Application of andrographolide in promoting growth of calves and relieving weaning stress based on microbial community regulation effect

Abstract

The invention provides application of andrographolide in promoting calf growth and relieving weaning stress based on a microbial community regulation effect, and belongs to the technical field of animal feeding. The andrographolide (AG) can significantly improve the growth performance and the health level of the calves, relieve the weaning stress of the calves and improve the respiratory tract health of the calves by inhibiting inflammation and oxidative stress and systematically regulating and controlling microbial communities at multiple parts. Under the low-dosage feeding level, AG is more prone to optimizing rumen microflora, rumen is promoted to turn to an efficient propionic acid type fermentation mode, and sustainable benefits are achieved; and under a high dosage level, AG exerts a stronger anti-inflammatory and anti-oxidation effect to realize short-term benefit maximization. AG is added, so that the cross-site interaction between microbial communities in the digestive tract and the nasal cavity is enhanced, the colonization of various potential pathogens in the nasal cavity is remarkably inhibited, and the respiratory tract health of the calf is improved.

Description

Technical Field

[0001] This invention belongs to the field of animal husbandry technology, specifically relating to the application of andrographolide in promoting calf growth and alleviating weaning stress based on the regulation of microbial communities. Background Technology

[0002] Weaning stress is a significant challenge for calves during their rapid growth and development. Calves are exposed to multiple stressors, including feed type transitions (from liquid to solid feed), social environment (isolation and regrouping), and physical environment. The cumulative effect of these stressors essentially induces inflammatory responses and oxidative stress in calves, ultimately leading to immune dysfunction, high morbidity, and growth retardation or arrest. During weaning, calves are prone to diarrhea and bovine respiratory disease (BRD). BRD is the most common disease during weaning, with sudden weaning causing a surge in morbidity and mortality. Diarrhea is often caused by feed type transitions, incomplete gastrointestinal development, impaired intestinal barrier function, and susceptibility to pathogens. Weaning stress not only directly affects calf growth performance, health, and welfare but also has the potential for long-term and profound impacts on future growth, reproductive, and milk production. This results in significant losses and soaring treatment costs for the calf farming industry. Furthermore, the prevention and treatment of health problems induced by weaning stress still rely heavily on antibiotics. The overuse of antibiotics has exacerbated the problem of antimicrobial resistance (AMR), ultimately endangering human and animal health. Therefore, finding safe and effective plant-based additives to replace antibiotics has become one of the key research areas in animal husbandry. Summary of the Invention

[0003] This invention provides the application of andrographolide (AG) in promoting calf growth and alleviating weaning stress based on the role of microbial community regulation. AG can significantly improve the growth performance and health of calves by inhibiting inflammation, oxidative stress and systematically regulating the microbial community in multiple sites.

[0004] This invention provides the application of andrographolide in the preparation of reagents for regulating the microbial community among calf organs.

[0005] This invention also provides the application of andrographolide in the preparation of drugs to alleviate weaning stress in calves.

[0006] This invention also provides the application of andrographolide in the preparation of calf growth promoters.

[0007] This invention also provides the use of andrographolide in the preparation of medicaments for the prevention and / or treatment of respiratory diseases in calves.

[0008] The present invention also provides a method for regulating the microbial community among calf organs, including feeding calves andrographolide after weaning.

[0009] In one specific embodiment of the present invention, the interorgan microbial community includes at least one of the following organs: rumen, rectum, and nasal cavity.

[0010] In one specific embodiment of the present invention, the dosage of andrographolide is not less than 8 mg / kg body weight.

[0011] The present invention also provides a method for alleviating the application of calf weaning, characterized by including feeding andrographolide to calves after weaning.

[0012] In one specific embodiment of the present invention, the dosage of andrographolide is not less than 8 mg / kg body weight.

[0013] The present invention also provides a method for promoting calf growth, comprising feeding calves andrographolide after weaning.

[0014] Beneficial Effects: This invention provides the application of andrographolide (AG) in alleviating weaning stress in calves, regulating the microbial community, improving calf growth performance, and improving calf respiratory health. In the embodiments of this invention, through the phased addition of AG, AG can significantly improve the growth performance and health level of calves by inhibiting inflammation, oxidative stress, and systematically regulating the microbial community in multiple sites. At low-dose feeding levels, AG tends to optimize the rumen microbiota, promoting the rumen to a highly efficient propionic acid fermentation mode, achieving sustainable benefits; while at high-dose levels, AG exerts a stronger anti-inflammatory and antioxidant effect to maximize short-term benefits. In addition, the addition of AG strengthens the cross-site interaction between the digestive tract and nasal cavity microbiota and significantly inhibits the colonization of various potential pathogens in the nasal cavity, improving the respiratory health of calves. Attached Figure Description

[0015] Figure 1 The figure shows the effects of AG on the rumen microbiome of calves. In the figure, A: Venn diagram of rumen ASVs; B: Alpha diversity analysis of rumen microbiota. P <0.05, P <0.01; C: PCoA plot of rumen microbiota based on Bray-Curtis distance; D: ANOSIM, PERMANOVA and PERMDISP analysis of rumen microbiota; Figure 2 The figure shows the results of the microbial composition in the rumen of calves. In the figure, A: the dominant phylum of bacteria in the rumen; B: the dominant genera of bacteria in the rumen; CE: the differential genera in the rumen. To ensure that the statistical results (FDR-adjusted P) are completely consistent with the trend of the visualization chart, a box plot was drawn based on the DESeq2 normalized count. P <0.05, P <0.01, P <0.001, P <0.0001; Figure 3 The figure shows the impact of AG on the rectal microbiome. In the figure, A: Venn diagram of rectal ASVs; B: Alpha diversity analysis of rectal microbiota. P <0.05; C: PCoA plot of rectal microbiota based on Bray-Curtis distance; D: ANOSIM, PERMANOVA and PERMDISP analysis of rectal microbiota; Figure 4 The diagram shows the composition of the rectal microbiota in calves. In the diagram, A represents the top 5 dominant phyla of bacteria in the rectum; B represents the top 10 dominant genera of bacteria in the rectum; and C represents the differentially expressed genera in the rectum, which is a box plot based on DEseq2 normalized counts. P <0.05, P <0.01; Figure 5 The figure shows the impact of AG on the nasal cavity microbiome. In the figure, A: Venn diagram of nasal ASVs; B: Alpha diversity analysis diagram of nasal microbiota. P <0.05; C: PCoA plot of nasal microbiota based on Bray-Curtis distance; D: ANOSIM, PERMANOVA and PERMDISP analysis of nasal microbiota; Figure 6 The diagram shows the composition of the nasal cavity microbiota. In the diagram, A represents the top 5 dominant phyla of bacteria in the nasal cavity; B represents the top 10 dominant genera of bacteria in the nasal cavity; and CD represents the differentially expressed genera of bacteria in the nasal cavity. The box plots are drawn based on DEseq2 normalized counts. P <0.05, P <0.01, P <0.001, P <0.0001; Figure 7The figure shows the results of db-RDA analysis of key physiological phenotypes and microbial community structure. In the figure, A: Rumen; B: Rectal; C: Nasal. Each model passed the PERMANOVA test of 999 permutations. The global significance P-value is shown in the upper right corner of the figure. Figure 8 To visualize the cross-domain connectivity network of microorganisms from different treatment groups, a global co-occurrence network was constructed based on the SpiecEasi algorithm. Edges and nodes that connect only different body parts were then selected. Node size is proportional to degree, reflecting the core position of the genus in the cross-domain network; degree represents the number of connections between a genus and genus from different body parts. In the diagram, A: CON; B: L-AG; C: H-AG. Detailed Implementation

[0016] This invention provides the application of andrographolide in the preparation of reagents for regulating the microbial community among calf organs.

[0017] In this invention, staged feeding of AG to weaned calves significantly alters the microbial community structure of the rumen, rectum, hindgut, and nasal cavity. Furthermore, as verified by the examples, at a low feeding level (8 mg / kg), AG tends to optimize the rumen microbiota, promoting a shift towards a highly efficient propionic acid fermentation mode in the rumen, thus achieving sustainable benefits.

[0018] This invention also provides the application of andrographolide in the preparation of drugs to alleviate weaning stress in calves.

[0019] The embodiments of this invention demonstrate that the phased addition of AG is an effective strategy to alleviate weaning stress in calves. It can significantly improve the growth performance and health level of calves by inhibiting inflammation, oxidative stress and systematically regulating the microbial community in multiple parts of the body.

[0020] This invention also provides the application of andrographolide in the preparation of calf growth promoters.

[0021] The embodiments of this invention demonstrate that AG can significantly improve the growth performance and health of calves by inhibiting inflammation, oxidative stress, and systematically regulating multi-site microbial communities.

[0022] This invention also provides the use of andrographolide in the preparation of medicaments for the prevention and / or treatment of respiratory diseases in calves.

[0023] In this invention, it is demonstrated that at a high dose (16 mg / kg), AG exerts a stronger anti-inflammatory and antioxidant effect to maximize short-term benefits. The addition of AG enhances the cross-site interaction between the digestive tract and nasal cavity microbiota and significantly inhibits the colonization of various potential pathogens in the nasal cavity, thereby improving the respiratory health of calves.

[0024] The present invention also provides a method for regulating the microbial community among calf organs, including feeding calves andrographolide after weaning.

[0025] The interorgan microbial community described in this invention includes at least one of the following organs: rumen, rectum, and nasal cavity, and the dosage of andrographolide is not less than 8 mg / kg body weight.

[0026] The present invention also provides a method for alleviating the application of calf weaning, characterized by including feeding andrographolide to calves after weaning.

[0027] The dosage of andrographolide described in this invention is not less than 8 mg / kg body weight.

[0028] The present invention also provides a method for promoting calf growth, comprising feeding calves andrographolide after weaning.

[0029] To further illustrate the present invention, the application of andrographolide provided by the present invention in promoting calf growth and alleviating weaning stress based on the regulation of microbial communities is described in detail below with reference to the embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0030] Unless otherwise specified, the materials and methods used in the embodiments of this invention are conventional materials and methods in the art: 1. The experiment of this invention was conducted at Guangshunhai Food Co., Ltd. in Meizhou City, Guangdong Province from October to December 2024. During the three-day preliminary observation period, information such as the frequency of coughing, diarrhea, and medical history was recorded to select 18 calves of similar weight (BW=88.25±4.98kg) as experimental subjects. The formal experiment began on the predetermined weaning date (90±3 days of age), and after weighing, they were transferred to an independent pen for weaning.

[0031] 2. The experimental design adopted a completely randomized block design, dividing the calves into three treatment groups: (1) CON group (oral administration of empty capsules); (2) L-AG group (8 mg / kg AG); (3) H-AG group (16 mg / kg AG). Andrographolide (AG) was filled into empty capsules and administered orally before morning feeding. On the 21st day of the experiment, the calves were weighed again and the AG dosage was adjusted according to their body weight. The experimental period lasted for 42 days. During this period, the calves had free access to water and alfalfa hay. The amount of starter feed was based on maintaining 5%-10% of the remaining amount the next day (feeding twice a day at 08:00 and 16:00). The calves had free access to water during the experiment, and the water troughs were cleaned twice a day. The AG used in the experiment was provided by Shaanxi Haochen Biotechnology Co., Ltd., and was a white fine powder with a purity of ≥98%. The chemical composition of the starter feed for calves is detailed in Table 1.

[0032] Table 1. Nutritional composition of calf starter feed and alfalfa hay (% dry weight)

[0033] 3. Growth performance testing and health parameter assessment Before morning feeding on days 0, 21, and 42 of the formal trial, calves in each group were weighed, and their height and chest circumference were measured. Average daily gain (ADG) was calculated as the ratio of the increase in body weight to the number of days between feedings.

[0034] Calf feces were scored using a 4-point scale: 0 points: normal and relatively hard; 1 point: soft or pasty feces; 2 points: loose and watery; 3 points: watery feces.

[0035] A simplified version of the calf respiratory scoring system was developed based on the method described in the article (Mcguirk SM, Peek SF. Timely diagnosis of dairy calfrespiratory disease using a standardized scoring system. Anim Health Res Rev2014;15(2):145-7. https: / / doi.org / 10.1017 / S1466252314000267.). The scoring system consisted only of cough and nose scores. The cough score was based on a 4-point scale: 0 points: no cough; 1 point: single cough; 2 points: intermittent cough; 3 points: continuous or repetitive cough. The nose score was also based on a 4-point scale: 0 points: normal; 1 point: small amount of unilateral discharge; 2 points: bilateral cloudy mucus; 3 points: large amount of bilateral purulent nasal discharge. Two trained veterinarians scored the calf feces and respiratory system daily. The respiratory score was observed when the calves were resting between 2:00 PM and 4:00 PM.

[0036] 4. Serum sample collection and analysis Before morning feeding on days 21 and 42, 20 ml of blood was collected from the tail vein of calves using disposable lancets and placed in two procoagulant blood collection tubes. The blood was then centrifuged at 3000 rpm for 10 min to obtain the supernatant. The supernatant was aliquoted into six 1.5 mL cryovials and temporarily stored in liquid nitrogen. Subsequent serum biochemical, antioxidant, and immunological marker analyses were conducted with the assistance of the Beijing Huaying Biotechnology Institute.

[0037] The kits used for measuring total protein (TP), albumin (ALB), cholesterol (CHO), triglycerides (TG), creatinine (CREA), urea (BUN), uric acid (Ua), glucose (GLU), alanine aminotransferase (ALT), and total bilirubin (TBIL) were provided by Sinopharm Group Co., Ltd. The kits used for measuring SOD, MDA, GSH-Px, GSH, T-AOC, CAT, and MPO were provided by Beijing Huaying Biotechnology Research Institute. Measurements were performed according to the kit instructions, using a Mindray BS-420 biochemical analyzer (Mindray Bio-Medical Electronics Co., Ltd., Shenzhen, China).

[0038] Immunoglobulin A (IgA), immunoglobulin G (IgG), and immunoglobulin M (IgM) were measured using immunoturbidimetry. Interferon-gamma (IFN-γ), interleukin-1β (IL-1β), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL-10), transforming growth factor-β1 (TGF-β1), growth hormone (GH), insulin-like growth factor-1 (IGF-1), and C-reactive protein (CRP) were measured using enzyme-linked immunosorbent assay (ELISA). Kits were provided by Beijing Huaying Biotechnology Research Institute, and the instruments used were DR-200BS ELISA analyzers (Hiwell-Diatek Instruments Co., Ltd., Wuxi, China).

[0039] 5. Microbial sample collection and VFA determination On the last day of the experiment, before morning feeding, the area around the calves' noses was cleaned and disinfected. A 15cm sterile swab was used to collect deep nasal samples from the left nostril of each calf. Specifically, the swab was inserted horizontally about 7-9cm along the bottom of the nasal cavity, and rotated for 10 seconds to ensure full contact with the mucous membrane, thus obtaining a nasal swab. Similarly, after cleaning and disinfecting the area around the anus, a sterile swab was inserted into the calf's anus and rotated several times along the anal wall to obtain an anal swab. Five nasal and anal swabs were collected from each calf, broken into 1.5mL cryovials, and stored in liquid nitrogen for analysis of the bacterial community in the calf's upper respiratory tract and hindgut.

[0040] Rumen fluid was collected from calves via an oral gastric tube. The first 30 mL of rumen fluid was discarded to avoid saliva contamination. The collected 50 mL of rumen fluid was filtered through four layers of sterile gauze. After thorough shaking, approximately 45 mL of rumen fluid was stored in liquid nitrogen for subsequent determination of volatile fatty acids (VFAs). 5 mL of rumen fluid was aliquoted into three 1.5 mL cryovials and stored in liquid nitrogen for subsequent analysis of the rumen bacterial community.

[0041] VFA in rumen fluid was determined by gas chromatography. After centrifugation at 5400 rpm for 10 min to remove impurities, 0.2 mL of 25% metaphosphate solution containing the internal standard 2EB was accurately added to 1 mL of rumen fluid using a pipette. After incubation in an ice-water bath for 30 min, the solution was centrifuged at 10000 rpm for 10 min to remove protein precipitates. The supernatant was then used for analysis. The instrument used was an Agilent 6890N gas chromatograph (Agilent Technologies, Avondale, PA, USA).

[0042] 6. DNA isolation and 16S rRNA amplicon library preparation and sequencing DNA was extracted from the samples (rumen fluid, anal swab, nasal swab) using the MagBeads FastDNA Kit for Soil (MP Biomedicals, LLC, Solon, OH, USA) according to the manufacturer's instructions. PCR amplification was performed using primers specific to the bacterial 16S rDNA V3-V4 region. 338F (SEQ ID No.1): 5'-barcode+ACTCCTACGGGAGGCAGCA-3'; 806R (SEQ ID No. 2): 5'-GGACTACHVGGGTWTCTAAT-3'.

[0043] PCR was performed using NEB Q5 DNA high-fidelity polymerase (New England Biolabs, Ipswich, MA, USA) for 27 cycles. The target fragment was purified and recovered using an Axygen gel extraction kit (Axygen, Tewksbury, MA, USA), and the PCR products were quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Libraries were constructed using the Illumina TruSeq Nano DNALT Library Prep Kit (Illumina, San Diego, CA, USA). After passing Agilent 2100 quality control (Agilent Technologies, Santa Clara, CA, USA), the libraries were sequenced at 2×250 bp on the Illumina MiSeq platform.

[0044] 7. Bioinformatics Analysis The original sequences were analyzed using QIIME 2 v2019.4 (see Boylyen E, Rideout JR, Dillon MR, Bokulich NA, Abnet CC, Al-Ghalith GA, et al. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat Biotechnol 2019;37(8):852-7. https: / / doi.org / ). After primer removal, the DADA2 plugin was used to perform quality filtering, denoising, splicing, and chimera removal on the sequences (Callahan BJ, Mcmurdie PJ, Rosen MJ, Han AW, Johnson AJA, Holmes SP. DADA2: High-resolution sample inference from Illumina amplicon data. Nat Methods 2016;13(7):581-3. https: / / doi.org / 10.1038 / nmeth.3869.), generating an ASVs table. Taxonomical annotation of ASV signature sequences was performed using the Greengenes2 2022.10 database (Mcdonald D, Jiang Y, Balaban M, Cantrell K, Zhu Q, Gonzalez A, et al. Greengenes2 unifies microbial data in a single reference tree. Nat Biotechnol 2024;42(5):715-8. https: / / doi.org / 10.1038 / s41587-023-01845-1.). Alpha and Beta diversity indices were calculated using QIIME2. Principal coordinate analysis (PCoA) based on Bray-Curtis distance was used to visualize clustering among samples, and the matrices were statistically tested using PERMANOVA (999 permutations), Anosim, and Permdisp. Likelihood ratio tests (LRT) were performed using DESeq2 to screen for differentially expressed genera among the three groups. The Wald test was used for pairwise comparisons of differentially expressed genera among the three groups. All p-values ​​were corrected for False Discovery Rate (FDR) using the Benjamini-Hochberg method. PA value <0.05 was considered significant. To facilitate the interpretation and visualization of differentially expressed bacterial genera, relevant functions of these genera were inferred through searches of PubMed, Web of Science, and the GTDB database.

[0045] 8. Multi-site microbial correlation analysis To explore the association between calf phenotype and the microbial community structure in multiple locations, distance-based redundancy analysis (db-RDA) was performed on the rumen, rectal, and nasal microbiota. All analyses were performed in the vegan package of R software (version 4.5.1) (Dixon P. VEGAN, a package of R functions for community ecology. J Veg Sci 2003;14(6):927-30. https: / / doi.org / https: / / doi.org / 10.1111 / j.1654-1103.2003.tb02228.x.). Taxonomic units with a prevalence of less than 10% were removed, and the Bray-Curtis distance matrix for each location was calculated. To construct a stable final model that avoids collinearity, a hypothesis-driven variable selection strategy was adopted. The individual explanatory power of the 11 phenotypic variables was evaluated one by one using the adonis2 function (based on Bray-Curtis distance, 999 permutations). Based on biological function classification (growth / health score, pro-inflammatory factors, anti-inflammatory factors, and antioxidants), the four most representative phenotypic variables (with the smallest p-value in the adonis2 test) for each microbial site were selected for the final analysis. A db-RDA model was constructed using the capscale function, with the Bray-Curtis distance matrix as the response variable and the above four variables as explanatory variables. The global significance of the model was assessed using the anova.cca function with 999 permutations (PERMANOVA).

[0046] To investigate the interactions among microorganisms in different treatment groups, a microbial co-occurrence network analysis was constructed. Prevalence filtering was used to exclude taxa with a prevalence of less than 20%. Microbial interactions were inferred using SpiecEasi (v1.99.0) in R software (Kurtz ZD, Müller CL, Miraldi ER, Littman DR, Blaser MJ, Bonneau RA. Sparse and compositionally robust inference of microbial ecological networks. PLoS Comput Biol 2015;11(5):e1004226. https: / / doi.org / 10.1371 / journal.pcbi.1004226.). The Meinshausen-Bühlmann neighborhood selection method (method='mb') was used, and the model parameters (lambda.min.ratio=1e-2, nlambda=20, pulsar.params=list(rep.num=50)) were determined by StARS stability selection. The topological properties (number of nodes, number of edges, network density, average path length, and modularity) of each network were calculated using the igraph package (v2.1.4) (Csardi G, Nepusz T, Traag V, Horvát S, Zanini F, Noom D, et al. Igraph: Network Analysis and Visualization in R. R package version 2024;2(3).). Cross-site network subgraphs containing only cross-site connections were extracted, and their edge count and proportion were calculated. Key genera in the network were identified by calculating the degree, betweenness, and closeness centrality of nodes. Network visualization was performed using the ggraph package (v2.2.2) (Pedersen TL. Ggraph: An implementation of grammar of graphics for graphs and networks. R package version 2020;2(3):1. https: / / doi.org / ).

[0047] 9. Statistical Analysis All data were analyzed using SAS 9.4 (SAS Institute Inc.). The normality of the experimental data was assessed using the Shapiro-Wilk test. For data that were approximately normal or normally distributed after data transformation, the MIXED program was used for analysis. For data that could not be transformed to approximately normal distribution (such as GLU, MDA, etc.), the GLIMMIX program was used for generalized linear mixed model (GLMM) analysis.

[0048] In the analysis of calf growth performance and VFA concentration, the MIXED program was used, with fixed effects as treatments and individual calves as random effects. The model was tested for linear and quadratic effects of andrographolide (AG) using orthogonal polynomial comparisons. The results were corrected for Tukey-Kramer using multiple comparisons.

[0049] For the analysis of serum markers in calves, the fixed effects included treatment, time, and their interactions. Random effects are incorporated into the individual calf intercepts and nested within the treatment groups to control for repeated measures correlation and inter-group heterogeneity. The covariance structure is ultimately determined to be optimal using the first-order autoregressive (AR1) structure based on minimizing the AIC / BIC criterion. For non-normally distributed variables (such as GLU, MDA, etc.), the GLMM process is used to accommodate data deviations and reduce the impact of outliers. P Values ​​≤ 0.05 are considered significant, and values ​​< 0.05 are considered significant. P A value ≤0.10 is considered a trend of difference and does not reach a significant level.

[0050] Example 1: The effect of supplemental AG on calf growth performance As shown in Table 2, all calves had similar body weights on the first day of the experiment, and by day 42, the calf weights showed a linear increase with increasing AG dosage. P =0.036). Throughout the entire experimental period (days 1-42), the ADG of H-AG was significantly higher than that of the CON group ( P =0.028); however, in the later stages of the experiment, from day 21 to 42, the ADG in the L-AG group was significantly higher than that in the CON group ( P =0.031), and shows a trend of quadratic effect ( P =0.078). From 21 to 42 days, calf feed intake showed a linear increase with increasing AG supplementation dosage ( P =0.044), but there was no significant difference between groups ( P >0.05). The stool and cough scores of the L-AG group were significantly lower than those of the CON group ( P =0.03 and P =0.018). Furthermore, there were no significant differences in height and chest circumference among the groups ( ). P >0.05).

[0051] Table 2. Effects of Andrographolide Supplementation on Growth Performance and Health Parameters in Calves

[0052] Example 2: Effects of AG supplementation on serum biochemical, immune, antioxidant, and hormonal indicators in calves The effects of different concentrations of AG on calf serum biochemistry are shown in Table 3. There were no significant differences in TP, ALB, TG, BUN, and ALT among the three groups. P >0.05). The processing of AG makes GLU ( P =0.03) concentration significantly increased, Ua ( P =0.003), CREA ( P The concentration of TC (0.002) decreased significantly with increasing AG feeding amount. P =0.05), TBIL ( P =0.044) both showed interaction effects in terms of treatment and time.

[0053] Regarding serum immune markers, feeding AG can increase serum IgA ( P =0.009), IL-4 ( P <0.001), IL-10 ( P =0.009) significantly increased, while IL-1β ( P =0.02), IFN-γ ( P =0.003) decreased significantly with increasing AG concentration. Furthermore, IL-2 ( P =0.01), IL-6 ( P =0.008), TGF-β1 ( P =0.02), CRP ( P =0.01) has an interaction effect on treatment and time.

[0054] Regarding serum antioxidant activity, serum MDA ( P =0.003), MPO ( P =0.006) decreased linearly with increasing AG concentration. Meanwhile, SOD ( P =0.02), GSH-Px ( P =0.022), CAT ( P =0.002), GSH ( P =0.04) There is an interaction between treatment and time.

[0055] Regarding serum hormone levels, GH ( ) in serum was higher after feeding AG. P =0.02) showed a significant linear increase, and IGF-1 (P =0.004) There is an interaction between treatment and time.

[0056] Table 3. Effects of Andrographolide Supplementation on Serum Biochemical, Immunological, Antioxidant, and Hormonal Indications in Calves

[0057] Example 3: Effect of supplemental AG on rumen fluid fermentation parameters As shown in Table 4, AG treatment significantly affected the acetic acid concentration and the acetic acid to propionic acid ratio (A / P), both of which exhibited significant secondary effects. P =0.03 and P =0.027); compared with the CON group, the L-AG group showed a significant decreasing trend in acetic acid and A / P ( ). P =0.072 and P =0.088). Furthermore, the AG treatment had no significant effects on propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, or total VFA. P >0.05).

[0058] Table 4. Effects of Andrographolide Supplementation on Rumen Fermentation Parameters

[0059] Example 4: Effects of AG on the rumen microbiome Eighteen calf rumen fluid samples were sequenced for the V3-V4 region of 16S rRNA, yielding 1,336,128 raw reads. After quality control, noise reduction, chimera and singleton removal, 811,961 high-quality valid sequences were obtained. Based on the DADA2 algorithm, 8245 ASVs were obtained. This method provides higher resolution than traditional 97% similarity OTU clustering, can distinguish single nucleotide variants, and thus more accurately characterizes the microbial community structure. Venn diagram analysis results are shown below. Figure 1 As shown, the three treatment groups have a total of 392 ASVs, while the CON, L-AG, and H-AG groups hold 2355, 2631, and 2440 unique ASVs, respectively. Figure 1 (A)

[0060] In Alpha diversity analysis ( Figure 1 (B) There was no significant difference in the Chao1 index among the three groups. P >0.05). Compared with the CON group, there were no significant differences in Simpson and Shannon indices between the L-AG and H-AG groups ( P >0.05), but the Simpson and Shannon indices of the L-AG group were significantly lower than those of the H-AG group ( P<0.05). The Pielou_e index of both the CON and L-AG groups was significantly lower than that of the H-AG group ( P <0.05).

[0061] Beta diversity is based on PCoA analysis using Bray-Curtis distance, such as Figure 1 According to C, PC1 and PC2 explained 33.8% and 17.8% of the total community variation, respectively, and a clear separation trend was observed among the three groups. Subsequent multivariate statistical analysis was then performed. Figure 1 (D), PERMANOVA analysis showed that the CON group and the L-AG group (R) 2 =0.482, P =0.001) and H-AG group (R 2 =0.325, P =0.001) showed a significant difference; PERMDISP analysis showed no significant difference in dispersion among all groups ( P >0.05). This indicates that AG treatment significantly altered the community structure of the rumen microbiota in calves.

[0062] Taxonomic analysis showed that in calf rumen fluid Firmicutes_A (Firmwallis_A) Actinobacteriota (Actinomycetes) Bacteroidota (Bacteroidetes) Proteobacteria (Proteobacteria) and Firmicutes_C Firmicutes (C) is the dominant phylum of bacteria. Figure 2 (A). At the genus level ( Figure 2 (B) The ten dominant bacterial genera are: CAG-793 , UBA1711 , Parafannyhessea (Parafni Hercetella) Prevotella (Prevotella) Pseudoscardovia (Pseudomonas spp.) Succinivibrio (Vibrio succinate) Tractidigestivibacter (Gastrointestinal Bacteria) Intestinibaculum (Enterozoella genus) Dialister (Dialectae) and CAG-791 .

[0063] Based on the LRT test of DESeq2, a total of 26 differentially expressed genera were identified (FDR-adjusted). P <0.05. To facilitate interpretation and visualization, based on the inferred functions of the differentially expressed genera (VFA generation, methanogenesis, immunity and inflammation), 20 major differentially expressed genera were visualized. Figure 2 (CE). Among the genera associated with VFA production ( Figure 2(C), L-AG group significantly inhibited Limimorpha , Pseudoramibacter (Pseudomonas) Eubacterium_T (Eubacterium_T) UBA1711 , Tractidigestivibacter (Gastrointestinal Bacteria) and Parafannyhessea (Parannehexenes). Meanwhile, the L-AG group was significantly enriched with... Pseudoscardovia (Pseudomonas spp.) CAG-238 , RUG12438 , CAG-127 , Sodaliphilus (Bacterium spp.) and Pseudobutyrivibrio (Pseudomonas butyricum). Among the genera associated with methane production ( Figure 2 (D), L-AG group significantly inhibited Tenuifilum (Filamentous fungi), and Intestinibaculum (Enterobacteria spp.) were significantly enriched in the L-AG group. Among the genera associated with immunity and inflammation ( Figure 2 (E) Allisonella (Aristolochic acid bacteria) and CAG-793 Significantly enriched in the L-AG group, while Desulfovibrio_R (Desulfovibrio spp._R) was significantly enriched in the H-AG group. Unlike the L-AG group, the H-AG group was significantly enriched with Limimorpha , UBA1711 and Parafannyhessea (Parfannie Hercetella), significantly inhibited Unclassified_f_Muribaculaceae (Unclassified - Rhizoctoniaceae family) CAG-269 , Allisonella (Aristolochic acid bacteria) and CAG-793 .

[0064] Example 5: Effects of AG on the rectal microbiome The analysis of rectal microbiota followed the same procedure as described above. Eighteen calf anal swabs were sequenced for the V3-V4 region of 16S rRNA, yielding 1,332,852 raw reads. After quality control, noise reduction, chimera and singleton removal, 743,867 high-quality valid sequences were obtained. Venn diagram analysis showed that there were 926 ASVs in total across the three treatment groups, with the CON, L-AG, and H-AG groups holding 6177, 4633, and 7057 unique ASVs, respectively. Figure 3 (A)

[0065] In Alpha diversity analysis ( Figure 3 In the B group, the Chao1, Simpson, Shannon, and pielou_e indices of the L-AG and H-AG groups were not significantly different from those of the CON group. P>0.05). However, the Chao1, Simpson, and Shannon levels in the L-AG group were significantly lower than those in the H-AG group ( P <0.05).

[0066] Beta diversity is based on PCoA analysis using Bray-Curtis distance. For example... Figure 3 C indicates that PC1 and PC2 explained 13.4% and 11.4% of the total community variation, respectively. The CON, L-AG, and H-AG groups showed some overlap, but a certain trend of separation was observed. In the multivariate statistical analysis ( Figure 3 (D), PERMANOVA analysis showed that the CON group and the L-AG group (R) 2 =0.121, P =0.033) and H-AG group (R 2 =0.146, P =0.006) showed a significant difference; PERMDISP analysis showed no significant difference in dispersion among all groups ( p >0.05).

[0067] Taxonomic analysis shows that at the phylum level ( Figure 4 The dominant bacterial phyla in the hindgut of calves (A) are: Firmicutes_A (Firmwallis_A) Bacteroidota (Bacteroidetes) Spirochaetota ( Spirochete phylum) Firmicutes_C (Firmwallis_C) and Actinobacteriota (Actinomycetes). At the genus level ( Figure 4 (B) The ten dominant bacterial genera are: Cryptobacteroides , Faecousia , RF16 , UBA737 , Paraprevotella (Paraplevobacter spp.) Treponema_D (Breospirolium genus_D) Prevotella (Prevotella) G11 , SFTJ01 and Phocaeicola_A (Maritime City Coccidia genus_A).

[0068] Based on the LRT test using DESeq2, only one differentially expressed genus was identified (FDR-adjusted). P <0.05, Figure 4 (C) Compared with the CON group, the H-AG group significantly inhibited [the activity]. Phascolarctobacterium_A ( Koala bacteria (A).

[0069] Example 6: The effect of AG on nasal cavity microbiota Eighteen calf nasal swabs were sequenced for the V3-V4 region of 16S rRNA, yielding 1,259,065 raw reads. After quality control, noise reduction, chimera and singleton removal, 1,061,478 high-quality valid sequences were obtained. Venn diagram analysis showed that there were 579 ASVs in the three treatment groups, with the CON, L-AG, and H-AG groups holding 1825, 1671, and 1979 unique ASVs, respectively. Figure 5 (A)

[0070] Alpha diversity analysis showed no significant differences in Chao1, Shannon, and Pielou_e indices among the three groups. P >0.05), while the Simpson index of the L-AG group was significantly higher than that of the CON group ( P <0.05, Figure 5 (B)

[0071] Beta diversity analysis was performed using PCoA based on Bray-Curtis distance. For example... Figure 5 The results showed that PC1 and PC2 explained 33.9% and 19.6% of the total community variation, respectively. The CON group was completely separated from the L-AG group and partially overlapped with the H-AG group. Subsequent multivariate statistical analysis was performed. Figure 5 (D), PERMANOVA analysis showed that the CON group and the L-AG group (R) 2 =0.413, P =0.001) and H-AG group (R 2 =0.331, P =0.003) showed a significant difference; PERMDISP analysis showed no significant difference in dispersion among all groups ( p >0.05).

[0072] Taxonomic analysis indicates that in the upper respiratory tract Proteobacteria (Proteobacteria) Firmicutes_D (Firmwallis_D) Actinobacteriota (Actinomycetes) Bacteroidota (Bacteroidetes) and Firmicutes_A Firmicutes (A) is the dominant phylum of bacteria. Figure 6 (A). At the genus level ( Figure 6 (B) The ten dominant bacterial genera are: Moraxella_C (Morax genus_C) Pasteurella (Pasteurella) Mycoplasmopsis_A (Similar to Mycoplasma genus_A) Mesomycoplasma (Intermediate Mycoplasma) Histophilus (Organophilic bacteria) Mannheimia (Mannheimia) Caviibacter (Caviella spp.) Faecousia, Prevotella (Prevotella) and Streptococcus (Streptococcus).

[0073] Based on the LRT test of DESeq2, a total of 12 potential pathogenic microorganisms and 37 genera related to the digestive tract were identified. Among the 12 potential pathogenic microorganisms ( Figure 6 The C, L-AG and H-AG groups significantly inhibited [the activity]. Brevundimonas (Shortwave Monoclonal bacteria) Exiguobacterium_A (Microbacteria genus_A) Stenotrophomonas_A (Oligotrophomonas genus_A) 、 Malacoplasma_A (A) and Dietzia (Dizzyella spp.), but significantly enriched Acholeplasma_A (Acholestapeptide genus A) and Erysipelothrix (Erysipelothrix). L-AG group Corynebacterium (Corynebacterium) and Pasteurella (Pasteurella spp.) showed stronger inhibitory activity, while the H-AG group showed less inhibition. Moraxella_C (Morax spp._C) and Ureaplasma (Ureaplasma) phenotype showed a stronger inhibitory effect. Among 37 differentially expressed bacterial genera related to the digestive tract ( Figure 6 (D) Several key functional bacterial communities were significantly enriched in the nasal cavity, such as Succinivibrio (Vibrio succinate) and Bifidobacterium (Bifidobacterium spp.), etc. Among them, Unclassified_f_Muribaculaceae (Unclassified_Rhizoctoniaceae) and CAG-269 It is significantly enriched not only in the nasal cavity but also in the rumen.

[0074] Example 7: Multi-site microbial association analysis 7.1 Association Analysis of Calf Phenotype and Multisite Microbial Community Structure To explore the association between physiological and health indicators of calves and the overall structure of microbial communities in multiple sites, and to construct a robust final model that avoids collinearity, this invention performs redundancy analysis based on Bray-Curtis distance (db-RDA) on the most representative phenotypes.

[0075] The results are as follows Figure 7 As shown, calf phenotype can significantly explain rumen ( Figure 7 A; PERMANOVA P =0.004) and nasal cavity ( Figure 7 B; PERMANOVA P The microbial community variation was 0.033, but the effect on the rectal microbiota was not significant. Figure 7C; PERMANOVA P =0.338).

[0076] In the rumen, the model's db-RDA1 and db-RDA2 metrics together explained 38.37% of the total microbial community variation. Clear separation was observed among the three groups: the CON group was significantly separated from the L-AG and H-AG groups along the db-RDA1 axis. The L-AG and H-AG groups differentiated along the db-RDA2 axis. Vector analysis showed that the Cough score and IL-6 were strongly correlated with the CON group, while IL-4 and CAT were strongly correlated with the H-AG group.

[0077] In the nasal cavity model, db-RDA1 and db-RDA2 together explained 28.94% of the total variation in the microbial community. Ordination plots showed that the CON group samples clustered on the left, exhibiting a clear separation trend from the L-AG and H-AG groups along the db-RDA1 axis, while the L-AG and H-AG groups showed partial overlap. Vector analysis revealed that the Cough score and IL-2 pointed to the clustered region of the CON group samples, while IL-4 and GSH-Px pointed to the AG treatment group samples.

[0078] 7.2 Co-occurrence and Topological Analysis of Multi-site Microbial Networks To investigate the interaction patterns of microorganisms in different body parts, this invention conducted a global microbial network co-occurrence analysis of all genera. Topological analysis results (Table 5) show that AG (aggregate-aggregate) enhanced the complexity of the microbial network in a dose-dependent manner. Compared to the CON group (total connections 2827), the total number of connections in the L-AG (3695) and H-AG groups (4026) increased by 30.7% and 42.4%, respectively. In cross-site connections connecting different body parts, compared to the 529 cross-site connections in the CON group, the number of cross-site connections in the L-AG (741) and H-AG groups (866) increased by 39.9% and 63.7%, respectively.

[0079] To visually demonstrate the structural differences in cross-part connections, this invention presents a co-occurrence diagram of cross-part connections among three groups. Figure 8 ); and based on the ranking of connectivity (Degree) in the network, the top 20 key bacterial genera were labeled.

[0080] Table 5. Topological characteristics of multi-site microbial symbiotic networks in different treatment groups

[0081] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of andrographolide in the preparation of reagents for regulating the microbial community among calf organs.

2. Application of andrographolide in the preparation of drugs to alleviate weaning stress in calves.

3. Application of andrographolide in the preparation of calf growth promoters.

4. Application of andrographolide in the preparation of drugs for the prevention and / or treatment of respiratory diseases in calves.

5. A method for regulating the interorgan microbial community in calves, characterized in that, This includes feeding andrographolide to calves after weaning.

6. The method according to claim 5, characterized in that, The interorgan microbial community includes at least one of the following organs: rumen, rectum, and nasal cavity.

7. The method according to claim 5, characterized in that, The dosage of the andrographolide is not less than 8 mg / kg body weight.

8. A method for alleviating the application of weaning techniques in calves, characterized in that, This includes feeding andrographolide to calves after weaning.

9. The method according to claim 8, characterized in that, The dosage of the andrographolide is not less than 8 mg / kg body weight.

10. A method for promoting calf growth, characterized in that, This includes feeding andrographolide to calves after weaning.

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