Macleaya cordata extract improves the growth performance and intestinal health of American eels (Anguilla rostrata) farmed in intensive system

Abstract The present study was conducted to evaluate the effects of dietary Macleaya cordata extract (MCE) supplementation on growth performance and intestinal health of American eels (Anguilla rostrata) farmed in intensive system. A total of six cement tanks of fish were randomly divided into a control group fed a commercial diet and an MCE group fed the commercial diet with 100 mg/kg MCE, respectively. There were three replicates in each group. The results suggested that 100 mg/kg MCE could improve the growth performance and intestinal health of the American eels by strengthening the barrier function and antioxidative ability in the intestine and beneficially modulating intestinal microbiota with the higher relative abundance and more species of the potential probiotics and the lower relative abundance of potentially pathogenic bacteria. Graphical Abstract


Introduction
Nowadays, the use of antibiotics and chemotherapeutics is the main strategy in commercial aquaculture for controlling diseases; however, the development of drug-resistant pathogens, drug residues, and environmental pollution have aroused great concerns. Recently, growing interest has arisen in the utilization of natural products of the medical plant as an eco-friendly measure to ensure the sustainability of aquaculture Fazio et al. 2022). The natural products of the medical plant usually possess multiple biological activities such as antimicrobial, anti-inflammatory, antioxidant, immunostimulatory, and appetite-stimulating effects.
Macleaya cordata extract (MCE) is mostly composed of quaternary benzo[c]phenanthridine alkaloids, mainly sanguinarine and chelerythrine. The MCE has been standardized to 1.5% w/w sanguinarine (Zdarilova et al. 2008;Hou and Zeng 2018). Because of its strong anti-inflammatory, antibacterial, and appetite-enhancing effect, MCE is becoming popularly utilized as a feed additive to promote growth and health status in animal production for husbandry animals and poultry nowdays (Hou and Zeng 2018). There were also some reports about the positive effects of MCE on growth performance and intestinal health of aquatic animals including grass carp (Ctenopharyngodon idellus) (Liu et al. 2020), red tilapia (Oreochromis niloticus) (Rawling et al. 2009), and Pacific white shrimp (Litopenaeus vannamei) (Bussabong et al. 2021). However, the research about the beneficial effects of MCE on those aquatic animal species was conducted under laboratory conditions, little information is available regarding the effects of dietary MCE supplementation on growth and intestinal health status of fish under the practical culture condition.
This study aimed to evaluate the effects of 100 mg/kg MCE supplementation on growth performance and intestinal health of American eels (Anguilla rostrata) farmed in intensive system. To our knowledge, this is the first study to research the biological activities of MCE in eel under practical culture condition, which could provide a useful guideline for MCE utilization effectively in fish diet.

Growth performance parameters
The effects of dietary MCE supplementation on growth performance parameters in American eels were shown in Table S1.
Compared with the CON group, the WGR、SGR, and FE in the MCE group were significantly increased (P < 0.05), which indicated that 100 mg/kg supplementation could improve the growth performance of American eels. Similar results were reported in grass carp and red tilapia fed the diets with MCE (sanguinarine) levels being 75 mg/kg to 100 mg/kg (Rawling et al. 2009;Liu et al. 2020).

Parameters related to barrier function and antioxidative ability in the intestine
The D-lactate level and the DAO activity in the serum of American eels were shown in Table S2. The intestinal morphology of American eels was shown in Figure S1. The antioxidative parameters in the intestine of American eels were shown in Table S3.
Compared with the CON group, the D-lactate level and the DAO activity in the serum of the MCE group were significantly decreased (P < 0.05), and the density of intestinal microvilli was increased obviously in the MCE group. The D-lactate and DAO in serum are usually used as indirect measures for evaluation of the intestinal mucosal barrier function, and their elevated values mirror the impairment of mucosal barrier function (Rahimnejad et al. 2021). The decreased D-lactate level and DAO activity in serum by MCE supplementation in this study indicated that mucosal barrier function might be improved. Those might be related to MCE increasing the expression of ZO-1 and claudin-1 (Liu et al. 2016). The higher density of intestinal microvilli indicated that the mucosal surface area would increase to improve the ability of intestinal digestion and absorption.
The activities of SOD, GSH-PX, and CAT in the MCE group were significantly increased with the decreased MDA level (P < 0.05). These parameters were considered as the prime monitors of antioxidative ability to scavenge free radicals in the organism. Similar results have been reported in grass carp fed the diets with MCE (sanguinarine) supplementation (Liu et al. 2020). It was found that the sanguinarine might exert its antioxidant function by inhibiting the activity of NADPH oxidase and scavenging free radicals directly (Varga et al., 2001;Hou and Zeng 2018).

Intestinal microbiota analysis
The indexes of alpha diversity and beta diversity of intestinal microbiota were shown in Figure S2 and Figure S3, respectively. The intestinal microbiota composition at the phylum level was shown in Figure S4, and the LefSe analysis of intestinal microbiota was shown in Figure S5.
There was no significant difference in the alpha diversity indexes of the intestinal bacteria community between the CON group and MCE group (P > 0.05), and it indicated that MCE supplementation could not affect the abundance and diversity of bacteria in the intestine of American eels. The result of beta diversity analyzed by the Principal Coordinates Analysis method showed that the difference in the intestinal bacterial community was not separated between the CON group and MCE group. While some difference in the beta diversity of intestinal bacteria community between the CON group and MCE group was shown by Partial Least Squares Discrimination Analysis method.
Compared with the CON group, the relative abundance of Proteobacteria in the MCE group was decreased, and the relative abundances of Bacteroidota and Actinobacteriota were slightly increased. It was found that the decreased prevalence of Proteobacteria might be a potential diagnostic signature to lower the dysbiosis and risk of disease (Shin et al. 2015), while the higher relative abundance of some Bacteroidota and Actinobacteriota might be beneficial to the mucosal immune system in the intestine .
The relative abundances of Lactococcus and Aeromonas in the MCE group were significantly higher than those of the CON group (P < 0.05), and the relative abundances of Aurantimicrobium, Alsobacter, and Kurthia in the CON group were significantly higher than those of MCE group (P < 0.05). Some strains of Lactococcus could inhibit harmful gut bacteria and modulate the immune system (Kimoto et al. 2002), and the Aeromonas might promote the carbohydrate utilization of the host (Huang et al. 2018). While some strains of Kurthia have been demonstrated as an opportunistic pathogen (Lozica et al. 2022), it might be a potential pathogen in the intestine of American eels. Little information is available about the function of Aurantimicrobium and Alsobacter in the fish intestine. The beneficial effects of MCE on intestinal microbiota were also reported in Pacific white shrimp (Bussabong et al. 2021). The antibacterial effect of MCE might be associated with perturbing the FtsZ assembly dynamics in the Z ring and reducing the bundling of FtsZ protofilaments (Beuria et al. 2005). MCE also could prevent cell division of pathogen by initiating the release of membrane-bound cell wall autolytic enzymes, which eventually resulted in lysis of the cell (Obiang-Obounou et al. 2011).

Conclusion
In conclusion, the results of the present study indicated that dietary 100 mg/kg MCE supplementation might promote the growth performance by improving the intestinal health status of American eels farmed in intensive system.