Dietary medium-chain fatty acid and Bacillus in combination alleviate weaning stress of piglets by regulating intestinal microbiota and barrier function

Abstract

The present study evaluated the effects of dietary medium-chain fatty acid (MCFA) and Bacillus on growth performance, nutrient digestibility, antioxidant capacity, colonic fermentation, and microbiota of weaning piglets. A total of 400 weaned piglets were randomly divided into 4 treatments, with 10 replicates per treatment and 10 pigs per replicate. The treatment included: basal diet (control, CON), basal diet with 0.588 g/kg MCFA (MCF), basal diet with 1.3 × 10⁹ CFU/kg Bacillus (BAC), and basal diet with 0.588 g/kg MCFA and 1.3 × 10⁹ CFU/kg Bacillus (SYN). Compared with CON group, the average daily gain of MCF and SYN in the early (1 to 9 d) and whole stage (1 to 36 d) of trail were improved (P < 0.05), the feed to gain ratio of MCF in later (10 to 36 d) and whole stage of trial were decreased (P < 0.05), and the diarrhea rate of SYN in the early stage (1 to 9 d) of trial decreased (P < 0.05). The digestibility of dry matter, ether extract, acid detergent fiber digestibility of MCF were decreased (P < 0.05) compared with CON. The serum d-lactic acid in MCF, BAC, and SYN were lower (P < 0.05) compared with CON group. Compared with CON group, the contents of total antioxidant capacity, superoxide dismutase, and glutathione peroxidase were greater (P < 0.05), whereas the content of malondialdehyde and the contents of colonic isobutyrate and isovalerate were lower (P < 0.05) in MCF. The microbial Shannon and Simpson diversity was lower in MCF (P < 0.05) than that in BAC and SYN. The relative abundance of Prevotella was greater (P < 0.05), whereas the Treponema and Oscillibacter were lower (P < 0.05) in MCF than that in BAC and SYN. In addition, the metabolic pathways of bacteria such as pentose phosphate pathway, adenosine nucleotides degradation II were enhanced (P < 0.05), whereas the pathways such as incomplete reductive TCA cycle, and TCA cycle IV (2-oxoglutarate decarboxylase) were decreased (P < 0.05) in MCF compared with BAC. The results indicated that dietary MCFA and Bacillus in combination improved the intestinal barrier function of piglets by changing the intestinal microbiota and its metabolic function, and finally alleviated the diarrhea rate in early weaning stage and improved growth performance in whole trial period. In addition, MCFA was effective in improving feed efficiency and antioxidant capacity of piglets.

Dietary medium-chain fatty acid and Bacillus in combination improved the intestinal barrier function of piglets by changing the intestinal microbial community and their metabolic function, and finally alleviated the weaning stress and improved the growth performance of piglets.

Introduction

Weaning is the most stressful period during the growth process of piglets, in which the piglets face challenges in production environment, feed nutrition, community struggle, and feeding management (Kim et al., 2012). Weaning stress can reduce the feed intake of piglets, destroy the intestinal barrier function, change the balance of intestinal microflora, cause diarrhea and even death of piglets, and finally result in economic losses to the livestock (Boudry et al., 2004). Recently, it has become a research hotspot to find alternatives to ensure the safety and stability of animal production after the prohibition of antibiotics in feed in China.

Medium-chain fatty acid (MCFA) is a carbon-chain fatty acid with carbon atoms ranging between 6 and 12. MCFA can inhibit Escherichia coli and Salmonella and enhance epithelial barrier function and immune function (Lopez-Colom et al., 2019). The addition of low concentration MCFA (0.1% to 0.5%, mass ratio) to the diet improved the intestinal epithelial structure by regulating the intestinal flora, improved the digestibility of crude protein (CP) and ether extract (EE), and feed conversion rate, and promoted the growth performance of weaned piglets (Hanczakowska et al., 2016; Gebhardt et al., 2020). Bacillus, including Bacillus subtilis and Bacillus licheniformis, are widely used in animal husbandry. It can inhibit pathogenic bacteria through oxygen consumption and secretion of antibacterial substances (Guo et al., 2006; Carlin, 2011). It can also secrete digestive enzymes to improve nutrient digestibility (Yanbo and Zirong, 2006). Studies showed that supplementation with 0.1% B. subtilis improved growth performance and intestinal health of piglets (Deng et al., 2020). Dietary supplementation of a mixture of spray-dried B. licheniformis and B. subtilis improved the growth and feed to gain ratio from weaning to slaughter and exerted a beneficial effect on apparent digestibility of fat and phosphorus during the growing period (Jørgensen et al., 2016). It is well known that both MCFA and Bacillus can inhibit intestinal pathogenic bacteria (Carlin, 2011; Lopez-Colom et al., 2019). However, previous studies (Deng et al., 2020; Gebhardt et al., 2020) mainly focused on the effect of MCFA or Bacillus alone on performance of weaned piglets, the combined application of both was rarely reported. Whether they have synergistic or antagonistic effect in alleviating weaning stress in practice has not been reported. The effects of MCFA and Bacillus in diet on intestinal microbiota and barrier function of piglets are also unclear. We hypothesized that dietary MCFA and Bacillus in combination could improve gastrointestinal health and alleviate weaning stress of piglets. This study was conducted to investigate the effects of MCFA and/or Bacillus on growth performance, apparent nutrient digestibility, antioxidant capacity, colonic fermentation parameters, and microbiota of weaned piglets.

Materials and Methods

This experiment was conducted in a commercial farm located in Shunyi district, Beijing, China. The experimental protocol was approved by the Animal Ethics Committee of the Institute of Feed Research of Chinese Academy of Agricultural Sciences (Protocol number: IFR-CAAS20220613), and humane animal care and handling procedures were implemented throughout the experiment.

Animals and treatments

Four hundred 28-d-old weaned pigs (Duroc × [Landrace × Yorkshire]) weighing 8.44 ± 1.52 kg were randomly divided into 10 blocks with 40 pigs each. Pigs in each block were randomly assigned to 4 treatments, with 10 pigs per treatment housed in one pen. The four treatments included: 1) control group (CON), feeding basal diet; 2) MCFA group (MCF), feeding basal diet with 0.588 g/kg MCFA; 3) Bacillus group (BAC), feeding basal diet with 1.3 × 10⁹ CFU/kg Bacillus; and 4) Synergistic group (SYN), feeding basal diet with 0.588 g/kg MCFA and 1.3 × 10⁹ CFU/kg Bacillus.

At the end of the experiment, 10 pigs were randomly selected from each treatment (1 pig per pen), fasted for 12 h, and then transported to a commercial slaughterhouse for slaughter. All pigs were slaughtered by bloodletting after electric shock. The abdominal cavity of piglets was immediately opened, and then the intestine tissue was ligated and taken out. The colonic content was collected, and its pH was measured immediately after collection using a digital pH meter (PB-10; Sartorius, Goettingen, Germany). The collected colonic contents were placed in liquid nitrogen until further treatment (fermentation parameters and microbial community analysis).

Diets, feeding, and management

The MCFA used in this trial was a mixture of high-purity active MCFAs with silica as a carrier, in which caproic acid = 2.2%, octanoic acid = 31.0%, decanoic acid = 22.0%, lauric acid = 6.8%, crude protein = 2.11%, moisture = 4.11%, and crude ash = 22.9%. It was tested by the Institute of Quality Standard and Testing Technology for Agro-Products, Chinese Academy of Agricultural Sciences. The Bacillus additive consisted of B. licheniformis-3809 and B. subtilis-3810 with the ratio of 1: 1, and the viable count was ≥3.2 × 10⁹ CFU/g. The experiment included two stages. Pigs were fed with starter feed from day 1 to 9 and fed with nursery pig feed from days 10 to 36. The basal diets were formulated according to NRC (2012) and the ingredients and chemical composition are shown in Table 1.

Table 1.

Composition and nutrient levels of basal diets (% of DM)

Ingredients	Formula		Nutrients1	Contents
	Days 1 to 9	Days 10 to 36		Days 1 to 9	Days 10 to 36
Corn		46.60	DM	91.99	88.71
Fish meal, CP 65%		1.45	NE, MJ/kg	10.52	10.32
Extruded corn	36.40	8.00	ME, MJ/kg	15.14	14.40
Barley	12.50	14.00	CP	17.21	16.83
Oatmeal	2.50		EE	6.99	5.77
Soybean meal	16.00	18.50	NDF	11.26	13.91
Soybean oil	4.50	3.00	ADF	4.17	4.77
Dextrose	5.00		Ca	0.58	0.64
Sucrose	2.00	3.00	P	0.55	0.55
Whey powder	4.35		Lys	1.25	1.15
Whey protein	7.50
Yeast	5.00	1.25
Flavoring agent	0.15
NaCl		0.10
Antioxidants	0.05	0.05
Emulsifier	0.05	0.05
Premix2	4.00	4.00
Total	100.00	100.00

DM, dry matter; NE, net energy; ME, metabolizable energy; CP, crude protein; EE, ether extract; NDF, neutral detergent fiber; ADF, acid detergent fiber; Ca, calcium; P, phosphorus; Lys, lysine.

¹Nutrient levels were calculated values.

²The premix provided per kg of diets: vitamin A, 8,000 IU; vitamin D₃, 2,000 IU; vitamin E, 25.0 IU; Fe, 100 mg; Cu, 100 mg; Mn, 4 mg; Zn, 100 mg; Se, 0.35 mg; I, 0.3 mg; Ca, 4.0 g; P, 3.0 g.

The temperature of the enclosure was controlled at 25 °C to 26 °C and the relative humidity was controlled at 50% to 70%. The enclosure was cleaned regularly and disinfected alternately to maintain ventilation and hygiene. All piglets were raised in the same environment and management conditions, with free accessed to feed and water. During the trail, the pigs were immunized and dewormed according to the normal procedures of pig farms, and the health status was recorded in detail every day.

Sampling and analysis

The offers and residuals were recorded daily. The piglets were weighed on the morning of days 1, 10, and 37 d of the trial. The dry matter intake (DMI), average daily gain (ADG), and feed-to-gain ratio (F/G) were calculated accordingly. During the feeding period, each pen was scored once every afternoon, and piglets’ feces were scored on a scale of 0 to 3 points: 0, normal; 1, mild; 2, moderate; and 3, serious.

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Antibiotics used during the test: antonidine for fever (a compound preparation composed of antipyrine, aminopyrine, and barbiturate sodium, with antipyretic, analgesic, and antispasmodic effects), gentamicin for diarrhea.

From days 32 to 36 of the trial, approximately 100 g of uncontaminated fecal samples were collected from each replicate every morning and afternoon. After each collection, the samples were treated with 10% sulfuric acid. After 5-d ­collection, fecal samples from each replicate were mixed and frozen at −20 °C for the determination of the apparent digestibility of nutrients, which were carried out using 4 mol/L hydrochloric acid insoluble ash (AIA) as an endogenous indicator. Dry matter (DM), CP, EE, neutral detergent fiber (NDF), acid detergent fiber (ADF), organic matter (OM), and AIA in feed and feces were measured according to AOAC (2000).

Before the morning feeding of day 37 of the trial, one piglet was randomly selected from each replicate, and blood sample was collected by jugular venipuncture into a 10-mL vacutainer tube without anticoagulants. After centrifugation at 3,000 rpm for 10 min, the supernatant was transferred to 1.5-mL Eppendorf tubes and stored at −20 °C until further analysis. Serum antioxidant indexes, including total antioxidant capacity (TAOC), glutathione peroxidase (GSH-Px) activity, superoxide dismutase (SOD) activity and malondialdehyde (MDA), serum immune indexes, including immunoglobulin G (IgG) and immunoglobulin M (IgM), serum growth indexes, including growth hormone (GH) and insulin-like growth factor-1 (IGF-1), intestinal permeability indexes, including diamine oxidase (DAO) and d-lactic acid, were determined with assay kits, referring to the specific detection methods instructed by manufacture (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

The colon content samples were thawed at 4 °C, and the VFA concentration was determined by gas chromatography (GC) using methyl valerate as the internal standard in an Agilent 6890 series GC equipped with a capillary column (HP-FFAP19095F-123, 30 m, 0.53 mm diameter, and 1 cm thickness) as described by Lv et al. (2020).

Microbial DNA was extracted from colonic contents using a commercial DNA kit (Omega Bio-tek, Norcross, GA, USA). Thermo NanoDrop 2000 UV spectrophotometer and 1% agarose gel electrophoresis were used to analyze the concentration and integrity of total DNA. The V3-V4 region of 16S rDNA was selected as the amplification region for PCR amplification. The 5ʹ end of the universal primer was added with the appropriate Illumina Miseq PE250 (Illumina, USA) sequencing index sequence and joint sequence to complete the design of specific primers, Forward primer (341F: CCTACGGRSGCAGCAG) and Reverse primer (806R: GGACTACVVGGTATCTAATC). After the genomic DNA template was diluted with water, the KAPA HiFi Hotstart ReadyMix PCR kit high fidelity enzyme (KAPA Biosystems, USA) was used for PCR to ensure the accuracy and efficiency of amplification. The PCR products were recovered by AxyPrep DNA gel recovery kit (AXYGEN, USA). The library quality was detected by Thermo NanoDrop 2000 ultraviolet spectrophotometer and 2% agarose gel electrophoresis. After quality inspection, Illumina Miseq PE250 was used for sequencing. A 16S-specific primer was designed to amplify the specific region and a 425 bp fragment was obtained. Add the connector, using Illumina platform, sequencing PE250 Paired-End data, by splicing to get a longer sequence, thus 16S analysis. After QC of the sequences, the data were analyzed and filtered by Usearch software. DNA sequences with similarity greater than 97% were clustered to form operational taxonomic unit (OTU). The abundance and diversity of flora were analyzed based on OTU, and the flora structure was analyzed at the taxonomic level of phylum and genus.

Statistical analysis

The data were analyzed using the general linear model procedure of SPSS V26.0 statistical software, using the pen as an experimental unit. All data were analyzed using a model that included the fixed effects of treatments, as well as the random effect of blocks. Duncan’s multiple range test was used to identify differences between specific treatments when a significant difference existed. P < 0.05 was considered to indicate a statistically significant difference.

Alpha-diversity was tested using the Kruskal–Wallis’ test with boxplots made in R (“ggpubr” packages). Beta diversity was visualized with a PCoA plot. The Linear discriminant analysis Effect Size (LEfSe) was used to identify the bacterial biomarkers of different treatments. The Random Forest was performed to identify the top microbiomes. Variables importance plot was then generated based on the importance scores (mean decrease in accuracy, MDA) in R (v3.6.0).

Results

Growth performance and health status

The effects of MCFA and Bacillus on the growth performance and health of weaned piglets are shown in Table 2. The body weight (BW) of piglets at days 10 and 37 were greater (P < 0.05) in MCF and SYN than that in CON. From days 1 to 9, the ADG and DMI of piglets in MCF and SYN were greater (P < 0.05) than those in CON, whereas the diarrhea rate of SYN was lower (P < 0.05) than that in CON. From d10 to d36, the ADG and feed-to-gain ratio of piglets in MCF were greater (P < 0.05) than those in CON. During the whole trial period, the ADG of piglets in MCF and SYN were greater (P < 0.05) than those in CON, and the feed-to-gain ratio of MCF was greater (P < 0.05) than that in CON.

Table 2.

Effect of MCFA and Bacillus on growth and health performance of weaned piglets

Items	CON	MCF	BAC	SYN	SEM	P-value
BW on day 1, kg	8.28	8.31	8.28	8.32	0.015	0.104
BW on day 10, kg	11.27a	11.58b	11.38a	11.62b	0.063	0.001
BW on day 37, kg	24.74a	25.73c	25.11ab	25.51bc	0.170	0.002
Mortality, %	2.11	0.00	1.11	1.11	0.998	0.533
Treated with antibiotics, %	0.29	0.12	0.09	0.12	0.055	0.063
Phase 1, days 1 to 9
ADG, g/d	332.87a	363.34b	344.54a	366.92b	6.436	0.002
DMI, g/d	454.65a	476.85b	460.65a	485.96b	5.570	0.002
F/G	1.37	1.32	1.34	1.33	0.020	0.400
Diarrhea rate, %	7.03a	5.46ab	5.14ab	4.38b	0.641	0.048
Phase 2, days 10 to 36
ADG, g/d	498.82a	524.04b	508.59ab	514.59ab	5.547	0.025
DMI, g/d	840.01	865.66	852.56	855.34	9.265	0.296
F/G	1.68b	1.65a	1.68ab	1.66ab	0.010	0.043
Diarrhea rate, %	3.61	3.22	2.63	3.06	0.383	0.355
Whole period, days 1 to 36
ADG, g/d	457.33a	483.86c	467.58ab	477.68bc	4.681	0.002
DMI, g/d	743.00	768.46	754.09	762.74	7.310	0.099
F/G	1.63b	1.59a	1.61ab	1.60ab	0.010	0.020
Diarrhea rate, %	4.46	3.78	3.25	3.39	0.320	0.055

BW, body weight; ADG, average daily gain; DMI, dry matter intake; F/G, feed to gain ratio; CON, basal diet; MCF, basal diet with 0.588 g/kg MCFA; BAC, basal diet with 1.3 × 10⁹ CFU/kg Bacillus; SYN, basal diet with 1.3 × 10⁹ CFU/kg Bacillus and 0.588 g/kg MCFA.

^a,b,cValues in the same row with no common letter superscripts mean significant difference (P < 0.05).

Apparent digestibility

The apparent digestibility of DM and EE in MCF was lower (P < 0.05) than that in CON (Table 3). The apparent ­digestibility of ADF in MCF, BAC, and SYN were lower (P < 0.05) than that in CON.

Table 3.

Effect of MCFA and Bacillus on the apparent nutrient digestibility of weaned piglets

Items	CON	MCF	BAC	SYN	SEM	P-value
DM, %	90.96b	89.39a	90.33b	90.47b	0.593	0.003
CP, %	88.09	86.27	87.83	87.28	1.465	0.058
EE, %	83.74b	81.20a	82.84ab	83.82b	0.396	0.018
NDF, %	76.85	74.10	76.61	76.62	2.194	0.203
ADF, %	80.05a	75.31b	76.86b	77.19b	0.643	0.009

DM, dry matter; CP, crude protein; EE, ether extract; NDF, neutral detergent fiber; ADF, acid detergent fiber; CON, basal diet; MCF, basal diet with 0.588 g/kg MCFA; BAC, basal diet with 1.3 × 10⁹ CFU/kg Bacillus; SYN, basal diet with 1.3 × 10⁹ CFU/kg Bacillus and 0.588 g/kg MCFA.

^a,bValues in the same row with no common letter superscripts mean significant difference (P < 0.05).

Serum index

As shown in Table 4, The d-lactic acid contents in MCF, BAC, and SYN were lower (P < 0.05) than that in CON. Compared with the CON, the contents of SOD, TAOC, and GHS-P_X in MCF were increased (P < 0.05), and the MDA content was decreased (P < 0.05). There was no difference (P > 0.05) in serum GH, IGF-1, IgG, IgM, and DAO contents among groups.

Table 4.

Effect of MCFA and Bacillus on serum index of weaned piglets

Items	CON	MCF	BAC	SYN	SEM	P-value
Growth index
GH, ng/mL	3.93	4.46	4.06	3.94	0.208	0.260
IGF-1, ng/mL	86.32	90.27	87.17	86.35	1.154	0.071
Immune index
IgG, g/L	6.48	6.49	6.30	6.66	0.104	0.140
IgM, g/L	0.87	0.86	0.87	0.91	0.017	0.172
Intestinal permeability index
DAO, U/L	6.54	6.75	6.82	6.62	0.177	0.692
d-lactic acid, mmol/mL	9.25a	8.41b	8.60b	8.42b	0.164	0.003
Antioxidant index
TAOC, U/mL	9.46b	10.24a	9.62b	9.42b	0.160	0.004
SOD, U/mL	109.22b	118.27a	111.19b	109.13b	1.808	0.004
GHS-Px, μmol/mL	771.71b	811.59a	780.45b	771.54b	8.207	0.005
MDA, nmol/mL	5.05b	4.50a	4.93b	5.05b	0.106	0.003

GH, growth hormone; IGF-1, insulin-like growth factors-1; IgG, immunoglobulin G; IgM, immunoglobulin M; DAO, diamine oxidase; SOD, superoxide dismutase; MDA, malondialdehyde; TAOC, total antioxidant capacity; GHS-P_X, glutathione peroxidase; CON, basal diet; MCF, basal diet with 0.588 g/kg MCFA; BAC, basal diet with 1.3 × 10⁹ CFU/kg Bacillus; SYN, basal diet with 1.3 × 10⁹ CFU/kg Bacillus and 0.588 g/kg MCFA.

^a,bValues in the same row with no common letter superscripts mean significant difference (P < 0.05).

Colonic fermentation parameters

There was no difference in colon pH and VFAs among groups except the contents of isobutyrate and isovalerate, which in MCF were lower (P < 0.05) than that in CON (Table 5).

Table 5.

Effects of MCFA and Bacillus on colonic fermentation parameters of weaned piglets

Items	CON	MCF	BAC	SYN	SEM	P-value
pH	6.13	5.93	6.28	6.13	0.11	0.162
TVFA, mmol/g	51.25	54.30	46.21	48.32	2.86	0.232
Acetate, mmol/g	22.82	25.75	22.05	22.36	1.69	0.403
Propionate, mmol/g	15.97	17.17	13.60	14.47	1.07	0.111
Butyrate, mmol/g	7.43	7.84	6.36	6.99	0.63	0.402
Valerate, mmol/g	2.50	2.08	1.90	1.93	0.25	0.299
Isobutyrate, mmol/g	1.67b	1.02a	1.53b	1.68b	0.16	0.020
Isovalerate, mmol/g	0.86b	0.43a	0.77b	0.88b	0.11	0.025

TVFA, total volatile fatty acids; CON, basal diet; MCF, basal diet with 0.588 g/kg MCFA; BAC, basal diet with 1.3 × 10⁹ CFU/kg Bacillus; SYN, basal diet with 1.3 × 10⁹ CFU/kg Bacillus and 0.588 g/kg MCFA.

^a,bValues in the same row with no common letter superscripts mean significant difference (P < 0.05).

Colonic microbiome

The 16S rDNA of the colon contents of 40 weaned piglets was amplified in the V3-V4 region, and then Illumina high-throughput sequencing was performed. A total of 1,398,963 effective sequences were obtained after quality control. The average effective sequences of each sample were 34,974 ± 2,148, and the average sequence length was 146 bp, of which 66.5% were between 420 and 440 bp. Based on the principle of similarity greater than 97%, the effective sequences were clustered, and the chimeras were filtered to obtain a total of 1,177 OUTs for species classification. 710 OUTs were shared among the four groups (Figure 1B). The number of OUTs in the CON, BAC, MCF, and SYN groups were 951, 1,072, 824, and 991, respectively. Observed species and Chao1 did not differ (P > 0.05) between groups, but Shannon and Simpson of SYN and BAC were greater (P < 0.05) than that of MCF (Figure 1A). Anosim analysis (R = 0.15, P = 0.007) indicating that the difference between groups was greater than that within groups (Figure 1C). PCoA method was used to show the difference among groups. In the PCoA1 (55.36%) direction, BAC and MCF, BAC and CON, and MCF and SYN were separated (Figure 1D).

Figure 1.

Effects of dietary MCFA and Bacillus on colon microbial diversity of weaned piglets. (A) Alpha diversity, (B) OTU Venn diagram, (C) weighted UniFrac Anosim analysis, and (D) PCoA diagram among groups. CON, control group; MCF, basal diet with 0.588 g/kg MCFA; BAC, basal diet with 1.3 × 10⁹ CFU/kg Bacillus; SYN, basal diet with 1.3 × 10⁹ CFU/kg Bacillus and 0.588 g/kg MCFA. *P < 0.05; **P < 0.01.

Next, the core microbiome among treatments was examined (Figure 2). A total of 18 phyla were observed, 4 of them were abundant over 1% of the total sequences in at least one group. The dominant bacteria at the phylum level were Bacteroidetes, Firmicutes, Proteobacteria, and Spirochaetes, which accounted for more than 95% of the total abundance. A total of 134 genera were observed, 36 of them were abundant over 0.1% of the total sequences in at least one group. The top 20 relative abundances at genus level were Paraprevotella, Alloprevotella, Preovtella of Bacteroidetes, ­Clostridium IV, etc.

Figure 2.

Effects of dietary MCFA and Bacillus on phylum and genus level composition of colonic microbiome. CON, basal diet; MCF, basal diet with 0.588 g/kg MCFA; BAC, basal diet with 1.3 × 10⁹ CFU/kg Bacillus; SYN, basal diet with 1.3 × 10⁹ CFU/kg Bacillus and 0.588 g/kg MCFA. Phylum levels with relative abundance greater than 1% and genus levels with the top 20 relative abundances are listed on the right.

To confirm the differences of bacteria among groups, LEfSe was carried out for taxon at all levels to find the featured genera among groups, and random forest prediction was carried out for the top 20 genera of relative abundance to explore the importance of genera (Figure 3). Phylum Lentisphaerae, and genera Oligosphaera, Romboutsia were featured bacteria for BAC group, Phylum Spirochaetes, and genera Treponema, Sphaerochaeta were featured bacteria for SYN group (Figure 3A). The genus Oscillibacter was the most important taxa among the top 20 genera (Figure 3B). The genus Treponema, which was the feature bacteria of SYN group, and Prevotella, which was the most abundant genus of all genera, was also important according to random forest prediction. The genus Oscillibacter in SYN group was greater (P < 0.05) than that of CON and MCF groups. The genus Treponema in BAC and SYN groups was greater (P < 0.05) than that of MCF group. Whereas the genus Prevotella in MCF was greater (P < 0.05) than that of BAC (Figure 3C).

Figure 3.

Effects of MCFA and Bacillus on the featured taxon of weaning piglets. (A) Selected taxa based on LEfSe analysis. (B) Selected taxa based on random forest analysis of the top 20 abundant genera. (C) Relative abundance of the genus Oscillibacter, Treponema, and Prevotella. CON, basal diet; MCF, basal diet with 0.588 g/kg MCFA; BAC, basal diet with 1.3 × 10⁹ CFU/kg Bacillus; SYN, basal diet with 1.3 × 10⁹ CFU/kg Bacillus and 0.588 g/kg MCFA. *Means significant difference (P < 0.05).

PICRUSt was used to analyze functional genes of colonic bacteria, and the function prediction information of bacteria in different samples was obtained. MetaCyc database was used to compare the sequencing data. We selected the MetaCycs with the top 80% of the total abundances and found 24 featured MetaCycs among groups with LEfSe (Figure 4A). The P105-PWY (TCA cycle IV [2-oxoglutarate decarboxylase]) was featured pathway of SYN group. The metabolic pathways specifically associated with the microbiota in MCF were notably enriched in nucleotide degradation and amino acid biosynthesis, as exemplified by SALVADEHYPOX-PWY (adenosine nucleotides degradation II), PWY-6608 (guanosine nucleotides degradation III), PWY-4984 (urea cycle), and HSERMETANA-PWY (l-methionine biosynthesis III). Whereas pathways related to biosynthesis were enriched in BAC group, such as P42-PWY (incomplete reductive TCA cycle), PWY-6897 (thiamin salvage II), and THISYN-PWY (super pathway of thiamin diphosphate biosynthesis I). The MetaCyc PENTOSE-P-PWY (pentose phosphate pathway), PWY-5913 (TCA cycle VI [obligate autotrophs]), and PWY-6897 (thiamin salvage II) were important according to random forest prediction (Figure 4B). The relative abundance of PENTOSE-P-PWY and SALVADEHYPOX-PWY metabolic pathways in MCF was greater (P < 0.05) than that in BAC group. and the relative abundance of P42-PWY was greater in BAC than that in MCF group, whereas the relative abundance of P105-PWY in MCF group was lower than that in SYN and CON groups (Figure 4C).

Figure 4.

Effects of MCFA and Bacillus on the featured MetaCycs of weaning piglets. (A) Featured MetaCycs among groups based on LEfSe analysis. (B) Selected pathways based on random forest analysis of the featured MetaCycs. (C) The relative abundance of PENTOSE-P-PWY, SALVADEHYPOX-PWY, P42-PWY, and P105-PWY. CON, basal diet; MCF, basal diet with 0.588 g/kg MCFA; BAC, basal diet with 1.3 × 10⁹ CFU/kg Bacillus; SYN, basal diet with 1.3 × 10⁹ CFU/kg Bacillus and 0.588 g/kg MCFA. *Means significant difference (P < 0.05).

Discussion

The hypothesis that dietary MCFA and Bacillus in combination could improve gastrointestinal health and alleviate weaning stress of piglets was supported by the results. MCFA and ­Bacillus in combination improved the intestinal barrier function of piglets and alleviated the diarrhea rate in the early weaning stage and improved the growth performance in whole trial period. Furthermore, MCFA was effective in improving feed efficiency and antioxidant capacity of piglets. It is supposed that MCFA and Bacillus in combination can positively affect the intestinal health, and the MCFA will be a more promising feed additive for piglets in this study. Therefore, this study allows us to deeply understand the combination application of additive in regulating the intestinal health of piglets, which provides insights into the importance of intestinal barrier function on weaning stress alleviation of piglets.

In this study, MCFA, or MCFA and Bacillus in combination increased ADG and morbidity of weaned piglets. Similar to our results, Hanczakowska et al. (2016) found that the dietary supplementation of 0.2% caprylic acid and capric acid alone or in combination increased ADG and reduced mortality in weaned piglets. Gebhardt et al. (2020) also found positive effect on ADG and feed efficiency of piglets using caproic acid, caprylic acid, and capric acid with different proportions (0.25% to 1.5%). It is suggested that combination with MCFA and Bacillus in this study possibly be a promoting method to cope with the weaning stress of weaned piglets. However, the MCFA decreased the apparent digestibility of DM, EE, and ADF compared with other groups. It is supposed that the increase of ADG in MCF group could be a result of other factors such as the antibacterial, antioxidant, and enhancing epithelial barrier function (Lopez-Colom et al., 2019). On the other hand, in this study, the Bacillus alone did not affect the ADG and DMI of weaned piglets. In the same way, Kritas and Morrison (2005) showed that adding B. subtilis and B. licheniformis had no effect on the growth of piglets. Whereas Deng et al. (2020) found that 100 mg/kg B. subtilis in diet increased ADG of piglets. The variations in results on growth performance are likely associated with variations in the administration level of probiotic products, health status within herds, and diet compositions, which need be confirmed by further research.

The stable antioxidant system can help animals maintain homeostasis and perform normal physiological functions (Tolmacheva and Nevinsky, 2022). In this study, MCFA has a positive effect on serum antioxidant capacity of piglets. The content of TAOC, SOD, GHS-Px increased, and MDA decreased compared with the CON group. It indicated that MCFA can inhibit lipid peroxidation by increasing the activity of antioxidant enzymes in vivo, thereby further enhancing the antioxidant capacity of animals. The results of this study were similar to those of Long et al. (2018), who found that the mixed organic acids tended to increase the level of TAOC, and the content of hydroxyl radical decreased by 20% compared with control group. The combination of MCFA and Bacillus did not improve the antioxidant capacity, which was surprising. Whether there is an antagonistic mechanism between MCFA and Bacillus to eliminate the improvement of MCFA on antioxidant capacity still needs further study.

Having a complete intestinal barrier is the basis for the body to maintain normal function and health (Yi et al., 2018). Once the intestinal barrier is damaged, pathogens or endotoxin cannot be prevented from entering the blood and organs (Wang et al., 2019). The results of this study showed that the d-lactic acid contents in the MCF, BAC, and SYN were lower than that of CON group, indicating that the dietary addition of MCFA and Bacillus alone or in combination could reduce the intestinal permeability and improve the intestinal barrier function of piglets. The enhancement of intestinal barrier function also provided an explanation for the improvement of growth performance of MCF and SYN group in this study. Interestingly, the results in this study showed that the contents of isobutyriate and isovaleriate in colon of MCF were lower compared with CON group. The isobutyrate and isovalerate were formed by Valine and Leucine degradation, respectively (Vitali et al., 2022). And the isovalerate was shown to cause relaxation of colon smooth muscle, thereby inhibiting the formation of intestinal barrier (Blakeney et al., 2019). Therefore, the decrease of isobutyrate and isovalerate in this study further proved that MCFA can enhance the intestinal barrier function.

Microbial diversity in the gastrointestinal tract is the result of host active selection and co-evolution in vivo (Backhed et al., 2005). Studies shown that feeding B. subtilis to weaned piglets improved the intestinal microbial diversity, affected the gastrointestinal tract flora of weaned piglets, and positively affected gastrointestinal health (Deng et al., 2020). Similarly, adding Bacillus to diet in this study improved the diversity of colon microbes. The increased bacterial diversity meant that the bacterial community was more stable, which improved the host’s response to interference (Pluske et al., 2018). Compatible with previous studies evaluating microbiome of the gut (Yang et al., 2017; Li et al., 2018), the largest proportion of microbes in this study were from the phyla Bacteroidetes and Firmicutes, and genus Prevotella. In this study, we found that Prevotella was greater, and Treponema and Oscillibacter were lower in MCF group compared with BAC group. Yang et al. (2017) clustered all samples into two enterotype-like groups. Enterotype-like group 1 and 2 were dominated by Treponema and Prevotella, respectively. It was found that the feed efficiency of pigs clustered in enterotype-like group 1 was greater than that of pigs clustered in enterotype-like group 2. The reduced apparent digestibility of nutrients and the lower content of isobutyrate and isovalerate in MCF group should be related the decrease of Treponema and Oscillibacter, which may be due to the certain antibacterial properties of MCFA (Zhou et al., 2019).

From our results, featured pathways included nucleotide degradation, amino acid biosynthesis, tricarboxylic acid cycle, and others, which were widely known to be involved in energy metabolism, synthesis of essential substances, and transmission of chemical information, and were of great significance to the growth, development (Yang et al., 2021). In this study, we observed greater adenosine nucleotide degradation in MCF group. Adenosine nucleotide is an extracellular signaling molecule that transmits signals through four different adenosine receptors, thereby regulating the physiological functions of tissues (Garcia-Gil et al., 2021). Amino acid regulation by gut bacteria is postulated to be correlated with the phenotype of growth and health (Kaur et al., 2017). We found that l-methionine synthesis pathway was more active at MCF group, which was closely correlated with growth of piglets (Kaur et al., 2017). In addition, TCA cycle IV (2-oxoglutarate decarboxylase) was greater in SYN group. TCA is a series of chemical reactions used in aerobic organisms, which generate energy by oxidation-derived acetyl coenzyme A (CoA) from carbohydrates, fatty acids and proteins (Choi et al., 2021).

Conclusions

In conclusion, this study investigated the application of MCFA or Bacillus and their combinations in piglets, which provides insights into the importance of intestinal barrier function on weaning stress alleviation. The results verified that MCFA was effective in improving feed efficiency and antioxidant capacity of piglets. But adding Bacillus or the combination of Bacillus and MCFA did not achieve the same effect. In addition, we identified the microbial biomarkers in the colon microbiome that may be used as targets for modulating growth and health of piglets. In terms of microbial composition and function prediction, dietary MCFA and Bacillus in combination improved the intestinal barrier function of piglets by changing the intestinal microbiota and its metabolic function, such as nucleotide metabolism, and amino acid metabolism, and finally alleviated the diarrhea rate in early weaning stage and improved growth performance in whole trial period. However, the causal relationship between the detected microbial signatures and phenotypes requires further study. In future, studies with omics techniques are needed to better understand the roles microbes on the hosts when MCFA and/or Bacillus are added to diet.

Glossary

Abbreviations

ADF

acid detergent fiber

ADG

average daily gain

BW

body weight

CP

crude protein

DM

dry matter

DMI

dry matter intake

DAO

diamine oxidase

EE

ether extract

F/G

feed to gain ratio

GH

growth hormone

GHS-Px

glutathione peroxidase

IgG

immunoglobulin G

IgM

immunoglobulin M

IGF-1

insulin-like growth factors-1

MDA

malondialdehyde

MCFA

medium-chain fatty acid

NDF

neutral detergent fiber

SOD

superoxide dismutase

TAOC

total antioxidant capacity

TVFA

total volatile fatty acids

Conflict of Interest Statement

The authors declare that there is no conflict of interest.