Use of clostridium herbivorans strains to improve farrowing performance in mammals

Abstract

The application discloses application of Clostridium herbivorans in improving litter performance of mammals, and a probiotic preparation containing Clostridium herbivorans, wherein the viable bacterial count of Clostridium herbivorans in the probiotic preparation is 10 6 ~10 10 million / mL. After the Clostridium herbivorans colonizes in the intestinal tract of mice, the number of litters and the number of live litters of the mice can be improved.

Description

Technical Field

[0001] This invention relates to microecological preparations, specifically to the application of a Clostridium herbivorans strain in improving the reproductive performance of mammals. Background Technology

[0002] In the fields of mammalian breeding and life science research, the number of live piglets produced is a core indicator for measuring reproductive performance, directly related to the economic benefits of breeding and the efficiency of experimental research. Its optimization and improvement have always been a key focus of the industry. Whether it is mice as model organisms or economically important animals such as sows in livestock production, the number of live piglets produced not only affects the rate of population expansion but also has a crucial impact on the stability of experimental data and the profitability of the breeding industry. Traditional methods to increase the number of live piglets produced often rely on nutritional additives, hormone regulation, or the use of antibiotics. While these methods can achieve short-term effects, long-term use can easily lead to problems such as hormone residues, gut microbiota imbalance, and drug resistance, threatening animal health and product safety and contradicting the industry's development trend of green breeding. With the large-scale and intensive development of the livestock industry and the increasing demands of consumers for food safety, there is an urgent need to develop safe, efficient, and side-effect-free reproductive performance regulation technologies. In recent years, the gut microbiota has been considered an integrated endocrine organ with profound influences on distant organs of the host and multiple metabolic pathways, including immunity and energy metabolism (Qi et al 2021, Davey et al 2023). Gut microbiota also regulate host reproductive health (Ashonibare et al. 2024). Significant changes occur in gut microbiota during pregnancy, and they play a crucial role in the metabolic state and reproductive endocrinology of mammals, revealing a significant correlation between reproductive performance and the gut microbiome (Ye et al. 2024). Notably, significant differences in reproductive capacity exist even among sows of the same breed, despite identical feed and environmental conditions (Li et al. 2022). This variability suggests that factors beyond genetics and management contribute to reproductive differences, highlighting the potential role of the gut microbiome (Nathalie et al. 2022). Gut microbiota are deeply involved in regulating reproductive performance by modulating processes such as nutrient absorption, immune homeostasis, and metabolic homeostasis (Wang et al. 2024). Studies have shown that gut microbiota can influence hormones and reproductive metabolic processes essential for reproductive organ function. In male reproduction, they affect sperm production and motility by influencing vitamin A metabolism (Zhang et al 2021), while in female reproduction, they participate in follicle and oocyte maturation, embryo migration, implantation, and the regulation of estrogen, androgens, and other hormones (Franasiak and Scott 2015). For example, gut microbiota can stimulate leptin secretion from gWAT around the ovary through gut microbiota-derived SCFAs, promoting mammalian follicle maturation and increasing clutch size (Xu et al 2025), thereby regulating mammalian reproductive performance. Maintaining a healthy microbiota can protect the host from pathogens, enhance reproductive potential, and reduce the incidence of adverse pregnancy outcomes (Li et al 2022).Studies have shown that adding probiotics to the diet can improve the immunity of unvaccinated sows and their reproductive performance after infection with swine fever virus (Takamitsu et al 2018).

[0003] However, despite significant progress in elucidating the role of the microbiome in reproductive biology, research on effectively addressing reproductive challenges remains limited. Existing studies largely focus on the correlation between the overall structure of the gut microbiota and reproductive traits, with insufficient screening and identification of specific functional strains, particularly lacking specific probiotic resources that can be directly applied to improve the number of live offspring in mammals. Reported probiotic preparations are mostly concentrated in areas such as improving gut health and enhancing growth performance; dedicated preparations for reproductive performance are scarce and suffer from issues such as weak strain specificity and unstable effects. Probiotic preparations are simple to prepare and cost-effective, solving the safety and stability problems of traditional regulatory methods. They also align with the trends of standardized laboratory animals and green development in the livestock industry. They can be directly applied to optimize the reproductive performance of mice and can be extended to the breeding of economically important animals such as sows through technology transfer, possessing broad application prospects and market value, and are of great significance in promoting technological upgrades in related fields.

[0004] Therefore, there is an urgent need to develop a safe, efficient, and targeted probiotic preparation for increasing the number of offspring in mammals. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide an application of Clostridium herbivorans strain in improving the reproductive performance of mammals. The Clostridium herbivorans strain of this invention can be used to increase the number of live piglets born in mammals. To explore the role of gut microbiota in the live piglet birth trait in mammals, this invention screened and identified key candidate strains of Clostridium herbivorans that regulate the number of live piglets born in sows by comparing high-producing and low-producing sows and using 16S rDNA sequencing technology. This invention prepares a probiotic formulation of Clostridium herbivorans strain for use in increasing the number of live piglets born.

[0006] To achieve the above objectives, the technical solution designed by the present invention is as follows: This invention provides an application of Clostridium herbivorans strain in improving mammalian reproductive performance. This invention also provides the application of Clostridium herbivorans strain in the preparation of products that promote mammalian reproductive performance.

[0007] The mammal in question is either a sow or a mouse.

[0008] The Clostridium herbivorans strain mentioned above, Clostridium herbivorans DSM-14428, was purchased from the German Culture Collection of Microorganisms (DSMZ, https: / / www.dsmz.de / collection / catalogue / details / culture / DSM-14428), and can also be isolated from pig feces or large intestine feces.

[0009] This invention also provides a probiotic preparation for promoting reproductive performance in mammals, wherein the probiotic preparation contains the Clostridium herbivorans strain, and the viable count of the Clostridium herbivorans strain in the probiotic preparation is 10. 6 ~10 10 per ml.

[0010] Furthermore, the viable count of the Clostridium herbivorans strain is 10. 8 ~10 9 per ml.

[0011] Furthermore, the viable count of the Clostridium herbivorans strain is 10. 8 per ml.

[0012] This invention also provides a method for preparing the above-mentioned probiotic preparation, wherein the method involves inoculating Clostridium herbivorans strain into DSMZ339a medium, culturing it in an anaerobic incubator, and obtaining a fresh bacterial solution as the probiotic preparation, wherein the viable count of Clostridium herbivorans strain in the probiotic preparation is 10. 6 ~10 10 per ml.

[0013] The formulation of the DSMZ339a culture medium of the present invention is as follows: Furthermore, the inoculum size of the Clostridium herbivorans strain is 1-2% of the volume of the DSMZ339a culture medium; the gas environment of the anaerobic incubator is 90% N2, 5% CO2, and 5% H2; and the incubation temperature is 37℃.

[0014] Furthermore, the inoculum size of the Clostridium herbivorans strain is 1% of the volume of the DSMZ339a culture medium.

[0015] The strain is inoculated into the liquid culture medium at a volume percentage of 1-2%, preferably 1%. This inoculation amount ensures rapid growth of the strain in the culture medium while avoiding the problem of intense nutrient competition caused by an excessively high initial bacterial count.

[0016] The specific incubation time can be adjusted according to the growth of the strain, until the culture medium becomes turbid and the number of viable bacteria reaches 10. 7 ~10 11 Stop culturing when the number of cells / mL reaches a certain threshold.

[0017] Furthermore, the probiotic preparation contains 10 live bacteria of the Clostridium herbivorans strain. 8 ~10 9 per ml.

[0018] Furthermore, the probiotic preparation contains 10 live bacteria of the Clostridium herbivorans strain. 8 per ml.

[0019] The present invention also provides an application of the above-mentioned probiotic preparation in improving the litter size and live birth rate of mammals. The method of application is as follows: the probiotic preparation is administered orally to the stomach of 8-week-old female mice at a dose of 200 microliters per dose, once every other day until the 6th day of pregnancy.

[0020] For long-distance transportation or long-term storage, the following methods can be used to maintain the activity of the bacteria in the probiotic preparation: Glycerol was added to the prepared probiotic formulation to obtain a bacterial cryopreservation solution (glycerol volume fraction 10%), which was then stored in liquid nitrogen at low temperature. Before use, the bacterial suspension was rapidly thawed in a 37°C water bath, and then centrifuged (4°C, 5000 rpm, 10 min) to obtain a bacterial pellet. The bacterial pellet was resuspended in phosphate-buffered saline (PBS) to obtain the probiotic formulation. The total number of viable bacteria per milliliter of the probiotic formulation was 10. 6 ~10 10 10, preferably 10 8 ~10 9 10, the optimal number is 10. 8 indivual.

[0021] The beneficial effects of this invention are: The probiotic preparation of this invention has a simple manufacturing process, low production cost, and good application prospects. Experimental results show that this probiotic preparation can increase the litter size of mammals. Furthermore, the strains selected in this invention are all beneficial strains naturally occurring in the intestines of sows or isolated from healthy animals, exhibiting high safety and no side effects. Attached Figure Description

[0022] Figure 1 A comparison chart of litter size between high-producing sows and low-producing sows; In the picture, express: P <0.001, ns means: P >0.05; Figure 2 Venn plots and principal component analysis (PCoA) plots of species composition for high-producing and low-producing sow groups; In the figure, A is the Venn diagram of species composition for the high-producing sow group and the low-producing sow group; B is the principal component analysis PCoA plot; Figure 3 A graph showing the correlation between sow litter size and bacterial abundance; Figure 4 A comparison of the abundance of key gut bacteria between high-producing and low-producing sow groups; Figure 5 The graph shows the effect of the probiotic preparation 1 prepared in Example 2 on the reproductive performance of mice. Detailed Implementation

[0023] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.

[0024] Example 1 Fresh feces were collected from 73 Large White × Landrace crossbred sows after farrowing and frozen at -80℃. The number of piglets born and the number of live piglets were recorded. Sows with more than 14 live piglets (n=10) were selected as the high-producing sow group, and sows with fewer than 10 live piglets were selected as the low-producing sow group. The total number of piglets born and the number of live piglets in the high-producing sow group were significantly higher than those in the low-producing sow group. Figure 1 Fecal samples from high- and low-producing sows were subjected to 16S rDNA sequencing to analyze the gut microbial species composition of the two groups. The results showed that the microbial composition of the two groups was not significantly different, with a large number of shared OTUs and a small percentage of OTUs specific to each group. Figure 2A); PCoA analysis of the weighted unifrac distance matrix was used, and the differences in β-diversity of microbial composition at the OUT level were assessed based on the Adonis test. The results showed that the principal coordinate components PC1 and PC2 at the OUT level contributed 45.06% and 14.78% to the differences in sample composition, respectively. The microbial communities of the high-producing sow group and the low-producing sow group were relatively separated in terms of evolutionary relationships and composition. Figure 2 B). Correlation analysis of total litter size, live litter size, healthy litter size, weak litter size, stillbirth size, and species abundance of sows revealed a highly significant negative correlation between Clostridium abundance and stillbirth size in sows. Figure 3 Simultaneously, Metagenomeseq was used to analyze the differentially dominant bacteria between the two groups at the species level and to screen for biomarkers. Metagenomeseq uses Cumulative-Sum Scaling (CSS) normalization and zero-inflated log-normal (ZIL) or zero-inflated Gaussian (ZIG) models to identify differentially dominant species, which can overcome the interference problems on the accuracy of results caused by data normalization and sparsity (sparse data, sequencing data usually has many zero values).

[0025] The results showed that one of the biomarkers in the high-producing sow group was Clostridium herbivorans, and the abundance of Clostridium herbivorans in the high-producing sow group was higher than that in the low-producing sow group. Figure 4 The Clostridium herbivorans strain was Clostridium herbivorans DSM-14428, purchased from the German Culture Collection of Microorganisms (DSMZ, https: / / www.dsmz.de / collection / catalogue / details / culture / DSM-14428).

[0026] Example 2 In probiotic preparation 1 for improving reproductive performance in mammals, the viable count of Clostridium herbivorans strain was 10. 8 per ml.

[0027] The Clostridium herbivorans strain mentioned above is Clostridium herbivorans DSM-14428, purchased from the German Culture Collection of Microorganisms (DSMZ, https: / / www.dsmz.de / collection / catalogue / details / culture / DSM-14428), and can also be isolated from pig feces or large intestine feces according to the method in Example 1.

[0028] The above-mentioned probiotic preparation 1 is obtained by the following method: 1) Clostridium herbivorans strain was inoculated into DSMZ339a medium and cultured in an anaerobic incubator with a gas environment of 90% N2, 5% CO2, and 5% H2 at a temperature of 37℃. Viable cell counts were performed to determine the viable cell count in the culture, and the count was adjusted to 10⁻⁶ cells / mL based on the results. 6 ~10¹ 0 Bacterial count / mL; then glycerol was added to obtain a bacterial cryopreservation solution, which was stored in liquid nitrogen at low temperature.

[0029] 2) The bacterial culture, rapidly thawed from liquid nitrogen cryopreservation using a 37°C water bath, was then inoculated into fresh liquid culture medium for enrichment culture. Once the liquid culture medium became turbid, an appropriate amount of bacterial culture was streaked onto solid culture medium using an inoculation loop. After single colonies grew in the solid culture medium, these colonies were picked and inoculated into fresh liquid culture medium for enrichment culture. Once the liquid culture medium became turbid, bacterial genomic DNA was extracted from the bacterial culture, and the full-length 16S rDNA was amplified by PCR and purified. The recovered 16S rDNA fragment was sent to a sequencing company for sequencing. The sequencing results were compared with the NCBI database to verify whether the picked single colonies were the original strain. The successfully sequenced bacterial culture was then inoculated into fresh liquid culture medium for expansion culture. Viable cell counts were performed to determine the viable cell count in the bacterial culture, and the viable cell count was adjusted to 10-1 based on the count results. 6 ~10¹ 0 Bacterial count / mL; then glycerol was added to obtain a bacterial cryopreservation solution, which was stored in liquid nitrogen at low temperature.

[0030] 3) Before use, rapidly thaw the bacterial suspension in a 37°C water bath, then centrifuge the resulting bacterial suspension to obtain a bacterial pellet. Resuspend the bacterial pellet in phosphate-buffered saline (PBS) to obtain probiotic preparation 1. Each milliliter of probiotic preparation 1 contains 10 live bacteria. 8 indivual.

[0031] Mouse efficacy test to verify probiotic formulation 1 Six-week-old ICR female mice were randomly divided into a control group and a Clostridium herbivorans oral administration group (probiotic preparation 1), and acclimatized for one week in an SPF-grade breeding center. Seven-week-old ICR female mice were treated with a mixed antibiotic in their drinking water for one week. After the antibiotic treatment ended, starting from the eighth week of age, mice were orally administered 200 μL of PBS (control group) or 200 μL of probiotic preparation 1 suspension (Clostridium herbivorans oral administration group) every other day for one week. Mice were then paired, and the presence of a vaginal plug the following morning indicated successful mating. After mating, the control group and Clostridium herbivorans oral administration group continued to be orally administered 200 μL of PBS and 200 μL of probiotic preparation 1 suspension every other day for a total of three times until day 6 of gestation. Each group initially consisted of 30 mice, and the number of pups born and the number of live pups were recorded at the time of parturition.

[0032] like Figure 5 As shown, the number of litters and the number of live pups in the gavage group increased, indicating that probiotic preparation 1 can improve reproductive performance.

[0033] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of a Clostridium herbivorans strain in improving mammalian reproductive performance.

2. Application of a Clostridium herbivorans strain in the preparation of products that promote mammalian reproductive performance.

3. A probiotic preparation for promoting reproductive performance in mammals, characterized in that: The probiotic preparation contains Clostridium herbivorans strain, wherein the viable count of Clostridium herbivorans strain in the probiotic preparation is 10. 6 ~10 10 per milliliter.

4. The probiotic preparation according to claim 3, characterized in that: The viable count of the Clostridium herbivorans strain was 10. 8 ~10 9 per milliliter.

5. The probiotic preparation according to claim 3 or 4, characterized in that: The viable count of the Clostridium herbivorans strain was 10. 8 per milliliter.

6. A method for preparing the probiotic preparation according to claim 3, characterized in that: The method involves inoculating Clostridium herbivorans strain into DSMZ339a medium, culturing it in an anaerobic incubator, and obtaining a fresh bacterial solution, which is the probiotic preparation. The inoculation amount of Clostridium herbivorans strain is 1-2% of the volume of DSMZ339a medium. The probiotic preparation contains 10 live bacteria of the Clostridium herbivorans strain. 6 ~10 10 per milliliter.

7. The preparation method according to claim 6, characterized in that: The inoculum size of the Clostridium herbivorans strain was 1% of the volume of the DSMZ339a culture medium. The gas environment of the anaerobic incubator was 90% N2, 5% CO2, and 5% H2; the incubation temperature was 37℃.

8. The preparation method according to claim 6 or 7, characterized in that: The probiotic preparation contains 10 live bacteria of the Clostridium herbivorans strain. 8 ~10 9 per milliliter.

9. The preparation method according to claim 8, characterized in that: The probiotic preparation contains 10 live bacteria of the Clostridium herbivorans strain. 8 per milliliter.

10. The application of the probiotic preparation according to any one of claims 3 to 5 in improving litter size and litter live birth rate in mammals, characterized in that: The method of application is as follows: the probiotic preparation is administered orally to the stomach of 8-week-old female mice at a dose of 200 microliters per dose, once every other day until the 6th day of pregnancy.

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