2.4. Sample collection

At the end of the aquaculture, the spotted sea bass were fasted for 24 hours. After fasting, the fish were anesthetized with eugenol in each tank, and after anesthesia, the fish were retrieved, counted, and weighed to obtain the total weight. The intestines of six fish were randomly selected from each tank, rinsed with 0.86% ice saline to wash away the attached adipose tissue, put into liquid nitrogen for rapid freezing, and then transferred to a -80°C refrigerator for storage for digestive enzyme activity, intestinal flora, and related gene detection. The intestinal (approximately 1 cm) of three fish in each group were taken and stored in 4% paraformaldehyde for 24 h, transferred to 75% ethanol for hematoxylin and eosin staining, and used for morphological observation of the intestines.

The fish growth performance index includes final weight (W_T), weight gain (WG), specific growth rate (SGR), feed coefficient rate (FCR), and condition factor (CF), which are calculated as follows:

WG = (W _t − W ₀)/W ₀×100%

SGR = (ln W _t − ln W ₀)/t×100%

FCR= F/(W _t − W ₀)

CF = W _b/L ³×100

Where W _t is final mean weight (g); W ₀ is initial mean mass (g); t is number of days (d); F is food intake (g); W_b is fish body mass (g); and L is fish body length (cm).

The samples of each group of spotted sea bass were dissected, and the intestine was isolated and added to pre-cooled saline (0.86%) in appropriate proportions (w/v) and homogenized by an adjustable high-speed centrifuge. The prepared homogenate was centrifuged at 4°C, 2500 r/min for 10 min. Then, the supernatant was collected for measurement. The amylase (AMS) activity was determined by α-amylase test kit, trypsin (TRS) by trypsin test kit, and lipase (LPS) by lipase kit, all from Nanjing Jiancheng Bio-Engineering Institute, China (Catalog No: AMS: C016-1-1, LPS: A054-1-1, TRS: A080-2).

Morphological changes in the intestinal tract of flowering bass were observed between groups using hematoxylin-eosin staining according to Shinde (17). Paraffin section preparation was first performed: tissue samples fixed in Bouin fixation were removed, washed three times with 70% alcohol, trimmed flat with scissors, and placed at the bottom of the tissue embedding box. The tissues were sequentially immersed in 70%, 85%, and 95% ethanol for 20 min each, then in 100% ethanol I and ethanol II for 10 min each. Then, 15 s of draining was required for each change of dehydrating agent. After dehydration, the tissues were immersed in alcohol-benzene solution for 10 min and then placed into xylene solution for 10 min to ensure the tissues were transparent. Immersion in xylene-paraffin wax equivalent mixture was done at 63°C. After the wax immersion was completed, the embedding cassette was stored in a 63°C oven to complete the embedding as soon as possible. After curing the paraffin wax, the slides were sliced by a microtome with a thickness of 4 μm. Structurally intact tissue spreads were selected, after which the labeled slides were baked in a 60°C oven. Next, H-E staining was performed: the slides were put into xylene I and II for 20 min each, anhydrous ethanol I and II for 5 min each, and 75% ethanol for 5 min and rinsed with tap water. Hematoxylin staining was performed, followed by eosin staining. After staining, the sections were sequentially transferred into anhydrous ethanol (I, II, and III) for 5 min each and sealed with neutral gum. Finally, the length and width of the intestinal villi and the thickness of the muscle layer were measured with the WT1000GM system.

Polymerase Chain Reaction was used to extract total RNA from the whole intestine of fish using a commercial kit (RC112, Nanjing Vazyme Biotech Co., Ltd, Nanjing, China) according to the manufacturer’s requirements and was electrophoresed on a 1.2% denaturing agarose gel to detect the quality. Then, extracted RNA was assessed by a Nano-800+ spectrophotometer (Shanghai Jiapeng Technology Co., LTD, China) to test the concentration. The cDNA was obtained by reverse transcription using a commercially available package (RC112-50, Nanjing Vazyme Biotech Co., Ltd, Nanjing, China). cDNA was quantified by fluorescence PCR using SYBR Green I chimeric fluorometric method, and the kit (Q711) was purchased from Nanjing Vazyme Biotech Co. β-actin was used as an internal reference gene, and the relative expression of individual genes between groups was calculated using the 2^-ΔΔCt method. The primers used were similar to those used in the previous study of the spotted sea bass (18) ( Table 2 ).

Primer sequences for RT-qPCR.

F means forward primer, while R means reverse primer.

The total bacterial genomic DNA of the intestinal samples was extracted by the TGuide S96 magnetic bead method, and the extracted genomic DNA was detected by 1.5% agarose gel electrophoresis. The upstream and downstream primers 338F (5’-ACTCCTACGGGAGGCAGCA-3’) and 806R (5’- GGACTACHVGGGTWTCTAAT-3’) were designed for the high mutation region V3-V4 of the 16S rRNA gene and PCR amplification was performed. PCR amplicons were quantitatively detected using 1.8% agarose gel and Image J software, and the quality of PCR amplicons was detected using the Qsep-400 method. Libraries were then created, and qualified libraries were sequenced using Illumina NovaSeq 6000. After pre-processing the sequencing data, bioinformatics analysis was performed. The products were sequenced on the Illumina NovaSeq by BMKCloud (www.biocloud.net) (Beijing, China).

All relevant data obtained were statistically analyzed using SPSS22.0 software and analyzed by one-way analysis of variance (ANOVA), followed by Duncan’s multiple comparisons if the differences were significant, and all data were expressed as mean ± standard deviation (Mean ± SD), with P<0.05 selected as the significant level.