Generally, the growth performance of broilers is highly dependent on their intestinal health, which is influenced by a variety of factors, such as diet, stress and infection. These factors may increase the intestinal permeability of broilers and compromise the structural integrity of their intestinal epithelium, resulting in an increase in inflammatory responses and ultimately the restriction of normal growth (1). In recent years, because of the ban on antibiotics in poultry feed, unspecified intestinal diseases have emerged in poultry production (2). Therefore, new alternatives are urgently required to reduce the incidence of intestinal disease and increase the production efficiency of broilers. Among these potential alternatives, probiotics, owing to their ecofriendly and pollution-free characteristics, are typically fed directly to inhibit the growth of pathogens and regulate their intestinal function of broilers (2, 3). Studies have demonstrated that probiotics could prevent pathogenic bacteria from adhering to the intestinal mucosa and hence improve the microecological environment of the gastrointestinal tract in order to optimize the intestinal function of broilers, thereby enhancing their growth performance (4, 5). Hence, dietary supplementation with probiotics is regarded as an effective method for enhancing the growth performance and enteric diseases resistance of broilers.

Clostridium butyricum CBM 588 is a typical probiotic strain that produces spores that are resistant to high temperature, humidity, stomach acid and bile salts, indicating its high adaptability to the intestinal environment (6). C. butyricum metabolites contain digestive enzymes that can break down large molecules of nutrients into small molecules that can be easily absorbed, thereby enhancing the utilization efficiency of these nutrient substances and ultimately promoting the growth performance of the host (6). As a source of energy, short-chain fatty acids (SCFA), another metabolite of C. butyricum, not only retard the growth of pathogenic enteric bacteria, but also promote intestinal development. Studies have indicated that the addition of C. butyricum to the diet of broilers could increase their intestinal digestive capacity and improve their intestinal morphology, which in turn enhances their growth performance (7, 8). Liao et al. (9) reported that C. butyricum treatment increased the concentrations of polyunsaturated fatty acids in the meat and improved the meat quality of broilers. In addition, C. butyricum was found to positively affect the immune response of broilers, which could promote the secretion of anti-inflammatory factors and immunoglobulins (10, 11). A recent study on Pekin ducks revealed that dietary C. butyricum supplementation increased the SCFA content in the gut and optimized the intestinal flora (12). Although the effects of C. butyricum treatment on the growth performance and intestinal health have been extensively documented in the literature on poultry production, the results remain inconsistent. Therefore, the further research on more targeted indicators is necessary to accurately evaluate the benefits of C. butyricum and explore the mechanism of through which C. butyricum regulates intestinal health. In addition, because of C. butyricum and its metabolites showing certain immuno–antioxidant properties in poultry (9, 13), we hypothesize that C. butyricum could regulate the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway (a key signaling pathway for regulating antioxidant capacity) and nuclear factor-κB (NF-κB) signaling pathway (a key signaling pathway for regulating inflammatory response). Accordingly, the present study investigated the effects of C. butyricum on the growth performance, meat quality and intestinal health of broilers to elucidate the potential mechanism though which C. butyricum regulates intestinal health.

The development of intensive production has predisposed poultry to diseases, which in turn inhibits their growth and development. Including probiotics such as C. butyricum in their diets is therefore regarded as an effective solution to this problem (16, 17). As the most reliable indicator of broiler growth and development, growth performance is the key to increase economic efficiency. According to a previous study, AA broilers fed with 1 × 10⁹ cfu/kg C. butyricum exhibited an increase in ADG and a decrease in F/G at 1–21 and 1–42 days, respectively (18). Nevertheless, Yang et al. (11) and Liao et al. (16) discovered that different doses of C. butyricum addition significantly increased the ADG of broilers at 22–42 d but did not considerably affect their feed conversion ratio. In the present study, we investigated the effect of C. butyricum on the growth performance of AA broilers. Our results indicate that although C. butyricum did not considerably affect the growth performance of the broilers at 1–21 days, it significantly improved their final BW and ADG at 42 and 1–42 days, respectively, which is consistent with the results of Yang et al. (11). These results suggest that C. butyricum treatment improves the growth performance of broilers, which may be due to the production of butyric acid, vitamin B, and amylase by C. butyricum to enhance the utilization of feed nutrients (19). In addition, different effects of C. butyricum supplementation have been observed in various studies, which could be attributed to some essential factors such as additive dosage, broiler breed and physiological stage, diet composition, and feeding management.

With the rise in living standards, consumers have become increasingly concerned with meat quality, especially in China. Meat quality is an accurate indicator of the physical and chemical properties of meat. In general, L^*, a^*, and b^* are used to evaluate the color of meat, which is a crucial indicator of the meat's freshness and quality (20). After broilers are slaughtered, the lactic acid is produced by glycolysis accumulates in the muscle tissue, resulting in a decrease in the pH value, protein denaturation, and a decline in the ability of muscle protein to bind water (21). Thus, the final pH and rate of pH decline in meat have a direct effect on the drip loss and ultimately on the water retention capacity and tenderness of the meat. Liu et al. (20) reported that C. butyricum treatment increased the pH_45min and a^* values but reduced the L^* value, drip loss and shear force of in Peking duck breast meat. In another study, Li et al. (22) dicovered that Partridge shank chickens fed with a synbiotic diet including C. butyricum exhibited lower breast muscle drip loss and cooking loss and higher a24h* and pH_24h values. In the present study, we discovered that C. butyricum increased the a24h* and pH_24h values but reduced the drip loss and shear force of breast meat, indicating an improvement of the freshness, tenderness and water retention capacity of the meat. These favorable results indicate that C. butyricum can enhance the meat quality of broilers.

Antioxidant enzyme activity and oxidation product concentration are reliable biomarkers for assessing the antioxidant status of animals. T-SOD is a crucial component of the enzyme system, which effectively scavenges free radicals and inhibits peroxidation. MDA is one of the final peroxidation products derived from membrane lipids. Its level is regarded as a crucial index for quantifying the degree of peroxidation (14). According to previous studies, C. butyricum could mitigate peroxidation and improve the antioxidant enzyme activity in vivo (13, 16, 23). Li et al. (18) reported that the addition of C. butyricum to the diets of broilers enhanced the activities of their serum T-AOC, GSH-Px and SOD. Zhan et al. (13) discovered that C. butyricum supplementation resulted in the improvement of T-SOD and GSH?Px activities in the serum of laying hens. Similarly, Han et al. (23) found that C. butyricum addition enhanced serum GSH-Px activity in weaned piglets. In the present study, we discovered that C. butyricum supplementation significantly decreased the serum MDA content in broilers at 42 d, indicating the lower level of lipid peroxidation. In addition, C. butyricum addition significantly increased the activities of T-SOD and T-AOC at 42 d, indicating an improvement of antioxidant capacity. Generally, an improvement in antioxidant capacity may reduce the degree of protein and lipid oxidative damage in muscles and prolong the shelf life of meat (14), further confirming that C. butyricum improves the quality of meat.

Digestive enzymes are important for the digestion of nutrient materials and their activities have a substantial effect on the feed conversion and growth performance of broilers. Previous studies have recorded that probiotics can produce digestive enzymes and enhance their activities in the intestine. According to a previous study, Cobb broilers fed with C. butyricum exhibited elevated jejunal amylase, lipase, and protease activity (10). Wang and Gu (24) discovered that the addition of Bacillus coagulans NJ0516 to the diets of broilers resulted in a significant increase in their duodenal protease and amylase activities. However, Rodjan et al. (25) reported that the supplementation of Bacillus spores did not significantly affect the digestive enzyme activity of Ross 308 broilers. Similar to the findings of Zhang et al. (10), we found that C. butyricum increased the jejunal lipase and trypsin activities in the broilers. These favorable results suggest that C. butyricum significantly improves the digestive function of broilers; this is attributable to their metabolism of SCFA, which may stimulate the secretion of digestive enzymes by digestive glands. However, the currently available information is inconsistent and limited, necessitating further evaluation of the underlying mechanism.

The intestine is an essential conduit for the exchange of materials between the external environment and the host environment. Intestinal morphology is a major factor in the evaluation of the intestinal status and is closely linked with the development of the intestinal epithelial structure, which is typically evaluated though the VH, CD and VH/CD. A decrease in CD and an increase in VH and VH/CD indicate a natural increase in epithelial cell turnover and a well-differentiated intestinal mucosa, suggesting an enhanced digestive and absorptive capacity (26). A previous study reported that C. butyricum treatment significantly improved the duodenal VH and VH/CD and reduced the duodenal CD in broilers (27). Cao et al. (28) found that broilers fed with C. butyricum exhibited an increased VH and a decreased CD in the ileum. Furthermore, a recent study revealed that the addition of C. butyricum to the diet of broilers infected with Clostridium perfringens increased their intestinal VH/CD and ameliorated the degree of their intestinal damage (29). In the present study, we discovered that C. butyricum considerably improved the duodenal VH and jejunal VH and VH/CD of the broilers, indicating an improvement in intestinal morphology. These positive results suggest that C. butyricum has a promotive effect on the development of the intestinal epithelial structure and thereby improves the intestinal morphology.

The intestinal barrier is crucial for protecting the host against infection and other diseases and maintaining normal physiological function. In addition to preventing the entry of luminal bacteria and dietary allergens into the mucosa, an intact intestinal barrier selectively regulates the entry of nutrients, ions, and water into the body. ZO-1 and occludin, the main tight junction proteins of the intestinal barrier, play a major role in regulating the integrity and permeability of the gut barrier by binding to the actin cytoskeleton and contributing to the formation of a tight junction structure. According to Li et al. (18), the addition of C. butyricum to the diets of broilers increased the relative expression levels of claudin-1 and ZO-1 in the gut. Liu et al. (30) reported that treatment with C. butyricum significantly increased the expression of occludin and ZO-1 in the jejunum of broilers at 42 d. A similar result was observed in our study; that is, after 42 d of feeding broilers with a diet containing C. butyricum, we discovered that the jejunal mRNA levels of ZO-1 increased, indicating an improvement in the broilers' intestinal barrier integrity.

With the exception of the intestinal barrier, intestinal health is inseparable from intestinal antioxidant status and immune function. As a major regulator, Nrf2 is a basic leucine zipper protein that regulates the expression of genes related to antioxidants or detoxifying enzymes and protects the body against oxidative stress (31, 32). In general, Nrf2 exists in an inactive form in the cytoplasm binding to its inhibitor: Kelch-like epichlorohydrin-related protein 1 (Keap1). In response to oxidative stress, the Keap1–Nrf2 complex dissociates, allowing Nrf2 to translocate into the nucleus and bind to the antioxidant response element. This results in the transcription of antioxidant genes such as heme oxygenase-1, SOD and CAT. Hence, activating the Nrf2 signaling pathway may increase the expression levels of antioxidant genes (33). In our work, we first found that C. butyricum supplementation in broilers resulted in a significant increase in the expression levels of Nrf2 and SOD1 in their jejunal mucosa, indicating an improvement in their intestinal antioxidant capacity. This positive finding may be attributable to the activation of the Nrf2 signaling pathway, which effectively protects the intestinal mucosa against oxidative stress damage (33). Moreover, NF-κB, a key transcription factor involved in immunity, can recognize, rapidly respond to, and transcribe a variety of proinflammatory cytokines and inflammatory mediators, such as TNF-α and ILs, resulting in an inflammatory crisis (34). Reduced expression of genes encoding proinflammatory cytokines, such as interferon-γ, IL-1β and TNF-α, may retard the activation of the NF-κB signaling pathway. Li et al. (18) demonstrated that C. butyricum increased the expression levels of IL-1β and TNF-α in the ileum of broilers. Besides, a study on weaned piglets challenged with lipopolysaccharides revealed that C. butyricum downregulated the expression of IL-1β, TNF-α and NF-κB, thereby mitigating inflammatory damage (35). Similarly, in the present study, we discovered that C. butyricum treatment significantly downregulated the mRNA expression of NF-κB and TNF-α in the jejunal mucosa of the broilers. Moreover, we first observed that C. butyricum significantly increased the mRNA expression of IL-10 in their mucosa. These positive results indicate the lower inflammation and the improved immune status, which is by likely inhibiting NF-κB signaling pathway. Indeed, a potential cross-link exists between the Nrf2 and NF-κB signaling pathways (36), and inhibiting the Nrf2 signaling pathway reduces the antioxidant capacity and increases the likelihood of oxidative stress damage to the body, ultimately leading to inflammation through NF-κB signaling pathway activation (37, 38). Therefore, improvements in intestinal health, including the intestinal morphology, intestinal barrier, antioxidant capacity and immune status, induced by C. butyricum may be closely related to the activation of the Nrf2 signaling pathway and inhibition of the NF-κB signaling pathway.

The addition of C. butyricum to the diets of broilers can improve their growth performance and meat quality. C. butyricum can also improve intestinal health, which is likely to be linked with the activation of the Nrf2 signaling pathway and inhibition of the NF-κB signaling pathway. However, additional research is necessary to determine the exact mechanism of action of C. butyricum.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

The animal study was reviewed and approved by the Animal Ethics Committee of Anhui Agricultural University.

Conceptualization: ZL and XJ. Methodology: LL and YL. Investigation and writing—original draft preparation: ZL and LL. Data curation: QW and YL. Formal analysis: XJ and QW. Writing—review and editing: CZ and XC. Supervision: ZG and CZ. Funding acquisition: CZ. All authors contributed to the article and approved the submitted version.