Application of frozen diluent containing sargassol in improving the effect of frozen semen preservation of sheep

Abstract

The application discloses application of a frozen diluent containing enhalidol in improving the freezing preservation effect of sheep semen. The application adds enhalidol with different concentrations into the freezing preservation diluent to improve the protection effect of the quality and antioxidant performance of sperm after freezing and thawing, and the effect of adding 1 muM enhalidol is the best. Therefore, adding enhalidol into the freezing diluent can quickly remove oxidative free radicals, significantly improve the antioxidant capacity, reduce lipid peroxidation, avoid oxidative damage, and then improve the freezing preservation effect of sheep semen, prolong the preservation time, and the preparation method of the freezing preservation diluent is simple and inexpensive, the freezing preservation procedure is simple and easy to popularize, and the freezing preservation diluent has high application value in artificial insemination.

Description

Technical Field

[0001] This invention belongs to the field of animal reproduction technology, specifically relating to the application of a cryodiluent containing sennaol in improving the cryopreservation effect of sheep semen. Background Technology

[0002] With the modernization of livestock and poultry farming, artificial insemination technology has been widely applied. As a key technology for efficient reproduction, it not only improves conception rates and prevents disease transmission, but also increases the utilization rate of superior breeding rams, thereby reducing costs and bringing significant economic benefits. The widespread adoption of this technology is crucial for maximizing the genetic potential of rams, and semen preservation, especially cryopreservation, is a vital aspect of artificial insemination. Frozen semen breaks the limitations of time and space, further improving the utilization efficiency of breeding rams, saving costs, enhancing livestock profitability, and promoting breed improvement. Therefore, semen freezing technology has significant practical implications for the sustainable development of my country's livestock industry and the stable supply of livestock products.

[0003] However, compared to fresh semen, thawed semen accumulates a large amount of reactive oxygen species (ROS) in the diluent and sperm during the dilution, equilibration, freezing, and thawing process, leading to varying degrees of oxidative stress in the sperm. Oxidative stress disrupts the balance between intracellular ROS and antioxidant mechanisms. While adequate ROS is crucial for maintaining cell signal transduction and immune responses, excessive ROS causes oxidative damage. Oxidative stress in sperm damages lipids, DNA, and proteins, thereby compromising sperm integrity, leading to lipid peroxidation, DNA strand breaks, protein misfolding, and ultimately sperm death. This manifests as a loss of sperm motility, plasma membrane function, and acrosome integrity, resulting in a severe reduction in quality, decreased sperm vitality, and reduced fertilization capacity. Consequently, conception rates in female animals decrease, leading to a decline in farrowing rates.

[0004] Sperm plasma membranes are rich in unsaturated fatty acids, which are excellent targets for reactive oxygen species (ROS) attacking sperm. The plasma membrane plays a crucial role in the acrosome reaction and fusion with the egg membrane, and its integrity directly affects sperm viability and fertilization capacity. Furthermore, mature sperm have less cytoplasm and fewer endogenous antioxidant enzymes, making them less resistant to ROS attack. Therefore, adding exogenous antioxidants to the basal diluent can improve semen quality, reduce ROS damage to sperm, and extend storage time while maintaining fertilization capacity. Addressing the critical issues of ram semen's sensitivity to low temperatures and susceptibility to oxidative stress, leading to low and unstable frozen-thawed semen quality and conception rates, improving cryopreservation diluents is an important direction for advancing artificial insemination in livestock.

[0005] Marine active antioxidants refer to natural compounds with significant antioxidant activity extracted from marine organisms such as algae, fish, and shellfish. However, there is currently no publicly available technology that discloses the addition of marine active antioxidants to the basic diluent for semen cryopreservation to improve the quality of frozen-thawed semen. Summary of the Invention

[0006] The purpose of this invention is to address the problem of oxidative damage to sperm during the cryopreservation of sheep semen. It proposes adding a marine-active antioxidant to the basal cryopreservation diluent to reduce oxidative stress and improve the quality of frozen-thawed semen. Specifically, addressing the critical issues of ram semen's sensitivity to low temperatures and susceptibility to oxidative damage, leading to low quality, low conception rates, and instability in frozen-thawed semen, this invention incorporates the marine-active antioxidant diosgenin (di-senna) into the cryopreservation basal diluent. This effectively reduces ROS-induced lipid peroxidation, improves sheep semen quality and fertilization capacity, and aims to provide theoretical and practical basis for the widespread application of cryopreserved sheep semen in artificial insemination.

[0007] Cryopreservation of semen effectively inhibits sperm metabolism, and the sperm retains a certain level of motility after thawing, achieving long-term preservation. However, the freeze-thaw process generates a large amount of reactive oxygen species (ROS). Sperm plasma membranes, rich in unsaturated fatty acids, are more susceptible to ROS attack, leading to membrane damage and causing sperm to lose their fertilization ability or even die. This manifests as a decrease in sperm viability and motility, resulting in a lower conception rate and consequently reduced reproductive efficiency.

[0008] Marine-derived antioxidants mainly consist of polyphenols, carotenoids, bioactive peptides, and polyunsaturated fatty acids, and are found in various marine organisms such as algae, fish, shrimp, and shellfish. This invention adds marine bioactive antioxidants to a basic diluent for sperm cryopreservation, which is then applied to the cryopreservation of sheep semen. This effectively neutralizes reactive oxygen species (ROS) generated during freezing, reduces ROS accumulation, enhances the antioxidant properties of sperm, effectively prevents oxidative damage, improves the quality of frozen sheep semen, maintains sperm fertilization capacity, and increases conception rate.

[0009] The first objective of this invention is to provide a semen cryopreservation diluent containing sennaol.

[0010] Preferably, the concentration of discholenol in the semen cryopreservation diluent is 0.25 μM-2 μM when cryopreserving semen.

[0011] Preferably, the semen cryopreservation diluent comprises semen cryopreservation diluent I and semen cryopreservation diluent II; The semen cryopreservation diluent I, calculated by volume percentage, comprises: 2.9% Tris-base, 0.4% fructose, 1.59% citric acid, 0.2% amikacin, 20% egg yolk, 0.5 μM-4 μM scheffleraol, with the remainder being water; wherein the concentrations of scheffleraol are 0.5 μM, 1 μM, 2 μM, and 4 μM. The semen cryopreservation diluent II, calculated by volume percentage, consists of: 12% glycerol, 2% OEP, and the remainder is cryopreservation diluent I without discholenol; When the semen cryopreservation diluent is used to preserve semen, the volume ratio of semen cryopreservation diluent I, semen, and semen cryopreservation diluent II is 9:1:10, so that the concentration of diserodium is 0.25 μM-2 μM, specifically 0.25 μM, 0.5 μM, 1 μM, and 2 μM.

[0012] Preferably, the semen cryopreservation diluent I, calculated by volume percentage, comprises: 2.9% Tris-base, 0.4% fructose, 1.59% citric acid, 0.2% amikacin, 20% egg yolk, 2 μM scheffleraol, and the remainder being water; when the semen cryopreservation diluent is used to preserve semen, the volume ratio of semen cryopreservation diluent I, semen, and semen cryopreservation diluent II is 9:1:10, resulting in a scheffleraol concentration of 1 μM.

[0013] A second objective of this invention is to provide the application of the aforementioned semen cryopreservation diluent in cryopreserved sheep semen.

[0014] Preferably, the application includes the following steps: S1. Fresh sheep semen was diluted with cryopreservation diluent I at the same temperature, with a volume ratio of fresh sheep semen to cryopreservation diluent I of 1:9, and equilibrated at 4℃ for 2 h. S2. Mix the mixed solution obtained in step S1 with the semen cryopreservation diluent II at a volume ratio of 1:1 and place it at 4℃ for equilibration for 3-4 hours. S3. The mixed solution obtained in step S2 is drawn into a cryogenic capillary and sealed. After being fumigated with liquid nitrogen for 15 minutes, it is stored in liquid nitrogen.

[0015] Preferably, the sheep semen is a diluted sheep semen obtained by mixing fresh sheep semen with a buffer solution at a volume ratio of 1:1; the buffer solution, calculated by volume percentage, comprises: 2.75% fructose, 0.69% trisodium citrate dihydrate, 0.1% NaHCO3, 0.235% EDTA-2Na, 0.29% citric acid, 0.565% Tris-base, 0.2% amikacin, and the balance being water.

[0016] Preferably, the goat semen is Leizhou black goat semen.

[0017] Preferably, the cryocapsule is a 0.25 mL cryocapsule.

[0018] The beneficial effects of this invention are: This invention combines marine active antioxidants with a basic diluent for cryopreservation of sheep semen, effectively improving the quality and fertilization capacity of cryopreserved semen. It significantly improves antioxidant properties, reduces ROS levels, alleviates lipid peroxidation, and enhances the cryopreservation effect of sheep semen, achieving the purpose of long-term cryopreservation and artificial insemination. The diluent preparation method is simple and inexpensive, the cryopreservation procedure is simple and easy to promote, and it significantly improves the quality and fertilization capacity of frozen-thawed sheep semen, which has high application value in artificial insemination practice. Attached Figure Description

[0019] Figure 1 Roadmap for cryopreservation technology of sheep semen.

[0020] Figure 2 The effect of different concentrations of sennaol on the total sperm motility in frozen-thawed sheep semen; 0 μM was the control, and *** indicates significant difference (0.0001 ≤ p < 0.001).

[0021] Figure 3 The effect of different concentrations of sennaol on the progressive motility of sperm in frozen-thawed sheep semen was investigated; 0 μM was the control, and *** indicates significant difference (0.0001 ≤ p < 0.001).

[0022] Figure 4 The effect of different concentrations of discholenol on the velocity curve (VCL) of sperm in frozen-thawed sheep semen; 0 μM was the control.

[0023] Figure 5 The effect of different concentrations of discholenol on the linear velocity (VSL) of sperm in frozen-thawed sheep semen; 0 μM was the control, and ** indicates significant difference (0.001 ≤ P < 0.01).

[0024] Figure 6 The effect of different concentrations of discholenol on the mean path velocity (VAP) of sperm in frozen-thawed sheep semen; 0 μM was the control.

[0025] Figure 7 The effect of different concentrations of discholenol on the lateral swing amplitude (ALH) of sperm in frozen-thawed sheep semen; 0 μM was the control.

[0026] Figure 8The effect of different concentrations of sennaol on the plasma membrane integrity of sperm in frozen-thawed sheep semen; 0 μM was the control, and **** indicates significant difference (P<0.0001).

[0027] Figure 9 The effect of different concentrations of discholenol on the acrosome integrity of sperm in frozen-thawed sheep semen; 0 μM was the control.

[0028] Figure 10 The effect of discholenol on the CAT enzyme content in frozen-thawed sheep semen; CT is the control, and * indicates significant difference (0.01 ≤ P<0.05).

[0029] Figure 11 The effect of discholenol on the SOD enzyme content of frozen-thawed sheep semen; CT is the control, ** indicates significant difference (0.001 ≤ P<0.01).

[0030] Figure 12 The effect of discholenol on the total antioxidant properties of frozen-thawed sheep semen was investigated; CT was the control, and ** indicates significant difference (0.001 ≤ P < 0.01).

[0031] Figure 13 The effect of discholenol on MAD content in frozen-thawed sheep semen; CT is the control, * indicates significant difference (0.01 ≤ P<0.05).

[0032] Figure 14 The effect of discholenol on ROS content in frozen-thawed sheep semen; CT is the control, ** indicates significant difference (0.001 ≤ P<0.01). Detailed Implementation

[0033] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0034] In the following examples, the marine active antioxidant is dieckol, a phenolic compound extracted from brown algae.

[0035] Example 1 I. Materials and Methods 1. Preparation of diluent The buffer solution was prepared with the following composition: 2.75% fructose, 0.69% trisodium citrate dihydrate, 0.1% NaHCO3, 0.235% EDTA-2Na, 0.29% citric acid, 0.565% Tris-base, 0.2% amikacin, and the remainder being ultrapure water.

[0036] The cryopreservation dilution buffer I was prepared as follows: 2.9% Tris-base, 0.4% fructose, 1.59% citric acid, 0.2% amikacin, 20% egg yolk, 0 μM-2 μM scheffleraol, and the remainder being ultrapure water. The concentrations of scheffleraol were 0 μM, 0.5 μM, 1 μM, 2 μM, and 4 μM.

[0037] The cryopreservation diluent II was prepared by using 12% glycerol, 2% OEP, and the remainder being cryopreservation diluent I with a concentration of 0 μM of sennaol.

[0038] In this embodiment, the percentage concentration (%) of each component refers to the percentage of the volume of that component relative to the total volume ((v / v) %).

[0039] After mixing cryopreservation diluent I, semen, and cryopreservation diluent II in a volume ratio of 9:1:10, the final concentrations of the antioxidants were approximately 0 μM (control, CT), 0.25 μM, 0.5 μM, 1 μM, and 2 μM.

[0040] In the cryopreservation diluent, each component plays a different protective role: Tris-base maintains and regulates the pH of the diluent, ensuring the preservation of sperm cell structure and physiological integrity; citric acid has good pH regulation and buffering properties; glucose provides energy for sperm metabolism; pyruvate plays a crucial role as an important intermediate in sperm cell sugar metabolism and the interconversion of various intracellular substances; egg yolk contains a large amount of vitamins, minerals, proteins, and lecithin, which can provide sufficient nutrition and protect the sperm plasma membrane; glycerol, as a permeability protectant, can reduce ice crystal formation, alleviate free radical damage to cells, and change cell membrane permeability; and amikacin sulfate prolongs sperm survival time through its bactericidal effect.

[0041] The sheep semen in this embodiment was collected from Leizhou black goats.

[0042] 2. Cryopreservation of sheep semen 2.1 Semen Collection and Processing Ram semen was collected using an electrostimulation method. It was then mixed with a buffer solution at a 1:1 volume ratio to appropriately dilute the semen (to stabilize sperm pH and osmotic pressure, and reduce the impact of environmental factors). The diluted semen was then stored in a thermos at 30℃-35℃ and transported to the laboratory for testing and processing within 1 hour. Fresh semen, appearing milky white and cloudy with no abnormal odor, and exhibiting a motility higher than 80% and a deformity rate lower than 10%, was suitable for experimental use.

[0043] 2.2 Semen cryopreservation Technical routes for cryopreservation of ram semen, such as Figure 1 As shown.

[0044] ① After semen collection, it was diluted with buffer and brought back. After centrifugation, it was resuspended and diluted with cryopreservation diluent I at the same temperature. The volume ratio of sheep semen to cryopreservation diluent I was 1:9, and the sperm concentration was adjusted to 2×10⁻⁶. 8 Sperm / mL: Diluted semen was aliquoted into centrifuge tubes. Initial incubation of the samples was achieved by placing the centrifuge tubes in a pre-set 38°C water bath. Subsequently, the apparatus, along with the warm water, was transferred to a 4°C refrigerator for 2 hours to equilibrate. This gradient cooling process was designed to allow the semen samples to slowly adapt to the low-temperature environment and reach the equilibrium state required for the experiment.

[0045] ② After equilibration, the mixed solution obtained in step ① is mixed thoroughly with an equal volume of cryopreservation diluent II on ice water. The final sperm concentration is 1×10⁻⁶. 8 Sperm / mL, along with ice water, were placed in a 4°C refrigerator for 20 minutes to equilibrate.

[0046] ③ After equilibration, mix the solution obtained in step ② evenly, draw it into a 0.25 mL cryogenic capillary tube and seal it with sealing powder. Fumigate with liquid nitrogen (-30℃) and place it in liquid nitrogen for storage after 10 min.

[0047] ④ After storage for 1 week, thaw the product in 37°C warm water for 15 seconds and evaluate the freezing effect.

[0048] In this example, the control group (CT) was the one without added antioxidants (i.e., the concentration of diserodium was 0 μM).

[0049] 2.3 Quality assessment of frozen-thawed sheep semen (1) Detection of sperm motility Sperm motility was assessed using a computer-aided sperm analysis (CASA) system. For testing, 10 μL of thawed sheep semen was added to 90 μL of preheated PBS (-) at 37°C and incubated in a 37°C water bath for 10 min. Subsequently, 5 μL of the sample was added to a sperm counting plate and observed using a sperm analyzer. Specific indicators observed included total motility (TM), progressive motility (PM), v-cliffe velocity (VCL), v-slip velocity (VSL), v-path velocity (VAP), and lateral swing amplitude (ALH), with progressive motility (PM) being the primary indicator for evaluating sperm motility. Each sample was tested three times, with three fields of view observed each time, ensuring a minimum of 300 sperm were detected. The entire experiment was repeated four times.

[0050] (2) Plasma membrane integrity detection The integrity of the sperm plasma membrane was assessed using the hypotonic swelling method. First, a hypotonic aqueous solution (containing 9 mg / mL fructose and 4.9 mg / mL sodium citrate dihydrate) was prepared. For testing, sperm were mixed with this hypotonic solution at a volume ratio of 1:9 and incubated in a 37°C water bath for 30 min. After incubation, each sample was mixed with eosin staining solution (Sigma Aldrich, P4170) at a volume ratio of 1:1 and smeared. After the smears were air-dried, the sperm tail curling was observed under a 400x microscope, and the tail curling rate was counted. At least 200 sperm were observed each time, and the entire experiment was repeated four times.

[0051] (3) Acrosome integrity inspection Sperm acrosome integrity was assessed using Coomassie Brilliant Blue G-250 staining. The procedure was as follows: 100 μL of sperm dilution buffer was centrifuged at 2000 r / min for 3 min, followed by washing twice with 200 μL of DPBS. The washed sperm were then fixed in 1 mL of 4% paraformaldehyde for 10 min, centrifuged at 2000 r / min for 10 min, the supernatant was discarded, and the sperm were resuspended in 1 mL of DPBS, centrifuged again, and the supernatant was discarded. The sperm were resuspended again in 20 μL of DPBS, smeared, and stained in a Coomassie Brilliant Blue staining jar for 30 min. After staining, the smear was rinsed with tap water, allowed to air dry, and then observed and counted under a 400x microscope to determine the acrosome membrane integrity rate. Sperm with a blue acrosome region and smooth edges were considered to have an intact acrosome, while sperm with an unstained or lightly stained acrosome region were considered to have a damaged acrosome. At least 200 sperm were observed each time, for a total of 3 observations, resulting in a total count of 600 sperm.

[0052] 2.4 Evaluation of the antioxidant properties of frozen-thawed sheep semen (1) Detection of antioxidant enzyme activity CAT enzyme content detection: Sperm samples were processed according to the instructions of the catalase (CAT) test kit (Sigma Aldrich, CAT100), a standard curve was plotted, and the absorbance at 405 nm was detected using an enzyme-linked immunosorbent assay (ELISA) reader.

[0053] T-SOD enzyme content detection: The samples were processed according to the instructions of the T-SOD kit (Sigma Aldrich, 19160) and a standard curve was plotted. Finally, the absorbance was measured at 450 nm.

[0054] (2) Detection of total antioxidant activity Total antioxidant activity was determined using the T-AOC kit (Sigma Aldrich, MAK187). A standard curve was constructed and its formula was obtained. The absorbance (OD value) at 405 nm was measured using a microplate reader. The measured OD value was substituted into the formula to obtain the result, which is expressed in mM.

[0055] (3) MDA content detection Sperm samples were processed according to the instructions of the malondialdehyde (MDA) test kit (Nanjing Jiancheng Bioengineering Institute, A003-1) and a standard curve was plotted. 0.25 mL of the sample was taken and the absorbance at 532 nm was measured using an ELISA reader.

[0056] (4) ROS content detection The reactive oxygen species (ROS) content was determined according to the instructions of the reactive oxygen species (ROS) test kit (Shanghai Beyotime Biotechnology Co., Ltd., S0033S). Sperm samples were analyzed using flow cytometry.

[0057] II. Results and Analysis 1. The effect of the marine active antioxidant discholenol on frozen-thawed sheep semen. 1.1 Effects of the marine active antioxidant sennaol on the quality of frozen-thawed sheep semen Cryopreservation diluent I containing different concentrations of scheffleraol was mixed with semen and cryopreservation diluent II at a volume ratio of 9:1:10, resulting in final scheffleraol concentrations of approximately 0 μM (control, CT), 0.25 μM, 0.5 μM, 1 μM, and 2 μM. The effects of adding scheffleraol to the cryopreservation diluent at final concentrations of 0 μM (control group, CT), 0.25 μM, 0.5 μM, 1 μM, and 2 μM on the quality of frozen-thawed sheep semen were investigated. Figure 2 , Figure 3 As shown, compared with the control group (CT), the addition of scheffleraol significantly improved the total sperm motility and progressive motility. The effect of adding scheffleraol at a concentration of 1 μM was the most significant (0.0001 ≤ P < 0.001), therefore 1 μM was selected as the optimal concentration of scheffleraol. Figures 4-7 As shown, compared with the control group (CT), the addition of di-spinol improved sperm motility. Specifically, di-spinol increased the curve velocity (VCL) and mean path velocity (VAP) of sperm, but the difference was not significant (P>0.05), and significantly increased the linear velocity (VSL) (0.001 ≤ P<0.01), but had no significant effect on the lateral swing amplitude (ALH) of sperm. Figures 8-9 This indicates the effect of di-spinol on sperm plasma membrane integrity and acrosome integrity. The experimental results show that di-spinol can significantly improve sperm plasma membrane integrity (P<0.0001), while there is no significant difference in its effect on sperm acrosome (P>0.05).

[0058] Sperm motility is the first hurdle in evaluating semen quality, and post-thawing sperm motility is the most direct indicator of the effectiveness of cryoprotectants and the quality of the freezing process. Higher motility indicates less lethal damage to sperm during freezing and thawing. Sperm motility is a crucial indicator of whether sperm can find an egg and complete fertilization. Higher levels of VCL, VSL, VAP, and ALH indicate a greater likelihood of successful fertilization. These results demonstrate that discholenol can significantly improve the quality of frozen-thawed sheep semen, with an optimal concentration of 1 μM.

[0059] 1.2 Effect of marine active antioxidant sennaol on antioxidant properties of frozen-thawed sheep semen As a marine active antioxidant, sennaol has a strong ability to scavenge free radicals and reduce their production, thus protecting cells from oxidative stress. Therefore, to verify whether sennaol can maintain sperm motility and fertilization capacity by improving antioxidant properties, the contents of CAT enzyme, SOD enzyme, T-AOC and MDA were measured.

[0060] Antioxidant performance results are as follows Figures 10-13 As shown, schizocarpine significantly improved the antioxidant properties of sperm compared to the control group (CT). Figure 10 As shown, in the detection of CAT enzyme content, compared with the control group (CT), the CAT enzyme content in the 1 μM scholenol treatment group was significantly increased (0.01 ≤ P < 0.05). During the freeze-thaw process of sheep semen, hydrogen peroxide is produced. CAT is an enzyme specifically responsible for rapidly decomposing hydrogen peroxide into harmless water and oxygen in organisms. If not cleared in time, hydrogen peroxide will directly trigger a lipid peroxidation chain reaction, destroying sperm. High CAT content or strong activity indicates a strong ability to clear hydrogen peroxide, and the sperm are in a relatively safe low-oxidative stress environment; if the CAT content is low, hydrogen peroxide will accumulate, meaning that the sperm face a higher risk of lipid peroxidation damage. Measuring CAT enzyme content is used to assess the sperm's ability to clear the most destructive hydrogen peroxide, thereby determining whether the sperm can effectively resist oxidative damage and maintain functional integrity.

[0061] like Figure 11The results showed that, compared with the control group (CT), the 1 μM scholenol treatment group significantly increased the content of SOD enzyme (0.001 ≤ P < 0.01). During the semen freeze-thaw process, the first and most numerous free radicals produced are superoxide anion free radicals. Superoxide anions generate substances such as hydrogen peroxide, which can cause lipid peroxidation. SOD is the only enzyme that can specifically scavenge superoxide anions. It catalyzes the reaction of two superoxide anions to generate hydrogen peroxide, and works synergistically with CAT for antioxidant activity. The SOD content in sperm largely reflects the antioxidant defense reserves acquired during sperm production. The higher the SOD activity, the stronger the ability to scavenge free radicals at the source, and the stronger the first line of defense against oxidative damage in sperm.

[0062] like Figure 12 The results showed that, compared with the control group (CT), the 1 μM sennaol treatment group significantly increased the total antioxidant activity of sperm (0.001 ≤ P < 0.01). Unlike the detection of antioxidant enzyme activity, the detection of total antioxidant activity provides a systematic evaluation of the comprehensive defense capacity of sperm against oxidative damage. Even if individual enzyme indicators are normal, a low T-AOC indicates that there are still deficiencies in the overall antioxidant system. A high T-AOC, on the other hand, directly indicates an improvement in antioxidant performance.

[0063] like Figure 13 The results showed that, compared with the control group (CT), the MDA content in the 1 μM senna-treated group was significantly lower (0.01 ≤ P < 0.05). MDA is the final product of lipid peroxidation and can directly reflect the occurrence of lipid peroxidation. The higher the MDA content, the more severe the lipid peroxidation; the lower the MDA content, the milder the lipid peroxidation and the more significant the effect of the antioxidant.

[0064] like Figure 14 The results showed that, compared with the control group, the 1 μM scheffleraol treatment group significantly reduced ROS levels (0.001 ≤ P < 0.01). Excessive ROS is a major cause of decreased sperm function. Detecting ROS levels can assess the ability of antioxidants to scavenge excess free radicals. The more significant the effect of the antioxidant, the stronger its ability to scavenge free radicals, and the lower the ROS level.

[0065] The results of this experiment indicate that the marine active antioxidant discholenol can significantly improve the antioxidant performance of sperm.

[0066] In summary, the sheep semen cryopreservation diluent and freezing method provided in this embodiment are low-cost and easy to operate. The diol in the cryopreservation diluent can significantly improve antioxidant capacity by rapidly scavenging free radicals, reducing lipid peroxidation, and preventing oxidative damage. This, in turn, improves the cryopreservation effect of sheep semen, extends its preservation time, and gives it greater application value in artificial insemination.

Claims

1. A semen cryopreservation diluent, characterized in that, It contains sennaol.

2. The semen cryopreservation diluent according to claim 1, characterized in that, The concentration of discholenol in the semen cryopreservation diluent is 0.25 μM-2 μM when cryopreserving semen.

3. The semen cryopreservation diluent according to claim 2, characterized in that, The semen cryopreservation diluent includes semen cryopreservation diluent I and semen cryopreservation diluent II; The semen cryopreservation diluent I, calculated by volume percentage, consists of: 2.9% Tris-base, 0.4% fructose, 1.59% citric acid, 0.2% amikacin, 20% egg yolk, 0.5 μM-4 μM scheffleraol, with the remainder being water; The semen cryopreservation diluent II, calculated by volume percentage, consists of: 12% glycerol, 2% OEP, and the remainder is cryopreservation diluent I without discholenol; When the semen cryopreservation diluent is used to preserve semen, the volume ratio of semen cryopreservation diluent I, semen, and semen cryopreservation diluent II is 9:1:10, so that the concentration of diserodium is 0.25 μM-2 μM.

4. The semen cryopreservation diluent according to claim 3, characterized in that, The semen cryopreservation diluent I, calculated by volume percentage, consists of: 2.9% Tris-base, 0.4% fructose, 1.59% citric acid, 0.2% amikacin, 20% egg yolk, 2 μM scheffleraol, and the remainder being water. When the semen cryopreservation diluent is used to preserve semen, the volume ratio of semen cryopreservation diluent I, semen, and semen cryopreservation diluent II is 9:1:10, resulting in a scheffleraol concentration of 1 μM.

5. The application of the semen cryopreservation diluent according to claim 3 in the cryopreservation of sheep semen.

6. The application according to claim 5, characterized in that, Includes the following steps: S1. Sheep semen was diluted with cryopreservation diluent I at the same temperature. The volume ratio of fresh sheep semen to cryopreservation diluent I was 1:

9. The mixture was then placed at 4°C for 2 hours to equilibrate. S2. Mix the mixed solution obtained in step S1 with the semen cryopreservation diluent II at a volume ratio of 1:1 and place it at 4℃ for equilibration for 3-4 hours. S3. The mixed solution obtained in step S2 is drawn into a cryogenic capillary and sealed. After being fumigated with liquid nitrogen for 15 minutes, it is stored in liquid nitrogen.

7. The application according to claim 5, characterized in that, The sheep semen is a diluted sheep semen obtained by mixing fresh sheep semen with a buffer solution at a volume ratio of 1:1; the buffer solution, calculated by volume percentage, consists of: 2.75% fructose, 0.69% trisodium citrate dihydrate, 0.1% NaHCO3, 0.235% EDTA-2Na, 0.29% citric acid, 0.565% Tris-base, 0.2% amikacin, and the balance being water.

8. The application according to claim 7, characterized in that, The goat semen mentioned is from Leizhou black goats.

9. The application according to claim 6, characterized in that, The cryocapsule is a 0.25 mL cryocapsule.

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