Calf diarrhea is considered one of the most serious diseases of livestock from an economic standpoint (Cho and Yoon 2014; Qu et al., 2023). Calf diarrhea can be caused by various factors. For example, environmental pollution and improper feeding management can lead to infections with bacteria, viruses and parasites in calves (Ngeleka et al., 2019; Pansri et al., 2022). To a certain extent, antibiotics have emerged as the predominant pharmaceuticals (Shukla et al., 2022) and feed additives employed for the prevention of diarrhea in calves. Nonetheless, it is crucial to consider the potential implications associated with this practice, including the emergence of antibiotic resistance and the persistence of medicine residues over an extended period, which generated a serious concern for the safety of livestock products (Ali et al., 2021). Previous research demonstrated that Chinese herbal medicines can provide beneficial effects for the prevention and treatment of animal diseases (Chen et al., 2024) and have the potential to be used as effective substitutes for antibiotics (Millar et al., 2021; Guo et al., 2022).
       
Our previous studies have shown that SKQ exhibits remarkable antidiarrheal, anti-inflammatory and analgesic effects against gastrointestinal tract disorders (Yu et al., 2019) and was not toxic to the central nervous system, cardiovascular system, or respiratory system (Yu et al., 2018). The Chinese medicine SKQ is made of Pomegranate rind, sophora flavescens and cortex fraxini by hot water extraction.  Chinese medicine SKQ, the three main active ingredients of traditional Chinese medicine are ellagic acid, matrine, oxymatrine, aesculin and aesculetin. Pomegranate rind is featured with beneficial properties such as carcinopreventive effects, hemostasis activity, anti-inflammatory effects and antibacterial activities (Yassin et al., 2021; Rajamani et al., 2026). Sophora flavescens, rich in alkaloids and flavonoids, has multiple pharmacological activities, including anti-bacterial, anti-inflammatory, analgesic and antineoplastic effects (He et al., 2015). Cortex Fraxini has been therapeutically used in clinical applications owing to its functions of antiperspiratory, antirheumatic, astringency-inducing and anti-dysenteric effects (Guo et al., 2017). In this study, SKQ was applied as a therapeutic intervention for diarrhea,exploring its potential as an alternative to antibiotics.
               
Rumen bacteria function as a pivotal organ regulating host digestion, nutrient absorption and metabolism by generating bacterial metabolites and microbial-associated molecular patterns to maintain calf health and to prevent various diseases. However, the variation of rumen bacteria remains largely unknown when feeding calves supplemented with the Chinese medicine SKQ. The present study focused on the impact of SKQ on the distribution of the rumen flora in calves and the possible outcomes of feeding calves SKQ as a supplement. We investigated the effects of SKQ on the rumen bacteria by metagenomics sequencing, by which the species distribution was comprehensively analyzed and functional annotation was performed to identify the physiological changes in calves. 

Preparation of Chinese medicine SKQ
 
The Chinese medicine SKQ was developed by the Veterinary Research and Animal Husbandry Institute, Guizhou Academy of Agricultural Sciences. The compound was extracted and concentrated by water at a ratio of 3 : 3 : 2 from pomegranate peel, Sophora flavescens and Fraxini Cortex. The concentrate was mixed with lactose at a ratio of 1:1 and dried by spray drying to make pellet feed. Concentrated feedstuff was purchased from Huirui Animal Husbandry Technology Ltd. in Linyi, Shandong Province. Corn, stems and silage were obtained from local cattle farms.
 
Analysis by HPLC
 
The effective components in the peel of the pomegranate, namely  ellagic acid, the effective components in Sophora flavescens the, namely  matrine and oxymatrine and the contents of aesculetin and aesculin in the cortex fraxini were detected according to the Chinese Pharmacopoeia (2020 Edition) Volume II. The detection was carried out using high-performance liquid chromatography (HPLC).

Animal care and experimental design
 
Twelve healthy calves approximately 5 monthes of age were chosen from a cattle farm in Tongren City (China,Guizhou yellow cattle). Their weights were around 80-100 kg. Animals (6 per group) were divided into treatment (Group A) and control (Group B) groups and housed separately. The calves were vaccinated and dewormed before the experiment. All calves were fed with same amount of feed using a single tank. The pre-trial period lasted for 10 days and the trial period lasted for 60 days. The calves were fed at 8:00 a.m. and 4:00 p.m. each day with free access to water. The feed formula of experimental animals is shown in Table 1. Calves in the control group were fed with  forage only and calves in the experimental group were fed with forage containing 0.3% SKQ.


 
Sample collection
 
After the trial period, samples of rumen fluid from three calves randomly selected from each group were collected by using an orally administered stomach tube, for a total of six samples. The extracted rumen fluid was filtered through six layers of gauze. Approximately 50 mL of ruminal fluid from each sample was then placed in liquid nitrogen for later analysis.
 
Metagenomic sequencing
 
The rumen fluid samples were used to measure the variation in rumen microorganisms by metagenomic sequencing. The process of database building was divided into seven stages: DNA extraction of the sample, DNA tests, library construction, library detection, sequencing, quality control and information analysis. The sequencing was performed by Novogene Co., Ltd. In addition, the databases used to analyze genetic data were the Kyoto Encyclopedia of Genes and Genomes (KEGG), Version: 2018.01, Evolutionary genealogy of genes: Non-supervised Orthologous Groups (eggNOG), Version: 4.5 and the Carbohydrate-Active enzymes Database (CAZy), Version: 2018.01.

Composition of SKQ
 
HPLC analysis indicated that the SKQ contained matrine and oxymatrine at 4.21 mg/g, aesculin and aesculetin at 2.19 mg/g and ellagic acid at 1.17 mg/g (Table 2). The content of active ingredients in the traditional Chinese medicine compound meets the requirements of the Chinese Pharmacopoeia (2020 Edition) Volume II.


 
Metagenomics sequencing
 
Analysis of gene number differences
 
To investigate the differences in the number of genes among the various groups, the sequencing company created a bar chart showing the differences in the number of genes between the groups based on the data results. To analyze the common and specific information of genes between different samples (groups), the Venn diagram was drawn (Fig 1) It showed that the total number of genes identified in the study was 764,896, with 50,180 genes exclusive to group A and 140,868 genes exclusive to group B. The results showed that the experimental group was  lower than the control group.


 
Species annotation
 
Analysis of the relative abundance of species in each group. The species based on annotation and abundance were compared between group A and group B. To be specific, the top ten most abundant species at the genus level were Prevotella, Bacteroides, Acetobacter, Fibrobacter, Paraprevotella, Clostridium, Succiniclasticum, Alistipes, Ruminococcus and Klebsiella in group A and Prevotella, Bacteroides, Klebsiella, Succiniclasticum, Clostridium, Alistipes, Paraprevotella, Ruminococcus, Fibrobacter and Acetobacter in group B. But the results clearly show that the content of Prevotella, Klebsiella, Alistipes in group A was significantly lower than that in group B (Fig 2). The literature indicates that infected frogs that were exposed to both Klebsiella pneumoniae and Elizabethkingia miricola exhibited faster and higher mortality rates compared to frogs infected with each bacterium separately (Li et al., 2023), revealing the pathogenicity of Elizabethkingia miricola. Studies have shown that the microbial composition of the SKQ-fed group is better because there is less Elizabethkingia, reducing the risk of negative effects. Studies have shown that Klebsiella is involved in several human infections (Wyres and Holt, 2016) and has multidrug-resistant ability (Rodríguez-Medina et al., 2019). Studies have shown that Klebsiella is involved in several human infections (Wyres and Holt, 2016) and has multidrug-resistant ability (Rodríguez-Medina et al., 2019). The genus Alistipes are a kind of anaerobic bacteria reported to be involved in inflammation, cancer and other diseases (Parker et al., 2020). In this study, the SKQ group contained fewer harmful bacteria.


 
Metastats analysis of different species between groups
 
Metastats (White et al., 2009) was used to analyze species abundance data. As shown in Fig 3, the genus Angelakisella from family Ruminococcaceae and genus Actinophytocola from Pseudonocardiaceae in group A were significantly lower than that in group B  (p<0.05). Additionally, the family Candidatus, genus Methanoperedenaceae in Group A was  extremely lower than that of Group B (p<0.01). (Fig 3). The study demonstrated that the relative abundance of the genus Angelakisella was negatively correlated to the levels of gastrointestinal active peptides (Chai et al., 2021). Although notable alterations have been found in the present study, bacteria such as Methanoperedenaceae and Actinophytocola were not discussed here due to the lack of literature reports.


 
LEfSe analysis of different  species between groups
 
LEfSe analysis of different species between groups: LEfSe analysis was used to screen for significant differences between groups. The rank sum test was used to detect the differences in species among groups and LDA (linear discriminant analysis) was used to achieve dimension reduction and evaluate the impact of the differential species (Segata et al., 2011). The LEfSe analysis included three parts, an LDA value distribution histogram, an evolutionary branching diagram (phylogenetic distribution) and a comparison diagram of abundance with biomarkers having significant differences between groups (Fig 4).


       
The histograms show that the genera Klebsiella, Pseudoxanthomonas, Alistipes and Succiniclasticum had significant reductions in group A  (Fig 5). The analysis results are consistent with Species annotation.


 
Annotation of common functions
 
Sketch of annotated gene numbers
 
The microbial gene function annotation based on KEGG indicated that the genes associated with metabolism were the highest abundant. those with the number of genes above 10000 being carbohydrate, amino acid, cofactors and vitamins, nucleotide metabolism and energy and glycan biosynthesis metabolism.
       
The annotation based on eggNOG showed that the functions with the number of genes above 20000 were cell wall/membrane/envelope biogenesis, carbohydrate transport and metabolism, replication-recombination-repair, amino acid transport and metabolism, translation-ribosomal structure and biogenesis, inorganic ion transport and metabolism and signal transduction mechanisms.
       
The annotation based on CAZy illustrated that the main enzymes were glycoside hydrolases involved with 27384 genes, glycosyl transferases with 9228 genes, carbohydrate-binding modules with 4384 genes, carbohydrate esterases with 3590 genes and polysaccharide lyases with 1280 genes.
 
Metastats analysis of different functions between groups
 
Metastats analysis was used to research the functions similar to the above analysis for the species between groups. The results showed that GT64 belonging to the class of glycosyltransferases, was highly abundant in group A compared with group B (p<0.01) (Fig 6). At present, the GT64 glycosyltransferase family has been studied in animals, including humans and mice and the human GT64 members are also known as the Exostosio family, consisting of five members, respectively exostosin 1 (EXT1), exostosin 2 (EXT2) and exostosin-like 1-3 (EXTL1–3), which play an important role in the synthesis of heparan sulfate (Wilson et al., 2022). Research has demonstrated that heparan sulfate is widely distributed and structurally similar in animal tissues. Additionally, heparan sulfate has a variety of biological activities and functions, including cell adhesion, regulation of cell growth and proliferation, developmental processes, blood coagulation and tumor metastasis (Rabenstein, 2002). Whether this has any impact on the growth and reproduction of the calves still needs further study.


 
LEfSe analysis of different function between groups
 
LEfSe analyses revealed that the LDA score of Amino acid transport and metabolism from the eggNOG database at level 1 was significantly different in Group B.The LDA scores of CE1 and GH29 from CAZy families at level 2 were much higher in Group B, while GH24, GH13, CBM20 and GH31 had higher scores in Group A (Fig 7).


       
GH24 is one of the glycoside hydrolase family.which includes a lysozyme gene firstly found in chicken egg white (Blake et al., 1967). SKQ may show a potential antimicrobial activity by upregulation of lysozyme. The GH13 family contains nearly 30 enzymes that are involved in starch degradation, hydrolyzing glycosidic bonds, polysaccharide degradation and glycosyl transfer. The GH31 family contains enzymes to hydrolyze oligosaccharides as well as to transfer mannose,which can cleave glucosidic bonds of glucose, galactose and mannose (Brun et al., 2020). CBM20 contains starch-binding domains (SBD) that can interact with cyclodextrins (Jia et al., 2017). It has been reported that CBM20 can boost catalytic efficiency combined with cyclodextrin glycosyltransferase.
               
The GH29 family shows its ability to hydrolyze practically all fucose-containing compounds via hemicellulolytic and a-fucosidase activity (Curci et al., 2021). The CE1 family contains acetyl xylan esterase, cinnamoyl esterase and feruloyl esterase, which are associated with cellulose and polysaccharide degradation and lipid metabolism (Lombard et al., 2014).

From the analysis of metagenomic sequencing, the abundance of beneficial bacteria in the experimental group increased (such as Prevotella and Bacteroides) and the abundance of inflammatory bacteria decreased (such as genus Alistipes and Angelakisella), suggesting a potential shift toward an anti-inflammatory microbial profile. At the same time, drug resistance and pathogenic microorganisms increased in the control group, such as Elizabethkingia, Klebsiella, Pseudoxanthomonas, which in turn caused changes in the contents of various carbohydrate active enzymes, especially the contents of sugar-degrading enzymes. This suggests that the supplementation of Chinese herbal additive Shikuqin may enhance the degradation of dietary carbohydrates, which could promote animal protein synthesis and potentially improve immune function, indicating that the degradation of sugar after the addition of Chinese herbal additive Shikuqin may promote animal protein synthesis and improve host immunity.

The present study was supported by Beef Cattle Modern Agricultural Technology System of Guizhou Province (No.GZRNCYJSTX-05), the Science and Technology Support Program of Guizhou Province (Research Project No. QKHZC-2023-021), Guizhou Provincial Key Laboratory of Livestock and Poultry Genetic Resources Innovation and Utilization (ZSYS[2025]034).
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by the University of Animal Care Committee.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsor- ship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.