A method for improving quality of frozen semen of lanzhou big-tail-han sheep based on ergothioneine

Abstract

This invention belongs to the field of animal husbandry technology and relates to a method for improving the quality of frozen semen from Lanzhou fat-tailed sheep based on ergothioneine. This application, through optimized combination of diluent, 8 layers of gauze wrapping for cooling, 4 cm fumigation height, and 8 mM ergothioneine, achieved a sperm motility rate of 45.80±3.62%, an acrosome integrity rate of 52.32±2.28%, and a plasma membrane integrity rate of 55.38±3.55% after thawing. All indicators are significantly better than those of the traditional single-optimization group.

Description

Technical Field

[0001] This invention belongs to the field of animal husbandry technology and relates to a method for improving the quality of frozen semen from Lanzhou fat-tailed sheep based on ergothioneine. Background Technology

[0002] Sperm cryopreservation is an important means of protecting the genetic diversity of species. Combined with artificial insemination, it can improve production efficiency and reduce the risk of disease transmission. Sperm structure varies significantly among different species, and the effectiveness of cryopreservation is affected by various factors, including the diluent, freezing method, and the altitude of liquid nitrogen fumigation. Therefore, traditional diluents often contain added egg yolk to mitigate cryopreservation damage, but this method suffers from inconsistent quality and carries the risk of disease transmission.

[0003] Cryopreservation of bovine and swine semen has been commercialized, but sheep sperm membranes are high in polyunsaturated fatty acids and low in cholesterol, which reduces membrane stability and adhesion, making them susceptible to damage during freezing. After thawing, their motility is only 55%-60% at most, and the technology is still in the experimental research stage.

[0004] During the freezing and thawing process, sheep sperm will produce a large amount of reactive oxygen species (ROS) due to low temperature stress. Excessive ROS will attack the sperm plasma membrane, damage DNA integrity, inhibit the activity of antioxidant enzymes, and cause abnormal changes in the sperm proteome, ultimately resulting in decreased sperm motility and loss of fertilization ability, which seriously affects the quality and application effect of frozen semen.

[0005] Ergothioneine, a rare natural chiral amino acid, possesses potent yet mild antioxidant properties. It can scavenge various reactive oxygen species, form redox-free complexes with divalent metal cations, inhibiting the reaction of these metal ions with ROS, and also reacts with Cu. 2+ The resulting complex can prevent Cu-induced oxidative damage to DNA and proteins. Furthermore, ergothioneine can regulate the expression of sperm metabolism-related proteins, participate in DNA damage repair, and provide multiple protective effects on sperm structure and function.

[0006] Currently, research on ergothioneine in semen preservation is still insufficient. Existing technologies lack mature and efficient ergothioneine-based cryopreservation methods for sheep semen, limiting its widespread application in sheep breeding. Therefore, developing a method for cryopreserving sheep semen with ergothioneine at a clearly defined concentration and with significant protective effects is of great significance for improving the quality of frozen sheep semen and promoting the efficient development of animal husbandry. Summary of the Invention

[0007] The purpose of this invention is to provide a method for improving the quality of frozen sheep semen.

[0008] The method for improving the quality of frozen sheep semen according to the present invention includes the following steps: The semen of Lanzhou fat-tailed sheep was isothermally diluted in a buffer solution containing 8 mM ergothioneine using a two-stage dilution method. The buffer solution was formulated as follows: 0.5044 g / 100 mL glucose, 1.8252 g / 100 mL citric acid, 3.6342 g / 100 mL Tris, 10 mL penicillin (100 IU / mL), 10 mL streptomycin (100 IU / mL), 20% egg yolk (v / v), 6% glycerol (v / v), pH (7.0±0.2). Equilibrate at 4°C by wrapping it in 8 layers of gauze. After equilibration, the semen was placed 4 cm above the liquid nitrogen surface and fumigated for 8 minutes before being frozen and stored in liquid nitrogen.

[0009] According to the method for improving the quality of frozen sheep semen of the present invention, 8 mM ergothioneine is added to the semen cryopreservation diluent for Lanzhou fat-tailed sheep, and the semen and diluent are diluted using a two-stage diluent method at a ratio of 1:3. Beneficial technical effects of this application

[0010] 1. Significantly improved the quality of Lanzhou fat-tailed sheep semen after thawing: The optimized combination scheme of this application (E diluent (Tris-citric acid-glucose system), 8 layers of gauze wrapping for cooling, 4 cm fumigation height, 8 mM ergothioneine) resulted in a sperm motility rate of 45.80±3.62%, an acrosome integrity rate of 52.32±2.28%, and a plasma membrane integrity rate of 55.38±3.55% after thawing. All indicators were significantly better than the traditional single optimization group.

[0011] 2. This application achieved a significant synergistic protective effect in the cryopreservation of Lanzhou fat-tailed sheep semen by combining E-diluent, slow cooling with 8 layers of gauze, fumigation height of 4 cm, and 8 mM ergothioneine. Experimental results show that this combined scheme not only surpasses any single-factor optimization group in each individual indicator, but also exhibits a systemic synergistic effect of "1+1>2" in terms of overall sperm viability, structural integrity, and functional activity. This synergistic effect stems from the complementary mechanisms of each technical step: E-diluent provides a stable metabolic environment for sperm; slow cooling and fumigation control reduce physical damage; and ergothioneine significantly improves sperm antifreeze properties and post-thawing motility through synergistic effects on multiple pathways such as energy metabolism regulation and DNA repair. Proteomics data further confirm that ergothioneine can upregulate RAD50 and CHSY1, forming a multi-layered protective network with cooling and buffering measures. The cryopreservation process generates excessive ROS that damage sperm cell membranes, leading to DNA damage and thus reducing sperm quality and quantity. It also exposes DNA to an oxidative environment, resulting in high-frequency single-strand and double-strand DNA breaks. RAD50, located in the non-homologous end junction repair and homologous recombination pathways, possesses a coiled-coil domain. The ATPase domains at both ends of RAD50 are crucial for maintaining the structural stability of the MRN complex and its DNA-binding ability. Ergothioneine reduces sperm damage caused by reactive oxygen species by activating the DNA damage repair factor MRN complex. Chondroitin sulfate synthase 1 (CHSY1) is a glycosyltransferase involved in the biosynthesis of chondroitin and dermatan sulfate glycosaminoglycans. It regulates cell signaling pathways, inhibits apoptosis, scavenge free radicals, and reduces oxidative stress-induced cell damage. Simultaneously, it elongates the chondroitin sulfate chain, and chondroitin sulfate covalently links with the core protein to form a chondroitin sulfate proteoglycan glycoconjugate. This chondroitin sulfate proteoglycan improves sperm motility and velocity, inhibits spontaneous acrosome reactions, and enhances sperm capacitation.

[0012] 3. Ergothioneine, as a natural antioxidant, is highly safe, has no toxic side effects, and is easy to add without changing the existing conventional process of semen cryopreservation. It is easy to promote and apply in production, and can effectively reduce reproductive losses caused by poor semen quality and improve the economic benefits of sheep farming. Attached Figure Description

[0013] Figure 1 This study shows the effect of different concentrations of ergothioneine on MDA in thawed semen. Figure 2 This study shows the effect of different concentrations of ergothioneine on SOD in thawed semen. Figure 3 This study shows the effects of different concentrations of ergothioneine on GSH-Px in thawed semen. Figure 4 This shows the effect of different concentrations of ergothioneine on CAT in thawed semen; Figure 5 This study shows the effect of different concentrations of ergothioneine on the T-AOC of semen after thawing. Figure 6 This study shows the effects of different concentrations of ergothioneine on AKP in thawed semen. Figure 7 This shows the effect of different concentrations of ergothioneine on ACP in thawed semen; Figure 8 Immunoblots showing sperm protein abundance in different treatment groups. Detailed Implementation

[0014] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0015] Experimental animals used: Based on the breeding, pregnancy and lambing records of Lanzhou fat-tailed sheep, five medium-sized, sexually active 2-3-year-old Lanzhou fat-tailed sheep were selected in Yongjing County, Gansu Province. They were kept in single pens during the semen collection period and grazed in the morning. Each ram was supplemented with 0.5-1.0 kg of carrots and 2 eggs.

[0016] Experimental materials: glucose, citric acid and sodium citrate, penicillin and streptomycin, tricarboxymethylaminomethane and Giemsa staining solution.

[0017] Semen collection and processing: Semen collection: Inject 2 / 3 volume of warm water at 38-40°C (simulating the temperature of a ewe's vagina) into the injection port of the artificial vagina. After closing the injection port, squeeze the artificial vagina to make the penile insertion end form a full triangle to ensure a good fit. Apply a thin layer of medical petroleum jelly (lubricant) evenly to the 1 / 3 of the insertion end of the artificial vagina to avoid damaging the ram's penis. Select a healthy ewe in estrus as the host ewe. The semen collector squats on the right rear side of the host ewe, holding the prepared artificial vagina with both hands and adjusting the angle to align with the direction of the ram's penis insertion. When the ram mounts the host ewe, quickly insert its penis into the artificial vagina. After the ram ejaculates by raising its head and lunging forward and slides off the host ewe, immediately remove the artificial vagina and keep it upright, allowing the semen to flow naturally into a pre-sterilized collection cup. Place the collection cup containing the semen in a 37°C insulated box and transport it to the laboratory for testing within 10 minutes.

[0018] Semen processing: Under constant temperature water bath conditions of 37 ℃, initial screening was performed by visual observation combined with microscopic examination, selecting semen that was milky white in color and appeared cloudy to the naked eye; sperm density and motility were detected by blood cell counting and smear method, respectively, and sperm with a density ≥1.5×10⁻⁶ were selected. 9 Semen with a viability of ≥0.8 mL and a viability of ≥0.8 was selected. Semen from five rams was mixed in equal volumes to reduce errors caused by individual differences.

[0019] Methods for evaluating the quality of frozen semen Viability determination: Take 10 μL of the intermediate layer semen and place it at the bottom of a preheated centrifuge tube. Dilute it with isothermal physiological saline at a ratio of 1:4. Place a drop of the intermediate layer semen onto a preheated glass slide, and cover it with a coverslip from left to right, taking care to avoid air bubbles. Observe the proportion of sperm with linear motility to the total sperm count under a 400x microscope.

[0020] Determination of acrosome integrity: After thawing, 5 μL of the middle layer of semen was dropped onto the left end of a glass slide to prepare a smear. After air drying at room temperature, it was fixed with formalin-phosphate fixative for 15 min, washed with distilled water, and air-dried. The smear was completely covered with Giemsa stain, and after 2 h, it was rinsed and air-dried. The acrosomes of the sperm were observed under a 1000x oil immersion microscope, and the number of sperm with intact acrosomes was counted.

[0021] Determination of plasma membrane integrity: 10 μL of the middle layer of thawed semen was added to 100 μL of HOST solution and incubated at 37 ℃ for 30 min. A drop of semen was placed on a glass slide, smeared with a coverslip at a 35° angle, and allowed to air dry for 7 min. The damage to the plasma membrane of 200 sperm cells was observed under a 400x microscope, and the proportion of sperm cells with curved tails was counted.

[0022] Antioxidant capacity indicators were determined using commercial reagent kits from Nanjing Jiancheng Bioengineering Institute. The malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, glutathione peroxidase (GPx) activity, catalase (CAT) activity, total antioxidant capacity (T-AOC), alkaline phosphatase (AKP) activity, and acid phosphatase (ACP) activity were measured.

[0023] TMT technology was used to perform proteomic sequencing on fresh sperm, frozen sperm, and frozen sperm with added ergothioneine to screen key proteins that lead to the decline in quality of cryopreserved sperm, and Western blot was used to verify the expression levels of ACAA2, HADHB, RAD50, and CHSY1 proteins.

[0024] Statistical analysis: Data were processed using Excel 2010, and one-way ANOVA was performed using SPSS 25.0. Results are expressed as mean ± standard error. P <0.05 indicates a significant difference. Example 1: Effect of different diluents on the quality of Lanzhou fat-tailed sheep semen after thawing

[0025] Semen diluents include basal solution, dilution I, and dilution II. Dilution I consists of 80% basal solution and 20% egg yolk. Dilution II is dilution I with 6% glycerol added. See Table 1 below for details.

[0026] Effects of different diluents on the quality of Lanzhou fat-tailed sheep semen after thawing: Semen was divided into 6 equal portions and diluted with different diluents (A, B, C, D, E, F). After dilution with solution I, the semen was wrapped in 8 layers of gauze and placed in a 4 ℃ freezer for 2 h to equilibrate. It was then diluted with isothermal solution II and equilibrated for another 2 h. The equilibrated semen was then filled into 0.25 mL frozen semen tubes at 4 ℃. For freezing, a float was first placed 4 cm above the liquid nitrogen surface for 8 min of fumigation, followed by fumigation of the frozen semen tubes on the float for 8 min, and then immersing them in liquid nitrogen for cryopreservation. Two tubes from each sample were thawed in a 39.5 ℃ water bath for 20 s, and relevant indicators were analyzed after thawing.

[0027] Table 1 Semen diluent formulation (100 mL) .

[0028] As shown in Table 2, the sperm motility of semen cryopreserved with dilution E was the highest after thawing, significantly higher than that of dilutions A, C, D, and F. P <0.05); while the sperm motility of dilution B was not significantly different from that of any of the experimental groups ( P >0.05); Semen cryopreserved with dilution E showed the highest acrosome integrity rate after thawing, and the acrosome integrity rate of semen cryopreserved with dilution E was significantly higher than that of dilution A. P <0.05), and there were no significant differences among the other groups ( P >0.05); the sperm plasma membrane integrity rate of semen cryopreserved with dilution E was significantly higher than that of semen with dilution A after thawing. P <0.05), and there were no significant differences among the other groups ( P >0.05). This indicates that using E diluent for cryopreservation of Lanzhou fat-tailed sheep semen is the most effective method.

[0029] Table 2. Effects of different diluent formulations on semen quality after thawing (n=5)

[0030] Note: Data in the same column with the same lowercase superscript indicates that the difference is not significant. P > 0.05 Different lowercase letters indicate significant differences. (P < 0.05) , the same as the table below.

[0031] The core function of semen diluents is to maintain sperm viability and fertilization activity in vitro by mimicking the in vivo microenvironment of sperm, reducing sperm density, and providing essential physiological support. The cryopreservation effect of sheep semen is significantly affected by breed differences, and specific cryopreservation procedures for local breeds are still insufficient. Lanzhou fat-tailed sheep, as a typical long-fat-tailed local sheep, may have differences in sperm plasma membrane lipid composition, metabolic patterns, and cryosensitivity compared to commonly used commercial sheep breeds, making it difficult to achieve stable thawing results with universal diluent systems. Therefore, this application optimizes the effects of different combinations of energy substrates and buffer systems on the quality of thawed semen. The results show that among the six diluents compared, diluent E, with a Tris-citric acid buffer system and glucose as the main energy substrate, significantly improved sperm viability, acrosome integrity, and plasma membrane integrity after thawing. This result indicates that for Lanzhou fat-tailed sheep, the diluent not only needs to provide energy support but also needs to maintain the stability of the sperm plasma membrane and enzyme system during cryopreservation stress. Glucose, as a readily available energy substrate in sheep sperm metabolism, can provide immediate energy for sperm motility and membrane repair via glycolysis, exhibiting superior energy supply efficiency compared to fructose during cryopreservation. In this application, the Tris-glucose combination is superior to fructose. The core function of semen diluent is to maintain sperm viability and fertilization activity in vitro by mimicking the in vivo microenvironment of sperm, reducing sperm density, and providing essential physiological support. The cryopreservation effect of sheep semen is significantly affected by breed differences, and specific cryopreservation procedures for local breeds are still clearly insufficient. Lanzhou fat-tailed sheep, as a typical long-fat-tailed local sheep, may have differences in sperm plasma membrane lipid composition, metabolic pathways, and cryosensitivity compared to commonly used commercial sheep breeds, making it difficult to achieve stable thawing results with a general diluent system. Therefore, this application optimizes the effects of different combinations of energy substrates and buffer systems on the quality of thawed semen. The results showed that, among the six diluents compared, diluent E, using a Tris-citric acid buffer system with glucose as the main energy substrate, significantly improved sperm viability, acrosome integrity, and plasma membrane integrity after thawing. This indicates that for Lanzhou fat-tailed sheep, the diluent not only needs to provide energy support but also needs to maintain the stability of the sperm plasma membrane and enzyme system during cryopreservation stress. Glucose, as a rapidly available energy substrate in sheep sperm metabolism, can provide immediate energy for sperm motility and membrane repair through glycolysis, and its effect is superior to that of fructose in energy supply efficiency during cryopreservation. In this application, the Tris-glucose combination is superior to fructose. The core function of semen diluents is to maintain sperm viability and fertilization activity in vitro by simulating the in vivo microenvironment of sperm, reducing sperm density, and providing necessary physiological support. The cryopreservation effect of sheep semen is significantly affected by breed differences, and specific cryopreservation procedures for local breeds are still significantly insufficient.Lanzhou fat-tailed sheep, a typical local breed, may exhibit differences in sperm plasma membrane lipid composition, metabolic pathways, and cryosensitivity compared to commonly used commercial sheep breeds. Universal diluent systems are insufficient to achieve stable thawing results. Therefore, this application optimizes the effects of different energy substrate and buffer system combinations on post-thawing semen quality. Results show that among the six diluents compared, diluent E, using a Tris-citric acid buffer system with glucose as the primary energy substrate, significantly improved post-thawing sperm viability, acrosome integrity, and plasma membrane integrity. This indicates that for Lanzhou fat-tailed sheep, the diluent not only needs to provide energy support but also needs to maintain the stability of the sperm plasma membrane and enzyme system during cryostress. Glucose, as a rapidly usable energy substrate in sheep sperm metabolism, can provide immediate energy for sperm motility and membrane repair through glycolysis, exhibiting superior energy supply efficiency compared to fructose during cryogenic freezing. In this application, the Tris-glucose combination is superior to fructose.

[0032] Furthermore, the Tris-citrate composite buffer system exhibited good protective effects in this application, which is related to its ability to maintain a stable pH and inhibit excessive sperm metabolic activation under low-temperature conditions. According to the buffer formulation of this application, excessively high or low pH can damage sperm enzyme activity and membrane stability. When the pH is too high, sperm metabolic activity increases, leading to rapid energy depletion and shortening survival time. During storage, acidic substances such as lactic acid and carbon dioxide produced by sperm metabolism gradually lower the pH value, causing sperm acidosis. This application optimizes the pH of the environment by constructing a buffer system in the diluent, reducing sperm energy consumption and extending storage time. Sodium citrate reduces excessive acidity or alkalinity caused by environmental changes, creating a relatively stable acid-base environment and preventing sperm death. In this application, the buffering capacity constructed using sodium citrate alone is limited, severely damaging sperm motility and integrity. The Tris-citrate combined buffer significantly improves sperm quality after thawing. Example 2: Effects of different freezing methods on the quality of Lanzhou fat-tailed sheep semen after thawing

[0033] After diluting the semen with diluent E, it was cooled to equilibrium using different methods (wrapped in 8 layers of gauze, cooled in a 170 mL water bath at 37 °C, cooled in a 200 mL water bath, cooled in a 230 mL water bath, and cooled in a 260 mL water bath). The equilibrated semen was then filled into 0.25 mL frozen semen tubes at 4 °C. The steps for freezing the frozen semen tubes in liquid nitrogen and thawing the samples were the same as in Example 1.

[0034] As shown in Table 3, the sperm motility was highest in the group treated with the 8-layer gauze wrapping cooling method after thawing, significantly higher than that in the 170 mL water bath cooling group, the 230 mL water bath cooling group, and the 260 mL water bath cooling group.P <0.05), but the survival rate was not significantly different from that of the 200 mL water bath cooling group ( P >0.05); the group treated with the 8-layer gauze wrapping cooling method had the highest acrosome integrity rate after semen thawing, which was significantly higher than the water bath cooling group. P <0.05); Among the water bath cooling groups, the 260 mL water bath cooling group had the lowest acrosome integrity rate, while the differences among the other groups were not significant. P >0.05); the plasma membrane integrity rate was highest in the 8-layer gauze wrapping cooling method group after thawing, significantly higher than that in the 170 mL water bath cooling method group, the 230 mL water bath cooling method group, and the 260 mL water bath cooling method group. P <0.05). Note: The 8-layer gauze wrapping and cooling method showed the best cryopreservation effect, followed by the 200 mL water bath cooling method.

[0035] Table 3. Effects of different cooling methods on the quality of thawed semen (n=5) .

[0036] It is worth noting that sheep sperm membranes have a low cholesterol content and a high ratio of polyunsaturated to saturated fatty acids. This characteristic makes sperm susceptible to damage from reactive oxygen species (ROS). Specifically, ROS combine with unsaturated fatty acids to form malondialdehyde (MDA), which can lead to structural and functional damage to the sperm membrane. Therefore, semen must undergo a period of acclimatization to relatively low temperatures before freezing, i.e., a semen equilibration process. Substances in the diluent enter the cells through the cell membrane, achieving ion balance and thus improving the sperm's tolerance to low-temperature freezing. The timing of this equilibration process is crucial: excessively long equilibration can cause sperm motility to naturally decline with prolonged in vitro storage, while excessively rapid equilibration can cause irreversible damage to sperm metabolism, motility, and fertilization capacity due to sudden temperature changes.

[0037] Currently, commonly used cooling and equilibration methods mainly include gauze wrapping cooling and water bath gradient cooling. Traditional methods often involve wrapping the sperm in gauze and placing it in a 4℃ refrigerator for 2-3 hours to allow for slow cooling and smooth transition of sperm across the sensitive low-temperature zone of 0-15℃. Water bath cooling achieves gradient cooling by adjusting the water bath volume. Existing technologies have compared cooling with different water bath volumes and 8-layer gauze wrapping methods, finding that 200 mL and 230 mL water baths significantly improved sperm motility and acrosome integrity after thawing compared to 150 mL, 100 mL, and 50 mL water baths and 8-layer gauze wrapping. However, in this application, the 8-layer gauze wrapping slow cooling method demonstrated the best results in freezing Lanzhou fat-tailed sheep semen, with significantly higher sperm motility, acrosome integrity, and plasma membrane integrity after thawing compared to most water bath gradient cooling treatments. These results indicate that, compared to exogenous cooling methods that regulate water bath volume, the gauze wrapping method provides a smoother and more continuous temperature transition, allowing the sperm plasma membrane sufficient time to complete lipid rearrangement and adapt to the low-temperature environment. This application found that Lanzhou fat-tailed sheep sperm have a narrow tolerance range for cooling rates; excessively rapid or uneven temperature changes are more likely to induce shock damage. Therefore, the optimal choice of cooling method is clearly breed-dependent. Example 3: Effect of different fumigation heights on the quality of Lanzhou fat-tailed sheep semen after thawing

[0038] Semen was diluted with E diluent and then equilibrated using an 8-layer gauze wrapping method for 4 hours. The equilibrated semen was then filled into 0.25 mL cryopreservation tubes at 4 °C. For freezing, floats were first placed at different heights (1 cm, 2.5 cm, 4 cm, 5.5 cm, 7 cm) above the liquid nitrogen surface for 8 minutes of fumigation. The cryopreservation tubes were then laid flat on the floats for 8 minutes of fumigation before being immersed in liquid nitrogen for cryopreservation. Two tubes from each sample group were thawed in a 39.5 °C water bath for 20 seconds, and relevant indicators were analyzed after thawing.

[0039] Table 4 shows that the sperm motility, acrosome integrity, and plasma membrane integrity were highest after thawing when the fumigation height was 4 cm, significantly higher than those of the fumigation height groups of 2.5 cm and 7 cm. P <0.05), with no significant difference compared to other groups ( P >0.05); Note: The optimal fumigation height for semen cryopreservation is 4 cm.

[0040] Table 4. Effects of different fumigation heights on semen quality after thawing. .

[0041] After equilibration, the freezing phase becomes even more critical in its impact on sperm survival due to temperature changes. Studies have shown that when the temperature drops rapidly from -5°C to -50°C during semen freezing, water molecules form ice crystals. While microcrystals cause minimal damage to sperm, large ice crystals directly disrupt the integrity of the sperm plasma membrane, exposing organelles to harmful external substances. This leads to mechanical damage such as chromosome breakage and enzyme inactivation, ultimately causing sperm death. Furthermore, the outward permeation of intracellular water molecules during cooling causes continuous sperm cell contraction and dehydration, further damaging the cell membrane structure. Therefore, controlling the cooling rate to reduce the formation of large ice crystals is crucial for minimizing freezing damage.

[0042] After reaching equilibrium, the semen enters a rapid cooling phase, during which it is highly susceptible to cell damage caused by intracellular and extracellular ice crystal formation and cell dehydration. Liquid nitrogen fumigation is currently the main method for cryopreservation of livestock and poultry semen. Its cooling rate is primarily determined by the fumigation height and time on the liquid nitrogen surface. The fumigation height and time during semen freezing should be controlled within specific ranges. According to the technical solution of this application, a fumigation height of 4 cm and a fumigation time of 8 minutes result in the best cooling rate. This result indicates that the cooling rate achieved at this height can reduce the formation of large intracellular ice crystals while avoiding excessive dehydration damage caused by slow cooling. If the fumigation height is too low, the cooling rate is too fast, easily causing mechanical damage from intracellular ice crystals; while if the fumigation height is too high, insufficient cooling may lead to irreversible hypothermic damage to the sperm before it enters the liquid nitrogen. Therefore, this application further determines that for Lanzhou fat-tailed sheep, a lower liquid nitrogen fumigation height is not necessarily better. For Lanzhou fat-tailed sheep, a fumigation height of 4 cm achieves a better balance between cooling rate and cell protection.

[0043] Preparation of diluent: The semen was diluted at a constant temperature in the basal diluent, which consisted of glucose (0.5044 g), citric acid (1.8252 g), tris(hydroxymethyl)aminomethane (Tris, 3.6342 g), penicillin and streptomycin (100,000 IU), 20% (volume fraction) egg yolk and 6% (volume fraction) glycerol per 100 mL.

[0044] Screening of ergothioneine concentration and dilution of semen: 0 mM, 4 mM, 6 mM and 8 mM of ergothioneine were added to the basal diluent. The ergothioneine was mixed with the basal diluent and stirred thoroughly to dissolve, thus preparing a cryo-diluent containing ergothioneine.

[0045] Semen dilution: The semen and the cryo-diluent with added ergothioneine were diluted twice at a ratio of 1:3. The diluted semen was wrapped in 8 layers of gauze and placed in a 4°C refrigerator for 4 hours to equilibrate. Then, a diluent with 6% glycerol content was added for further dilution.

[0046] Semen freezing and preservation: After secondary dilution, the semen was placed in a refrigerator for a period of time to equilibrate. Then, at 4 ℃, the semen was aliquoted into 0.25 mL cryopreservation tubes using a pipette. First, a float was placed 4 cm above the liquid nitrogen surface for 8 min of fumigation. Then, the cryopreservation tubes were laid flat on the float and fumigated for another 8 min before being immersed in liquid nitrogen for freezing and preservation.

[0047] Thawing of frozen semen: After 7 days, take 5 capillary tubes and thaw them in a water bath at 39.5 ℃ for 20 s. Take a drop of semen and observe its indicators under a microscope.

[0048] Determination of sperm antioxidant capacity: The levels of MDA, SOD, GSH-Px, CAT, T-AOC, AKP, and ACP enzymes in semen were determined using a kit, and the measurement procedures were performed according to the relevant kit instructions.

[0049] Protein expression: TMT technology was used to perform proteomic sequencing on fresh sperm, frozen sperm and frozen sperm with added ergothioneine, and the expression levels of ACAA2, HADHB, RAD50 and CHSY1 proteins were verified by Western blot.

[0050] This invention fully utilizes the mechanism of action of ergothioneine. Screening of ergothioneine concentrations showed that adding 8 mM ergothioneine to the diluent resulted in the best cryopreservation effect for Lanzhou fat-tailed sheep semen. 8 mM ergothioneine was added to the diluent for cryopreservation of Lanzhou fat-tailed sheep semen. The semen and diluent were diluted using a two-stage diluent method at a ratio of 1:3. At 4 ℃, the semen was pipetted into 0.25 mL frozen semen tubes, wrapped with 8 layers of gauze, and cooled for 4 h. The tubes were then placed on a floating plate 4 cm above the liquid nitrogen surface for 8 min of fumigation. Afterward, the tubes were cryopreserved in liquid nitrogen. After thawing, the sperm motility (45.80±3.62%), acrosome integrity (55.38±3.55%), and plasma membrane integrity (52.32±2.28%) in the experimental group were significantly higher than those in the control group. P <0.05); SOD, GPx activity and T-AOC were significantly increased ( P <0.05%, MDA content decreased; proteomics studies were conducted on sperm samples from the fresh semen group, the control frozen semen group, and the 8 mM ergothioneine frozen semen group. Results showed that cryopreservation caused significant changes in the sperm proteome of Lanzhou fat-tailed sheep. A total of 2958 differentially expressed proteins were identified between the fresh semen group and the control frozen semen group. P <0.05), GO and KEGG enrichment analyses revealed that the differentially expressed proteins were mainly enriched in metabolic processes, biological regulation, and JAK-STAT-related pathways; a total of 3002 differentially expressed proteins were identified between the fresh sperm group and the 8 mM ergothioneine frozen sperm group. P<0.05), GO and KEGG enrichment analyses revealed that the differentially expressed proteins were mainly enriched in metabolic processes, biological regulation, and AMPK-related pathways; a total of 46 differentially expressed proteins were identified between the control frozen sperm group and the 8 mM ergothioneine frozen sperm group. P <0.05), GO and KEGG enrichment analyses revealed that the differentially expressed proteins were mainly enriched in pathways related to metabolic processes, biological regulation, glycosaminoglycan biosynthesis, and homologous recombination. Bioinformatics analysis showed that the expression levels of RAD50 and CHSY1 proteins were significantly higher than those in the control group (…). P CHSY1 (<0.05) is located in the glycosaminoglycan biosynthesis-chondroitin sulfate pathway, reducing damage caused by reactive oxygen species. RAD50 is located in the non-homologous end joining repair and homologous recombination pathway, thus repairing sperm DNA loss. The coordinated action of RAD50 and CHSY1 reduces irreversible damage caused by sperm DNA breaks due to oxidative stress, reduces sperm death, and effectively protects sperm structure and function. Western blot validation showed that four differentially expressed proteins—ACAA2, HADHB, RAD50, and CHSY1—may serve as potential marker proteins, playing an important role in the regulation of sperm motility and quality after freeze-thaw cycles.

[0051] As shown in Table 5, the sperm motility was highest after thawing when the ergothioneine concentration in the diluent was 8 mM, significantly higher than that when the ergothioneine concentrations were 0 mM, 2 mM, 4 mM, and 6 mM. P The survival rate of <0.05 mM was not significantly different among 0 mM, 2 mM, 4 mM and 6 mM. p >0.05; the sperm acrosome integrity rate was highest after thawing when the ergothioneine concentration was 8 mM, significantly higher than that when the ergothioneine concentration was 0 mM and 2 mM. P＜0.05 The acrosome integrity rate was not significantly different from that at ergothioneine concentrations of 4 mM and 6 mM. P＞0.05 The sperm plasma membrane integrity rate was highest when the ergothioneine concentration was 8 mM, which was significantly higher than that when the ergothioneine concentrations were 0 mM, 2 mM, and 6 mM. （P＜0.05） The difference in plasma membrane integrity between the two concentrations was not significant when the ergothioneine concentration was 4 mM. P＞0.05） This indicates that the optimal sperm quality for cryopreservation is achieved when the ergothioneine concentration in the diluent is 8 mM. Adding 8 mM ergothioneine to the semen cryopreservation diluent can significantly improve the semen viability, acrosome integrity, and plasma membrane integrity of frozen semen from Lanzhou fat-tailed sheep after thawing, thus improving the semen preservation effect.

[0052] Table 5. Effects of different concentrations of ergothioneine on sperm quality after thawing. .

[0053] Through systematic optimization and combination of diluent formulation, cooling method, fumigation height, and ergothioneine concentration, this application not only achieves the superposition of the technical effects of each individual factor, but also generates a significant synergistic enhancement effect through complementary mechanisms. Specifically, this is manifested in: Synergistic improvement in structural integrity indicators: Sperm viability, acrosome integrity, and plasma membrane integrity were all significantly higher in the combined scheme than in any single-factor optimization group. P <0.05 indicates that different freezing methods have a positive interaction in protecting sperm structure.

[0054] Synergistic activation of the antioxidant system: The addition of ergothioneine combined with a gentle, slow cooling method involving wrapping the product in eight layers of gauze significantly enhanced the activity of sperm endogenous antioxidant enzymes (SOD, GPx) and total antioxidant capacity (T-AOC), while reducing the content of lipid peroxidation products (MDA), indicating that low temperature provides a better environment for the action of antioxidants.

[0055] Synergistic regulation of metabolic and repair pathways: Proteomics analysis showed that the combined approach could simultaneously regulate the expression of RAD50 and CHSY1 proteins, thereby activating the DNA damage repair factor MRN complex. The ATPase domains at both ends of RAD50 are crucial for maintaining the structural stability of the MRN complex and its ability to bind DNA, reducing oxidative stress damage to sperm. Chondroitin sulfate synthase 1 (CHSY1) participates in the biosynthesis of osteoin and dermatan sulfate glycosaminoglycans, assisting RAD50 in scavenging free radicals in the body, reducing oxidative stress damage to cells, improving sperm viability, sperm motility and velocity, and inhibiting spontaneous acrosome reactions to enhance sperm capacitation.

[0056] Therefore, the technical solution of this application does not simply add up the technical effects, but rather achieves a synergistic effect through systematic integration, with the triple synergistic effect of cryogenic environment control, metabolic support and oxidative protection.

[0057] like Figure 1 As shown, the MDA content gradually decreased with increasing ergothioneine concentration, and the sperm MDA content was lowest at an ergothioneine concentration of 8 mM, but the difference between the two groups was not significant. P >0.05). This indicates that ergothioneine added to the semen cryopreservation diluent has a tendency to reduce the MDA content in sperm.

[0058] like Figure 2 As shown, SOD activity gradually increased with increasing ergothioneine concentration, with the highest SOD activity in sperm at an ergothioneine concentration of 8 mM, significantly higher than that at an ergothioneine concentration of 0 mM. P<0.05), but the difference in SOD activity in sperm was not significant compared with that at ergothioneine concentrations of 2 mM, 4 mM and 6 mM ( P >0.05). This indicates that adding 8 mM ergothioneine to the semen cryopreservation diluent can significantly improve the SOD motility of frozen semen sperm. like Figure 3 As shown, the GPx motility in sperm was highest at an ergothioneine concentration of 8 mM, significantly higher than that at an ergothioneine concentration of 0 mM. P <0.05), and the activity of GPx was not significantly different from that of ergothioneine at concentrations of 2 mM, 4 mM, and 6 mM. P >0.05). This indicates that adding 8 mM ergothioneine to the semen cryopreservation diluent can significantly improve the motility of sperm GPx in frozen semen. like Figure 4 As shown, the addition of ergothioneine can increase the activity of CAT in sperm, with the highest CAT activity observed at a concentration of 8 mM, but the difference between the two groups was not significant. P >0.05). This indicates that ergothioneine added to the semen cryopreservation diluent has a tendency to enhance sperm CAT motility.

[0059] like Figure 5 As shown, the T-AOC activity of sperm after thawing was highest when the ergothioneine concentration was 8 mM, which was significantly higher than that of T-AOC at ergothioneine concentrations of 0 mM, 2 mM, and 4 mM. P <0.01), and the activity of T-AOC was not significantly different from that at a ergothioneine concentration of 6 mM. P >0.05); the activity of T-AOC at a ergothioneine concentration of 6 mM was significantly higher than that at ergothioneine concentrations of 0 mM and 2 mM. P <0.01); the activity of T-AOC at a 4 mM ergothioneine concentration was significantly higher than that at 0 mM and 2 mM ergothioneine concentrations (T-AOC activity <0.01); P <0.05); T-AOC activity was significantly higher at ergothioneine concentration of 2 mM than at ergothioneine concentration of 0 mM ( P <0.05). This indicates that adding 2-8 mM ergothioneine to the semen cryopreservation diluent can significantly improve the T-AOC motility of sperm after thawing of frozen semen from Lanzhou fat-tailed sheep.

[0060] Depend on Figure 6 It was found that the sperm AKP motility was highest at an ergothioneine concentration of 8 mM, but the difference between the two groups was not significant. P>0.05). This indicates that ergothioneine concentration of 8 mM tends to increase AKP motility in sperm.

[0061] Depend on Figure 7 It was found that the sperm ACP motility was highest at an ergothioneine concentration of 4 mM, and there was no significant difference compared with ergothioneine concentrations of 2 mM, 6 mM, and 8 mM. P >0.05), significantly higher than the ergothioneine concentration of 0 mM ( P <0.05). This indicates that the ACP activity in sperm cryopreserved at an ergothioneine concentration of 4 mM is the highest.

[0062] like Figure 8 As shown, the expression levels of ACAA2, HADHB, RAD50, and CHSY1 proteins were detected by Western blot, with β-actin as the internal reference protein. Relative quantification of the target proteins was performed. The expression levels of ACAA2 and HADHB proteins in the fresh sperm group were significantly higher than those in the control frozen sperm group and the 8 mM ergothioneine frozen sperm group. P <0.05), there was no significant difference between the two frozen sperm groups ( P >0.05); the expression levels of RAD50 and CHSY1 proteins in the 8 mM ergothioneine frozen sperm group were significantly higher than those in the control frozen sperm group and the fresh sperm group. P <0.05), the frozen semen group was significantly higher than the fresh semen group ( P <0.05), consistent with the proteomics detection results.

[0063] The above description is merely a preferred embodiment of the present invention, and the scope of protection of the present invention is not limited thereto. Any simple changes or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the scope of the technology disclosed in the present invention shall fall within the scope of protection of the present invention.

Claims

1. A method for improving the quality of frozen sheep semen, characterized in that, The method includes the following steps: The semen of Lanzhou fat-tailed sheep was isothermally diluted in a diluent containing 8 mM ergothioneine using a two-stage dilution method. The diluent was formulated as follows: 0.5044 g / 100 mL glucose, 1.8252 g / 100 mL citric acid, 3.6342 g / 100 mL Tris, 10 mL penicillin, 10 mL streptomycin, 20 v / v% egg yolk, 6 v / v% glycerol, pH 7.0 ± 0.

2. The concentration of penicillin and streptomycin was 100 IU / mL. Equilibrate at 4°C by wrapping it in 8 layers of gauze. After equilibration, the semen was placed 4 cm above the liquid nitrogen surface and fumigated for 8 minutes before being frozen for storage.

2. The method for improving the quality of frozen sheep semen according to claim 1, characterized in that, The semen was diluted with the diluent at a ratio of 1:3.

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