China is a large aquaculture country, intensification of aquaculture is an important part of Chinese fisheries, it has become a major growth point for fishermen to increase their income and rural revitalization (Hou et al., 2021; Hu et al., 2021). Yet, the exacerbation of the aquaculture process is affected by factors such as parasites, bacteria, and fungi, which leads to outbreaks of diseases in aquatic animals and causes mass mortality, seriously threatening aquaculture industries (Valladão et al., 2015; Yarahmadi et al., 2016). Therefore, enhancing the immunity and disease resistance of fish in aquaculture has become increasingly crucial. The gut is the biggest immune organ of fish (Fan and Pedersen, 2021). And plays a vital role in various functions such as nutrient digestion and absorption, excretory functions, resistance to pathogenic microorganisms, participation in antimicrobial peptides and secretion of ingestion-regulating hormones. If the function of the intestine has abnormalities, it will affect the growth of the fish (Bischoff et al., 2014; Celi et al., 2019; Zhao et al., 2021). In traditional aquaculture, antibiotics commonly used to treatment disease in animals, but the misuse of antibiotics not only created the problem of drug resistance, but use of these antibiotics could have numerous adverse impacts on the water system, animals and humans, and this treatment method has been criticized (Sapkota et al., 2008; Milić et al., 2013; Kovalakova et al., 2020). Therefore, fermented herbs have a good prospect as a green feed additive after the era of antibiotics. Fermenting Chinese herbs involves the use of enzymes and microorganisms to increase their inherent properties and create new benefits (Hussain et al., 2016; Li et al., 2020a). The fermentation could degrade cellulose, lignin and other substances in the cell wall, the active ingredients were released, and the active ingredients in herbs were dismantled to small moieties to improve the effectiveness of the medicines (Mandebvu et al., 1999; Chang et al., 2012; Hussain et al., 2016; Wang et al., 2016). Studies have shown that fermented herbs are commonly employed in farming due to their antioxidative, antidiarrheal, antiviral, and hepatoprotective properties (Hussain et al., 2016). Feeding Bacillus subtilis fermented citrus by-products to juvenile olive flounder Could exert salutary influences on inborn immunity, thus improving the ability of juvenile olive flounder to survive and resist disease (Lee et al., 2013). In addition to enhancing the hematologic and physiological traits of catfish, fermented herbs have positively influenced the survival, specific growth, and feed conversion rates of catfish (Syawal et al., 2021). Incorporating probiotic fermented herbs into the diet of grouper may enhance their Growth capacity and anti-oxidation ability, while also decreasing foregut lipase levels (Nie et al., 2022). Furthermore, researchers have revealed that fermented herbs could modify the intestinal oxidative stress and innate immunity in fish by altering the gut microbiota, regulating the gut barrier, and inflammatory response (Zhang et al., 2015; Park et al., 2016; Wang et al., 2021b; Zhang et al., 2022).

Largemouth bass (Micropterus salmoides) is native to the United States and it’s a freshwater fish species. During the 20th century, it was introduced to China because of its rapid growth, adaptability, fresh and tender meat, and has since become a primary species for freshwater aquaculture in the country (Li et al., 2020b; Xu et al., 2021). In order to reduce usage of antibiotics and guarantee the quality and security of aquatic animals, fermented Chinese herbs have emerged as a viable solution for improving the gut health of largemouth bass and mitigating intestinal diseases. Our study aimed to explore the impact of incorporating fermented Chinese herbs into the diets of juvenile largemouth bass. Specifically, we examined the effects on their intestinal barrier function, immunity, and resistance to A.hydrophila. The goal was to offer effective strategies and insights for promoting the healthy farming of largemouth bass.

Chinese herbal medicine contained many biologically active ingredients, which could stimulate the appetite or digestive function of animals, increase feed intake and growth performance (Awad and Awaad, 2017; Pu et al., 2017). In addition, fermented Chinese herbal medicine also contained many probiotics, which could improve the intestinal health and morphology of animals, increase the digestibility and absorption rate of feed in the intestine, promote the metabolism of nutrients, and improve the production performance of animals (Melara et al., 2022; Chen et al., 2023). Our research found that supplementing FCHM improved the WGR and SGR of juvenile largemouth bass, possibly because FCHM promoted their digestion and absorption of nutrients, thereby promoting growth.

Bacterial infection was frequently used as a fish health indicator at the end of culture trials (Shan et al., 2018). Freshwater fish commonly suffer from bacterial infections in their intestines, and A. hydrophila is often considered a primary pathogen among the various disease-causing bacteria (Maulu et al., 2021; Xv et al., 2021). Our study revealed that largemouth bass fed with FCHM displayed a greater ability to survive after being infected with A.hydrophila. This indicates that FCHM could potentially improve the immunity of largemouth bass and enhance their resistance to A.hydrophila.

When fish undergo metabolic processes during their life activities, oxygen radicals are generated. To counteract their harmful effects, antioxidant enzymes like CAT, GSH-Px, and SOD form the fish’s antioxidant defense system. The SOD can convert the excess reactive oxygen radicals in the body into hydrogen peroxide, while the CAT can cooperate with the SOD to convert the hydrogen peroxide into water, thus slowing down the oxidative stress (Harikrishnan and Balasundaram, 2005; Hongling Zhang et al., 2020a). T-AOC level is a crucial measure of the anti-oxidant capacity in animals and is directly indicative of the anti-oxidant ability of fish (Ge et al., 2021). When the body exposed to adverse conditions will produce a lot of oxygen radicals, which can attack the cell membrane and produce lipid peroxidation reactions, converted into MDA, thus causing oxidative damage to the body (Hoseinifar et al., 2021). When the body is exposed to adverse conditions will produce many oxygen radicals, which can attack the cell membrane and produce lipid peroxidation reactions, converted into MDA, thus causing oxidative damage to the body (Wei Zhao et al., 2022). Hydrogen peroxide and lipid peroxides can also be scavenged by GSH-Px, while GSH can synergize with GSH-Px to scavenge free radicals (Taso et al., 2019). Therefore, the increase in antioxidant enzyme activity could improve the disease resistance of fish. Feeding fermented moringa oleifera lam could improve the serum SOD and CAT activity, reduce the content of serum MDA and improve the resistance to A.hydrophila of crucian carp (Liu et al., 2019). Lactobacillus fermented Artemia could improve antioxidant capacity and antibacterial ability of fish and has potential as a feed additive (Li et al., 2021). Our study revealed that administering FCHM to juvenile largemouth bass resulted in heightened activity of SOD, GSH-PX, and CAT while reducing MDA concentration in their intestines. The inclusion of FCHM in their diet significantly enhanced the antioxidant capacity of the fish. This can be attributed to FCHM’s ability to scavenge reactive oxygen radicals in animals and minimize lipid peroxidation reactions.

Research has demonstrated that AKP, ACP, and LZM are crucial enzymes in the fish immune defense system, indicating the organism’s immune condition (Zhang et al., 2020b). The utilization of LZM can serve as a crucial measure for assessing non-specific immunity in fish, and it holds a significant function in regulating inflammatory processes, repairing, and regenerating tissues, as well as exterminating bacteria within the organism (Chen et al., 2021). AKP and ACP are crucial hydrolytic enzymes in the immune defense system of fish and are also important metabolic regulatory enzymes, which mainly catalyze the hydrolysis of phosphate esters to form inorganic phosphate and thus promote the production of ATP during the immune reaction. Meanwhile, phosphatase activity in aquatic animals can be used as an indicator to determine the immunity of animals (Shi et al., 2021; Wang et al., 2022). The conclusions of this study indicate that administering FCHM to juvenile largemouth bass led to an increase in AKP, ACP, and LZM levels in their intestines both pre-infection and post-infection with A.hydrophila. This indicates that FCHM has the potential to enhance non-specific immunity and bolster the largemouth bass’s ability to resist diseases. The overall health of fish intestines is influenced by factors like the morphology of the gut villi, physical barriers, and the activity of digestive enzymes. By improving the morphology of gut tissues in fish, including the width and length of villi and thickness of the muscle layer, the digestibility of nutrients can be enhanced, ultimately contributing to the maintenance of fish health (Yu et al., 2020; Dawood, 2021; Wang et al., 2021a). Fermented herbs improve the physical barrier of the gut, mainly in the height of the gut villus and the enhanced muscle layer thickness (Nawaz et al., 2018; Abdel-Latif et al., 2020). Adding fermented astragalus to the diets could enhance the height of gut villi, thus increasing the digestive and absorption capacity of carp for nutrients and improving growth performance (Chen et al., 2023). It was found that fermented lemon peel could improve the intestinal morphology of Lates calcarifer and can be utilized as a functional feed supplement to maintain intestinal health (Shi et al., 2022). In line with prior research, FCHM sustained the gut health of largemouth bass by enhancing their gut morphology.

We assessed the expression levels of gut barrier genes in largemouth bass to gain more insight into how FCHM controls intestinal health in largemouth bass. In the intestine, the intestinal tight junctions are a dynamic, changing structure that allows for the reabsorption of water, ions, and nutrients to regulate intercellular permeability, thus preventing external pathogens and toxins from entering the intestinal lumen (Zhuo et al., 2021). Claudin mainly constitutes a tight junction protein that forms intercellular pores in intestinal epithelial and endothelial cells that affect intestinal permeability (Assimakopoulos et al., 2011). Occludin is another cell adhesive component and a part of tight junction proteins, its main role is to participate in the closure of the paracellular gap and to regulate the signals generated in tight junctions, the increased or decreased adhesion proteins will be directly reflected in the intestinal permeability, resulting in changes in intestinal permeability (Günzel and Yu, 2013). ZO-1 is a crucial component of tight junction proteins that serve to safeguard and uphold the integrity of the intestinal mucosa (Schneeberger and Lynch, 2004). Assessing the expressed of these 3 tight junction proteins provides a clearer picture of how FCHM supports the gut mucosal integrity. As such, we discovered that the expression of Occludin, Claudin and ZO-1 in the intestines of young largemouth bass was boosted after an 8-weeks FCHM diet. This suggests that FCHM is advantageous in preserving the integrity of the gut mucosa and improving the physical barrier function of the gut of largemouth bass. Inflammatory responses mediated by cytokines are critical to the immunity of fish organisms and can regulate immune responses by increasing or decreasing cytokine production (Coyne et al., 2002). Cytokines in fish are divided into anti-inflammatory biomarkers (e.g., TGF-β and IL-10) and pro-inflammatory biomarkers (e.g., IL-8, IL-15, TNF-α, and IL-1β) that are crucial in immune responses. Enteritis could reduce the expression of anti-inflammatory biomarkers and increase pro-inflammatory biomarkers, which enable them to react to molecular-level inflammatory damage (Safari et al., 2016; Fehrmann-Cartes et al., 2019). Research has shown that the innate immunity of fish intestines can be regulated by herbs and probiotics, which can boost the production of anti-inflammatory biomarkers and reduce the expression of pro-inflammatory biomarkers. This enhances the fish’s resilience against pathogenic bacteria, thereby reduce inflammation (Gil, 2002; Song et al., 2014; Giri et al., 2015; Safari et al., 2016). The research demonstrated that feeding FCHM to juvenile largemouth bass effectively modulated the expression of gut inflammatory factors following infection with A.hydrophila. FCHM was observed to alter the inflammatory response in the fish’s gut, thereby decreasing the incidence of gut disease caused by A.hydrophila.

The study revealed that supplementing a specific quantity of FCHM enhanced the resistance of juvenile largemouth bass to A.hydrophila and growth performers by improving the physical barrier, immune enzyme activity, anti-oxidant capacity, and inflammatory response of the intestines. The most effective concentration was observed to be 1%.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by College of Life Science and Engineering, Southwest University of Science and Technology.

LJ: Supervision, Methodology, Conceptualization, Investigation, Writing – original draft. XZ: Conceptualization, Methodology, Data curation, Formal analysis, Writing – original draft. SB: Data curation, Investigation, Formal analysis. QW: Data curation, Investigation. JL: Investigation. YW: Supervision, Methodology, Investigation, BL: Supervision, Writing – review and editing. All authors contributed to the article and approved the submitted version.