Application of lactobacillus reuteri in preparation of product for improving intestinal health and salpingitis of laying hens

Abstract

The invention belongs to the technical field of microorganisms and medicines, and particularly relates to application of lactobacillus reuteri in preparation of products for improving intestinal health and salpingitis of laying hens. The lactobacillus reuteri is a lactobacillus reuteri BNCC192190 strain, and the lactobacillus reuteri is a lactobacillus reuteri BNCC192190 strain. Research results show that by supplementing the lactobacillus reuteri, the colonization of harmful bacteria in intestinal tracts and reproductive tracts can be reduced by improving the reproductive tract injury and inflammatory response of the laying hens and regulating the flora composition of the intestinal tracts and the reproductive tracts, the inflammatory injury of fallopian tubes of the laying hens is relieved, and the production performance of the laying hens and the egg quality are further improved. The invention provides application of lactobacillus reuteri in preparation of a product for improving intestinal health and salpingitis of laying hens, and solves the problem that the lactobacillus reuteri in the prior art is indefinite in research on the genital tract.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and medicine, and particularly relates to application of Lactobacillus reuteri in preparing products for improving intestinal health and oviduct inflammation in laying hens. Background Art

[0002] Under large-scale farming, laying hens are in a sub-healthy state for a long time, which is easy to induce laying hen salpingitis. The main clinical features of laying hen salpingitis are decreased egg production, no or short egg production peak period, and decreased egg quality. When the inflammation spreads to the abdominal cavity of laying hens, it will cause large-scale sudden death. The factors that cause this disease are very complex, often causing immeasurable economic losses to the poultry industry.

[0003] In the laying hen industry, oviduct inflammation is one of the important diseases that affects the production performance of laying hens and the quality of eggs. At present, the treatment and management of oviduct inflammation mainly adopt antibiotic therapy, environmental improvement and nutritional regulation. Although antibiotic therapy can quickly control inflammation, long-term use can easily lead to drug resistance and drug residues, affecting food safety and human health. Although environmental improvement measures such as increasing ventilation and reducing stress can help prevent it, the effect is limited and it is difficult to cure the inflammation that has occurred. In terms of nutritional regulation, although some nutrients have been shown to enhance the body's immunity, the specific treatment effect for oviduct inflammation is not yet obvious.

[0004] Lactobacillus reuteri belongs to the Firmicutes, Bacillus, Lactobacillaceae, and Lactobacillus. It is a catalase-negative, Gram-positive, non-motile, non-spore-forming, and obligately heterofermentative bacterium. Among them, the Latin name of Lactobacillus reuteri is Lactobacillus reuteri; the Latin name of Firmicutes is Firmicutes; the Latin name of Bacillus is Bacilli; the Latin name of Lactobacillales is Lactobacillales; the Latin name of Lactobacillaceae is Lactobacillaceae; and the Latin name of Lactobacillus is Lactobacillus. When administered as a probiotic, Lactobacillus reuteri helps to restore the balance of intestinal flora and maintain the health of the host body through various mechanisms. For example, it produces metabolites such as organic acids, bacteriocins, hydrogen peroxide, and other antagonistic substances to exert antibacterial and immunomodulatory effects. At present, most of the research on Lactobacillus reuteri is focused on the intestine, while research on Lactobacillus reuteri on the reproductive tract remains to be clarified. Therefore, research on Lactobacillus reuteri on the reproductive tract is needed. Summary of the invention

[0005] In order to carry out a study on the effects of Lactobacillus reuteri on reproductive tract, the present invention provides an application of Lactobacillus reuteri in preparing a product for improving intestinal health and oviduct inflammation in laying hens.

[0006] To achieve the above purpose, the present invention adopts the following technical solution.

[0007] The present invention provides an application of Lactobacillus reuteri in preparing a product for improving intestinal health and oviduct inflammation in laying hens, so as to solve the problem that the prior art Lactobacillus reuteri has unclear research on reproductive tract. The Lactobacillus reuteri is a strain of Lactobacillus reuteri BNCC192190.

[0008] At the same time, the present invention provides a new application of Lactobacillus reuteri and a method for improving intestinal health and oviduct inflammation in laying hens. The method provided by the present invention can alleviate oviduct inflammation in laying hens, improve the production performance and egg quality of laying hens, and reduce the use of antibiotics to ensure food safety.

[0009] Preferably, the product is an animal feed additive prepared using the bacterial liquid of Lactobacillus reuteri as an active ingredient.

[0010] Preferably, the animal feed additive is a bacterial liquid of Lactobacillus reuteri obtained by inoculating the Lactobacillus reuteri into a liquid MRS culture medium and culturing at 36° C. to 38° C.

[0011] Preferably, the number of colonies in the bacterial solution of Lactobacillus reuteri is ≥10 9 CFU / mL.

[0012] Preferably, the animal feed additive is used to improve the production performance and egg quality of laying hens.

[0013] Preferably, the animal feed additive is used to alleviate inflammatory damage to the oviduct of laying hens, thereby improving the production performance of laying hens and the quality of eggs.

[0014] Preferably, the animal feed additive is used to improve reproductive tract damage and inflammatory response in laying hens, thereby improving production performance and egg quality of laying hens.

[0015] Preferably, the method for preparing the bacterial solution of Lactobacillus reuteri comprises the following steps:

[0016] The Lactobacillus reuteri BNCC192190 strain was inoculated into a liquid MRS culture medium, and cultured at 36°C to 38°C for 23 to 25 hours, and then a single colony was picked for purification and culture until the number of colonies in the bacterial solution reached 10 9 CFU / mL or above, the bacterial solution is centrifuged and the supernatant is collected to obtain the bacterial solution of Lactobacillus reuteri.

[0017] Preferably, the activated Lactobacillus reuteri BNCC192190 strain is inoculated into a liquid MRS culture medium, and the activation method is: after thawing the Lactobacillus reuteri BNCC192190 strain, inoculate it into a liquid MRS culture medium at a volume ratio of 1:25 for activation culture.

[0018] Preferably, the activation culture condition is culturing at 36° C. to 38° C. for 23 hours to 25 hours.

[0019] Preferably, the purification culture conditions are: shaking culture at 36°C to 38°C.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The present invention provides an application of Lactobacillus reuteri in the preparation of a product for improving the intestinal health and oviduct inflammation of laying hens, solving the problem of unclear research on the reproductive tract of Lactobacillus reuteri in the prior art.

[0022] 2. The present invention provides a new application of Lactobacillus reuteri, specifically exploring the effects of Lactobacillus reuteri on the oviduct barrier and immune regulation. The mechanism and effect of Lactobacillus reuteri on the health of the oviduct of laying hens are revealed, and the present invention aims to provide a scientific basis and important reference for the prevention and treatment of oviduct-related diseases.

[0023] 3. The research results of the present invention show that supplementation with Lactobacillus reuteri can improve reproductive tract damage and inflammatory response in laying hens, alleviate inflammatory damage to the oviduct of laying hens, and further improve the production performance and egg quality of laying hens.

[0024] 4. The application method provided by the present invention has the following advantages: safe, no side effects, suitable for long-term application in laying hen farming. Strong pertinence, significant relief effect on oviduct inflammation. Environmentally friendly and economical, reducing farming costs and improving economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a diagram showing the effect of Lactobacillus reuteri of the present invention on improving oviduct bleeding and decreased egg production rate caused by phenol colloid; wherein, Figure 1 A represents the experimental process and experimental grouping. The experiment was divided into four groups: control group, fallopian tube inflammation group, Lactobacillus reuteri treatment group, and heat-killed Lactobacillus reuteri treatment group, with 8 replicates in each group; Figure 1 B represents the eggs laid by laying hens in each experimental group during the modeling period; Figure 1 C represents the egg production rate of laying hens in each experimental group during the modeling period; Figure 1 D represents the egg weight of eggs laid by laying hens in each experimental group during the modeling period; Figure 1 E represents the feed intake of laying hens in each experimental group during the modeling period; Figure 1F represents the eggs produced by laying hens in each experimental group in the first week of gavage; Figure 1 G represents the egg production rate of laying hens in each experimental group in the first week of gavage; Figure 1 H represents the egg weight of laying hens in each experimental group in the first week of gavage; Figure 1 I represents the feed intake of laying hens in each experimental group in the first week of gavage; Figure 1 J represents the eggs produced by laying hens in each experimental group in the second week of gavage; Figure 1 K represents the egg production rate of laying hens in each experimental group in the second week of gavage; Figure 1 L represents the egg weight of eggs laid by laying hens in each experimental group in the second week of gavage; Figure 1 M represents the feed intake of laying hens in each experimental group in the second week of gavage. The results are shown as mean ± standard error of the mean, n = 8; *, P < 0.05, **, P < 0.01, ***, P < 0.001.

[0026] Figure 2 The development of follicles of laying hens in different test groups; Figure 2 A in the figure represents the original state of the follicle; Figure 2 B shows the follicles separated and arranged by size.

[0027] Figure 3 The effects of different experimental groups on the oviduct tissue morphology and pathological changes of laying hens; Figure 3 A in the figure indicates H&E staining of the ampulla of the fallopian tube; Figure 3 B in the figure indicates AB staining of the ampulla of the fallopian tube; Figure 3 C in the figure indicates H&E staining of the shell gland of the fallopian tube; Figure 3 D in the figure shows AB staining of the shell gland of the oviduct.

[0028] Figure 4 The results of the determination of serum and shell gland inflammatory factors in laying hens in different test groups are shown in the figure; Figure 4 A in the figure represents the result of determination of IL-1β in serum; Figure 4 B in the figure shows the result of measuring IL-6 in serum; Figure 4 C in the figure represents the measurement result of IL-8 in serum; Figure 4 D in the figure represents the measurement result of IL-10 in serum; Figure 4 E in the figure represents the result of determination of TNF-α in serum; Figure 4 F in the figure shows the measurement result of IL-1β in the shell gland; Figure 4 G in the figure represents the measurement result of IL-6 in the shell gland; Figure 4 H in the figure represents the measurement result of IL-8 in the shell gland; Figure 4 I in the figure represents the measurement result of IL-10 in the shell gland; Figure 4J in the figure indicates the results of the measurement of TNF-α in the shell gland. The results are shown in the table as mean ± standard error of the mean, n = 8; *, P < 0.05, **, P < 0.01, ***, P < 0.001.

[0029] Figure 5 The effect of different experimental groups on the expression of oviduct inflammation genes in laying hens; Figure 5 A in the figure shows the result of determination of TNF-α in the putamen; Figure 5 B in the figure shows the measurement result of IL-1β in the shell gland;

[0030] Figure 5 C in the figure shows the results of the measurement of IL-6 in the shell gland; Figure 5 D in the figure shows the measurement result of tight junction protein Claudin in the shell gland; Figure 5 E in the figure shows the measurement result of tight junction protein Occludin in the shell gland; Figure 5 F in the figure shows the result of determination of TNF-α in ampulla; Figure 5 G in the figure indicates the measurement result of IL-1β in the ampulla; Figure 5 H in the figure represents the measurement result of IL-6 in the ampulla; Figure 5 I in the figure represents the measurement result of the tight junction protein Claudin in the ampulla; Figure 5 J in the figure indicates the determination result of tight junction protein Occludin in ampulla. The result table is mean ± standard error of the mean, n = 8; *, P < 0.05, **, P < 0.01, ***, P < 0.001.

[0031] Figure 6 The effect of Lactobacillus reuteri on the composition of intestinal flora; Figure 6 A in it represents the cecal microbial composition at the phylum level; Figure 6 B in it is the cecal microbial composition at the genus level; Figure 6 C in is the abundance of Lactobacillus in the cecum of each experimental group; Figure 6 D in is the abundance of Erysipelotrichaceae in norank f of each experimental group; Figure 6 E in is the abundance of Veillonella in each experimental group; Figure 6 F in the figure is the abundance of Intestinimonas in each experimental group.

[0032] in, Figures 1 to 6 CON in the table are all control groups; Figures 1 to 6 The SALP in all of them are in the fallopian tube inflammation group; Figures 1 to 6 SALP-LR in all were Lactobacillus reuteri treatment groups; Figures 1 to 6The SALP-HLR in the group were all treated with heat-killed Lactobacillus reuteri. DETAILED DESCRIPTION

[0033] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0034] The strains used in the following examples are as follows:

[0035] Lactobacillus reuteri BNCC192190 strain was purchased from Beina Biotechnology Co., Ltd.

[0036] The present invention provides an application of Lactobacillus reuteri in the preparation of a product for improving the intestinal health and oviduct inflammation of laying hens. The present invention aims to carry out research on the reproductive tract of Lactobacillus reuteri, explore the role of Lactobacillus reuteri in the oviduct health of laying hens, and specifically explore its effects on the oviduct barrier, immune regulation and infection prevention. The mechanism and effect of Lactobacillus reuteri on the oviduct health of laying hens are revealed, and the present invention aims to provide a scientific basis and important reference for the prevention and treatment of oviduct-related diseases.

[0037] Example 1

[0038] 1. Test methods

[0039] 1. Animal testing

[0040] The animal experiment in this embodiment selected 32 laying hens of Jingfen No. 6 aged 56 weeks and randomly divided them into four experimental groups: a control group, a fallopian tube inflammation group, a Lactobacillus reuteri treatment group, and a heat-killed Lactobacillus reuteri treatment group. Each group had 8 laying hens, which were raised in a single cage, and the production performance of the laying hens in each experimental group was recorded every day.

[0041] Among them, the control group is also called CON group. The fallopian tube inflammation group is also called SALP group. The Lactobacillus reuteri treatment group is also called SALP-LR group. The heat-killed Lactobacillus reuteri treatment group is also called SALP-HLR group.

[0042] In the first week of the experiment, phenol glue was used to establish the oviduct inflammation model of laying hens.

[0043] Among them, the steps of establishing the oviduct inflammation model of laying hens are as follows: the present invention adopts the oviduct inflammation group chemical modeling method. First, 5 mL of liquid phenol, 1 g of tragacanth gum, and 4 mL of glycerol are measured in a fume hood, and ultrapure water is added to 20 mL. The mixture is heated and stirred in a water bath to mix well, and the mixture is placed in a glass beaker and sealed for later use.

[0044] Infusion method: One person grabs the laying hens out of the chicken cage, and grabs the thigh roots of two laying hens with his right hand at the same time, with his abdomen facing the guard, so that he cannot move. At the same time, use the thumb, index finger and middle finger of the left hand to press and turn the cloaca outward. After the oviduct is turned out, another person immediately uses a sperm gun to inject 1mL of phenol glue into the oviduct. After the injection is completed, the guard slowly releases his left hand to ensure that the cloaca is turned back. At the same time, to ensure that the infused liquid enters the oviduct, after the infusion is completed, the heads and chests of the laying hens in the experimental group are placed against the trough, and their legs are hung on the fence with Velcro. After 20 minutes, put them back in the original breeding cage.

[0045] In the second week of the experiment, the four experimental groups were gavaged with equal amounts of MRS culture medium, Lactobacillus reuteri liquid, and heat-killed Lactobacillus reuteri liquid every other day for 2 weeks. Sampling began on the 22nd day.

[0046] Among them, the method for obtaining the bacterial liquid of Lactobacillus reuteri is as follows: take out the Lactobacillus reuteri BNCC192190 strain from the -80℃ refrigerator, and after thawing, inoculate it into 5mL liquid MRS culture medium at a volume ratio of 1:25, and shake culture it at 37℃ for 24 hours to activate it. After activation, inoculate it into solid MRS culture medium by streaking method, and after constant temperature culture at 37℃ for 24 hours, pick a single colony and inoculate it into 100mL liquid MRS culture medium, and shake culture it at 37℃ until the number of colonies in the bacterial liquid reaches 10 9 CFU / mL, and dispensed into 50 mL sterile centrifuge tubes to obtain a bacterial solution of Lactobacillus reuteri, which was stored at 4°C for later use.

[0047] The preparation method of heat-killed Lactobacillus reuteri liquid is as follows: taking out the bacterial liquid of Lactobacillus reuteri obtained above, boiling it in a boiling water bath for 15 minutes, and obtaining the heat-killed Lactobacillus reuteri liquid.

[0048] Liquid MRS medium was purchased from Beijing Aoboxing Biotechnology Co., Ltd. Lactobacillus reuteri BNCC192190 strain was purchased from Beina Biotechnology Co., Ltd.

[0049] 2. Sample collection and preparation

[0050] Disinfect the sampling room with ultraviolet light in advance, wipe and disinfect the sampling table and clean bench with alcohol, and use a vertical steam pressure sterilizer to sterilize sampling tools such as surgical scissors, scalpels, tweezers, etc. at 121°C for 30 minutes.

[0051] After the test period, the venous blood under the wings of laying hens in each test group was collected in a procoagulant blood collection tube, placed in a 4°C refrigerator for about 4 hours, and centrifuged at 4°C, 1500r / min, for 15 minutes. The separated serum was stored in an 80°C refrigerator.

[0052] After blood collection, the laying hens in the control group, the oviduct inflammation group, the Lactobacillus reuteri treatment group, and the heat-killed Lactobacillus reuteri treatment group were immediately killed by neck dislocation. The cecum, complete oviduct, and ovary of the laying hens in each experimental group were collected by dissection. 2 After the ampulla and shell gland tissues of the fallopian tube were rinsed with PBS, they were placed in a 4% formalin fixative and stored at room temperature. Then, from the remaining part after cutting, 1 cm × 2 cm ampulla and shell gland tissues were taken and placed in a sterile enzyme-free cryopreservation tube. At the same time, the contents of the cecum, shell gland, and ampulla were collected in a sterile enzyme-free cryopreservation tube next to the alcohol lamp on the clean bench. After the samples were collected in the above cryopreservation tubes, they were immediately closed and placed in liquid nitrogen. After freezing, they were transferred to a -80°C refrigerator for subsequent analysis of related indicators.

[0053] Among them, the vertical steam pressure sterilizer was purchased from BKQ-B50II, Jinan.

[0054] 3. Determination method

[0055] (1) Egg quality testing: During the experiment, eggs laid by laying hens in each experimental group were collected at 2:00 p.m. every day, and the egg production rate was calculated. Egg quality testing was also performed on the eggs on the last day of each experimental week. The egg weight, eggshell strength, Haugh unit, albumen height, and yolk color of the eggs were tested using a fully automatic egg quality tester, and the eggshell thickness was measured and recorded using a vernier caliper.

[0056] (2) Production performance statistics: The number of eggs laid and broken by each experimental group was recorded at 2:00 p.m. each day, and the egg weight was weighed. After the experiment, the daily egg production rate, egg production rate per day, average egg weight and feed-egg ratio were calculated.

[0057] Among them, egg production rate = total number of eggs produced during the test period / (number of laying hens * total number of test days) * 100%;

[0058] The unit of egg production rate is %.

[0059] Daily egg weight = total number of eggs produced during the trial period / (total number of eggs produced*number of laying hens in stock);

[0060] Among them, the unit of daily egg weight is g.

[0061] Feed-egg ratio = total feed consumption during the trial period / total egg weight;

[0062] The units of total feed consumption and total egg weight during the trial period are both kg.

[0063] (3) Inflammatory factors in serum and tissues

[0064] The concentrations of interleukin-6, interleukin-8, interleukin-10, interleukin-1β, and tumor necrosis factor in serum and tissues were detected according to the kit instructions.

[0065] Among them, the test kits were all purchased from Shanghai Yuanmu Technology Co., Ltd.

[0066] (4) Tissue sections of the shell gland and ampulla, pathological evaluation, and H&E and AB staining analysis

[0067] H&E and AB staining were used to observe the tissue morphology and mucosal status of the fallopian tube shell gland and ampulla, respectively. The embedded wax blocks of the fallopian tube shell gland and ampulla were placed in a microtome for slicing, further dewaxed to water, stained and sealed, and the sections were observed and photographed using a Nikon Eclipse E100 upright optical microscope. Each sample had at least two slices. Case viewer software was used for observation and analysis.

[0068] Among them, microtome: Shanghai Leica Instruments, RM2016.

[0069] (5) Real-time PCR relative quantification of differentially expressed gene mRNA levels

[0070] RNA was extracted using the FastPure cell / Tissue Total RNA Isolation Kit V2 tissue RNA extraction kit, and real-time fluorescence quantitative PCR was performed using a real-time PCR detection system.

[0071] Among them, the real-time PCR detection system is Bio-Rad, CFX Connect TM .

[0072] The reaction system of PCR assay was based on the instructions of Taq Pro Universal SYBR qPCR Master Mix kit, which was purchased from Nanjing Novozyme Biotech Co., Ltd. with the catalog number Q712-02.

[0073] The reaction procedure of PCR assay was: 42°C for 90 min, 95°C for 5 min, and 25°C for 20 min. The obtained cDNA was diluted 10 times and stored at -20°C for later use.

[0074] (6) Determination of microbial composition of cecal contents

[0075] On the 22nd day after the experiment officially started, 32 samples of cecal food were collected from laying hens, with 8 samples from each experimental group. The total genomic DNA of the microbial community was extracted according to the instructions of soil DNA kit, the quality of the extracted genomic DNA was detected by 1% agarose gel electrophoresis, and the DNA concentration and purity were determined using NanoDrop2000.

[0076] The extracted DNA was used as a template, and the upstream primer 338F and the downstream primer 806R carrying the barcode sequence were used to amplify the variable region of 16S rRNA gene V3-V4 by PCR, and the purified PCR product was library constructed using the NEXTFLEX Rapid DNA-Seq Kit. Sequencing was performed using the Miseq PE300 platform of Illumina.

[0077] in, soil DNA kit: Omega Bio-tek, Norcross, GA, US.

[0078] NanoDrop2000: Thermo Scientific, USA.

[0079] Miseq PE300 platform: Shanghai Migi Biopharmaceutical Technology Co., Ltd.

[0080] The nucleotide sequence of the upstream primer 338F is shown in SEQ ID NO.1:

[0081] 5′-ACTCCTACGGGAGGCAGCAG-3′.

[0082] The nucleotide sequence of downstream primer 806R is shown in SEQ ID NO.2:

[0083] 5′-GGACTACHVGGGTWTCTAAT-3′.

[0084] 2. Test results

[0085] 1. Clinical manifestations after modeling

[0086] The test process is as follows Figure 1 As shown in A.

[0087] The eggs collected from each experimental group during the salpingitis modeling period were as follows Figure 1 As shown in B. Figure 1 B in Figure 1 F and Figure 1 It can be clearly seen in the J that compared with the eggs collected from the control group, the surface of the eggs collected from the oviduct inflammation group was covered with blood stains, indicating that the phenol glue treatment of laying hens significantly damaged the health of the reproductive tract and caused hemorrhagic damage. Figure 1 As shown in Figures F and J, one week after intragastric administration of Lactobacillus reuteri, the blood stains on the eggshell surface of the Lactobacillus reuteri-treated group were significantly reduced compared with the fallopian tube inflammation group.

[0088] The above results show that the oviduct inflammation model can be well constructed by treating laying hens with phenol slurry.

[0089] 2. Effect of Lactobacillus reuteri on production performance of laying hens with oviduct inflammation

[0090] The results of egg production rate, egg weight and feed intake of laying hens after phenol glue modeling are as shown in C, Figure 1 D and Figure 1 As shown in Figure E, the egg production rate of laying hens significantly decreased after phenol glue modeling, the egg weight decreased, and the feed intake decreased. The results of egg production rate, egg weight, and feed intake after gavage with Lactobacillus reuteri for one week are shown in Figure E. Figure 1 G in Figure 1 H and Figure 1 As shown in Figure 1, compared with the oviduct inflammation group, the treatment with Lactobacillus reuteri bacterial solution significantly increased the egg production rate, egg weight and restored feed intake of laying hens. However, compared with the oviduct inflammation group, the treatment with heat-killed Lactobacillus reuteri bacterial solution did not improve the decrease in egg weight and feed intake caused by oviduct inflammation.

[0091] 3. Effect of Lactobacillus reuteri on follicular development in laying hens with oviduct inflammation

[0092] The development of ovarian follicles Figure 2 As shown, compared with the control group, the oviduct development of laying hens induced by phenol glue was significantly inhibited, while the treatment with Lactobacillus reuteri bacterial solution alleviated this phenomenon, but this phenomenon was not improved after treatment with heat-killed Lactobacillus reuteri bacterial solution.

[0093] The control group is also called the CON group. The fallopian tube inflammation group is also called the SALP group. The Lactobacillus reuteri treatment group is also called the SALP-LR group. The heat-killed Lactobacillus reuteri treatment group is also called the SALP-HLR group.

[0094] 4. Effects of Lactobacillus reuteri on the morphological and pathological changes of oviduct tissues in laying hens with oviduct inflammation

[0095] The results of H&E staining and AB staining of the ampulla and shell gland of the fallopian tube are as follows Figure 3 shown. Figure 3 A in Figure 3 B in Figure 3 C and Figure 3 D in the figure analyzed the tissue morphology, pathological changes and mucus secretion of the ampulla and shell gland of laying hens in the control group, oviduct inflammation group, Lactobacillus reuteri treatment group and heat-killed Lactobacillus reuteri treatment group.

[0096] Among them, the results of HE in the ampulla of laying hens were as follows Figure 3As shown in A, compared with the laying hens in the oviduct inflammation group, the treatment with the bacterial liquid of Lactobacillus reuteri effectively improved the blurred wall of the ampulla, unclear stratification, loose structure, shedding of oviduct epithelial cells, and infiltration of inflammatory cells in laying hens with oviduct inflammation.

[0097] also, Figure 3 Figure B is the result of Alcian blue staining of the ampulla of laying hens. The results showed that the number of goblet cells in the ampulla of laying hens in the oviduct inflammation group was significantly lower than that in the control group, while the number of goblet cells was significantly improved after treatment with the bacterial solution of Lactobacillus reuteri.

[0098] At the same time, the oviduct shell gland of laying hens was stained and analyzed. Figure 3 As shown in C, compared with the laying hens in the fallopian tube inflammation group, the treatment with the bacterial liquid of Lactobacillus reuteri effectively improved the structural disorder of the mucosal layer and muscular layer of the shell gland of laying hens with fallopian tube inflammation, the shedding of mucosal epithelial cells accompanied by edema, and the unclear cell boundaries and the occurrence of cell rupture and disappearance.

[0099] Figure 3 As shown in D, the results of Alcian blue staining showed that the treatment with Lactobacillus reuteri bacterial solution significantly alleviated the phenomenon of reduced mucus secretion in the shell gland of laying hens with oviduct inflammation. The above phenomenon was not effectively improved after the treatment with heat-killed Lactobacillus reuteri bacterial solution.

[0100] 5. Effect of Lactobacillus reuteri on serum and shell gland inflammatory factors in laying hens with oviduct inflammation

[0101] The results of inflammatory factor expression in laying hen serum are as follows Figure 4 A in Figure 4 B in Figure 4 C in Figure 4 D and Figure 4 As shown in Figure E, compared with the control group, the levels of IL-1β, IL-6, IL-8 and TNF-α in the serum of laying hens in the oviduct inflammation group increased significantly (P<0.05), and the level of IL-10 decreased significantly (P<0.05). After the laying hens were treated with the bacterial solution of Lactobacillus reuteri, the levels of IL-1β, IL-6, IL-8 and TNF-α in the serum of laying hens in the oviduct inflammation group were significantly downregulated (P<0.05), and the level of the anti-inflammatory factor IL-10 was significantly upregulated (P<0.05).

[0102] The results of inflammatory factors in the shell gland of laying hens are as follows Figure 4 F in Figure 4 G in Figure 4 H in Figure 4 I and Figure 4J, compared with the control group, the shell gland of the laying hens in the oviduct inflammation group significantly increased the levels of IL-1β, IL-6, IL-8 and TNF-α in the laying hens' serum (P<0.05), and significantly decreased the level of IL-10 (P<0.05). After the laying hens were treated with the bacterial solution of Lactobacillus reuteri, the levels of IL-1β, IL-6, IL-8 and TNF-α in the oviduct inflammation group were significantly reduced (P<0.05), and the expression level of the anti-inflammatory factor IL-10 was significantly increased (P<0.05).

[0103] 6. Effect of Lactobacillus reuteri on the expression of genes in oviduct inflammation in laying hens

[0104] To further determine the protective effect of Lactobacillus reuteri on oviduct inflammation in laying hens, this experiment further measured the expression of genes related to inflammatory response and reproductive tract barrier in the oviduct. Figure 5 A in Figure 5 B in Figure 5 C in Figure 5 D and Figure 5 As shown in E. Compared with the control group, the oviduct inflammation group induced a significant increase in the levels of inflammatory factors IL-6 and TNF-α in the serum of laying hens (P<0.05), while the treatment of laying hens with the bacterial solution of Lactobacillus reuteri significantly downregulated the levels of IL-6 and TNF-α induced by the oviduct inflammation group (P<0.05).

[0105] The results of the ampulla are as follows Figure 5 F in Figure 5 G in Figure 5 H in Figure 5 I and Figure 5 As shown in Figure 1, the oviduct inflammation group induced a significant increase in the levels of inflammatory factors IL-6 and TNF-α in the serum of laying hens (P<0.05), while the treatment of laying hens with the bacterial solution of Lactobacillus reuteri significantly downregulated the levels of IL-6 and TNF-α in the oviduct inflammation group (P<0.05).

[0106] 7. Effect of Lactobacillus reuteri on the structure and composition of intestinal flora in laying hens with oviduct inflammation

[0107] The results of the bacterial flora composition at the cecal level and genus level of laying hens with oviduct inflammation by Lactobacillus reuteri were as follows: Figure 6 The abundances of Lactobacillus, norankfErysipelotrichaceae, Veillonella, and Intestinimonas are shown in Figure 1. Figure 6As shown in C, D, E, and F in Figure 2. At the genus level, the abundance of Lactobacillus in the intestines of laying hens in the Lactobacillus reuteri treatment group was significantly increased compared with the control group, and the abundance of Lactobacillus in the intestines of laying hens in the Lactobacillus reuteri treatment group was also significantly increased compared with the oviduct inflammation group, while no increase in the abundance of Lactobacillus was detected in the intestines of laying hens treated with heat-killed Lactobacillus reuteri liquid. At the same time, a significant increase in the abundance of norankfErysipelotrichaceae was observed in the oviduct inflammation group. The content of Veillonella also showed an increasing trend in the Lactobacillus reuteri treatment group and the heat-killed Lactobacillus reuteri treatment group.

[0108] Based on the oviduct inflammation model of laying hens, the results showed that after the treatment with Lactobacillus reuteri, the cecum had a higher abundance of Lactobacillus and Veillonella, and a lower abundance of norankfErysipelotrichaceae at the genus level. Lactobacillus, whose English name is Lactobacillus, is a recognized probiotic that has been shown to improve bacterial vaginosis and maintain reproductive tract homeostasis. Veillonella, whose English name is Veillonella, is a common genus in the oral cavity. Studies have found that Veillonella can induce TLR2 signal transduction, promote the production of anti-inflammatory factor IL-12, and downregulate the inflammatory content of IL-8, IL-6, IL-10 and TNF-α. The abundance of norankf Erysipelotrichaceae was increased in hosts with colorectal cancer. Similarly, in a study investigating chronic HIV-infected patients receiving suppressive antiretroviral therapy and HIV-uninfected controls, the relative abundance of Erysipelotrichaceae was positively correlated with TNF-α levels, causing host metabolic disorders.

[0109] In summary, the above experiments show that oral supplementation with Lactobacillus reuteri can improve inflammatory damage to the epithelial cells of the shell glands of laying hens, improve the structure of intestinal flora, maintain intestinal health and homeostasis of the internal environment of laying hens, and thus improve production performance.

[0110] The present invention provides the use of Lactobacillus reuteri in preparing a product for improving intestinal health and oviduct inflammation in laying hens, wherein the product is an animal feed additive prepared by using the metabolites of the Lactobacillus reuteri BNCC192190 strain as active ingredients.

[0111] The present invention aims to carry out research on the effects of Lactobacillus reuteri on the reproductive tract and develop a new application of Lactobacillus reuteri on the reproductive tract. The bacterial liquid of Lactobacillus reuteri provided by the present invention can be used as an animal feed additive and combined with a basic diet for daily feeding of laying hens. The basic diet can be a basic diet for conventional laying hens.

[0112] The animal feed additive provided by the invention can be used to improve the production performance and egg quality of laying hens and to improve reproductive tract damage and inflammatory response of laying hens.

[0113] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0114] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they understand the basic inventive concepts, and all such changes and modifications fall within the scope of the present invention.

Claims

1. Application of Lactobacillus reuteri in preparing a product for improving intestinal health and oviduct inflammation in laying hens, characterized in that: The Lactobacillus reuteri is the Lactobacillus reuteri BNCC192190 strain.

2. The use of Lactobacillus reuteri according to claim 1 in preparing a product for improving intestinal health and oviduct inflammation in laying hens, characterized in that: The product is an animal feed additive prepared by taking the bacterial liquid of Lactobacillus reuteri as the active ingredient.

3. The use of Lactobacillus reuteri according to claim 2 in preparing a product for improving intestinal health and oviduct inflammation in laying hens, characterized in that: The animal feed additive is a bacterial liquid of Lactobacillus reuteri obtained by inoculating the Lactobacillus reuteri into a liquid MRS culture medium and culturing the culture medium at 36° C. to 38° C.

4. The use of Lactobacillus reuteri according to claim 3 in preparing a product for improving intestinal health and oviduct inflammation in laying hens, characterized in that: The number of colonies in the bacterial solution of Lactobacillus reuteri is ≥10 9 CFU / mL.

5. The use of Lactobacillus reuteri according to claim 2 in preparing a product for improving intestinal health and oviduct inflammation in laying hens, characterized in that: The animal feed additive is used for improving the production performance of laying hens and the quality of eggs.

6. The use of Lactobacillus reuteri according to claim 5 in preparing a product for improving intestinal health and oviduct inflammation in laying hens, characterized in that: The animal feed additive is used for improving reproductive tract damage and inflammatory response of laying hens.

7. The use of Lactobacillus reuteri according to claim 4 in preparing a product for improving intestinal health and oviduct inflammation in laying hens, characterized in that: The preparation method of the bacterial liquid of Lactobacillus reuteri comprises the following steps: The Lactobacillus reuteri BNCC192190 strain was inoculated into a liquid MRS culture medium, and cultured at 36°C to 38°C for 23 to 25 hours, and then a single colony was picked for purification and culture until the number of colonies in the bacterial solution reached 10 9 CFU / mL or above, the bacterial solution is centrifuged and the supernatant is collected to obtain the bacterial solution of Lactobacillus reuteri.

8. The use of Lactobacillus reuteri according to claim 7 in preparing a product for improving intestinal health and oviduct inflammation in laying hens, characterized in that: The activated Lactobacillus reuteri BNCC192190 strain is inoculated into a liquid MRS culture medium. The activation method is: after thawing the Lactobacillus reuteri BNCC192190 strain, it is inoculated into a liquid MRS culture medium at a volume ratio of 1:25 for activation culture.

9. The use of Lactobacillus reuteri in preparing a product for improving intestinal health and oviduct inflammation in laying hens according to claim 8, characterized in that: The activation culture condition is 36° C. to 38° C. for 23 to 25 hours.

10. The use of Lactobacillus reuteri in preparing a product for improving intestinal health and oviduct inflammation in laying hens according to claim 7, characterized in that: The purification culture conditions are: shaking culture at 36°C to 38°C.

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