Lactobacillus reuteri for improving ovarian function of laying hens and application of lactobacillus reuteri

Abstract

The invention discloses lactobacillus reuteri, which is characterized in that the lactobacillus reuteri can be used for preparing lactobacillus reuteri, and the lactobacillus reuteri can be used for preparing lactobacillus reuteri. The lactobacillus reuteri strain is preserved in the General Microbiological Culture Collection Center (GCMCC) of the China Committee for Culture Collection of Microorganisms, and the preservation number is CGMCC No. 34177; the strain can penetrate through the intestinal tract-fallopian tube-ovary of a laying hen, improve microbiota of each part and improve the ovarian function of the laying hen. The invention also discloses an application of the feed containing Lactobacillus reuteri in the feed for increasing the laying rate of laying hens. Experimental results show that the laying rate of laying hens is obviously increased.

Description

Technical Field

[0001] The present invention relates to the technical field of animal husbandry, and particularly relates to a Lactobacillus mucosae Royae that can penetrate the intestine - oviduct - ovary, improve the microbiota of each part, and thus enhance the ovarian function of laying hens, and its application. Background Art

[0002] As laying hens age, oxidative stress in their bodies intensifies, and their antioxidant and immune functions decline, leading to the aging of the ovary and oviduct, the decline of physiological functions, poor follicle development, an increase in atretic follicles, and a decrease in the ovulation rate, which will in turn affect the egg production rate. The late peak egg production period is the turning point for the egg production rate of laying hens to decline from high to low. Therefore, improving the ovarian function of laying hens and delaying ovarian decline are particularly important for prolonging the egg production performance in the late peak egg production period of laying hens and increasing breeding benefits. The microbiota of the reproductive system (including the intestine, oviduct, and ovary) of laying hens is closely related to the health and physiological functions of laying hens. Therefore, finding a safe and effective method to improve the microbiota of the reproductive system of laying hens and enhance the ovarian function of laying hens is of great significance.

[0003] Lactobacillus mucosae Royae is a common probiotic with various functions such as regulating the intestinal microbiota and enhancing immunity. Therefore, the present invention provides a Lactobacillus mucosae Royae that can penetrate the intestine - oviduct - ovary of laying hens and improve the microbiota of each part, thereby significantly enhancing the ovarian function of laying hens. Summary of the Invention

[0004] The purpose of the present invention is to provide a Lactobacillus mucosae Royae that can penetrate the intestine - oviduct - ovary of laying hens and improve the microbiota of each part, thereby significantly enhancing the ovarian function of laying hens.

[0005] A Lactobacillus mucosae Royae ( Limosilactobacillus reuteri ), characterized in that the strain preservation number is CGMCC NO.34177, and the strain was preserved in the China General Microbiological Culture Collection Center on April 14, 2025; Preservation address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. This strain can penetrate the intestine - oviduct - ovary of laying hens, improve the microbiota of each part, and enhance the ovarian function of laying hens.

[0006] The strain of the present invention can colonize in the intestine of laying hens and enter the oviduct through the cloaca; specifically, the strain reaches the oviduct and ovary through the cloaca and colonizes in the oviduct and ovary; at the same time, it improves the microbiota structure of the intestine, oviduct, and ovary, increases the number of beneficial microorganisms, and reduces the number of harmful microorganisms. The physiological and biochemical characteristics of this Lactobacillus mucosae Royae are as follows: After being cultured on a solid medium, Lactobacillus mucosae Royae forms round, convex, and smooth - surfaced colonies.

[0007] Lactobacillus mucosae Roy has the following characteristics simultaneously: 1. It can colonize in the intestine of laying hens and enter the oviduct through the cloaca.

[0008] 2. It can reach the oviduct and ovary through the cloaca and colonize in the oviduct and ovary.

[0009] Improve the microbiota structure in the intestine, oviduct and ovary, increase the number of beneficial microorganisms, and reduce the number of harmful microorganisms.

[0010] 3. Regulate the ovarian microbiota of laying hens and improve the ovarian function of laying hens.

[0011] The present invention also provides a fermented feed containing the above Lactobacillus mucosae Roy.

[0012] The present invention also discloses the application of Lactobacillus mucosae Roy in regulating the ovarian microbiota of laying hens and improving the ovarian function of laying hens. In particular, its application in feed for improving the egg production rate of laying hens containing Lactobacillus mucosae Roy. Experimental results show that the egg production rate of laying hens has increased significantly.

[0013] The Lactobacillus mucosae Roy of the present invention can penetrate the intestine-oviduct-ovary of laying hens, improve the microbiota of each part, and thus significantly improve the ovarian function of laying hens. Compared with the traditional methods for improving the ovarian function of laying hens, it has the following advantages simultaneously: (1) High safety: Lactobacillus mucosae Roy is a natural probiotic, which is harmless to laying hens and the environment and will not cause problems such as drug residues.

[0014] (2) Regulate the microbiota of the reproductive system: Lactobacillus mucosae Roy can colonize in the reproductive system of laying hens, increase the number of beneficial bacteria, and inhibit the growth of harmful bacteria, thus improving the balance of the microbiota in the reproductive system.

[0015] (3) Improve ovarian function: By regulating the microbiota, this strain can increase the secretion level of ovarian hormones and the number of dominant ovarian follicles, and thus improve the ovarian function of laying hens. Description of the Drawings

[0016] Figure 1 Composition of the microbiota at the phylum level in the reproductive system of laying hens; Figure 2 Composition of the microbiota at the genus level in the reproductive system of laying hens; Figure 3SourceTracker analysis of the microbial community in the reproductive system of laying hens; Note: Figure A is the SourceTracker analysis of the control group; Figure B is the SourceTracker analysis of the fermented feed group; Figure 4 The intestinal microbiota is transmitted to the ovary through the oviduct; Figure 5 Morphological structure of the ovarian tissue of laying hens; Note: Figure A is the hematoxylin / eosin staining of the ovary, scale bar = 900 µm; Figure B is the follicle morphology; Figure 6 mRNA expression of genes related to ovarian reproductive function; Figure 7 mRNA expression of genes related to ovarian apoptosis function; Figure 8 mRNA expression of genes related to ovarian antioxidant function; Figure 9 mRNA expression of genes related to ovarian immune function. Detailed implementation manners

[0017] The present invention will be described below through specific implementation examples. Unless otherwise specified, the technical means used in the present invention are all methods well known to those skilled in the art. In addition, the implementation examples should be understood as illustrative and not limiting the scope of the present invention. The essence and scope of the present invention are only defined by the claims. For those skilled in the art, various changes or modifications to the material components and dosages in these implementation examples also fall within the protection scope of the present invention. The feeds and various raw materials used in the present invention are all commercially available. Examples

[0018] The Lactobacillus mucosae used in the present invention ( Limosilactobacillus reuteri ) is a strain preserved in the Animal Nutrition Laboratory of Tianjin Agricultural University (the strain was deposited in the China General Microbiological Culture Collection Center on April 14, 2025, and the strain deposit number is CGMCC NO. 34177).

[0019] The Lactobacillus mucosae was inoculated into MRS broth medium and cultured at 37 °C for 10 - 12 hours. When the bacterial liquid concentration reached 10 8 -10 9 CFU / mL, it can be used for subsequent experiments or preservation. The formula of the MRS broth medium is as follows: peptone: 10 g / L, beef powder: 10 g / L, yeast powder: 5 g / L, glucose: 20 g / L, magnesium sulfate: 0.1 g / L, sodium acetate: 5 g / L, sodium citrate: 2 g / L, potassium dihydrogen phosphate: 2 g / L, manganese sulfate: 0.05 g / L, Tween 80: 1 g / L.

[0020] 1. Raw material preparation: The fermentation substrate is a corn-soybean meal-based diet, including 61.77% corn, 24.0% soybean meal, 8.30% limestone powder, 2.0% wheat bran, 1.5% calcium hydrogen phosphate, 1.0% soybean oil, 0.33% salt, 0.1% DL-methionine, and 1.0% premix (commercially available, Tianjin Modern Tianjiao Aquatic Feed Co., Ltd.).

[0021] 2. Feed-water mixing: According to the feed-water ratio of 1:1, sterile water is added to the feed supplemented with sucrose and enzyme preparation, and stirred thoroughly to make the feed and water evenly mixed.

[0022] 3. Inoculation with Lactobacillus mucosae: The cultured Lactobacillus mucosae bacterial liquid is inoculated into the feed treated above at 6% of the feed weight and mixed thoroughly.

[0023] 4. Fermentation condition control: The inoculated feed raw materials are packed into fermentation bags, sealed, and placed under suitable temperature and humidity conditions for fermentation. The fermentation temperature is controlled at 33 °C, and the fermentation time is 42 hours. The fermented feed is obtained after the fermentation time ends. Example

[0024] 112 laying hens at the late peak laying period of 56 weeks old are selected. The laying hens are randomly divided into 2 groups, with 4 replicates in each group and 14 in each replicate. The pre-trial lasts for 1 week and the formal trial lasts for 8 weeks. The test groups are the control group and the fermented feed group, namely: Control group: Basic feed; Fermented feed group: 70% basic diet + 30% fermented feed.

[0025] The composition and nutrient levels of the basic diet are shown in Table 1 Table 1 Composition and nutrient levels of the basic diet Items Contents corn 61.77 Soybean meal 24.00 Limestone 8.30 Wheat bran 2.00 Dicalcium phosphate 1.50 Soybean oil 1.00 NaCl 0.33 DL-Methionine 0.10 <![CDATA[Premix 1 > 1.0 Summation 100.00 <![CDATA[Nutrient levels 2 > MC / Kg 2.69 Crude protein (%) 17.87 Lysine (%) 0.80 Methionine (%) 0.35 Ca (%) 4.04 TP (%) 0.75 1The premix provides per kilogram of the diet: VA 8,000 IU, VD3 1,600 IU, VE 50 IU, VK 3 mg, VB1 3 mg, VB2 10 mg, pantothenic acid 10 mg, niacin 40 mg, pyridoxine 10 mg, biotin 0.15 mg, folic acid 0.8 mg, vitamin B12 0.02 mg, choline 1,000 mg, copper 10 mg, iron 70 mg, zinc 90 mg, manganese 70 mg, iodine 1.0 mg, selenium 0.30 mg. The premix contains a compound enzyme preparation. The main components and activities of the compound enzyme preparation are as follows: xylanase ≥ 4,000 U / g, β-glucanase ≥ 6,000 U / g, protease ≥ 10,000 U / g, amylase ≥ 6,000 U / g, cellulase ≥ 1,500 U / g, arabinoxylanase = 1,000 U / g.

[0026] 2 Except for the metabolizable energy, other indicators are measured values (commercially available, Tianjin Modern Tianjiao Aquatic Feed Co., Ltd.).

[0027] Sample collection On the last day of the 8th week of the experiment, after fasting the laying hens for 12 hours, 1 laying hen (close to the average body weight) was selected and sacrificed from each replicate (4 in each group). One portion of the laying hen ovaries was separated and stored in 4% paraformaldehyde for morphological determination; 2 tubes of ovarian gene samples were collected and placed in EP tubes, snap-frozen in liquid nitrogen and then transferred to -80 °C for storage. Cecal contents were collected for microbial 16s sequencing. Microbial samples from the cloaca, vagina, uterus, magnum, and ovaries were collected using sterile swabs, and then the swabs were snap-frozen in liquid nitrogen and transferred to -80 °C for storage, for microbial 16s sequencing.

[0028] Determination of the microbiota of the reproductive system of laying hens The microbial samples of the cecal contents, cloaca, vagina, uterus, magnum, and ovaries were sent to Beijing Biomarker Technologies Co., Ltd. for 16s rRNA sequencing.

[0029] Ovarian function determination (1) Ovarian morphological structure The ovarian samples fixed in 4% paraformaldehyde solution were dehydrated, embedded, sectioned into 5 μm cross-sections, then stained with hematoxylin-eosin (H.E), and subsequently, ovarian morphology was observed.

[0030] (2) Expression of mRNA of genes related to ovarian function Genes related to ovarian function were determined using real-time fluorescence quantitative method. The genes measured included: genes related to reproductive function: anti-Müllerian hormone ( AMH ), luteinizing hormone receptor ( LHR), follicle-stimulating hormone receptor ( FSHR ), and estrogen receptor 1 ( ESR1 ); Genes related to antioxidant function: nuclear factor E2-related factor 2 ( Nrf2 ), heme oxygenase-1 ( HO-1 ), superoxide dismutase ( SOD1 ), catalase ( CAT ), and glutathione peroxidase 3 ( GPX3 ); Genes related to apoptosis factors: B-cell lymphoma-2 ( Bcl-2 ), caspase3 ), caspase8 ; Genes related to immune function: interleukin-10 ( IL-10 ), interleukin-6 ( IL-6 ), tumor necrosis factor-α ( TNF-α ), and interferon-γ ( IFN-γ ).

[0031] From Figure 1 and Figure 2 it can be seen that the dominant phyla of the cecal microbiota of laying hens in the control group and the fermented feed group were both Bacteroidota and Firmicutes at the phylum level. At the genus level, the dominant genera of laying hens in the control group were Bacteroides ( Bacteroides ), and Rikenellaceae RC9 gut group ( Rikenellaceae_RC9_gut_group ). The dominant genera of laying hens in the fermented feed group were Bacteroides, Lactobacillus reuteri ( Limosilactobacillus ), and Rikenellaceae RC9 gut group.

[0032] The dominant phyla of the cloacal microbiota of laying hens in the control group and the fermented feed group were both Firmicutes and Bacteroidota, with no significant difference. At the genus level, compared with the control group, the relative abundance of Bacteroides increased in the fermented feed group, while the relative abundance of Actinomyces ( Actinomyces ) decreased.

[0033] There were differences in the vaginal microbiota composition between the control group and the fermented feed group of laying hens. Specifically, at the phylum level, compared with the control group, the relative abundance of Firmicutes increased and the relative abundance of Actinobacteria decreased in the fermented feed group. At the genus level, the relative abundances of Lactobacillus, Romboutsia ( Romboutsia ), and Lactobacillus reuteri were higher in the fermented feed group, while Corynebacterium ( Corynebacterium ) was the dominant genus in the control group.

[0034] There were differences in the uterine microbiota composition between the control group and the fermented feed group of laying hens. At the phylum level, the dominant phyla of the uterine microbiota in the control group of laying hens were Actinobacteria, Firmicutes, and Proteobacteria; while in the fermented feed group, Firmicutes and Bacteroidetes were the dominant phyla. At the genus level, compared with the control group, the fermented feed group increased the relative abundances of Bacteroides, Lactobacillus reuteri, and Rikenellaceae RC9 gut group, and decreased the relative abundance of Brevibacillus ( Brevibacillus ).

[0035] The microbiota compositions of the magnum of the two groups of laying hens were different. At the phylum level, the fermented feed group increased the relative abundance of Bacteroidetes and decreased the relative abundance of Actinobacteria. At the genus level, the fermented feed group of laying hens increased the relative abundances of Bacteroides and Rikenellaceae RC9 gut group; and decreased the relative abundance of Brevibacillus.

[0036] The microbiota compositions of the ovaries of laying hens were significantly different. At the phylum level, compared with the control group, the fermented feed group significantly increased the relative abundance of Firmicutes and decreased the relative abundance of Bacteroidetes. At the genus level, compared with the control group, the fermented feed group increased the relative abundances of Lactobacillus, Lactobacillus reuteri, and Romboutsia, and decreased the relative abundances of Corynebacterium and Bacteroides. The fermented feed of the present invention can improve the microbiota compositions of the cecum, vagina, uterus, magnum, and ovaries of laying hens, and the abundance of Lactobacillus reuteri in each part increases.

[0037] SourceTracker analysis was used to reveal that cecal microbiota affects ovarian microbiota through transmission via the oviduct. Figure 3 A is the SourceTracker analysis of the control group. In the laying hens of the control group, when the ovary was the target sample, a total of 52.25% of the microbiota originated from the reproductive tract, among which the magnum microbiota accounted for 27.99%, the uterine microbiota accounted for 11.96%, the vaginal microbiota accounted for 10.3%, and the cloacal microbiota accounted for 2%; while the cecal microbiota contributed 7.06%, and other microbiota contributed 40.69%. When the magnum was the target sample, the microbiota of the magnum mainly originated from the uterine microbiota (55.13%). When the uterus was the target sample, its reproductive tract microbiota contributed 59.04%. When the vagina was the target sample, the main sources of the vaginal microbiota were the cloacal and cecal microbiota, which contributed 37.37% and 26.24% respectively. When the cloaca was the target sample, the vaginal and cecal microbiota were the main contributors. When the cecum was the target sample, the cloacal and vaginal microbiota were the main contributors.

[0038] The laying hens in the fermented feed group also presented similar results ( Figure 3B). When the ovary was used as the target sample, 55.31% of the microbiota originated from the reproductive tract (25.6% from the magnum, 12.71% from the uterus, 12.03% from the vagina, and 4.97% from the cloaca), and 10.79% of the microbiota originated from the cecum. When the vagina was used as the target sample, the main sources of the vaginal microbiota were the microbiota of the cloaca and cecum, which contributed 31.57% and 24.21%, respectively. When the cloaca and cecum were used as the target samples, the microbiota of the vagina and cecum were the main contributors. When the cecum was used as the target sample, the microbiota of the cloaca and vagina were the main contributors. Through SourceTracker analysis, it was found that the microbiota of the laying hen oviduct was susceptible to the influence of the microbiota from the intestine and cloaca, while the microbiota of the ovary was susceptible to the influence of the oviduct microbiota.

[0039] From the above results, it was found that the abundance of Lactobacillus reuteri in the cecum, oviduct, and ovary microbiota of laying hens fed with fermented feed increased, while the abundance of Lactobacillus reuteri in the control group was relatively low. The cecal microbiota (Lactobacillus reuteri) of laying hens appeared in the oviduct and ovary, indicating that Limosilactobacillus intestinal microbiota could pass through the oviduct to reach the ovary. By ingesting fermented feed, the fermenting strain Lactobacillus reuteri colonized in the gastrointestinal tract of laying hens, and the intestinal microbiota affected the oviduct microbiota. Therefore, fermented feed could regulate the intestinal, oviduct, and ovary microbiota of laying hens through Lactobacillus reuteri. Thus, the intestine-oviduct-ovary was used as the microbial transmission model for laying hens. The transmission path of Lactobacillus reuteri is shown as

[0040] The terminal parts of the reproductive tract and digestive tract of poultry are both the cloaca, which forms the basis for the intestinal microbiota to affect the reproductive tract microbiota, and the cloaca is the bridge connecting the two. Through SourceTracker analysis, it was found that the microbiota of the laying hen oviduct was susceptible to the influence of the microbiota from the intestine and cloaca, while the microbiota of the ovary was susceptible to the influence of the oviduct microbiota. The relatively high abundance of Lactobacillus and Lactobacillus reuteri in the reproductive tract of laying hens was because the fermenting strain of the fermented feed in this study was Lactobacillus reuteri. By ingesting fermented feed every day, Lactobacillus reuteri colonized in the intestine of laying hens, affecting the microbiota of the oviduct and ovary, resulting in a relatively high abundance of this genus in the reproductive tract. Figure 4 The fermenting strain Lactobacillus reuteri of the present invention will reach the oviduct through the cloaca and then reach the ovary.

[0041] The morphological structure of the ovary is shown as

[0042] Figure 5 ​As shown. From the perspective of follicle morphology, dominant follicles (>12 mm in diameter) and pre-rank yellow follicles (6-12 mm) were densely arranged in the ovaries of the fermented feed group; while the number of follicles in the control group was less ( Figure 5 B). The follicle development on the H.E. stained sections of the ovaries in the fermented feed group was more mature than that in the control group, and there were more atretic follicles on the ovaries of the control group ( Figure 5 A).

[0043] (1)mRNA expression of genes related to the reproductive function of laying hen ovaries The effects of fermented feed on the mRNA expression of genes related to the reproductive function of laying hen ovaries are as Figure 6 shown. The mRNA expression levels of AMH and FSHR in the ovaries of the fermented feed group were significantly higher than those in the control group. It shows that the fermented feed of Lactobacillus reuteri can improve the quality and quantity of dominant follicles in the ovaries of laying hens and promote the maturation of follicles.

[0044] The effects of fermented feed on the mRNA expression of genes related to apoptosis factors in laying hen ovaries are as Figure 7 shown. The mRNA expression level of the anti-apoptosis gene Bcl-2 in the ovaries of laying hens in the fermented feed group was significantly higher than that in the control group; the mRNA expression levels of the pro-apoptosis genes caspase3 and caspase8 were both significantly lower than those in the control group. It indicates that the fermented feed of Lactobacillus reuteri can inhibit cell apoptosis and increase the number of normally developing follicles.

[0045] The effects of fermented feed on the mRNA expression of genes related to the antioxidant function of laying hen ovaries are as Figure 8 shown. The mRNA expression levels of Nrf2 , SOD1 , CAT , GPX3 and HO-1 in the ovaries of the fermented feed group were significantly higher than those in the control group. It shows that the fermented feed of Lactobacillus reuteri can improve the antioxidant capacity of the ovaries, relieve the oxidative stress of the ovaries, and thus delay the decline of ovarian function.

[0046] The effects of fermented feed on the mRNA expression of genes related to the immune function of laying hen ovaries are as Figure 9 shown. The mRNA expression level of the anti-inflammatory factor IL-10 in the ovaries of the fermented feed group was significantly higher than that in the control group, and the mRNA expression levels of the pro-inflammatory factors IL-6 and TNF-α in the ovaries of the control group were significantly higher than those in the fermented feed group. It shows that the fermented feed of Lactobacillus reuteri can improve the immune capacity of the ovaries and reduce the impact of inflammation on the ovaries.

[0047] Conclusion: In summary, Lactobacillus mucosae can reach the fallopian tubes and ovaries from the intestine through the cloaca, improve the microbial community structure in the intestine, fallopian tubes and ovaries, increase the number of beneficial microorganisms, reduce the number of harmful microorganisms, and can improve the antioxidant capacity and immune capacity of the ovaries, delay the decline of ovarian function, inhibit apoptosis, reduce follicular atresia, thereby improving ovarian function.

[0048] Effect of Fermented Feed on Laying Rate of Laying Hens As can be seen from Table 2, the fermented feed containing Lactobacillus mucosae can improve the laying rate of laying hens.

[0049]

[0050] No letter or the same letter in the superscript of peer data indicates no significant difference (P>0.05), and different lowercase letters indicate significant difference (P<0.05).

Claims

1. A Lactobacillus reuteri mucilage Limosilactobacillus reuteri ), characterized in that The Limosilactobacillus reuteri strain is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms (GCMCC), with the deposit number CGMCC No. 34177.

2. The Lactobacillus mucosae Royae according to claim 1, characterized in that, The strain can colonize in the intestine of laying hens and enter the oviduct through the cloaca; specifically, the strain reaches the oviduct and ovary through the cloaca and colonizes in the oviduct and ovary; at the same time, it improves the microbial community structure in the intestine, oviduct and ovary, increases the number of beneficial microorganisms and reduces the number of harmful microorganisms.

3. Use of the Limosilactobacillus reuteri according to claim 1 for regulating the ovarian microbiota of laying hens and improving the ovarian function of laying hens.

4. Use of a feed containing the Limosilactobacillus reuteri according to claim 1 in a feed for increasing the egg production rate of laying hens.

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