A cooling diluent for 5°C cryopreservation of boar semen and its application.

Abstract

This invention discloses a cooling and diluting solution for preserving bovine semen at 5°C. Each liter contains: 27.5g D-glucose, 6.89g sodium citrate, 1g sodium bicarbonate, 2.35g EDTA disodium dihydrate, 2.9g citric acid, 5.65g Tris, 0.8g potassium chloride, 0.9g polyvinyl alcohol, 1g cholesterol-loaded cyclodextrin, 3.23g L-carnitine, and 11.4mg resveratrol. The pH is 7.2-7.3, and the osmotic pressure is 300-320 mOsm / kg. It is antibiotic-free. Semen is diluted with this solution and then stored at 5°C. The three components synergistically maintain sperm membrane structure, mitochondrial function, and antioxidant capacity, resulting in sperm motility ≥80% after 7 days and ≥70% after 14 days at 5°C. This solution is suitable for long-distance transport and artificial insemination.

Description

Technical Field

[0001] This invention relates to the field of low-temperature preservation technology for bovine semen, specifically to a cooling diluent for low-temperature preservation of bovine semen at 5°C and its application. Background Technology

[0002] Artificial insemination in pigs is one of the most widely used reproductive techniques in modern large-scale pig farming. The quality of boar semen directly affects sow conception rate, litter size, and farm productivity. Currently, boar semen is mainly preserved in liquid form, including room temperature (15-18℃), cryopreservation (4-5℃), and cryopreservation (-196℃). Cryopreservation can reduce sperm metabolic rate, decrease energy consumption, and effectively inhibit bacterial proliferation in semen, offering potential advantages such as extended preservation time, reduced risk of microbial contamination, and reduced reliance on antibiotics. However, boar sperm are highly sensitive to low temperatures. Their plasma membrane has a low cholesterol / phospholipid ratio and a high content of unsaturated fatty acids, making them prone to membrane lipid phase transitions and cold shock damage during cooling, leading to a significant decrease in sperm motility, membrane integrity, and fertilization capacity.

[0003] A search revealed that patent application number CN201610633712.6 discloses a diluent for the in vitro cryopreservation of boar semen and its preparation method. This diluent includes D-fructose, 2-amino-2-hydroxymethyl-1,3-propanediol, trisodium citrate, sodium bicarbonate, citric acid, disodium EDTA, N-acetylcysteine, skim milk powder, freeze-dried egg yolk powder, and broad-spectrum antibiotics and antibacterial agents such as amikacin sulfate. This technical solution is suitable for the in vitro preservation of fresh boar semen at 4-5℃. By adding protective components such as skim milk powder and freeze-dried egg yolk powder, as well as broad-spectrum antibiotics, it aims to inhibit damage to sperm structure during cryopreservation, maintain the glycogen metabolism and oxidative phosphorylation processes of boar sperm, and thus extend the survival time of sperm during cryopreservation.

[0004] However, this diluent still has the following shortcomings: First, it relies on antibiotics to control bacterial proliferation, which may lead to problems such as antibiotic dosage reduction and drug resistance; second, it mainly relies on dairy products and egg yolk components for protection, and lacks a systematic protective strategy targeting the lipid homeostasis, fatty acid oxidation metabolism, and oxidative stress of porcine sperm membranes; third, the overall preservation effect is limited, and sperm motility decreases significantly in the later stages of preservation, making it difficult to achieve long-term effective preservation for more than 7 days at 5°C. Summary of the Invention

[0005] (a) Technical problems to be solved To address the shortcomings of existing technologies, this invention provides a cooling diluent for the low-temperature preservation of bovine semen at 5°C and its application.

[0006] (II) Technical Solution This invention provides a cooling diluent for the low-temperature preservation of bovine semen at 5°C and its application.

[0007] On the one hand, the present invention provides a cooling diluent for the low-temperature preservation of bovine semen at 5°C.

[0008] The cooling diluent consists of the following components: D-glucose 27.5 g / L, sodium citrate 6.89 g / L, sodium bicarbonate 1 g / L, disodium EDTA dihydrate 2.35 g / L, citric acid 2.9 g / L, Tris 5.65 g / L, potassium chloride 0.8 g / L, polyvinyl alcohol 0.9 g / L, cholesterol-loaded cyclodextrin 1 g / L, L-carnitine 3.23 g / L, resveratrol 11.4 mg / L.

[0009] The pH value of the cooling diluent is 7.2-7.3, and the osmotic pressure is 300-320 mOsm / kg.

[0010] No antibiotics were added to the cooling diluent.

[0011] The cooling diluent is sterilized by filtration through a 0.22 μm filter membrane, dispensed, and stored at 4°C for later use.

[0012] Furthermore, the cholesterol-loaded cyclodextrin is a complex formed by cholesterol and methyl-β-cyclodextrin, and its preparation method includes: dissolving cholesterol in chloroform, dissolving methyl-β-cyclodextrin in methanol, mixing and drying with nitrogen to obtain crystalline powder.

[0013] The L-carnitine mentioned is L-carnitine.

[0014] The resveratrol is a natural extract or chemically synthesized product with a purity of ≥98%.

[0015] The degree of polymerization of the polyvinyl alcohol is 1700-1800.

[0016] Furthermore, the cholesterol-loaded cyclodextrin, as a membrane lipid stabilizing component, supplements cholesterol by embedding into the sperm plasma membrane, reduces the membrane lipid phase transition temperature and cold shock damage, and maintains the integrity of the sperm plasma membrane, the cholesterol / phospholipid ratio, and membrane fluidity.

[0017] The L-carnitine, as a fatty acid oxidation support component, promotes β-oxidation by transporting long-chain fatty acids into the mitochondrial matrix, and maintains mitochondrial membrane potential and ATP production levels under low temperature conditions.

[0018] Resveratrol, as an antioxidant protective component, protects sperm membrane lipids and proteins from oxidative damage by scavenging reactive oxygen free radicals and reducing the level of lipid peroxidation products.

[0019] The three components work synergistically to ensure that the total sperm motility of the cooled diluent is ≥80% after 7 days of storage at 5°C and ≥70% after 14 days. Furthermore, the sperm acrosome integrity, mitochondrial membrane potential, and ATP content are significantly better than those of the conventional diluent under the same low-temperature conditions during storage.

[0020] On the other hand, the present invention provides a method for preserving boar semen at 5°C using the above-mentioned cooling diluent, comprising the following steps: Step (1) Semen collection: Semen from healthy adult boars is collected by hand gripping. During the collection process, the gelatinous substance is completely removed, and urine, blood, purulent secretions or other impurities are strictly avoided from causing contamination. The fresh semen is immediately placed in a constant temperature environment of 37°C for temporary storage, and the preliminary quality assessment is completed within 30 minutes after collection. Step (2) Quality assessment: Fresh semen was tested for sperm motility, sperm density and abnormality rate. Sperm motility was objectively assessed using a computer-aided sperm analysis system, and abnormality rate was examined morphologically using eosin-aniline black staining. Only semen with total sperm motility ≥90%, sperm abnormality rate ≤20%, and a milky white appearance without obvious odor or abnormal color was selected for subsequent low-temperature preservation. Step (3) Isothermal dilution: After removing the above-mentioned cooling diluent from its 4℃ storage environment, preheat it in a 37℃ water bath to the same temperature as fresh semen, ensuring that the temperature difference between the diluent and the semen does not exceed 0.5℃. Then, slowly add the preheated cooling diluent along the container wall to the fresh semen, mixing gently by rotating as you add it, avoiding violent shaking that could generate bubbles or mechanical shear force, to accurately dilute the semen density to 3.0 × 10⁻⁶. 7 ~3.5×10 7 The concentration is calculated per mL, where the dilution factor is determined based on the actual density of fresh semen. Step (4) Programmed cooling: The diluted semen sample is first placed in a constant temperature chamber pre-equilibrated to 25°C and left to stand for 2 hours to allow the sperm to gradually adapt to the mild cooling environment; then the semen is transferred to a constant temperature chamber pre-equilibrated to 17°C and left to stand for 1 hour to complete the transition from room temperature to 17°C; then the semen is placed in a constant temperature device with an ambient temperature of 10°C and cooled to 10°C at a uniform rate within 3 hours, at which time the cooling rate is controlled at 2-3°C per hour; finally, the semen is transferred to a 5°C refrigerator and cooled to 5°C at a uniform rate within 4 hours, at which time the cooling rate is controlled at 1-1.5°C per hour; after the entire cooling process is completed, the semen is continuously stored in a constant temperature environment of 5°C ± 0.5°C for subsequent long-term preservation or transportation; Step (5) Management during storage: During the entire cooling process described in step (4), the semen is gently inverted and mixed once every 1 hour to prevent the sperm from settling naturally and causing excessive local density; During long-term storage at 5℃, the semen is gently inverted and mixed once every 12 to 24 hours, and the mixing action lasts for 30 to 60 seconds to ensure that the sperm are evenly suspended; No antibiotics are added, no liquid is changed, and no centrifugation is performed during the entire cooling and storage process. Step (6) Warming operation: Before artificial insemination or experimental testing, take the cryopreserved semen out of the 5°C environment and immediately place it in a 37°C water bath for constant temperature incubation for 15-20 minutes. After incubation, invert and mix 3-5 times to fully restore the semen temperature to 37°C. The warmed semen should be used within 1 hour. Vigorous shaking or high-speed mixing is prohibited during the warming process.

[0021] Furthermore, in step (3), the cooling diluent is used for dilution immediately after being preheated to 37°C, and the entire dilution operation is completed within 2 hours after semen collection; after the cooling diluent is prepared, it is sealed and stored in the dark at 4°C for no more than 7 days. Diluent exceeding the storage period shall not be used for semen preservation operations; before use, the diluent must be checked for any precipitation, turbidity or discoloration, and it can only be used after confirming that there are no abnormalities.

[0022] Furthermore, the cooling process described in step (4) is carried out entirely in a constant temperature device, and the temperature deviation of the constant temperature device is controlled within ±0.5℃. During the cooling process from 17℃ to 10℃, the cooling rate is strictly controlled at 2 to 3℃ per hour, and sudden drops or temperature rebounds are prohibited. During the cooling process from 10℃ to 5℃, the cooling rate is strictly controlled at 1 to 1.5℃ per hour. This stage adopts a gradual cooling method, and the actual temperature is recorded every 30 minutes to ensure that the cooling curve is smooth and continuous. If abnormal temperature fluctuations occur during the cooling process, the cooling should be stopped immediately and the operating status of the constant temperature device should be checked. The cooling operation should be continued after the temperature stabilizes.

[0023] Furthermore, in steps (4) and (5), the 5℃ long-term storage environment is a medical-grade refrigerator or constant temperature incubator, with the storage temperature set at 5℃±0.5℃. The storage environment has real-time temperature monitoring and alarm functions. During storage, the semen sample is placed in a sealed sterile container, with a small amount of air space reserved at the top of the container to maintain gas exchange. The mixing operation is performed manually and gently inverted every 12 to 24 hours, with each mixing lasting 30 to 60 seconds. After mixing, the semen is immediately returned to the 5℃ storage environment to minimize the exposure time of the semen at room temperature. No antibiotics are added during storage, no liquid is changed, no centrifugation is performed, and no additional oxygen or carbon dioxide gas is added.

[0024] Further, in step (6), the rewarming operation is initiated within 30 minutes before artificial insemination or experimental testing. The rewarming process is carried out in a constant temperature water bath at 37°C, and the temperature deviation of the water bath is controlled within ±0.5°C. The semen container is kept in a semi-submerged state during the incubation process to ensure that the semen is heated evenly. The incubation time is 15 to 20 minutes. After the rewarming is completed, the sperm motility is immediately tested using a computer-aided sperm analysis system. Only after ensuring that the total sperm motility is ≥70% can the semen be used for artificial insemination. If the total sperm motility is lower than 70% after rewarming, the batch of semen shall not be used for artificial insemination. The rewarmed semen shall be used up within 1 hour. Any remaining semen that is not used within 1 hour shall be discarded and shall not be repeatedly cooled and stored.

[0025] Furthermore, when the above method is used to preserve bovine semen at 5°C for 7 days, the total sperm motility is ≥80%, the progressive sperm motility is ≥60%, the sperm plasma membrane integrity is ≥70%, the sperm acrosome integrity is ≥65%, and the normal rate of sperm mitochondrial membrane potential is ≥60%. When preserved for 14 days, the total sperm motility is ≥70%, the progressive sperm motility is ≥50%, the sperm plasma membrane integrity is ≥60%, the sperm acrosome integrity is ≥55%, and the normal rate of sperm mitochondrial membrane potential is ≥50%. Compared with conventional Modena basal dilution at 17°C, when the method of this invention is used to preserve semen at 5°C for 14 days, there is no significant difference in total sperm motility and progressive sperm motility, while the bacterial proliferation level in the semen is reduced by at least two orders of magnitude, and no antibiotics are required.

[0026] In another aspect, the present invention provides the application of the above-mentioned cooling diluent in the low-temperature preservation of boar semen at 5°C, characterized in that the application enables the total sperm motility of boar semen to be ≥80% after 7 days of preservation at 5°C and ≥70% after 14 days of preservation, and no antibiotics need to be added during the preservation process.

[0027] (III) Beneficial Effects Compared with the prior art, the present invention provides a cooling diluent for the low-temperature preservation of bovine semen at 5°C and its application, which has the following beneficial effects: 1. This invention significantly inhibits bacterial proliferation in porcine semen by using a low-temperature environment of 5°C, reducing bacterial levels during storage by at least two orders of magnitude compared to storage at room temperature of 17°C. This effectively controls microbial risks without the need to add any antibiotics, thus reducing reliance on antimicrobial drugs.

[0028] 2. This invention stabilizes the cholesterol homeostasis of the sperm plasma membrane by loading cholesterol cyclodextrin, reduces the membrane lipid phase transition temperature and cold shock damage, significantly maintains the integrity of the sperm plasma membrane, cholesterol / phospholipid ratio and membrane fluidity during low-temperature preservation, and effectively protects the sperm membrane structure.

[0029] 3. This invention promotes the entry of long-chain fatty acids into mitochondria for β-oxidation by L-carnitine, maintaining mitochondrial membrane potential and ATP production levels under low temperature conditions, effectively solving the problems of sperm energy metabolism disorders and decreased motility during cryopreservation.

[0030] 4. This invention uses resveratrol to scavenge reactive oxygen free radicals and reduce the level of lipid peroxidation products, which significantly reduces oxidative stress damage induced by low temperature preservation, protects sperm membrane lipids and proteins from oxidative damage, and improves sperm antioxidant capacity.

[0031] 5. Through the synergistic effect of the three components, this invention enables the total sperm motility of boar semen to be ≥80% after 7 days of storage at 5℃ and ≥70% after 14 days of storage, significantly extending the low-temperature storage period of boar semen and meeting the needs of long-distance transportation and expanding the reach of artificial insemination. Attached Figure Description

[0032] Figure 1 This is a comparison chart of the detection results of membrane lipid-related indicators in porcine semen from different treatment groups according to the present invention. In the chart, A is the detection result of the proportion of live sperm with acrosome integrity, B is the detection result of the cholesterol / phospholipid ratio, and C is the detection result of the membrane fluidity.

[0033] Figure 2 This is a comparison chart of the detection results of bacterial proliferation levels in bovine semen from different treatment groups according to the present invention.

[0034] Figure 3 This is a comparison chart of the detection results of oxidative stress levels in bovine semen from different treatment groups according to the present invention, where A is the detection result of reactive oxygen species level and B is the detection result of lipid peroxidation level.

[0035] Figure 4 This is a comparison of the detection results of mitochondrial function and energy levels in porcine sperm from different treatment groups according to the present invention. In the figure, A is the detection result of ATP level and B is the detection result of high mitochondrial membrane potential level.

[0036] Figure 5 This is a comparison chart of the changes in sperm motility in bovine semen from different treatment groups after 1 to 14 days of preservation, where A is the trend of total sperm motility and B is the trend of progressive sperm motility. Detailed Implementation

[0037] The present invention will be further described below through specific embodiments, but the present invention is not limited to the following embodiments. The boar semen used was all from a pig farm in Jimo District, Qingdao City, Shandong Province. Semen was collected from a total of 10 healthy adult Landrace boars aged 2-3 years using the hand-holding method. After quality assessment, only semen with total sperm motility ≥90% and sperm deformity rate ≤20% was selected for the experiment.

[0038] Example 1 The cooling dilution solution and its preservation method of this invention correspond to group C. 1. Preparation of cooling diluent (1) Preparation of cholesterol-loaded cyclodextrin: Weigh 200 mg of cholesterol and add it to 1 mL of chloroform. Shake thoroughly to dissolve it completely to obtain a cholesterol chloroform solution. Weigh 1 g of methyl-β-cyclodextrin and add it to 2 mL of methanol. Shake thoroughly to dissolve it completely to obtain a methyl-β-cyclodextrin methanol solution. Take 0.45 mL of the cholesterol chloroform solution and slowly add it dropwise to the methyl-β-cyclodextrin methanol solution. Shake continuously during the dropwise addition to ensure that the cholesterol and methyl-β-cyclodextrin are fully mixed and form a complex. Place the mixture in a fume hood and use a nitrogen blower to slowly dry it with nitrogen gas to remove chloroform and methanol until crystals precipitate. Store the prepared cholesterol-loaded cyclodextrin powder in a dark and dry environment.

[0039] (2) Preparation of base solution: Weigh 27.5g of D-glucose, 6.89g of sodium citrate, 1g of sodium bicarbonate, 2.35g of disodium ethylenediaminetetraacetate dihydrate, 2.9g of citric acid, 5.65g of Tris and 0.8g of potassium chloride, add them to about 800mL of ultrapure water and stir thoroughly until completely dissolved.

[0040] (3) Addition of functional protective ingredients: Add 0.9g of polyvinyl alcohol, 1g of cholesterol-loaded cyclodextrin, 3.23g of L-carnitine and 11.4mg of resveratrol to the base solution and mix thoroughly.

[0041] (4) pH and osmotic pressure adjustment: Adjust the pH to 7.2~7.3 with citric acid or sodium citrate, bring the volume to 1L with ultrapure water, and test and adjust the osmotic pressure to 300~320mOsm / kg.

[0042] (5) Filtration and sterilization: The prepared cooling dilution solution is filtered through a 0.22μm filter membrane for sterilization, and then packaged and sealed and stored in the dark at 4℃.

[0043] 2. Method for preserving boar semen at 5°C (1) Semen collection: Semen from healthy adult boars was collected by hand gripping. During the collection process, the gelatinous substance was completely removed, and urine, blood, purulent secretions or other impurities were strictly avoided from causing contamination. The fresh semen was immediately placed in a constant temperature environment of 37°C for temporary storage. The preliminary quality assessment was completed within 30 minutes after collection.

[0044] (2) Quality assessment: Fresh semen was tested for sperm motility, sperm density, and sperm abnormality rate. Sperm motility was objectively assessed using a computer-aided sperm analysis system, and sperm abnormality rate was examined morphologically using the eosin-aniline black staining method. Semen with total sperm motility ≥90%, sperm abnormality rate ≤20%, and a milky white appearance without obvious odor or abnormal color was selected for subsequent low-temperature preservation.

[0045] (3) Isothermal dilution: Remove the above-mentioned cooling diluent from its 4℃ storage environment and preheat it in a 37℃ water bath to the same temperature as fresh semen, ensuring that the temperature difference between the diluent and the semen does not exceed 0.5℃. Slowly add the preheated cooling diluent along the container wall to the fresh semen, mixing gently by rotating as you add it, avoiding violent shaking that could generate air bubbles or mechanical shear force, to accurately dilute the semen density to 3.0 × 10⁻⁶. 7 ~3.5×10 7 The concentration of semen samples was 1 / mL, and the dilution operation was completed within 2 hours after semen collection.

[0046] (4) Programmed cooling: The diluted semen sample was first placed in a constant temperature incubator pre-equilibrated to 25°C and left to stand for 2 hours to allow the sperm to gradually adapt to the mild cooling environment. Then, the semen was transferred to a constant temperature incubator pre-equilibrated to 17°C and left to stand for 1 hour to complete the transition from room temperature to 17°C. Subsequently, the semen was placed in a constant temperature device with an ambient temperature of 10°C and cooled to 10°C at a uniform rate over 3 hours, with the cooling rate controlled at 2-3°C per hour. Finally, the semen was transferred to a 5°C refrigerator and cooled to 5°C at a uniform rate over 4 hours, with the cooling rate controlled at 1-1.5°C per hour. Throughout the cooling process, the semen was gently inverted and mixed once every 1 hour to prevent sperm sedimentation. After the cooling process was completed, the semen was continuously stored in a constant temperature environment of 5°C ± 0.5°C.

[0047] (5) Management during storage: During long-term storage at 5℃, the semen should be gently inverted and mixed every 12-24 hours, each time for 30-60 seconds, to ensure uniform suspension of sperm. No antibiotics should be added, no liquid should be changed, and no centrifugation should be performed during the entire cooling and storage process.

[0048] (6) Warming operation: Before artificial insemination or experimental testing, take the low-temperature preserved semen out of the 5℃ environment and immediately place it in a 37℃ water bath for constant temperature incubation for 15~20min. After incubation, invert and mix 3~5 times to completely restore the semen temperature to 37℃. The warmed semen should be used within 1 hour.

[0049] Comparative Example 1 Use Modena diluent and store at 17°C (Group A) 1. Preparation of diluent: Weigh 27.5g D-glucose, 6.89g sodium citrate, 1g sodium bicarbonate, 2.35g disodium EDTA dihydrate, 2.9g citric acid, 5.65g Tris, and 0.8g potassium chloride. Add to ultrapure water and bring the volume to 1L. Adjust the pH to 7.2-7.3 and the osmotic pressure to 300-320 mOsm / kg. Add conventional antibiotics, filter through a 0.22μm filter membrane for sterilization, and store at 4℃. This diluent is free of polyvinyl alcohol, cholesterol-loaded cyclodextrin, L-carnitine, and resveratrol.

[0050] 2. Preservation method: Semen collection and quality assessment standards are the same as in Example 1. The above-mentioned diluent was preheated to 37°C and then diluted isothermally with fresh semen to a semen density of 3.0 × 10⁻⁶. 7 ~3.5×10 7 The diluted semen was stored in a 17°C incubator, and mixed every 12-24 hours. No programmed cooling was performed during storage.

[0051] Comparative Example 2 Use Modena diluent and store at 5°C, corresponding to group B. 1. Preparation of diluent: Same as comparative example 1.

[0052] 2. Preservation method: Semen collection and quality assessment standards are the same as in Example 1. The above-mentioned diluent was preheated to 37°C and then diluted isothermally with fresh semen to a semen density of 3.0 × 10⁻⁶. 7 ~3.5×10 7 The diluted semen was stored directly in a 5°C refrigerator without programmed cooling, and mixed every 12-24 hours.

[0053] Effect comparison test The semen samples obtained from Example 1, Comparative Example 1, and Comparative Example 2 were labeled as Group C, Group A, and Group B, respectively. During the storage period, various indicators were tested, and the results are as follows.

[0054] 1. Detection of membrane lipid-related indicators, such as... Figure 1 As shown; On day 7 of storage, the acrosome integrity, cholesterol / phospholipid ratio, and membrane fluidity of sperm in each group were measured. The results showed that group B had significantly reduced acrosome integrity, cholesterol / phospholipid ratio, and membrane fluidity, indicating that simply using the conventional diluent for 5°C cryopreservation significantly disrupted the membrane lipid homeostasis of porcine sperm and caused acrosome structural damage. Group C, under 5°C storage conditions, maintained a high acrosome integrity, cholesterol / phospholipid ratio, and membrane fluidity, with significantly higher results than group B and close to or higher than group A. These results demonstrate that the diluent of this invention can effectively alleviate the disruption of porcine sperm membrane lipid structure and acrosome damage during cryopreservation, and has a significant effect on maintaining sperm membrane structural stability.

[0055] 2. Detection of bacterial proliferation levels, such as... Figure 2 As shown; On day 7 of storage, the bacterial proliferation level in the semen of each group was measured. The results showed that the bacterial proliferation level in group A semen (calculated as log10 CFU / mL) was significantly higher than that in groups B and C, while groups B and C were at lower levels. This indicates that low-temperature storage at 5℃ can effectively inhibit bacterial proliferation in porcine semen. Compared with room temperature storage at 17℃, low-temperature storage can significantly reduce the risk of microbial growth and is beneficial in reducing the damage of bacterial metabolites to sperm.

[0056] 3. Oxidative stress level detection, such as Figure 3 As shown; On day 7 of storage, the levels of reactive oxygen species (ROS) and lipid peroxidation in sperm were measured in each group. The results showed that the levels of ROS and lipid peroxidation in group B porcine sperm were significantly elevated, indicating that ordinary cryopreservation induces significant oxidative stress and membrane lipid peroxidation damage. The levels of ROS and lipid peroxidation in group C were significantly lower than those in group B and approached those in group A. These results demonstrate that resveratrol in the diluted solution of this invention can effectively scavenge or reduce excess ROS generated during cryopreservation and alleviate membrane lipid peroxidation damage.

[0057] 4. Mitochondrial function and energy level testing, such as... Figure 4 As shown; On day 7 of storage, the ATP levels and high mitochondrial membrane potential ratios of sperm in each group were measured. The results showed that the ATP levels and high mitochondrial membrane potential ratios of porcine sperm in group B were significantly lower than those in group A, indicating that storage at 5°C using the basal Modena diluent leads to decreased mitochondrial function and insufficient energy supply in porcine sperm. The ATP levels and high mitochondrial membrane potential ratios of porcine sperm in group C were significantly higher than those in group B, and were also higher than or close to those in group A. These results demonstrate that the L-carnitine in the diluent of this invention can effectively maintain mitochondrial function and improve ATP production capacity in porcine sperm during cryopreservation.

[0058] 5. Sperm motility and progressive motility testing, such as... Figure 5 As shown; The total sperm motility and progressive motility of each group were measured on days 1, 3, 5, 7, 10, and 14 of storage. The results showed that the total sperm motility and progressive motility of all three groups of boar sperm decreased with prolonged storage. Group B showed the most significant decrease, with both total motility and progressive motility significantly lower than groups A and C in the later stages of storage. Group C maintained good total sperm motility and progressive motility during storage at 5℃ for 1–14 days, with total sperm motility ≥80% on day 7 and ≥70% on day 14. Its motility was significantly better than group B, indicating that the diluent of this invention can effectively delay the decline in sperm motility during low-temperature storage and improve the stability of boar semen during low-temperature storage.

[0059] In summary, while using the basic Modena diluent for cryopreservation at 5°C can reduce bacterial proliferation, it also disrupts sperm membrane lipid homeostasis, decreases acrosome integrity, increases reactive oxygen species and lipid peroxidation levels, reduces mitochondrial membrane potential, and leads to insufficient ATP supply, ultimately resulting in a significant decrease in overall sperm motility and forward motility. In contrast, the novel cryopreservation diluent of this invention, under 5°C storage conditions, can simultaneously maintain sperm membrane lipid structure stability, reduce oxidative stress damage, protect mitochondrial function, and increase ATP levels, thus significantly improving the cryopreservation effect of porcine semen. This results in sperm motility ≥80% after 7 days of storage at 5°C and ≥70% after 14 days, without the need for the addition of any antibiotics during the storage process.

[0060] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A cooling diluent for preserving bovine semen at 5°C, characterized in that, The cooling diluent consists of the following components: D-glucose 27.5 g / L, sodium citrate 6.89 g / L, sodium bicarbonate 1 g / L, disodium EDTA dihydrate 2.35 g / L, citric acid 2.9 g / L, Tris 5.65 g / L, potassium chloride 0.8 g / L, polyvinyl alcohol 0.9 g / L, cholesterol-loaded cyclodextrin 1 g / L, L-carnitine 3.23 g / L, resveratrol 11.4 mg / L; The pH value of the cooling diluent is 7.2-7.3, and the osmotic pressure is 300-320 mOsm / kg; No antibiotics were added to the cooling diluent; The cooling diluent is sterilized by filtration through a 0.22 μm filter membrane, dispensed, and stored at 4°C for later use.

2. The cooling and diluting solution for preserving bovine semen at 5°C according to claim 1, characterized in that: The cholesterol-loaded cyclodextrin is a complex formed by cholesterol and methyl-β-cyclodextrin, and its preparation method includes: Cholesterol was dissolved in chloroform, and methyl-β-cyclodextrin was dissolved in methanol. The mixture was then dried under nitrogen to obtain a crystalline powder. The L-carnitine mentioned is L-carnitine; The resveratrol is a natural extract or chemically synthesized product with a purity of ≥98%; The degree of polymerization of the polyvinyl alcohol is 1700-1800.

3. The cooling and diluting solution for preserving bovine semen at 5°C according to claim 2, characterized in that: The cholesterol-loaded cyclodextrin, as a membrane lipid stabilizing component, supplements cholesterol by embedding into the sperm plasma membrane, reduces the membrane lipid phase transition temperature and cold shock damage, and maintains the integrity of the sperm plasma membrane, the cholesterol / phospholipid ratio, and membrane fluidity. The L-carnitine, as a fatty acid oxidation support component, promotes β-oxidation by transporting long-chain fatty acids into the mitochondrial matrix, and maintains mitochondrial membrane potential and ATP production levels under low temperature conditions. The resveratrol, as an antioxidant protective component, protects sperm membrane lipids and proteins from oxidative damage by scavenging reactive oxygen free radicals and reducing the level of lipid peroxidation products. The three components work synergistically to ensure that the total sperm motility of the cooled diluent is ≥80% after 7 days of storage at 5°C and ≥70% after 14 days. Furthermore, the sperm acrosome integrity, mitochondrial membrane potential, and ATP content are significantly better than those of the conventional diluent under the same low-temperature conditions during storage.

4. A method for cryopreserving boar semen at 5°C using the cooling diluent according to any one of claims 1 to 3, characterized in that, Includes the following steps: Step (1) Semen collection: Semen from healthy adult boars is collected by hand gripping. During the collection process, the gelatinous substance is completely removed, and urine, blood, purulent secretions or other impurities are strictly avoided from causing contamination. The fresh semen is immediately placed in a constant temperature environment of 37°C for temporary storage, and the preliminary quality assessment is completed within 30 minutes after collection. Step (2) Quality assessment: Fresh semen was tested for sperm motility, sperm density and abnormality rate. Sperm motility was objectively assessed using a computer-aided sperm analysis system, and abnormality rate was examined morphologically using eosin-aniline black staining. Only semen with total sperm motility ≥90%, sperm abnormality rate ≤20%, and a milky white appearance without obvious odor or abnormal color was selected for subsequent low-temperature preservation. Step (3) Isothermal dilution: After removing the cooling diluent according to any one of claims 1 to 3 from the 4°C storage environment, preheat it in a 37°C water bath to the same temperature as fresh semen, ensuring that the temperature difference between the diluent and the semen does not exceed 0.5°C. Then, slowly add the preheated cooling diluent along the container wall to the fresh semen, mixing gently by rotating as you add it, avoiding violent shaking that could generate bubbles or mechanical shear force, to accurately dilute the semen density to 3.0 × 10⁻⁶. 7 ~3.5×10 7 The concentration is calculated per mL, where the dilution factor is determined based on the actual density of fresh semen. Step (4) Programmed cooling: The diluted semen sample is first placed in a constant temperature chamber pre-equilibrated to 25°C and left to stand for 2 hours to allow the sperm to gradually adapt to the mild cooling environment; then the semen is transferred to a constant temperature chamber pre-equilibrated to 17°C and left to stand for 1 hour to complete the transition from room temperature to 17°C; then the semen is placed in a constant temperature device with an ambient temperature of 10°C and cooled to 10°C at a uniform rate within 3 hours, at which time the cooling rate is controlled at 2-3°C per hour; finally, the semen is transferred to a 5°C refrigerator and cooled to 5°C at a uniform rate within 4 hours, at which time the cooling rate is controlled at 1-1.5°C per hour; after the entire cooling process is completed, the semen is continuously stored in a constant temperature environment of 5°C ± 0.5°C for subsequent long-term preservation or transportation; Step (5) Management during storage: During the entire cooling process described in step (4), the semen is gently inverted and mixed once every 1 hour to prevent the sperm from settling naturally and causing excessive local density; During long-term storage at 5℃, the semen is gently inverted and mixed once every 12 to 24 hours, and the mixing action lasts for 30 to 60 seconds to ensure that the sperm are evenly suspended; No antibiotics are added, no liquid is changed, and no centrifugation is performed during the entire cooling and storage process. Step (6) Warming operation: Before artificial insemination or experimental testing, take the cryopreserved semen out of the 5°C environment and immediately place it in a 37°C water bath for constant temperature incubation for 15-20 minutes. After incubation, invert and mix 3-5 times to fully restore the semen temperature to 37°C. The warmed semen should be used within 1 hour. Vigorous shaking or high-speed mixing is prohibited during the warming process.

5. The method according to claim 4, characterized in that: In step (3), the cooling diluent is used for dilution immediately after being preheated to 37°C. The entire dilution operation is completed within 2 hours after semen collection. After the cooling diluent is prepared, it is sealed and stored in the dark at 4°C for no more than 7 days. Diluent exceeding the storage period shall not be used for semen preservation. Before use, the diluent must be checked for any precipitation, turbidity or discoloration. It can only be used after confirming that there are no abnormalities.

6. The method according to claim 4, characterized in that: The cooling process described in step (4) is carried out in a constant temperature device, and the temperature deviation of the constant temperature device is controlled within ±0.5℃. During the cooling process from 17℃ to 10℃, the cooling rate is strictly controlled at 2 to 3℃ per hour, and sudden drops or temperature rebounds are prohibited. During the cooling process from 10℃ to 5℃, the cooling rate is strictly controlled at 1 to 1.5℃ per hour. This stage adopts a gradual cooling method, and the actual temperature is recorded every 30 minutes to ensure that the cooling curve is smooth and continuous. If abnormal temperature fluctuations occur during the cooling process, cooling should be stopped immediately and the operating status of the constant temperature equipment should be checked. Cooling should only be resumed after the temperature has stabilized.

7. The method according to claim 4, characterized in that: In steps (4) and (5), the 5℃ long-term storage environment is a medical-grade refrigerator or constant temperature incubator, with the storage temperature set at 5℃±0.5℃. The storage environment has real-time temperature monitoring and alarm functions. During storage, the semen sample is placed in a sealed sterile container with a small amount of air space reserved at the top of the container to maintain gas exchange. The mixing operation is performed manually and gently inverted every 12 to 24 hours, and each mixing lasts for 30 to 60 seconds. After mixing, the semen is immediately returned to the 5℃ storage environment to minimize the exposure time of the semen at room temperature. No antibiotics are added during storage, no liquid is changed, no centrifugation is performed, and no additional oxygen or carbon dioxide gas is added.

8. The method according to claim 4, characterized in that: In step (6), the rewarming operation is initiated within 30 minutes before artificial insemination or experimental testing. The rewarming process is carried out in a constant temperature water bath at 37℃, and the temperature deviation of the water bath is controlled within ±0.5℃. The semen container is kept in a semi-submerged state during the incubation process to ensure that the semen is heated evenly. The incubation time is 15-20 minutes. After the rewarming is completed, the sperm motility is immediately tested using a computer-aided sperm analysis system. Only after ensuring that the total sperm motility is ≥70% can the semen be used for artificial insemination. If the total sperm motility is lower than 70% after rewarming, the batch of semen shall not be used for artificial insemination. The rewarmed semen shall be used up within 1 hour. Any remaining semen that is not used within 1 hour shall be discarded and shall not be repeatedly cooled and stored.

9. The method according to any one of claims 4 to 8, characterized in that: When bovine semen is preserved at 5°C for 7 days using the method described above, the total sperm motility is ≥80%, the progressive sperm motility is ≥60%, the sperm plasma membrane integrity is ≥70%, the sperm acrosome integrity is ≥65%, and the normal rate of sperm mitochondrial membrane potential is ≥60%. After 14 days of preservation, the total sperm motility is ≥70%, the progressive sperm motility is ≥50%, the sperm plasma membrane integrity is ≥60%, the sperm acrosome integrity is ≥55%, and the normal rate of sperm mitochondrial membrane potential is ≥50%. Compared with conventional Modena basal dilution at 17°C, when the semen is preserved at 5°C for 14 days using the method of this invention, there is no significant difference in total sperm motility and progressive sperm motility, while the bacterial proliferation level in the semen is reduced by at least two orders of magnitude, and no antibiotics are required.

10. The application of the cooling diluent according to any one of claims 1 to 3 in the low-temperature preservation of boar semen at 5°C, characterized in that, The application ensures that the total sperm motility of bovine semen is ≥80% after 7 days of storage at 5°C and ≥70% after 14 days of storage, without the need to add antibiotics during the storage process.

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