Application of L-proline oligomer 8 in preparation of bovine semen freezing diluent

Abstract

The invention belongs to the technical field of cryopreservation of sperms, and particularly discloses application of an L-proline oligomer 8 in preparation of a bovine semen freezing diluent. The invention provides application of the L-proline oligomer 8 in preparation of the bovine semen freezing diluent, and further provides the bovine semen freezing diluent which comprises the L-proline oligomer 8 and a basic diluent. The invention discloses an application of an L-proline oligomer 8 in preparation of a bovine semen freezing diluent, and the L-proline oligomer 8 is added into the freezing diluent, so that sperm motility, motility rate, plasma membrane integrity and acrosome integrity can be effectively improved, the late apoptosis proportion of sperms is inhibited, and bovine sperm quality is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of ultra-low temperature cryopreservation of sperm, and particularly relates to application of L-proline oligomer 8 in preparing a bovine semen freezing diluent. Background Art

[0002] Semen cryopreservation technology plays a key role in animal husbandry breeding, artificial insemination and endangered species protection. Its core lies in maintaining sperm activity through ultra-low temperature preservation. However, in the existing technology, the freezing-thawing process can induce ice crystal damage and oxidative stress, leading to sperm plasma membrane rupture, acrosome damage, and decreased mitochondrial function, significantly reducing sperm motility, and thus reducing sperm vitality and viability. In order to solve the above problems, existing freezing diluents usually add antioxidants and ice-inhibiting antifreeze protectants as antifreeze protectants. However, since the mode of action of traditional antioxidant cryoprotectants is post-damage repair, their efficiency in scavenging reactive oxygen species is limited, making it difficult to completely inhibit freezing-induced oxidative damage, and their inhibition of structural damage is limited.

[0003] Ice-inhibiting cryoprotectants can reduce the growth rate of ice crystals during the freezing process, reducing the mechanical damage of ice crystals to sperm cell membranes and organelles during the freezing process. However, the extraction and synthesis costs of traditional ice-inhibiting cryoprotectants are high, and some permeable ice-inhibiting cryoprotectants penetrate into the cells and cause certain toxicity to the cells. L-Proline oligomer 8 (L-Pro8) is a small molecule non-permeable cryoprotectant that does not penetrate the cell membrane into the cell. Its oligomeric structure can stably bind to the outer surface of the cell membrane, inhibiting the growth of ice crystals around the cell membrane, thereby protecting the cells, and its synthesis cost is relatively low.

[0004] Currently available bovine semen cryo-dilution solutions lack highly effective, non-permeable ice-inhibiting ingredients, making it difficult to effectively inhibit ice crystal growth without damaging cells. The limitations of existing technologies significantly reduce sperm motility, viability, and membrane integrity after freezing and thawing, failing to meet the requirements for high-quality semen preservation. Summary of the Invention

[0005] The present invention aims to provide the use of L-proline oligomer 8 in the preparation of bovine semen freezing diluent. By adding L-proline oligomer 8 to the freezing diluent, sperm motility, viability, plasma membrane integrity and acrosome integrity can be effectively improved, the proportion of late sperm apoptosis can be inhibited, and the quality of bovine sperm can be improved.

[0006] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0007] Application of L-proline oligomer 8 in the preparation of bovine semen cryo-dilution solution.

[0008] The present invention also provides a bovine semen freezing diluent, comprising the L-proline oligomer 8 and a basic diluent.

[0009] Preferably, the concentration of L-proline oligomer 8 in the bovine semen freezing diluent is 16 mg / mL.

[0010] Preferably, the basic diluent comprises one or more of Tris buffer, glucose, citric acid, glycerol, antibiotics and egg yolk.

[0011] The present invention also provides a method for freezing and preserving bovine semen, comprising the following steps:

[0012] S1. Mix the bovine semen with the bovine semen freezing diluent to make the final sperm concentration be 1×10 9 / mL, and a mixed solution was obtained;

[0013] S2, equilibrate the mixed solution obtained in S1 at 37°C and then gradually cool it down;

[0014] S3. The mixed solution treated in S2 is fumigated and frozen with liquid nitrogen and then stored in liquid nitrogen.

[0015] Preferably, in S2, the gradient cooling is achieved by placing the mixed solution in a 37°C water bath, and transferring the mixed solution and the water bath container to a 4°C environment and maintaining the gradient cooling for 3 hours.

[0016] Preferably, in S3, during the liquid nitrogen fumigation, the frozen fine tube is 4 cm away from the liquid nitrogen surface, and the fumigation time is 7 minutes.

[0017] The present invention also provides the use of L-proline oligomer 8 in improving the quality of bovine sperm after freezing and thawing.

[0018] Preferably, the L-proline oligomer 8 improves the quality of bovine sperm by increasing sperm motility, viability, plasma membrane integrity and acrosome integrity, inhibiting the proportion of late sperm apoptosis.

[0019] The present invention also provides a method for improving the quality of bovine sperm after freezing and thawing, wherein the bovine semen is treated with L-proline oligomer 8 and then cryopreserved.

[0020] Compared with the prior art, the present invention has the following advantages and technical effects:

[0021] The present invention discloses an application of L-proline oligomer 8 in preparing a bovine semen freezing diluent, clarifies the effect of L-proline oligomer 8 on the quality of bovine frozen semen after thawing, and shows that L-proline oligomer 8 can significantly improve the quality of bovine semen after freezing and thawing. After conventional freezing and thawing, the sperm motility of the frozen group was 53.60%, and the sperm motility was 85.58%. After 16 mg / mL of L-proline oligomer 8 was added to the freezing diluent, the sperm motility was 76.17%, and the sperm motility was 96.98%. Both the motility and the motility were significantly improved compared with those of the frozen group.

[0022] The present invention provides a new method for improving the quality of frozen semen, which can be widely used to improve the freezing efficiency of bull semen, reduce the cost of artificial insemination, and extend the preservation time of endangered cattle breeds' genetic resources.

[0023] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The results of the effects of freezing on sperm quality in Example 1 are as follows: Figure 1 A in the figure is a histogram of sperm motility statistics of fresh and frozen groups. Figure 1 B is a histogram of sperm motility statistics of fresh and frozen groups. Figure 1 C in the figure is a bar graph showing the statistical analysis of the average sperm motility velocity in the fresh and frozen groups. Figure 1 D in the figure is a bar graph showing the statistical analysis of the average sperm movement speed in the fresh and frozen groups. Figure 1 E in the figure is a bar graph showing the statistical analysis of the average linear motion velocity of sperm in the fresh and frozen groups. Figure 1 F in the figure is the statistical analysis bar graph of the lateral swing amplitude of sperm heads in fresh and frozen groups. Figure 1 G in the figure is a histogram of the statistical analysis of sperm whipping frequency in fresh and frozen groups. Figure 1 H in the figure is a representative picture of sperm apoptosis detected by flow cytometry in the fresh group. Figure 1 I in the figure is a representative picture of sperm apoptosis detected by flow cytometry in the frozen group. Figure 1 J in the figure is the statistical chart of quantitative analysis of normal sperm in fresh and frozen groups. Figure 1 K in the figure is the statistical chart of quantitative analysis of late apoptotic sperm in fresh and frozen groups. Figure 1 L in the figure is a representative picture of the integrity of the acrosome and plasma membrane of the fresh sperm detected by flow cytometry. Figure 1 The M in the figure is a representative image of the sperm acrosome and plasma membrane integrity detected by flow cytometry in the frozen group. Figure 1 N in the figure is the statistical chart of quantitative analysis of sperm with intact plasma membrane in fresh and frozen groups. Figure 1 The O in the figure is the statistical chart of quantitative analysis of sperm with intact acrosome in fresh and frozen groups. Figure 1The P in the figure is a representative picture of mitochondrial membrane potential of fresh sperm detected by flow cytometry. Figure 1 The Q in the figure is a representative picture of mitochondrial membrane potential of sperm in the frozen group detected by flow cytometry. Figure 1 R in the figure is the statistical chart of quantitative analysis of mitochondrial membrane potential of sperm in fresh and frozen groups. Figure 1 The S in the figure is a representative picture of the reactive oxygen species level in sperm of fresh and frozen groups detected by flow cytometry. Figure 1 T in the figure is a statistical chart of quantitative analysis of sperm reactive oxygen species levels in fresh and frozen groups;

[0025] Figure 2 The results of the effect of adding L-proline oligomer 8 on sperm quality in Example 1 are as follows, Figure 2 A in the figure is a bar graph showing the statistical analysis of sperm motility in the frozen group and the frozen group with L-proline oligomer 8 added. Figure 2 B is a bar graph showing the statistical analysis of sperm motility in the frozen group and the frozen group with L-proline oligomer 8 added. Figure 2 C in the figure is a bar graph showing the statistical analysis of the average sperm motility velocity in the frozen group and the group with L-proline oligomer 8 added. Figure 2 D in the figure is a bar graph showing the statistical analysis of the average sperm movement speed in the frozen group and the group with L-proline oligomer 8 added. Figure 2 E in the figure is a bar graph showing the statistical analysis of the average linear motion velocity of sperm in the frozen group and the frozen group with L-proline oligomer 8 added. Figure 2 F in the figure is a bar graph showing the statistical analysis of the lateral swing amplitude of the sperm head in the frozen group and the frozen group with L-proline oligomer 8 added. Figure 2 G is a bar graph of statistical analysis of sperm whipping frequency in the frozen group and the group with L-proline oligomer 8 added. Figure 2 H in the figure is a representative picture of sperm apoptosis detected by flow cytometry in the frozen group. Figure 2 I in the figure is a representative picture of sperm apoptosis detected by flow cytometry in the frozen group with L-proline oligomer 8 added. Figure 2 J in the figure is the statistical diagram of the quantitative analysis of normal sperm in the frozen group and the frozen group with L-proline oligomer 8. Figure 2 K in the figure is the statistical graph of quantitative analysis of late apoptotic sperm in the frozen group and the frozen group with L-proline oligomer 8. Figure 2 L in the figure is a representative image of the sperm acrosome and plasma membrane integrity detected by flow cytometry in the frozen group. Figure 2 The M in the figure is a representative image of the integrity of the acrosome and plasma membrane of sperm in the frozen group 8 after the addition of L-proline oligomers by flow cytometry. Figure 2 N in the figure is the statistical chart of quantitative analysis of sperm with intact plasma membrane in 8 frozen groups and those with L-proline oligomers added. Figure 2 The O in the figure is the statistical chart of the quantitative analysis of sperm with intact acrosomes in the frozen group and the group with L-proline oligomer added. Figure 2The P in the figure is a representative picture of mitochondrial membrane potential of sperm in the frozen group detected by flow cytometry. Figure 2 Q in the figure is a representative image of mitochondrial membrane potential of sperm in the frozen group with L-proline oligomer 8 detected by flow cytometry. Figure 2 The R in the figure is the statistical diagram of quantitative analysis of sperm mitochondrial membrane potential in the frozen group and the frozen group with L-proline oligomer 8 added. Figure 2 The S in the figure is a representative image of the reactive oxygen species level in spermatozoa detected by flow cytometry after freezing and freezing with L-proline oligomer 8. Figure 2 T in the figure is a statistical diagram of quantitative analysis of sperm reactive oxygen species levels in the frozen group and the frozen group supplemented with L-proline oligomer 8;

[0026] Figure 3 The results of the effect of adding proanthocyanidin B2 on sperm quality in Example 1 are shown, among which, Figure 3 A in the figure is a bar chart showing the statistical analysis of sperm motility in the frozen group and the frozen group with added proanthocyanidin B2. Figure 3 B in the figure is the statistical analysis bar graph of sperm motility in the frozen group and the frozen group with added proanthocyanidin B2. Figure 3 C in the figure is a bar graph showing the statistical analysis of the average sperm motility velocity in the frozen group and the frozen group with proanthocyanidin B2 added. Figure 3 D in the figure is a bar graph showing the statistical analysis of the average sperm movement speed in the frozen group and the frozen group with added proanthocyanidin B2. Figure 3 E in the figure is a bar chart showing the statistical analysis of the average linear motion velocity of sperm in the frozen group and the frozen group with added proanthocyanidin B2. Figure 3 F in the figure is the statistical analysis bar graph of the lateral swing amplitude of sperm heads in the frozen group and the frozen group with proanthocyanidin B2 added. Figure 3 G in the figure is a bar graph showing the statistical analysis of sperm whipping frequency in the frozen group and the frozen group with added proanthocyanidin B2. Figure 3 H in the figure is a representative picture of sperm apoptosis detected by flow cytometry in the frozen group. Figure 3 I in the figure is a representative picture of sperm apoptosis detected by flow cytometry in the frozen group with added proanthocyanidin B2. Figure 3 J in the figure is the statistical chart of quantitative analysis of normal sperm in the frozen group and the frozen group with proanthocyanidin B2 added. Figure 3 K in the figure is the statistical chart of quantitative analysis of late apoptotic sperm in the frozen group and the frozen group with proanthocyanidin B2 added. Figure 3 L in the figure is a representative image of the sperm acrosome and plasma membrane integrity detected by flow cytometry in the frozen group. Figure 3 The M in the figure is a representative image of the integrity of the sperm acrosome and plasma membrane in the frozen group supplemented with proanthocyanidin B2 detected by flow cytometry. Figure 3 N in the figure is the statistical chart of quantitative analysis of sperm with intact plasma membrane in the frozen group and the frozen group with added proanthocyanidin B2. Figure 3 The O in the figure is the statistical chart of the quantitative analysis of sperm with intact acrosomes in the frozen group and the frozen group with added proanthocyanidin B2. Figure 3The P in the figure is a representative picture of mitochondrial membrane potential of sperm in the frozen group detected by flow cytometry. Figure 3 The Q in the figure is a representative picture of the mitochondrial membrane potential of sperm in the frozen group with added proanthocyanidin B2 detected by flow cytometry. Figure 3 R in the figure is the statistical chart of quantitative analysis of sperm mitochondrial membrane potential in the frozen group and the frozen group with proanthocyanidin B2 added. Figure 3 The S in the figure is a representative picture of the reactive oxygen species level in sperm of the frozen group and the frozen group with proanthocyanidin B2 detected by flow cytometry. Figure 3 T in the figure is a statistical chart showing the quantitative analysis of the reactive oxygen species levels in sperm of the frozen group and the frozen group with added proanthocyanidin B2. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0028] Unless otherwise defined, technical or scientific terms used in the present invention shall have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0029] Source of test materials:

[0030] In the present invention, unless otherwise specified, other test materials and instruments and equipment are conventional test materials in this field and can be purchased through commercial channels.

[0031] Example 1

[0032] Determination of the effect of conventional freezing system on the motility of frozen bovine sperm

[0033] 1. Experimental Methods

[0034] 1.1 Freezing and Thawing of Bovine Semen

[0035] Semen collection and dilution: Semen samples were collected from cattle using the pseudovaginal method. The samples were transferred to preheated 15 mL centrifuge tubes. 20 μL of semen sample was diluted with diluent at a ratio of 1:3 for initial dilution and pressing. Sperm motility, viability, and motility were observed and recorded using a computer-assisted semen analysis system. Semen samples were tested for density using a sperm density meter and diluted to 1 × 10 using the frozen diluents listed in Table 1. 9 The semen samples were then pressed and the sperm motility was observed. The motility of fresh and frozen semen samples collected was tested, and the motility of all fresh semen samples used for freezing was above 80%.

[0036] Table 1 Semen freezing diluent

[0037] Element dose Tris 1.8g Citric acid 1g glucose 0.5g Dual antibody 0.6mL glycerin 3mL Yolk 7.5mL Ultrapure water 38.9mL

[0038] Semen Freezing and Thawing: Thoroughly mix the diluted semen sample with the diluent. Add 230-250 mL of 37°C water to a 500 mL beaker. Insert the centrifuge tube containing the semen sample into the float and place it in the beaker filled with 37°C water, ensuring that the centrifuge tube does not touch the wall of the beaker. The beaker containing the sample is then placed in a 4°C constant-temperature refrigerator to cool for 2 hours. After cooling, mix the semen sample at 4°C and quickly transfer it to a pre-chilled 0.25 mL frozen sperm tube. Seal the tube with a steel ball and equilibrate it in a 4°C constant-temperature refrigerator for 1 hour. After equilibration, place the frozen sperm tube on a pre-chilled iron rack and transfer it to a foam box 4 cm above the liquid nitrogen surface for liquid nitrogen fumigation for 7 minutes. Immediately plunge the frozen sperm tube into the liquid nitrogen after fumigation.

[0039] Before thawing the semen, preheat the centrifuge tubes and slides on a warming table. Take the frozen sperm tubes out of the liquid nitrogen, shake them in the air for 3 seconds to remove the liquid nitrogen on the surface, and thaw them in a 37°C water bath for 30 seconds. After thawing, wipe the moisture on the sperm tubes with a paper towel, cut off the seal, and allow the semen to flow into the preheated centrifuge tubes for subsequent experimental operations.

[0040] To determine bovine semen quality, place 5 μL of semen onto a glass slide and cover with a coverslip (avoid air bubbles). Remove excess liquid with filter paper to form a uniform thin layer (approximately 20-30 μm thick). Post-freeze-thaw sperm motility, viability, and motility parameters were recorded using a computer-assisted semen analysis system: average curve line (VCL), average path velocity (VAP), average linear velocity (VSL), lateral head amplitude (ALH), and beat cross frequency (BCF).

[0041] Sperm cell apoptosis detection: Use the FITC-Annexin V / PI apoptosis kit and prepare the staining solution according to the kit instructions. Preheat the staining solution in a 37°C incubator. Take 50 μL of the diluted semen sample, add 800 μL of Dulbecco's phosphate-buffered saline (DPBS), centrifuge at 1500 rpm for 5 minutes, and remove the supernatant. Repeat this procedure twice. Add 100 μL of 1× Annexin V binding buffer to the sperm pellet to resuspend the sperm. Add 10 μL of FITC-Annexin and 5 μL of PI staining solution, mix well, and incubate at room temperature in the dark for 10-15 minutes. Resuspend the sample 2-3 times during the incubation period to ensure uniform staining. After the incubation period, add 400 μL of 1× Annexin V and analyze the sample using flow cytometry.

[0042] Sperm acrosome integrity testing: Peanut agglutinin (FITC-PNA) and propidium iodide (PI) double staining was used to detect the integrity of the sperm acrosome. 50 μL of diluted semen sample was added to 200 μL of 10 μg / mL PNA staining solution, followed by 5 μL of PI staining solution. Mix thoroughly and incubate in a 37°C incubator protected from light for 20 minutes. Resuspend the sample 2-3 times during incubation to ensure uniform staining. After incubation, resuspend the sperm in 250 μL of DPBS and analyze using a flow cytometer.

[0043] Sperm Reactive Oxygen Species (ROS) Level Assay: 2',7'-DCFH-DA fluorescent probe was used to detect ROS levels in sperm. The staining solution was prepared according to the kit instructions and preheated in a 37°C incubator. 80 μL of diluted semen sample was added to 1 mL of DPBS and centrifuged at 1500 rpm for 5 minutes. The supernatant was removed. This procedure was repeated twice. The sperm were resuspended in 1 mL of DPBS. 80 μL of the sperm suspension was transferred to a 1.5 mL centrifuge tube. 80 μL of the 2',7'-DCFH-DA staining solution was added. Gently pipette to mix thoroughly. The tube was incubated in a 37°C incubator protected from light for 20 minutes. Resuspend the tube 2-3 times during the incubation period to ensure uniform staining. After incubation, 800 μL of DPBS was added to the tube and centrifuged at 1500 rpm for 5 minutes. The supernatant was removed. This procedure was repeated twice. The sperm were resuspended in 500 μL of DPBS and analyzed by flow cytometry.

[0044] Sperm mitochondrial membrane potential (MMP) assay: JC-1 fluorescent probe was used to detect changes in sperm mitochondrial membrane potential. Prepare the staining solution according to the kit instructions and preheat in a 37°C incubator. Take 80 μL of diluted semen sample, add 1 mL of DPBS, centrifuge at 1500 rpm for 5 minutes, remove the supernatant, repeat this procedure twice, and resuspend the sperm in 1 mL of DPBS. Pipette 50 μL of the sperm suspension into a 1.5 mL centrifuge tube, add 50 μL of JC-1 working staining solution, and incubate in a 37°C incubator protected from light for 15 minutes. Resuspend the tube 2-3 times during incubation to ensure uniform staining. After incubation, centrifuge at 1500 rpm for 5 minutes, remove the supernatant, add 100 μL of JC-1 staining buffer (1×), centrifuge at 1500 rpm for 5 minutes, remove the supernatant, repeat this procedure twice, and resuspend the sperm in 500 μL of DPBS for analysis using flow cytometry.

[0045] 1.2 Experimental Grouping

[0046] Experimental group: bovine semen frozen in conventional freezing system; control group: fresh bovine semen.

[0047] 2. Experimental Results

[0048] 1. Conventional freezing system leads to a decline in the quality of frozen semen: the effects of freezing on sperm motility, viability and movement parameters are as follows: Figure 1 A- Figure 1 As shown in G.

[0049] Depend on Figure 1 A- Figure 1 The results showed that freezing could lead to the decrease of sperm motility (53.60±0.47% vs.96.07±0.40% P<0.05), viability (85.58±4.95% vs.99.81±0.09% P<0.05), VCL (38.07±3.97 vs.64.72±6.56μm / s P<0.05), VAP (26.51±4.11vs. s.46.90±2.60μm / sP<0.05.), VSL (20.77±4.50vs.35.87±1.64μm / sP<0.05.), ALH (1.70±0.19vs.2.43±0.25μm.P<0.05.) and BCF (6.43±0.59vs.9.09±0.64Hz.P<0.05.) were significantly decreased.

[0050] 2. Conventional freezing system leads to an increase in the apoptosis rate of frozen sperm: FITC-Annexin V and PI were used to detect the apoptosis level of sperm in each group. The results are as follows: Figure 1 H- Figure 1 As shown in K.

[0051] Depend on Figure 1 H and Figure 1 As can be seen from the I in the figure, normal sperm are distributed in the lower left quadrant, and late apoptotic sperm are distributed in the upper right quadrant. The distribution ratio of normal sperm in fresh sperm is higher than that in frozen sperm (63.50 vs. 26.30%); the distribution ratio of late apoptotic sperm in fresh sperm is lower than that in frozen sperm (23.20 vs. 58.40%). Figure 1 J and Figure 1 As shown in the K, freezing reduced the proportion of normal sperm (26.57±1.24% vs. 63.93±0.68%. P<0.001.) and increased the proportion of late apoptotic sperm (57.83±1.02% vs. 22.60±1.37%. P<0.001.).

[0052] 3. Conventional freezing system leads to decreased sperm membrane integrity: FITC-PNA and PI were used to detect the integrity of sperm acrosome and plasma membrane in each group. The results are as follows: Figure 1 L- Figure 1 The O in the.

[0053] Depend on Figure 1 L and Figure 1 As can be seen from the M in the figure, sperm with intact plasma membranes are distributed in the lower left and lower right quadrants, and sperm with intact acrosomes are distributed in the upper left and lower left quadrants. The distribution ratio of sperm with intact plasma membranes in fresh sperm is higher than that in frozen sperm (74.75 vs. 35.06%); the distribution ratio of sperm with intact acrosomes in fresh sperm is higher than that in frozen sperm (90.00 vs. 79.80%).

[0054] Depend on Figure 1 N and Figure 1 As shown in the O, freezing reduced the sperm plasma membrane integrity (34.67±0.28% vs. 77.02±1.94%. P<0.001.) and acrosome integrity (79.97±0.31% vs. 91.37±1.41%. P<0.001.).

[0055] 4. Effect of conventional freezing system on ROS and mitochondrial membrane potential of frozen sperm: JC-1 fluorescent probe and 2',7'-DCFH-DA were used to detect sperm MMP in each group. Figure 1 P- Figure 1 As shown in Q, the ROS level results are as follows Figure 1 As shown in S,

[0056] Depend on Figure 1 P and Figure 1 As can be seen from the Q in the figure, sperm with high mitochondrial membrane potential are distributed in the upper right quadrant, and the distribution ratio of sperm with high mitochondrial membrane potential in fresh sperm is higher than that in frozen sperm (99.30 vs. 43.50%).

[0057] Depend on Figure 1 As shown in S, the peak area represents the ROS level, and the ROS level in fresh sperm is lower than that in frozen sperm.

[0058] Depend on Figure 1 R and Figure 1 As shown in T, freezing significantly reduced the sperm MMP level (44.73±1.82% vs. 98.90±0.57%. P<0.001.) and significantly increased the ROS level (1.88±0.37% vs. 1.00±0.03%, P<0.05.).

[0059] Example 2

[0060] Determination of the effect of the addition of L-proline oligomer 8 on the motility of frozen bovine sperm: The freezing and thawing methods of bovine semen were similar to those of Example 1, except that this example was divided into a conventional freezing system group and an L-proline oligomer 8-modified freezing system group. Among them, the L-proline oligomer 8-modified freezing system group was a conventional freezing system supplemented with 16 mg / mL L-proline oligomer 8.

[0061] The methods for semen quality determination, sperm cell apoptosis detection, sperm acrosome plasma membrane integrity detection, sperm reactive oxygen species (ROS) content detection, and sperm mitochondrial membrane potential (MMP) detection were the same as those in Example 1.

[0062] The experimental results are as follows:

[0063] 1. L-proline oligomer 8 improves the quality of frozen semen: Effects of freezing on sperm motility, viability and motility parameters Figure 2 A- Figure 2 As shown in G.

[0064] Depend on Figure 2 A- Figure 2 C and Figure 2 E and Figure 2 As shown in G, L-proline oligomer 8 significantly improved the motility (76.17±1.56% vs. 53.60±0.47%, P<0.05), viability (96.98±0.94% vs. 85.58±4.95%, P<0.05), VCL (51.90±2.87 vs. 38.07±3.97 μm / s, P<0.05), VSL (27.79±2.92 vs. 20.77±4.50 μm / s, P<0.05), and BCF (7.90±0.49 vs. 6.43±0.59 Hz, P<0.05). Figure 2 D and Figure 2 It can be seen from F in that there is no significant effect on VAP and ALH.

[0065] 2. L-proline oligomer 8 reduces the level of sperm apoptosis in frozen sperm: FITC-Annexin V and PI were used to detect the level of sperm apoptosis in the frozen group and the frozen group supplemented with L-proline oligomer 8. The results are as follows: Figure 2 H- Figure 2 As shown in K.

[0066] Depend on Figure 2 H and Figure 2As can be seen from I in the figure, normal sperm are distributed in the lower left quadrant, and late apoptotic sperm are distributed in the upper right quadrant. The distribution ratio of normal sperm in frozen sperm is lower than that in sperm frozen with L-proline oligomer 8 (26.30 vs. 38.70%). The distribution ratio of late apoptotic sperm in fresh sperm is higher than that in sperm frozen with L-proline oligomer 8 (58.40 vs. 49.50%).

[0067] Depend on Figure 2 J and Figure 2 It can be seen from the K in the results that L-proline oligomer 8 can significantly increase the proportion of normal sperm (41.77±2.62% vs. 26.57±1.24%. P<0.001.) and reduce the proportion of late apoptotic sperm (46.47±2.45% vs. 57.83±1.02%. P<0.05.).

[0068] 3. Effect of L-proline oligomer 8 on the integrity of frozen sperm membrane: FITC-PNA and PI were used to detect the integrity of sperm membrane in the frozen group and the frozen group supplemented with L-proline oligomer 8. The results are as follows: Figure 2 L- Figure 2 As shown in O.

[0069] Depend on Figure 2 L and Figure 2 As can be seen from the M in the figure, sperm with intact plasma membranes are distributed in the lower left and lower right quadrants, and sperm with intact acrosomes are distributed in the upper left and lower left quadrants. The distribution ratio of sperm with intact plasma membranes in frozen sperm is lower than that in sperm frozen with the addition of L-proline oligomer 8 (35.06 vs. 74.16%); the distribution ratio of sperm with intact acrosomes in frozen sperm is lower than that in sperm frozen with the addition of L-proline oligomer 8 (79.80 vs. 90.70%).

[0070] Depend on Figure 2 N and Figure 2 It can be seen from the O in the figure that L-proline oligomer 8 can also significantly improve the sperm plasma membrane integrity (75.03±0.68% vs. 34.67±0.28%. P<0.001.) and acrosome integrity (91.00±0.29% vs. 79.97±0.31%. P<0.001.).

[0071] 4. Effect of L-proline oligomer 8 on ROS and mitochondrial membrane potential in frozen sperm: JC-1 fluorescent probe and 2',7'-DCFH-DA were used to detect sperm MMP in the frozen group and the frozen group supplemented with L-proline oligomer 8. Figure 2 P- Figure 2 As shown in Q, the ROS level results are as follows Figure 2 As shown in S.

[0072] Depend on Figure 2 P and Figure 2 As can be seen from the Q in the figure, sperm with high mitochondrial membrane potential are distributed in the upper right quadrant, and the distribution ratio of sperm with high mitochondrial membrane potential in frozen sperm is equivalent to that in sperm frozen with L-proline oligomer 8.

[0073] Depend on Figure 2 As shown in S, the peak area represents the ROS level. The ROS level in frozen sperm is equivalent to that in sperm frozen with L-proline oligomer 8.

[0074] Depend on Figure 2 R and Figure 2 As shown in T, L-proline oligomer 8 had no significant effect on the MMP level and ROS level of frozen sperm (1.88±0.37% vs. 1.00±0.03%, P<0.05.).

[0075] Example 3

[0076] Determination of the effect of adding proanthocyanidin B2 on the motility of bovine frozen sperm: The bovine semen freezing and thawing method refers to Example 1, except that this example is divided into a conventional freezing system group and a proanthocyanidin B2 improved freezing system group, wherein the proanthocyanidin B2 improved freezing system group is a conventional freezing system with 15 μg / mL proanthocyanidin B2 added.

[0077] The methods for semen quality determination, sperm cell apoptosis detection, sperm acrosome plasma membrane integrity detection, sperm reactive oxygen species (ROS) content detection, and sperm mitochondrial membrane potential (MMP) detection were the same as those in Example 1.

[0078] Experimental results:

[0079] 1. Effect of proanthocyanidin B2 addition on the quality of frozen semen: Effect of freezing on sperm motility, viability and motility parameters Figure 3 A- Figure 3 As shown in G.

[0080] Depend on Figure 3 A in Figure 3 E and Figure 3 As shown in G, proanthocyanidin B2 significantly improved the motility of frozen sperm (63.85±0.51% vs. 53.60±0.47%. P<0.05.), VSL (27.53±2.19 vs. 20.77±4.50μm / sP<0.05.) and BCF (7.86±0.66 vs. 6.43±0.59Hz. P<0.05.). Figure 3 C- Figure 3It can be seen from F in that there is no significant effect on VCL, VAP and ALH.

[0081] 2. Effect of proanthocyanidin B2 addition on the apoptosis level of frozen sperm: FITC-Annexin V and PI were used to detect the apoptosis level of sperm in the frozen group and the frozen group supplemented with proanthocyanidin B2. Figure 3 H- Figure 3 As shown in K.

[0082] Depend on Figure 3 H and Figure 3 As can be seen from the I in the figure, normal sperm are distributed in the lower left quadrant, and late apoptotic sperm are distributed in the upper right quadrant. The distribution ratio of normal sperm in frozen sperm is lower than that in frozen sperm with added procyanidin B2 (26.30 vs. 41.60%). The distribution ratio of late apoptotic sperm in fresh sperm is higher than that in frozen sperm with added procyanidin B2 (58.40 vs. 46.50%). Figure 3 J and Figure 3 It can be seen from the K in the results that proanthocyanidin B2 can significantly increase the proportion of normal sperm (40.87±0.71% vs. 26.57±1.24%. P<0.001.) and reduce the proportion of late apoptotic sperm (46.80±0.42% vs. 57.83±1.02%. P<0.05.).

[0083] 3. Effect of proanthocyanidin B2 addition on the integrity of frozen sperm membrane: FITC-PNA and PI were used to detect the integrity of sperm membrane in the frozen group and the group with proanthocyanidin B2 added. Figure 3 L- Figure 3 The O in the.

[0084] Depend on Figure 3 L and Figure 3 As can be seen from the M in the figure, sperm with intact plasma membranes are distributed in the lower left and lower right quadrants, and sperm with intact acrosomes are distributed in the upper left and lower left quadrants. The distribution ratio of sperm with intact plasma membranes in fresh sperm is lower than that in sperm frozen with procyanidin B2 (35.06 vs. 69.35%). The distribution ratio of sperm with intact acrosomes in fresh sperm is lower than that in sperm frozen with procyanidin B2 (79.80 vs. 88.50%).

[0085] Depend on Figure 3 N and Figure 3 It can be seen from the O in the figure that proanthocyanidin B2 can also significantly improve the sperm plasma membrane integrity (69.41±0.99% vs. 34.67±0.28%. P<0.001.) and acrosome integrity (88.83±0.34% vs. 79.97±0.31%. P<0.001.).

[0086] 4. Effect of proanthocyanidin B2 addition on ROS and mitochondrial membrane potential of frozen sperm: JC-1 fluorescent probe and 2',7'-DCFH-DA were used to detect sperm MMP in the frozen group and the proanthocyanidin B2-added frozen group. Figure 3 P- Figure 3 As shown in Q, the ROS level results are as follows Figure 3 As shown in S,

[0087] Depend on Figure 3 P- Figure 3 As can be seen from the Q in the figure, sperm with high mitochondrial membrane potential are distributed in the upper right quadrant, and the distribution ratio of sperm with high mitochondrial membrane potential in frozen sperm is lower than that in frozen sperm with added proanthocyanidin B2 (43.50 vs. 99.80%).

[0088] Depend on Figure 3 As can be seen from the S in the figure, the peak area represents the ROS level. The ROS level in frozen sperm is lower than that in frozen sperm with added procyanidin B2.

[0089] Depend on Figure 3 R and Figure 3 As shown in T, proanthocyanidin B2 significantly increased the MMP level (99.00±1.13% vs. 44.73±1.82%. P<0.001.) and significantly decreased the ROS level in frozen sperm (0.62±0.17% vs. 1.88±0.37%, P<0.05.).

[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. Application of L-proline oligomer 8 in the preparation of bovine semen freezing diluent.

2. A bovine semen freezing diluent, characterized in that: The method comprises the L-proline oligomer 8 as claimed in claim 1 and a basic diluent.

3. The bovine semen freezing diluent according to claim 2, characterized in that The concentration of L-proline oligomer 8 in the bovine semen freezing diluent is 16 mg / mL.

4. The bovine semen freezing diluent according to claim 2, characterized in that The basic diluent includes one or more of Tris buffer, glucose, citric acid, glycerol, antibiotics and egg yolk.

5. A method for freezing and preserving bovine semen, characterized in that: The following steps are involved: S1, mixing the bovine semen with the bovine semen freezing diluent according to claim 2, so that the final sperm concentration is 1×10 9 / mL, and a mixed solution was obtained; S2, equilibrate the mixed solution obtained in S1 at 37°C and then gradually cool it down; S3. The mixed solution treated in S2 is fumigated and frozen with liquid nitrogen and then stored in liquid nitrogen.

6. The method according to claim 5, characterized in that In S2, the gradient cooling is achieved by placing the mixed solution in a 37°C water bath, and transferring the mixed solution and the water bath container to a 4°C environment and maintaining the gradient cooling for 3 hours.

7. The method according to claim 5, characterized in that In S3, during the liquid nitrogen fumigation, the frozen fine tube was kept 4 cm away from the liquid nitrogen surface, and the fumigation time was 7 minutes.

8. Application of L-proline oligomer 8 in improving bovine sperm quality after freezing and thawing.

9. The application according to claim 8, characterized in that: The L-proline oligomer 8 improves the quality of bovine sperm by increasing sperm motility, viability, plasma membrane integrity and acrosome integrity, inhibiting the proportion of late sperm apoptosis.

10. A method for improving the quality of bovine sperm after freezing and thawing, characterized in that: Bovine semen was treated with L-proline oligomer 8 and cryopreserved.

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