2.3.3. Intestinal Digestive Enzyme Activity Measurements

Protein concentrations in intestinal tissues were determined by the Komas Brilliant Blue method. Intestinal α-amylase (α-AMS) and lipase (LPS) activities were determined via the enzyme starch colorimetric method and enzyme colorimetric method, respectively. All samples were assayed according to the operating instructions of the kit (Jiancheng Bioengineering Institute, Nanjing, China).

#### 2.3.4. Fixation and Observation of Intestinal Section

The foregut, midgut, and hindgut of the experimental fish were fixed in 10% formalin for 24 h, dehydrated in 70%, 80%, 90, 95%, and anhydrous alcohol in that order, transplanted into xylene, embedded in paraffin, sectioned (5 μm) with a microtome (Leica RM2235, Wetzlar, Germany), then stained with hematoxylin and eosin, and sealed with neutral gum. A microscope (Leica DM1000, Wetzlar, Germany) was used to observe the intestinal tissues (magnification of intestinal tissues was 10). Image J software was used to measure the intestinal muscular thickness (MS), intestinal villus length (VL), and villus thickness (VT).

## *2.4. Statistical Analysis*

Excel 2019 software was used for data processing. All statistical analyses were performed with SPSS 26.0 software. All figures were drawn using the Prism GraphPad 9 software. The data obtained in the text were subjected to one-way ANOVA followed by Duncan's multiple comparison analysis to determine if there were any significant differences (*p* < 0.05), and the experimental results are expressed as mean ± standard deviation (mean ± SD). Assumptions of normality, equality of variances, and outliers were confirmed prior to statistical analysis.
