Comparison and Evaluation of the Performance of the Mineral Mix Supplemented Diet for Asian Seabass (Lates calcarifer) Culture in Fresh Water-based Recirculating Aquaculture System

Common dietary macro and micro minerals are calcium, phosphorus, manganese, sodium, chloride, potassium, chlorine, sulfur, selenium and magnesium. These minerals regulate osmotic balance and aid in bone formation and integrity. Common microminerals are iron, copper, chromium, iodine, manganese, zinc and selenium. These trace minerals are required in small amounts as components in enzyme and hormone systems. The experiment was carried out in a Recirculating Aquaculture System with a flow rate of 20,000 LPH.

The experimental duration was 60 days. The experiment was carried out in 36 Nos. of rectangular polyethylene (PE) tanks with 30 nos of seabass fry each. All the tanks were connected to a recirculating aquaculture system with a drum filter (20,000 LPH), sand filter (20,000 LPH), carbon filter (20,000 LPH), biofilter (5 tonnes with biofilter media of 400 m²/m³), cartridge filter (20,000 LPH), bag filter (20,000 LPH) and UV filter (20,000 LPH). Approximately 2000 nos. of Asian Seabass seeds of 2g size were procured from Canares Aquaculture, Kumta, Karnataka. The experiment included 2 controls and 10 treatments. The 10 treatments consisted of are formulated experimental diets prepared with 0.1%, 0.2%, 0.3%, 0.4%, 0.5%,0.6%, 0.7%, 0.8%, 0.9% and 1.0% of major mineral premix supplemented diets in triplicates. The experiment was conducted in a recirculating aquaculture system with flow rate of 20,000 LPH.

Experimental diets
 
The experimental diets were prepared with 45% protein and 7% lipid along with 1% supplemented calcium and phosphorus minerals based on previous studies. The experimental diet was prepared with a mineral mix containing major minerals namely magnesium, sodium, potassium, iron, copper, zinc, manganese and selenium (Table 1) in addition to calcium and phosphorus in the same concentrations in all treatments, Control 1, control 2 diets were prepared without any supplemented minerals. The percentage composition of wheat flour was adjusted to increase the mineral premix inclusion without affecting the proximate composition (Table 2).  Samplings were done once every 15 days and the feeding rates were revised according to the body weight. The fish were fed @ 5% of their body weight during the experimental period.
 

 

 
Water quality parameters
 
Temperature, dissolved oxygen (DO) and pH were measured in the experimental site itself using a YSI meter. Water pH was measured using a portable pH meter (Hanna Instruments, Mumbai) and salinity was measured using a hand-held refractometer (RHS 10 ATC). The total alkalinity and total hardness of the water were estimated titrimetric ally using phenolphthalein, methyl orange and Eriochrome Black-T indicators following the standard methods (APHA, 2005). Total Ammonia-N, Nitrite-Nitrogen and Nitrate-Nitrogen of the water samples were estimated using standard protocols (APHA, 2005). During the experimental period, the temperature (26.6±2°C), dissolved oxygen (4.8±1.5 mg/L), pH (7.8±0.75), total alkalinity (56±15 mg CaCO₃), hardness (246±28 mgCaCO₃), ammonia (0.05±0.01 mg/L), nitrite (0.01±0.01 mg/L) and nitrate (0.1±0.06 mg/L) were maintained within the optimum level.
 
Growth parameters
 
The growth parameters, such as percentage weight gain, specific growth rate, feed conversion ratio, feed efficiency ratio, protein efficiency ratio, mean growth rate, thermal growth coefficient and condition factor, were estimated according to the standard formulas.
 
Mineral analysis
 
Calcium
 
The standards prepared were measured for absorbance. An unknown sample was loaded and run in AAS to determine known concentration of calcium.
 
Phosphorus
 
Phosphorus was estimated using the spectroscopic method described by Baird et al., (2017).
 
Other minerals
 
Sample for the analysis of magnesium, iron, copper, selenium, manganese and chromium measured by Instrument Atomic Absorption Spectroscopy (Systronics, India). Sodium and potassium of the samples were estimated using a Flame Photometer (Systronics, India).
 
Proximate composition
 
The protein was estimated using the Kjedhal method Latimer (2016).
        The fat composition was estimated using an Automated Soxhlet Apparatus (Pelican equipment).
        The total moisture content of the whole fish body was calculated according to Latimer (2016).
        The determination of ash content was carried out in a muffle furnace.
        The crude fiber was estimated using Fibra Plus (Pelican Equipment).
 
Nitrogen Free Extraction (NFE)
 
The calculation for nitrogen free extract is:
   
Biochemical parameter
 
All the biochemical parameters mentioned below were estimated using Alpha chem biochemistry analyzer model number 100. In the software of the Alpha chem100 selection of appropriate tests was done and the desired biochemical parameters were displayed. For all the parameters except ALP, the standard reagent was fed to the machine which aspirates the samples and measures the OD value and the factor value was calculated automatically. This was followed by washing with distilled water. Then the sample was fed to the machine which aspirated it measured the OD value and provided the direct results.
· Glucose
· Cholesterol
· Triglycerides
· Lipase
· Urea
· Uric acid
· Albumin
· High Density Lipid
· Calcium
· Antistreptolysin O
· Alkaline Phosphatase
· Micro Albumin
· Total protein
· C- reactive protein
 
Hematological parameters
 
The sample is collected in a 2 ml K2 EDTA tube. It was  mixed to avoid microclots. The sample was analysed in a complete blood cell analyzer. Before analyzing, the sample was mixed and aspirated within 1 minute, the result will be displayed on the analyzer.
       
The following Hematological parameters were analyzed using cell Tech Model No.380
· White Blood Cell.
· Lymphocytes.
· Mid-range absolute count.
· Granulocyte.
· Haemoglobin.
· Haematocrit.
· Means Corpuscular Volume.
· Means Corpuscular Haemoglobin.
· Red cells distribution width.
· Platelet Count.
· Mean Platelet Count.
· Platelet Distribution Width.
 
Digestive enzymes
 
The amylase activity was estimated using the Rick and Stegbauer (1974) method.
 
Protease
 
The protease activity was determined using the Moore and Stein (1948) method.
 
Lipase
 
The lipase activity was assayed by following the Cherry and Crandal (1932) method.
 
Total protein
 
The total protein content in the liver was determined using the Bradford (1976) method.
 
SOD (Superoxide dismutase)
 
Superoxide’s dismutase activity was determined by means of inhibition of Pyrogallo auto-oxidation by the enzymes.
 
NBT (Nitro blue tetrazolium)
 
It was carried out using nitro blue tetrazolium (NBT) assay by following the modified Anderson and Siwicki (1995) method.
 
Serum protein and glutathione activity
 
The serum protein test and glutathione activity was determined by spectrophotometer.
 
Peroxides
 
The samples were subjected to enzyme assay as described by Civello et al., (1995) for the determination of peroxidase activity.
 
Glutathione peroxidase
 
GPx reaction was performed as per the protocol of Wendel, 1981.
 
Alkaline phosphatase
 
The Alkaline Phosphates activity (ALP) was determined using the Garen and Levinthal (1960) method.
 
Statistical analysis
 
The IBM SPSS software was used to calculate the one-way analysis of variance at a 95% level of significance for all the treatments. The mean and standard deviation were also calculated using the same software.
       
The experiments were carried out in the wet laboratory of Dr. M.G.R. Fisheries College and Research Institute, Ponneri during the year 2021-2022 in freshwater and water exchange was performed at 10% once in two days to maintain optimum TDS. All water quality parameters were monitored regularly and maintained in optimum condition. The experiment was conducted in accordance with the rules and regulations of the Animal Welfare Board of India.

Even though there are studies on Vitamin C supplementation in European seabass KOP Aysun (2019) studies on mineral incorporation are limited in fish even though they are well-studied in broiler chicken (Boussouar, 2024) and hence this study was taken up. The maximum mean growth rate, percentage weight gain and specific growth rate of asian seabass with an extreme of as 50±2.00 g, 1566.22±24% and 78.10±4.004 g, were obtained in T8 and the least mean growth value of 36.96±2.05 g, 1133.11±15%, 56.20±4.015 g were obtained in C1 respectively. The feed conversion ratio of asian seabass with a maximum of 3.1±0.25 was obtained in T10 and the minimum mean growth value of 2.8±0.5 was obtained in C2. The feed efficiency ratio, protein efficiency ratio and mean growth rate of asian seabass with the highest of 0.166±0.01, 1.11±0.04 and 29±3 g were obtained in T8 and the value of 0.150±0.05 was obtained in T10, 0.80±0.04 was obtained in C1 and 28±1 g was obtained in C1, C2 and T10 respectively. The thermal unit growth coefficient and condition factor of asian seabass with a maximum of 0.87±0.01, 949.64±13 were obtained in T8 and T1 and the minimum of 0.62±0.05, 862.96±20 were obtained in C1 and T7 respectively. 
 
Blood parameter
 
The White blood cell, lymphocyte and mid-range absolute count values with the extreme of 40±5 10⁹/l, 46.5±3 10⁹/l and 4.5±0.5 10⁹/l were observed in T8 and moderate of 30.22±2 10⁹/l, 20.25±2 10⁹/l and 2.05±2 10⁹/l in T1.  The granulocyte, red blood cell and haemoglobin values with the highest of 5.3±0.17 10⁹/l, 2.8±0.2 10⁹/l and 4±0.5 g/dl were observed in T8 and the lowest of 2.2±0.3 10⁹/l, 0.87±0.13 10⁹/l and 3.1±0.1 g/dl were observed in T1. The hematocrit, Means corpuscular volume and Mean corpuscular haemoglobin with a maximum of 23±0.5%, 100±5 fl and 44±4 pg were observed in T8 and a minimum of 10.05±1.0%, 60±5fl and 32±2pg were observed in T1. The red cells distribution width, with greater 52±3%, 69±1 10⁹/l and 5±2% were observed in T10, T4 and T10 respectively and the lowest of 15.2±0.2%, 12±3 10⁹/l and 0.01 ±0.01 % were observed in T7, C1 and C1, C2 and T7 respectively. The mean platelet count, platelet distribution width and platelet large cell count with a maximum of 12.5 ±0.5 fl, 40±2.3% and 52±3 mg/dl were observed in T1, T4, T10 and a minimum of 8.3±1 fl, 9.7±1.1 %, 1±0.6 mg/dl and 14.38±3 mg/dl were observed in T9, T1, T7 and T10 respectively. Similar research work on hematological parameters was also performed in RAS culture under different stocking densities by Ezhilmathi et al., (2022).
 
Biochemical parameters
 
The Glucose, Cholesterol level, Triglycerides level and Lipase were at the extreme of 75.56±2.1mg/dl, 238.31±3.1mg/dl, 133.65±3 mg/dl and 186.78±3mg/dl were observed in T8, T1, T1 and T1 respectively. A moderate of 45.85±0.9 mg/dl, 134.85±2.6 mg/dl, 125.23±2.8 mg/dl and 125.23±2.8mg/dl were observed in T1, T8, T8 and T8 respectively. The urea, uric acids, albumin and high-density lipoprotein with a maximum of 240.76±3.1 mg/dl, 12.02±2.1 mg/dl, 8.76±3.1 mg/dl and 133.05±1 mg/dl were observed in T8 and a minimum of 120.87±1.1 mg/dl, 4.22±0.4 mg/dl, 4.33±2.9 mg/dl and 60.34±3 mg/dl were observed in T1. The Calcium, A-Streptolysin – O and Alkaline phosphatase with the top most 6.40 ±2 mg/dl, 120.44±3.1 mg/dl and 87.76±3.06 mg/dl were observed in T8 and the bottom least of 2.38 ±2 mg/dl, 40.25±3.01 mg/dl and 51.72±1.52 mg/dl were observed in T1, T1 and C1 respectively. The microalbumin, total protein and C-reactive protein with greater of 9.20 ±0.5 mg/dl, 4.20 ±1 mg/dl and 6.33±2 mg/dl were observed in T8 and a lower of 3.45 ±0.4 mg/dl, 1.80±0.7 mg/dl and 2.00±2 mg/dl were observed in T1 respectively. 
       
The amylase, protease and lipase in the liver with greater than 90.06±2.1 Umg^-1 protein^-1 min^-1, 84.07±0.8 Umg^-1 protein^-1 min^-1 and 84.07±0.8 Umg^-1 protein^-1 min^-1, were observed in T8 and a lower of 48.06±0.4 Umg^-1 protein  min^-1, 23.09±0.9 Umg^-1 protein min^-1 and 49.03±0.4 Umg^-1 protein^-1 min^-1 were observed in C1, T1 and C1 respectively. The Amylase, Protease and lipase in the Intestine with a maximum of 92.06±2.1 Umg^-1 protein min^-1, 88.07±0.8 Umg^-1 protein min^-1 and 92.58±2.1 Umg^-1 protein min^-1 were observed in T8 and a minimum of 52.06±0.4 Umg^-1 protein min^-1, 42.06±0.9 Umg^-1 protein min^-1 and 52.03±0.4 Umg^-1 protein min^-1 were observed in C1. 
       
The Amylase, Protease andlipase in Kidney were 86.06±2.1 Umg^-1 protein min^-1, 88.07±0.8 Umg^-1 protein  min^-1 and 91.58±2.1 Umg^-1 protein min^-1 in T8, while the level 58.06±0.4 Umg^-1 protein min^-1, 91.58±2.1 Umg^-1 protein  min^-1 and 56.03±0.4 Umg^-1 protein min^-1 in C1. In the stomach Amylase, protease and lipase with the highest 86.78±2.1 Umg^-1 protein min^-1, 80.27±0.9 Umg^-1 protein min^-1and 86.63±2.1 Umg^-1 protein min^-1 were observed in T8 and the lowest of 62.08±0.4 Umg^-1protein min^-1, 52.16±0.9 Umg^-1 protein min^-1 and 58.13±0.4 Umg^-1 protein min^-1 were observed in C1. 
       
The Superoxide Dismutase, Nitro blue tetrazolium and serum protein levels were at a maximum of 94.68±1.5 mg/dl, 69.46±1.9 mg/dl and 92.08±4.3 mg/dl in T8 while the minimum level 49.98±1.55 mg/dl, 32.46±1.5mg/dl and 36.08±1.7 mg/dl were observed in C1. The glutathione and peroxidase values were extreme levels at 66.47±5.1 mg/dl and 76.22±2.9 mg/dl in T8, while moderate levels of 25.18±1.87mg/dl and 46.00±2.5 mg/dl were observed in C1. 
 
Mineral composition
 
The overall supplementation of calcium and phosphorus was studied by Moses et al., (2024) which is the first study in the Asian Seabass on mineral supplementation of both calcium and phosphorus at a 1% level of supplementation. In the present study, 0.2% supplemented magnesium gives better results. Similar research was carried out by Shiau and Hsieh (2001) in tilapia and found that 0.002% gives optimum growth. Further studies by Liang et al., (2012) in grass carp also found that 0.08% is required for optimum growth. However, Wang et al., (2011) reported that 0.07% of magnesium is required for grass carp. 0.2% of supplemented sodium is giving better results similar research work was carried out by Fernandes et al., (2001) in tilapia and found that 10% is required. Further studies by Duncan et al., (2011) in red drum also proved that 0.92% is required. However, Shiau et al., (2007), reported that 0.2% of sodium is required for tilapia in fresh water.  Mzengereza and Kang (2015) also report that 1.7% of Sodium is required for tilapia. In the present study, 0.28% supplemented potassium gave the best result. In the present study, 0.21% of supplemented Iron has given better results. Similar research work was performed by Rigos et al., (2010) in gilthead seabream and found that 0.02%.is required for optimum growth. Further studies by Shiau and Su (2003) on tilapia also proved that 0.016% of dietary supplementation is needed for optimum growth. 0.01% of supplemented manganese has given better results. Similar research work was carried out by Ishac and Dollar (1968) for dietary supplementation in tilapia and found that 0.0007% is required for optimum growth. 0.12% of supplemented zinc has given good results. Studies by Liang et al., (2012) in Grass carp also proved that 0.0055% is the requirement 0.0007% of supplemented Selenium has given good results. Similar research work was carried out by Zhu et al., (2011) in large mouth bass and found that 0.0001% has given optimum results. Further studies by Wang et al., (1997) in Catfish also proved that 0.0005% is the requirement. 0.03% of supplemented copper is the requirement found by this study. Similar research work was carried out by Lin et al., (2008) in Epinephelus malabaricus and found that 20.05mg Cu/Kg is giving optimum results. Further studies by Ling et al., (2010) in Common carp also proved that 4.37 mg Cu/kg is required. The biochemical parameters were also found to be in the optimum range in treatment 8 containing a 0.8% level of mineral mix in the diet. The haematological parameters also proved the results.

The inclusion of calcium and phosphorus at the rate of 1% each in the diet of Asian seabass along with the mineral mix of 0.8% containing minerals such as manganese, sodium, chloride, potassium, chlorine, sulfur, selenium and magnesium for culture in a freshwater-based RAS system significantly increased the growth rate and survival rate compared to other percentages of inclusions. Therefore, it is concluded that mineral nutrition is one the vital components for fish nutrition, especially in a recirculating aquaculture system.

We sincerely acknowledge the funding support from the NADP-RKVY(TN/RKVY-FISH/2018/813) 2018-2021 project for conducting the experiment as given in the research paper.

We declare that there is no conflict of interest.