Optimized diluent composite additive and application method thereof in cryopreservation of sex-controlled frozen semen

Abstract

The invention belongs to the technical field of cryopreservation, and discloses an optimized diluent composite additive and an application method thereof in sex control frozen sperm cryopreservation, according to the invention, through an orthogonal test, quercetin and vitamin E antioxidants with different proportioning concentrations are compared, and the quercetin and vitamin E antioxidants are grouped as follows: I (3.02 mu g / mL quercetin and 0.1 mg / mL vitamin E), II (15.1 mu g / mL quercetin and 0.1 mg / mL vitamin E), and III (15.1 mu g / mL quercetin and 0.1 mg / mL vitamin E). The antioxidant is added into the control group, III is (3.02 mu g / mL of quercetin and 0.5 mg / mL of vitamin E), IV is (15.1 mu g / mL of quercetin and 0.5 mg / mL of vitamin E), and 0 is (no antioxidant is added, and a blank control group). The dye JC1 in the experiment is an ideal fluorescent probe which is widely used for detecting the mitochondrial membrane potential and can be used for detecting the mitochondrial membrane potential of cells, tissues or purified. When the mitochondrial membrane potential is relatively high, JC1 is gathered in a matrix of mitochondria to form a polymer, and red fluorescence can be generated; when the mitochondrial membrane potential is low, JC1 cannot be gathered in a matrix of mitochondria, and at the moment, JC1 is a monomer and can generate green fluorescence.

Description

Technical Field

[0001] This invention belongs to the field of cryopreservation technology, and particularly relates to an optimized diluent compound additive and its application method in the cryopreservation of sex-controlled frozen semen. Background Technology

[0002] In the field of cryopreservation for sex-controlled sperm, the activity, functional integrity, and post-cryopreservation quality of sperm cells are key factors affecting their subsequent applications (such as the success rate of artificial insemination). Mitochondria, as important organelles of sperm cells, have membrane potential states closely related to sperm energy metabolism and motility. The level of mitochondrial membrane potential reflects the energy reserves of mitochondria; maintaining a high mitochondrial membrane potential helps ensure the normal physiological function of sperm during cryopreservation and subsequent use. Currently, during cryopreservation, sperm cells are affected by various adverse factors (such as low temperature, toxicity of cryoprotectants, and changes in osmotic pressure), leading to impaired mitochondrial function and decreased membrane potential, thereby affecting sperm motility and fertilization capacity.

[0003] Based on the above analysis, the problems and shortcomings of the existing technology are as follows:

[0004] During cryopreservation, sperm cells are affected by a variety of adverse factors (such as low temperature, toxicity of cryoprotectants, changes in osmotic pressure, etc.), which can lead to impaired mitochondrial function and decreased membrane potential, thereby affecting sperm motility and fertilization capacity. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides an optimized diluent compound additive and its application method in the cryopreservation of sex-controlled frozen semen.

[0006] This invention is achieved as follows: an optimized diluent compound additive and its application in the cryopreservation of sex-controlled frozen semen include:

[0007] Step 1: Transfer the collected semen to an incubator at 18–20°C. Add appropriate antibiotics according to the semen volume. Then, determine the amount of semen to be added, as well as the amount of Hoechst 33342 dye and XYstangding diluent, based on the collected semen density.

[0008] Step 2: First, add the dye and XYstangding diluent and vortex for 20 seconds. Then, add the semen and mix by inverting. Place the mixture in a 34°C constant temperature metal bath for 50 minutes. Add the 4% EggYork solution, which has been preheated in a water bath for 10 minutes, to the diluent containing the dye and semen and mix well. Filter the mixture through a 50μm filter into a new test tube.

[0009] Step 3: Adjust the machine's separation parameters to the required settings, then load the stained sperm sample into the separation stage and begin the separation process; separate and collect X and Y sperm into different receiving tubes containing BUFF buffer.

[0010] Step 4: Place the receiving tube at 4°C and allow it to cool slowly to 4°C. Add glycerol in two batches. Add 6.25 mL first and equilibrate for 15 min, then add another 6.25 mL and equilibrate for 15 min. Place the receiving tube containing glycerol into a centrifuge at 4°C and centrifuge at 2080 g for 20 minutes. Discard the supernatant and add 300 μL of cryo-diluent to the sperm precipitate at the bottom. Shake and mix well.

[0011] Step 5: Dispense the dispensing using a capillary filling machine at 4°C. Place the dispensed capillary tubes into a programmable freezer and fumigate them with liquid nitrogen. When the temperature reaches 196°C, quickly remove them and store them in a liquid nitrogen tank.

[0012] Furthermore, the XYstangding diluent comprises the following raw materials:

[0013] Hepes buffer 9.52 mg / mL

[0014] Magnesium chloride hexahydrate (MgCl2·6H2O) 0.08 mg / mL

[0015] Sodium chloride (NaCl) 5.518 mg / mL

[0016] Potassium chloride (KCl) 0.224 mg / mL

[0017] Sodium bicarbonate (NaHCO2) 0.84 mg / mL

[0018] Sodium pyruvate (C3H3NaO3) 0.22 mg / mL

[0019] Sugar (C6H) 12 O6) 0.90 mg / mL

[0020] Bovine serum albumin (BSA) 3.00 mg / mL

[0021] Disodium hydrogen phosphate (Na₂HPO₄) 0.04 mg / mL

[0022] Liquid sodium lactate (Nalactate) 3.61 μL / mL

[0023] Quercetin 3.02 μg / mL

[0024] Vitamin E 0.1 mg / mL.

[0025] Furthermore, the method for preparing the XYstangding diluent is as follows:

[0026] (1) Take a 250mL beaker and fill it with 187.5mL of ultrapure water. Place the beaker on a thermostatic magnetic stirrer and put in the sterilized stir bar.

[0027] (2) Add the measured amounts of the above-mentioned drugs in sequence, and adjust the pH value to 7.40 after the drugs are completely dissolved;

[0028] (3) Pour the solution in the beaker into a 250mL volumetric flask, rinse the beaker three times, add water to the mark of the volumetric flask and pour it in at an angle to prevent air bubbles.

[0029] (4) After filtering the solution, pour it into a new volumetric flask, add antibiotics, seal and store in the refrigerator. It is valid for 10 days.

[0030] Another object of the present invention is to provide an optimized diluent compound additive and its application system in the cryopreservation of sex-controlled frozen semen, comprising:

[0031] The heat preservation module is used to transfer the collected semen to a heat preservation box at 18-20℃. Appropriate antibiotics are added according to the semen volume, and the amount of semen added, as well as the amount of Hoechst33342 dye and XYstangding diluent, are determined according to the collected semen density.

[0032] The mixing module is used to first add the dye and XYstangding diluent, vortex mix for 20 seconds, then add the semen and mix by inverting, place in a 34°C constant temperature metal bath for 50 minutes, add 4% EggYork solution preheated in a water bath for 10 minutes to the diluent containing the dye and semen and mix, and filter into a new test tube using a 50μm filter.

[0033] The separation module is used to adjust the machine's separation parameters to the required state, then load the stained sperm sample into the separation sample stage and start the separation process; X sperm and Y sperm are separated and collected into different receiving tubes containing BUFF buffer.

[0034] The cooling module is used to place the receiving tube at 4°C and allow it to cool slowly to 4°C. Glycerin is added in two parts: 6.25 mL is added first and equilibrated for 15 min, and then another 6.25 mL is added and equilibrated for 15 min. The receiving tube containing glycerin is then placed in a centrifuge at 4°C and centrifuged at 2080 g for 20 minutes. The supernatant is discarded, and 300 μL of cryo-diluent is added to the sperm precipitate at the bottom and shaken to mix.

[0035] The storage module is used to dispense the filler tubes at 4°C using a capillary filling machine. The dispensed capillary tubes are placed in a programmable freezer and fumigated with liquid nitrogen. When the temperature is frozen to 196°C, they are quickly removed and stored in a liquid nitrogen tank.

[0036] Based on the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solution to be protected by this invention are as follows:

[0037] First, an orthogonal experiment was conducted to compare different concentrations of quercetin and vitamin E antioxidants, grouped as follows: I (3.02 μg / mL quercetin and 0.1 mg / mL vitamin E), II (15.1 μg / mL quercetin and 0.1 mg / mL vitamin E), III (3.02 μg / mL quercetin and 0.5 mg / mL vitamin E), IV (15.1 μg / mL quercetin and 0.5 mg / mL vitamin E), and 0 (no antioxidant added, blank control group).

[0038] The dye JC1 used in the experiment is an ideal fluorescent probe widely used to detect mitochondrial membrane potential, which can be measured in cells, tissues, or purified mitochondrial membranes. At high mitochondrial membrane potentials, JC1 aggregates in the mitochondrial matrix, forming polymers and producing red fluorescence; at low mitochondrial membrane potentials, JC1 cannot aggregate in the mitochondrial matrix, remaining as monomers and producing green fluorescence. Currently, flow cytometry is used to analyze the red-to-green fluorescence ratio (JC1Red / Green Ratio) released by stained sperm cells. A higher red fluorescence ratio indicates more abundant energy reserves in the sperm mitochondria. Results showed that the red-to-green fluorescence ratios in the experimental groups with added concentrations of compound antioxidants were significantly higher than those in the blank control group without antioxidants. However, the differences between the experimental groups were not significant. Therefore, the relatively low addition amounts of 3.02 μg / mL quercetin and 0.5 mg / mL vitamin E were selected as the optimal dosage.

[0039] Secondly, over the years, with the continuous innovation and advancement of flow cytometry, separation efficiency and accuracy have been continuously improved, and production costs have been continuously reduced. Although some species, such as bovine semen, have been successfully applied to commercial production, there are still many limiting factors in their application in production. The fertilization capacity of sex-controlled frozen semen is far lower than that of conventional frozen semen and fresh semen, which has always been the biggest obstacle to the practical application of sex-controlled frozen semen. The sex-controlled semen sorting process involves multiple steps and quite drastic environmental changes, including high pressure, strong electric field stimulation, pH of the diluent, penetration of the dye Hoechst 33342, and stimulation from the components of the sheath fluid and cryoprotectant. All of these factors cause additional stress and mechanical damage to sperm, which are the main reasons for the low quality and fertilization capacity of sex-controlled sperm compared to conventional frozen semen. Numerous studies have shown that the low fertilization capacity of sex-controlled frozen semen is closely related to the large amount of reactive oxygen species (ROS) generated by the oxidative stress during its preparation process. ROS can cause oxidative stress damage to sperm, disrupt the balance of the sperm's internal antioxidant defense system, and alter sperm structure and function. However, the application of single antioxidants often has limitations, making it difficult to comprehensively and continuously combat the oxidative stress challenges faced by sperm in complex sex-sorting environments. In the context of the fact that the cryopreservation performance of sex-controlled frozen semen is affected by multiple factors, and the mechanisms by which these factors affect sperm freezing effects are not yet fully understood, this invention utilizes the different effects of quercetin and vitamin E in inhibiting oxidative stress, and screens out the optimal ratio to effectively improve the quality of frozen and thawed sex-controlled frozen semen from beef cattle. Compared to commercial diluents without added compound additives, sperm motility is significantly improved. Attached Figure Description

[0040] Figure 1 This is a flowchart illustrating the optimized diluent composite additive and its application method in the cryopreservation of sex-controlled frozen semen, as provided in this invention.

[0041] Figure 2 This is a block diagram of the optimized diluent composite additive and its application in the cryopreservation of sex-controlled frozen semen provided in the embodiments of the present invention.

[0042] Figure 3 This is a sperm mitochondrial membrane potential detection diagram provided in an embodiment of the present invention.

[0043] Figure 4 This is a pH change graph of different levels of the antioxidant XYstanding solution provided in the embodiments of the present invention. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0045] like Figure 1 As shown in the figure, the optimized diluent compound additive and its application method in the cryopreservation of sex-controlled frozen semen provided by the present invention include the following steps:

[0046] S101, the collected semen is transferred to an incubator at 18-20℃, and the appropriate antibiotics are added according to the semen volume. The amount of semen added, as well as the amount of Hoechst33342 dye and XYstangding diluent, are determined according to the collected semen density.

[0047] S102, first add the dye and XYstangding diluent, vortex and mix for 20 seconds, then add the semen and mix by inversion, place in a 34°C constant temperature metal bath for 50 minutes, add the 4% EggYork solution preheated in a water bath for 10 minutes to the diluent containing the dye and semen and mix well, filter through a 50μm filter into a new test tube.

[0048] S103, adjust the machine's separation parameters to the required state as instructed, then load the stained sperm sample into the separation sample stage and begin the separation process; separate and collect X sperm and Y sperm into different receiving tubes containing BUFF buffer;

[0049] S104, place the receiving tube at 4℃ and allow it to cool slowly to 4℃. Add glycerol in two portions. Add 6.25 mL first and equilibrate for 15 min, then add another 6.25 mL and equilibrate for 15 min. Place the receiving tube containing glycerol into a centrifuge at 4℃ and centrifuge at 2080g for 20 minutes. Discard the supernatant and add 300 μL of cryo-diluent to the sperm precipitate at the bottom. Shake and mix well.

[0050] S105 is packaged using a capillary filling machine at 4°C. The packaged capillary tubes are then placed in a programmable freezer and fumigated with liquid nitrogen. When the temperature is frozen to -196°C, they are quickly removed and stored in a liquid nitrogen tank.

[0051] The XYstangding diluent provided in this embodiment of the invention comprises the following raw materials:

[0052] Hepes buffer 9.52 mg / mL

[0053] Magnesium chloride hexahydrate (MgCl2·6H2O) 0.08 mg / mL

[0054] Sodium chloride (NaCl) 5.518 mg / mL

[0055] Potassium chloride (KCl) 0.224 mg / mL

[0056] Sodium bicarbonate (NaHCO2) 0.84 mg / mL

[0057] Sodium pyruvate (C3H3NaO3) 0.22 mg / mL

[0058] Sugar (C6H) 12 O6) 0.90 mg / mL

[0059] Bovine serum albumin (BSA) 3.00 mg / mL

[0060] Disodium hydrogen phosphate (Na₂HPO₄) 0.04 mg / mL

[0061] Liquid sodium lactate (Nalactate) 3.61 μL / mL

[0062] Quercetin 3.02 μg / mL

[0063] Vitamin E 0.1 mg / mL.

[0064] The method for preparing the XYstangding diluent provided in this embodiment of the invention is as follows:

[0065] (1) Take a 250mL beaker and fill it with 187.5mL of ultrapure water. Place the beaker on a thermostatic magnetic stirrer and put in the sterilized stir bar.

[0066] (2) Add the measured amounts of the above-mentioned drugs in sequence, and adjust the pH value to 7.40 after the drugs are completely dissolved;

[0067] (3) Pour the solution in the beaker into a 250mL volumetric flask, rinse the beaker three times, add water to the mark of the volumetric flask and pour it in at an angle to prevent air bubbles.

[0068] (4) After filtering the solution, pour it into a new volumetric flask, add antibiotics, seal and store in the refrigerator. It is valid for 10 days.

[0069] like Figure 2 As shown, the present invention provides an optimized diluent compound additive and its application system in the cryopreservation of sex-controlled frozen semen, comprising:

[0070] The heat preservation module is used to transfer the collected semen to a heat preservation box at 18-20℃. Appropriate antibiotics are added according to the semen volume, and the amount of semen added, as well as the amount of Hoechst33342 dye and XYstangding diluent, are determined according to the collected semen density.

[0071] The mixing module is used to first add the dye and XYstangding diluent, vortex mix for 20 seconds, then add the semen and mix by inverting, place in a 34°C constant temperature metal bath for 50 minutes, add 4% EggYork solution preheated in a water bath for 10 minutes to the diluent containing the dye and semen and mix, and filter into a new test tube using a 50μm filter.

[0072] The separation module is used to adjust the machine's separation parameters to the required state, then load the stained sperm sample into the separation sample stage and start the separation process; X sperm and Y sperm are separated and collected into different receiving tubes containing BUFF buffer.

[0073] The cooling module is used to place the receiving tube at 4°C and allow it to cool slowly to 4°C. Glycerin is added in two parts: 6.25 mL is added first and equilibrated for 15 min, and then another 6.25 mL is added and equilibrated for 15 min. The receiving tube containing glycerin is then placed in a centrifuge at 4°C and centrifuged at 2080 g for 20 minutes. The supernatant is discarded, and 300 μL of cryo-diluent is added to the sperm precipitate at the bottom and shaken to mix.

[0074] The storage module is used to dispense the filler tubes at 4°C using a capillary filling machine. The dispensed capillary tubes are placed in a programmable freezer and fumigated with liquid nitrogen. When the temperature is frozen to -196°C, they are quickly removed and placed in a liquid nitrogen tank for storage.

[0075] Orthogonal experiments were conducted to compare different concentrations of quercetin and vitamin E antioxidants, and the groups were as follows: I (3.02 μg / mL quercetin and 0.1 mg / mL vitamin E), II (15.1 μg / mL quercetin and 0.1 mg / mL vitamin E), III (3.02 μg / mL quercetin and 0.5 mg / mL vitamin E), IV (15.1 μg / mL quercetin and 0.5 mg / mL vitamin E), and 0 (no antioxidant added, blank control group).

[0076] The dye JC1 used in the experiment is an ideal fluorescent probe widely used to detect mitochondrial membrane potential, and can detect the membrane potential of cells, tissues, or purified mitochondria. When the mitochondrial membrane potential is high, JC1 aggregates in the mitochondrial matrix, forming a polymer that produces red fluorescence; when the mitochondrial membrane potential is low, JC1 cannot aggregate in the mitochondrial matrix, and in this case, JC1 exists as a monomer, producing green fluorescence. Currently, the ratio of red to green fluorescence released by stained sperm cells (JC1Red / GreenRatio) is analyzed by flow cytometry. A higher proportion of red light indicates a more abundant energy reserve in the sperm mitochondria. Results ( Figure 3 The results showed that the proportions of red and green fluorescence in the experimental groups with added compound antioxidants at various concentrations were significantly higher than those in the blank control group without added antioxidants. However, the differences between the experimental groups were not significant. Therefore, the optimal dosage was selected as 3.02 μg / mL quercetin and 0.5 mg / mL vitamin E, which have relatively low addition levels.

[0077] The specific application areas or related products of this invention.

[0078] 1. Select 5 healthy Australian Wagyu cattle and collect semen in 5 separate collections. Test the motility of the collected sperm each time. Select sperm with a motility greater than 80%, add an appropriate amount of antibiotics, and store in an 18°C ​​constant temperature incubator.

[0079] 2. Add antibiotics according to the semen volume. Then, based on the collected semen density, determine the amount of Hoechst 33342 dye, the amount of semen added, and the diluent (containing 3.02 μg / mL quercetin and 0.1 mg / mL vitamin E). First, add the dye and diluent and vortex for 20 seconds. Then, add the semen and invert to mix. Place in a 34°C constant temperature metal bath for 50 minutes. Remove from the bath for 10 minutes to preheat the 4% EggYork solution. After the water bath, add the preheated 4% EggYork solution to the diluent containing the dye and semen and mix well. Filter through a 50 μm filter into a new test tube. Adjust the machine's separation parameters to the required settings. Then, load the stained sperm sample into the separation sample stage and begin the separation process. Separate and collect X and Y sperm into different receiving tubes containing BUFF buffer.

[0080] 3. Place the receiving tube at 4°C and allow it to cool slowly to 4°C. Add glycerol in two batches: first add 6.25 mL, equilibrate for 15 minutes, then add another 6.25 mL, equilibrate for another 15 minutes. Centrifuge the receiving tube containing glycerol at 2080g for 20 minutes at 4°C. Discard the supernatant. Add 300 μL of cryo-diluent to the sperm precipitate at the bottom and mix thoroughly. Mix X and Y sperm at the same level. Dispense the sperm using a capillary filling machine at 4°C. Place the dispensed capillary tubes in a programmed freezer and fumigate with liquid nitrogen. When the temperature reaches -196°C, quickly remove them and store them in a liquid nitrogen tank.

[0081] Sampling and testing were conducted to determine the sperm motility and movement parameters (such as linear motion speed, curvilinear motion speed, etc.) after revival.

[0082] Figure 4 Investigating pH changes in prepared XYstanding solutions with different levels of antioxidants

[0083] Safety analysis was performed on XYstanding solutions with different concentrations of antioxidants, and the results are as follows: Figure 1 As shown, there was no significant difference in pH value between the experimental groups and the control group, indicating that the pH of the diluted solution had no significant effect within the experimental concentration range.

[0084]

[0085] Product quality control analysis was performed on XYstanding solutions with different concentrations of antioxidants. The results are shown in Figure 1. There was no significant difference in the purity of X and Y sperm between the experimental groups and the control group, indicating that the addition of quercetin and vitamin E did not affect the purity of sperm sorting.

[0086] Semen was collected from healthy, sexually active Australian Wagyu bulls aged 5-6 years (raised at the bull depot). Semen was collected using a prosthetic vagina method. Good samples with a fresh semen motility of 0.65 or higher and a sperm density of 1 billion / ml or higher were used for subsequent cryopreservation production, following the same procedures as above.

[0087] A computer-aided semen analyzer was used to analyze sperm motility, mortality, vitality, linear velocity (VSL), curved velocity (VCL), average velocity (VAP), lateral swing amplitude (ALH), wobble amplitude (WOB), whiplash frequency (BCF), linearity (LIN), movement angle (MAD), and antegrade spurt (STR). The results are shown in the table below.

[0088]

[0089]

[0090] The above results indicate that formulations II and III achieve good semen preservation effects, outperforming commercial diluents without added compound antioxidants in multiple indicators. In particular, formulation III shows more significant effects in terms of sperm viability and linear motility, while its safety is no different from that of commercial diluents, effectively improving the quality of frozen semen after thawing.

[0091] Example 1 (Treatment of Bovine Semen)

[0092] Perform the following procedures as described in steps S101–S105: Collect 75 mL of Holstein bull semen (density 1.1 × 10^9 sperm / mL) and incubate at 18°C; add 0.25 mg / mL of a mixed antibiotic of streptomycin and penicillin. Set the final concentration of Hoechst 33342 to 5 μg / mL and add XYstangding diluent at a 1:1 volume ratio. After incubating at the same temperature for 20 s, add more semen and invert to mix thoroughly. Incubate at 34°C in a metal bath for 50 min; then add an equal volume of preheated 4% EggYolk solution and mix thoroughly. Filter through a 50 μm filter. Set the separator flow rate to 15 kHz and the pressure to 40 psi to separate X and Y sperm into two tubes (each containing BUFF buffer).

[0093] The receiving tubes were equilibrated twice with 6.25 mL of glycerol each time at 4°C, centrifuged at 2080 g for 20 min, the supernatant was discarded, and the cells were resuspended in 300 μL of cryopreservation dilution buffer. The cells were then filled into capillary tubes at 4°C, programmed to cool to -196°C, and stored in liquid nitrogen for long-term preservation. Recovery testing showed that X sperm purity was 90%, Y sperm purity was 88%, total motility was 62%, and progressive motility was 55%, demonstrating good cryopreservation protection.

[0094] Example 2 (Boar Semen Treatment)

[0095] Sixty mL of Duroc boar semen (density 1.5 × 10^9 sperm / mL) was collected and transported at 18°C ​​with 0.30 mg / mL antibiotic added. Hoechst 33342 was added at a final concentration of 4 μg / mL, and XYstangding diluent was added at a volume ratio of 1:0.8. After co-staining for 20 s and incubation at 34°C for 50 min, an equal volume of 4% EggYolk solution was added, mixed thoroughly, and filtered. X and Y sperm were successfully collected using a sorter at a flow rate of 12 kHz and a pressure of 35 psi.

[0096] Two-step glycerol equilibration (5 mL each) was performed at 4℃, followed by centrifugation at 2080 g for 20 min. The supernatant was discarded, and the mixture was resuspended in 300 μL of cryogenic dilution buffer. The suspension was then aliquoted into capillary tubes and frozen to -196℃. Recovery testing revealed X sperm purity of 87% and Y sperm purity of 85%. The total motility rate after recovery was 58%, and the progressive motility rate was 51%, verifying the broad applicability and stability of this compound additive in semen from different species.

[0097] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for applying an optimized diluent compound additive in the cryopreservation of sex-controlled frozen semen, characterized in that, Includes the following steps: S1. Place the collected semen in an incubator at 18-20℃, add antibiotics according to the semen volume, and determine the amount of Hoechst33342 dye and XYstangding diluent to be added based on the semen density. S2. Mix the dye with XYstangding diluent, vortex for 20 seconds, add semen, invert and mix, place in a 34°C metal bath for 50 minutes, then add the solution that has been preheated in a 4% EggYork solution water bath for 10 minutes and mix well, then filter using a 50μm filter. S3. Place the processed sample into the separation device, and separate X sperm and Y sperm by adjusting the separation parameters. Collect them into receiving tubes containing BUFF buffer. S4. Cool the receiving tube slowly at 4°C, add glycerol in two portions of 6.25 mL each time, with an interval of 15 minutes, centrifuge to remove the supernatant, and then add 300 μL of cryogenic dilution to the precipitate and mix well. S5. Semen samples were aliquoted at 4°C, frozen to 196°C using a programmed cryostat with liquid nitrogen fumigation, and then transferred to a liquid nitrogen tank for long-term storage.

2. A diluent for cryopreservation of sex-controlled frozen sperm, characterized in that, It is made according to the following proportions: Hepes buffer 9.52 mg / mL Magnesium chloride hexahydrate (MgCl2·6H2O) 0.08 mg / mL Sodium chloride (NaCl) 5.518 mg / mL Potassium chloride (KCl) 0.224 mg / mL, Sodium bicarbonate (NaHCO2) 0.84 mg / mL Sodium pyruvate (C3H3NaO3) 0.22 mg / mL Glucose (C6H) 12 O6) 0.90 mg / mL, Bovine serum albumin (BSA) 3.00 mg / mL Disodium hydrogen phosphate (Na₂HPO₄) 0.04 mg / mL Liquid sodium lactate (NaLactate) 3.61 μL / mL Quercetin 3.02 μg / mL Vitamin E 0.1 mg / mL.

3. The XYstangding diluent as described in claim 2, wherein the preparation method comprises: Add ultrapure water to a 250mL beaker; Add each component of the medicine in sequence and dissolve it completely under constant temperature magnetic stirring; Adjust the pH to 7.40; Transfer the solution to a 250 mL volumetric flask and dilute to volume. After being filtered through a sterile membrane and treated with antibiotics, it is sealed and stored in a refrigerator. The shelf life is 10 days.

4. A system for the application of optimized diluent compound additives in the cryopreservation of sex-controlled frozen semen, characterized in that, include: A heat preservation module is used for mixing and adding antibiotics, diluents, and dyes at 18-20℃. The mixing module is used for the sequential mixing and filtration of semen with diluent and EggYork solution; The separation module is used to separate sperm samples and collect X sperm and Y sperm separately; Cooling module for slow cooling at 4°C, glycerol equilibration, and centrifugation; The preservation module is used for thin-tube dispensing and liquid nitrogen fumigation cryopreservation in a programmed freezer.

5. The method as described in claim 1, characterized in that, In step S2, the EggYork solution added after staining is at a concentration of 4%, and the solution is preheated in a water bath at 37°C for 10 minutes.

6. The method as described in claim 1, characterized in that, Quercetin added to the XYstangding diluent is used to reduce sperm DNA damage during freezing.

7. The method as described in claim 1, characterized in that, After adding glycerol, the centrifugation conditions were 4℃, 2080g, and 20 minutes.

8. The XYstangding diluent as described in claim 2, characterized in that, This diluent contains dual antioxidant protection components, namely quercetin and vitamin E, to improve the viability of sex-controlled frozen sperm after freezing and thawing.

9. The system as described in claim 4, characterized in that, The cooling module further includes a precision temperature control unit for controlling temperature fluctuations within ±0.5°C during the glycerol equilibration process.

10. The system as described in claim 4, characterized in that, The preservation module includes a programmed freezer and a liquid nitrogen tank. The programmed freezer controls the freezing rate so that the sample cools down at a rate of 0.5 to 1.0°C per minute to avoid freezing damage.

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