Coated tannin supplementation improves growth performance, nutrients digestibility, and intestinal function in weaned piglets

Abstract

To explore the effect of coated tannin (CT) on the growth performance, nutrients digestibility, and intestinal function in weaned piglets, a total of 180 piglets Duroc × Landrace × Yorkshire (28 d old) weighing about 8.6 kg were randomly allotted to three treatments: 1) Con: basal diet (contains ZnSO₄); 2) Tan: basal diet + 0.15% CT; and 3) ZnO: basal diet + ZnO (Zn content is 1,600 mg/kg). The results showed that 0.15% CT could highly increase the average daily gain and average daily feed intake of weaned piglets compared with the control group, especially decreasing diarrhea incidence significantly (P < 0.05). Compared with the control group, crude protein apparent digestibility and digestive enzyme activity of the piglets fed with 0.15% CT were enhanced obviously (P < 0.05). Meanwhile, the intestinal villi and microvilli arranged more densely, while the content of serum diamine oxidase was decreased, and the protein expressions of zonula occludens-1 (ZO-1) and claudin-1 were significantly upregulated (P < 0.05). In addition, CT altered the structure of intestinal microbiota and augmented some butyrate-producing bacteria such as Ruminococcaceae and Megasphaera. PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) analysis also showed that the abundances of pathways related to butyrate metabolism and tryptophan metabolism were increased; however, the function of lipopolysaccharide biosynthesis proteins was significantly decreased. The results demonstrated that 0.15% CT could improve growth performance, digestibility, and intestinal function of weaned piglets, and it had the potential to replace ZnO applied to farming.

Lay Summary

Studies in recent years have shown that tannic acid has various biological functions such as astringency, anti-inflammatory effect, and anti-oxidation property, which has good potential to improve diarrhea and intestinal health of animals. However, it can also lead to oxidative moisture absorption, poor palatability, and feed intake reduction when added to feed. Fortunately, coating treatment can effectively solve these problems. Under the above background, we hypothesized that tannic acid can repair the above shortcomings and improve growth and gut health parameters in weaned piglets with the help of coatings. Therefore, this study explored the effects of coated tannin (CT) on the growth performance, nutrients digestibility, and intestinal function in weaned piglets, which aimed to provide a scientific basis for CT replacing ZnO as a green and safe additive in farming and simultaneously also provide a reference for the application of other polyphenols in animals’ health.

Coated tannin could improve growth performance, digestibility, and intestinal function of weaned piglets, and it had the potential to replace ZnO applied to farming.

Introduction

In the early stage of weaning, the digestive and immune organs of piglets are not fully developed and mature, leading to stress symptoms such as diarrhea and growth retardation, which have usually caused huge economic losses to the pig industry (Hedemann and Jensen, 2004). In current animal production practices, pharmacological doses of zinc oxide (ZnO) are usually added to piglets’ diets to prevent postweaning diarrhea (Walk et al., 2015). However, high doses of ZnO affect the absorption of copper and iron, and the unabsorbable Zn released into the environment increases the risk of heavy metal pollution (Bednorz et al., 2013; Ma et al., 2021a). Besides, long-term feeding of weaned piglets with high doses of ZnO diets can cause intestinal mucosal lesions and microbial drug resistance (Roselli et al., 2003; Vahjen et al., 2015). According to the announcement of the Ministry of Agriculture and Rural Affairs of China, the maximum amount of zinc in the feed of piglets is 1,600 mg/kg in the first 2 wk after weaning. Therefore, it is of great necessity to find a safe and practical alternative to ZnO.

Tannin is a general term for a class of polyphenols, divided into hydrolyzed tannin and condensed tannin. Condensed tannin may reduce the nutrient digestibility and hinder the growth performance of monogastric animals, which is mostly regarded as anti-nutritional factor (Smulikowska et al., 2001). Hydrolyzed tannin (tannic acid) is highly water-soluble and has a wide range of sources that are found in many plants and fruits. The hydroxyl structure of polyphenols makes it easier to react with proteins, metal ions, etc. and has the functions of scavenging free radicals and activating immune responses (Frasca et al., 2012). The studies of Van Parys et al. (2010) and Redondo et al. (2015) reported that chestnut tannic acid has a strong antibacterial ability. Studies in recent years have shown that tannic acid has various biological functions such as astringency, anti-inflammatory effect, and anti-oxidation property (Ma et al., 2016; Juan María et al., 2018). In short, tannic acid has good potential to improve diarrhea and intestinal health of animals (Choy et al., 2014; Brus et al., 2018). However, it can also lead to oxidative moisture absorption, poor palatability, and feed intake reduction when added to feed (Ani et al., 2021; Guo et al., 2021). Fortunately, coating treatment can effectively solve them. The coating treatment adds a special structure to the surface of tannin, which overcomes the disadvantage such as its high decomposability and oxidizability. Simultaneously, it reduces the destruction of tannin by gastric acid, enzymes, and other substances, thus improving the effective utilization rate of tannin and reducing the usage amount and cost.

Under the above background, we hypothesized that tannic acid could repair the above shortcomings and improve growth and gut health parameters in weaned piglets with the help of coatings. Therefore, this study explored the effect of coated tannin (CT) on the growth performance, nutrients digestibility, and intestinal function in weaned piglets. The aim of the present study was to provide a scientific basis for CT replacing ZnO as a green and safe additive in farming and also a reference for the application of other polyphenols in animals’ health.

Materials and Methods

The feeding experiment was conducted at Nanʹan Daling Breeding Co., Ltd. (Jiangxi, China) and approved by the Animal Care and Use Committee of Zhejiang University (Zhejiang, China). CT (25% tannin content) was provided by King Techina Feed Co., Ltd. (Zhejiang, China), which was derived from chestnut and mainly coated with hydrogenated palm oil.

Experimental design and treatments

A total of 180 Duroc × Landrace × Yorkshire hybrid healthy weaned barrows (28 d old, average weight about 8.6 kg) were randomly allotted to 3 treatments with 4 replications of 15 piglets in each pen. The dietary treatments were: 1) Con: basal diet (containing ZnSO₄); 2) Tan: basal diet + 1,500 mg/kg CT; and 3) ZnO: basal diet + ZnO (Zn content was 1,600 mg/kg). All piglets were immunized according to the routine immunization program and farmed in clean pens with the same fence specifications. The feeding trial period lasted for 20 d, and all piglets had free access to feed and water. The basal diet was formulated to meet the nutrient requirements of the National Research Council (2012). The composition and nutrient levels of the basal diet are presented in Table 1.

Table 1.

Composition of the basal diet

Ingredients	Content, %	Nutrient levels2	Content
Corn	59.1	Digestive energy, MJ/kg	13.3
Fermented soybean meal	15.0	Crude protein, %	19.3
Extruded soybean	13.0	Calcium, %	0.9
Whey powder	5.0	Phosphor, %	0.7
Bran	2.0	Crude fiber, %	2.5
Fish meal	3.0	Crude fat, %	5.1
Salt	0.3	Crude ash, %	3.5
Dicalcium phosphate	1.0	Lysine, %	1.3
Stone powder	0.6
Premix1	1.0

Premix provided the following per kilogram diet: vitamin A, 5,000 IU; vitamin B1, 3 mg; vitamin B2, 6.5 mg; vitamin B6, 2.4 mg; vitamin D, 2,000 IU; vitamin E, 20 IU; vitamin K, 1 mg; biotin, 0.4 mg; folic acid, 1.45 mg; pantothenic acid, 23 mg; niacin, 1 mg; Fe (ferrous sulfate), 200 mg; Cu (copper sulfate), 100 mg; Mn (manganese sulfate), 30 mg; Zn (zinc sulfate), 100 mg; I (calcium iodate), 20 mg; Se (sodium selenite), 10 mg.

The crude fat in nutrient composition was actual measured value, while the rest were calculated value.

Growth performance and diarrhea incidence

During the experiment, diarrhea of piglets was recorded every day. Feed and individual body weight (BW) were recorded on days 0 and 21 of the experimental period, and weighing was carried out after 12 h fasting (freely drinking). Average daily gain (ADG), average daily feed intake (ADFI), feed to gain ratio (F/G), and diarrhea incidence were calculated based on the above values. The calculation of diarrhea incidence was as follows: Diarrhea incidence = the total number of diarrheal piglets/(number of piglets × number of days tested) × 100%.

Sample collection

Samples of all diets feed and approximately 100 g of fresh feces were collected in the last 3 d of the experiment. Feces samples were added 10% hydrochloric acid in a volume ratio of 5:1 to fix, put into a sample bag, and stored in a sealed container at −20 °C before analysis.

At the end of the feeding trial, a total of 24 piglets selected randomly from the Con, Tan, and ZnO groups (4 replications and 2 piglets per pen) were slaughtered. Serum samples were collected by centrifugation at 3,000 r/min for 10 min, and samples were stored at −80 °C for analysis. The jejunum and colon tissue samples about 0.1 × 0.3 cm² were flushed with ice-cold saline and immersed in 2.5% glutaraldehyde fixative solution, stored at 4 °C for electron microscope observation. Fresh contents of the duodenum and colon were sampled into sterile tubes separately. At the same time, mucosa of the jejunum and colon were scraped by a sterile glass microscope slide at 4 °C. Then, all samples were rapidly frozen in liquid nitrogen and stored at −80 °C until analyses.

Apparent nutrient digestibility

According to AOAC (2012), diets and fecal samples were analyzed for crude protein (CP), crude fat (Dry Matter [DM], method 934.01), crude ash, Ca (method 985.01), and P (method 985.01). Apparent nutrient digestibility was calculated as follows: Nutrient digestibility = (nutrient intake − nutrient voided)/nutrient intake × 100%, Nutrient intake = nutrient in diet × feed intake, and Nutrient voided = nutrient in feces × amount of feces voided.

Digestive enzyme activities

Approximately 0.2 g of duodenal contents and jejunal mucosa were rinsed with phosphate buffer solution (PBS) respectively, and dried with filter paper. After that, weighed again and added PBS equal to 9 times the mass of contents or mucosa. With the help of physiological saline, the tissue was quickly cut into pieces and poured into a homogenization tube. So, the tissue was mashed with a homogenizer to obtain a 10% tissue homogenate. At last, the tissue homogenate was centrifuged at 4,000 r/min for 15 min. The supernatant of duodenal contents was taken to determine duodenal trypsin, lipase, and amylase activities in different groups. The supernatant of jejunal mucosa was taken to determine disaccharidases such as lactase, maltase, and sucrase activities of piglets. All kits were purchased from Nanjing Jiancheng Bioengineering Institute (Jiangsu China), and the assays were carried out in accordance with instructions.

Intestinal morphology and permeability

The jejunum and colon tissues were rinsed three times with 0.1 M PBS and fixed in 1% osmium acid solution for 1 to 2 h. After rinsing three times with PBS, the jejunum tissues were dehydrated by ethanol solution and then treated with ethanol and isoamyl acetate, dried, and coated. Finally, the morphology of jejunal villi and microvilli was observed by scanning electron microscope (SEM). The colon tissues were treated with embedding agent and acetone, heated and sliced, and then stained with lead citrate solution and 50% ethanol saturated solution of uranyl acetate. Eventually, the morphology of colon microvilli was observed with a transmission electron microscope (TEM).

To detect the contents of d-lactate, diamine oxidase (DAO), and endotoxin in the serum, the determination kits were purchased from Nanjing Jiancheng Bioengineering Institute (Jiangsu, China), and the assays were carried out in accordance with instructions.

Western blot detection of intestinal tight junction protein

The relative protein expressions in the colon were determined by Western blot according to the previous study (Hu et al., 2017). The primary antibodies used in our study included anti-ZO-1 antibody (Thermo fisher 40-2200), anti-Claudin-1 antibody (Abcam ab129119), anti-Occludin antibody (Abcam ab222691), anti-GAPDH antibody (Abcam ab181602), Goat anti-Mouse IgG (H + L) secondary antibody (Thermo Pierce), and Goat anti-Rabbit IgG (H + L) secondary antibody (Thermo Pierce).

Colonic lumen microbiome analysis and functional prediction

DNA was extracted from about 100 mg of colon content in each sample by the method of adsorption column and then detected and diluted. According to the different sequencing regions to select the amplification primers of the corresponding regions, DNA was amplified by the polymerase chain reaction (PCR) of bacterial 16S rRNA genes. After that, PCR products were quantified and identified, then mixed, and purified (GeneJET Gel Extraction Kit). The NEB Next Ultra DNA Library Prep Kit (Illumina, USA) was used to construct the library, which was evaluated on Qubit 2.0 fluorometer (Life Technologies, CA, USA) and Agilent Bioanalyzer 2100 system. Finally, the Illumina MiSeq platform formed a 250-bp paired-end sequence. Data obtained by sequencing were filtered to produce a credible target sequence for subsequent analysis. The PEAR sequence splicing algorithm was used for splicing, and sequences with similarity ≥97% were selected and classified as a class of operational taxonomic units (OTUs). Species annotation analysis was performed by the RDP Classifier method and GreenGene database. The dilution curve display diagrams (Shannon, Chao1, Observed species, and Simpson) were calculated and drawn using QIIME (version 1.8.0) and R software (version 3.2.5). β-Diversity was determined using principal coordinate analysis (PCoA) and nonmetric multidimensional scaling (NMDS). Linear discriminant analysis (LDA) and effect size (LEfSe) analysis were performed using an online LEfSe tool. PICRUSt (phylogenetic investigation of communities by reconstruction of unobserved states) was used to generate predictive functional profiling of the intestinal microbiota. STAMP was used to analyze the functional differences between groups.

Statistical analysis

Data in the article are expressed as mean ± SD, and P < 0.05 indicates a significant difference. Statistical analysis was performed by SPSS 20.0 (Chicago, IL, USA). Student’s t-test and one-way analysis of variance (ANOVA) followed by DUNCAN’s multiple comparisons were used to test the significance of differences.

Results

Growth performance and diarrhea incidence

The results of growth performance and diarrhea incidence of weaned piglets are presented in Table 2. Compared with the Con group, piglets fed a tannin diet had greater ADG which increased by 22.2% (P < 0.05), whereas they had lower F/G which decreased by 10.3% (P < 0.05), and there was no significant difference between the Tan group and ZnO group (P > 0.05). As for diarrhea incidence, the Tan group and ZnO group were significantly lower than the Con group (P < 0.05).

Table 2.

Effects of coated tannin on growth performance and diarrhea incidence of weaned piglets

Item1	Con2	Tan2	ZnO2
Initial BW, kg	8.56 ± 0.43	8.78 ± 0.32	8.67 ± 0.49
Final BW, kg	15.77 ± 2.55b	17.59 ± 2.89a	17.91 ± 2.45a
ADG, g/d	360 ± 18.7b	440 ± 25.6a	462 ± 21.5a
ADFI, g/d	525 ± 35.2b	576 ± 40.1b	623 ± 49.7a
F/G	1.46 ± 0.03a	1.31 ± 0.03b	1.34 ± 0.02b
Diarrhea incidence, %	10.16 ± 0.11a	4.95 ± 0.05b	4.86 ± 0.05b

ADFI, average daily feed intake; ADG, average daily gain; BW, body weight; F/G, ratio of feed to gain.

Con, basal diet; Tan, basal diet adds 1,500 mg/kg coated tannin; ZnO, basal diet adds ZnO (contains Zn 1,600 mg/kg).

Different superscripts within a row indicate significant differences (P < 0.05).

Apparent nutrient digestibility

As presented in Table 3, compared with the Con group, the digestibility of CP in the Tan group was significantly improved (P < 0.05), while there were no significant differences in the crude fat, crude ash, calcium, and phosphorus between the two groups. Compared with the ZnO group, the digestibility of crude fat in the Tan group was significantly reduced, while no significant difference was observed in other indicators.

Table 3.

Effects of coated tannin on apparent nutrient digestibility of weaned piglets

Item, %	Con1	Tan1	ZnO1
Crude protein	67.14 ± 1.12b	70.85 ± 0.46a	71.62 ± 0.64a
Crude fat %	46.12 ± 0.21b	45.53 ± 0.73b	50.72 ± 0.54a
Crude ash	51.24 ± 0.81	51.30 ± 1.02	53.38 ± 0.87
Calcium	58.62 ± 0.69	59.21 ± 0.98	58.01 ± 1.12
Phosphorus	64.88 ± 1.14	65.47 ± 1.02	65.97 ± 0.94

Con, basal diet; Tan, basal diet adds 1,500 mg/kg coated tannin; ZnO, basal diet adds ZnO (contains Zn 1,600 mg/kg).

Different superscripts within a row indicate significant differences (P < 0.05).

Digestive enzyme activities

Figure 1 shows the results of digestive enzyme activities in the duodenal and jejunal mucosa. Compared with the Con group, the activities of duodenal trypsin in Tan and ZnO groups were significantly improved (P < 0.05), while activities of lipase and amylase were not significantly changed (Figure 1A). Meanwhile, we also tested three kinds of disaccharidases in the jejunal mucosa of piglets (Figure 1B). Results showed that maltase and sucrase in the Tan and ZnO groups had higher activities than those in the Con group (P < 0.05). There was no obvious difference in lactase among the three groups.

Figure 1.

Effects of coated tannin on the digestive enzyme activities of weaned piglets. (A) Duodenal trypsin, lipase, and amylase activities in different groups. (B) Disaccharidases, such as lactase, maltase, and sucrose, activities of piglets in different groups. Con, basal diet; Tan, basal diet adds 1,500 mg/kg coated tannin; ZnO, basal diet adds ZnO (contains Zn 1,600mg/kg). *P < 0.05.

Intestinal morphology and mucosal barrier

Jejunum villi are finger shaped. In the Con group, they were much damaged, while they were more intact in Tan and ZnO groups (Figure 2A). Jejunum microvilli are arranged in a cylindrical shape. Compared with the Con group, those in the Tan and ZnO groups were arranged more orderly and densely (Figure 2B). Meanwhile, the TEM also showed that colonic microvilli of the Tan and ZnO groups were more neatly, densely arranged and connected tightly, while the intercellular junctions in the control group were broken (Figure 2C).

Figure 2.

Coated tannin improved the intestinal morphology of weaned piglets. (A) Representative jejunal villi scanning electron microscope (SEM) images (scale bars = 300 µm). (B) Representative jejunum microvilli SEM images (scale bars = 1.00 µm). (C) Representative colon microvilli transmission electron microscopy images (scale bars = 1 µm).

Based on SEM and TEM images of the jejunum and colonic tissue, we further measured the indexes of intestinal permeability (Figure 3A) and the expression of epithelial integrity-associated tight junction proteins (Figure 3B). Results showed that serum DAO and endotoxin content in the Tan and ZnO groups were significantly lower than those in the Con group (P < 0.05), while no significant difference was observed in d-lactate content among the groups (P > 0.05) (Figure 3A). As for the expression of epithelial integrity-associated tight junction proteins, we tested claudin-1, occludin, and ZO-1 of the colon among three groups (Figure 3B). The results revealed that the relative expressions of ZO-1, claudin-1, and occludin in the Tan group were significantly higher than those in the Con group (P < 0.05), and diet contained ZnO upregulated colonic relative protein expressions of ZO-1 and claudin-1 (P < 0.05).

Figure 3.

Coated tannin improved the intestinal mucosal barrier of weaned piglets. (A) The concentration of serum diamine oxidase (DAO), endotoxin, and d-lactate. (B) Relative protein expression of epithelial integrity-associated tight junctions (claudin-1, occludin, and zona occludens-1 [(ZO-1]) in the colon among groups. Con, basal diet; Tan, basal diet adds 1,500 mg/kg coated tannin; ZnO, basal diet adds ZnO (contains Zn 1,600 mg/kg). **P < 0.01; *P < 0.05.

Structure of intestinal microbiota and functional prediction

CT affected the diversity of intestinal microbiota in the colon of piglets

Figure 4 shows the results of α- and β-diversity indexes of intestinal microbiota. Among three groups, the microbiota evenness indexes such as Chao1 and Observed species were not significantly different (P > 0.05), whereas the microbiota abundance indexes such as Simpson and Shannon of the Con and Tan groups were significantly higher than those in the ZnO group (P < 0.05) (Figure 4A). It suggested that ZnO reduced the diversity of the intestinal microbiota in weaned piglets.

Figure 4.

Coated tannin affected the diversity of intestinal microbiota in the colon of piglets. (A) α-diversity index: Observed species, Chao1, Simpson, and Shannon. (B) Principal coordinate analysis (PCoA) of piglets’ intestinal microbiota among different groups. (C) Nonmetric multidimensional scaling (NMDS) plots to show the difference of bacterial community in three groups. C, T, and Z represent the control group, 0.15% coated tannin group, and ZnO group, respectively. **P < 0.01; *P < 0.05.

Obviously, contributions of the main components principal component (PC)1 and PC2 were 58.59% and 16.29%, respectively (Figure 4B). The results of PCoA analysis showed that there was no significant difference in the structure of the microbiota among the three groups, while they could be distinguished well. And further, LEfSe analysis is needed.

NMDS analysis was applied to reflect the species information contained in the sample with a form of points. The distances between points were used to reflect the degrees of difference within and between the sample groups. The Stress value was 0.124, which was less than 0.2, indicating that NMDS can accurately reflect the degree of difference between samples. Based on the results of NMDS analysis of OTU levels, it could be seen that the samples within each group were similar, while the differences between the groups were not significant (Figure 4C).

CT altered structure of intestinal microbiota in the colon of piglets

The relative abundance of the 15 most abundant microbes in different groups at phylum (Figure 5A), family (Figure 5B), and genus (Figure 5C) levels is shown, respectively. At the phylum level, Firmicutes was the most abundant phylum, followed by Bacteroidetes. The most abundant family in the Tan and ZnO groups was Ruminococcaceae, followed by Lachnospiraceae, which was opposite to the Con group.

Figure 5.

Coated tannin altered the structure of intestinal microbiota in the colon of piglets. (A) Relative abundance of the microbial community in the colon at the phylum level. (B) Relative abundance of the microbial community in the colon at the family level. (C) Relative abundance of the microbial community in the colon at the genus level. (D) Linear discriminant analysis (LDA) value distribution histogram between coated tannin and control groups. (E) LDA value distribution histogram between coated tannin and ZnO groups. C, T, and Z represent the control group, 0.15% coated tannin group, and ZnO group, respectively.

Histograms of LDA values of the Tan group compared with the Con and ZnO groups are shown in Figure 5D and E, respectively. Abscissa represents the LDA score value above 2 of the species, and each column stands for a species with a significant difference in abundance. It could be seen that Faecalibacterium, Faecalitalea, Acidaminococcus, Methanobrevibacter smithii, and Turicibacter in the Tan group were significantly increased compared with the Con group at the genus level. And compared with ZnO, CT improved the relative abundance of Faecalibacterium, Alloprevotella, Roseburia, and Megacoccus significantly, while it decreased Terrisporobacter and Bilophila significantly.

Functional prediction of intestinal microbiota between CT and control groups

The STAMP software was used to analyze the difference in function prediction between CT and control groups, and the results are shown in Figure 6. Compared with the control group, CT obviously promoted the abundance of butanoate metabolism, propanoate metabolism, tryptophan metabolism, tight junction, pyruvate metabolism, fatty acid metabolism, ABC transporter and replication, recombination, and repair protein but restrained the function of lipopolysaccharide biosynthesis proteins significantly.

Figure 6.

Functional prediction of intestinal microbiota between coated tannin and control group. Difference analysis diagram of KEGG third-level pathway. C, control group; T, 0.15% coated tannin group.

Discussion

Affected by weaning stress, piglets often have a poor appetite and feed intake reduction, which can easily cause weight loss, diarrhea, and growth retardation in the first few days after weaning (Hao et al., 2015). Previous studies have shown that dietary 1,000 mg/kg and 1,500 mg/kg chestnut tannin supplementation had no influence on ADG and ADFI, while it could decrease the diarrhea incidence of piglets significantly (Myrie et al., 2008; Liu et al., 2020). Meanwhile, the inclusion in the diets of a medium tannin dose of 1% tannin did not affect BW and feed efficiency ratio (Girard et al., 2018). However, the study also reported that tannin supplementation at 2% showed a positive effect on ADFI and ADG (Girard et al., 2020, preprint). Our previous study had demonstrated that 0.1%, 0.15%, and 0.2% CT all increased the ADFI of piglets. Comprehensively, 0.15% CT had the best effect, indicating that 0.15% CT could increase the feed palatability and feed intake significantly. Maybe the protective effect of the coated treatment on tannin reduces its consumption in the stomach and improves the utilization rate (Cappai et al., 2013). Girard et al. (2018) found that adding 0.54% chestnut tannin could reduce the quantities of diarrhea and bacterial infection of weaning piglets. In our present study, 0.15% CT supplementation has the same result. We supposed that was related to tannin’s interference to the integrity of bacterial cell walls and inhibitation of the reproduction of pathogenic microorganisms (Dong et al., 2018). Matsumura et al. (2017) showed that 2% persimmon-derived tannin could reduce the expression of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), thereby inhibiting the inflammatory response. And meanwhile, dietary supplementation of tannin significantly decreased disease activity and colon inflammation, and hydrolysate of tannin directly suppressed expression of inflammatory genes in macrophages in vitro (Kitabatake et al. 2021). These implied that tannin could effectively increase the ability to produce immune substances to improve animal health. The above shows that 0.15% CT could effectively alleviate the stress of weaning, reduce the rate of diarrhea, and promote the growth performance of piglets, which has an equal effect to ZnO (Zn content is 1,600 mg/kg) supplementation.

High-dose hydrolyzed tannin can combine with digestive enzymes and proteins, which reduces digestive enzymes activities and protein digestibility (Chung and Reed, 2012). Tércia Cesária et al. (2019) reported that a higher level of tannins negatively affected the apparent ileal digestibility of dry matter, energy and CP. Similarly, Antongiovanni et al. (2007) found that 0.25% and 0.5% of water-soluble chestnut tannin significantly depressed the apparent digestibility of both dietary dry matter and protein, which decreased 0.5% compared with the control group. Our experiment showed that the digestibility of CP in the CT group increased significantly and other indicators had not been affected. The result showed that 0.15% CT of chestnut did not adversely affect the apparent digestibility of nutrients, even though increased the digestibility of CP. On the one hand, these results had similar conclusions with Biagia et al. (2010) that adding 0.113%, 0.225%, and 0.45% uncoated chestnut tannin significantly improved feed efficiency. On the other hand, the result corresponds to the enhancement of the growth performance of piglets.

To verify the above conclusions, we determined the activities of duodenal digestive enzymes and jejunal mucosal disaccharidase. We know that disaccharides cannot be directly absorbed and utilized by the small intestine, so intestinal disaccharidase plays a vital role in the utilization of carbohydrates. In a previous study conducted several years ago, the author reported that tannin could positively affect digestive enzyme activity and microbial fermentation in the gut (Scalbert, 1991). Yao et al. (2019) also reported that Galla tannin could promote the pancreatic secretion of trypsin and increase trypsin activity. In our experiment, CT could significantly increase the activities of duodenal trypsin and jejunal mucosal maltase and sucrase, which was equivalent to ZnO. It can be seen that CT could promote the activity of digestive enzymes and play a positive role in the digestibility of nutrients.

Intestinal morphology is an important indicator of the digestion and absorption of nutrients. Weaning stress in piglets will cause diarrhea symptoms, which were accompanied by damage to intestinal villi, microvilli, and intercellular connections (Fiesel et al., 2014). A previous study found that tannin could significantly increase the villus height of jejunum and ileum in Hu sheep (Zhao et al., 2019). Meanwhile, high doses of tannins (1,000 mg/kg) significantly reduced the number of Escherichia coli and altered great variability of microbial populations in the small intestine and colon of 28-d-old chickens (Jamroz et al., 2009). In the researches of tannin influencing the intestinal morphology, most of them focused on the height of intestinal villi and the ratio of villus height to crypt depth. For example, Wang et al. (2020) found that 1,000 mg/kg tannin treatment improved the duodenal morphology and modulated the colonic bacteria composition. In this experiment, we observed the intestinal microvilli and cell morphology under an electron microscope and found that the microvilli were arranged more neatly and densely in the Tan than those in the ZnO group. In addition, it significantly enhanced the tight junctions between intestinal epithelial cells and maintained the integrity of the mitochondrial structure. We speculated that it may be related to the ability of tannin to change the permeability and physiological functions of cells or the ability to improve the antioxidant capacity of piglets (Liu et al., 2017; Meng et al., 2018). In order to figure out the above conjecture, we further tested the intestinal permeability and tight junction protein expression. The results revealed that CT could upregulate the protein expression of ZO-1 and Claudin-1 and downregulate the concentration of serum DAO and endotoxin significantly. Studies showed that long-term feeding of ZnO could cause zinc poisoning and damage the intestinal mucosa of piglets (Buff et al., 2005; Schulte et al., 2016). High-zinc diets would reduce the number of beneficial intestinal bacteria and affect the composition and biological activity of intestinal microbes (Starke et al., 2014). At the same time, it will also destroy the intestinal microbiota ecological environment and stability of piglets (Janczyk et al., 2015). The above results showed that tannin could improve the intestinal barrier function of piglets in a green and safe way, which was consistent with the results of nutrient digestion and intestinal morphology.

Intestinal microbiota can regulate the epigenetic modification of the intestine to mediate the interaction between microbiota and mucosal barrier, which is closely related to a variety of intestinal and immune diseases of animals (Wu et al., 2020)^. In our study, 16S rRNA sequencing analysis showed that the microbial diversity indexes (Simpson and Shannon index) of the Con and Tan groups were significantly higher than the ZnO group, indicating that ZnO reduced the intestinal microbiota diversity of weaned piglets. What’s more, CT increased the diversity of intestinal microbiota while playing anti-diarrhea and growth-promoting functions.

Firmicutes and Bacteroidetes are the absolute dominant microbiota in the intestine of piglets, whose relative abundance is higher than 96% (He et al., 2017; Ma et al., 2021b). Firmicutes can maintain intestinal health by producing short-chain fatty acids, inhibiting inflammation, and providing energy for intestinal epithelial cells (Gophna et al., 2017), whereas Bacteroides may become conditional pathogens under certain conditions (Turnbaugh et al., 2009; Yatsunenko et al., 2012). The ratio of Firmicutes/Bacteroidetes can be used as an important indicator for evaluating the status of intestinal microbes (Mueller et al., 2006; Liu et al., 2018). In our study, the ratio in the Tan group showed an upward trend compared with the Con group. Ruminococcaceae is the main microorganism that converts primary bile acids into secondary bile acids and can prevent ulcerative colitis (Sinha et al., 2020). Meanwhile, we found that CT could significantly increase its quantity. Linear effect size analysis also showed that there were a variety of butyrate-producing bacteria in the different bacterial genera between each group. Among them, Megasphaera was an important bacterium, which is of great significance to the host (Maia et al., 2007; Louis and Flint, 2009). Lactic acid and butyric acid are the main metabolites of Faecalitalea (Sanna et al., 2019). And it is well known that butyric acid can provide energy for colonic epithelial cells, improve the integrity of the intestinal epithelial cell barrier, and regulate the type and quantity of intestinal microbiota (Geirnaert et al., 2017). Faecalibacterium prausnitzii of Faecalibacterium and Methanobrevibacter smithii both have anti-inflammatory effects and can induce inflammatory cytokine responses (Bang et al., 2014; Ruaud et al., 2020). Especially, the supernatant of F. prausnitzii can induce IL-10 secretion in mice (Sokol et al., 2008). In summary, CT could improve intestinal function and maintain the integrity of the intestinal barrier by increasing the relative abundance of butyrate-producing bacteria and other beneficial bacteria.

The changes in microbiota structure will directly alter its metabolic function, so we adopted the function prediction analysis to further detect how the metabolic function of microbiota changed. The results showed that the relative abundance of butyrate metabolism and tryptophan metabolism pathways in the Tan group was significantly increased, which was consistent with the results of the LEfSe analysis. In addition, CT decreased the function of lipopolysaccharide biosynthesis. Butyrate is an important mediator in the regulation of intestinal microbiota and host metabolism. It can be utilized by the colon and extra colon tissue cells, which are involved in regulating various metabolic functions (Zhang et al., 2021). Tryptophan is metabolized under the action of intestinal microbiota to produce indole derivatives, which can act as ligands for aromatic hydrocarbon receptors and promote the production of IL-22, thereby protecting the colon from inflammation (Scott et al., 2020). Tryptophan metabolites also lead to the decrease of inflammation and central nervous system autoimmunity caused by Nuclear Factor-kappa B (NF-kB) (Marsland et al., 2016). However, lipopolysaccharides can exert pro-inflammatory effects through Toll-like receptor 4, which will destroy the physiological homeostasis and barrier integrity of intestines (Farhadi et al., 2010). These results suggested that CT could affect the metabolic function of microbiota by altering its structure and then improve the morphology and barrier function of the intestine, which was consistent with the aforementioned results.

CT supplementation could increase growth performance and decrease diarrhea incidence of weaned piglets. In addition, 0.15% CT significantly improved apparent nutrient digestibility and digestive enzyme activities of piglets, which altered the structure of intestinal microbiota and increased related gene abundance to improve intestinal morphology and barrier function. To sum up, CT had the potential as a green and safe additive to replace ZnO.

Glossary

Abbreviations

ADFI

average daily feed intake

ADG

average daily gain

CP

crude protein

CT

coated tannin

DAO

diamine oxidase

F/G

feed to gain ratio

LDA

linear discriminant analysis

NMDS

nonmetric multidimensional scaling

PCoA

principal coordinate analysis

PCR

polymerase chain reaction

SEM

scanning electron microscopy

TEM

transmission electron microscopy

ZO-1

zonula occludens-1

Conflict of interest statement

The authors declare no real or perceived conflicts of interest.

Data Availability

The original contributions presented in the study are publicly available. These data can be found here: National Center for Biotechnology Information (NCBI) repository, https://dataview.ncbi.nlm.nih.gov/object/PRJNA771778?reviewer=ip65i6sh5vu4dc04afuc6rasg2.