A probiotic compound for improving male animal fertility

Abstract

The present invention relates to the field of probiotics, and in particular to a probiotic compound for improving the fertility of male animals. The compound of the present invention can better colonize Lactobacillus reuteri LRY15 in the animal body, and has a stronger tolerance to artificial bile salts, artificial gastric acid, and artificial intestinal fluid; it can better improve intestinal health, better improve intestinal immunity, and increase the sperm motility, concentration, pregnancy rate, and live birth number of male animals; the compound can improve the metabolism of the animal body, increase beneficial metabolites, thereby increasing testicular beneficial metabolites such as retinoic acid (regulating the start of spermatogenesis), thereby improving the sperm quality and fertility of male animals.

Description

Technical Field

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

[0002] How to improve the reproductive performance of male animals is a question that researchers have been exploring recently. However, there is no good solution to effectively improve semen quality and treat male infertility. Existing technical studies have found that intestinal flora is very important to the health of animals, and the relationship between intestinal flora and male reproductive health is also very relevant. It has been found that intestinal flora can improve or reduce the semen quality of male animals. Beneficial intestinal bacteria can improve the semen quality and fertility of animals, while harmful bacteria may reduce semen quality and fertility. However, it is unknown whether intestinal flora and some known prebiotics such as yak milk oligosaccharides and red hair algae oligosaccharides can better improve the semen quality and fertility of male animals. Summary of the invention

[0003] The purpose of the present invention is to provide a probiotic compound for improving the fertility of male animals.

[0004] Another object of the present invention is to provide the use of the above-mentioned probiotic compound for improving the fertility of male animals.

[0005] The probiotic compound for improving male animal fertility according to the present invention comprises Lactobacillus reuteri ( Lactobacillus reuteri ) LRY15, yak milk oligosaccharides and red hair algae oligosaccharides, the Lactobacillus reuteri ( Lactobacillus reuteri ) The deposit number of LRY15 is CGMCC No. 30814, and the deposit date is May 30, 2024.

[0006] According to the probiotic compound for improving male animal fertility of the present invention, the compound can be a liquid bacterial agent or a solid bacterial agent, which is prepared by conventional technical means and adding auxiliary materials permitted in the field of microbial preparations.

[0007] The present invention provides the use of the above-mentioned probiotic compound for improving male animal fertility for preparing preparations for the following purposes:

[0008] (1) Improve intestinal health;

[0009] (2) Improve blood and testicular metabolome;

[0010] (3) Increase sperm concentration in male animals;

[0011] (4) Increase sperm motility in male animals;

[0012] (5) Increase the mating success rate of male animals and the pregnancy rate of mating female animals; and / or

[0013] (6) Increase the number of live offspring born from mating between male animals and female animals.

[0014] 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 intestinal health, better enhance intestinal immunity, and increase sperm motility, concentration, pregnancy rate, and live birth number of male animals; the composite agent can improve animal body metabolism, increase beneficial metabolites, thereby increasing beneficial testicular metabolites such as retinoic acid (regulating the initiation of spermatogenesis), thereby improving sperm quality and fertility of male animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the growth curve of Lactobacillus reuteri LRY15 of the present invention (MRS medium);

[0016] Figure 2 The weight change of male mice in Example 3 of the present invention during the 35-day test period is shown;

[0017] Figure 3 The sperm concentration of the male mice tested in Example 3 of the present invention is shown. a, b, c, d, and e indicate significant differences. The same letters indicate no significant differences. Different letters indicate significant differences. P<0.05.

[0018] Figure 4 Shows the sperm motility of the male mice tested in Example 3 of the present invention, a, b, c, d indicate significant differences, the same letters are not significantly different, different letters are significantly different, p<0.05;

[0019] Figure 5 The figure shows the sperm acrosome integrity of the male mice tested in Example 3 of the present invention. a, b, and c indicate significant differences. The same letters indicate no significant differences, while different letters indicate significant differences, p<0.05;

[0020] Figure 6 The acrosome malformation rate of sperm in male mice tested in Example 3 of the present invention is shown. a, b, c, d indicate significant differences. The same letters indicate no significant differences. Different letters indicate significant differences. P<0.05.

[0021] Figure 7 The pregnancy rate of male mice tested in Example 3 of the present invention is shown. a, b, c, d, and e indicate significant differences. The same letters indicate no significant differences. Different letters indicate significant differences. P<0.05.

[0022] Figure 8The numbers of live offspring of male mice tested in Example 3 of the present invention are shown. a, b, c, and d indicate significant differences. The same letters indicate no significant differences. Different letters indicate significant differences. P<0.05.

[0023] Fig. 9 The semen collection amount of the test boars in Example 4 of the present invention is shown. a, b, c indicate significant differences. The same letters are not significantly different. Different letters are significantly different. P<0.05;

[0024] Fig.10 The concentration of the test boar sperm in Example 4 of the present invention is shown. a, b, and c indicate significant differences. The same letters are not significantly different, and different letters are significantly different, p<0.05;

[0025] Fig.11 The motility of the test boar sperm in Example 4 of the present invention is shown. a, b, and c indicate significant differences. The same letters are not significantly different, and different letters are significantly different, p<0.05;

[0026] Fig.12 The acrosome integrity of the test boar sperm in Example 4 of the present invention is shown. a and b indicate significant differences. The same letters are not significantly different, and different letters are significantly different, p<0.05;

[0027] Fig.13 The deformity rate of the test boar sperm in Example 4 of the present invention is shown. a, b, c represent significant differences. The same letters are not significantly different, and different letters are significantly different, p<0.05;

[0028] Fig.14 The concentration of spermatozoa of the male goat tested in Example 5 of the present invention is shown. a, b, c, and d indicate significant differences. The same letters indicate no significant differences, while different letters indicate significant differences, p<0.05;

[0029] Fig.15 The figure shows the motility of spermatozoa of male goats tested in Example 5 of the present invention. a, b, and c indicate significant differences. The same letters indicate no significant differences, while different letters indicate significant differences, p<0.05;

[0030] Fig.16 The acrosome integrity of the male goat sperm tested in Example 5 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;

[0031] Fig.17 The figures show the deformity rate of sperm of the test goats in Example 5 of the present invention. a, b, c, d indicate significant differences. The same letters indicate no significant differences, while different letters indicate significant differences, p<0.05.

[0032] 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

[0033] The present invention verifies the beneficial effect of the Yijingsheng compound on the fertility of male animals, establishes a mouse model of asthenospermia and oligospermia with busulfan (Busulfan; abbreviated as Bu), and finds that the compound can improve sperm concentration, motility, and acrosome integrity more than the single strain LRY15, more effectively reduce sperm deformity rate, and further improve the success rate of mating between male mice and normal female mice, and increase the pregnancy rate and live piglets of female mice after mating. High temperature and high humidity climate will reduce the semen quality of boars and rams, resulting in a decrease in the pregnancy rate and live piglets of sows and ewes. The compound can further improve the sperm concentration and motility, acrosome integrity of boars and rams under high temperature and high humidity in summer, and better reduce the sperm deformity rate.

[0034] Example 1 Isolation and cultivation of Lactobacillus reuteri LRY15 in Yijingsheng compound, preparation of yak milk oligosaccharides and red hair algae oligosaccharides

[0035] 1. Isolation and cultivation of Lactobacillus reuteri LRY15

[0036] (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 the plate for purification and culture for 48 hours. A total of 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 male animals. This application also provides research data of another strain of Lactobacillus reuteri LRY07 (as a control).

[0037] (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.

[0038] (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 bacteria that had been determined in the database at NCBI. It was found that these strains were similar to Lactobacillus reuteri. Lactobacillus reuteri The 16S rDNA sequence of HKU070513 has the highest homology similarity, with a similarity greater than or equal to 99.9%, confirming that this strain is Lactobacillus reuteri. Lactobacillus reuteri .

[0039] 2. Preparation of Yak milk oligosaccharide (YOS)

[0040] (1) Defatting and deproteinizing yak milk to obtain crude yak milk oligosaccharides: centrifuge the yak milk at 4°C and 10,000 rpm for 30 min to remove the fat on the upper layer and a small amount of protein on the bottom. Take out the middle layer and add anhydrous ethanol (1:4) to react at 4°C for 12 h. After the reaction, centrifuge at 4°C and 10,000 rpm for 30 min. Take the supernatant and concentrate and dry it with a vacuum concentrator to obtain crude yak milk oligosaccharides.

[0041] (2) Dissolving crude yak milk oligosaccharides in ultrapure water, adding glucose degrading enzyme to remove glucose from the crude yak milk oligosaccharides, and obtaining a further yak milk oligosaccharide solution.

[0042] (3) The solution obtained in step (2) is desalted using a strong acid styrene-based cation exchange resin or a macroporous weakly basic anion exchange resin with a polyacrylic acid skeleton until the conductivity of the concentrate is less than 350 μs / cm, thereby removing unnecessary impurities.

[0043] (4) The yak milk oligosaccharide powder obtained in step (3) is tested, and the yak milk oligosaccharide powder having a yak milk oligosaccharide content of ≥95% is qualified and stored at 4°C for later use.

[0044] 3. Red hair algae oligosaccharide ( Bangia fusco-purpurea Preparation of oligosaccharide (BOS)

[0045] (1) Preparation of red hair algae: wash and weigh the red hair algae, add water at a solid-liquid ratio of 120 g / L, and evenly crush it with a homogenizer, then add sodium carbonate (concentration of 4.5% w / v), and degrade it at a constant temperature of 95°C for 8 h to obtain red hair algae;

[0046] (2) adding 1x phosphate buffer to the red hair algae obtained in step (1), heating to 60°C to dissolve the red hair algae, preparing a 0.5% w / v substrate, then adjusting the pH to 7.5, adding 0.56 IU / mL of a specific red hair algae oligosaccharide degrading enzyme, mixing thoroughly, and then enzymolyzing at 40°C for 12 h;

[0047] (3) The enzymatic hydrolysate obtained in step (2) was heated to boiling for 10 min to inactivate the enzyme, and the protein in the solution was repeatedly removed by the Sevage method, followed by centrifugation at 12000 rpm / min for 30 min, and the supernatant was recovered and concentrated by a rotary evaporator until 70% of the supernatant was evaporated;

[0048] (4) precipitating the concentrated solution obtained in step (3) with alcohol: isopropanol in a ratio of 1:1, 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 red hair algae oligosaccharide powder;

[0049] (5) The red hair algae oligosaccharide powder obtained in step (4) is tested, and the red hair algae oligosaccharide powder with a content of ≥95% is qualified and stored at 4° 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 LRY07) + YOS + BOS, and 100 grams of the composite bacterial agent includes 25-45 grams (dry bacteria of strain LRY15 or LRY07), 15-25 grams of yak milk oligosaccharide YOS, and 15-25 grams of red hair algae oligosaccharide BOS. When the composite bacterial agent solution or oligosaccharide solution is added to the experimental treatment, 1 gram of the composite bacterial agent or the dry bacteria or oligosaccharides equivalent to the content in 1 gram of the bacterial agent is dissolved in 100 ml of physiological saline. In the same experimental setting, the mass of the dry bacteria of strain LRY15 or LRY07, YOS, and BOS used in each parallel treatment (for example, YOS+BOS group, LRY15 group, LRY15+YOS+BOS group, LRY07 group, LRY09+LOS+COS group) is the same.

[0052] For example, for the composite bacterial agent treatment group (LRY15+YOS+BOS group), 1 gram of composite bacterial agent was fed every day, while the amount of active ingredients fed to the parallel strain LRY15 or LRY07 group and YOS+BOS group was the amount of dry bacterial agent or YOS+BOS 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] For the composite inoculum (strain LRY15+LOS+COS), 100 g of the composite inoculum included 25-45 g of strain LRY15, 20-25 g of wolfberry oligosaccharide LOS, and 10-30 g of chestnut oligosaccharide COS; when the composite inoculum solution or oligosaccharide solution was added to the experimental treatment, 1 g of the composite inoculum was dissolved in 100 ml of normal saline, or the dry bacteria or oligosaccharides equivalent to 1 g of the inoculum were dissolved in 100 ml of normal saline. In the same experiment, the mass of dry bacteria of strain LRY15, LOS, and COS used in each parallel treatment (e.g., LOS+COS group, LRY15 group, LRY15+LOS+COS group) was the same.

[0054] 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 and LOS+COS groups 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.

[0055] Example 2 Detection of the properties of Lactobacillus reuteri Y15 in the Yijingsheng compound

[0056] 1. The microbiological characteristics of Lactobacillus reuteri LRY15 (similar to LRY07) in Yijingsheng compound are as follows:

[0057] (1) Colony morphology: round single colony, about 1.1-1.9 mm in diameter, white, opaque, LRY15 colony surface is smooth with slight protrusions and regular edges;

[0058] (2) After staining, the morphology appears short rod-shaped, without flagella, and cannot move;

[0059] (3) Growth characteristics: LRY15 was inoculated alone (0.5 ml of 1.0×10 9 CFU / mL) in MRS liquid medium and placed at 37°C for static culture. It entered the logarithmic growth phase after 4 hours and entered the plateau phase after 14 hours. The maximum number of viable bacteria was 2.92×10 9 CFU / mL. Yijingsheng compound (LRY15+YOS+BOS) (100 ml of culture medium inoculated with 0.5 ml of 1.0×10 9 CFU / mL) were inoculated into MRS liquid medium and placed in static culture at 37°C. After 2 hours, it entered the logarithmic growth phase and 20 hours later, it entered the plateau phase. The maximum number of viable bacteria was 3.80×10 9 CFU / mL.

[0060] At the same time, chitosan oligosaccharide (ChOS) and mannooligosaccharide (MOS) were used as controls, and LRY15+ChOS+MOS were inoculated (100 ml of culture medium was inoculated with 0.5 ml of 1.0×10 9 CFU / mL) in MRS liquid medium and placed at 37°C for static culture. It entered the logarithmic growth phase after 2 hours and the plateau phase after 20 hours. The maximum number of viable bacteria was 3.42×10 9 CFU / mL.

[0061] LRY07 was inoculated alone (0.5 ml of 1.0 × 10 9 CFU / mL) in MRS liquid medium and placed at 37°C for static culture. It entered the logarithmic growth phase after 4 hours and entered the plateau phase after 14 hours. The maximum number of viable bacteria was 2.68×10 9 CFU / mL. The complex (containing LRY15) was inoculated in (100 mL of culture medium with 0.5 mL of 1.0×10 9 CFU / mL) in MRS liquid medium and placed at 37°C for static culture. After 2 hours, it entered the logarithmic growth phase and 20 hours later, it entered the plateau phase. The maximum number of viable bacteria was 3.22×10 9 CFU / mL.

[0062] 2. Bile salt tolerance test

[0063] The activated bacterial solution with a concentration of 1×10 8 CFU / mL of bacterial solution, 1mL of bacterial solution containing only bacteria (LRY15 or LRY07) or 1mL of bacterial solution of Yijingsheng compound (LRY15+YOS+BOS), LRY15+ChOS+MOS, or LRY07+YOS+BOS was inoculated into 9mL of sterile saline containing porcine bile at a concentration of 0.3g / 100mL, and the number of viable bacteria was determined after 1h by the plate spreading method.

[0064] The results showed (Table 1) that at a bile salt concentration of 0.3 g / 100 mL, Lactobacillus reuteri LRY15 and LRY07 could grow, with a survival rate of about 25.00%; the Yijingsheng compound LRY15+YOS+BOS could grow better and show better bile salt tolerance, with a survival rate of about 29.32%. LRY07 was similar to LRY15. LRY15+YOS+BOS and LRY07+YOS+BOS were similar to the Yijingsheng compound.

[0065] Table 1 Survival rate of different samples at 0.3% concentration of strong bile salts

[0066] sample Survival rate LRY15 <![CDATA[24.55±0.38 a ]]> LRY15+YOS+BOS <![CDATA[29.32±0.54 b ]]> LRY15+ChOS+MOS <![CDATA[28.25±0.47 b ]]> LRY07 <![CDATA[25.35±0.41 a ]]> LRY07+YOS+BOS <![CDATA[28.55±0.35 b ]]>

[0067] a, b indicate significant differences. The same letters are not significantly different, and different letters are significantly different, p < 0.05.

[0068] 3. Artificial gastric juice resistance test

[0069] The activated bacterial solution with a concentration of 1×10 8 CFU / mL of bacterial solution, 1mL of bacterial solution containing only bacteria (LRY15 or LRY07) or 1mL of bacterial solution of Yijingsheng compound (LRY15+YOS+BOS), LRY15+ChOS+MOS, and LRY07+YOS+BOS were inoculated into 9mL of artificial gastric juice with pH=1.5, and the number of viable bacteria was determined by plate spreading method after 1h.

[0070] The results showed (Table 2) that after 1 hour in artificial gastric juice with pH = 1.5, Lactobacillus reuteri LRY15 and LRY07 had good resistance to artificial gastric juice, with a survival rate of about 21.00%; the Yijingsheng compound LRY15+YOS+BOS was able to grow better and showed better resistance to artificial gastric juice, with a survival rate of about 30.44%. LRY07 was similar to LRY15. LRY15+YOS+BOS and LRY07+YOS+BOS were similar to the Yijingsheng compound.

[0071] Table 2 Survival rate of different samples in artificial gastric juice at pH=1.5%

[0072] Strain number Survival rate LRY15 <![CDATA[21.21±0.48 a ]]> LRY15+YOS+BOS <![CDATA[30.44±0.53 b ]]> LRY15+ChOS+MOS <![CDATA[28.57±0.56 b ]]> LRY07 <![CDATA[21.22±0.35 a ]]> LRY07+YOS+BOS <![CDATA[29.47±0.35 b ]]>

[0073] a, b indicate significant differences. The same letters are not significantly different, and different letters are significantly different, p < 0.05.

[0074] 4. Artificial intestinal fluid resistance test

[0075] The activated bacterial solution with a concentration of 1×10 8 CFU / mL of bacterial solution, 1mL of bacterial solution containing only bacteria (LRY15 or LRY07) or 1mL of bacterial solution of Yijingsheng compound (LRY15+YOS+BOS), LRY15+ChOS+MOS, and LRY07+YOS+BOS were 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.

[0076] The results showed (Table 3) that after 4 hours in artificial intestinal fluid, Lactobacillus reuteri LRY15 and LRY07 had good resistance to artificial intestinal fluid, with a survival rate of about 23.00%; the Yijingsheng compound LRY15+YOS+BOS was able to grow better and showed better resistance to artificial gastric fluid, with a survival rate of about 31.51%. LRY07 was similar to LRY15. LRY15+YOS+BOS and LRY07+YOS+BOS were similar to the Yijingsheng compound.

[0077] Table 3 Survival rate of different samples in artificial intestinal fluid%

[0078] Strain number Survival rate LRY15 <![CDATA[23.42±0.57 a ]]> LRY15+YOS+BOS <![CDATA[31.51±0.49 b ]]> LRY15+ChOS+MOS <![CDATA[30.21±0.51 b ]]> LRY07 <![CDATA[23.54±0.54 a ]]> LRY07+YOS+BOS <![CDATA[30.23±0.42 b ]]>

[0079] a, b indicate significant differences. The same letters are not significantly different, and different letters are significantly different, p < 0.05.

[0080] Example 3 Effect of Yijingsheng Compound on the Fertility of Male Mice in Busulfan Model

[0081] After confirming that the Yijingsheng compound has excellent probiotic properties, the Yijingsheng compound was used in subsequent experiments.

[0082] 1. Experimental Methods

[0083] 1. 180 21-day-old ICR male mice were selected and raised 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.

[0084] 2. All male mice were randomly divided into 9 groups (20 mice in each group): control group (Con), busulfan group (Bu), Bu + YOS + BOS group (YOS + BOS), Bu + ChOS + MOS group (ChOS + MOS), Bu + LRY15 group (LRY15), Bu + Yijingsheng compound LRY15 + YOS + BOS group (LRY15 + YOS + BOS), Bu + LRY15 + ChOS + MOS group (LRY15 + ChOS + MOS), Bu + LRY07 group (LRY07), Bu + LRY07 + YOS + BOS group (LRY07 + YOS + BOS). On the first day of the experiment, each animal in the Bu group, YOS + BOS group, ChOS + MOS group, LRY15 group, LRY15 + YOS + BOS group, LRY15 + ChOS + MOS group, LRY07 group, and LRY07 + YOS + BOS group was injected with 25 mg / kg body weight of busulfan. Starting from the first day, the Con group: gavage with 0.1 mL of normal saline once a day for 21 days; the B group: gavage with 0.1 mL of normal saline once a day for 21 days; the YOS+BOS group: gavage with 0.1 mL of YOS+BOS solution (YOS+BOS dissolved in 100 mL of normal saline) once a day for 21 days; the ChOS+MOS group: gavage with 0.1 mL of Yijingsheng compound ChOS+MOS solution (ChOS+MOS dissolved in 100 mL of normal saline) once a day for 21 days; the LRY15 group: gavage with 0.1 mL of LRY15 bacterial solution (LRY15 dry bacteria dissolved in 100 mL of normal saline) once a day for 21 days; the Yijingsheng compound LRY15+YOS+BOS group: gavage with 0.1 mL of Yijingsheng compound LRY15+YOS+BOS solution (1 gram of compound dissolved in 100 ml of saline) for 21 days; LRY15+ChOS+MOS group: gavage once a day with 0.1 mL of LRY15+ChOS+MOS solution for 21 days; LRY07 group: gavage once a day with 0.1 mL of LRY07 solution (LRY07 dry bacterial agent dissolved in 100 ml of saline) for 21 days; LRY07+YOS+BOS group: gavage once a day with 0.1 mL of LRY07+YOS+BOS solution (1 gram of compound dissolved in 100 ml of saline) for 21 days. Then all male mice were raised normally for 21 days (3 weeks) without any treatment. During the experiment, the weight of male mice was measured every week, and the status, survival, and clinical abnormal symptoms of male mice were observed and recorded. After a total of 42 days (21 days + 21 days), on the 43rd day, 10 male mice were randomly selected from each group and samples were collected for analysis. The remaining 10 male mice in each group mated with normal female mice, with each male mouse mating with 2 female mice for 6 days.Six days after mating, the female mice were kept in a single cage, and the male mice were sampled for testing and analysis. Twenty-one days after mating, the pregnancy rate of the female mice and the number of live pups were counted.

[0085] 2. Testing indicators

[0086] 1. During the experiment, the weight of male mice was counted every 7 days;

[0087] 2. Measure sperm concentration, motility, acrosome integrity and deformity rate of male mice;

[0088] 3. 21 days after mating, the pregnancy rate and number of live pups of female mice were counted;

[0089] 4. And detect the blood indicators and testicular metabolites of male mice.

[0090] 3. Experimental Results

[0091] 1. Growth: The growth changes of male mice during the whole experiment are as follows: Figure 2 As shown, busulfan caused the weight gain of male mice to slow down. From the 7th day of treatment, the body weight of the busulfan group (B) was lower than that of the control group, and until the end of the experiment, the body weight of the busulfan group was significantly lower than that of the control group. The body weight of male mice in the two groups treated with busulfan and fed with LRY15 or Yijingsheng compound was higher than that of the busulfan group or the control group; the body weight of other groups was similar to that of the control group or Bu group.

[0092] 2. Sperm concentration: Figure 3 As shown in the figure, the sperm concentration of male mice in the busulfan model group was much lower than that in the control group. The sperm concentration of the group fed with LRY15 after busulfan treatment was higher than that in the busulfan group, and the sperm concentration of the Yijingsheng compound LRY15+YOS+BOS group was significantly higher than that in the LRY15 group. The sperm concentrations of the YOS+BOS group, ChOS+MOS group, LRY07 group, and LRY07+YOS+BOS group were higher than those of the Bu group, but lower than those of the LRY15 group and the Yijingsheng compound LRY15+YOS+BOS group. LRY15 and YOS+BOS had a synergistic effect, with a synergistic coefficient of 570%. However, LRY15 had no synergistic effect with ChOS+MOS, and LRY07 had no synergistic effect with YOS+BOS.

[0093] 3. Sperm motility: Figure 4As shown in the figure, the sperm motility of male mice in the busulfan model group was much lower than that in the control group. The sperm motility of the group fed with LRY15 after busulfan treatment was higher than that in the busulfan group, and the sperm motility of the Yijingsheng compound LRY15+YOS+BOS group was significantly higher than that in the LRY15 group. The sperm motility of the YOS+BOS group, ChOS+MOS group, LRY07 group and LRY07+YOS+BOS group was higher than that in the Bu group, but lower than that in the LRY15 group and the Yijingsheng compound LRY15+YOS+BOS group. LRY15 and YOS+BOS had a synergistic effect, with a synergistic coefficient of 548%. However, LRY15 and ChOS+MOS had no synergistic effect, and LRY07 and YOS+BOS had no synergistic effect.

[0094] 4. Sperm acrosome integrity: Figure 5 As shown in the figure, the sperm acrosome integrity of male mice in the busulfan model group deteriorated, far lower than that of the control group. The sperm acrosome integrity of the group fed with LRY15 after busulfan treatment was higher than that of the busulfan group, while the sperm acrosome integrity of the Yijingsheng compound LRY15+YOS+BOS group was better than that of the LRY15 group. The YOS+BOS group, ChOS+MOS group, LRY07 group and LRY07+YOS+BOS group were similar to the Bu group. LRY15 and YOS+BOS had a synergistic effect, with a synergistic coefficient of 213%. However, LRY15 and ChOS+MOS did not have a synergistic effect, and LRY07 and YOS+BOS did not have a synergistic effect.

[0095] 5. Sperm deformity rate: Figure 6 As shown in the figure, the sperm deformity rate of male mice in the busulfan model group was higher than that in the control group, the sperm deformity rate of the group fed with LRY15 after busulfan treatment was lower than that in the busulfan group, and the sperm deformity rate of the Yijingsheng compound LRY15+YOS+BOS group was significantly lower than that in the LRY15 group. The YOS+BOS group, ChOS+MOS group, LRY07 group and LRY07+YOS+BOS group were lower than those in the Bu group, but higher than those in the LRY15 group. LRY15 and YOS+BOS had a synergistic effect, with a synergistic coefficient of 313%. However, LRY15 had no synergistic effect with ChOS+MOS, and LRY07 had no synergistic effect with YOS+BOS.

[0096] 6. Pregnancy rate: Figure 7As shown, the pregnancy rate of male mice in the busulfan model group after mating with normal female mice was much lower than that of the control group. The pregnancy rate of mice in the group treated with busulfan and fed with LRY15 after mating with normal female mice was much higher than that of the busulfan group, and the pregnancy rate of the Yijingsheng compound LRY15+YOS+BOS group was higher than that of the LRY15 group. The YOS+BOS group, ChOS+MOS group, LRY07 group and LRY07+YOS+BOS group were higher than the Bu group, but lower than the LRY15 and LRY15+YOS+BOS groups. LRY15 and YOS+BOS had a synergistic effect, with a synergistic coefficient of 506%. However, LRY15 had no synergistic effect with ChOS+MOS, and LRY07 had no synergistic effect with YOS+BOS.

[0097] 7. Number of piglets born alive: Figure 8 As shown in the figure, after the male mice in the busulfan model group mated with normal female mice, the number of live pups born by the female mice was much less than that in the control group. After the male mice in the busulfan-treated group fed with LRY15 mated with normal female mice, the number of live pups born by the female mice was much more than that in the busulfan group, and the number of live pups born by the female mice in the Yijingsheng compound LRY15+YOS+BOS group was more than that in the LRY15 group. The YOS+BOS group, ChOS+MOS group, LRY07 group and LRY07+YOS+BOS group were more than the Bu group, but less than the LRY15 and LRY15+YOS+BOS groups. LRY15 and YOS+BOS had a synergistic effect, with a synergistic coefficient of 367%. However, LRY15 had no synergistic effect with ChOS+MOS, and LRY07 had no synergistic effect with YOS+BOS.

[0098] 8. In addition, the analysis of blood and testicular metabolites of male mice treated with liquid chromatography-mass spectrometry (LC / MS / MS) revealed that busulfan increased harmful metabolites in the blood and testicles and reduced beneficial metabolites. The group fed with LRY15 or Yijingsheng compound LRY15+YOS+BOS after busulfan treatment increased beneficial metabolites in the blood and testicles, such as melatonin, unsaturated fatty acids, glutathione, and retinoic acid. The effects of other groups were not significant.

[0099] Table 4 Blood metabolites (ng / g protein)

[0100] Melatonin Unsaturated fatty acids Glutathione Retinoic acid Con <![CDATA[25.23±0.43 b ]]> <![CDATA[267.41±6.58 b ]]> <![CDATA[20.11±0.45 c ]]> <![CDATA[15.21±0.32 b ]]> Bu <![CDATA[4.56±0.36 a ]]> <![CDATA[20.24±1.84 a ]]> <![CDATA[0.76±0.56 a ]]> <![CDATA[1.43±0.24 a ]]> YOS+BOS <![CDATA[5.65±0.37 a ]]> <![CDATA[21.25±1.65 a ]]> <![CDATA[2.32±0.76 b ]]> <![CDATA[2.43±0.54 a ]]> ChOS+MOS <![CDATA[6.11±0.57 a ]]> <![CDATA[22.32±1.43 a ]]> <![CDATA[2.12±0.32 b ]]> <![CDATA[2.41±0.34 a ]]> LRY15 <![CDATA[26.67±0.75 b ]]> <![CDATA[333.43±3.98 c ]]> <![CDATA[30.45±0.32 d ]]> <![CDATA[14.64±0.43 b ]]> LRY15+YOS+BOS <![CDATA[40.32±0.35 c ]]> <![CDATA[425.21±4.87 d ]]> <![CDATA[44.54±0.78 e ]]> <![CDATA[24.11±0.54 c ]]> LRY15+ChOS+MOS <![CDATA[26.98±0.46 b ]]> <![CDATA[329.43±4.45 c ]]> <![CDATA[31.15±0.87 d ]]> <![CDATA[14.34±0.32 b ]]> LRY07 <![CDATA[5.88±0.26 a ]]> <![CDATA[22.43±0.54 a ]]> <![CDATA[2.45±0.65 b ]]> <![CDATA[2.12±0.32 a ]]> LRY07+YOS+BOS <![CDATA[6.98±0.32 a ]]> <![CDATA[23.54±0.57 a ]]> <![CDATA[2.01±0.57 b ]]> <![CDATA[2.23±0.43 a ]]>

[0101] a, b, c, d, e indicate significant differences. The same letters are not significantly different, and different letters are significantly different, p < 0.05.

[0102] Table 5 Testicular metabolites (ng / g protein)

[0103] Melatonin Unsaturated fatty acids Glutathione Retinoic acid Con <![CDATA[11.23±0.21 b ]]> <![CDATA[214.54±3.45 b ]]> <![CDATA[18.36±0.49 c ]]> <![CDATA[20.25±0.43 b ]]> Bu <![CDATA[3.43±0.54 a ]]> <![CDATA[13.04±1.54 a ]]> <![CDATA[0.21±0.11 a ]]> <![CDATA[1.23±0.22 a ]]> YOS+BOS <![CDATA[3.35±0.22 a ]]> <![CDATA[13.24±1.45 a ]]> <![CDATA[1.35±0.32 b ]]> <![CDATA[1.56±0.54 a ]]> ChOS+MOS <![CDATA[3.45±0.44 a ]]> <![CDATA[14.14±1.28 a ]]> <![CDATA[1.12±0.34 b ]]> <![CDATA[1.34±0.54 a ]]> LRY15 <![CDATA[12.43±0.43 b ]]> <![CDATA[243.32±5.43 c ]]> <![CDATA[18.03±0.65 c ]]> <![CDATA[19.43±0.42 b ]]> LRY15+YOS+BOS <![CDATA[19.22±0.76 c ]]> <![CDATA[298.27±5.21 d ]]> <![CDATA[31.32±0.54 d ]]> <![CDATA[35.53±0.46 c ]]> LRY15+ChOS+MOS <![CDATA[12.57±0.32 b ]]> <![CDATA[244.32±6.46 c ]]> <![CDATA[19.12±0.77 c ]]> <![CDATA[19.98±0.65 b ]]> LRY07 <![CDATA[3.52±0.35 a ]]> <![CDATA[14.32±0.52 a ]]> <![CDATA[1.56±0.32 b ]]> <![CDATA[1.24±0.1 a ]]> LRY07+YOS+BOS <![CDATA[3.42±0.78 a ]]> <![CDATA[12.34±0.54 a ]]> <![CDATA[1.45±0.32 b ]]> <![CDATA[1.67±0.32 a ]]>

[0104] a, b, c, d indicate significant differences. The same letters are not significantly different, and different letters are significantly different, p < 0.05.

[0105] Example 4 Effect of Yijingsheng Compound on Boar Semen Quality

[0106] 1. Experimental Methods

[0107] 80 healthy boars (2-3 years old) weighing about 300 kg were selected from the boar station. All boars 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.

[0108] All boars were randomly divided into 8 groups: control group (Con); YOS+BOS group (YOS+BOS), ChOS+MOS group (ChOS+MOS), LRY15 group (LRY15), Yijingsheng compound LRY15+YOS+BOS group (LRY15+YOS+BOS), LRY15+ChOS+MOS group (LRY15+ChOS+MOS), LRY07 group (LRY07), and LRY07+YOS+BOS group (LRY07+YOS+BOS); there were 10 boars in each group. Con group: fed with commercial feed; YOS+BOS group: fed with commercial feed + YOS+BOS for two weeks (14 days); ChOS+MOS group: fed with commercial feed + ChOS+MOS for two weeks (14 days); LRY15 group: fed with commercial feed + Lactobacillus LRY15 for two weeks (14 days); Yijingsheng compound LRY15+YOS+BOS group: fed with commercial feed + LRY15+YOS+BOS (1 gram of compound per pig per day for two weeks (14 days); LRY15+ChOS+MOS group: fed with commercial feed +LRY15+ChOS+MOS (1 gram of compound per pig per day), fed for two weeks (14 days); LRY07 group: fed commercial feed + Lactobacillus LRY07, fed for two weeks (14 days); LRY07+YOS+BOS group: fed commercial feed + LRY07+YOS+BOS (1 gram of compound per pig per day), fed for two weeks (14 days). Then all groups were fed commercial feed for 56 days (total experimental period of 70 days), fed twice a day, and fed in a fixed amount. During the experiment, the status of the boars was observed and recorded. After 70 days, the semen of the boars was collected and tested and analyzed.

[0109] 2. Testing indicators

[0110] 1. During the trial, the boars' physical signs were counted every day;

[0111] 2. Start testing the semen quality of boars from the 71st day.

[0112] 3. Experimental Results

[0113] 1. Semen collection amount: Fig. 9 As shown in the figure, the semen collection of the group fed with LRY15 was higher than that of the control group; the group of Yijingsheng compound LRY15+YOS+BOS was significantly higher than that of the LRY15 group; the groups of YOS+BOS, ChOS+MOS, LRY07 and LRY07+YOS+BOS were similar to the Con group, but less than the LRY15 and LRY15+YOS+BOS groups. LRY15 and YOS+BOS had a synergistic effect, with a synergistic coefficient of 145%. However, LRY15 and ChOS+MOS had no synergistic effect, and LRY07 and YOS+BOS had no synergistic effect.

[0114] 2. Sperm concentration: Fig.10 As shown in the figure, the sperm concentration of the group fed with LRY15 was higher than that of the control group; the sperm concentration of the Yijingsheng compound LRY15+YOS+BOS group was significantly higher than that of the LRY15 group; the YOS+BOS group, ChOS+MOS group, LRY07 group and LRY07+YOS+BOS group were similar to the Con group, but less than the LRY15 and LRY15+YOS+BOS groups. LRY15 and YOS+BOS had a synergistic effect with a synergistic coefficient of 219%. However, LRY15 and ChOS+MOS did not have a synergistic effect, and LRY07 and YOS+BOS did not have a synergistic effect.

[0115] 3. Sperm motility: Fig.11 As shown in the figure, the sperm motility of the group fed with LRY15 was better than that of the control group; the sperm motility of the Yijingsheng compound LRY15+YOS+BOS group was significantly higher than that of the LRY15 group; the YOS+BOS group, ChOS+MOS group, LRY07 group and LRY07+YOS+BOS group were similar to the Con group, but less than the LRY15 and LRY15+YOS+BOS groups. LRY15 and YOS+BOS had a synergistic effect, with a synergistic coefficient of 182%. However, LRY15 and ChOS+MOS did not have a synergistic effect, and LRY07 and YOS+BOS did not have a synergistic effect.

[0116] 4. Sperm acrosome integrity: Fig.12As shown in the figure, the sperm acrosome integrity of the group fed with LRY15 was higher than that of the control group; the Yijingsheng compound LRY15+YOS+BOS group was significantly higher than the LRY15 group; the YOS+BOS group, ChOS+MOS group, LRY07 group and LRY07+YOS+BOS group were similar to the Con group, but less than the LRY15 and LRY15+YOS+BOS groups. LRY15 and YOS+BOS had a synergistic effect with a synergistic coefficient of 167%. However, LRY15 and ChOS+MOS did not have a synergistic effect, and LRY07 and YOS+BOS did not have a synergistic effect.

[0117] 5. Sperm deformity rate: Fig.13 As shown in the figure, the sperm deformity rate of the group fed with LRY15 was lower than that of the control group; the sperm deformity rate of the Yijingsheng compound LRY15+YOS+BOS group was significantly lower than that of the LRY15 group; the YOS+BOS group, ChOS+MOS group, LRY07 group and LRY07+YOS+BOS group were similar to the Con group, but higher than the LRY15 and LRY15+YOS+BOS groups. LRY15 and YOS+BOS had a synergistic effect, with a synergistic coefficient of 223%. However, LRY15 and ChOS+MOS had no synergistic effect, and LRY07 and YOS+BOS had no synergistic effect.

[0118] Example 5 Effect of Yijingsheng Compound on Semen Quality of Male Goats

[0119] 1. Experimental Methods

[0120] 80 healthy male goats (2-3 years old) weighing about 100 kg were selected. All goats 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.

[0121] All male goats were randomly divided into 8 groups: control group (Con); YOS+BOS group (YOS+BOS), ChOS+MOS group (ChOS+MOS), LRY15 group (LRY15), Yijingsheng compound LRY15+YOS+BOS group (LRY15+YOS+BOS), LRY15+ChOS+MOS group (LRY15+ChOS+MOS), LRY07 group (LRY07), and LRY07+YOS+BOS group (LRY07+YOS+BOS); there were 10 male goats in each group.

[0122] Con group: fed with commercial feed; YOS+BOS group: fed with commercial feed + YOS+BOS for two weeks (14 days); ChOS+MOS group: fed with commercial feed + ChOS+MOS for two weeks (14 days); LRY15 group: fed with commercial feed + Lactobacillus LRY15 for two weeks (14 days); Yijingsheng compound LRY15+YOS+BOS group: fed with commercial feed + LRY15+YOS+BOS (1 gram compound per sheep per day) The LRY15+ChOS+MOS group was fed with commercial feed + LRY15+ChOS+MOS (1 gram of compound per goat per day) for two weeks (14 days); the LRY07 group was fed with commercial feed + Lactobacillus LRY07 for two weeks (14 days); the LRY07+YOS+BOS group was fed with commercial feed + LRY07+YOS+BOS (1 gram of compound per goat per day) for two weeks (14 days). Then all groups were fed with commercial feed for 56 days (total experimental period of 70 days) and quantitative feeding. During the experiment, the status of the male goats was observed and recorded. After 70 days, the semen of the male goats was collected and tested and analyzed.

[0123] 2. Testing indicators

[0124] 1. During the test, the rams' physical signs were counted every day;

[0125] 2. Start testing the semen quality of rams from the 71st day.

[0126] 3. Experimental Results

[0127] 1. Sperm concentration: Fig.14 As shown, the sperm concentration of the group fed with LRY15 was higher than that of the control group; the sperm concentration of the Yijingsheng compound LRY15+YOS+BOS group was significantly higher than that of the LRY15 group; the YOS+BOS group, ChOS+MOS group, LRY07 group and LRY07+YOS+BOS group were similar to the Con group, but less than the LRY15 and LRY15+YOS+BOS groups. LRY15 and YOS+BOS had a synergistic effect with a synergistic coefficient of 235%. However, LRY15 and ChOS+MOS did not have a synergistic effect, and LRY07 and YOS+BOS did not have a synergistic effect.

[0128] 2. Sperm motility: Fig.15As shown, the sperm motility of the group fed with LRY15 was higher than that of the control group; the sperm motility of the Yijingsheng compound LRY15+YOS+BOS group was significantly higher than that of the LRY15 group; the YOS+BOS group, ChOS+MOS group, LRY07 group and LRY07+YOS+BOS group were similar to the Con group, but less than the LRY15 and LRY15+YOS+BOS groups. LRY15 and YOS+BOS had a synergistic effect with a synergistic coefficient of 167%. However, LRY15 and ChOS+MOS did not have a synergistic effect, and LRY07 and YOS+BOS did not have a synergistic effect.

[0129] 3. Sperm acrosome integrity: Fig.16 As shown in the figure, the sperm acrosome integrity of the group fed with LRY15 was higher than that of the control group; the Yijingsheng compound LRY15+YOS+BOS group was significantly higher than the LRY15 group; the YOS+BOS group, ChOS+MOS group, LRY07 group and LRY07+YOS+BOS group were similar to the Con group, but less than the LRY15 and LRY15+YOS+BOS groups. LRY15 and YOS+BOS had a synergistic effect with a synergistic coefficient of 200%. However, LRY15 and ChOS+MOS had no synergistic effect, and LRY07 and YOS+BOS had no synergistic effect.

[0130] 4. Sperm deformity rate: Fig.17 As shown in the figure, the sperm deformity rate of the group fed with LRY15 was lower than that of the control group; the sperm deformity rate of the Yijingsheng compound LRY15+YOS+BOS group was significantly lower than that of the LRY15 group; the YOS+BOS group, ChOS+MOS group, LRY07 group and LRY07+YOS+BOS group were similar to the Con group, but higher than the LRY15 and LRY15+YOS+BOS groups. LRY15 and YOS+BOS had a synergistic effect with a synergistic coefficient of 247%. However, LRY15 and ChOS+MOS had no synergistic effect, and LRY07 and YOS+BOS had no synergistic effect.

[0131] In conclusion, compared with LRY15, the Yijingsheng compound LRY15+YOS+BOS has better characteristics in improving animal reproductive performance, and LRY15 and YOS+BOS have a synergistic effect. Other combinations do not have such an effect. Moreover, in the Yijingsheng compound LRY15+YOS+BOS, LRY15 has a stronger tolerance to artificial bile salts, artificial gastric acid, and artificial intestinal fluid.

[0132] 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. A probiotic compound for improving the fertility of male animals, characterized in that: The probiotic compound comprises Lactobacillus reuteri ( Lactobacillus reuteri ) LRY15, yak milk oligosaccharides and red hair algae oligosaccharides, the Lactobacillus reuteri ( Lactobacillus reuteri )The deposit number of LRY15 is CGMCC No. 30814.

2. The probiotic compound for improving male animal fertility according to claim 1, characterized in that: The probiotic compound is a liquid bacterial agent or a solid bacterial agent.

3. The probiotic compound for improving male animal fertility according to claim 1, characterized in that: Based on 100 g of probiotic complex, including Lactobacillus reuteri ( Lactobacillus reuteri ) LRY15 dry fungus powder 20-50 grams, yak milk oligosaccharide LOS 15-25 grams, red hair algae oligosaccharide COS 15-25 grams, and the rest are auxiliary materials.

4. The probiotic compound for improving male animal fertility according to claim 1 is used for preparing a preparation for the following purposes: (1) Improve intestinal health; (2) Improve blood and testicular metabolome; (3) Increase sperm concentration in male animals; (4) Increase sperm motility in male animals; (5) Increase the mating success rate of male animals and the pregnancy rate of mating female animals; and / or (6) Increase the number of live offspring born from mating between male animals and female animals.

Images