This research was funded by Zhanjiang Science 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Chemists (AOAC), 1995 Of cial methods of analysis of Official Analytical Chemists International, 16th edition Association of Official Analytical Chemists, Arlington, VA, USA Bonamusa, L., Garcia... proportion of dietary lipid was deposited as lipid not glycogen in soft body Protein content in soft body significantly increased with increase of the dietary starch level, it indicated dietary carbohydrate. .. utilize dietary carbohydrate as an energy source depends on digestibility, endogenous metabolic enzymes, and assimilation of different dietary carbohydrates (Stone et al., 2003) The incorporation of