Application of ursolic acid in the cryopreservation of goat semen

Abstract

The present application relates to the field of animal breeding technology, in particular to the application of ursolic acid in the frozen semen preservation of sheep. The present application applies ursolic acid in the frozen semen preservation of sheep. The addition of 0.5-2.0 micromole / L ursolic acid in the frozen diluent can improve the quality of frozen semen preservation of sheep and goats, and can improve the sperm motility and vitality after thawing, which helps the thawed sperm to pass through the vagina, uterus and enter the oviduct to complete fertilization, improves the insemination success rate of frozen sperm, and provides technical support for strengthening the protection of local sheep breed resources.

Description

Technical Field

[0001] This invention relates to the field of animal reproductive technology, specifically to the application of ursolic acid in the cryopreservation of sheep semen. Background Technology

[0002] my country boasts abundant sheep breed resources, but some local breeds still face significant survival crises. Effective protection and utilization of these resources is a core aspect of national seed industry security. Semen cryopreservation is an important auxiliary technique for protecting livestock genetic resources and artificial insemination. However, the results of deep vaginal insemination with frozen sheep semen are currently unsatisfactory, mainly due to two reasons: firstly, the structure of the ewe's reproductive tract is not conducive to inserting the insemination gun into the uterus; secondly, the quality of frozen semen needs improvement. Current cryopreservation technologies cannot guarantee that thawed sperm will pass through the vagina and uterus to the oviduct for fertilization. Therefore, improving the quality of frozen semen, especially its motility and movement within the vagina, uterus, and oviduct after artificial insemination, is a key solution. Strategies for improving frozen semen quality mainly include screening cryoprotectants and optimizing cryo-thawing procedures (primarily freezing and thawing speeds). Common cryoprotectants include ice crystal inhibitors, sugars, antioxidants, and apoptosis inhibitors. Among these, the cryoprotective effect of antioxidants is increasingly attracting research attention.

[0003] Mammalian sperm cells are highly sensitive to cryogenic damage. The freezing and thawing process can cause severe oxidative damage to sperm, ultimately leading to impairment of cell structure and function. The conventional practice in this field is to add a certain concentration of antioxidants to the semen cryopreservation solution to reduce oxidative damage to sperm cells during freezing and thawing. However, the sensitivity of sperm cells to cryogenic damage varies considerably among different animal species, and the extent of damage differs. Mature sperm cells and other somatic cells, such as fibroblasts, exhibit significant differences in morphology and structure. Slight variations in the compatibility and dosage of various chemical reagents can result in vastly different freezing outcomes. Therefore, even though it is theoretically sound to use antioxidants to improve frozen semen quality, there are too many types of antioxidants, and not all antioxidants are effective for all animal semen.

[0004] Yunnan Province boasts over 20 sheep breeds, including two bred breeds. Due to chaotic crossbreeding, extensive feeding and management practices, and weak awareness of resource conservation, most breeds face varying degrees of survival crisis. How to effectively protect and utilize these local resources is a significant scientific and social issue. Globally, semen cryopreservation is widely recognized as a major method for protecting local sheep breed resources. However, in practice, it has been found that thawed cryopreserved semen results in low insemination quality, a problem that urgently needs to be addressed. The applicant's prior patent (patent number 201610086372.X) uses inositol compounds as ice crystal inhibitors combined with resveratrol as an antioxidant for the cryopreservation of sheep semen. This reduces the mechanical damage to cell membranes and mitochondria caused by extracellular ice crystals and effectively reduces oxidative damage to sperm at 0-5℃ low-temperature equilibrium. However, in recent years, the effectiveness of this method for sheep semen cryopreservation has become increasingly unsatisfactory, possibly due to breed differences, environmental changes, and nutritional imbalances. Therefore, it is necessary to optimize and upgrade the sheep semen cryopreservation technology system.

[0005] Ursolic acid is a natural pentacyclic triterpenoid carboxylic acid compound with a molecular weight of 456.70 g / mol. Its structure is ursane-type, containing one C-3 hydroxyl group and one C-28 carboxyl group. Ursolic acid was long considered biologically inactive; however, recent studies have revealed its beneficial pharmacological effects and low cytotoxicity, attracting widespread attention. Research in plants and mammals has demonstrated that ursolic acid possesses a wide range of biological properties, including neuroprotection, antioxidant, anti-inflammatory, anticancer, antibacterial, and ability to counteract endogenous and exogenous biostimuli, thus making it widely used as a pharmaceutical and cosmetic ingredient. Currently, there are no reports of ursolic acid being used for the cryopreservation of sheep semen. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides the application of ursolic acid in the cryopreservation of sheep semen, thus solving the problem of poor cryopreservation effect of sheep semen in the prior art mentioned in the background section.

[0007] This invention provides the application of ursolic acid in the cryopreservation of sheep semen. By adding ursolic acid to the semen cryopreservation diluent at a concentration of 0.5-2 μM, the quality of semen cryopreservation is improved.

[0008] Furthermore, each 100 mL of the semen cryopreservation diluent comprises the following components: 2.71 g of tris(hydroxymethyl)aminomethane, 1.4 g of citric acid, 1.0 g of glucose, 20 mL of fresh egg yolk, 50,000 IU of penicillin, 50,000 IU of streptomycin, 7 mL of glycerol, 300 µg of vitamin B12, 0.5-2 μM of ursolic acid, and the balance being ultrapure water.

[0009] Preferably, the concentration of ursolic acid added is 0.5 μM.

[0010] Furthermore, the sheep in question is either a sheep or a goat.

[0011] Furthermore, the sheep in question is a Yunnan semi-fine wool sheep.

[0012] Furthermore, improving the quality of semen cryopreservation means improving sperm viability and motility after thawing.

[0013] Furthermore, 100 mM trehalose was added to the frozen dilution of the sheep semen.

[0014] The beneficial effects of this invention are as follows:

[0015] This invention is the first to apply ursolic acid to the cryopreservation of sheep semen. It discloses that supplementing the cryopreservation diluent with 0.5-2.0 μmol / L ursolic acid can improve the quality of cryopreserved sheep and goat semen, enhancing sperm viability and motility after thawing. This helps thawed sperm pass through the sheep's vagina and uterus into the oviduct for fertilization, increasing the success rate of fertilization and providing technical support for strengthening the protection of local sheep breeds. The preferred supplementation concentration is 0.5 μmol / L. Specifically, supplementing with 0.5 μmol / L ursolic acid significantly improves the cryopreservation effect on sheep semen compared to goats, especially on Yunnan semi-fine wool sheep semen, where the effect is significantly better than that on Yunnan black goats.

[0016] The present invention also discloses that adding ursolic acid or a combination of ursolic acid and trehalose as an antifreeze agent to the cryo-diluent of semen from sheep breeds raised in Yunnan can significantly improve the viability and motility of sperm after thawing. Detailed Implementation

[0017] The technical solutions of the present invention are further illustrated below through specific embodiments, but the technical solutions of the present invention are not limited to these embodiments.

[0018] Example 1: Comparison of the effects of ursolic acid and resveratrol on the cryopreservation of Yunnan semi-fine wool sheep semen:

[0019] At Laishishan Livestock Farm in Qiaojia County, Zhaotong City, six healthy adult rams (2-3 years old) with normal breeding records were randomly selected from the same flock of Yunnan semi-fine wool sheep. Semen was collected from each ram using an artificial vaginal method. Semen was collected twice from each ram, with a 10-minute interval between collections. The two semen samples were then mixed and placed in a 37°C water bath. The fresh semen quality requirements for each ram were as follows: ejaculate volume ≥ 1.0 mL, sperm motility ≥ 75%, and sperm concentration > 2.0 × 10⁻⁶. 9 per mL.

[0020] At room temperature (25°C), the fresh semen was slowly added to the cryogenic diluent until a final concentration of 2.0 × 10⁻⁶ was achieved. 8 sperm count / mL. The cryodiluents are of two types: ursolic acid cryodiluent and resveratrol cryodiluent, with the following formulations:

[0021] Ursolic acid cryo-diluent: Each 100 mL of ursolic acid cryo-diluent contains 2.71 g of tris(hydroxymethyl)aminomethane, 1.4 g of citric acid, 1.0 g of glucose, 20% (v / v) fresh egg yolk, 50,000 IU of penicillin, 50,000 IU of streptomycin, 7% (v / v) glycerol, 3 µg / mL vitamin B12, and different concentrations of ursolic acid (concentrations of 0 μmol / L, 0.5 μmol / L, 2.0 μmol / L, 5.0 μmol / L, 15.0 μmol / L, and 30.0 μmol / L, respectively), with the balance being ultrapure water.

[0022] Resveratrol cryopreservation buffer: Each 100 mL of resveratrol cryopreservation buffer contains 2.71 g of tris(hydroxymethyl)aminomethane, 1.4 g of citric acid, 1.0 g of glucose, 20% (v / v) fresh egg yolk, 50,000 IU of penicillin, 50,000 IU of streptomycin, 7% (v / v) glycerol, 3 µg / mL vitamin B12, and different concentrations of resveratrol (0 μmol / L, 10 μmol / L, 50 μmol / L, 100 μmol / L, and 250 μmol / L), with the balance being ultrapure water. All buffers should be prepared fresh before use.

[0023] The diluted semen was aliquoted into 0.25 mL cryovials, with three replicates per experimental group. The samples were equilibrated at 4°C for 3.0 h, then the capillary tubes were placed 3.5 cm above liquid nitrogen (-80°C) and frozen for 10 min. Immediately afterwards, the capillary tubes were immersed in liquid nitrogen for at least 72 h. For thawing, the frozen semen was incubated in a 37°C water bath for 30 seconds. Sperm motility and movement parameters were measured using the CASA sperm analysis system, including sperm motility (TM), sperm viability (PR), velocity curve (VCL), velocity path (VAP), velocity linear (VSL), lateral swing amplitude (ALH), and whiplash frequency (BCF). The results are shown in Table 1.

[0024] Table 1. Comparison of the effects of ursolic acid and resveratrol on the cryopreservation of Yunnan semi-fine wool sheep semen:

[0025]

[0026] Note: Only vertical column comparisons are made (different protective agents are compared separately). a vs c indicates extremely significant difference (P < 0.01), and a vs b indicates significant difference (P < 0.05). The uppercase letters indicate comparisons between the optimal concentration groups of various protective agents, and AVS B indicates significant difference (P < 0.05).

[0027] The results in Table 1 show that:

[0028] 1-1. When the concentration of ursolic acid in the semen cryopreservation diluent was 0.5 μM, the sperm viability, motility, and curvilinear motility after thawing were significantly higher than those in the control group and other treatment groups (P<0.05). However, compared with the control group, ursolic acid did not significantly improve path speed, linear motility, lateral swing amplitude, and whiplash frequency (P>0.05).

[0029] 1-2. When the resveratrol concentration in the semen cryopreservation diluent was 10 μM and 50 μM, the sperm viability, motility and curvilinear motility were significantly better than those in the control group after thawing (P<0.05), but resveratrol could not significantly improve path speed, linear motility, lateral swing amplitude and whiplash frequency (P>0.05).

[0030] 1-3. Compared with the cryoprotective effects of 10 μM and 50 μM resveratrol, 0.5 μM ursolic acid can significantly improve sperm viability and motility (P<0.05).

[0031] Example 2: Comparison of the effects of ursolic acid and butylated hydroxytoluene (BHT) on the cryopreservation of semen from Zhaotong sheep:

[0032] At the Dadi Breeding Cooperative in Ludian County, nine healthy adult rams (2-3 years old) with normal breeding records were randomly selected from the same Zhaotong sheep flock. Semen was collected from each ram using an electrostimulation method. Semen was collected twice from each ram, with a 10-minute interval between collections. The two collections were then mixed and placed in a 37°C water bath. The fresh semen quality requirements for each ram were as follows: ejaculate volume ≥ 1.0 mL, sperm motility ≥ 75%, and sperm concentration > 2.0 × 10⁻⁶. 9 per mL.

[0033] At room temperature (25°C), the fresh semen was gently added to the cryogenic diluent until a final concentration of 2.0 × 10⁻⁶ was achieved. 8 sperm / mL. The cryodiluent is divided into two types: ursolic acid cryodiluent and butylated hydroxytoluene (BHT) cryodiluent, with the following formulations:

[0034] Ursolic acid cryo-diluent: Each 100 mL of ursolic acid cryo-diluent contains 2.71 g of tris(hydroxymethyl)aminomethane, 1.4 g of citric acid, 1.0 g of glucose, 1% (w / v) soybean lecithin, 50,000 IU of penicillin, 50,000 IU of streptomycin, 7% (v / v) glycerol, 3 µg / mL vitamin B12, and different concentrations of ursolic acid (0 μmol / L, 0.5 μmol / L, 2.0 μmol / L, 5.0 μmol / L, 15.0 μmol / L, and 30.0 μmol / L, respectively), with the balance being ultrapure water.

[0035] BHT cryo-diluent: Each 100 mL of BHT cryo-diluent contains 2.71 g of tris(hydroxymethyl)aminomethane, 1.4 g of citric acid, 1.0 g of glucose, 1% (w / v) soybean lecithin, 50,000 IU of penicillin, 50,000 IU of streptomycin, 7% (v / v) glycerol, 3 µg / mL vitamin B12 and different concentrations of BHT (0 μmol / L, 500 μmol / L, 1000 μmol / L and 2000 μmol / L, respectively), with the balance being ultrapure water.

[0036] The diluent used must be prepared fresh each time it is used.

[0037] The diluted semen was aliquoted into 0.25 mL cryovials, with three replicates per experimental group. The samples were equilibrated at 4°C for 3.0 h, then the capillary tubes were placed 3.5 cm above liquid nitrogen (-80°C) and frozen for 10 min. Immediately afterwards, the capillary tubes were immersed in liquid nitrogen for at least 72 h. Upon thawing, the frozen semen was incubated in a 37°C water bath for 30 seconds. Sperm motility and movement parameters were measured using the CASA sperm analysis system. The measurements included sperm motility (TM), sperm viability (PR), velocity curve (VCL), velocity path (VAP), velocity linear (VSL), lateral swing amplitude (ALH), and whiplash frequency (BCF). The results are shown in Table 2.

[0038] Table 2. Comparison of the effects of ursolic acid and butylated hydroxytoluene (BHT) on the cryopreservation of semen from Zhaotong sheep:

[0039]

[0040] Note: Only vertical column comparisons are made (different protective agents are compared separately). a vs c indicates extremely significant difference (P < 0.01), and a vs b indicates significant difference (P < 0.05). The uppercase letters indicate comparisons between the optimal concentration groups of various protective agents, and AVS B indicates significant difference (P < 0.05).

[0041] The results in Table 2 show that:

[0042] 2-1. When the ursolic acid concentration was 0.5 μM, the sperm motility (TM), sperm viability (PR), and velocity (VCL) after thawing were significantly higher than those in the control group and other treatment groups (P<0.05). However, compared with the control group, ursolic acid did not significantly improve path velocity (VAP) and linear velocity (VSL) (P>0.05). In addition, ursolic acid also failed to improve lateral swing amplitude (ALH) and whiplash frequency (BCF) (P>0.05).

[0043] 2-2. When the BHT concentration was 500 μM, the sperm TM and PR were significantly better than those in the control group after thawing (P<0.05), but BHT could not significantly improve VCL, VAP, VSL, ALH and BCF (P>0.05).

[0044] 2-3. Compared with the cryoprotective effect of 500μM BHT, 0.5μM ursolic acid can significantly improve sperm motility indicators (P<0.05).

[0045] Example 3: Comparison of the effects of ursolic acid and sodium pyruvate on the cryopreservation of Yunnan semi-fine wool sheep semen:

[0046] At Kunming Yixingheng Livestock Technology Co., Ltd., eight healthy adult rams (2-3 years old) with normal breeding records were randomly selected from the same Yunnan semi-fine wool sheep herd. Semen was collected from each ram using the artificial vagina method. Semen was collected twice from each ram, with a 20-minute interval between collections. The collected semen was immediately placed in a 37°C water bath, and then the two collections were mixed. The fresh semen quality requirements for each ram were as follows: ejaculate volume ≥ 1.0 mL, sperm motility ≥ 75%, and sperm concentration > 2.0 × 10⁻⁶. 9 per mL.

[0047] At room temperature (25°C), the fresh semen was gently added to the cryogenic diluent until a final concentration of 2.0 × 10⁻⁶ was achieved. 8 sperm / mL. The cryodiluent is divided into two types: ursolic acid cryodiluent and sodium pyruvate cryodiluent, with the following formulations.

[0048] Ursolic acid cryo-diluent: Each 100 mL of ursolic acid cryo-diluent contains 2.71 g of tris(hydroxymethyl)aminomethane, 1.4 g of citric acid, 1.0 g of glucose, 15% (V / V) fresh egg yolk, 50,000 IU of penicillin, 50,000 IU of streptomycin, 7% (V / V) glycerol, 3 µg / mL vitamin B12, and different concentrations of ursolic acid (0 μmol / L, 0.5 μmol / L, 2.0 μmol / L, 5.0 μmol / L, 15.0 μmol / L, and 30.0 μmol / L, respectively), with the balance being ultrapure water.

[0049] Sodium pyruvate cryo-diluent: Each 100 mL of sodium pyruvate cryo-diluent contains 2.71 g of tris(hydroxymethyl)aminomethane, 1.4 g of citric acid, 1.0 g of glucose, 15% (v / v) fresh egg yolk, 50,000 IU of penicillin, 50,000 IU of streptomycin, 7% (v / v) glycerol, 3 µg / mL vitamin B12, and different concentrations of sodium pyruvate (0 μmol / L, 5 μmol / L, 10 μmol / L, and 20 μmol / L). The diluent should be prepared fresh for each use.

[0050] The diluted semen was aliquoted into 0.25 mL cryovials, with three replicates per experimental group. The mixture was equilibrated at 4°C for 3.0 h, then the capillary tubes were placed 3.5 cm above liquid nitrogen (-80°C) and frozen for 10 min. Immediately afterwards, the capillary tubes were immersed in liquid nitrogen for at least 72 h. Upon thawing, the frozen semen was incubated in a 37°C water bath for 30 seconds. Sperm motility and movement parameters were measured using the CASA sperm analysis system. The measurements included sperm motility (TM), sperm vitality (PR), velocity curve (VCL), velocity path (VAP), velocity linear (VSL), lateral swing amplitude (ALH), and whiplash frequency (BCF). The results are shown in Table 3.

[0051] Table 3. Comparison of the effects of ursolic acid and sodium pyruvate on the cryopreservation of Yunnan semi-fine wool sheep semen:

[0052]

[0053] Note: Only vertical column comparisons are made (different protective agents are compared separately). a vs c indicates extremely significant difference (P < 0.01), and a vs b indicates significant difference (P < 0.05). The uppercase letters indicate comparisons between the optimal concentration groups of various protective agents, and AVS B indicates significant difference (P < 0.05).

[0054] The results in Table 3 show that:

[0055] 3-1. When the ursolic acid concentration was 0.5 μM, the sperm motility (TM), sperm viability (PR), and velocity (VCL) after thawing were significantly higher than those in the control group and other treatment groups (P<0.05). However, compared with the control group, ursolic acid did not significantly improve path velocity (VAP) and linear velocity (VSL) (P>0.05). In addition, ursolic acid also failed to improve lateral swing amplitude (ALH) and whiplash frequency (BCF) (P>0.05).

[0056] 3-2. When the sodium pyruvate concentration was 5 μM, the sperm TM, PR and VCL were significantly better than those in the control group after thawing (P<0.05), but sodium pyruvate could not significantly improve VAP, VSL, ALH and BCF (P>0.05).

[0057] 3-2. Compared with the cryoprotective effect of 5 μM sodium pyruvate, 0.5 μM ursolic acid can significantly improve sperm motility indicators (P<0.05).

[0058] Example 4: Study on the effect of ursolic acid on cryopreservation of semen from Yunshang black goats:

[0059] At the Wuding Matoushan breeding base of Yunnan Xingmu Livestock Co., Ltd., we randomly selected four healthy adult rams (2-3 years old) from the same flock and collected their semen using an artificial vagina method. Semen was collected twice from each ram, with a 10-minute interval between collections. The two semen samples were then mixed and placed in a 37°C water bath. The fresh semen quality was analyzed using a sperm quality auxiliary analysis system (CASA, Barcelona, ​​Spain). The fresh semen quality requirements for each ram were: ejaculation volume ≥ 0.8 mL, total sperm motility ≥ 75%, and sperm concentration > 3.0 × 10⁻⁶. 9 Fresh semen samples with a concentration of [number] cells / mL were divided into 6 equal portions. At 25°C, these fresh semen samples were gently added to a cryogenic diluent until a final concentration of 3 × 10⁻⁶ was achieved. 8 sperm / mL. The cryo-diluent formulation consists of a base solution and ursolic acid:

[0060] The base solution consisted of 20% (v / v) AndroMed® (Minitube, Germany), 80% (v / v) ddH2O, 5 mg / mL lactose (Sigma-Aldrich, USA), and 3 µg / mL vitamin B12 (Chenxin Pharmaceutical, China). Ursolic acid was prepared by dissolving ursolic acid reagent (Sigma-Aldrich, USA) in dimethyl sulfoxide (Sigma-Aldrich, USA). Based on different ursolic acid concentrations (0 μmol / L, 0.5 μmol / L, 2.0 μmol / L, 5.0 μmol / L, 15.0 μmol / L, 30.0 μmol / L), six experimental groups were formed, with three replicates per group. Diluents should be prepared fresh before use.

[0061] The diluted semen was loaded into 0.25 mL cryovials and equilibrated at 4°C for 3.0 h. The capillary was then placed 3.5 cm above liquid nitrogen (-80°C) and frozen for 10 min. Immediately afterwards, the capillary was placed in liquid nitrogen for storage for at least 72 h. To thaw, the frozen semen was incubated in a 37°C water bath for 30 seconds.

[0062] Sperm motility and movement parameters were measured using the CASA sperm analysis system. The measurements included sperm motility (TM), sperm viability (PR), velocity of curvilinear motility (VCL), velocity of path (VAP), velocity of linear motility (VSL), lateral swing amplitude (ALH), and whiplash frequency (BCF). The results are shown in Table 4.

[0063] Table 4. Effects of different concentrations of ursolic acid on cryopreservation of Yunshang Black Goat semen:

[0064]

[0065] Note: Only the vertical columns are compared. Different uppercase letters indicate extremely significant differences (P < 0.01), and different lowercase letters indicate significant differences (P < 0.05).

[0066] Table 4 shows that supplementing the cryodiluent with 0.5 μmol / L ursolic acid helped improve the total motility, forward motility, and motility rate (including curve rate (VCL), linear rate (VSL), and average rate (VAP)) of frozen-thawed sperm from Yunshang Black Goats, but the effect was not significant (P > 0.05). When the ursolic acid concentration reached 5.0–30.0 μmol / L, it was detrimental to the total motility, forward motility, and motility rate of frozen-thawed sperm (P > 0.05). Supplementing the cryodiluent with 0.5–30.0 μmol / L ursolic acid had little effect on the lateral swing amplitude (ALH) of frozen-thawed sperm, while supplementing with 15.0–30.0 μmol / L ursolic acid significantly altered the whiplash frequency of frozen-thawed sperm (P < 0.05).

[0067] Based on Examples 1 to 4, it is evident that supplementing the cryopreservation solution with 0.5-2.0 μmol / L ursolic acid can improve the cryopreservation quality of sheep and goat semen, enhancing sperm viability and motility after thawing. The preferred supplementation concentration is 0.5 μmol / L. Specifically, supplementing with 0.5 μmol / L ursolic acid significantly improves the cryopreservation effect on sheep semen compared to goat semen, especially on Yunnan semi-fine wool sheep semen, where the effect is significantly better than that on Yunnan black goat semen.

[0068] Example 5: Comparison of the effects of cryoprotectants and trehalose on the cryopreservation of Romney sheep semen:

[0069] Trehalose, as an important cryoprotectant, is widely used to improve sperm survival rate and functional integrity during freezing and thawing. Trehalose is a non-reducing disaccharide with a unique ability to stabilize cell membranes and proteins, effectively reducing sperm damage during freezing. Based on a comparison of the cryoprotective effects of ursolic acid with three other antioxidants (resveratrol, butylated hydroxytoluene (BHT), and sodium pyruvate), the research team further confirmed that the combination of ursolic acid and trehalose can significantly improve frozen sperm motility and motility parameters. The combination of these two cryoprotectants not only reduces oxidative damage to sheep sperm during freezing and thawing but also effectively reduces the physicochemical damage to the sperm cell membrane during the freezing and thawing process.

[0070] At the Yunnan Provincial Sheep Breeding and Extension Center, six healthy adult rams (2-3 years old) with normal mating records were randomly selected from the same Romney sheep flock. Semen was collected from each ram using the artificial vagina method. Semen was collected twice from each ram, with a 20-minute interval between collections. The collected semen was immediately placed in a 37°C water bath, and then the two collections were mixed. The fresh semen quality requirements for each ram were as follows: ejaculate volume ≥ 1.0 mL, sperm motility ≥ 75%, and sperm concentration > 2.0 × 10⁻⁶. 9 per mL.

[0071] At room temperature (25°C), the fresh semen was gently added to the cryogenic diluent until a final concentration of 2.0 × 10⁻⁶ was achieved. 8 sperm / mL. The cryodiluent is divided into four groups, with the following formulation:

[0072] Ursolic acid group: Each 100mL of frozen dilution contained 2.71g of tris(hydroxymethyl)aminomethane, 1.4g of citric acid, 1.0g of glucose, 20% (V / V) fresh egg yolk, 50,000 IU of penicillin, 50,000 IU of streptomycin, 7% (V / V) glycerol, 3µg / mL vitamin B12, 0.5μmol / L ursolic acid + 100mM trehalose, with the balance being ultrapure water.

[0073] Resveratrol group: Each 100 mL of frozen dilution contained 2.71 g of tris(hydroxymethyl)aminomethane, 1.4 g of citric acid, 1.0 g of glucose, 20% (V / V) fresh egg yolk, 50,000 IU of penicillin, 50,000 IU of streptomycin, 7% (V / V) glycerol, 3 µg / mL vitamin B12, 10 μmol / L resveratrol + 100 mM trehalose, with the balance being ultrapure water.

[0074] Butylated hydroxytoluene (BHT) group: Each 100 mL of cryopreservation dilution contains 2.71 g of tris(hydroxymethyl)aminomethane, 1.4 g of citric acid, 1.0 g of glucose, 20% (V / V) fresh egg yolk, 50,000 IU of penicillin, 50,000 IU of streptomycin, 7% (V / V) glycerol, 3 µg / mL vitamin B12, 500 μmol / L BHT + 100 mM trehalose, with the balance being ultrapure water.

[0075] Sodium pyruvate group: Each 100 mL of frozen diluent contains 2.71 g of tris(hydroxymethyl)aminomethane, 1.4 g of citric acid, 1.0 g of glucose, 20% (v / v) fresh egg yolk, 50,000 IU of penicillin, 50,000 IU of streptomycin, 7% (v / v) glycerol, 3 µg / mL vitamin B12, 5 μmol / L sodium pyruvate + 100 mM trehalose, with the balance being ultrapure water. The diluent should be prepared fresh for each use.

[0076] The diluted semen was aliquoted into 0.25 mL cryovials, with three replicates per group. The samples were equilibrated at 4°C for 3.0 h, then the capillary tubes were placed 3.5 cm above liquid nitrogen (-80°C) and frozen for 10 min. Immediately afterwards, the capillary tubes were immersed in liquid nitrogen for at least 72 h. For thawing, the frozen semen was incubated in a 37°C water bath for 30 seconds. Sperm motility and motility parameters were measured using the CASA sperm analysis system. The results are shown in Table 5.

[0077] Table 5. Comparison of the effects of cryoprotectants and trehalose combinations on the cryopreservation of Romney sheep semen:

[0078]

[0079] Note: Only vertical comparisons are made (different protective agents are compared separately), a vs b indicates a significant difference (P < 0.05).

[0080] The results in Table 5 show that when ursolic acid and trehalose were added to the diluent, the sperm motility (TM), sperm viability (PR), path velocity (VAP), and linear velocity (VSL) after thawing were significantly higher than those in the other three treatment groups (P<0.05). However, there were no significant differences in VCL among the four treatment groups (P>0.05).

[0081] Based on Examples 1 to 5, it is evident that ursolic acid, as an antioxidant, significantly improves the cryopreservation quality of sheep and goat semen in the semen cryopreservation diluent, and significantly enhances sperm viability and motility after thawing. 0.5 μM is the preferred ursolic acid supplementation concentration. This technical solution shows significantly better cryopreservation effects on sheep semen than goat semen, with the best results observed in Yunnan semi-fine wool sheep semen. The combination of ursolic acid and another cryoprotectant, trehalose, shows significantly better effects than other antioxidants. Therefore, when cryopreserving semen from sheep breeds raised in Yunnan, adding ursolic acid or a combination of ursolic acid and trehalose as a cryoprotectant to the semen cryopreservation diluent can significantly improve sperm viability and motility after thawing. This helps thawed sperm pass through the sheep's vagina and uterus into the oviduct for fertilization, increasing the success rate of fertilization and providing technical support for strengthening the protection of local sheep breed resources.

[0082] The above embodiments are for illustrative purposes only and do not constitute any limitation on the scope of protection of the present invention. This document has described the principles and implementation methods of the present invention through specific embodiments. The above description is only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

[0083] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. The application of ursolic acid in the cryopreservation of sheep semen, characterized in that, Adding ursolic acid to the cryopreservation diluent of sheep semen at a concentration of 0.5-2 μM can improve the quality of semen cryopreservation.

2. The application of ursolic acid in the cryopreservation of sheep semen according to claim 1, characterized in that, The frozen dilution of sheep semen per 100 mL comprises the following components: 2.71 g of tris(hydroxymethyl)aminomethane, 1.4 g of citric acid, 1.0 g of glucose, 20 mL of fresh egg yolk, 50,000 IU of penicillin, 50,000 IU of streptomycin, 7 mL of glycerol, 300 µg of vitamin B12, 0.5-2 μM of ursolic acid, and the balance being ultrapure water.

3. The application of ursolic acid in the cryopreservation of sheep semen according to claim 1 or 2, characterized in that, The concentration of ursolic acid added is 0.5 μM.

4. The application of ursolic acid in the cryopreservation of sheep semen according to claim 1, characterized in that, The sheep in question is either a sheep or a goat.

5. The application of ursolic acid in the cryopreservation of sheep semen according to claim 4, characterized in that, The sheep in question are Yunnan semi-fine wool sheep.

6. The application of ursolic acid in the cryopreservation of sheep semen according to claim 1, characterized in that, Improving the quality of semen cryopreservation means increasing sperm viability and motility after thawing.

7. The application of ursolic acid in the cryopreservation of sheep semen according to claim 1, characterized in that, 100 mM trehalose was added to the frozen dilution of the sheep semen.

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