Experimental design
The present study was conducted at the Sindh fish hatchery near Hawkes Bay town from March 2019 to Jan 2020. A total of 598 uniform sized (1.24±1.08 grams) sea bass juveniles were collected from the coastal area of Sindh Sakro and stocked (166 fish/Tank) in five treatments (T1, T2, T3, T4 and T5). Each treatment tank was 30×6×4 feet containing 0, 5, 22, 35 and 42 ppt salinity by following the methods of Azodi et al. (2016) with some modifications. Tank water was regulated by blending filtered water from the ocean and freshwater. Water was exchanged for the maintenance of the water quality by up to 50% every 2 days. Fish were fed at 6% of body weight per day (BW day-1) for 40 days and the feeding rate adjusted every week. The extracted pellet (42.0% crude protein) was used for the manual feeding at two distinct satiation levels twice daily. After 3 hours of feeding, the extra feed was removed. The total consumption of food was reported daily in each treatment. A 42% CP-level diet was formed using various components (Table 1), referred to as the entire diet/ration structure. This diet was offered regularly based on the daily wet bodyweight of the individuals.
Feed formulation and preparation
Feed ingredients were collected from Karachi's local market and formulated as previously methods described by Ghosh et al. (2011). The major source of protein used in diets was fishmeal, and the levels of protein were preserved at 42%. The feed ingredients have been combined to form a dough with fish oil and water. The palletizer was carried through the dough to create the necessary pellet diet. Under dry conditions, the feed was stored in a freezer until used. Proximate dry matter, crude fat, crude protein, crude fibres, and moisture levels were determined in a percentage of the dried feed specimens (Table 1).
Samples collection and Biochemical analysis
The biochemical analysis of feed and fish carcass samples was carried out based on (AOAC 2000). At the end of the experiment, five fish were removed from each tank and then dissected to weigh the liver and viscera, then HSI and VSI were determined. Techniques (AOAC 2000) were used to analyze crude lipid (CL), moisture and crude protein (CP). Moisture was measured at 105o Celsius in the oven for 24 h (Labostar-LG122 Tabia Espec, Osaka, Japan). Chloroform/methanol (2:1v/v) extraction process predicted for crude lipids. (Folch et al. 1957) The CP analyzed using the automatic processing of Kjeldahl (Buchi430/) Using the Kjeltec method (N 6.25) with the automatic Kjeldahl system (Buchi 430/323) Model 1265, Moline IL, USA). Furthermore, for the determination of ash, the sample was burned at 550 C in a muffle furnace for 3 hours. Gross energy (GE) has been approximated for formulated diets factors 23.62, 39.5 and 17.56 KJ/g for CP, EE and carbohydrate respectively were used (NRC 1993).
Water quality parameters
Water quality parameters such as temperature, salinity, dissolved oxygen (DO), and pH were recorded daily using Celsius glass thermometer, Handheld Refractometer, mobile digital DO-meter (Model: HI9146) and Digital pH meter, respectively. The concentrations of ammonia, alkalinity, nitrate, and nitrite were measured by following the methods of APHA (1995). Details of the water quality parameters recorded during the study period are given in Table 2.
Growth performance analysis
The wet weight gain, average daily weight gain (ADWG), specific growth rate (SGR), feed conversion ratio (FCR), hepatosomatic index (HSI), viscerosomatic index (VSI), Fulton’s condition factor (CF), survival rate (SR) and total biomass were calculated with the following formula adopted by Hassan et al. (2020b):
WG=Final weight-initial weight
ADWG= {(Final weight-Initial weight)/Days}
SGR (%) = [(Ln FBW – Ln IBW) / day] × 100
FCR=Food given (g)/Weight gain (g)
Fulton’s condition factor (K) = (Weight/Length3) ×100
VSI = {weight of visceral organs and associated fat tissue (g)/wet body weight} ×100
HSI= weight of liver (g)/empty fish weight (g) ×100
SR (%) = (No. of fish survived/ No. of fish released) ×100
Cannibalism (%) = 100 × [(LS – M – LC)/LS]
Where, LS stocked at the beginning of sea bass, LC is the number of sea bass collected at the end of the study and M is natural mortality
Biomass = Average bodyweight × No of fish
Statistical analysis
All data were analyzed using STATISTICA V.13 software (StatSoft Inc., Tulsa, OK, USA). Residuals were tested for normality (Shapiro–Wilk test) and homogeneity of variance (plot of residuals versus predicted values). The percentages data were natural log (LN)-transformed before analysis. The effects of different salinity group on the growth performance and health indices of Asian seabass juveniles at different culture periods were analyzed using a repeated-measures ANOVA model containing culture period (fixed repeated factor) and different salinity groups (fixed factor) main effects as well the culture period × different salinity groups interaction term. Then, the model was decomposed into a series of one-way ANOVA was run to test the effects of different salinity levels on the wet weight gain, average daily weight gain, specific growth rate, hepatosomatic index, viscerosomatic index, Fulton’s condition factor, feed conversion ratio, survival rate and biomass for different culture periods. A posteriori analysis was performed using Tukey’s multiple comparisons procedures. A significance level of p < 0.05 was used for all statistical tests.