Diluent preparation capable of prolonging normal-temperature survival time of boar semen and preparation method of diluent preparation

Abstract

The invention relates to the technical field of semen preservative preparation, in particular to a diluent preparation capable of prolonging the normal-temperature survival time of boar semen and a preparation method of the diluent preparation. Glucose, L-glutamine, an antioxidant and polysaccharide components are scientifically proportioned, and an innovative polyethyleneimine-polysaccharide copolymer is combined, so that the normal-temperature survival time of boar semen is prolonged; the dual effects of sperm metabolism regulation and cell membrane protection are realized. The polyethyleneimine-polysaccharide copolymer enhances the biocompatibility and protective ability of the diluent through stable cross-linking of amino and carboxyl, and effectively relieves the problem of sudden change of osmotic pressure and acidity of the traditional preservative. In the formula, antioxidant components, such as reduced glutathione, vitamin E and coenzyme Q10, synergistically reduce oxidative stress and delay sperm senescence. According to the whole formula, proper pH and osmotic pressure are guaranteed, the sperm motility and survival time are remarkably prolonged, the requirement of pig industry for long-term preservation of sperm is met, and the feed additive has remarkable application value and popularization potential.

Description

Technical Field

[0001] This invention relates to the field of semen preservation agent preparation technology, specifically to a diluent preparation that can prolong the survival period of porcine semen at room temperature and its preparation method. Background Technology

[0002] With the rapid development of modern pig farming, the collection of high-quality boar semen and artificial insemination techniques have become key means to improve the genetic quality of breeding pigs and increase farming efficiency. The widespread application of artificial insemination has not only increased the utilization rate of superior boars but also promoted the large-scale and standardized management of the pig industry. However, boar semen has a short shelf life outside the body, especially at room temperature, which limits the transport distance and flexibility of its use, becoming a significant bottleneck restricting the promotion of artificial insemination and the improvement of farming efficiency.

[0003] Currently, pig farms commonly use glucose-based diluents to dilute and preserve fresh boar semen at room temperature. While these traditional preservatives can temporarily meet the basic metabolic needs of semen, they have significant drawbacks: as storage time increases, nutrients are continuously consumed, leading to the depletion of energy substrates such as glucose in the semen. The accumulation of cellular metabolic products causes changes in osmotic pressure and acidity, resulting in drastic fluctuations in the sperm environment and a rapid decrease in sperm motility. The preservation period is typically only 2 to 3 days, which is insufficient to meet the modern livestock industry's demand for long-term semen preservation.

[0004] Furthermore, differences in the genetic background and breeding environment of breeding pigs result in inconsistent effectiveness of existing diluents and preservatives, making it difficult to achieve stable semen quality. With the increasing demands for semen preservation technology in the pig industry, developing a novel diluent that can effectively slow down sperm metabolism, stabilize the preservation environment, and extend the survival time of semen at room temperature has become an urgent technical challenge.

[0005] To overcome the shortcomings of existing technologies, the present invention aims to provide a diluent formulation and its preparation method that can extend the survival period of porcine semen at room temperature. This invention achieves the dual effects of semen metabolic regulation and cell membrane protection through a scientifically proportioned blend of glucose, L-glutamine, antioxidants, and polysaccharides, combined with an innovative polyethyleneimine-polysaccharide copolymer. The polyethyleneimine-polysaccharide copolymer, through stable cross-linking of amino and carboxyl groups, enhances the biocompatibility and protective capacity of the diluent, effectively alleviating the problems of drastic changes in osmotic pressure and acidity associated with traditional preservatives. Antioxidant components in the formulation, such as reduced glutathione, vitamin E, and coenzyme Q10, synergistically reduce oxidative stress and delay sperm aging. The overall formulation, while maintaining suitable pH and osmotic pressure, significantly extends sperm motility and survival time, meeting the needs of the pig farming industry for long-term semen preservation, and possesses significant application value and promotion potential.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A diluent preparation for prolonging the survival period of porcine semen at room temperature comprises the following components by weight: 2-3.5 parts glucose, 0.5-1 part L-glutamine, 0.15-0.6 parts polyethyleneimine-polysaccharide copolymer, 90-95 parts phosphate buffer, 0.01-0.02 parts reduced glutathione, 0.01-0.02 parts vitamin E, 0.05-0.12 parts calcium lactate, 0.03-0.06 parts sodium alginate, 0.01-0.03 parts triazine gum, 0.005-0.01 parts coenzyme Q10, and 0.02-0.05 parts β-glucan.

[0007] Preferably, the polyethyleneimine-polysaccharide copolymer is prepared by the following steps: S11. Weigh out the natural polysaccharide and add it to deionized water to prepare a uniform and transparent polysaccharide aqueous solution; S12. Weigh out polyethyleneimine powder and dissolve it in phosphate buffer solution to prepare a polyethyleneimine solution; S13. The polyethyleneimine solution is added dropwise to the polysaccharide aqueous solution, and the reaction is stirred at room temperature for 2-4 hours. During the reaction, the solution is kept uniform to promote the formation of a stable copolymer between the amino groups of polyethyleneimine and the carboxyl groups of polysaccharide. S14. After the reaction in step S13 is completed, the reaction solution is transferred to a dialysis bag for dialysis to remove unreacted small molecule impurities and buffer salts. The dialysis water is changed every 4-6 hours and dialysis is continued for 48-72 hours. The purified solution is then sterile filtered to remove microorganisms and suspended impurities. S15. The purified reaction solution is placed in a freeze dryer for vacuum freeze drying to obtain the polyethyleneimine-polysaccharide copolymer.

[0008] Preferably, in step S11, the concentration of the polysaccharide aqueous solution is 1-2 mg / mL; in step S12, the concentration of the polyethyleneimine solution is 2-5 mg / mL; and in step S13, the mass ratio of the polyethyleneimine solution to the polysaccharide aqueous solution is 1:10-15.

[0009] Preferably, in step S12, the molecular weight of the polyethyleneimine is 2000-10000 Da.

[0010] Preferably, in step S14, the molecular weight cutoff of the dialysis bag is 3000-3500 kDa; the sterile filtration is achieved through a 0.22 μm filter membrane.

[0011] Preferably, in step S15, the process parameters for the vacuum freeze-drying process are set as follows: temperature range of -30℃ to -20℃, vacuum degree ≤100 mTorr, and processing time of 8-12h.

[0012] Preferably, the pH range of the phosphate buffer solution is 7.2-7.4.

[0013] Preferably, the molecular weight of the sodium alginate is 100,000-250,000 Da; and the molecular weight of the β-glucan is 50,000-200,000 Da.

[0014] A method for preparing a diluent formulation that can prolong the survival period of bovine semen at room temperature, comprising the following steps: S1. Add glucose, L-glutamine, calcium lactate, and vitamin E to the phosphate buffer solution in the correct proportions, and stir for 10-20 minutes. S2. Add sodium alginate and triazine gum to the solution obtained in S1 in sequence, stir for 5-10 min, autoclave at 110-120℃ for 10-15 min, and cool for later use. S3. Add L-glutamine, coenzyme Q10, β-glucan, and polyethyleneimine-polysaccharide copolymer sequentially to the solution obtained in S2, and stir for 5-10 minutes to obtain the diluent preparation that can prolong the survival period of porcine semen at room temperature.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: This diluent formulation, through a scientifically and rationally combined combination of multiple functional components, effectively prolongs the survival time of porcine semen at room temperature. Glucose and L-glutamine, as the main energy substrates, provide continuous and stable metabolic support for sperm, delaying energy depletion and maintaining motility. Phosphate buffer adjusts and stabilizes the pH of the solution within the range of 7.2 to 7.4, ensuring that sperm are in a suitable acid-base environment and avoiding cell damage caused by pH fluctuations. Polyethyleneimine-polysaccharide copolymer, as the core protective agent, forms a stable structure through the cross-linking of the amino groups of polyethyleneimine and the carboxyl groups of polysaccharides, enhancing the protective effect of the diluent on sperm cell membranes, effectively preventing membrane structure damage and osmotic pressure abnormalities, and improving sperm tolerance and survival rate. The synergistic effect of highly effective antioxidants such as reduced glutathione, vitamin E, and coenzyme Q10 significantly reduces oxidative stress, slows down free radical damage to sperm cells, and delays the cellular aging process. Sodium alginate, triacin, and β-glucan, among other natural polysaccharides, endow the diluent with suitable viscosity and film-forming properties, optimizing microenvironmental stability and further protecting sperm from mechanical and chemical stresses. Simultaneously, it regulates the osmotic pressure of the solution, preventing cell water loss or swelling. The addition of calcium lactate stabilizes ion balance and promotes sperm function maintenance. The overall formula works synergistically through multiple mechanisms, addressing energy supply, antioxidant protection, membrane structure stabilization, and microenvironment regulation, systematically improving sperm physiological state and survival time. This significantly overcomes the technical bottlenecks of traditional diluents, such as short preservation time and rapid motility decline, meeting the practical needs of the pig farming industry for long-term, room-temperature preservation of bovine semen. Attached Figure Description

[0016] Figure 1 This is a process flow diagram of the preparation of the diluent formulation for prolonging the survival period of bovine semen at room temperature, as described in this invention. Figure 2 This is a flow chart of the preparation process of the polyethyleneimine-polysaccharide copolymer described in this invention. Detailed Implementation

[0017] The present invention will now be clearly and completely described in conjunction with embodiments thereof. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0018] Please see Figure 1-2 The present invention provides a technical solution: Example 1: This example provides a formulation for a diluent preparation that can prolong the survival period of bovine semen at room temperature, comprising the following components by weight (one part by weight is defined as 100g):

[0019] The pH range of the phosphate buffer solution is 7.2-7.4; The molecular weight of the sodium alginate is 100,000 Da; The molecular weight of the β-glucan is 50,000 Da; The polyethyleneimine-polysaccharide copolymer is prepared by the following steps: S11. Weigh the natural polysaccharide and add it to deionized water to prepare a uniform and transparent polysaccharide aqueous solution with a concentration of 2 mg / mL; S12. Weigh out polyethyleneimine powder and dissolve it in phosphate buffer to prepare a polyethyleneimine solution with a concentration of 5 mg / mL; S13. Add 10 parts by weight of the polyethyleneimine solution to 100 parts by weight of the polysaccharide aqueous solution dropwise, stir and react at room temperature for 2 hours, keep the solution uniform during the reaction, and promote the formation of a stable copolymer between the amino groups of polyethyleneimine and the carboxyl groups of polysaccharide. S14. After the reaction in step S13 is completed, the reaction solution is transferred to a dialysis bag for dialysis to remove unreacted small molecule impurities and buffer salts. The dialysis water is changed every 4 hours and dialysis is continued for 48 hours. The purified solution is then aseptically filtered through a 0.22μm filter membrane to remove microorganisms and suspended impurities. S15. The purified reaction solution is placed in a freeze dryer for vacuum freeze drying to obtain the polyethyleneimine-polysaccharide copolymer; In step S12, the molecular weight of the polyethyleneimine is 10000 Da; In step S14, the molecular weight cutoff of the dialysis bag is 3500 kDa; In step S15, the process parameters for the vacuum freeze-drying process are set as follows: temperature -30℃, vacuum degree ≤100 mTorr, and processing time 8h.

[0020] This embodiment also provides a preparation method for preparing the diluent formulation that can prolong the survival period of porcine semen at room temperature, comprising the following steps: S1. Add glucose, L-glutamine, calcium lactate, and vitamin E to the phosphate buffer solution in the specified proportions, and stir for 10 minutes. S2. Add sodium alginate and triazine gum to the solution obtained in S1 in sequence, stir for 10 min, autoclave at 110℃ for 15 min, and cool for later use; S3. Add L-glutamine, coenzyme Q10, β-glucan, and polyethyleneimine-polysaccharide copolymer sequentially to the solution obtained in S2, and stir for 5 minutes to obtain the diluent preparation that can prolong the survival period of porcine semen at room temperature.

[0021] Example 2: Example 2 differs from Example 1 in that, in Example 2, the formulation of the diluent preparation for prolonging the survival period of bovine semen at room temperature includes the following components by weight (one part by weight is defined as 100g):

[0022] The remaining steps are exactly the same in Examples 1 and 2.

[0023] Example 3: Example 3 differs from Example 1 in that, in Example 3, the formulation of the diluent preparation for prolonging the survival period of bovine semen at room temperature includes the following components by weight (one part by weight is defined as 100g):

[0024] The remaining steps are exactly the same as in Examples 1 and 3.

[0025] Comparative Example, Comparative Example 1: The only difference between Comparative Example 1 and Example 1 is that the polyethyleneimine-polysaccharide copolymer, reduced glutathione, vitamin E and coenzyme Q10, which were originally present in Example 1, were omitted in Comparative Example 1. The remaining steps are exactly the same in Comparative Example 1 and Example 1.

[0026] Comparative Example 2: The only difference between Comparative Example 2 and Example 1 is that sodium alginate, trisaccharide and β-glucan, which were originally present in Example 1, were omitted in Comparative Example 2. The remaining steps are exactly the same in Comparative Example 2 and Example 1.

[0027] Performance testing Fresh semen (initial motility ≥85%, density 2×10⁻⁶) was collected from the same healthy boar (Duroc, 3 years old). 8 ( / mL), diluted 1:9 with the diluents used in different examples and comparative examples, homogenized, dispensed into sterile tubes, and stored at 17°C. Sampling times were 0, 24, 48, and 72 hours, according to GB / T 25172-2020 "Technical Specifications for Production and Preservation of Porcine Semen at Room Temperature" and GB 23238-2021. The methods specified in "Ugly Boar Semen at Room Temperature" were used to determine sperm motility (%), viability (%), and abnormality rate (%). The test results are shown below:

[0028] As can be seen from the above, the diluents of Examples 1-3 showed significantly better results than Comparative Examples 1 and 2 in prolonging the survival time of bovine semen at room temperature. This difference mainly stems from the mechanism of action and synergistic effect of the key components in the diluents, which played a decisive role in the protection and maintenance of sperm cell function.

[0029] Polyethyleneimine-polysaccharide copolymer, a unique composite component in this example, forms a stable copolymer structure through the polycationic amino groups of polyethyleneimine and the carboxyl groups of polysaccharides, thus constructing a protective membrane at the molecular level. This protective membrane effectively resists damage to sperm cell membranes caused by changes in osmotic pressure and mechanical stress in the external environment, maintaining the integrity and fluidity of the cell membrane and preventing cell edema or dehydration. Because sperm cell membranes are rich in polyunsaturated fatty acids, they are highly susceptible to oxidative stress. Once the membrane structure is damaged, cell function is impaired, and viability and survival rate decrease. The presence of polyethyleneimine-polysaccharide copolymer significantly improves membrane stability and reduces the risk of membrane lipid oxidation and rupture. Reduced glutathione, vitamin E, and coenzyme Q10 play crucial free radical scavenging roles in the diluent. During semen preservation, metabolic activity and changes in the external environment lead to the generation of large amounts of reactive oxygen species (ROS), which directly attack sperm membrane lipids and DNA, inducing lipid peroxidation, damaging cell structure, and inducing apoptosis or loss of function. Reduced glutathione, as a major intracellular antioxidant, can efficiently reduce peroxides and maintain intracellular redox balance. Vitamin E, as a fat-soluble antioxidant, can embed itself in the cell membrane to block lipid peroxidation chain reactions. Coenzyme Q10 not only participates in the mitochondrial electron transport chain and promotes energy metabolism, but also has antioxidant functions, protecting mitochondrial structure and function. These three components work synergistically to significantly reduce oxidative damage and ensure sperm motility and structural stability. Finally, natural polysaccharide components (sodium alginate, triacinol, and β-glucan) give the diluent suitable viscosity and film-forming properties, optimizing the sperm microenvironment and stabilizing pH and osmotic pressure. The colloidal structure formed by sodium alginate and triacinol buffers external physical shocks, reducing mechanical damage, and simultaneously prevents morphological changes in sperm cells due to sudden changes in osmotic pressure by regulating the solution's osmotic pressure. β-glucan has immunomodulatory and antioxidant capabilities, further enhancing the protective performance of the diluent. The moderate viscosity of the diluent is beneficial for maintaining normal sperm motility during storage, reducing energy consumption and cellular stress.

[0030] Comparative Example 1 lacked polyethyleneimine-polysaccharide copolymer and antioxidants, resulting in a weakened sperm membrane protection mechanism, ineffective inhibition of reactive oxygen species damage, exacerbated membrane lipid peroxidation, and impaired cell membrane integrity, thus affecting sperm motility and survival rate. Comparative Example 2 lacked natural polysaccharide components, had insufficient viscosity of the diluent, poor physical protection performance, and made sperm susceptible to mechanical and chemical stimulation. It also reduced osmotic pressure regulation capacity, increased the risk of cell membrane damage, and resulted in poor preservation performance.

[0031] In summary, the multifunctional components in Examples 1-3, through a triple mechanism of physical coating, chemical antioxidation, and microenvironment regulation, synergistically ensured the stability and functional integrity of sperm cell membranes, delayed oxidative stress and cell senescence, and significantly improved the motility and survival rate of porcine semen under room temperature storage conditions. In contrast, the comparative group, lacking key protective components, could not effectively resist multiple damaging factors during storage, leading to a rapid decline in sperm function. This fully demonstrates that the rational integration of membrane protectants, antioxidants, and natural polysaccharides in the diluent formulation design is a key factor in improving the room temperature storage performance of porcine semen.

[0032] 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 variations 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. A diluent formulation for prolonging the survival period of boar semen at room temperature, characterized in that, By mass parts, the following components are included: 2-3.5 parts of glucose, 0.5-1 part of L-glutamine, 0.15-0.6 part of polyethyleneimine-polysaccharide copolymer, 90-95 parts of phosphate buffer, 0.01-0.02 part of reduced glutathione, 0.01-0.02 part of vitamin E, 0.05-0.12 part of calcium lactate, 0.03-0.06 part of sodium alginate, 0.01-0.03 part of triose gum, 0.005-0.01 part of coenzyme Q10, and 0.02-0.05 part of β-glucan.

2. The diluent formulation for prolonging the survival period of boar semen at room temperature according to claim 1, characterized in that, The polyethyleneimine-polysaccharide copolymer is prepared by the following steps: S11. A natural polysaccharide is weighed and added to deionized water to prepare a uniform and transparent polysaccharide aqueous solution; S12. Polyethyleneimine powder is weighed and dissolved in a phosphate buffer to prepare a polyethyleneimine solution; S13. The polyethyleneimine solution is added dropwise to the polysaccharide aqueous solution, and stirred at room temperature for 2-4 hours. The solution is kept uniform during the reaction to promote the formation of stable copolymer between the amino group of polyethyleneimine and the carboxyl group of polysaccharide; S14. After the reaction in step S13 is completed, the reaction solution is transferred to a dialysis bag for dialysis to remove unreacted small molecular impurities and buffer salts. The dialysis water is changed every 4-6 hours, and the dialysis is continued for 48-72 hours. The purified solution is subjected to sterile filtration to remove microorganisms and suspended impurities; S15. The purified reaction solution is placed in a freeze dryer for vacuum freeze drying treatment to prepare the polyethyleneimine-polysaccharide copolymer.

3. The diluent formulation for prolonging the survival period of boar semen at room temperature according to claim 2, characterized in that, In step S11, the concentration of the polysaccharide aqueous solution is 1-2 mg / mL; in step S12, the concentration of the polyethyleneimine solution is 2-5 mg / mL; and in step S13, the mass ratio of the polyethyleneimine solution to the polysaccharide aqueous solution is 1:10-15.

4. The diluent formulation for prolonging the survival period of boar semen at room temperature according to claim 3, characterized in that, In step S12, the molecular weight of the polyethyleneimine is 2000-10000 Da.

5. The diluent formulation for prolonging the survival period of boar semen at room temperature according to claim 2, characterized by, In step S14, the molecular weight cut-off of the dialysis bag is 3000-3500 KDa; and the sterile filtration is achieved by a 0.22 μm filter membrane.

6. The diluent formulation for prolonging the survival period of boar semen at room temperature according to claim 2, characterized by, In step S15, the process parameters of the vacuum freeze drying treatment are set as follows: temperature range of -30℃~-20℃, vacuum degree of ≤100 mTorr, and treatment time of 8-12 hours.

7. The diluent formulation of claim 1, wherein the diluent formulation is capable of extending the survival period of boar semen at room temperature. The pH of the phosphate buffer ranges from 7.2 to 7.

4.

8. The diluent formulation for prolonging the survival period of boar semen at room temperature according to claim 1, characterized by, The molecular weight of the sodium alginate is 100000-250000 Da; and the molecular weight of the β-glucan is 50000-200000 Da.

9. A method for preparing a diluent formulation for extending the survival period of boar semen at room temperature, for preparing the diluent formulation for extending the survival period of boar semen at room temperature according to any one of claims 1 to 8, characterized in that, The following steps are included: S1. Glucose, L-glutamine, calcium lactate, and vitamin E are sequentially added to the phosphate buffer in proportion, and stirred for 10-20 minutes; S2. Sodium alginate and triose gum are sequentially added to the solution prepared in S1, stirred for 5-10 minutes, and autoclaved at 110-120℃ for 10-15 minutes. After cooling, it is ready for use; S3. To the solution prepared in S2, L-glutamine, coenzyme Q10, β-glucan, polyethyleneimine-polysaccharide copolymer are sequentially added, and stirred for 5-10 min to prepare the diluent preparation for prolonging the survival period of pig sperm at room temperature.

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