Process Optimization of Compound Fermented Traditional Chinese Medicine and Its Feeding Effect on Dairy Cows

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Five TCMs and three probiotics were selected from 25 candidate herbs and six probiotics based on in vitro antibacterial activity against Staphylococcus aureus and Escherichia coli using agar well diffusion and paper disc diffusion methods. The five TCMs were formulated according to the “Jun-Chen-Zuo-Shi” principle, and the optimal solid-state fermentation process was identified through single-factor and orthogonal experiments. The results showed that the optimal conditions included a Bacillus subtilis : Bacillus coagulans : Lactobacillus plantarum ratio of 1:3:1, a moisture content of 32%, an inoculation rate of 10%, a temperature of 31°C, and a fermentation period of 5 d. HPLC was used to examine changes in bioactive compound levels before and after fermentation. Fermentation significantly increased the concentrations of Resveratrol , Polydatin , and Quercetin , whereas the Polydatin content decreased. To determine in vivo effects, the fermented TCM was fed to dairy cows, and the indices of production performance, milk quality, serum biochemical levels, immune function, antioxidant capacity, and hormone levels were evaluated, and the changes in intestinal flora were analyzed by sequencing rectal contents using the 16S rRNA. The results showed that fermentation TCM signiffcantly improved the production performance of dairy cows as well as the milk quality, enhanced humoral immunity, strengthened antioxidant capacity, favorable modulation of gut microbiota, and metabolic regulation. This study provides strong experimental support for developing and applying compound fermented TCMs as functional feed additives in dairy production. Compound fermented traditional Chinese medicine Dairy cows Milk quality Microbial flora Figures Figure 1 Figure 2 Figure 3 Introduction Dairy farming, regarded as one of the main industries within China animal husbandry, ensures a steady supply of high-quality animal protein for the population. The efficiency of dairy production and the quality of dairy products are closely linked to the economic return of the industry and national public health (Li et al. 2025 a). In recent years, with the rollout of policies such as the National Action Plan for Containing Antibiotic Resistance in Animals (2021 to 2025), the reduction of antibiotic use has become a central goal in upgrading livestock and poultry farming. However, the traditional farming model that depended on antibiotics to maintain dairy cow health and raise production output has created favorable conditions for the spread of antibiotic resistance in pathogenic bacteria, including Staphylococcus aureus and Escherichia coli . This approach also increased the likelihood of antibiotic residues in milk. Continued antibiotic exposure further disrupts intestinal microecology in dairy cows, diminishes microbial diversity, and contributes to impaired digestion and nutrient absorption. These issues collectively hinder sustainable gains in farming productivity (Dong et al. 2022 ; Niu et al. 2023 ). Therefore, the development of green, efficient, and residue-free strategies that can replace antibiotics has become a key requirement for advancing the high-quality development of the dairy sector. Chinese herbal medicine, a core component of traditional Chinese medicine (TCM), has increasingly shown value in livestock and poultry systems because of its natural origin, diverse chemical composition, relatively low toxicity, and minimal adverse reactions (Wang et al. 2024 ). Research has demonstrated that dietary supplementation with compound herbal formulations can support mastitis and ketosis management, reduce heat stress, enhance milk yield and fat content, and strengthen immune-related functions, thereby improving overall productivity in dairy cows (Cui et al. 2022 ; Li et al. 2023 ; Saleh et al. 2023 ). Modern pharmacological investigations confirm that alkaloids, flavonoids, and polysaccharides in these herbal materials regulate immune pathways, improve antioxidant activity, and suppress pathogenic bacterial growth (Liu et al. 2020 ; Tong et al. 2022 ; Zeng et al. 2020 ). Consequently, the use of compound herbal preparations as nutritional supplements offers promising potential for improving dairy production. However, raw herbal materials often show limited release of active components, poor bioavailability, and weaker palatability, which restrict their stable and large-scale adoption in modern dairy farming. The emergence of probiotic fermentation technology offers a promising strategy to overcome these limitations. With the advancement of probiotic fermentation research, it has been confirmed that probiotics such as Bacillus subtilis and Lactobacillus plantarum secrete cellulase, hemicellulase, and related enzyme systems during fermentation, which break down the cell walls of herbal matrices. This process facilitates the release, modification, and improved absorption of active ingredients by enhancing their solubility and bioactivity. In addition, these bioactive compounds can act directly on the host or exert their effects through secondary metabolites produced by intestinal flora metabolism, including short-chain fatty acids (SCFAs), amino acids, and vitamins (Guo et al. 2022 ; Liu et al. 2020 ; Zhou et al. 2025 ). Research has also shown that metabolites produced by probiotics, such as organic acids and antimicrobial peptides, can work together with herbal bioactives to strengthen antibacterial and anti-inflammatory activities, lower systemic endotoxin and C-reactive protein levels, and support intestinal barrier integrity. Concurrently, fermentation improves the palatability of herbal preparations, reduces potential toxicities, and enhances feed intake in dairy cows (Ma et al. 2025 ; Zhang et al. 2023 ). Overall, combining herbal medicine with probiotic fermentation as a feed additive strategy shows strong potential to support sustainable and efficient development within the animal feed industry. Current studies have confirmed that fermented single Chinese herbal medicines or herbal formulas show marked effects in improving the immunity of livestock and poultry and enhancing their production performance (Chen et al. 2023 ; Li et al. 2025 ). However, systematic research on selecting suitable multi-herbal Chinese medicine combinations, optimizing probiotic consortia, and defining appropriate fermentation parameters remains limited. In particular, there is a clear shortage of studies that incorporate the physiological characteristics and production requirements of dairy cows to refine the preparation of compound fermented Chinese medicines and explore their regulatory mechanisms relating to milk quality, metabolic status, immune responses, and intestinal microecology. To address this gap, the present study aimed to increase the application value of compound Chinese medicines in dairy cow production. First, in vitro antibacterial assays were conducted to select Chinese herbal ingredients and probiotic strains with inhibitory activity against common dairy cow pathogens such as S. aureus and E . coli , thereby identifying the optimal composition of the herbal formula and probiotic combination. Second, single-factor and orthogonal experiments were used to optimize solid-state fermentation parameters and clarify the contribution of probiotics to transforming bioactive components within the herbal mixture. Finally, feeding trials were performed to evaluate how the optimized compound fermented Chinese medicine influences production performance, milk quality, blood biochemical indices, immune activity, antioxidant capacity, and gut microbiota structure in dairy cows, thereby providing a scientific basis for its application in modern animal husbandry. Materials and methods Materials TCM and probiotics The twenty-five types of TCMs, including Atractylodes macrocephala , Rheum officinale, Salvia miltiorrhiza , Angelica sinensis , Codonopsis pilosula , Wolfiporia cocos , Glycyrrhiza uralensis , Phellodendron amurense , Coptis chinensis , Scutellaria baicalensis , Astragalus membranaceus , Reynoutria japonica , Lysimachia christinae , Lonicera japonica , Sophora flavescens , Gynostemma pentaphyllum , Forsythia suspensa , Akebia quinata , Ligustrum lucidum , Taraxacum mongolicum , Fraxinus rhynchophylla , Dioscorea opposita, Houttuynia cordata , Vaccaria segetalis , and Viola philippica , were sourced from the Chinese Medicine Procurement and Supply Station (Yuzhou, China). The eight bacterial strains, including E. coli (WDCM NO:827), S. aureus ༈CGMCC 1.12409༉, B. subtilis ༈CGMCC 1.108༉, B. coagulans ༈GDMCC NO:66306༉, Pediococcus pentosaceus , Enterococcus faecium Lactobacillus rhamnosus , and Lactobacillus plantarum ༈CGMCC NO:1.557༉were obtained from the Henan Microbial Biotransformation Engineering Research Center. Chemicals and reagents The ELISA kits were purchased from Yuanju Biotechnology Center (Shanghai, China). MRS broth, LB broth, and nutrient broth were provided by Haibo Biotechnology Co., Ltd (Qingdao, China). HPLC-grade methanol and acetonitrile were procured from Sigma-Aldrich (St. Louis, MO, USA). Phosphoric acid and acetic acid were supplied by Xilong Chemical Co., Ltd (Hubei, China). Reference standards of Polydatin , Resveratrol , Salvianolic acid B , and Quercetin were obtained from Yuanye Bio-Technology Co., Ltd (Shanghai, China). Experimental methods Culture activation Eight bacterial strains stored at − 80°C were thawed before activation. Four lactic acid bacteria strains ( Pediococcus pentosaceus , Enterococcus faecium , Lactobacillus rhamnosus , and Lactobacillus plantarum ) were inoculated into 6 mL MRS broth tubes. Two spore-forming strains ( B. subtilis and B. coagulans ) were transferred into 6 mL nutrient broth tubes, and two pathogenic strains ( E. coli and S. aureus ) were inoculated into 6 mL LB broth tubes. All tubes were incubated at 37°C for 24 to 48 h. The activated lactic acid bacteria were then inoculated at a 1% ratio into 100 mL MRS broth and incubated statically at 37°C for 48 h. One milliliter of B. and B. coagulans cultures was added to 100 mL of nutrient broth, while E. coli and S. aureus strains were inoculated into 100 mL LB broth and shaken at 37°C for 24 h. All cultures were subsequently stored at 4°C for later use. Antibacterial activity of probiotics and TCM against S. aureus and E. coli The paper disc method (Tan et al. 2016 ) was used to assess the antibacterial activity of probiotics against S. aureus and E. coli . In brief, 100 µL of E. coli (1.25 × 10⁶ CFU/mL) and S. aureus (9.10 × 10⁶ CFU/mL) suspensions were spread evenly onto LB agar plates and allowed to dry completely. Sterile blank antibiotic discs were immersed in suspensions of B. subtilis (1.25 × 10⁸ CFU/mL), B. coagulans (1.96 × 10⁸ CFU/mL), Pediococcus pentosaceus (1.70 × 10⁸ CFU/mL), Enterococcus faecium (3.40 × 10⁸ CFU/mL), Lactobacillus rhamnosus (2.60 × 10⁸ CFU/mL), and Lactobacillus plantarum (1.14 × 10⁸ CFU/mL), and then placed carefully onto the inoculated agar surfaces. Each probiotic strain was tested twice on two independent plates to ensure reproducibility. After incubation at 37°C for 24 h, inhibition zone diameters were recorded using a vernier caliper. Five grams of each of the 25 TCMs were weighed, placed into individual conical flasks, and mixed with 50 mL ultrapure water. Samples were soaked for 30 min, brought to a boil, and simmered for 30 min. The mixture was filtered, followed by the addition of 40 mL ultrapure water, and simmered again for 30 min. The two filtrates were combined and concentrated to 5 mL to obtain extracts at 1 g/mL. The extracts were centrifuged at 3000 g/min for 10 min, and the supernatant was collected for further testing. The antibacterial activity of the TCMs against S. aureus and E. coli was assessed using the hole-punching method (Tan et al. 2016 ). A volume of 100 µL of E. coli (1.25 × 10⁶ CFU/mL) and S. aureus (9.10 × 10⁶ CFU/mL) suspensions was spread on LB agar plates. Wells of 5 mm diameter were punched into the agar, and 50 µL of each TCM extract was added to the wells. Every TCM was tested on two plates, with four wells per plate. After incubation at 37°C for 24 h, inhibition zone diameters were measured using a vernier caliper. Fermentation of TCM and optimization of process parameters Five TCMs ( Polygonum cuspidatum , Salvia miltiorrhiza , Taraxacum officinale , Houttuynia cordata , and Gynostemma pentaphyllum ) were selected as raw materials and combined according to the traditional “Jun-Chen-Zuo-Shi” principle (Ke 2023 ). A single-factor experimental approach was used to optimize the fermentation parameters. First, five experimental groups were prepared with different inoculation ratios of B. subtilis , B. coagulans , and Lactobacillus plantarum (1:3:1, 1:1:3, 3:1:1, 1:1:1, and 2:1:2), each tested in triplicate. Fermentation was conducted using a 15% inoculation volume, 41% moisture content, and 37°C for 3 d. Colony-forming units (CFUs) were enumerated via plate counting to identify the ratio that yielded the highest microbial growth. Next, the three strains were mixed at a 1:3:1 ratio, and moisture content was evaluated at 29%, 32%, 35%, 38%, and 41%, with other variables held constant (15% inoculation volume, 37°C, 3 d incubation). In the subsequent step, inoculation volume (15%, 20%, 25%, 30%, and 35%) was assessed under fixed conditions (1:3:1 strain ratio, 41% moisture content, and 37°C for 3 d). Temperature optimization was then performed at 28°C, 31°C, 34°C, 37°C, and 40°C, using the 1:3:1 ratio, 15% inoculation volume, and 41% moisture content for 3 d. Finally, fermentation duration was tested at 1, 2, 3, 4, and 5 d, under standardized parameters (1:3:1 ratio, 15% inoculation volume, 41% moisture content, 37°C), with viable counts determined by the plate count method. Based on the single-factor results, a five-factor four-level (L 16 4⁵) orthogonal experimental design was implemented to evaluate the effects of strain ratios (1:1:3, 1:3:1, 3:1:1, 2:1:2), moisture levels (29%, 32%, 35%, 38%), inoculation volumes (10%, 15%, 20%, 25%), fermentation temperatures (28°C, 31°C, 34°C, 37°C), and fermentation durations (2, 3, 4, and 5 d) on total viable bacterial counts. The goal was to determine the optimal fermentation conditions. The experimental layout is presented in additional Table 1 . HPLC-based analysis of compositional changes in TCM before and after fermentation Accurately 6.10 mg of standard Polydatin , 6.80 mg of Resveratrol , 4.80 mg of Salvianolic acid B , and 8.80 mg of Quercetin were weighed, dissolved, and diluted with HPLC-grade methanol to the calibration mark. The mixture was then mixed thoroughly, resulting in a stock solution containing 0.244 mg/mL Polydatin , 0.272 mg/mL Resveratrol , 0.192 mg/mL Salvianolic acid B , and 0.352 mg/mL Quercetin , which was reserved for further analyses. One gram each of unfermented TCM and fermented TCM (collected at 3, 5, and 7 d post-fermentation) was weighed and transferred into 50 mL beakers. Each sample was combined with 50 mL of 70% methanol and subjected to reflux extraction for 30 min. After extraction, the samples were filtered through 0.22 µm membranes and concentrated to dryness. Pure methanol was then added to a fixed volume of 10 mL to prepare the final sample solution. Chromatographic separation was performed using a Hega Technologie C18 column (460 mm × 4.6 mm, 5 µm). For Polydatin and Resveratrol , the mobile phase consisted of acetonitrile (A) and 0.01% acetic acid water (B), operated under isocratic elution (23:77). The flow rate was 1 mL/min, with a detection wavelength of 306 nm, column temperature of 30°C, and injection volume of 10 µL. For Salvianolic acid B , the mobile phase was acetonitrile (A) and 0.02% phosphoric acid water (B) at 22:78, with a 1.2 mL/min flow rate, 286 nm detection wavelength, 20°C column temperature, and 10 µL injection volume. For Quercetin , the mobile phase consisted of methanol (A) and 0.02% phosphoric acid water (B) at 50:50, with a 1.0 mL/min flow rate, 360 nm detection wavelength, 30°C column temperature, and 10 µL injection volume. Standard solutions of Polydatin , Resveratrol , Salvianolic acid B , and Quercetin were prepared, and aliquots of 0.5, 1, 2, 3, 4, and 5 mL were transferred into 10 mL volumetric flasks to generate six concentrations of each compound using methanol. All samples were analyzed under the specified chromatographic conditions. Peak areas were measured, and regression equations were constructed for quantitative analysis. Animal diets and experimental design This experiment was conducted at a commercial dairy farm in Zhongmu County, Zhengzhou City, Henan Province. The dietary composition and nutritional levels are listed in additional Table 2 . Eighteen healthy Holstein dairy cows with similar body weight, parity, days in lactation, and milk yield were selected and randomly assigned to two groups, with three replicates per group and three cows per replicate. The control group (Group B) received a basal diet without additives, whereas the experimental group (Group A) was fed the basal diet supplemented with 200 g/cow/d of compound fermented TCM. Dairy cows were fed three times daily at 07:00, 15:00, and 21:00. The experimental period lasted for 30 d. All animals were maintained under standard farm conditions using a free-stall housing system and milked three times daily at 05:30, 13:30, and 19:30. Milk yield and milking times were recorded automatically using the Waikato Milking System. Animals had ad libitum access to feed and water. All procedures were approved by the Animal Care and Use Committee of Henan Agricultural University (Approval No. HNND2024092901). Composition and nutrient levels of the basal diet Milk sampling and analysis Milk samples were collected from each dairy cow before the experiment and on day 30. For each cow, milk obtained from the three daily milkings (05:30, 13:30, 19:30) was mixed thoroughly. Potassium dichromate was added to the mixed samples for milk quality assessment. After collection, 50 mL of each sample was sent immediately to Henan Provincial Dairy Cow Production Performance Testing Co., Ltd. for the determination of milk fat percentage, milk protein percentage, lactose, total solids, milk urea nitrogen (MUN), and somatic cell count (SCC). Blood sampling and analysis Blood samples were collected from each dairy cow via the caudal vein into evacuated tubes without anticoagulant before morning feeding on day 30. Samples were allowed to stand at room temperature for 30 min, followed by centrifugation at 3000 rpm and 4°C for 15 min to obtain serum. The serum was stored at − 20°C for subsequent analyses. Biochemical indicators included total protein (TP), albumin (ALB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (Cr), glucose (Glu), and blood urea nitrogen (BUN). Immune indicators included immunoglobulin A (IgA), immunoglobulin G (IgG), immunoglobulin M (IgM), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-8 (IL-8), and tumor necrosis factor-α (TNF-α). Antioxidant indicators included total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA). Hormonal indicators included growth hormone (GH), estradiol (E2), and prolactin (PRL). All parameters were measured after storage. Gut microbiota analysis On day 30, fecal samples were collected from experimental dairy cows using the rectal method. Samples were placed into sterile, enzyme-free 50 mL centrifuge tubes and transported immediately to Shanghai Paisenno Biotechnology Co., Ltd. for high-throughput gut microbiota sequencing. Statistical analysis IBM SPSS 27.0 software was used for statistical analyses. One-way Analysis of Variance (ANOVA) with Tukey test was applied to evaluate inter-group differences in milk yield, milk indicators, serum indicators, and other measured parameters. Data are expressed as mean ± standard deviation. A P value < 0.01 was considered highly significant, P < 0.05 was considered significant, and 0.05 ≤ P < 0.1 was regarded as a trend. Graphs were prepared using Origin and GraphPad Prism 8. Results Inhibitory screening identifies key TCMs and probiotic strains with antibacterial activity Among the 25 TCM extracts screened, 15 showed varying levels of inhibition against S . aureus (Table 1 ). Rheum officinale , Salvia miltiorrhiza , Codonopsis pilosula , Glycyrrhiza uralensis , Coptis chinensis , Scutellaria baicalensis , and Reynoutria japonica all produced measurable inhibition zones. Coptis chinensis demonstrated the strongest activity with a 30.00 mm inhibition zone, followed by Codonopsis pilosula (28.50 mm) and Salvia miltiorrhiza (27.00 mm). Rheum officinale and Glycyrrhiza uralensis also showed notable inhibition of 21.00 mm and 22.00 mm, respectively. Scutellaria baicalensis and Polygonum cuspidatum produced moderate zones of 15.50 mm. Several other TCMs, including Phellodendron amurense , Lysimachia christinae , Forsythia suspensa , Ligustrum lucidum , Taraxacum mongolicum , Fraxinus rhynchophylla , Houttuynia cordata , and Viola philippica , displayed limited inhibition, with zone diameters too small to be considered significant. Against E. coli , only three TCMs exhibited measurable antibacterial effects: Angelica sinensis , Coptis chinensis , and Ligustrum lucidum , with inhibition zones of 7.00 mm, 10.00 mm, and 7.50 mm, respectively. As shown in Table 2 , among the six probiotic strains assessed, Lactobacillus plantarum , B. subtilis , and B. coagulans demonstrated moderate inhibition of both S. aureus (11.00 mm, 11.50 mm, and 13.50 mm) and E. coli (10.00 mm, 10.50 mm, and 12.50 mm). Enterococcus faecium inhibited S. aureus with a 9.00 mm zone but showed no inhibition toward E. coli . Lactobacillus rhamnosus and Pediococcus pentosaceus produced no measurable inhibitory activity against either pathogen. Table 1 Average inhibition zone diameter of Staphylococcus aureus and Escherichia coli by 25 Chinese herbal extracts (mm) TCM/Strain S.aureus E. coli Atractylodes macrocephala - - Rheum officinale 21.00 ± 1.41 - Salvia miltiorrhiza 27.00 ± 1.41 - Angelica sinensis - 7.00 ± 2.83 Codonopsis pilosula 28.50 ± 0.71 - Wolfiporia cocos - - Glycyrrhiza uralensis 22.00 ± 1.41 - Phellodendron amurense 9.50 ± 0.71 - Coptis chinensis 30.00 ± 1.41 10.00 ± 0.00 Scutellaria baicalensis 15.50 ± 0.71 - Astragalus membranaceus - - Reynoutria japonica 15.50 ± 0.71 - Lysimachia christinae 7.50 ± 2.12 - Lonicera japonica - - Sophora flavescens - - Gynostemma pentaphyllum - - Forsythia suspensa 6.50 ± 0.71 - Akebia quinata - - Ligustrum lucidum 13.50 ± 0.71 7.50 ± 0.71 Taraxacum mongolicum 6.00 ± 1.41 - Fraxinus rhynchophylla 9.00 ± 1.41 - Dioscorea opposita - - Houttuynia cordata 7.50 ± 0.71 - Vaccaria segetalis - - Viola philippica 7.00 ± 2.83 - Note: The punch diameter was 5 mm. The inhibition zone was classified as follows: ≥20 mm, extreme sensitivity; 15–19 mm, high sensitivity; 10–14 mm, moderate sensitivity; <10 mm, low sensitivity. The symbol “–” indicates no inhibition zone. Table 2 Average inhibition zone diameter of six probiotic strains (mm) Strain S. aureus E. coli Enterococcus faecium 9.00 ± 1.41 - Lactobacillus rhamnosus - - Pediococcus pentosaceus - - Lactobacillus plantarum 11.00 ± 1.41 10.00 ± 1.41 Bacillus subtilis 11.50 ± 3.54 10.50 ± 0.71 Bacillus coagulans 13.50 ± 0.71 12.50 ± 0.71 Note: The antimicrobial susceptibility disc had a diameter of 6 mm. Inhibition zones were classified as follows: ≥20 mm, extremely sensitive; ≥15 mm, highly sensitive; ≥10 mm, moderately sensitive; ≥6 mm, slightly sensitive. The symbol “–” indicates no inhibition zone. Single-factor and orthogonal optimization identify ideal solid-state fermentation conditions Based on antibacterial activity patterns and formulation principles, the compound TCM was prepared using Reynoutria japonica , Salvia miltiorrhiza , Gynostemma pentaphyllum , Taraxacum mongolicum , and Houttuynia cordata . The single-factor experiment outcomes for solid-state fermentation are presented in Fig. 1 . Probiotic addition ratios, moisture levels, inoculation amounts, temperature, and fermentation duration all influenced the viable bacterial count. The highest viable count, 2.10 × 10⁸ CFU/g, occurred when B. subtilis , B. coagulans , and Lactobacillus plantarum were combined at 3:1:1 (Fig. 1 A). A moisture level of 35% produced the maximum count of 5.55 × 10⁸ CFU/g (Fig. 1 B). With a 20% mixed inoculum, the viable count reached 2.33 × 10⁸ CFU/g, but further increases in inoculation amount caused a downward trend (Fig. 1 C). Fermentation at 34°C generated the highest viable count of 9.45 × 10⁸ CFU/g (Fig. 1 D). The viable count peaked on day 3 at 5.05 × 10⁸ CFU/g (Fig. 1 E). Based on these results, an orthogonal experiment was conducted (additional Table 3 ). Range (R) and mean (K) analyses showed that inoculation amount had the most significant effect on viable count ( P < 0.05), followed by moisture level, fermentation time, temperature, and probiotic ratio. The optimal combination was A₂B₂C₁D₂E₄, corresponding to a strain ratio of 1:3:1 ( B. subtilis , B. coagulans , and Lactobacillus plantarum ), 32% moisture level, 10% inoculation amount, 31°C temperature, and a 5 d duration. Under these optimized parameters, the final product yielded a viable count of 2.50 × 10⁸ CFU/g, a spore count of 5.15 × 10⁷ CFU/g, and a pH of 4.79. Fermentation drives major shifts in key bioactive compounds of the TCM formulation HPLC analysis revealed substantial biotransformation of major active compounds during fermentation (Fig. 2 ). Polydatin declined steadily, decreasing from the initial level to 0.41 µg/mL by day 7, representing a 78.59% reduction ( P < 0.05). In contrast, Resveratrol increased significantly, reaching 1.70 µg/mL on day 7, a 74.90% rise relative to the unfermented preparation ( P < 0.05). Salvianolic acid B showed a decrease to 4.22 µg/mL on day 3, followed by a gradual increase, reaching 5.41 µg/mL on day 7. This value was 4.99% higher than the unfermented level. Quercetin was undetectable before fermentation but became detectable after day 3, rising consistently to 0.87 µg/mL by day 7 ( P < 0.05). These results indicate that microbial fermentation reduces macromolecular components and increases small-molecule bioactives, promoting the biotransformation of complex compounds and enhancing their potential absorption and utilization in animals. Fermented TCM improves milk yield, milk fat, lactose, and udder health indicators The effects of supplementing the diet with compound fermented TCM on milk production and composition are summarized in Table 3 . Compared with Group B (control), Group A showed a 6.66% increase in milk yield, indicating an upward trend (P = 0.083). Among milk components, milk fat percentage in Group A increased significantly by 20.97% ( P < 0.05), and lactose content also showed an upward trend (P = 0.095). MUN in Group A decreased significantly by 13.93% ( P < 0.05). In addition, SCC declined by 34,400/mL, corresponding to a 54.00% reduction ( P 0.05). Overall, the addition of compound fermented TCM enhanced milk quality and reduced inflammatory cell counts, contributing to improved production performance in Holstein dairy cows. Table 3 Effects of compound fermented TCM on production performance and milk quality in dairy cows Items Groups P-value A B Milk yield (kg/d) 31.57 ± 1.23 a 29.6 ± 0.82 a 0.083 Milk fat (%) 4.50 ± 0.55 b 3.72 ± 0.90 a 0.031 Milk protein (%) 3.47 ± 0.19 a 3.38 ± 0.11 a 0.202 Lactose (%) 5.15 ± 0.05 a 5.05 ± 0.16 a 0.095 Total solids (%) 14.94 ± 0.83 a 14.36 ± 1.06 a 0.194 MUN (mg/dL) 14.52 ± 1.72 b 16.87 ± 2.26 a 0.017 SCC (10 4 /个) 2.93 ± 1.39 b 6.37 ± 3.75 a 0.014 Fermented TCM elevates protein metabolism markers without altering liver function indicators The changes in blood biochemical profiles are shown in Table 4 . After compound fermented TCM supplementation, TP increased significantly, rising by 5.94% ( P < 0.05). ALB reached 16.48 g/L, reflecting a 4.97% increase versus Group B ( P 0.05). In summary, dietary supplementation with compound fermented TCM enhanced protein metabolism, reduced nitrogen metabolite accumulation, and improved nitrogen utilization efficiency without adversely affecting liver function in dairy cows. Table 4 Effects of compound fermented TCM on serum biochemical indices of dairy cows Items Groups P-value A B TP (µg/mL) 944.47 ± 27.90 b 891.53 ± 55.66 a 0.015 ALB (g/L) 16.48 ± 0.73 b 15.70 ± 0.87 a 0.045 ALT (pg/mL) 85.32 ± 4.60 a 87.94 ± 5.75 a 0.275 AST (pg/mL) 35.96 ± 2.37 a 37.38 ± 2.15 a 0.179 Cr (mmol/L) 22.91 ± 1.85 a 23.00 ± 1.49 a 0.912 Glu (ng/mL) 42.77 ± 1.11 a 41.89 ± 1.59 a 0.171 BUN (mmol/L) 24.09 ± 0.55 b 25.04 ± 1.59 a 0.090 Fermented TCM enhances key humoral immune indicators in dairy cows Immune-related changes are presented in Table 5 . Compared with Group B, cows in Group A exhibited significant increases in IgG, IgM, and IL-4 by 5.55%, 3.44%, and 3.70%, respectively ( P 0.05). Overall, compound fermented TCM elevated major humoral immune markers, suggesting improved immune responsiveness in dairy cows. Table 5 Effects of compound fermented TCM on the immune performance of dairy cows Items Groups P-value A B IgA (µg/mL) 1198.02 ± 45.48 a 1164.58 ± 67.15 a 0.209 IgG (g/L) 24.92 ± 1.56 b 23.61 ± 0.70 a 0.025 IgM (µg/mL) 2766.69 ± 108.13 b 2674.65 ± 80.76 a 0.045 IL-2 (pg/mL) 292.25 ± 9.82 a 302.20 ± 12.30 a 0.061 IL-4 (pg/mL) 63.92 ± 2.09 b 61.64 ± 1.84 a 0.019 IL-8 (pg/mL) 241.26 ± 12.29 a 244.39 ± 10.95 a 0.568 TNF-α (pg/mL) 170.24 ± 7.25 a 172.73 ± 10.93 a 0.556 Fermented TCM enhances antioxidant capacity and lowers oxidative stress markers in dairy cows Serum antioxidant indicators, including T-AOC, SOD, GSH-Px, and MDA, were measured after 30 days of supplementation. As shown in Table 6 , cows in Group A exhibited significantly higher T-AOC and SOD levels, with increases of 3.70% and 6.34%, respectively ( P < 0.05). MDA concentration in Group A reached the lowest value of 7.98 nmol/mL, representing a significant reduction compared with Group B ( P 0.05). Collectively, dietary supplementation with compound fermented TCM reduced oxidative damage and improved serum antioxidant enzyme activities, thereby strengthening the overall antioxidant status of dairy cows. Table 6 Effects of compound fermented TCM on antioxidant properties of dairy cows Items Groups P-value A B T-AOC (pg/mL) 179.95 ± 5.56 b 173.53 ± 7.67 a 0.046 SOD (ng/mL) 22.80 ± 1.21 b 21.44 ± 1.45 a 0.035 GSH-Px (mIU/mL) 810.48 ± 25.16 a 794.85 ± 23.58 a 0.169 MDA (nmol/mL) 7.98 ± 0.35 b 8.35 ± 0.40 a 0.041 Fermented TCM increases circulating GH levels without altering PRL or E2 Serum hormone profiles are summarized in Table 7 . Group A recorded a GH concentration of 15.43 ng/mL, significantly higher than Group B ( P < 0.05). PRL in Group A was 3.73% higher than in Group B, showing an upward trend, although the difference did not reach significance (P = 0.072). No significant changes were observed in E2 levels between the two groups ( P > 0.05). These findings indicate that compound fermented TCM elevated circulating GH, which may support improved production outcomes in dairy cows. Table 7 Effects of compound fermented TCM on blood hormones levels of dairy cows Items Groups P-value A B GH (ng/mL) 15.43 ± 0.90 b 14.19 ± 1.18 a 0.016 E 2 (pg/mL) 77.83 ± 1.83 a 76.83 ± 2.62 a 0.335 PRL (mIU/L) 716.03 ± 30.15 a 690.31 ± 30.13 a 0.072 Fermented TCM alters microbial richness and shifts key gut bacterial taxa in dairy cows 16S rDNA sequencing was used to analyze the intestinal microbiota of Group A and Group B. Sequences were clustered into operational taxonomic units (OTUs) based on sequence similarity, yielding 39,672 OTUs in total. Among these, 19,963 OTUs were unique to Group B, 16,052 were unique to Group A, and 3,657 were shared between both groups (Fig. 3 A). The microbiota were classified into 15 phyla, 20 classes, 49 orders, 91 families, 265 genera, and 209 species. Dilution curves for samples from Group A and Group B (Fig. 3 B) showed an initial rapid rise followed by a plateau. This indicated that once sequencing depth exceeded 40,000 reads, coverage and species richness were sufficient for reliable downstream diversity analyses. Alpha and Beta diversity analyses were performed to assess microbial richness and community variation. As shown in Fig. 3 C, significant differences were detected in the Chao1 and observed species indices ( P 0.05), suggesting comparable overall microbial diversity. These results indicate that compound fermented TCM reduced rectal microbial richness without markedly altering overall diversity. To further examine compositional differences, principal coordinate analysis (PCoA) based on Bray–Curtis distances and non-metric multidimensional scaling (NMDS) based on the Bray–Curtis matrix were conducted and visualized in two dimensions. The PCoA and NMDS plots (Fig. 3 D) showed that microbial communities in both groups were closely distributed, indicating high similarity in microbial composition and richness. However, the Group B samples were more dispersed, suggesting higher inter-individual variation under normal feeding conditions. At the phylum level, the top ten dominant taxa were analyzed. In Group A, the predominant phyla were Firmicutes_A (61.58%), Bacteroidota (29.38%), Firmicutes_D (3.11%), and Actinobacteriota (3.02%). In Group B, the dominant phyla were Firmicutes_A (65.30%), Bacteroidota (26.13%), Firmicutes_D (2.99%), and Actinobacteriota (2.44%). In both groups, Firmicutes_A and Bacteroidota accounted for more than 90% of total microbial abundance. Compared with Group B, Group A showed decreasing trends in Firmicutes_A and Proteobacteria ( P > 0.05), while Bacteroidota , Firmicutes_D , and Actinobacteriota showed increasing trends ( P > 0.05). These findings suggest that fermented TCM modulated major phyla by reducing Firmicutes_A and Proteobacteria and promoting Bacteroidota , Firmicutes_D , and Actinobacteriota (Fig. 3 E). Further genus-level analysis (Fig. 3 G) identified shifts in microbial composition. In Group A, dominant genera included Faecousia (16.88%), Cryptobacteroide s (8.71%), Phocaeicola_A (5.28%), PeH17 (4.66%), RUG13077 (3.04%), Alistipes_A (2.70%), UBA737 (2.37%), Bifidobacterium (2.51%), RF16 (2.23%), CAG-41 (2.33%), Paraprevotella_A (2.18%), Paraclostridium (2.08%), Romboutsia_B (1.44%), SFMI01 (1.20%), and Prevotella (1.08%). In Group B, the dominant genera were Faecousia (14.20%), Cryptobacteroide s (7.98%), Phocaeicola_A (4.44%), PeH17 (4.08%), RUG13077 (2.72%), Alistipes_A (2.09%), UBA737 (2.25%), Bifidobacterium (1.98%), RF16 (2.26%), CAG-41 (2.13%), Paraprevotella_A (2.22%), Paraclostridium (2.24%), Romboutsia_B (1.64%), SFMI01 (1.37%), and Prevotella (0.63%). Compared with Group B, Group A showed higher relative abundances of Faecousia , Cryptobacteroides , Phocaeicola_A , Bifidobacterium , and Prevotella , although differences were not significant ( P > 0.05). This suggests that compound fermented TCM may promote beneficial microbial genera. Linear discriminant analysis effect size (LefSe) (Fig. 3 F) revealed significant structural differences in intestinal microbiota. Group A was enriched with Phascolarctobacterium_A , f_Acidaminococcaceae , g_Erysipelothrix , g_CAG_632 , g_Eubacterium_F , g_Limosilactobacillus , and g_Faecalibacterium , whereas Group B was enriched with f_Enterobacteriaceae , g_Streptococcus , and f_Streptococcaceae . In conclusion, fermented TCM restructured the intestinal microbiota by inhibiting pathogenic groups and enhancing beneficial taxa, indicating a positive modulation of gut microbial ecology in dairy cows. Discussion TCM contains a wide range of bioactive substances, including polysaccharides, alkaloids, glycosides, organic acids, tannins, polyphenols, pigments, and volatile oils. These components can interact synergistically through several biological pathways. This multi-target activity supports broad bacteriostatic effects. As a result, TCM has been applied widely in animal husbandry for many years. Different TCMs, however, show clear differences in antibacterial range and strength. These variations mainly reflect differences in the types and levels of their active constituents (Cao et al. 2010; Wong et al. 2010 ). In this study, the TCM formula was developed through a structured screening process and scientific evaluation of herbal compatibility. The agar well diffusion method was used to analyze aqueous extracts from 25 TCMs. The findings indicated that Rheum officinale , Salvia miltiorrhiza , Reynoutria japonica , and Glycyrrhiza uralensis had strong inhibitory effects against S. aureus . Their inhibition of E. coli was weak. Several factors may explain this pattern. High-temperature decoction or high-pressure extraction can damage heat-sensitive compounds. Volatile components may also evaporate under these conditions. In addition, some antibacterial molecules are poorly soluble in water, which reduces their extraction efficiency. These factors can lower the measured in vitro activity of TCM extracts. The form of TCM used in production also affects its antibacterial effect. In livestock systems, herbs are typically provided as powders or fermented preparations rather than aqueous extracts. Studies have shown that fermentation by lactic acid bacteria can enhance inhibitory zones. For example, fermented extracts increased the inhibition of S. aureus by more than 20%, and Salmonella by more than 12% compared with non-fermented products (Wang et al. 2023 ). Some TCM components also require host metabolic enzymes to convert them into active forms. In vitro assays lack this metabolic environment. As a result, they may not reflect the true antibacterial potential of compounds that depend on in vivo transformation. Therefore, while in vitro bacteriostatic tests offer useful preliminary information, they cannot fully represent the complex pharmacological effects observed in animals. A combined evaluation of in vitro and in vivo results is required to accurately determine the antibacterial efficacy and practical application value of TCM formulations. Medication compatibility is a central principle of TCM. Proper combinations of herbs can enhance therapeutic effects, reduce required dosages, and lower treatment costs. Traditional studies have often focused on single herbs that show strong bacteriostatic activity. These herbs are usually selected as the core for compatibility. However, herbs with weak or undetectable bacteriostatic effects are frequently overlooked. Such herbs may still contribute through synergistic interactions when combined correctly. For example, Chen ( 2009 ) reported that Terminalia chebula Retz had a minimum inhibitory concentration (MIC) of 250.00 mg/mL against Salmonella typhimurium . Phellodendron chinense Schneid showed no measurable inhibition. When both herbs were combined, the MIC decreased to 62.50 mg/mL, which demonstrated a strong synergistic effect. Following this concept, the formula in this study included Gynostemma pentaphyllum , even though it did not form a bacteriostatic circle in vitro. This herb has functions related to clearing heat, detoxifying, and supporting leukocyte recovery. It was therefore combined with four herbs that showed measurable bacteriostatic effects: Reynoutria japonica , Salvia miltiorrhiza , Taraxacum mongolicum , and Houttuynia cordata . This combination was designed to act on pathogenic bacteria through multiple pathways, thereby improving the overall antimicrobial potential of the formulation. Probiotics are widely used as feed microecological agents. They produce various enzymes that support digestion and absorption in the host. Their metabolic products also include several bacteriostatic substances, which help strengthen the host's antimicrobial defenses. In vitro antibacterial assays offer a direct way to test the activity of these probiotic metabolites. In this study, the disk diffusion method was used to evaluate four lactic acid bacteria strains and two Bacillus strains against S. aureus and E. coli . The results showed that Lactobacillus plantarum , B. subtilis , and B. coagulans had clear inhibitory activity against both pathogens. Among them, B. coagulans produced the largest inhibition zones against S. aureus and E. coli . This was followed by B. subtilis and Lactobacillus plantaru m. The variation in bacteriostatic performance among these strains may reflect differences in their antimicrobial metabolites and mechanisms. Lactobacillus plantarum and Bacillus species are gram-positive bacteria, but they rely on different antimicrobial strategies. Lactobacillus plantarum produces organic acids, such as lactic and acetic acid (Qiao 2020 ). These acids lower the surrounding pH and suppress the growth of pathogenic microorganisms. B. subtilis and B. coagulans , in contrast, produce antimicrobial peptides or bacteriocins. These compounds can inhibit cell wall synthesis or form pores in the bacterial membrane (Pei et al. 2017 ). Both actions compromise membrane integrity and eventually cause cell lysis. During solid-state fermentation of TCM with probiotics, the control of parameters such as probiotic ratios, moisture levels, inoculum size, temperature, and fermentation time plays a decisive role. These factors directly influence microbial growth and reproduction. Optimizing such parameters can therefore improve both the quality and functional activity of the final fermented product (Chen 2022 ; Hu et al. 2023 ). In this study, the viable bacterial count was selected as the main evaluation index. The results showed clear responses to each fermentation parameter. When the ratio of B. subtilis , B. coagulans , and Lactobacillus plantarum was 3:1:1, the viable count reached 2.10 × 10⁸ CFU/g. At a moisture level of 35%, the count increased to 5.55 × 10⁸ CFU/g. A mixed inoculum size of 20% produced a maximum count of 2.33 × 10⁸ CFU/g, while larger inoculum sizes caused a decline in viable bacteria. Under incubation at 34°C, the viable count reached 9.45 × 10⁸ CFU/g. On the third fermentation day, the count peaked at 5.05 × 10⁸ CFU/g. Zhao ( 2019 ) examined similar fermentation optimization using Lactobacillus plantarum . Their orthogonal experiments evaluated temperature, fermentation time, and inoculum size. The concentration of fermented TCM extract was 0.2 mg/mL. Based on single-factor testing and orthogonal design, the ideal parameters were identified as follows: a microbial ratio of B. subtilis : B. coagulans : Lactobacillus plantarum = 1:3:1, moisture content 32%, inoculum size 10%, temperature 31°C, and a fermentation duration of 5 days. TCM contains many macromolecular organic compounds, including polysaccharides, pectin, proteins, polypeptides, and starch. These compounds usually have molecular weights above 1.5 kDa, and their large size limits absorption and utilization by animals and humans. Microbial fermentation can break down these macromolecules into smaller, more absorbable units. Fermentation also introduces enzymatic modification, which can convert inactive compounds into biologically active forms. In Reynoutria japonica, key flavonoids include Polydatin and Resveratrol . Polydatin has a molecular weight of 390.38 Da and shows antitussive, hypolipidemic, and anti-shock activities. Resveratrol , with a molecular weight of 228.24 Da, has strong antioxidant, anti-inflammatory, anticancer, and immunomodulatory properties. Polydatin can be enzymatically converted into Resveratrol (Meng et al. 2020 ). Several studies support this conversion. Fermenting Polygonum cuspidatum with lactic acid bacteria allows clear detection of Polydatin -to- Resveratrol biotransformation by HPLC (Tian 2024 ). Jin et al. ( 2013 ) reached a conversion rate of 96.70% by co-biotransforming Polydatin with Aspergillus niger and yeast . In this study, HPLC was used to quantify Polydatin and Resveratrol before and after fermentation. The results showed a marked decline in Polydatin levels with longer fermentation time and a corresponding increase in Resveratrol . This pattern aligns with earlier studies. During fermentation, cellulase breaks down the cell walls of Reynoutria japonica and releases free Polydatin and Resveratrol . β-glucosidase then catalyzes the hydrolysis of the β-D-glucosidic bond at the non-reducing end of Polydatin . This reaction releases glucose and generates the aglycone form, which is further converted into Resveratrol (Wang 2016 ). Salvianolic acid B is the most abundant phenolic acid compound in Salvia miltiorrhiza and has shown strong antioxidant activity in both in vitro and in vivo studies. This compound not only removes oxygen free radicals effectively but also limits lipid peroxidation. In this study, the content of Salvianolic acid B after fermentation was significantly higher than before fermentation, and it showed an initial decline followed by an increase with longer fermentation time. This dynamic pattern may reflect several mechanisms. During the early fermentation stage, B. subtilis , B. coagulans , and Lactobacillus plantarum secrete hydrolases such as β-glucosidase, esterase, and polyphenol oxidase, which can break down Salvianolic acid B into smaller phenolic molecules, including danshensu, protocatechualdehyde, and caffeic acid (Gong et al. 2017 ). At the same time, during active microbial growth, probiotics may use phenolic acid components in TCM as supplementary carbon sources, leading to a temporary drop in Salvianolic acid B levels. However, as fermentation continues, Salvianolic acid B concentration begins to rise again. Two mechanisms may explain this rebound. First, microbial secondary metabolism may promote the reassembly of phenolic acid precursors. Under anaerobic conditions, Lactobacillus plantarum can catalyze glycosylation through glycosyltransferases, while Bacillus species may drive esterification via acetyltransferases, potentially forming Salvianolic acid B or structural derivatives (Pan et al. 2013 ). Second, microorganisms release cell wall-degrading enzymes such as cellulase and pectinase, which disrupt the plant matrix and release bound Salvianolic acid B or its biosynthetic intermediates. In addition, organic acids produced by lactic acid bacteria lower the pH of the TCM matrix, which accelerates early degradation of Salvianolic acid B (Nuria and Bent 2001 ). However, at later fermentation stages, the stable acidic environment may improve the structural stability of Salvianolic acid B and support conditions that favor its resynthesis. Collectively, the enzymatic reactions triggered by probiotic fermentation create a complex interconversion network among phenolic acids, indicating the need for further detailed mechanistic studies. Quercetin is a natural antioxidant widely found in plant epidermis, rhizomes, and leaves. Gynostemma pentaphyllum contains several bioactive components that show hypoglycemic, anti-aging, immunoenhancing, and hepatoprotective properties (Mastinu et al. 2021 ). This study indicated that Quercetin was not detectable in Gynostemma pentaphyllum before fermentation, but its content increased significantly after solid-state fermentation, reaching its highest level on day 7. This increase may be related to the fact that many bioactive components in TCM are embedded within plant cell walls and cytoplasm, making them difficult to degrade with the digestive enzymes of livestock and poultry. This limited degradation is a major reason for the relatively weak efficacy of TCM in animal production. Fermentation is considered one of the simplest and most effective methods to disrupt cell wall structure. On one hand, Bacillus and lactic acid bacteria produce hydrolases such as cellulase, which break down cell wall structures and release free flavonoids, including Quercetin . On the other hand, proteases and ligninases hydrolyze cellulose polysaccharides, oxidatively decompose aromatic ring polymers, and break glycoprotein or peptide bonds in the cell membrane, thereby liberating bound forms of Quercetin . In the later stages of fermentation, cellulase activity gradually decreases, while the activity of modifying enzymes such as glycosidase remains relatively stable. Glycosidase can catalyze the modification of bioactive components such as flavonoids and phenolic acids. As fermentation continues, Quercetin is progressively released, resulting in higher concentrations and improved pharmacological activity. Milk yield is the most intuitive indicator for evaluating production performance and milk quality, and it serves as a key parameter in pasture management and overall assessment of dairy cow performance (Rojas Canadas et al. 2023 ). Previous studies have shown that increased rumen propionic acid concentration can promote milk yield in dairy cows (Lehloenya et al. 2008 ). In this study, although no significant difference in milk yield was observed between Group A and Group B, a numerically higher yield was recorded in Group A. This suggests that the compound fermented Chinese medicine may have influenced the rumen microbial community by enhancing the abundance of fiber-degrading microorganisms. This shift may have increased propionic acid production in the rumen and subsequently supported milk synthesis and secretion. Additionally, Gynostemma pentaphyllum and Taraxacum mongolicum in the formulation are rich in rutin, which may provide a potential lactogenic effect in dairy cows (Cui et al. 2015 ). Milk components in raw milk (milk fat, milk protein, lactose, and SCC) not only reflect the nutritional status and lactation efficiency of dairy cows but also affect the nutritional and economic value of raw and processed dairy products. The results of this study indicated a significant increase in milk fat percentage and significant reductions in MUN and SCC in Group A, with no notable changes in milk protein, lactose, or total solids. These findings may be explained by a dual mechanism involving rumen metabolic optimization and inflammation suppression. On one hand, probiotics such as B. subtilis and Lactobacillus plantarum in the compound fermented Chinese medicine may increase the abundance of fiber-degrading bacteria (e.g., Bacteroidetes ) in the rumen. This change may promote the breakdown of dietary cellulose into acetic acid (a precursor for milk fat synthesis) and propionic acid (a substrate for gluconeogenesis). Propionic acid may increase blood glucose levels through hepatic glucose synthesis and provide energy for lactation, while acetic acid may directly support milk fat synthesis in mammary tissue. This is consistent with the conclusion of Niu et al. ( 2016 ), who reported that changes in milk fat depend on rumen volatile fatty acid metabolism. On the other hand, the antimicrobial properties of bioactive components in Chinese medicinal herbs may inhibit the growth of mastitis-causing pathogens such as Staphylococcus aureus (Fu et al. 2025 ), thereby reducing mammary inflammation and lowering SCC. Concurrently, improved nitrogen utilization by rumen microbes due to probiotic activity may reduce ammonia accumulation and decrease MUN levels. Blood acts as a direct mirror of physiological function, and its biochemical indicators shift in response to nutrition, metabolism, management conditions, and environmental stressors. These changes collectively reflect the functional state of organs and tissues in animals. Serum TP and ALB serve as key markers of protein digestion, absorption, and overall utilization. ALT and AST are used to evaluate hepatocellular integrity because hepatocyte injury increases their leakage into circulation (Zhou et al. 2012 ). BUN provides an indirect measure of protein utilization, and a marked rise in BUN suggests disrupted nitrogen metabolism or reduced efficiency of amino acid synthesis (Ren et al. 2012 ). Glu is mainly derived from hepatic gluconeogenesis and usually remains stable under neural and hormonal regulation. Cr is produced through a non-enzymatic dehydration reaction during muscle metabolism. It enters the bloodstream and is excreted in urine, making serum Cr highly dependent on renal filtration capacity. In this study, cows receiving compound fermented TCM showed higher TP and ALB levels and lower BUN concentrations, which is consistent with findings by Luo ( 2022 ). These results indicate enhanced protein breakdown, improved utilization efficiency, and greater nitrogen retention. No significant changes were observed in ALT, AST, Cr, or Glu, likely because lactating cows possess a mature rumen and robust homeostatic mechanisms that stabilize these indices. Together, these findings demonstrate that compound fermented TCM does not impair hepatic or renal function and supports normal protein and energy metabolism. Immunoglobulins are fundamental components of humoral immunity, and different types dominate in specific physiological environments. IgG is the major antibody class, accounting for nearly three-quarters of total immunoglobulins and widely distributed in serum and tissues. Its primary immune functions include opsonizing antigens to support phagocytosis, promoting antigen clumping, and neutralizing viral particles. IgM is concentrated mainly in the bloodstream, where it activates the complement cascade to lyse invading bacteria and represents the earliest antibody produced during an initial immune challenge. IgA provides essential mucosal and skin surface protection, acting as a frontline defense against external pathogens. Zhang et al. ( 2016 ) reported that compound fermented TCM preparations promote the development of immune organs and elevate antibody synthesis in broilers. Guo et al. ( 2025 ) similarly observed that Astragalus residue supplementation raises serum IgA, IgG, and IgM levels in finishing pigs, thereby improving their overall immune capacity. In this study, dairy cows in Group A showed markedly higher concentrations of IgG and IgM than those in Group B, consistent with previous research. Cytokines are critical markers reflecting immune activation during host responses to microbial invasion. After immune stimulation, IL-2, IL-4, IL-8, and TNF-α function together to coordinate inflammatory and regulatory pathways. IL-2 is a multifunctional cytokine produced mainly by activated Th1 cells, and Sun et al. (2015) confirmed its significant upregulation in inflamed mammary gland tissue. IL-4 is released from Th2 helper cells, promotes eosinophil activation, and assists B cells in increasing IgG subtype synthesis (Mollaoglu et al. 2024 ). IL-8 acts as a strong chemotactic and activating signal for neutrophils, and is secreted by monocytes-macrophages, endothelial cells, and platelets. Liu et al. ( 2017 ) demonstrated that serum IL-8 can function as a diagnostic indicator of mastitis in dairy cows. TNF-α is a major endogenous pro-inflammatory cytokine, and elevated TNF-α levels in milk can trigger apoptosis of mammary epithelial cells (Yang 2010 ). Ma ( 2020 ) found that dietary Lonicera japonica increased IL-4 and IgG while suppressing IL-2 in heat-stressed dairy cows. In the current study, IL-2, IL-4, IL-8, and TNF-α were examined. A decline in IL-2 and an elevation in IL-4 were detected in cows receiving the fermented TCM diet. These results indicate that fermented TCM helps rebalance Th1/Th2 responses and strengthens immune regulation in dairy cows. T-AOC functions as a key indicator of overall antioxidant capacity, reflecting the ability of the organism to convert reactive peroxides into non-toxic metabolites. SOD and GSH-Px are critical enzymatic antioxidants, and their activities are closely linked to the efficiency of free-radical scavenging; reductions in these enzymes often signal compromised hepatic function. MDA, produced during lipid peroxidation, serves as an indirect biomarker of oxidative damage and reflects the extent of ROS-mediated injury to cellular lipids. Zhao et al. ( 2022 ) reported that fermented Chinese herbal preparations markedly elevate serum T-AOC, SOD, and CAT levels in cows. Likewise, Xu ( 2022 ) found that administering 90 g/d of astragalus polysaccharides significantly lowers MDA concentrations. In this study, T-AOC and SOD showed significant increases, accompanied by a clear reduction in MDA, whereas GSH-Px activity remained unchanged. These improvements in antioxidant profiles may be from the greater accessibility and higher concentrations of active constituents such as flavonoids (for example, Quercetin ) and polyphenols (for example, Salvianolic acid B ) generated through fermentation, which collectively strengthen the antioxidant defense system. Additionally, the polyphenolic antioxidants present in the herbal mixture may further contribute to the removal of excess free radicals and help reduce oxidative stress. Estrogen levels in dairy cows are widely used as an important indicator of reproductive status, and lactation performance is jointly regulated by several hormones, including GH, E2, and PRL. GH primarily enhances lactation by stimulating the development and functional activity of mammary tissue. Liu et al. ( 2025 ) reported that compound Lianxian Powder significantly elevates serum GH concentrations in 42-day-old broilers. E2 contributes to lactation by promoting the mitotic activity of mammary epithelial cells, thereby supporting tissue growth. PRL plays a central role in mammary gland maturation, and upon binding to its receptors, it activates the cAMP and cGMP signaling pathways, which drive the synthesis and secretion of milk components (Lacasse et al. 2011 ). Liu et al. ( 2025 ) also showed that an optimized Bazhen Powder formulation increases PRL and E2 levels in hypogalactic mice, thereby improving sex hormone balance and enhancing lactation function. In this study, GH levels increased significantly, PRL showed a clear upward trend, and E2 remained unchanged. These outcomes may be associated with herbs such as Taraxacum mongolicum and Gynostemma pentaphyllum in the formulation, which could support follicular development and stimulate follicle-stimulating hormone secretion, thereby elevating circulating hormone levels in dairy cows. Another possibility is that the fermented herbal preparation improved gut health and nutrient absorption, indirectly enhancing endocrine function and promoting GH and PRL secretion. The intestinal microbiota constitutes a fundamental component of gut health, and their composition, richness, and diversity are influenced by a wide range of factors, including diet, management practices, antibiotic use, environmental exposure, genetic background, and age (Gacesa et al. 2022 ; Huang et al. 2022 ; Zhernakova et al. 2024 ). In the assessment of microbial α-diversity, Chao1 and Observed species indices primarily reflect species richness- the higher the value, the greater the number of distinct taxa present. The results of this study indicate that supplementation with the compound fermented Chinese herbal medicine reduced bacterial species richness in the rectal contents of dairy cows. The Shannon and Simpson indices are commonly used to assess community diversity. No significant differences were observed between the two groups for these indices. This indicated that the compound fermented herbal medicine had little impact on the bacterial community diversity in rectal contents. Beta diversity analysis evaluates the dissimilarity of microbial communities across multiple samples and reflects how microbial composition varies under different conditions. In this study, beta diversity revealed high similarity in community structure between the two groups, indicating that dietary supplementation with the compound fermented Chinese herbal medicine did not induce significant shifts in the composition of the microbial community. Notably, these findings contrast with those of Fan et al. ( 2024 ), who reported that probiotic-fermented BanQi significantly altered intestinal microbial communities. This discrepancy may be attributed to differences in the specific probiotic strains and herbal constituents employed in the respective formulations. Characteristically, the six dominant bacterial phyla in the gastrointestinal tract are Firmicutes , Bacteroidetes , Actinobacteria , Proteobacteria , Fusobacteria , and Verrucomicrobia . Among these, Firmicutes and Bacteroidetes are the most abundant and prevalent about 90% of the gut microbiota (Rinninella et al. 2019 ). Cluster analysis in this study revealed that the dominant bacterial phyla in both groups of dairy cows were Firmicutes, Bacteroidetes , Actinobacteria , and Proteobacteria . Firmicutes contain numerous fiber-utilizing genera, such as Ruminococcus and Lachnospira , which contribute directly to structural carbohydrate degradation and shape the rumen’s enzyme profile (Pushpanathan et al. 2019 ). Bacteroidetes form a major backbone of the gut community, harboring abundant cellulose-degrading bacteria that participate in complex polysaccharide turnover, support metabolite production, and regulate immune responses. Bacteroidetes often exceeds that of Firmicutes in the gut (Min et al. 2024 ), a finding consistent with the results of this study, suggesting more cellulose-degrading bacteria in the rumen than in the gut. Proteobacteria are a marker of gut microbiota imbalance. A significant increase in Proteobacteria abundance indicates a disparity in the gastrointestinal microecological balance (Pitta et al. 2016 ). This study showed that adding the compound fermented Chinese herbal medicine to the diet lowered the relative abundance of Proteobacteria , thereby helping stabilize microbial homeostasis. Actinobacteria , one of the four major phyla of the gut microbiota, although present in lower proportions, play an essential role in maintaining intestinal homeostasis (Binda et al. 2018 ). In this study, while there was no significant change in the composition of dominant bacterial phyla between the two groups, there were significant differences in the proportions of different phyla between the groups. Specifically, in the compound fermented Chinese herbal medicine group, the abundances of Firmicutes_D , Bacteroidetes , and Actinobacteria in dairy cows increased, while the abundances of Firmicutes_A and Proteobacteria decreased. This indicates that the compound fermented Chinese herbal medicine may have the effect of maintaining the balance of gastrointestinal microbiota. At the genus level, Oscillospira belongs to Firmicutes and is widely distributed in the gastrointestinal tracts of humans and animals. It possesses a butyrate kinase-mediated pathway, enabling butyrate production, and is involved in immune regulation and various physiological metabolic processes (Molino et al. 2022 ). Prevotella , a member of Bacteroidetes , can utilize diverse fermentation substrates and degrade plant polysaccharides such as pectin, starch, and xylan, often acting synergistically with fiber-degrading bacteria (Flint et al. 2008 ). Cryptobacterium and Phocaeicola , members of Bacteroidaceae , exhibit probiotic-like functions, enhancing dietary fiber breakdown, stimulating short-chain fatty acid generation, and supporting both microbial equilibrium and host immune activity. In this study, the abundance of Bifidobacterium was elevated in Group A. Metabolites produced by Bifidobacterium can inhibit colonization of certain pathogenic bacteria, promote immune regulation, and strengthen the defensive function of rumen epithelial cells. The increased abundance of Prevotella may enhance nutrient degradation from the diet, thereby supporting improved lactation performance in dairy cows. Linear Discriminant Analysis (LDA) results showed that the relative abundances of Phascolarctobacterium , Acidaminococcaceae , Lachnospiraceae CAG-632 , Eubacterium , Lactobacillus , and Faecalibacterium were significantly higher in Group A than in Group B. These findings indicate that the compound fermented Chinese herbal medicine significantly enriches the abundance of microorganisms related to milk fat synthesis in the rectal contents, thereby increasing the milk fat yield of dairy cows. Conclusion This study identified the optimal fermentation parameters for the compound TCM formula ( Reynoutria japonica , Salvia miltiorrhiza , Taraxacum mongolicum , Houttuynia cordata , and Gynostemma pentaphyllum ) as a B. subtilis : B. coagulans : Lactobacillus plantarum ratio of 1:3:1, 32% moisture content, 10% inoculum, and fermentation at 31°C for 5 days. Under these conditions, fermentation markedly increased the concentrations of key bioactive metabolites, including Resveratrol , Salvianolic acid B , and Quercetin . Supplementation of 200 g/day of the fermented preparation improved milk quality, enhanced immune and antioxidant functions, supported favorable hormonal responses, and beneficially reshaped rectal microbiota in dairy cows. These outcomes indicate that the fermented TCM blend serves as an effective functional feed additive that promotes intestinal health and production traits. Overall, this study provides a scientific basis for optimizing multi-herb TCM fermentation and highlights its practical potential in improving dairy cow performance. Abbreviations TCM, traditional Chinese medicine; HPLC, high-performance liquid chromatography; ELISA, enzyme-linked immunosorbent assay; S. aureus, Staphylococcus aureus; E. coli, Escherichia coli ; MUN, Milk Urea Nitrogen; SCC, Somatic Cell Count; TP, Total Protein; ALB, Albumin; BUN, Blood Urea Nitrogen; ALT, Alanine Aminotransferase; AST, Aspartate Aminotransferase; Cr, Creatinine; Glu, Glucose; IgA, Immunoglobulin A; IgG, Immunoglobulin G; IgM， Immunoglobulin M; IL-4, Interleukin-4; IL-2, Interleukin-2; IL-8, Interleukin-8; TNF-α, Tumor Necrosis Factor-α; T-AOC, Total Antioxidant Capacity; SOD, Superoxide Dismutase; MDA, Malondialdehyde; GH, Growth Hormone; PRL, Prolactin; E2, Estradiol. Declarations Data availability statement All data generated during this study are included in this article. All 16S rRNA sequences were deposited in the NCBI Short Read Archive under the bioproject number PRJNA1394483 (https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA1394483) CRediT authorship contribution statement Man Zhang : Writing – original draft, Software, Methodology, Investigation, Data curation. Zhiyi Zao : Validation, Software, Investigation. Zhewei Zhang, Yanting Sun, Yu Kang : Methodology, Investigation. Hui Ma, Yuchang Ning : Software, Data curation. Xiaozhan Zhang : Investigation, Data curation. Fayin Tang and Zhanyong Wei : Supervision, Formal analysis. Chuanzhou Bian, Hongxing Qiao : Writing – review & editing, Supervision, Resources, Project administration, Funding acquisition, Conceptualization. Disclosures The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This study was funded by Henan Province Modern Agriculture Pig System Traditional Chinese Medicine Antibiotic Substitution Position Expert Project (HARS-22-12-G2); Key Research and Development Project of Henan Province (241111113400, 251111110700); Henan Province Key Discipline Project (312); Postdoctoral research funds of Henan University of Animal Husbandry and Economy (M4080004). References Binda C, Lopetuso LR, Rizzatti G, Gibiino G, Cennamo V, Gasbarrini A (2018) Actinobacteria: A relevant minority for the maintenance of gut homeostasis. Dig Liver Dis 50(5):421–428. https://doi.org/10.1016/j.dld.2018.02.012 Cao YQ, Wei DD, Zhang S, Zeng F, Guo S, Guo JM, Duan JA (2020) Evaluation of in vitro antibacterial activity of isoprenylated flavonoids from Sophora flavescens combined with flavonoids from Glycyrrhiza uralensis and study on their effect against mastitis in mice (in Chinese). J Nanjing Univ Chin Med 36(3):331–338 Chen G, Li ZQ, Liu SL, Tang T, Chen QH, Yan ZM, Peng J, Yang ZK, Zhang GF, Liu YT, Zheng ML (2023) Fermented Chinese herbal medicine promoted growth performance, intestinal health, and regulated bacterial microbiota of weaned piglets. Anim (Basel) 13(3):476. https://doi.org/10.3390/ani13030476 Chen H (2009) In vitro antibacterial test of Chinese herbal medicines against 8 kinds of livestock and poultry intestinal pathogens (in Chinese). Dissertation, Gansu Agricultural University Chen YW (2022) Study on process optimization of jujube-corn flour fermented by three probiotic strains and effect of fermented products on feeding laying hens in late laying period (in Chinese). Dissertation, Shanxi Agricultural University Cui K, Guo XD, Tu Y, Zhang NF, Ma T, Diao QY (2015) Effect of dietary supplementation of rutin on lactation performance, ruminal fermentation and metabolism in dairy cows. J Anim Physiol Anim Nutr 99(6):1065–1073. https://doi.org/10.1111/jpn.12334 Cui YZ, Shan ZR, Hou LT, Wang QJ, Loor JJ, Xu C (2022) Effect of natural chinese herbal supplements (TCMF4) on lactation performance and serum biomarkers in peripartal dairy cows. Front Vet Sci 8:801418. https://doi.org/10.3389/fvets.2021.801418 Dong L, Meng L, Liu HM, Wu HM, Schroyen M, Zheng N, Wang JQ (2022) Effect of cephalosporin treatment on the microbiota and antibiotic resistance genes in feces of dairy cows with clinical mastitis. Antibiot (Basel) 11(1):117. https://doi.org/10.3390/antibiotics11010117 Fan JY, Cui HY, Mu ZY, Yao CX, Yang MF, Jin Y, Ning CS, Zhang HY (2024) Non-targeted metabolomics analysis of fermented traditional Chinese medicine and its impact on growth performance, serum biochemistry, and intestinal microbiome of weaned lambs. Sci Rep 14(1):20385. https://doi.org/10.1038/s41598-024-71516-x Flint HJ, Bayer EA, Rincon MT, Lamed R, White BA (2008) Polysaccharide utilization by gut bacteria: Potential for new insights from genomic analysis. Nat Rev Microbiol 6(2):121–131. https://doi.org/10.1038/nrmicro1817 Fu SD, Yang SY, Fu SY, Hu QY, Xia YL, Wang HH, Qiu YW, Xu YY, Miao JF (2025) Synergistic antimicrobial effects of polyphenolic Chinese medicine active compounds-loaded photothermal nanoparticles against multidrug-resistant Staphylococcus aureus infections. Phytomedicine 148:157498. https://doi.org/10.1016/j.phymed.2025.157498 Gacesa R, Kurilshikov A, Vich Vila A, Sinha T, Klaassen MAY, Bolte LA, Andreu-Sánchez S, Chen L, Collij V, Hu S, Dekens JAM, Lenters VC, Björk JR, Swarte JC, Swertz MA, Jansen BH, Gelderloos-Arends J, Jankipersadsing S, Hofker M, Vermeulen RCH, Sanna S, Harmsen HJM, Wijmenga C, Fu J, Zhernakova A, Weersma RK (2022) Environmental factors shaping the gut microbiome in a Dutch population. Nature 604(7907):732–739. https://doi.org/10.1038/s41586-022-04567-7 Gong XC, Huang SC, Pan JY, Qu HB (2017) Modeling of degradation kinetics of salvianolic acid B at different temperatures and pH values. Chin J Chem Eng 25(1):68–73 Guo Q, Wang JY, Wang XH, Zhang HY, Xing JJ (2025) Supplemented with Astragalus dregs improves growth performance, immunity, and antioxidant capacity in fattening pigs. J Nutr Sci 14:e5. https://doi.org/10.1017/jns.2024.95 Guo X, Yan ZP, Wang JX, Fan XF, Kang J, Niu RY, Sun ZL (2022) Effect of traditional chinese medicine (TCM) and its fermentation using Lactobacillus plantarum on ceftriaxone sodium-induced dysbacteriotic diarrhea in mice. Chin Med 17(1):20. https://doi.org/10.1186/s13020-022-00575-x Hu MZ, Yu JS, Chen M, Zhang HQ, Liu Y (2023) Preliminary study on microbial fermentation method of Houttuynia cordata (in Chinese). Heilongjiang Anim Sci Vet Med 66(12):125–129 Huang C, Feng SY, Huo FJ, Liu HL (2022) Effects of four antibiotics on the diversity of the intestinal microbiota. Microbiol Spectr 10(2):e0190421. https://doi.org/10.1128/spectrum.01904-21 Jin S, Luo M, Wang W, Zhao CJ, Gu CB, Li CY, Zu YG, Fu YJ, Guan Y (2013) Biotransformation of polydatin to resveratrol in Polygonum cuspidatum roots by highly immobilized edible Aspergillus niger and Yeast . Bioresour Technol 136:766–770. https://doi.org/10.1016/j.biortech.2013.03.027 Ke SX (2023) The principles of health, illness and treatment - The key concepts from The Yellow Emperor's Classic of Internal Medicine. J Ayurveda Integr Med 14(1):100637. https://doi.org/10.1016/j.jaim.2022.100637 Lacasse P, Lollivier V, Bruckmaier RM, Boisclair YR, Wagner GF, Boutinaud M (2011) Effect of the prolactin-release inhibitor quinagolide on lactating dairy cows. J Dairy Sci 94(3):1302–1309. https://doi.org/10.3168/jds.2010-3649 Lehloenya KV, Stein DR, Allen DT, Selk GE, Jones DA, Aleman MM, Rehberger TG, Mertz KJ, Spicer LJ (2008) Effects of feeding yeast and propionibacteria to dairy cows on milk yield and components, and reproduction. J Anim Physiol Anim Nutr 92(2):190–202. https://doi.org/10.1111/j.1439-0396.2007.00726.x Li H, Kang Y, Sun YT, Bian CZ, Fan MX, Zhang H, Zhao SZ, Song XH, Qiao HX (2025a) The role of Lactobacterium plantarum in solid-state fermentation of Astragalus membranaceus for broiler chicken feed. AMB Express 15(1):26. https://doi.org/10.1186/s13568-025-01823-7 Li K, Zhu SW, Cai GF, Wu ZY, Chang GJ, Li WZ, Wang CL, Ma XW, Wang DY (2023) Observation on the therapeutic effect of traditional Chinese medicine compound on dairy cow ketosis. Anim Husb Vet Med 55(3):125–129 Li SL, Yao K, Liu CQ, Wang YC, Wang ZH, Li JX, Wang JQ, Zhang HP, Cao ZJ, Tang WX (2025) 2024 Annual report on the development of dairy cow industry and technology. Chin J Anim Sci 61(3):395–398 Liu BJ, Liu GW, Che YP, Gao L (2017) Experiment on Fe activity in dairy cows with different degrees of mastitis (in Chinese). Chin J Vet Med 53(5):40–41 Liu J, Wang Y, Liu L, Ma GM, Zhang YG, Ren J (2023) Effect of Moringa leaf flavonoids on the production performance, immune system, and rumen fermentation of dairy cows. Vet Med Sci 9(2):917–923. https://doi.org/10.1002/vms3.993 Liu Q, Yu ZM, Tian FW, Zhao JX, Zhang H, Zhai QX, Chen W (2020) Surface components and metabolites of probiotics for regulation of intestinal epithelial barrier. Microb Cell Fact 19(1):23. https://doi.org/10.1186/s12934-020-1289-4 Liu SQ (2025) Formulation optimization of modified Bazhen Powder and its effect on reproductive performance of sows. Dissertation, Northeast Agricultural University Liu ZC, Liu MJ, Lin YL, Qu Q, Ren XX, Gong XH, Guo SN (2025) Effects of Compound Lianxian Powder on growth and development as well as antioxidant function of broilers. Chin J Tradit Vet Med 44(5):12–21 Luo DQ (2022) Effects of fermented compound traditional Chinese medicine on growth performance, nutrient digestion and blood indicators of beef cattle (in Chinese). Dissertation, Inner Mongolia Agricultural University, Hohhot Ma FT (2020) Effects of Lonicera japonica extract on growth performance, antioxidant capacity and rumen microbial flora structure of dairy cows under heat stress (in Chinese). Dissertation, Chinese Academy of Agricultural Sciences Ma J, Wang JR, Wan YJ, Wang SH, Jiang CQ (2025) Probiotic-fermented traditional Chinese herbal medicine, a promising approach to maintaining the intestinal microecology. J Ethnopharmacol 337(Pt 1):118815. https://doi.org/10.1016/j.jep.2024.118815 Mastinu A, Bonini SA, Premoli M, Maccarinelli G, Mac Sweeney E, Zhang LL et al (2021) Protective effects of Gynostemma pentaphyllum (var. Ginpent) against Lipopolysaccharide - Induced inflammation and motor alteration in Mice. Molecules 26(3):570. https://doi.org/10.3390/molecules26030570 Meng X, Zhou J, Zhao CN, Gan RY, Li HB (2020) Health benefits and molecular mechanisms of resveratrol: A narrative review. Foods 9(3):340. https://doi.org/10.3390/foods9030340 Min LZ, Ablitip A, Wang R, Luciana T, Wei MX, Ma XD (2024) Effects of exercise on gut microbiota of adults: A systematic review and meta-analysis. Nutrients 16(7):1070. https://doi.org/10.3390/nu16071070 Molino S, Lerma-Aguilera A, Jiménez-Hernández N, Rufián Henares JÁ, Francino MP (2022) Evaluation of the effects of a short supplementation with tannins on the gut microbiota of healthy subjects. Front Microbiol 13:848611. https://doi.org/10.3389/fmicb.2022.848611 Mollaoglu G, Tepper A, Falcomatà C, Potak HT, Pia L, Amabile A, Mateus-Tique J, Rabinovich N, Park MD, LaMarche NM, Brody R, Browning L, Lin JR, Zamarin D, Sorger PK, Santagata S, Merad M, Baccarini A, Brown BD (2024) Ovarian cancer-derived IL-4 promotes immunotherapy resistance. Cell 187(26):7492–7510. https://doi.org/10.1016/j.cell.2024.10.006 Niu CY, Yan ML, Yao ZL, Dou JH (2023) Antibiotic residues in milk and dairy products in China: occurrence and human health concerns. Environ Sci Pollut Res Int 30(53):113138–113150. https://doi.org/10.1007/s11356-023-30312-2 Niu M, Appu Hamy JADRN, Ley Tem AB, Dungan RS, Kebreab E (2016) Effect of dietary crude protein and forage contents on enteric methane emissions and nitrogen excretion from dairy cows simultaneously. Anim Prod Sci 56(3):312–321 Nuria C, Bent BJ (2001) Fermented liquid feed—microbial and nutritional aspects and impact on enteric diseases in pigs. Anim Feed Sci Technol 83(1–2):17–40. https://doi.org/10.1016/j.anifeedsci.2011.12.021 Pan JY, Gong XC, Qu HB (2013) Quantitative 1 H NMR method for hydrolytic kinetic investigation of salvianolic acid B. J Pharm Biomed Anal 85:28–32. https://doi.org/10.1016/j.jpba.2013.06.020 Pei J, Li XS, Han H, Tao Y (2017) Purification and characterization of plantaricin SLG1, a novel bacteriocin produced by L. plantarum isolated from yak cheese. Food Control 84:111–117 Pitta DW, Pinchak WE, Indugu N, Vecchiarelli B, Sinha R, Fulford JD (2016) Metagenomic analysis of the rumen microbiome of steers with wheat-induced frothy bloat. Front Microbiol 7:689. https://doi.org/10.3389/fmicb.2016.00689 Pushpanathan P, Mathew GS, Selvarajan S, Seshadri KG, Srikanth P (2019) Gut microbiota and its mysteries. Indian J Med Microbiol 37(2):268–277. https://doi.org/10.4103/ijmm.IJMM_19_373 Qiao HX (2020) Metabolomics of Astragalus membranaceus biotransformed by Lactobacillus plantarum and its feeding effect on laying hens (in Chinese). Dissertation, Huazhong Agricultural University Ren CY, Zhou LY, Bu DP, Ma H, Zhao XW, Li FD, Hu RX (2012) Main influencing factors of milk urea nitrogen and its application in dairy cow production (in Chinese). China Anim Husb Vet Med 39(9):129–133 Rinninella E, Raoul P, Cintoni M, Franceschi F, Miggiano GAD, Gasbarrini A, Mele MC (2019) What is the healthy gut microbiota composition? A changing ecosystem across age, environment, diet, and diseases. Microorganisms 7(1):14. https://doi.org/10.3390/microorganisms7010014 Rojas Canadas E, Herlihy MM, Kenneally J, Kearney F, Lonergan P, Butler ST (2023) Postpartum vaginal discharge score is associated with genetic traits, postpartum fertility phenotypes, metabolic status and overall reproductive performance in seasonal - calving pasture - based dairy cows. J Dairy Sci 106(12):9778–9792. https://doi.org/10.3168/jds.2023-23324 Saleh AA, Soliman MM, Yousef MF, Eweedah NM, El-Sawy HB, Shukry M, Wadaan MAM, Kim IH, Cho S, Eltahan HM (2023) Effects of herbal supplements on milk production quality and specific blood parameters in heat-stressed early lactating cows. Front Vet Sci 10:1180539. https://doi.org/10.3389/fvets.2023.1180539 Sun HQ (2015) Expression and significance of IL-2 and IL-4 in plasma cell mastitis tissue (in Chinese). Dissertation, Shihezi University Tan CD, Zhu MJ, Du SX, Yao YF (2016) Comparative study on the inhibition zone method in antibacterial tests (in Chinese). Food Ind 37(11):122–125 Tian ZK (2024) Study and application of a fermented traditional Chinese medicine preparation for preventing gout in goslings (in Chinese). Dissertation, Henan Agricultural University Tong JJ, Sun Y, Wang ZY, Cui DF, Jiang LS (2022) Evaluation of biological mechanisms of artemisinin on bovine mammary epithelial cells by integration of network pharmacology and TMT-based quantitative proteomics. Front Pharmacol 13:968149. https://doi.org/10.3389/fphar.2022.968149 Wang BG, Yang LJ, Cao PL, Liang XK, Wang JP, Chen Q (2023) Effects of different treatment methods on in vitro antibacterial and antioxidant activities of Chinese herbal medicines (in Chinese). China Feed 34(3):71–76 Wang M, Fu RJ, Xu DQ, Chen YY, Yue SJ, Zhang S, Tang YP (2024) Traditional chinese medicine: A promising strategy to regulate the imbalance of bacterial flora, impaired intestinal barrier and immune function attributed to ulcerative colitis through intestinal microecology. J Ethnopharmacol 318(Pt A):116879. https://doi.org/10.1016/j.jep.2023.116879 Wang SS (2016) Exploration on biotransformation pathway and study on bioactivity of resveratrol (in Chinese). Dissertation, Shenzhen University Wong RWK, Hägg U, Samaranayake L, Yuen MKZ, Seneviratne CJ, Kao R (2010) Antimicrobial activity of Chinese medicine herbs against common bacteria in oral biofilm: A pilot study. Int J Oral Maxillofac Surg 39(6):599–605. https://doi.org/10.1016/j.ijom.2010.02.024 Xu LY (2022) Effects of astragalus polysaccharide gavage on production performance, serum biochemical indicators and gastrointestinal microbial flora of dairy cowsn (in Chinese). Dissertation, Xinjiang Agricultural University Yang L (2010) Detection of three cytokines in milk of dairy cows with mastitis and effect of TNF-α on apoptosis and proliferation of mammary gland cells (in Chinese). Dissertation, Inner Mongolia Agricultural University Zeng HF, Xi YM, Li YQ, Wang ZD, Zhang L, Han ZY (2020) Analysis of astragalus polysaccharide intervention in heat-stressed dairy cows' serum metabolomics. Anim (Basel) 10(4):574. https://doi.org/10.3390/ani10040574 Zhang GZ, Xing QY, Jin SX (2016) Effects of compound traditional Chinese medicine mixed bacteria fermentation preparation on immune function of AA broilers (in Chinese). Chin J Vet Med 52(7):52–55 Zhang XL, Miao Q, Pan CX, Yin J, Wang LL, Qu LB, Yin YL, Wei YJ (2023) Research advances in probiotic fermentation of Chinese herbal medicines. iMeta 2(2):e93. https://doi.org/10.1002/imt2.93 Zhao SP, Shan CH, Wu ZJ, Feng M, Song LJ, Wang YA, Wang C (2022) Fermented Chinese herbal preparation: Impacts on milk production, nutrient digestibility, blood biochemistry, and antioxidant capacity of late-lactation cows under heat stress. Anim Feed Sci Technol 292:115448. https://doi.org/10.1016/j.anifeedsci.2022.115448 Zhao T (2019) Study on process optimization of traditional Chinese medicine compound fermented by Lactobacillus plantarum and its effect on intestinal flora of mice (in Chinese). Dissertation, Lanzhou University Zhernakova DV, Wang DM, Liu L, Andreu-Sánchez S, Zhang Y, Ruiz-Moreno AJ, Peng H, Plomp N, Del Castillo-Izquierdo Á, Gacesa R, Lopera-Maya EA, Temba GS, Kullaya VI, van Leeuwen SS, Lifelines Cohort Study, Xavier RJ, de Mast Q, Joosten LAB, Riksen NP, Rutten JHW, Netea MG, Sanna S, Wijmenga C, Weersma RK, Zhernakova A, Harmsen HJM, Fu J (2024) Host genetic regulation of human gut microbial structural variation. Nature 625(7996):813–821. https://doi.org/10.1038/s41586-023-06893-w Zhou MT, Zhang YG, Song ZY, Tang SP, Liu ZX, Pang MX, Zhang D, Wu X, Yu X, Wang P, Wei Y (2025) Enhanced bioactivity of honeysuckle-Cassia seeds extracts through Lactobacillus acidophilus and Bacillus subtilis co-fermentation: Impact on alcoholic liver disease and gut microbiota. Food Chem 486:144463. https://doi.org/10.1016/j.foodchem.2025.144463 Zhou YX, Lü YL, Wang J, Ji S, Hou PX (2012) Application overview of blood biochemical indicators in animal production and nutrition regulation research (in Chinese). Anim Husb Feed Sci 33(S1):72–74 Additional Declarations No competing interests reported. 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19:31:09","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":39540,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinegroupimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8476572/v1/16a64de1340dd5a7f33679f3.png"},{"id":99837153,"identity":"1258b27b-e3c1-4a61-bc8f-03ffcde44496","added_by":"auto","created_at":"2026-01-08 19:31:09","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":25635,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinegroupimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8476572/v1/9940f79263bb6b14fd18a7dc.png"},{"id":99837152,"identity":"315b4c07-1cb0-43ae-bb90-cf142c27ec91","added_by":"auto","created_at":"2026-01-08 19:31:09","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":33250,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinegroupimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8476572/v1/2929844cc5be8bedc7342618.png"},{"id":99837156,"identity":"d4c6c7d5-212a-461b-9fdf-ff48c05ef471","added_by":"auto","created_at":"2026-01-08 19:31:09","extension":"xml","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":235796,"visible":true,"origin":"","legend":"","description":"","filename":"e32ec4b1904f44fdb71717f7c939860e1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8476572/v1/c0a710136cd877d6b7d3a3c9.xml"},{"id":99837155,"identity":"5919bb23-e6a6-4a1e-935b-0bf1372f6272","added_by":"auto","created_at":"2026-01-08 19:31:09","extension":"html","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":258168,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8476572/v1/a4dde648699503a960bca9c1.html"},{"id":99837137,"identity":"e7077966-9419-4c3a-987a-c25a28608223","added_by":"auto","created_at":"2026-01-08 19:31:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":401895,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of different fermentation conditions on viable bacterial count. (A) Probiotic addition ratio, (B) moisture content, (C) inoculation volume, (D) fermentation temperature, and (E) fermentation duration.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8476572/v1/aeaf302d3e1e9fe3d9829f2f.png"},{"id":100356686,"identity":"99647b9c-6334-4844-98c9-241e15fbb769","added_by":"auto","created_at":"2026-01-16 07:16:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":517264,"visible":true,"origin":"","legend":"\u003cp\u003eDissolution rates of \u003cem\u003ePolydatin\u003c/em\u003e, \u003cem\u003eResveratrol\u003c/em\u003e, \u003cem\u003eSalvianolic acid B\u003c/em\u003e, and \u003cem\u003eQuercetin\u003c/em\u003e before and after fermentation. (A) Unfermented compound TCM, (B) fermented for 3 days, (C) fermented for 5 days, and (D) fermented for 7 days. Chromatographic peaks: 1 = \u003cem\u003ePolydatin\u003c/em\u003e, 2 = \u003cem\u003eResveratrol\u003c/em\u003e, 3 = \u003cem\u003eSalvianolic acid B\u003c/em\u003e, 4 = \u003cem\u003eQuercetin\u003c/em\u003e. (E) The content of \u003cem\u003ePolydatin\u003c/em\u003e, \u003cem\u003eResveratrol\u003c/em\u003e, \u003cem\u003eSalvianolic acid B\u003c/em\u003e, and \u003cem\u003eQuercetin\u003c/em\u003e before and after fermentation. Different lowercase letters above the values indicate a significant difference (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05). Identical letters or no letters indicate no significant difference (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05). This rule applies to all similar tables below.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8476572/v1/3429874dc283248c4fd90074.png"},{"id":100356697,"identity":"3a881ab6-e022-4d47-866b-5c623432218f","added_by":"auto","created_at":"2026-01-16 07:17:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1300036,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of compound fermented TCM on the microbial composition of rectal contents.\u003c/p\u003e\n\u003cp\u003e(A) OTU distribution Venn diagram; (B) dilution curve; (C) alpha diversity indices (Chao1, Observed species, Shannon, Simpson) for Groups A and B; (D) Bray–Curtis distance–based NMDS, PCA, and PCoA analyses (each point represents a sample, the distance shows community differences, and colors represent groups; when Stress \u0026lt; 0.2, NMDS reliably reflects sample variation); (E) relative abundance of the top 10 bacterial phyla (E1 = Firmicutes_A; E2 = Bacteroidetes; E3 = Firmicutes_D; E4 = Actinobacteria; E5 = Proteobacteria); (F) LefSe differential abundance analysis; (G) relative abundance of the top 30 genera (G1 = Faecousia; G2 = Cryptobacteroides; G3 = Phocaeicola_A; G4 = Bifidobacterium; G5 = Prevotella).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8476572/v1/67bd1e8bd5eb1da66023ecfc.png"},{"id":105755911,"identity":"57a49424-9e55-4109-ba97-56d65790df58","added_by":"auto","created_at":"2026-03-30 16:32:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3769110,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8476572/v1/c7ced4a8-7471-49a1-8000-b93cfc08aaa6.pdf"},{"id":100357462,"identity":"7a1b8889-007c-4fc5-bbce-6b3c12176c1c","added_by":"auto","created_at":"2026-01-16 07:19:53","extension":"doc","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":64094,"visible":true,"origin":"","legend":"","description":"","filename":"AdditionalTable13.doc","url":"https://assets-eu.researchsquare.com/files/rs-8476572/v1/e4d6d78667f781662968a9a4.doc"}],"financialInterests":"No competing interests reported.","formattedTitle":"Process Optimization of Compound Fermented Traditional Chinese Medicine and Its Feeding Effect on Dairy Cows","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDairy farming, regarded as one of the main industries within China animal husbandry, ensures a steady supply of high-quality animal protein for the population. The efficiency of dairy production and the quality of dairy products are closely linked to the economic return of the industry and national public health (Li et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2025\u003c/span\u003ea). In recent years, with the rollout of policies such as the National Action Plan for Containing Antibiotic Resistance in Animals (2021 to 2025), the reduction of antibiotic use has become a central goal in upgrading livestock and poultry farming. However, the traditional farming model that depended on antibiotics to maintain dairy cow health and raise production output has created favorable conditions for the spread of antibiotic resistance in pathogenic bacteria, including \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003eEscherichia coli\u003c/em\u003e. This approach also increased the likelihood of antibiotic residues in milk. Continued antibiotic exposure further disrupts intestinal microecology in dairy cows, diminishes microbial diversity, and contributes to impaired digestion and nutrient absorption. These issues collectively hinder sustainable gains in farming productivity (Dong et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Niu et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Therefore, the development of green, efficient, and residue-free strategies that can replace antibiotics has become a key requirement for advancing the high-quality development of the dairy sector.\u003c/p\u003e \u003cp\u003eChinese herbal medicine, a core component of traditional Chinese medicine (TCM), has increasingly shown value in livestock and poultry systems because of its natural origin, diverse chemical composition, relatively low toxicity, and minimal adverse reactions (Wang et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Research has demonstrated that dietary supplementation with compound herbal formulations can support mastitis and ketosis management, reduce heat stress, enhance milk yield and fat content, and strengthen immune-related functions, thereby improving overall productivity in dairy cows (Cui et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Saleh et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Modern pharmacological investigations confirm that alkaloids, flavonoids, and polysaccharides in these herbal materials regulate immune pathways, improve antioxidant activity, and suppress pathogenic bacterial growth (Liu et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Tong et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zeng et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Consequently, the use of compound herbal preparations as nutritional supplements offers promising potential for improving dairy production. However, raw herbal materials often show limited release of active components, poor bioavailability, and weaker palatability, which restrict their stable and large-scale adoption in modern dairy farming.\u003c/p\u003e \u003cp\u003eThe emergence of probiotic fermentation technology offers a promising strategy to overcome these limitations. With the advancement of probiotic fermentation research, it has been confirmed that probiotics such as \u003cem\u003eBacillus subtilis\u003c/em\u003e and \u003cem\u003eLactobacillus plantarum\u003c/em\u003e secrete cellulase, hemicellulase, and related enzyme systems during fermentation, which break down the cell walls of herbal matrices. This process facilitates the release, modification, and improved absorption of active ingredients by enhancing their solubility and bioactivity. In addition, these bioactive compounds can act directly on the host or exert their effects through secondary metabolites produced by intestinal flora metabolism, including short-chain fatty acids (SCFAs), amino acids, and vitamins (Guo et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zhou et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Research has also shown that metabolites produced by probiotics, such as organic acids and antimicrobial peptides, can work together with herbal bioactives to strengthen antibacterial and anti-inflammatory activities, lower systemic endotoxin and C-reactive protein levels, and support intestinal barrier integrity. Concurrently, fermentation improves the palatability of herbal preparations, reduces potential toxicities, and enhances feed intake in dairy cows (Ma et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Overall, combining herbal medicine with probiotic fermentation as a feed additive strategy shows strong potential to support sustainable and efficient development within the animal feed industry.\u003c/p\u003e \u003cp\u003eCurrent studies have confirmed that fermented single Chinese herbal medicines or herbal formulas show marked effects in improving the immunity of livestock and poultry and enhancing their production performance (Chen et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). However, systematic research on selecting suitable multi-herbal Chinese medicine combinations, optimizing probiotic consortia, and defining appropriate fermentation parameters remains limited. In particular, there is a clear shortage of studies that incorporate the physiological characteristics and production requirements of dairy cows to refine the preparation of compound fermented Chinese medicines and explore their regulatory mechanisms relating to milk quality, metabolic status, immune responses, and intestinal microecology. To address this gap, the present study aimed to increase the application value of compound Chinese medicines in dairy cow production. First, in vitro antibacterial assays were conducted to select Chinese herbal ingredients and probiotic strains with inhibitory activity against common dairy cow pathogens such as \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e, thereby identifying the optimal composition of the herbal formula and probiotic combination. Second, single-factor and orthogonal experiments were used to optimize solid-state fermentation parameters and clarify the contribution of probiotics to transforming bioactive components within the herbal mixture. Finally, feeding trials were performed to evaluate how the optimized compound fermented Chinese medicine influences production performance, milk quality, blood biochemical indices, immune activity, antioxidant capacity, and gut microbiota structure in dairy cows, thereby providing a scientific basis for its application in modern animal husbandry.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eTCM and probiotics\u003c/p\u003e \u003cp\u003eThe twenty-five types of TCMs, including \u003cem\u003eAtractylodes macrocephala\u003c/em\u003e, \u003cem\u003eRheum officinale, Salvia miltiorrhiza\u003c/em\u003e, \u003cem\u003eAngelica sinensis\u003c/em\u003e, \u003cem\u003eCodonopsis pilosula\u003c/em\u003e, \u003cem\u003eWolfiporia cocos\u003c/em\u003e, \u003cem\u003eGlycyrrhiza uralensis\u003c/em\u003e, \u003cem\u003ePhellodendron amurense\u003c/em\u003e, \u003cem\u003eCoptis chinensis\u003c/em\u003e, \u003cem\u003eScutellaria baicalensis\u003c/em\u003e, \u003cem\u003eAstragalus membranaceus\u003c/em\u003e, \u003cem\u003eReynoutria japonica\u003c/em\u003e, \u003cem\u003eLysimachia christinae\u003c/em\u003e, \u003cem\u003eLonicera japonica\u003c/em\u003e, \u003cem\u003eSophora flavescens\u003c/em\u003e, \u003cem\u003eGynostemma pentaphyllum\u003c/em\u003e, \u003cem\u003eForsythia suspensa\u003c/em\u003e, \u003cem\u003eAkebia quinata\u003c/em\u003e, \u003cem\u003eLigustrum lucidum\u003c/em\u003e, \u003cem\u003eTaraxacum mongolicum\u003c/em\u003e, \u003cem\u003eFraxinus rhynchophylla\u003c/em\u003e, \u003cem\u003eDioscorea opposita, Houttuynia cordata\u003c/em\u003e, \u003cem\u003eVaccaria segetalis\u003c/em\u003e, \u003cem\u003eand Viola philippica\u003c/em\u003e, were sourced from the Chinese Medicine Procurement and Supply Station (Yuzhou, China). The eight bacterial strains, including \u003cem\u003eE. coli\u003c/em\u003e(WDCM NO:827), \u003cem\u003eS. aureus\u003c/em\u003e༈CGMCC 1.12409༉, \u003cem\u003eB. subtilis\u003c/em\u003e༈CGMCC 1.108༉, \u003cem\u003eB. coagulans\u003c/em\u003e༈GDMCC NO:66306༉, \u003cem\u003ePediococcus pentosaceus\u003c/em\u003e, \u003cem\u003eEnterococcus faecium Lactobacillus rhamnosus\u003c/em\u003e, and \u003cem\u003eLactobacillus plantarum\u003c/em\u003e༈CGMCC NO:1.557༉were obtained from the Henan Microbial Biotransformation Engineering Research Center.\u003c/p\u003e \u003cp\u003eChemicals and reagents\u003c/p\u003e \u003cp\u003eThe ELISA kits were purchased from Yuanju Biotechnology Center (Shanghai, China). MRS broth, LB broth, and nutrient broth were provided by Haibo Biotechnology Co., Ltd (Qingdao, China). HPLC-grade methanol and acetonitrile were procured from Sigma-Aldrich (St. Louis, MO, USA). Phosphoric acid and acetic acid were supplied by Xilong Chemical Co., Ltd (Hubei, China). Reference standards of \u003cem\u003ePolydatin\u003c/em\u003e, \u003cem\u003eResveratrol\u003c/em\u003e, \u003cem\u003eSalvianolic acid B\u003c/em\u003e, and \u003cem\u003eQuercetin\u003c/em\u003e were obtained from Yuanye Bio-Technology Co., Ltd (Shanghai, China).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExperimental methods\u003c/h3\u003e\n\u003cp\u003eCulture activation\u003c/p\u003e \u003cp\u003eEight bacterial strains stored at \u0026minus;\u0026thinsp;80\u0026deg;C were thawed before activation. Four lactic acid bacteria strains (\u003cem\u003ePediococcus pentosaceus\u003c/em\u003e, \u003cem\u003eEnterococcus faecium\u003c/em\u003e, \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e, and \u003cem\u003eLactobacillus plantarum\u003c/em\u003e) were inoculated into 6 mL MRS broth tubes. Two spore-forming strains (\u003cem\u003eB. subtilis\u003c/em\u003e and \u003cem\u003eB. coagulans\u003c/em\u003e) were transferred into 6 mL nutrient broth tubes, and two pathogenic strains (\u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e) were inoculated into 6 mL LB broth tubes. All tubes were incubated at 37\u0026deg;C for 24 to 48 h. The activated \u003cem\u003elactic acid bacteria\u003c/em\u003e were then inoculated at a 1% ratio into 100 mL MRS broth and incubated statically at 37\u0026deg;C for 48 h. One milliliter of \u003cem\u003eB.\u003c/em\u003e and \u003cem\u003eB. coagulans\u003c/em\u003e cultures was added to 100 mL of nutrient broth, while \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e strains were inoculated into 100 mL LB broth and shaken at 37\u0026deg;C for 24 h. All cultures were subsequently stored at 4\u0026deg;C for later use.\u003c/p\u003e \u003cp\u003eAntibacterial activity of probiotics and TCM against S. \u003cem\u003eaureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e \u003cp\u003eThe paper disc method (Tan et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) was used to assess the antibacterial activity of probiotics against \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e. In brief, 100 \u0026micro;L of \u003cem\u003eE. coli\u003c/em\u003e (1.25 \u0026times; 10⁶ CFU/mL) and \u003cem\u003eS. aureus\u003c/em\u003e (9.10 \u0026times; 10⁶ CFU/mL) suspensions were spread evenly onto LB agar plates and allowed to dry completely. Sterile blank antibiotic discs were immersed in suspensions of \u003cem\u003eB. subtilis\u003c/em\u003e (1.25 \u0026times; 10⁸ CFU/mL), \u003cem\u003eB. coagulans\u003c/em\u003e (1.96 \u0026times; 10⁸ CFU/mL), \u003cem\u003ePediococcus pentosaceus\u003c/em\u003e (1.70 \u0026times; 10⁸ CFU/mL), \u003cem\u003eEnterococcus faecium\u003c/em\u003e (3.40 \u0026times; 10⁸ CFU/mL), \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e (2.60 \u0026times; 10⁸ CFU/mL), and \u003cem\u003eLactobacillus plantarum\u003c/em\u003e (1.14 \u0026times; 10⁸ CFU/mL), and then placed carefully onto the inoculated agar surfaces. Each probiotic strain was tested twice on two independent plates to ensure reproducibility. After incubation at 37\u0026deg;C for 24 h, inhibition zone diameters were recorded using a vernier caliper.\u003c/p\u003e \u003cp\u003eFive grams of each of the 25 TCMs were weighed, placed into individual conical flasks, and mixed with 50 mL ultrapure water. Samples were soaked for 30 min, brought to a boil, and simmered for 30 min. The mixture was filtered, followed by the addition of 40 mL ultrapure water, and simmered again for 30 min. The two filtrates were combined and concentrated to 5 mL to obtain extracts at 1 g/mL. The extracts were centrifuged at 3000 g/min for 10 min, and the supernatant was collected for further testing. The antibacterial activity of the TCMs against \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e was assessed using the hole-punching method (Tan et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). A volume of 100 \u0026micro;L of \u003cem\u003eE. coli\u003c/em\u003e (1.25 \u0026times; 10⁶ CFU/mL) and \u003cem\u003eS. aureus\u003c/em\u003e (9.10 \u0026times; 10⁶ CFU/mL) suspensions was spread on LB agar plates. Wells of 5 mm diameter were punched into the agar, and 50 \u0026micro;L of each TCM extract was added to the wells. Every TCM was tested on two plates, with four wells per plate. After incubation at 37\u0026deg;C for 24 h, inhibition zone diameters were measured using a vernier caliper.\u003c/p\u003e \u003cp\u003eFermentation of TCM and optimization of process parameters\u003c/p\u003e \u003cp\u003eFive TCMs (\u003cem\u003ePolygonum cuspidatum\u003c/em\u003e, \u003cem\u003eSalvia miltiorrhiza\u003c/em\u003e, \u003cem\u003eTaraxacum officinale\u003c/em\u003e, \u003cem\u003eHouttuynia cordata\u003c/em\u003e, \u003cem\u003eand Gynostemma pentaphyllum\u003c/em\u003e) were selected as raw materials and combined according to the traditional \u0026ldquo;Jun-Chen-Zuo-Shi\u0026rdquo; principle (Ke \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). A single-factor experimental approach was used to optimize the fermentation parameters. First, five experimental groups were prepared with different inoculation ratios of \u003cem\u003eB. subtilis\u003c/em\u003e, \u003cem\u003eB. coagulans\u003c/em\u003e, and \u003cem\u003eLactobacillus plantarum\u003c/em\u003e (1:3:1, 1:1:3, 3:1:1, 1:1:1, and 2:1:2), each tested in triplicate. Fermentation was conducted using a 15% inoculation volume, 41% moisture content, and 37\u0026deg;C for 3 d. Colony-forming units (CFUs) were enumerated via plate counting to identify the ratio that yielded the highest microbial growth. Next, the three strains were mixed at a 1:3:1 ratio, and moisture content was evaluated at 29%, 32%, 35%, 38%, and 41%, with other variables held constant (15% inoculation volume, 37\u0026deg;C, 3 d incubation). In the subsequent step, inoculation volume (15%, 20%, 25%, 30%, and 35%) was assessed under fixed conditions (1:3:1 strain ratio, 41% moisture content, and 37\u0026deg;C for 3 d). Temperature optimization was then performed at 28\u0026deg;C, 31\u0026deg;C, 34\u0026deg;C, 37\u0026deg;C, and 40\u0026deg;C, using the 1:3:1 ratio, 15% inoculation volume, and 41% moisture content for 3 d. Finally, fermentation duration was tested at 1, 2, 3, 4, and 5 d, under standardized parameters (1:3:1 ratio, 15% inoculation volume, 41% moisture content, 37\u0026deg;C), with viable counts determined by the plate count method. Based on the single-factor results, a five-factor four-level (L\u003csub\u003e16\u003c/sub\u003e 4⁵) orthogonal experimental design was implemented to evaluate the effects of strain ratios (1:1:3, 1:3:1, 3:1:1, 2:1:2), moisture levels (29%, 32%, 35%, 38%), inoculation volumes (10%, 15%, 20%, 25%), fermentation temperatures (28\u0026deg;C, 31\u0026deg;C, 34\u0026deg;C, 37\u0026deg;C), and fermentation durations (2, 3, 4, and 5 d) on total viable bacterial counts. The goal was to determine the optimal fermentation conditions. The experimental layout is presented in additional Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eHPLC-based analysis of compositional changes in TCM before and after fermentation\u003c/p\u003e \u003cp\u003eAccurately 6.10 mg of standard \u003cem\u003ePolydatin\u003c/em\u003e, 6.80 mg of \u003cem\u003eResveratrol\u003c/em\u003e, 4.80 mg of \u003cem\u003eSalvianolic acid B\u003c/em\u003e, and 8.80 mg of \u003cem\u003eQuercetin\u003c/em\u003e were weighed, dissolved, and diluted with HPLC-grade methanol to the calibration mark. The mixture was then mixed thoroughly, resulting in a stock solution containing 0.244 mg/mL \u003cem\u003ePolydatin\u003c/em\u003e, 0.272 mg/mL \u003cem\u003eResveratrol\u003c/em\u003e, 0.192 mg/mL \u003cem\u003eSalvianolic acid B\u003c/em\u003e, and 0.352 mg/mL \u003cem\u003eQuercetin\u003c/em\u003e, which was reserved for further analyses. One gram each of unfermented TCM and fermented TCM (collected at 3, 5, and 7 d post-fermentation) was weighed and transferred into 50 mL beakers. Each sample was combined with 50 mL of 70% methanol and subjected to reflux extraction for 30 min. After extraction, the samples were filtered through 0.22 \u0026micro;m membranes and concentrated to dryness. Pure methanol was then added to a fixed volume of 10 mL to prepare the final sample solution.\u003c/p\u003e \u003cp\u003eChromatographic separation was performed using a Hega Technologie C18 column (460 mm \u0026times; 4.6 mm, 5 \u0026micro;m). For \u003cem\u003ePolydatin\u003c/em\u003e and \u003cem\u003eResveratrol\u003c/em\u003e, the mobile phase consisted of acetonitrile (A) and 0.01% acetic acid water (B), operated under isocratic elution (23:77). The flow rate was 1 mL/min, with a detection wavelength of 306 nm, column temperature of 30\u0026deg;C, and injection volume of 10 \u0026micro;L. For \u003cem\u003eSalvianolic acid B\u003c/em\u003e, the mobile phase was acetonitrile (A) and 0.02% phosphoric acid water (B) at 22:78, with a 1.2 mL/min flow rate, 286 nm detection wavelength, 20\u0026deg;C column temperature, and 10 \u0026micro;L injection volume. For \u003cem\u003eQuercetin\u003c/em\u003e, the mobile phase consisted of methanol (A) and 0.02% phosphoric acid water (B) at 50:50, with a 1.0 mL/min flow rate, 360 nm detection wavelength, 30\u0026deg;C column temperature, and 10 \u0026micro;L injection volume. Standard solutions of \u003cem\u003ePolydatin\u003c/em\u003e, \u003cem\u003eResveratrol\u003c/em\u003e, \u003cem\u003eSalvianolic acid B\u003c/em\u003e, and \u003cem\u003eQuercetin\u003c/em\u003e were prepared, and aliquots of 0.5, 1, 2, 3, 4, and 5 mL were transferred into 10 mL volumetric flasks to generate six concentrations of each compound using methanol. All samples were analyzed under the specified chromatographic conditions. Peak areas were measured, and regression equations were constructed for quantitative analysis.\u003c/p\u003e \u003cp\u003eAnimal diets and experimental design\u003c/p\u003e \u003cp\u003eThis experiment was conducted at a commercial dairy farm in Zhongmu County, Zhengzhou City, Henan Province. The dietary composition and nutritional levels are listed in additional Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Eighteen healthy Holstein dairy cows with similar body weight, parity, days in lactation, and milk yield were selected and randomly assigned to two groups, with three replicates per group and three cows per replicate. The control group (Group B) received a basal diet without additives, whereas the experimental group (Group A) was fed the basal diet supplemented with 200 g/cow/d of compound fermented TCM. Dairy cows were fed three times daily at 07:00, 15:00, and 21:00. The experimental period lasted for 30 d. All animals were maintained under standard farm conditions using a free-stall housing system and milked three times daily at 05:30, 13:30, and 19:30. Milk yield and milking times were recorded automatically using the Waikato Milking System. Animals had ad libitum access to feed and water. All procedures were approved by the Animal Care and Use Committee of Henan Agricultural University (Approval No. HNND2024092901).\u003c/p\u003e \u003cp\u003eComposition and nutrient levels of the basal diet\u003c/p\u003e \u003cp\u003eMilk sampling and analysis\u003c/p\u003e \u003cp\u003eMilk samples were collected from each dairy cow before the experiment and on day 30. For each cow, milk obtained from the three daily milkings (05:30, 13:30, 19:30) was mixed thoroughly. Potassium dichromate was added to the mixed samples for milk quality assessment. After collection, 50 mL of each sample was sent immediately to Henan Provincial Dairy Cow Production Performance Testing Co., Ltd. for the determination of milk fat percentage, milk protein percentage, lactose, total solids, milk urea nitrogen (MUN), and somatic cell count (SCC).\u003c/p\u003e \u003cp\u003eBlood sampling and analysis\u003c/p\u003e \u003cp\u003eBlood samples were collected from each dairy cow via the caudal vein into evacuated tubes without anticoagulant before morning feeding on day 30. Samples were allowed to stand at room temperature for 30 min, followed by centrifugation at 3000 rpm and 4\u0026deg;C for 15 min to obtain serum. The serum was stored at \u0026minus;\u0026thinsp;20\u0026deg;C for subsequent analyses. Biochemical indicators included total protein (TP), albumin (ALB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (Cr), glucose (Glu), and blood urea nitrogen (BUN). Immune indicators included immunoglobulin A (IgA), immunoglobulin G (IgG), immunoglobulin M (IgM), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-8 (IL-8), and tumor necrosis factor-α (TNF-α). Antioxidant indicators included total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA). Hormonal indicators included growth hormone (GH), estradiol (E2), and prolactin (PRL). All parameters were measured after storage.\u003c/p\u003e \u003cp\u003eGut microbiota analysis\u003c/p\u003e \u003cp\u003eOn day 30, fecal samples were collected from experimental dairy cows using the rectal method. Samples were placed into sterile, enzyme-free 50 mL centrifuge tubes and transported immediately to Shanghai Paisenno Biotechnology Co., Ltd. for high-throughput gut microbiota sequencing.\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eIBM SPSS 27.0 software was used for statistical analyses. One-way Analysis of Variance (ANOVA) with Tukey test was applied to evaluate inter-group differences in milk yield, milk indicators, serum indicators, and other measured parameters. Data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. A \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.01 was considered highly significant, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant, and 0.05\u0026thinsp;\u0026le;\u0026thinsp;\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.1 was regarded as a trend. Graphs were prepared using Origin and GraphPad Prism 8.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eInhibitory screening identifies key TCMs and probiotic strains with antibacterial activity\u003c/h2\u003e \u003cp\u003eAmong the 25 TCM extracts screened, 15 showed varying levels of inhibition against \u003cem\u003eS\u003c/em\u003e. \u003cem\u003eaureus\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). \u003cem\u003eRheum officinale\u003c/em\u003e, \u003cem\u003eSalvia miltiorrhiza\u003c/em\u003e, \u003cem\u003eCodonopsis pilosula\u003c/em\u003e, \u003cem\u003eGlycyrrhiza uralensis\u003c/em\u003e, \u003cem\u003eCoptis chinensis\u003c/em\u003e, \u003cem\u003eScutellaria baicalensis\u003c/em\u003e, \u003cem\u003eand Reynoutria japonica\u003c/em\u003e all produced measurable inhibition zones. \u003cem\u003eCoptis chinensis\u003c/em\u003e demonstrated the strongest activity with a 30.00 mm inhibition zone, followed by \u003cem\u003eCodonopsis pilosula\u003c/em\u003e (28.50 mm) and \u003cem\u003eSalvia miltiorrhiza\u003c/em\u003e (27.00 mm). \u003cem\u003eRheum officinale\u003c/em\u003e and \u003cem\u003eGlycyrrhiza uralensis\u003c/em\u003e also showed notable inhibition of 21.00 mm and 22.00 mm, respectively. \u003cem\u003eScutellaria baicalensis\u003c/em\u003e and \u003cem\u003ePolygonum cuspidatum\u003c/em\u003e produced moderate zones of 15.50 mm. Several other TCMs, including \u003cem\u003ePhellodendron amurense\u003c/em\u003e, \u003cem\u003eLysimachia christinae\u003c/em\u003e, \u003cem\u003eForsythia suspensa\u003c/em\u003e, \u003cem\u003eLigustrum lucidum\u003c/em\u003e, \u003cem\u003eTaraxacum mongolicum\u003c/em\u003e, \u003cem\u003eFraxinus rhynchophylla\u003c/em\u003e, \u003cem\u003eHouttuynia cordata\u003c/em\u003e, and \u003cem\u003eViola philippica\u003c/em\u003e, displayed limited inhibition, with zone diameters too small to be considered significant. Against \u003cem\u003eE. coli\u003c/em\u003e, only three TCMs exhibited measurable antibacterial effects: \u003cem\u003eAngelica sinensis\u003c/em\u003e, \u003cem\u003eCoptis chinensis\u003c/em\u003e, and \u003cem\u003eLigustrum lucidum\u003c/em\u003e, with inhibition zones of 7.00 mm, 10.00 mm, and 7.50 mm, respectively. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, among the six probiotic strains assessed, \u003cem\u003eLactobacillus plantarum\u003c/em\u003e, \u003cem\u003eB. subtilis\u003c/em\u003e, and \u003cem\u003eB. coagulans\u003c/em\u003e demonstrated moderate inhibition of both \u003cem\u003eS. aureus\u003c/em\u003e (11.00 mm, 11.50 mm, and 13.50 mm) and \u003cem\u003eE. coli\u003c/em\u003e (10.00 mm, 10.50 mm, and 12.50 mm). \u003cem\u003eEnterococcus faecium\u003c/em\u003e inhibited \u003cem\u003eS. aureus\u003c/em\u003e with a 9.00 mm zone but showed no inhibition toward \u003cem\u003eE. coli\u003c/em\u003e. \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e and \u003cem\u003ePediococcus pentosaceus\u003c/em\u003e produced no measurable inhibitory activity against either pathogen.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAverage inhibition zone diameter of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003eEscherichia coli\u003c/em\u003e by 25 Chinese herbal extracts (mm)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTCM/Strain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS.aureus\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAtractylodes macrocephala\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eRheum officinale\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSalvia miltiorrhiza\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAngelica sinensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.83\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCodonopsis pilosula\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eWolfiporia cocos\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eGlycyrrhiza uralensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePhellodendron amurense\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCoptis chinensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eScutellaria baicalensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAstragalus membranaceus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eReynoutria japonica\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLysimachia christinae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.50\u0026thinsp;\u0026plusmn;\u0026thinsp;2.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLonicera japonica\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSophora flavescens\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eGynostemma pentaphyllum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eForsythia suspensa\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAkebia quinata\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLigustrum lucidum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTaraxacum mongolicum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFraxinus rhynchophylla\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eDioscorea opposita\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHouttuynia cordata\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eVaccaria segetalis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eViola philippica\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eNote: The punch diameter was 5 mm. The inhibition zone was classified as follows: \u0026ge;20 mm, extreme sensitivity; 15\u0026ndash;19 mm, high sensitivity; 10\u0026ndash;14 mm, moderate sensitivity; \u0026lt;10 mm, low sensitivity. The symbol \u0026ldquo;\u0026ndash;\u0026rdquo; indicates no inhibition zone.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAverage inhibition zone diameter of six probiotic strains (mm)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEnterococcus faecium\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePediococcus pentosaceus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLactobacillus plantarum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBacillus subtilis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.50\u0026thinsp;\u0026plusmn;\u0026thinsp;3.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBacillus coagulans\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eNote: The antimicrobial susceptibility disc had a diameter of 6 mm. Inhibition zones were classified as follows: \u0026ge;20 mm, extremely sensitive; \u0026ge;15 mm, highly sensitive; \u0026ge;10 mm, moderately sensitive; \u0026ge;6 mm, slightly sensitive. The symbol \u0026ldquo;\u0026ndash;\u0026rdquo; indicates no inhibition zone.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSingle-factor and orthogonal optimization identify ideal solid-state fermentation conditions\u003c/h2\u003e \u003cp\u003eBased on antibacterial activity patterns and formulation principles, the compound TCM was prepared using \u003cem\u003eReynoutria japonica\u003c/em\u003e, \u003cem\u003eSalvia miltiorrhiza\u003c/em\u003e, \u003cem\u003eGynostemma pentaphyllum\u003c/em\u003e, \u003cem\u003eTaraxacum mongolicum\u003c/em\u003e, and \u003cem\u003eHouttuynia cordata\u003c/em\u003e. The single-factor experiment outcomes for solid-state fermentation are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Probiotic addition ratios, moisture levels, inoculation amounts, temperature, and fermentation duration all influenced the viable bacterial count. The highest viable count, 2.10 \u0026times; 10⁸ CFU/g, occurred when \u003cem\u003eB. subtilis\u003c/em\u003e, \u003cem\u003eB. coagulans\u003c/em\u003e, and \u003cem\u003eLactobacillus plantarum\u003c/em\u003e were combined at 3:1:1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). A moisture level of 35% produced the maximum count of 5.55 \u0026times; 10⁸ CFU/g (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). With a 20% mixed inoculum, the viable count reached 2.33 \u0026times; 10⁸ CFU/g, but further increases in inoculation amount caused a downward trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Fermentation at 34\u0026deg;C generated the highest viable count of 9.45 \u0026times; 10⁸ CFU/g (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). The viable count peaked on day 3 at 5.05 \u0026times; 10⁸ CFU/g (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on these results, an orthogonal experiment was conducted (additional Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Range (R) and mean (K) analyses showed that inoculation amount had the most significant effect on viable count (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), followed by moisture level, fermentation time, temperature, and probiotic ratio. The optimal combination was A₂B₂C₁D₂E₄, corresponding to a strain ratio of 1:3:1 (\u003cem\u003eB. subtilis\u003c/em\u003e, \u003cem\u003eB. coagulans\u003c/em\u003e, and \u003cem\u003eLactobacillus plantarum\u003c/em\u003e), 32% moisture level, 10% inoculation amount, 31\u0026deg;C temperature, and a 5 d duration. Under these optimized parameters, the final product yielded a viable count of 2.50 \u0026times; 10⁸ CFU/g, a spore count of 5.15 \u0026times; 10⁷ CFU/g, and a pH of 4.79.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFermentation drives major shifts in key bioactive compounds of the TCM formulation\u003c/h3\u003e\n\u003cp\u003eHPLC analysis revealed substantial biotransformation of major active compounds during fermentation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). \u003cem\u003ePolydatin\u003c/em\u003e declined steadily, decreasing from the initial level to 0.41 \u0026micro;g/mL by day 7, representing a 78.59% reduction (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In contrast, \u003cem\u003eResveratrol\u003c/em\u003e increased significantly, reaching 1.70 \u0026micro;g/mL on day 7, a 74.90% rise relative to the unfermented preparation (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). \u003cem\u003eSalvianolic acid B\u003c/em\u003e showed a decrease to 4.22 \u0026micro;g/mL on day 3, followed by a gradual increase, reaching 5.41 \u0026micro;g/mL on day 7. This value was 4.99% higher than the unfermented level. \u003cem\u003eQuercetin\u003c/em\u003e was undetectable before fermentation but became detectable after day 3, rising consistently to 0.87 \u0026micro;g/mL by day 7 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These results indicate that microbial fermentation reduces macromolecular components and increases small-molecule bioactives, promoting the biotransformation of complex compounds and enhancing their potential absorption and utilization in animals.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eFermented TCM improves milk yield, milk fat, lactose, and udder health indicators\u003c/h3\u003e\n\u003cp\u003eThe effects of supplementing the diet with compound fermented TCM on milk production and composition are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Compared with Group B (control), Group A showed a 6.66% increase in milk yield, indicating an upward trend (P\u0026thinsp;=\u0026thinsp;0.083). Among milk components, milk fat percentage in Group A increased significantly by 20.97% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and lactose content also showed an upward trend (P\u0026thinsp;=\u0026thinsp;0.095). MUN in Group A decreased significantly by 13.93% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In addition, SCC declined by 34,400/mL, corresponding to a 54.00% reduction (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating improved udder health. No significant differences were observed in milk protein percentage or total solids between groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Overall, the addition of compound fermented TCM enhanced milk quality and reduced inflammatory cell counts, contributing to improved production performance in Holstein dairy cows.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of compound fermented TCM on production performance and milk quality in dairy cows\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eItems\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eP-value\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMilk yield (kg/d)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e31.57\u0026thinsp;\u0026plusmn;\u0026thinsp;1.23\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.083\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMilk fat (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.90\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.031\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMilk protein (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.202\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLactose (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.095\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal solids (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.36\u0026thinsp;\u0026plusmn;\u0026thinsp;1.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.194\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMUN (mg/dL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14.52\u0026thinsp;\u0026plusmn;\u0026thinsp;1.72\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.87\u0026thinsp;\u0026plusmn;\u0026thinsp;2.26\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.017\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSCC (10\u003csup\u003e4\u003c/sup\u003e/个)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.93\u0026thinsp;\u0026plusmn;\u0026thinsp;1.39\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.37\u0026thinsp;\u0026plusmn;\u0026thinsp;3.75\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.014\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eFermented TCM elevates protein metabolism markers without altering liver function indicators\u003c/h2\u003e \u003cp\u003eThe changes in blood biochemical profiles are shown in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. After compound fermented TCM supplementation, TP increased significantly, rising by 5.94% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). ALB reached 16.48 g/L, reflecting a 4.97% increase versus Group B (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). BUN in Group A decreased by 3.94%, showing a downward trend (P\u0026thinsp;=\u0026thinsp;0.090). Levels of ALT, AST, Cr, and Glu did not differ significantly between groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In summary, dietary supplementation with compound fermented TCM enhanced protein metabolism, reduced nitrogen metabolite accumulation, and improved nitrogen utilization efficiency without adversely affecting liver function in dairy cows.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of compound fermented TCM on serum biochemical indices of dairy cows\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eItems\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eP-value\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTP (\u0026micro;g/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e944.47\u0026thinsp;\u0026plusmn;\u0026thinsp;27.90\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e891.53\u0026thinsp;\u0026plusmn;\u0026thinsp;55.66\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.015\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eALB (g/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.045\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eALT (pg/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e85.32\u0026thinsp;\u0026plusmn;\u0026thinsp;4.60\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e87.94\u0026thinsp;\u0026plusmn;\u0026thinsp;5.75\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.275\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAST (pg/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e35.96\u0026thinsp;\u0026plusmn;\u0026thinsp;2.37\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e37.38\u0026thinsp;\u0026plusmn;\u0026thinsp;2.15\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.179\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCr (mmol/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22.91\u0026thinsp;\u0026plusmn;\u0026thinsp;1.85\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.49\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.912\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlu (ng/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e42.77\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e41.89\u0026thinsp;\u0026plusmn;\u0026thinsp;1.59\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.171\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBUN (mmol/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25.04\u0026thinsp;\u0026plusmn;\u0026thinsp;1.59\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.090\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eFermented TCM enhances key humoral immune indicators in dairy cows\u003c/h2\u003e \u003cp\u003eImmune-related changes are presented in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Compared with Group B, cows in Group A exhibited significant increases in IgG, IgM, and IL-4 by 5.55%, 3.44%, and 3.70%, respectively (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). IL-2 decreased by 3.40%, showing a declining trend (P\u0026thinsp;=\u0026thinsp;0.061). No significant differences were observed for IgA, IL-8, or TNF-α (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Overall, compound fermented TCM elevated major humoral immune markers, suggesting improved immune responsiveness in dairy cows.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of compound fermented TCM on the immune performance of dairy cows\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eItems\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eP-value\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIgA (\u0026micro;g/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1198.02\u0026thinsp;\u0026plusmn;\u0026thinsp;45.48\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1164.58\u0026thinsp;\u0026plusmn;\u0026thinsp;67.15\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.209\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIgG (g/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24.92\u0026thinsp;\u0026plusmn;\u0026thinsp;1.56\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.025\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIgM (\u0026micro;g/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2766.69\u0026thinsp;\u0026plusmn;\u0026thinsp;108.13\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2674.65\u0026thinsp;\u0026plusmn;\u0026thinsp;80.76\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.045\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIL-2 (pg/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e292.25\u0026thinsp;\u0026plusmn;\u0026thinsp;9.82\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e302.20\u0026thinsp;\u0026plusmn;\u0026thinsp;12.30\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.061\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIL-4 (pg/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e63.92\u0026thinsp;\u0026plusmn;\u0026thinsp;2.09\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e61.64\u0026thinsp;\u0026plusmn;\u0026thinsp;1.84\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.019\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIL-8 (pg/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e241.26\u0026thinsp;\u0026plusmn;\u0026thinsp;12.29\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e244.39\u0026thinsp;\u0026plusmn;\u0026thinsp;10.95\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.568\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTNF-α (pg/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e170.24\u0026thinsp;\u0026plusmn;\u0026thinsp;7.25\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e172.73\u0026thinsp;\u0026plusmn;\u0026thinsp;10.93\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.556\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eFermented TCM enhances antioxidant capacity and lowers oxidative stress markers in dairy cows\u003c/h2\u003e \u003cp\u003eSerum antioxidant indicators, including T-AOC, SOD, GSH-Px, and MDA, were measured after 30 days of supplementation. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, cows in Group A exhibited significantly higher T-AOC and SOD levels, with increases of 3.70% and 6.34%, respectively (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). MDA concentration in Group A reached the lowest value of 7.98 nmol/mL, representing a significant reduction compared with Group B (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Although GSH-Px showed a mild upward trend in Group A, the difference was not significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Collectively, dietary supplementation with compound fermented TCM reduced oxidative damage and improved serum antioxidant enzyme activities, thereby strengthening the overall antioxidant status of dairy cows.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of compound fermented TCM on antioxidant properties of dairy cows\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eItems\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eP-value\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT-AOC (pg/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e179.95\u0026thinsp;\u0026plusmn;\u0026thinsp;5.56\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e173.53\u0026thinsp;\u0026plusmn;\u0026thinsp;7.67\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.046\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSOD (ng/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22.80\u0026thinsp;\u0026plusmn;\u0026thinsp;1.21\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21.44\u0026thinsp;\u0026plusmn;\u0026thinsp;1.45\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.035\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGSH-Px (mIU/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e810.48\u0026thinsp;\u0026plusmn;\u0026thinsp;25.16\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e794.85\u0026thinsp;\u0026plusmn;\u0026thinsp;23.58\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.169\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMDA (nmol/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.041\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eFermented TCM increases circulating GH levels without altering PRL or E2\u003c/h2\u003e \u003cp\u003eSerum hormone profiles are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. Group A recorded a GH concentration of 15.43 ng/mL, significantly higher than Group B (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). PRL in Group A was 3.73% higher than in Group B, showing an upward trend, although the difference did not reach significance (P\u0026thinsp;=\u0026thinsp;0.072). No significant changes were observed in E2 levels between the two groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). These findings indicate that compound fermented TCM elevated circulating GH, which may support improved production outcomes in dairy cows.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of compound fermented TCM on blood hormones levels of dairy cows\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eItems\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eP-value\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGH (ng/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.90\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.19\u0026thinsp;\u0026plusmn;\u0026thinsp;1.18\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.016\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE\u003csub\u003e2\u003c/sub\u003e (pg/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e77.83\u0026thinsp;\u0026plusmn;\u0026thinsp;1.83\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e76.83\u0026thinsp;\u0026plusmn;\u0026thinsp;2.62\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.335\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePRL (mIU/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e716.03\u0026thinsp;\u0026plusmn;\u0026thinsp;30.15\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e690.31\u0026thinsp;\u0026plusmn;\u0026thinsp;30.13\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.072\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eFermented TCM alters microbial richness and shifts key gut bacterial taxa in dairy cows\u003c/h2\u003e \u003cp\u003e16S rDNA sequencing was used to analyze the intestinal microbiota of Group A and Group B. Sequences were clustered into operational taxonomic units (OTUs) based on sequence similarity, yielding 39,672 OTUs in total. Among these, 19,963 OTUs were unique to Group B, 16,052 were unique to Group A, and 3,657 were shared between both groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The microbiota were classified into 15 phyla, 20 classes, 49 orders, 91 families, 265 genera, and 209 species. Dilution curves for samples from Group A and Group B (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) showed an initial rapid rise followed by a plateau. This indicated that once sequencing depth exceeded 40,000 reads, coverage and species richness were sufficient for reliable downstream diversity analyses. Alpha and Beta diversity analyses were performed to assess microbial richness and community variation. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, significant differences were detected in the Chao1 and observed species indices (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Compared with Group B (control), Group A displayed lower microbial richness but greater community stability. No significant differences were observed in Shannon or Simpson indices (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), suggesting comparable overall microbial diversity. These results indicate that compound fermented TCM reduced rectal microbial richness without markedly altering overall diversity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further examine compositional differences, principal coordinate analysis (PCoA) based on Bray\u0026ndash;Curtis distances and non-metric multidimensional scaling (NMDS) based on the Bray\u0026ndash;Curtis matrix were conducted and visualized in two dimensions. The PCoA and NMDS plots (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) showed that microbial communities in both groups were closely distributed, indicating high similarity in microbial composition and richness. However, the Group B samples were more dispersed, suggesting higher inter-individual variation under normal feeding conditions. At the phylum level, the top ten dominant taxa were analyzed. In Group A, the predominant phyla were \u003cem\u003eFirmicutes_A\u003c/em\u003e (61.58%), \u003cem\u003eBacteroidota\u003c/em\u003e (29.38%), \u003cem\u003eFirmicutes_D\u003c/em\u003e (3.11%), and \u003cem\u003eActinobacteriota\u003c/em\u003e (3.02%). In Group B, the dominant phyla were \u003cem\u003eFirmicutes_A\u003c/em\u003e (65.30%), \u003cem\u003eBacteroidota\u003c/em\u003e (26.13%), \u003cem\u003eFirmicutes_D\u003c/em\u003e (2.99%), and \u003cem\u003eActinobacteriota\u003c/em\u003e (2.44%). In both groups, \u003cem\u003eFirmicutes_A\u003c/em\u003e and \u003cem\u003eBacteroidota\u003c/em\u003e accounted for more than 90% of total microbial abundance. Compared with Group B, Group A showed decreasing trends in \u003cem\u003eFirmicutes_A\u003c/em\u003e and \u003cem\u003eProteobacteria\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), while \u003cem\u003eBacteroidota\u003c/em\u003e, \u003cem\u003eFirmicutes_D\u003c/em\u003e, and \u003cem\u003eActinobacteriota\u003c/em\u003e showed increasing trends (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). These findings suggest that fermented TCM modulated major phyla by reducing \u003cem\u003eFirmicutes_A\u003c/em\u003e and \u003cem\u003eProteobacteria\u003c/em\u003e and promoting \u003cem\u003eBacteroidota\u003c/em\u003e, \u003cem\u003eFirmicutes_D\u003c/em\u003e, and \u003cem\u003eActinobacteriota\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eFurther genus-level analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG) identified shifts in microbial composition. In Group A, dominant genera included \u003cem\u003eFaecousia\u003c/em\u003e (16.88%), \u003cem\u003eCryptobacteroide\u003c/em\u003es (8.71%), \u003cem\u003ePhocaeicola_A\u003c/em\u003e (5.28%), \u003cem\u003ePeH17\u003c/em\u003e (4.66%), \u003cem\u003eRUG13077\u003c/em\u003e (3.04%), \u003cem\u003eAlistipes_A\u003c/em\u003e (2.70%), \u003cem\u003eUBA737\u003c/em\u003e (2.37%), \u003cem\u003eBifidobacterium\u003c/em\u003e (2.51%), \u003cem\u003eRF16\u003c/em\u003e (2.23%), \u003cem\u003eCAG-41\u003c/em\u003e (2.33%), \u003cem\u003eParaprevotella_A\u003c/em\u003e (2.18%), \u003cem\u003eParaclostridium\u003c/em\u003e (2.08%), \u003cem\u003eRomboutsia_B\u003c/em\u003e (1.44%), \u003cem\u003eSFMI01\u003c/em\u003e (1.20%), and \u003cem\u003ePrevotella\u003c/em\u003e (1.08%). In Group B, the dominant genera were \u003cem\u003eFaecousia\u003c/em\u003e (14.20%), \u003cem\u003eCryptobacteroide\u003c/em\u003es (7.98%), \u003cem\u003ePhocaeicola_A\u003c/em\u003e (4.44%), \u003cem\u003ePeH17\u003c/em\u003e (4.08%), \u003cem\u003eRUG13077\u003c/em\u003e (2.72%), \u003cem\u003eAlistipes_A\u003c/em\u003e (2.09%), \u003cem\u003eUBA737\u003c/em\u003e (2.25%), \u003cem\u003eBifidobacterium\u003c/em\u003e (1.98%), \u003cem\u003eRF16\u003c/em\u003e (2.26%), \u003cem\u003eCAG-41\u003c/em\u003e (2.13%), \u003cem\u003eParaprevotella_A\u003c/em\u003e (2.22%), \u003cem\u003eParaclostridium\u003c/em\u003e (2.24%), \u003cem\u003eRomboutsia_B\u003c/em\u003e (1.64%), \u003cem\u003eSFMI01\u003c/em\u003e (1.37%), and \u003cem\u003ePrevotella\u003c/em\u003e (0.63%). Compared with Group B, Group A showed higher relative abundances of \u003cem\u003eFaecousia\u003c/em\u003e, \u003cem\u003eCryptobacteroides\u003c/em\u003e, \u003cem\u003ePhocaeicola_A\u003c/em\u003e, \u003cem\u003eBifidobacterium\u003c/em\u003e, and \u003cem\u003ePrevotella\u003c/em\u003e, although differences were not significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). This suggests that compound fermented TCM may promote beneficial microbial genera.\u003c/p\u003e \u003cp\u003eLinear discriminant analysis effect size (LefSe) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF) revealed significant structural differences in intestinal microbiota. Group A was enriched with \u003cem\u003ePhascolarctobacterium_A\u003c/em\u003e, \u003cem\u003ef_Acidaminococcaceae\u003c/em\u003e, \u003cem\u003eg_Erysipelothrix\u003c/em\u003e, \u003cem\u003eg_CAG_632\u003c/em\u003e, \u003cem\u003eg_Eubacterium_F\u003c/em\u003e, \u003cem\u003eg_Limosilactobacillus\u003c/em\u003e, and \u003cem\u003eg_Faecalibacterium\u003c/em\u003e, whereas Group B was enriched with \u003cem\u003ef_Enterobacteriaceae\u003c/em\u003e, \u003cem\u003eg_Streptococcus\u003c/em\u003e, \u003cem\u003eand f_Streptococcaceae\u003c/em\u003e. In conclusion, fermented TCM restructured the intestinal microbiota by inhibiting pathogenic groups and enhancing beneficial taxa, indicating a positive modulation of gut microbial ecology in dairy cows.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eTCM contains a wide range of bioactive substances, including polysaccharides, alkaloids, glycosides, organic acids, tannins, polyphenols, pigments, and volatile oils. These components can interact synergistically through several biological pathways. This multi-target activity supports broad bacteriostatic effects. As a result, TCM has been applied widely in animal husbandry for many years. Different TCMs, however, show clear differences in antibacterial range and strength. These variations mainly reflect differences in the types and levels of their active constituents (Cao et al. 2010; Wong et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In this study, the TCM formula was developed through a structured screening process and scientific evaluation of herbal compatibility. The agar well diffusion method was used to analyze aqueous extracts from 25 TCMs. The findings indicated that \u003cem\u003eRheum officinale\u003c/em\u003e, \u003cem\u003eSalvia miltiorrhiza\u003c/em\u003e, \u003cem\u003eReynoutria japonica\u003c/em\u003e, and \u003cem\u003eGlycyrrhiza uralensis\u003c/em\u003e had strong inhibitory effects against \u003cem\u003eS. aureus\u003c/em\u003e. Their inhibition of \u003cem\u003eE. coli\u003c/em\u003e was weak. Several factors may explain this pattern. High-temperature decoction or high-pressure extraction can damage heat-sensitive compounds. Volatile components may also evaporate under these conditions. In addition, some antibacterial molecules are poorly soluble in water, which reduces their extraction efficiency. These factors can lower the measured in vitro activity of TCM extracts. The form of TCM used in production also affects its antibacterial effect. In livestock systems, herbs are typically provided as powders or fermented preparations rather than aqueous extracts. Studies have shown that fermentation by \u003cem\u003elactic acid bacteria\u003c/em\u003e can enhance inhibitory zones. For example, fermented extracts increased the inhibition of \u003cem\u003eS. aureus\u003c/em\u003e by more than 20%, and \u003cem\u003eSalmonella\u003c/em\u003e by more than 12% compared with non-fermented products (Wang et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Some TCM components also require host metabolic enzymes to convert them into active forms. In vitro assays lack this metabolic environment. As a result, they may not reflect the true antibacterial potential of compounds that depend on in vivo transformation. Therefore, while in vitro bacteriostatic tests offer useful preliminary information, they cannot fully represent the complex pharmacological effects observed in animals. A combined evaluation of in vitro and in vivo results is required to accurately determine the antibacterial efficacy and practical application value of TCM formulations.\u003c/p\u003e \u003cp\u003eMedication compatibility is a central principle of TCM. Proper combinations of herbs can enhance therapeutic effects, reduce required dosages, and lower treatment costs. Traditional studies have often focused on single herbs that show strong bacteriostatic activity. These herbs are usually selected as the core for compatibility. However, herbs with weak or undetectable bacteriostatic effects are frequently overlooked. Such herbs may still contribute through synergistic interactions when combined correctly. For example, Chen (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) reported that \u003cem\u003eTerminalia chebula Retz\u003c/em\u003e had a minimum inhibitory concentration (MIC) of 250.00 mg/mL against \u003cem\u003eSalmonella typhimurium\u003c/em\u003e. \u003cem\u003ePhellodendron chinense Schneid\u003c/em\u003e showed no measurable inhibition. When both herbs were combined, the MIC decreased to 62.50 mg/mL, which demonstrated a strong synergistic effect. Following this concept, the formula in this study included \u003cem\u003eGynostemma pentaphyllum\u003c/em\u003e, even though it did not form a bacteriostatic circle in vitro. This herb has functions related to clearing heat, detoxifying, and supporting leukocyte recovery. It was therefore combined with four herbs that showed measurable bacteriostatic effects: \u003cem\u003eReynoutria japonica\u003c/em\u003e, \u003cem\u003eSalvia miltiorrhiza\u003c/em\u003e, \u003cem\u003eTaraxacum mongolicum\u003c/em\u003e, and \u003cem\u003eHouttuynia cordata\u003c/em\u003e. This combination was designed to act on pathogenic bacteria through multiple pathways, thereby improving the overall antimicrobial potential of the formulation.\u003c/p\u003e \u003cp\u003eProbiotics are widely used as feed microecological agents. They produce various enzymes that support digestion and absorption in the host. Their metabolic products also include several bacteriostatic substances, which help strengthen the host's antimicrobial defenses. In vitro antibacterial assays offer a direct way to test the activity of these probiotic metabolites. In this study, the disk diffusion method was used to evaluate four \u003cem\u003elactic acid\u003c/em\u003e bacteria strains and two \u003cem\u003eBacillus\u003c/em\u003e strains against \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e. The results showed that \u003cem\u003eLactobacillus plantarum\u003c/em\u003e, \u003cem\u003eB. subtilis\u003c/em\u003e, and \u003cem\u003eB. coagulans\u003c/em\u003e had clear inhibitory activity against both pathogens. Among them, \u003cem\u003eB. coagulans\u003c/em\u003e produced the largest inhibition zones against \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e. This was followed by \u003cem\u003eB. subtilis\u003c/em\u003e and \u003cem\u003eLactobacillus plantaru\u003c/em\u003em. The variation in bacteriostatic performance among these strains may reflect differences in their antimicrobial metabolites and mechanisms. \u003cem\u003eLactobacillus plantarum\u003c/em\u003e and \u003cem\u003eBacillus\u003c/em\u003e species are gram-positive bacteria, but they rely on different antimicrobial strategies. \u003cem\u003eLactobacillus plantarum\u003c/em\u003e produces organic acids, such as lactic and acetic acid (Qiao \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These acids lower the surrounding pH and suppress the growth of pathogenic microorganisms. \u003cem\u003eB. subtilis\u003c/em\u003e and \u003cem\u003eB. coagulans\u003c/em\u003e, in contrast, produce antimicrobial peptides or bacteriocins. These compounds can inhibit cell wall synthesis or form pores in the bacterial membrane (Pei et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Both actions compromise membrane integrity and eventually cause cell lysis.\u003c/p\u003e \u003cp\u003eDuring solid-state fermentation of TCM with probiotics, the control of parameters such as probiotic ratios, moisture levels, inoculum size, temperature, and fermentation time plays a decisive role. These factors directly influence microbial growth and reproduction. Optimizing such parameters can therefore improve both the quality and functional activity of the final fermented product (Chen \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Hu et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In this study, the viable bacterial count was selected as the main evaluation index. The results showed clear responses to each fermentation parameter. When the ratio of \u003cem\u003eB. subtilis\u003c/em\u003e, \u003cem\u003eB. coagulans\u003c/em\u003e, and \u003cem\u003eLactobacillus plantarum\u003c/em\u003e was 3:1:1, the viable count reached 2.10 \u0026times; 10⁸ CFU/g. At a moisture level of 35%, the count increased to 5.55 \u0026times; 10⁸ CFU/g. A mixed inoculum size of 20% produced a maximum count of 2.33 \u0026times; 10⁸ CFU/g, while larger inoculum sizes caused a decline in viable bacteria. Under incubation at 34\u0026deg;C, the viable count reached 9.45 \u0026times; 10⁸ CFU/g. On the third fermentation day, the count peaked at 5.05 \u0026times; 10⁸ CFU/g. Zhao (\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) examined similar fermentation optimization using \u003cem\u003eLactobacillus plantarum\u003c/em\u003e. Their orthogonal experiments evaluated temperature, fermentation time, and inoculum size. The concentration of fermented TCM extract was 0.2 mg/mL. Based on single-factor testing and orthogonal design, the ideal parameters were identified as follows: a microbial ratio of \u003cem\u003eB. subtilis\u003c/em\u003e: \u003cem\u003eB. coagulans\u003c/em\u003e: \u003cem\u003eLactobacillus plantarum\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1:3:1, moisture content 32%, inoculum size 10%, temperature 31\u0026deg;C, and a fermentation duration of 5 days.\u003c/p\u003e \u003cp\u003eTCM contains many macromolecular organic compounds, including polysaccharides, pectin, proteins, polypeptides, and starch. These compounds usually have molecular weights above 1.5 kDa, and their large size limits absorption and utilization by animals and humans. Microbial fermentation can break down these macromolecules into smaller, more absorbable units. Fermentation also introduces enzymatic modification, which can convert inactive compounds into biologically active forms. In Reynoutria japonica, key flavonoids include \u003cem\u003ePolydatin\u003c/em\u003e and \u003cem\u003eResveratrol\u003c/em\u003e. \u003cem\u003ePolydatin\u003c/em\u003e has a molecular weight of 390.38 Da and shows antitussive, hypolipidemic, and anti-shock activities. \u003cem\u003eResveratrol\u003c/em\u003e, with a molecular weight of 228.24 Da, has strong antioxidant, anti-inflammatory, anticancer, and immunomodulatory properties. \u003cem\u003ePolydatin\u003c/em\u003e can be enzymatically converted into \u003cem\u003eResveratrol\u003c/em\u003e (Meng et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Several studies support this conversion. Fermenting Polygonum cuspidatum with lactic acid bacteria allows clear detection of \u003cem\u003ePolydatin\u003c/em\u003e-to-\u003cem\u003eResveratrol\u003c/em\u003e biotransformation by HPLC (Tian \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Jin et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) reached a conversion rate of 96.70% by co-biotransforming \u003cem\u003ePolydatin\u003c/em\u003e with \u003cem\u003eAspergillus niger\u003c/em\u003e and \u003cem\u003eyeast\u003c/em\u003e. In this study, HPLC was used to quantify \u003cem\u003ePolydatin\u003c/em\u003e and \u003cem\u003eResveratrol\u003c/em\u003e before and after fermentation. The results showed a marked decline in \u003cem\u003ePolydatin\u003c/em\u003e levels with longer fermentation time and a corresponding increase in \u003cem\u003eResveratrol\u003c/em\u003e. This pattern aligns with earlier studies. During fermentation, cellulase breaks down the cell walls of Reynoutria japonica and releases free \u003cem\u003ePolydatin\u003c/em\u003e and \u003cem\u003eResveratrol\u003c/em\u003e. β-glucosidase then catalyzes the hydrolysis of the β-D-glucosidic bond at the non-reducing end of \u003cem\u003ePolydatin\u003c/em\u003e. This reaction releases glucose and generates the aglycone form, which is further converted into \u003cem\u003eResveratrol\u003c/em\u003e (Wang \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eSalvianolic acid B\u003c/em\u003e is the most abundant phenolic acid compound in \u003cem\u003eSalvia miltiorrhiza\u003c/em\u003e and has shown strong antioxidant activity in both in vitro and in vivo studies. This compound not only removes oxygen free radicals effectively but also limits lipid peroxidation. In this study, the content of \u003cem\u003eSalvianolic acid B\u003c/em\u003e after fermentation was significantly higher than before fermentation, and it showed an initial decline followed by an increase with longer fermentation time. This dynamic pattern may reflect several mechanisms. During the early fermentation stage, \u003cem\u003eB. subtilis\u003c/em\u003e, \u003cem\u003eB. coagulans\u003c/em\u003e, and \u003cem\u003eLactobacillus plantarum\u003c/em\u003e secrete hydrolases such as β-glucosidase, esterase, and polyphenol oxidase, which can break down \u003cem\u003eSalvianolic acid B\u003c/em\u003e into smaller phenolic molecules, including danshensu, protocatechualdehyde, and caffeic acid (Gong et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). At the same time, during active microbial growth, probiotics may use phenolic acid components in TCM as supplementary carbon sources, leading to a temporary drop in \u003cem\u003eSalvianolic acid B\u003c/em\u003e levels. However, as fermentation continues, \u003cem\u003eSalvianolic acid B\u003c/em\u003e concentration begins to rise again. Two mechanisms may explain this rebound. First, microbial secondary metabolism may promote the reassembly of phenolic acid precursors. Under anaerobic conditions, \u003cem\u003eLactobacillus plantarum\u003c/em\u003e can catalyze glycosylation through glycosyltransferases, while \u003cem\u003eBacillus\u003c/em\u003e species may drive esterification via acetyltransferases, potentially forming \u003cem\u003eSalvianolic acid B\u003c/em\u003e or structural derivatives (Pan et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Second, microorganisms release cell wall-degrading enzymes such as cellulase and pectinase, which disrupt the plant matrix and release bound \u003cem\u003eSalvianolic acid B\u003c/em\u003e or its biosynthetic intermediates. In addition, organic acids produced by \u003cem\u003elactic acid\u003c/em\u003e bacteria lower the pH of the TCM matrix, which accelerates early degradation of \u003cem\u003eSalvianolic acid B\u003c/em\u003e (Nuria and Bent \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). However, at later fermentation stages, the stable acidic environment may improve the structural stability of \u003cem\u003eSalvianolic acid B\u003c/em\u003e and support conditions that favor its resynthesis. Collectively, the enzymatic reactions triggered by probiotic fermentation create a complex interconversion network among phenolic acids, indicating the need for further detailed mechanistic studies.\u003c/p\u003e \u003cp\u003e \u003cem\u003eQuercetin\u003c/em\u003e is a natural antioxidant widely found in plant epidermis, rhizomes, and leaves. \u003cem\u003eGynostemma pentaphyllum\u003c/em\u003e contains several bioactive components that show hypoglycemic, anti-aging, immunoenhancing, and hepatoprotective properties (Mastinu et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This study indicated that \u003cem\u003eQuercetin\u003c/em\u003e was not detectable in Gynostemma pentaphyllum before fermentation, but its content increased significantly after solid-state fermentation, reaching its highest level on day 7. This increase may be related to the fact that many bioactive components in TCM are embedded within plant cell walls and cytoplasm, making them difficult to degrade with the digestive enzymes of livestock and poultry. This limited degradation is a major reason for the relatively weak efficacy of TCM in animal production. Fermentation is considered one of the simplest and most effective methods to disrupt cell wall structure. On one hand, \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003elactic acid\u003c/em\u003e bacteria produce hydrolases such as cellulase, which break down cell wall structures and release free flavonoids, including \u003cem\u003eQuercetin\u003c/em\u003e. On the other hand, proteases and ligninases hydrolyze cellulose polysaccharides, oxidatively decompose aromatic ring polymers, and break glycoprotein or peptide bonds in the cell membrane, thereby liberating bound forms of \u003cem\u003eQuercetin\u003c/em\u003e. In the later stages of fermentation, cellulase activity gradually decreases, while the activity of modifying enzymes such as glycosidase remains relatively stable. Glycosidase can catalyze the modification of bioactive components such as flavonoids and phenolic acids. As fermentation continues, \u003cem\u003eQuercetin\u003c/em\u003e is progressively released, resulting in higher concentrations and improved pharmacological activity.\u003c/p\u003e \u003cp\u003eMilk yield is the most intuitive indicator for evaluating production performance and milk quality, and it serves as a key parameter in pasture management and overall assessment of dairy cow performance (Rojas Canadas et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Previous studies have shown that increased rumen propionic acid concentration can promote milk yield in dairy cows (Lehloenya et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). In this study, although no significant difference in milk yield was observed between Group A and Group B, a numerically higher yield was recorded in Group A. This suggests that the compound fermented Chinese medicine may have influenced the rumen microbial community by enhancing the abundance of fiber-degrading microorganisms. This shift may have increased propionic acid production in the rumen and subsequently supported milk synthesis and secretion. Additionally, Gynostemma pentaphyllum and Taraxacum mongolicum in the formulation are rich in rutin, which may provide a potential lactogenic effect in dairy cows (Cui et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Milk components in raw milk (milk fat, milk protein, lactose, and SCC) not only reflect the nutritional status and lactation efficiency of dairy cows but also affect the nutritional and economic value of raw and processed dairy products. The results of this study indicated a significant increase in milk fat percentage and significant reductions in MUN and SCC in Group A, with no notable changes in milk protein, lactose, or total solids. These findings may be explained by a dual mechanism involving rumen metabolic optimization and inflammation suppression. On one hand, probiotics such as \u003cem\u003eB. subtilis\u003c/em\u003e and \u003cem\u003eLactobacillus plantarum\u003c/em\u003e in the compound fermented Chinese medicine may increase the abundance of fiber-degrading bacteria (e.g., \u003cem\u003eBacteroidetes\u003c/em\u003e) in the rumen. This change may promote the breakdown of dietary cellulose into acetic acid (a precursor for milk fat synthesis) and propionic acid (a substrate for gluconeogenesis). Propionic acid may increase blood glucose levels through hepatic glucose synthesis and provide energy for lactation, while acetic acid may directly support milk fat synthesis in mammary tissue. This is consistent with the conclusion of Niu et al. (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), who reported that changes in milk fat depend on rumen volatile fatty acid metabolism. On the other hand, the antimicrobial properties of bioactive components in Chinese medicinal herbs may inhibit the growth of mastitis-causing pathogens such as Staphylococcus aureus (Fu et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), thereby reducing mammary inflammation and lowering SCC. Concurrently, improved nitrogen utilization by rumen microbes due to probiotic activity may reduce ammonia accumulation and decrease MUN levels.\u003c/p\u003e \u003cp\u003eBlood acts as a direct mirror of physiological function, and its biochemical indicators shift in response to nutrition, metabolism, management conditions, and environmental stressors. These changes collectively reflect the functional state of organs and tissues in animals. Serum TP and ALB serve as key markers of protein digestion, absorption, and overall utilization. ALT and AST are used to evaluate hepatocellular integrity because hepatocyte injury increases their leakage into circulation (Zhou et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). BUN provides an indirect measure of protein utilization, and a marked rise in BUN suggests disrupted nitrogen metabolism or reduced efficiency of amino acid synthesis (Ren et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Glu is mainly derived from hepatic gluconeogenesis and usually remains stable under neural and hormonal regulation. Cr is produced through a non-enzymatic dehydration reaction during muscle metabolism. It enters the bloodstream and is excreted in urine, making serum Cr highly dependent on renal filtration capacity. In this study, cows receiving compound fermented TCM showed higher TP and ALB levels and lower BUN concentrations, which is consistent with findings by Luo (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These results indicate enhanced protein breakdown, improved utilization efficiency, and greater nitrogen retention. No significant changes were observed in ALT, AST, Cr, or Glu, likely because lactating cows possess a mature rumen and robust homeostatic mechanisms that stabilize these indices. Together, these findings demonstrate that compound fermented TCM does not impair hepatic or renal function and supports normal protein and energy metabolism.\u003c/p\u003e \u003cp\u003eImmunoglobulins are fundamental components of humoral immunity, and different types dominate in specific physiological environments. IgG is the major antibody class, accounting for nearly three-quarters of total immunoglobulins and widely distributed in serum and tissues. Its primary immune functions include opsonizing antigens to support phagocytosis, promoting antigen clumping, and neutralizing viral particles. IgM is concentrated mainly in the bloodstream, where it activates the complement cascade to lyse invading bacteria and represents the earliest antibody produced during an initial immune challenge. IgA provides essential mucosal and skin surface protection, acting as a frontline defense against external pathogens. Zhang et al. (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) reported that compound fermented TCM preparations promote the development of immune organs and elevate antibody synthesis in broilers. Guo et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) similarly observed that \u003cem\u003eAstragalus\u003c/em\u003e residue supplementation raises serum IgA, IgG, and IgM levels in finishing pigs, thereby improving their overall immune capacity. In this study, dairy cows in Group A showed markedly higher concentrations of IgG and IgM than those in Group B, consistent with previous research. Cytokines are critical markers reflecting immune activation during host responses to microbial invasion. After immune stimulation, IL-2, IL-4, IL-8, and TNF-α function together to coordinate inflammatory and regulatory pathways. IL-2 is a multifunctional cytokine produced mainly by activated Th1 cells, and Sun et al. (2015) confirmed its significant upregulation in inflamed mammary gland tissue. IL-4 is released from Th2 helper cells, promotes eosinophil activation, and assists B cells in increasing IgG subtype synthesis (Mollaoglu et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). IL-8 acts as a strong chemotactic and activating signal for neutrophils, and is secreted by monocytes-macrophages, endothelial cells, and platelets. Liu et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) demonstrated that serum IL-8 can function as a diagnostic indicator of mastitis in dairy cows. TNF-α is a major endogenous pro-inflammatory cytokine, and elevated TNF-α levels in milk can trigger apoptosis of mammary epithelial cells (Yang \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Ma (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) found that dietary \u003cem\u003eLonicera japonica\u003c/em\u003e increased IL-4 and IgG while suppressing IL-2 in heat-stressed dairy cows. In the current study, IL-2, IL-4, IL-8, and TNF-α were examined. A decline in IL-2 and an elevation in IL-4 were detected in cows receiving the fermented TCM diet. These results indicate that fermented TCM helps rebalance Th1/Th2 responses and strengthens immune regulation in dairy cows.\u003c/p\u003e \u003cp\u003eT-AOC functions as a key indicator of overall antioxidant capacity, reflecting the ability of the organism to convert reactive peroxides into non-toxic metabolites. SOD and GSH-Px are critical enzymatic antioxidants, and their activities are closely linked to the efficiency of free-radical scavenging; reductions in these enzymes often signal compromised hepatic function. MDA, produced during lipid peroxidation, serves as an indirect biomarker of oxidative damage and reflects the extent of ROS-mediated injury to cellular lipids. Zhao et al. (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) reported that fermented Chinese herbal preparations markedly elevate serum T-AOC, SOD, and CAT levels in cows. Likewise, Xu (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) found that administering 90 g/d of astragalus polysaccharides significantly lowers MDA concentrations. In this study, T-AOC and SOD showed significant increases, accompanied by a clear reduction in MDA, whereas GSH-Px activity remained unchanged. These improvements in antioxidant profiles may be from the greater accessibility and higher concentrations of active constituents such as flavonoids (for example, \u003cem\u003eQuercetin\u003c/em\u003e) and polyphenols (for example, \u003cem\u003eSalvianolic acid B\u003c/em\u003e) generated through fermentation, which collectively strengthen the antioxidant defense system. Additionally, the polyphenolic antioxidants present in the herbal mixture may further contribute to the removal of excess free radicals and help reduce oxidative stress.\u003c/p\u003e \u003cp\u003eEstrogen levels in dairy cows are widely used as an important indicator of reproductive status, and lactation performance is jointly regulated by several hormones, including GH, E2, and PRL. GH primarily enhances lactation by stimulating the development and functional activity of mammary tissue. Liu et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) reported that compound Lianxian Powder significantly elevates serum GH concentrations in 42-day-old broilers. E2 contributes to lactation by promoting the mitotic activity of mammary epithelial cells, thereby supporting tissue growth. PRL plays a central role in mammary gland maturation, and upon binding to its receptors, it activates the cAMP and cGMP signaling pathways, which drive the synthesis and secretion of milk components (Lacasse et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Liu et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) also showed that an optimized Bazhen Powder formulation increases PRL and E2 levels in hypogalactic mice, thereby improving sex hormone balance and enhancing lactation function. In this study, GH levels increased significantly, PRL showed a clear upward trend, and E2 remained unchanged. These outcomes may be associated with herbs such as Taraxacum mongolicum and Gynostemma pentaphyllum in the formulation, which could support follicular development and stimulate follicle-stimulating hormone secretion, thereby elevating circulating hormone levels in dairy cows. Another possibility is that the fermented herbal preparation improved gut health and nutrient absorption, indirectly enhancing endocrine function and promoting GH and PRL secretion.\u003c/p\u003e \u003cp\u003eThe intestinal microbiota constitutes a fundamental component of gut health, and their composition, richness, and diversity are influenced by a wide range of factors, including diet, management practices, antibiotic use, environmental exposure, genetic background, and age (Gacesa et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Huang et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhernakova et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In the assessment of microbial α-diversity, Chao1 and Observed species indices primarily reflect species richness- the higher the value, the greater the number of distinct taxa present. The results of this study indicate that supplementation with the compound fermented Chinese herbal medicine reduced bacterial species richness in the rectal contents of dairy cows. The Shannon and Simpson indices are commonly used to assess community diversity. No significant differences were observed between the two groups for these indices. This indicated that the compound fermented herbal medicine had little impact on the bacterial community diversity in rectal contents. Beta diversity analysis evaluates the dissimilarity of microbial communities across multiple samples and reflects how microbial composition varies under different conditions. In this study, beta diversity revealed high similarity in community structure between the two groups, indicating that dietary supplementation with the compound fermented Chinese herbal medicine did not induce significant shifts in the composition of the microbial community. Notably, these findings contrast with those of Fan et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), who reported that probiotic-fermented BanQi significantly altered intestinal microbial communities. This discrepancy may be attributed to differences in the specific probiotic strains and herbal constituents employed in the respective formulations.\u003c/p\u003e \u003cp\u003eCharacteristically, the six dominant bacterial phyla in the gastrointestinal tract are \u003cem\u003eFirmicutes\u003c/em\u003e, \u003cem\u003eBacteroidetes\u003c/em\u003e, \u003cem\u003eActinobacteria\u003c/em\u003e, \u003cem\u003eProteobacteria\u003c/em\u003e, \u003cem\u003eFusobacteria\u003c/em\u003e, and \u003cem\u003eVerrucomicrobia\u003c/em\u003e. Among these, \u003cem\u003eFirmicutes\u003c/em\u003e and \u003cem\u003eBacteroidetes\u003c/em\u003e are the most abundant and prevalent about 90% of the gut microbiota (Rinninella et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Cluster analysis in this study revealed that the dominant bacterial phyla in both groups of dairy cows were \u003cem\u003eFirmicutes, Bacteroidetes\u003c/em\u003e, \u003cem\u003eActinobacteria\u003c/em\u003e, and \u003cem\u003eProteobacteria\u003c/em\u003e. \u003cem\u003eFirmicutes\u003c/em\u003e contain numerous fiber-utilizing genera, such as \u003cem\u003eRuminococcus\u003c/em\u003e and \u003cem\u003eLachnospira\u003c/em\u003e, which contribute directly to structural carbohydrate degradation and shape the rumen\u0026rsquo;s enzyme profile (Pushpanathan et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). \u003cem\u003eBacteroidetes\u003c/em\u003e form a major backbone of the gut community, harboring abundant cellulose-degrading bacteria that participate in complex polysaccharide turnover, support metabolite production, and regulate immune responses. \u003cem\u003eBacteroidetes\u003c/em\u003e often exceeds that of \u003cem\u003eFirmicutes\u003c/em\u003e in the gut (Min et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), a finding consistent with the results of this study, suggesting more cellulose-degrading bacteria in the rumen than in the gut. \u003cem\u003eProteobacteria\u003c/em\u003e are a marker of gut microbiota imbalance. A significant increase in Proteobacteria abundance indicates a disparity in the gastrointestinal microecological balance (Pitta et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). This study showed that adding the compound fermented Chinese herbal medicine to the diet lowered the relative abundance of \u003cem\u003eProteobacteria\u003c/em\u003e, thereby helping stabilize microbial homeostasis. \u003cem\u003eActinobacteria\u003c/em\u003e, one of the four major phyla of the gut microbiota, although present in lower proportions, play an essential role in maintaining intestinal homeostasis (Binda et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In this study, while there was no significant change in the composition of dominant bacterial phyla between the two groups, there were significant differences in the proportions of different phyla between the groups. Specifically, in the compound fermented Chinese herbal medicine group, the abundances of \u003cem\u003eFirmicutes_D\u003c/em\u003e, \u003cem\u003eBacteroidetes\u003c/em\u003e, and \u003cem\u003eActinobacteria\u003c/em\u003e in dairy cows increased, while the abundances of \u003cem\u003eFirmicutes_A\u003c/em\u003e and \u003cem\u003eProteobacteria\u003c/em\u003e decreased. This indicates that the compound fermented Chinese herbal medicine may have the effect of maintaining the balance of gastrointestinal microbiota. At the genus level, \u003cem\u003eOscillospira\u003c/em\u003e belongs to \u003cem\u003eFirmicutes\u003c/em\u003e and is widely distributed in the gastrointestinal tracts of humans and animals. It possesses a butyrate kinase-mediated pathway, enabling butyrate production, and is involved in immune regulation and various physiological metabolic processes (Molino et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). \u003cem\u003ePrevotella\u003c/em\u003e, a member of \u003cem\u003eBacteroidetes\u003c/em\u003e, can utilize diverse fermentation substrates and degrade plant polysaccharides such as pectin, starch, and xylan, often acting synergistically with fiber-degrading bacteria (Flint et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). \u003cem\u003eCryptobacterium\u003c/em\u003e and \u003cem\u003ePhocaeicola\u003c/em\u003e, members of \u003cem\u003eBacteroidaceae\u003c/em\u003e, exhibit probiotic-like functions, enhancing dietary fiber breakdown, stimulating short-chain fatty acid generation, and supporting both microbial equilibrium and host immune activity. In this study, the abundance of \u003cem\u003eBifidobacterium\u003c/em\u003e was elevated in Group A. Metabolites produced by \u003cem\u003eBifidobacterium\u003c/em\u003e can inhibit colonization of certain pathogenic bacteria, promote immune regulation, and strengthen the defensive function of rumen epithelial cells. The increased abundance of \u003cem\u003ePrevotella\u003c/em\u003e may enhance nutrient degradation from the diet, thereby supporting improved lactation performance in dairy cows. Linear Discriminant Analysis (LDA) results showed that the relative abundances of \u003cem\u003ePhascolarctobacterium\u003c/em\u003e, \u003cem\u003eAcidaminococcaceae\u003c/em\u003e, \u003cem\u003eLachnospiraceae CAG-632\u003c/em\u003e, \u003cem\u003eEubacterium\u003c/em\u003e, \u003cem\u003eLactobacillus\u003c/em\u003e, and \u003cem\u003eFaecalibacterium\u003c/em\u003e were significantly higher in Group A than in Group B. These findings indicate that the compound fermented Chinese herbal medicine significantly enriches the abundance of microorganisms related to milk fat synthesis in the rectal contents, thereby increasing the milk fat yield of dairy cows.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study identified the optimal fermentation parameters for the compound TCM formula (\u003cem\u003eReynoutria japonica\u003c/em\u003e, \u003cem\u003eSalvia miltiorrhiza\u003c/em\u003e, \u003cem\u003eTaraxacum mongolicum\u003c/em\u003e, \u003cem\u003eHouttuynia cordata\u003c/em\u003e, and \u003cem\u003eGynostemma pentaphyllum\u003c/em\u003e) as a \u003cem\u003eB. subtilis\u003c/em\u003e: \u003cem\u003eB. coagulans\u003c/em\u003e: \u003cem\u003eLactobacillus plantarum\u003c/em\u003e ratio of 1:3:1, 32% moisture content, 10% inoculum, and fermentation at 31\u0026deg;C for 5 days. Under these conditions, fermentation markedly increased the concentrations of key bioactive metabolites, including \u003cem\u003eResveratrol\u003c/em\u003e, \u003cem\u003eSalvianolic acid B\u003c/em\u003e, and \u003cem\u003eQuercetin\u003c/em\u003e. Supplementation of 200 g/day of the fermented preparation improved milk quality, enhanced immune and antioxidant functions, supported favorable hormonal responses, and beneficially reshaped rectal microbiota in dairy cows. These outcomes indicate that the fermented TCM blend serves as an effective functional feed additive that promotes intestinal health and production traits. Overall, this study provides a scientific basis for optimizing multi-herb TCM fermentation and highlights its practical potential in improving dairy cow performance.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cp\u003eTCM, traditional Chinese medicine; HPLC, high-performance liquid chromatography; ELISA, enzyme-linked immunosorbent assay; \u003cem\u003eS. aureus, Staphylococcus aureus; E. coli, Escherichia coli\u003c/em\u003e; MUN, Milk Urea Nitrogen; SCC, Somatic Cell Count; TP, Total Protein; ALB, Albumin; BUN, Blood Urea Nitrogen; ALT, Alanine Aminotransferase; AST, Aspartate Aminotransferase; Cr, Creatinine; Glu, Glucose; IgA, Immunoglobulin A; IgG, Immunoglobulin G; IgM， Immunoglobulin M; IL-4, Interleukin-4; IL-2, Interleukin-2; IL-8, Interleukin-8; TNF-\u0026alpha;, Tumor Necrosis Factor-\u0026alpha;; T-AOC, Total Antioxidant Capacity; SOD, Superoxide Dismutase; MDA, Malondialdehyde; GH, Growth Hormone; PRL, Prolactin; E2, Estradiol.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated during this study are included in this article. All 16S rRNA sequences were deposited in the NCBI Short Read Archive under the bioproject number PRJNA1394483 (https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA1394483)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMan Zhang\u003c/strong\u003e: Writing \u0026ndash; original draft, Software, Methodology, Investigation, Data curation. \u003cstrong\u003eZhiyi Zao\u003c/strong\u003e: Validation, Software, Investigation. \u003cstrong\u003eZhewei Zhang, Yanting Sun, Yu Kang\u003c/strong\u003e: Methodology, Investigation. \u003cstrong\u003eHui Ma, Yuchang Ning\u003c/strong\u003e: Software, Data curation. \u003cstrong\u003eXiaozhan Zhang\u003c/strong\u003e: Investigation, Data curation.\u003cstrong\u003e\u0026nbsp;Fayin Tang and Zhanyong Wei\u003c/strong\u003e: Supervision, Formal analysis. \u003cstrong\u003eChuanzhou Bian, Hongxing Qiao\u003c/strong\u003e: Writing \u0026ndash; review \u0026amp; editing, Supervision, Resources, Project administration, Funding acquisition, Conceptualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by Henan Province Modern Agriculture Pig System Traditional Chinese Medicine Antibiotic Substitution Position Expert Project (HARS-22-12-G2); Key Research and Development Project of Henan Province (241111113400, 251111110700); Henan Province Key Discipline Project (312); Postdoctoral research funds of Henan University of Animal Husbandry and Economy (M4080004).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBinda C, Lopetuso LR, Rizzatti G, Gibiino G, Cennamo V, Gasbarrini A (2018) Actinobacteria: A relevant minority for the maintenance of gut homeostasis. Dig Liver Dis 50(5):421\u0026ndash;428. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.dld.2018.02.012\u003c/span\u003e\u003cspan address=\"10.1016/j.dld.2018.02.012\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCao YQ, Wei DD, Zhang S, Zeng F, Guo S, Guo JM, Duan JA (2020) Evaluation of in vitro antibacterial activity of isoprenylated flavonoids from Sophora flavescens combined with flavonoids from Glycyrrhiza uralensis and study on their effect against mastitis in mice (in Chinese). J Nanjing Univ Chin Med 36(3):331\u0026ndash;338\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen G, Li ZQ, Liu SL, Tang T, Chen QH, Yan ZM, Peng J, Yang ZK, Zhang GF, Liu YT, Zheng ML (2023) Fermented Chinese herbal medicine promoted growth performance, intestinal health, and regulated bacterial microbiota of weaned piglets. Anim (Basel) 13(3):476. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ani13030476\u003c/span\u003e\u003cspan address=\"10.3390/ani13030476\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen H (2009) In vitro antibacterial test of Chinese herbal medicines against 8 kinds of livestock and poultry intestinal pathogens (in Chinese). Dissertation, Gansu Agricultural University\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen YW (2022) Study on process optimization of jujube-corn flour fermented by three probiotic strains and effect of fermented products on feeding laying hens in late laying period (in Chinese). Dissertation, Shanxi Agricultural University\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCui K, Guo XD, Tu Y, Zhang NF, Ma T, Diao QY (2015) Effect of dietary supplementation of rutin on lactation performance, ruminal fermentation and metabolism in dairy cows. J Anim Physiol Anim Nutr 99(6):1065\u0026ndash;1073. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/jpn.12334\u003c/span\u003e\u003cspan address=\"10.1111/jpn.12334\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCui YZ, Shan ZR, Hou LT, Wang QJ, Loor JJ, Xu C (2022) Effect of natural chinese herbal supplements (TCMF4) on lactation performance and serum biomarkers in peripartal dairy cows. Front Vet Sci 8:801418. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fvets.2021.801418\u003c/span\u003e\u003cspan address=\"10.3389/fvets.2021.801418\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDong L, Meng L, Liu HM, Wu HM, Schroyen M, Zheng N, Wang JQ (2022) Effect of cephalosporin treatment on the microbiota and antibiotic resistance genes in feces of dairy cows with clinical mastitis. Antibiot (Basel) 11(1):117. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/antibiotics11010117\u003c/span\u003e\u003cspan address=\"10.3390/antibiotics11010117\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFan JY, Cui HY, Mu ZY, Yao CX, Yang MF, Jin Y, Ning CS, Zhang HY (2024) Non-targeted metabolomics analysis of fermented traditional Chinese medicine and its impact on growth performance, serum biochemistry, and intestinal microbiome of weaned lambs. Sci Rep 14(1):20385. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-024-71516-x\u003c/span\u003e\u003cspan address=\"10.1038/s41598-024-71516-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFlint HJ, Bayer EA, Rincon MT, Lamed R, White BA (2008) Polysaccharide utilization by gut bacteria: Potential for new insights from genomic analysis. Nat Rev Microbiol 6(2):121\u0026ndash;131. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nrmicro1817\u003c/span\u003e\u003cspan address=\"10.1038/nrmicro1817\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFu SD, Yang SY, Fu SY, Hu QY, Xia YL, Wang HH, Qiu YW, Xu YY, Miao JF (2025) Synergistic antimicrobial effects of polyphenolic Chinese medicine active compounds-loaded photothermal nanoparticles against multidrug-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e infections. Phytomedicine 148:157498. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.phymed.2025.157498\u003c/span\u003e\u003cspan address=\"10.1016/j.phymed.2025.157498\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGacesa R, Kurilshikov A, Vich Vila A, Sinha T, Klaassen MAY, Bolte LA, Andreu-S\u0026aacute;nchez S, Chen L, Collij V, Hu S, Dekens JAM, Lenters VC, Bj\u0026ouml;rk JR, Swarte JC, Swertz MA, Jansen BH, Gelderloos-Arends J, Jankipersadsing S, Hofker M, Vermeulen RCH, Sanna S, Harmsen HJM, Wijmenga C, Fu J, Zhernakova A, Weersma RK (2022) Environmental factors shaping the gut microbiome in a Dutch population. Nature 604(7907):732\u0026ndash;739. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41586-022-04567-7\u003c/span\u003e\u003cspan address=\"10.1038/s41586-022-04567-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGong XC, Huang SC, Pan JY, Qu HB (2017) Modeling of degradation kinetics of salvianolic acid B at different temperatures and pH values. Chin J Chem Eng 25(1):68\u0026ndash;73\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo Q, Wang JY, Wang XH, Zhang HY, Xing JJ (2025) Supplemented with Astragalus dregs improves growth performance, immunity, and antioxidant capacity in fattening pigs. J Nutr Sci 14:e5. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1017/jns.2024.95\u003c/span\u003e\u003cspan address=\"10.1017/jns.2024.95\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo X, Yan ZP, Wang JX, Fan XF, Kang J, Niu RY, Sun ZL (2022) Effect of traditional chinese medicine (TCM) and its fermentation using \u003cem\u003eLactobacillus plantarum\u003c/em\u003e on ceftriaxone sodium-induced dysbacteriotic diarrhea in mice. Chin Med 17(1):20. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s13020-022-00575-x\u003c/span\u003e\u003cspan address=\"10.1186/s13020-022-00575-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu MZ, Yu JS, Chen M, Zhang HQ, Liu Y (2023) Preliminary study on microbial fermentation method of Houttuynia cordata (in Chinese). Heilongjiang Anim Sci Vet Med 66(12):125\u0026ndash;129\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang C, Feng SY, Huo FJ, Liu HL (2022) Effects of four antibiotics on the diversity of the intestinal microbiota. Microbiol Spectr 10(2):e0190421. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/spectrum.01904-21\u003c/span\u003e\u003cspan address=\"10.1128/spectrum.01904-21\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJin S, Luo M, Wang W, Zhao CJ, Gu CB, Li CY, Zu YG, Fu YJ, Guan Y (2013) Biotransformation of polydatin to resveratrol in \u003cem\u003ePolygonum cuspidatum\u003c/em\u003e roots by highly immobilized edible \u003cem\u003eAspergillus niger\u003c/em\u003e and \u003cem\u003eYeast\u003c/em\u003e. Bioresour Technol 136:766\u0026ndash;770. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biortech.2013.03.027\u003c/span\u003e\u003cspan address=\"10.1016/j.biortech.2013.03.027\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKe SX (2023) The principles of health, illness and treatment - The key concepts from The Yellow Emperor's Classic of Internal Medicine. J Ayurveda Integr Med 14(1):100637. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jaim.2022.100637\u003c/span\u003e\u003cspan address=\"10.1016/j.jaim.2022.100637\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLacasse P, Lollivier V, Bruckmaier RM, Boisclair YR, Wagner GF, Boutinaud M (2011) Effect of the prolactin-release inhibitor quinagolide on lactating dairy cows. J Dairy Sci 94(3):1302\u0026ndash;1309. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3168/jds.2010-3649\u003c/span\u003e\u003cspan address=\"10.3168/jds.2010-3649\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLehloenya KV, Stein DR, Allen DT, Selk GE, Jones DA, Aleman MM, Rehberger TG, Mertz KJ, Spicer LJ (2008) Effects of feeding yeast and propionibacteria to dairy cows on milk yield and components, and reproduction. J Anim Physiol Anim Nutr 92(2):190\u0026ndash;202. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1439-0396.2007.00726.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1439-0396.2007.00726.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi H, Kang Y, Sun YT, Bian CZ, Fan MX, Zhang H, Zhao SZ, Song XH, Qiao HX (2025a) The role of \u003cem\u003eLactobacterium plantarum\u003c/em\u003e in solid-state fermentation of \u003cem\u003eAstragalus membranaceus\u003c/em\u003e for broiler chicken feed. AMB Express 15(1):26. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s13568-025-01823-7\u003c/span\u003e\u003cspan address=\"10.1186/s13568-025-01823-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi K, Zhu SW, Cai GF, Wu ZY, Chang GJ, Li WZ, Wang CL, Ma XW, Wang DY (2023) Observation on the therapeutic effect of traditional Chinese medicine compound on dairy cow ketosis. Anim Husb Vet Med 55(3):125\u0026ndash;129\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi SL, Yao K, Liu CQ, Wang YC, Wang ZH, Li JX, Wang JQ, Zhang HP, Cao ZJ, Tang WX (2025) 2024 Annual report on the development of dairy cow industry and technology. Chin J Anim Sci 61(3):395\u0026ndash;398\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu BJ, Liu GW, Che YP, Gao L (2017) Experiment on Fe activity in dairy cows with different degrees of mastitis (in Chinese). Chin J Vet Med 53(5):40\u0026ndash;41\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu J, Wang Y, Liu L, Ma GM, Zhang YG, Ren J (2023) Effect of Moringa leaf flavonoids on the production performance, immune system, and rumen fermentation of dairy cows. Vet Med Sci 9(2):917\u0026ndash;923. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/vms3.993\u003c/span\u003e\u003cspan address=\"10.1002/vms3.993\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Q, Yu ZM, Tian FW, Zhao JX, Zhang H, Zhai QX, Chen W (2020) Surface components and metabolites of probiotics for regulation of intestinal epithelial barrier. Microb Cell Fact 19(1):23. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12934-020-1289-4\u003c/span\u003e\u003cspan address=\"10.1186/s12934-020-1289-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu SQ (2025) Formulation optimization of modified Bazhen Powder and its effect on reproductive performance of sows. Dissertation, Northeast Agricultural University\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu ZC, Liu MJ, Lin YL, Qu Q, Ren XX, Gong XH, Guo SN (2025) Effects of Compound Lianxian Powder on growth and development as well as antioxidant function of broilers. Chin J Tradit Vet Med 44(5):12\u0026ndash;21\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuo DQ (2022) Effects of fermented compound traditional Chinese medicine on growth performance, nutrient digestion and blood indicators of beef cattle (in Chinese). Dissertation, Inner Mongolia Agricultural University, Hohhot\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa FT (2020) Effects of Lonicera japonica extract on growth performance, antioxidant capacity and rumen microbial flora structure of dairy cows under heat stress (in Chinese). Dissertation, Chinese Academy of Agricultural Sciences\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa J, Wang JR, Wan YJ, Wang SH, Jiang CQ (2025) Probiotic-fermented traditional Chinese herbal medicine, a promising approach to maintaining the intestinal microecology. J Ethnopharmacol 337(Pt 1):118815. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jep.2024.118815\u003c/span\u003e\u003cspan address=\"10.1016/j.jep.2024.118815\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMastinu A, Bonini SA, Premoli M, Maccarinelli G, Mac Sweeney E, Zhang LL et al (2021) Protective effects of \u003cem\u003eGynostemma pentaphyllum\u003c/em\u003e (var. Ginpent) against Lipopolysaccharide - Induced inflammation and motor alteration in Mice. Molecules 26(3):570. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/molecules26030570\u003c/span\u003e\u003cspan address=\"10.3390/molecules26030570\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeng X, Zhou J, Zhao CN, Gan RY, Li HB (2020) Health benefits and molecular mechanisms of resveratrol: A narrative review. Foods 9(3):340. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/foods9030340\u003c/span\u003e\u003cspan address=\"10.3390/foods9030340\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMin LZ, Ablitip A, Wang R, Luciana T, Wei MX, Ma XD (2024) Effects of exercise on gut microbiota of adults: A systematic review and meta-analysis. Nutrients 16(7):1070. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/nu16071070\u003c/span\u003e\u003cspan address=\"10.3390/nu16071070\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMolino S, Lerma-Aguilera A, Jim\u0026eacute;nez-Hern\u0026aacute;ndez N, Rufi\u0026aacute;n Henares J\u0026Aacute;, Francino MP (2022) Evaluation of the effects of a short supplementation with tannins on the gut microbiota of healthy subjects. Front Microbiol 13:848611. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fmicb.2022.848611\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2022.848611\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMollaoglu G, Tepper A, Falcomat\u0026agrave; C, Potak HT, Pia L, Amabile A, Mateus-Tique J, Rabinovich N, Park MD, LaMarche NM, Brody R, Browning L, Lin JR, Zamarin D, Sorger PK, Santagata S, Merad M, Baccarini A, Brown BD (2024) Ovarian cancer-derived IL-4 promotes immunotherapy resistance. Cell 187(26):7492\u0026ndash;7510. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cell.2024.10.006\u003c/span\u003e\u003cspan address=\"10.1016/j.cell.2024.10.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNiu CY, Yan ML, Yao ZL, Dou JH (2023) Antibiotic residues in milk and dairy products in China: occurrence and human health concerns. Environ Sci Pollut Res Int 30(53):113138\u0026ndash;113150. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11356-023-30312-2\u003c/span\u003e\u003cspan address=\"10.1007/s11356-023-30312-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNiu M, Appu Hamy JADRN, Ley Tem AB, Dungan RS, Kebreab E (2016) Effect of dietary crude protein and forage contents on enteric methane emissions and nitrogen excretion from dairy cows simultaneously. Anim Prod Sci 56(3):312\u0026ndash;321\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNuria C, Bent BJ (2001) Fermented liquid feed\u0026mdash;microbial and nutritional aspects and impact on enteric diseases in pigs. Anim Feed Sci Technol 83(1\u0026ndash;2):17\u0026ndash;40. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.anifeedsci.2011.12.021\u003c/span\u003e\u003cspan address=\"10.1016/j.anifeedsci.2011.12.021\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePan JY, Gong XC, Qu HB (2013) Quantitative \u003csup\u003e1\u003c/sup\u003eH NMR method for hydrolytic kinetic investigation of salvianolic acid B. J Pharm Biomed Anal 85:28\u0026ndash;32. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jpba.2013.06.020\u003c/span\u003e\u003cspan address=\"10.1016/j.jpba.2013.06.020\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePei J, Li XS, Han H, Tao Y (2017) Purification and characterization of plantaricin SLG1, a novel bacteriocin produced by L. plantarum isolated from yak cheese. Food Control 84:111\u0026ndash;117\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePitta DW, Pinchak WE, Indugu N, Vecchiarelli B, Sinha R, Fulford JD (2016) Metagenomic analysis of the rumen microbiome of steers with wheat-induced frothy bloat. Front Microbiol 7:689. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fmicb.2016.00689\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2016.00689\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePushpanathan P, Mathew GS, Selvarajan S, Seshadri KG, Srikanth P (2019) Gut microbiota and its mysteries. Indian J Med Microbiol 37(2):268\u0026ndash;277. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.4103/ijmm.IJMM_19_373\u003c/span\u003e\u003cspan address=\"10.4103/ijmm.IJMM_19_373\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQiao HX (2020) Metabolomics of Astragalus membranaceus biotransformed by Lactobacillus plantarum and its feeding effect on laying hens (in Chinese). Dissertation, Huazhong Agricultural University\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRen CY, Zhou LY, Bu DP, Ma H, Zhao XW, Li FD, Hu RX (2012) Main influencing factors of milk urea nitrogen and its application in dairy cow production (in Chinese). China Anim Husb Vet Med 39(9):129\u0026ndash;133\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRinninella E, Raoul P, Cintoni M, Franceschi F, Miggiano GAD, Gasbarrini A, Mele MC (2019) What is the healthy gut microbiota composition? A changing ecosystem across age, environment, diet, and diseases. Microorganisms 7(1):14. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/microorganisms7010014\u003c/span\u003e\u003cspan address=\"10.3390/microorganisms7010014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRojas Canadas E, Herlihy MM, Kenneally J, Kearney F, Lonergan P, Butler ST (2023) Postpartum vaginal discharge score is associated with genetic traits, postpartum fertility phenotypes, metabolic status and overall reproductive performance in seasonal - calving pasture - based dairy cows. J Dairy Sci 106(12):9778\u0026ndash;9792. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3168/jds.2023-23324\u003c/span\u003e\u003cspan address=\"10.3168/jds.2023-23324\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaleh AA, Soliman MM, Yousef MF, Eweedah NM, El-Sawy HB, Shukry M, Wadaan MAM, Kim IH, Cho S, Eltahan HM (2023) Effects of herbal supplements on milk production quality and specific blood parameters in heat-stressed early lactating cows. Front Vet Sci 10:1180539. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fvets.2023.1180539\u003c/span\u003e\u003cspan address=\"10.3389/fvets.2023.1180539\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun HQ (2015) Expression and significance of IL-2 and IL-4 in plasma cell mastitis tissue (in Chinese). Dissertation, Shihezi University\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTan CD, Zhu MJ, Du SX, Yao YF (2016) Comparative study on the inhibition zone method in antibacterial tests (in Chinese). Food Ind 37(11):122\u0026ndash;125\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTian ZK (2024) Study and application of a fermented traditional Chinese medicine preparation for preventing gout in goslings (in Chinese). Dissertation, Henan Agricultural University\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTong JJ, Sun Y, Wang ZY, Cui DF, Jiang LS (2022) Evaluation of biological mechanisms of artemisinin on bovine mammary epithelial cells by integration of network pharmacology and TMT-based quantitative proteomics. Front Pharmacol 13:968149. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fphar.2022.968149\u003c/span\u003e\u003cspan address=\"10.3389/fphar.2022.968149\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang BG, Yang LJ, Cao PL, Liang XK, Wang JP, Chen Q (2023) Effects of different treatment methods on in vitro antibacterial and antioxidant activities of Chinese herbal medicines (in Chinese). China Feed 34(3):71\u0026ndash;76\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang M, Fu RJ, Xu DQ, Chen YY, Yue SJ, Zhang S, Tang YP (2024) Traditional chinese medicine: A promising strategy to regulate the imbalance of bacterial flora, impaired intestinal barrier and immune function attributed to ulcerative colitis through intestinal microecology. J Ethnopharmacol 318(Pt A):116879. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jep.2023.116879\u003c/span\u003e\u003cspan address=\"10.1016/j.jep.2023.116879\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang SS (2016) Exploration on biotransformation pathway and study on bioactivity of resveratrol (in Chinese). Dissertation, Shenzhen University\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWong RWK, H\u0026auml;gg U, Samaranayake L, Yuen MKZ, Seneviratne CJ, Kao R (2010) Antimicrobial activity of Chinese medicine herbs against common bacteria in oral biofilm: A pilot study. Int J Oral Maxillofac Surg 39(6):599\u0026ndash;605. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ijom.2010.02.024\u003c/span\u003e\u003cspan address=\"10.1016/j.ijom.2010.02.024\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu LY (2022) Effects of astragalus polysaccharide gavage on production performance, serum biochemical indicators and gastrointestinal microbial flora of dairy cowsn (in Chinese). Dissertation, Xinjiang Agricultural University\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang L (2010) Detection of three cytokines in milk of dairy cows with mastitis and effect of TNF-α on apoptosis and proliferation of mammary gland cells (in Chinese). Dissertation, Inner Mongolia Agricultural University\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZeng HF, Xi YM, Li YQ, Wang ZD, Zhang L, Han ZY (2020) Analysis of astragalus polysaccharide intervention in heat-stressed dairy cows' serum metabolomics. Anim (Basel) 10(4):574. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ani10040574\u003c/span\u003e\u003cspan address=\"10.3390/ani10040574\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang GZ, Xing QY, Jin SX (2016) Effects of compound traditional Chinese medicine mixed bacteria fermentation preparation on immune function of AA broilers (in Chinese). Chin J Vet Med 52(7):52\u0026ndash;55\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang XL, Miao Q, Pan CX, Yin J, Wang LL, Qu LB, Yin YL, Wei YJ (2023) Research advances in probiotic fermentation of Chinese herbal medicines. iMeta 2(2):e93. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/imt2.93\u003c/span\u003e\u003cspan address=\"10.1002/imt2.93\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao SP, Shan CH, Wu ZJ, Feng M, Song LJ, Wang YA, Wang C (2022) Fermented Chinese herbal preparation: Impacts on milk production, nutrient digestibility, blood biochemistry, and antioxidant capacity of late-lactation cows under heat stress. Anim Feed Sci Technol 292:115448. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.anifeedsci.2022.115448\u003c/span\u003e\u003cspan address=\"10.1016/j.anifeedsci.2022.115448\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao T (2019) Study on process optimization of traditional Chinese medicine compound fermented by Lactobacillus plantarum and its effect on intestinal flora of mice (in Chinese). Dissertation, Lanzhou University\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhernakova DV, Wang DM, Liu L, Andreu-S\u0026aacute;nchez S, Zhang Y, Ruiz-Moreno AJ, Peng H, Plomp N, Del Castillo-Izquierdo \u0026Aacute;, Gacesa R, Lopera-Maya EA, Temba GS, Kullaya VI, van Leeuwen SS, Lifelines Cohort Study, Xavier RJ, de Mast Q, Joosten LAB, Riksen NP, Rutten JHW, Netea MG, Sanna S, Wijmenga C, Weersma RK, Zhernakova A, Harmsen HJM, Fu J (2024) Host genetic regulation of human gut microbial structural variation. Nature 625(7996):813\u0026ndash;821. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41586-023-06893-w\u003c/span\u003e\u003cspan address=\"10.1038/s41586-023-06893-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou MT, Zhang YG, Song ZY, Tang SP, Liu ZX, Pang MX, Zhang D, Wu X, Yu X, Wang P, Wei Y (2025) Enhanced bioactivity of \u003cem\u003ehoneysuckle-Cassia seeds\u003c/em\u003e extracts through \u003cem\u003eLactobacillus acidophilus\u003c/em\u003e and \u003cem\u003eBacillus subtilis\u003c/em\u003e co-fermentation: Impact on alcoholic liver disease and gut microbiota. Food Chem 486:144463. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.foodchem.2025.144463\u003c/span\u003e\u003cspan address=\"10.1016/j.foodchem.2025.144463\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou YX, L\u0026uuml; YL, Wang J, Ji S, Hou PX (2012) Application overview of blood biochemical indicators in animal production and nutrition regulation research (in Chinese). Anim Husb Feed Sci 33(S1):72\u0026ndash;74\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"amb-express","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ambe","sideBox":"Learn more about [AMB Express](http://amb-express.springeropen.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/AMBE/default.aspx","title":"AMB Express","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Compound fermented traditional Chinese medicine, Dairy cows, Milk quality, Microbial flora","lastPublishedDoi":"10.21203/rs.3.rs-8476572/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8476572/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study evaluated the efficacy of a compound fermented traditional Chinese medicine (TCM) preparation in dairy cows. Five TCMs and three probiotics were selected from 25 candidate herbs and six probiotics based on in \u003cem\u003evitro\u003c/em\u003e antibacterial activity against \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003eEscherichia coli\u003c/em\u003e using agar well diffusion and paper disc diffusion methods. The five TCMs were formulated according to the \u0026ldquo;Jun-Chen-Zuo-Shi\u0026rdquo; principle, and the optimal solid-state fermentation process was identified through single-factor and orthogonal experiments. The results showed that the optimal conditions included a \u003cem\u003eBacillus subtilis\u003c/em\u003e: \u003cem\u003eBacillus coagulans\u003c/em\u003e: \u003cem\u003eLactobacillus plantarum\u003c/em\u003e ratio of 1:3:1, a moisture content of 32%, an inoculation rate of 10%, a temperature of 31\u0026deg;C, and a fermentation period of 5 d. HPLC was used to examine changes in bioactive compound levels before and after fermentation. Fermentation significantly increased the concentrations of \u003cem\u003eResveratrol\u003c/em\u003e, \u003cem\u003ePolydatin\u003c/em\u003e, and \u003cem\u003eQuercetin\u003c/em\u003e, whereas the \u003cem\u003ePolydatin\u003c/em\u003e content decreased. To determine \u003cem\u003ein vivo\u003c/em\u003e effects, the fermented TCM was fed to dairy cows, and the indices of production performance, milk quality, serum biochemical levels, immune function, antioxidant capacity, and hormone levels were evaluated, and the changes in intestinal flora were analyzed by sequencing rectal contents using the 16S rRNA. The results showed that fermentation TCM signiffcantly improved the production performance of dairy cows as well as the milk quality, enhanced humoral immunity, strengthened antioxidant capacity, favorable modulation of gut microbiota, and metabolic regulation. This study provides strong experimental support for developing and applying compound fermented TCMs as functional feed additives in dairy production.\u003c/p\u003e","manuscriptTitle":"Process Optimization of Compound Fermented Traditional Chinese Medicine and Its Feeding Effect on Dairy Cows","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-08 19:31:04","doi":"10.21203/rs.3.rs-8476572/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-15T08:37:43+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-28T17:36:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"177400515490155713987998429405816311813","date":"2026-01-28T17:30:24+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-07T07:25:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-30T07:24:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-30T05:22:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"AMB Express","date":"2025-12-30T02:06:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"amb-express","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ambe","sideBox":"Learn more about [AMB Express](http://amb-express.springeropen.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/AMBE/default.aspx","title":"AMB Express","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"eba613f2-f840-4b92-a777-e6ac3f34a7f0","owner":[],"postedDate":"January 8th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-30T16:31:06+00:00","versionOfRecord":{"articleIdentity":"rs-8476572","link":"https://doi.org/10.1186/s13568-026-02038-0","journal":{"identity":"amb-express","isVorOnly":false,"title":"AMB Express"},"publishedOn":"2026-03-25 16:10:41","publishedOnDateReadable":"March 25th, 2026"},"versionCreatedAt":"2026-01-08 19:31:04","video":"","vorDoi":"10.1186/s13568-026-02038-0","vorDoiUrl":"https://doi.org/10.1186/s13568-026-02038-0","workflowStages":[]},"version":"v1","identity":"rs-8476572","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8476572","identity":"rs-8476572","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}