Ovum extraction liquid for live pig egg collection and preparation method of egg extraction liquid

Abstract

The invention discloses an egg extraction liquid for live pig egg collection and a preparation method of the egg extraction liquid. The egg extraction liquid is prepared from a basic culture medium, an antioxidant system, an osmotic pressure regulator and an energy metabolism accelerant according to the volume ratio of (50-70): (10-20): (5-15): (5-15). The pH value of the egg extraction liquid is 7.2-7.6, and the osmotic pressure of the egg extraction liquid is 280-320 mOsm / kg; the basic culture medium is selected from one or a mixture of more of DMEM / F12, MEM or RPMI 1640, and bovine serum albumin with the mass concentration of 0.1%-0.5% is added. The invention relates to the technical field of safety control. According to the egg extraction liquid for live pig egg collection and the preparation method of the egg extraction liquid, oxidative damage of pig oocytes in the egg collection process can be remarkably reduced by scavenging free radicals and inhibiting lipid peroxidation reaction through the anti-oxidation system, the osmotic pressure of the egg extraction liquid is matched with the physiological environment of the pig oocytes due to the addition of the osmotic pressure regulator, and the egg collection efficiency is improved. The cell swelling or shrinking phenomenon caused by osmotic pressure imbalance is avoided, and the energy supply capacity of the porcine oocytes in the egg collecting process is enhanced.

Description

Technical Field

[0001] This invention relates to the field of safety control technology, specifically to an oocyte extraction fluid for live pig oocyte collection and its preparation method. Background Technology

[0002] The live oocyte retrieval technique for pigs involves surgically removing oocytes from the fallopian tubes through an open abdominal surgery. It is suitable for animals such as pigs, sheep, and rabbits. The procedure requires opening the abdominal cavity, pulling out the fallopian tubes, and then flushing them in either the retrograde or forward direction to obtain the oocytes. However, it is highly invasive, has a long recovery period, and is difficult to reuse live animals.

[0003] The patent publication number “CN111280163B” discloses an oocyte extraction fluid for live bovine oocyte retrieval, its preparation method and uses. The oocyte extraction fluid contains: 15-25 mg sodium pyruvate, 300-400 mg 4-hydroxyethylpiperazine ethanesulfonic acid, 5-15 mL TCM199, 10-20 mg L-glutamine, 10-20 mg sodium bicarbonate, 0.5-1.5 mg heparin, 5-7 mg penicillin, 10-15 mg streptomycin, 1-3 mL fetal bovine serum, 20-30 mg / L taurine, 0.5-1.5 mg / L zinc lactate, and water up to 100 mL.

[0004] As shown in the above technology, the existing technical solutions are mainly designed for bovine oocytes, and their component ratios and functions may not be fully applicable to porcine oocytes. In addition, the oocyte extraction fluid does not fully consider the specific nutritional factors required by porcine oocytes, which may lead to oxidative stress or mechanical damage to porcine oocytes during oocyte retrieval, reducing the survival rate and developmental potential of oocytes after oocyte retrieval. Therefore, it is necessary to develop an oocyte extraction fluid and its preparation method that are more suitable for the characteristics of porcine oocytes in order to improve the effect of live oocyte retrieval from pigs and the subsequent developmental capacity of oocytes. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides an oocyte extraction fluid for live pig oocyte collection and its preparation method, solving the problem of insufficient compatibility of existing oocyte extraction fluids with live pigs.

[0006] To achieve the above objectives, the present invention provides the following technical solution: an oocyte extraction fluid for live pig oocyte collection, wherein the oocyte extraction fluid is prepared by a basal culture medium, an antioxidant system, an osmotic pressure regulator, and an energy metabolism promoter in a volume ratio of 50-70:10-20:5-15:5-15; the pH value of the oocyte extraction fluid is 7.2-7.6, and the osmotic pressure is 280-320 mOsm / kg; the basal culture medium is selected from one or more mixtures of DMEM / F12, MEM, or RPMI 1640, and bovine serum albumin with a mass concentration of 0.1%-0.5% is added.

[0007] Preferably, the antioxidant system consists of reduced glutathione, vitamin C and vitamin E in a mass ratio of 2-5:1-3:1-2.

[0008] Preferably, the antioxidant system accounts for 15%-20% of the volume of the egg extraction fluid.

[0009] Preferably, the osmotic pressure regulator is selected from one or more combinations of sodium chloride, potassium chloride, or magnesium chloride, and the mass concentration of the osmotic pressure regulator is 0.5%-1.0%, wherein sodium chloride accounts for 60%-80% of the total mass of the osmotic pressure regulator.

[0010] Preferably, the energy metabolism promoter is composed of sodium pyruvate, sodium lactate and glucose in a mass ratio of 1-3:1-2:1-2.

[0011] Preferably, the sodium pyruvate has a mass concentration of 0.2%-0.5%, the sodium lactate has a mass concentration of 0.1%-0.3%, and the glucose has a mass concentration of 0.1%-0.4%.

[0012] This invention also discloses a method for preparing oocyte extraction fluid for live pig oocyte collection, specifically including the following steps: Step 1: Add the basal culture medium to a sterile container at a volume ratio of 50-70, then add the antioxidant system, osmotic pressure regulator and energy metabolism promoter in sequence and stir. Step 2: Use a 0.22μm microporous membrane to filter and sterilize the preliminary mixture to obtain a sterile mixture; Step 3: Store the aseptic mixture at 4°C for no more than 24 hours, or freeze it at -20°C for no more than 3 months.

[0013] Preferably, in step one, the stirring is performed at a speed of 100-500 r / min for 10-30 minutes to obtain a preliminary mixture.

[0014] Beneficial effects

[0015] This invention provides an oocyte extraction fluid for live pig oocyte collection and its preparation method. Compared with the prior art, it has the following advantages: 1. The oocyte extraction fluid and its preparation method for live oocyte retrieval from pigs, through an antioxidant system, can significantly reduce oxidative damage to pig oocytes during oocyte retrieval by scavenging free radicals and inhibiting lipid peroxidation. The addition of an osmotic pressure regulator makes the osmotic pressure of the oocyte extraction fluid match the physiological environment of pig oocytes, avoiding cell swelling or shrinkage caused by osmotic pressure imbalance. The energy metabolism promoter enhances the energy supply capacity of pig oocytes during oocyte retrieval by providing a direct energy source and metabolic intermediates.

[0016] 2. The oocyte extraction fluid and its preparation method for live oocyte retrieval from pigs demonstrate good stability and biocompatibility in practical applications, effectively maintaining the biological structural characteristics and developmental potential of pig oocytes. Experimental data show that the survival rate and developmental potential of pig oocytes retrieved using the oocyte extraction fluid of this invention are significantly higher than those of existing technologies.

[0017] 3. The oocyte extraction fluid and its preparation method used for live oocyte retrieval from pigs were optimized based on the characteristics and needs of pig oocytes. By introducing an antioxidant system, osmotic pressure regulator and energy metabolism promoter, the effects of oxidative stress and mechanical damage on pig oocytes during oocyte retrieval were effectively reduced. Attached Figure Description

[0018] Figure 1 This is a flowchart illustrating the preparation process of the oocyte extraction solution for this invention. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Please see Figure 1 The following technical solution is provided for the oocyte extraction fluid used in live pig oocyte collection and its preparation method: The basal culture medium, as the main component, is first added to a sterile container, followed by the addition of an antioxidant system, an osmotic pressure regulator, and an energy metabolism promoter. These components are prepared in a volume ratio of 50-70:10-20:5-15:5-15 and thoroughly mixed at 100-500 r / min for 10-30 minutes to ensure uniform distribution. Careful control of the mixing speed and time is crucial to avoid uneven mixing and deterioration of solution performance. The preliminary mixture is then filtered through a 0.22 μm microporous membrane in one or multiple stages to remove potential microbial contamination, resulting in a sterile mixture. Finally, the filtered sterile mixture is stored at 4°C for no more than 24 hours or frozen at -20°C for no more than 3 months in step 7. Before practical application, if the oocyte extraction fluid is stored at 4°C, it must be brought to room temperature and gently shaken to ensure uniform distribution of components within the solution.

[0021] The basal culture medium is the core carrier of the oocyte extraction fluid. It uses one or more mixtures of DMEM / F12, MEM, or RPMI 1640, with the addition of 0.1%-0.5% bovine serum albumin (BSA) to enhance solution stability and cell protection. The antioxidant system consists of reduced glutathione, vitamin C, and vitamin E in a mass ratio of 2-5:1-3:1-2, accounting for 15%-20% of the oocyte extraction fluid volume. Its main function is to scavenge free radicals and inhibit lipid peroxidation, thereby reducing oxidative stress damage to porcine oocytes during oocyte retrieval. The osmolarity regulator consists of one or more combinations of sodium chloride, potassium chloride, or magnesium chloride, with a mass concentration of 0.5%-1.0%, with sodium chloride being the main component, accounting for 60%-80% of the total mass of the osmolarity regulator. This component maintains the osmolarity of the oocyte extraction fluid within the range of 280-320 mOsm / kg by regulating the ion concentration of the solution, avoiding cell swelling or shrinkage caused by osmolarity imbalance. Energy metabolism promoter 4 is composed of sodium pyruvate, sodium lactate, and glucose in a mass ratio of 1-3:1-2:1-2, wherein the mass concentration of sodium pyruvate is 0.2%-0.5%, the mass concentration of sodium lactate is 0.1%-0.3%, and the mass concentration of glucose is 0.1%-0.4%. This component provides porcine oocytes with a direct energy source and metabolic intermediates, enhancing their energy supply capacity during oocyte retrieval.

[0022] In practical use, the application of the oocyte extraction fluid must be tailored to the specific needs of live oocyte retrieval in pigs. For example, in one experiment, healthy adult sows were selected as the oocyte retrieval subjects. Cumulo-oocyte complexes (COCs) were obtained using ultrasound-guided ovarian puncture. During oocyte retrieval, the prepared oocyte extraction fluid was injected into the retrieval needle, bringing it into contact with the COCs. Because the oocyte extraction fluid has a pH of 7.4, an osmotic pressure of 300 mOsm / kg, and contains sufficient antioxidants and energy metabolism promoters, it effectively maintains the integrity and activity of the COCs. Experimental results showed that the survival rate of pig oocytes retrieved using the oocyte extraction fluid of this invention reached over 90%, significantly higher than the approximately 70% of existing technologies. Furthermore, subsequent in vitro maturation culture experiments demonstrated a significant improvement in the developmental potential of the retrieved oocytes, further validating the practical application value of the oocyte extraction fluid of this invention.

[0023] In another embodiment, to verify the effect of different component ratios on the performance of oocyte extraction fluid, multiple comparative experiments were designed. For example, in one experiment, the volume ratio of the antioxidant system was adjusted to 10%, in another experiment, the mass concentration of the osmotic pressure regulator was adjusted to 0.4%, and in yet another experiment, the mass concentration of sodium pyruvate in energy metabolism promoter 4 was adjusted to 0.1%. Analysis of the results of each group of experiments revealed that when the volume ratio of the antioxidant system was less than 15%, the antioxidant capacity of the oocyte extraction fluid decreased significantly, leading to increased oxidative damage to oocytes during oocyte retrieval. When the mass concentration of osmotic pressure regulator 3 was less than 0.5%, the osmotic pressure of the oocyte extraction fluid could not match the physiological environment of porcine oocytes, easily causing cell swelling or shrinkage. When the mass concentration of sodium pyruvate in the energy metabolism promoter was less than 0.2%, the energy supply capacity of oocytes was insufficient, affecting their activity maintenance during oocyte retrieval. Therefore, the selection and ratio of each component in this invention have been verified by a large number of experiments and have scientific basis and practical application value.

[0024] In addition, to further improve the stability and biocompatibility of the oocyte extraction fluid, other auxiliary components can be introduced during the preparation process. For example, adding an appropriate amount of hyaluronic acid or collagen to the basal culture medium can enhance the adhesion and protective effect of the solution; adding a small amount of coenzyme Q10 to the antioxidant system can further enhance its antioxidant capacity; appropriately increasing the proportion of magnesium chloride in the osmotic pressure regulator can optimize the ion balance of the solution; and supplementing an appropriate amount of amino acids in the energy metabolism promoter can enrich the types of its metabolic intermediates. These improvements can be flexibly adjusted according to specific experimental needs, but it must be ensured that they do not have a negative impact on the overall performance of the oocyte extraction fluid.

[0025] The oocyte extraction fluid preparation method provided by this invention is simple, easy to implement, and moderately cost-effective. It is applicable to live oocyte retrieval in pigs and related research fields. By rationally designing the proportions and functions of each component, it achieves precise adaptation to the characteristics of pig oocytes, effectively reducing the impact of oxidative stress and mechanical damage during oocyte retrieval. Furthermore, the oocyte extraction fluid preparation process is clear and straightforward, facilitating large-scale promotion and application.

[0026] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0027] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. An oocyte extraction fluid for live pig oocyte collection, characterized in that: The oocyte extraction solution is prepared by mixing basal culture medium, antioxidant system, osmotic pressure regulator and energy metabolism promoter in a volume ratio of 50-70:10-20:5-15:5-15; the pH value of the oocyte extraction solution is 7.2-7.6, and the osmotic pressure is 280-320 mOsm / kg; the basal culture medium is selected from one or more of DMEM / F12, MEM or RPMI1640, and bovine serum albumin with a mass concentration of 0.1%-0.5% is added.

2. The oocyte extraction fluid for live pig oocyte collection according to claim 1, characterized in that: The antioxidant system is composed of reduced glutathione, vitamin C and vitamin E in a mass ratio of 2-5:1-3:1-2.

3. The oocyte extraction fluid for live pig oocyte collection according to claim 1, characterized in that: The antioxidant system accounts for 15%-20% of the volume of the egg extraction fluid.

4. The oocyte extraction fluid for live pig oocyte collection according to claim 1, characterized in that: The osmotic pressure regulator is selected from one or more combinations of sodium chloride, potassium chloride, or magnesium chloride, and the mass concentration of the osmotic pressure regulator is 0.5%-1.0%, wherein sodium chloride accounts for 60%-80% of the total mass of the osmotic pressure regulator.

5. The oocyte extraction fluid for live pig oocyte collection according to claim 1, characterized in that: The energy metabolism promoter is composed of sodium pyruvate, sodium lactate and glucose in a mass ratio of 1-3:1-2:1-2.

6. The oocyte extraction fluid for live pig oocyte collection according to claim 1, characterized in that: The sodium pyruvate has a mass concentration of 0.2%-0.5%, the sodium lactate has a mass concentration of 0.1%-0.3%, and the glucose has a mass concentration of 0.1%-0.4%.

7. A method for preparing oviduct fluid for live pig oviduct collection as described in claims 1-6, characterized in that: Specifically, the following steps are included: Step 1: Add the basal culture medium to a sterile container at a volume ratio of 50-70, then add the antioxidant system, osmotic pressure regulator and energy metabolism promoter in sequence and stir. Step 2: Use a 0.22μm microporous membrane to filter and sterilize the preliminary mixture to obtain a sterile mixture; Step 3: Store the aseptic mixture at 4°C for no more than 24 hours, or freeze it at -20°C for no more than 3 months.

8. The method for preparing oviposition fluid for live pig oviposition according to claim 1, characterized in that: In step one, the mixture is stirred at a speed of 100-500 r / min for 10-30 minutes to obtain a preliminary mixture.

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