In the poultry industry, antibiotics have been used for long years as a feed additive to promote growth and control disease. However, recently, its use has been banned due to the risk of antibiotic-resistant microbes, residues in animal products, and environmental pollution (1). Therefore, there is an urgent need for antibiotic substitutes that can replace its functions while surmounting its shortcomings. In this regard, dietary fibers such as polysaccharides are known to improve health and homeostasis by enhancing the intestinal immune response and gastrointestinal barrier function (2). However, human and animal enzymes are unable to digest dietary fibers and are subject to fermentation by the gut microbiota (3). The gut microbiota lives in a symbiotic relationship with the host, in which the host provides habitat and nutrients for their growth, while the microbiota provides essential nutrients via the fermentation of fibers. Therefore, the dynamic diet-microbiota interactions shape the health and immune response of the host (4).

In chicken, caecum is the main organ that harbors a vast diversity of microbes responsible for the fermentation of fiber (5). These microbes possess polysaccharide-degrading enzyme-encoding genes and pathways involved in the production of short-chain volatile fatty acids (SCFAs), which are beneficial to the host physiology and energy homeostasis (6). The microbiota-derived SCFAs play an important role in maintaining an intestinal immune response, barrier function, and immune metabolism via the activation of metabolite-sensing G-protein coupled receptors (GPCRs) (7). Apart from metabolites production, gut microbiota plays a decisive role in maintaining the homeostasis and health of the host via direct involvement in gut structure and morphology, regulating immune responses, and protection from luminal pathogens (8). Therefore, changes in the type of polysaccharides consumed are expected to alter the composition and function of the microbiota, thereby altering the host immune system. Thus, understanding how a given microbial population responds to polysaccharides diet, and the role and association of this microbiota and its metabolites with immune response of the host play crucial roles to develop polysaccharide-based functional foods to prevent and treat gut microbiota-related diseases.

Enteromorpha prolifera (E. prolifera) is a seaweed green alga with a long history of use as food and traditional medicine. A sulfated polysaccharide is one of the main biologically active substances in E. prolifera, which is responsible for the immunomodulating, hypolipidemic, antitumor, anti-aging, antibacterial, anticoagulant, antiviral, and anticancer activities of these algae (9–12). E. prolifera polysaccharides (EP) are made up of α- and β-(1, 4)- linked monosaccharides (rhamnose, xylose, and glucuronic acid) (13), where the sulfate group is attached at the C-3 position of rhamnose (14). Recent studies indicated increased production performance, breast muscle yield, egg quality, antioxidant capacity, and intestinal morphology of chickens fed seaweed polysaccharides (15–18). In addition, EP supplementation increased the weight and differentially regulates the gene expression at the transcriptome level in the bursa of Fabricius of Arbor Acres chickens (19). Similarly, EP supplementation has been shown to improve the growth performance, non-specific immunity, and intestinal function of banana shrimp F. merguiensis fish (20). Furthermore, administration of EP was found to regulate intestinal microbiota in mice (21) and fecal microbiota in humans (22). Our recent study showed that supplementation of diet with EP- zinc complex could reduce diarrhea rate and improved intestinal barrier function in piglets (23). Although the biological activities of EP have been well-established so far, their interactions with gut microbiota in broiler chickens are largely unknown.

Considering the above information and the fact that polysaccharides are digested by the intestinal microbiota, we designed this study to investigate the effects of EP supplementation on growth performance, immune response, intestinal integrity, and gut microbiota in broiler chickens.

Apart from its use as a food and traditional medicine, recent studies have shown that sulfated polysaccharides from marine algae exerted various biological activities, such as immunomodulation, antioxidant, antidiabetic, and hypolipidemic. In the present study, we supplemented the broiler diet with polysaccharides isolated from marine algae EP to evaluate its effect on growth performance, immune response, intestinal barrier function, and caecal microbiota.

Herein, chickens fed EP exhibited higher body weight and average daily gain than chickens fed only basal diet, suggesting that EP supplementation to broiler diet could improve growth performance. Similar conclusions were made by Liu et al. (16) and Li et al. (30), reported that algae-derived polysaccharide could enhance growth performance in chicken. Likewise, dietary supplementation of EP was found to improve growth performance in mice (31) and crucian carp (32). The growth-promoting effect of EP may be associated with its enhancement of nutrient absorption by improving intestinal function and morphology (31).

Research evidence has shown that polysaccharide from E. prolifera has an immunomodulatory activity (33). Cytokine profiling is a valuable tool for monitoring immune responses associated with inflammation and immunity. In this study, supplementation of diet with EP induced a profound change in the serum cytokine contents of broilers as evidenced by significantly higher IL-1β, IL-2, IFNα-, and IFN-γ levels in the supplemented group. This study is consistent with the previous study that reported EP could stimulate proinflammatory cytokine production in mice (34). The results of the present study also support the idea that EP has an immunomodulatory activity by stimulating proinflammatory cytokines.

To better understand whether the changes in cytokine levels were accompanied by changes in the expression of inflammatory factor genes, we detected mRNA expression in the jejunum and ileum tissues. We found upregulated mRNA expression levels of IL-1β and TNF-α in the ileum and jejunum, and IL-2 in the ileum in the EP group compared with the control group. In contrast, Liu et al. (35) reported that algae-derived polysaccharides down-regulated the mRNA expression of TNF-α, and IL-6 in the bursa of Fabricius of heat stress chicken. The discrepancy of our study with the previous study could attribute to the difference in immune status and stress condition of chicken.

More importantly, the pro-inflammatory pathway is transcriptionally regulated by NF-κB, and the up-regulation of NF-κB activates the immune response and cytokine production. The TLR4/MyD88 signaling pathway is the upstream gatekeeper of NF-κB (36). The present data showed that the expression of TLR4, MyD88, and NF-κB in ileum and jejunum were significantly higher in chicken-fed EP than in chicken-fed only basal diet. TLR4 is a family of pathogen recognition receptors (PRRs) that orchestrate the host immune system through MyD88 to induce pro-inflammatory cytokines via NF-κB (37, 38). As the downstream of the TLR4 and interleukin-1 (IL-1) receptor, MyD88 activates NF-κB, and thereby the inflammatory signaling pathways (39). Therefore, the present study indicated that EP supplementation activates the TLR4/MyD88/NF-κB signaling pathway, thereby induces an immune response. This observation further supported the viewpoint that EP has immunomodulatory activity. Similarly, Wei et al. (33) reported upregulation of NF-κB transcription factors in mice that received EP. In contrast, a study on oxygen-glucose deprivation-induced human cardiac microvascular endothelial cells showed that EP inhibits proinflammatory factors expression through the up-regulation of HIF-1α and inactivation of the NF-κB pathway (40). This may be due to EP might play both immunomodulatory and anti-inflammatory roles depending on the physiological condition of experimental animals or cells.

Intestinal morphology affects nutrient absorption in the body. The longer intestinal villi and lower crypt depth are an indicator of higher nutrient absorption (41). The present study demonstrated that EP supplementation increased villus height and reduced crypt depth in the ileum and jejunum of supplemented chicken. The longer villus height and higher villus height: crypt depth ratio is associated with active cell mitosis, improved nutrient digestibility, and absorption in chickens (42, 43). Thus, the present study suggests the beneficial effects of EP supplementation on improving intestinal morphology. These results are in agreement with the study by Liu et al. (16), who reported villus height and villus height: crypt depth was higher in chickens fed algae-derived polysaccharides. Similar results were obtained by Guo et al. (15), who showed that marine-derived polysaccharide enhanced jejunal villus height and villus height/crypt depth ratio in layer chickens.

The mucus layer of the epithelial cells is an essential first line of defense that forms a barrier to prevent the penetration of the epithelium by gut microorganisms (44, 45). In addition, epithelial barrier integrity is maintained by the tight junction proteins, including occludin, claudins, and zonula occludens. Herein, we examined the effects of EP supplementation on intestinal barrier function by measuring the mRNA expression of mucin-2 and tight junction proteins. Our data demonstrated that the mRNA expressions of mucin-2 and occludin 1 in the ileum and jejunum were upregulated in the EP supplemented chickens. mucin-2 plays an important role in the secretion of mucus, which is a layer that protects the epithelial cells from exposure to the microbiome (46). Occludin provides structural integrity and assembly of tight junction and knockdown of occludin induces an increase in paracellular permeability to macromolecules (47). Claudins are also playing an essential role in barrier formation and paracellular permeable selectivity (48). Therefore, an increase in the expression of mucin-2, occludin 1, and claudin 1 in the intestinal segments in this study, indicates EP supplementation improved intestinal integrity in broiler chickens via regulating mucin-2 and tight junction protein. This is partly explained by the fact that Enteromorpha polysaccharides may be directly recognized by the pattern recognition receptors of intestinal epithelial cells (31, 49), leading to the activation of mucin-2 and tight junction proteins, thereby improving intestinal integrity. This idea is reinforced by the mRNA expression results of TLR4 and TLR2 in jejunum and ileum. In addition, EP was fermented by caecal microtia and produced SCFA, which could stimulate intestinal goblet cells to secrete mucin-2 through their action on NOD-like receptor family pyrin domain containing 6 (NLRP6) and G protein-coupled receptors (GPCRs) (50).

In broiler chickens, TLR2 is the principal receptor for peptidoglycan from gram-negative and gram-positive bacteria (51). Toll-like receptor 2 signaling has been implicated in preserving intestinal barrier integrity and is considered to be a crucial target for therapeutic intervention of metabolic and inflammatory conditions (52). In the present study, we found a significant increment in the mRNA expression of TLR2 in the ileum tissue of chicken fed EP. This result indicates that apart from the mucin-2 and tight junction protein, TLR2 might also be involved in the improvement of intestinal integrity observed in this study.

The fermentation of fiber diet by gut microbiota produced metabolites such as short-chain volatile fatty acids. These microbiota-derived short-chain fatty acids play important roles in maintaining an intestinal immune response, barrier function, and immune metabolism either by modulating gene transcription or via the activation of metabolite sensing GPCRs (53). In the current study, we found that the SCFAs particularly acetate, butyrate, and propionate increased in the caecal content of EP supplemented chickens, suggesting that EP enhanced the ability of the microbiota to induce SCFAs. Furthermore, we found significant correlations of differentially abundant gut microbes with acetate, propionate, and butyrate production, which further confirmed that the increase of SCFA observed in this study was due to the microbiota compositional shift. In agreement with the current study, mice fed EP showed an increase in SCFAs production in the colon (21). The SCFAs, particularly butyrate and propionate, provide energy for the immune cell by activating intestinal gluconeogenesis, thereby improving inflammatory and effector cytokines production and antigen presentation (54, 55). Furthermore, microbial-derived butyrate was found to promote mucosal barrier integrity by stimulating the production of mucin-2, tight junction proteins, and antimicrobial peptides (56). Therefore, the immune-modulatory role of EP in the current study might be via enhancing the gut microbiota-derived SCFA that contribute to intestinal immune response and gut barrier function.

A growing body of evidence suggests that gut microbiota is involved in the digestion and utilization of fibers such as polysaccharides, which otherwise cannot be utilized by the host. It is well-established that diet and nutritional factors have a direct effect on the microbial colonization of the gut (57–59). In this study, we conducted 16S-RNA sequencing from the caecal content of chickens fed EP to investigate microbiota dysbiosis. We found that the abundances of phylum Bacteroidetes and genera Bacteroides were increased, whereas phylum Firmicutes and genus Faecalibacterium were decreased in chickens fed EP, suggesting that Bacteroides might involve in the digestion of E. prolifera polysaccharides. This may be explained by the fact that Bacteroides possess genes encoding carbohydrate-active enzymes (CAZymes) within their polysaccharide utilization loci (PUL) (60, 61), which could confer them with the strong ability to ferment diverse types of dietary polysaccharides (62). Apart from polysaccharide fermentation, Bacteroides can also affect host immune system development (63), and maintenance of gut microbial balance (64). The present study is concurrent with the previous study that showed E. prolifera polysaccharides supplementation caused microbiota dysbiosis in mice (21) and rabbitfish S. oramin (65). These findings suggest that EP supplementation shifts microbiota composition, particularly Bacteroides, which produce SCFA that involve in immune response and gut barrier function. Notably, the species Bacillus_licheniformis, Auraticoccus_monumenti, and Alkalibacillus_haloalkaliphilus were biomarkers in the EP group. Previous studies reported that B. licheniformis isolated from a human fecal sample can be used for manufacturing biochemicals, enzymes, antibiotics, and aminopeptidase (66, 67). Alkalibacillus_haloalkaliphilus is used in enzyme synthesis, organic acid, food biotechnology, biodegradation, and antibiotics (68) and can convert carbohydrates to organic acids such as acetic acid (69). However, further study is needed to confirm their specific roles in the fermentation of polysaccharides in the intestine.

We concluded that EP inclusion in chickens' diet improves growth performance, enhances intestinal immune response and integrity, and modulates the caecal microbiota of broilers. This study suggests the application of EP as an alternative to antibiotics in chicken and also provides insight to develop marine algae polysaccharide-based functional food and therapeutic agent.

The original contributions presented in the study are publicly available. This data can be found here: https://www.ncbi.nlm.nih.gov/bioproject/765947.

The animal study was reviewed and approved by Animal Care and Use Committee of the Institute of Subtropical Agriculture, Chinese Academy of Sciences, Hunan, China.

TW: conceptualization, investigation, data generation, curation, and original draft preparation. XD, KG, and ZY: data generation and curation and editing. ZL: methodology, investigation, data generation, curation, and reviewing. YY, CX, and XW: conceptualization, supervision, validation, reviewing, and editing. All authors contributed to the article and approved the submitted version.

We would like to acknowledge the earmarked fund for National Natural Science Foundation of China (31902196), China Agriculture Research System (CARS-35), China Postdoctoral Science Foundation-funded project (2021M693383 and 2019M662273), and Taishan industry leading talent blue talent project for their financial support.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.