Animals, housing and diet
The study was conducted from June 2022 to August 2022 at a large-scale farm in Hubei Province, China. A total of 100 weaned piglets (mean weight 7.03 ± 1.04 kg, mean weaning age 26 d) were randomly divided into two treatment groups on the weaning day with 5 replicates in each group and 10 piglets in each replicate. The dietary formulations of the piglets in the two treatment groups were shown in Table 1, which were used to feed experimental piglets 1 to 14 days after weaning and 15 to 28 days after weaning. The control group diet was the basal diet (control group, CON), and the experimental group diet was supplemented with 0.6% benzoic acid and 0.1% 1-monolaurin on the basis of the basal diet (Complex acid group, CA). The trial lasted for 28 days. During the experiment, the piglets drink freely and piglets were kept in a semi-enclosed nursery with cement floor, good ventilation and warmth measures, and other feeding management and immunization procedures were uniformly implemented in accordance with the regulations of the farm.
Sample collection
Serum sample
On the mornings of day 1, 15, and 29, 5 median weight piglets were randomly selected from each treatment for labeling and blood collection. 20 ml of blood was collected from the anterior vena cava in a non-heparinized vacuum tube, centrifuged at 3000 × g at 4℃ for 10 min, and serum was extracted. The serum samples were immediately stored at − 20°C for further analysis.
Sample of cecal contents
On the morning of day 1, 15 and 29, the piglets were euthanized by intravenous injection of chlorpromazine hydrochloride with 2 mg/kg piglet body weight (Shanghai Harvest Pharmaceutical Co., LTD., Shanghai, China) about 30 minutes after feeding. According to a previously published method [12], the whole intestine was removed, and the cecal contents samples of 5 piglets in each treatment were collected and placed in a sterile freezable tube and immediately frozen in liquid nitrogen for the analysis of microbial quantity and microbial metabolites.
Diet samples and fecal samples
Representative diet samples of about 2.0 kg were taken from each stage of each treatment group. In addition, representative faeces samples were collected to determine apparent total digestive digestibility of nutrients, according to the method described by Silva et al. (2020) [11]. Simply put, on days 12 to 14 and 26 to 28, fresh faeces were collected from each field using rectal palpation (also known as rectal swab collection), taking care to remove impurities such as pig hair and environmental grit, and the faeces were immediately frozen at−20°C. The feces collected over three days were combined by field, followed by a representative sample of about 400g, which was dried at 65°C for 72 hours. The fecal samples and diet samples were thoroughly crushed and passed through a 40-mesh screen for subsequent nutrient apparent digestibility analysis.
Determination of growth performance and diarrhea rate
On the morning of day 1, day 15 and day 29 after weaning, piglets were weighed by head, and the average BW of piglets at day 1, day 14 and day 28 after weaning was counted, and the ADG at day 1–14, day 15–28 and day 1–28 was calculated in the CON and CA group. Feed piglets a few times a day, record the total feed amount according to the field, and record the remaining feed amount in the feed tank at 17:00 every day. The ADFI and FCR (average daily feed intake/average daily gain) of piglets from 1 to 14 days, from 15 to 28 days and from 1 to 28 days were calculated.
During the experiment, the fecal situation of piglets was observed every day, and the anus of piglets was examined one by one to observe whether there was fecal contamination and redness. For fecal score, refer to the scoring criteria of Yuan et al. (1998) : 0 point, normal stool; 1 point, paste stool; 2 points, semi-fluid stool; 3 points, liquid feces. If the stool score is ≥ 2, it is considered diarrhea [13]. The proportion of diarrhea and diarrhea frequency of each group was calculated. Proportion of diarrhea (%) = number of diarrhea piglets/number of experimental piglets ×100; Diarrhea frequency (%) = (number of diarrhea piglets × number of diarrhea days)/total number of experimental piglets × number of experimental days ×100.
Apparent digestibility of nutrients
Use of indigestible hydrochloric insoluble ash (AIA) in diet and fecal samples as an endogenous indicator [14, 15] to determine the apparent digestibility of dry matter (DM), crude protein (CP) and crude fat (EE). Fecal samples and experimental diets were analyzed for DM (method 930.15), ash (method 942.05), CP (method 990.03) and EE (method 996.01) according to the Official Association of Analytical Chemists (AOAC, 2007). GE was determined by adiabatic bomb calorimeter (Parr 1281, automatic energy analyzer; Moline, IL, USA). Organic matter (OM) is calculated as DM minus ash. AIA concentrations in diet and fecal samples were determined according to the procedure described by Prawirodigdo et al. (2021) [15]. Apparent digestibility of a nutrient (%) = [1- (content of the nutrient in feces × AIA content in diet)/(content of the nutrient in diet × AIA content in feces) ×100.
Microbial composition in cecal digesta was analyzed by 16S rRNA sequencing
Total genomic DNA was extracted from each cecal digesta sample using QIAamp Fast DNA stool Minikit (Qiagen, Germany), according to the manufacturer’s instructions. The forward primer F (5'-ACTCCTACGGGAGGCAGCA−3') and the reverse primer R (5'-GGACTACHVGGGTWTCTAAT−3') were used for amplification of the V3-V4 hypervariable region of a 16S rRNA gene. PCR amplicons were purified and quantified, and the PCR products were used for the construction of the libraries and then paired-end sequenced on an MiSeq platform (Illumina, United States) at the Shanghai Penosen Biotechnology Co., LTD (Shanghai, China). Sequencing data were processed using QIIME 2 (version 2019.4), Alpha-diversity values of each sample were assessed based on the observed OTUs, Chao1, and Shannon index. Beta-diversity measures dependent on weighted-UniFrac distance were calculated using mothur. LDA Effect Size (LEfSe) was conducted to identify bacterial taxa differentially represented between different groups at the genus or higher taxonomy level [16].
Examination of short-chain fatty acid content in cecal digesta
The SCFAs concentrations of piglets cecal digesta was analysed by a gas chromatographic method. Specifically, approximately 20 mg of cecal digesta was first homogenised in 1.0 ml of 0.5% phosphoric acid solution, and the entire sample was centrifuged at 12 000×g at 4°C for 10 min. Take 100 µl supernatant and add it to the corresponding 1.5 mL centrifuge tube, add 500 µl MTBE solvent with inner target and swirl for 3 min. After ultrasound in ice bath for 5 min, and centrifuge at 12 000×g at 4°C for 10 min. The sample was injected into a gas chromatography-triple quadrupole mass spectrometry (GC-MS/MS, 8890−7000D, Agilent, USA) equipped with a DB-FFAP column (30m × 0.25mm × 0.25µm). The GC-MS/MS detection parameters were shown in Table 2, and the contents of acetic acid, butyric acid and valeric acid in cecal chymus were determined.
Detection of intestinal permeability markers and inflammatory factors in serum
Serum diamine oxidase activity (DAO), endotoxin (ET), immunoglobulin A (IgA), interleukin−1β (IL−1β), interleukin−6 (IL−6), interleukin−8 (IL−8) and tumor necrosis factor α (TNF-α) were measured by ELISA. The test kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China), and the test method was strictly operated in accordance with the kit instructions.
Statistical analyses
The data of growth performance indicators, apparent digestibility of nutrients, SCFAs content of cecum digesta and serum biochemical indicators were recorded by Excel and processed. Then SPSS 27.0 software was used to conduct one-way ANOVA and Duncan multiple comparison. Chi-square test was used to compare diarrhea rate indicators among groups. P < 0.05 indicated a significant difference between groups, and P < 0.01 indicated a very significant difference between groups.