Eggs are one of the most cost-effective sources of high biological value and well-balanced protein. The nutritional value and customer preferences are influenced by the eggshell and internal quality, which is crucial to the egg industry’s economic viability (Roberts, 2004). Antibiotic growth promoters (AGPs) are commonly used in food-animal feeds in modern animal husbandry to boost production performance and protect animals from diseases (Castanon, 2007). Although utilizing AGPs in laying hen diet increased laying production and egg quality, it also increased antibiotic residues in eggs and the potential for antibiotic-resistant strains of bacteria to cause health problems in humans. Thus, it is necessary to investigate alternative ways for improving laying performance, egg quality, and preventing laying hen diseases, to ensure healthy and sustainable laying hen production.

Natural plant (Chinese herbal medicine) extracts are known for their multi-functionality, no known bacteria resistance, and low toxicity. Recent studies indicate that natural plant extracts have positive effects on the laying performance and egg quality of laying hens (Abdelli et al., 2021; Righi et al., 2021). Natural antioxidants in the diet, such as tea polyphenols, may help to lay hens increase their performance, albumen quality, magnum shape, antioxidant status, and egg antioxidant capacity (Wang et al., 2018; Zhou et al., 2021). Wang et al. (2019) discovered that dietary tea polyphenol supplementation could mitigate the negative effects of high molybdenum exposure on performance, egg quality, and antioxidant status in laying hens. Also, dietary tea polyphenol supplementation differentially enriched microbial compositions in the cecum enhanced the enrichment of Bacilli, Lactobacillates, Lactobacillus, and Lactobacillus gasseri, and indicated that dietary tea polyphenols maintain eubiosis of the cecum microbiota in molybdenum-challenged layers (Wang et al., 2019).

Intestinal microbiota plays a vital role in maintaining gut health and influences the overall performance of laying hens. Gut microbiota is actively involved in the development of the immune system, can confer protection from pathogen infection via competitive exclusion and production of antimicrobial compounds, supplies micronutrients, amino acids, and short-chain fatty acids, and influences the development of intestinal epithelium (Khan et al., 2020). Throughout the laying hen production cycle, the gut microbiota composition is altered with age in distinct ways (Joat et al., 2021; Sun et al., 2021). Understanding the baseline and evolution of gut microbiota in laying hens throughout the course of their lives was crucial to obtaining optimal performance and gut health. According to new research, supplementing natural plant extracts enhances laying hen performance and egg quality while also modifying the gut microbiome (Kim et al., 2018; Abad et al., 2020; Dilawar et al., 2021).

Macleaya cordata (Chinese name “Bo-luo-hui”), also known as plume poppy, is a perennial traditional medicinal herb of the Papaveraceae family that is widely distributed in southern China. Its main active compounds are benzophenanthridine alkaloids (sanguinarine and chelerythrine) and protopine alkaloids (protopine and allocryptopine). Our previous studies showed that sanguinarine extracted from Macleaya cordata modulated the gut microbiome and intestinal morphology to enhance growth performance in broilers (Liu et al., 2020). Compounds comprising sanguinarine and chelerythrine derived from Macleaya cordata were recognized as feed additives in the European Union in 2005, and are widely utilized in poultry and livestock production to replace antibiotic growth promoters. In 2019, compounds containing protopine and allocryptopine isolated from Macleaya cordata were registered as veterinary medications in China as Bopu powder (Veterinary Drug No. 180415374), which can be used to treat chicken diarrhea caused by E. coli.

Propine and allocryptopine have a wide range of therapeutic biological actions, including anti-inflammatory, antibacterial, antiviral, liver repair, and neuroprotective properties (Vacek et al., 2010; Huang et al., 2021). However, there is limited research in the scientific literature on the effects of protopine and allotytopine on the health and egg quality of laying hens. As a result, the purpose of this study was to assess the effects of dietary supplementation with various amounts of Bopu powder on laying performance, egg quality, serum antioxidant capacity, and gut microbiota in laying hens.

When using new alkaloid resources for animal production, safety is critical. More than 290 components of Macleaya cordata have been discovered and/or isolated, with anti-inflammatory, anticarcinogenic, antibacterial, and insecticidal activities (Lin et al., 2018). In animal production, Isoquinoline alkaloids (sanguinarine and chelerythrine) were frequently employed as natural growth promoters with anti-inflammatory and antibacterial properties (Khadem et al., 2014; Ni et al., 2016; Liu et al., 2020). Furthermore, the main constituents of Bopu powder are protopine alkaloids (protopine and allocryptopine), which have anti-inflammatory properties and can be used to treat poultry E. coli diarrhea. The oral administration of LD₅₀ of protopine in ICR mice was reported to be 313.10 mg/kg, and large dosages of protopine induced brain and liver damage, showing that protopine was moderately hazardous (Hu et al., 2021). In the present study, we found no mortality or morbidity of laying hens, and there was no significant difference in production performances among the experimental groups, indicating that dietary 25–400 mg/kg Bopu powder supplementation had no adverse effects on laying hens. Similarly, a prior study indicated that supplementing broilers with benzophenanthridine and protopine in their drinking water enhanced their productive efficiency index (Previato do Amaral et al., 2016). Furthermore, daily consumption of 1.5 mg of benzophenanthridine alkaloids and protopine alkaloids reduced the unfavorable impacts of extreme heat on feedlot ewes’ growth performance (Estrada-Angulo et al., 2016). This study is the first, to our knowledge, to show that dietary Bopu powder supplementation of 25–400 mg/kg has no deleterious effect on laying hens in a 56-day feeding trial.

The liver function index includes AST and ALT, which were frequently employed as indications of liver health (Diaz et al., 1999; Limdi and Hyde, 2003; Lala and Minter, 2021). Dietary Bopu powder supplementation of 25–400 mg/kg showed no influence on serum AST and ALT activity in this investigation, showing that Bopu powder supplementation had no detrimental effects on the liver of laying hens. Furthermore, serum CHO was linked to liver lipid metabolism, which was significantly elevated in fatty liver-laying hens (Harms and Simpson, 1979; Dong and Tong, 2019; Lin et al., 2021). According to the findings of this study, dietary Bopu powder supplementation significantly reduced serum CHO concentrations, suggesting that Bopu powder supplementation may improve hepatic lipid metabolism and protect the liver against fatty liver disease. Previous research found that protopine extracted from Fumariaindica Pugsley has the same hepatoprotective effect as the conventional medication, silymarine (Rathi et al., 2008). Similarly, nutritional supplementation with the antioxidant of plant spices such as garlic and tamarind reduced serum cholesterol in laying hens (Chowdhury et al., 2002; Chowdhury et al., 2005). Furthermore, the liver produces globulin fraction, which contains hundreds of serum proteins, carrier proteins, enzymes, complements, and immunoglobulins, except immunoglobulins, which are produced by plasma cells. In this study, dietary Bopu powder supplementation significantly elevated serum GLB and ALB concentrations, suggesting that Bopu powder could improve liver health and immunological function. It could be because protopine, which contains anti-inflammatory and antioxidant properties, can regulate hepatic lipid metabolism and increase immune cell activation. A study in vitro demonstrated that protopine reduced the inflammatory response in lipopolysaccharide-stimulated murine macrophages (Bae et al., 2012). Furthermore, in a carrageenan-induced mouse model, protopine attenuated inflammatory symptoms through modulation of MAPKs/NF-κB signaling cascades (Alam et al., 2019). In this study, dietary Bopu powder supplementation significantly enhanced the serum GSH-Px and catalase activities, demonstrating that Bopu powder increased the antioxidant capacity of laying hens. In line with a previous study, pretreatment of protopine increased serum superoxide dismutase activity in the middle cerebral artery occlusion in rats (Xiao et al., 2007). Similarly, Xiao et al. (2008) demonstrated that protopine relieved H₂O₂-induced oxidative stress and apoptosis in PC12 cells. Therefore, supplementation of Bopu powder might ameliorate hepatic lipid metabolism and protect the laying hens from fatty liver disease via antioxidant and anti-inflammatory mechanisms.

Egg quality, including shell and interior quality, is critical to the global egg industry. The hen’s egg is made up of the yolk (30%–33%), albumen (about 60%), and the shell (9%–12%). The Haugh Units were calculated using the thickness of the albumen and the weight of an egg (Haugh, 1937). This was a key indicator of egg albumen quality and related to shelf life. In the present study, dietary 25–50 mg/kg Bopu powder supplementation significantly increased egg Haugh Units. We speculated that it might be attributed to the antioxidant activity of protopine. Meanwhile, similar results were found in a previous study, which demonstrated that dietary antioxidant tea polyphenol supplementation increased the egg HU in hens during the late laying period (Wang et al., 2018). The eggshell contained mainly calcium carbonate, which was formed in the shell gland pouch for more than 15 h. In this study, dietary Bopu powder increased eggshell thickness considerably. Propine administration may have improved the health of the gut and shell gland pouch through anti-inflammatory and antioxidant processes. Similarly, Guo et al. (2021) also demonstrated that dietary Macleaya cordata extract (consisting of 7.5% sanguinarine) significantly increased eggshell thickness in Xuefeng black-bone chicken. Hen eggs are healthy foods with balanced nutrition, which contain high-quality proteins and lipids, trace elements, and vitamins. Thus, eggs are easily oxidized by a series of oxidative reactions during storage, resulting in a negative effect on egg nutritional values. In this study, dietary Bopu powder enhanced the antioxidant capacity of the yolk, hence improving egg quality and shelf life. We also discovered that serum antioxidant capacity was positively correlated with yolk antioxidant capacity, implying that Bopu powder supplementation could minimize oxidative stress by regulating laying hen metabolism. A recent study found that dietary antioxidants like selenium-enriched yeast or natural astaxanthin improved the antioxidant capacity of the yolk (Gao et al., 2020; Lin et al., 2020). As a result, nutritional supplementation with Bopu powder may increase egg quality by enhancing laying hen antioxidant capacity.

The gut microbiome has been identified as one of the primary elements influencing laying hen productivity and health. Previous research has linked greater gut microbiota richness and diversity to improved health and productivity (Stanley et al., 2012; Yan et al., 2017). The current study found that dietary Bopu powder supplementation improved the diversity of microbiota in the foregut, implying that supplemented Bopu power may improve the health and productivity of laying hens. Similarly, we discovered that 50 mg/kg Bopu powder administration increased the diversity of microbiota in the foregut, which was consistent with increased serum antioxidant capacity and egg quality in laying hens. We also discovered that the distribution of microbial communities differed significantly between the foregut and hindgut in this investigation. Xiao et al. (2021) discovered that the dominating bacteria in the duodenum were Lactobacillus, however, the dominant microorganisms in the cecum and colorectum were more complex, primarily comprising Bacteroides, Odoribacter, and Clostridiales vadin BB60 group in laying hens. At the phylum level, Firmicutes was the main flora in the foregut, while Firmicutes and Bacteroidota were the dominant flora in the hindgut in this study. The addition of 50–400 mg/kg of Bopu powder to the diet increased the relative abundance of Bacteroidota in the foregut. Interestingly, Bacteroidota is commonly seen as a “generalist” degrader of dietary fiber, implying that supplementing with Bopu powder may boost fiber fermentation and short-chain fatty acid (SCFA) production. At genus level, dietary 25–100 mg/kg Bopu powder supplementation enriched Enterococcus in the foregut, Bacteroidaceae, Fusobacteriaceae, and Lachnospiraceae in the hindgut. Enterococcus was assumed to be a natural antibacterial probiotic capable of preventing diarrhea, improving feed efficiency, and promoting animal production growth (Franz et al., 2011). Bacteroidaceae was considered as a probiotic for its ability to degrade complex polysaccharides and produce acetate, propionate, or succinate (Polansky et al., 2016; Medvecky et al., 2018). Fusobacteriaceae could modulate the growth of other bacterial species through metabolic by-products, which was helpful for the maintenance of an overall microbial structure. Similarly, Wang et al. (2020) found that dietary probiotic Bacillus subtilis supplementation increased the abundances of Fusobacteria phylum, Fusobacteriia class, Fusobacteriaceae family, and Fusobacterium genus and improved the performance of breeding geese during the laying period. Lachnospiraceae was involved in the production of SCFAs (butyrate, propionate, and acetate), which were important energy sources for intestinal epithelial cells (Rychlik, 2020). In recent studies, Lachnospiraceae have been shown to be enriched in long-living Italian and Chinese populations (Kong et al., 2016). Given that egg layers have a 1-year lifespan of production, enrichment with Lachnospiraceae could have a significant favorable impact on health and productivity. In addition, LEfSe analysis showed that the relative abundance of Tannerellaceae and Muribaculaceae were significantly enriched in the BP50 group, while the BP0 group significantly enriched the abundance of Desulfovibrionaceae in the hindgut. Previous studies found that the Tannerellaceae were enriched in certain gastrointestinal disorders, such as Crohn’s disease, indicating that Tannerellaceae might be related to intestinal immunity (Hernández et al., 2019). Furthermore, Zhao et al. (2020) demonstrated that Tannerellaceae was negatively correlated with immune traits, whereas Muribaculaceae was positively correlated with immune traits. In the present study, Tannerellaceae and Muribaculaceae were both enriched in hindgut in the BP50 group and positively correlated with improving egg quality and antioxidant capacity of laying hens. Thus, we speculated that the enrichment abundance of Tannerellaceae and Muribaculaceae might improve the gut health of laying hens. Chang et al. (2022) discovered that orange corn (high carotenoids) diet significantly enriched Tannerellaceae in laying hens compared to white corn (low carotenoids) diet. Desulfovibrionaceae could release sulfate in the gut and produce hydrogen sulfide. A larger concentration of hydrogen sulfide, on the other hand, was hazardous and could cause inflammatory bowel disease in the colon (Guo et al., 2016). In laying hens with an average age of 64 weeks (Joat et al., 2021), the abundance of Desulfovibrionaceae increased significantly, showing that Desulfovibrionaceae was negatively correlated with intestinal health.

Surprisingly, dietary low-dose 25–50 mg/kg Bopu powder supplementation had a greater impact on egg quality and antioxidant status in laying hens but high-dose, 100–400 mg/kg, groups had no additive effects. The effects of Bopu powder supplementation were connected with the diversity of gut microbiota in the current investigation. It is possible that low-dose Bopu powder supplementation improved egg quality and antioxidant capacity of layers by increasing the diversity of gut microbiota and the abundance of beneficial bacteria, whereas high doses reduced the diversity of gut microbiota due to Bopu powder’s antibacterial effect. Furthermore, excessive Bopu powder supplementation may raise drug metabolism stress in laying hens, thereby counteracting the synergistic effects of Bopu powder.

In the 56-day trial, dietary Bopu power supplementation of 25–400 mg/kg had no negative effects on laying hens. Furthermore, supplementing laying hens with 50 mg/kg Bopu powder improved egg quality and antioxidant capacity, which could be attributed to an increase in intestinal microbiota richness and changes in microbial composition, particularly the enrichment of Enterococcus in the foregut and Bacteroidaceae, Fusobacteriaceae, and Lachnospiraceae in the hindgut. Thus, Bopu powder may be utilized in laying hens to improve egg quality and intestinal health, and we proposed that the ideal dietary dose of Bopu powder in laying hens was 25–50 mg/kg.