The effect of combined dietary supplementation of herbal additives on carcass traits, meat quality, immunity and cecal microbiota composition in Hungarian white geese

Introduction

To produce qualified livestock or poultry whose meat are low in fat and high in proteins is highly required to fulfill people’s growing need in recent years. (Li et al., 2019). Goose meat is considered as a healthy food with high content in amino acids and minerals exerting beneficial effects on human health (Simopoulos, 2008). Several diseases, including cardiovascular problems, inflammatory and autoimmune disorders, are associated with the imbalance intake of n-6 and n-3 polyunsaturated fatty acids in the diet (Qi et al., 2010). The high concentration of polyunsaturated fatty acids in goose flesh makes it a healthier choice for consumers. For the moment, goose production in China has accounted for approximately 94% of the gross production of global industry (Liu & Zhou, 2013; Yu et al., 2020). In the past few decades, antibiotic growth promoters (AGPs) have been extensively applied in poultry diets to prevent and treat disease and improve goose growth, as antibiotic feed utilization could maximize profits and efficiency. However, the abuse of antibiotics has led to an increasing number of bacterial drug-resistant strains in animal products, posing a serious threat to human and animal health. Therefore, AGPs were banned by the European Union in 2006 (Villanueva, 2012), North America in 2017, and China in 2020, successively. It is urgent to develop safe and effective additives to serve as AGPs alternatives in the animal production industry.

A growing body of research has been conducted on Chinese herbal medicines and their purified components and indicated they could serve as novel growth enhancers and antibiotic alternatives (Seidavi et al., 2021). Chinese herbal medicine could improve immunity, reduce inflammation, provide antibacterial and antioxidant properties (Hernandez et al., 2004; Acamovic & Brooker, 2005; Giannenas et al., 2018). Consequently, they could protect the intestinal mucosal structure in poultry and affect the fowl gut microbiota (Abd El-Hack et al., 2022; Khalaji et al., 2011; Abolfathi et al., 2019). Pulsatilla, Gentian, Rhizoma coptidis, Codonopsis pilosula, Atractylodes, Poria cocos and Licorice were selected with the such properties and were utilized in this research (Zhong et al., 2022; Mirzaee et al., 2017; Wang et al., 2019; Fu et al., 2018; Bailly, 2021b; Tian et al., 2019).

The large-scale production and intensification of animal husbandry could shorten the feeding cycle, at the same time generate severe metabolic burden, leading to the decline of meat quality (Xing et al., 2019). Chinese herbal medicine exerted antioxidant activity and potential beneficial effects on poultry meat quality and immunity (Qaid et al., 2022; Xie et al., 2022). Gentian, as a bittering agent, could affect digestion by increasing appetite in rats, stimulating bitter receptor cells in gastrointestinal tracts and thus improving nutrient absorption and muscle water retention (Mirzaee et al., 2017). Licorice could improve the meat quality both in broilers and fattening pigs. Flavonoids and triterpene saponins are the main active substances of licorice (Ahmed et al., 2016; Qiao et al., 2022) Licorice extract is known to improve immune response by increasing interferon and globulin levels in animals (Toson et al., 2022). Moreover, Poria cocos polysaccharide has also been demonstrated to perform multiple immune effects by promoting antibody production in B lymphocytes and the spleen, increasing serum IgG levels, and enhancing the phagocytosis of macrophages (Tian et al., 2019; Pu et al., 2019). Polysaccharides from Codonopsis pilosula have been reported to protect the intestinal mucosal immune barrier, to maintain intestinal homeostasis (Li et al., 2022).

Previous studies indicated that Pulsatilla, Rhizoma coptidis, Codonopsis pilosula and Atractylodes could regulate gut microbiota composition (Li et al., 2020; Wang et al., 2019; Bailly, 2021a; Bailly, 2021b). Pulsatilla chinensis saponins (PRS) are the main active component of Pulsatilla and its antioxidant and immunomodulatory functions have been extensively studied (Li et al., 2020). In a rat model of dextran sodium sulfate (DSS)-induced ulcerative colitis (UC), the administration of PRS regulated the composition and biodiversity of the gut microbiota, significantly improving UC symptoms and reducing inflammation (Liu et al., 2021). In addition, Rhizoma coptidis has various pharmacological effects containing alkaloids such as berberine, coptisine and palmatine, which could exert antioxidant, and anti-viral effects (Lyu et al., 2021; Zhang et al., 2021; Wang et al., 2018). Studies have shown that Codonopsis polysaccharides could enhance intestinal mucosal immune function by stimulating the secretion of sIgA (Fu et al., 2018; Zou et al., 2019). According to previous studies, sesquiterpene lactams and lactones are the main active components of Atractylodes macrocephala, which perform excellent anti-inflammatory and antioxidant activities (Bailly, 2021b). Atractylodes are commonly used to heal gastrointestinal disorders due to their potential role in regulating intestinal microbiota (Feng et al., 2020; Wang et al., 2022). Previous studies have indicated that several herbal medicines are usually combined to achieve the synergetic effect better than single herb based on their similar therapeutic properties (Xu et al., 2022; Zhou et al., 2016; Li et al., 2021). In our study, CHAA (Pulsatilla, Gentian and Rhizoma coptidis) and CHAB (Codonopsis pilosula, Atractylodes, Poria cocos and Licorice) were prepared respectively. However, most research on herbal supplementations has been performed in mammals rather than in poultry (Li et al., 2016; Huang et al., 2021; Li et al., 2021). Therefore, this study aims to investigate the effects of CHAA and CHAB on carcass traits, meat quality, immunity standard and intestinal microbiome in Hungarian white goose.

Materials & Methods

Schematic overview of the experimental program

CON group: geese were fed with the basal diet for 70 days. HS groups: geese were fed with the basal diet supplemented with CHAA (day 0-42) and CHAB (day 43-70). On day 42 and 70, serum was collected to determine serum IgA, IgM, IgG, and the contents of the cecum were collected for 16S sequencing. On day 70, carcass traits and meat quality were detected after slaughter experiments (Fig. 1).

Results

Serum IgM, IgG and IgA concentrations

The effects of the herbal mixtures on IgG, IgA and IgM in geese serum were shown in Fig. 2. The results indicated that adding CHAA and CHAB in basal diet had no effect on levels of serum IgA and IgM on day 43 (ns), and promoted significantly the secretion levels of IgG (P = 0.0027, F = 1.088, R-sq =0.4027). On day 70, there were a significant increased concentration of serum IgA (P < 0.0001, F = 7.443, R-sq =0.9245), IgM (P < 0.0001, F = 2.204, R-sq =0.6930) and IgG (P = 0.0005, F = 4.322, R-sq =0.5032) in the HS group than CON group (P < 0.01).

Carcass traits

The impact of CHAA and CHAB powders on the carcass traits was shown in Fig. 3. The SR, HCR, ER and BMR indexes in the HS group were slightly higher than those in the CON group on day 70 respectively (P > 0.05).

Muscle chemical composition

The chemical compositions of the meat were detected and the results were shown in Fig. 4. There was no significant effect of dietary treatment on the moisture or protein levels between the two groups. Besides, significant increased levels in the carbohydrate content (P < 0.0001, F = 2.333, R-sq =0.9894), fat content (P = 0.0009, F = 9.333, R-sq =0.9509) and energy (P = 0.0005, F = 2.154, R-sq =0.9626) were observed and significant decreased level in cholesterol content (P < 0.0001, F = 1.105, R-sq =0.9933) were observed in the HS group than the CON group (Fig. 4A). For the contents of Zn (P = 0.0005, F = 3.083, R-sq =0.9626), P (P = 0.0124, F = 12.18, R-sq =0.9626), Fe (P < 0.0001, F = 12, R-sq =0.9999), Na (P < 0.0001, F = 1.333, R-sq =0.9978), Ca (P < 0.0001, F = 33.33, R-sq =0.9880) and Se(P < 0.0001, F = 204.8, R-sq =0.9984) (Fig. 4B) in the muscle, significant decreased levels were found in the HS group in comparison to the CON group.

Gut microbial diversity and composition

As shown in Fig. 5A, we found that the Shannon sparsity curve was relatively flat. In addition, the rank abundance curves (Fig. 5B) showed that the abundance difference of OTUs in the community was small enough. There were no significant differences in the Chao1 index, the observed-species index, or the Simpson index between the HS and CON groups (Fig. 5D). Principal coordinates analysis (PCoA) showed that the microbial structure of the HS and CON groups was similar on days 43 and 70, separately. Moreover, the cecal microbial community structure in the HS group on day 43 were significantly different from the HS group on day 70 (Fig. 5C).

Microbial taxa analysis

The study analyzed the relative abundance of the two groups at the phylum and genus levels respectively. At the phylum level, Firmicutes, Proteobacteria and Bacteroidetes were the dominant phyla in the HS group. The cecal microbiota of the geese was dominated by the Firmicutes (52.46%), Proteobacteria (25.61%) and Bacteroidetes (5.47%) in the HS group on day 70 (Fig. 6A). At the genus level, the taxonomic unit with the highest abundance was Lactobacillus (5.73%) in the HS group on day 70. What is more, the CON group had a significantly higher abundance of Escherichia-Shigella (30.89%), Desulfovibrio (5.37%) and Fusobacterium (6.61%) and a lower abundance of Paraclostridium (9.56%) than the HS group on day 70 (Fig. 6B). A heatmap was constructed using R language software to determine the relative abundance of the specific gut microbes. The results were illustrated in Fig. 7. The populations of Lactobacillus increased remarkably in the HS group on day 70. On day 70, Escherichia-Shigella and Desulfovibrio had lower abundance in the HS group compared with the CON group.

Linear discriminant analysis effect size (LEfSe) analysis

A LEfSe analysis was performed to identify the specific bacterial taxa that may be responsible for the significant differences in community composition associated with CHAA and CHAB treatments. The cladograms revealed 41 potential microbial biomarkers with significant statistical differences. Among these, the abundance of three genera were significantly increased in the HS group than CON group on day 43, including Lactobacillus sharpeae, Streptococcus_parauberis and Corynebacteriales. Epulopiscium, Lachnospiraceae bacterium mt14, Bacteria, and Paraclostridium also showed significantly increased abundance in the HS group on day 70 compared with the CON group (Fig. 8).

Discussion

In recent years, the emergence of antibiotic resistance and antibiotic residues in food has become serious issues. Antibiotics are gradually banned as growth promoters in animal husbandry. Therefore, to develop antibiotic alternatives is of great interest and urgent for livestock industry. Herbal feed additives have become more common in the poultry industry nowadays (Gao et al., 2022). Several studies have revealed that combined herbal mixtures were more effective in improving animal health than single herbal extracts (Xu et al., 2022). However, the effect of Chinese herbal medicine mixtures on poultry production at different growth stages has not yet explored. This study aims to investigate the impact of CHAA, used at the starter phase and CHAB, used at the growth phase, on the meat quality, immunity and intestinal microbiota composition in Hungarian white geese.

Since the gosling has a short digestive tract, poor digestive gland function and weak immunity during the starter phase period (0-42), it is crucial to enhance digestion function and resistance against pathogens. CHAA contains pulsatilla, gentian and Rhizoma coptidis, which are widely known to have beneficial functions in antioxidant action, immune regulation, maintenance of the balance in intestinal microbiota, and the promotion of gastric juice secretion. Therefore, using CHAA can boost geese low resistance to pathogens and improve digestive system functions in the early stages (Lyu et al., 2021; Zhang et al., 2021; Wang et al., 2018; Mirzaee et al., 2017). In the growth stage, digestion ability and immunity are improved and strong. However, bones, muscles, and feathers grow faster, it is crucial to supply enough energy to enhance spleen and stomach functions. Codonopsis pilosula, Atractylodes and Poria cocos can regulate spleen and stomach function, which are demonstrated by the Qi-blood theory of Chinese medicine (Gong & Hu, 2022; Cheng et al., 2009; Zhang et al., 2021; Yan et al., 2021). As a result, CHAB was added to the basal diet during the grower phase (day 43 -70). Moreover, supplement of CHAA at 0.2% and CHAB at 0.15% to the basal diet promoted the growth performance of geese in our previous study. Therefore, we designed the experimental program (Fig. 1).

Immunoglobulins are essential indicators of the immune system and play an important role in the immune response (Mikocziova, Greiff & Sollid, 2021). Studies have shown that plant polysaccharides could improve the immune function of poultry (Long et al., 2021; Yu et al., 2022) and activate macrophages to exert immunomodulatory effects by recognizing and binding to specific receptors (Zhang et al., 2021). In our study, several herbs of CHAA and CHAB are rich in polysaccharides, such as Codonopsis pilosula, Atractylodes and Poria cocos. Besides, some other chemical components in herbal medicines also showed immune-enhanced effects, such as flavonoids in Rhizoma coptidis and glycyrrhizic acid in Licorice (Zhang, (Liu et al., 2021). Previous research showed that Codonopsis pilosula, Poria cocos, and Licorice supplementation could improve secretion of IgG, IgA and IgM (Zhan, Yang & Xiao, 2015; Xie et al., 2013; Yin et al., 2022). Our study found that supplementation with CHAA and CHAB enhanced humoral immune responses which was in agreement with these researches (Fig. 2).

PH value, filtration rate, shear force, fat content and flavor are indicators to evaluate meat quality traits. The accumulation of lactic acid caused by anaerobic respiration after slaughter will reduce meat’s pH and affect meat water-holding capacity (Ding et al., 2020). The pH value of normal muscle was around 7, and decreased rapidly after slaughter (Nkukwana et al., 2016). In this study, there were no significant differences in the pH of goose meat among different treatments. This may be because these experimental geese were under similar management and nutritional conditions, including the isocaloric and isonitrogenous diet. Similarly, several studies have argued that Anacardium occidentale leaf and Moringa oleifera leaf supplementation did not influence on meat pH (Adeyemi et al., 2021; Cui et al., 2018). On the other hand, shear force and water loss rate are closely related to meat tenderness (Guo et al., 2022). Some studies have found that adding Astragalus and Glycyrrhiza complex polysaccharides could improve broiler meat quality by reducing muscle water loss and shear force (Qiao et al., 2022). In fact, CHAA and CHAB did not enhance the meat’s tenderness, and there was no significant difference in HS group and the CON group in our study (Table 2). (Yu et al., 2020), also found that the supplementation of cottonseed meal to the feed had no effects on goose meat tenderness (Yu et al., 2020).

The composition and content of amino acids are important factors in meat quality and are frequently used to predict the nutritional value and flavor of meat (Guo et al., 2022). The composition and content of flavor amino acids, including Gly, Ala, Asp, Glu, Phe and Tyr, directly affect meat’s freshness and flavor (Liu et al., 2018). Interestingly, the diet with CHAA and CHAB significantly increased the total amino acid levels, especially Glu, Lys and Asp levels. As a result, these findings proved that herb additives could improve the quality of goose meat by increasing the concentration of flavor amino acids and total amino acids (Fig. 4C). Similarly, adding resveratrol increases the concentration of flavor-enhancing amino acid content in Pekin ducks, thereby improving the meat flavor. Cholesterol levels are also closely related to meat quality (Omojola et al., 2015). However, the increase in cholesterol content induces related diseases, such as cardiovascular diseases. In this study, cholesterol concentrations were significantly lower in geese fed with herb additives treatment than those in the CON group (Fig. 4A). In addition, studies have found that Licorice can exert anti-atherosclerotic effects by reducing cholesterol content (Markina et al., 2022; Innih, Eze & Omage, 2022). In our experiment, CHAB contains Licorice, which may explain the reduction of cholesterol content by supplementing CHAB. Furthermore, a certain amount of moisture contributes to the juiciness and tenderness of the meat and improve its quality. This study indicated that protein and moisture were not affected by the treatment of herbs additives. Intriguingly, we observed a higher muscle fat content in the HS group compared with that in the CON group (Fig. 4A). Fat content also could increase the juiciness and tenderness to improve the meat quality (Yu et al., 2020). Our data demonstrated that CHAA and CHAB increased the meat tenderness and flavor to some extent. The trace elements in the muscle of the HS group were significantly lower than those in the CON group (Fig. 4B). The exact mechanism will be needed to explore in the future.

Carcass traits are important indicators for evaluating animal production performance. Our results showed there were no difference in the percentage of tested carcass traits or weight for all treatments between the two groups (Fig. 3). These findings are consistent with previous studies (Adeyemi et al., 2021; Yu et al., 2021; Miao et al., 2020). This may be related to the fact that during avian development, carcass traits grow at a similar rate to body weight. The CHAA and CHAB used in our study did not increase the protein and energy content in the feed, resulting in similar carcass weights between the two groups.

Intestine is the primary site for food digestion and nutrient absorption, whose microbiota composition is important for homeostasis maintenance (Zou et al., 2021). In this study, we analyzed the microbiota of the caecum contents of Hungarian white geese by 16S rRNA sequencing. The results revealed that the predominant phyla were Firmicutes, followed by Proteobacteria and Bacteroidetes (Fig. 5, Fig. 6), which was in accordance with previous studies (Li et al., 2017; Liu et al., 2018). The Firmicutes/Bacteroidetes ratio (F/B) was significantly correlated with the capacity to obtain energy (Kasai et al., 2015). The present study found that dietary CHAA and CHAB supplementation increased this ratio. Moreover, Lactobacillus, as a probiotic, can improve poultry production performance and immunity by regulating gut microbiota (Bian et al., 2016). Our study showed that the addition of Chinese herbs increased the levels of Lactobacillus.

Furthermore, Bacteroides and Desulfovibrio have been reported to be important producers of LPS (Diling et al., 2017). At the genus level, we found that the abundance of Bacteroides and Desulfovibrio in the HS group decreased on day 70 (Fig. 7, Fig. 8), indicating that CHAB may inhibit the proliferation of these bacteria. Escherichia-Shigella is a pathogenic bacterium that causes diarrheal diseases, such as bacillary dysentery and hemorrhagic colitis (Lee, Yoon & Tesh, 2020). Studies have shown that flavonoids and saponins could inhibit the growth of pathogens such as Escherichia coli and Pseudomonas (Shu et al., 2020; Niu et al., 2023). In addition, Polysaccharides inhibited the levels of pathogenic bacteria by promoting the production of short-chain fatty acids and organic acids in the intestine, significantly increasing the abundance of dominant bacteria (Shi et al., 2020; Li, Zhao & Wang, 2009). In our research, CHAA and CHAB decreased the abundance of Escherichia-Shigella. Taken together, CHAA and CHAB could shape the intestinal microbiota by increasing the abundance of beneficial bacteria and reducing the abundance of pathogenic bacteria.

Conclusion

In conclusion, our data indicate that dietary supplementation of CHAA and CHAB significantly improved the meat quality and flavor by increasing the total amino acid content (Glu, Lys and Asp) and reducing the cholesterol content. Additionally, adding CHAA and CHAB could significantly stimulate and improve humoral immunity by increasing the levels of IgG, IgA and IgM in serum. CHAA and CHAB both showed an influence on increasing the beneficial microbiota abundance and decreasing the pathogenic microbiota abundance in the ceacum. Generally, these results provide valuable information that CHAA and CHAB could improve the meat quality, regulate immunity and shape the intestinal microbiota composition in Hungarian white geese. This study lays a foundation for developing and applying Chinese herbal medicine as a functional feed additive. The impact of the precise ingredient of Chinese herbal medicine on the growth performance and meat quality in geese will be investigated in the future.

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