1. Introduction
Clostridium perfringens (
C. perfringens) can decompose nutrients and produce large amounts of gas, resulting in tissue aerocele and extensive necrosis. It is the main pathogen that causes necrotic enteritis (NE) in poultry [
1]. NE, known as enterotoxemia, is mainly caused by
C. perfringens type A, which often accompanies or occurs successively with coccidia in production, considerably impacting the economy of the poultry breeding industry, with an estimated annual loss of up to USD 6 billion [
2,
3,
4]. Antibiotic growth promoters (AGPs) can be used to protect broilers against intestinal diseases and improve their productivity. In light of the emergence of bacterial resistance and concerns about antibiotic residues in animal products, however, numerous countries and regions have implemented restrictions and bans on the use of AGPs in livestock and poultry feeds. Consequently, there is a pressing need for research investigating suitable alternatives to AGPs to effectively manage the rising occurrence of NE in chickens.
A range of polyphenols are present in plants. Among them, tannic acid, a secondary metabolite produced by plants, has potential as a feed additive [
5]. In monogastric animals, supplementation of different concentrations of tannins has had positive effects owing to their antioxidant, antimicrobial, and anti-inflammatory properties [
6,
7,
8]. Studies have shown that the addition of tannic acid to diets reduces the colonization of intestinal pathogenic bacteria, including
Escherichia coli and
Salmonella, and promotes intestinal health [
9,
10]. In vitro studies have reported that tannic acid can inhibit the reproduction of
C. perfringens [
11,
12]. Although there has been progress in the application of tannic acid in broiler feed, its effects on the intestinal health and antioxidant function of broilers with NE have not been fully elucidated. Several investigations have examined alterations in cytokine concentrations in broilers afflicted with NE [
13,
14,
15]. However, there is a need for more robust and dependable data regarding the immunomodulatory impacts of tannins on NE-infected broilers. Therefore, comprehending the effects of tannins on intestinal health, immune response, and antioxidant activity in NE-infected broilers is a crucial effort due to the promise tannins hold as an alternative to antibiotics.
In this study, we have investigated the effects of adding different concentrations of tannic acid on the fecal water content, intestinal barrier integrity, chyme pH, intestinal and serum antioxidant capacity and immunity, and intestinal microorganisms in broilers infected with NE. This study was conducted to verify whether the addition of appropriate doses of tannic acid could reduce the negative effects of NE by increasing the antioxidant capacity and improving the intestinal health of broilers, and provides new scientific data for the application of tannic acid in broilers.
2. Materials and Methods
2.1. Experimental Design
The experiment was conducted at the poultry experiment base of China Agricultural University, located in Zhuozhou City, China. In the experiment, 630 one-day-old Cobb500 male chicks were randomly divided into six treatment groups, with seven replicate cages and fifteen birds in each cage. Broilers were housed in two-tiered battery cage units, with the battery cage as the experimental unit. The treatment groups were as follows: negative control group (NC), unchallenged and untreated; positive control group (PC), challenged with NE and untreated; PTA1, challenged with NE and administered control diet supplemented with 250 mg/kg tannic acid; PTA2, challenged with NE and administered control diet supplemented with 500 mg/kg tannic acid; PTA3, challenged with NE and administered control diet supplemented with 750 mg/kg tannic acid; and PTA4, challenged with NE and administered control diet supplemented with 1000 mg/kg tannic acid. Broilers were given ad libitum access to feed and water during the experiment. All chickens were vaccinated and subjected to standard immunization and management protocols for Cobb broilers. The test diets were in pelleted form with reference to the Chinese Chicken Feeding Standard (NY/T 33-2004) (
http://down.foodmate.net/standard/yulan.php?itemid=7410 (accessed on 20 June 2023)) and were formulated as a corn-soybean meal-based diet. The formulation of the basic diet is shown in
Table 1.
2.2. Challenging Broilers with Necrotic Enteritis
Broilers were challenged with NE, as described in a previous study (co-infection of broilers with coccidia and
C. perfringens) with slight modifications [
16]. Compared with previous studies, we adjusted the dose and duration of infection for
C. perfringens and coccidia in the current study accordingly. The process employed to establish the NE model is illustrated in
Figure 1. With the exception of the NC group, all groups were administered a 1 mL oral dose of attenuated quadrivalent coccidia vaccine suspension (Foshan Zhengdian Biotechnology Co., Ltd., Foshan, China) at a 25-fold dose on d 19. The NC group received an equivalent volume of saline via oral gavage. During d 22–28, the chickens in the NC group were gavaged 1 mL of sterile fluid medium, whereas the birds in the other groups were gavaged 1 mL of
C. perfringens type A CVCC52 (3 × 10
8 CFU/mL).
2.3. Fecal Water Content
The 24 h fecal samples of broilers were collected on d 25, 26, 27, 28, and 35 of the experiment; approximately 400 g of feces was collected after kneading, and was weighed, and dried in an oven at 65 °C for 3 days. The weight was recorded after constant weight and the initial water content of feces was calculated. The average value of the water content in d 25–28 manure was calculated to represent the initial water content of manure at d 25–28. The initial water content of fecal samples (%) was calculated as follows: The initial water content of fecal samples = (fresh weight of fecal samples − dry weight of fecal samples)/fresh weight of fecal samples × 100%.
2.4. Determination of Ileum and Cecum Chyme pH
Chyme pH was determined according to the method described by Burnell et al. [
17]. In brief, the entire contents of the ileum and cecum were collected and thawed on ice. Then, approximately 1.5 g of chyme was added to a 15 mL centrifuge tube and diluted with deionized water at a ratio of 1:8 (
w:
v). The solution was shaken and mixed with a vortex shaker for 10 min. Finally, the pH was measured using a pH meter (METTLER TOLEDO, Shanghai, China).
2.5. Determination of Serum Diamine Oxidase and D-Lactic Acid Concentrations
The concentrations of diamine oxidase (DAO) and D-lactic acid (D-LA) in blood were measured using the DAO kit (A088-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and D-LA kit (E-BC-K002-M, Wu Han Elabscience Biotechnology Co., Ltd., Wuhan, China) according to the manufacturer’s instructions.
2.6. Determination of Cytokines in Ileum and Serum
A 0.1 g sample of middle ileum tissue was collected and homogenized with ten times the volume of pre-cooled PBS. The supernatant was then subjected to thorough homogenization and the protein content was subsequently measured. The concentrations of TNF-α (ml002790, Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China), IL-4 (ml059838, Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China), and IL-10 (ml059830, Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China) in the ileal tissue homogenate and serum were determined using enzyme-linked immunoassay kits. The determination procedures were conducted following the instructions provided by the manufacturer.
2.7. Determination of Antioxidant Indexes in Serum and Ileal Mucosa
Blood collected from the wing vein was centrifuged at 1500× g for 10 min at 4 °C to separate the serum, which was stored at −80 °C. The total antioxidant capacity (T-AOC), malondialdehyde (MDA) content, and superoxide dismutase (SOD) activity were measured using the collected serum. MDA (A003-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) was determined using the serum stock solution. For the determination of SOD, one tube was prepared by diluting the serum volume with a 1:19 ratio of saline volume (A001-3, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). A ratio of serum volume:saline volume of 1:4 was used for determining T-AOC (A015-2-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The middle ileal mucosa collected on d 28 and 35 was prepared into a 10% homogenate using precooled saline, and mucosal MDA (A003-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and total protein (TP, A045-2-2, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) were measured. Precooled normal saline was added to the homogenate and diluted according to the ratio of 10% homogenate volume:normal saline volume = 1:19 for SOD determination (A001-3, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The T-AOC (A015-2-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) of the ileal mucosa was measured according to the ratio of 10% homogenate volume:normal saline volume = 1:4. Serum enzyme activity was expressed as U/mL and ileal mucosal enzyme activity was expressed as U/mg of protein. All assay procedures were performed according to the manufacturer’s instructions.
2.8. Sequencing of Cecal Microorganisms
Cecal microbial sequencing was performed according to the method described by Zhang et al. [
18]. Bacterial genomic DNA was extracted from 0.2 g cecal chyme samples using the QIAamp DNA Stool Mini Kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer’s instructions. DNA was examined using 1% agarose gel electrophoresis. After dilution, the genomic DNA served as a template for amplifying bacterial DNA using the universal primers 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′), targeting the V3-V4 region of the 16S rDNA gene. Subsequently, the PCR products were purified, quantified, and homogenized to create a sequencing library. The resulting libraries were assessed for quantity using Qubit and Q-PCR before being sequenced on a NovaSeq6000 platform. Sequencing procedures were conducted by Beijing Novogene Co., Ltd. (Beijing, China). The number of operational taxonomic units (OTUs), Shannon index, Simpson index, abundance-based coverage estimator index, and Chao1 index were calculated for each sample using Qiime software (Qiime2-2019.7, Nature Biotechnology, USA). Bacterial populations with differential abundance were analyzed using the linear discriminant analysis effect size (LEfSe) method.
2.9. Statistical Analysis
The data were analyzed using SPSS 26.0 (SPSS, Inc., Chicago, IL, USA) and compared using one-way analysis of variance (ANOVA) and Duncan’s multiple comparisons. The challenged groups (PC, PTA1, PTA2, PTA3, and PTA4) were analyzed using contrast tests for the linear and quadratic effects of different doses of tannic acid. Results are expressed as mean ± standard error of mean (SEM). A p-value of <0.05 was considered a significant difference. Pearson’s correlation analysis was employed to assess the relationships between the microbiota and various indicators of antioxidation, intestinal health, immunity, and chyme pH in broilers at 28 and 35 days of age.
4. Discussion
This study aimed to investigate the effects of tannic acid on the antioxidant function, immunity and intestinal health of broiler chickens infected with NE. Oxidative stress plays an important role in NE pathogenesis [
19]. NE can increase MDA concentrations in the body [
20,
21] and decrease the activities of antioxidant enzymes such as catalase [
22]. In the present study, we observed that NE reduced the antioxidant capacity of the body by decreasing T-AOC and increasing MDA concentrations; these results are consistent with those of previous studies [
23,
24]. Oxidative damage is caused by an imbalance between antioxidant defense and free radical generation systems [
25]. Throughout evolution, most organisms have developed innate enzymatic defense mechanisms, non-enzymatic antioxidant defenses, and repair systems to safeguard against oxidative stress-induced damage. However, these natural antioxidant systems often fall short in providing adequate protection. Hence, the search for antioxidants to counteract oxidative damage is of the utmost importance. Numerous studies have substantiated the antioxidant properties of tannic acid [
26,
27,
28,
29,
30,
31]. Tannins are rich in hydroxyl groups; the hydrogen released from these hydroxyl groups exhibits a strong antioxidant capacity by scavenging free radicals. Further, tannic acid may reduce oxidative stress by activating the Nrf2-Keap1 pathway [
28]. However, in the current investigation tannic acid did not exhibit outstanding antioxidant capacity. Nevertheless, it dose-dependently elevated the T-AOC of both the serum and ileal mucosa, indicating a dose-dependent antioxidant effect of tannic acid. A previous study has reported that the antioxidant function of tannic acid is related to its structure and polymerization degree [
32]; this could be the reason why tannic acid exhibited no prominent antioxidant capacity in the current study.
The rate of diarrhea was significantly higher in broiler chickens with intestinal bacterial infection [
33,
34], as demonstrated by the significant increase in fecal water content found in this study. We observed that addition of tannic acid to the diet linearly reduced fecal water content and was effective in suppressing the increase in fecal water content caused by NE; this result is consistent with that of Yang et al. [
35], Girard et al. [
36], and Choi et al. [
37], who showed that addition of tannic acid to the diet alleviated diarrhea in animals and reduced fecal water content and the duration of diarrhea. The potential antidiarrheal effect of tannic acid can be attributed to the following factors: First, tannic acid acts as an astringent when it enters the animal’s intestine, which leads to a slowdown in intestinal peristalsis and an enhancement in water reabsorption. Second, tannic acid has the ability to improve intestinal health and enhance the intestinal barrier in chickens by promoting favorable changes in intestinal morphology and upregulating the expression of genes related to intestinal tight junction proteins. Lastly, tannic acid exhibits selective antibacterial properties, and the reduction of cytotoxin production by bacteria is one of the contributing factors to the alleviation of diarrhea symptoms in broilers. Tannic acid has inhibitory effects on pathogenic bacteria such as
C. perfringens and
Salmonella, thereby reducing the occurrence of diarrhea [
12,
38,
39].
Maintaining optimal intestinal pH is important in order to maximize nutrient utilization and maintain a steady state of intestinal microbiota. Any changes in the intestinal flora may affect chyme pH. In this study, broiler chickens infected with NE exhibited a notable reduction in ileal and cecum chyme pH. Several potential factors could contribute to this observation, including alterations in intestinal flora caused by NE infection, diminished digestive capacity, and the fermentation of undigested nutrients serving as substrates for intestinal microorganisms. Intestinal pH has been found to be reduced under unfavorable conditions such as heat stress and NE [
40,
41,
42], which is consistent with the results of the present study. Tannic acid addition did not significantly affect ileal and cecum chyme pH in broilers with NE infection. The effects of NE and tannic acid on dynamic metabolites of the intestinal flora (e.g., short-chain fatty acids) need further investigation.
The intestine is an effective barrier against pathogenic bacterial infection [
43]. The intestinal mucosa and tight junctions are disrupted during intestinal inflammation, thereby increasing intestinal permeability [
44,
45]. In the present study, serum D-LA and DAO concentrations were higher in broiler chickens with NE, suggesting that NE impairs intestinal barrier integrity. This is consistent with the results of a previous study [
23,
46,
47]. The decline in serum D-LA and DAO concentrations observed in broilers after the addition of tannic acid, indicating the potential positive impact of tannic acid on maintaining intestinal barrier integrity in broilers with NE. Similarly, a recent study demonstrated that the inclusion of chestnut tannins in the diet significantly reduced serum DAO concentrations in broilers subjected to heat stress [
48]. Liu et al. found that dietary addition of different tannins could increase gene expression of
zonula occludens-1,
Claudin-1, and
Occludin in broiler jejunum [
31]. Yu et al. revealed that the addition of tannic acid to the diet of weaned piglets increased the mRNA expression concentrations of
Occludin and
zonula occludens-1, leading to enhanced intestinal barrier function and reduced concentrations of serum DAO and D-LA [
49]. These studies have demonstrated the positive effects of tannic acid on intestinal barrier function; however, the specific mechanisms need to be further investigated.
In order to investigate the impact of tannic acid on immunity in broiler chickens with NE, we assessed the expression of relevant pro- and anti-inflammatory factors in both the intestine and serum. Proinflammatory and anti-inflammatory cytokines interact with each other, and their dynamic balance plays a crucial role in the development and outcome of inflammation. Previous studies have demonstrated that NE can induce inflammation in the intestine and the whole organism, leading to impaired intestinal health and reduced productivity [
50,
51,
52,
53,
54]. Consistent with these findings, our study revealed that NE triggered inflammatory responses in both the intestine and the organism. Following NE infection in broiler chickens, the proinflammatory factor TNF-α increased and the anti-inflammatory factors IL-4 and IL-10 decreased, disrupting the dynamic balance and promoting inflammation. However, we observed that the addition of tannins decreased the concentrations of proinflammatory factors and increased the concentrations of anti-inflammatory factors, suggesting a potential anti-inflammatory effect of tannins. In support of this, Park et al. demonstrated that condensed tannins extracted from blackberry seeds inhibited nitric oxide production in lipopolysaccharide-induced macrophages [
55]. Liu et al. reported that hydrolyzed tannins could reduce ear swelling and inflammatory responses in mouse models of arthritis [
56]. Peng et al. demonstrated that tannin improved the immune function of broilers and inhibited liver inflammation by blocking the TLR4/NF-κB pathway [
57]. Furthermore, our previous study indicated that the addition of tannins reduced the concentrations of MPO and CRP in serum [
58]. The immunomodulatory effects of tannins may be attributed to their complex structure. Additionally, our previous research indicates that tannic acid may influence the immune response in the intestine by inhibiting the concentrations of
Clostridium perfringens, modulating the intestinal microbiota, and reducing intestinal pathogen-induced stimulation [
58]. The protective effects of tannins after NE infection in broiler chickens may be associated with the enhancement of anti-inflammatory function; however, the exact underlying mechanism requires further investigation.
Complex microorganisms in the gut play a key role in nutrition and intestinal health. NE is an intestinal disease that can disrupt intestinal microbial homeostasis and lead to intestinal ecological dysbiosis. To elucidate the mechanism by which tannic acid improves intestinal health, we further analyzed the microbes in the cecum. We observed that cecum microbial α-diversity was significantly decreased and β-diversity was significantly altered after NE infection compared with the NC group. These results are consistent with those of previous studies [
59,
60,
61], and indicate that NE significantly alters the gut microbiota. In our experiment, the relative abundance of Clostridia as well as
Clostridium sp. in the cecum flora of NE broilers was elevated, which may be caused by NE-inducing changes in the gut microbiota [
60]. Although the relative abundance of Clostridia was elevated, due to the limitations of the 16S assay we could not determine whether this was
C. perfringens CVCC52. In our previous study [
58] we quantified the abundance of
C. perfringens in the cecum and found that its abundance was significantly elevated in NE-infected broilers. In addition,
Oscillospira is a dominant genus in the PC group, which was observed by Tang et al. as well [
21]. The increased abundance of
Oscillospira is possibly associated with intestinal inflammation [
62]; its increased abundance has additionally been positively correlated with reduced body weight [
63,
64].
Streptococcus is a major strain of colorectal cancer [
65]; we found that its abundance was significantly increased in broilers with NE. Therefore, NE is likely to elevate the relative abundance of Clostridia,
Streptococcus, and
Oscillospira in the cecal microbiota of broiler chickens. These findings indicate the significance of these bacteria in contributing to the adverse consequences associated with NE. In contrast, the NC group exhibited a predominance of beneficial bacteria such as
Lactococcus and Rikenella, which are known for their production of short-chain fatty acids, in comparison to the PC group [
66].
Ruminococcaceae are part of the natural intestinal flora of chickens; they helps with sugar absorption by cells, produce short-chain fatty acids, and inhibit the growth of
C. perfringens bacteriocins [
67], which is beneficial in maintaining intestinal health and may lead to weight gain [
68,
69,
70]. Studies have reported that abundance of
Faecalibacterium is reduced in the intestinal flora of broiler chickens with coccidial infection or heat stress [
71,
72].
Faecalibacterium can produce butyrate, which is associated with the maintenance of intestinal health [
73].
Bacteroides_dorei reduces lipopolysaccharide production in the intestine and effectively inhibits the proinflammatory response [
74]; simultaneously, it can promote the proliferation of
Lactobacillus and
Bifidobacterium [
75]. Our study suggests that the increased abundance of
Ruminococcaceae,
Faecalibacterium, and
Bacteroides_dorei following the addition of tannic acid is a contributing factor to the observed improvement intestinal health. Our findings indicate that NE disrupts the balance of intestinal flora and that the enhancement of beneficial bacterial growth associated with tannic acid supplementation may contribute to the amelioration of NE symptoms and restoration of intestinal health.