Application of alpha-ketoglutaric acid or salt of alpha-ketoglutaric acid in preparation of normal-temperature sheep semen preservation diluent
By adding α-ketoglutarate or its salt to sheep semen diluent, a suitable room-temperature storage diluent is formed, which solves the problem of plasma membrane and acrosome damage during room-temperature storage of goat sperm, improves sperm motility and conception rate, and achieves efficient semen preservation and utilization.
Patent Information
- Application Number
- CN202511546718.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-27
- Publication Date
- 2026-01-16
AI Technical Summary
During the storage of goat semen at room temperature, the sperm plasma membrane and acrosome are easily damaged, making it difficult for the sperm to combine with the oocyte and reducing the conception rate. Existing diluents cannot effectively improve sperm motility and performance, thus affecting the utilization efficiency and conception rate of breeding rams.
Using α-ketoglutarate or its salt as the main component, a room-temperature preservation diluent for sheep semen was prepared with a concentration range of 5-200 μg/mL. Combined with buffering substances, energy substances and antibacterial substances, a suitable preservation diluent system was formed.
It significantly improved sperm viability, motility, motility, survival rate, plasma membrane and acrosome integrity, increased the pregnancy rate of artificial insemination, extended the semen preservation time, reduced the cost of ram feeding, and reduced the spread of diseases.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of animal reproductive technology, specifically relating to the application of α-ketoglutarate or its salts in the preparation of a diluent for the preservation of sheep semen at room temperature. Background Technology
[0002] Livestock semen preservation is mainly divided into room temperature preservation, low temperature preservation, and cryopreservation. In actual production, room temperature preservation is highly significant for widespread application due to its simple operation, easy preservation method, low requirements, and lack of special temperature control conditions. Goat sperm plasma membranes contain a large amount of polyunsaturated fatty acids, making them highly susceptible to oxidative damage. Simultaneously, the sperm plasma membrane and acrosome are also easily stimulated and damaged. Sperm with damaged plasma membranes and acrosomes have difficulty combining with oocytes to achieve a normal acrosome reaction, leading to a reduced conception rate.
[0003] Diluting goat semen at room temperature not only prolongs its storage time and improves sperm motility and performance, but also enhances the utilization efficiency of breeding rams. Combined with estrus synchronization and artificial insemination techniques, this allows for the full utilization of high-quality ram semen, reducing ram feeding costs, minimizing disease transmission, and maximizing the scale of goat farming. Therefore, it is necessary to develop a goat semen diluent for room temperature storage. Summary of the Invention
[0004] The first aspect of the present invention is to provide the use of α-ketoglutaric acid or its salts.
[0005] The second objective of this invention is to provide a diluent for preserving sheep semen at room temperature.
[0006] The third objective of this invention is to provide the application of the diluent for preserving sheep semen at room temperature, as described in the second aspect of this invention.
[0007] The fourth aspect of this invention aims to provide a method.
[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of the invention provides the use of α-ketoglutaric acid or a salt thereof in any one of a1)-a3): a1) Prepare a diluent for storing sheep semen at room temperature; a2) Store and / or dilute sheep semen at room temperature; a3) Improve the quality of sheep semen when stored at room temperature and / or diluted.
[0009] In some embodiments, the working concentration of α-ketoglutarate or its salt in the room temperature preservation diluent for sheep semen described in a1) is 5-200 μg / mL, for example: it can be 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 μg / mL or any value between them; further, it is 7-110 μg / mL; even further, it is 7-9 μg / mL or 75-85 μg / mL.
[0010] In some embodiments, the content of α-ketoglutarate or its salt in the room temperature preservation diluent for sheep semen described in a1) is sufficient to make its working concentration in the range of 5-200 μg / mL, for example: it can be 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 μg / mL or any value between them; further, it is 7-110 μg / mL; even further, it is 7-9 μg / mL or 75-85 μg / mL.
[0011] In some embodiments, the concentration of α-ketoglutarate or its salt described in a2) in the system of storing and / or diluting sheep semen at room temperature is 5-200 μg / mL, for example: it can be 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 μg / mL or any value between them; further, it is 7-110 μg / mL; even further, it is 7-9 μg / mL or 75-85 μg / mL.
[0012] In some embodiments, the concentration of α-ketoglutaric acid or its salt described in a3) in the system of sheep semen stored and / or diluted at room temperature is 5-200 μg / mL, for example: it can be 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 μg / mL or any value between them; further, it is 7-110 μg / mL; even further, it is 7-9 μg / mL or 75-85 μg / mL.
[0013] In some embodiments, the semen quality described in a3) includes at least one of sperm viability, sperm motility, sperm morphology, sperm survival ability, sperm plasma membrane integrity rate, sperm acrosome integrity rate, and artificial insemination pregnancy rate; further, it includes sperm viability, sperm motility, sperm morphology, sperm survival ability, sperm plasma membrane integrity rate, sperm acrosome integrity rate, or artificial insemination pregnancy rate.
[0014] In some embodiments, the sperm motility performance includes at least one of the following: average curvilinear motility rate, average linear motility rate, average path rate, linear motility ratio, head oscillation amplitude, and sperm oscillation ratio; further, it includes average curvilinear motility rate, average linear motility rate, average path rate, linear motility ratio, head oscillation amplitude, or sperm oscillation ratio.
[0015] In some implementations, the sperm viability is referred to as the sperm survival index.
[0016] A second aspect of the present invention provides a diluent for preserving sheep semen at room temperature, comprising: a base solution for preserving sheep semen at room temperature; and α-ketoglutarate or a salt thereof.
[0017] A diluent for storing sheep semen at room temperature, comprising a base solution for storing sheep semen at room temperature containing α-ketoglutarate or its salt.
[0018] In some embodiments, the working concentration of α-ketoglutarate or its salt in the room-temperature preservation diluent for sheep semen is 5-200 μg / mL, for example: it can be 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 μg / mL or any value between them; further, it is 7-110 μg / mL; even further, it is 7-9 μg / mL or 75-85 μg / mL.
[0019] In some embodiments, the content of α-ketoglutarate or its salt in the room-temperature preservation diluent of sheep semen is sufficient to make its working concentration in the range of 5-200 μg / mL, for example: it can be 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 μg / mL or any value between them; further, 7-110 μg / mL; even further, 7-9 μg / mL or 75-85 μg / mL.
[0020] In some embodiments, the diluent for preserving sheep semen at room temperature includes: a buffer substance, an energy substance, citric acid or its salt, and an antibacterial substance.
[0021] In some embodiments, the buffering substance may include, but is not limited to, at least one of HEPES, Tris, TES, MOPS, and TAPSO; further, Tris.
[0022] In some embodiments, the working concentration of Tris in the room temperature preservation diluent for sheep semen is 20-32 g / L, for example: it can be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 g / L or any value between them; further, it is 24-25 g / L; even further, it is 24.16-24.96 g / L; and still further, it is 24.16-24.32 g / L.
[0023] In some embodiments, the Tris content in the room-temperature preservation diluent for sheep semen is sufficient to make its working concentration in the range of 20-32 g / L, for example: it can be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 g / L or any value between them; further, 24-25 g / L; even further, 24.16-24.96 g / L; and still further, 24.16-24.32 g / L.
[0024] In some embodiments, the energy substance may include, but is not limited to, at least one of fructose and glucose; more specifically, glucose.
[0025] In some embodiments, the working concentration of glucose in the diluent for preserving sheep semen at room temperature is 5-15 g / L, for example: it can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 g / L or any value between them; further, it is 8-10 g / L; even further, it is 8-9.6 g / L; and still further, it is 8-9.08 g / L.
[0026] In some embodiments, the glucose content in the diluent for preserving sheep semen at room temperature is sufficient to make its working concentration in the range of 5-15 g / L, for example: it can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 g / L or any value between them; further, 8-10 g / L; even further, 8-9.6 g / L; and still further, 8-9.08 g / L.
[0027] In some embodiments, the working concentration of citric acid or its salt in the room temperature preservation diluent for sheep semen is 10-20 g / L, for example: it can be 11, 12, 13, 14, 15, 16, 17, 18, 19 g / L or any value between them; further, it is 12-16 g / L; even further, it is 12.72-15.92 g / L; and still further, it is 12.72-13.52 g / L.
[0028] In some embodiments, the content of citric acid or its salt in the room temperature preservation diluent of sheep semen is sufficient to make its working concentration in the range of 10-20 g / L, for example: it can be 11, 12, 13, 14, 15, 16, 17, 18, 19 g / L or any value between them; further, 12-16 g / L; even further, 12.72-15.92 g / L; and still further, 12.72-13.52 g / L.
[0029] In some embodiments, the antibacterial substance may include, but is not limited to, at least one of gentamicin or its salt, penicillin or its salt, streptomycin or its salt; further including streptomycin or its salt (preferably streptomycin sulfate), and penicillin or its salt (preferably penicillin sodium).
[0030] In some embodiments, the working concentration of streptomycin or its salt in the room temperature preservation diluent for sheep semen is 0.6-1.2 g / L, for example: it can be 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2 g / L or any value between them; further, it is 0.7-0.9 g / L; even further, it is 0.72-0.88 g / L; and still further, it is 0.72-0.8 g / L.
[0031] In some embodiments, the content of streptomycin or its salt in the room temperature preservation diluent for sheep semen is sufficient to make its working concentration in the range of 0.6-1.2 g / L, for example: it can be 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2 g / L or any value between them; further, 0.7-0.9 g / L; even further, 0.72-0.88 g / L; and still further, 0.72-0.8 g / L.
[0032] In some embodiments, the working concentration of penicillin or its salt in the room temperature preservation diluent for sheep semen is 700,000 to 1,100,000 IU / L, for example: it can be 700,000, 720,000, 750,000, 780,000, 820,000, 850,000, 880,000, 900,000, 920,000, 950,000, 1,000,000, 1,100,000 IU / L or any value between them; further, it is 700,000 to 900,000 IU / L; even further, it is 720,000 to 880,000 IU / L; and still further, it is 720,000 to 800,000 IU / L.
[0033] In some embodiments, the content of penicillin or its salt in the room-temperature preservation diluent for sheep semen is sufficient to make its working concentration in the range of 700,000 to 1,100,000 IU / L, for example: it can be 700,000 to 1,100,000 IU / L or any value between them; further, it is 700,000 to 900,000 IU / L; even further, it is 720,000 to 880,000 IU / L; and still further, it is 720,000 to 800,000 IU / L.
[0034] A third aspect of the present invention provides the application of the diluent for preserving sheep semen at room temperature according to the second aspect of the present invention in any one of a2)-a3): a2) Store and / or dilute sheep semen at room temperature; a3) Improve the quality of sheep semen when stored at room temperature and / or diluted.
[0035] In some embodiments, the semen quality described in a3) includes at least one of sperm viability, sperm motility, sperm morphology, sperm survival ability, sperm plasma membrane integrity rate, sperm acrosome integrity rate, and artificial insemination pregnancy rate; further, it includes sperm viability, sperm motility, sperm morphology, sperm survival ability, sperm plasma membrane integrity rate, sperm acrosome integrity rate, or artificial insemination pregnancy rate.
[0036] In some embodiments, the sperm motility performance includes at least one of the following: average curvilinear motility rate, average linear motility rate, average path rate, linear motility ratio, head oscillation amplitude, and sperm oscillation ratio; further, it includes average curvilinear motility rate, average linear motility rate, average path rate, linear motility ratio, head oscillation amplitude, or sperm oscillation ratio.
[0037] In some implementations, the sperm viability is referred to as the sperm survival index.
[0038] A fourth aspect of the present invention provides a method comprising the step of using a diluent for preserving sheep semen at room temperature according to a second aspect of the present invention; The method is any one of b1)-b2): b1) Methods for storing and / or diluting sheep semen at room temperature; b2) Methods to improve the quality of sheep semen when stored at room temperature and / or diluted.
[0039] In some embodiments, the method includes the following step: mixing sheep semen with a sheep semen diluent stored at room temperature according to the second aspect of the present invention.
[0040] In some embodiments, the concentration of α-ketoglutarate or its salt in the room-temperature preserved diluent of sheep semen in the mixed system is 5-200 μg / mL, for example: it can be 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 μg / mL or any value between them; further, it is 7-110 μg / mL; even further, it is 7-9 μg / mL or 75-85 μg / mL.
[0041] In some embodiments, the concentration of Tris in the diluent for preserving sheep semen at room temperature in the mixed system is 20-32 g / L, for example: it can be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 g / L or any value between them; further, it is 24-25 g / L; even further, it is 24.16-24.96 g / L; and still further, it is 24.16-24.32 g / L.
[0042] In some embodiments, the concentration of glucose in the diluent for preserving sheep semen at room temperature in the mixed system is 5-15 g / L, for example: it can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 g / L or any value between them; further, it is 8-10 g / L; even further, it is 8-9.6 g / L; and still further, it is 8-9.08 g / L.
[0043] In some embodiments, the concentration of citric acid or its salt in the diluent for preserving sheep semen at room temperature in the mixed system is 10-20 g / L, for example: it can be 11, 12, 13, 14, 15, 16, 17, 18, 19 g / L or any value between them; further, it is 12-16 g / L; even further, it is 12.72-15.92 g / L; and still further, it is 12.72-13.52 g / L.
[0044] In some embodiments, the concentration of streptomycin or its salt in the diluent for preserving sheep semen at room temperature in the mixed system is 0.6-1.2 g / L, for example: it can be 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2 g / L or any value between them; further, it is 0.7-0.9 g / L; even further, it is 0.72-0.88 g / L; and still further, it is 0.72-0.8 g / L.
[0045] In some embodiments, the concentration of penicillin or its salt in the room-temperature preserved diluent of sheep semen in the mixed system is 700,000 to 1,100,000 IU / L, for example: it can be 700,000 to 1,100,000 IU / L or any value between them; further, it is 700,000 to 900,000 IU / L; even further, it is 720,000 to 880,000 IU / L; and still further, it is 720,000 to 800,000 IU / L.
[0046] In some embodiments, the semen quality described in b2) includes at least one of sperm viability, sperm motility, sperm morphology, sperm survival ability, sperm plasma membrane integrity rate, sperm acrosome integrity rate, and artificial insemination pregnancy rate; further, it includes sperm viability, sperm motility, sperm morphology, sperm survival ability, sperm plasma membrane integrity rate, sperm acrosome integrity rate, or artificial insemination pregnancy rate.
[0047] In some embodiments, the sperm motility performance includes at least one of the following: average curvilinear motility rate, average linear motility rate, average path rate, linear motility ratio, head oscillation amplitude, and sperm oscillation ratio; further, it includes average curvilinear motility rate, average linear motility rate, average path rate, linear motility ratio, head oscillation amplitude, or sperm oscillation ratio.
[0048] In some implementations, the sperm viability is referred to as the sperm survival index.
[0049] In some implementations, the mixing time is 0-170h, for example, it can be 0, 0.1, 5, 10, 20, 22, 24, 26, 46, 48, 50, 70, 72, 74, 94, 96, 98, 118, 120, 122, 142, 144, 146, 148, 166, 168, 170h or any value in between.
[0050] In some embodiments, the mixing temperature is 15-40°C, for example, it can be 16, 17, 18, 19, 20, 25, 30, 35, 36, 37, 38, 39°C or any value between them.
[0051] In this invention, the sheep may include, but is not limited to, goats, sheep, blue sheep, and tar sheep; more specifically, goats.
[0052] In this invention, room temperature refers to 15-25°C; more specifically, 16-20°C.
[0053] In some embodiments, the salt includes at least one of a metal salt, an ammonium salt, a salt formed with an organic base, a salt formed with an inorganic acid, a salt formed with an organic acid, a salt formed with a basic amino acid, and a salt formed with an acidic amino acid.
[0054] In some embodiments, the metal salt includes at least one of alkali metal salts (e.g., sodium salts, potassium salts, etc.), alkaline earth metal salts (e.g., calcium salts, magnesium salts, barium salts, etc.), and aluminum salts.
[0055] In some embodiments, the salt formed with an organic base includes salts formed with one or more of the following organic bases: trimethylamine, triethylamine, pyridine, methylpyridine, 2,6-dimethylpyridine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine.
[0056] In some embodiments, the salt formed with the inorganic acid includes salts formed with one or more of the following inorganic acids: hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid.
[0057] In some embodiments, the salt formed with the organic acid includes salts formed with one or more of the following organic acids: formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.
[0058] In some embodiments, the salt formed with the basic amino acid includes salts formed with one or more of the following basic amino acids: arginine, lysine, ornithine.
[0059] In some embodiments, the salt formed with the acidic amino acid includes a salt formed with one or more of the following acidic amino acids: aspartic acid, glutamic acid.
[0060] The beneficial effects of this invention are: This invention discloses for the first time the application of α-ketoglutarate or its salt in the preservation and / or dilution of sheep semen at room temperature, which can improve the quality of sheep semen when preserved at room temperature and / or diluted, such as sperm viability, sperm motility, sperm performance, sperm survival ability, sperm plasma membrane integrity rate, sperm acrosome integrity rate, and / or artificial insemination pregnancy rate. It can be used to prepare a diluent for the preservation of sheep semen at room temperature. Attached Figure Description
[0061] Figure 1 The effect of adding α-ketoglutarate to the diluted basal solution stored at room temperature on sperm viability was shown.
[0062] Figure 2 The effect of adding α-ketoglutarate to the diluted base solution stored at room temperature on sperm plasma membrane integrity was shown.
[0063] Figure 3 The effect of adding α-ketoglutarate to the diluted base solution stored at room temperature on sperm acrosome integrity was shown.
[0064] Figure 4 The effect of adding α-ketoglutarate to the diluted base solution stored at room temperature on the pregnancy rate of artificial insemination was shown. Detailed Implementation
[0065] definition Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.
[0066] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.
[0067] The present invention will be further described in detail below through specific embodiments.
[0068] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.
[0069] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in these examples are commercially available. For reagents whose manufacturers are listed, similar products from other manufacturers are substituted.
[0070] Unless otherwise specified, the quantitative experiments in the following examples were performed in five replicates, and the results were averaged.
[0071] Example I. Test Reagents and Consumables Main instruments and supplies: Beijing Tianyuan Aorui sperm quality analysis system (2001532), fluorescence microscope (Nikon, Y-TV55), semiconductor hot and cold box (Fuwei, F-W12SA), electric thermostatic water bath (Beijing Changfeng Instruments, HW-SY11-K).
[0072] Main reagents and drugs: α-ketoglutarate (Sigma), glucose (Sinopharm Chemical Reagent Co., Ltd.), citric acid (Sinopharm Chemical Reagent Co., Ltd.), Tris (Research reagents), sodium citrate (Sinopharm Chemical Reagent Co., Ltd.), fructose (Beijing Solarbio Co., Ltd.), modified Giemsa staining solution 20X (Beyotime), streptomycin sulfate for injection (North China Pharmaceutical Group), sodium penicillin for injection (North China Pharmaceutical Group).
[0073] II. Specific Operating Procedures The preparation method of the goat room temperature storage diluted base solution used in this embodiment is as follows: 3.04 g of tris(hydroxymethyl)aminomethane (Tris), 1.136 g of anhydrous glucose, 1.69 g of citric acid, 0.1 g of streptomycin sulfate, and 100,000 IU of penicillin sodium were accurately weighed using a balance and dissolved in 100 mL of sterilized ultrapure water. The solution was then filtered through a 0.22 μm filter membrane for sterilization and stored in a refrigerator at 4°C after sterilization.
[0074] The preparation method of the goat room temperature preservation diluent used in this embodiment is as follows: different masses of α-ketoglutaric acid were added to the above-mentioned goat room temperature preservation diluent base solution to obtain goat room temperature preservation diluents with final concentrations of α-ketoglutaric acid of 10 μg / mL and 100 μg / mL, respectively.
[0075] (a) The process of collecting and diluting semen When using a prosthetic vagina to collect goat semen, the prosthetic vagina and the collection cup should be cleaned, disinfected and dried in advance. When collecting semen, the water temperature of the prosthetic vagina should be controlled at 40~45℃. The collected goat semen should be collected into a 2.5 mL cryovial and placed in a water bath at 37℃ for dilution within 30 minutes. Collected goat semen was diluted 2-fold using a preheated (37°C) goat room-temperature preservation diluent. Sperm motility was measured using a computer-assisted sperm analysis (CASA) system. Semen with a motility less than 0.8 was discarded. Semen that met the requirements was mixed in 15 mL centrifuge tubes to eliminate individual differences. Preheated (37°C) goat room-temperature preservation diluents with final concentrations of 10 μg / mL and 100 μg / mL (the control group used a 0 μg / mL goat room-temperature preservation diluent) were slowly added along the wall of the centrifuge tubes. The semen was diluted at a ratio of 1:4 (v / v) of the pre-treated semen (undiluted): goat room-temperature preservation diluent (the control group used a 0 μg / mL goat room-temperature preservation diluent) = 1:4.
[0076] (II) Semen quality testing Diluted semen was stored at room temperature in a water bath at 18°C. Every 12 hours, the centrifuge tubes containing the semen were gently shaken and inverted to prevent sperm precipitation and the accumulation of metabolites that could have a toxic effect on sperm. Every 24 hours, parameters such as sperm motility, morphology, and survival index were measured using a computer-aided sperm analysis system (CASA). The integrity of the sperm plasma membrane and acrosome was measured using a modified Giemsa stain and a hypotonic swelling test (HOST). Semen diluted with goat room temperature storage diluent (the control group used goat room temperature storage diluent, 0 μg / mL) was used for artificial insemination via cervical insemination. Pregnancy was diagnosed using a veterinary ultrasound machine 45 days after insemination.
[0077] (III) Evaluation of Semen Preservation Effectiveness Sperm viability, motility, and movement parameters were measured using CASA every 24 hours. 10 μL of semen was aspirated into a sperm test plate for testing. After 24 h, 48 h, and 72 h of sperm storage at room temperature, modified Giemsa staining and hypotonic swelling assay were used to detect the acrosome integrity rate and plasma membrane integrity rate.
[0078] Survival index test: The average value of sperm TM (sperm viability) within each test interval is multiplied by the cumulative sum of the corresponding time intervals.
[0079] Plasma membrane integrity test: Weigh 0.9 g fructose and 0.49 g sodium citrate using a balance and dissolve them in 100 mL of sterile ultrapure water to prepare a hypotonic swelling solution. Take 100 μL of semen and add 1.9 mL of the hypotonic swelling solution. Incubate in a 37°C water bath for 30 minutes. Count the curvature of sperm tails under a 400 X microscope. The total number of sperm counted each time should not be less than 200. Sperm plasma membrane integrity rate = (number of curvature sperm / total number of sperm counted) × 100%.
[0080] Acrosome integrity test: 10 μL of semen was smeared onto a slide, air-dried, and then fixed in 4% paraformaldehyde solution for 15 minutes. After rinsing with distilled water, the slide was air-dried again. The air-dried slide was stained with 1X modified Giemsa stain for 45 minutes. After washing away the excess stain with distilled water, the slide was air-dried again. The slide was observed under 1000X oil immersion, and the acrosome condition of the sperm was counted. The total number of sperm in each field of view was no less than 200. The acrosome integrity rate = (number of sperm with intact acrosomes / total number of sperm counted) × 100%.
[0081] Scheduled insemination: Young ewes were treated with progesterone sponge suppositories on day 0, and the suppositories were removed at 19:00 on day 14, simultaneously with an injection of pregnant mare serum gonadotropin (250 IU / ewe) and cloprostenol sodium (0.1 g / ewe). On day 16, 0.5 mL of insemination was administered through the cervix at 7:00 AM, and 0.3 mL at 18:00 PM. Pregnancy was diagnosed and the pregnancy rate was calculated using veterinary ultrasound 45 days after insemination.
[0082] (iv) Evaluation results of semen preservation effect 1. Effects of adding α-ketoglutaric acid to the diluted basal solution at room temperature on sperm motility and viability. As shown in Table 1, the sperm motility of goat diluents with final α-ketoglutarate concentrations of 10 μg / mL and 100 μg / mL stored at room temperature for 144 h was significantly higher than that of the goat basal solution group (control group) stored at room temperature.
[0083] Table 1. Effect of α-ketoglutarate on sperm motility (%) of goat semen stored at room temperature
[0084] Note: Different lowercase letters in the same row indicate significant differences (P<0.05), while the same lowercase letter indicates no significant differences (P>0.05).
[0085] As shown in Table 2, compared with the control group, the sperm motility of the goat dilution with a final α-ketoglutarate concentration of 100 μg / mL at room temperature was significantly improved after 72 h of storage at room temperature; compared with the control group, the sperm motility of the goat dilution with a final α-ketoglutarate concentration of 10 μg / mL at room temperature was significantly improved after 168 h of storage at room temperature.
[0086] Table 2 Effect of α-ketoglutarate on sperm motility (%) in goat semen stored at room temperature
[0087] Note: Different lowercase letters in the superscript of peer data indicate significant differences. P <0.05, the same lowercase letter indicates no significant difference ( P >0.05).
[0088] 2. Effects of adding α-ketoglutarate to the diluted basal solution at room temperature on sperm motility. Table 3 shows that, compared with the control group, the goat room-temperature storage diluent group with a final α-ketoglutarate concentration of 100 μg / mL significantly increased sperm VCL, VSL, and VAP at room temperature for 24 h, 72 h, and 96 h, significantly increased sperm LIN at 24 h, 72 h, 96 h, and 144 h, significantly increased sperm WOB at 72 h, 96 h, and 144 h, and significantly increased sperm ALH at 72 h. The goat room-temperature storage diluent group with a final α-ketoglutarate concentration of 10 μg / mL significantly increased sperm VCL at 72 h, 96 h, and 168 h, significantly increased sperm VSL at 24 h, 72 h, 96 h, and 168 h, significantly increased sperm VAP at 72 h, 96 h, 120 h, and 168 h, significantly increased sperm LIN at 96 h, and significantly increased sperm ALH at 72 h. Sperm WOB at 96 h and 168 h significantly increased sperm ALH after 72 h and 168 h storage at room temperature.
[0089] Table 3. Effects of α-ketoglutarate on sperm motility in goat semen after room temperature dilution and preservation.
[0090] Note: Different lowercase letters in the superscript of peer data indicate significant differences. P <0.05, the same lowercase letter indicates no significant difference ( P>0.05). Abbreviations: VCL, mean curvilinear velocity; VSL, mean linear velocity; VAP, mean path velocity; LIN, linear velocity ratio; ALH, head oscillation amplitude; WOB, sperm oscillation ratio.
[0091] 3. Effects of adding α-ketoglutaric acid to the diluted basal solution at room temperature on sperm viability like Figure 1 As shown, the goat room temperature storage dilution group with a final concentration of 100 μg / mL of α-ketoglutarate had the highest sperm survival index (424.64), which was significantly higher than that of the control group (405.42).
[0092] 4. Effect of adding α-ketoglutaric acid to the diluted basal solution at room temperature on sperm plasma membrane integrity. like Figure 2 As shown, compared with the control group, the sperm cell membrane integrity rate was significantly improved in the goat room temperature preservation dilution group with a final α-ketoglutarate concentration of 100 μg / mL after 24 h, 48 h, and 72 h of room temperature preservation; and the sperm cell membrane integrity rate was significantly improved in the goat room temperature preservation dilution group with a final α-ketoglutarate concentration of 10 μg / mL after 72 h of room temperature preservation.
[0093] 5. Effect of adding α-ketoglutarate to the diluted basal solution at room temperature on sperm acrosome integrity. like Figure 3 As shown, compared with the control group, the acrosome integrity rate of sperm was significantly improved in the goat room temperature preservation dilution groups with α-ketoglutarate final concentrations of 10 μg / mL and 100 μg / mL after 24 h, 48 h and 72 h of room temperature preservation.
[0094] 6. Effect of α-ketoglutarate on the pregnancy rate of artificial insemination using goat semen stored at room temperature Semen diluted with room-temperature storage diluent (semen (not diluted 2 times): goat room-temperature storage diluent = 1:4 (v / v)) was immediately subjected to an AI test at the cervix using a vaginal speculum. The results were as follows: Figure 4 As shown, the pregnancy rate of the goat room temperature dilution group (α-KG) with a final concentration of 100 μg / mL α-ketoglutarate was significantly higher than that of the control group (40.87 ± 1.60% vs 30.08 ± 1.12%), with a pregnancy rate increase of 10.79 percentage points and an increase of approximately 35.87%.
[0095] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.
Claims
1. Application of α-ketoglutaric acid or its salts in any of a1)-a3): a1) Prepare a diluent for storing sheep semen at room temperature; a2) Store and / or dilute sheep semen at room temperature; a3) Improve the quality of sheep semen when stored at room temperature and / or diluted.
2. The application according to claim 1, characterized in that, The working concentration of α-ketoglutarate or its salt in the diluent for preserving sheep semen at room temperature as described in a1) is 5-200 μg / mL; more specifically, it is 7-110 μg / mL. Preferably, the concentration of α-ketoglutaric acid or its salt described in a2) in the system for storing and / or diluting sheep semen at room temperature is 5-200 μg / mL; more preferably 7-110 μg / mL; Preferably, the concentration of α-ketoglutaric acid or its salt described in a3) in the system of sheep semen stored and / or diluted at room temperature is 5-200 μg / mL; more preferably 7-110 μg / mL.
3. A diluent for preserving sheep semen at room temperature, comprising: Dilute the base solution for sheep semen at room temperature; And, α-ketoglutaric acid or its salts.
4. The diluent for preserving sheep semen at room temperature according to claim 3, characterized in that, The working concentration of α-ketoglutarate or its salt in the diluent for room temperature preservation of sheep semen is 5-200 μg / mL; more specifically, it is 7-110 μg / mL.
5. The diluent for preserving sheep semen at room temperature according to any one of claims 3-4, characterized in that, The diluent for preserving sheep semen at room temperature includes: buffering substances, energy substances, citric acid or its salts, and antibacterial substances; Preferably, the buffering material is Tris; Preferably, the energy substance is glucose; Preferably, the antibacterial substance includes streptomycin or a salt thereof, and penicillin or a salt thereof.
6. The diluent for preserving sheep semen at room temperature according to claim 5, characterized in that, The working concentration of Tris in the diluent for preserving sheep semen at room temperature is 20-32 g / L; more specifically, it is 24-25 g / L. Preferably, the working concentration of glucose in the diluent for preserving sheep semen at room temperature is 5-15 g / L; more preferably, it is 8-10 g / L. Preferably, the working concentration of citric acid or its salt in the room temperature preservation diluent for sheep semen is 10-20 g / L; more preferably, it is 12-16 g / L. Preferably, the working concentration of streptomycin or its salt in the room temperature preservation diluent for sheep semen is 0.6-1.2 g / L; more preferably 0.7-0.9 g / L. Preferably, the working concentration of penicillin or its salt in the room temperature preservation diluent for sheep semen is 700,000 to 1,100,000 IU / L; more preferably, it is 700,000 to 900,000 IU / L.
7. The use of the sheep semen room temperature preservation diluent according to any one of claims 3-6 in any one of a2)-a3): a2) Store and / or dilute sheep semen at room temperature; a3) Improve the quality of sheep semen when stored at room temperature and / or diluted.
8. A method comprising the step of using the sheep semen room temperature preservation diluent according to any one of claims 3-6; The method is any one of b1)-b2): b1) Methods for storing and / or diluting sheep semen at room temperature; b2) Methods to improve the quality of sheep semen when stored at room temperature and / or diluted.
9. The method according to claim 8, characterized in that, The method includes the following steps: mixing sheep semen with the sheep semen room temperature preservation diluent according to any one of claims 3-6; Preferably, the concentration of α-ketoglutarate or its salt in the diluent for preserving sheep semen at room temperature in the mixed system is 5-200 μg / mL; more preferably 7-110 μg / mL. Preferably, the concentration of Tris in the diluent for preserving sheep semen at room temperature in the mixed system is 20-32 g / L; more preferably, it is 24-25 g / L. Preferably, the concentration of glucose in the diluent for preserving sheep semen at room temperature in the mixed system is 5-15 g / L; more preferably, it is 8-10 g / L. Preferably, the concentration of citric acid or its salt in the diluent for preserving sheep semen at room temperature in the mixed system is 10-20 g / L; more preferably 12-16 g / L. Preferably, the concentration of streptomycin or its salt in the room-temperature preserved diluent of sheep semen in the mixed system is 0.6-1.2 g / L; more preferably 0.7-0.9 g / L. Preferably, the concentration of penicillin or its salt in the diluent for preserving sheep semen at room temperature in the mixed system is 700,000 to 1,100,000 IU / L; more preferably, it is 700,000 to 900,000 IU / L.
10. The application or method according to any one of claims 1-2 and 7-9, characterized in that, The semen quality includes at least one of the following: sperm motility, sperm vitality, sperm motility performance, sperm viability, sperm plasma membrane integrity, sperm acrosome integrity, and artificial insemination pregnancy rate; Preferably, the sperm motility performance includes at least one of the following: average curvilinear motility rate, average linear motility rate, average path rate, linear motility ratio, head oscillation amplitude, and sperm oscillation ratio. Preferably, the sperm viability is the sperm survival index.