Research materials and methods
This research was carried out on November to December of 2016 at the Aquaculture Department at the Behbahan Khatam Alanbia University of Technology, Iran. In this regard, 12 fiberglass cylindrical tanks of 300 L for treatments and 2 tanks of 500 L were used. Then, the tanks were placed in four treatments, each with 3 triplicates. Aerated tubes fitted into each of the air stones were placed inside each tank. In this experiment, a high pressure air pump of all the tanks was used. It was aerated for 48 h to remove urban water chlorine.
Fish preparation
In this study, young common carp with a weight approximately 20 to 23 g and a relative length of 8 to 9 cm were considered. Therefore, the carp fishes transferred from the Shahid Maleki Native Fish Center. Large double-glazed plastic bags used for this purpose. The 500 common carp with weight between 20 and 23 g were bought. After dividing the fishes into 5 bags, one third of the bag was filled with water and two thirds of it was filled with oxygen. At the beginning of the work, bags containing the fish placed in a pre-prepared tank, so that the water in the bags was temporarily tempered by the reservoir's water, otherwise the fish will get heat shock and die. During this time, air pipes in bags are necessary, so that the fish do not have oxygen deficiency. After two h, the bags slowly opened and the fish stored in the tank with water.
Fish adaptation period
By entering the fish into the storage tanks, the period of adaptation began. Adaptation time considered for one week. In this period, a special diet used, with commercial name Fera Deneh. Before the end of the adaptation period, the fish were categorized, after the biometry 360 fish with an average weight of 20 g and a standard length of 9 cm which selected and randomly divided into 12 ready-for-use tanks.
Cultivation of fish (treatments)
On November 23, 2017, the main phase of the project, preparation of treatments, began with fish fed daily with the desired diet. The whole period was 42 days.
Feeding
The fish feed was carried out twice a day with 3% of the body weight of the fish. Total daily feed intake=Biomass of the fish per tank ×fish feed percentage. Fish biomass calculated from multiplying the number of fish by the average weight of fish. Daily meals handled manually at 2 times.
Sampling and harvesting
The fish feed was cut for 48 h to empty the digestive system, then, 6 fish randomly removed from each tank and their weight and length calculated. The liver and viscera are used to calculate the weight. Then, the skin was removed and the fish tissue was separated to determine the chemical composition.
Parameters reviewed
Nutritional indices
Feed conversion ratio (FCR)
FCR = Average diet (g) /Average fresh body weight gain (g)
Food conversion ratio = amount of food consumed during the period / weight gain of fish×100
Feed Conversion Efficiency
FCE = Feed rate/Average dry body weight gain (g)
Protein conversion efficiency= fish weight gain (g) / consumed crude protein (g)×100
Fat-to-performance ratio and Protein efficiency ratio.
Compositional analysis
Prior to samples storage at refrigerated temperature, composite samples of fish fillets were analyzed for moisture, protein, ash, and lipids based on the AOAC (8) method of analysis. Analyses were conducted in triplicate and all reagents were of analytical grade.
Standard method (8) used for biochemical analysis of samples. All experiments performed in triplicates for the moisture, crude fat, crude protein, and crude ash. The samples transferred to central laboratory of the General Veterinary Office of Ahvaz for testing. The 200 g of prepared fillet placed in an oven at 65 ° C for 24 h to dry completely. The samples then, removed from the apparatus and after being cooled, milled and powdered and labeled in separate containers and transferred to the laboratory for testing.
Moisture measurement
The moisture content determined by drying 10 g of the powdered sample in the oven at 105 °C for 24 h to reach a constant weight (8). After removing from the oven for 30 min, it placed in the desiccator to cool, then, the sample weighed. The difference of the dry weight of the sample with wet weight indicated the amount of moisture remaining.
Moisture content = sample initial weight (g)_secondary sample weight (g) × 100/sample initial weight (g)
Measurement of crude fat
Total fat content determined by the Soxhlet method (13). To do this, The 5 g of the homogenate sample into Erlenmeyer, added 35 ml of concentrated HCL and 6 ml of distilled water and heated it. Erlen content was filtered through the filter paper and then the filter paper was washed with hot water with the filtered material on it. To dry the filter paper, it inserted into the oven and cooled in the dishwasher. After drying, it was extracted in cardboard and the fat extracted using a Doptrol ether solvent by Soxtec. Finally, the extraction balloons were separated from the apparatus and the solvent residue was evaporated with a water bath. Then, to dry the balloon until it reaches its final weight, it is then placed in the oven, after which it was cooled in the desiccator to obtain the total fat content of the sample. The fat percentage of the sample was determined using the following formula:
Fat percentage = Secondary balloon weight - Balloon primary weight / Sample weight
Measurement of crude protein
Protein content assay performed using Kjeldal method (13). The 1 g of the homogenated sample with 20 ml of concentrated sulfuric acid and 8 g of catalyst in a special tube, transferred to a Kjeldahl digestive tract. Digestion for 30 min at 250 °C and 45 min at 410 °C carried out. The sample then placed in a distillation machine. 80 ml of distilled water and 80 ml of 32% NaOH added automatically to the sample. Distillation vapors were introduced into a 2% boric acid container with a few drops of reagent and finally titrated to 0.1 HCl. The nitrogen content of the sample calculated by the following formula and multiplied by the number obtained at 6.25 the amount of sample protein.
Nitrogen percentage = HCL intake volume × 1.4007 ×0.1
Ash measurement
The ash content of the sample determined by an electric furnace [8]. The 10 g of dried sample placed into a Chinese bush and placed in an electric oven at a temperature of 550 ° C, when it turned gray out of the oven. The residual material in the Chinese plant indicated the amount of sample ash.
Ash percentage = (Secondary plant weight- initial plant weight ×100 / sample weight).
Statistical analysis of data
To analyze the data, using the SPSS software and the Kolmogorov-Smirnov test, the normal distribution of the data was determined and then by using one way ANOVA, the presence or absence of differences between the treatments determined. And after observing a significant difference, Duncan test at 95% confidence level was used to check the difference between treatments. Excel software was used to draw charts.