*2.4. Sampling and Analysis*

The body length and weight of the fish were measured at the beginning and the end of the feeding experiment. Then the specific growth rate, weight gain percentage, feed coefficient and condition factor were calculated. The calculation formulas are as follows:

Weight gain percentage (%) = (final average weight − initial average weight)/initial average weight × 100

Specific growth rate (%/d) = (Ln final average weight − Ln initial average weight)/breeding test days × 100

Condition factor = body weight/body length3 × <sup>100</sup>

Feed coefficient = feed weight/fish weight gain

In the formula, the unit of body length is cm, the unit of final average weight and initial average weight is g, and the unit of breeding test days is d.

At the two sampling times, fish blood was taken from the tail vein with a 1-mL syringe and was left to clot at room temperature for 2 h, then separated by centrifugation (3000× *g*, 10 min). Serum was collected and kept at −80 ◦C until analysis. Serum was used to detect enzyme activity. Total cholesterol (TC) was measured by the CHOD-PAP enzymaticcolorimetric method. Triglycerides (TG) were determined enzymatically by the Triglyceride method [39]. Albumin (ALB) was analyzed according to the method described by Doumas et al., (1971) [40]. Aspartate aminotransferase (AST/GOT) and alanine aminotransferase (ALT/GPT) were evaluated using a colorimetric method according to Reitman and Frankel (1957) [41]. Lysozyme (LZM) activity was determined using a turbidimetric assay as in Ref. [42].

In addition, the liver, head kidney, intestine, gill and spleen of fish were sampled and put into centrifuge tubes separately, followed by immediate storage at −80 ◦C until analyzed. A nine-fold volume of 0.86% saline solution equal to weight was added in tissue samples. The tissue was then homogenized using the handheld homogenizer (Gloucester, Prima PB100, England) on ice. The homogenate was centrifuged for 20 min at 2500× *g*, and the supernatant was used for the further analysis. Amylase, Lipase and Trypsin were analyzed according to the method described by Reimer (1982) [43]. Na+K+ adenosinetriphosphatase (Na+/K+–ATPase) activity determination was performed according to Ay et al., (1999) [44]. Creatine kinase (CK) activity was determined using the method of Weng et al., (2002) [45]. γ-glutamyl transpeptidase (γ-GT) activities were measured using the method of Rosalki et al., (1970) [46].

All assays were determined using commercial assay kits (Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China) according to the manufacturer's instruction.

#### *2.5. Gene Expression*

All fish tissue samples were taken to extract total RNA, according to the manufacturer's protocol for Trizol reagent (Invitrogen, Thermo Fisher Scientific Co., Ltd., Shanghai, China). A micro ultraviolet spectrophotometer (ND5000, Bioteke Corporation, Beijing, China) and agarose gel electrophoresis were used to test the concentration and purity of RNA. RNA was considered valid when OD260/280 (Optical density) was in the range of 1.9~2.1. 1 μg total RNA, drawn off for the reverse transcription process following the manufacturer's instruction for One-Step gDNA Removal and cDNA Synthesis SuperMix (EasyScript, Beijing TransGen Biotech Co., Ltd., Beijing, China).

Quantitative real-time PCR analysis was run by Real-Time PCR System (Q1000, Hangzhou LongGene Scientific Instruments Co., Ltd., Hangzhou, China). The thermal cycling program was performed as follows: initial denaturation at 95 ◦C for 15 min, 40 cycles of denaturation at 95 ◦C for 10 s, annealing at 60 ◦C for 20 s, extension at 72 ◦C for 30 s. All PCR amplifications were performed in triplicate on 20 μL of reaction mixture containing 10 μL of 2×RealUniversal PreMix, 0.6 μL (10 μmol/L) of each primer, 2 μL of template cDNA, and 6.8 μL of RNase-free ddH2O. EF1 α and β-actin were used as the reference genes. A dissociation analysis was performed to determine the absence of nonspecific products at the end of each PCR reaction. A single peak was seen on the melt curve analysis, which indicated a single PCR product. A standard curve was established for 10-fold serial dilutions of cDNA for each primer pair. PCR reaction efficiency reached the range of 90–110%. The information on primers used for the analysis is provided in Table 2. The relative expression of HSP70 and HSP90 were determined in liver, head kidney, gill and

spleen tissues. The relative expression of NF-κB1, TNF-α, IL-1β, IL-8, TGF-β1, C3, C4, IgT, Hepc, IFN-γ, and Mx were determined in intestine tissue.


**Table 2.** Primer sequence table.

<sup>1</sup> Primers of TNF-α, IL-1β, IL-8 and β-Actin were based on the study of [47]. <sup>2</sup> Primers of IgT and Hep were based on the study of [48]. <sup>3</sup> EF1-α and β-actin were used as reference genes.
