2.3 Growth parameters
Nine samples per sink were used for each sampling period. At the end of the experiment, body length (cm3), body weight (g), and total feed weight (g) were used to calculate final mean body weight (g), weight gain rate (WGR, %), specific growth rate (SGR, %), feed conversion ratio (FCR, %), and condition factor (CF, g/cm3), as follows:
SGR = (ln(Wt) − ln(W0)) / t × 100,
where W0 is initial biomass of the fish, Wt is the total biomass of the fish at the end of the test cycle, and t is the experimental cycle;
FCR = total feed quantity / (Wt − W0);
CF = Wt / body length; and
WGR= (Wt − W0) / W0 × 100.
2.4 Physiological and biochemical analysis
Within 1 h of collection, total blood hemoglobin (Hb) and methemoglobin were directly measured with using a kit (#A102-1, Nanjing Jiancheng Bioengineering Research Institute, China), according to the manufacturer’s instructions.
Catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX) levels were determined using the corresponding kits (A007-1-1, visible light method; A001-3-2, WST-1 method; A005-1-2, colorimetric method; Nanjing Jiancheng Institute of Biological Engineering, Nanjing, China), according to the relevant instructions. Plasma lysozyme was determined using the turbidimetric method (A050-1-1 kit, Nanjing Jiancheng Institute of Biological Engineering, Nanjing, China).
2.5 Health performance
The cumulative survival (CS, %), hepatosomatic index (HSI, %), spleen somatic index (SSI, %), and kidney somatic index (KSI, %) were calculated using the number of experimental fish, as follows (weights given in grams):
CS = (N1 / N0) × 100,
where N1 and N0 are the final and initial numbers of fish, respectively;
HSI = WH / WA × 100,
where WH and WA are the liver and final body weights of individuals, respectively;
SSI = WS / WA × 100,
where WS is the spleen weight; and
KSI = WK / WA × 100,
where WK is the kidney weight.
2.6 Gene molecular analysis
Total RNA was extracted from tissue according to the operating instructions of SPARKeasy Tissue/Cell RNA Rapid Extraction Kit with Genomic DNA Clean Column (Sparkjade, Shandong, China), followed by qPCR using the Evo M-MLV RT Mix Kit with gDNA Clean for qPCR (AG, China). The reaction system (20 µL) was reverse transcribed to cDNA, and total RNA concentration was determined using a Nanodrop 2000 spectrophotometer(GENE COMPANY LIMITED,Hong Kong, China ). Sample purity was assessed by determining the 260 nm / 280 nm optical density ratio.
Primers (Table 1) were designed using Primer Premier 6.2, using a SYBR Green Premix Pro Taq HS qPCR Kit (AG, China), with a 20 µL reaction solution. qRT-PCR was conducted using a CFX Connet Real-Time PCR System (Bio-Rad, China), as follows: 95 °C for 15 min, followed by 35 cycles of 15 s at 95 °C and 60 s at 60 °C.
2.7 Statistical analysis
Results are expressed as the mean ± standard deviation (SE). Statistical analysis using IBM SPSS included one-way ANOVA, followed by the Tukey test to check for significant differences between groups. A significance level of P < 0.05 was used.