2.1 Experimental design and feeding trial The feeding trial was conducted at the Fisheries Biology and Genetics laboratory, Hajee Mohammad Danesh Science and Technology University, Dinajpur. Reba carp fry were collected from the hatchery complex of Caritas, Bochagonj, Dinajpur, Bangladesh which were produced under the research project of NATP-CRG 488 funded by USAID and Ministry of Agriculture, Government of Bangladesh. The fish were transported in oxygenated plastic bags filled with freshwater. The fish were acclimatized for two weeks prior to the initiation of the study and were fed with a commercial feed. Thereafter, the fish (average weight 0.25g) were randomly distributed into nine glass aquaria measuring (18 cm × 10 cm × 10 cm) with a stocking density of 10 fish per aquarium. Three replicate aquaria for each treatment were established. During the experimental period, the reba fry were fed the respective experimental diets based on a percentage of body weight (10%) twice a day for a period of 12 weeks. Throughout the experiment, water was supplied from a deep water tube well. Water quality parameters such as temperature, DO and pH were monitored regularly. Faeces were regularly siphoned out. Aquaria were cleaned weekly to reduce the risk of accumulation of nitrogenous waste.
2.2 Diet preparation Three experimental diets were prepared. Each contained a different dose of MOS 0 (control), 0.2% and 0.4% MOS (International Food Grade, Laboratory of USA, Purity > 90%). The concentrations of MOS were carefully chosen on the basis of previous studies. Fish meal and soybean meal were used as protein sources, while soybean oil and fish oil were used as lipid sources. To ensure similar energy levels in all experimental diets carbohydrate sources such as corn starch and wheat flour were used. Diets were inclined by thoroughly mixing the feed ingredients in a food mixer for 30 minutes at a capacity of 1 kg. The feed ingredients were also mixed for another 10 minutes after adding of soybean oils and fish oil. An adequate amount of water was then added to make it dough which was then extruded through a pelletizing machine to make 3 mm diameter pellets. The pellets were air-dried overnight and then broken into smaller pieces by hand. The resultant pellets were then packed separately in plastic bags in order to store in a freezer at -20 oC throughout the feeding trial.
2.3 Water quality assessment In the present study, water samples were collected from each aquarium. Recording on the spot data and collection of samples were made between 9.00 to 11.30 A.M. Water temperatures, pH, dissolved oxygen (DO) were recorded every 10 days interval between 9.00 am to 11.30 am. The temperature of water was taken from each aquarium by using a standard mercury thermometer. pH of water was taken from each tank by using a pH meter. The DO was measured by using a digital DO meter.
2.4 Sampling procedure The weight of each fish was determined at the start and end of the experiment; the combined weight of the fish in each aquarium was measured fortnightly to monitor somatic growth and to adjust the amount of feed to be given. Survival of the fish was monitored throughout the experimental period. After 12 weeks, three fish from each replicate aquarium were randomly selected and analysed separately for whole body proximate composition and body indices. The selected fishes were killed by keeping them in a refrigerator. After being killed, the individual fishes were weighed immediately and kept in an oven for drying. Thereafter, the dried fishes were then grinned using a blender machine in order to determine the moisture, crude protein, crude lipid and ash content of whole fish body. The weight of the whole fish body, liver, intraperitoneal fat, and viscera were recorded to obtain the hepatosomatic index (HSI), intraperitoneal fat (IPF) and viscerosomatic index (VSI).
2.5 Growth performance, survival, feed utilization and body indices The following growth performances, survival, feed utilization parameters and body indices were evaluated using the following equations: 2.5.1 Growth parameters, survival and feed utilization parameters Weight gain (g) Weight gain (g) = Final weight (g) –Initial weight (g) Specific growth rate (SGR % day-1) The SGR is the momentary change in weight of fish calculated as the percent increase in body weight per day over a given time interval.
2.6 Proximate Composition Analysis Whole body fish composition was determined by using the standard protocols as mentioned by the Association of Official Analytical Chemists. The moisture content of the sample was evaluated by drying the samples in the hot air oven at 1050 for 12 hours until constant weight was obtained. The moisture free samples were then used in order to determine the crude protein, lipid and ash content. The crude protein content of the fish samples was calculated indirectly by determining the total nitrogen content of the sampled fish by a Kjeldahl method using kjeldhal apparatus. The crude lipid content of the samples were determined by removing the lipid from the samples after homogenizing it in 60 ml of chloroform and methanol solution in a ratio of 2:1 and thereafter the solvent was evaporated by heating in a oven at 80 0C. The moisture-free samples were taken in porceline basin made crucible and weighed. Thereafter, the ash content was measured by igniting the samples in a muffle furnace at a temperature of 550 °C for 6 hours.
2.7 Data Analysis All data were tested using one-way analysis of variance (ANOVA). Significant results (P<0.05) were further tested using one-way ANOVA followed by Duncan’s Multiple Range Test to identify a significant differences between means. The data were expressed as average ± SE and statistical analysis was performed using SPSS version 22 and Microsoft Office Excel for the window.