Intestinal bacteria for improving semen quality and application thereof

Abstract

The invention discloses an intestinal bacterium for improving semen quality and application thereof, and belongs to the field of microbial technology and biological medicine. In order to solve the problem that in the prior art, specific probiotics are not used for improving male reproductive injury induced by high fat diet, the invention provides a method for applying the allobacter faecalis strain (A.s) or an inactivated form (P-A.s) of the allobacter faecalis strain (A.s) to the male reproductive injury. Animal experiments show that compared with a high fat diet model group, supplementation of A.s or P-A.s can significantly inhibit abnormal weight growth, improve testis index, improve sperm density, vitality, movement speed and testicular testosterone level, and can effectively repair tissue form damage of testis and epididymis and abnormal sperm ultrastructure. Wherein the improvement effect of the inactivated bacterium form P-A.s is particularly prominent.

Description

Technical Field

[0001] This invention relates to the fields of microbial technology and biomedicine, specifically to a particular intestinal strain—*Isobacterium faecalis* strain. Allobaculum stercoricanis The application of its inactivated form in improving male reproductive damage, and probiotic preparations containing this strain. Background Technology

[0002] The information disclosed in this background section is intended only to enhance some understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art.

[0003] Improving metabolism using gut microbiota strains is a cutting-edge research area in the biomedical field. Related research has gradually progressed from observing the correlation between obesity and gut microbiota to verifying the causal relationship between specific strains, mechanisms of action, and metabolic efficacy, as well as developing products.

[0004] As a potential probiotic, *Isobacterium faecalis* has been shown to play a positive role in diseases such as obesity, hypertension, and arthritis. Allobaculum stercoricanis (hereinafter referred to as) A.s This strain is a highly regarded core species in the genus. It was successfully isolated in 2004, and studies have found that it is more abundant in healthy, lean animals, and its abundance increases significantly after diet-induced weight loss in obese animals, suggesting that this strain is closely related to the body's healthy metabolic state.

[0005] Currently, *Ischemicum* spp. ( Allobaculum Especially strains A.s The research primarily focuses on the gut microbiome. This bacterium has been identified as an important short-chain fatty acid producer in the mammalian gut, particularly notable for its butyrate production. Butyrate plays a crucial role in anti-inflammation, maintaining the intestinal barrier, and regulating immunity; therefore, it has been the subject of numerous studies. Allobaculum Increased abundance is often associated with improved metabolic status; for example, in animal models of obesity, type 2 diabetes, and inflammatory bowel disease, increased abundance often predicts improved health indicators. However, most existing studies remain at the level of correlational observation. The specific mechanism of action of this bacterium and its causal role in host physiology remain unclear, and the vast majority of discussions are limited to the gut environment and have not yet been extended to other physiological systems.

[0006] However, in research on semen quality and male reproductive health, A.sThe role of this strain remains largely unexplored. Currently, there is no direct evidence or published research exploring the association between this strain and semen parameters such as sperm concentration, motility, morphology, or DNA integrity. Behind this gap lies the traditional view of gut microbiota and reproductive health as relatively independent systems. However, with the rise of the "gut-testis axis" concept, the scientific community has begun to focus on the possibility that gut microbiota may remotely influence testicular function and spermatogenesis through pathways such as immune regulation, metabolite secretion, and endocrine disruption.

[0007] However, so far, no information has been found regarding... Allobaculum stercoricanis There are no reports on whether strains or their inactivated forms can be directly used to improve, in particular repair, damage to the male reproductive system caused by metabolic stress such as high-fat diets, and there is a lack of technical inspiration for developing them into related products. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a specific intestinal strain—a strain of the genus *Ischemicum*. Allobaculum stercoricanis The application of its inactivated form in improving male reproductive damage, and probiotic preparations containing this strain.

[0009] The technical solution adopted in this invention is as follows: In a first aspect, the present invention provides a probiotic preparation for improving male reproductive damage. The active ingredient of this preparation comprises a strain of *Isobacterium faecalis*. Allobaculum stercoricanis ( A.s ) or the inactivated form of the strain (P- A.s ), as well as pharmaceutically acceptable excipients (such as probiotic protectants, carriers, excipients, etc.).

[0010] Preferably, the formulation contains As strain or P- A.s The daily effective dose shall not be less than 1×10 9 CFU or an equivalent amount of bacterial cells. This preparation can be formulated as a probiotic preparation, functional food, or dietary supplement.

[0011] Secondly, this invention provides strains of the genus *Isobacterium*. Allobaculum stercoricanis ( A.s ) or its inactivated form (P- A.s Its application in the preparation of products for improving male reproductive damage.

[0012] Preferably, the male reproductive injury is induced by a high-fat diet.

[0013] Preferably, the "improvement of male reproductive damage" specifically includes, but is not limited to, at least one of the following beneficial effects: Inhibit abnormal weight gain caused by a high-fat diet; Increase testicular index (testicular weight / body weight); Improving sperm quality includes increasing sperm density, motility, velocity of curvilinear movement (VCL), and velocity of linear movement (VSL). Increase the relative testosterone content in testicular tissue; Repairing histological damage to the seminiferous tubules and epididymal ducts of the testes; Repair damage to the ultrastructures of sperm, such as the acrosome and mitochondrial sheath.

[0014] The products may include, but are not limited to, probiotic preparations, functional foods, dietary supplements, or feed additives.

[0015] Compared with the related technologies known to the inventors, one of the technical solutions of the present invention has the following beneficial effects: This invention is the first to discover and validate specific gut strains. A.s and its inactivated form P- A.s The proven efficacy of this strain in improving high-fat diet-induced male reproductive damage fills a technological gap in the application of this strain in reproductive health.

[0016] The strains of this invention can not only improve metabolic indicators (such as inhibiting abnormal weight gain), but also act directly on the reproductive system, including restoring testicular function, improving sperm quality, and repairing morphological and ultrastructural damage to reproductive tissues and sperm, with comprehensive effects.

[0017] The present invention unexpectedly discovered that the inactivated bacterial form (P- A.s The present invention demonstrates superior improvement over live bacteria, which avoids the potential biosafety risks of live bacteria applications, making the product safer, more stable, easier to store and transport, and broadening its application scenarios in food, health products and other fields.

[0018] This invention expands the application scope of probiotics and provides a safe and effective new approach for the intervention of male reproductive damage related to high-fat diets. The probiotics involved can be used in the preparation of health products, feed additives and other products, and have broad market application potential.

[0019] This invention provides specific bacterial strain examples and experimental data to support the "gut-testis axis," promoting the development of this theory and laying a solid experimental foundation for the development of male reproductive health products based on microbial intervention. Attached Figure Description

[0020] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0021] Figure 1Effects of different diets / treatments on body weight-related indicators, testicular function, and sperm quality in mice; A: Experimental grouping and timeline design; B: Mouse body weight change curve over time; C: Statistical analysis of mouse body weight gain; D: Observation of the morphology of mouse testes and epididymis; E: Statistical analysis of mouse testicular index; F: Statistical analysis of sperm curvilinear motility; G: Statistical analysis of sperm linear motility; H: Statistical analysis of sperm density; I: Statistical analysis of sperm motility; J: Statistical analysis of relative testosterone content in the testes; K: Observation of HE staining morphology of testicular and epididymal tissues; L: Electron microscopy observation of sperm longitudinal and cross-sectional ultrastructure.

[0022] Figure 2 Effects of different treatments on the integrity of mouse sperm plasma membrane; A: SYBR staining (green, marking all sperm); PI staining (red, marking sperm with damaged plasma membrane); Merge (overlay image); B: Sperm plasma membrane integrity statistics (percentage). Detailed Implementation

[0023] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0024] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0025] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0026] Example 1: Based on Allobaculum stercoricanis Probiotic strains improve male reproductive damage induced by a high-fat diet. 1. Experimental Materials and Methods Strains: Allobaculum stercoricanis ( A.s The standard strain used in this experiment was deposited at the German Society for the Collection of Microbial Cultures (DSMZ) with accession number DSM 13633. The strain used in this experiment was derived from this accession number and was provided by Wuhan Gray Algae Biotechnology Co., Ltd.

[0027] Experimental animals: 60 SPF-grade male C57BL / 6 mice (6-8 weeks old, weighing 18-22g).

[0028] Feed: Normal control diet (NCD, 10% fat energy) and high-fat diet (HFD, 60% fat energy).

[0029] Formulation preparation: [The following appears to be a separate, unrelated sentence:] ...cultured to the logarithmic growth phase... A.s The bacterial culture was centrifuged to collect the bacterial cells. A portion was resuspended in physiological saline to prepare a live bacterial preparation. A.s Another portion, after high-temperature inactivation, was resuspended in physiological saline as an inactivating agent (P-). A.s Both concentrations were adjusted to an oral gavage dose of 1×10⁻⁶. 9 CFU / animal / day (inactivated bacteria are calculated based on an equal amount of bacterial cells).

[0030] Experimental grouping: After one week of adaptive feeding, participants were randomly divided into 4 groups (n=15), as follows: Figure 1 A: Normal diet group (NCD group): fed NCD and administered an equal volume of physiological saline by gavage daily.

[0031] High-fat diet model group (HFD group): fed HFD and administered an equal volume of physiological saline by gavage daily.

[0032] High-fat diet + active A.s strain group (HFD+) A.s Group 1): Feed with HFD, daily by gavage. A.s Live bacteria preparation.

[0033] High-fat diet + inactivation A.s strain group (HFD+P- A.s Group 1): fed HFD, daily gavage with P- A.s Inactivated bacterial preparations.

[0034] Experiment duration: 8 weeks of continuous intervention.

[0035] Detection indicators and methods: Body weight: The mice were weighed at fixed times each week during the experiment, and the weight gain during the experimental period was calculated.

[0036] Reproductive organ observation and testicular index: After the experiment, the mice were sacrificed, the testes and epididymis were removed, their appearance was photographed and recorded, and the testicular index was calculated by weighing (testicular weight / mouse body weight × 100%).

[0037] Sperm quality analysis: Sperm from the epididymal tail were separated and measured using a computer-aided sperm analysis system (CASA) to detect sperm density, motility, velocity of curvilinear movement (VCL), and velocity of linear movement (VSL).

[0038] Testosterone levels: Testicular tissue homogenate was collected and the relative testosterone content in the testicular tissue was detected by enzyme-linked immunosorbent assay (ELISA).

[0039] Histopathology: After fixation, paraffin embedding, and sectioning, the testicular and epididymal tissues were stained with hematoxylin and eosin (HE) and their morphology was observed under an optical microscope.

[0040] Ultrastructural observation: Epididymal sperm were collected, fixed, dehydrated, embedded, and ultrathinly sectioned. The longitudinal and cross-sectional ultrastructure of the sperm was observed using a transmission electron microscope (TEM).

[0041] Sperm plasma membrane integrity testing: The SYBR-14 / PI double staining method was used. Fresh epididymal sperm suspension was taken, and SYBR-14 staining solution (for marking live and dead sperm) and PI staining solution (for marking sperm with damaged plasma membranes only) were added. After incubation in the dark, the sperm were observed under a fluorescence microscope. Sperm with green fluorescence (intact membrane) and red fluorescence (damaged membrane) were counted separately, and the percentage of sperm with intact plasma membranes was calculated.

[0042] 2. Experimental Results The results are as follows Figure 1 As shown, the specific data comparison is as follows: Weight control ( Figure 1 B, C): Compared with the NCD group, the body weight of mice in the HFD group increased significantly by 35.2% at the end of the experiment (P<0.001). A.s and P- A.s After the intervention, the weight gain was reduced by 18.7% and 29.4% compared to the HFD group (P<0.001), respectively, and P- A.s The inhibitory effect of the group was significantly better than that of the group. A.s Group.

[0043] Testicular appearance and index ( Figure 1 D, E): The testis volume of mice in the HFD group was reduced, the epididymis was darker, and the testis index decreased by 22.5% compared with the NCD group (P<0.001). A.s and P- A.s After the intervention, the testicular index increased by 12.3% and 20.1% respectively, and the appearance of the reproductive organs basically recovered to the level of the NCD group.

[0044] Sperm function and testosterone levels ( Figure 1 FJ: Sperm density in the HFD group ( Figure 1 H), vitality ( Figure 1 I), VCL ( Figure 1 F), VSL ( Figure 1 G) decreased by 38.6%, 42.1%, 29.3%, and 31.5% respectively compared to the NCD group, and the relative testosterone content in the testes ( Figure 1 J) decreased by 35.7% (all P<0.05); A.sAfter the intervention, the above indicators rebounded by 19.2%, 21.5%, 15.7%, 16.3%, and 18.4%, respectively, P- A.s After intervention, the rates rebounded by 30.5%, 33.2%, 24.1%, 25.6%, and 29.8% (all P < 0.05), P- A.s It has a more significant effect on improving sperm quality.

[0045] Tissue morphology repair ( Figure 1 K): In the HFD group, the seminiferous tubules of the testes were dilated, the spermatogenic cells were arranged in a disordered manner, and the epididymal duct epithelial cells were atrophied. A.s and P- A.s After intervention, HFD+ A.s Group and HFD+P- A.s In this group, the seminiferous tubule structure of the testes and the morphology of the epididymal epithelium have basically returned to normal, the spermatogenic cells are arranged in an orderly manner, and the morphology of the epididymal epithelium has been repaired, including P- A.s The tissue repair effect of the group was closer to that of the NCD group.

[0046] Ultrastructure repair ( Figure 1 L): Under electron microscopy, the ultrastructure of sperm in the HFD group showed abnormalities such as acrosome damage and disordered mitochondrial sheath arrangement. A.s and P- A.s After intervention, the damage to the sperm ultrastructure was significantly repaired, and the mitochondrial sheaths became more orderly.

[0047] Sperm plasma membrane integrity ( Figure 2 The integrity of the sperm plasma membrane in the HFD group was significantly lower than that in the NCD group (approximately 25.2% decrease, P<0.001). (P-) A.s After intervention, integrity improved; A.s The improvement was more significant after the intervention (P<0.05). A.s The improvement effect of the group was significantly better than that of P- A.s Group.

[0048] 3. Conclusion This embodiment fully demonstrates that a high-fat diet significantly induces abnormal weight gain in mice, and causes testicular dysfunction, decreased sperm quality, and damage to the morphology and ultrastructure of reproductive tissues; supplementation with probiotics A.s and its inactivated form P- A.s It can effectively alleviate metabolic disorders (excessive weight gain) in mice induced by a high-fat diet and significantly improve their reproductive system damage, including restoring testicular function, increasing sperm count and motility, raising testosterone levels, and repairing reproductive tissue and sperm ultrastructure. Particularly noteworthy is the inactivated P- A.s It outperformed live bacteria in multiple indicators. A.sThe effectiveness of these findings provides a new direction and strong experimental evidence for developing safer and more stable products to improve male reproductive health. Notably, live bacteria (As) showed superior efficacy compared to inactivated forms (PA.s) in improving sperm membrane integrity, suggesting that live bacteria may play an irreplaceable role in maintaining sperm cell structural integrity through their metabolic activity or dynamic interactions with the host.

[0049] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A probiotic preparation for improving male reproductive damage, characterized in that, The active ingredient of the formulation contains strains of the genus *Ischemicum*. Allobaculum stercoricanis ( As ) or the inactivated form of the strain (P- As ), and pharmaceutically acceptable excipients.

2. The probiotic preparation according to claim 1, characterized in that, The formulation As strain or P- As The effective daily dose is not less than 1×10 9 CFU or an equivalent amount of bacterial cells.

3. The probiotic preparation according to claim 1 or 2, characterized in that, The formulation is in the form of a probiotic preparation, a functional food, or a dietary supplement.

4. *Ischemicum faecalis* strains Allobaculum stercoricanis ( As ) or its inactivated form (P- As Its application in the preparation of products for improving male reproductive damage.

5. The application according to claim 4, characterized in that, The male reproductive damage was induced by a high-fat diet.

6. The application according to claim 4 or 5, characterized in that, The improvement of male reproductive damage includes at least one of the following: inhibiting abnormal weight gain caused by a high-fat diet; increasing testicular index; It improves sperm quality; increases the relative testosterone content in testicular tissue; repairs the histomorphological damage to the seminiferous tubules and epididymal ducts of the testes; and repairs the ultrastructural damage to the sperm acrosome and mitochondrial sheath.

7. The application according to claim 6, characterized in that, The improvement of sperm quality includes increasing at least one of sperm density, motility, curvilinear motility, and linear motility.

8. The application according to claim 4, characterized in that, The products are probiotic preparations, functional foods, dietary supplements, or feed additives.

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