Effects of dietary indole-3-acetate sodium on intestinal morphology, nutrient absorption, and inflammatory responses in weaned piglets

TL;DR

Adding indole-3-acetate sodium to piglet diets improves intestinal health and nutrient absorption, possibly by reducing inflammation and boosting cell renewal.

Contribution

This study is the first to investigate the effects of dietary indole-3-acetate sodium on intestinal health in weaned piglets.

Findings

Dietary indole-3-acetate sodium increased jejunal villus width and nutrient digestibility in piglets.

Supplementation reduced proinflammatory cytokines and enhanced epithelial cell renewal in the intestine.

Organoid experiments confirmed improved epithelial renewal with indole-3-acetate sodium treatment.

Abstract

Weaning stress can severely damage the piglets’ intestines. Microbial tryptophan catabolites play a vital role in maintaining the health of the intestinal mucosa. Indole-3-acetic acid (IAA), an indole derivative with known anti-inflammatory properties, has not yet been studied for its impact on piglets’ intestinal health. Twenty-four weaned crossbred piglets (Duroc × Yorkshire × Landrace, weighing 6.58 ± 0.07 kg) were randomly allocated to receive diets containing 0, 120, or 240 mg/kg indole-3-acetate sodium (IAA-Na). Although dietary IAA-Na did not significantly impact growth performance or diarrhea incidence (P > 0.05), the 240 mg/kg IAA-Na elevated jejunal villus width (P < 0.05), tended to increase villus surface area (P < 0.10), and enhanced apparent nutrient digestibility alongside upregulating the mRNA expression of transporters (P < 0.05). Furthermore, dietary IAA-Na promoted intestinal epithelial cell proliferation and reduced secretory cell numbers (P < 0.05). Transcriptomic analysis of the jejunum of the 240 mg/kg group revealed significant modulation of pathways related to the “inflammatory response” and “immune system processes.” Consistent with this, dietary supplementation with 240 mg/kg IAA-Na downregulated the content and expression of proinflammatory cytokines while upregulating the content and expression of anti-inflammatory cytokines in the jejunum. To further elucidate the effect of IAA on epithelial renewal, piglet jejunal organoids were employed as an in vitro model. Treatment with 0.5 and 2 μM IAA-Na significantly increased the organoids budding rate on d 3 (P < 0.01), indicating enhanced epithelial renewal capacity. In conclusion, although dietary IAA did not improve overall growth performance, 240 mg/kg IAA-Na promoted nutrient absorption in weaned piglets, potentially through enhancing anti-inflammatory responses and epithelial renewal.

Dietary indole-3-acetate sodium enhances intestinal nutrient absorption and immune function in weaned piglets, offering a potential approach to improving intestinal health during the stressful transition to weaning.

Graphical Abstract

Figures

Intestinal morphology of weaned piglets fed with different IAA-Na concentrations. The results of the quantitative analysis of jejunum morphology are shown as the A) villus height, B) crypt depth, C) villus width, D) VH: CD, E) and villus surface area (ANOVA was performed). F) Representative images of jejunal morphology. VH: CD, ratio of villus height: crypt depth. ns indicates not significant and P < 0.01. scale bar = 200 μm (magnification 100×), n = 8.

Effect of dietary IAA-Na on the apparent total tract digestibility of nutrients in weaned piglets. Quantitative analysis of apparent total tract digestibility of nutrients shown in A) dry matter, B) crude protein, C) crude ash, and D) gross energy. The data were analyzed by ANOVA, the gross energy values were transformed, and the Kruskal–Wallis test was used for their analysis. ns indicates not significant, P < 0.01, *P < 0.001, n = 8.

Effect of IAA-Na supplementation on the activities of digestive enzymes in the jejunal mucosa of weaned piglets. Quantitative analysis of the activities of digestive enzymes in the jejunal mucosa, including A) sucrase activity, B) maltase activity, and C) ALP activity. The data were analyzed by ANOVA, the maltase activity values were transformed, and the Kruskal–Wallis test was used for their analysis. The Welch’s test was used for alkaline phosphatase activity analysis. ns indicates not significant and *P < 0.05, n = 8.

Effect of dietary IAA-Na on the mRNA expression of jejunal nutrient transporters in weaned piglets. Quantitative analysis of the mRNA expression of nutrient transporters in the jejunal mucosa, namely, A) glucose transporters, B) neutral amino acid transporters, C) cationic amino acid transporters, D) anionic AA transporters, and E) peptide transporters. The data were analyzed by ANOVA, and Welch’s test was performed in the relative mRNA expression of SLC7A1 and SLC38A5 analyses, and the Brown–Forsythe test was performed in IL-22 analysis. SLC5A1, also known as SGLT1, solute carrier family 5 member 1; SLC2A2, also known as GLUT2, solute carrier family 2 member 2; SLC6A19, solute carrier family 6 member 19; SLC38A5, solute carrier family 38 member 5; SLC7A1, solute carrier family 7 member 1; SLC7A9, solute carrier family 7 member 9; SLC1A1, solute carrier family 1 member 1; SLC15A1, solute carrier family 15 member 1. ns indicates not significant, *P < 0.05, P < 0.01, n = 8.

Effect of dietary supplementation with IAA-Na on the number of proliferating cells and secretory cell lineages in the jejunum of weaning piglets. Quantitative analysis of cell proliferation and differentiation in the jejunum is shown in A–E) (ANOVA was performed). F) Representative images of cell proliferation, G) enteroendocrine cells, and H) goblet cells are shown. EECs, enteroendocrine cells; GCs, goblet cells. ns indicates not significant, *P < 0.05, P < 0.01, *P < 0.001. Scale bar = 200 μm (magnification 100×), n = 8.

Jejunal organoid growth activity in piglets treated with IAA-Na. A) The organoid budding rate, B) number of crypts per organoid, and C) crypt depth were determined by ANOVA, and the Welch’s test was used for the crypt depth of organoids. D) Representative images of jejunal organoids. ns indicates not significant, *P < 0.05, P < 0.01. Scale bar, 100 μm (magnification 50×), n = 3.

Transcriptome analysis and related mRNA expression levels in the jejunal mucosa of piglets fed dietary IAA-Na. A) Scatter plot of differentially expressed genes (DEGs) in jejunal mucosa between control and 240 mg/kg IAA-Na-treated piglets. B) Heatmap analysis of DEGs between control and 240 mg/kg IAA-Na-treated piglets. C) GO enrichment analysis of DEGs between control and 240 mg/kg IAA-Na-treated piglets. D) DEGs enriched in KEGG pathways between control and 240 mg/kg IAA-Na-treated piglets. E) Heatmap analysis of upregulated (red) or downregulated genes (blue) related to inflammatory cytokines, F) nutrient transporters, and H) IL-17 and I) TNF signaling pathways in the jejunum. G and J) GSEA of KEGG pathways in 240 mg/kg IAA-Na-treated piglets compared with control piglets. GO, Gene Ontology. n = 3.

Effect of IAA-Na on the expression of intestinal inflammatory cytokines of piglets. A–F) The contents of intestinal inflammatory cytokines of piglets were normalized by the protein concentration of each sample. G–L) Relative mRNA expression levels of intestinal inflammatory cytokines in piglets. The data were analyzed by ANOVA. IL-17, interleukin 17; IL-17R, interleukin 17 receptor; TGF-β, transforming growth factor-β; TNF-α, tumor necrosis factor α; IL-10, interleukin 10; IL-22, interleukin 22; IL-10RB, interleukin 10 receptor subunit beta. ns indicates not significant, *P < 0.05, P < 0.01, *P < 0.001. n = 8.

Tables

Table 1. Effect of dietary IAA-Na on the growth performance of weaned piglets.1

Items	Dietary IAA-Na, mg/kg			SEM	P-value
	0	120	240
BW, kg
Day 0	6.58	6.58	6.58	0.07	0.999
Day 28	10.24	10.53	9.86	0.23	0.496
ADG, g
Day 0-14	43.30	82.14	55.81	8.13	0.149
Day 15-28	218.31	229.59	178.13	11.16	0.140
Day 0-28	130.80	155.87	116.97	7.53	0.107
ADFI, g
Day 0-14	228.3	280.6	282.0	10.87	0.090*
Day 15-28	500.6	528.8	484.4	18.52	0.645
Day 0-28	364.5	404.7	383.2	12.04	0.423
F: G, g/g
Day 0-14	3.58	4.00	5.00	0.34	0.231
Day 15-28	2.48	2.34	2.54	0.08	0.639
Day 0-28	2.68	2.67	3.29	0.14	0.122

Table 2. Effect of dietary IAA-Na on diarrhea in weaned piglets.1

Items	Dietary IAA-Na, mg/kg			SEM	P-value
	0	120	240
Fecal score
Day 0-14	1.9	1.9	1.5	1.55	0.235
Day 15-28	1.4	1.7	1.3	0.07	0.099*
Day 0-28	1.7	1.8	1.4	0.07	0.084*
Diarrhea rates,2 %
Day 0-14	30.36	33.93	17.86	3.86	0.209
Day 15-28	14.29	22.32	9.82	2.74	0.859
Day 0-28	22.32	28.13	13.84	2.80	0.107

Table 3. Effect of dietary IAA-Na on the organ indices of weaned piglets.1

Items2	Dietary IAA-Na, mg/kg			SEM	P-value
	0	120	240
Liver
Total weight, g	245.9	278.9	267.9	7.76	0.234
Relative weight, g/kg	24.03b	26.41a	26.35a	0.43	0.025
Spleen
Total weight, g	23.7	26.6	28.3	1.23	0.313
Relative weight, g/kg	2.31	2.52	2.87	0.11	0.080*
Double kidney
Total weight, g	64.8b	71.8a,b	75.6a	1.87	0.049
Relative weight, g/kg	6.35b	6.86	7.66a	0.15	<0.001
Small intestine
Total weight, g	503.6	567.5	497.6	17.16	0.251
Relative weight, g/kg	49.03	53.93	49.97	1.03	0.109
Total length, m	11.5	12.1	11.3	0.28	0.518
Relative length, m/kg	1.12	1.11	1.14	0.02	0.843
Large intestine
Total weight, g	169.8	178.8	170.4	6.01	0.809
Relative weight, g/kg	16.58	16.96	17.95	0.34	0.273
Total length, m	2.1	2.0	2.1	0.03	0.474
Relative length, m/kg	0.21a	0.18b	0.21a	0.00	0.013

Full text

Introduction

Weaning is a critical period in swine production that directly impacts economic benefits. However, piglets are susceptible to environmental, physiological, and nutritional stresses. These factors can impair intestinal morphology and mucosal function, reduce digestion and absorption capacity, and disrupt intestinal barriers. This can lead to an increased diarrhea rate, gut microbiota disturbance, and poor growth performance (Moeser et al 2017; Tang et al 2022a). Consequently, significant efforts are being made to develop nutritional regulation strategies that alleviate weaning stress and improve intestinal function and growth performance in piglets. The gut is a complex ecosystem inhabited by diverse microbial communities, collectively termed the intestinal microbiota. This microbiota plays a crucial role in host physiology through metabolites produced by microbes or derived from the host (Agus et al 2018; Zhang et al 2021). Mounting evidence suggests that intestinal microbial metabolites are critical mediators of diet-induced host-microbiota crosstalk. Tryptophan metabolites have received growing attention for their essential regulatory roles in intestinal immune homeostasis (Dodd et al 2017).

Intestinal tryptophan metabolism follows three major pathways: 1) direct conversion by intestinal microbiota into indole and its derivatives, including indole propionic acid (IPA), indole-3-acetic acid (IAA), indoleacrylic acid (IA), and indole-3-aldehyde (I3A). These metabolites are ligands for the aryl hydrocarbon receptor (AHR); 2) the serotonin pathway through tryptophan hydroxylase 1 (TPH1) in enterochromaffin cells; and 3) the kynurenine pathway through indoleamine 2,3-dioxygenase 1 (IDO1) in epithelial and immune cells (Agus et al 2018). These metabolites affect various physiological processes. They bind to the AHR, playing an anti-inflammatory role, activating the immune system, strengthening intestinal epithelial barrier function, secreting gut hormones, stimulating gastrointestinal motility, exerting antioxidative effects, and regulating the intestinal flora (Roager and Licht 2018). Although IAA is classically recognized as a plant growth hormone, it is also an important microbial-derived indole metabolite produced by intestinal microbiota. There, it plays a role in regulating host intestinal function and immune responses. Furthermore, IAA has been found to improve the efficacy of chemotherapy for human pancreatic cancer in gnotobiotic mouse models through a mechanism involving neutrophil-derived myeloperoxidase (Tintelnot et al 2023). Additionally, orally administered IAA has been shown to alleviate steatosis and nonalcoholic steatohepatitis in mice (Ding et al 2024), reduce intestinal inflammation, restore barrier function, and correct colonic microbiota disturbance in mice challenged with Salmonella typhimurium (Fan et al 2024). Moreover, IAA reduces dextran sulfate sodium salt (DSS)-induced colitis by increasing the abundance of Bifidobacterium pseudopodium in mice (Li et al 2024). Besides, IAA reportedly plays a significant role in regulating gut motility through the AHR signaling pathway in mice (Chen et al 2023). A previous study demonstrated that IAA-Na improved the laying performance of Danzhou chickens (Chen et al 2021). However, the effect of IAA, an intestinal microbiota-derived tryptophan metabolite, on the intestinal health of piglets has yet to be reported. We hypothesize that IAA improves intestinal health in weaning pigs primarily through enhancing intestinal morphology, promoting nutrient absorption, and modulating intestinal immune responses.

Intestinal health is closely associated with intestinal morphology, epithelial renewal, nutrient transport, and immune balance (Tang et al 2022a). The structural integrity of the intestine is essential for the efficient digestion and absorption of nutrients in piglets. Intestinal epithelium renewal is driven by intestinal stem cells (ISCs) and depends on their proliferation and differentiation. Efficient nutrient absorption is mediated by specific transporters, such as glucose transporters (SGLT1 and GLUT2) and amino acid (AA) and peptide transporters (PEPT1 and neutral AA transporters). Meanwhile, intestinal immune homeostasis is maintained by balancing pro-inflammatory (such as IL-1β, TNF-α, and IL-17) and anti-inflammatory (such as IL-10, IL-22, and TGF-β) cytokines, which collectively regulate inflammatory responses, epithelial integrity, and host-microbiota interactions. This study aimed to evaluate the effects of dietary IAA-Na on intestinal morphology, epithelial renewal, nutrient absorption, and inflammation in weaned piglets. These findings provide a theoretical basis for the application of IAA to improve intestinal health in piglet production.

Materials and methods

Animal welfare statement

All animal experimental procedures were approved by the Animal Care and Use Committee of Hunan Normal University (Changsha, Hunan, China, approval number 2020-039).

Animal experimental design

Twenty-four crossbred boar piglets (Duroc × Yorkshire × Landrace, weaned on d 21) were randomly assigned to three treatments according to their similar initial body weights (IBW) of 6.58 ± 0.07 kg. Each group had eight piglets. This study used a fully randomized block design with eight experimental units per group. Pigs were labeled from 1 to 24, sorted, and divided into three groups (named A, B, and C) via Excel according to body weights. Then, adjustments were made to ensure a similar average body weight in each group. Blinding was used throughout the experimental process, which was performed by different independent investigators. Previous studies have demonstrated that orally administering 20 or 40 mg/kg IAA to mice alleviates intestinal inflammation and maintains intestinal epithelial homeostasis (Wang et al 2024a). In our preliminary trial, we added 10, 20, and 50 mg/kg IAA-Na to the drinking water of Sprague-Dawley rats. We found that 10 mg/kg IAA-Na increased the rats’ average daily gain (ADG, unpublished data). Therefore, IAA-Na (S18032, 98% high purity, Shanghai Yuanye Bio-Technology Co., Ltd., Shanghai, China) was added to the basal diet at concentrations of 120 and 240 mg/kg. All the piglets were raised individually, and they had access to food and water. The experimental unit was the piglet. Pigs were individually housed to measure individual feed intake, body weight, diarrhea incidence, intestinal morphology, nutrient absorption, and the inflammatory responses of each piglet to dietary IAA-Na supplementation. Room temperature was maintained at around 28 °C using lamps. The diet formulation (see Table S1) satisfied the piglets’ nutritional requirements, which were divided into two phases and changed on d 15. The nutrition levels were analyzed and provided. Feed intake, body weight, and diarrhea were recorded and calculated throughout the 28-d experiment, which was conducted at the Yong’an Branch Pig Farm of Hunan New Wellful Co., Ltd. Pigs used to establish intestinal organoids were separate from the 24 piglets involved in the dietary IAA feeding experiment. The donor piglets were of the same breed, age, and housing conditions as the experimental pigs, but they were euthanized specifically for the isolation of ISCs.

Fecal score

During the experimental period, the diarrhea of the piglets was recorded five times a day at 8:00, 11:00, 14:00, 17:00, and 20:00. The health status and behavioral observations of the pigs were also recorded at these times. Health status assessments included daily monitoring of clinical signs of illness, such as diarrhea, lethargy, abnormal respiration, and reduced feed intake, as well as mortality. Behavioral observations included general activity, posture, social interaction, and feeding behavior to identify signs of discomfort or abnormal behavior. Two independent investigators scored fecal consistency blindly according to a 5-point scale (1 = dry and hard stool, 2 = wet stool, 3 = mild diarrhea, 4 = severe diarrhea, and 5 = watery diarrhea). A stool score of 3 or higher indicated diarrhea (Trevisi et al 2011).

Sample collection

Fecal samples were collected over three consecutive days prior to the end of the experiment. The collected samples were then treated with 10% tartaric acid for nitrogen fixation. Piglets were administered a 4% sodium pentobarbital solution (40 mg/kg BW) via jugular vein injection to induce general anesthesia and were then euthanized. The abdominal cavity was opened immediately, and the internal organs were removed. The small intestine was separated from the large intestine. After removing the intestinal contents, the weight and length of the intestine were measured. The other organs were weighed to calculate the relative organ indices. After removing the mesentery and fat, the following were excised: the 3-cm proximal duodenum, the middle jejunum, the terminal ileum, the cecum, and the colon. They were rinsed with physiological saline to clear the intestinal contents and were quickly fixed in 4% neutral-buffered formalin for further experiments. An adjacent 5 cm jejunal segment was opened longitudinally and rinsed gently with physiological saline. The mucosa was carefully scraped with a sterile glass slide. The collected jejunum mucosal samples and the 2-cm duodenum, jejunum, ileum, cecum, and colon were wrapped in tin foil and immediately snap-frozen in liquid nitrogen. They were then stored in a −80 °C refrigerator until analysis.

Intestinal morphology analysis

All intestinal samples were embedded in paraffin, sectioned (4 μm), stained with hematoxylin and eosin (H&E), and photographed using an optical microscope (Leica Microsystems, Germany). Two independent investigators measured the images in a blinded manner using Image-Pro Plus version 6.0 (Media Cybernetics, San Diego, CA, USA). Each value was calculated as the mean of 30 complete villus-crypt structures.

Determination of apparent total tract digestibility

The calculated nutritional levels of the feed were derived from data provided by the China Feed Database (Tian et al 2023). Apparent total tract digestibility (ATTD) analyses of crude ash, dry matter (DM), crude protein (CP), and gross energy (GE) were conducted in duplicate using the acid-insoluble ash method (Mccarthy et al 1974). In this method, acid-insoluble ash serves as an indigestible marker. Feed and fecal samples were analyzed for crude ash (method 942.05), CP (method 976.06), and DM (method 930.15) according to the AOAC (2007) guidelines. The DM content of the feed was determined by weighing approximately 2.00 g of the sample, drying it at 105 °C for 12 h until it reached a constant weight, and calculating the weight change. The CP was measured using an automatic determination of nitrogen analyzer KDN-103F (Shanghai Xianjian Instruments Co., Ltd., China) after digesting the dried samples. The GE in diets and feces was detected using benzoic acid as the calibration standard in an isothermal automatic calorimeter (5E-AC8018, Changsha Kaide Instrument Co., Ltd., China). The ATTD (%) of nutrients was calculated using the following equation (Wang et al 2024b): ATTD (%) = [(Nutrients_in—Nutrients_out)/Nutrients_in_] × 100, where Nutrients_in_ and Nutrients_out_ represent the nutrient intake in the diet and output in feces, respectively, both in grams on a DM basis. The ATTD of DM, CP, GE, and crude ash was determined based on the chemical analysis of the actual experimental diets and fecal samples collected in this study, rather than using database values. All experimental diets were prepared in a single batch for each treatment to minimize variation between batches. Representative samples of each diet were collected during feed preparation and analyzed for nutrient composition. The analyzed nutrient values were then used to calculate ATTD. Therefore, differences in ATTD among treatments were attributed to dietary IAA-Na supplementation rather than to differences in ingredient composition. Digestibility calculations were performed separately for each dietary treatment.

Digestive enzyme activity analysis

For the enzyme activity assay, the jejunal mucosa of the piglets was ground into a powder and homogenized with 0.9% NaCl solution. Then, it was centrifuged, and the resulting supernatant was collected for further analysis. Enzyme activities, such as those of alkaline phosphatase (ALP, A059-2-2), maltase (Nanjing Jiancheng Bioengineering Institute, Nanjing, China, A082-3-1), and sucrase (Suzhou Comin Biotechnology Co., Ltd., Suzhou, China, ZTM-1-Y), were detected with commercial kits according to the manufacturers’ instructions. All standards and samples were run in duplicate. Enzyme activity values were normalized to the total protein content, which was determined using a protein assay kit (Beyotime Biotechnology Co., Ltd., China, P0398S).

RNA extraction and RT-qPCR

Total RNA was extracted from the jejunal mucosa as previously described (Yin et al 2023). Complementary DNA (cDNA) was generated using the RT Reagent Kit and gDNA Eraser (TaKaRa, Dalian, China, RR047A) according to the manufacturer’s protocol. Real-time quantitative PCR (RT-qPCR) analysis was conducted using TB Green^®^ Premix Ex Taq™ (TaKaRa, RR420A), and all samples were run in triplicate. Relative mRNA expression was normalized to the expression of the internal control β-actin. The primers used for RT-qPCR are listed in Table S2.

Immunohistochemistry analysis

Jejunal sections were deparaffinized with xylene and gradually rehydrated in ethanol. Endogenous peroxidase was blocked with 3% H_2_O_2_ in methanol. Then, the slides were boiled in 0.01 M sodium citrate buffer (pH = 6) at 100 °C for 10 min for antigen retrieval. Then, the intestinal sections were incubated with 5% BSA at 37 °C for 30 min to block nonspecific binding sites. Then, the slides were incubated with a primary antibody at 37 °C for 1 h, washed with PBS, and incubated with a secondary antibody (ZSGB-BIO, Beijing, China, PV-9001) at 37 °C for 30 min. Positive cells were visualized using a diaminobenzidine (DAB) kit (ZSGBBIO, Beijing, China, ZLI-9018). The slides were then stained with hematoxylin, gradually dehydrated, and cover-slipped for further analysis. Thirty well-oriented crypt-villus structures were randomly selected by two independent investigators for counting the number of positive cells. The antibodies used for immunohistochemistry were as follows: Ki67 (a cell proliferation marker; Abcam, ab15580; diluted 1:800) and Chromogranin A (ChgA, an enteroendocrine cell marker; Abcam, ab45179; diluted 1:600). These antibodies were used to evaluate intestinal epithelial cell renewal.

Alcian blue-periodic acid-Schiff staining

Goblet cells were detected with an Alcian Blue-Periodic Acid-Schiff (AB-PAS) kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China, D033-1-2) according to the manufacturer’s instructions. In brief, the jejunal sections were deparaffinized, rehydrated, washed with distilled water, stained with Alcian blue dye, and washed with distilled water. Then, they were stained with periodic acid dye, washed again with distilled water, stained with Schiff reagent, washed again with distilled water, dried, and cover-slipped for observation and calculation. The number of goblet cells in at least 30 villi and crypts was counted for each piglet.

Piglet jejunal organoid culture and treatment

The ISCs used for organoid culture were isolated from the jejunum of healthy piglets (Duroc × Yorkshire × Landrace, weaned at d 21). These piglets were not part of the current in vivo feeding trial. The donor piglets were housed under standard conditions and euthanized for reasons unrelated to this study. The ISCs, which are located at the base of the crypts, were isolated, embedded in Matrigel (Corning, 356231), cultured, and passaged as previously described (Yin et al 2022). Briefly, a 3-cm segment of the proximal jejunum was separated immediately and rinsed with ice-cold PBS (Meilunbio) to clear the intestinal contents. The segments were then cut into 2- to 3-mm-thick sections, washed several times with cold PBS, and incubated with ethylene diamine tetraacetic acid (EDTA; Sigma, E9884) on a rotator at 4 °C for 1 h. The supernatant containing crypts was collected after incubation, filtered through a 100-μm cell strainer (BD, 352360), and spun at 150× g for 5 min. The isolated crypt pellets were suspended in Matrigel and embedded in a prewarmed 24-well plate. After the Matrigel solidified, we added 500 μL of intestinal organoids culture medium to each well. This medium was supplemented with Wnt3a, R-spondin1, or Noggin (WRN)-conditioned medium, DMEM/F12 (Gibco, 10565018), 100 U/mL penicillin, 100 mg/mL streptomycin (Gibco, 15140122), 10 mM HEPES (Gibco, 15630080), 1× GlutaMAX Supplement (Gibco, 35050061), B27 supplement (1:50; Gibco, 17504044), N2 supplement (1:100; Gibco, 17502048), 50 ng/mL EGF (Sigma, SRP3196), 1 mM N-acetyl cysteine (MCE, HY-B0215), 10 mM nicotinamide (Sigma, 481907), 0.5 μM A8301 (TGF-β inhibitor; Tocris, 2939), 3 μM SB202190 (p38 MAPK inhibitor; Sigma, S7067), and 2.5 μM CHIR99021 (GSK3β inhibitor; Sigma, SML1046). Porcine intestinal organoids from multiple donors were pooled, expanded, and cryopreserved. Primary organoids at passages 3–5 were cryopreserved in a cell freezing medium and stored in liquid nitrogen for long-term preservation. For the present study, the vial was rapidly warmed in a 37 °C water bath, resuspended in DMEM/F12 medium, and then centrifuged to remove the cell freezing medium. The organoids were embedded in Matrigel. The thawed organoids were allowed to recover and expand for two passages to ensure stable growth and phenotypic consistency before treatment. According to previous studies, IAA is detected in human serum and feces of healthy adults at the mean concentrations of 1.3 μM and 5 μM, respectively (Roager and Licht 2018). Consequently, different concentrations of IAA-Na (0.5, 2, and 5 μM) were added to the organoids culture medium. Photos were taken on day 3 after treatment, and organoid activity was measured by two independent investigators using Image-Pro Plus version 6.0 (Media Cybernetics, San Diego, California, USA) in a blinded manner. The organoid budding rate was calculated as the ratio of budding organoids to total organoids. Organoids touching the edge of the images were excluded from the analysis, and the statistical analyses of budding rate, crypt depth (CD), and crypt domains per organoid were based on individual wells (Lindemans et al 2015).

Transcriptomic sequencing and analysis

To identify differentially expressed genes (DEGs) between the control (0 mg/kg IAA-Na) and high-dose (240 mg/kg IAA-Na) groups, transcriptomic analysis was conducted using jejunal mucosa samples from piglets. Total RNA was extracted, its quality was verified, libraries were prepared, and sequencing was conducted at Shanghai Majorbio Biopharm Biotechnology Co., Ltd. (Shanghai, China). Clean reads were aligned to the reference genome. Differential expression analysis was performed using DESeq2, a P-adjusted value ≤ 0.05 indicated significant differential expression. Functional enrichment analysis of the DEGs (|fold change| ≥ 2; P-adjusted value ≤ 0.05) was performed, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Gene set enrichment analysis (GSEA) was conducted on all gene sets included in the present study.

Inflammatory cytokines of the jejunal mucosa measurement

The homogenized intestinal tissues of pigs were centrifuged at 4 °C, 5000× g for 10 min. The concentrations of the following cytokines in the jejunal mucosa were detected with enzyme-linked immunosorbent assay (ELISA) kits (JianglaiBio, Shanghai, China): IL-17A (JL21870), IL-17F (JL21872), TNF-α (JL13203), TGF-β (JL22001), IL-10 (JL21866), and IL-22 (JL21877). The assays were performed according to the manufacturers’ instructions. The intra- and inter-assay coefficients of variation for all assays were less than 10%, as specified by the manufacturer. The inflammatory cytokine content was normalized to the protein concentration of each sample.

Statistical analysis

The data were analyzed with IBM SPSS Statistics version 20 (SPSS Inc., Chicago, IL, USA). Blinding was used during data collection and analysis, which were performed by different investigators. We performed a residual normality test of data distribution with the Shapiro-Wilk test. One-way ANOVA was used for all statistical analyses to test for differences when the data followed a normal distribution and homogeneity of variance. Differences between groups were estimated via Duncan’s multiple comparisons (LSD). If the residuals were not normally distributed, the values were transformed before statistical analysis. Nonparametric tests (Kruskal-Wallis tests) were performed when a normal distribution could not be assumed. If the homogeneity of variance could not be satisfied, Welch’s test or Brown-Forsythe test was performed, and differences between groups were estimated by the Games-Howell test. Data are expressed as the mean and standard error of the mean (SEM). Differences with *P *< 0.05 were considered statistically significant, while 0.05 ≤ *P *< 0.10 indicates a trend toward significance. *, **, and *** indicate P values < 0.05, < 0.01, and < 0.001, respectively, while “ns” indicates “not significant.”

Results

Growth performance

Table 1 displays the growth performance of the piglets. No significant differences in growth performance were observed during the experimental period (*P *> 0.05). However, compared to the control group, dietary supplementation with 120 mg/kg IAA-Na resulted in higher, though not statistically significant, ADFI and ADG during the first two weeks, with increases of 47.29% and 18.62%, respectively. The fecal score decreased more in the 240 mg/kg IAA-Na group than in the 120 mg/kg IAA-Na group from days 15 to 28 and days 0 to 28 (Table 2, *P *< 0.10). Collectively, these findings suggest that dietary supplementation with IAA-Na did not significantly improve growth performance or reduce diarrhea in piglets.

Organ index

Table 3 shows the effect of dietary IAA-Na on the organ indices of pigs. Compared to the control diet, a diet containing 240 mg/kg IAA-Na increased the relative weights of the liver (*P *< 0.05), spleen (*P *< 0.10), and kidney (*P *< 0.001). The relative length of the large intestine decreased significantly in the 120 mg/kg IAA-Na group (*P *< 0.05). Overall, these results suggest that dietary IAA-Na stimulated intestinal and organ development.

Intestinal morphology

To evaluate the effect of dietary IAA-Na on intestinal morphology, the morphology of the small and large intestines was examined. No significant differences were observed in the ileum, cecum, or colon (see Table S3, *P *> 0.05). Compared to the control diet, a diet containing 240 mg/kg IAA-Na significantly reduced the duodenal CD (see Table S3, *P *< 0.05). Compared to the 120 mg/kg group, the 240 mg/kg group tended to have increased jejunal villus height of pigs (VH; Figure 1A, *P *< 0.10). Compared to the 0 and 120 mg/kg groups, the 240 mg/kg group had increased the jejunal villus width (VW; Figure 1C, *P *< 0.05) and villus surface area of pigs (VSA; Figure 1E, *P *< 0.10). Representative images are presented in Figure 1F. In conclusion, these findings suggest that dietary IAA-Na improved the morphology of the jejunum in weaned piglets.

*Intestinal morphology of weaned piglets fed with different IAA-Na concentrations. The results of the quantitative analysis of jejunum morphology are shown as the A) villus height, B) crypt depth, C) villus width, D) VH: CD, E) and villus surface area (ANOVA was performed). F) Representative images of jejunal morphology. VH: CD, ratio of villus height: crypt depth. ns indicates not significant and *P < 0.01. scale bar = 200 μm (magnification 100×), n = 8.

Apparent nutrient digestibility and digestive enzyme activities

To evaluate the effect of dietary IAA-Na on nutrient absorption, we measured apparent nutrient digestibility and intestinal digestive enzyme activities. The apparent digestibility of nutrients in the piglets is presented in Figure 2. Apparent digestibility increased significantly in the 240 mg/kg IAA-Na group for DM (Figure 2A, *P *< 0.001), CP (Figure 2B, *P *< 0.01), crude ash (Figure 2C, *P *< 0.001), and GE (Figure 2D, *P *< 0.001). Digestive enzyme activities in the jejunal mucosa are displayed in Figure 3. Compared to the control group, the dietary intake of 120 mg/kg IAA-Na significantly increased the sucrase activity in weaned piglets (Figure 3A, *P *< 0.05). No significant differences in maltase or ALP activity were detected (Figure 3C and D, *P *> 0.05). In conclusion, these findings suggest that dietary IAA-Na increased intestinal nutrient absorption in weaned piglets.

*Effect of dietary IAA-Na on the apparent total tract digestibility of nutrients in weaned piglets. Quantitative analysis of apparent total tract digestibility of nutrients shown in A) dry matter, B) crude protein, C) crude ash, and D) gross energy. The data were analyzed by ANOVA, the gross energy values were transformed, and the Kruskal–Wallis test was used for their analysis. ns indicates not significant, **P < 0.01, **P < 0.001, n = 8.

*Effect of IAA-Na supplementation on the activities of digestive enzymes in the jejunal mucosa of weaned piglets. Quantitative analysis of the activities of digestive enzymes in the jejunal mucosa, including A) sucrase activity, B) maltase activity, and C) ALP activity. The data were analyzed by ANOVA, the maltase activity values were transformed, and the Kruskal–Wallis test was used for their analysis. The Welch’s test was used for alkaline phosphatase activity analysis. ns indicates not significant and P < 0.05, n = 8.

Relative mRNA expression of nutrient transporters

To further explore the effect of dietary IAA-Na on nutrient absorption, the expression of relevant genes of nutrient transporters was measured. Figure 4 illustrates the effect of dietary IAA-Na on the mRNA expression of nutrient transporters. The relative mRNA expression of the glucose transporters, GLUT2 and SGLT1, was significantly higher in the 240 mg/kg IAA-Na treatment group than in the control group (Figure 4A, *P *< 0.01). The relative mRNA expression of the neutral AA transporters, SLC6A19 (Figure 4B, *P *< 0.10) and SLC38A5 (Figure 4B, *P *< 0.05), increased in response to the 120 mg/kg IAA-Na treatment. The expression of SLC7A9, a cationic AA transporter, was significantly upregulated (Figure 4C, *P *< 0.05) in the 240 mg/kg IAA-Na treatment group. Dietary intake of 240 mg/kg IAA-Na significantly upregulated the relative mRNA expression levels of the intestinal anionic AA transporter SLC1A1 (Figure 4D, *P *< 0.05) and the peptide transporter SLC15A1 (Figure 4E, *P *< 0.01). Overall, these findings suggest that high dietary concentrations of IAA-Na promoted the mRNA expression of intestinal nutrient transporters.

*Effect of dietary IAA-Na on the mRNA expression of jejunal nutrient transporters in weaned piglets. Quantitative analysis of the mRNA expression of nutrient transporters in the jejunal mucosa, namely, A) glucose transporters, B) neutral amino acid transporters, C) cationic amino acid transporters, D) anionic AA transporters, and E) peptide transporters. The data were analyzed by ANOVA, and Welch’s test was performed in the relative mRNA expression of SLC7A1 and SLC38A5 analyses, and the Brown–Forsythe test was performed in IL-22 analysis. SLC5A1, also known as SGLT1, solute carrier family 5 member 1; SLC2A2, also known as GLUT2, solute carrier family 2 member 2; SLC6A19, solute carrier family 6 member 19; SLC38A5, solute carrier family 38 member 5; SLC7A1, solute carrier family 7 member 1; SLC7A9, solute carrier family 7 member 9; SLC1A1, solute carrier family 1 member 1; SLC15A1, solute carrier family 15 member 1. ns indicates not significant, *P < 0.05, *P < 0.01, n = 8.

Intestinal epithelium cell proliferation and differentiation

To investigate the impact of dietary IAA-Na on intestinal epithelial cell renewal, we measured the proliferation and differentiation in the jejunum. As is illustrated in Figure 5, dietary supplementation with IAA-Na significantly increased the number of proliferating cells in the crypts of weaned piglets (Figure 5A, *P *< 0.001). Dietary IAA-Na significantly reduced the number of enteroendocrine cells per villus (Figure 5B, *P *< 0.01). Additionally, 120 mg/kg IAA-Na tended to increase the number of enteroendocrine cells per crypt compared to the control group (Figure 5C, *P *< 0.10). Compared to the 0 and 120 mg/kg groups, the 240 mg/kg group had fewer goblet cells in the villi (Figure 5D, *P *< 0.10) and significantly decreased the number of goblet cells in the crypts (Figure 5E, *P *< 0.05). Representative images are presented in Figure 5F–H. In conclusion, these findings suggest that dietary supplementation with IAA-Na promoted intestinal epithelial cell proliferation and reduced the number of secretory cell lineages.

*Effect of dietary supplementation with IAA-Na on the number of proliferating cells and secretory cell lineages in the jejunum of weaning piglets. Quantitative analysis of cell proliferation and differentiation in the jejunum is shown in A–E) (ANOVA was performed). F) Representative images of cell proliferation, G) enteroendocrine cells, and H) goblet cells are shown. EECs, enteroendocrine cells; GCs, goblet cells. ns indicates not significant, *P < 0.05, **P < 0.01, **P < 0.001. Scale bar = 200 μm (magnification 100×), n = 8.

Intestinal organoid growth activity in vitro

To further explore the influence of IAA-Na on intestinal epithelial cell renewal, different concentrations of IAA-Na were added to the jejunal organoid culture medium to measure stem cell activity in vitro. As shown in Figure 6, 0.5 and 2 μM IAA-Na significantly increased the organoid budding rate on day 3 compared to that of the control (Figure 6A, *P *< 0.01). The CD was shorter in the 2 μM IAA-Na treatment group than in the control group (Figure 6C, *P *< 0.05). Representative images are presented in Figure 6D. These results indicate that low concentrations of IAA-Na promoted the growth of piglets’ jejunal organoids.

*Jejunal organoid growth activity in piglets treated with IAA-Na. A) The organoid budding rate, B) number of crypts per organoid, and C) crypt depth were determined by ANOVA, and the Welch’s test was used for the crypt depth of organoids. D) Representative images of jejunal organoids. ns indicates not significant, *P < 0.05, *P < 0.01. Scale bar, 100 μm (magnification 50×), n = 3.

Transcriptome and inflammatory responses analysis of piglet jejunal mucosa

To investigate whether high levels of IAA-Na improved nutrient absorption capacity, we performed an RNA-sequencing analysis of piglet jejunal mucosa. We compared piglets fed a basal diet (control, 0 mg/kg IAA-Na) with those fed a high-IAA-Na diet (240 mg/kg IAA-Na) to identify DEGs. A scatter plot of the DEGs revealed 59 upregulated genes and 36 downregulated genes (Figure 7A). Cluster analysis showed that IAA-Na significantly altered the transcriptomic profile of the piglets’ intestines (Figure 7B). Functional enrichment analysis showed that the DEGs were primarily enriched in biological processes, such as “inflammatory response” and “immune system process,” as well as molecular functions, including “transporter activity” (Figure 7C). KEGG pathway analysis suggested that the DEGs were enriched in signaling pathways, including the “IL-17 signaling pathway” and “PPAR signaling pathway” (Figure 7D). GSEA revealed that the IL-17 signaling pathway (Figure 7G) and the TNF signaling pathway (Figure 7J) were downregulated in the intestines of piglets treated with high concentrations of IAA-Na. Additionally, heatmap analysis demonstrated that dietary supplementation with 240 mg/kg IAA-Na substantially inhibited the expression of related inflammatory cytokines (Figure 7E) and promoted the expression of nutrient transporters (Figure 7F). Furthermore, heatmap analysis revealed that the expression levels of the IL-17 and TNF signaling pathway target genes (Figure 7H and I) were downregulated in the high IAA-Na group. Moreover, ELISA analysis demonstrated that dietary 240 mg/kg IAA-Na reduced the content of TNF-α (Figure 8C, *P *< 0.05) and increased anti-inflammatory cytokine production (TGF-β and IL-10; Figure 8D and E, *P *< 0.05). Additionally, IL-22 levels increased in pigs fed 120 mg/kg IAA-Na (Figure 8F, *P *< 0.10). Furthermore, RT-qPCR analysis revealed that a dietary intake of 240 mg/kg IAA-Na tended to decrease the relative mRNA expression of proinflammatory cytokines in the jejunum, especially IL-17 (Figure 8G, *P *< 0.10). The relative mRNA expression of anti-inflammatory cytokines, including TGF-β, IL-10, IL-22, and IL-10RB (Figure 8I–L), was significantly increased in piglets that received 240 mg/kg IAA-Na. In summary, these findings suggest that IAA-Na exerted anti-inflammatory effects and stimulated the expression of intestinal nutrient transporters.

Transcriptome analysis and related mRNA expression levels in the jejunal mucosa of piglets fed dietary IAA-Na. A) Scatter plot of differentially expressed genes (DEGs) in jejunal mucosa between control and 240 mg/kg IAA-Na-treated piglets. B) Heatmap analysis of DEGs between control and 240 mg/kg IAA-Na-treated piglets. C) GO enrichment analysis of DEGs between control and 240 mg/kg IAA-Na-treated piglets. D) DEGs enriched in KEGG pathways between control and 240 mg/kg IAA-Na-treated piglets. E) Heatmap analysis of upregulated (red) or downregulated genes (blue) related to inflammatory cytokines, F) nutrient transporters, and H) IL-17 and I) TNF signaling pathways in the jejunum. G and J) GSEA of KEGG pathways in 240 mg/kg IAA-Na-treated piglets compared with control piglets. GO, Gene Ontology. n = 3.

*Effect of IAA-Na on the expression of intestinal inflammatory cytokines of piglets. A–F) The contents of intestinal inflammatory cytokines of piglets were normalized by the protein concentration of each sample. G–L) Relative mRNA expression levels of intestinal inflammatory cytokines in piglets. The data were analyzed by ANOVA. IL-17, interleukin 17; IL-17R, interleukin 17 receptor; TGF-β, transforming growth factor-β; TNF-α, tumor necrosis factor α; IL-10, interleukin 10; IL-22, interleukin 22; IL-10RB, interleukin 10 receptor subunit beta. ns indicates not significant, *P < 0.05, **P < 0.01, **P < 0.001. n = 8.

Discussion

IAA, a metabolite of tryptophan metabolism, plays a critical role in maintaining intestinal homeostasis. Nevertheless, the intrinsic mechanism involved is still unclear. Here, we demonstrate that IAA significantly promotes intestinal nutrient digestion and absorption, thereby regulating the immune response. Consistent with its known roles as an AHR ligand and immunomodulator, we found that dietary IAA-Na supplementation significantly influenced intestinal nutrient absorption and modulated the immune response. These results largely align with our initial hypothesis.

IAA is a common and crucial natural auxin that is important for plant growth and development (Zhang et al 2022). As an indole derivative of tryptophan metabolism, IAA affects intestinal health and host immunity (Lamas et al 2018). Whether IAA affects the intestinal health of pigs remains unknown. One of the greatest challenges for piglets throughout their life is weaning, which can lead to diarrhea, intestinal epithelium damage, intestinal dysfunction, decreased brush border enzyme activity, growth retardation, and even death (Moeser et al 2017; Tang et al 2022a). There have been more efforts to develop nutritional strategies that alleviate weaning stress and improve the growth of weaned piglets. In the present study, we investigated the effects of dietary IAA-Na on the growth performance, intestinal digestion and absorption capacity, and intestinal epithelium renewal of weaned piglets. Dietary intake of 200 mg/kg IAA-Na improved the laying performance of Danzhou chickens (Chen et al 2021). IAA significantly reversed DSS-induced weight loss in mice (Wang et al 2024a). Although no significant differences were observed in IAA-Na piglets, those fed 120 mg/kg IAA-Na exhibited a numerical increase in ADFI (47.29%) and ADG (18.62%) during the initial 2 wk compared to controls, suggesting the potential of dietary IAA supplementation to improve piglet growth performance. Piglets were housed individually to allow for the measurement of dietary intake, which may reduce postweaning stress caused by social stressors, such as competition and aggression. Additionally, this setup allows for the assessment of the growth performance, intestinal development, and intestinal function of each pig in response to dietary IAA-Na supplementation. However, individual differences among the pigs existed in the individual housing setup. Therefore, large-scale group housing experiments with weaned piglets are necessary for future studies on the application of IAA in pig production.

Furthermore, dietary supplementation with 240 mg/kg IAA-Na tended to decrease the fecal score, which is consistent with the anti-inflammatory effects of tryptophan metabolites (Lavelle and Sokol 2020). Interestingly, dietary supplementation with IAA-Na increased the relative weights of the liver, spleen, and kidney, suggesting that it promoted organ development. These results indicate that dietary IAA plays a crucial role in promoting organ development in weaned piglets.

Intestinal morphology reflects the integrity of the intestinal epithelium, which guarantees intestinal function. Nutrient digestion and absorption primarily occur in the small intestine. Weaning stress impairs the intestinal structure, causing dysfunction in nutrient digestion and absorption. Generally, a reduction in VH suggests impaired intestinal mucosal function and reduced digestion and absorption in the intestine. Conversely, a greater intestinal VH and a lower CD value indicate better intestinal function (Tang and Xiong 2022a). To verify the role of IAA in intestinal epithelial integrity, we measured the morphology of the small and large intestines. A diet containing 240 mg/kg IAA-Na resulted in greater jejunal villus width and lower duodenal CD, suggesting that it could improve the intestinal morphology of weaned piglets.

Furthermore, ATTD was chosen as the primary measure of nutrient utilization in the present study because it is a well-established, non-invasive, and practical indicator of overall dietary nutrient availability under practical feeding conditions. ATTD also allows for the evaluation of the impact of dietary IAA-Na supplementation on the entire gastrointestinal tract. A diet containing 240 mg/kg IAA-Na increased the apparent digestibility of DM, CP, GE, and crude ash. Intestinal digestive enzymes modulate animal growth and development, improve feed digestibility, and regulate nutrient metabolism (Liu et al 2021). Small intestine disaccharidases (sucrase, maltase, and lactase) are the critical enzymes for piglet digestion and carbohydrate absorption (Tang et al 2022b). Disaccharidase activity in piglets decreased significantly after weaning (Marion et al 2005). Additionally, ALP is essential for nutrient uptake and transport in the intestine (Tang and Xiong 2022a). Early weaning decreased ALP activity in the small intestine of piglets (Lackeyram et al 2010), indicating that weaning stress impaired intestinal digestion and absorption. Therefore, small intestinal digestive enzyme activities are important indicators for evaluating digestive capacity and intestinal development in weaned pigs. Intestinal morphology is related to changes in digestive enzyme activities (Wang et al 2019). The present study revealed that dietary IAA-Na enhanced piglets’ jejunal mucosa sucrase activity, which was consistent with increased villus width and villus surface area.

Furthermore, the mRNA expression of the primary transporters of glucose, AAs, and small peptides in the jejunal mucosa was assessed. Upregulated expression of GLUT2, SGLT1, SLC38A5, SLC7A9, SLC1A1, and SLC15A1 was observed in piglets fed 240 mg/kg IAA-Na. In conclusion, high dietary concentrations of IAA improved jejunal morphology, increased nutrient digestion and absorption, and alleviated weaning stress.

The structural and functional integrity of the intestinal epithelium depends on the dynamic balance between proliferation and apoptosis (Tang and Xiong 2022b). As a self-renewing tissue, the intestinal epithelium is completely renewed every 3 to 5 d. This process is driven by ISCs located at the base of the crypts. Subsequently, these cells divide into progenitors and differentiate into mature functional intestinal epithelial cells, which perform primary intestinal functions. Accumulating evidence suggests that the expression of genes involved in cell proliferation and differentiation is downregulated in the jejunum of weaned piglets (Yang et al 2016), while the expression of genes associated with apoptosis is upregulated (Zhu et al 2014). In the present study, cell proliferation and differentiation were measured to evaluate change in the intestinal epithelium. In our current study, dietary IAA-Na promoted intestinal epithelial cell proliferation and reduced the differentiation of secretory cell lineages, including goblet cells and enteroendocrine cells. Goblet cells secrete mucus to protect the intestinal epithelium. Coadministration of IPA and IAA increased the number of colonic goblet cells in the DSS-induced colitis in mice (Wang et al 2024a). Additionally, IAA administration had no significant effect on the goblet cell number. Enteroendocrine cells constitute less than 1% of the epithelium and release hormones into the bloodstream upon stimulation (Gehart and Clevers 2019). Further experiments are needed to investigate the potential mechanism underlying the inhibition of secretory cell lineage differentiation after IAA administration, which may be due to the upregulation of intestinal transporters in absorptive intestinal epithelial cells. Enterocytes, which make up to 80% of the intestinal epithelium, are responsible for the selective uptake of ingested molecules, including ions, water, sugars, peptides, and lipids (Gehart and Clevers 2019). These findings suggest that dietary IAA promoted intestinal epithelium renewal in piglets.

Self-renewal in the intestine is driven by ISCs. Recently, intestinal organoids isolated from adult ISCs that contain crypt-villus structures and mature intestinal epithelial cells have been used as an in vitro model to evaluate the effects of nutrients on intestinal epithelial cell turnover and development (Yin et al 2023). In this study, IAA-Na was added to the organoid culture medium to investigate its effect on the growth activity of intestinal organoids in piglets. The organoid budding rate increased significantly after treatment with 0.5 and 2 μM IAA-Na on d 3; however, the CD decreased at 2 μM IAA-Na. There were more crypts per organoid in the 0.5 μM IAA-Na group than in the other groups on d 3. Organoid buds undergo ISCs expansion and new crypt formation via crypt fission (Yin et al 2021). A greater CD of organoids indicates rapid ISCs proliferation and differentiation, simulating the self-renewal of the intestinal epithelium along the crypt-villus axis (Yin et al 2019). Our study revealed that IAA stimulated the renewal of intestinal epithelial cells, and reduced CD may be related to the differentiation into the secretory cell lineage. These findings are consistent with those observed in the jejunum of piglets. This hypothesis needs to be verified. These results imply that IAA increased the growth of jejunal organoids.

Furthermore, IAA reportedly plays a significant role in regulating gut motility in mice through the AHR signaling pathway (Chen et al 2023). Additionally, IAA has been found to enhance the efficacy of chemotherapy for human pancreatic cancer in gnotobiotic mouse models via a mechanism involving neutrophil-derived myeloperoxidase (Tintelnot et al 2023). To investigate how high levels of IAA affect intestinal nutrient absorption, we compared the transcriptomic profiles of the jejunal mucosa between control and 240 mg/kg IAA-Na piglets using RNA sequencing. We observed a significant alteration in the intestinal transcriptomic profile, with notable enrichment of inflammatory response and immune system processes were significantly enriched in the piglet intestine. Furthermore, the KEGG pathway enrichment and GSEA analyses revealed that high dietary concentrations of IAA-Na downregulated inflammation-related pathways, including the IL-17 and TNF signaling pathways. Consistent with previous findings (Wang et al 2024a), Lactobacillus metabolizes tryptophan to indole-3-lactic acid, which leads to the synthesis of other indole derivatives, including IPA and IAA. L. reuteri administration also downregulated inflammation-related pathways, such as TNF and NF-κB signaling, as well as type 17 T helper (Th17) cell differentiation in mice with DSS-induced colitis. Furthermore, orally administered IAA relieved intestinal inflammation and restored barrier function in mice challenged with Salmonella typhimurium (Fan et al 2024). Additionally, IAA mitigated DSS-induced colitis by increasing the abundance of Bifidobacterium pseudopodium in mice (Li et al 2024). Indole and its derivatives promoted IL-22 expression and strengthened intestinal mucosa integrity (Wang et al 2024a). IAA reportedly acts as an AHR agonist that secretes IL-22, strengthening intestinal mucosa integrity in type 3 innate lymphoid cells (ILC3s) (Roager and Licht 2018; Zhang et al 2021). Consistent with these previous studies, the relative mRNA expression levels of IL-22 and its receptor, IL-10RB, were significantly upregulated in piglets with high IAA-Na. IAA-Na suppresses intestinal inflammation by reducing the expression of proinflammatory cytokines (TNF-α and IL-17) and promoting the production of anti-inflammatory cytokines (TGF-β, IL-10, and IL-22). It also downregulated the immune signaling pathways, such as the IL-17 and TNF signaling pathways. Additionally, RNA-sequencing data revealed that transporter activity was predominantly enriched in high-IAA-Na piglets. High IAA-Na substantially promoted the expression of nutrient transporters, consistent with the results of intestinal nutrient transporter expression. These findings suggest that IAA enhances intestinal mucosal immunity and stimulates nutrient digestion and absorption.

The present study used boar piglets at an early postweaning stage prior to sexual maturation. These piglets are widely used in nutritional and intestinal physiology studies because they provide a consistent and well-controlled model for investigating intestinal development, nutrient absorption, and immune responses during the critical weaning period. Although IAA can serve as an intermediate in microbial skatole synthesis under specific anaerobic conditions, these findings do not support a significant role for skatole in mediating the observed intestinal benefits of dietary IAA. This is evident from the consistent improvements in gut morphology and nutrient absorption. Skatole production increases during postnatal development in boars and is influenced by the composition of the gut microbiome. Therefore, quantification of IAA and skatole levels in porcine feces or serum is recommended for future studies.

Conclusion

Dietary supplementation with 240 mg/kg IAA-Na improved jejunal morphology and elevated the apparent nutrient digestibility and increased the relative mRNA expression levels of nutrient transporters. Additionally, 240 mg/kg of dietary IAA-Na promoted intestinal epithelial cell proliferation. This was verified by in vitro experiments showing that IAA-Na treatment enhanced the jejunal organoid budding rate. Furthermore, 240 mg/kg of dietary IAA-Na strongly altered the intestinal transcriptomic profile; the most enriched biological processes were “inflammatory response” and “immune system process.” Dietary 240 mg/kg IAA-Na reduced TNF-α content and increased the anti-inflammatory cytokine production in pigs (TGF-β, IL-10, and IL-22). The relative mRNA expression levels of the pro-inflammatory cytokine (IL-17) were downregulated, while the levels of the anti-inflammatory cytokines (IL-10, IL-22, and TGF-β) were upregulated in the jejunum of piglets fed 240 mg/kg IAA-Na. These results suggest that dietary IAA plays an important role in improving intestinal health and intestinal function by modulating the immune response. This provides a theoretical basis for the application of IAA in piglet production.

Supplementary Material

skag048_Supplementary_Data