Materials and methods

Diet preparation

Basal diet formulation and proximate composition analysis (AOAC, 1995) are shown in Table 1. Casein (Sigma Chemical Co., St. Louis, MO, USA), fish oil (semi-refined fish oil, Oleaginosa Victoria S.A., Peru) and corn oil (Tai-Tang Industrial, Taiwan), and corn starch (Sigma Chemical) were used as dietary protein, lipid and carbohydrate sources, respectively. All diets were kept isoenergetic at 15.05 MS kg^-1 diet which was recommended by Shiau & Lan (1996). An attractant that had a similar chemical composition to squid mantle tissue (Mackie & Mitchell 1985) was added at 60 g kg^-1 diet to all diets to increase palatability and diet acceptance. In Experiment 1, NT mixture (mixed-NT, Sigma Chemical) of IMP, AMP, GMP, UMP and CMP at ratio of 1:1:1:1:1 was added to the basal diet at concentrations of 0.5, 1.0, 1.5 and 2.0 g mixed-NT kg^-1 diet. Experiment 2 was a follow-up based on the results of the Experiment 1 in which 1.5 g kg^-1 diet (result of the Experiment 1) of either IMP, AMP, GMP, UMP or CMP was added to the basal diet. Basal diet without NT supplementation (NT-free) was used as control in both Experiments. In Experiment 2, 1.5 g mixed-NT kg^-1 was also included for comparison. The dry ingredients were mixed with oil and then cold water was added until a stiff dough resulted. This was then passed through a mincer with die, and the resulting strands were dried using an electrical fan at 20 °C. After drying, the diets were broken up and sieved into pellets (1.2 mm in diameter) and stored at -20°C until feeding.

¹ Vitamin mixture (mg g^-1 mixture): thiamin hydrochloride, 2.5; riboflavin, 10; calcium pantothenate, 25; nicotinic acid, 37.5; pyri-doxine hydrochloride, 2.5; folic acid, 0.75; inositol, 100; L-ascorbyl-2-monophosphate Mg, 5; choline chloride, 250; menadione, 2; alpha-tocopheryl acetate, 5; retinyl acetate, 1; cholecalciferol, 0.0025; biotin, 0.25; vitamin B₁₂, 0.05. All ingredients were diluted with alpha-cellulose to 1 g.
² Mineral mixture (mg g^-1 mixture): calcium lactate, 327; K₂PO₄, 239.8; CaHPO₄·2H₂O, 135.8; MgSO₄·7H₂O, 132; Na₂HPO₄·2H₂O, 87.2; NaCl, 43.5; ferric citrate, 29.7; ZnSO₄·7H₂O, 3; CoCl₂·6H₂O, 1; MnSO₄·H₂O, 0.8; KI, 0.15; AlCl₃·6H₂O, 0.15.
³ As mg kg^-1 diet: L-aspartic acid, 180; L-threonine, 440; L-serine, 330; L-glutamic acid, 530; L-valine, 360; L-methionine, 360; L-iso-leucine, 290; L-leucine, 550; L-tyrosine, 220; L-phenylalanine, 290; L-lysine-HCl, 290; L-histidine-HCl, 150; L-proline, 14,560; L-alanine, 2,730; L-arginine, 2,280; taurine, 3,370; glycine, 8,920; betain-HCl, 9,100; trimethylamine-HCl, 910; trimethylamine n-oxide HCl, 11,380; hypoxanthine, 470; L-(+)-lactic acid, 910; alpha-cellulose, 1,450 (Mackie & Mitchell 1985).

Experimental procedure

E. malabaricus juveniles obtained from a local hatchery (Pingtung, Taiwan) were used in the study. Upon arrival, they were acclimated to laboratory conditions for 4 weeks in a 1000 L-plastic tank and fed a commercial diet (Uni-Presi-dent Enterprise Corp., Tainan, Taiwan). The proximate composition (g kg^-1) of the commercial diet was as follows: moisture, 117; crude protein, 433; lipid, 88; ash, 93. At the beginning of the experiment, 10 fish (mean initial weight: 5.90 ± 0.01 g, Experiment 1; 10.33 ± 0.01 g, Experiment 2) were stocked in each aquarium (0.305 · 0.610 · 0.555 m³). Each experimental diet was fed to fish in three aquaria. The fish were chosen for the experiment and the diets were assigned to the respective groups randomly. Each aquarium was part of a closed recirculating system with a common reservoir of water at 29-32 g L^-1 salinity. The water was circulated at 2 L min^-1 through two separate biofilters to remove impurities and reduce ammonia concentrations. Half of the water in the system was exchanged daily. The fish were fed 30 g kg^-1 of their body weight per day. This amount was close to the maximal daily ration for grouper according to feed consumption during the acclimation period of the study. The daily ration was divided into two equal meals fed at 08:30 and 16:30 h. Grouper finished their ration within 1-2 min after feeding, thereby minimizing the leaching of NT into water was very low and negligible. Fish were weighed once every 2 weeks and the daily ration adjusted accordingly. The remaining feed and feces were removed by a siphon immediately after the feeding. A photo-period of 12 h light (08:00 to 20:00 h), 12 h dark was used. Any dead fish were removed and not replaced during the experiment. The fish were given the test diets for an 8-week period. At the end of the feeding trial, percentage of body weight gain (WG) in each aquarium [100 х (final body weight ) initial body weight)/initial body weight], feed efficiency (FE) [(final body weight ) initial body weight)/feed intake] and survival [100 х (initial fish number/initial body weight] were calculated.