A method for improving quality of frozen semen of lanzhou big-tail sheep based on soybean lecithin

Abstract

The application relates to the field of agricultural biotechnology, in particular to a method for improving the quality of frozen semen of Lanzhou big-tailed sheep based on soybean lecithin. According to the technical scheme of the application, Tris-citric acid buffer containing 2% soybean lecithin is diluted with semen at a ratio of 1:3, then the semen is wrapped with 8 layers of gauze, cooled and balanced, placed at a distance of 4 cm from the liquid nitrogen surface, fumigated for 8 min, and then put into liquid nitrogen for cryopreservation, and the sperm quality is best after thawing.

Description

Technical Field

[0001] This invention belongs to the field of animal husbandry technology, specifically relating to a method for improving the quality of frozen semen from Lanzhou fat-tailed sheep based on soybean lecithin. 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 intensity of liquid nitrogen fumigation. Therefore, traditional diluents often contain added egg yolk to mitigate cryopreservation damage, but this method suffers from instability and carries the risk of disease transmission. Plant-derived soybean lecithin, due to its standardized composition and stable performance, offers a solution. ] Its advantages make it an effective substitute for egg yolk and help maintain the integrity of the sperm plasma membrane.

[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. Summary of the Invention

[0004] The purpose of this application is to provide a method for improving the quality of frozen semen from Lanzhou fat-tailed sheep.

[0005] The method for improving the quality of frozen semen from Lanzhou fat-tailed sheep according to this application includes the following steps; The semen of Lanzhou fat-tailed sheep was isothermally diluted in a buffer solution containing 2% soybean lecithin 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, 10 mL streptomycin, 20% egg yolk (v / v), 6% glycerol (v / v), and pH (7.0±0.2). The concentration of penicillin was 100 IU / mL, and the concentration of 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 and stored in liquid nitrogen.

[0006] The method for improving the quality of frozen semen from Lanzhou fat-tailed sheep according to this application is characterized in that the buffer solution containing 2% soybean lecithin is diluted with the semen from Lanzhou fat-tailed sheep at a ratio of 1:3. Beneficial technical effects of this application

[0007] 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 + 2% soybean lecithin) resulted in a sperm motility rate of 38.40±2.14%, an acrosome integrity rate of 45.60±1.21%, and a plasma membrane integrity rate of 50.00±2.28% after thawing. All indicators were significantly better than the traditional single optimized group, especially the motility rate, which was about 8% higher than the optimal single diluent group (E diluent). 2. This application achieves synergistic effects through system optimization and complementary mechanisms: E-diluent (Tris-citric acid-glucose system) provides stable pH and immediate energy; 8 layers of gauze wrapping for cooling achieve a smooth temperature transition and reduce cold shock; 4 cm fumigation height balances the cooling rate and reduces ice crystal damage; 2% soybean lecithin enhances membrane stability and reduces lipid peroxidation. The combined effect significantly improves all indicators compared to the sum of the individual optimization groups, especially in data stability and resistance to freezing damage. 3. This application addresses the structural characteristics of Lanzhou fat-tailed sheep semen membranes, which are characterized by low cholesterol and high polyunsaturated fatty acid content. By optimizing the buffer system, cooling curve, and membrane preservation method, it overcomes the bottleneck of unstable effects in traditional sheep semen cryopreservation methods, providing a repeatable and efficient technical solution for the cryopreservation of semen from local sheep breeds. Detailed Implementation

[0008] The technical solution of this application is described below with reference to specific embodiments.

[0009] In the following examples, the experimental animals used were: five medium-sized, sexually active Lanzhou fat-tailed sheep aged 2-3 years were selected in Yongjing County, Gansu Province, based on their breeding, pregnancy and lambing records. 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.

[0010] Experimental materials: glucose, citric acid and sodium citrate, penicillin and streptomycin, tricarboxymethylaminomethane and Giemsa staining solution.

[0011] 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.

[0012] Semen processing: Under constant temperature water bath conditions of 37 ℃, initial screening was carried out by visual observation combined with microscopic examination, selecting semen that was milky white in color and appeared cloudy to the naked eye; the density and motility of sperm were tested by blood cell counting and smear method, respectively, and qualified semen with sperm density ≥1.5×109 / mL and motility ≥0.8 was selected. The qualified semen from 5 rams were mixed in equal volume proportions to reduce the error caused by individual differences.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] 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

[0017] 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.

[0018] 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.

[0019] Table 1 Semen diluent formulation (100 mL) .

[0020] 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.

[0021] Table 2. Effects of different diluent formulations on semen quality after thawing (n=5)

[0022] 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.

[0023] 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, and its energy supply efficiency is superior to that of fructose during cryogenic freezing. In this application, the Tris-glucose combination is superior to fructose.

[0024] 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

[0025] 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.

[0026] 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 that of 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.

[0027] Table 3. Effects of different cooling methods on the quality of thawed semen (n=5) .

[0028] 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.

[0029] 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

[0030] 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.

[0031] 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.

[0032] Table 4. Effects of different fumigation heights on semen quality after thawing. .

[0033] 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.

[0034] 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. Example 4: Effects of different concentrations of soybean lecithin on the quality of Lanzhou fat-tailed sheep semen after thawing.

[0035] Different concentrations of soybean lecithin (0, 0.5%, 1%, 1.5%, 2%, 2.5%) were added to the E-base solution to dilute the semen. The semen was then equilibrated using an 8-layer gauze wrapping method for 4 hours. The equilibrated semen was then filled into 0.25 mL cryopreservation seminiferous tubes at 4 °C. The procedures for freezing the seminiferous tubes in liquid nitrogen and thawing the samples were the same as in Example 1.

[0036] As shown in Table 5, the semen viability of the group with 2% soybean lecithin added after thawing was the highest, significantly higher than that of the other experimental groups. P <0.05), among which the 0.5% soybean lecithin group had the lowest viability after thawing; the 2% soybean lecithin group had the highest acrosome integrity rate after thawing, which was not significantly different from the 1.0% soybean lecithin group and the 1.5% soybean lecithin group. P >0.05), significantly higher than the other experimental groups ( P<0.05), the acrosome integrity rate in the 0.5% soybean lecithin group; the semen plasma membrane integrity rate was highest in the 2% soybean lecithin group after thawing, and there was no significant difference compared with the 20% egg yolk, 1.0% soybean lecithin and 1.5% soybean lecithin groups. P >0.05), indicating that the sperm motility, acrosome integrity, and plasma membrane integrity were highest in the 2% soybean lecithin group after thawing.

[0037] Table 5. Effects of different concentrations of soybean lecithin on semen quality after thawing (n=5) .

[0038] As a non-permeable cryoprotectant, soy lecithin can increase the extracellular solution concentration, causing intracellular water molecules to flow out and reducing the formation of intracellular ice crystals. Simultaneously, its hydrophilic groups bind to water molecules within the sperm, forming a protective film on the sperm surface and increasing extracellular osmotic pressure, thus enhancing the sperm's resistance to freezing. During cryopreservation, the polyunsaturated fatty acids in the sperm plasma membrane bind with soy lecithin, serving as energy and bioactive substances to improve sperm viability. Therefore, adding soy lecithin during semen cryopreservation can prevent cryodamage and provide the nutrients needed for sperm metabolism.

[0039] However, this application found that soybean lecithin leads to increased viscosity of the diluent, increasing sperm motility resistance. At the same time, incompletely dispersed phospholipid particles also cause mechanical damage to the sperm plasma membrane. Therefore, when the concentration of soybean lecithin was further increased to 2.5%, the sperm viability, acrosome integrity rate and plasma membrane integrity rate decreased significantly, even lower than the experimental group without added soybean lecithin, indicating that soybean lecithin is negatively correlated with sperm viability.

[0040] According to the technical solution of this application, and in conjunction with the aforementioned technical improvements, the problem of a negative correlation between soybean lecithin and sperm motility can be solved. Adding 2% soybean lecithin to the E-basal solution significantly improves sperm motility and acrosome integrity after thawing, but has no significant effect on plasma membrane integrity. Soybean lecithin can reduce the risk of intracellular ice crystal formation by increasing extracellular osmotic pressure and promoting intracellular water outflow; simultaneously, its phospholipid components can embed into the sperm plasma membrane, enhancing membrane structural stability and mitigating lipid peroxidation damage caused by freezing stress.

[0041] The technical effect achieved in this application is not the result of a single independent factor, but rather the synergistic effect of multiple factors: "E diluent (Tris-citric acid-glucose system) + 8 layers of gauze wrapping for cooling + 4 cm fumigation height + 2% soybean lecithin". Functionally, the E diluent provides energy and buffering, the 8 layers of gauze wrapping provides gentle cooling, the 4 cm fumigation height optimizes the cooling rate, and the 2% soybean lecithin enhances membrane protection. Furthermore, the addition of 2% soybean lecithin resulted in more stable acrosome integrity. The freezing regimen of "E diluent + 8 layers of gauze wrapping for cooling + 4 cm fumigation height + 2% soybean lecithin" outperforms any single-factor group in terms of viability, acrosome integrity, and plasma membrane integrity.

[0042] The above embodiments are only used to understand the technical solutions of this application and do not limit the scope of protection of this application.

Claims

1. A method for improving the quality of frozen semen from Lanzhou fat-tailed sheep, characterized in that, The method includes the following steps; The semen of Lanzhou fat-tailed sheep was isothermally diluted in a buffer solution containing 2% soybean lecithin 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, 10 mL streptomycin, 20 v / v% egg yolk, 6 v / v% glycerol, pH 7.0 ± 0.2, with a penicillin concentration of 100 IU / mL and a streptomycin concentration of 100 IU / mL. Wrap it in 8 layers of gauze and equilibrate it at 4℃; 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 semen from Lanzhou fat-tailed sheep according to claim 1, characterized in that, The buffer solution containing 2% soybean lecithin was diluted with the semen of Lanzhou fat-tailed sheep at a ratio of 1:3.

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