Application of carvacrol in preparation of feed additive and / or feed for repairing intestinal injury of piglets

Abstract

The invention provides application of carvacrol in preparation of a feed additive and / or feed for repairing intestinal injury of piglets, and belongs to the technical field of animal feed. The invention discloses application of carvacrol in preparation of a feed additive and / or feed for repairing intestinal injury of piglets. Experiments show that weaning stress causes piglet growth performance reduction and intestinal barrier injury; the feed added with CAR can effectively repair intestinal injuries of piglets, maintain the integrity of intestinal barriers and regulate the intestinal microbial structure, and carvacrol also has the activity of resisting the characteristics of porcine epidemic diarrhea virus and porcine delta coronavirus. The invention provides a reasonable feeding strategy and theoretical support for piglet intestinal injury caused by weaning stress.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal feed, and particularly relates to the application of carvacrol in preparing a feed additive and / or feed for repairing intestinal damage in piglets. Background Art

[0002] Numerous studies have shown that weaning activates the immune system in the piglet's gastrointestinal tract, producing excessive pro-inflammatory cytokines and triggering intestinal inflammation and dysfunction. This inflammatory response increases intestinal permeability, allowing pathogens and cells present in the intestinal contents to enter the subcutaneous tissue and cause mucosal damage. Increased intestinal permeability leads to leakage, further triggering imbalances in the intestinal ecology and immune response, as well as systemic diseases. Alleviating weaning-induced intestinal damage is crucial for the healthy growth of piglets.

[0003] Carvacrol (CAR) is widely used as a botanical feed additive to replace antibiotics in animal feed. For example, when added to broiler chicken feed, CAR can significantly improve meat quality, enhance antioxidant capacity, and reduce intestinal damage caused by Clostridium perfringens. Furthermore, in rumen-containing animal feed, CAR reduces in vitro digestibility of dry matter, crude protein, and neutral detergent fiber to a greater extent than monensin, increases butyrate content, and reduces acetate content, making it a potential rumen fermentation regulator. However, there are currently no reports on the use of carvacrol for treating intestinal damage in piglets. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide the use of carvacrol in the preparation of feed additives and / or feed for repairing intestinal damage in piglets. Carvacrol maintains the integrity of the piglet's intestinal barrier and regulates the balance of intestinal microbial flora, thereby achieving the purpose of repairing intestinal damage in piglets.

[0005] The present invention provides the use of carvacrol in preparing a feed additive and / or feed for repairing intestinal damage in piglets.

[0006] Preferably, the intestinal damage of the piglets is caused by weaning stress and / or porcine pathogenic viruses;

[0007] The porcine pathogenic viruses include porcine epidemic diarrhea virus (PEDV) and / or porcine delta coronavirus (PDCoV).

[0008] Preferably, the repair of intestinal damage in piglets includes at least one of the following: maintaining intestinal barrier function, promoting intestinal cell proliferation and renewal, alleviating small intestinal inflammatory response, and improving intestinal microbial flora structure and abnormal metabolites thereof.

[0009] Preferably, the maintenance of intestinal barrier function includes at least one of the following: repairing intestinal morphological damage, reducing intestinal permeability changes, reducing the expression level of key intestinal barrier proteins, and reversing the decrease in the amount of colon mucus layer and goblet cells.

[0010] Preferably, the promotion of intestinal cell proliferation and renewal includes upregulating the expression of intestinal stem cell proliferation-related factors, the expression of the Wnt pathway or an effective enhancer of the Wnt pathway, alleviating the abnormal secretion of small intestinal mucosal goblet cells and mucin, and upregulating the expression level of the Lyz1 gene encoding lysozyme in small intestinal Paneth cells.

[0011] Preferably, the alleviating small intestinal inflammatory response comprises inhibiting the level of inflammatory factors by inhibiting the TLR4 / Myd88 pathway.

[0012] Preferably, the improvement of the intestinal microbial flora structure and the abnormality of its metabolites includes increasing the relative abundance of intestinal Eubacterium coprostanoligenes group and / or Lactobacillus and / or reducing the relative abundance of Escherichia coli.

[0013] Preferably, the application also includes the use of carvacrol in the preparation of feed additives and / or feeds for alleviating piglet diarrhea, promoting small intestinal development and promoting piglet growth performance.

[0014] Preferably, the feed additive further comprises auxiliary materials;

[0015] The auxiliary materials include protein nutrients, fillers, and adhesives;

[0016] The mass ratio of the carvacrol, the protein nutrient, the filler and the adhesive is 3.158-15:18-22:7-9:30-35.

[0017] Preferably, the concentration of carvacrol in the feed is not less than 50 mg / kg.

[0018] Preferably, the piglets are 26 to 32 days old.

[0019] The present invention provides the use of carvacrol in the preparation of feed additives and / or feed for repairing intestinal damage in piglets. The present invention uses 4-week-old weaned piglets as experimental animals to evaluate the effect of adding carvacrol to feed on the repair of intestinal damage in piglets. The results show that the addition of CAR to feed can effectively increase the height of small intestinal villi, reduce the depth of crypts, and improve the morphology and density of intestinal microvilli; increase the expression levels of tight junction proteins (ZO-1, Claudin1, Occludin, Claudin5) and adhesion junction proteins (E-cadherin), and reduce the concentrations of intestinal permeability markers D-lactic acid (D-Lactate, D-LA), diamine oxidase (Diamine oxidase, DAO), and endotoxin (Endotoxin, ET) in serum; increase the number of intestinal goblet cells and the expression of goblet cell marker protein MUC2, which indicates that the addition of CAR to feed alleviates the damage of weaning stress to the intestinal barrier of piglets. At the same time, dietary CAR significantly reduced serum inflammatory cytokine concentrations (TNF-α, IL-1β, and IL-6); reduced the transcriptional levels of TNF-α, IL-1β, and IL-6, and the expression of TLR4 pathway-related proteins (TLR4, Myd88, and p-p65) in the intestinal mucosa. Molecular docking revealed that CAR formed hydrogen bonds with TLR4, Myd88, and NF-κB p65, providing intuitive theoretical support for CAR's ability to block activation of the TLR4-Myd88-NF-κB signaling pathway. Furthermore, dietary CAR significantly increased the protein expression levels of Wnt1, β-catenin, C-Myc, and Cyclin D1. Activating the Wnt1 / β-catenin pathway also increased the proportion of Lgr5-positive cells, a marker for intestinal stem cells (ISCs), the number of goblet cells, the expression of the Paneth cell Lyz1 gene, and Ki67 fluorescence intensity, indicating that dietary CAR can effectively alleviate the abnormal intestinal epithelial cell differentiation caused by weaning stress in piglets.In addition, the addition of CAR to feed significantly improved the diversity and abundance of the intestinal flora of weaned piglets; increased the abundance of the beneficial marker bacteria Lactobacillus johnsonii, reduced the abundance of the conditionally pathogenic bacteria Escherichia coli, and improved the intestinal microenvironment; KEGG functional prediction analysis found that the signaling pathways of "environmental adaptation", "nucleotide metabolism", and "energy metabolism and tissue repair" were significantly enriched after the addition of CAR; analysis of intestinal fecal metabolites found that metabolites with anti-inflammatory, antioxidant, and sedative effects, such as 3-dehydroquinate, 11-methoxyyangonin, and alectrol, were significantly upregulated; functional enrichment analysis of differential metabolites found that α-linolenic acid metabolism, methyl butyrate metabolism, and carbohydrate digestion and absorption metabolic pathways were significantly enriched, indicating that the addition of CAR to feed alleviates the intestinal flora structure of piglets induced by weaning stress and increases the abundance of beneficial metabolites; at the same time, the CAR also has activity against PEDV and / or PDCoV. It can be seen that CAR effectively alleviates the effect of weaning stress on intestinal damage in piglets and maintains the integrity of the intestinal barrier; it downregulates the relative abundance of pathogenic bacteria in the intestine, providing a reasonable feeding strategy and theoretical support for adding CAR to alleviate intestinal damage in piglets caused by weaning stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The results of the effects of CAR on growth performance and diarrhea in piglets, where A: body weight; B: total feed intake (per head); C: feed-to-meat ratio; D: average daily gain; E: diarrhea index; F: diarrhea rate; G: small intestine length;

[0021] Figure 2 The results of the effect of CAR on the intestinal pH of piglets;

[0022] Figure 3 The results of the effect of CAR on the intestinal morphology of piglets; A: scanning electron microscopy (SEM) results (300×); B: HE results;

[0023] Figure 4 The results of the effects of CAR on the villi and crypts in the intestine of piglets, where A: villus height; B: crypt depth; C: villus-crypt ratio;

[0024] Figure 5 The results show the effect of CAR on intestinal permeability of piglets;

[0025] Figure 6The results of the effects of CAR on small intestinal barrier-related factors in weaned piglets, where A: mRNA level of intestinal barrier-related proteins (duodenum); B: mRNA level of intestinal barrier-related proteins (jejunum); C: mRNA level of intestinal barrier-related proteins (ileum);

[0026] Figure 7 The results of the effects of CAR on key intestinal barrier proteins in weaned piglets, where A: intestinal barrier-related protein levels; B: ZO-1 immunofluorescence results;

[0027] Figure 8 The results of the effect of CAR on the colon protein levels of weaned piglets, where A: HE and PAS images of the colon; B: Fecal MUC2 levels; C: Expression levels of colon barrier-related proteins;

[0028] Figure 9 The results of the effect of CAR on the colon barrier of weaned piglets, where A: colon ZO-1 immunofluorescence results; B: colon Claudin5 immunofluorescence results;

[0029] Figure 10 The results show the effect of CAR on inflammatory factors in the serum of weaned piglets;

[0030] Figure 11 The results show the effect of CAR on the mRNA level of TLR4 / Myd88 pathway in the small intestine of piglets;

[0031] Figure 12 The results show the effect of CAR on TLR4 / Myd88 pathway in piglet small intestine;

[0032] Figure 13 The results are for genes related to the Wnt / β-catenin pathway and ISC proliferation;

[0033] Figure 14 The expression results of proteins related to Wnt / β-catenin pathway and ISC proliferation are shown.

[0034] Figure 15 The fluorescence expression results of proteins related to the Wnt / β-catenin pathway and ISC proliferation, where A: Ki67 immunofluorescence; B: β-catenin immunofluorescence;

[0035] Figure 16 The results of the effect of CAR on the protein levels of goblet cells in the small intestine of piglets, where A: PAS result diagram; B: goblet cell number analysis; C: MUC2 protein level;

[0036] Figure 17 The results show the effect of CAR on the fluorescence intensity of goblet cells in the small intestine of piglets;

[0037] Figure 18 The results of the effect of CAR on intestinal Paneth cells in weaned stressed piglets, where A: Lyz1 mRNA expression level; B: Lyz immunofluorescence image; C: Lyz fluorescence intensity;

[0038] Figure 19 This is the Venn diagram of the piglet microbiome;

[0039] Figure 20 The results of the effect of CAR on the α and β diversity of intestinal flora in weaned piglets, where A: Shannon diversity index dilution curve; B: PCoA analysis;

[0040] Figure 21 The results of the effect of CAR on the Chao1 index and Shannon index of intestinal flora in weaned piglets, where A: Chao1 index; B: Shannon index;

[0041] Figure 22 This is the ternary phase diagram of the effect of CAR on the intestinal flora structure of weaned piglets;

[0042] Figure 23 The results of the effect of CAR on the abundance of intestinal flora in weaned piglets, where A: relative abundance of flora (phylum); B: relative abundance of flora (genus); C: relative abundance of flora (species);

[0043] Figure 24 The results show the effect of CAR on the intestinal flora LEfSe of weaned piglets;

[0044] Figure 25 The results show the effects of CAR on intestinal flora markers in weaned piglets;

[0045] Figure 26 KEGG function prediction analysis results;

[0046] Figure 27 The results of principal component analysis (PCA) are as follows;

[0047] Figure 28 is the result of orthogonal partial least squares discriminant analysis (OPLS-DA);

[0048] Figure 29 OPLS-DA test results of each group, where A: OPLS-DA model permutation test diagram (Con vs X1); B: OPLS-DA model permutation test diagram (Con vs X1); C: OPLS-DA model permutation test diagram (Con vs X1);

[0049] Figure 30 Volcano plot of differential metabolites;

[0050] Figure 31KEGG enrichment map of differential metabolites;

[0051] Figure 32 KEGG enrichment map of differential metabolites;

[0052] Figure 33 The results are for the effect of CAR on differential classification of intestinal metabolites;

[0053] Figure 34 The results of CAR on the differential classification of intestinal metabolites, where A: Classification diagram of differential metabolite pathways in each group (Con vs X3); B: Differential metabolites;

[0054] Figure 35 The results of the effect of CAR on the replication stage of PEDV, where A: CCK8 test; B: qRT-PCR results; C: Western blotting results;

[0055] Figure 36 The results of the effect of CAR on the replication stage of PDCoV, where A: CCK8 test; B: qRT-PCR results; C: protein immunoblotting results; D: grayscale analysis results. DETAILED DESCRIPTION

[0056] The present invention provides the use of carvacrol in preparing a feed additive and / or feed for repairing intestinal damage in piglets.

[0057] In the present invention, the intestinal damage of the piglets preferably includes that caused by weaning stress. The age of the piglets is preferably 26 to 32 days, and can be 28 to 30 days. The present invention has no special restrictions on the breed of the piglets, and the pig breeds with the numerical values ​​described in the art can be used. In an embodiment of the present invention, the piglets are a hybrid of Large White pigs and Landrace pigs. Weaning stress causes diarrhea in piglets, which is manifested as an increase in diarrhea rate and diarrhea index, and a decrease in body weight, daily weight gain and feed intake, which leads to reduced growth performance. In addition, weaning stress also leads to intestinal barrier dysfunction in piglets, intestinal inflammatory response, decreased intestinal proliferation and self-renewal ability, and abnormal intestinal microbial flora structure and its metabolites.

[0058] In the present invention, the repair of intestinal damage in piglets preferably includes at least one of the following: maintaining intestinal barrier function, promoting intestinal cell proliferation and renewal, alleviating small intestinal inflammatory response, and improving intestinal microbial flora structure and its abnormal metabolites. The maintenance of intestinal barrier function preferably includes at least one of the following: repairing intestinal morphological damage, reducing changes in intestinal permeability, reducing the expression level of key intestinal barrier proteins, and reversing the decrease in colonic mucus layer and goblet cell quantity. The repair of intestinal morphological damage is to increase intestinal villus density, increase villus height, and reduce crypt depth. The promotion of intestinal cell proliferation and renewal preferably includes upregulating the expression of intestinal stem cell proliferation-related factors, the expression of the Wnt pathway or an effective enhancer of the Wnt pathway, alleviating abnormal secretion of small intestinal mucosal goblet cells and mucin, and upregulating the expression level of the Lyz1 gene encoding lysozyme in small intestinal Paneth cells. The alleviation of small intestinal inflammatory response preferably includes inhibiting the level of inflammatory factors by inhibiting the TLR4 / Myd88 pathway. The inflammatory factors include TNF-α, IL-1β, and IL-6. The improvement of the intestinal microbial flora structure and its metabolite abnormalities preferably includes increasing the relative abundance of Eubacterium coprostanoligenes group and / or Lactobacillus and / or reducing the relative abundance of Escherichia coli. The dominant species of Lactobacillus preferably includes Lactobacillus johnsonii.

[0059] In the present invention, since the immunity of piglets decreases after weaning, they are easily infected with pathogenic bacteria or pathogenic viruses. The pig pathogenic viruses preferably include porcine epidemic diarrhea virus and / or porcine delta coronavirus. The porcine epidemic diarrhea virus and / or porcine delta coronavirus are common pathogenic viruses for piglets, which cause diarrhea and digestive system damage in piglets and then lead to intestinal damage. Based on this, the present invention also studies the activity of carvacrol against porcine pathogenic viruses. Experiments show that the carvacrol has activity against porcine epidemic diarrhea virus and / or porcine delta coronavirus. In the concentration range of 1.5 to 25 μM, carvacrol significantly reduces the protein level and nucleic acid level of PEDV, and exerts an antiviral effect. In the concentration range of 6.25 to 50 μM, carvacrol significantly reduces the protein level and nucleic acid level of PDCoV, and exerts an antiviral effect.

[0060] In the present invention, the use preferably also includes the use of carvacrol in the preparation of a feed additive and / or feed for alleviating piglet diarrhea, promoting small intestinal development, and promoting piglet growth performance. The alleviating piglet diarrhea preferably includes reducing the piglet diarrhea rate and / or diarrhea index. The promoting piglet growth performance preferably includes increasing piglet weight.

[0061] In the present invention, the feed additive preferably also includes auxiliary materials. The auxiliary materials preferably include protein nutrients, fillers and adhesives. The mass ratio of the carvacrol, protein nutrients, fillers and adhesives is preferably 3.158-15:18-22:7-9:30-35, and can be 6.67:20:8:32. The protein nutrients preferably include whey protein concentrate, soy protein isolate and / or casein. The filler preferably includes maltodextrin, corn starch and / or microcrystalline cellulose. The adhesive preferably includes pectin, sodium carboxymethyl cellulose and / or sodium alginate. The concentration of carvacrol in the feed is preferably not less than 50 mg / kg, and can be 100-200 mg / kg. When the feed additive is used to prepare feed, the added mass percentage of the feed additive is 0.08%-0.12%, and can be 0.1%.

[0062] The application of carvacrol provided by the present invention in the preparation of feed additives and / or feed for repairing intestinal damage in piglets is described in detail below with reference to the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0063] Example 1

[0064] Preparation method of CAR feed additive

[0065] Whey protein concentrate was dissolved in distilled water to form a 5% (w / v) protein solution. Pectin was dissolved in distilled water with stirring to obtain a 2% (w / v) pectin solution. Maltodextrin was also dissolved in distilled water to form an 8% (w / v) maltodextrin solution. These three solutions were mixed in a 1:1:1 volume ratio and then fully hydrated at room temperature to form a premixed carrier, namely the whey protein concentrate-pectin complex. The pH of the premixed carrier was then adjusted to 6 using HCl and NaOH. Under ultrasonication, 3.51g, 7.41g, and 16.67g of 90% pure CAR were added to 1200mL of the premixed carrier, mixed uniformly, and dried at low temperature. The dried mixture was then coated and granulated. After screening through a 40-60 mesh sieve, CAR products with concentrations of 5%, 10%, and 20% were obtained. 1 kg of these products was added to each ton of feed, and the final concentration of CAR was 50 mg / kg, 100 mg / kg and 200 mg / kg.

[0066] Example 2

[0067] Animal experiments

[0068] 1. Experimental Methods

[0069] 1.1 Experimental Animal Grouping and Treatment

[0070] This study was conducted at a pig farm in Heilongjiang Province. Two hundred castrated piglets (Large White x Landrace) weaned at 28 days of age and weighing similar weight (8.51 ± 0.51 kg) were randomly assigned to four groups, each containing five pens containing 10 piglets. Suitable temperature and humidity levels were maintained in the housing, and pigs were housed with free access to feed and water. The control group was fed a standard diet (see Table 1), the X1 group was fed a standard diet supplemented with 50 mg / kg CAR, the X2 group was fed a diet supplemented with 100 mg / kg CAR, and the X3 group was fed a diet supplemented with 200 mg / kg CAR. All diets met the NRC (2012) nutritional standards for pigs. Feed intake and weight changes were recorded weekly. Six pigs were randomly selected from each group and slaughtered on the 21st day of the study.

[0071] Table 1 Nutritional composition of feed

[0072] Diet composition and ratio 0-14d 14-28d 28-42d corn 53.80 57.10 66.00 whey powder 10.00 10.00 10.00 fermented beans 4.00 4.00 4.00 yeast 3.00 2.00 - soybean meal 24.70 27.90 30.00 Stone powder 0.92 1.19 1.16 fishmeal 4.00 - - Salt 0.65 0.62 0.59 soybean oil 1.50 0.64 0.16 calcium bicarbonate 0.79 0.65 0.62 Threonine 0.16 0.15 0.14 Diet composition and ratio 0-14d 14-28d 28-42d L-Tryptophan 0.07 0.06 0.05 Lysine 0.51 0.55 0.41 Vitamin premix 1 0.05 0.05 0.05 <![CDATA[Trace element premix 2 > 0.15 0.15 0.15 Crude protein, % 89.9 90.2 90.8 Crude fiber, % 21.61 20.21 19.70 Ash content, % 5.74 5.61 5.83 Dry matter, % 89.90 90.20 90.80

[0073] 1 Each kilogram of feed provides the following trace elements: 44 mg Mn as MnSO4, 23 mg Fe as FeSO4, 165 mg Zn as ZnSO4, and 17 mg Zn as CuSO4;

[0074] 2 Each kilogram of feed provides the following nutrients: 0.04 mg vitamin B12, 4.4 mg menadione, 55 mg niacin, 3.3 mg pyridoxine, 0.2 mg biotin, 61 mg pantothenic acid, 3.3 mg thiamine, 1.1 mg folic acid, 1101 IU vitamin A, 55 IU vitamin E, and 1652 IU vitamin D.

[0075] 2.2.3 Statistical methods for growth performance and diarrhea rate

[0076] To monitor changes in weight and appetite, weekly piglet feed intake and weight were recorded, and dynamic changes in weight and feed intake were calculated at different stages of the feeding process. Total body weight, total feed intake, average daily feed intake, average daily weight gain, and feed-to-weight ratio were calculated based on the recorded data.

[0077] To record diarrhea, fecal condition and tail cleanliness of piglets were observed at fixed times daily during the trial. The number of piglets with diarrhea was counted, and the diarrhea rate and diarrhea index were calculated based on the fecal condition score. Fecal condition scoring was as follows: 0 = normal stool (firm stool); 1 = moist stool (medium consistency); 2 = mild diarrhea (watery stool); 3 = severe diarrhea (watery stool).

[0078] Diarrhea rate (%) = number of diarrheal pigs / (number of experimental piglets × number of experimental days) × 100%.

[0079] Diarrhea index = total score of piglet feces during the trial period / total number of piglets.

[0080] 1.2 Sample collection method

[0081] After 21 days of feeding, 6 piglets were randomly selected from each group and anesthetized with propofol intravenously and then killed. Different colored strings were used to distinguish the intestinal contents of each intestinal segment. After careful rinsing with saline, tissue samples and intestinal mucosa were collected. Tissue samples were chopped and collected in centrifuge tubes, and the intestinal mucosa was gently scraped in one direction using a disposable glass slide tilted 45°, also collected in a tube, and stored at -80°C. Blood was collected through the jugular vein into a vacuum blood collection tube and centrifuged. The supernatant was also stored at -80°C for analysis. Samples for histological observation and scanning electron microscopy observation were stored in paraformaldehyde and glutaraldehyde, respectively.

[0082] 1.3 Intestinal pH detection method

[0083] Use a calibrated handheld pH meter to measure pH in different intestinal segments. Use surgical scissors to make a small incision in the designated intestinal segments (5-10 cm from the upper duodenum, 5-10 cm from the upper jejunum, 5-10 cm from the upper ileum, 5-10 cm from the ileocecal junction, and 5-10 cm from the upper colon). Once calibrated, the pH meter is quickly inserted and the pH of the intestinal contents measured. Three replicates are performed at a time and recorded to prevent pH changes over time.

[0084] 1.4 Histological and morphological analysis methods

[0085] Collected intestinal and lung tissues were fixed in 4% paraformaldehyde for at least 48 hours. Subsequently, the tissues were dehydrated using various concentrations of alcohol (75%, 90%, 95%, and 100%). Paraffin-embedded intestinal and lung sections were sectioned at 5 μm thickness and stained with hematoxylin and eosin. Intestinal goblet cells were stained using post-transcriptional scintigraphy (PAS), and villus height, crypt depth, and goblet cell number were analyzed using Image J.

[0086] 1.5 Scanning electron microscopy observation method

[0087] Cut the sample to be tested into 2×5cm size and place it in the fixative at 4℃ for 24h. Rinse it three times with 0.1molpH7.2PBS, 10min each time, and then use different concentrations (50%, 70%, 90%) of ethanol for gradient dehydration. Dehydrate it twice with 100% ethanol, and then replace it with ethanol and tert-butanol. Then freeze it at -20℃ for 30min, and then place the sample in ES-2030 (HITACH) freeze dryer for drying. After drying, take out the sample with the observation side facing up, stick the bottom to the conductive glue sample stage and use ion sputtering coating instrument to coat the metal film on the surface of the sample. Finally, put the sample into the test and observe it and save the image.

[0088] 1.6 RNA extraction and reverse transcription

[0089] The steps for extracting total RNA are as follows:

[0090] (1) Mix tissues (duodenum, jejunum, ileum, lung, trachea) with Trizol and grind thoroughly;

[0091] (2) After centrifugation, the RNA is in the upper aqueous phase. Transfer the upper liquid to a new centrifuge tube and add the same volume of isopropanol.

[0092] (3) Mix, transfer to an adsorption column, and centrifuge to discard the liquid at the bottom of the tube;

[0093] (4) Add ice-cold ethanol (4°C) and centrifuge again;

[0094] (5) Centrifuge again without adding any liquid, and place the adsorption column in a new tube;

[0095] (6) Add 30 μL DEPC water to elute RNA.

[0096] The obtained RNA was then reverse transcribed into cDNA using a reverse transcription kit for storage and detection.

[0097] 1.7 qPCR detection

[0098] mRNA levels in samples were measured using a real-time fluorescence quantitative detector using SYBR qPCR SuperMix Plus reagent (Jinan). The reaction system is shown in Table 2, and the primers are shown in Table 3.

[0099] Table 2 Real-time fluorescence quantitative PCR reaction system

[0100] Reagents volume 2×SYBR qPCR SuperMix Plus 10 μL Upstream primer (10 μM) 0.5μL Downstream primer (10 μM) 0.5μL cDNA 4 μL <![CDATA[RNase-free H2O]]> 5μL

[0101] Table 3 Primer sequences

[0102]

[0103]

[0104] Add the above system to an 8-tube tube strip, mix thoroughly, centrifuge, and place in a fluorescence quantitative analyzer. The reaction program is: 95°C (30 s); 95°C (30 s), 60°C (30 s), 72°C (30 s), 40 cycles; 95°C (5 s), 60°C (1 min), 95°C (continued); 50°C (30 s). After the reaction is complete, read the CT value and analyze the data.

[0105] 1.8 Immunofluorescence detection method

[0106] (1) Dewaxing of paraffin sections: Wash the sections in xylene I (5 min) - xylene II (15 min) - anhydrous ethanol I (5 min) - anhydrous ethanol II (5 min) - 85% alcohol (5 min) - 75% alcohol (5 min) - distilled water.

[0107] (2) Repair: After the sections are slightly dried, use an immunohistochemistry pen to draw a circle around the tissue (to prevent the liquid from flowing away). Add proteinase K working solution to the circle to cover the tissue and incubate in a warm oven for 25 minutes. Place the slide in PBS (pH 7.4) and shake and wash three times for a total of 15 minutes.

[0108] (3) Membrane permeabilization: After the slices are slightly dried, add membrane permeabilization working solution in the circle to cover the tissue, incubate at room temperature for 20 minutes, and place the slides in PBS (pH 7.4) and shake to wash.

[0109] (4) Staining: Add primary antibody and corresponding secondary antibody and incubate in the dark for a fixed time.

[0110] (5) DAPI counterstaining of cell nuclei: Wash sections with PBS (pH 7.4) three times for 5 min each time. After removing the PBS, add DAPI staining solution to the circle and incubate at room temperature for 10 min in the dark.

[0111] (6) Mounting: Place the slides in PBS (pH 7.4) and wash them three times on a decolorizing shaker, each time for 5 minutes. After the sections are slightly dried, mount them with anti-fluorescence quenching mounting medium.

[0112] (7) Microscopic examination and photography: The sections were observed and images were collected under a fluorescence microscope (DAPI ultraviolet excitation wavelength 330-380 nm, emitting blue light; FITC excitation wavelength 465-495 nm, emitting green light; CY3 excitation wavelength 510-560 nm, emitting red light).

[0113] 1.9 Western Blotting Method

[0114] To determine changes in intestinal protein expression levels after CAR addition, weighed tissue was first ground in a prepared protein lysis buffer, incubated on ice for 0.5 h, and the supernatant collected after centrifugation. A defined amount of 5× protein loading buffer was added to the supernatant and boiled for 15 min. Proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane by SDS-PAGE electrophoresis. The membrane was then soaked in 5% skim milk at room temperature or 4°C for a defined period of time. Primary and secondary antibodies were then added for reaction, followed by elution and exposure to light for quantitative analysis of expressed proteins.

[0115] 1.1016S rRNA Sequencing Analysis Method

[0116] Total genomic DNA was extracted from 24 collected stool samples. The quality and quantity of the extracted DNA were assessed by 1.8% agarose gel electrophoresis, and the DNA concentration and purity were determined using a NanoDrop 2000 UV-visible spectrophotometer. The full-length 16S rRNA gene was amplified using primer pairs F: AGRGTTTGATYNTGGCTCAG (SEQ ID NO: 37) and R: TASGGHTACCTTGTTASGACTT (SEQ ID NO: 38). Both the forward and reverse 16S primers were tailed with sample-specific PacBio barcode sequences to facilitate multiplex sequencing. SMRTbell libraries were prepared from the amplified DNA using the SMRTbell Express Template Prep Kit 2.0. The pooled and barcoded sample-purified SMRTbell libraries were sequenced on the PacBio Sequel II platform using the Sequel II Binding Kit 2.0. Eligible sequences with a similarity threshold greater than 97% were assigned to an operational taxonomic unit (OTU) using USEARCH (version 10.0). Based on the Bayesian classifier in QIIME2, OTUs / asv were classified and annotated using the SILVA database (release 138.1) with a confidence threshold of 70%. Alpha analysis of species diversity complexity of each sample was performed using QIIME2 software. Beta diversity was calculated using principal coordinate analysis (PCoA) to assess species complexity. Bacterial abundance and diversity were compared using one-way analysis of variance. Linear discriminant analysis (LDA) combined with effect size (LEfSe) was used to evaluate differentially abundant taxa. Sequencing data were analyzed using the online platform BMKCloud (https: / / www.biocloud.net).

[0117] 1.11 Metabolomics Analysis Methods

[0118] The process for processing stool samples to be tested is as follows:

[0119] (1) Weigh 50 mg of sample, add 1000 μL of extract containing internal standard (methanol:acetonitrile:water volume ratio = 2:2:1, internal standard concentration 20 mg / L), and vortex mix for 30 seconds; (2) Add steel beads, grind at 45 Hz for 10 minutes, and sonicate for 10 minutes (ice-water bath); (3) Let it stand at -20°C for 1 hour; (4) Centrifuge the sample at 4°C, 12000 rpm for 15 minutes; (5) Carefully remove 500 μL of supernatant and place it in a centrifuge tube; (6) Dry the extract in a vacuum concentrator; (7) Add 160 μL of extract (acetonitrile:water volume ratio: 1:1) to the dried metabolites for re-dissolution; (8) Vortex mix for 30 seconds, and sonicate for 10 minutes in an ice-water bath; (9) Centrifuge the sample at 4°C, 12000 rpm for 15 minutes; (10) Carefully remove 120 μL of supernatant and place it in a 2 mL injection vial, and take 10 μL of each sample to mix as QC samples for detection on the instrument.

[0120] Metabolomics analysis of processed samples was performed using a LC / MS system consisting of a Waters Acquity I-Class PLUS ultra-high-performance liquid chromatography coupled to a Waters Xevo G2-XS QTof high-resolution mass spectrometer. Raw data were acquired using MassLynx V4.2 and processed using Progenesis QI software, including peak extraction and alignment. Identification was performed using the Progenesis QI online METLIN database and a Biomark library. Theoretical fragment identification and mass deviation were both within 100 ppm. Raw peak areas were normalized to the total peak area before subsequent analysis. Principal component analysis and Spearman correlation analysis were used to assess reproducibility within the group and for quantitative controls. Identified compounds were classified and pathway-based searched using the KEGG, HMDB, and lipidmaps databases. Fold differences were calculated and compared based on group information, and P-values ​​for compound significance were calculated using a T-test. OPLS-DA modeling was performed using the R package ropls, and 200 permutation tests were performed to validate the model's reliability. The VIP value of the model was calculated through multiple cross-validation. Differential metabolites were screened using a combination of the fold difference, P value, and VIP value from the OPLS-DA model. The screening criteria were FC > 1, P value < 0.05, and VIP > 1. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of differentially expressed metabolites was calculated using the hypergeometric distribution test.

[0121] 1.12 Test data statistics and analysis

[0122] All experimental data were verified for normal distribution and homogeneity of variance using the Shapiro-Wilk test using SPSS software. Data were analyzed and plotted using STAMP and GraphPad Prism 9.0 (GraphPad Inc., San Diego, CA, USA). One-way ANOVA was used to determine differences between groups. *P < 0.05, **P < 0.01, and ***P < 0.001 indicate significant changes.

[0123] 2. Results and Analysis

[0124] 2.1 CAR alleviates the reduced growth performance of piglets caused by weaning stress

[0125] 2.1.1 Test results of piglet growth performance

[0126] After feeding different doses of CAR, the weight, feed intake, and diarrhea of ​​piglets were tracked and recorded for 42 days, and the obtained data were analyzed. Figure 1 As shown, with increasing CAR dosage, body weight and feed intake showed a clear dose-dependent effect. From 14 to 28 days, the feed-to-meat ratio decreased in the X1 and X3 groups, with a highly significant difference compared to the control group (P < 0.001). On days 7, 14, 21, and 28, body weight in the X3 group increased, with highly significant differences compared to the control group (P < 0.05, P < 0.001). On days 14 and 21, body weight in the X2 group increased significantly (P < 0.05). Only on day 28 did the X1 group show a significant upward trend. These results indicate that the critical period for weight gain within the first 42 days after weaning occurs primarily within 28 days, and there is no significant difference in weight gain among the groups after 28 days.

[0127] Furthermore, all three concentrations of CAR significantly improved piglet diarrhea. After weaning, piglets fed CAR showed a downward trend in diarrhea rate (P<0.05, P<0.01) and diarrhea index (P<0.05, P<0.001), both significantly correlated with the added CAR concentration. Small intestine length also showed significant changes during the experimental period, with a significant increase in length observed in the X3 group compared to the control group (P<0.05). This suggests that supplementing CAR with feed alleviates diarrhea caused by weaning stress in piglets, promotes small intestinal development, and improves piglet growth performance.

[0128] 2.1.2 Results of piglet intestinal pH test

[0129] In order to explore whether CAR would affect the pH of each intestinal tract, the pH of each intestinal segment of the piglets in each experimental group was tested. Figure 2As shown in the figure, the results showed that the pH in the small intestine (duodenum, jejunum, and ileum) of the piglets in the CAR-added experimental group showed a downward trend. Except for the pH in the cecum and colon of the X2 group, which was significantly different from that of the control group (P<0.01), the differences between the other groups were not significant (P>0.05), indicating that the addition of CAR to the feed had little effect on the intestinal pH.

[0130] 2.2 Results of CAR alleviating intestinal barrier function damage induced by weaning stress in piglets

[0131] 2.2.1 Results of piglet small intestine morphology test

[0132] In order to explore the effect of CAR on intestinal morphology, the changes in the ultrastructure of the small intestine were first observed by SEM. Figure 3 and Figure 4 As shown in the figure, the arrangement of microvilli on the intestinal surface was observed. The microvilli in each intestinal segment of the Con group were lower in density than those in the CAR-added test group, and the arrangement was disorderly and not neat and dense. HE staining of the small intestinal villi and crypts revealed that villus height in the duodenum of the X2 and X3 groups was significantly increased compared with the control group, while crypt depth in the X3 group was significantly decreased. The villus-crypt ratio (VH:CD) in the X2 and X3 groups also showed an upward trend (P<0.001). In the jejunum, villus height in the X1, X2, and X3 groups was significantly increased (P<0.001), while crypt depth in the X2 and X3 groups was significantly decreased (P<0.001). The VH:CD ratio in the X2 and X3 groups was significantly increased (P<0.05, P<0.001). In the ileum, villus height in the X2 and X3 groups was significantly increased, while crypt depth in the X2 and X3 groups was significantly decreased. VH:CD ratio in the X2 and X3 groups was significantly increased (P<0.01, P<0.001). This suggests that weaning stress causes small intestinal morphological damage, and that CAR supplementation can alleviate this damage and have a certain repair effect.

[0133] 2.2.2 Results of piglet intestinal permeability test

[0134] The intestinal damage caused by weaning stress in piglets can be analyzed by the concentration of intestinal permeability markers D-LA, DAO, and ET in the blood. Figure 5 After adding CAR to the feed, it was found that as the CAR dosage increased, the levels of D-LA, DAO, and ET in the serum of piglets in all experimental groups showed a downward trend. The concentrations of these three markers in the X3 group were significantly different from those in the control group (P<0.05, P<0.001). This indicates that weaning stress causes increased intestinal permeability, and that adding CAR can reduce the changes in intestinal permeability caused by weaning stress.

[0135] 2.2.3 Detection results of piglet small intestinal barrier-related proteins

[0136] The intestinal barrier is mainly composed of tight junction proteins (TJ) and adherens junction proteins (AJ). After adding CAR to the feed. Figure 6 and Figure 7 As shown, the mRNA expression levels of TJ proteins Occludin and Claudin-1 in the jejunum of piglets in group X3 were significantly increased compared with those in the control group (P<0.001); the mRNA levels of Claudin-1 and Claudin-5 in the ileum of piglets in group X3 were significantly increased compared with those in the control group (P<0.05); the mRNA level of AJ protein E-cadherin in the jejunum of piglets in groups X2 and X3 were significantly increased compared with those in the control group (P<0.001), and only the mRNA level of AJ protein E-cadherin in the jejunum of piglets in groups X3 showed significant changes compared with those in the control group (P<0.01).

[0137] Analysis of their protein levels showed that the levels of ZO-1, Claudin1, Occludin and E-cadherin proteins in the small intestine (duodenum, jejunum and ileum) in the X3 group were significantly increased compared with the control group (P<0.001); the levels of Claudin1, Occludin and E-cadherin proteins in the duodenum of the X2 group were significantly increased compared with the control group (P<0.05, P<0.01), and the levels of ZO-1, Claudin1, E-cadherin in the jejunum were significantly decreased compared with the control group (P<0.05, P<0.01). The protein level of -cadherin in group X1 was significantly increased compared with that in the control group (P<0.01, P<0.001), and the protein levels of Claudin1 and ZO-1 in the ileum were significantly increased compared with those in the control group (P<0.05, P<0.001). The protein level of Claudin1 in group X1 was significantly increased in all three segments of the intestine compared with that in the control group (P<0.05, P<0.001), and the protein level of E-cadherin was significantly increased only in the jejunum compared with that in the control group (P<0.01).

[0138] These results demonstrate that CAR significantly impacts the expression levels of TJ and AJ proteins, key intestinal barrier proteins, after weaning in piglets. Furthermore, a comparison of protein and mRNA levels across different intestinal segments revealed that protein expression levels varied significantly in the jejunum, while changes were less pronounced in the duodenum and ileum. Similarly, the fluorescence intensity of ZO-1 showed a significant upward trend following CAR supplementation, suggesting that CAR supplementation alleviates weaning stress, leading to decreased expression levels of key intestinal barrier proteins in piglets.

[0139] 2.2.4 Results of the test on the physical and chemical barriers of the piglet colon

[0140] After detecting the expression levels of small intestinal barrier-related proteins in weaned piglets, the changes in the physical and chemical barriers of the colon were also detected to clarify whether the effect of CAR on the intestinal barrier is related to the intestinal segment distribution. The results are as follows: Figure 8 and Figure 9 Goblet cells, as key cells that secrete mucin MUC2, play an important role in the composition of the intestinal mucus layer. The mucus layer, as the first chemical barrier to protect the intestine from pathogen invasion, is affected by the number of intestinal goblet cells and their ability to secrete mucin. After HE staining of the colon, it was found that the control group had obvious inflammatory cell infiltration in the crypts (marked by red arrows), while this phenomenon did not occur in the experimental group after adding CAR to the feed. PAS staining of the number of goblet cells in the colon showed that, except for the control group, the goblet cells in the colon were evenly distributed and the difference in number was not significant after adding CAR. In addition, ELISA detection of MUC2 concentration in feces found that the MUC2 concentration in the X3 group was significantly increased compared with the control group (P<0.05). Subsequent analysis of the physical barrier TJ and AJ protein levels revealed that TJ protein levels in the X1, X2, and X3 groups significantly increased after CAR addition compared to the control group (P<0.05, P<0.01, and P<0.001). E-cadherin protein levels were also significantly increased in the X2 and X3 groups compared to the control group (P<0.01). This suggests that weaning stress can lead to colonic barrier damage in piglets, and that CAR supplementation can alleviate this damage, potentially repairing the physical and chemical barriers.

[0141] 2.3 CAR alleviates the disorder of TLRs pathway regulation in the small intestine of piglets caused by weaning stress

[0142] 2.3.1 Results of inflammatory factor levels in piglet serum

[0143] Multiple stress factors during weaning may cause imbalance in the levels of inflammatory factors in piglets. Figure 10 As shown in the data, in the serum of the experimental group of piglets supplemented with CAR, the concentrations of TNF-α, IL-1β, and IL-6 showed a significant downward trend compared with the control group. In particular, the levels of the three inflammatory factors in the X3 group were significantly different from those in the control group (P<0.001). In addition, the IL-1β in the serum of the X2 group also showed a significant downward trend compared with the control group (P<0.01).

[0144] 2.3.2 Detection results of TLR4 pathway expression levels in piglet small intestine

[0145] Since the inflammatory factors in the serum of piglets changed significantly after adding CAR, the TLR4 / Myd88 related proteins of the classic inflammatory pathway in small intestine tissue were detected, such as Figure 11 and Figure 12As shown, the inflammatory factors and TLR4, Myd88, and NF-κB p65 in the small intestine (duodenum, jejunum, and ileum) were first detected and their mRNA levels showed a downward trend after the addition of CAR. After analyzing their protein levels, it was found that the CAR-added experimental group significantly reduced the expression levels of TLR4 (P<0.05, P<0.001), Myd88 (P<0.05, P<0.001), and p-p65 (P<0.001) proteins compared with the control group, and the expression levels were negatively correlated with the amount of CAR added. These results demonstrate that CAR alleviates the small intestinal inflammatory response induced by weaning stress by inhibiting the TLR4 / Myd88 pathway.

[0146] CAR alleviates weaning stress-induced abnormal proliferation and differentiation of intestinal stem cells (ISCs) in piglets

[0147] 2.4.1 Results of mRNA expression of Wnt / β-catenin pathway and ISC proliferation-related factors in piglet small intestine

[0148] As a key driver of most tissue stem cells, the Wnt pathway plays a huge role in the growth and development of the body. In order to explore the specific mechanism of CAR repair of intestinal damage, the mRNA levels of genes related to the small intestine Wnt / β-catenin pathway and ISC proliferation-related genes were analyzed. It was found that the mRNA expression levels of genes related to the Wnt / β-catenin pathway (Wnt1, c-Myc, Lgr5, Lrp5), ISC activity marker genes (Olfm4, Ascl2) and ISC static marker genes (Bmi1, Msi1) changed significantly with the increase of CAR concentration. In addition, it was found that the mRNA levels of R-spondin1 (Rspo1), R-spondin2 (Rspo2), and R-spondin3 (Rspo3), which are effective enhancers of the Wnt pathway, also changed significantly in the CAR groups with different concentrations (see Figure 13 These results indicate that the effect of CAR on ISCs depends on the activation of the Wnt pathway and the sustained enhancement effect of R-spondin on the Wnt pathway to maintain intestinal proliferation and self-renewal.

[0149] 2.4.2 Detection results of proteins related to the Wnt / β-catenin pathway and ISC proliferation in the piglet small intestine

[0150] After analyzing the mRNA levels of Wnt / β-catenin pathway and ISC proliferation genes, the protein levels were detected by Western blot. Figure 14 and Figure 15As shown, in addition to Wnt1 (P<0.05, P<0.001) and β-catenin (P<0.05, P<0.01, P<0.001), the downstream target genes of the Wnt pathway, C-Myc (P<0.01, P<0.001) and CyclinD1 (P<0.01, P<0.001), which are related to the cell cycle, increased to varying degrees in the CAR-supplemented experimental group compared with the control group. Furthermore, the expression of the intestinal stem cell marker Lgr5 (P<0.05, P<0.01, P<0.001) and the intestinal proliferation marker Ki67 (P<0.001) gradually increased with the activation of the Wnt pathway. These results suggest that CAR activates the Wnt pathway to promote ISC proliferation and repair intestinal damage caused by weaning stress.

[0151] 2.4.3 Detection results of goblet cell-related factors in the epithelial cells of the small intestinal mucosa of piglets

[0152] Goblet cells, as key factors in secreting mucin, play an important role in maintaining the intestinal barrier. Figure 16 . According to the PAS results, it can be seen that the number of goblet cells in the duodenum and ileum of the X2 and X3 groups increased significantly compared with the control group (P<0.05, P<0.001); the number of goblet cells in the ileum of the X3 group increased significantly compared with the control group (P<0.01). Analysis of the protein level of MUC2, the main marker of goblet cells, found that the addition of CAR was beneficial to the expression of MUC2 protein. In addition, the feeding of CAR significantly increased the mRNA levels of Muc1, Muc2, Muc4, Muc13 and Muc20 in the small intestine, and the immunofluorescence results also confirmed this phenomenon (P<0.05, P<0.01) (see Figure 17 These results indicate that CAR can alleviate the abnormal secretion of small intestinal goblet cells and mucin caused by weaning stress.

[0153] 2.4.4 Detection results of Paneth cell-related factors in piglet small intestine

[0154] In addition to goblet cells, Paneth cells, as important elements in maintaining the intestinal immune system, are also affected by CAR. Figure 18 As shown in the results, in the experimental group supplemented with CAR, the mRNA level of the Lyz1 gene in Paneth cells, which encodes lysozyme, was significantly increased compared with that in the control group (P<0.05, P<0.01, P<0.001), and its fluorescence intensity in the small intestine was also found to have an upward trend compared with that in the control group (P<0.01, P<0.001), indicating that the addition of CAR improved the levels of Paneth cells and their related transcription factors in the small intestine.

[0155] 2.5 CAR alleviates the abnormalities in intestinal microbial flora structure and function in weaned piglets induced by weaning stress

[0156] 2.5.1 OTU analysis of piglet microbiota

[0157] Draw a Venn diagram based on the OTUs of the four groups of samples Con, X1, X2, and X3, as shown in the following example: Figure 19 As shown, 622, 535, 721, and 728 OTUs were obtained from the colon contents of the four groups, respectively, of which 404 were identical. In addition, there were 22 unique OTUs in the Con group, 8 unique OTUs in the X1 group, 37 unique OTUs in the X2 group, and 32 unique OTUs in the X3 group. This indicates that the addition of CAR alleviates the decrease in the number of OTUs in the intestine of piglets caused by weaning stress.

[0158] 2.5.2 Results of piglet intestinal flora diversity test

[0159] To investigate the effects of different doses of CAR on the diversity of intestinal flora in weaned piglets, the results of 16S rRNA analysis were as follows: Figure 20 and Figure 21 As shown in the figure, the Shannon diversity index dilution curve gradually tends to be flat, indicating that the amount of sequencing data is sufficient to fully reflect the vast majority of bacterial community information in the sample, and the characteristic species almost cover all bacterial groups in the sample as the sequencing amount increases. The β diversity (PCoA) results show that there are significant differences in the bacterial community composition between the CAR-added group and the control group fed with a normal diet. In the X2 and X3 groups, the α diversity (Chao1 and Shannon index) (P<0.05, P<0.01) increased significantly compared with the control group, indicating that the addition of CAR to the feed alleviates the abnormal intestinal flora structure caused by weaning stress.

[0160] 2.5.3 Results of piglet intestinal flora abundance detection

[0161] See the results Figure 22 and Figure 23Analysis of bacterial abundance at the phylum, genus, and species levels revealed that Firmicutes, Bacteroidota, Proteobacteria, Actinobacteriota, and Spirochaetes dominated at the phylum level. Comparative analysis of species composition across groups using ternary phase diagrams and bar charts provides an intuitive view of the proportions and relationships of different species within each group. At the phylum level, Firmicutes and Bacteroids were significantly more abundant in the X2 and X3 groups, showing a significant increase compared to the control group. Proteobacteria and Actinobacteriota were more abundant in the Con group, but did not shift toward the CAR-supplemented group. At the genus level, the abundance of the Eubacterium coprostanoligenes group increased in the CAR-supplemented group, while Lactobacillus increased significantly and correlated with the CAR dosage. The relative abundances of the Lachnospiraceae NK4A136 group and uncultured rumen bacteria decreased significantly. At the species level, the relative abundance of Lactobacillus johnsonii increased significantly, while the relative abundance of Escherichia coli decreased significantly. These results suggest that CAR ameliorates the abnormal gut microbiota structure in piglets induced by weaning stress.

[0162] 2.5.4 Detection results of key strains in piglet intestine

[0163] See the results Figure 24LEfSe discriminant analysis (LDA) of colonic microbial markers in weaned piglets was performed (>3.5). In the Con group, Shigella (Shigella) was more abundant at the genus level, and Escherichia (coli) was more abundant at the species level. In the CAR supplementation group, Lactobacillus and Eubacterium (coprostanoligenes) were more abundant at the genus level, and Lactobacillus johnsonii was more abundant at the species level. The abundance of Lactobacillus in the X1, X2, and X3 groups was significantly increased compared with the control group (P < 0.05, P < 0.001), while the abundance of Escherichia (coli) was significantly decreased in the X2 and X3 groups compared with the control group (P < 0.001). This indicates that the addition of CAR made Lactobacillus johnsonii the dominant bacterial species, while suppressing the abundance of the opportunistic pathogen Escherichia coli, creating a relatively healthy microbial environment for the intestines of piglets suffering from weaning stress.

[0164] 2.5.5 KEGG prediction results of microbial community functions

[0165] PICRUSt2 was used to align the reference sequences of the microbial genome database (Aliign) to construct an evolutionary tree, and the gene information of unknown species was predicted based on the gene types and abundance information of known species, thereby combining the KEGG pathway information of the genes to predict the pathway status of the entire community. Through the composition and difference analysis of the KEGG metabolic pathway, the differences and changes in the functional genes of the microbial communities in different groups were observed in the metabolic pathways, such as Figure 25 As shown, analysis of the combined data from groups X1, X2, and X3 revealed that significantly enriched signaling pathways after CAR addition included "environmental adaptation," "nucleotide metabolism," and "energy metabolism and tissue repair," while "drug resistance" was significantly enriched in the Con group. These results suggest that CAR improves gut microbiome function by regulating it, enabling it to repair intestinal damage and enhance environmental adaptability. Furthermore, it improves drug resistance in the Con group, a characteristic that is inconsistent with the current "antibiotic alternative" landscape.

[0166] 2.6 Results of CAR alleviating the changes in metabolites of colonic microbiota in piglets induced by weaning stress

[0167] 2.6.1 Principal Component Analysis

[0168] By performing principal component analysis on samples of different groups, we can understand the overall difference between the two groups of samples and the degree of variation between samples within the group. The results of principal component analysis are as follows: Figure 27 As shown, the first principal component (PC1) between the Con and X1 groups explained 27.62% of the variation, the second principal component (PC2) explained 20.40% of the variation, and PC1 and PC2 together explained 48.02% of the variation. Between the Con and X2 groups, PC1 explained 31.09% of the variation, PC2 explained 22.27% of the variation, and PC1 and PC2 together explained 53.36% of the variation. Between the Con and X3 groups, PC1 explained 29.76% of the variation, PC2 explained 21.78% of the variation, and PC1 and PC2 together explained 51.54% of the variation. Overall, these results indicate that the metabolites in the X1, X2, and X3 groups differed significantly from those in the control group after CAR addition.

[0169] 2.6.2OPLS-DA analysis

[0170] OPLS-DA filters irrelevant noise information under the background of known sample grouping, performs orthogonal transformation correction, obtains inter-group difference information, and performs discriminant analysis on the regression results. Figure 28 and Figure 29 As shown, the horizontal axis represents the difference between groups, and the vertical axis represents the difference within the group. In addition, the closer the prediction parameters R2Y and Q2Y of the evaluation model are to 1, the more stable and reliable the model is, that is, this model can be used to screen differential metabolites. It can be seen in the figure that Con has significant differences with the X1, X2, and X3 groups respectively. In order to check the reliability of the OPLS-DA model, a permutation test was performed. OPLS-DA modeling was performed according to the permuted grouping and its R2Y and Q2Y were calculated. The results of multiple modeling were plotted as a scatter plot. The X-axis in the figure represents the correlation between the permuted grouping and the original model grouping, the Y-axis represents the value of R2Y or Q2Y, the blue dots and red dots represent the R2Y and Q2Y of the permuted model, respectively, and the two dotted lines are the regression lines of R2Y and Q2Y fitting. If the slope of the Q2Y fitting regression line is positive, it means that the model is meaningful. The blue dots are generally above the red dots, which means that the independence of the modeling training set and the test set is good, indicating that the differential metabolites screened by the model have high credibility.

[0171] 2.6.3 Screening of differential metabolites

[0172] In order to quickly view the overall trend of the difference in metabolite content between the two groups, VIP>1 and P<0.05 were used as the thresholds for screening differentially expressed metabolites, e.g. Figure 30As shown, there were 1358 differential metabolites in the X1 group compared with the Con group, including 651 up-regulated metabolites and 707 down-regulated metabolites. Among them, N-Methylcalystegine C1, an alkaloid with anti-inflammatory and antioxidant effects, was the most up-regulated, and 3,6,8-Trimethylallantoin was significantly down-regulated; there were 1960 differential metabolites in the X2 group compared with the Con group, including 901 up-regulated metabolites and 1059 down-regulated metabolites. Among them, 2-hydroxynicotine, a nicotinic compound, 3-dehydroquinate, an alkaloid with antibacterial effects, and 11-methoxyyangonin, an alkaloid with sedative and relaxing effects, were significantly up-regulated, and CDP-DG (6keto-PGF1alpha / 22:3(10Z,13Z,16Z)) and others were significantly downregulated; the X3 group had a total of 1,278 differential metabolites compared to the Con group, of which 527 were upregulated and 751 were downregulated. It was also found that the alkaloid 11-methoxyyangonin was significantly upregulated, as were protionamide, which has antibacterial effects, and alectrol, which has anti-inflammatory and antioxidant effects. However, the ursolic acid-derived compound 11-oxoursolic acid acetate was significantly downregulated. These results indicate that the abundance of metabolites with antibacterial, anti-inflammatory, and antioxidant effects increased significantly after the addition of CAR, and that metabolites with sedative effects can better relieve the anxiety and tension of piglets when facing weaning stress.

[0173] 2.6.4 Functional enrichment analysis of differential metabolites

[0174] In order to further study the specific effects of differential metabolites on metabolic pathways, we used the KEGG annotation analysis method to identify the metabolic pathways in which they participated. Figure 31 and Figure 32Among the top 20 metabolic pathways enriched with differential metabolites between the Con and X1 groups, ubiquinone and other terpenoid-quinone biosynthesis was significantly enriched (P<0.05); among the top 20 metabolic pathways enriched with differential metabolites between the Con and X2 groups, alpha-linolenic acid metabolism was significantly enriched (P<0.05); and among the top 20 metabolic pathways enriched with differential metabolites between the Con and X3 groups, butanoate metabolism and carbohydrate digestion and absorption were significantly enriched (P<0.05). These results indicate that CAR supplementation has an anti-inflammatory, digestive, and SCFA metabolism-affecting physiological effect on piglets.

[0175] 2.6.5 Functional classification analysis of differential metabolites

[0176] Since differential metabolites interact with each other in the body and form different pathways, the KEGG database was used to annotate the differential metabolites. The top 20 entries with the most differential metabolites annotated in the pathway were selected. The column length represents the number of differential metabolites annotated in the pathway. Figure 33 Classification of the top 20 metabolic pathways annotated with differential metabolites between the Con and different doses of X1, X2, and X3 groups revealed that a large number of amino acid metabolisms changed in each group, such as arginine and proline metabolism, histidine metabolism, tryptophan metabolism, and tyrosine metabolism. In the digestive system category, changes in bile secretion and protein digestion and absorption occurred in the X1 and X2 groups. In the lipid metabolism category, multiple differential metabolites appeared in the X1 and X2 groups, including arachidonic acid metabolism, unsaturated fatty acid biosynthesis, and α-linolenic acid metabolism.

[0177] In the carbohydrate metabolism category, multiple differential metabolites were found in amino sugar and nucleotide sugar metabolism and butanoate metabolism between the X3 group and the Con group. Figure 34. Among them, the metabolic process of methyl butyrate is metabolized into metabolites such as acetone and butyric acid in the body. In the metabolic process of intestinal microorganisms, methyl butyrate can also be fermented by microorganisms as a substrate to produce SCFAs, such as acetic acid, propionic acid and butyric acid. The production of SCFAs can affect the metabolism of α-linolenic acid. SCFAs can provide it with energy and substrates, and promote the metabolism of α-linolenic acid into longer-chain unsaturated fatty acids. When screening SCFAs, it was found that acetic acid (P<0.01) and butyric acid (P<0.05) showed significant changes, while propionic acid (P>0.05) did not change significantly.

[0178] Example 2

[0179] Because weaned piglets have reduced immunity and are highly susceptible to viral infection, porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) are common pathogenic viruses in the pig farming industry, easily causing diarrhea and digestive system diseases in piglets, leading to intestinal damage. Therefore, this example also conducted an experiment to test the resistance of carvacrol to porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV).

[0180] 1. Effect of carvacrol on porcine epidemic diarrhea virus (PEDV)

[0181] To determine the optimal dosage of carvacrol in porcine intestinal epithelial cells (IPEC-J2), the cytotoxicity of carvacrol was detected by CCK-8 assay. 3 cells), and carvacrol at different concentrations of 400 μM, 300 μM, 200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, and 3.125 μM was added for treatment for 24 hours, and then CCK-8 reagent was added for incubation for 2 hours. The absorbance at 450 nm was measured using a microplate reader, and the cell viability was calculated. When the cell viability was above 90% after the addition of carvacrol, the carvacrol concentration was non-cytotoxic, and the maximum non-toxic dose of carvacrol was determined based on this.

[0182] To determine whether carvacrol inhibits PEDV proliferation, a cytotoxicity assay was performed to determine the maximum nontoxic dose of carvacrol. IPEC-J2 cells were cultured in 6-well plates and inoculated with PEDV (MOI = 0.1). After adsorption at 37°C for 1 hour, the maintenance medium was replaced with carvacrol at concentrations of 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, and 1.5 μM. Samples were collected at 24 hours, based on the viral replication cycle. RT-qPCR and Western blot were used to examine changes in PEDV nucleic acid and protein expression in IPEC-J2 cells following carvacrol treatment.

[0183] See the results Figure 35 The results showed that within the concentration range of 1.5 to 25 μM, carvacrol significantly reduced the levels of PEDV protein and nucleic acid, exerting an antiviral effect.

[0184] 2. Effect of carvacrol on porcine delta coronavirus (PDCoV)

[0185] To determine the optimal dosage of carvacrol in porcine testicular cells (ST), the CCK-8 assay was used to detect the cytotoxicity of carvacrol. 3 Cells were treated with different concentrations of carvacrol for 24 hours, and then CCK-8 reagent was added to incubate for 2 hours. The absorbance at 450 nm was measured using a microplate reader to calculate the cell survival rate. When the cell survival rate was above 90% after the addition of carvacrol, the concentration of carvacrol was non-cytotoxic. Based on this, the maximum non-toxic dose of carvacrol was determined.

[0186] To determine whether carvacrol inhibits PDCoV proliferation, a cytotoxicity assay was performed to determine the maximum nontoxic dose of carvacrol. ST cells were cultured in 6-well plates and inoculated with PDCoV (MOI = 0.1). After adsorption at 37°C for 1 hour, the maintenance medium was replaced with carvacrol at concentrations of 50 μM, 25 μM, 12.5 μM, 6.25 μM, and 3.125 μM. Cell samples were collected at 24 hours, based on the viral replication cycle. qRT-PCR and Western blot were used to examine changes in PEDV nucleic acid and protein expression in IPEC-J2 cells following carvacrol treatment. Changes in protein and nucleic acid levels were analyzed.

[0187] See the results Figure 36 The results showed that within the concentration range of 6.25 to 50 μM, carvacrol significantly reduced the protein and nucleic acid levels of PDCoV, exerting an antiviral effect.

[0188] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Application of carvacrol in the preparation of feed additives and / or feed for repairing intestinal damage in piglets.

2. The application according to claim 1, characterized in that The intestinal damage of the piglets includes that caused by weaning stress and / or porcine pathogenic viruses; The porcine pathogenic viruses include porcine epidemic diarrhea virus and / or porcine delta coronavirus.

3. The use according to claim 1 or 2, characterized in that: The repair of piglet intestinal damage includes at least one of the following: maintaining intestinal barrier function, promoting intestinal cell proliferation and renewal, alleviating small intestinal inflammatory response, and improving intestinal microbial flora structure and abnormal metabolites thereof.

4. The application according to claim 3, characterized in that The maintenance of intestinal barrier function includes at least one of the following: repairing intestinal morphological damage, reducing intestinal permeability changes, reducing the expression level of key intestinal barrier proteins, and reversing the decrease in the amount of colon mucus layer and goblet cells.

5. The application according to claim 3, characterized in that: The promotion of intestinal cell proliferation and renewal includes upregulating the expression of intestinal stem cell proliferation-related factors, the expression of the Wnt pathway or an effective enhancer of the Wnt pathway, alleviating the abnormal secretion of small intestinal mucosal goblet cells and mucin, and upregulating the expression level of the Lyz1 gene encoding lysozyme in small intestinal Paneth cells.

6. The application according to claim 3, characterized in that: The relief of small intestinal inflammatory response includes suppressing the level of inflammatory factors by inhibiting the TLR4 / Myd88 pathway.

7. The use according to claim 3, characterized in that: The improvement of the intestinal microbial flora structure and the abnormal metabolites thereof includes increasing the relative abundance of the intestinal Eubacterium coprostanoligenes group and / or the Lactobacillus genus and / or reducing the relative abundance of Escherichia coli.

8. The application according to claim 1, characterized in that: The application also includes the application of carvacrol in preparing feed additives and / or feeds for alleviating piglet diarrhea, promoting small intestine development and promoting piglet growth performance.

9. The use according to any one of claims 1 to 8, characterized in that: The feed additive also includes auxiliary materials; The auxiliary materials include protein nutrients, fillers, and adhesives; The mass ratio of carvacrol, protein nutrient, filler and binder is 3.158-15:18-22:7-9:30-35; The concentration of carvacrol in the feed is not less than 50 mg / kg.

10. The use according to any one of claims 1 to 8, characterized in that: The piglets are 26 to 32 days old.

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