CN117286080B - Probiotics for improving sperm motility and application thereof - Google Patents
Probiotics for improving sperm motility and application thereof Download PDFInfo
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- CN117286080B CN117286080B CN202311567115.4A CN202311567115A CN117286080B CN 117286080 B CN117286080 B CN 117286080B CN 202311567115 A CN202311567115 A CN 202311567115A CN 117286080 B CN117286080 B CN 117286080B
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
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Abstract
The invention relates to probiotics for improving sperm motility and application thereof, belongs to the technical field of microorganisms, and particularly relates to a method for analyzing the correlation between intestinal flora difference and cock semen quality by using a 16S rRNA sequencing technology by taking cock with high sperm motility and low sperm motility as a research object; 7 lactic acid bacteria, namely Pediococcus pentosaceus, lactobacillus johnsonii, lactobacillus crispatus, lactobacillus salivarius, lactobacillus reuteri, pediococcus acidilactici and enterococcus faecium, are obtained by separation, screening and identification in the content of the cock ileum, wherein the Lactobacillus salivarius (with the preservation number of CGMCC No. 28410) is a differential probiotic discovered by sequencing of 16S rRNA. Then, experiment verification is carried out on 24-week-old cock with low sperm motility and model mice with low sperm motility by using different probiotics, and the result shows that the lactobacillus salivarius obtained by the invention can effectively improve sperm motility.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to probiotics for improving sperm motility and application thereof.
Background
Currently, artificial insemination techniques play a very important role in the development of animal husbandry. The fertility of male animals depends on the yield and quality of semen. The sperm motility is used as a key index for measuring semen quality, can influence sperm fertility, and has important significance for evaluating male animal fertility. In the breeding cock breeding process, the artificial insemination technology is adopted to improve the utilization rate of the breeding cock and the fertilization rate of the hatching eggs, so that the breeding cost is reduced, the breeding process is quickened, and the semen quality requirement in insemination is higher. However, most roosters with lower fertilization rates were found in the survey data and were also lower in sperm motility. How to improve the sperm quality of cock is always a great problem which puzzles the chicken raising industry in China, so improving the sperm motility is the current research focus.
At present, researches show that probiotics (the main components are bacillus, lactobacillus and saccharomycetes) are added into the diet of the breeder cock to improve the sperm motility, and the feed has a certain improvement effect on improving the reproductive capacity of the breeder cock; the combined administration of lactobacillus rhamnosus and bifidobacterium longum can improve the sperm motility of men with weak sperm; the bacillus probiotic supplement also has a certain effect on improving the sperm motility of the cock. However, efficient means for improving sperm motility have yet to be developed, and although some studies have demonstrated that probiotics have the potential to improve sperm motility, there is still a lack of probiotic products that significantly improve sperm motility. Although the prior art CN116144536A discloses a composite bacterium which is used for fermenting a traditional Chinese medicine composition, the fermentation liquor can effectively improve the sperm motility of men, but the function of the composite bacterium on the sperm motility is not verified; the prior art CN113975301a and CN115478029a disclose the potential of lactobacillus microcystis Teng Senshi and lactobacillus reuteri LRB5, respectively, in sperm motility enhancement, but it has not yet been explored whether other probiotics and complex probiotics can function. Therefore, the development of probiotics for improving sperm motility is of great importance.
Disclosure of Invention
Aiming at the defects and actual demands of the prior art, the invention aims to improve the sperm motility and provides a probiotic.
The invention aims at realizing the following technical scheme:
the invention provides a Lactobacillus salivarius, which is preserved in China general microbiological culture Collection center CGMCC (China general microbiological culture Collection center) at the date of 09 and 08 of 2023, wherein the address is 1 to 3 of North Chenxi Lu in the Korean area of Beijing, the post code is 100101 of the institute of microorganisms of the national academy of sciences of China, and is classified and named as Lactobacillus salivarius,Lactobacillus Salivariusthe preservation number is CGMCC No.28410.
In one aspect, the invention provides a composition comprising the lactobacillus salivariusLactobacillus Salivarius) Is a microbial agent.
The microbial agent is a liquid microbial agent or a solid microbial agent, and is prepared by adding auxiliary materials allowed in the field of microbial preparations by adopting a conventional technical means.
The invention provides a probiotics for improving sperm motility, which comprises lactobacillus salivarius, the strain is preserved in China general microbiological culture Collection center (CGMCC) of China general microbiological culture Collection center (CGMCC) in the 09/08/2023, and is classified and named as lactobacillus salivariusLactobacillus SalivariusThe preservation number is CGMCC No.28410; wherein the viable count is not less than 1×10 9 CFU/mL, treatment time is more than 4 weeks.
In one aspect, the invention provides a probiotic composition, comprising lactobacillus salivarius or a lactobacillus salivarius inoculant, in combination with other probiotics, for improving sperm motility; wherein the viable count of Lactobacillus salivarius is not less than 1×10 9 CFU/mL, treatment time is more than 4 weeks.
In one aspect, the invention provides a microbial agent, a probiotic for improving sperm motility, and the use of a probiotic composition, including the use of improving sperm motility in a product.
On the one hand, the invention utilizes the ileum content of the cock with high sperm motility to obtain 7 lactic acid bacteria, namely Pediococcus pentosaceus, lactobacillus johnsonii, lactobacillus crispatus, lactobacillus salivarius, lactobacillus reuteri, pediococcus acidilactici and enterococcus faecium, through separation, screening and identification.
Wherein lactobacillus salivarius (with the preservation number of CGMCC No. 28410) is a differential probiotic which is discovered when the correlation between the intestinal flora difference and the cock semen quality is analyzed by 16S rRNA sequencing.
On one hand, the invention uses different probiotics to test the low sperm motility 24-week old cock and the low sperm motility model mouse, and verifies that the obtained lactobacillus salivarius can effectively improve sperm motility, wherein the lactobacillus salivarius CGMCC No.28410 has the most obvious effect of improving sperm motility.
The invention also provides application of the probiotic composition in preparing a product for improving sperm motility, wherein the probiotic composition comprises the probiotics.
Compared with the prior art, the invention has the beneficial effects that:
(1) The invention screens and obtains a probiotics, which is identified as lactobacillus salivarius with the preservation number of CGMCC No.28410 and is preserved in China industry microbiological culture Collection center (China center for type culture Collection) at the 09 th year of 2023.
(2) The invention discovers that 10 is adopted 9 The CFU/mL lactobacillus salivarius CGMCC No.28410 and lactobacillus reuteri carry out 4-week treatment test on 24-week-old cock with low sperm motility, and the result shows that the probiotics have remarkable effect on improving the cock sperm motility, wherein the effect of improving the sperm motility of the lactobacillus salivarius CGMCC No.28410 is most remarkable, and is improved by 55% compared with a control group.
(3) Further experiments prove that the lactobacillus salivarius CGMCC No.28410 and lactobacillus reuteri have the capability of improving the sperm motility of the low sperm model mice.
Drawings
FIG. 1 effect of different strains on cock sperm motility. CRT as control, poc as pediococcus pentosaceus group, lac as lactobacillus salivarius group, lrt as lactobacillus reuteri group, pac as pediococcus acidilactici group.Representing a statistically significant difference (P < 0.001). />The difference was shown to be statistically significant (P < 0.01). # indicates a statistically significant difference (P < 0.05).
FIG. 2 therapeutic effect of different strains on sperm motility in mice after gossypol treatment. CRT is a control, gy is a gossypol treated group, gy+poc is a gossypol and pediococcus pentosaceus group, gy+lac is a gossypol and lactobacillus salivarius group, gy+lrt is a gossypol and lactobacillus reuteri group, gy+pac is a gossypol+pediococcus acidi group.Representing a statistically significant difference (P < 0.001). />The difference was shown to be statistically significant (P < 0.01). />Indicating statistically significant differences (P < 0.05).
Detailed Description
The present invention will be described in detail by examples. It should be understood that the following examples are illustrative only of the present invention and are not intended to limit the present invention.
In the following examples, pathogenic E.coli was derived from laboratory deposited bacteria; 16S rRNA sequencing work was done by Shanghai Paeno Biotech Co., ltd; 16S DNA sequencing was performed by Jilin Kumei biotechnology technologies Co., ltd; MRS medium was from Beijing Cool Lei technology Co., ltd; the bacterial genome DNA extraction kit is from Tiangen Biochemical technology Co., ltd; amplification polymerase (NEB), agarose, saline, and other chemicals were purchased from regular suppliers.
Example 1 cock sperm motility assay and grouping
The cock is subjected to semen collection training in the first 2 weeks of the beginning of the formal experiment, and cock semen is collected by adopting an abdomen-back combined massage method. Semen samples collected during the formal test were sent to the laboratory within 30 min for testing the sperm motility of all test cocks. The method comprises the steps of sucking 10 mu L of fresh semen in a 1.5 mL test tube by using a pipette, slowly adding isothermal physiological saline along the wall of the test tube, diluting the semen to 500 mu L, and then lightly rotating, and uniformly mixing to enable the semen to be 50 times of diluted semen. And (3) taking 10 mu L of diluted middle-layer semen on a glass slide, detecting under a high-power microscope after covering, and recording the number of sperm moving in a straight line in a visual field to obtain the sperm motility.
30 roosters were selected and separated into high sperm motility groups (IH group) and low sperm motility groups (IL group) of 15 each according to sperm motility. Wherein the average sperm motility of the cock with high sperm motility is 66.67+ -1.60%, and the average sperm motility of the cock with low sperm motility is 31.33+ -2.56%. The results are shown in table 1, where the effective sperm count of the high sperm motility group is significantly higher than that of the low sperm motility group (P < 0.01).
TABLE 1 semen quality index of roosters with different sperm motility
Note that: the number is expressed as average value + -standard error, and the difference of the upper-case letters in the same row is extremely remarkableP<0.01）。
Example 2 differences in intestinal flora of different sperm motility groups
The high and low sperm motility cock screened in example 1 was used as a study object, the ileum and cecum contents of the high and low sperm motility cock were collected, and the flora structures of the ileum and cecum of the two groups of cock were studied by 16S rRNA sequencing, and the species composition of the intestinal flora of the high and low sperm motility cock was analyzed and differential probiotics were screened.
The specific process is as follows: 6 cock with high sperm motility and cock with low sperm motility are randomly slaughtered, the ileum and cecum contents are collected in a sterile freezing tube, and the frozen liquid nitrogen is placed at-80 ℃ for storage for standby. The collected ileum and cecum content samples were sent to the Sharpe North Biotech Co., ltd., subjected to total DNA extraction by microorganisms, PCR amplification, and double-ended sequencing of the DNA fragments using the Illumina platform. The original data is subjected to steps such as primer removal, quality filtration, chimera examination and the like, and then bioinformatics analysis including sequence processing analysis, alpha diversity analysis, beta diversity analysis, species difference analysis, species composition analysis and the like is carried out. The analysis results show that 1) the bacterial colony diversity of the male chicken ileum and the cecum with high and low sperm motility is not different; the flora structure composition of the ileum is more different between the two groups, while the cecum flora structure composition is less different; 2) In the ileum level, the relative abundance of the high sperm motility cock firmicutes is significantly higher than that of the low sperm motility cock; at the genus level, lactobacillus is taken as dominant bacteria of the cock with high sperm motility, the relative abundance of the lactobacillus is extremely higher than that of the cock with low sperm motility, and the lactobacillus and the sperm motility are positively correlated; at the species level, the relative abundance of lactobacillus helveticus and lactobacillus salivarius is extremely reduced in the low sperm motility group, and the relative abundance of lactobacillus vaginalis is remarkably reduced; 3) In cecal levels, the thick-walled mycota of the two groups of roosters were not significantly different; at the level, the relative abundance of the lactobacillus species of the cock with high sperm motility is extremely higher than that of the cock with low sperm motility, and is positively correlated with sperm motility; at the seed level, the relative abundance of lactobacillus vaginalis is extremely reduced in the low sperm motility group and enterococcus cecum is significantly increased.
Wherein Lactobacillus, enterococcus, helicobacter and Shigella are the dominant genera of the IH group, the relative abundance of these four genera being up to 85.66% in total in the ileum content of the IH group, wherein Lactobacillus comprises 69.60%, enterococcus comprises 7.44%, helicobacter comprises 4.39% and Shigella comprises 4.23%.
Example 3 isolation, screening and identification of probiotics
The ileum content of the cock with high sperm motility is taken as a study object, bacteria in the intestinal content are separated by adopting an MRS culture medium, screening is carried out through a bacteriostasis test, and 16s rDNA is utilized for strain identification, so that a test material is provided for improving the sperm quality of the cock for subsequent probiotic feeding.
Firstly, 6 cock with high sperm activity are randomly slaughtered in the test process, the body surface of an animal is disinfected by alcohol, the abdominal cavity of the animal is cut off by using sterile surgical scissors, the ileum is taken out, and the ileum content is collected by forceps in a 10 mL centrifuge tube containing normal saline in a sterile manner. The contents were thoroughly mixed with physiological saline using a vortex oscillator to prepare a bacterial liquid.
Then, the evenly mixed bacterial liquid is dipped by an inoculating loop and evenly streaked on MRS solid culture medium, and the bacterial liquid is put into a constant temperature incubator at 37 ℃ for inverted culture of 18-24 h. Single colonies with different forms are picked up on the culture medium to a 5 mL centrifuge tube containing MRS liquid culture medium, and are evenly mixed by using a shaker and then placed into a constant temperature shaker for culture of 18-24 h. After mixing, the purified bacterial liquid is dipped and taken by an inoculating loop, and is evenly streaked on an MRS solid culture medium, and is inversely cultured for 18-24 h in a constant temperature incubator at 37 ℃, and the process is repeated for three times.
Performing a bacteriostasis test on the obtained bacteria, namely naturally thawing frozen pathogenic escherichia coli at room temperature, uniformly mixing by using a shaker, adding the mixed bacteria into an LB liquid culture medium, uniformly mixing again, and culturing in a constant-temperature shaker for 18-24 h to obtain pathogenic bacteria liquid; the inhibition ability of the separated and purified bacteria to pathogenic escherichia coli is detected by adopting an oxford cup method. 100 mu L of pathogenic bacteria liquid is added on an LB solid culture medium, a triangular glass rod is used for coating uniformly, 3 oxford cups are clamped by forceps and uniformly placed on the culture medium, 240 mu L of purified bacteria liquid is added into the oxford cups, and the mixture is placed into a 37 ℃ constant temperature incubator for culturing 18-24 h after marking. After the cultivation, the diameter of the inhibition zone is measured by using a vernier caliper.
The above test results showed that 133 bacteria were isolated and purified from the ileum contents of 6 high sperm cell motility roosters. The bacteriostasis effect is observed by measuring the diameter of the bacteriostasis ring, wherein the diameter of the bacteriostasis ring is more than or equal to 20mm, the diameter of the bacteriostasis ring is more than or equal to 15mm, the bacteriostasis ring is highly bacteriostasis, the diameter of the bacteriostasis ring is more than or equal to 15mm, the diameter of the bacteriostasis ring is more than or equal to 10mm, the bacteriostasis ring is moderately bacteriostasis, and the diameter of the bacteriostasis ring is less than or equal to 10mm, and the bacteriostasis ring has no bacteriostasis effect. The results are shown in Table 2, 9 strains of bacteria extremely inhibit bacteria, 21 strains of bacteria highly inhibit bacteria, 46 strains of bacteria moderately inhibit bacteria, and 57 strains of bacteria have no antibacterial effect.
Table 2 antibacterial effect of the strain on E.coli
According to the bacteriostasis test, 40 bacteria with better bacteriostasis effect are selected, bacterial genome DNA is extracted and PCR amplification is carried out, and 16s universal primers 27F and 1492R are used for amplification. PCR product sequencing was performed by Jilin Cookimei Biotechnology Co., ltd, and comparison analysis was performed with BLAST in NCBI. The 39 strains are all lactic acid bacteria by strain identification, and the total strains are 3 strains, wherein the Pediococcus is 12, the Lactobacillus is 26 and the enterococcus is 1. The specific identification results are shown in Table 3. According to the identification result, the lactobacillus in the ileum content is rich in species and mainly comprises lactobacillus. Wherein lactobacillus salivarius is consistent with the differential probiotics found in 16S rRNA sequencing in example 2. The lactobacillus salivarius is identified, and the lactobacillus salivarius is preserved in China industry microbiological culture collection center (CGMCC) No.28410 at the date of 2023 and 09 and 08.
TABLE 3 identification of the ileal content bacteria
Example 4 Effect of probiotics on cock sperm motility
100 24-week-old cocks (randomly divided into 5 groups, control group, pediococcus pentosaceus group, lactobacillus salivarius group, lactobacillus reuteri group, pediococcus acidilactici group. 20 chickens per group) were screened for low sperm motility, and sperm motility was examined after four weeks of feeding. The treatment group adopts a perfusion mode, and the concentration is 1 multiplied by 10 9 CFU/Kg×body。
The results in fig. 1 show that the lactobacillus salivarius group and lactobacillus reuteri group significantly improved sperm motility compared to the control group, but there was a significant difference therebetween. There was no significant difference between the Pediococcus pentosaceus and Pediococcus acidilactici groups. The sperm motility of lactobacillus salivarius is improved from 43.14% to 66.95% after feeding, and the sperm motility of lactobacillus reuteri is improved from 43.14% to 57.5% after feeding. In conclusion, the lactobacillus salivarius has the most remarkable effect of improving sperm motility, and is improved by 55% compared with a control group.
Example 5 influence of probiotics on mouse sperm motility
The 60 mice of 6 weeks of age were randomly divided into 6 groups of 10 mice each, the control group was fed with a standard diet, and the treated group was fed with gossypol for one month to construct a low sperm motility model. The gossypol treatment adopts a mode of gastric lavage, and the concentration of the gastric lavage is 15 mg/kg once every two days.
The total of 6 groups in the experimental process are respectively normal diet groups; a gossypol group; gossypol + pediococcus pentosaceus group; gossypol + lactobacillus salivarius group; gossypol + lactobacillus reuteri group; gossypol + pediococcus acidilactici group. The gastric lavage concentration of probiotics is 4×10 9 CFU/mL, each mouse was perfused with 0.25. 0.25 mL, followed by four weeks of intragastric administration. After four weeks of stomach irrigation, semen of the mice was collected, bilateral epididymis of the mice was taken out, transferred to a centrifuge tube containing 2mL of physiological saline, sheared with tissue, incubated in a thermostatic water bath at 37 ℃ for 15 min, 10 μl of sperm suspension was taken out on a sperm counting plate, and sperm motility and kinematic parameters were detected with an automatic sperm analyzer. Results as shown in fig. 2, after gossypol treatment was used to construct a low sperm model, the lactobacillus salivarius group had a significant therapeutic effect with the lactobacillus reuteri group, and there was no significant difference between pediococcus pentosaceus and pediococcus acidilactici. The sperm motility after lactobacillus salivarius treatment is improved from 25.3% to 45%, and the sperm motility after lactobacillus reuteri treatment is improved from 25.3% to 37.5%. Consistent with the previous results, the lactobacillus salivarius treatment effect is best, and the activity is improved by 77% compared with the control group.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (8)
1. A lactobacillus salivarius is characterized in that the strain is preserved in China general microbiological culture Collection center CGMCC (China general microbiological culture Collection center) at 09/08 of 2023 and is classified and named as lactobacillus salivariusLactobacillus SalivariusThe preservation number is CGMCC No.28410.
2. A microbial agent comprising the lactobacillus salivarius of claim 1.
3. The microbial agent according to claim 2, wherein the microbial agent is a liquid microbial agent or a solid microbial agent, and is prepared by adding auxiliary materials allowed in the field of microbial agents by adopting a conventional technical means.
4. A probiotic bacterium for improving sperm motility, comprising the Lactobacillus salivarius according to claim 1, wherein the strain is deposited in China general microbiological culture Collection center CGMCC (China general microbiological culture Collection center), designated as Lactobacillus salivarius by classification, at the date of 09/08 of 2023Lactobacillus SalivariusThe preservation number is CGMCC No.28410.
5. The probiotic bacterium according to claim 4, characterized in that the viable count is not less than 1 x 10 9 CFU/mL, treatment time is more than 4 weeks.
6. A probiotic composition comprising the lactobacillus salivarius of claim 1 or the microbial agent of claim 2.
7. The probiotic composition according to claim 6, characterized in that the viable count is not less than 1 x 10 9 CFU/mL, treatment time is more than 4 weeks.
8. Use of a probiotic composition according to claim 6 or 7 for the preparation of a product for improving sperm motility.
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