Experimental system Juveniles of red sea bream were collected from a local hatchery, in Miyazaki prefecture, Japan, and transported to the Kamoike Marine Production Laboratory, faculty of Fisheries, Kagoshima University, Japan. The feeding trial using juveniles' average initial body weight of 3.35g was carried out in 100 L polycarbonated tanks (filled with 80 of water) at 15 fishes/tank where each tank was equipped with an inlet, outlet and continuous aeration. Each treatment having triplicates. All fish were fed twice daily up to apparent satiation. Uneaten diets were collected, oven dried and weighed for getting real feed intake. Periodic sampling was conducted every 2 weeks to monitor instantaneous growth and mortality in tanks. The tanks were maintained under a natural light and dark regime. The seawater was pumped from the deep basin of Kagoshima Bay, Japan; gravel filtered and supplied to the system. A flow rate of 1.5l/ min was maintained throughout the experimental period (42 days). Initial sample from stock tank (20 fishes) were taken for body chemical composition. In order to minimize error in body weight data, fish were starved 24 h before final (42 days) sampling. Total number of fish and individual body weight of fish from each tank was recorded. Two fish from each tank were randomly taken and keep at -20? for body chemical composition. Using heparinized syringes, blood was collected from the caudal vein of five in each replicate tank and polled. Plasma samples were obtained by centrifugation at 3000 ×g for 15 minutes using a high speed refrigerated microcentrifuge (MX-160; Tomy Tech USA Inc., Tokyo, Japan) kept at -80?. Fish were dissected for liver and muscle and weighed and store in -800 c for measuring the hepato-somatic index and other analysis.
Test diets summarized the composition of experimental diets. All the dietary components were obtained commercially except SDBP. SDBP obtained from Biochemistry and feed chemistry lab. So in this study, we used condensed liquid SDBP. The liquid part of SDBP was first separated by a decanter and the liquid part was condensed (Hayashi et al., 2009. Six isonitrogenous (more than 44% crude protein), isolipidic (10-11% total lipid) diets were formulated; where diet 1 was a 100% fishmeal (FM) based control diet (D1). Diets 2 to 5 were prepared as follows by replacing FM protein with SDBP 2% or D2; SDBP 2% and KS meal (Krill+Squid meal) or D3; SDBP 16% or D4; SDBP 16% and KS meal (Krill+Squid meal) or D5 and SDBP 20% or D6. Here fish meal and soybean meal is the source of protein, wheat meal is the source of carbohydrate, pollack liver oil is the source of lipid and SDBP itself is functional ingredients. Krill and squid meal is the source of protein. The diets were prepared by mixing all ingredients in food processor for 30 min. Pellet size was 1.5 and 1.9 mm and pellets was oven (DK 400 Yamato Scientific, Tokyo, Japan) dried for 2 h at 600. The diets were stored in a cold room.
Proximate analysis of whole body Whole body in each treatment was analyzed for moisture, crude protein, total lipid and ash, in triplicate, using standard AOAC methods (AOAC, 1990). Amino acid analysis Amino acid analysis of diets samples was analyzed using high performance liquid chromatography (HPLC, Shimadzu Corp.) according to Teshima et al., 1986. To quantify free amino acid, 40 mg of sample was mixed with 100μl norleucine as internal standard (0.6mg), 900μl cold distilled water and 2.5 ml of cold 10% trichloroacetic acid (TCA) and was homogenized by using polytron homogenizer (Kinematica, Gmbh LITTAU, Lucerne, Switzerland). Samples were then centrifuged at 3000×g for 15 minutes at 40 and washed with diethyl ether to remove TCA from the homogenate. The PH of the homogenate was then adjusted to 2.2 and diluted to 5 ml sodium citrate, filtered (0.45μ) and stored at 40 for HPLC injection. Health condition or blood parameter and oxidative stress To measure the health condition we used plasma. So plasma chemical parameters were measured spectrophotometrically with an automated analyzer (SPOTCHEMTM EZ model SP4430, Arkray, Inc. Kyoto, Japan). Biological antioxidant potential (BAP) and reactive oxygen metabolites (d-ROMs) were also measured spectrophotometrically from blood plasma with an automated analyzer FRAS4, Diacron International s.r.l., Grosseto, Italy by following Morganti et al.(2002).
Statistic analysis All data were tested using one-way analysis of variance (Package Super-ANOVA, version 1.11; Abacus Concepts, Berkery, CA, USA). The level of significance between individual treatments (p<0.05) was evaluated by Tukey Kramer test. Results were presented as means± standard deviations.