Sheep semen freezing diluent, its preparation method and application

Abstract

The application discloses a water-containing silybin sheep semen freezing diluent and a preparation method and application thereof, and belongs to the technical field of animal reproduction. The sheep semen freezing diluent provided by the application contains silybin as an antioxidant functional additive, can effectively remove excessive active oxygen generated in the process of semen freezing and thawing, reduce lipid peroxidation reaction, protect sperm plasma membrane and DNA integrity, and significantly improve the survival rate and quality of thawed sperm. The application further provides a preparation method of the freezing diluent and application of the freezing diluent in sheep semen cryopreservation.

Description

Technical Field

[0001] This invention belongs to the field of livestock semen cryopreservation technology, specifically relating to a sheep semen cryopreservation diluent, its preparation method, and its application. Background Technology

[0002] Xinjiang, as one of my country's four major livestock producing areas, boasts unique natural resources and abundant local sheep breeds, making sheep farming one of the region's most advantageous and distinctive traditional industries. To fully utilize the genetic potential of superior rams, accelerate sheep breed improvement, and enhance reproductive efficiency, artificial insemination technology has been widely promoted and applied in modern sheep farming due to its advantages such as accelerating genetic progress, significantly reducing ram feeding costs, and effectively preventing the spread of reproductive tract diseases.

[0003] However, the practical effectiveness of artificial insemination technology largely depends on the quality and storage time of the semen. The length of time semen can maintain its normal fertilization capacity has become a key bottleneck limiting the widespread adoption and efficient utilization of artificial insemination. Currently, artificial insemination of sheep in my country mainly uses fresh semen, but the effective survival time of fresh semen under a 37°C water bath is usually no more than 30 minutes. This characteristic greatly limits the application radius and scope of artificial insemination, especially in Xinjiang, where the vast territory and scattered pastoral areas present significant challenges to timely delivery and use of semen. Therefore, extending the time semen retains its fertilization capacity is a crucial step in promoting the widespread application of artificial insemination technology in pastoral areas.

[0004] Semen cryopreservation, a core biotechnology enabling long-term and even ultra-long-term preservation of semen, involves storing semen in an ultra-low temperature environment (liquid nitrogen, -196℃). The complete technical process includes key steps such as quality inspection after semen collection, preparation and isothermal dilution of the diluent, temperature equilibration, filling and dispensing, and programmed freezing, ultimately achieving long-term stable preservation of the semen in liquid nitrogen. Numerous studies have shown that ultra-low temperature environments reduce sperm metabolic activity to extremely low levels, bringing sperm life activities to a near standstill. After scientifically and rationally thawing, sperm activity can be effectively restored. This technology not only overcomes the time and space limitations of semen preservation, making the cross-regional and cross-seasonal use of superior ram semen possible, but also significantly improves the utilization efficiency of superior rams, providing strong technical support for the construction of modern livestock breeding systems and breed genetic improvement.

[0005] However, semen cryopreservation technology still faces many challenges in practical applications. During the complete freezing and thawing process, sperm cells are subjected to a series of intense physicochemical stressors, including dilution stress, sudden temperature drops, drastic changes in osmotic pressure, ice crystal formation and recrystallization, and warming upon thawing. This complex stress process leads to the massive production and rapid accumulation of reactive oxygen species (ROS) within sperm cells. When the rate of ROS production exceeds the clearance capacity of the cell's own antioxidant defense system, severe oxidative stress occurs. Sheep sperm membranes are rich in long-chain polyunsaturated fatty acids, which are crucial for maintaining sperm membrane fluidity and functional integrity, but are also highly sensitive to ROS attacks. Excessive ROS accumulation attacks the polyunsaturated fatty acids on the sperm membrane, triggering a severe lipid peroxidation cascade reaction. This directly damages the integrity and selective permeability of the sperm plasma membrane and acrosome membrane, leading to serious consequences such as abnormal acrosome shedding and loss of sperm motility. Simultaneously, ROS also attack the mitochondrial structure of sperm, causing mitochondrial membrane potential collapse and severe impairment of ATP synthesis, leading to sperm motility loss due to energy deficiency. More seriously, ROS can penetrate the sperm cell nucleus, attacking the bases and deoxyribose backbone of nuclear DNA, causing DNA strand breaks and fragmentation, directly affecting the fertilization process and normal early embryonic development. These cellular and molecular-level oxidative damages manifest macroscopically as a sharp decline in sperm viability after thawing, a significant reduction in linear motility, a marked decrease in acrosome and plasma membrane integrity, and an abnormally high rate of sperm abnormalities—a series of quality problems that ultimately result in unsatisfactory conception rates in ewes during artificial insemination, severely hindering the widespread application of frozen sheep semen technology in the vast pastoral areas of Xinjiang.

[0006] To effectively address the common technical challenge of oxidative stress damage during cryopreservation, adding exogenous high-efficiency antioxidants to semen cryopreservation diluents is currently recognized as one of the most direct, effective, and cost-effective strategies in domestic and international research. At present, in the field of sheep semen cryopreservation, commonly used diluents mainly fall into two categories: one is diluents formulated by individual laboratories according to classic recipes, typically based on a tris(hydroxymethyl)aminomethane-citric acid-fructose buffer system, with added egg yolk and glycerol as cryoprotectants; the other is commercially available pre-packaged diluents. These products are manufactured and quality-controlled by professional biotechnology companies according to standardized formulas, offering significant advantages such as stable composition, minimal batch-to-batch variation, ready-to-use, and convenient operation.

[0007] Minitüb diluents (e.g., product codes 13512 / 0001 or 13512 / 0010) are commercially available sheep semen cryopreservation diluents manufactured by Minitüb GmbH, Germany, and widely used internationally. Their formulation has undergone long-term optimization and extensive practical verification, demonstrating reliable effectiveness in maintaining osmotic pressure, buffering pH, providing nutrients, and offering basic cryoprotection. However, conventional commercial diluents, including Minitüb, still share the common problem of insufficient antioxidant protection against the severe oxidative stress that occurs during freezing. Scientifically adding exogenous high-efficiency antioxidants to the finished diluent not only compensates for this deficiency in antioxidant function but also simplifies the process, eliminating the need for individual preparation from raw materials. This significantly reduces the technical difficulty and human error associated with the process, making it highly suitable for widespread application in grassroots production units.

[0008] Dolan sheep is a well-known local breed of dual-purpose (meat and fat) sheep in my country, mainly produced in Maigaiti County and its surrounding areas on the northwestern edge of the Tarim Basin in Xinjiang. It is characterized by its large size, rapid growth and development, outstanding meat performance, and strong adaptability to desert and semi-desert ecological environments, making it one of the main sheep breeds raised by farmers and herders in southern Xinjiang. However, because Dolan sheep have a very high fat deposition capacity, their sperm plasma membrane has a relatively high content of polyunsaturated fatty acids. Compared with other lean-type sheep breeds, their sperm is more sensitive to oxidative stress damage during freezing, resulting in low sperm motility and low conception rates after freezing. Therefore, there is an urgent practical need to develop a special diluent formula for freezing sheep semen, based on commercially available diluents and supplemented with highly effective antioxidant protective components, specifically tailored to the characteristics of the Dolan sheep breed. Summary of the Invention

[0009] In view of this, the present invention provides a sheep semen cryopreservation diluent containing silybin. This diluent is based on commercially available Minito diluent, with the addition of high-purity silybin co-soluble with dimethyl sulfoxide. Utilizing the multi-target synergistic antioxidant protection mechanism of silybin, it compensates for the deficiencies in antioxidant function of the commercially available diluent, effectively reducing oxidative stress damage during the freezing-thawing process, thereby significantly improving the overall quality of the thawed sperm.

[0010] To achieve the above objectives, the present invention provides the following technical solution: The present invention provides a sheep semen cryo-diluent, characterized in that the sheep semen cryo-diluent comprises a base diluent and silymarin.

[0011] Silymarin is the core antioxidant active ingredient in this invention. Silymarin exerts its sperm cryopreservation protective effect through the following multi-target synergistic mechanisms: First, the phenolic hydroxyl groups in its molecular structure can directly and efficiently scavenge excess reactive oxygen species generated in sperm cells during the freezing-thawing process, blocking the lipid peroxidation chain reaction at its source; Second, silymarin can effectively stabilize mitochondrial membrane potential and maintain ATP synthesis efficiency; Third, silymarin can inhibit the oxidative stress-induced mitochondrial apoptosis pathway, reducing the sperm apoptosis rate. Systematic concentration gradient screening experiments confirmed that a silymarin concentration of 15 μM provided the best protective effect.

[0012] Preferably, the sheep semen cryopreservation diluent is a commercially available sheep semen cryopreservation base diluent.

[0013] As a preferred option, the commercially available base diluent for cryopreservation of sheep semen is the ready-made Minito diluent.

[0014] The finished Minito diluent serves as the base diluent for this invention. This product is a commercially available sheep semen cryopreservation diluent that has undergone long-term optimization and extensive practical verification. Its formula is scientifically sound, containing salts and sugars to maintain suitable osmotic pressure, a buffer system to stabilize pH, fermentable sugar substrates to provide metabolic energy for sperm, and basic cryopreservation components such as egg yolk and glycerol. The finished Minito diluent meets the basic requirements for semen cryopreservation, is convenient and quick to use, and maintains consistent quality between batches. It is an ideal carrier for silymarin, the antioxidant active ingredient of this invention.

[0015] Furthermore, the finished Minitu diluent is a product of Minitüb, catalog number 13512 / 0001 or 13512 / 0010. Its formulation has been professionally optimized, exhibiting reliable performance in osmotic pressure maintenance, pH buffering, nutrient substrate supply, and basic cryoprotection. Silymarin is one of the main active ingredients in milk thistle extract, with CAS number 22888-70-6, a molecular weight of 482.44 g / mol, and a purity of ≥98% by HPLC.

[0016] Preferably, the concentration of silymarin in the sheep semen cryo-diluent is 5-20 µM; more preferably, the concentration of silymarin is 15 µM.

[0017] Preferably, the silymarin is added after being solubilized with dimethyl sulfoxide.

[0018] Dimethyl sulfoxide (DMSO) is used as a co-solvent for silybin. Silybin has very low solubility in water and is difficult to dissolve directly in aqueous Minito base working solution. DMSO is a widely used, low-toxicity organic solvent that can efficiently dissolve silybin, forming a homogeneous and stable solution that is easy to measure accurately and disperse uniformly in the diluent. In this invention, the volume percentage of DMSO in the final cryopreservation diluent is extremely low (not exceeding 0.1%, i.e., less than one-thousandth). This volume concentration is far below the threshold concentration at which DMSO causes cytotoxicity to sperm reported in the literature. At this concentration, DMSO itself will not have a detectable negative impact on sperm survival and function.

[0019] Adding the working solution using a stock solution method ensures the consistency and repeatability of the working solution concentration for each experiment, while avoiding errors and waste caused by repeated weighing.

[0020] The present invention also provides a method for preparing the above-mentioned frozen dilution solution of sheep semen, characterized by comprising the following preparation steps: S1. Prepare the base diluent into a base diluent working solution and refrigerate for later use; S2. Silybin was prepared into a 20 mM silybin stock solution using dimethyl sulfoxide; S3. Weigh the Minito base working solution and silymarin stock solution according to the final concentration, mix them well to obtain sheep semen cryo-diluted solution.

[0021] The present invention also provides applications of the above-mentioned sheep semen cryo-diluent in the following aspects: To improve sperm motility after freezing and thawing sheep semen; To improve the acrosome integrity rate of sheep semen after freezing and thawing; To improve the sperm plasma membrane integrity rate after freezing and thawing of sheep semen; Extend the effective survival time of sheep semen after freezing and thawing; Reduce the sperm abnormality rate after freezing and thawing sheep semen.

[0022] Preferably, the method for preserving sheep semen with the sheep semen cryo-diluent includes the following steps: 1) Semen collection and quality inspection: Fresh semen was collected from Dolan sheep rams using the artificial vagina method. After collection, the semen was quickly placed in a 37°C constant temperature water bath for insulation. Under the constant temperature of 37°C, routine quality inspection of the semen was carried out. Semen samples with sperm motility greater than 0.7 and sperm density greater than 1.5 billion / mL were selected. To eliminate individual differences, the qualified semen samples from multiple rams were mixed in equal volumes at 37°C to form a mixed semen sample. 2) Dilution and equilibration: Mix the mixed semen obtained in step 1) with sheep semen cryo-diluent at a volume ratio of 1:5 to 1:10 under a 37°C water bath and gently shake to mix. After dilution, place the diluted semen in a 4°C refrigerator for low-temperature equilibration treatment for 3 to 5 hours, gently shaking it once every 30 minutes to keep the sperm in suspension. 3) Filling: Place the 0.25 mL plastic capillary tubes and filling machine in a constant temperature environment of 4℃ for full pre-cooling. Then, fill the semen after the equilibrium in step 2) into the 0.25 mL plastic capillary tubes at 4℃. After filling, seal the tubes and neatly stack them on the freezer rack. 4) Programmed freezing: Turn on the programmed freezing machine and stabilize the freezing chamber temperature at 4℃; quickly place the freezing rack with the thin tubes arranged in step 3) into the freezing chamber and start the freezing program; the freezing program is as follows: cool down from 4℃ to -10℃ at a cooling rate of 4℃ / min, and then cool down from -10℃ to -100℃ at a cooling rate of 40℃ / min; after freezing to -100℃, quickly immerse the entire freezing rack into liquid nitrogen, ensuring that the liquid nitrogen completely submerges all the thin tubes, thus completing the cryopreservation of sheep semen.

[0023] Preferably, in step 2), the volume ratio of the mixed semen to the silymarin cryo-diluted solution is 1:8; and the low-temperature equilibration treatment time is 4 hours.

[0024] This invention also provides a method for thawing frozen sheep semen, comprising the following steps: removing the frozen semen tube from liquid nitrogen, immediately immersing it in a 37°C constant temperature water bath, and gently shaking it continuously for 30 seconds to thaw; after removing it, wiping the surface of the tube dry with sterile absorbent paper, cutting open the sealed end of the tube, collecting the semen into a centrifuge tube preheated to 37°C, and gently shaking it to mix, thus completing the thawing process.

[0025] It contains at least the following beneficial technical effects: This invention marks the first application of silymarin as an antioxidant additive in sheep semen cryopreservation diluent. Silymarin effectively reduces the effects of cryopreservation through multiple mechanisms, including scavenging reactive oxygen species and protecting sperm membrane and DNA integrity. The thawing process causes oxidative stress damage to sheep sperm. Studies have shown that silymarin can significantly improve sperm viability and mitochondrial membrane potential, while reducing sperm lipid peroxidation, DNA breakage, and apoptosis; it can increase superoxide dismutase (SOD) activity and total antioxidant capacity (TAC), and reduce malondialdehyde (MDA) levels; it can also regulate the expression of apoptosis-related genes, reducing sperm apoptosis.

[0026] The silymarin-containing cryodiluent for sheep semen provided by this invention can significantly improve the total motility, progressive motility, survival rate, plasma membrane integrity, and DNA integrity of thawed sheep sperm, effectively improving the quality of frozen sheep semen and providing a new technical solution for the commercial production of frozen sheep semen. The preparation method of this invention is simple, cost-controllable, and suitable for industrial production and widespread application. Detailed Implementation

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0030] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0031] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0032] Unless otherwise specified, "room temperature" and "normal temperature" in this invention refer to 25±2℃.

[0033] Unless otherwise specified, all raw materials or instruments used in the following embodiments of the present invention are commercially available.

[0034] Example 1: Preparation of cryo-diluted sheep semen containing silymarin This embodiment provides a specific process for preparing frozen diluents of sheep semen with four concentration gradients of silymarin at 5, 10, 15, and 20 μM, based on commercially available Minitu diluent.

[0035] 1. Preparation of finished Minito base working fluid: Commercially available Minitu diluent (manufactured by Minitüb GmbH, Germany; product code can be found in the specific product catalog) is a concentrated solution, with each bottle capable of preparing 1000 mL of working solution. Prepare according to the product instructions: Take one bottle of Minitu concentrate and place it in a 20 mL glass beaker. Accurately measure approximately 80 mL of sterile double-distilled water (preheated to 35°C to accelerate dissolution) using a graduated cylinder and add it to the beaker. Place a clean magnetic stir bar in the beaker and place it on the magnetic stirrer. Stir at a medium speed (400 rpm) at room temperature for 25 minutes until the solution is a homogeneous and fine emulsion with no visible layering. Transfer the solution from the beaker to a 100 mL volumetric flask. Rinse the beaker walls with a small amount of sterile double-distilled water and transfer the rinse water to the volumetric flask. Stopper the flask and invert it repeatedly 15 times to obtain the basic Minitu working solution. Transfer the working solution to a sterile glass reagent bottle, seal it, and pre-cool it in a 4°C refrigerator. Use it within 48 hours of preparation.

[0036] 2. Preparation of silymarin stock solution: Accurately weigh 9.65 mg of silybin standard (CAS No. 22888-70-6, HPLC ≥ 98%) and place it in a sterile centrifuge tube. Add 1.0 mL of dimethyl sulfoxide (DMSO) and vortex thoroughly to dissolve, preparing a 20 mM silybin stock solution. After aliquoting, wrap the stock solution in aluminum foil to protect it from light and store it at -20°C. Before use, remove it and allow it to reach room temperature. Alternatively, the preparation volume can be increased proportionally. For example, to prepare a 10 mL stock solution, weigh 96.5 mg of silybin and dissolve it in 10 mL.

[0037] 3. Preparation of dilution solutions for each concentration gradient: Taking the preparation of 100 mL of cryodiluent as an example, calculate the required volume of 20 mM silybin stock solution to be added based on the target concentration: 5 μM treatment group: Add 25 μL of stock solution to every 100 mL of basic working solution; 10 μM treatment group: Add 50 μL of stock solution to every 100 mL of basic working solution; 15 μM treatment group (optimal concentration group): Add 75 μL of stock solution to every 100 mL of basic working solution; 20 μM treatment group: Add 100 μL of stock solution to every 100 mL of basic working solution.

[0038] After adding the stock solution, stir gently until homogeneous, seal, and pre-cool in a 4°C refrigerator for later use. Add an equal volume (75 μL) of pure DMSO (equal volume to the 15 μM group) to the blank control group. All dilutions should be used within 24 hours of preparation.

[0039] Example 2: Cryopreservation and Thawing of Dolan sheep Semen In this embodiment, the silymarin cryopreservation solution prepared in Example 1 was used to standardize the cryopreservation of Dolan sheep semen, and the thawing and recovery were carried out according to the standard procedure.

[0040] 1. Semen collection and initial quality inspection: At the Wuzheng National Dolan Sheep Breeding Farm in Maigaiti County, four healthy adult Dolan sheep (3-4 years old, weighing 90-110 kg) with normal reproductive function were selected. Fresh semen samples were collected from each sheep in the early morning using the artificial vagina method. Immediately after collection, the samples were placed in a 37℃ water bath for incubation, and routine quality checks were completed within 10 minutes. Testing showed that all four semen samples were milky white in appearance, with a pH of 6.5-6.8, sperm motility of 0.80-0.90, and sperm density of 2-3 billion / mL, all meeting the standards. The four semen samples were then mixed in equal volumes at 37℃ to obtain approximately 6.0 mL of mixed semen sample.

[0041] 2. Semen grouping and experimental design: The experiment consisted of five treatment groups: 5 μM silymarin, 10 μM silymarin, 15 μM silymarin, and 20 μM silymarin, as well as a blank control group (prepared Minitu working solution containing only DMSO). Each group had eight replicates, for a total of 40 frozen fine tubes.

[0042] 3. Semen dilution and low-temperature equilibration: Both the mixed semen and the corresponding cryodiluents for each treatment group were preheated to a stable temperature in a 37°C water bath. At a volume ratio of 1:8, the cryodiluents for each treatment group were slowly added along the wall of the centrifuge tube to the corresponding aliquoted mixed semen, and the centrifuge tube was gently rotated to ensure thorough and uniform mixing. After dilution, each group of diluted semen was aliquoted into 15 mL sterile centrifuge tubes, clearly labeled with the group number. The centrifuge tubes were placed in a 4°C freezer for equilibration for 4 hours. During equilibration, each centrifuge tube was removed every 30 minutes and gently shaken for approximately 15 seconds to ensure the sperm remained uniformly suspended and to guarantee sufficient contact between the sperm and active ingredients such as silymarin.

[0043] 4. Filling and programmed freezing: Before filling, pre-cool the 0.25 mL plastic frozen capillary tubes and the capillary filling machine in a 4°C constant temperature refrigerator for 1.5 hours. Under constant temperature conditions (4°C), fill each group of balanced diluted semen into the capillary tubes, controlling the filling speed evenly to avoid air bubbles. Immediately after filling, seal the ends with capillary sealing powder and neatly stack the sealed capillary tubes on a stainless steel freezer rack, marking the edges of the rack with information about each processing group.

[0044] Turn on the program-controlled freezer and stabilize the freezer temperature at 4°C for 20 minutes. Quickly place the freezer rack with the thin tubes in place into the freezer, close the door, and start the preset freezing program. The freezing program is set as follows: Stage 1: 4°C → -10°C, cooling rate 4°C / min; Stage 2: -10°C → -100°C, cooling rate 40°C / min. The program will automatically end when the temperature reaches -100°C. The operator should then quickly immerse the entire freezer rack into a wide-mouth liquid nitrogen tank filled with liquid nitrogen, ensuring that all thin tubes are completely submerged. After the liquid nitrogen stops boiling violently, group the thin tubes into thumb tubes and transfer them to a large liquid nitrogen storage tank for long-term storage.

[0045] 5. Semen thawing: After cryopreservation for at least 24 hours, three frozen seminiferous tubes were randomly selected from each group in the liquid nitrogen tank for thawing and quality testing. The constant temperature water bath was precisely adjusted to 37°C and stabilized for at least 30 minutes. The frozen seminiferous tubes were picked up from the liquid nitrogen with long-handled tweezers, and quickly checked for any damage in the air (within 2 seconds). They were then immediately and completely immersed in the 37°C constant temperature water bath, and gently shaken continuously for 30 seconds to rapidly thaw and revive. After thawing, the tubes were immediately removed, and the surface moisture was wiped dry with sterile absorbent paper. The sealed end was cut open with sterile scissors, and the semen was squeezed into a sterile 1.5 mL centrifuge tube preheated to 37°C. The tubes were gently shaken to mix, and the thawing was completed. Subsequent quality tests were then immediately performed.

[0046] Example 3: Detection and Results of Sperm Quality Indicators After Thawing This embodiment conducts systematic quality testing on the thawed Dolan sheep frozen semen of each group in Example 2, and compares and analyzes the protective effects of different concentrations of silymarin.

[0047] 1. Detection method: Sperm motility and motility parameters: These were detected using a computer-aided semen analysis system (CASA). 10 μL of thawed semen was placed on a preheated (37°C) counting slide. At least five fields of view were randomly selected at each time point for each group, with at least 200 sperm in each field. Analyses were performed at 0, 2, 4, and 6 hours post-thawing. Key analytical parameters included total sperm motility, percentage of progressively motile sperm, curvilinear motility, and linear motility.

[0048] Sperm plasma membrane integrity: The hypotonic swelling test (HOST) was used for detection. 50 μL of thawed semen was added to 500 μL of preheated hypotonic solution and incubated at 37°C for 30 minutes. The sperm were observed and counted under a 400x phase contrast microscope. Sperm with curled tails were counted as having intact plasma membranes. At least 200 sperm were counted per sample.

[0049] Sperm acrosome integrity: Detected using FITC-PNA fluorescence staining. After fixing sperm smears with methanol, FITC-PNA working solution was added, and the smears were incubated at 37°C in the dark for 30 minutes. Sperm with intact acrosomes and bright green fluorescence at the anterior part of the acrosome were observed under a fluorescence microscope.

[0050] Sperm deformity rate: Detected using Giemsa staining. Sperm smears were fixed with methanol, stained with Giemsa, and then observed under a 1000x oil immersion microscope. Deformities of the head, neck, and tail were classified and recorded, with at least 200 sperm counted per sample.

[0051] 2. Test Results: The sperm quality indicators of each group at 0 hours after thawing are shown in Table 1.

[0052] Table 1. Sperm quality indicators of Dolan sheep frozen semen at 0 hours after thawing in different silymarin treatment groups. Note: Different lowercase letters in the superscript of the same data indicate significant differences between groups (P < 0.05), different uppercase letters indicate extremely significant differences between groups (P < 0.01), and the same letters indicate no significant differences (P > 0.05). The same applies below.

[0053] As shown in Table 1, the treatment groups with added silymarin showed significantly better results than the control group in terms of plasma membrane integrity, acrosome integrity, and abnormality rate (P < 0.05), fully demonstrating that silymarin has a clear and significant protective effect on the cryopreservation of Dolan sheep sperm. The protective effect increased in a dose-dependent manner as the silymarin concentration increased from 5 µM to 15 µM: the plasma membrane integrity rate (75.48%) and acrosome integrity rate (77.26%) in the 10 µM group were significantly higher than those in the 5 µM group (P < 0.05), while the abnormality rate (16.87%) was significantly lower (P < 0.05); the 15 µM group showed further improvements in all indicators, with a plasma membrane integrity rate of 85.36%, an acrosome integrity rate of 86.14%, and an abnormality rate reduced to 13.52%, all showing highly significant differences compared to the 10 µM group (P < 0.01). When the silymarin concentration was further increased to 20 μM, the protective effect decreased significantly compared to the 15 μM group: the plasma membrane integrity rate decreased to 77.36%, the acrosome integrity rate decreased to 78.64%, and the abnormality rate increased to 16.18%, all of which showed highly significant differences compared to the 15 μM group (P < 0.01). This indicates that excessively high concentrations of silymarin may have certain cytotoxic or metabolic interference effects on sperm, leading to a decrease in the protective effect. Therefore, the optimal protective concentration of silymarin for cryopreservation of Dolan sheep semen is 15 µM.

[0054] (2) Sperm motility parameters 0 hours after thawing: To further evaluate the protective effect of silymarin on sperm motility, the sperm motility parameters of each group were comprehensively tested at 0 hours after thawing using the CASA system. The results are shown in Table 2.

[0055] Table 2. CASA motility parameters of Dolan sheep frozen semen at 0 hours after thawing in different silymarin treatment groups. Note: Different lowercase letters in the superscript of the same data indicate significant differences between groups (P < 0.05), different uppercase letters indicate extremely significant differences between groups (P < 0.01), and the same letters indicate no significant differences (P > 0.05). The same applies below.

[0056] As shown in Table 2, all treatment groups with added silymarin were superior to the blank control group in all motility parameters, exhibiting a dose-response trend consistent with the baseline quality indicators. The 15 µM group was significantly superior to the 10 µM group and other groups in all motility parameters, including total viability (61.28%), progressive motility rate (50.63%), VCL (118.75 μm / s), VSL (43.00 μm / s), VAP (60.50 μm / s), LIN (40.50%), and STR (72.75%) (P < 0.01), fully demonstrating that 15 µM is the optimal protective concentration.

[0057] (3) Dynamic changes in sperm motility at different time points after thawing: Table 3 shows the dynamic changes in sperm viability at different time points (0, 1, 2, and 4 hours) after thawing of sperm from Dolan sheep in each group and incubation at 37°C.

[0058] Table 3. Dynamic changes in sperm motility (%) at different time points after thawing of Dolan sheep frozen semen in different treatment groups. Note: Different lowercase letters in the superscript of the same data indicate significant differences between groups (P < 0.05), different uppercase letters indicate extremely significant differences between groups (P < 0.01), and the same letters indicate no significant differences (P > 0.05). The same applies below.

[0059] As shown in Table 3, after thawing, the sperm viability of each group gradually decreased with the extension of the constant temperature incubation time at 37℃. However, there were significant differences in the rate of decline and the duration of maintenance of sperm viability among the groups.

[0060] As shown in Table 3, the sperm viability of the 15 μM group was still 37.48% 4 hours after thawing, which was significantly higher than 30% (the basic viability standard usually required for artificial insemination), indicating that its effective survival time (viability ≥30%) could reach more than 4 hours; the viability of the blank control group dropped to 25.73% 1 hour after thawing, which could not meet the requirements for insemination.

[0061] The above results indicate that 15 μM silymarin dilution can not only significantly improve sperm motility immediately after thawing, but also effectively slow down the rate of decline in sperm motility after thawing. It significantly extends the effective in vitro survival time of sperm to meet the quality requirements of artificial insemination from less than 1 hour in the blank control group to more than 4 hours, providing more time guarantee for the flexible use and long-distance delivery of frozen sperm in actual artificial insemination operations.

[0062] Based on all the above test results, it is fully confirmed that the sheep semen cryopreservation diluent containing 15 μM silybin provided by this invention, through a simple preparation method—using commercially available Minito diluent as a base and quantitatively adding silybin stock solution dissolved in DMSO—significantly enhances the antioxidant protection capacity of the diluent, comprehensively improves the overall quality of Dolan sheep frozen semen after thawing, significantly increases sperm motility, plasma membrane integrity, and acrosome integrity, effectively prolongs the effective survival time in vitro, and greatly reduces the deformity rate. This invention provides a clear and reliable technical solution and a directly applicable product formula for the optimization and standardized promotion of semen cryopreservation technology for Dolan sheep and other sheep breeds.

[0063] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A cryo-diluent for sheep semen, characterized in that, The sheep semen cryo-diluent contains a base diluent and silymarin.

2. The sheep semen cryo-diluent according to claim 1, characterized in that, The sheep semen cryopreservation diluent is a commercially available basic diluent for the cryopreservation of sheep semen.

3. The sheep semen cryo-diluent according to claim 2, characterized in that, The base diluent for commercially available sheep semen cryopreservation is the ready-made Minitu diluent.

4. The sheep semen cryo-diluent according to claim 1, characterized in that, The concentration of silymarin in the frozen dilution of sheep semen is 5-20 µM.

5. The sheep semen cryo-diluent according to claim 1, characterized in that, The silymarin was added after being solubilized with dimethyl sulfoxide.

6. The method for preparing the sheep semen cryo-diluent according to any one of claims 1-4, characterized in that, The preparation steps include the following: S1. Prepare the base diluent into a base diluent working solution and refrigerate for later use; S2. Silybin was prepared into a 20 mM silybin stock solution using dimethyl sulfoxide; S3. Weigh the Minito base working solution and silymarin stock solution according to the final concentration, mix them well to obtain sheep semen cryo-diluted solution.

7. The use of the sheep semen cryo-diluent according to any one of claims 1 to 5 in the following aspects: To improve sperm motility after freezing and thawing sheep semen; To improve the acrosome integrity rate of sheep semen after freezing and thawing; To improve the sperm plasma membrane integrity rate after freezing and thawing of sheep semen; Extend the effective survival time of sheep semen after freezing and thawing; Reduce the sperm abnormality rate after freezing and thawing sheep semen.

8. The application according to claim 7, characterized in that, The method for preserving sheep semen with the described sheep semen cryo-diluent includes the following steps: 1) Semen collection and quality inspection: Fresh semen was collected from Dolan sheep rams using the artificial vagina method. After collection, the semen was quickly placed in a 37°C constant temperature water bath for insulation. Under the constant temperature of 37°C, routine quality inspection of the semen was carried out. Semen samples with sperm motility greater than 0.7 and sperm density greater than 1.5 billion / mL were selected. To eliminate individual differences, the qualified semen samples from multiple rams were mixed in equal volumes at 37°C to form a mixed semen sample. 2) Dilution and equilibration: Mix the mixed semen obtained in step 1) with sheep semen cryo-diluent at a volume ratio of 1:5 to 1:10 under a 37°C water bath and gently shake to mix. After dilution, place the diluted semen in a 4°C refrigerator for low-temperature equilibration treatment for 3 to 5 hours, gently shaking it once every 30 minutes to keep the sperm in suspension. 3) Filling: Place the 0.25 mL plastic capillary tubes and filling machine in a constant temperature environment of 4℃ for full pre-cooling. Then, fill the semen after the equilibrium in step 2) into the 0.25 mL plastic capillary tubes at 4℃. After filling, seal the tubes and neatly stack them on the freezer rack. 4) Programmed freezing: Turn on the programmed freezing machine and stabilize the freezing chamber temperature at 4℃; quickly place the freezing rack with the thin tubes arranged in step 3) into the freezing chamber and start the freezing program; the freezing program is as follows: cool down from 4℃ to -10℃ at a cooling rate of 4℃ / min, and then cool down from -10℃ to -100℃ at a cooling rate of 40℃ / min; after freezing to -100℃, quickly immerse the entire freezing rack into liquid nitrogen, ensuring that the liquid nitrogen completely submerges all the thin tubes, thus completing the cryopreservation of sheep semen.

9. The method for cryopreserving sheep semen according to claim 8, characterized in that, In step 2), the volume ratio of the mixed semen to the cryo-diluted solution containing silymarin is 1:8; the low-temperature equilibration treatment time is 4 hours.

10. A method for thawing frozen sheep semen, characterized in that, Includes the following steps: Remove the frozen semen tubes from the liquid nitrogen and immediately immerse them in a 37°C constant temperature water bath. Gently shake for 30 seconds to thaw. After removing them, wipe the surface of the tubes dry with sterile absorbent paper, cut open the sealed end of the tubes, collect the semen into a centrifuge tube preheated to 37°C, and gently shake to mix. Thawing is now complete.

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