The animal experiments were approved by the Institutional Animal Care and Use Committee of Hunan Agricultural University, Hunan, China.
Animal Protocol and Dietary Treatment
A total of 480 piglets (Duroc × Landrace × Large Yorkshire) weaned at 25-d of age were randomly assigned to 3 groups (8 pens per group and 20 piglets per pen) as follows: (1) Negative Control (NC), low-protein basal diet no antibiotics included; (2) Positive control (PC), low-protein Basal diet + antibiotics (75 ppm quinocetone, 20 ppm virginomycin and 50 ppm aureomycin); (3) EUF treatment (EUF), low-protein Basal diet + 0.1‰ EUF. The low-protein basal diet was formulated in two phases (phase 1, days 0-15; phase 2, days 15-35) according to the nutrient requirements for weanling piglets (NRC, 2012) and the previous studies  (Table 1). EUF powder contained 83.61% total flavones was prepared at the department of medicine, Jishou University (Jishou, Hunan, China), which has been used in the previous study by Yuan et al . Additives were added to the negative control diet at the expense of corn.
The piglets were housed in an environmentally controlled nursery with hard plastic slatted flooring, and had free access to drinking water. Piglets were fed their respective diets 3 times per day at 8:00, 13:00 and 18:00 for a 35-d period. On the morning at d 15 and 35, 24 piglets (1 piglet per pen) were randomly selected and blood samples were obtained aseptically from the jugular vein at 2 h after feeding. Serum samples were obtained by centrifugation at 2000 × g for 10 min at 4 oC and then immediately stored at -80 oC for further analysis. Piglets were anesthetized with sodium pentobarbital and killed by jugular puncture. And the intestine samples were collected after rinsing thoroughly with ice-cold physiological saline solution. About 2 cm segments of the jejunum and ileum were cut and fixed in 4% formaldehyde for observation of the morphology of intestinal mucosa. Ileal and colonic contents were collected for bacteria counting and DNA extraction.
Growth Performance, Diarrhea Index
Body weight and feed intake were measured at day 15 and 35. Average daily gain, average daily feed intake and gain : feed ratio were calculated. The number of pigs with diarrhea was recorded every day. Diarrhea index (%) was calculated as the number of diarrhea piglets × diarrhea days / the total number of piglets × experiment days.
Intestinal Morphology Evaluation
The jejunal and ileal morphology were analyzed using hemotoxylin eosin staining according to Xiao et al. . Villous height and crypt depth were measured with computer-assisted microscopy (Micrometrics TM; Nikon ECLIPSE E200, Tokyo, Japan).
Serum Biochemical Parameters Determination
Total protein (TP), Albumin (ALB), blood urea nitrogen (BUN), glucose (GLU), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), immunoglobulin G (IgG) and IgM in the serum were measured using Biochemical Analytical Instrument (Cobas c311, F. Hoffmann-La Roche Ltd., Basel, Switzerland) and commercial kits (F. Hoffmann-La Roche Ltd., Basel, Switzerland).
Microbiota Composition Analysis
Bacteria counting were performed according to the previous studies [5, 19]. 0.2 g of ileal and colonic contents were collected and immediately diluted with 9 mL of 0.9% sterilizing saline and homogenized. Then, 10-fold dilutions of homogenate were performed (ranging from 10−1 to 10−8) and then cultivated onto MacConkey Agar Medium for the enumeration of Escherichia coli, and GM17 Medium for the enumeration of Lactic acid bacteria. The GM17 medium were then incubated for 48 h at 30 °C under anaerobic conditions, while the MacConkey agar plates were incubated for 24 h at 37 °C. The coliform bacteria and lactic acid bacteria colonies were counted immediately after removal from the incubator. Values were reported as log10 colony-forming units per gram.
DNA was extracted from ileul and colonic contents with the Tiangen stool mini kit (TianGen) according to the instructions of the manufacturer. DNA concentration was determined by spectrophotometry (Nanodrop). The DNA obtained from the intestinal luminal content was used as the template to analyze intestinal bacteria by qRT-PCR. Primers (Lactobacillus spp. (F) 5’-CACCGCTACACATGGAG-3’ (R) 5’-TGGAAGATTCCCTACTGCT-3’, Escherichia coli (F) 5’-CATGCCGCGTGTATGAAGAA-3’ (R) 5’-TTTGCTCATTGACGTTACCCG-3’, total bacteria (F) 5’-ACTCCTACGGGAGGCAGCAG-3’ (R) 5’-ATTACCGCGGCTGCTGG-3’) were synthesized according to the previous study . Relative expression of genes in the treatment group was normalized to the values for the NC.
All data were subjected to ANOVA analysis using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). The differences among treatments were evaluated using Tukey’s test. Probability values p < 0.05 were taken to indicate statistical significance.