A large number of microorganisms live in the intestinal tract of sheep and are closely involved in host metabolism, immunity, tissue homeostasis, and intracellular homeostasis (Su et al., 2022). The composition and diversity of microorganisms are influenced by diet composition, host genetics, and external environment. For example, parasitic infections increase pathogenic bacteria and decrease beneficial bacteria in sheep (Chatanga et al., 2021; Liu and Yin, 2021). Studies have shown that the addition of oregano essential oil to diets increases the abundance of Rumenococci, Bifidobacteria, and Enterococci (Jia et al., 2022). Rumen degradable starch as a ration alters intestinal bacterial communities and carbohydrate digestibility in dairy goats (Han et al., 2021). In addition, the microbial communities in different intestinal segments of sheep have unique characteristics. According to the similarity of community composition determined by principal coordinate analysis, the microbial community of the entire intestine can be divided into two groups: small intestine and large intestine (Wang et al., 2017). Dominant microbiota from the foregut to hindgut of Aohan wool sheep has changed from Proteobacteria to Bacteroidota (Ma et al., 2022).

PAS is the seed left behind by the ripening, drying, and filtration of Chinese prickly ash. Which are rich in α- Linolenic acid plays an important role in anti-asthma and anti-thrombosis (Tang et al., 2014; Yang et al., 2014). In addition, the anti-inflammatory ability of prickly ash extract has been proven multiple times (Hou et al., 2021). An antimicrobial peptide named NP-6 in PAS inhibited all tested strains (Hou et al., 2019). PAS may inhibit lung inflammation and tissue damage during asthma (Wang et al., 2016). The research shows that Prickly Ash oil extracted from PAS has inhibited osteoclastogenesis and anticancer effects against malignant melanoma (Pang et al., 2019; Zhang B. et al., 2023). The addition of PAS to the diet was effective in improving the fatty acid composition of the longest muscle of the pig’s back (Song et al., 2020). Different flora produced by Prickly Ash oil fed to small-tailed frigid sheep affected on nutrient digestion and absorption and gut health of the host and significantly altered the abundance of rumen microbiota (Zhang et al., 2022a,b). Lamb diarrhea is a common disease caused by pathogenic microbial infection and digestive system dysfunction, which poses a significant threat to the sheep industry. Diarrhea in sheep is mainly caused by rotavirus, Escherichia coli, streptococcus, salmonella, clostridium welchii, and Parasitic disease. And PAS has potential anti-inflammatory, antibacterial, and antioxidant properties (Fortuoso et al., 2019; Zhong et al., 2022).

The inflammatory response promotes tissue repair and an immune response, but when the immune response is excessive or persistent, it can trigger a range of serious diseases (Neurath, 2014). Inflammatory factor refers to a class of signaling factors produced by cells in the process of inflammatory response, which can stimulate immune cells to further release other inflammatory factors (Kong et al., 2021). Among inflammatory cytokines, IL-1β is considered a typical multifunctional cytokine that affects virtually all types of cells, whether acting alone or in combination with other cytokines. IL-1β is essential for cellular defense and tissue repair in almost all tissues, and has been implicated in pain, inflammation, and autoimmunity (Zhou et al., 2019). IL-1β is also involved in neuroprotection, tissue remodeling and repair (Tran et al., 2023). TNF-α enhances macrophage activity and killing function, and enhances the ability of macrophages to promote immune response; it also has a promoting effect on the activity and aggregation of neutrophils localized in inflammation (Zhang et al., 2021). IL-10 is also an important immunomodulator, which mainly inhibits and terminates inflammatory immune responses by inhibiting monocyte and macrophage activation (Bouquet et al., 2020).

In this study, a total of 60 samples from the intestinal tract (duodenum, jejunum, ileum, cecum, and colon) of normally fed and PAS-intervened sheep were studied for a comparative study of bacterial community structure and distribution by using 16 s rRNA full-length sequencing. Relevant inflammatory factors were then validated using ELISA kits. The aim was to complement the incomplete study of PAS on the intestinal flora of sheep and to provide a theoretical basis for the application of PAS to the regulation of intestinal health in ruminants.

Increased α-diversity of the gut microbiota is thought to be closely associated with intestinal homeostasis and health. A high diversity of intestinal flora can effectively resist the attack of harmful bacteria, improve intestinal immunity, and promote food digestion and absorption. A high diversity of flora also contributes to the maintenance of gut microbial balance and overall health. In this study, the diversity and richness of gut microorganisms were increased by the addition of PAS. It indicates that PAS helps to improve intestinal immunity and maintain intestinal microbial balance in sheep lambs. The Ace index was lower in the LZS group than in the CK group, probably because ACE considered a wider range of rare species and made corrections for coefficient of variation and sample coverage. Both PCoA and NMDS analysis results showed that PAS altered the community composition of gut microbiota in Hu sheep. Heat map analysis shows significant differences in the microbiota of the small intestine and large intestine. Comparing the species distribution of LZS group and CK group at the phylum level, it was found that Firmicutes were the most important flora in the intestine, which was also found in other mammals (Chang et al., 2020; Minozzi et al., 2020). Firmicutes is an efficient energy harvesting bacterium, which is closely related to the energy acquisition and immune response regulation of the body. The increased abundance of Firmicutes is to meet the energy demand of muscle growth (Du et al., 2022). PAS increased the number of Verrucomicrobiota and Cyanobacteria in the intestine. Verrucomicrobia is a newly classified bacterium, which exists in the intestinal mucosa of healthy people, and has also been found in the Gut microbiota of Tan sheep, blue sheep (Pseudois nayaur) and other groups (Wang et al., 2023; Zhao et al., 2023). Research has found that Verrucomimicrobia has anti-inflammatory effects and can maintain glucose homeostasis in the host microbiota (Wang X. et al., 2022). Christensenellaceae_R_7_group as highly efficient hydrogen-producing bacteria, and when enriched with methanogens at the same time, they were able to work synergistically to improve the efficiency of enteric fermentation of starch and other polysaccharides (Li et al., 2023). Cyanobacteria have a number of specialized functions, including vitamin B speciali K synthesis, specialized anaerobic fermentation, coprophilic H2 production y and nitrogen fixation. It was also found that cyanobacteria are one of the main gates of gut microorganisms in sheep (Di Rienzi et al., 2013). At the genus level, PAS increased the number of Gut microbiota Christensenellaceae_R_7_group and Romboutsia. Romboutsia was an ileum-specific genus of bacteria. Romboutsia has effects on the inflammatory process and is inversely related to inflammatory bowel disease (Schirmer et al., 2019). Christensenellaceae_R_7_group is a highly efficient hydrogen producing bacterium. When it is enriched with Methanogen, they can synergistically improve the intestinal fermentation efficiency of starch and other polysaccharides (Li et al., 2023). These results are basically consistent with previous studies, indicating that adding prickly ash products can have beneficial effects on the gut of sheep (Zhang et al., 2022b). LEfSe analysis showed that the LZS group was significantly enriched with bacterial communities such as Verrucomimicrobiota, Kiritimatiellae, and WCHBI_41. Research has shown that Kiritimatellae plays an important role in the digestion process of herbivores (Wang X. et al., 2022), while Akkermansia and Kiritimatellaeota are involved in the biosynthesis pathway of arginine and fatty acids (Baniel et al., 2021). WCHB1-41 is significantly affected by lifestyle changes (Zhang C. et al., 2023), Akkermannia and WCHB1-41 encoded arginine and Fatty acid synthesis metabolic pathway can effectively improve energy and nitrogen utilization efficiency of yaks (Guo et al., 2021). By analyzing the differences in KEGG metabolic pathways, it was found that the proportions of metabolic pathways of Excretory system, Folding, sorting and degradation, and Immune system in the LZS group were significantly higher than those in the CK group (p < 0.05), and that the proportions of metabolic pathways of Digestive system and Infectious diseases:parasitic were significantly higher than those in the CK group (p < 0.05). The proportion of metabolic pathways in Digestive system and Infectious diseases:parasitic was significantly higher than that in LZS group (p < 0.05). It shows that PAS can indeed improve the immunity of Hu sheep lambs, reduce the Parasitic disease of Hu sheep lambs (He et al., 2018; Patil and Mallya, 2022), and at the same time, PAS also has a certain impact on the digestion and excretion of sheep. However, PICRUSt2 still has shortcomings in predicting potential functions. Therefore, it is necessary to further study the impact of PAS on the metabolic ability of Hu sheep lambs.

After performing ELISA analysis of inflammatory factors, the results showed a significant increase in IL-10 levels (p < 0.05) and a significant decrease in IL-1β and TNF-α levels (p < 0.05) in the LZS group compared to the CK group. IL-10 prevents colitis by eliminating dysfunctional mitochondria and inhibiting mTOR signaling and inflammatory vesicle activation in macrophages (Ip et al., 2017). Elevated levels of IL-10 indicated an improvement in the immune mechanism of Hu sheep by PAS. In the context of chronic inflammation, high circulating expression of IL-1β promotes the production of oncogenic mediators to induce tumorigenesis, but IL-1β can also promote T cell-mediated adaptive immune responses (Kallapur et al., 2011). TNF-α is closely associated with intestinal flora disorders. It can modulate immunity by promoting IL-10 production by B cells, inducing T cell apoptosis, altering T cell receptor (TCR) signaling, inhibiting T helper 17 (Th17) cell differentiation, and enhancing the number and function of regulatory T (Treg) cells (Galinsky et al., 2020). Elevated levels of IL-1β and TNF-α similarly indicated a positive effect of PAS on the immunocompetence of Hu sheep (Zhang et al., 2021). After conducting correlation heat map analysis, we found very interesting phenomena. Verrucomimicrobiota, as a differential phylum of gut microbiota, has certain anti-inflammatory effects, IL-10 is a recognized inflammatory and immunosuppressive factor (Zheng et al., 2019). We intuitively found them to be positively correlated through heat maps, which has also been confirmed; IL-1β and TNF-α belong to the TNF ligand superfamily and are both pleiotropic pro-inflammatory cytokines (Barichello et al., 2010). Streptococcuis an important pathogen that causes bacterial meningitis. Actinobacteriota and Aeriscardovia may also have certain pro-inflammatory effects (Liu et al., 2023). It was inferred that there may be some connection between inflammatory factors and intestinal flora composition and that PAS has a positive effect on resistance to inflammatory bowel disease in Hu sheep.

Because of the differences in the action functions of the small and large intestine (Yang et al., 2021), after exploring the effects of PAS on intestinal microorganisms, we focused our analysis on individual intestinal segments. It was found that the number of features unique to the small intestine segments of the CK group and the small intestine segments of the LZS group were both greater than that of the large intestine segments, and that the small intestine had a higher abundance and diversity of intestinal microorganisms than the large intestine. PCoA showed that the gut microbial population structure in the large intestine segments of the test and control groups was essentially the same. For the large intestinal segments, the microbial communities of the cecum and colon changed almost identically after feeding PAS. This is basically the same as other scholarly studies (Ma et al., 2022). At the phylum and genus level, the predominant colony in the cecum and colon was Verrucomicrobiota, and this colony increased considerably in both the cecum and colon with the addition of PAS. Proteobacteria are positively correlated with fiber intake (Zhao et al., 2022); Akkermansia, in the cecum and colon, was also significantly increased by PAS, which promotes intestinal barrier integrity, modulates immune responses, suppresses inflammation, and supports an anti-inflammatory gut microbiota (Xi et al., 2020; Hagi and Belzer, 2021; Kalia et al., 2022). There was also an increase in UCG-005, a member of the Ruminococcaceae family, which is associated with cellulose and starch digestion (Sun et al., 2019; Liu and Yin, 2021). LEfSe analysis showed a shift in the dominant flora of the large intestine segment from Bacteroidetes and Gammaproteobacteria to Akkermansiaceae and Verrucomicrobiae. Bacteroidetes live in the intestines of animals, and studies have found that Bacteroidetes promote digestion and increased utilization of complex carbohydrates, and are associated with obesity levels (Hales et al., 2017; Zhang et al., 2018). Gammaproteobacteria, belonging to Pseudomonadota in the bacterial kingdom, usually exists in the digestive tract and can metabolize cancer drugs, drive immunosuppression and tumorigenesis. Gammaproteobacteria can induce cell damage by infecting pancreatic cells, even hiding within pancreatic cells, causing double stranded DNA breakage (Halimi et al., 2021). Interestingly, the microbial communities in the duodenum, jejunum and ileum of the small intestine were changed after feeding PAS. We then found that at the phylum level, the dominant flora in small intestine is Firmicutes, which has been demonstrated in multiple studies to be the dominant intestinal phylum for the breakdown of complex nutrients and the production of beneficial metabolites (Gebeyew et al., 2021); At the genus level, the dominant genus in the small intestine is uncultured_rumen_bacterium, which is in general agreement with other scholarly studies (Kumar et al., 2022). Also ureaplasma is a harmful bacterium that can cause inflammation of the fetal membranes (Snyder et al., 2013; Heiter et al., 2023). LEfSe analysis result showed that Kiritimatiellae and WCHB1_41 significantly enriched in the LZS group while Lactobacillales and Aeriscardovia significantly enriched in the CK group. Lactobacillales belongs to the class Bacilli and can improve sheep manure composting efficiency and final product quality and can influence mitochondrial biogenesis to improve meat quality in sheep (Li et al., 2020, Wang C. et al., 2022). Aeriscardovia belongs to the family Bifidobacteriaceae and is found in omnivorous organisms that consume a wide range of nutrients and are capable of expanding their metabolism of different carbon sources present in the host (Lugli et al., 2017). In summary, adding PAS to the diets can increase beneficial flora content such as Verrucomicrobia, Firmicutes, Kiritimatiellae, Christensenellaceae_R_7_group, WCHBI_41, UCG-005 and Akkermansia, which in turn improves the sheep intestinal tract, and provides a new view on the improvement of sheep immunity.

This study revealed the effect of PAS addition on intestinal bacteria in Hu sheep lambs and validated the associated inflammatory factors. It was found that the addition of PAS could increase the diversity and abundance of intestinal microorganisms, influence the composition of intestinal community in sheep, and increase the content of beneficial intestinal bacteria. Moreover, the excretory system and immune system were significantly enhanced. The ELISA test showed that PAS has a certain impact on the levels of inflammatory factors. Then we explored the effects of PAS on the small and large intestines, respectively, and found that for both CK and LZS groups, they showed that the number of unique features of the small intestinal segment was more than that of the large intestinal segment, and that the gut microbial abundance and diversity in the small intestines were higher than that in the large intestines. Moreover, the addition of PAS significantly increased Akkermansiaceae and Verrucomicrobiae in the large intestine segment, and Kiritimatiellae and WCHB1 41 in the small intestine segment. Thus, it can be confirmed that feeding PAS promotes changes in the abundance of intestinal microorganisms in sheep, affects the levels of inflammatory factors and thus improves immunity in sheep lambs. This study provides new insights into improving the stress resistance and intestinal health of sheep by adding green additives, and provides a theoretical basis for understanding how ruminant gut microbiota are affected by PAS.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: NCBI - PRJNA1003863.

The animal studies were approved by Animal Care Committee of Gansu Agricultural University (GSAU-AEW-2020-0057). The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from the owners for the participation of their animals in this study.

DL: Methodology, Writing – original draft, Writing – review & editing. HY: Writing – review & editing. QL: Writing – review & editing. KM: Writing – review & editing. HW: Writing – review & editing. CW: Writing – review & editing. TL: Writing – original draft, Writing – review & editing. YM: Methodology, Writing – original draft, Writing – review & editing.