ABSTRACT

The effects of fish meal (FM) replacement with graded level of soybean meal (SBM) on growth performance, feed utilization, survival rate and body composition were investigated in juvenile tiger grouper, Epinephelus fuscoguttatus (initial body weight 13.9 ± 0.65 g). Six experimental diets were formulated to contain 50% crude protein, 16% crude lipid and 365.8 kcal/100g feed with SBM replacing FM protein at 0% (SM 0), 10% (SM 0), 20% (SM 20), 30% (SM 30), 40% (SM 40) and 20% with phytase (SM 20P) replacement levels. At the end of the ten-week feeding trial, there were no significant differences detected in terms of growth performances (weight gain and specific growth rate), feed conversion ratio (FCR) and survival rates of fish fed with the control diet (SM 0), SM 20, SM 30 and SM 20P. Net protein utilization of fish fed SM 20P was higher than those fed with other diets suggesting an improved utilization of nutrients with phytase addition in the diet. Replacements of FM protein with SBM at 10% and 40% have resulted in significantly lower growth and poorer FCR than other replacement levels. Survival rates remain high (≥90%) throughout the trial. Whole-body proximate composition of the fish was significantly affected by the inclusion of SBM in the diets. It can be concluded that 20% - 30% of FM protein replacement with SBM is recommended and addition of phytase in the SBM-based diet should be considered to improve nutrient utilization of tiger grouper juvenile.

1. INTRODUCTION

Fish meal (FM) is still widely used as the main protein source in commercial diets for the cultured marine fishes all over the world due to its high nutritional value, balanced amino acid and fatty acid profiles, good palatability and digestibility [1]. Unfortunately, the use of FM in fish diets is always associated with sustainability issue and high cost due to its limited supplies. Research on diet development using alternative dietary ingredients is one of the current focuses in the field of aquaculture nutrition. For several years, continuing research has been intensively conducted in order to identify the suitable alternative protein sources to replace FM in aquaculture diets.

Among all the plant protein ingredients, soybean (SBM) meal has been identified as one of the most promising candidates to replace FM. It has been viewed as an ideal alternative due to its availability, lower price compared to FM and high protein content [2]. Studies conducted on several marine fish species such as spotted rose snapper, (Lutjanus guttatus), Asian seabass (Lates calcarifer), tiger puffer (Takifugu rubripes), sea bream (Sparus aurata), and Japanese flounder (Paralichthys olivaceus) showed that SBM can replaced FM at a level of up to 45% [3-7]. However, not much information is available on the potential of soybean meal as a source of protein in the diets of grouper species.

It is well reported that the constraint in utilizing SBM as a dietary protein source in fish diet is associated with the presence of anti-nutritional factors, especially phytate. Phytate disables the protein in SBM to be fully digested and utilized by the fish. Supplementation of phytase, an enzyme that helps improve nutrient efficiency in plant protein, can be implemented in fish feed. Therefore, the present study intends to determine the effects of FM replacement with SBM on the growth performance, survival, feed utilization, body composition of juvenile tiger grouper and to investigate if the phytase supplementation is able to improve the utilization of SBM-based diets in tiger grouper.

2. MATERIALS AND METHODS

2.1. Experimental Diets

Danish FM and defatted SBM were used as the source of protein in this feeding trial. For dietary lipid source, industrial grade fish oil and soybean oil were used. Diets were formulated with 50% protein, 16% lipid and energy content of 365.8 kcal/100g diet where FM protein was replaced with SBM protein at 0% (SM 0), 10% (SM 10), 20% (SM 20), 30% (SM 30), 40% (SM 40) and 20% plus phytase (SM 20P) replacement levels. Diets were prepared by mixing the dry homogenized ingredients in a mixer followed by dietary lipid and water. For SM 20P diet, phytase (Natuphos^® 10,000 G) dose of 2000 FTU/kg enzyme was dissolved with water and added to the mixture of ingredients [8]. The dough was screw-pressed through a 3 mm die and oven dried at 40˚C for 6 hours. Formulation and proximate composition of experimental diets are shown in Table 1.

2.2. Experimental Fish and Culture Condition

Tiger grouper juveniles were obtained from a local fish farm in Kuala Penyu, Sabah. Upon arrival, the fish were acclimated to hatchery condition for 2 weeks and fed with control diet (SM 0). After the acclimation period, fish with an average initial body weight of 13.9 ± 0.65 g were randomly distributed into 18 tanks and stocked at 10 individual per tank. Fiberglass tanks (150 L) supplied with aeration using flow-through seawater system (3 L·min⁻¹) was used in this trial. Fish were hand-fed twice a day at apparent satiation level. The feeding trial was lasted for 10 weeks.

2.3. Sample Collection and Chemical Analysis

At the beginning of the feeding trial, 8 fish from an initial pool of fish were sampled and ground into powder form for analysis of whole body composition. Prior to measurements, fish were starved for 24 hrs to ensure there was no food in the digestive tract. An overdose of commercial anesthetic (a-methylquinoline) was used to

anaesthetize the fish before taking weight and length measurements. At the end of experiment, fish liver and viscera from each tank were removed and weighed to assess the hepatosomatic index (HSI) and visceralsomatic index (VSI), respectively, and adequate amount of fish samples were stored at −86 for final body proximate analysis.

Proximate composition of diets, whole-body fish, and feces were determined following AOAC [9] method. Crude protein was determined using the Kjeldahl method using an automatic system (Kjeltec 2300). Crude lipid was determined gravimetrically using ether-extraction method in a soxhlet extraction unit (Soxtec 2043). Ash content was determined as the residue remaining after incineration of samples at 550˚C in a muffle furnace for 6 hours.

Amino acid composition of experimental diets was determined by hydrolysing the samples with 6 N HCl at 110˚C for 24 hr and then derivatized with AccQ reagent (6-aminoquinolyl-N-hydroxysuccinimdyl carbamite) before chromatographic separation using an AccQ Tag^TM reversed phase (3.9 × 150 mm) analytical column (Waters^®). The amino acid analysis was performed on a HPLC system which consisted of Waters 1525 Binary HPLC Pump, 717 Plus auto-sampler (Waters^®) and Waters 2475 Multi λ Fluorescence detector (wavelength excitation 250 nm, emission 395 nm). Chromatographic peaks were integrated, identified and quantified with Breeze^TM software, version 3.20 by comparing to known standards (Amino acid standard H, Pierce, Rockford, Illinois, USA). Methionine and cystine were determined from the same method of acid hydrolysis after treatment with performic acid oxidation.

2.4. Calculation

Growth performances, feed utilization efficiency and body indices were calculated as follows:

% weight gain = percentage of initial body weight at the end of trial;

Specific growth rate (SGR) = [(ln final weight − ln initial weight)/days] × 100;

Feed conversion ratio (FCR) = dry feed consumed (kg)/wet weight gain (kg);

Condition factor (CF) = [fish weight/(total length)³] × 100;

Hepatosomatic index (HSI) = (liver weight/fish weight) × 100;

Viserosomatic index (VI) = (viseral weight/body weight) × 100.

2.5. Statistical Analysis

The fish growth performances, survival, feed utilization, whole body proximate composition and body indices were analyzed by One-way ANOVA of variance (ANOVA) for significant differences. The homogeneity of variances was tested using Levene’s test, and Duncan’s multiple range test was used to compare the individual treatment mean differences at the 0.05 significance level. The statistical package IBM SPSS Statistics 19 for Windows was used for all statistical analyses.

3. RESULTS

3.1. Nutrient Composition of Experimental Diets

Protein and lipid levels in the experimental diets corresponded to the calculated values (49.7% - 50.6% and 15.8% - 16.8%, respectively). Crude ash and crude fiber contents of experimental diets ranged from 10.1% to 11.5%, and 0.1% to 1.34%, respectively (Table 1). There was a trend of reduced crude ash content as SBM inclusion increased in the diets, opposed to the trend of crude fiber. The amino acids composition of the experimental diets with different SBM inclusion is presented in Table 2. In general, essential amino acid such as methionine and lysine were observed to be reduced with increasing SBM levels in the diets. Methionine and lysine values ranged from 0.92% to 1.53% and 3.28% to 3.75%, respectively.

3.2. Growth Performance, Survival Rates and Feed Utilization of Tiger Grouper Fed Graded Levels of Soybean Meal

FBW of fish fed SM 0 (30.44 g), SM 20 (29.10 g) SM 30 (30.11 g), and SM 20P (30.31 g) were not significantly different. FBW of fish fed diets SM 10 and SM 40 (26.13 - 26.72 g) were significantly lower (P < 0.05) than FBW of fish in other groups. The BWG of tiger grouper fed experimental diets ranged from 89.75% (SM 40) to 119.51% (SM 0). Similarly, fish fed SM 10 and SM 40 attained significantly lower (P < 0.05) BWG than the fish fed those of the other treatments. The SGR of tiger grouper ranged from 1.12% to 1.38% and followed the trend of BWG. All treatments showed high survival rates (90.0% - 96.7%). There was no significant difference (P < 0.05) observed among the survival of fish in all treatments (Table 3).

Table 4 shows the feed utilization of tiger grouper in the 10 weeks feeding trial. Total feed intake values ranged from 21.80 g to 24.84 g per individual. The lowest feed intake was observed in fish fed SM 40 (21.80 g/fish) and this value was significantly lower (P < 0.05)