Mutton sheep frozen semen protective agent as well as preparation method and application method thereof

Abstract

The invention relates to a mutton sheep frozen semen protective agent and a preparation method and application method thereof, and the mutton sheep frozen semen protective agent comprises a multi-pillar synergistic protection system, the protection system comprises a basic buffer system, an energy and impermeable protection system, a penetrable cryoprotection system, a mitochondrial targeting protection system, an epigenetic regulation system, a biological nano composite system, a membrane stabilization and cell survival signal regulation system and an auxiliary system.

Description

Technical Field

[0001] This invention relates to the field of animal reproductive biotechnology, and in particular to a preservative for frozen semen of mutton sheep, as well as its preparation and application methods. Background Technology

[0002] Cryopreservation of sheep semen is an important method in modern animal husbandry for long-term preservation of superior breeds and for remote breeding. However, compared to cattle and other livestock, ram semen is more sensitive to cryogenic damage, and conventionally frozen semen shows a significant decrease in sperm motility and fertilization rate after thawing. Ice crystal formation and osmotic pressure changes during ultra-low temperature freezing can damage sperm cell membranes and structures, leading to impaired sperm motility, acrosome integrity, and DNA integrity. Oxidative stress induced by cryopreservation is one of the main causes of sperm damage: the freeze-thaw process significantly increases the level of reactive oxygen species (ROS), weakening sperm's antioxidant capacity. Therefore, adding antioxidants and other protective agents to the cryopreservation medium to mitigate ROS damage is a necessary strategy.

[0003] Traditional cryoprotectants for sheep semen are mostly based on Tris (tris(hydroxymethyl)aminomethane)-egg yolk diluent, but they have problems such as biosafety risks (e.g., pathogen transmission) and large batch-to-batch variations. In recent years, some improved solutions have emerged. For example, some commercially available "four-branch" systems have added cholesterol-loaded cyclodextrin, resveratrol + nano-selenium, and apoptosis inhibitors to the base diluent in order to improve the freezing effect from aspects such as membrane lipid stabilization, anti-oxidation, and inhibition of programmed cell death. However, these solutions still have shortcomings: (1) The protection mechanism is relatively simple, mainly focusing on increasing membrane cholesterol content and scavenging free radicals, failing to fully cover all aspects of cryodamage; (2) There is insufficient understanding of the deep cellular molecular damage mechanism caused by freezing. For example, in addition to apoptosis (programmed cell death), iron-dependent lipid peroxidation cell death (ferroptosis) and other mechanisms have not been fully considered during freezing; (3) The potential impact of freezing stress on the genetic information of sperm expression has not been fully considered, which may affect the health of offspring; (4) Some additives are expensive and have limited sources, which limits their large-scale application. Summary of the Invention

[0004] In view of the above analysis, the present invention aims to provide a preservative for frozen semen of mutton sheep, as well as its preparation method and application method, in order to at least solve one of the above-mentioned problems.

[0005] On one hand, the present invention provides a preservative for frozen sheep semen, comprising a multi-pillar synergistic protection system, wherein the protection system includes:

[0006] The basic buffer system is configured to maintain the liquid environment at a suitable pH and ionic strength in order to stabilize the extracellular environment.

[0007] An energy and non-osmotic protection system is configured to provide energy substrates during freezing and protect cells from osmotic stress.

[0008] A penetrating cryoprotection system is configured to allow moisture to penetrate into sperm cells, lowering the freezing point to prevent intracellular ice crystal formation.

[0009] The mitochondrial targeted protection system is configured to maintain sperm mitochondrial function, enhance ATP function, and reduce the production of mitochondrial-related ROS.

[0010] The epigenetic regulatory system is configured to mitigate the damage to sperm epigenetic markers caused by freezing and protect sperm DNA and chromatin structure.

[0011] The bio-nanocomposite system is configured to utilize nanomaterials and biomacromolecules to synergistically protect sperm structure and release functional factors.

[0012] The membrane stability and cell survival signaling regulation system is configured to maintain sperm plasma membrane integrity, reduce cold shock damage, and activate cell survival pathways.

[0013] The auxiliary system is configured to ensure the microbial safety of the diluent and maintain the environment required for normal sperm activity.

[0014] Furthermore, the basic buffer system comprises trihydroxyaminomethane, 4-hydroxyethylpiperazine ethanesulfonic acid, sodium citrate, and disodium ethylenediaminetetraacetate, in a ratio of 200–300: 20–40: 60–100: 0.3–0.8, respectively.

[0015] Furthermore, the energy and non-permeability protection system includes fructose, trehalose, and betaine, with a weight ratio of 40–80:40–80:20–50.

[0016] Furthermore, the penetrating cryoprotection system comprises glycerol and dimethyl sulfoxide in a ratio of 6–10:1–3.

[0017] Furthermore, the mitochondrial targeted protection system includes a mitochondrial protectant, cyclosporine A, and nicotinamide ribose, wherein the mitochondrial protectant is one of MitoQ or SS-31 tetrapeptide.

[0018] When the mitochondrial protectant is MitoQ, the ratio of MitoQ, cyclosporine A, and nicotinamide ribose is 0.05–0.15: 0.1–0.5: 100–300, respectively.

[0019] When the mitochondrial protectant is SS-31 tetrapeptide, the ratio of SS-31 tetrapeptide, cyclosporine A and nicotinamide ribose is 6-12:0.1-0.5:100-300.

[0020] Furthermore, the epigenetic regulatory system includes sodium butyrate and epigallocatechin gallate, with a ratio of 1000-5000:10-50.

[0021] Furthermore, the bio-nanocomposite system comprises water-soluble chitosan derivative nanoparticles, hyaluronic acid, and recombinant human lactoferrin;

[0022] The auxiliary system includes antibiotics and pH-regulating and balancing salts, wherein the antibiotics include penicillin G and streptomycin.

[0023] On the other hand, the present invention provides a method for preparing a frozen semen protectant for mutton sheep, which can at least be used to prepare the above-mentioned frozen semen protectant for mutton sheep, including the following steps:

[0024] S100: Prepare the basic buffer system solution;

[0025] S200: Energy and non-osmotic protection system are added to the basic buffer system solution to obtain the first preparative solution;

[0026] S300: Prepare a colloidal solution for the bio-nanocomposite system, then add the first preparative solution to obtain the second preparative solution;

[0027] S400: Add the epigenetic regulation system to the second preparative solution to obtain the third preparative solution;

[0028] S500: First, pre-dissolve each component of the membrane stability and cell survival signal regulation system, then add the third preparative solution to obtain the fourth preparative solution;

[0029] S600: First, a portion of the mitochondrial targeted protection system is added to the fourth preparative solution to obtain the fifth preparative solution; then, the remaining mitochondrial targeted protection system is prepared into a solution and added to the fifth preparative solution to obtain the sixth preparative solution.

[0030] S700: Add the sixth preparative solution to near the preset volume, add the components of the auxiliary system, adjust the pH to 7.0-7.4, and bring the volume to the preset volume to obtain the precursor solution of frozen semen protectant for mutton sheep;

[0031] S800: Prepare a penetrating cryoprotection system solution. When using the mutton sheep frozen semen protectant, the mutton sheep frozen semen protectant precursor solution and the penetrating cryoprotection system solution are mixed with the semen sequentially at a volume ratio of 1:1.

[0032] Furthermore, the preparation method includes the following steps:

[0033] S100: Dissolve 3.03g Tris base, 0.71g HEPES, 2.35g sodium citrate, and 0.019g EDTA-2Na in approximately 80mL of ultrapure water to form a basic buffer system solution, and adjust the pH to 7.2.

[0034] S200: Add 1.08g of fructose, 2.05g of trehalose, and 0.36g of betaine to the basic buffer system solution and stir to dissolve, to obtain the first preparative solution;

[0035] S300: Prepare a suspension with a particle size of 100nm by 0.02g of carboxymethyl chitosan nanoparticles, add 0.10g of hyaluronic acid and 0.01g of recombinant human lactoferrin to the suspension, and stir magnetically for 1 hour at room temperature to fully disperse it into a uniform colloidal solution. Then add the colloidal solution to the first preparative solution to obtain the second preparative solution.

[0036] S400: After sterilizing sodium butyrate 0.022g and EGCG 0.009g through a 0.22μm filter membrane, add them to the second preparative solution and mix gently to obtain the third preparative solution;

[0037] S500: First, pre-dissolve 0.0023g of melatonin and 0.00068g of sphingosine-1-phosphate in a small amount of sterile ethanol and water respectively, and then add them to the third preparative solution; then pre-dissolve 0.0835g of P188 in a 37℃ water bath and slowly add it to the third preparative solution to obtain the fourth preparative solution;

[0038] S600: First, add 0.006g of nicotinamide ribose and 0.000024g of cyclosporine A to the fourth preparative solution and stir in the dark to fully dissolve them to obtain the fifth preparative solution; then, transfer 10μL of commercial MitoQ stock solution, pre-dilute it with double-distilled water or ultrapure water, add it to the fifth preparative solution and mix quickly to ensure uniform distribution to obtain the sixth preparative solution;

[0039] S700: Add sterile ultrapure water to the sixth preparative solution to approximately 100 mL, adjust the pH of the solution to the range of 7.0 to 7.4, add a mixture of penicillin G 500 IU / mL and streptomycin 500 μg / mL antibiotics, bring the volume to 100 mL, filter to sterilize and obtain a clear precursor solution for frozen semen protectant in mutton sheep.

[0040] S800: Prepare 100 mL of a penetrating cryoprotection system solution containing 14% glycerol and 4% DMSO by volume. When using the sheep frozen semen protectant, mix 100 mL of the sheep frozen semen protectant precursor solution and 100 mL of the penetrating cryoprotection system solution with the semen sequentially.

[0041] Furthermore, this invention provides a method for applying a mutton sheep frozen semen preservative. The aforementioned mutton sheep frozen semen preservative or the mutton sheep frozen semen preservative prepared above can be used for semen cryopreservation, comprising the following steps:

[0042] A100: Semen collection;

[0043] A200: Dilute the semen with a precursor solution for frozen sheep semen preservation agent;

[0044] A300: Programmable cooling of diluted semen;

[0045] A400: After equilibration with a penetrating cryoprotection system solution, it is tube-sealed, frozen, and stored in liquid nitrogen;

[0046] The precursor solution for frozen semen protectant in mutton sheep includes a basic buffer system, an energy and non-osmotic protection system, a mitochondrial targeted protection system, an epigenetic regulation system, a bio-nanocomposite system, a membrane stability and cell survival signal regulation system, and an auxiliary system.

[0047] In this invention, the above-described technical solutions can be combined with each other to achieve more preferred combinations. Other features and advantages of this invention will be set forth in the following description, and some advantages may become apparent from the description or be learned by practicing the invention. The objectives and other advantages of this invention can be realized and obtained through the specific points highlighted in the description. Detailed Implementation

[0048] The preferred embodiments of the present invention are described in detail below. These embodiments are used to illustrate the principles of the present invention and are not intended to limit the scope of the present invention.

[0049] A specific embodiment of the present invention discloses a preservative for frozen sheep semen, comprising a multi-pillar synergistic protection system, the protection system comprising:

[0050] The basic buffer system is configured to maintain the liquid environment at a suitable pH and ionic strength in order to stabilize the extracellular environment.

[0051] An energy and non-osmotic protection system is configured to provide energy substrates during freezing and protect cells from osmotic stress.

[0052] A penetrating cryoprotection system is configured to allow moisture to penetrate into sperm cells, lowering the freezing point to prevent intracellular ice crystal formation.

[0053] The mitochondrial targeted protection system is configured to maintain sperm mitochondrial function, enhance ATP (adenosine triphosphate) function, and reduce mitochondrial-related ROS production.

[0054] The epigenetic regulatory system is configured to mitigate the damage to sperm epigenetic markers caused by freezing and protect sperm DNA and chromatin structure.

[0055] The bio-nanocomposite system is configured to utilize nanomaterials and biomacromolecules to synergistically protect sperm structure and release functional factors.

[0056] The membrane stability and cell survival signaling regulation system is configured to maintain sperm plasma membrane integrity, reduce cold shock damage, and activate cell survival pathways.

[0057] The auxiliary system is configured to ensure the microbial safety of the diluent and maintain the environment required for normal sperm activity.

[0058] Preferably, when using the sheep frozen semen protectant, a precursor solution for the sheep frozen semen protectant is first prepared, consisting of a basic buffer system, an energy and non-osmotic protection system, a mitochondrial targeted protection system, an epigenetic regulation system, a bio-nanocomposite system, a membrane stability and cell survival signal regulation system, and an auxiliary system. Then, a penetrating cryoprotection system solution is prepared. When processing fresh semen, the fresh semen is first mixed with the sheep frozen semen protectant precursor solution, and then mixed with the penetrating cryoprotection system solution to better protect the sperm.

[0059] The basic buffer system comprises trihydroxyaminomethane (Tris), 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), sodium citrate, and disodium EDTA (disodium ethylenediaminetetraacetate), in a ratio of 200–300:20–40:60–100:0.3–0.8, with a preferred ratio of 250:30:80:0.5. Tris provides the buffer base, HEPES synergistically buffers Tris to stabilize pH at low temperatures, sodium citrate provides osmotic pressure buffering and chelates small amounts of metal ions to protect the membrane structure, and disodium EDTA chelates trace amounts of heavy metal ions to prevent metal-induced peroxidation damage.

[0060] For example, taking the preparation of 100 mL of pretreatment solution for frozen semen of mutton sheep as an example, the concentration of Tris is 200-300 mM, preferably 250 mM; the concentration of HEPES is 20-40 mM, preferably 30 mM; the concentration of sodium citrate is 60-100 mM, preferably 80 mM; and the concentration of disodium EDTA is 0.3-0.8 mM, preferably 0.5 mM.

[0061] The energy and non-osmotic protection system comprises fructose, trehalose, and betaine, in a ratio of 40–80:40–80:20–50, preferably 60:60:30. Fructose can be used as an energy source by sperm and is less likely to participate in the heroni reaction at low temperatures; trehalose, as a non-osmotic protectant, can replace intracellular water, inhibit ice crystal formation, and stabilize membrane protein structures; betaine, as a natural osmotic protectant, helps maintain cell volume, reduces cryogenic stress, and, as a methyl donor, helps protect the methylation state of DNA.

[0062] For example, taking the preparation of 100 mL of pretreatment solution for frozen semen protectant in mutton sheep as an example, the concentration of fructose is 40-80 mM, preferably 60 mM; the concentration of trehalose is 40-80 mM, preferably 60 mM; and the concentration of betaine is 20-50 mM, preferably 30 mM.

[0063] The penetrating cryoprotection system comprises glycerol and dimethyl sulfoxide (DMSO) in a ratio of 6–10:1–3, preferably 7:2. Glycerol acts as a penetrating protectant, replacing intracellular water to inhibit ice crystal formation. The glycerol concentration is controlled at a low level to reduce its toxicity to sperm. DMSO works synergistically with glycerol, penetrating cells rapidly, protecting the internal structure of sperm during the initial cooling phase, and allowing for a suitable reduction in the amount of glycerol used.

[0064] For example, in preparing a 100 mL breakthrough cryoprotection system solution, the volume percentage of glycerol is 6-10% (v / v), preferably 7%. The volume percentage of DMSO is 1-3% (v / v), preferably 2%.

[0065] The mitochondrial-targeted protection system includes a mitochondrial protectant, cyclosporine A (CsA), and nicotinamide ribose (NR). The mitochondrial protectant is one of the following: MitoQ (mitochondrial-targeted coenzyme Q10), SS-31 tetrapeptide (Elamipretide), etc.

[0066] When MitoQ is used as the mitochondrial protectant, the ratio of MitoQ, cyclosporine A, and nicotinamide ribose is 0.05–0.15:0.1–0.5:100–300, preferably 0.1:0.2:200. MitoQ delivers the antioxidant coenzyme Q10 to the inner mitochondrial membrane via its triphenylphosphine cationic group, clearing excess ROS generated during mitochondrial respiration and preventing a decrease in mitochondrial membrane potential. The addition of MitoQ can significantly improve the mitochondrial motility and viability of frozen sperm.

[0067] When the mitochondrial protectant is SS-31 tetrapeptide (Elamipretide), the component ratio of SS-31 tetrapeptide, cyclosporine A, and nicotinamide ribose is 6–12:0.1–0.5:100–300, preferably 9:0.2:200. SS-31 tetrapeptide is a mitochondrial-targeting antioxidant peptide that can embed into the inner mitochondrial membrane to stabilize cardiolipin and scavenge free radicals. At low concentrations, it can improve sperm motility and mitochondrial membrane potential after thawing, and reduce ROS and lipid peroxidation levels.

[0068] CsA specifically inhibits the opening of the mitochondrial permeability transition pore (mPTP), protects mitochondrial membrane potential, and reduces the initiation of apoptosis pathways caused by increased mitochondrial membrane permeability. Low-dose CsA can maintain mitochondrial function during cryo-stress.

[0069] As a precursor to NAD+, NR increases intracellular NAD+ levels, promotes mitochondrial energy metabolism and DNA repair, and helps restore sperm ATP and stabilize genetic material after freezing.

[0070] For example, taking the preparation of 100 mL of pretreatment solution for frozen sheep semen as an example, when the mitochondrial protectant is MitoQ, its concentration is 50-150 nM, preferably 100 nM; when the mitochondrial protectant is SS-31, its concentration is 6-12 μM, preferably 9 μM; the concentration of CsA is 0.1-0.5 μM, preferably 0.2 μM; and the concentration of NR is 100-300 μM, preferably 200 μM.

[0071] The epigenetic regulatory system comprises sodium butyrate and epigallocatechin gallate (EGCG), with a ratio of 1000–5000:10–50, preferably 2000:25. Sodium butyrate, as a histone deacetylase (HDAC) inhibitor, can maintain histone acetylation levels during freezing, preventing excessive chromatin aggregation and facilitating the maintenance of sperm chromatin accessibility and integrity. It can also promote sperm DNA deagglutination after thawing, improving pronucleus formation efficiency during fertilization. EGCG from green tea catechins possesses both antioxidant and epigenetic regulatory functions. It can inhibit DNA methyltransferase activity, thereby reducing abnormal methylation, and protect sperm DNA from oxidative damage by scavenging free radicals. EGCG can also stabilize cell membranes and improve sperm motility and mitochondrial function after thawing frozen sperm.

[0072] For example, taking the preparation of 100 mL of pre-treatment solution for frozen semen protectant in mutton sheep as an example, the concentration of sodium butyrate is 1-5 mM, preferably 2 mM; and the concentration of EGCG is 10-50 μM, preferably 25 μM.

[0073] In practical applications, epigenetic regulatory systems can also be composed of other natural molecules, such as resveratrol, which exerts epigenetic and metabolic protective effects by activating the SIRTI / AMPK pathway. For example, when preparing a 100mL solution of frozen sheep semen protectant, the concentration of resveratrol is 50μM.

[0074] The bio-nanocomposite system comprises water-soluble chitosan derivative (carboxymethyl chitosan, CMC) nanoparticles, hyaluronic acid, and recombinant human lactoferrin (rhLF). Carboxymethyl chitosan possesses excellent biocompatibility and antioxidant properties. Its nanoparticles can adsorb onto the sperm membrane surface to form a protective layer, mitigating mechanical damage and osmotic stress during freezing. Furthermore, CMC nanoparticles can bind free radicals and release protective agents, enhancing overall antioxidant capacity. Appropriate concentrations of CMC can improve sperm motility parameters and acrosome integrity after thawing; however, excessively high concentrations may have side effects, and dosage optimization is necessary. Hyaluronic acid (HA), a natural high-molecular-weight polysaccharide, can mimic the cervical mucus environment of ewe cervical mucus, improving sperm osmotic tolerance during cooling and rewarming. HA molecules increase the viscosity of the solution at low temperatures, inhibiting ice crystal growth, while their antioxidant properties help scavenge free radicals. HA can also bind to receptors on the sperm membrane, stabilizing the membrane structure and providing the ability for sperm to bind to cumulus granules after thawing. Lactoferrin is a multifunctional globulin with highly efficient iron ion chelating ability and antibacterial and antioxidant effects. Adding lactoferrin to the dilution solution can capture free iron, block the Fenton reaction, reduce ROS generation and membrane lipid peroxidation catalyzed by iron, thereby preventing freeze-induced ferroptosis damage. Simultaneously, lactoferrin can inhibit bacterial growth and improve the biosafety of frozen sperm. Using genetically engineered recombinant sources ensures the absence of pathogen contamination and stable activity.

[0075] For example, taking the preparation of 100 mL of pretreatment solution for frozen sheep semen as an example, the concentration of CMC nanoparticles is 0.01-0.05 mM, preferably 0.02 mM, and the particle size is 50-150 nm; the concentration of HA is 0.05-0.2 mM, preferably 0.1 mM; and the concentration of recombinant human lactoferrin is 50-200 μg / mL, preferably 100 μg / mL.

[0076] The membrane stability and cell survival signaling regulation system comprises Poloxamer 188 (P188), sphingosine-1-phosphate (S1P), and melatonin, in a ratio of 500–1000:0.5–2:50–200, preferably 1000:1:100. P188 is a nonionic surfactant block copolymer that can insert and repair damaged sperm membranes during freezing / thawing. Appropriate amounts of P188 can reduce freeze-induced membrane rupture and enzyme leakage, allowing more sperm to maintain an intact membrane structure after thawing. Adding approximately 0.75–1 mM of P188 to the cryopreservation solution for ram epididymal sperm can improve total sperm viability and forward motility after thawing and help reduce glycerol usage. S1P is an endogenous sphingosine metabolite with a strong pro-cell survival signaling effect. Adding low concentrations of SIP can enhance sperm's tolerance to freezing by binding to SIP receptors on sperm cell membranes, activating survival pathways such as PI3K-Akt, and inhibiting the apoptosis cascade induced during freezing. SIP can also affect membrane lipid raft structure, enhancing membrane stability and reducing the damage to sperm motility caused by cold shock. Melatonin is a highly effective antioxidant molecule that can also regulate various cellular functions through receptor-mediated regulation. Adding melatonin to frozen sperm diluent can scavenge large amounts of ROS, reducing lipid peroxidation and DNA damage. Simultaneously, melatonin can improve mitochondrial function and upregulate the expression of anti-apoptotic proteins, thereby reducing apoptosis during freezing and promoting sperm motility recovery after thawing. Melatonin is inexpensive and highly safe, making it an ideal broad-spectrum protective agent.

[0077] For example, taking the preparation of 100 mL of pre-treatment solution for frozen semen protectant in mutton sheep as an example, the concentration of P188 is 0.5 to 1.0 mM, preferably 1.0 mM; the concentration of SIP is 0.5 to 2 μM, preferably about 1 μM; and the concentration of melatonin is 50 to 200 μM, preferably 100 μM.

[0078] The auxiliary system includes antibiotics and pH adjustment and balancing salts. The antibiotics include penicillin G and streptomycin, used to inhibit bacterial growth and prevent semen contamination during processing. Amphotericin B can also be added as needed to prevent fungal contamination. pH adjustment and balancing salts: Adjust the diluted solution to pH 7.0–7.4 using appropriate amounts of NaOH or HCl. Supplement with appropriate amounts of Na... + K + Cl - Plasma is used to maintain an isotonic (approximately 300 mOsm / kg) environment, with NaCl controlled at 0.6–0.7% and KCl controlled at 0.05–0.15%.

[0079] For example, taking the preparation of 100 mL of pre-treatment solution for frozen semen of mutton sheep as an example, the amount of penicillin G is 500 IU / mL, the concentration of streptomycin sulfate is 500 μg / mL, and optionally, amphotericin B is added at a concentration of 100 μg / mL as needed.

[0080] The sheep semen cryopreservation agent of this invention, through the synergistic interaction of the aforementioned eight systems, provides multi-layered protection for sperm cells. During the cooling and freezing stages, the basic buffer system and the energy and non-osmotic protection system maintain extracellular homeostasis and reduce ice crystal damage; the mitochondrial targeted protection system and the membrane stability and cell survival signal regulation system jointly mitigate oxidative stress and cell death; and the epigenetic regulation system and the bio-nanocomposite system ensure the integrity of sperm genetic material and functional structure. This multi-pillar system significantly improves the sperm's cryopreservation resistance.

[0081] This invention also discloses a method for preparing a cryopreservative for sheep semen, comprising the following steps:

[0082] S100: Prepare the basic buffer system solution;

[0083] S200: Energy and non-osmotic protection system are added to the basic buffer system solution to obtain the first preparative solution;

[0084] S300: Prepare a colloidal solution for the bio-nanocomposite system, then add the first preparative solution to obtain the second preparative solution;

[0085] S400: Add the epigenetic regulation system to the second preparative solution to obtain the third preparative solution;

[0086] S500: First, pre-dissolve each component of the membrane stability and cell survival signal regulation system, then add the third preparative solution to obtain the fourth preparative solution;

[0087] S600: First, a portion of the mitochondrial targeted protection system is added to the fourth preparative solution to obtain the fifth preparative solution; then, the remaining mitochondrial targeted protection system is prepared into a solution and added to the fifth preparative solution to obtain the sixth preparative solution.

[0088] S700: Add the sixth preparative solution to near the preset volume, add the components of the auxiliary system, adjust the pH to 7.0-7.4, and bring the volume to the preset volume to obtain the precursor solution of frozen semen protectant for mutton sheep;

[0089] S800: Prepare a penetrating cryoprotection system solution. When using the mutton frozen semen protectant, the mutton frozen semen protectant precursor solution and the penetrating cryoprotection system solution are mixed with fresh semen at a volume ratio of 1:1.

[0090] Adding components step-by-step in the above sequence helps each component dissolve and function effectively under suitable conditions. For example, first preparing a basic buffer system (S100) to establish a stable pH and ionic strength environment, then adding an energy source and non-osmotic protectant (S200) to provide energy and osmotic pressure protection, ensures the initial solution's stability and prevents subsequent additives from precipitating or becoming inactive. Subsequently, preparing a separate colloid of the bio-nanocomposite system in S300 and adding it ensures the nanomaterials are fully and uniformly dispersed, avoiding aggregation when directly mixed with large amounts of solute. Step-by-step addition of epigenetic regulators (S400) and pre-dissolved membrane stabilizers / cell survival regulators (S500) prevents these active molecules from prematurely contacting other components under adverse conditions, thus preventing interference or degradation. For example, P188 needs to be pre-dissolved at 37°C before slow addition to ensure complete dissolution and avoid precipitation caused by sudden cooling. Sequential operation ensures that the system conditions (temperature, pH, solvent) are optimized at each step, facilitating the efficacy of each component.

[0091] The eight protective systems are introduced sequentially according to the steps described above, each performing its specific function and working synergistically. For example, the basic buffer and non-permeable components first reduce ion imbalance and osmotic stress during freezing; bio-nanoparticles and epigenetic regulators protect sperm structure and gene integrity; then membrane stabilizers and survival signaling molecules alleviate cold shock and activate survival pathways; finally, mitochondrial-targeted antioxidants (such as MitoQ / SS-31 peptides) reduce ROS damage. This multi-pronged approach covers all aspects of freezing damage, achieving synergistic effects. This invention is the first to organically combine multiple novel components, optimizing concentrations to avoid toxic side effects while generating synergistic benefits. Its comprehensive protective effect is superior to any single solution. Adding them in the predetermined order ensures that each system performs its function without interference, ultimately improving the sperm cryopreservation survival rate.

[0092] The above sequence is not only chemically necessary in the preparation process, but also significantly contributes to the effectiveness of frozen semen. According to the invention, semen prepared using the formula of this invention exhibits significantly better motility and integrity indicators after thawing compared to traditional methods. The synergistic effect of the multi-pillar system results in a total sperm viability of over 80-85% after thawing, far exceeding the less than 50% level of traditional Tris-egg yolk diluent. This demonstrates that introducing each protective system step-by-step maximizes its protective effect and prevents any deficiency in any step from affecting the overall result.

[0093] In summary, preparing cryoprotectants according to the above steps has significant beneficial effects: ensuring that each component is fully dissolved and stable, exerts its optimal efficacy, and forms a synergistic protective mechanism, thereby improving the antifreeze properties of semen and the quality of sperm after thawing from multiple levels.

[0094] Specifically, in step S100, Tris base, HEPES, sodium citrate, and disodium EDTA are dissolved in ultrapure water to form a basic buffer system solution, and the pH is adjusted to 6.9–7.2. For example, to prepare 100 mL of a pretreatment solution for frozen sheep semen, 250 mM Tris base (approximately 3.03 g), 30 mM HEPES (approximately 0.71 g), 80 mM sodium citrate (approximately 2.35 g), and 0.5 mM EDTA-2Na (approximately 0.019 g) are dissolved in approximately 80 mL of ultrapure water to form a basic buffer system solution, and the pH is adjusted to 7.2.

[0095] In step S200, fructose, trehalose, and betaine are added to the basic buffer system solution to obtain the first preparative solution. For example, to prepare 100 mL of a preparative solution for frozen sheep semen, 60 mM fructose (approximately 1.08 g), 60 mM trehalose (approximately 2.05 g), and 30 mM betaine (approximately 0.36 g) are added to the basic buffer system solution and stirred until dissolved to obtain the first preparative solution.

[0096] In step S300, CMC nanoparticles are first prepared into a suspension. Hyaluronic acid and recombinant human lactoferrin are added to the suspension, and then the mixture is magnetically stirred at room temperature to fully disperse it and form a uniform colloidal solution of the bio-nanocomposite system. If necessary, ultrasonic treatment for several minutes can be performed to ensure uniform dispersion of the nanoparticles. For example, taking the preparation of 100 mL of preservative solution for frozen sheep semen as an example, 0.02 g of CMC nanoparticles are prepared into a suspension with a particle size of approximately 100 nm. 0.10 g of hyaluronic acid and 0.01 g of recombinant human lactoferrin are added to the suspension, and the mixture is magnetically stirred at room temperature for 1 hour to obtain a colloidal solution of the bio-nanocomposite system.

[0097] In step S400, sodium butyrate and EGCG are added to the second preparative solution after being sterilized by passing them through a filter membrane. The filter membrane has a pore size of 0.22 μm. For example, to prepare 100 mL of preparative solution for frozen sheep semen, 2 mM sodium butyrate (approximately 0.022 g) and 25 μM EGCG (approximately 0.009 g) are sterilized by passing them through a 0.22 μm filter membrane and then added to the second preparative solution, and gently mixed.

[0098] In step S500, melatonin and sphingosine-1-phosphate are pre-dissolved in small amounts (e.g., about 0.5 mL, including 0.5 mL) of sterile ethanol and water, respectively. P188 is pre-dissolved in a 37°C water bath. The pre-dissolved solutions of melatonin and sphingosine-1-phosphate are first added to the third preparative solution, and then the pre-dissolved solution of P188 is slowly added to the third preparative solution to obtain the fourth preparative solution. For example, taking the preparation of 100 mL of pre-treatment solution for frozen semen protectant in sheep as an example, the amount of melatonin used is 100 μM (about 0.0023 g), the amount of sphingosine-1-phosphate used is 1 μM (about 0.00068 g), and the amount of P188 used is 1 mM (about 0.0835 g).

[0099] In step S600, NR and cyclosporine are first added to the fourth preparatory solution and stirred in the dark to dissolve them completely, thus obtaining the fifth preparatory solution. Then, mitochondrial protectant (MitoQ or SS-31) is added to obtain the sixth preparatory solution. For example, taking the preparation of 100 mL of sheep frozen semen protectant precursor solution as an example, the amount of NR is 200 μM (about 0.006 g), the amount of cyclosporine is 0.2 μM (about 0.000024 g), and the mitochondrial protectant is MitoQ (100 nM). 10 μL of commercial MitoQ stock solution (typical concentration 1 mM ethanol solution) is transferred, pre-diluted with an appropriate amount (e.g., 1 mL) of double-distilled water or ultrapure water, added to the fifth preparatory solution, and quickly mixed to ensure uniform distribution to obtain the sixth preparatory solution.

[0100] In step S700, sterile ultrapure water is added to approximately the preset volume, and the pH is adjusted to the range of 7.0–7.4 (measured at 25°C). Then, penicillin and streptomycin are added, and the volume is brought to the preset volume. The mixture is then filtered to obtain a clear precursor solution for frozen sheep semen protectant. For example, to prepare 100 mL of the precursor solution, sterile ultrapure water is added to approximately 100 mL, the pH is adjusted to the range of 7.0–7.4 (measured at 25°C), and a mixture of penicillin G 500 IU / mL and streptomycin 500 μg / mL (0.5 mL of 100,000 IU / mL penicillin solution and 0.5 mL of 100 mg / mL streptomycin solution) is added. The volume is brought to 100 mL, and the mixture is filtered to obtain a clear precursor solution for frozen sheep semen protectant (using a 0.22 μm filter). The precursor solution of frozen semen protectant for mutton sheep can be stably stored for 2 weeks under 4C light-protected conditions. For long-term storage, it is advisable to repackage and freeze at -20℃ or freeze-dry.

[0101] In step S800, the sheep frozen semen protectant precursor solution needs to be added to a penetrating cryoprotection system solution to the working concentration before use, i.e., glycerol and DMSO are added to the working concentration. Because high concentrations of glycerol are toxic to sperm, a two-step dilution method is used, adding glycerol on-site to improve sperm tolerance. First, a glycerol-free sheep frozen semen protectant precursor solution is prepared. Separately, a penetrating cryoprotection system solution (containing glycerol and DMSO) with a high concentration of glycerol is prepared. The sheep frozen semen protectant precursor solution and the penetrating cryoprotection system solution are mixed with the semen in a 1:1 volume ratio. For example, taking the preparation of 200 mL of sheep frozen semen protectant as an example, the volume percentage of glycerol in the penetrating cryoprotection system solution is 14%, and the volume percentage of DMSO is 4%. After the sheep frozen semen protectant precursor solution is mixed with the semen in a 1:1 volume ratio with the penetrating cryoprotection system solution, the final volume percentage of glycerol is 7%, and the volume percentage concentration of DMSO is 2%.

[0102] This invention discloses an example of a method for preparing a cryoprotectant for frozen sheep semen. Taking the preparation of 200 mL of cryoprotectant for sheep as an example, the method includes the following steps:

[0103] S100: Dissolve 250 mM Tris base (approximately 3.03 g), 30 mM HEPES (approximately 0.71 g), 80 mM sodium citrate (approximately 2.35 g), and 0.5 mM EDTA-2Na (approximately 0.019 g) in approximately 80 mL of ultrapure water to form a basic buffer system solution, and adjust the pH to 7.2;

[0104] S200: Add 60mM fructose (approximately 1.08g), 60mM trehalose (approximately 2.05g), and 30mM betaine (approximately 0.36g) to the basic buffer system solution and stir to dissolve, thus obtaining the first preparative solution;

[0105] S300: 0.02g of carboxymethyl chitosan (CMC) nanoparticles (prepared as a suspension with a particle size of approximately 100nm), 0.10g of hyaluronic acid and 0.01g of recombinant human lactoferrin are added to the suspension. The mixture is magnetically stirred at room temperature for 1 hour to fully disperse and form a uniform colloidal solution. This colloidal solution is then added to the first preparative solution to obtain the second preparative solution. If necessary, ultrasonic treatment for several minutes can be performed to ensure uniform dispersion of the nanoparticles.

[0106] S400: After sterilizing sodium butyrate 2mM (approximately 0.022g) and EGCG 25μM (approximately 0.009g) through a 0.22μm filter membrane, add them to the second preparative solution and mix gently to obtain the third preparative solution;

[0107] S500: First, melatonin 100μM (approximately 0.0023g) and sphingosine-1-phosphate 1μM (approximately 0.00068g) are pre-dissolved in small amounts of sterile ethanol and water, respectively, and then added to the third preparative solution; then P188 1mM (approximately 0.0835g) is pre-dissolved in a 37°C water bath and then slowly added to the third preparative solution to obtain the fourth preparative solution;

[0108] S600: First, add 200 μM nicotinamide ribose (NR) (approximately 0.006 g) and 0.2 μM cyclosporine A (approximately 0.000024 g) to the fourth preparative solution and stir in the dark to ensure complete dissolution and obtain the fifth preparative solution; then, transfer 10 μL of commercial MitoQ stock solution (typical concentration 1 mM ethanol solution), pre-dilute it with 1 mL of double-distilled water or ultrapure water, add it to the fifth preparative solution and mix quickly to ensure uniform distribution and obtain the sixth preparative solution;

[0109] S700: Add sterile ultrapure water to the sixth preparatory solution to approximately 100 mL, adjust the pH of the solution to the range of 7.0–7.4 (measured at 25°C), add a mixture of penicillin G 500 IU / mL and streptomycin 500 μg / mL (0.5 mL of 100,000 units / mL penicillin solution and 0.5 mL of 100 mg / mL streptomycin solution), bring the volume to 100 mL, and filter sterilize (using a 0.22 μm filter) to obtain a clear precursor solution for frozen semen protectant in mutton sheep;

[0110] S800: Prepare a 100mL solution of penetrating cryoprotection system containing 14% glycerol and 4% DMSO by volume. When using the sheep frozen semen protectant, mix 100mL of the sheep frozen semen protectant precursor solution and 100mL of the penetrating cryoprotection system solution with the semen sequentially. The glycerol in the sheep frozen semen protectant has a volume percentage of 7% and the DMSO has a volume percentage of 2%.

[0111] This invention also discloses a method for applying a cryopreservative agent for sheep semen, which can be used for the cryopreservation of semen, specifically including the following steps:

[0112] A100: Semen collection;

[0113] A200: Dilute the semen with a precursor solution for frozen sheep semen preservation agent;

[0114] A300: Programmable cooling of diluted semen;

[0115] A400: After equilibration with a penetrating cryoprotection system solution, it is tube-sealed, frozen, and stored in liquid nitrogen.

[0116] In step A100, the collection of raw semen is as follows: Select healthy rams with high genetic value and collect semen using an artificial vagina method. Place the fresh ejaculate in a 37°C thermos and immediately assess density and motility. Sperm density is determined using a hemocytometer; the initial sperm concentration is generally 30 × 10⁻⁶. 8 / mL or higher. If the semen contains impurities or clots, it can be diluted 1:1 with isotonic sodium citrate solution and centrifuged at 800×g for 10 minutes to remove impurities. Then discard the supernatant and keep the sperm precipitate for later use.

[0117] In step A200, the precursor solution of the frozen semen preservative for mutton sheep is equilibrated in a 37°C water bath. The semen collected in A100 is then added dropwise, with a volume ratio of semen to the precursor solution of the frozen semen preservative for mutton sheep of 1:3 to 4. The initial sperm concentration is adjusted to achieve a final diluted sperm concentration of 4 × 10⁻⁶. 8 The ideal concentration is 1 / mL. Mix the diluent slowly and gently, avoiding violent shaking that could cause cold shock. Let the diluted semen stand at room temperature for 5–10 minutes to allow the osmotic pressure to gradually equalize.

[0118] In step A300, the diluted semen is gradually cooled to 5°C, preferably using a programmed cooler or in a refrigerator for staged cooling. For example, it can be cooled to 15°C at a rate of -0.25°C per minute and maintained for five minutes, then cooled to 5°C at a rate of -0.25°C per minute. The entire cooling process takes approximately 2 hours. Slow cooling allows the sperm membrane lipid phase transition to reach equilibrium, reducing cold shock damage. After reaching 5°C, the mixture is allowed to stand at 4–5°C for 30 minutes to equilibrate.

[0119] In step A400, an equal volume of the pre-concentration cryoprotectant solution for frozen sheep semen is slowly added to the diluted and cooled semen. The addition is done in stages, drop by drop, with each addition being 1 / 4 of the total volume (i.e., added evenly in four separate additions), with 10-minute intervals between each addition. This gradual addition of glycerol allows the sperm to gradually adapt to the hypertonic environment, avoiding osmotic shock caused by sudden exposure to high glycerol levels. After the cryoprotectant solution is added, the mixture is the final cryo-diluted semen. The semen is then allowed to equilibrate at 5°C for 30–60 minutes to allow glycerol to fully penetrate the sperm cells and for the various protective systems to function effectively. During this period, the test tube is inverted several times to maintain a homogeneous sperm suspension. The equilibrated semen sample is then transferred to 0.25 mL French caps (the small inner diameter of the pipette facilitates heat exchange) using a sterilized pipette, with each cap filled with 0.25 mL of semen (containing approximately 50 million sperm). Polyvinyl alcohol powder or heat sealing can be used to ensure a good seal on the caps. Label the capillary tubes with their numbers and ram information. Freezing process: Arrange the capillary tubes containing semen neatly on a 5°C pre-cooled liquid nitrogen vapor phase cryopreservation rack, and freeze them using either liquid nitrogen vapor phase freezing or a programmed cooling system. Preferred method: Suspend the capillary tubes approximately 4cm above the liquid nitrogen surface and pre-freeze them for 7 minutes in a liquid nitrogen vapor phase environment at approximately -120°C to rapidly lower the semen temperature to below -100°C. Immediately afterwards, immerse the capillary tubes completely in liquid nitrogen to achieve ultra-low temperature storage of the semen. The temperature gradient is uniform throughout the freezing process, requiring no additional manual intervention. If a programmable cooling system is available, it can be set to cool from 5°C to -80°C at a rate of -20°C per minute before direct immersion in liquid nitrogen. Both methods yield frozen semen with high survival rates. After freezing, quickly transfer the capillary tubes to a liquid nitrogen tank for long-term storage, maintaining a liquid nitrogen temperature of -196°C. To ensure quality, one or two samples can be taken after freezing for 24 hours for thawing and testing to verify whether the quality of the frozen semen batch meets the expected standards.

[0120] When using semen treated with the frozen semen preservative of this invention, a cryopreservation process is first performed. Specifically, the frozen capillary tube is removed from liquid nitrogen and quickly placed in a 37°C constant temperature water bath with shaking for 30 seconds until the ice is completely melted. If faster thawing is required, a 7-second thawing in a 50°C water bath can also be used, but the time must be strictly controlled to prevent overheating. Immediately after thawing, wipe off surface water droplets with a clean, dry cotton towel, cut open the capillary tube, and transfer the semen into a pre-warmed centrifuge tube at 37°C. Post-thawing processing and evaluation: Take an appropriate amount of thawed semen to assess motility and integrity. Specifically, measure sperm motility parameters using a computer-aided sperm analysis system (CASA), including total motility rate, forward motility rate, and various motility trajectories; use the HOS test to detect sperm membrane functional integrity rate; use fluorescence staining (PI / FITC-PSA double staining) to assess acrosome integrity rate and early apoptosis rate; and use acidic Aniline Blue or SCSA methods to detect chromatin integrity. According to the present invention, the frozen semen exhibits a sperm forward motility rate >70%, total viability ≥80%, membrane integrity ≥80%, acrosome integrity >85%, mitochondrial activity (proportion of high membrane potential sperm) >60%, and significantly lower early apoptosis and mortality rates compared to the control. For example, compared to conventional Tris-egg yolk glycerol dilution, the proportion of forward motility sperm in the frozen semen of the present invention increased by approximately 20 percentage points, demonstrating a statistically significant difference (P<0.01).

[0121] After thawing, the semen can be directly used for artificial insemination. Under the cryoprotection of this invention, each 0.25 mL thin tube of semen contains approximately 50 million live sperm, and due to the improved quality, it meets the requirements for deep vaginal insemination or intrauterine insemination. In field application, the thawed semen is drawn into an insemination gun and inseminated 12 hours after the ewe comes into estrus. Field trial results show that the conception rate using the cryoseed of this invention can reach over 75%, significantly higher than the control group using traditional cryoseed (approximately 55%). This confirms the superiority of the formula of this invention in actual production.

[0122] The table below compares the quality parameters of frozen semen with the multi-mechanism synergistic protectant of this invention, the traditional Tris-egg yolk diluent, and the commercial modified diluent (whose main components include the Tris buffer system and glycerol, and are modified with cholesterol-loaded cyclodextrin, resveratrol, nano-selenium, and apoptosis inhibitors). Each group has 10 samples, and the data are expressed as averages. The results show that the protectant of this invention increases the forward sperm motility rate by about 20 percentage points compared with the traditional Tris-egg yolk diluent (P < 0.01). The protectant of this invention is significantly superior to existing products in all key indicators (P < 0.01).

[0123]

[0124]

[0125] Semen samples from the same batch of rams were cryopreserved using the preservative of this invention and a commercial preservative (e.g., a modified Tris diluent containing cholesterol cyclodextrin and antioxidants). Sperm quality after thawing was compared. Results showed that the total sperm viability (82.4%) and progressive motility (72.5%) in the invention group were significantly higher than those in the commercial preservative group (60.3% and 45.8%, respectively, P<0.01); the acrosome integrity rate (86.7%) and membrane integrity rate (81.0%) in the invention group were also superior to those in the control group (70.2% and 62.5%, respectively); kinematic parameters such as sperm mean path velocity (VAP) and linear velocity (VSL) increased by approximately 15–20%; sperm ROS levels (measured by DCFH-DA method) decreased by approximately 30%, and MDA product concentration decreased by more than 25%; the proportion of apoptotic positive sperm decreased by approximately 40%. These data are consistent with or better than those reported in the literature regarding the effects of single additives, such as resveratrol improving motility and mitochondrial function, nano-selenium enhancing integrity, and Fer-1 reducing ROS. This invention achieves a more comprehensive protective effect through the cumulative effect of multiple mechanisms. In actual breeding, the fertilization rate and lambing rate of the frozen semen in this invention group were also significantly higher than those in the control group. Therefore, this invention represents a qualitative leap in sperm cryopreservation compared to existing technologies and has significant application value.

[0126] Compared with the prior art, the mutton semen cryopreservative of the present invention also includes at least the following beneficial effects:

[0127] (1) Multiple protective mechanisms to comprehensively improve sperm quality: This invention integrates multiple mechanisms such as membrane stabilization, ice crystal inhibition, anti-oxidation, anti-apoptosis / anti-ferroptosis, mitochondrial function maintenance, and epigenetic protection. It provides three-dimensional protection for sperm throughout the freezing process, overcoming the shortcomings of traditional cryopreservatives that focus on only one aspect. For example, it repairs / strengthens cell membranes through P188 and carboxymethyl chitosan nanoparticles, protects mitochondrial energy supply and clears ROS through MitoQ / SS-31 and melatonin, and inhibits multiple cell death pathways using Z-VAD substitutes (such as endogenous signaling SIP) and lactoferrin. Therefore, all key indicators of sperm during cryopreservation are optimized.

[0128] (2) Innovative Components and Synergistic Effects: While drawing on existing effective ingredients, this invention organically introduces a series of innovative components to achieve synergistic effects. For example, carboxymethyl chitosan nanoparticles and SIP signaling molecules are applied to semen cryopreservation for the first time, leveraging their membrane protection and survival-promoting advantages; SS-31 mitochondrial antioxidant peptide is introduced to synergize with classic antioxidants; nicotinamide ribose is used to increase sperm NAD+ levels in conjunction with sodium butyrate to maintain epigenetic stability. The concentrations of each component have been optimized to produce synergistic benefits while avoiding toxic side effects and overlapping component functions. This novel "combination punch" formulation is different from any existing single patent solution, possessing uniqueness and advancement;

[0129] (3) Significantly improves sperm motility and integrity after freeze-thaw: The semen from mutton treated with this invention exhibits quality indicators far superior to existing products after thawing. The expected total sperm viability after thawing is over 80-85%, with the proportion of progressively motile sperm remaining stable at over 70%, significantly higher than the typically less than 50% level of traditional Tris-egg yolk diluent. Sperm membrane integrity exceeds 80%, acrosome integrity is no less than 85%, and DNA fragmentation is controlled below 5% (DNA integrity approximately 95%), all superior to commercially available high-end cryopreservation solutions. Adding key components significantly improves sperm survival rate and functional parameters after freezing. For example, 1 μg / mL nano-selenium significantly improves sperm survival rate and progressive motility after thawing; 50 μM resveratrol effectively reduces acrosome damage and membrane rupture caused by freezing; simultaneously, the combination of multiple antioxidants lowers ROS levels and significantly reduces the content of malondialdehyde (MDA), a lipid peroxidation product.

[0130] (4) Improved fertilization capacity and reproductive efficiency: This invention not only improves sperm quality indicators in vitro but also focuses on preserving sperm function. Improved acrosome and membrane integrity enhance sperm binding to and penetration into oocytes; good mitochondrial function ensures capacitation motility and sperm survival time in the female reproductive tract. Frozen sperm treated with this invention can achieve an artificial insemination success rate of over 75-80%, significantly higher than the approximately 50-60% success rate of conventional frozen sperm. Sperm performance in in vitro fertilization (IVF) will also be improved, with significantly increased fertilization and embryo development rates. This will bring higher reproductive efficiency and economic benefits to sheep farms.

[0131] (5) Simple and scalable process: This invention does not contain animal-derived ingredients (such as egg yolk, milk, etc.), and all components are chemically synthesized or recombinant, ensuring batch stability and high biosafety. The diluent can be formulated into a concentrated solution or lyophilized powder for easy storage and transportation. The semen processing procedure is compatible with conventional frozen semen processes (two-step dilution, programmed cooling, and liquid nitrogen freezing), requiring no special equipment and easy to implement in production. Furthermore, because this formula enhances the tolerance of thawed sperm, it can maintain sperm activity under a wider range of thawing conditions, making it more user-friendly.

[0132] In summary, this invention, through an innovative multi-target synergistic formulation, significantly improves the thawing quality and fertilization effect of frozen sheep semen, addresses the weaknesses of existing technologies, and has significant practical value and promising prospects for promotion.

[0133] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A preservative for frozen semen of mutton sheep, characterized in that, This includes a multi-pillar collaborative protection system, which comprises: The basic buffer system is configured to maintain the liquid environment at a suitable pH and ionic strength in order to stabilize the extracellular environment; An energy and non-osmotic protection system is configured to provide energy substrates during freezing and protect cells from osmotic stress. The penetrating cryoprotection system is configured to allow moisture to penetrate into sperm cells, lowering the freezing point to prevent intracellular ice crystal formation. The mitochondrial targeted protection system is configured to maintain sperm mitochondrial function, enhance ATP function, and reduce the production of mitochondrial-related ROS. The epigenetic regulatory system is configured to mitigate the damage to sperm epigenetic markers caused by freezing and protect sperm DNA and chromatin structure. The bio-nanocomposite system is configured to utilize nanomaterials and biomacromolecules to synergistically protect sperm structure and release functional factors. The membrane stability and cell survival signaling regulation system is configured to maintain sperm plasma membrane integrity, reduce cold shock damage, and activate cell survival pathways. The auxiliary system is configured to ensure the microbial safety of the diluent and maintain the environment required for normal sperm activity.

2. The sheep semen cryopreservative according to claim 1, characterized in that, The basic buffer system comprises trihydroxyaminomethane, 4-hydroxyethylpiperazine ethanesulfonic acid, sodium citrate, and disodium ethylenediaminetetraacetate, in a ratio of 200–300: 20–40: 60–100: 0.3–0.

8.

3. The sheep semen cryopreservative according to claim 1, characterized in that, The energy and non-permeable protection system includes fructose, trehalose and betaine, in a ratio of 40-80:40-80:20-50.

4. The sheep semen cryopreservative according to claim 1, characterized in that, The penetrating cryoprotection system comprises glycerol and dimethyl sulfoxide in a ratio of 6–10:1–3.

5. The sheep semen cryopreservative according to claim 1, characterized in that, The mitochondrial targeted protection system includes a mitochondrial protectant, cyclosporine A, and nicotinamide ribose, wherein the mitochondrial protectant is one of MitoQ or SS-31 tetrapeptide. When the mitochondrial protectant is MitoQ, the ratio of MitoQ, cyclosporine A, and nicotinamide ribose is 0.05–0.15: 0.1–0.5: 100–300, respectively. When the mitochondrial protectant is SS-31 tetrapeptide, the ratio of SS-31 tetrapeptide, cyclosporine A and nicotinamide ribose is 6-12:0.1-0.5:100-300.

6. The sheep semen cryopreservative according to claim 1, characterized in that, The epigenetic regulatory system comprises sodium butyrate and epigallocatechin gallate in a ratio of 1000–5000:10–50.

7. The sheep semen cryopreservative according to claim 1, characterized in that, The bio-nanocomposite system includes water-soluble chitosan derivative nanoparticles, hyaluronic acid, and recombinant human lactoferrin. The auxiliary system includes antibiotics and pH-regulating and balancing salts, wherein the antibiotics include penicillin G and streptomycin.

8. A method for preparing a preservative for frozen sheep semen, characterized in that, It can be used at least to formulate a frozen semen protectant for mutton sheep as described in any one of claims 1 to 7, comprising the following steps: S100: Prepare the basic buffer system solution; S200: Energy and non-osmotic protection system are added to the basic buffer system solution to obtain the first preparative solution; S300: Prepare a colloidal solution for the bio-nanocomposite system, then add the first preparative solution to obtain the second preparative solution; S400: Add the epigenetic regulation system to the second preparative solution to obtain the third preparative solution; S500: First, pre-dissolve each component of the membrane stability and cell survival signal regulation system, then add the third preparative solution to obtain the fourth preparative solution; S600: First, a portion of the mitochondrial targeted protection system is added to the fourth preparative solution to obtain the fifth preparative solution; then, the remaining mitochondrial targeted protection system is prepared into a solution and added to the fifth preparative solution to obtain the sixth preparative solution. S700: Add the sixth preparative solution to near the preset volume, add the components of the auxiliary system, adjust the pH to 7.0-7.4, and bring the volume to the preset volume to obtain the precursor solution of frozen semen protectant for mutton sheep; S800: Prepare a penetrating cryoprotection system solution. When using the mutton sheep frozen semen protectant, the mutton sheep frozen semen protectant precursor solution and the penetrating cryoprotection system solution are mixed with the semen sequentially at a volume ratio of 1:

1.

9. The preparation method according to claim 8, characterized in that, Includes the following steps: S100: Dissolve 3.03g Tris base, 0.71g HEPES, 2.35g sodium citrate, and 0.019g EDTA-2Na in approximately 80mL of ultrapure water to form a basic buffer system solution, and adjust the pH to 7.

2. S200: Add 1.08g of fructose, 2.05g of trehalose, and 0.36g of betaine to the basic buffer system solution and stir to dissolve, to obtain the first preparative solution; S300: Prepare a suspension with a particle size of 100nm by 0.02g of carboxymethyl chitosan nanoparticles, add 0.10g of hyaluronic acid and 0.01g of recombinant human lactoferrin to the suspension, and stir magnetically for 1 hour at room temperature to fully disperse it into a uniform colloidal solution. Then add the colloidal solution to the first preparative solution to obtain the second preparative solution. S400: After sterilizing sodium butyrate 0.022g and EGCG 0.009g through a 0.22μm filter membrane, add them to the second preparative solution and mix gently to obtain the third preparative solution; S500: First, pre-dissolve 0.0023g of melatonin and 0.00068g of sphingosine-1-phosphate in a small amount of sterile ethanol and water respectively, and then add them to the third preparative solution; then pre-dissolve 0.0835g of P188 in a 37℃ water bath and slowly add it to the third preparative solution to obtain the fourth preparative solution; S600: First, add 0.006g of nicotinamide ribose and 0.000024g of cyclosporine A to the fourth preparative solution and stir in the dark to fully dissolve them to obtain the fifth preparative solution; then, transfer 10μL of commercial MitoQ stock solution, pre-dilute it with double-distilled water or ultrapure water, add it to the fifth preparative solution and mix quickly to ensure uniform distribution to obtain the sixth preparative solution; S700: Add sterile ultrapure water to the sixth preparative solution to approximately 100 mL, adjust the pH of the solution to the range of 7.0 to 7.4, add a mixture of penicillin G 500 IU / mL and streptomycin 500 μg / mL antibiotics, bring the volume to 100 mL, filter to sterilize and obtain a clear precursor solution for frozen semen protectant in mutton sheep. S800: Prepare 100 mL of a penetrating cryoprotection system solution containing 14% glycerol and 4% DMSO by volume. When using the sheep frozen semen protectant, mix 100 mL of the sheep frozen semen protectant precursor solution and 100 mL of the penetrating cryoprotection system solution with the semen sequentially.

10. A method for applying a preservative to frozen semen of mutton sheep, characterized in that, The mutton semen cryopreservative according to any one of claims 1 to 7 or the mutton semen cryopreservative prepared according to claim 8 or 9 can be used for semen cryopreservation, comprising the following steps: A100: Semen collection; A200: Dilute the semen with a precursor solution for frozen sheep semen preservation agent; A300: Programmable cooling of diluted semen; A400: After equilibration with a penetrating cryoprotection system solution, it is tube-sealed, frozen, and stored in liquid nitrogen; The precursor solution for frozen semen protectant in mutton sheep includes a basic buffer system, an energy and non-osmotic protection system, a mitochondrial targeted protection system, an epigenetic regulation system, a bio-nanocomposite system, a membrane stability and cell survival signal regulation system, and an auxiliary system.

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