In this study, we investigated the modulating effects of different tannic acid additive levels on the intestinal health of broiler chickens co-infected with coccidia and C. perfringens. NE infection was found to significantly reduce the body weight and F/G of broilers, and their small intestines showed remarkable pathological changes, such as intestinal congestion, red bruises, thinning of the intestinal wall, and intestinal distension. The intestinal section analysis further showed damage to the villi structure in the PC group, which indicated the successful establishment of the necrotizing enteritis model and was consistent with the results of previous studies [15, 16]. Regarding the intestinal barrier genes as well as nutrient transporters, most mRNA expression in the PC group showed negative effects. Previous studies have reported that NE reduces growth performance and elevated serum ET [15], which is consistent with our findings. In addition, the serum levels of zonulin levels were elevated, further indicating the successful establishment of NE model. Tannic acid addition to the feed had beneficial effects on the broilers with NE and resulted in reduced intestinal lesion scores, increased villus height, and improved intestinal morphology.
Effects of tannic acid addition to the diet on the growth performance of broiler chickens with NE
Results showed that tannic acid in the feed could ameliorate the growth performance degradation caused by NE, which is consistent with the results of previous studies. Yang et al. observed better F/G ratios when broilers were fed 7.5 to 15 mg/kg proanthocyanidins [17]. Wang et al. investigated the effects of addition of 5–80 mg/kg grape seed extracts to the coccidia-challenged diet and found that 10–20 mg/kg significantly improved the growth performance [18]; these results were consistent with those of this study. However, some studies have shown that adding 20 g/kg tannic acid significantly reduces body weight gain and the feed conversion efficiency in broilers [10].Jamroz et al. added 0.025–0.1% sweet chestnut tannin to the diet and found that < 0.05% had no effect on growth performance, whereas 0.1% significantly reduced growth performance [19]. These findings suggest that the biological effects of tannins are highly dose-dependent, and the inconsistent effects of tannins in the literature may be due to differences in their source, dose, extraction process, encapsulation, and basal diets. In the present study, the growth performance showed a quadratic curve that increased with the addition of tannic acid; 500–750 mg/kg tannic acid was found to be the optimal concentration for improving the growth performance. The reason dietary tannic acid improves the growth performance of NE may be that it enhances the intestinal barrier, improves intestinal morphology, balances microbiota, and improves nutrient absorption capacity.
Effects Of Tannic Acid On The Intestinal Morphological Structures Of Broiler Chickens With Ne Infection
A normal villus morphological structure is a prerequisite for the intestine to perform its absorption function. An increase in villus height may lead to enhanced nutrient absorption, and a lower crypt depth indicates a decrease in the metabolic cost of intestinal epithelial renewal. Our findings suggested that NE significantly increased crypt depth, indicating that there was faster tissue renewal for the villi, as needed in response to the inflammation caused by pathogens or their toxins, which is consistent with previous findings [11, 20]. The addition of tannic acid significantly reduced the increase in the depth of intestinal crypts caused by NE and improved the V/C ratio, demonstrating the beneficial effects of tannic acid on the intestinal morphology. The V/C showed a quadratic curve change with the addition of tannic acid, which is consistent with our results regarding the growth performance, as 500–1000 mg/kg tannic acid was highly effective at ameliorating the intestinal morphology.
Effects of tannic acid on the fecal coccidia oocyst excretion in broiler chickens with NE infection
Examining coccidia oocyst shedding is an effective method to determine the level of coccidia infection [21]. The addition of tannic acid reduced the number of coccidia oocysts shed compared to that in the PC group, which is consistent with the results of previous reports [18, 22, 23]. The reduction in the number of oocysts in the feces indicated an increase in coccidia resistance in broilers. In the present study, the amount of excreted coccidia oocysts decreased linearly with an increase in tannic acid concentration. The decrease in the number of coccidia oocysts after tannic acid addition could be attributed to the modulation of intestinal microorganisms by tannic acid and its own antiparasitic biological properties [24, 25]. Taken together, tannic acid supplementation reduced coccidia proliferation, significantly reduced the number of coccidia oocysts excreted in feces, accelerated coccidia clearance, and improved intestinal health.
Effect Of Tannic Acid On The Immune Indexes In Broiler Chickens With Ne
In this study, NE infection resulted in impaired intestinal morphology and elevated lesion scores, whereas the addition of tannic acid to the feed alleviated these changes. This process was accompanied by changes in the serum levels of IgM, MPO, CRP and C. perfringens-specific antibodies in broilers. IgM is a circulating antibody secreted by B cells that first responds to initial encounters with foreign antigens; however, IgM concentrations in the blood rapidly decline due to clearance, which is consistent with our study findings, as IgM levels were found to be elevated during the first days of infection with NE and did not change significantly seven days after the infection. CRP is an acute temporal protein that activates complement and enhances phagocytosis, and MPO is mainly found in myeloid cells; both of these are markers of the inflammatory response. In this study, the increased MPO and CRP activities in the serum of the PC group indicated that NE infection could activate immune cells in the blood and promote inflammation. The addition of tannic acid to the feed reduced the inflammatory response caused by NE and the concentration of C. perfringens-specific antibodies in the serum, which may be related to the anti-inflammatory and proliferation inhibitory properties of tannic acid [4, 7]. The results of this experimental study showed that NE infection exerted long-term effects on broiler chickens, including reduced intestinal barrier function seven days after infection, continued high levels of MPO and CRP in the serum, and inferior growth performance compared to those in the NC group.
In innate immunity, the intestinal mucosa is considered the first line of defense against pathogen infection, and mucosal immunity plays an important role in NE. The results of the present study showed that NE infection significantly damaged the ileum and stimulated the expression of sIgA in the intestinal mucosa, which is consistent with the results of a previous study [11]. We found that tannic acid reduced the sIgA secretion in the ileal mucosa induced by NE, which might be because tannic acid significantly reduced the number of coccidian merozoites and the colonization of C. perfringens. In addition, we observed that TLR2 was activated and inflammatory factor expression was increased in NE, consistent with previous studies [26, 27]. We found that tannic acid addition reduced the number of C. perfringens and coccidia in the intestinal tract by stimulating TLR2 and thus activating the immune system, on the one hand, and by inhibiting the number of C. perfringens and coccidia, on the other hand, thus reducing the inflammatory response.
Effects of tannic acid addition to the feed on mRNA expression of the gut barrier genes and nutrient transport carriers in chickens with NE
The intestine plays key roles in defense against pathogens, host nutrient digestion, and absorption [28]. Numerous studies have shown that the mRNA expression of tight junctions and nutrient transport carriers decreases and nutrient digestion and absorption are diminished during intestinal inflammation [29–32]. Intestinal inflammation results in a reduction in mucin synthesis and the number of cupped cells, increasing the chances of further intestinal infection and bacterial translocation. In this study, NE led to a reduction in the mRNA expression of Occludin, ZO-1, and MUC2, causing an increase in intestinal permeability. However, the mRNA expression of intestinal tight junctions and MUC2 was increased with the addition of tannic acid compared to that in the PC group, indicating the positive effects of tannic acid in protection of chickens from NE.
Zonulin is the only known regulator of tight intercellular junctions and is involved in the regulation of intestinal barrier function. Studies have also shown that zonulin levels in the serum are remarkably elevated in patients with irritable bowel syndrome or Crohn’s disease [33]. However, the changes in the sensitivity to zonulin in poultry intestinal diseases require further investigation. In the present study, NE led to a significant increase in zonulin levels in the serum, indicating that the intestinal barrier permeability was increased. ET is a gram-negative bacterial cell wall component, and serum ET levels are elevated in broiler chickens suffering from NE [34, 35], which was also confirmed in our study. The addition of tannic acid to the feed increased the mRNA expression of Occludin, ZO-1, and MUC2 and improved the integrity of the intestinal barrier, thereby reducing zonulin and ET levels. Nevertheless, the upregulation of intestinal tight junction-related pathways by tannins requires further investigation.
I-FABP is a cytoplasmic protein located in mature cells at the top of intestinal villi, and it plays an important role in fatty acid uptake and metabolism. We observed that the mRNA expression of I-FABP was reduced in broiler chickens infected with NE, which was consistent with the results of a previous study [36]. However, it has also been shown that the mRNA expression of I-FABP does not change significantly in the dexamethasone-induced intestinal disorder model or infection with Campylobacter jejuni [37, 38], which might be due to the different disease models and severity. In addition, we observed a decrease in the mRNA expression of PEPT1 and SGLT1 in broiler chickens infected with NE, which is consistent with the results of a previous study [39]. Addition of tannic acid improved the mRNA expression of nutrient transporter carriers and reduced the negative effects of NE on nutrient absorption in chickens. A comprehensive analysis revealed that growth performance, intestinal villus height, crypt depth, V/C, and nutrient transporter expression showed relatively consistent quadratic curve changes after tannic acid was added to the diet. The reason for this is that lower doses of tannins can reduce gastrointestinal peristalsis, thereby increasing the residence time of chyme in the gastrointestinal tract and improving nutrient digestibility and intestinal health. However, high doses of tannins exert a negative impact on growth performance by binding starch and protein in the feed, reducing digestive enzyme activity, and binding to intestinal wall proteins [40, 41].
Effect Of Tannic Acid On The Ileal Microflora Of Broilers With Ne
The intestinal microbiota forms a highly complex microecosystem. To further investigate the mechanisms by which tannic acid alleviates the intestinal damage caused by NE, we analyzed the microflora structure of the ileal microorganisms using 16S rRNA sequencing. The results of this study showed that the challenge did not have a significant effect on the microbial α-diversity, which is consistent with the results of previous studies [42, 43]. We speculated that the possible reason for this result was that NE infection inhibited the proliferation of minor microorganisms in the broiler intestinal flora, which resulted in a convergence with the α-diversity of the NC group microbiota. However, NE has also been shown to result in lower or higher gut microbial α diversity [15, 44], which may be related to the collection of different parts of the chyme. In the present study, addition of tannic acid significantly reduced the α-diversity but did not affect the balance abundance of the flora, which is consistent with a previous study [45]. It has been shown that low-abundance microbes lead to the formation of simpler metabolic networks, resulting in increased concentrations of specific components used to support host energy requirements [45]. Shabat et al. reported that a reduced microbiome abundance may be closely associated with an increased feed conversion efficiency, with specific enrichment and metabolic pathways of microbes leading to better energy and carbon delivery to the animal body [46], which could be one of the reasons tannic acid improves growth performance. The significant difference in β-diversity among the three groups indicated that the addition of tannins or challenges significantly altered the microbial community structures. Bacteroidetes are an important contributor to intestinal health, and this phylum plays an important role in breaking down complex molecules into simpler compounds and producing short-chain fatty acids that are beneficial for increased growth performance [47, 48]. We found that NE led to a decrease in the abundance of Bacteroides, which is consistent with the results of a previous study [43]. The fermentation products of Bacteroidetes, such as Bacteroides fragilis, inhibit C. perfringens spore formation, and the decrease in Bacteroidetes may predispose the animal intestine to C. perfringens infection and gastroenteritis [49], which may be one of the reasons for the decrease in Bacteroidetes abundance in NE. Lately, several studies have suggested that stressed birds present a significantly increasing trend for the Firmicutes/Bacteroides ratio [50, 51]. In this study, we found that the high Firmicutes/Bacteroidetes ratio caused by NE may be one of the reasons for dysbiosis of the gut microbial community.
The results also showed that at the genus level, NE significantly reduced the abundance of the beneficial bacterium Ligilactobacillus, suggesting that NE infection inhibited the growth of beneficial intestinal bacteria. At the species level, NE significantly reduced the relative abundance of Lactobacillus salivarius, which is a probiotic in poultry that dates back more than 15 years. Furthermore, this bacterium can re-establish the proper microbial balance of bacteria by forming lactic and propionic acid, stimulate butyric acid-producing butyric acid production, and inhibit the production of pro-inflammatory cytokines [52]. In broilers, Lactobacillus_salivarius was found to improve growth performance, reduce the expression of inflammatory factors, and improve intestinal health [9, 53, 54]. In conclusion, in this study, we found that the intestinal flora of broiler chickens infected with NE was disturbed and the abundance of beneficial bacteria was reduced, leaving the intestine in an unhealthy state. The addition of tannic acid after the challenge significantly increased the relative abundance of the genus Anaerocolumna, which belongs to the Lachnospiraceae and can be metabolized by the fermentation of carbohydrates into acetate [55]. In addition, the relative abundance of Thermoanaerobacterium, which can secrete xylanase, degrade hemicellulose and even cellulose, and produce acetate and butyric acid via fermentation using xylan and others, was increased [56]. The genus Thermosinus includes gram-negative bacteria that can convert CO to carbon dioxide through a series of chemical reactions and can decompose organic substrates (glucose, sucrose, or lactose) to produce acetic acid as well as acetate, H2, and CO2 during glucose fermentation [57]. Thus, it regulates the intestinal pH, nourishes butyric acid-producing bacteria, protects against pathogens, and contributes to intestinal health. In conclusion, the addition of tannins increases the abundance of short-chain fatty acid-producing-related flora, which may be one of the reasons for their beneficial impacts on intestinal health.
In the PICRUSt analysis, we found that differences in the metabolic pathways were the most common in this study, especially in the PC group, where there were large metabolic differences when compared with the other two groups. The present study showed that nutrient metabolism in the intestinal flora of broilers with NE was generally downregulated, and the pathways related to intestinal flora metabolism were more enriched in the NC group, which was consistent with previous findings [43]. Bacteroidetes are the main carbohydrate-degrading bacteria in the intestine that contribute to the degradation of complex pectins. Therefore, the enrichment of the carbohydrate metabolic pathway in the NC group may be due to the high abundance of Bacteroidetes, which degrade complex polysaccharides and indigestible carbohydrates to generate short-chain fatty acids and maintain intestinal health [58]. Microbial catabolism of amino acids produces a large number of byproducts, including amines, phenols, indoles, and short-chain fatty acids [59], which can have beneficial or detrimental effects on epithelial cells, depending on the concentration of metabolic byproducts in the lumen. For example, aromatic amino acids are metabolized by colonies to produce tryptamine and indoles. Indoles play an important role in host defense by enhancing intestinal barrier function and downregulating the proinflammatory cytokines [60]. We speculated that the healthier gut in the NC group may be related to the products of carbohydrate and amino acid metabolism formed by the intestinal flora. Bacteroides, Propionibacterium, Fusobacterium, Lactobacillus, and Streptococcus play important roles in the protein hydrolysis process [61], and the downregulation of amino acid metabolic pathways in the PC group of microorganisms may be due to a decline in Bacteroides and Lactobacillus abundance. In the PC group, genes related to disease immunity, metabolic diseases, and energy metabolism were upregulated, which suggested that during the infection of chickens with C. perfringens, the intestinal flora competed with the host for energy, and more energy allocation was required for stimulation of the immune system, which may have resulted in the reduction in chicken growth performance. According to this study, the microbial communities in normal chickens were relatively healthier, and the ileal flora structure and function of the tannic acid groups were predicted to be similar to those of the NC group chickens. Therefore, we conclude that the addition of tannic acid could alleviate ileal microflora disorder in broiler chickens with NE. Taken together, the mechanism underlying the effect of tannic acid on intestinal health may involve changes in the intestinal microflora; however, relevant metabolomic analyses are still required to explore the effects of tannic acid on metabolites, which can further help us to establish the relationship among tannic acid, intestinal flora, metabolites, and intestinal health.
To investigate the correlation between intestinal flora and growth performance, immunity, and mRNA expression of intestinal barrier genes and nutrient transport carriers, simple correlation analysis was performed. Correlation analysis can indicate the correlation between indicators but cannot determine a causal relationship. The results suggested that the abundance of Lactobacillus salivarius was positively correlated with growth performance and significantly negatively correlated with MPO, CRP, ET, and zonulin, which may be because Lactobacillus salivarius can inhibit inflammation [62, 63] and improve intestinal barrier function [41]. The increase in the abundance of Thermosinus carboxydivorans and Thermosinus after the addition of tannic acid was significantly and positively correlated with the mRNA expression of GLUT2, which suggested that the regulation of intestinal flora by tannic acid may be related to nutrient translocation and absorption. However, the specific mechanisms require further investigation.