The two commercial MOA products used in our research were produced in the Netherlands (Nutreco N.V., Amsterdam, Netherlands). MOA1 is a synergetic compound of free and buffered SCFAs (mainly formic acid, acetic acid, and propionic acid) combined with a small percentage of MCFA (mainly C8:0 and C10:0). MOA2 is a synergetic compound of a phenolic blend, mainly contain monounsaturated fatty acids and MCFA (mainly C10:0 and C12:0), target release butyrate and sorbic acid. The carrier for MOA1 and MOA2 is silica.

Six crossbred barrows (Duroc×Landrace×Yorkshire), with an average body weight 78.8±4.21 kg, were allotted to a double 3×3 Latin square design with 3 periods and 3 diets. Pigs were surgically fitted with a T-cannula in the distal ileum using procedures adapted from Stein et al [18]. Each period consisted of a 5-d adjustment period followed by a 2-d total collection of feces and then a 2-d collection of ileal digesta. The dietary treatments included a corn-soybean-wheat bran basal diet (CTR), mixed organic acid 1 diet (MOA1; CTR+3,000 mg/kg MOA1), and mixed organic acid 2 diet (MOA2; CTR+2,000 mg/kg MOA2). Vitamins and minerals were supplemented to meet or exceed the estimated nutrient requirements for pigs as recommended by the NRC [19]. Chromic oxide (2,500 mg/kg) was included in all diets as an inert marker. The ingredients composition of the experimental diets is shown in Table 1.

Pigs were housed in individual pens in an environmentally controlled room (22°C±2°C). A feeder and a nipple drinker were installed in each pen. Body weight was recorded at the beginning of the experiment. Feed allowance was equivalent to 40 g/kg of body weight and divided into 2 equal meals fed at 08:00 and 17:00 h each day. Water was available at all times throughout the experiment periods.

Each period lasted 9 days. Feces were collected via grab sampling in the morning of d 6 and 7 in each period. The fecal samples were stored at −20°C quickly after collection. At the end of the experiment, feces were dried at 65°C for 72 h. Samples were ground through a 1mm screen after drying and then were stored at −20°C until used for chemical analysis. Ileal digesta was collected continuously for 12 h from 08:00 to 20:00 h on d 8 and 9 according to procedures described by Zeng et al [20]. In brief, a 200 mL plastic bag was attached to the open cannula using a cable tie. Bags were removed whenever they were filled with digesta or at least every 30 min and stored at −20°C to prevent bacterial degradation of the amino acids in the digesta.

Feed and fecal samples were analyzed for CP, dry matter (DM), ash, neutral detergent fiber (NDF), and acid detergent fiber (ADF). Ileal digesta samples were analyzed for CP, DM, and ash according to the methods of AOAC [21]. The NDF and ADF were determined using fiber bags and Fiber Analyzer (Ankom Technology, Macedon, NY, USA). The GE was determined by an Automatic Energy Analyzer (Parr 1281, Moline, IL, USA). Amino acids in diets and digesta were analyzed using the methods described by Zeng et al [20].

The chromium content in the diets and feces was measured using an atomic absorption spectrophotometer (Z-5000; Hitachi, Tokyo, Japan) according to the procedure of Williams et al [22]. Organic matter (OM) was calculated as 1–ash content (DM-base). Nutrient digestibility was determined by the equation as follows:

Fresh fecal samples collected on d 28 were used for analy sis of VFAs. Frozen samples were thawed at room temperature and approximately 1.5 g of sample was placed into a centrifuge tube, and mixed with 1.5 mL of sterile water. The content was thoroughly mixed and centrifuged at 15,000×g for 10 minutes at 4°C. The supernatant (1 mL) was transferred into a gas chromatograph sample bottle, and 200 μL meta-phosphoric acid was added. The bottle was immersed in ice for 30 minutes and then centrifuged at 15,000×g for 10 minutes at 4°C. A Hewlett Packard 5890 gas chromatograph (HP, Palo Alto, CA, USA) was used to determine VFA concentrations.

The VFA concentrations of frozen digesta of cecum and colon were determined using a modified method. A 1.0 g sample was diluted with 2.0 mL of 0.10 mol/L HCl solution and put on ice for 30 min and then centrifuged at 12,000×g at 4°C for 15 min. Exactly 1.0 mL of the supernatant was passed through a 0.22 mm Nylon Membrane Filter (Millipore, Bedford, OH, USA) and then 5.0 L of the solution was injected into a gas chromatographic system (Agilent HP 6890 Series, Santa Clara, CA, USA).

Total bacteria genomic DNA extraction was performed from cecal specimens by use of the E.Z.N.A. Stool DNA Kits (Omega Biotek, Norcross, GA, USA). The microbial 16S rDNA was amplified with indexes and adaptors-linked universal primers (341F: ACTCCTACGGGRSGCAGCAG, 806R: GGA CTACVVGGGTATCTAATC) targeting the V3-V4 region, polymerase chain reaction (PCR) was performed using KAPA HiFi Hotstart ReadyMix PCR kit high fidelity enzyme to ensure the accuracy and efficiency of the expansion. The PCR products were detected w 20 g/kg agarose gel electrophoresis, and then recovered by AxyPrep DNA gel Recovery Kit (AXYGEN, San Francisco, CA, USA). Library quality inspection was quantified by Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) to pool into even concentration, and then sequenced on Illumina HiSeq PE250 platform (Illumina, San Diego, CA, USA) for paired-end reads of 250 bp. The paired-end reads were assembled into longer tags and quality-filtered to remove tags with length of <220 nt, average quality score of <20, and tags containing >3 ambiguous bases by PANDAseq for Clean Reads. All Clean Reads were compared to operational taxonomic unit (OTU) sequences to obtain the final Mapped Reads [23]. After discarding the singletons, the high-quality tags were clustered into OTUs for species taxonomy by using UPARSE with a similarity threshold of 0.97. And the OTUs were further subjected to the taxonomy-based analysis with the ribosomal database project (RDP) database, using the RDP classifier at a 0.80 confidence level. A heat map was created using R. Cluster analysis was done with Cluster 3.0 (Sun Microsystems, Inc., Santa Clara, CA, USA). Alpha diversity (Simpson and Shannon) and beta diversity were analyzed using QIIME (University of California, San Diego, CA, USA). Linear discriminant analyses and effect size (LEfSe) analyses were performed with the LEfSe tool [24]. The relative abundance of bacteria was expressed as the percentage.

All data were analyzed using the Proc-Mixed procedure of SAS (SAS Inst. Inc., Cary, NC, USA). The statistical model for all the values had treatment and period as fixed effects and pig as a random effect. Statistical differences among treatments were separated by Student-Neuman-Keul’s multiple range tests. Values were presented as least square means with standard error of the mean. Statistically significant differences were declared at p<0.05, and differences at 0.05≤p<0.10 were considered a trend toward significance.

Compared to CTR, pigs fed diets supplemented with MOA tended to increase (p = 0.08) the AID of DM. The ATTD of GE, DM, OM was improved (p<0.05) in pigs supplemented with diets containing MOA, while ATTD of NDF was improved (p<0.05) in pigs fed a diet supplemented with MOA2 compared to CTR (Table 2). There was no significant difference between the CTR diet and the MOA diet for the AID of amino acids (Table 3).

Compared to CTR, the pH value in ileum was decreased (p< 0.05) in pigs fed diets supplemented with MOA (MOA1 or MOA2). The concentrations of lactic acid and TVFA in ileum were improved (p<0.01) in pigs supplemented with diets containing MOA compared to CTR (Table 4). In feces, pigs fed diets supplemented with MOA showed higher (p<0.05) concentration of acetic acid and a tendency (p = 0.06) for an improved content of lactic acid, while these pigs had lower (p<0.05) content of formic acid. The concentration of TVFA in feces was increased (p<0.05) in diets supplemented with of MOA1 of pigs, while pigs fed diet containing MOA1 showed higher (p<0.05) concentration of butyric acid in feces compared to CTR (Table 5).

No significant differences of the intestinal microbiota concentration were observed in feces of pigs fed CTR and MOA. But a noticeable increase was observed in Lactobacillus by the MOA1 treatment, from 1.3% in the CTR pigs to 7.6% in the MOA1 pigs (data not shown). The decreases in Escherichia abundance with exposure to the MOA1 were significant: from 12.0% in the feces of the CTR pigs to 5.7% with the MOA1 treatment (Figure 1). An obvious increase was observed in Firmucutes by the MOA1 treatment, from 39.1% in the CTR pigs to 70.4% in the MOA1 pigs (Figure 2). A noticeable decrease was observed in Bacteroidetes by the MOA1 treatment, from 14.5% in the CTR pigs to 5.7% in the MOA1 pigs (data not shown). For the Firmicutes/Bacteroidetes ratio, we observed significant differences between CTR (2.7) and MOA1 (12.4). There is a clear distinction between CTR, MOA1, and MOA2 (Figure 3).