Application of lactobacillus johnsonii in promoting in-vitro maturation of porcine oocytes

Abstract

The invention discloses an application of lactobacillus johnsonii in promoting in-vitro maturation of porcine oocytes, and a synergistic regulation system is constructed by optimizing formulas of an oocyte maturation culture solution, an electric activation solution and an embryo culture solution. The application can effectively promote the in-vitro maturation rate of the porcine oocytes to be increased from 68.84% to 77.60%, promote the discharge of first polar bodies, reduce the apoptosis rate of the oocytes and improve the development potential of the oocytes after nuclear transplantation. Therefore, the in-vitro maturation efficiency of the oocytes is improved, the problems of low maturation efficiency, high apoptosis ratio and long culture period at present are solved, and the technical system can provide more high-quality oocytes for somatic cell nuclear transplantation, manual cloning, in-vitro fertilization, transgenic cloning, gene editing and the like, and has a wide application prospect in actual production.

Description

Technical Field

[0001] This invention relates to the field of animal embryo engineering technology, specifically to the application of Lactobacillus johnsonii in promoting the in vitro maturation of porcine oocytes. Background Technology

[0002] In vitro maturation of oocytes (IVM) is a core component of animal embryo biotechnology, widely used in in vitro fertilization of livestock, somatic cell nuclear transfer, transgenic animal production, and gene editing. It is of great significance for the breeding of superior livestock and the construction of disease models in biomedicine. As an important economic livestock, the development of embryo engineering technology for pigs plays a crucial role in improving breeding efficiency and protecting superior breeds. Furthermore, pigs share a high degree of physiological similarity with humans, making their oocytes and embryos important experimental materials for biomedical research. However, current in vitro maturation technology for pig oocytes still faces many bottlenecks, hindering the development of related fields.

[0003] In current technologies, the in vitro maturation rate of porcine oocytes is generally low, typically between 60% and 70%, and the quality of mature oocytes varies considerably, with a high apoptosis rate. This leads to insufficient cleavage rate, blastocyst rate, and developmental potential in subsequent nuclear transfer embryos. One of the core reasons for this problem is the significant difference between the in vitro culture environment and the in vivo physiological environment, especially the influence of oxidative stress. During oocyte in vitro culture, the relatively high oxygen concentration in the culture system, coupled with the lack of a complex antioxidant defense network in vivo, easily leads to a large accumulation of reactive oxygen species (ROS). ROS attack oocyte DNA, proteins, lipids, and other biomolecules, causing oxidative damage, resulting in oocyte maturation arrest, abnormal meiosis, and even apoptosis. Simultaneously, ROS also affect lipid droplet metabolism, mitochondrial function, and signaling pathway regulation within oocytes, further reducing the developmental potential of oocytes.

[0004] To address oxidative stress, existing technologies attempt to add antioxidants, such as vitamin C and glutathione, to the culture medium. However, the effects of a single antioxidant are limited, and they are difficult to synergistically improve multiple aspects of oocyte maturation, including nutrient metabolism and signal transduction. Furthermore, some antioxidants exhibit concentration-dependent toxicity; excessively high concentrations can damage oocytes. Simultaneously, the nutritional composition of existing oocyte maturation culture media is relatively simple, primarily consisting of basal culture medium combined with serum and hormones, lacking specific regulatory components for key oocyte maturation processes (such as lipid droplet breakdown, energy metabolism, and cell cycle regulation), resulting in poor oocyte maturation quality.

[0005] Probiotics, as beneficial live microorganisms, have attracted widespread attention for their antioxidant and metabolic regulatory effects. Existing studies have shown that some Lactobacillus probiotics can exert antioxidant effects by scavenging free radicals, activating the host's antioxidant enzyme system, and regulating cell signaling pathways, thereby improving the developmental state of germ cells. For example, reports indicate that feeding zebrafish Lactobacillus can significantly promote follicle development, increase the number of oocytes released, and improve embryo quality; adding Bifidobacterium metabolites to mouse oocyte in vitro culture can reduce apoptosis rate and increase maturation rate. However, research on the application of probiotics in the in vitro maturation of porcine oocytes is still in its early stages. Currently, there are no reports of applying Lactobacillus johnsonii to porcine oocyte in vitro maturation culture media, and existing probiotic applications mostly focus on the addition of single probiotics without combining them with other functional components to form a synergistic system, resulting in limited effectiveness.

[0006] Furthermore, there is room for improvement in existing electroactivation solutions and embryo culture media. Electroactivation is a crucial step in activating reconstructed embryos for nuclear transfer, and its formulation directly affects activation efficiency and subsequent embryonic development. Existing electroactivation solutions mainly use mannitol as an osmotic pressure regulator, binding calcium and magnesium ions, but lack auxiliary regulatory components for cell membrane stability and intracellular signal transduction, resulting in unstable activation success rates. As a nutrient source for the early development of nuclear transfer embryos, existing embryo culture media formulations can meet basic nutritional needs, but they lack specificity in areas such as antioxidants, energy supply, and cell proliferation regulation, making it difficult to effectively improve blastocyst quality and total cell count.

[0007] Meanwhile, existing in vitro maturation culture parameters for porcine oocytes largely borrow from those of other mammals, failing to fully consider the specificities of porcine oocytes. Porcine oocytes contain numerous lipid droplets in their cytoplasm, and their energy metabolism primarily relies on lipid breakdown. However, current culture parameters such as temperature, gas environment, and culture time are not fully adapted to the lipid metabolism characteristics of porcine oocytes, leading to abnormal lipid droplet metabolism and further affecting maturation efficiency. Furthermore, oocytes from different sources (such as ovaries from pigs of different ages and breeds) exhibit significant differences in adaptability to culture conditions, making it difficult for existing universal culture systems to meet diverse needs and resulting in insufficient technical stability.

[0008] In summary, current in vitro maturation technology for porcine oocytes suffers from problems such as low maturation rate, high apoptosis rate, insufficient embryonic developmental potential, poor targeting of culture systems, and non-optimized process parameters. Existing technologies lack a dedicated culture medium system and supporting process that can synergistically address multiple issues such as oxidative stress, nutrient supply, and signal regulation. Therefore, developing a novel application scheme based on Lactobacillus johnsonii, optimizing the culture medium formulation, and improving the culture process are of significant practical importance and application value for improving the quality of in vitro maturation of porcine oocytes and promoting the development of embryo engineering technology. Summary of the Invention

[0009] The purpose of this invention is to overcome the shortcomings of the prior art and provide the application of Lactobacillus johnsonii in promoting the in vitro maturation of porcine oocytes. By optimizing the special culture medium system (oocyte maturation culture medium, electroactivation medium, embryo culture medium) and supporting culture process, it synergistically solves problems such as oxidative stress, nutritional metabolism, and signal regulation in the in vitro maturation process of porcine oocytes, significantly improves the in vitro maturation rate of oocytes, reduces the apoptosis rate, and enhances the developmental potential of nuclear transfer embryos.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] The application of Lactobacillus johnsonii in promoting the in vitro maturation of porcine oocytes is characterized in that the promotion of in vitro maturation of porcine oocytes is achieved through a special culture medium system containing Lactobacillus johnsonii, wherein the special culture medium system includes oocyte maturation culture medium, electroactivation medium and embryo culture medium.

[0012] Furthermore, the oocyte maturation culture medium includes culture medium A and culture medium B.

[0013] Further, the culture medium A contains: 70% TCM199, 12% porcine follicular fluid, 18% fetal bovine serum, 0.1 mg / mL glutamine, 0.6 g / L glucose, 2.3 g / L NaHCO3, 10 IU / mL human chorionic gonadotropin, 15 IU / mL pregnant mare serum gonadotropin, 0.75 g / L penicillin, 0.5 g / L streptomycin, and 10 9 CFU / g Lactobacillus johnsonii 0.5%, 0.05g / L Vitamin E, 0.03g / L Melatonin, 0.15g / L L-Carnitine.

[0014] Further, the B culture medium contains: 70% TCM199, 12% porcine follicular fluid, 18% fetal bovine serum, 0.1 mg / mL glutamine, 0.6 g / L glucose, 2.3 g / L NaHCO3, 0.75 g / L penicillin, 0.5 g / L streptomycin, and 10 9 CFU / g Lactobacillus johnsonii 0.5%, 0.05g / L Vitamin E, 0.03g / L Melatonin, 0.15g / L L-Carnitine.

[0015] Furthermore, in culture medium A and culture medium B, the purity of vitamin E is ≥99%, melatonin is a natural extract, and L-carnitine is an L-type optically pure isomer.

[0016] Further, the electroactivating solution comprises: 0.3 mol / L mannitol, 0.1 mmol / L CaCl2, 0.1 mmol / L MgSO4, 1 mmol / L Hepes, 0.01% (w / v) PVA, 0.05 g / L penicillin, 0.02 g / L streptomycin, 0.08 mmol / L EGTA, 0.02 g / L BSA, and 0.1 mmol / L L-arginine.

[0017] Furthermore, in the electro-activation solution, EGTA is of anhydrous purity, BSA is of fatty acid-free type, and L-arginine is of biological reagent grade.

[0018] Furthermore, the embryo culture medium contains: 108 mmol / L NaCl, 10 mmol / L KCl, 0.35 mmol / L KH2PO4, 0.3 mmol / L MgSO4·7H2O, 24.65 mmol / L NaHCO3, 0.2 mmol / L sodium pyruvate, 2.0 mmol / L calcium lactate, 1.0 mmol / L L-glutamine, 5.0 mmol / L taurine, 20 ml / L essential amino acids, 15 ml / L non-essential amino acids, 5 g / L BSA, 0.04 g / L vitamin C, 0.02 g / L coenzyme Q10, and 0.015 g / L β-carotene.

[0019] Furthermore, in the embryo culture medium, vitamin C is L-ascorbic acid, coenzyme Q10 is a fat-soluble natural extract, and β-carotene has a purity of ≥95%.

[0020] This invention also provides a method for using *Lactobacillus johnsonii* in promoting in vitro maturation of porcine oocytes, comprising the following steps:

[0021] (1) Isolation and screening of oocytes: Ovaries of healthy sows were collected from the slaughterhouse and placed in sterile physiological saline containing penicillin and streptomycin. They were returned to the laboratory within 2-3 hours. The surface of the ovary was wiped with sterile gauze to remove blood, mesangium and connective tissue. 2-5 mm of clear follicular fluid was drawn with a 10 mL sterile syringe and injected into a sterile centrifuge tube. The tube was placed on a 37℃ constant temperature heating plate for 15-20 min. The supernatant was discarded and the precipitate was resuspended with DMEM culture medium. The suspension was transferred to a sterile glass dish. Under a Nikon stereomicroscope, COCs with more than three layers of granulosa cells, uniform cytoplasm and good refractive properties were selected and washed 3 times with PBS solution for later use.

[0022] (2) In vitro culture of oocytes: The selected COCs were transferred into culture medium A preheated to 38.5℃ and cultured in an incubator with 5% CO2, saturated humidity and 38.5℃ for 20-22h. Then they were transferred into culture medium B preheated to 38.5℃ and cultured under the same conditions for another 20-22h. The total culture time was 40-44h.

[0023] (3) Culture of donor cells: The tissue block adherence method was used to culture donor cells; healthy fetal pig thigh muscle tissue was isolated, washed 3-5 times with PBS solution containing double antibiotics, cut into 3-4 mm tissue blocks, spread evenly on the bottom of a 60 mm sterile culture dish, and DMEM culture medium containing 10% FBS was added. The culture dish was inverted in a 5% CO2, saturated humidity, 37℃ incubator for 24 h. After that, the culture dish was upright, an appropriate amount of DMEM culture medium containing 10% FBS was added, and the floating tissue blocks were removed. The culture medium was changed once after 72 h. When the fibroblasts grew to about 90% confluence, they were digested and passaged with 0.25% trypsin. Fibroblasts cultured to the 3rd-5th generation were used as nuclear transfer donor cells.

[0024] (4) Construction and activation of nuclear transfer embryos: Mature oocytes cultured for 40-44 h were transferred into PBS solution containing 1 mg / mL hyaluronidase. The granulosa cells were gently blown off. Mature oocytes with the first polar body, intact zona pellucida, clear perivitelline space, and uniform cytoplasm were selected. The oocytes were washed three times with PBS solution and enucleated by blind aspiration: Under an inverted microscope, 1 / 3 of the cytoplasm around the polar body was aspirated with a 28 µm diameter enucleation needle. Then, a single well-dispersed and regularly shaped donor cell was aspirated with the same needle and injected into the perivitelline space of the enucleated oocyte to complete the enucleation operation. The reconstructed body was transferred into an electroactivation solution preheated to 38.5 °C for equilibration for 10 min. Then, it was transferred to a fusion tank with electroactivation solution. The electroactivation parameters were set as follows: electric field strength 60 V / mm, pulse width 50 μs, and pulse count 3 times. Electroactivation treatment was performed.

[0025] (5) In vitro culture of nuclear transfer embryos: The reconstructed embryos after electro-activation were washed three times with embryo culture medium, transferred into microdroplets of embryo culture medium preheated to 39°C, covered with sterile mineral oil, and cultured in an incubator with 5% CO2, saturated humidity, and 39°C for 144~168h.

[0026] (6) Detection indicators and methods:

[0027] Oocyte maturation rate detection: After culturing for 40-44 hours, observe and count the number of oocytes that have expelled the first polar body, and calculate the maturation rate;

[0028] Oocyte apoptosis rate detection: The apoptosis rate was detected by TUNEL assay combined with DAPI staining. Mature oocytes were collected, washed three times with PBS solution, fixed in 4% paraformaldehyde for 30 min, washed three times with PBS / PVA solution, and treated with 0.1% Triton X-100 in PBS permeabilization buffer at room temperature for 10 min. Then, they were incubated with PBS blocking buffer containing 1% BSA for 20 min. The oocytes were then immersed in TUNEL staining solution and incubated at 37°C in the dark for 1.5 h. After washing three times with PBS / PVA solution, DAPI staining solution was added and incubated at room temperature in the dark for 5 min. Finally, the slides were mounted with anti-fluorescence quenching mounting solution and observed under a laser confocal microscope with excitation wavelengths of 488 nm and 405 nm. The number of apoptotic cells was counted and the apoptosis rate was calculated.

[0029] Detection of developmental indicators of nuclear transfer embryos: After 44 h of culture, the 2-cell cleavage rate was counted; after 168 h of culture, the blastocyst rate was counted. Blastocysts were stained with 10 μg / mL Hoechst 33342 dye for 15 min, mounted with anti-fluorescence quenching mounting solution, and observed and counted under a fluorescence microscope.

[0030] Compared with the prior art, the technical advantages of this invention are:

[0031] Advantage 1: Constructing a multi-dimensional synergistic antioxidant system to completely solve the core problem of oxidative stress.

[0032] In existing technologies, the main bottleneck in the in vitro maturation of porcine oocytes is oxidative stress. Traditional methods often use single antioxidants (such as vitamin C and glutathione), which can only target a certain type of ROS or a specific step, resulting in limited antioxidant effects and difficulty in addressing the metabolic needs during oocyte maturation. This invention overcomes this limitation by constructing a multidimensional synergistic antioxidant system consisting of "probiotic metabolites + lipid-soluble antioxidants + water-soluble antioxidants + endogenous antioxidant activators": Lactobacillus johnsonii metabolites (short-chain fatty acids, polysaccharides) scavenge extracellular ROS and activate endogenous antioxidant enzymes; vitamin E (lipid-soluble) protects cell membrane lipid structure and scavenge lipid peroxidation free radicals; melatonin (lipid-soluble + water-soluble) directly scavenge ROS and induces antioxidant gene expression; vitamin C (water-soluble) scavenge intracellular water-soluble ROS and promote GSH synthesis; coenzyme Q10 (lipid-soluble) scavenge mitochondrial ROS and enhance energy metabolism; and β-carotene (lipid-soluble) scavenge ROS and regulate developmental genes. This system covers the entire process and all regions (extracellular, intracellular, cell membrane, and mitochondria) of oocyte maturation and embryonic development. Compared with solutions that only add a single antioxidant, the synergistic system of this invention can not only more effectively inhibit oxidative stress, but also avoid the concentration toxicity that may exist with a single antioxidant through the synergistic effect of antioxidant and metabolic regulatory components, significantly improving the survival quality of oocytes and embryos. This is a technical effect that cannot be achieved by existing technologies.

[0033] Advantage 2: Targeted optimization of lipid metabolism regulation, adapted to the specific physiological characteristics of porcine oocytes

[0034] The core physiological characteristic of porcine oocytes is the presence of numerous lipid droplets in their cytoplasm, with energy metabolism primarily dependent on the β-oxidation of these droplets. However, existing culture media generally lack specific lipid metabolism regulators, leading to abnormal lipid droplet metabolism and insufficient energy supply, which in turn affects maturation rate and developmental potential. This invention addresses this specific need by adding L-carnitine to the oocyte maturation culture medium and optimizing the ratio of energy substrates such as glucose and calcium lactate. L-carnitine, as a coenzyme for acyl-CoA transferase, promotes the breakdown of lipid droplets into fatty acids and their transport to mitochondria for β-oxidation, increasing lipid droplet metabolism efficiency by over 35% and providing sufficient energy for oocyte meiosis. Simultaneously, L-carnitine reduces cytoplasmic heterogeneity caused by excessive lipid droplet accumulation, increasing oocyte cytoplasmic uniformity by 28%. Furthermore, the optimized calcium lactate concentration (2.0 mmol / L) in the embryo culture medium is adapted to the lactate-dependent metabolism of early porcine embryos. Sodium pyruvate and glucose serve as supplementary energy substrates, forming a multi-energy supply system of "lipid metabolism + glucose metabolism + lactate metabolism," meeting the energy needs of oocyte maturation and different stages of embryonic development. Compared with existing universal culture media, the lipid metabolism regulation scheme of this invention increases the porcine oocyte maturation rate from 68.84% to over 77.60%, and the nuclear transfer embryo blastocyst rate from 21.36% to over 24.56%, fully demonstrating the precise adaptation to the specific physiological characteristics of porcine oocytes and solving the technical pain point of "universal formulas not matching species specificity" in existing technologies.

[0035] Advantage 3: Optimized matching of electro-activation solution and embryo culture medium enhances the continuity of embryo development in nuclear transfer embryos.

[0036] In existing technologies, the design of electroactivation solutions and embryo culture media is often independent. Electroactivation solutions focus only on activation efficiency, while embryo culture media focuses only on nutrient supply, lacking coordinated regulation of the "activation-development" transition process. This results in reconstructed organisms struggling to adapt quickly to the embryo culture environment after activation, leading to impaired developmental potential. This invention overcomes this limitation by achieving optimized matching of electroactivation solutions and embryo culture media: the newly added L-arginine in the electroactivation solution promotes cell repair and signal transduction after reconstructed organism activation, laying the foundation for embryo development; fatty acid-free BSA protects cell membrane integrity, enabling reconstructed organisms to quickly adapt to the embryo culture environment after activation; the newly added vitamin C and coenzyme Q10 in the embryo culture media promptly remove ROS generated during activation, preventing the accumulation of oxidative damage; simultaneously, the consistent buffer system (Hepes / NaHCO3) and sterile system (penicillin + streptomycin) in both the electroactivation solution and embryo culture media reduce environmental stress during the transfer of reconstructed organisms from the electroactivation solution to the embryo culture medium. Furthermore, the synergistic effect of electroactivation parameters (60V / mm, 50μs, 3 pulses) and embryo culture temperature (39℃) promotes rapid genomic activation in the reconstructed embryos after activation, increasing the 2-cell cleavage rate from 80.58% to over 83.33%. This optimized "activation-development" process solves the problem of disconnect between activation and culture in existing technologies, significantly improving the developmental continuity of nuclear transfer embryos and increasing the total number of blastocyst cells from 33.67 to over 37.45.

[0037] Advantage 4: Precise adaptation of process parameters to achieve standardization and high stability of the cultivation process.

[0038] Existing culture techniques often borrow from other mammals such as mice and cattle, failing to adequately consider the specificity of porcine oocytes and embryos. This results in poor technical stability, with maturation rates fluctuating by more than 10% between different batches. This invention, through extensive experimental optimization, has determined process parameters precisely suited to porcine oocytes and embryos: oocyte culture temperature (38.5℃), two-step culture time (20-22h each), electroactivation parameters (60V / mm, 50μs, 3 pulses), embryo culture temperature (39℃), and culture medium replacement frequency (half replacement every 48h). These parameters are not simply numerical choices, but rather precise matches based on physiological characteristics such as the meiotic process of porcine oocytes, lipid metabolism enzyme activity, and embryonic genome activation time: a culture temperature of 38.5℃ maintains lipid metabolism enzyme activity at optimal levels (a 22% increase compared to 37℃); the two-step culture time of 20-22 hours perfectly matches the meiotic process of porcine oocytes from the GV stage to the MII stage (with an error of no more than 1 hour); replacing half of the culture medium every 48 hours replenishes nutrients while avoiding environmental mutations, keeping batch-to-batch maturation rate fluctuations within 3%. Furthermore, this invention clarifies key operational specifications such as oocyte selection criteria (encapsulated in three or more layers of granulosa cells, uniform cytoplasm, and good refractive index) and donor cell culture passages (3-5 passages), forming a standardized culture process. Compared with the non-standardized processes of existing technologies, the process parameters of this invention are highly precise and adaptable, significantly improving technical stability and meeting the consistency and reliability requirements of industrial production, laying the foundation for the large-scale application of embryo engineering technology.

[0039] Advantage 5: Non-toxic and highly biosafe, meeting the requirements of green farming and biomedical applications.

[0040] Some existing technologies, in order to enhance efficacy, may add chemically synthesized antioxidants or hormone analogs, posing a potential risk of toxic residues and limiting their application in biomedicine (such as the construction of human disease models) and green farming. All added components in this invention are of natural origin or are substances with extremely high biosafety: *Lactobacillus johnsonii* is a recognized probiotic, and its metabolites are non-toxic to animals and humans; Vitamin E, melatonin, Vitamin C, and β-carotene are all natural nutrients, widely found in food, and their safety has been fully verified; L-carnitine and L-arginine are essential amino acid derivatives in humans and animals, with no toxic side effects; EGTA and fatty acid-free BSA are biological reagent-grade products with a purity ≥99%, posing no residue risk. Simultaneously, this invention optimizes the concentration of antibiotics (penicillin, streptomycin), controlling the concentration to the lowest effective dose while ensuring sterility, reducing the potential impact of antibiotic residues on oocytes and embryos. Safety testing revealed no toxic residues in the oocytes and embryos cultured in the medium of this invention. Recipient sows showed no abnormal reactions after embryo transfer, and the health rate of offspring piglets exceeded 98%. Compared to existing technologies that may involve the addition of chemically synthesized substances, this invention offers significant advantages in biosafety. It is not only suitable for breeding superior livestock but also for constructing pig embryo models in the biomedical field, expanding its application scenarios and aligning with the development trends of green agriculture and biomedicine. Attached Figure Description

[0041] Figure 1 Oocyte maturation in vitro (A: 0.5% Lactobacillus johnsonii treatment for 22 h; B: 0.5% Lactobacillus johnsonii treatment for 44 h, Bar = 50 μm);

[0042] Figure 2 Nuclear transfer embryo development (A: 0.5% Lactobacillus johnsonii treatment group; B: blank control group, Bar = 50 μm);

[0043] Figure 3 Cell counts in blastocyst stage embryos after nuclear transfer (A: 0.5% Lactobacillus johnsonii treatment group; B: blank control group, Bar = 50 μm). Detailed Implementation

[0044] The specific embodiments of the present invention will be further described below with reference to examples.

[0045] In this embodiment of the invention, the following technical solution is adopted:

[0046] I. Formula for Special Culture Medium System

[0047] 1. Oocyte maturation culture medium (including culture medium A and culture medium B)

[0048] Culture medium A: 70% TCM199, 12% porcine follicular fluid, 18% fetal bovine serum, 0.1 mg / mL glutamine, 0.6 g / L glucose, 2.3 g / L NaHCO3, 10 IU / mL human chorionic gonadotropin (hCG), 15 IU / mL pregnant mare serum gonadotropin (PMSG), 0.75 g / L penicillin, 0.5 g / L streptomycin, 10 9 CFU / g Lactobacillus johnsonii 0.5%, 0.05g / L Vitamin E, 0.03g / L Melatonin, 0.15g / L L-Carnitine.

[0049] Culture medium B: 70% TCM199, 12% porcine follicular fluid, 18% fetal bovine serum, 0.1 mg / mL glutamine, 0.6 g / L glucose, 2.3 g / L NaHCO3, 0.75 g / L penicillin, 0.5 g / L streptomycin, 10 9 CFU / g Lactobacillus johnsonii 0.5%, 0.05g / L Vitamin E, 0.03g / L Melatonin, 0.15g / L L-Carnitine.

[0050] 2. Electro-activation solution: 0.3 mol / L mannitol, 0.1 mmol / L CaCl2, 0.1 mmol / L MgSO4, 1 mmol / L Hepes, 0.01% (w / v) PVA, 0.05 g / L penicillin, 0.02 g / L streptomycin, 0.08 mmol / L EGTA, 0.02 g / L BSA (fatty acid-free type), 0.1 mmol / L L-arginine.

[0051] 3. Embryo culture medium: 108 mmol / L NaCl, 10 mmol / L KCl, 0.35 mmol / L KH2PO4, 0.3 mmol / L MgSO4·7H2O, 24.65 mmol / L NaHCO3, 0.2 mmol / L sodium pyruvate, 2.0 mmol / L calcium lactate, 1.0 mmol / L L-glutamine, 5.0 mmol / L taurine, 20 ml / L essential amino acids, 15 ml / L non-essential amino acids, 5 g / L LSA, 0.04 g / L vitamin C (L-ascorbic acid), 0.02 g / L coenzyme Q10, 0.015 g / L β-carotene.

[0052] II. Supporting Cultivation Process

[0053] 1. Oocyte isolation and screening: Ovaries from healthy sows were collected from the slaughterhouse and placed in sterile saline containing penicillin (0.1 g / L) and streptomycin (0.075 g / L). The ovaries were returned to the laboratory within 2-3 hours. The ovarian surface was wiped with sterile gauze to remove blood, mesangium, and connective tissue. 2-5 mm of clear follicular fluid was drawn using a 10 mL sterile syringe (12-gauge needle) and injected into a sterile centrifuge tube. The tube was incubated on a 37°C heated plate for 15-20 minutes. The supernatant was discarded, and the precipitate was resuspended in DMEM culture medium. The suspension was transferred to a sterile glass dish. Under a Nikon stereomicroscope, cumulus-oocyte complexes (COCs) with three or more layers of granulosa cells, uniform cytoplasm, and good refractive properties were selected and washed three times with PBS solution for later use.

[0054] 2. In vitro culture of oocytes: Selected COCs were transferred to culture medium A preheated to 38.5℃ and cultured in an incubator with 5% CO2, saturated humidity, and 38.5℃ for 20-22 hours. Then they were transferred to culture medium B preheated to 38.5℃ and cultured for another 20-22 hours under the same conditions. The total culture time was 40-44 hours.

[0055] 3. Donor Cell Culture: Donor cells were cultured using the tissue block adherence method. Muscle tissue from the thigh of healthy fetal pigs was isolated and washed 3-5 times with PBS solution containing penicillin (0.1 g / L, streptomycin 0.075 g / L). The tissue was cut into 3-4 mm pieces and evenly spread at the bottom of a 60 mm sterile culture dish. A small amount of DMEM culture medium containing 10% FBS was added. The dish was then inverted and incubated in a 5% CO2, saturated humidity, 37°C incubator for 24 h. Afterward, the dish was inverted, and an appropriate amount of DMEM culture medium containing 10% FBS was added. Floating tissue blocks were removed. The culture medium was changed after 72 h. When the fibroblasts reached approximately 90% confluence, they were passaged using 0.25% trypsin. Fibroblasts cultured to passages 3-5 were used as nuclear transfer donor cells.

[0056] 4. Construction and Activation of Nuclear Transfer Embryos: Mature oocytes cultured for 40-44 hours were transferred to PBS solution containing 1 mg / mL hyaluronidase. Granulosa cells were gently detached by pipetting. Mature oocytes with an intact first polar body, intact zona pellucida, clear perivitelline space, and homogeneous cytoplasm were selected and washed three times with PBS solution. Blind enucleation was performed: Under an inverted microscope, the cytoplasm (including the nucleus) surrounding the polar body was aspirated using an enucleation needle with a diameter of approximately 28 µm. Then, a single, well-dispersed, and regularly shaped donor cell was aspirated using the same needle and injected into the perivitelline space of the enucleated oocyte, completing the enucleation operation. The reconstructed embryos were transferred to an electroactivation solution preheated to 38.5 °C for equilibration for 10 min, and then transferred to a fusion tank lined with electroactivation solution. Electroactivation parameters were set as follows: electric field strength 60 V / mm, pulse width 50 μs, and 3 pulses.

[0057] 5. In vitro culture of nuclear transfer embryos: The reconstructed embryos after electro-activation were washed three times with embryo culture medium and transferred into microdrops of embryo culture medium preheated to 39°C (20 μL per drop, containing 10-15 reconstructed embryos). They were covered with sterile mineral oil and cultured in an incubator with 5% CO2, saturated humidity, and 39°C for 144-168 h.

[0058] 6. Detection indicators and methods:

[0059] Oocyte maturation rate detection: After culturing for 40-44 hours, observe and count the number of oocytes that have expelled the first polar body, and calculate the maturation rate (maturation rate = number of oocytes that have expelled the first polar body / total number of cultured oocytes × 100%).

[0060] Oocyte apoptosis rate detection: The apoptosis rate was detected using the TUNEL method combined with DAPI staining. Mature oocytes were collected, washed three times with PBS solution, fixed in 4% paraformaldehyde for 30 min, washed three times with PBS / PVA solution, and treated with 0.1% Triton X-100 in PBS permeabilization buffer at room temperature for 10 min. Then, they were incubated with PBS blocking buffer containing 1% BSA for 20 min. Oocytes were then immersed in TUNEL staining solution and incubated at 37°C in the dark for 1.5 h. After washing three times with PBS / PVA solution, DAPI staining solution was added and incubated at room temperature in the dark for 5 min. Finally, the slides were mounted with anti-fluorescence quenching mounting solution and observed under a laser confocal microscope with excitation wavelengths of 488 nm (TUNEL) and 405 nm (DAPI). The number of apoptotic cells was counted, and the apoptosis rate was calculated (apoptosis rate = number of apoptotic oocytes / total number of oocytes detected × 100%).

[0061] Developmental markers of nuclear transfer embryos were measured: After 44 hours of culture, the 2-cell cleavage rate was calculated (2-cell cleavage rate = number of 2-cell embryos / total number of reconstructed embryos × 100%); after 168 hours of culture, the blastocyst rate was calculated (blastocyst rate = number of blastocysts / total number of reconstructed embryos × 100%). Blastocysts were stained with 10 μg / mL Hoechst 33342 dye for 15 min, mounted with anti-fluorescence quenching mounting medium, and observed and counted under a fluorescence microscope.

[0062] Technical principle of the invention:

[0063] The core innovation of this invention lies in constructing a specialized culture medium system for the synergistic effect of Lactobacillus johnsonii and multiple functional components. Combined with optimized culture processes, it improves the in vitro maturation microenvironment of porcine oocytes from multiple dimensions, including oxidative stress regulation, nutritional metabolism optimization, and signaling pathway regulation, thereby enhancing oocyte maturation quality and embryonic developmental potential. The specific technical principles are as follows:

[0064] 1. The synergistic effect mechanism of oocyte maturation culture medium

[0065] (1) Core functions of Lactobacillus johnsonii: As a probiotic, Lactobacillus johnsonii's metabolites (such as short-chain fatty acids, polysaccharides, vitamins, etc.) can reduce oxidative stress damage by clearing ROS in oocytes and activating the activity of antioxidant enzymes (such as superoxide dismutase and glutathione peroxidase). At the same time, Lactobacillus johnsonii can also regulate the level of inflammatory factors in oocytes, inhibit the activation of apoptosis signaling pathways (such as the Caspase pathway), and reduce the apoptosis rate of oocytes. In addition, Lactobacillus johnsonii metabolites can also promote the secretion of gonadotropin receptors and growth factors by granulosa cells, improve paracrine signal transduction between oocytes and granulosa cells, and provide a good paracrine environment for oocyte maturation.

[0066] (2) Functions and synergistic effects of the newly added components:

[0067] Vitamin E: As a fat-soluble antioxidant, it can penetrate the oocyte membrane and enter the cell to scavenge lipid peroxidation free radicals, protect the integrity of the cell membrane lipid structure, and form a synergistic effect with the water-soluble antioxidant metabolites of Lactobacillus johnsonii to scavenge ROS from different levels and enhance the antioxidant effect. At the same time, vitamin E can also promote the synthesis and metabolism of cholesterol in oocytes, provide raw materials for the production of sex hormones, and assist hormone regulation of oocyte maturation.

[0068] Melatonin: Possesses potent antioxidant activity, directly scavenging ROS and nitrogen free radicals, while simultaneously inducing the expression of antioxidant genes (such as SOD and GSH-Px) in oocytes, enhancing endogenous antioxidant capacity; melatonin can also regulate calcium in oocytes. 2+The concentration promotes normal meiosis and synergistically inhibits apoptosis signaling pathways with Lactobacillus johnsonii, further reducing the oocyte apoptosis rate.

[0069] L-Carnitine: As a key regulator of lipid metabolism, it can promote the breakdown of lipid droplets in the cytoplasm of porcine oocytes, transport fatty acids to mitochondria for β-oxidation, and provide sufficient energy for oocyte maturation. At the same time, L-Carnitine can also reduce the toxic effects of excessive lipid droplet accumulation on oocytes, improve cytoplasmic uniformity, and synergistically regulate the metabolic balance of oocytes with Lactobacillus johnsonii, thereby improving maturation quality.

[0070] (3) Role of basic components: TCM199 serves as the basic culture medium, providing essential nutrients (such as amino acids and minerals) required for oocyte maturation; porcine follicular fluid contains natural growth factors, hormones, and nutrients, simulating the in vivo follicular microenvironment; fetal bovine serum provides albumin, growth factors, etc., promoting granulosa cell growth and oocyte maturation; glutamine is an intermediate product of energy metabolism, supplementing energy supply; glucose provides basic energy for oocytes; NaHCO3 maintains the acid-base balance of the culture medium; hCG and PMSG synergistically promote the recovery and maturation of oocyte meiosis; penicillin and streptomycin inhibit contamination by miscellaneous bacteria, ensuring the sterility of the culture system.

[0071] 2. The synergistic effect principle of electro-activating liquid

[0072] (1) The complete role of the basic components: Mannitol, as the core osmotic pressure regulator, can precisely maintain the osmotic pressure balance inside and outside the reconstructed cell membrane, avoiding cell membrane rupture caused by drastic fluctuations in osmotic pressure, and providing a stable physical environment for the electroactivation process; CaCl2 provides Ca 2+ It is a key signaling molecule for the activation of the reconstructed body; under the action of an electrical pulse, Ca... 2+ Influx can trigger a series of activation reactions, such as meiosis recovery and pronucleus formation in the reconstructed body, and is a core signal for initiating embryonic development; MgSO4 provides Mg 2+ Participates in various intracellular enzymatic reactions, regulates the activity of ion channels on the cell membrane, and interacts with Ca2+. 2+ The system works synergistically to maintain intracellular ion homeostasis, preventing damage to the reconstructed organisms caused by excessively high concentrations of a single ion; Hepes acts as a buffer, stabilizing the pH of the electroactivation solution outside of a CO2 culture environment, preventing pH fluctuations from affecting cell membrane potential and activation signal transduction; PVA (polyvinyl alcohol) replaces albumin in serum, reducing non-specific adhesion between cells and culture vessels during electroactivation, while also reducing interference from unknown components in serum on the activation effect; penicillin and streptomycin form a dual antibacterial system, inhibiting potential bacterial contamination in the electroactivation solution and ensuring a sterile environment for the reconstructed organisms.

[0073] (2) Functions and synergistic mechanisms of the newly added components:

[0074] 2. EGTA (ethylene glycol bis(2-aminoethyl ether)tetraacetic acid): As a specific calcium chelating agent, it can precisely regulate the free Ca in the electro-activation solution. 2+ Concentration. During electro-activation, Ca... 2+ Influx is key to activation, but excessive Ca... 2+ This can lead to abnormal cell membrane permeability, mitochondrial damage, and even apoptosis in reconstructed somatic cells. EGTA works by reversibly binding to some Ca2+ cells. 2+ This forms a "buffer pool" to prevent Ca from being affected by electrical pulses. 2+ A sudden excessive influx of intracellular calcium... 2+ The concentration was maintained within the optimal range required for activation (100-300 nmol / L) to ensure effective transduction of the activation signal while protecting cells from calcium overload damage. Simultaneously, EGTA and Mg... 2+ Synergistic effect, further stabilizing Ca on the cell membrane 2+ -Mg 2+ The activity of ATPase-dependent enzymes promotes the restoration of intracellular ion balance and enhances the survival rate of the reconstructed cells after activation.

[0075] 3. Fatty acid-free BSA (bovine serum albumin): Compared with ordinary BSA, fatty acid-free BSA avoids the damage of fatty acids to the reconstructed cell membrane. At the same time, its hydrophobic groups can bind to the phospholipid bilayer of the cell membrane, enhancing the fluidity and stability of the cell membrane and reducing cell membrane perforation damage caused by electrical pulses. In addition, BSA can bind to trace toxic substances that may be present in the electroactivation solution, reducing their impact on the reconstructed body. It also works synergistically with PVA to form a cell membrane protection network, improving the safety and effectiveness of the electroactivation process.

[0076] 4. L-Arginine: As a substrate for nitric oxide (NO) synthase, it can synthesize NO in the reconstituted organism. NO, as an important signaling molecule, can regulate the activity of guanylate cyclase on the cell membrane, promote cGMP production, and thus regulate intracellular calcium. 2+ Concentration enhances the efficiency of activation signal transduction; simultaneously, NO has mild antioxidant activity, which can scavenge the small amount of ROS generated during electroactivation and reduce oxidative damage; in addition, L-arginine can provide energy metabolism raw materials for the reconstructed body, promote cell repair and metabolic recovery after activation, and interact with Ca... 2+ Mg 2+ Synergistically optimize the activated intracellular environment and enhance the potential of the reconstructed organism to develop into a normal embryo.

[0077] 3. The synergistic effect mechanism of embryo culture medium

[0078] (1) Roles of basic components: NaCl and KCl maintain the osmotic pressure and cell membrane potential of embryonic cells, providing a basic ionic environment for cell metabolism; KH2PO4 and MgSO4·7H2O provide minerals such as phosphorus and magnesium, participating in key metabolic processes such as nucleic acid synthesis and enzymatic reactions; NaHCO3, as the main buffer, maintains the pH of the culture medium at 7.2-7.4 in a 5% CO2 culture environment, which is suitable for the acid-base tolerance range of embryonic cells; sodium pyruvate is an intermediate product of sugar metabolism, which can be quickly absorbed and utilized by embryonic cells to supplement energy supply; calcium lactate provides lactic acid for the embryo as an early... Essential substrates for embryonic energy metabolism (early pig embryos mainly rely on lactate metabolism for energy); L-glutamine can be converted into glutamate and ammonia, providing nitrogen and energy for embryonic cells, while also participating in protein synthesis; taurine, as an antioxidant, scavenges ROS generated during embryonic development, protecting embryonic cells from oxidative damage; essential and non-essential amino acids provide raw materials for embryonic cell proliferation and differentiation, promoting embryonic genome activation and inhibiting apoptosis; BSA provides nutrient carriers and protection, binding nutrients and toxic substances in the culture medium, regulating nutrient absorption and metabolic balance in embryonic cells.

[0079] (2) Functions and synergistic mechanisms of the newly added components:

[0080] 5. Vitamin C (L-ascorbic acid): As a potent water-soluble antioxidant, it can penetrate embryonic cell membranes and directly scavenge intracellular ROS. At the same time, it promotes the synthesis of glutathione (GSH) in embryonic cells, enhancing endogenous antioxidant capacity. Vitamin C can also promote the synthesis of collagen and extracellular matrix, improve adhesion and signal transduction between embryonic cells, and promote the formation and expansion of the blastocyst cavity. In addition, vitamin C can regulate the expression of embryonic development-related genes (such as Oct4, Nanog and other pluripotency genes), enhance the developmental potential of the embryo, and synergistically form a "water-soluble + endogenous" dual antioxidant network with taurine, comprehensively inhibiting the damage of oxidative stress to the embryo.

[0081] 6. Coenzyme Q10: As a fat-soluble antioxidant and a key component of the mitochondrial respiratory chain, it can be located in the mitochondria of embryonic cells. On the one hand, it removes ROS produced by mitochondria (mitochondria are the main site of ROS production), protecting the integrity of mitochondrial DNA and structure. On the other hand, it participates in the ATP synthesis process, improving the energy supply efficiency of mitochondria and meeting the high energy demand of early embryonic development (especially the blastocyst stage). Coenzyme Q10 works synergistically with BSA, which promotes its dispersion in the culture medium and absorption by embryonic cells, enhancing its bioavailability. It forms a "water-soluble + fat-soluble" antioxidant synergy with vitamin C, covering oxidative stress protection in different regions of the cell, while optimizing energy metabolism and providing sufficient energy for embryonic development.

[0082] 7. β-Carotene: As a precursor to vitamin A, it can be converted into retinoic acid in embryonic cells. Retinoic acid is a key regulator of embryonic development, regulating the expression of development-related genes such as the Hox gene family, and promoting the proliferation, differentiation, and tissue formation of embryonic cells. At the same time, β-carotene has strong antioxidant activity, which can scavenge ROS and inhibit lipid peroxidation, protecting embryonic cell membranes and organelles. In addition, β-carotene can enhance the absorption efficiency of nutrients by embryonic cells, and work synergistically with essential and non-essential amino acids to provide sufficient raw materials for embryonic cell proliferation, increase the total number and quality of blastocyst cells, and work synergistically with vitamin C and coenzyme Q10 to construct a multidimensional embryonic development support system of "antioxidant + gene regulation + nutrient absorption".

[0083] 4. Technical principles of process parameter selection

[0084] (1) Oocyte culture temperature and time: The present invention selects 38.5±0.5℃ as the oocyte culture temperature, which is highly consistent with the follicular environment temperature in pigs (38.3-38.7℃), avoiding the temperature stress caused by the commonly used 37℃ or 39℃ in the prior art. Pig oocytes have a high lipid droplet content in their cytoplasm. Temperature deviation from the physiological range will lead to a decrease in the activity of lipid droplet metabolic enzymes and abnormal lipid droplet accumulation, which will affect the meiotic process. At the same time, a two-step culture method of "20-22h A culture medium + 20-22h B culture medium" is adopted. Culture medium A contains hCG and PMSG, which can promote the recovery of oocyte meiosis (from GV phase to MI phase). The 20-22h culture time is exactly matched with the time process of the recovery of oocyte meiosis. Culture medium B does not contain gonadotropins, which can promote the completion of oocyte meiosis (from MI phase to MII phase) and avoid the excessive activation of oocytes caused by the continuous presence of gonadotropins. The two-step culture method, combined with hormone regulation, increases the oocyte maturation rate by more than 8% compared to traditional single-medium culture, and significantly improves the cytoplasmic uniformity of mature oocytes.

[0085] (2) Electroactivation parameters: The combination of 60 V / mm electric field strength, 50 μs pulse width, and 3 pulses was selected based on the optimized results of the zona pellucida thickness (approximately 15-20 μm) and the characteristics of the reconstructed somatic cell membrane in porcine oocytes. Too low an electric field strength (<50 V / mm) cannot effectively penetrate the zona pellucida and cell membrane, leading to Ca2+ activation. 2+ Insufficient influx leads to activation failure; excessively high electric field strength (>70 V / mm) causes irreversible damage to the cell membrane, resulting in cell death. A pulse width of 50 μs can create transient channels on the cell membrane, ensuring both Ca2+ and... 2+ Inflow is achieved while avoiding excessively large pores that could lead to loss of cellular contents; the 1-second interval between the three pulses allows for partial repair of the cell membrane after each pulse, reducing cumulative damage, and simultaneously gradually increasing intracellular calcium. 2+Concentration, simulating activated Ca in vivo 2 + Oscillating signals promote complete activation of the reconstructed organisms. This parameter combination increases the activation rate of the reconstructed organisms by 12% and reduces the apoptosis rate by 5% compared to traditional parameters (50V / mm, 60μs, 2 pulses).

[0086] (3) Embryo culture conditions: The embryo culture temperature of 39℃ is suitable for the physiological temperature of early development of pig embryos, which is 0.5℃ higher than the oocyte culture temperature, and can promote the activation of the embryo genome (pig embryo genome activation occurs in the 4-8 cell stage); 5% CO2 concentration can maintain the pH stability of the NaHCO3 buffer system and avoid pH fluctuations affecting the enzyme activity of embryo cells; saturated humidity prevents the osmotic pressure increase caused by the evaporation of culture medium and protects embryo cells; replacing 1 / 2 of the fresh embryo culture medium every 48 hours can replenish nutrients and remove metabolic waste (such as lactic acid, ammonia, etc.) in time, avoid the toxic effects of metabolic waste accumulation on the embryo, and at the same time reduce the environmental stress caused by complete replacement of culture medium, so that the blastocyst rate is increased by more than 7% compared with no replacement of culture medium.

[0087] To make the present invention more fully disclosed, more specific embodiments are described below.

[0088] Example 1: A dedicated culture medium system containing Lactobacillus johnsonii promotes in vitro maturation of porcine oocytes.

[0089] 1. Preparation of special culture medium

[0090] (1) Oocyte maturation culture medium

[0091] Culture medium A: Accurately weigh each component according to volume ratio and mass concentration: 70% TCM199, 12% porcine follicular fluid (extracted from 36mm porcine follicles, sterilized by 0.22μm filter membrane), 18% fetal bovine serum (low endotoxin type), 0.1mg / mL glutamine, 0.6g / L glucose, 2.3g / L NaHCO3, 10IU / mL hCG, 15IU / mL PMSG, 0.75g / L penicillin, 0.5g / L streptomycin, 10 9 The following ingredients were added sequentially to a sterile beaker: 0.5% CFU / g Lactobacillus johnsonii, 0.05 g / L Vitamin E, 0.03 g / L Melatonin, and 0.15 g / L L-Carnitine. The mixture was stirred with a magnetic stirrer for 30 minutes until completely dissolved. The pH was adjusted to 7.2-7.4. The mixture was then filtered through a 0.22 μm membrane for sterilization. It was stored at 4°C for later use, and preheated from 37°C to 38.5°C before use.

[0092] Culture medium B: The formula is the same as that of culture medium A, except that it does not contain hCG and PMSG, and the preparation method is the same.

[0093] (2) Electro-activation solution: Accurately weigh each component: 0.3 mol / L mannitol, 0.1 mmol / L CaCl2, 0.1 mmol / L MgSO4, 1 mmol / L Hepes, 0.01% (w / v) PVA, 0.05 g / L penicillin, 0.02 g / L streptomycin, 0.08 mmol / L LEGTA, 0.02 g / L BSA, and 0.1 mmol / L L-arginine. Dissolve in deionized water, adjust the pH to 7.2-7.3, adjust the osmotic pressure to 280-300 mOsm / kg, filter through a 0.22 μm filter membrane for sterilization, and store at 4℃ for later use. Preheat to 38.5℃ and equilibrate for 20 min before use.

[0094] (3) Embryo culture medium: Accurately weigh each component: 108 mmol / L NaCl, 10 mmol / L KCl, 0.35 mmol / L KH2PO4, 0.3 mmol / L MgSO4·7H2O, 24.65 mmol / L NaHCO3, 0.2 mmol / L sodium pyruvate, 2.0 mmol / L calcium lactate, 1.0 mmol / L L-glutamine, 5.0 mmol / L taurine, 20 ml / L essential amino acids, 15 ml / L non-essential amino acids, 5 g / L LSA, 0.04 g / L vitamin C (L-ascorbic acid, purity ≥99%), 0.02 g / L coenzyme Q10, 0.015 g / L β-carotene (purity ≥95%). Dissolve in deionized water, adjust the pH to 7.2-7.4, adjust the osmotic pressure to 270-290 mOsm / kg, filter with a 0.22μm filter membrane for sterilization, refrigerate at 4℃ for later use, and preheat to 39℃ before use.

[0095] 2. Oocyte isolation and culture

[0096] Healthy large white pig ovaries were collected from the slaughterhouse and placed in sterile saline containing 0.1 g / L penicillin and 0.075 g / L streptomycin. They were returned to the laboratory within 2 hours. The ovarian surface was wiped with sterile gauze to remove blood and mesentery. 25 mm of clear follicular fluid was drawn using a 10 mL sterile syringe (12-gauge needle) and injected into a sterile centrifuge tube. The tube was incubated at 37°C for 18 minutes, the supernatant was discarded, and the precipitate was resuspended in DMEM culture medium and transferred to a sterile glass dish. Under a Nikon stereomicroscope, cocci with three or more layers of granulosa cells, uniform cytoplasm, and good refractive properties were selected. The cells were washed three times with PBS solution and transferred to culture medium A preheated to 38.5°C. The cells were then incubated in a 5% CO2, saturated humidity, 38.5°C incubator for 22 hours (see [link to in vitro oocyte maturation information]). Figure 1 A), then transferred to culture medium B and cultured for another 22 hours (see section A for oocyte maturation status). Figure 1 B).

[0097] 3. Donor cell culture

[0098] Thigh muscle tissue was isolated from healthy fetal pigs (30 days of gestation), washed four times with PBS solution containing antibiotics, and cut into 3-4 mm tissue blocks. The blocks were evenly spread at the bottom of a 60 mm culture dish, and a small amount of DMEM culture medium containing 10% FBS was added. The dish was then inverted and incubated at 37°C with 5% CO2 for 24 hours. The dish was then upright, and 5 mL of DMEM culture medium containing 10% FBS was added. Floating tissue blocks were removed, and the culture medium was replaced after 72 hours. When the fibroblast confluence reached 90%, the cells were digested with 0.25% trypsin and passaged to passage 4 for use as donor cells.

[0099] 4. Construction and activation of nuclear transfer embryos

[0100] After 44 hours of culture, oocytes were transferred to PBS solution containing 1 mg / mL hyaluronidase. Granulosa cells were gently removed by pipetting, and mature oocytes with intact first polar bodies and zona pellucida were selected. The oocytes were washed three times with PBS. Under an inverted microscope, the cytoplasm surrounding the polar body was blindly aspirated using a 28 µm enucleation needle (1 / 3 of the cytoplasm was removed), and a single donor cell was injected into the periovarian space. The reconstructed oocytes were transferred to electroactivation solution and equilibrated for 10 min. They were then transferred to a fusion tank, and the parameters were set as follows: 60 V / mm, 50 μs, 3 pulses. After electroactivation, the oocytes were washed three times with embryo culture medium.

[0101] 5. Nuclear transfer embryo culture and detection

[0102] The reconstructed embryos were transferred into microdroplets of embryo culture medium (20 μL / drop, containing 12 reconstructed embryos), covered with mineral oil, and incubated at 39°C, 5% CO2, and saturated humidity for 168 h. Half of the embryo culture medium was replaced every 48 h. The development of embryos after nuclear transfer was observed. Figure 2 A. After 44 hours of culture, the oocyte maturation rate and apoptosis rate were calculated; after 44 hours of culture, the 2-cell cleavage rate was calculated; after 168 hours of culture, the blastocyst rate and total number of blastocyst cells were calculated. Blastocysts were stained with 10 μg / mL Hoechst 33342 dye for 15 min. The stained blastocysts were then transferred to anti-quenching droplets on a glass slide for pressing and mounting. Cell nuclear staining was then observed and cell counts were performed under a fluorescence microscope at 460 nm excitation wavelength. Cell counts at the blastocyst stage of nuclear transfer embryos are shown in [the table below]. Figure 3 A.

[0103] Example 2: Effects of different concentrations of Lactobacillus johnsonii on in vitro maturation of porcine oocytes

[0104] The difference between this embodiment and Example 1 is that the concentration of *Lactobacillus johnsonii* in the oocyte maturation culture medium is 0.1% (10 9 (CFU / g), other formulations and process parameters are the same as in Example 1.

[0105] Example 3: Effects of different concentrations of Lactobacillus johnsonii on in vitro maturation of porcine oocytes

[0106] The difference between this embodiment and Example 1 is that the concentration of *Lactobacillus johnsonii* in the oocyte maturation culture medium is 1.0% (10). 9 (CFU / g), other formulations and process parameters are the same as in Example 1.

[0107] Comparative Example 1: Standard culture medium without Lactobacillus johnsonii and the added components

[0108] Compared to Example 1, the amount of *Lactobacillus johnsonii* used was 0%, while other aspects remained the same as in Example 1. The development of the embryos after nuclear transfer culture is shown in [the original text]. Figure 2 B, Cell counts at the blastocyst stage of nuclear transfer embryos are shown in [see section B]. Figure 3 B.

[0109] Comparative Example 2: Lactobacillus rhamnosus replacing Lactobacillus johnsonii

[0110] The difference between this comparative example and Example 1 is that 0.5% (10) was used in the oocyte maturation culture medium. 9 Lactobacillus rhamnosus (CFU / g) was used to replace Lactobacillus johnsonii, and other formulations and process parameters were the same as in Example 1.

[0111] Effect verification and data statistics:

[0112] 1. Detection Indicators and Methods

[0113] (1) Oocyte maturation rate: After 44 hours of culture, the number of oocytes that have extruded the first polar body was observed and counted under a stereomicroscope. Maturation rate = (number of oocytes that have extruded the first polar body / total number of cultured oocytes) × 100%.

[0114] (2) Oocyte apoptosis rate: The TUNEL method combined with DAPI staining was used, and the detection steps in Example 1 were followed. The apoptosis rate was observed under a laser confocal microscope. The apoptosis rate = (number of apoptotic oocytes / total number of oocytes detected) × 100%.

[0115] (3) 2-cell cleavage rate: After 44 hours of culture, the number of embryos that developed into the 2-cell stage was counted. Cleavage rate = (number of 2-cell embryos / total number of reconstructed embryos) × 100%.

[0116] (4) Blastocyst rate: After 168 hours of culture, the number of embryos that developed into blastocysts was counted. Blastocyst rate = (number of blastocysts / total number of reconstructed embryos) × 100%.

[0117] (5) Total number of blastocyst cells: After blastocysts are stained with Hoechst33342, the number of cell nuclei is counted under a fluorescence microscope, and the average value of three replicates is taken.

[0118] 2. Data Statistics and Analysis

[0119] All experiments were repeated three times. Data are expressed as mean ± standard deviation. One-way ANOVA was performed using software. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant.

[0120] 3. Test Results

[0121] The experimental results are shown in Tables 1 and 2:

[0122]

[0123] Note: Different letters on the superscript of data in the same column indicate significant differences (P<0.05), while no letter indicates no significant differences (P>0.05).

[0124]

[0125] Note: Different letters in the superscript of data in the same column indicate significant differences (P<0.05), while no letter or the same letter indicates no significant differences (P>0.05).

[0126] 4. Results Analysis

[0127] The conclusions based on the data in Tables 1 and 2 are as follows:

[0128] (1) The regulatory effect of Lactobacillus johnsonii on the in vitro maturation of porcine oocytes

[0129] 1) Significant effect on improving maturity rate

[0130] Table 1 shows that the first polar body expulsion rate (maturation rate) of porcine oocytes in all example groups (Examples 1-3) with added *Lactobacillus johnsonii* was significantly higher than that in Comparative Example 1 without added *Lactobacillus johnsonii* and Comparative Example 2 with *Lactobacillus rhamnosus* (P<0.05). Among them, Example 1 (0.5% *Lactobacillus johnsonii* concentration) had the highest maturation rate, reaching 77.60±1.65%, which was 12.73% higher than that in Comparative Example 1 (68.84±1.07%), and also higher than that in Example 2 (72.34±1.44%) and Example 3 (74.19±1.52%) (P<0.05). There was no significant difference in the maturation rate between Example 2 (0.1% concentration) and Example 3 (1.0% concentration) (P>0.05), but both were significantly better than those in Comparative Example 2 (72.82±1.13%) and Comparative Example 1 (P<0.05). This indicates that Lactobacillus johnsonii has a specific promoting effect on the in vitro maturation of porcine oocytes, and there is a concentration-dependent optimization effect. 0.5% is the optimal addition concentration, while too high or too low concentrations will weaken its maturation-promoting effect.

[0131] 2) Clear apoptosis inhibition effect

[0132] The oocyte apoptosis rate data showed an opposite trend to the maturation rate: the apoptosis rate in Example 1 was the lowest (9.89±0.58%), significantly lower than all other groups (P<0.05); the apoptosis rates in Example 2 (12.77±0.79%), Example 3 (12.90±0.84%), and Comparative Example 2 (12.82±1.15%) showed no significant difference (P>0.05), but were all significantly lower than Comparative Example 1 (14.07±1.06%) (P<0.05). This indicates that *Lactobacillus johnsonii* can effectively inhibit apoptosis in porcine oocytes during in vitro culture, and the inhibitory effect is strongest at a concentration of 0.5%; *Lactobacillus rhamnosus* does not possess the same anti-apoptotic activity as *Lactobacillus johnsonii*.

[0133] (2) The promoting effect of Lactobacillus johnsonii on the in vitro developmental potential of porcine nuclear transfer embryos

[0134] 1) Cleavage rate and blastocyst rate increase simultaneously

[0135] Table 2 shows that the addition of Lactobacillus johnsonii significantly improved the early developmental performance of porcine nuclear transfer embryos: the 2-cell cleavage rate (87.13±3.16%) and blastocyst rate (29.70±2.14%) in Example 1 were the highest among all groups, higher than those in Example 2 (83.96±2.45%, 25.47±1.31%), Example 3 (83.33±2.28%, 24.56±1.44%), Comparative Example 2 (81.25±2.31%, 25.00±1.62%), and Comparative Example 1 (80.58±2.06%, 21.36±1.06%) (P<0.05). There were no significant differences in cleavage rate and blastocyst rate between Examples 2 and 3 (P>0.05), but both were significantly better than Comparative Example 1 (P<0.05). While Comparative Example 2 had higher cleavage rate and blastocyst rate than Comparative Example 1, it was significantly lower than Examples 1-3 (P<0.05). This suggests that *Lactobacillus johnsonii* can optimize oocyte maturation quality, providing a good foundation for early cleavage and blastocyst formation in nuclear transfer embryos, and that a 0.5% addition concentration maximizes its development-promoting effect, while *Lactobacillus rhamnosus* cannot replace this function of *Lactobacillus johnsonii*.

[0136] 2) The total number of blastocyst cells increased significantly.

[0137] The total number of blastocyst cells is a key indicator reflecting embryonic development quality and implantation potential. In Example 1, the total number of blastocyst cells reached 46.19±3.05, which was higher than that in Example 2 (38.33±2.74), Example 3 (37.45±2.68), Comparative Example 2 (38.86±2.24), and Comparative Example 1 (33.67±1.89) (P<0.05). There was no significant difference in the total number of blastocyst cells between Examples 2 and 3 and Comparative Example 2 (P>0.05), but all were significantly higher than that in Comparative Example 1 (P<0.05). This result further confirms that *Lactobacillus johnsonii* not only increases the blastocyst formation rate but also significantly enhances the cell proliferation capacity of blastocysts, optimizing blastocyst quality, with the 0.5% concentration showing the most significant synergistic effect; the promoting effect of *Lactobacillus rhamnosus* on blastocyst cell proliferation is far less than that of *Lactobacillus johnsonii*.

[0138] (3) Overall Conclusion

[0139] 1) The special culture medium system containing Lactobacillus johnsonii can significantly improve the in vitro maturation rate of porcine oocytes, inhibit apoptosis, and promote the early development of porcine nuclear transfer embryos (increasing cleavage rate and blastocyst rate) and blastocyst quality (increasing the total number of blastocyst cells). Its effect is better than that of conventional culture medium and culture medium system replaced by Lactobacillus rhamnosus, which confirms that Lactobacillus johnsonii has a specific synergistic effect on porcine oocyte maturation and nuclear transfer embryo development.

[0140] 2) The concentration of *Lactobacillus johnsonii* added has a significant impact on the efficacy; 0.5% (10 9 The optimal concentration (CFU / g) is the concentration at which oocyte maturation quality and embryonic development potential reach their best levels. When the concentration is too high (1.0%) or too low (0.1%), its effects on promoting maturation, anti-apoptosis, and promoting embryonic development will be weakened, but it is still better than the non-Lactobacillus non-Joe's addition group.

[0141] The above description is a detailed description of the preferred embodiments of the present invention. However, the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modifications made under the technical spirit of the present invention should fall within the patent scope covered by the present invention.

Claims

1. The application of *Lactobacillus johnsonii* in promoting in vitro maturation of porcine oocytes, characterized in that... The in vitro maturation of porcine oocytes is promoted through a special culture medium system containing Lactobacillus johnsonii, which includes oocyte maturation culture medium, electroactivation medium, and embryo culture medium.

2. The application of *Lactobacillus johnsonii* according to claim 1 in promoting in vitro maturation of porcine oocytes, characterized in that, The oocyte maturation culture medium includes culture medium A and culture medium B.

3. The application of *Lactobacillus johnsonii* according to claim 2 in promoting in vitro maturation of porcine oocytes, characterized in that... Culture medium A contains: 70% TCM199, 12% porcine follicular fluid, 18% fetal bovine serum, 0.1 mg / mL glutamine, 0.6 g / L glucose, 2.3 g / L NaHCO3, 10 IU / mL human chorionic gonadotropin, 15 IU / mL pregnant mare serum gonadotropin, 0.75 g / L penicillin, 0.5 g / L streptomycin, and 10 9 CFU / g Lactobacillus johnsonii 0.5%, 0.05g / L Vitamin E, 0.03g / L Melatonin, 0.15g / L L-Carnitine.

4. The application of *Lactobacillus johnsonii* according to claim 2 in promoting in vitro maturation of porcine oocytes, characterized in that... The B culture medium consisted of: 70% TCM199, 12% porcine follicular fluid, 18% fetal bovine serum, 0.1 mg / mL glutamine, 0.6 g / L glucose, 2.3 g / L NaHCO3, 0.75 g / L penicillin, 0.5 g / L streptomycin, and 10... 9 CFU / g Lactobacillus johnsonii 0.5%, 0.05g / L Vitamin E, 0.03g / L Melatonin, 0.15g / L L-Carnitine.

5. The application of *Lactobacillus johnsonii* according to claim 2 in promoting in vitro maturation of porcine oocytes, characterized in that... In the culture medium A and culture medium B, the purity of vitamin E is ≥99%, melatonin is a natural extract, and L-carnitine is an L-type optically pure isomer.

6. The application of *Lactobacillus johnsonii* according to claim 1 in promoting in vitro maturation of porcine oocytes, characterized in that... The electro-activating solution consists of: 0.3 mol / L mannitol, 0.1 mmol / L CaCl2, 0.1 mmol / L MgSO4, 1 mmol / L Hepes, 0.01% (w / v) PVA, 0.05 g / L penicillin, 0.02 g / L streptomycin, 0.08 mmol / L EGTA, 0.02 g / L BSA, and 0.1 mmol / L L-arginine.

7. The application of *Lactobacillus johnsonii* according to claim 6 in promoting in vitro maturation of porcine oocytes, characterized in that... In the electro-activation solution, EGTA is of anhydrous purity, BSA is of fatty acid-free type, and L-arginine is of biological reagent grade.

8. The application of *Lactobacillus johnsonii* according to claim 1 in promoting in vitro maturation of porcine oocytes, characterized in that, The embryo culture medium contained: 108 mmol / L NaCl, 10 mmol / L KCl, 0.35 mmol / L KH2PO4, 0.3 mmol / L MgSO4·7H2O, 24.65 mmol / L NaHCO3, 0.2 mmol / L sodium pyruvate, 2.0 mmol / L calcium lactate, 1.0 mmol / L L-glutamine, 5.0 mmol / L taurine, 20 ml / L essential amino acids, 15 ml / L non-essential amino acids, 5 g / L BSA, 0.04 g / L vitamin C, 0.02 g / L coenzyme Q10, and 0.015 g / L β-carotene.

9. The application of *Lactobacillus johnsonii* according to claim 8 in promoting in vitro maturation of porcine oocytes, characterized in that... The embryo culture medium contains L-ascorbic acid for vitamin C, coenzyme Q10 for coenzyme Q10 as a fat-soluble natural extract, and β-carotene with a purity of ≥95%.

10. A method for using *Lactobacillus johnsonii* according to any one of claims 1-9 in promoting in vitro maturation of porcine oocytes, characterized in that, Includes the following steps: (1) Isolation and screening of oocytes: Ovaries of healthy sows were collected from the slaughterhouse and placed in sterile physiological saline containing penicillin and streptomycin. They were returned to the laboratory within 2-3 hours. The surface of the ovary was wiped with sterile gauze to remove blood, mesangium and connective tissue. 2-5 mm of clear follicular fluid was drawn with a 10 mL sterile syringe and injected into a sterile centrifuge tube. The tube was placed on a 37℃ constant temperature heating plate for 15-20 min. The supernatant was discarded and the precipitate was resuspended with DMEM culture medium. The suspension was transferred to a sterile glass dish. Under a Nikon stereomicroscope, COCs with more than three layers of granulosa cells, uniform cytoplasm and good refractive properties were selected and washed 3 times with PBS solution for later use. (2) In vitro culture of oocytes: The selected COCs were transferred into culture medium A preheated to 38.5℃ and cultured in an incubator with 5% CO2, saturated humidity and 38.5℃ for 20-22h. Then they were transferred into culture medium B preheated to 38.5℃ and cultured under the same conditions for another 20-22h. The total culture time was 40-44h. (3) Culture of donor cells: The tissue block adherence method was used to culture donor cells; healthy fetal pig thigh muscle tissue was isolated, washed 3-5 times with PBS solution containing double antibiotics, cut into 3-4 mm tissue blocks, spread evenly on the bottom of a 60 mm sterile culture dish, and DMEM culture medium containing 10% FBS was added. The culture dish was inverted in a 5% CO2, saturated humidity, 37℃ incubator for 24 h. After that, the culture dish was upright, an appropriate amount of DMEM culture medium containing 10% FBS was added, and the floating tissue blocks were removed. The culture medium was changed once after 72 h. When the fibroblasts grew to about 90% confluence, they were digested and passaged with 0.25% trypsin. Fibroblasts cultured to the 3rd-5th generation were used as nuclear transfer donor cells. (4) Construction and activation of nuclear transfer embryos: Mature oocytes cultured for 40-44 h were transferred into PBS solution containing 1 mg / mL hyaluronidase. The granulosa cells were gently blown off. Mature oocytes with the first polar body, intact zona pellucida, clear perivitelline space, and uniform cytoplasm were selected. The oocytes were washed three times with PBS solution and enucleated by blind aspiration: Under an inverted microscope, 1 / 3 of the cytoplasm around the polar body was aspirated with a 28 µm diameter enucleation needle. Then, a single well-dispersed and regularly shaped donor cell was aspirated with the same needle and injected into the perivitelline space of the enucleated oocyte to complete the enucleation operation. The reconstructed body was transferred into an electroactivation solution preheated to 38.5 °C for equilibration for 10 min. Then, it was transferred to a fusion tank with electroactivation solution. The electroactivation parameters were set as follows: electric field strength 60 V / mm, pulse width 50 μs, and pulse count 3 times. Electroactivation treatment was performed. (5) In vitro culture of nuclear transfer embryos: The reconstructed embryos after electro-activation were washed three times with embryo culture medium, transferred into microdroplets of embryo culture medium preheated to 39°C, covered with sterile mineral oil, and cultured in an incubator with 5% CO2, saturated humidity, and 39°C for 144-168h. (6) Detection indicators and methods: Oocyte maturation rate detection: After culturing for 40-44 hours, observe and count the number of oocytes that have expelled the first polar body, and calculate the maturation rate; Oocyte apoptosis rate detection: The apoptosis rate was detected by TUNEL assay combined with DAPI staining. Mature oocytes were collected, washed three times with PBS solution, fixed in 4% paraformaldehyde for 30 min, washed three times with PBS / PVA solution, and treated with 0.1% Triton X-100 in PBS permeabilization buffer at room temperature for 10 min. Then, they were incubated with PBS blocking buffer containing 1% BSA for 20 min. The oocytes were then immersed in TUNEL staining solution and incubated at 37°C in the dark for 1.5 h. After washing three times with PBS / PVA solution, DAPI staining solution was added and incubated at room temperature in the dark for 5 min. Finally, the slides were mounted with anti-fluorescence quenching mounting solution and observed under a laser confocal microscope with excitation wavelengths of 488 nm and 405 nm. The number of apoptotic cells was counted and the apoptosis rate was calculated. Detection of developmental indicators of nuclear transfer embryos: After 44 h of culture, the 2-cell cleavage rate was counted; after 168 h of culture, the blastocyst rate was counted. Blastocysts were stained with 10 μg / mL Hoechst 33342 dye for 15 min, mounted with anti-fluorescence quenching mounting solution, and observed and counted under a fluorescence microscope.

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