Murine combined lactobacillus capable of improving reproductive performance of livestock

Abstract

The invention discloses a strain of Ligilbacillus murinus PV211184 capable of improving the reproductive performance of livestock, which belongs to the field of agricultural animal husbandry, is preserved in Guangdong Province Microbial Culture Collection Center, has a preservation number of GDMCC No: 66039, has a bacterial colony in milk white forms such as round bulge, smoothness and the like, has a suitable growth temperature of 30-45 DEG C, a pH value of 4.0-6.5, preferably 37 DEG C and a pH value of 6.0, and is subjected to facultative anaerobic culture by using an MRS culture medium. The bacterium can be colonized in the uterus, has good endometrial epithelial cell adhesion capability, has no negative influence on sperm motility and IVF embryonic development, and can promote endometrial epithelial cell proliferation. Experiments show that the litter size per litter of a sow can be increased by 1.3, the litter size per litter can be increased by 1.0, the lambing rate of Hu sheep can be increased by 7.2%, the lambing rate per litter can be increased by 14.5%, and the feed can be used as a probiotic agent for improving the reproductive performance of livestock.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of agricultural and livestock applications, and particularly relates to a Ligilactobacillus murinus PV211184 with improved reproductive performance of livestock. BACKGROUND

[0002] Through screening of pig-derived uterine probiotics, the obtained probiotics can effectively improve the adverse effects on animal reproductive results caused by changes in the uterine microbial flora environment. Ligilactobacillus murinus is a kind of Lactobacillus, which widely exists in the intestinal tract and reproductive organs of animals as a potential probiotic. However, there are relatively few reports on the application of Ligilactobacillus murinus in assisted reproduction of livestock.

[0003] The uterine microbial flora environment is closely related to the reproductive results of livestock. A healthy uterine microbial flora environment provides protection for inhibiting pathogenic substances and ensuring the production quality of livestock. LANIEWSKI et al. found that there is microbial colonization in the uterus, and Lactobacillus is the key probiotic. Lactobacillus in the uterus regulates the pH of the uterine environment by secreting lactic acid, effectively inhibits the growth of pathogenic bacteria, and maintains the homeostasis of the uterine environment (Pawel L, Zehra EI, Melissa MH. The microbiome and gynaecological cancer development, prevention and therapy. Nat Rev Urol. 2020 Apr; 17(4): 232-250). He et al. found that Lactobacillus crispatus has the function of relieving the failure of embryo implantation caused by E. coli-induced mouse endometritis (He X, Chen W, Zhou X. The Therapeutic Potential of Lactobacillus crispatus for Chronic Endometritis: A Comprehensive Clinical Trial and Experimental Investigation. Probiotics Antimicrob Proteins. 2024 Aug; 22). However, there is no report on the isolation of Lactobacillus from the uterus of pigs, and the pig uterus-derived Lactobacillus with improved reproductive performance of livestock. Therefore, the pig uterus is selected as the main part for screening of probiotics, which can more directly show the function of probiotics in regulating the microecological balance of the uterine environment, regulating immune function, and affecting the reproductive results of livestock. Therefore, screening of probiotic strains with the function of affecting the reproduction of livestock from the uterine flora of pigs has practical production significance. SUMMARY

[0004] Based on the above actual problems and needs in the process of livestock breeding production, the present application aims to provide a Ligilactobacillus murinus PV211184 with improved livestock reproductive performance, which can colonize in the uterus of livestock and maintain the homeostasis of the uterus microbial environment, thereby helping to improve the reproductive performance of livestock.

[0005] The present application achieves the above-mentioned purpose by the following technical solution: a Ligilactobacillus murinus PV211184 with improved livestock reproductive performance, which is preserved in the Guangdong Microbial Culture Collection Center, located at 100, Martyrs' Road, Guangzhou, Guangdong Institute of Microbiology, and preserved on March 21, 2025, with the preservation number GDMCCNo:66039 and the classification name Ligilactobacillus murinus PV211184. Its biological characteristics include: the colony is round, convex, smooth, wet, and milky white.

[0006] The present application also has the following technical features:

[0007] As described above, the growth conditions of the Ligilactobacillus murinus are temperature 30-45℃, pH=4.0-6.5.

[0008] Further preferably, as described above, the growth conditions of the Ligilactobacillus murinus are temperature 37℃, pH=6.0.

[0009] The present application also provides a culture method of the Ligilactobacillus murinus as described above, as follows: take the seed liquid and inoculate it into the culture medium at an inoculation amount of 1%, and then incubate it at a constant temperature, with temperature 30-45℃, pH=4.0-6.5, and facultative anaerobic culture, and the culture medium is MRS medium.

[0010] The present application also provides the application of the Ligilactobacillus murinus as described above as a probiotic agent for improving the reproductive performance of livestock.

[0011] The present application also provides a bacterial agent containing the Ligilactobacillus murinus as described above.

[0012] The mouse combined lactobacillus strain provided by the application can be planted in the uterus; the cell ex vivo test proves that the mouse combined lactobacillus strain provided by the application has good adhesion capacity with the endometrial epithelial cells of domestic animals, further proving the characteristic that the mouse combined lactobacillus strain can be planted in the uterus; meanwhile, the mouse combined lactobacillus provided by the application can obviously improve and enhance the reproductive performance of domestic animals as probiotics. In conclusion, the mouse combined lactobacillus is a pig source probiotic with high application value, and has important significance for how to improve the reproductive performance of domestic animals in the animal husbandry. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 It is a colony morphology chart of the strain in the MRS culture medium;

[0014] Figure 2 It is a phylogenetic tree chart of the mouse combined lactobacillus;

[0015] Figure 3 It is a growth curve chart of the mouse combined lactobacillus;

[0016] Figure 4 It is a chart of the mouse combined lactobacillus promoting the proliferation of pig endometrial epithelial cells, wherein A, B and C are the mRNA expression amounts of the cell proliferation related genes CTNNB, PCNA and WNT2 detected by real-time quantitative PCR; D and E are the CTNNB protein expression amounts detected by Western Blot; F and G are the cell proliferation positive rates detected by Edu;

[0017] Figure 5 It is a statistical chart of the influence of the mouse combined lactobacillus on the sperm activity of pigs, wherein A is the sperm activity detection after the mouse combined lactobacillus and pig sperm are co-cultured under the condition of 17 DEG C; B is the sperm activity detection after the mouse combined lactobacillus and pig sperm are co-cultured under the condition of 37 DEG C;

[0018] Figure 6 It is a chart of the mouse combined lactobacillus supernatant and pig IVF embryo co-culture. DETAILED DESCRIPTION

[0019] The content of the application will be further illustrated by the following examples. It should be noted that the embodiments of the application and the features in each embodiment can be combined with each other without conflict.

[0020] The application will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0021] Example 1: Screening, identification and preservation of the mouse combined lactobacillus strain in the uterus

[0022] 1. Sample collection: collect the uterus tissue of the farrowing sow, adopt the cotton swab sampling method, and scrape the inner wall of the pig uterus.

[0023] 2. Isolation and purification of the strain: The cotton swab scraped from the inner wall of the pig uterus was placed in 1.5 mL of physiological saline and shaken thoroughly to prepare a suspension, which was diluted 10-fold. The suspension and the 10-fold dilution were spread on selective MRS plates containing 2% CaCO3, and each dilution was repeated twice. The plates were incubated at 37°C in an anaerobic incubator for 48 h, and single colonies were picked and streaked for anaerobic culture.

[0024] The selective MRS broth medium containing 2% CaCO3 (1 L) contains peptone 10.0 g, beef extract 10.0 g, yeast extract 5.0 g, glucose (C6H 12 O6·H2O) 20.0 g, manganese sulfate (MnSO4·H2O) 0.05 g, magnesium sulfate (MgSO4·7H2O) 0.1 g, sodium acetate (CH3COONa·3H2O) 5.0 g, diammonium hydrogen citrate [(NH4)2HC6H5O7] 2.0 g, potassium hydrogen phosphate (K2HPO4·3H2O) 2.0 g, Tween 80 1.0 g, and calcium carbonate (CaCO3) 20 g.

[0025] 3. Strain screening: After 48 h of culture, the strains capable of producing lactic acid were identified by the presence of transparent calcium-dissolving rings around the colonies on the MRS plates. Single colonies with obvious calcium-dissolving rings were picked and inoculated into MRS broth for 24 h of culture.

[0026] 4. Microscopic observation of the strain and 16S rRNA sequencing identification: The selected strain was subjected to Gram staining, and then its morphology was observed under a microscope. The colonies were purple after staining, and the bacteria were Gram-positive rods, straight or slightly curved, and arranged regularly.

[0027] 5. The strain with obvious transparent calcium-dissolving rings around the colony was selected for strain identification. The strain DNA was extracted using a bacterial genomic DNA extraction kit, 16S rRNA was amplified by PCR technology, sequencing analysis was performed, the species of the strain was identified, and the phylogenetic tree of the strain was drawn, as shown in Figure 1 .

[0028] 6. Experimental results: Multiple microbial strains capable of producing lactic acid were screened from the pig uterus. Through 16S rRNA sequencing analysis and morphological observation, strain L1 was identified as Ligilactobacillus murinus, and the sequence of its 16S rRNA (as shown in SEQ ID NO. 1) had a similarity of 99.73% with the sequence of the reported Ligilactobacillus murinus.

[0029] 7. Colony characteristics: The strain was inoculated into MRS medium and anaerobically cultured for 48 h, and the colony characteristics were observed.

[0030] 8. Experimental Results: The strain was anaerobically cultured on MRS solid medium for 2 days, such as... Figure 2 As shown, the colonies are round, raised, smooth, moist, and milky white, with a diameter of approximately 1 mm. This strain has been deposited at the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC No: 66039.

[0031] Example 2: Mouse-Lactobacillus Growth Curve Experiment

[0032] 1. Determination of growth curve: The third generation of *Lactobacillus simulans*, which was preserved in 30% glycerol tubes at -80℃, was revived and activated as seed culture. It was inoculated into MRS broth at an inoculation ratio of 1% and placed in a microbial incubator at 37℃. Every 2 hours, 200 μL was aspirated and the absorbance at OD600 nm was measured using an ELISA reader, and the growth curve was plotted.

[0033] 2. Experimental results are as follows Figure 3 The results showed that *Lactobacillus assemblica* entered the logarithmic growth phase after 4 hours, reached the plateau phase after 14 hours, and finally achieved an OD value of 1.9 at the plateau phase.

[0034] Example 3: Determination of the adhesion ability of mouse combined with Lactobacillus

[0035] 1. Strain activation: The *Lactobacillus murineis* strain preserved in 30% glycerol tubes at -80℃ was streaked onto MRS medium and placed in a 37℃ microbial incubator for 48 hours; then a single colony was picked and inoculated into MRS liquid medium and incubated at 37℃ for 12 hours.

[0036] 2. Autoagglutination ability test: The *Lactobacillus assemblica* strain cultured overnight was centrifuged at 12000 rpm / min for 1 min to collect the bacterial cells. The cells were washed twice with sterile 1×PBS solution, and the bacterial concentration was adjusted to 1×10⁻⁶. 7 CFU / mL. Then, 5 mL of the adjusted bacterial suspension was vortexed for 10 seconds and incubated at 37°C for 6 hours. To observe the self-aggregation ability of *Lactobacillus assemblica*, after 6 hours of incubation, the upper layer of the bacterial suspension was collected, and the absorbance (At) was measured at OD600 nm. Three replicates were performed each time, and the bacterial self-aggregation rate was calculated using the following formula:

[0037]

[0038] Where A0 represents the initial absorbance of the bacterial suspension at 0 hours, and At represents the absorbance of the upper layer of the bacterial suspension after 6 hours of incubation.

[0039] 3. Surface hydrophobicity determination: After centrifugation at 12000 rpm / min for 1 min, the cells of the overnight culture of L. jensenii strains were collected, washed twice with sterilized 1x PBS solution, and the concentration of the bacterial solution was adjusted to 1x10 7 CFU / mL. Then, 2 mL of the adjusted bacterial suspension was mixed with an equal volume of chloroform, vortexed for 10 min, and allowed to stand at 25°C for 40 min for two-phase separation. The lower aqueous phase was carefully collected. The absorbance was measured at OD600nm and recorded as Af, and three replicates were measured each time.

[0040] Surface hydrophobicity (%) = 1 - (Af / A0) x 100

[0041] wherein Af represents the final absorbance and A0 represents the initial absorbance.

[0042] 4. Porcine endometrial epithelial cell (PEECs) cell adhesion ability determination: After the PEECs cells were cultured to 70-80% confluence, they were digested with 0.25% trypsin, blown to single cells, and counted using a hemocytometer. The concentration of the living cells was adjusted to 1x10 5 CFU / mL. Then, 2 mL of the adjusted bacterial suspension was mixed with an equal volume of chloroform, vortexed for 10 min, and allowed to stand at 25°C for 40 min for two-phase separation. The lower aqueous phase was carefully collected. The absorbance was measured at OD600nm and recorded as Af, and three replicates were measured each time. 7 CFU / mL (V0) was added to each well, and the cells were incubated at 37°C in a 5% CO2 incubator for 2 hours. After incubation, the cells were washed three times with sterile PBS solution to elute the adhered lactic acid bacteria. Then, the cells were digested with 0.25% trypsin, and when the cells were completely digested, the cells were collected and diluted by a factor of ten. The dilutions were spread on MRS solid medium, and the number of adhered lactic acid bacteria (V) was determined by plate colony counting. The experiment was repeated three times. The adhesion rate of lactic acid bacteria to PEECs cells was calculated according to the following formula:

[0043]

[0044] wherein V represents the final absorbance and V0 represents the initial absorbance.

[0045] 5、Strain adhesion capacity determination results: strain self-aggregation capacity, surface hydrophobicity and PEECs cell adhesion capacity are shown in Table 1. L. murinus showed hydrophobicity to solvent, self-aggregation capacity and adhesion capacity to PEECs cells. The results showed that L. murinus could adhere, colonize and survive in uterine epithelium.

[0046] Table 1 Strain adhesion capacity determination

[0047]

[0048] Example 4: Determination of the effect of L. murinus on the proliferation of porcine endometrial epithelial cells (PEECs)

[0049] 1. Strain activation: L. murinus stored in 30% glycerol tubes at -80°C was activated for three generations as seed liquid, inoculated into MRS broth at a ratio of 1%, and incubated in a 37°C microbiological incubator.

[0050] 2. Determination of the effect of L. murinus on the proliferation of porcine endometrial epithelial cells: After L. murinus was cultured to the logarithmic growth phase, it was centrifuged at high speed (12000 r / min), and the supernatant was discarded after centrifugation to collect the bacterial cells, which were resuspended with PBS solution sterilized by high temperature and high pressure. L. murinus (1 x 10 6 CFU / mL) was co-cultured with PEECs for 12 hours, and real-time quantitative PCR, Western Blot and Edu method were used to detect cell proliferation.

[0051] 3. Experimental results: The results of the effect of L. murinus on the proliferation of porcine endometrial epithelial cells are shown in Figure 4 After L. murinus and PEECs were co-cultured for 12 hours, the mRNA expression of cell proliferation-related genes CTNNB1, PCNA and WNT2 was significantly up-regulated (A, B, C); the expression of CTNNB protein was significantly up-regulated (D, E); and the positive rate of Edu detection of cells was significantly increased (F, G). The results showed that L. murinus could promote the proliferation of porcine endometrial epithelial cells. Figure 4 Figure 4 Figure 4

[0052] Example 5: Determination of the effect of L. murinus on the motility of porcine sperm

[0053] 1. Strain activation: L. murinus stored in 30% glycerol tubes at -80°C was activated for three generations as seed liquid, inoculated into MRS broth at a ratio of 1%, and incubated in a 37°C microbiological incubator.

[0054] ​​​2. Determination of the effect of L. gasseri on the activity of pig sperm: After L. gasseri was cultured to the logarithmic growth phase, high-speed centrifugation (12000 r / min) was performed, and the supernatant was discarded after centrifugation to collect the bacterial body. The bacterial body was resuspended with PBS solution sterilized by high temperature and high pressure. The bacterial-PBS suspension and pig semen were mixed and cultured at 17°C and 37°C, respectively. The control group was mixed with PBS solution and pig semen in the same way. Under the culture condition of 17°C, the sperm activity was determined and recorded every 24 hours; under the culture condition of 37°C, the sperm activity was determined and recorded every 6 hours.

[0055] 3. Experimental results: The effect of L. gasseri on the activity of pig sperm is shown in Table 1. After 168 hours of addition of L. gasseri under the culture condition of 17°C, the sperm activity can still reach 95.74%, and there is no significant difference between the control group (A). Figure 5 Figure 5 Under the culture condition of 37°C, after 30 hours of addition of L. gasseri, the sperm activity can reach 80.47%, and there is no significant difference between the control group (B). The results show that L. gasseri has no negative effect on the activity of pig sperm. Figure 5

[0056] Example 6: Determination of the co-culture of L. gasseri supernatant and pig IVF embryos

[0057] 1. Strain activation: L. gasseri stored in a 30% glycerol tube at -80°C was recovered and activated for three generations as a seed solution, which was inoculated into MRS broth at a ratio of 1%, and then placed in a 37°C microbiological incubator for constant temperature culture.

[0058] 2. Determination of the co-culture of L. gasseri supernatant and pig IVF embryos: After L. gasseri was cultured to the logarithmic growth phase, high-speed centrifugation (12000 r / min) was performed, and the supernatant was discarded after centrifugation to collect the bacterial body. The bacterial body was resuspended with PBS solution sterilized by high temperature and high pressure. The bacterial-PBS suspension and pig semen were mixed and cultured at 17°C and 37°C, respectively. The control group was mixed with PBS solution and pig semen in the same way. Under the culture condition of 17°C, the sperm activity was determined and recorded every 24 hours; under the culture condition of 37°C, the sperm activity was determined and recorded every 6 hours.

[0059] 3. Experimental results: The effect of L. gasseri on the activity of pig sperm is shown in Table 1. After 168 hours of addition of L. gasseri under the culture condition of 17°C, the sperm activity can still reach 95.74%, and there is no significant difference between the control group (A). Figure 6

[0060] Example 7: Determination of the effect of L. gasseri on increasing the number of live piglets born by sows

[0061] ​​​1. Strain activation: The third generation of *Lactobacillus simulans* preserved in 30% glycerol tubes at -80℃ was revived and activated as seed culture. The seed culture was then inoculated into MRS broth at an inoculation ratio of 1% and placed in a 37℃ microbial incubator for constant temperature culture.

[0062] 2. Determination of the effect of *Lactobacillus aspergillus* on increasing the live birth rate of sows: *Lactobacillus aspergillus* was cultured to the logarithmic growth phase and then centrifuged at high speed (12000 r / min). After centrifugation, the supernatant was discarded and the bacterial cells were collected. The bacterial cells were resuspended in PBS solution that had been sterilized by high temperature and high pressure. The bacterial-PBS suspension was then introduced into the uterus of sows that had reached the mating requirements after estrus detection.

[0063] 3. Inoculation of bacterial suspension into the uterus of sows: Forty healthy sows undergoing estrus synchronization treatment were randomly divided into a control group and an experimental group, with 20 sows in each group. Sows in the experimental group received 1 mL of bacterial-PBS suspension. During artificial insemination, the bacterial-PBS suspension was instilled into the uterine cavity along with the semen through an insemination tube. First, the bacterial-PBS suspension was mixed with the semen; then, the insemination tube was inserted deep into the cervix, and the semen-bacterial-PBS mixture was slowly injected. Sows in the control group received a semen-sterile PBS mixture during the same period. The mating methods and feeding management conditions were the same as those in the experimental group.

[0064] 4. Experimental Results: The results of the study on the effect of *Lactobacillus simulans* on increasing the number of live piglets born in sows are shown in Table 2. After *Lactobacillus simulans* colonized the sow uterus, the average litter size increased by 1.3 piglets, and the average number of live piglets born increased by 1.0 piglet. The results indicate that *Lactobacillus simulans* colonization in the sow uterus can increase the number of live piglets born in sows.

[0065] Table 2 Statistics on the number of piglets born to sows

[0066] Example 8: Determination of the effect of *Lactobacillus simulans* on improving the lambing rate of Hu sheep

[0067] 1. Strain activation: The third generation of *Lactobacillus simulans* preserved in 30% glycerol tubes at -80℃ was revived and activated as seed culture. The seed culture was then inoculated into MRS broth at an inoculation ratio of 1% and placed in a 37℃ microbial incubator for constant temperature culture.

[0068] 2. Determination of the effect of *Lactobacillus simulans* on improving the multi-lamb birth rate in Hu sheep: *Lactobacillus simulans* was cultured to the logarithmic growth phase and then centrifuged at high speed (12000 r / min). After centrifugation, the supernatant was discarded and the bacterial cells were collected. The bacterial cells were resuspended in PBS solution that had been sterilized by high temperature and high pressure. The bacterial-PBS suspension was then introduced into the uterus of ewes that had reached the mating requirements after estrus detection.

[0069] 3. Inoculation of bacterial suspension into the uterus of ewes: Forty healthy ewes undergoing estrus synchronization treatment were randomly divided into a control group and an experimental group, with 20 ewes in each group. Ewes in the experimental group received 1 mL of bacterial-PBS suspension. During artificial insemination, the bacterial-PBS suspension was instilled into the uterine cavity along with the semen using an insemination gun. First, the bacterial-PBS suspension was mixed with the semen; then, the insemination tube was inserted deep into the cervix, and the semen-bacterial-PBS mixture was slowly injected. Ewes in the control group received a semen-sterile PBS mixture during the same period. The mating methods and feeding management conditions were the same as those in the experimental group.

[0070] 4. Experimental Results: The results of the study on the effect of *Lactobacillus simulans* on the multi-lamb birth rate in Hu sheep are shown in Table 3. After *Lactobacillus simulans* colonized the uterus of Hu sheep, the multi-lamb birth rate increased by 7.2%, and the average birth rate per head increased by 14.5%. The results indicate that the colonization of *Lactobacillus simulans* in the uterus of Hu sheep can improve the multi-lamb birth rate.

[0071] Table 3. Statistics on the reproductive performance of Hu sheep

[0072]

Claims

1. A strain of *Ligilactobacillus murinus* PV211184, which is known to improve the reproductive performance of livestock, is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at the Institute of Microbiology, Guangdong Academy of Sciences, No. 100 Xianlie Middle Road, Guangzhou, on March 21, 2025. The accession number is GDMCC No. 66039, and the classification name is *Ligilactobacillus murinus* PV211184.

2. The *Lactobacillus murineis* strain that improves livestock reproductive performance according to claim 1, characterized in that: Its growth conditions are a temperature of 30℃-45℃ and a pH of 4.0-6.

5.

3. The *Lactobacillus murineis* strain for improving livestock reproductive performance according to claim 2, characterized in that: Its growth conditions are a temperature of 37℃ and a pH of 6.

0.

4. The method for culturing a strain of *Lactobacillus murineis* that improves livestock reproductive performance according to claim 1, characterized in that: The method is as follows: Take the seed liquid and inoculate it into the culture medium at a volume ratio of 1%, keep it at a constant temperature of 30℃-45℃, pH=4.0-6.5, and culture it in a facultative anaerobic environment. The culture medium is MRS medium.

5. The application of the strain of *Lactobacillus murineis* that improves livestock reproductive performance as described in claim 1 as a microbial agent for improving livestock reproductive performance.

6. A microbial agent, characterized in that: It includes a strain of *Lactobacillus simulans* as described in claim 1, which enhances the reproductive performance of livestock.

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