Experimental diet
Feed ingredients such as SBM, fish oil, fish hydrolysate, rice bran oil, mono calcium phosphate, vitamin and mineral premix were obtained from Growel feeds Pvt, Limited, Andhra Pradesh, India. SSM, common salt and cassava starch were purchased from local market. The ingredients were dried well and powdered. These major ingredients were mixed in the feed at three different concentration
viz., SSM 25%, 30% and 35%. The control feed was prepared without adding SSM. All the ingredients and feed additives except vitamin and mineral mixture were mixed well and made it as a ball and cooked in a pressure cooker for 10-15 minutes. Then each dough was pelletized by using the manual pelletizer. Then pelletized feeds were dried at 60°C for 12 h and stored in airtight containers. The ingredients composition of control feed and experimental diets and proximate composition of protein supplements were presented in Table 1 and 2
.
Proximate analysis and Amino acid analysis of experimental feed
The proximate analysis of experimental feed was estimated using standard method (
AOAC, 1995). The results were given in Table 3. The amino acid profile of the experimental feed were given in Table 4.
Experimental setup
The experimental set up comprised of three sets of treatments and one sets of control (Replicates) for a period of 90 days. Experiment was conducted in rectangular cement tanks (volume: 1000 l) installed with 14 fish hapa’s of the size 0.30 m
3 (0.73 m*0.59 m*0.71 m) (volume: 300 l) were used.
Experimental fishes
The experimental fish (Thai-Chitralada) were procured from Svara Biotechnovations fish farm, Madurai, Tamil Nadu. All the fish seeds were properly acclimatized in cement tanks. The fishes were graded according to their weight prior to the experiment. An average of 2.650 g size 460 Nos of Thai-Chitralada were selected for the experiment. The fishes were stocked at 30 numbers per hapa and capacity of the hapa is 300 l. Daily the fishes were fed @ 5% of their body weight. Fortnightly the sampling was carried out. The bio-growth parameters were calculated.
DNA/RNA ratio
Estimation of RNA and DNA in fish were done by method of
Buckley and Bulow (1987) and the results were presented in Table 5.
Extraction of nucleic acids
In Laboratory, larvae were thawed and measured (0.1 mm) under a dissecting microscope equipped with ocular micrometer. Conventional fluorimetric analysis (CFA) developed by
Clemmesen (1988, 1993) and further modified by
Chicharo (1996) modified ´ fluorimetric analysis (MFA) were used to quantify nucleic acids in individual fish larvae. Fish larvae were extracted in 0.15 ml of 1% sarcosine (sodium N-lauroylsarcosine) in Tris-EDTA buffer (pH 8.0) to give a final concentration of 0.1%. After centrifugation, aliquots of the supernatant were used for further analysis.
CFA
A 0.2 ml aliquot of extracted sample was combined with 0.4 ml of Tris-NaCl 21 (pH 7.5) and 0.05 ml of ethidium bromide (0.1 mg/ml). Another 0.2 ml aliquot of the same extracted sample was combined with 0.35 ml of Tris-NaCl 21 and 0.05 ml of ribonuclease A (Type-II A, 0.12 mg/ml). This mixture was incubated at 37.8oC for 30 min, allowed to reach room temperature for 15 min and stained with 0.05 ml of EB.
MFA
For purification of nucleic acids, a third 0.6 ml aliquot of the extracted sample was washed with 0.6 ml of phenol- chloroform-isoamyl alcohol (49.5:49.5:1, v/v) and then with 0.3 ml of chloroform-isoamyl alcohol (24:1, v/v). After these purification steps, 0.2 ml aliquots of the supernatant were treated as above for CFA.
Fluorescence assays
Calculations of nucleic acids concentration were identical for both procedures. Endogenous sample fluorescence (blank) was subtracted from total sample-EB dye fluorescence. The fluorescence due to total RNA, mainly ribosomal, was calculated as the difference between total fluorescence (RNA and DNA) and the fluorescence measured after ribonuclease treatment, which is assumed to be due to DNA. Fluorescence was determined by exciting at 365 nm and reading at 590 nm with a spectrofluorometer (Hitachi Model 650-10). Concentrations were determined by running standard curves of DNA-EB and RNA-EB every day with known concentrations of 21 21 l-DNA (0.25 mg/ml) and 16s-23s RNA (4 mg/ml ), in the appropriate range of values. All chemicals used in the procedures described above were analytical grade. The limit of detection,
i.e. the analyte concentration giving a signal equal to the blank signal plus 2 standard deviations of the blank (Miller and Miller, 1984), was 0.16 21 21 mg/ml for DNA and 0.46 mg/ml for RNA. Percent recovery of added l-DNA to eight larvae homogenates (DNA spike) was 95.3% for CFA and 88.8% for MFA and the recovery of added 16s 1 23s RNA (RNA spike) was 105.6% for CFA and 62.8% for MFA. Total amounts of nucleic acids were corrected based on these values. The coefficient of variation (
Zar, 1999) calculated for estimate from eight homogenate samples was: (1) 1.5% for DNA and 3.5% for RNA when using CFA and (2) 14.8% for DNA and 17.8% for RNA when using MFA.
AA profile analysis
The AA profile of experimental diets were analyzed by Ultra Pressure Liquid Chromatography (Model- Waters ACQUITY-UPLC, Waters, Massachusetts, USA), following the method of
Ishida et al., (1981) at TNJFU Referral Laboratory for Fish Quality Monitoring and Certification, FC and RI, Thoothukudi.
Waterquality parameters
Water quality parameters were estimated using standard procedures (
APHA, 2005). The average range of water quality parameters were presented in Table 6.
Statistical analysis
All the data of this study were examined to one-way analysis of variance (ANOVA) utilizing the statistical software program SPSS version 16.0 (SPSS Inc., IL, USA). Duncan’s posthoc test was used to compare the averages of data at significance level of p<0.05.
