Application of lactobacillus gasseri in preparation of medicine for improving salpingitis and intestinal health of laying hens

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of lactobacillus gasseri in preparation of a medicine for improving salpingitis and intestinal health of laying hens. The lactobacillus gasseri is a lactobacillus gasseri bacterial strain BNCC339385, and the lactobacillus gasseri is a lactobacillus gasseri bacterial strain The application effect of the lactobacillus gasseri in improving the fallopian tube inflammation injury of the laying hens is reported for the first time; in addition, the potential mechanism of improving the reproductive tract inflammation by inhibiting the PI3K / AKT signal channel of the lactobacillus gasseri is verified on the molecular level. The invention aims to explore the effect of lactobacillus gasseri on relieving female reproductive system diseases by taking laying hens as research objects, provides a new application of lactobacillus gasseri in improving intestinal health of laying hens and related products of fallopian tube inflammation, and expands the related research of probiotics on relieving intestinal health of laying hens and reproductive system diseases.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to the application of Lactobacillus gasseri in the preparation of drugs for improving oviduct inflammation and intestinal health of laying hens. Background Art

[0002] The female reproductive system not only plays an important role in human reproductive health, but also plays a crucial role in the reproductive performance of livestock and poultry. Female reproductive system diseases not only cause female infertility, but also lead to problems such as decreased production performance, reduced production income, and obstruction of the breeding process in livestock production. In laying hen production, due to the excessive pursuit of production efficiency and continuous improvement of production benefits, the production pressure of laying hens has gradually increased, and oviduct inflammation has become one of the high-incidence diseases in the late laying period of laying hens. Oviduct inflammation can not only lead to a decline in egg quality, increase the rate of cracked and soft eggs, reduce production performance, cause a decrease in laying rate and average egg weight, and increase the feed-to-egg ratio, increasing production costs, but also lead to problems such as pecking at the anus and feathers of laying hens, affecting the health of laying hens and even causing death, which results in a decrease in the utilization rate of laying hens and a significant drop in production efficiency. Therefore, extending the service life of laying hens, alleviating oviduct inflammation of laying hens, improving the production performance in the late laying period, and pursuing higher benefits have gradually become an industry necessity. In production, oviduct inflammation is commonly treated with antibiotics such as amoxicillin. However, the use of antibiotics not only leads to the generation of drug-resistant bacteria, but also easily causes drug residues, affecting egg quality. With the promulgation of the domestic feed antibiotic ban regulations, the prevention and treatment of oviduct inflammation have become more difficult. Therefore, finding alternative antibiotic products with high efficiency in preventing and treating bacterial infections is of great significance for the treatment and prevention of oviduct inflammation in laying hens, improving body inflammation, and increasing production benefits.

[0003] Currently, there are more and more studies on the use of Chinese herbal medicines and probiotics as antibiotic substitutes in the poultry industry. For example, Rhizobium is used to treat liver damage in chickens caused by aflatoxin. However, there are few studies on the effects and mechanism analysis of probiotics in alleviating intestinal health and reproductive system diseases of laying hens, especially Lactobacillus. Therefore, it is necessary to develop new uses of Lactobacillus in products related to improving intestinal health and oviduct inflammation of laying hens. Summary of the Invention

[0004] In order to develop a new use of Lactobacillus in products related to improving intestinal health and oviduct inflammation of laying hens, the present invention takes laying hens as the research object, aims to explore the effect of Lactobacillus gasseri in alleviating female reproductive system diseases, and provides an application of Lactobacillus gasseri in the preparation of drugs for improving oviduct inflammation and intestinal health of laying hens.

[0005] To achieve the above object, the present invention adopts the following technical solutions.

[0006] The present invention provides the application of Lactobacillus formosensis in the preparation of a drug for improving the intestinal health and oviduct inflammation of laying hens, and the Lactobacillus formosensis is the Lactobacillus formosensis strain BNCC339385. The present invention takes laying hens as the research object, aims to explore the effect of Lactobacillus gasseri in relieving female reproductive system diseases, provides a new use of Lactobacillus gasseri in products related to improving the intestinal health and oviduct inflammation of laying hens, and expands the related research on Lactobacillus in relieving the intestinal health and reproductive system diseases of laying hens.

[0007] Preferably, the drug uses the culture of the Lactobacillus formosensis as the active ingredient and is supplemented with pharmaceutically acceptable excipients.

[0008] Preferably, the preparation method of the culture of the Lactobacillus formosensis comprises the following steps:

[0009] Inoculate the Lactobacillus formosensis strain BNCC339385 into a solid MRS medium, and after incubating at a constant temperature of 36°C to 38°C for 23 hours to 25 hours, pick single colonies and then perform purification culture until the number of colonies in the bacterial liquid reaches 10 9 CFU / mL or more, then centrifuge the bacterial liquid and collect the supernatant to obtain the culture of the Lactobacillus formosensis.

[0010] Preferably, the activated Lactobacillus formosensis strain BNCC339385 is inoculated into the solid MRS medium, and the activation method is: thaw the Lactobacillus formosensis strain BNCC339385 and inoculate it into a liquid MRS medium for activation culture according to a volume ratio of 1:24 to 26.

[0011] Preferably, the conditions for the activation culture are: culture at 36°C to 38°C for 23 hours to 25 hours.

[0012] Preferably, the conditions for the purification culture are: shake culture at 36°C to 38°C.

[0013] Preferably, the drug is a solution. The excipient is a solvent. The solution is obtained by compounding the culture of the Lactobacillus formosensis and the solvent.

[0014] Preferably, the solvent is any one of DMSO, water, ethanol, and glycerol.

[0015] Among them, Lactobacillus gasseri is a Gram-positive bacterium and is widely used in the food processing industry. Lactobacillus gasseri is a non-motile, non-spore-forming, facultative aerobic, catalase-negative Gram-positive bacterium. Lactobacillus gasseri, as a dominant probiotic in the reproductive system, has biological functions such as antifungal, antioxidant, anti-apoptotic, regulating intestinal barrier, and regulating immune response. Through research, the present invention has found that Lactobacillus gasseri not only plays an improving role in intestinal diseases, but oral administration of it can effectively improve the anti-diarrhea ability of piglets, improve the intestinal flora, reduce Clostridium difficile in feces, and relieve obesity in mice caused by a high-fat diet.

[0016] Meanwhile, in the reproductive system, Lactobacillus gasseri has also been proven to have a regulatory effect. It can effectively kill Trichomonas vaginalis, treat HPV infection, improve the immune response of human cervical epithelial cells, and show an effective alleviating effect on reproductive system diseases. However, there is less research on its role in laying hen production. The present invention takes laying hens as the research object, aims to explore the effect of Lactobacillus gasseri in relieving female reproductive system diseases, and provides a new use of Lactobacillus gasseri in products related to improving the intestinal health and oviduct inflammation of laying hens.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. The present invention provides the application of Lactobacillus gasseri in the preparation of drugs for improving the oviduct inflammation and intestinal health of laying hens. The present invention reports for the first time the application effect of Lactobacillus gasseri in improving oviduct inflammation injury in laying hens. The present invention takes laying hens as the research object, aims to explore the effect of Lactobacillus gasseri in relieving female reproductive system diseases, provides a new use of Lactobacillus gasseri in products related to improving the intestinal health and oviduct inflammation of laying hens, and expands the related research on probiotics in relieving the intestinal health and reproductive system diseases of laying hens.

[0019] 2. The research results of the present invention show that: Lactobacillus gasseri can negatively regulate the gene expression of the PI3K / AKT signaling pathway, up-regulate the gene expression of the signal pathway related to anti-inflammation, down-regulate the gene expression of the pro-inflammatory related pathway, regulate the intestinal microbial community, relieve oviduct inflammation injury, and finally improve the production performance and egg quality of laying hens.

[0020] Among them, the Latin name of Lactobacillus gasseri is Lactobacillus gasseri.

[0021] 3. Salpingitis is highly prevalent in laying hens in the late laying period, often leading to a decrease in egg production rate and an increase in the production of cracked and soft-shelled eggs, causing significant economic losses to the egg poultry industry. Lactobacillus gasseri, as a probiotic colonized in the oviduct of healthy laying hens, has biological functions such as antibacterial, anti-inflammatory, and antioxidant. Some studies have found that the use of lactic acid bacteria can effectively improve the damage of the oviduct of laying hens, but whether Lactobacillus gasseri has a relieving effect on salpingitis in laying hens and its molecular mechanism of action are still unclear. Moreover, reproductive system diseases have gradually attracted attention in today's society. Diseases such as vaginitis, polycystic ovary syndrome, and endometritis have become high-incidence factors troubling women's lives. Clinically, drugs and surgeries are mostly used for treatment, but they have large side effects and cumbersome operations, bringing great inconvenience to life. Using probiotics for prevention and treatment has the advantages of convenience and small side effects. Based on the above research background, this invention designed 3 experiments to systematically explore the effect of Lactobacillus gasseri on female genital tract inflammation, providing a theoretical basis for alleviating and preventing salpingitis in laying hens and female reproductive system diseases in production. This study first designed an oral relieving experiment of Lactobacillus gasseri in laying hens, and explored its effect on the inflammatory damage of the oviduct of laying hens by observing production performance, egg quality, histology, serum tissue biochemical indexes, gene expression, microbiomics, and transcriptomics. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the experimental flow chart in the present invention.

[0023] Figure 2 It is for Lactobacillus gasseri relieving the decrease in production performance induced by phenol mucilage in Example 1 of the present invention; wherein, Figure 2 a in it is the experimental design of the experiment in Example 1; Figure 2 b in it is the photo of the eggs laid by laying hens in each treatment group on the 5th day of the experiment; Figure 2 c in it is the photo of the eggs laid by laying hens in each treatment group on the 21st day of the experiment; wherein, MRS is the control group; SALP is the salpingitis group; the treatment group is also called the SALP + L. gasseri group; the modeling + heat-inactivated Lactobacillus gasseri treatment group is also called the SALP + HeatKilled L. gasseri group.

[0024] Figure 3 It is the photo of the H&E staining section of the ampulla and shell gland of the oviduct in Example 1 of the present invention, wherein, the magnification is 5× and 35×; n = 4; Figure 3 Figure A in it is the photo of the H&E staining section of the ampulla in the MRS group; Figure 3 Figure B in it is the photo of the H&E staining section of the ampulla in the SALP group; Figure 3 Figure C in it is the photo of the H&E staining section of the ampulla in the SALP + L. gasseri group; Figure 3Figure D in it is the photo of the H&E staining section of the ampulla in the SALP+HeatKilledL.gasseri group; Figure 3 Figure E in it is the photo of the H&E staining section of the shell gland in the MRS group; Figure 3 Figure F in it is the photo of the H&E staining section of the shell gland in the SALP group; Figure 3 Figure G in it is the photo of the H&E staining section of the shell gland in the SALP+L.gasseri group; Figure 3 Figure H in it is the photo of the H&E staining section of the shell gland in the SALP+HeatKilledL.gasseri group; among them, MRS is the control group; the SALP group is the salpingitis group; SALP+L.gasseri is the modeling+Lactobacillus gasseri treatment group; SALP+HeatKilledL.gasseri is the modeling+heat-inactivated Lactobacillus gasseri treatment group; among them, the English name of the ampulla is Magnum; the English name of the shell gland is Shell Glands.

[0025] Figure 4 This is the effect of Lactobacillus gasseri on the body of laying hens with salpingitis and inflammatory factors in the oviduct tissue in Example 1 of the present invention; among them, Figure 4 a in it is the levels of inflammatory factors TNF-α, IL-1β, IL-6 and IL-10 in the serum measured by ELISA; Figure 4 b in it is the levels of inflammatory factors TNF-α, IL-1β, IL-6 and IL-10 in the shell gland of the oviduct measured by ELISA; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; n = 8.

[0026] Figure 5 This is the effect of Lactobacillus gasseri and its inactivated bacterial solution on the expression levels of inflammatory factors in the oviduct tissue of laying hens in Example 1 of the present invention; among them, Figure 5 a in it is the relative expression levels of inflammatory factors TNF-α, IL-1β, IL-6 and IL-10 in the ampulla of the oviduct measured by RT-qPCR; Figure 5 b in it is the relative expression levels of inflammatory factors TNF-α, IL-1β, IL-6 and IL-10 in the shell gland of the oviduct measured by RT-qPCR; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; n = 8.

[0027] Figure 6 This is the effect of Lactobacillus gasseri on the tight junction proteins of the oviduct of laying hens in Example 1 of the present invention; among them, Figure 6 a in it is the relative mRNA expression levels of tight junction proteins Occludin-1 and ZO-1 in the ampulla of the oviduct; Figure 6In which, b is the relative mRNA expression levels of tight junction proteins Occludin-1 and ZO-1 in the shell gland part of oviduct; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; n = 8.

[0028] Figure 7 This is the effect of Lactobacillus gasseri on the cecal microbiota composition of laying hens with salpingitis in Example 1 of the present invention; wherein, Figure 7 In which, a is the chao index of the cecal microbiota α-diversity of laying hens; Figure 7 In which, b is the shannon index of the cecal microbiota α-diversity of laying hens; Figure 7 In which, c is the bar graph of the cecal microbiota (at the genus level) composition of laying hens; Figure 7 In which, d~ Figure 7 In which, f are the PCoA analysis diagrams of the cecal microbiota of laying hens respectively: Figure 7 In which, d is the comparison between the MRS group and the SALP+L.gasseri group; Figure 7 In which, e is the comparison between the MRS group and the SALP group; Figure 7 In which, f is the comparison between the SALP+L.gasseri group and the SALP group; *P<0.05, **P<0.01, ***P<0.001; n = 8.

[0029] Figure 8 This is the effect of Lactobacillus gasseri on the cecal microbiota composition of laying hens with salpingitis in Example 1 of the present invention; wherein, Figure 8 In which, a~ Figure 8 In which, d are the LEfSe analysis diagrams of the cecal microbiota of laying hens (at the genus level): Figure 8 In which, a is the comparison between the control group and the Lactobacillus gasseri group; Figure 8 In which, b is the comparison between the control group and the SALP group; Figure 8 In which, c is the comparison between the SALP+L.gasseri group and the SALP group; Figure 8 In which, d is the comparison between the SALP+L.gasseri group and the SALP+Heat killedL.gasseri group; LDA>2.

[0030] Figure 9 This is the effect of Lactobacillus gasseri in the SALP+L.gasseri group vs the SALP group on the oviduct transcriptome in Example 1 of the present invention; wherein, Figure 9 In which, a is the number of differential genes; Figure 9 In which, b is the volcano plot of differential genes; Figure 9 In which, c is the heat map of differential genes.

[0031] Figure 10GO enrichment analysis of the oviduct transcriptome of the SALP+L. gasseri group vs the VSSALP group in Example 1 of the present invention; wherein, Figure 10 a in Figure 10 is GO database enrichment analysis; Figure 10 b in Figure 10 is GO enrichment analysis for up-regulated differential genes; Figure 10 c in Figure 10 is GO enrichment analysis for down-regulated differential genes.

[0032] Figure 11 KEGG enrichment analysis of the oviduct transcriptome of the SALP+L. gasseri group vs the VSSALP group in Example 1 of the present invention; wherein, Figure 11 a in Figure 11 is KEGG database enrichment analysis; Figure 11 b in Figure 11 is KEGG enrichment analysis for up-regulated differential genes; Figure 11 c in Figure 11 is KEGG enrichment analysis for down-regulated differential genes.

[0033] Figure 12 Effect of Lactobacillus gasseri on the PI3K / AKT signaling pathway in the shell gland of the oviduct of laying hens in Example 1 of the present invention; wherein, Figure 12 a to Figure 12 c in Figure 12 are the RT-qPCR results of genes related to the PI3K / AKT signaling pathway: Figure 12 a in Figure 12 is PI3K; Figure 12 b in Figure 12 is AKT; Figure 12 c in Figure 12 is IL-17A; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; n = 8. Detailed implementation manners

[0034] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0035] The strains used in the following embodiments are shown below:

[0036] Lactobacillus gasseri: Lactobacillus gasseri BNCC339385 strain, purchased from Beina Biotechnology Co., Ltd.

[0037] The present invention takes laying hens as the research object, and explores the effect of orally administered Lactobacillus gasseri on the uterine inflammation of laying hens from the following aspects by means of feeding experiments, determination of immune-related indexes, microbiome, and transcriptomics: the effect of Lactobacillus gasseri on alleviating the inflammatory damage of the oviduct of laying hens; the test flow chart is as Figure 1 shown and the test design of the test in Example 1 below is as Figure 2as shown in a of

[0038] Example 1

[0039] Thirty-two 53-week-old Jingfen No. 6 laying hens were randomly selected and randomly divided into 4 treatment groups, with 8 replicates in each group and 1 chicken in each replicate. After observing for 1 week, the experiment was carried out. The experiment lasted for 3 weeks. In the first week, a 25% phenol mucilage by volume was used to induce salpingitis in laying hens. From the second week to the third week, the perfusion experiment of Lactobacillus gasseri and its heat-inactivated bacterial solution was carried out. Sampling was carried out by slaughtering on the 21st day of the experiment. The specific methods are as follows:

[0040] (1) In the control group, 1 mL of sterile normal saline was used for treatment during the modeling period, and 1 mL of sterile liquid MRS medium was used for perfusion during the perfusion period. Among them, the control group is also called the MRS group, MRS. The liquid MRS medium was purchased from Beijing Aoboxing Biotechnology Co., Ltd., China.

[0041] (2) In the SALP group, 1 mL of 25% phenol mucilage by volume was used for treatment during the modeling period, and 1 mL of sterile liquid MRS medium was given during the perfusion period. Among them, the SALP group is also called SALP.

[0042] (3) In the modeling + Lactobacillus gasseri treatment group, 1 mL of 25% phenol mucilage by volume was used for treatment during the modeling period, and 1 mL of Lactobacillus gasseri bacterial solution was given during the perfusion period. Among them, the modeling + Lactobacillus gasseri treatment group is also called the SALP + L. gasseri group, SALP + L. gasseri.

[0043] The above-mentioned Lactobacillus gasseri bacterial solution refers to the culture of Lactobacillus gasseri. The preparation method of the culture of Lactobacillus gasseri is as follows:.

[0044] The Lactobacillus gasseri BNCC339385 strain was taken out from the -80 °C refrigerator preservation. After thawing, it was inoculated into 5 mL of liquid MRS medium at a ratio of 1:25 by volume and cultured with shaking at 37 °C for 24 hours to activate it. After activation, it was inoculated onto the solid MRS medium by the streaking method and cultured at a constant temperature of 37 °C for 24 hours. Then, a single colony was picked and inoculated into 100 mL of liquid MRS medium and cultured with shaking at 37 °C until the number of colonies in the bacterial solution reached 10 9 CFU / mL. After centrifugation, the supernatant was collected and aliquoted into sterile centrifuge tubes to obtain the culture of Lactobacillus gasseri. Among them, 50 mL was stored at 4 °C for later use, and the other 50 mL was placed in a sterile centrifuge tube, water-bathed at 100 °C for 30 min, and stored at 4 °C for later use.

[0045] The culture of Lactobacillus gasseri is the above-mentioned Lactobacillus gasseri bacterial solution.

[0046] (4) In the modeling + heat-inactivated Lactobacillus gasseri treatment group, during the modeling period, 1 mL of phenol mucilage with a volume percentage of 25% was used for treatment, and during the perfusion period, 1 mL of heat-boiled Lactobacillus gasseri bacterial solution was administered. Among them, the modeling + heat-inactivated Lactobacillus gasseri treatment group is also known as the SALP + Heat Killed L. gasseri group, SALP + Heat Killed L. gasseri. The heat-boiled Lactobacillus gasseri bacterial solution is also known as the heat-inactivated Lactobacillus gasseri bacterial solution.

[0047] 1. On the 5th day and the 21st day of the experiment, the eggs laid by the laying hens in each of the above treatment groups were photographed. The photos of the eggs laid by the laying hens in each treatment group on the 5th day of the experiment are as Figure 2 shown in b of

[0048] As can be seen from Figure 2 b in Figure 2 and c in

[0049] the photos of the eggs in the modeling + heat-inactivated Lactobacillus gasseri treatment group also showed a reduction in bloodstains on the eggshell surface caused by oviduct injury after treatment.

[0050] 2. During the 1st to 7th day and the 8th to the 21st day of the experiment, the production performance was measured, and the results are shown in Table 1. Among them, the production performance includes the laying rate, total feed consumption, average daily feed intake, daily egg production per hen, average egg weight, feed-to-egg ratio, and the percentage of cracked and soft eggs.

[0051] Among them, the laying rate refers to the percentage of laying hens that have laid eggs in the total number of laying hens; the total feed consumption refers to the amount of feed consumed by all laying hens; the average daily feed intake refers to the ratio of the amount of feed consumed by laying hens / the number of days; the daily egg production per hen refers to the amount of eggs laid by each laying hen per day; the average egg weight refers to the ratio of the total weight of all eggs / the number of all eggs; the feed-to-egg ratio refers to the ratio of daily feed consumption / daily egg weight; the percentage of cracked and soft eggs refers to the percentage of the number of cracked and soft eggs in the total number of eggs.

[0052] Table 1 Effects of Lactobacillus gasseri on the production performance of laying hens with salpingitis

[0053]

[0054] Note: All values are averages (n = 8); different lowercase letters in the same row with superscripts indicate significant differences (P < 0.05).

[0055] As can be seen from Table 1, from 1 day to 7 days after modeling in the modeling + Lactobacillus gasseri treatment group and the modeling + heat-inactivated Lactobacillus gasseri treatment group, the egg production rate decreased significantly from 98.25% in the MRS group to 46.50% - 66.25% (P < 0.05). Compared with the MRS group, the total feed consumption, average feed intake, and daily egg production per hen in the SALP group showed significant decreases (P < 0.05), and there was also an upward trend in the feed-to-egg ratio; after treatment with Lactobacillus gasseri solution, the daily egg production per hen (51.62 g) showed a significant increase compared with the SALP group (P < 0.05), and the feed-to-egg ratio decreased significantly (2.14) (P < 0.05). The heat-inactivated Lactobacillus gasseri solution also showed a significant improvement effect. Compared with the SALP group, the egg production rate and daily egg production per hen increased significantly (P < 0.05), and the feed-to-egg ratio decreased (P < 0.05).

[0056] As can be seen from the above results, Lactobacillus gasseri solution significantly improved the production performance of laying hens with salpingitis after treatment.

[0057] 3. During the 1st - 7th day, 8th - 14th day, and 15th - 21st day of the experiment, the egg quality was measured, and the results are shown in Table 2. Among them, the egg quality includes eggshell strength (measuring instrument), eggshell thickness (measuring instrument), thick albumen height (measuring instrument), egg yolk color, and Haugh unit.

[0058] Among them, the eggshell strength was measured using an eggshell strength measuring instrument. The eggshell thickness was measured using an eggshell thickness measuring instrument. The thick albumen height and egg yolk color can both be measured using an egg quality analyzer.

[0059] The Haugh unit was measured using a fully automatic egg quality measuring instrument, and the data could be directly measured.

[0060] Table 2 Effects of Lactobacillus gasseri on the egg quality of laying hens with salpingitis

[0061]

[0062] Note: All values are averages (n = 8); different lowercase superscripts in the same row indicate significant differences (P < 0.05).

[0063] As can be seen from the egg quality results in Table 2, Lactobacillus gasseri solution and heat-inactivated Lactobacillus gasseri solution also had a certain improvement effect on egg quality. In the modeling + Lactobacillus gasseri treatment group and the modeling + heat-inactivated Lactobacillus gasseri treatment group.

[0064] During the model establishment period, compared with the MRS group, the phenol mucilage significantly reduced the eggshell thickness (P<0.05), while the other indicators showed no significant changes. After one week of gavage following model establishment, there were no significant differences in eggshell thickness among the four treatment groups (P>0.05). However, in the SALP+Heat Killed L. gasseri group, the Haugh unit significantly decreased compared with the MRS group and the SALP group (P<0.05). After two weeks of gavage, there were no significant differences in egg quality indicators (P>0.05).

[0065] From the above results, it can be seen that the L. gasseri bacterial solution and the heat-inactivated L. gasseri bacterial solution also have a certain effect on improving egg quality.

[0066] 4. On the 22nd day of the experiment, H&E staining sections were made of the ampulla and shell gland of the oviduct, and the results are as Figure 3 shown.

[0067] Figure 3 The H&E staining sections of the ampulla and shell gland of the oviduct are shown. From the staining results, it can be seen that compared with the MRS group, the phenol mucilage caused inflammatory cell infiltration (black), glandular edema (green), and interstitial cell edema (red) in the ampulla tissue of the oviduct in the SALP group. The L. gasseri bacterial solution alleviated the tissue inflammatory infiltration, glandular edema, and interstitial cell edema caused by salpingitis, and the heat-inactivated L. gasseri bacterial solution also had similar results.

[0068] Moreover,[[]] Figure 3 in the shell gland of the oviduct shown in[[[]]], inflammatory infiltration and glandular edema also occurred in the SALP group, and the L. gasseri bacterial solution and the heat-inactivated L. gasseri bacterial solution alleviated the inflammatory infiltration and tissue edema caused by the phenol mucilage.

[0069] From the above results, it can be seen that the L. gasseri bacterial solution alleviated the tissue inflammatory infiltration, glandular edema, and interstitial cell edema caused by salpingitis, and the heat-inactivated L. gasseri bacterial solution also had similar results.

[0070] 5. On the 22nd day of the experiment, the effects of L. gasseri on the body of laying hens with salpingitis and inflammatory factors in the oviduct tissue were measured, and the results are as Figure 4 shown.

[0071] Among them, ELISA was used to measure the levels of inflammatory factors TNF-α, IL-1β, IL-6, and IL-10 in the serum of the wing vein under the chicken wing.

[0072] ELISA was used to measure the levels of inflammatory factors TNF-α, IL-1β, IL-6, and IL-10 in 0.5 square centimeters of the shell gland of the oviduct.

[0073] From[[[]]] Figure 4It can be seen that, compared with the MRS group, the phenol glue paste significantly (P<0.01) increased the levels of inflammatory cytokines TNF-α, IL-1β and IL-6 in the serum, and decreased (P<0.01) the level of the anti-inflammatory cytokine IL-10 in the serum. The Lactobacillus gasseri liquid and the heat-inactivated Lactobacillus gasseri liquid effectively reversed the phenomenon caused by the phenol glue paste, significantly (P<0.01) reducing TNF-α, IL-1β and IL-6 and increasing the level of IL-10.

[0074] Moreover, compared with the SALP+L. gasseri group, the heat-inactivated Lactobacillus gasseri liquid more effectively (P<0.01) reduced the levels of TNF-α and IL-6 and increased the level of IL-10 in the serum. Similarly, as shown in Figure 4 b of [reference], the levels of inflammatory factors in the shell gland part of the oviduct, the Lactobacillus gasseri liquid and the heat-inactivated Lactobacillus gasseri liquid treatment significantly (P<0.01) alleviated the increase in the levels of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 and the decrease in the anti-inflammatory cytokine IL-10 in the shell gland tissue caused by the phenol glue paste. And compared with the Lactobacillus gasseri liquid, the alleviating effect of the heat-inactivated Lactobacillus gasseri liquid was more obvious, significantly (P<0.01) reducing the levels of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) and increasing the level of the anti-inflammatory cytokine IL-10.

[0075] From the above results, it can be seen that the Lactobacillus gasseri liquid and the heat-inactivated Lactobacillus gasseri liquid treatment significantly (P<0.01) alleviated the increase in the levels of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 and the decrease in the anti-inflammatory cytokine IL-10 in the shell gland tissue caused by the phenol glue paste. And compared with the Lactobacillus gasseri liquid, the alleviating effect of the heat-inactivated Lactobacillus gasseri liquid was more obvious, significantly (P<0.01) reducing the levels of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 and increasing the level of the anti-inflammatory cytokine IL-10.

[0076] 6. On the 22nd day of the experiment, the effects of the Lactobacillus gasseri liquid and the heat-inactivated Lactobacillus gasseri liquid on the expression levels of inflammatory factors in the oviduct tissue of laying hens were measured, and the results are as shown in Figure 5 shown.

[0077] Among them, RT-qPCR was used to measure the relative expression levels of inflammatory factors TNF-α, IL-1β, IL-6 and IL-10 in the ampulla of the oviduct of laying hens.

[0078] RT-qPCR was used to measure the relative expression levels of inflammatory factors TNF-α, IL-1β, IL-6 and IL-10 in the shell gland part of the oviduct.

[0079] Among them, inflammatory factors in serum and tissues

[0080] (1) Detect the concentrations of interleukin-6, interleukin-8, interleukin-10, interleukin-1β, and tumor necrosis factor in serum and tissues according to the kit instructions.

[0081] Among them, all the kits were purchased from Shanghai Yuanmu Technology Co., Ltd.

[0082] (2) Histopathological evaluation and H&E and AB staining analysis of the shell gland and ampulla tissues

[0083] Use H&E and AB staining to observe the tissue morphology and mucosal status of the shell gland and ampulla of the oviduct respectively. Use the wax blocks of the shell gland and ampulla of the oviduct after embedding, put them into a microtome for sectioning, further dewax to water and stain and mount the slides. Use a Nikon Eclipse E100 upright optical microscope to observe and photograph the sections. Each sample has at least two sections. Use Case viewer software for observation and analysis.

[0084] Among them, the microtome: Shanghai Leica Instruments, RM2016.

[0085] (3) Real-time PCR relative quantification of the mRNA expression levels of differentially expressed genes

[0086] Extract RNA using the FastPure cell / Tissue Total RNA Isolation Kit V2 tissue RNA extraction kit, and perform real-time fluorescence quantitative PCR determination using a real-time PCR detection system.

[0087] Among them, the real-time PCR detection system is Bio-Rad, CFX Connect TM .

[0088] The reaction system for PCR determination refers to the instruction manual of the TaqPro Universal SYBR qPCR Master Mix kit. This kit is purchased from Nanjing Novoprotein Science and Technology Co., Ltd., and the product number is Q712-02.

[0089] The reaction program for PCR determination is: 42°C for 90 min, 95°C for 5 min, 25°C for 20 min. The obtained cDNA is diluted 10-fold and stored at -20°C for later use.

[0090] 7. Determination of the microbial composition of cecal contents

[0091] Collect a total of 32 samples of cecal digesta from laying hens on the 22nd day when the experiment officially starts, with 8 samples in each experimental group. According to Extract the total genomic DNA of the microbial community according to the instructions of the soil DNAkit. Detect the quality of the extracted genomic DNA using 1% agarose gel electrophoresis, and measure the DNA concentration and purity using NanoDrop2000.

[0092] Using the above-extracted DNA as a template, perform PCR amplification of the V3-V4 variable region of the 16S rRNA gene using the upstream primer 338F and the downstream primer 806R carrying the Barcode sequence. Use the NEXTFLEX Rapid DNA-Seq Kit to construct a library for the purified PCR products. Sequence using the Illumina Miseq PE300 platform.

[0093] Among them, soil DNAkit: Omega Bio-tek, Norcross, GA, U.S.

[0094] NanoDrop2000: Thermo Scientific, U.S.

[0095] Miseq PE300 platform: Shanghai Majorbio Bio-Pharm Technology Co., Ltd.

[0096] The nucleotide sequence of the upstream primer 338F is shown in SEQ ID NO.1:

[0097] 5’-ACTCCTACGGGAGGCAGCAG-3’.

[0098] The nucleotide sequence of the downstream primer 806R is shown in SEQ ID NO.2:

[0099] 5’-GGACTACHVGGGTWTCTAAT-3’.

[0100] From Figure 5 a in Figure 5As shown in b of , by detecting the relative expression levels of inflammatory cytokines TNF-α, IL-1β, IL-6, and IL-10 in the ampulla of the oviduct, the effect of Lactobacillus gasseri on salpingitis in laying hens was verified. The results showed that compared with the MRS group, phenol mucilage significantly (P<0.01) increased the relative expression levels of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in the ampulla and shell gland of the oviduct, while simultaneously increasing the relative expression level of the anti-inflammatory cytokine IL-10 in the tissue. However, after treatment with Lactobacillus gasseri bacterial solution and heat-inactivated Lactobacillus gasseri bacterial solution, the relative expression levels of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in the ampulla of the oviduct were significantly (P<0.05) down-regulated, while in the shell gland of the oviduct, they were extremely significantly (P<0.01) down-regulated. However, compared with the MRS group, Lactobacillus gasseri bacterial solution and heat-inactivated Lactobacillus gasseri bacterial solution had no significant (P>0.05) effect on the relative expression level of IL-10.

[0101] 8. On the 22nd day of the experiment, Wuhan Sevier Biotechnology Co., Ltd. was commissioned to measure the effect of Lactobacillus gasseri on the tight junction proteins of the oviduct of laying hens. The results are as Figure 6 shown.

[0102] As Figure 6 shown in a of , in the ampulla of the oviduct, compared with the SALP group, the relative expression level of the tight junction protein Occludin-1 was significantly decreased after treatment with Lactobacillus gasseri bacterial solution (P<0.05), while after treatment with heat-inactivated Lactobacillus gasseri bacterial solution, the expression level of Occludin-1 was significantly increased compared with the SALP+L. gasseri group (P<0.05).

[0103] And as Figure 6 shown in b of , in the shell gland of the oviduct, the expression level of Occludin-1 in the SALP group was significantly down-regulated compared with the MRS group, but the expression level of ZO-1 was up-regulated (P<0.05). And in the SALP+Heat Killed L. gasseri group, the mRNA expression level of the tight junction protein Occludin-1 was significantly up-regulated compared with the SALP group (P<0.05). However, for ZO-1 in the shell gland of the oviduct, Lactobacillus gasseri bacterial solution and heat-inactivated Lactobacillus gasseri bacterial solution significantly down-regulated its expression level compared with the SALP group (P<0.05).

[0104] 9. On the 22nd day of the experiment, Shanghai Majorbio Bio-Pharm Technology Co., Ltd. was commissioned to detect the effect of Lactobacillus gasseri on the microbial composition in the cecal digesta. The results are as Figure 7 shown.

[0105] As Figure 7As shown in a of , the SALP + L. gasseri group decreased the α-diversity - Chao index of cecal microbiota compared with the MRS group (P < 0.05), but had no significant effect (P > 0.05) on the Shannon index. The treatment with phenol mucilage alone also had no significant effect (P > 0.05) on the α-diversity (Chao index, Shannon index) of the cecal flora. As Figure 7 Shown in b of is the composition of cecal chyme flora at the genus level. Among the four treatment groups, Bacteroides (12 - 14%) accounted for a relatively large proportion. It is worth noting that in the SALP group, the content of Lactobacillus (12.65%) was significantly higher than that in the MRS group (7.61%). The level of Lactobacillus in the SALP + L. gasseri group (9.31%) was nearly the same as that in the MRS group, but there was a significant increase in the SALP + Heat Killed L. gasseri group (13.08%). Figure 7 As shown in c of ~ Figure 7 The principal coordinate analysis shown in f of demonstrates the differences in the flora composition among groups.

[0106] The results are as Figure 7 shown in c of . There were significant differences in the flora composition between the MRS group and the SALP + L. gasseri group (P < 0.05). As Figure 7 shown in d of , Figure 7 e of and Figure 7 f of , there were only certain differences (P > 0.05) in the cecal flora composition between the MRS group and the SALP group, between the SALP + L. gasseri group and the SALP group, and between the SALP + L. gasseri group and the SALP + Heat Killed L. gasseri group.

[0107] 10. On the 22nd day of the experiment, Shanghai Majorbio Bio - Pharm Technology Co., Ltd. was commissioned to analyze the differential microorganisms among groups by linear discriminant analysis. The results are as Figure 8 shown.

[0108] As Figure 8As can be seen from a in [reference], in the cecal microbiota of laying hens in the control group, compared with the SALP+L. gasseri group, Faecalibacterium, Bacteroidales, Blautia, Butyricicoccus, Christensenellaceae, Coriobacteriaceae, Oscillibacter, Enorma, Ruminococcaceae, Parabacteroides, CHKCI002, Alloprevotella, UCG-009, NK4A214 group, Barnesiellaceae, and Eubacterium coprostanoligenes were enriched.

[0109] And as can be seen from Figure 8 b in [reference], when comparing the MRS group with the SALP group, Lachnospiraceae, Lachnospiraceae FCS020, Subdoligranulum, Erysipelotrichaceae, Oscillibacter, Alloprevotella, Megamonas, GCA-900066575, and Enterococcus were enriched.

[0110] As can be seen from Figure 8As can be seen from c in [reference], compared with the SALP+L. gasseri group, the SALP group was enriched in the genera Erysipelatoclostridium, Eubacterium, Clostridium, and Veillonella; while compared with the SALP group, the SALP+L. gasseri group had an increased relative abundance of the following genera: Ruminococcus torques, Christensenellaceae, Clostridia vadinBB60, Ruminococcaceae, UCG-010, NK4A214, Eubacterium, Monoglobus, Saccharimonadales, Barnesiellaceae, and Ruminococcus.

[0111] As can be seen from Figure 8 d in [reference], by comparing the cecal microbiota of the SALP+L. gasseri group and the SALP+Heat killed L. gasseri group, it was found that the relative abundances of Enterococcus, Clostridium sensu stricto, and Clostridium innocuum were upregulated in the SALP+L. gasseri group compared with the SALP+Heat killed L. gasseri group; while the heat-inactivated Lactobacillus gasseri treatment group was enriched in Bacteroidales, Desulfovibrio, Barnesiella, Tannerellaceae, Barnesiellaceae, Shuttleworthia, Odoribacter, UCG-010, Lachnospiraceae UCG-002, and Rhodospirillales compared with the Lactobacillus gasseri group.

[0112] 11. On the 22nd day of the experiment, Shanghai Majorbio Bio-Pharm Technology Co., Ltd. was commissioned to detect the transcriptomics of the shell gland part of the oviduct in the SALP+L. gasseri group compared with the SALP group, and the results are as Figure 9 shown.

[0113] As can be seen fromFigure 9 It can be seen that a total of 21,174 expressed genes were detected in this analysis, including 20,687 known genes and 487 novel genes; a total of 78,346 expressed transcripts were detected, including 66,235 known transcripts and 12,111 novel transcripts. Based on the quantification results of expression levels, differential gene analysis between groups was performed, and a total of 230 genes (DEGs) with differential expression (P<0.05) were obtained between the two groups, including 77 up-regulated DEGs and 153 down-regulated DEGs.

[0114] 12. On the 22nd day of the experiment, a comparison with the GO database was commissioned from Shanghai Majorbio Bio-pharm Technology Co., Ltd., and the results are as Figure 10 shown.

[0115] Figure 10 a in Figure 10As shown in b of [reference], the oviduct shell gland tissue of the SALP+L. gasseri group was significantly enriched (P<0.05) compared with the SALP group in cell components such as extracellular matrix, external encapsulating structure, collagen containing extracellular matrix, fibrinogen complex, basement membrane, extracellular region, etc., as well as biological processes related to genes such as mononuclear cell migration, regulation of biological quality, cation homeostasis, inorganic ion homeostasis, ion homeostasis, lymphocyte migration, positive regulation of cell-substrate adhesion, defense response, substrate-dependent cell migration, regulation of cytosolic calcium ion concentration, reactive oxygen species metabolic process, cellular cation homeostasis, leukocyte migration, platelet activation, etc.Among them, genes related to biological processes such as defense response, response to other organism, response to external biotic stimulus, defense response to other organism, response to biotic stimulus, biological process involved in interspecies interaction between organisms, immune system process, immune response, response to external stimulus, chemotaxis, taxis, locomotion, response to bacterium, innate immune response, inflammatory response, response to stress, response to stimulus, myeloid cell differentiation, cell chemotaxis, lymphocyte chemotaxis were significantly up-regulated; significantly down-regulated (... Figure 10Molecular functions such as c) ion channel inhibitor activity, macrolide binding, FK506 binding, etc., cellular components such as integral component of presynaptic membrane, collagen trimer, basement membrane, fibrinogen complex, collagen-containing extracellular matrix, external encapsulating structure, extracellular matrix, etc., and genes related to biological processes such as endodermal cell differentiation, focal adhesion assembly, extracellular matrix organization, extracellular structure organization, external encapsulating structure organization, regulation of multicellular organismal process, positive regulation of cell-substrate adhesion, substrate-dependent cell migration, platelet activation, regulation of cytosolic calcium ion concentration, etc.

[0116] 13. On the 22nd day of the experiment, the transcriptome of the fallopian tube was compared with the KEGG database, and the results are as Figure 11 shown.

[0117] Figure 11It can be seen that compared with the SALP group, the oviduct shell gland tissue of the SALP+L.gasseri group significantly (P<0.05) enriched genes related to pathways such as Amoebiasis, Coronavirus disease-COVID-19, Rheumatoid arthritis, Staphylococcus aureus infection, Viral myocarditis, Asthma, PI3K-Akt signaling pathway, Transcriptional misregulation in cancer, Phospholipase D signaling pathway, Dilated cardiomyopathy, Allograft rejection, Epstein-Barr virus infection, NF-kappa B signaling pathway, African trypanosomiasis, IL-17 signaling pathway, Leishmaniasis, Hematopoietic cell lineage, Calcium signaling pathway, B cell receptor signaling pathway, Intestinal immune network for IgA production, etc. Among them, ((b)) Epstein-Barr virus infection, Rheumatoid arthritis, Coronavirus disease-COVID-19, NF-kappa B signaling pathway, Asthma, Staphylococcus aureusinfection), Viral myocarditis, Amoebiasis, Allograft rejection, Phospholipase D signaling pathway, Dilated cardiomyopathy, Leishmaniasis, Intestinal immunenetwork for IgA production, B cell receptor signaling pathway, African trypanosomiasis, Yersinia infection, Autoimmune thyroid disease, Hematopoietic cell lineage, Natural killer cell mediated cytotoxicity, Systemic lupus erythematosus and other signal pathway related gene levels; significantly down-regulated ((c)) Protein digestion and absorption, ECM-receptor interaction, Butanoate metabolism, Focal adhesion, Relaxin signaling pathway, AGE-RAGE signaling pathway in diabetic complications, Amoebiasis, Taste transduction, Platelet activation, Small cell lung cancer, Bacterial invasion of epithelial cells, PI3K-Akt signaling pathway, Estrogen signaling pathway, VascularGenes related to signaling pathways such as smooth muscle contraction, Coronavirus - COVID - 19, Fluid shear stress and atherosclerosis, Longevity regulating pathway - multiple species, Complement and coagulation cascades, Neuroactive ligand - receptor interaction, Prolactin signaling pathway, etc.

[0118] From the above results, it can be seen that Lactobacillus gasseri can exert an anti - inflammatory effect by inhibiting the PI3K / AKT signaling pathway.

[0119] The related gene expressions of the PI3K / AKT signaling pathway were further detected in the shell gland, and the results are as Figure 12 shown. In the case of salpingitis, the mRNA expression level of PI3K in the shell gland of the oviduct in the SALP group was significantly higher than that in the MRS group (P < 0.05), indicating the activation of PI3K. After treatment with Lactobacillus gasseri and its heat - killed bacterial solution, it was extremely significantly decreased (P < 0.01), indicating that Lactobacillus gasseri inhibited the activation of PI3K. The expression level of AKT increased in the SALP group, and Lactobacillus gasseri also showed a phenomenon of decreasing the AKT expression level, but the result was not significantly different (P > 0.05). The expression level of the PI3K / AKT downstream gene - IL - 17A increased significantly in the SALP group and decreased significantly in the SALP + L. gasseri group and the SALP + Heat - Killed L. gasseri group (P < 0.05). This also shows that Lactobacillus gasseri can exert an anti - inflammatory effect by inhibiting the PI3K / AKT signaling pathway.

[0120] Figure 12It can be seen that in the case of salpingitis, the mRNA expression level of PI3K in the shell gland part of the oviduct in the SALP group was significantly higher than that in the MRS group (P<0.05), indicating the activation of PI3K. After treatment with Lactobacillus gasseri suspension and heat-killed Lactobacillus gasseri suspension, it was extremely significantly decreased (P<0.01), indicating that Lactobacillus gasseri inhibited the activation of PI3K. The expression level of AKT increased in the SALP group, and Lactobacillus gasseri also showed a phenomenon of reducing the expression level of AKT, but the result was not significantly different (P>0.05). The expression level of the PI3K / AKT downstream gene - IL-17A increased significantly in the SALP group and decreased significantly in the SALP+L. gasseri group and the SALP+HeatKilledL. gasseri group (P<0.05).

[0121] The above results indicate that Lactobacillus gasseri can effectively alleviate the decline in production performance and egg quality of laying hens with salpingitis caused by phenol glue paste by inhibiting the PI3K / AKT pathway, and improve oviduct damage and inflammatory infiltration, down-regulate the content of pro-inflammatory cytokines in serum and shell gland tissue, and their expression levels in the shell gland part and ampulla, and up-regulate the content of IL-10. Lactobacillus gasseri can also improve the cecal flora composition and inhibit pro-inflammatory related pathways such as PI3K / AKT. Compared with live bacteria, heat-killed suspension is more effective in alleviating salpingitis, suggesting that Lactobacillus gasseri can secrete a certain substance to play an anti-inflammatory role.

[0122] It should be noted that all the omics experiments in the above examples were commissioned to Shanghai Majorbio Bio-pharm Technology Co., Ltd. to complete, and the section experiments were commissioned to Wuhan Servicebio Technology Co., Ltd. to complete.

[0123] When the numerical ranges are involved in the present invention, it should be understood that any one of the two endpoints of each numerical range and any value between the two endpoints can be selected. To prevent repetition, the preferred embodiments of the present invention are described.

[0124] Although the preferred embodiments of the present invention have been described, once those skilled in the art learn the basic creative concept, additional changes and modifications can be made to these embodiments, and all such changes and modifications fall within the scope of the present invention.

Claims

1. Use of Lactobacillus formatum in the preparation of a medicament for improving the intestinal health and oviduct inflammation of laying hens, characterized in that, The Lactobacillus in the format is the Lactobacillus strain BNCC339385 in the format.

2. The application according to claim 1, wherein The drug uses the culture of the Lactobacillus in the format as the active ingredient and is supplemented with pharmaceutically acceptable excipients.

3. The application according to claim 2, characterized in that, The preparation method of the culture of the Lactobacillus in the format includes the following steps: Inoculate the Lactobacillus formatum strain BNCC339385 into a solid MRS medium, and after culturing at a constant temperature of 36°C to 38°C for 23 hours to 25 hours, pick single colonies and then perform purification culture. After the colony count in the bacterial liquid reaches 10 9 CFU / mL or more, centrifuge the bacterial liquid and collect the supernatant to obtain the culture of the Lactobacillus formatum strain.

4. The application according to claim 3, wherein Inoculate the activated Lactobacillus strain BNCC339385 in the format into the solid MRS medium. The activation method is: thaw the Lactobacillus strain BNCC339385 in the format and inoculate it into the liquid MRS medium according to a volume ratio of 1:24 to 26 for activation culture.

5. The application according to claim 4, characterized in that The conditions for the activation culture are: culture at 36°C to 38°C for 23 hours to 25 hours.

6. The application according to claim 3, characterized in that The conditions for the purification culture are: shake culture at 36°C to 38°C.

7. The application according to claim 2, characterized in that The drug is a solution; the excipient is a solvent; The solution is obtained by compounding the culture of the Lactobacillus in the format and the solvent.

8. The application according to claim 7, wherein The solvent is any one of DMSO, water, ethanol, and glycerol.

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