A probiotic compound for improving the fertility of female animals and its application

Abstract

The present invention relates to the field of probiotics, and in particular to a probiotic compound and application thereof for improving the fertility of female animals. Lactobacillus reuteri ) LRY15, with a preservation number of CGMCC No.30814, probiotic compound LRY15 and wolfberry oligosaccharides and chestnut oligosaccharides. The compound can better colonize Lactobacillus reuteri LRY15 in animals, and has a stronger tolerance to artificial bile salts, artificial gastric acid, and artificial intestinal fluid; it can better improve female intestinal health, better improve intestinal immunity, increase female animal egg quality, in vitro fertilization rate, pregnancy rate, and live births; the compound can improve female animal body metabolism, increase beneficial metabolites, thereby increasing ovarian beneficial metabolites such as melatonin and retinoic acid (regulating the start of gametogenesis), thereby improving female animal egg quality and fertility.

Description

Technical Field

[0001] The invention relates to the field of probiotics, and in particular to a probiotic compound for improving the fertility of female animals and its application. Background Art

[0002] Female ovarian function is one of the important key factors affecting their reproductive ability, and female ovarian function is difficult to regulate. There is currently no solution to treat female infertility by effectively improving egg quality and the number of effective eggs. Intestinal flora is very important to the health of animals. In addition, existing technical studies have found that intestinal flora is closely related to female reproductive health. It has been found that harmful intestinal flora reduces the egg quality of female animals, thereby leading to a decrease in the reproductive ability of female animals. Little is known about whether beneficial bacteria improve egg quality and increase the number of effective eggs. In addition, it is unknown whether intestinal flora can better improve the egg quality and fertility of female animals together with some known prebiotics such as chestnut oligosaccharides and wolfberry oligosaccharides. Summary of the invention

[0003] The purpose of the present invention is to provide a Lactobacillus reuteri LRY15.

[0004] The purpose of the present invention is to provide a probiotic compound for improving the fertility of female animals.

[0005] Another object of the present invention is to provide the use of the above-mentioned probiotic compound for improving the fertility of female animals.

[0006] According to the present invention, Lactobacillus reuteri ( Lactobacillus reuteri )LRY15, the deposit number is CGMCC No. 30814, and the deposit date is May 30, 2024.

[0007] The probiotic compound for improving the fertility of female animals according to the present invention comprises the above-mentioned Lactobacillus reuteri ( Lactobacillus reuteri )LRY15 and wolfberry oligosaccharides (LOS) and chestnut oligosaccharides (COS).

[0008] According to the probiotic compound for improving the fertility of female animals of the present invention,

[0009] According to the probiotic compound for improving the fertility of female animals of the present invention, the compound can be a liquid bacterial agent or a solid bacterial agent, and is prepared by conventional technical means and by adding auxiliary materials permitted in the field of microbial preparations.

[0010] The present invention provides the use of the above-mentioned probiotic compound for improving the fertility of female animals for preparing preparations for the following purposes:

[0011] (1) Improve the intestinal health of female animals;

[0012] (2) Improve the blood and ovarian metabolome of female animals;

[0013] (3) Increase the quality of eggs in female animals;

[0014] (4) Increase the in vitro fertilization rate of female animal eggs;

[0015] (5) Improving the mating success rate and pregnancy rate of female animals; and / or

[0016] (6) Increase the number of live piglets born by female animals.

[0017] The composite agent of the present invention can better enable Lactobacillus reuteri LRY15 to colonize in the animal body, and has a stronger tolerance to artificial bile salts, artificial gastric acid, and artificial intestinal fluid; it can better improve the intestinal health of females, better enhance intestinal immunity, and increase the egg quality, in vitro fertilization rate, pregnancy rate, and live birth number of female animals; the composite agent can improve the metabolism of female animals, increase beneficial metabolites, thereby increasing beneficial ovarian metabolites such as melatonin and retinoic acid (regulating the initiation of gametogenesis), thereby improving the egg quality and fertility of female animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The growth curves of Lactobacillus reuteri LRY15 (and control strain LRY09) in the composite of the present invention under different conditions (MRS medium);

[0019] Figure 2 The weight changes of the female mice in the experiment in Example 3 of the present invention during the 35-day experiment period are shown;

[0020] Figure 3 The total number of follicles of the test mice in Example 3 of the present invention is shown. a, b, c, d indicate significant differences. The same letters are not significantly different, and different letters are significantly different, p<0.05;

[0021] Figure 4 The figure shows the 2-cell rate of in vitro fertilization of the oocytes of the test mother mice in Example 3 of the present invention. a, b, c, and d indicate significant differences. The same letters are not significantly different, and the different letters are significantly different, p<0.05;

[0022] Figure 5 The in vitro fertilization morula rate of the oocytes of the test female mice in Example 3 of the present invention is shown, and a, b, c, and d indicate significant differences. The same letters are not significantly different, and the different letters are significantly different, p<0.05;

[0023] Figure 6 The pregnancy rate of the test rats in Example 3 of the present invention is shown, and a, b, c, and d indicate significant differences. The same letters are not significantly different, and the different letters are significantly different, p<0.05;

[0024] Figure 7 The figure shows the number of live offspring born by the test mother mice in Example 3 of the present invention. a, b, and c indicate significant differences. The same letters are not significantly different, and the different letters are significantly different, p<0.05;

[0025] Figure 8 The total number of follicles of the test sows in Example 4 of the present invention is shown, and a, b, and c indicate significant differences. The same letters are not significantly different, and the different letters are significantly different, p<0.05;

[0026] Fig. 9 The 2-cell rate of in vitro fertilization of oocytes of the test sows in Example 4 of the present invention is shown, and a and b indicate significant differences. The same letters are not significantly different, and the different letters are significantly different, p<0.05;

[0027] Fig.10 The morula rate of in vitro fertilization of oocytes of the test sows in Example 4 of the present invention is shown, and a and b indicate significant differences. The same letters are not significantly different, and the different letters are significantly different, p<0.05;

[0028] Fig.11 The total number of follicles of the test ewes in Example 5 of the present invention is shown, and a and b indicate significant differences. The same letters are not significantly different, and the different letters are significantly different, p<0.05;

[0029] Fig.12 The figure shows the 2-cell rate of in vitro fertilization of the oocytes of the test ewes in Example 5 of the present invention, a and b indicate significant differences. The same letters are not significantly different, and the different letters are significantly different, p<0.05;

[0030] Fig.13 The figures show the morula rate of in vitro fertilization of the eggs of the test ewes in Example 5 of the present invention, and a and b indicate significant differences. The same letters are not significantly different, and the different letters are significantly different, p<0.05.

[0031] The Lactobacillus reuteri included in the present invention ( Lactobacillus reuteri ) LRY15, classified as Lactobacillus reuteri Lactobacillus reuteri The strain was deposited on May 30, 2024 in the General Microbiology Center of China Microorganism Culture Collection (CGMCC for short, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101), with the deposit number CGMCC No. 30814. DETAILED DESCRIPTION

[0032] The present invention verifies the beneficial effect of the oviposit compound on the fertility of female animals. Cisplatin is used to establish a model of premature ovarian damage in female mice. It is found that the oviposit compound can improve the quality of eggs and the in vitro fertilization rate of eggs more than the single strain LRY15, and further improve the success rate of mating, pregnancy rate, and number of live piglets of female mice. The high temperature and high humidity climate in summer will reduce the reproductive performance of sows and ewes, resulting in a decrease in the pregnancy rate and number of live piglets of sows and ewes. The compound can further improve the egg quality of sows and ewes under high temperature and high humidity in summer, and better improve the in vitro fertilization rate. This application isolated a total of 27 strains of Lactobacillus reuteri, named LRY01, LRY02, LRY03, ..., LRY27. Through research, it was found that only LRY15 can significantly improve the fertility of female animals.

[0033] Example 1 Isolation and cultivation of Lactobacillus reuteri LRY15 in the egg-benefiting compound, preparation of wolfberry oligosaccharides and chestnut oligosaccharides

[0034] 1. Isolation and cultivation of Lactobacillus reuteri LRY15

[0035] (1) Strain isolation: Lactobacillus reuteri ( Lactobacillus reuteri ) LRY15 was isolated from intestinal contents. Take the intestinal contents, dilute them 10 times with sterile saline, take 0.1 mL of the dilution with the appropriate dilution and evenly spread it on MRS agar. After culturing at 37°C for 48 hours, the grown colonies were inoculated into a new MRS solid medium by streaking on a plate for purification and culture for 48 hours. 27 strains of Lactobacillus reuteri were isolated and named LRY01, LRY02, LRY03, ..., LRY27. Through research, it was found that only LRY15 can significantly improve the fertility of female animals. This application also provides research data on another strain of Lactobacillus reuteri, LRY09.

[0036] (2) Strain enrichment: The purified strains were inoculated one by one into sterile MRS broth medium using an inoculation loop on a sterile operating table, placed in a shaker, and cultured at 37°C for 48 h. 16S rRNA identification was performed, and the remaining portion was mixed with physiological saline containing 25% glycerol and stored in a -80°C refrigerator.

[0037] (3) 16S rRNA identification: The enriched bacterial solution was amplified by colony PCR using universal bacterial primers, and then 16S rDNA sequencing was performed for identification. The 16S rDNA sequences of each strain were compared with the 16S rDNA sequences of all determined bacteria in the database on NCBI. It was found that strains LRY01-LRY27 were similar to Lactobacillus reuteri. Lactobacillus reuteriThe 16S rDNA sequence of HKU 070513 had the highest homology, with a similarity greater than or equal to 99.9%, confirming that these strains were Lactobacillus reuteri. Lactobacillus reuteri .

[0038] 2. Lycium barbarum oligosaccharide ( Lycium chinense Preparation of Miller oligosaccharide (LOS)

[0039] (1) Preparation of Lycium barbarum polysaccharide: Wash and weigh the Lycium barbarum, add water at a solid-liquid ratio of 100 g / L, and evenly crush it with a homogenizer. Then, add sodium carbonate (concentration of 6.5% w / v) and degrade it at a constant temperature of 90°C for 24 hours to obtain Lycium barbarum polysaccharide;

[0040] (2) Add 2 times phosphate buffer to the Lycium barbarum polysaccharide obtained in step (1), heat to 55°C to dissolve the Lycium barbarum polysaccharide, prepare a 0.6% w / v substrate, then adjust the pH to 7.0, add 0.45 IU / mL of specific Lycium barbarum oligosaccharide degrading enzyme, mix thoroughly, and then perform enzymolysis at 37°C for 12 hours;

[0041] (3) The enzymatic hydrolysate obtained in step (2) was heated to boiling for 15 min to inactivate the enzyme, and then centrifuged at 15,000 rpm / min for 30 min, the supernatant was recovered, and the supernatant was concentrated by a rotary evaporator until 80% of the supernatant was evaporated;

[0042] (4) precipitating the concentrated solution obtained in step (3) with alcohol: isopropanol in a ratio of 1:1.5, drying the precipitate using freeze-drying technology, and then dissolving the freeze-dried powder in ultrapure water, further purifying it through a gel resin column, and freeze-drying the purified liquid to obtain wolfberry oligosaccharide powder;

[0043] (5) The wolfberry oligosaccharide powder obtained in step (4) is tested, and the wolfberry oligosaccharide powder with a content of ≥96% is qualified and stored at -20°C for later use.

[0044] 3. Preparation of Castanea mollissima oligosaccharide (COS)

[0045] (1) Preparation of chestnut polysaccharide: Peel and wash the chestnut fruit, weigh it, add water at a solid-liquid ratio of 110 g / L, crush it evenly with a homogenizer, then add sodium carbonate (concentration of 5.5% w / v), and degrade it at a constant temperature of 97°C for 18 h to obtain chestnut polysaccharide;

[0046] (2) Add 1.5 times phosphate buffer to the chestnut polysaccharide obtained in step (1), heat to 60°C to dissolve the chestnut polysaccharide, prepare a 0.7% w / v substrate, then adjust the pH to 7.2, add 0.55 IU / mL of a specific chestnut oligosaccharide degrading enzyme, mix well, and then perform enzymolysis at 42°C for 18 hours;

[0047] (3) The enzymatic hydrolysate obtained in step (2) was heated to boiling for 20 min to inactivate the enzyme, and then centrifuged at 12000 rpm / min for 45 min, the supernatant was recovered, and the supernatant was concentrated by a rotary evaporator until 75% of the supernatant was evaporated;

[0048] (4) The concentrated solution obtained in step (3) is precipitated with alcohol: isopropanol in a ratio of 1:1.5, the precipitate is dried using freeze-drying technology, and the powder obtained by freeze-drying is dissolved in ultrapure water, further purified by a gel resin column, and the purified liquid is freeze-dried to obtain chestnut oligosaccharide powder;

[0049] (5) The chestnut oligosaccharide powder obtained in step (4) is tested, and the chestnut oligosaccharide powder with a content of ≥98% is qualified and stored at -20°C for later use.

[0050] 4. Preparation of composite bacterial agent

[0051] The composite bacterial agent used in the following examples includes: (strain LRY15 or LRY09) + LOS + COS, 100 grams of the composite bacterial agent includes 25-45 grams (dry bacteria of strain LRY15 or LRY09), 20-25 grams of wolfberry oligosaccharide LOS, and 10-30 grams of chestnut oligosaccharide COS; when the composite bacterial agent solution or oligosaccharide solution is added to the experimental treatment, 1 gram of the composite agent is dissolved in 100 milliliters of normal saline, or the dry bacteria or oligosaccharides equivalent to the content of 1 gram of the bacterial agent are dissolved in 100 milliliters of normal saline. In the same experiment, the mass of dry bacteria of strain LRY15 or LRY09, LOS, and COS used in each parallel treatment (for example, LOS+COS group, LRY15 group, LRY15+LOS+COS group, LRY09 group, LRY09+LOS+COS group) is the same.

[0052] For example, for the composite bacterial agent treatment group (LRY15+LOS+COS group), 1 gram of composite bacterial agent was fed every day, while the amount of active ingredients fed to the parallel strain LRY15 or LRY09 group and LOS+COS group was the amount of dry bacterial agent or LOS+COS contained in 1 gram of composite bacterial agent. That is, the amount of the same active ingredient used in the parallel treatment groups was the same.

[0053] Example 2 Detection of the characteristics of Lactobacillus reuteri LRY15 and another strain of Lactobacillus reuteri LRY09 in the egg-benefiting compound

[0054] 1. The microbiological characteristics of Lactobacillus reuteri LRY15 and LRY09 (the two strains have similar morphology) are as follows:

[0055] (1) Colony morphology: round single colony, diameter about 1.1~1.9mm, white, opaque, smooth surface with slight protrusions and regular edges;

[0056] (2) After staining, the morphology appears short rod-shaped, without flagella, and cannot move;

[0057] (3) Growth characteristics: Figure 1 LRY15 was inoculated alone in MRS liquid medium and placed in static culture at 37°C. It entered the logarithmic growth phase after 4 hours and the plateau phase after 14 hours. The maximum number of viable bacteria was 2.91×10 9 CFU / mL. The beneficial egg-producing compound (LRY15+LOS+COS) was inoculated in MRS liquid culture medium and placed in static culture at 37°C. It entered the logarithmic growth phase after 2 hours and the plateau phase after 20 hours. The maximum number of viable bacteria was 3.6×10 9 CFU / mL.

[0058] LRY09 was inoculated alone in MRS liquid medium and placed in static culture at 37°C. It entered the logarithmic growth phase after 4 hours and the plateau phase after 14 hours. The maximum number of viable bacteria was 2.76×10 9 CFU / mL. LRY09+LOS+COS were inoculated in MRS liquid medium and placed in static culture at 37°C. After 2 hours, they began to enter the logarithmic growth phase and the plateau phase after 20 hours. The maximum number of viable bacteria was 3.27×10 9 CFU / mL( Figure 1 ).

[0059] 2. Bile salt tolerance test

[0060] The activated bacterial solution with a concentration of 1×10 8 CFU / mL of bacterial solution, take 1mL of bacterial solution containing only LRY15 or LRY09, or 1mL of bacterial solution containing LRY15+LOS+COS, or 1mL of bacterial solution containing LRY09+LOS+COS and inoculate it into 9mL of sterile saline containing porcine bile salt with a concentration of 0.3g / 100mL, and determine the number of viable bacteria by plate spreading method after 1h.

[0061] The results showed (Table 1) that at a bile salt concentration of 0.3 g / 100 mL, Lactobacillus reuteri LRY15 could grow, with a survival rate of about 24.55%; LRY15 in the egg-benefitting compound could grow better, showing better bile salt tolerance, with a survival rate of about 30.56%. LRY09 was similar to LRY15.

[0062] Table 1 Survival rate of different samples at 0.3% concentration of strong bile salts

[0063] sample Survival rate LRY15 <![CDATA[24.55±0.38 a ]]> LRY15+LOS+COS <![CDATA[30.56±0.42 b <!-- 4 -->]]> LRY09 <![CDATA[24.78±0.41 a ]]> LRY09+LOS+COS <![CDATA[30.23±0.35 b ]]> .

[0064] a, b indicate significant differences. The same letters are not significantly different, and different letters are significantly different, p < 0.05.

[0065] 3. Artificial gastric juice resistance test

[0066] The activated bacterial solution with a concentration of 1×10 8 CFU / mL of bacterial solution, 1 mL of bacterial solution containing only LRY15 or LRY09, or 1 mL of bacterial solution containing beneficial oviposit compound (LRY15+LOS+COS), or LRY09+LOS+COS was inoculated into 9 mL of artificial gastric juice with pH=1.5, and the number of viable bacteria was determined by plate spreading method after 1 hour.

[0067] The results showed (Table 2) that after 1 hour in artificial gastric juice with pH = 1.5, Lactobacillus reuteri LRY15 had good resistance to artificial gastric juice, with a survival rate of about 21.21%; LRY15 in the beneficial egg-producing compound was able to grow better and showed better resistance to artificial gastric juice, with a survival rate of about 32.21%. LRY09 was similar to LRY15.

[0068] Table 2 Survival rate of different samples in artificial gastric juice at pH=1.5%

[0069] Strain number Survival rate LRY15 <![CDATA[21.21±0.48 a ]]> LRY15+LOS+COS <![CDATA[32.21±0.24 b ]]> LRY09 <![CDATA[22.19±0.35 a ]]> LRY09+LOS+COS <![CDATA[33.12±0.31 b ]]>

[0070] a, b indicate significant differences. The same letters are not significantly different, and different letters are significantly different, p < 0.05.

[0071] 4. Artificial intestinal fluid resistance test

[0072] The activated bacterial solution with a concentration of 1×10 8 CFU / mL of bacterial solution, 1mL of bacterial solution containing only LRY15 or LRY09, or 1mL of bacterial solution containing beneficial oviposit compound (LRY15+LOS+COS), or LRY09+LOS+COS was inoculated into 9mL of artificial gastric juice with pH=6.8, and the number of viable bacteria was determined by plate spreading method after 4h.

[0073] The results showed (Table 3) that after 4 hours in artificial intestinal fluid, Lactobacillus reuteri LRY15 had good resistance to artificial intestinal fluid, with a survival rate of about 23.42%; LRY15 in the Yi Ovum compound was able to grow better and showed better resistance to artificial gastric fluid, with a survival rate of about 33.65%. LRY09 was similar to LRY15.

[0074] Table 3 Survival rate of different samples in artificial intestinal fluid%

[0075] Strain number Survival rate LRY15 <![CDATA[23.42±0.57 a ]]> LRY15+LOS+COS <![CDATA[33.65±0.37 b ]]> LRY09 <![CDATA[23.42±0.57 a ]]> LRY09+LOS+COS <![CDATA[33.65±0.37 b ]]>

[0076] a, b indicate significant differences. The same letters are not significantly different, and different letters are significantly different, p < 0.05.

[0077] Example 3 Effect of Yilansheng Compound on the Fertility of Female Mice in Cisplatin Model

[0078] After confirming that the beneficial egg-growing compound has good beneficial properties, the beneficial egg-growing compound was used in subsequent experiments.

[0079] 1. Experimental Methods

[0080] 1. 210 21-day-old female CD-1 mice were selected and housed in the Animal Science Research Center of the Chinese Academy of Agricultural Sciences. During the experiment, the temperature of the mouse room was controlled at 22±2℃, the light / dark time was 12 hours, and the mice had free access to food and water.

[0081] 2. All female mice were randomly divided into 7 groups (30 female mice in each group): control group (Con group), cisplatin group (Cis), Cis + LOS + COS (LOS + COS), Cis + LRY15 group (LRY15), Cis + Yilansheng compound group (LRY15 + LOS + COS), Cis + LRY09 group (LRY09), Cis + LRY09 + LOS + COS (LRY09 + LOS + COS). In the first 3 days of the experiment, the Cis group, LOS + COS group, LRY15 group, LRY15 + LOS + COS group, LRY09 group, and LRY09 + LOS + COS group were injected with 5 mg / kg body weight of cisplatin every day (for 3 consecutive days). From the first day, the Con group: gavage with 0.1 mL of saline once a day for 21 days; the Cis group: gavage with 0.1 mL of saline once a day for 21 days; the LOS+COS group: gavage with 0.1 mL of LOS+COS solution once a day for 21 days; the LRY15 group: gavage with 0.1 mL of LRY15 bacterial solution once a day for 21 days; the LRY15+LOS+COS group: gavage with 0.1 mL of Yilansheng compound solution once a day for 21 days; the LRY09 group: gavage with 0.1 mL of LRY09 bacterial solution once a day for 21 days; the LRY09+LOS+COS group: gavage with 0.1 mL of LRY09+LOS+COS solution once a day for 21 days. Then all the female mice were raised normally for 21 days (3 weeks) without any treatment. During the experiment, the weight of the female mice was measured every week, and the status, survival, and clinical abnormal symptoms of the female mice were observed and recorded. After a total of 42 days (21 days + 21 days), on the 43rd day, 10 female mice were randomly selected from each group and their eggs were collected for in vitro fertilization. The remaining 20 female mice in each group were mated with normal male mice, with each male mouse mating with 2 female mice for 6 days. After 6 days of mating, the female mice were kept in a single cage. After 21 days of mating, the pregnancy rate of the female mice and the number of live pups were counted.

[0082] 2. Testing indicators

[0083] 1. During the experiment, the weight of the female mice was counted every 7 days;

[0084] 2. The number of follicles in the female mouse's ovaries;

[0085] 3. In vitro fertilization rate;

[0086] 2. 21 days after mating, the pregnancy rate and number of live pups of the female mice were counted.

[0087] 3. Experimental Results

[0088] 1. Growth: The growth changes of the female mice during the whole experiment are as follows: Figure 2As shown in the figure, cisplatin caused the weight gain of female mice to slow down. The weight of the cisplatin group (Cis) was lower than that of the control group from the 7th day of treatment and continued until the end of the experiment. The weight of the two groups fed with LRY15 or beneficial egg-producing compound (LRY15+LOS+COS) after cisplatin treatment was higher than that of the Cis group or the control group (Con). The three groups fed with LOS+COS, LRY09 or LRY09+LOS+COS were similar to the Cis group or the control group (Con).

[0089] 2. Number of follicles: Figure 3 As shown, the number of follicles in the cisplatin model group was far less than that in the control group. The number of follicles in the group fed with LRY15 after cisplatin treatment was more than that in the cisplatin (Cis) group, and the number of follicles in the beneficial egg compound group (LRY15+LOS+COS) was more than that in the LRY15 group; LRY15 and LOS+COS had a synergistic effect, with a synergistic coefficient of 325%. However, the two groups fed with LRY09 or LRY09+LOS+COS had a slightly higher number than the Cis group, but significantly less than the control group (Con), and LRY09 had no synergistic effect with LOS+COS.

[0090] 3. In vitro fertilization 2 cell rate: Figure 4 As shown, the rate of 2-cell development after IVF of oocytes of female mice in the Cis model group (Cis) was much lower than that of the control group (Con), the rate of 2-cell development after IVF of oocytes of the group fed with LRY15 after cisplatin treatment was higher than that of the Cis group, and the rate of 2-cell development after IVF of oocytes of the Yilansheng compound group (LRY15+LOS+COS) was significantly higher than that of the LRY15 group; LRY15 and LOS+COS had a synergistic effect, with a synergistic coefficient of 6.12%. However, the two groups fed with LRY09 or LRY09+LOS+COS were similar to the Cis group and had no synergistic effect.

[0091] 4. In vitro fertilization morula rate: Figure 5 As shown, the rate of oocytes developing into morulae after IVF in the cisplatin model group (Cis) was much lower than that in the control group (Con), the rate of oocytes developing into morulae after IVF in the group fed with LRY15 after cisplatin treatment was higher than that in the cisplatin group, and the rate of oocytes developing into morulae after IVF in the Yilansheng compound group (LRY15+LOS+COS) was higher than that in the LRY15 group; LRY15 and LOS+COS had a synergistic effect, with a synergistic coefficient of 781%. However, the two groups fed with LRY09 or LRY09+LOS+COS were similar to the Cis group and had no synergistic effect.

[0092] 5. Pregnancy rate: Figure 6As shown, the pregnancy rate of female mice in the cisplatin model group (Cis) after mating with normal male mice was lower than that in the control group (Con), the pregnancy rate of female mice fed with LRY15 after cisplatin treatment and mating with normal male mice was higher than that in the cisplatin group, and the pregnancy rate of female mice in the Yilansheng compound group (LRY15+LOS+COS) after mating with normal male mice was higher than that in the LRY15 group; LRY15 and LOS+COS had a synergistic effect, with a synergistic coefficient of 451%. However, the two groups fed with LRY09 or LRY09+LOS+COS were similar to the Cis group and had no synergistic effect.

[0093] 6. Number of piglets born alive: Figure 7 As shown, the number of live pups born after mating with normal male mice in the cisplatin model group (Cis) was less than that in the control group (Con), the number of live pups born after mating with normal male mice in the cisplatin-treated female mice fed with LRY15 was more than that in the cisplatin group, and the number of live pups born after mating with normal male mice in the Yilansheng compound group (LRY15+LOS+COS) was more than that in the LRY15 group; LRY15 and LOS+COS had a synergistic effect, with a synergistic coefficient of 176%. However, the two groups fed with LRY09 or LRY09+LOS+COS were similar to the Cis group and had no synergistic effect.

[0094] 7. In addition, the analysis of blood and ovarian metabolites of treated female mice using liquid chromatography-mass spectrometry (LC / MS / MS) revealed that cisplatin increased harmful metabolites in the blood and ovaries and reduced beneficial metabolites. However, feeding LRY15 or beneficial egg-producing compound (LRY15+LOS+COS) after cisplatin treatment increased beneficial metabolites in the blood and ovaries, such as melatonin, unsaturated fatty acids, glutathione, and retinoic acid. However, the two groups fed LRY09 or LRY09+LOS+COS did not significantly increase beneficial metabolites in the blood and ovaries, such as melatonin, unsaturated fatty acids, glutathione, and retinoic acid.

[0095] Table 4 Blood metabolites (ng / g protein)

[0096] Melatonin Unsaturated fatty acids Glutathione Retinoic acid Con <![CDATA[23.11±0.23 b ]]> <![CDATA[223.45±5.57 c ]]> <![CDATA[12.12±0.23 c ]]> <![CDATA[13.23±0.26 c ]]> Cis <![CDATA[5.42±0.11 a ]]> <![CDATA[21.06±1.43 a ]]> <![CDATA[0.56±0.12 a ]]> <![CDATA[1.65±0.43 a ]]> LOS+COS <![CDATA[6.26±0.25 a ]]> <![CDATA[23.43±1.57 a ]]> <![CDATA[2.41±0.35 b ]]> <![CDATA[2.54±0.35 a ]]> LRY15 <![CDATA[26.44±0.43 b ]]> <![CDATA[273.39±3.57 b ]]> <![CDATA[11.02±0.34 c ]]> <![CDATA[9.65±0.32 b ]]> LRY15+LOS+COS <![CDATA[35.44±0.65 c ]]> <![CDATA[326.46±4.21 c ]]> <![CDATA[22.36±0.26 d ]]> <![CDATA[14.32±0.51 c ]]> LRY09 <![CDATA[6.41±0.45 a ]]> <![CDATA[25.65±0.35 a ]]> <![CDATA[1.38±0.35 b ]]> <![CDATA[2.42±0.36 a ]]> LRY09+LOS+COS <![CDATA[6.48±0.57 a ]]> <![CDATA[26.34±0.64 a ]]> <![CDATA[2.01±0.57 b ]]> <![CDATA[2.56±0.26 a ]]>

[0097] a, b, c, d indicate significant differences. The same letters are not significantly different, and different letters are significantly different, p < 0.05.

[0098] Table 5 Ovarian metabolites (ng / g protein)

[0099] Melatonin Unsaturated fatty acids Glutathione Retinoic acid Con <![CDATA[11.23±0.32 b ]]> <![CDATA[187.41±3.48 c ]]> <![CDATA[10.32±0.45 c ]]> <![CDATA[18.35±0.36 b ]]> Cis <![CDATA[3.13±0.31 a ]]> <![CDATA[12.06±1.32 a ]]> <![CDATA[0.32±0.08 a ]]> <![CDATA[1.12±0.23 a ]]> LOS+COS <![CDATA[3.29±0.42 a ]]> <![CDATA[13.42±1.34 a ]]> <![CDATA[1.02±0.12 b ]]> <![CDATA[1.32±0.43 a ]]> LRY15 <![CDATA[11.35±0.29 b ]]> <![CDATA[155.32±4.45 b ]]> <![CDATA[9.04±0.52 c ]]> <![CDATA[16.65±0.56 b ]]> LRY15+LOS+COS <![CDATA[17.23±0.41 c ]]> <![CDATA[245.23±5.43 d ]]> <![CDATA[15.36±0.31 d ]]> <![CDATA[29.34±0.65 c ]]> LRY09 <![CDATA[3.41±0.34 a ]]> <![CDATA[15.34±0.43 a ]]> <![CDATA[1.12±0.33 b ]]> <![CDATA[1.42±0.26 a ]]> LRY09+LOS+COS <![CDATA[3.28±0.36 a ]]> <![CDATA[13.35±0.26 a ]]> <![CDATA[1.01±0.42 b ]]> <![CDATA[1.54±0.32 a ]]>

[0100] a, b, c, d indicate significant differences. The same letters are not significantly different, and different letters are significantly different, p < 0.05.

[0101] Example 4 Effect of the egg-benefiting compound on the quality of sow eggs

[0102] 1. Experimental Methods

[0103] 60 healthy gilts (2-3 years old) weighing about 110 kg were selected. All sows were housed in closed pig sties and provided with commercial feed and free drinking water. The pig sty environment was kept clean, ventilated, and disinfected regularly. The experiment was conducted in July and August in summer.

[0104] All sows were randomly divided into 6 groups: control group (Con), LOS+COS group, LRY15 group, beneficial egg-producing compound group (LRY15+LOS+COS) group, LRY09 group, and LRY09+LOS+COS group; each group had 10 sows. Con group: fed with commercial feed; LOS+COS group: fed with commercial feed + LOS+COS per pig every day; LRY15 group: fed with commercial feed + LRY15; beneficial egg-producing compound group (LRY15+LOS+COS): fed with commercial feed + LRY15+LOS+COS (1 gram of compound per pig per day); LRY09 group: fed with commercial feed + LRY09; LRY09+LOS+COS group: fed with commercial feed + LRY09+LOS+COS (1 gram of compound per pig per day). LRY15 (or LRY09) and the compound were fed for 3 weeks (21 days), and then all groups were fed with commercial feed for 49 days (total experimental period of 70 days), fed twice a day, and fed in fixed quantities. During the experiment, the state of the sows was observed and recorded. After 70 days, the sow follicles were collected and fertilized in vitro.

[0105] 2. Testing indicators

[0106] 1. During the experiment, the physical signs of sows were counted every day;

[0107] 2. Detect the quality of sow's follicles.

[0108] 3. Experimental Results

[0109] 1. Number of follicles: Figure 8 As shown, the number of follicles in the group fed with LRY15 was more than that in the control group (Con), and the number of follicles in the beneficial egg compound group (LRY15+LOS+COS) was significantly more than that in the LRY15 group; LRY15 and LOS+COS had a synergistic effect, with a synergistic coefficient of 177%. However, the two groups fed with LRY09 or LRY09+LOS+COS were similar to the control group (Con) and had no synergistic effect.

[0110] 2. In vitro fertilization 2 cell rate: Fig. 9As shown, the rate of 2-cell development after IVF in the group of sows fed LRY15 was higher than that in the control group, and the rate of 2-cell development after IVF in the group of beneficial egg-producing compound (LRY15+LOS+COS) was significantly higher than that in the LRY15 group; LRY15 and LOS+COS had a synergistic effect, with a synergistic coefficient of 400%. However, the two groups fed LRY09 or LRY09+LOS+COS were similar to the control group (Con), with no synergistic effect.

[0111] 3. In vitro fertilization morula rate: Fig.10 As shown, the rate of oocytes developing into morula after IVF in the group of sows fed LRY15 was higher than that in the control group, while the rate of oocytes developing into morula after IVF in the Yilansheng compound group (LRY15+LOS+COS) was higher than that in the LRY15 group; LRY15 and LOS+COS had a synergistic effect, with a synergistic coefficient of 175%. However, the two groups fed LRY09 or LRY09+LOS+COS were similar to the control group (Con) and had no synergistic effect.

[0112] Example 5 Effect of the egg-benefiting compound on the quality of female goat eggs

[0113] 1. Experimental Methods

[0114] 60 healthy female goats (2-3 years old) weighing about 100 kg were selected. All ewes were housed in closed sheep sheds and provided with commercial feed (concentrated feed and grass) and free drinking water. The sheep pen environment was kept clean, ventilated, and disinfected regularly. The experiment was conducted in July and August in summer.

[0115] All ewes were randomly divided into 6 groups: control group (Con), LOS+COS group, LRY15 group, beneficial egg-producing compound group (LRY15+LOS+COS) group, LRY09 group, and LRY09+LOS+COS group; each group had 10 ewes. Con group: fed with commercial feed; LOS+COS group: fed with commercial feed + LOS+COS; LRY15 group: fed with commercial feed + LRY15; beneficial egg-producing compound group (LRY15+LOS+COS): fed with commercial feed + LRY15+LOS+COS (1 gram of compound per sheep per day); LRY09 group: fed with commercial feed + LRY09; LRY09+LOS+COS group: fed with commercial feed + LRY09+LOS+COS (1 gram of compound per sheep per day). LRY15 (or LRY09) and the compound were fed for 3 weeks (21 days), and then all groups were fed with commercial feed for 49 days (total experimental period of 70 days), fed twice a day, and fed in a fixed amount. During the experiment, the status of the ewes was observed and recorded. After 70 days, the follicles of the ewes were collected and in vitro fertilization was performed.

[0116] 2. Testing indicators

[0117] 1. During the experiment, the physical signs of the ewes were counted every day;

[0118] 2. Detect the quality of follicles of ewes.

[0119] 3. Experimental Results

[0120] 1. Number of follicles: Fig.11 As shown, the number of follicles in the group fed with LRY15 was more than that in the control group (Con), and the number of follicles in the beneficial egg compound group (LRY15+LOS+COS) was significantly more than that in the LRY15 group; LRY15 and LOS+COS had a synergistic effect, with a synergistic coefficient of 233%. However, the two groups fed with LRY09 or LRY09+LOS+COS were similar to the control group (Con) and had no synergistic effect.

[0121] 2. In vitro fertilization 2 cell rate: Fig.12 As shown, the rate of 2-cell development after IVF in the group of ewes fed LRY15 was higher than that in the control group, while the rate of 2-cell development after IVF in the group of beneficial egg-producing compound (LRY15+LOS+COS) was higher than that in the LRY15 group; LRY15 and LOS+COS had a synergistic effect, with a synergistic coefficient of 200%. However, the two groups fed LRY09 or LRY09+LOS+COS were similar to the control group (Con), with no synergistic effect.

[0122] 3. In vitro fertilization morula rate: Fig.13 As shown, the rate of oocytes developing into morula after in vitro fertilization in the group fed with LRY15 was higher than that in the control group, while the rate of oocytes developing into morula after in vitro fertilization in the Yilansheng compound group (LRY15+LOS+COS) was higher than that in the LRY15 group; LRY15 and LOS+COS had a synergistic effect, with a synergistic coefficient of 200%. However, the two groups fed with LRY09 or LRY09+LOS+COS were similar to the control group (Con) and had no synergistic effect.

[0123] Compared with LRY15, the beneficial egg-producing compound (LRY15+LOS+COS) has the characteristics of better improving animal reproductive performance. Moreover, LRY15 in the beneficial egg-producing compound has stronger tolerance to artificial bile salts, artificial gastric acid, and artificial intestinal fluid. However, another strain of bacteria, LRY09, did not significantly improve the reproductive capacity of female animals. This shows that even if the same genus and species, but different strains of bacteria, the effects are different, and all strains have the same effect.

[0124] The above embodiments are only used to explain the technical solution of the present application and do not limit the protection scope of the present application.

Claims

1. Lactobacillus reuteri ( Lactobacillus reuteri )LRY15, characterized in that, The Lactobacillus reuteri ( Lactobacillus reuteri )The deposit number of LRY15 is CGMCC No. 30814.

2. A probiotic compound for improving the fertility of female animals, characterized in that: The probiotic compound comprises the Lactobacillus reuteri ( Lactobacillus reuteri )LRY15 and wolfberry oligosaccharides and chestnut oligosaccharides.

3. The probiotic compound for improving the fertility of female animals according to claim 2, characterized in that: Based on 100 grams of the probiotic complex, including the Lactobacillus reuteri ( Lactobacillus reuteri ) 25-45 grams of LRY15 dried bacteria, 20-25 grams of wolfberry oligosaccharide LOS, 10-30 grams of chestnut oligosaccharide COS, and the rest are auxiliary materials.

4. The probiotic compound for improving the fertility of female animals according to claim 2, characterized in that: The composite agent is a liquid bacterial agent or a solid bacterial agent.

5. The probiotic compound for improving the fertility of female animals according to claim 2 is used for preparing the following preparations: (1) Improve the blood and ovarian metabolome of female animals; (2) Increase the quality of eggs in female animals; (3) Increase the in vitro fertilization rate of female animal eggs; (4) Improving the mating success rate and pregnancy rate of female animals; and / or (5) Increase the number of live piglets born by female animals.

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