Sheep sperm endogenous ferroptosis inhibitor protein protective agent and protection method and application

Abstract

The present invention discloses a protective agent for endogenous iron death inhibitory protein in sheep sperm, a protective method and an application thereof. The protective agent for endogenous iron death inhibitory protein in sheep sperm comprises the following components: Tris, citric acid, glucose, chloroquine, glycerol, a mixture of penicillin-streptomycin, egg yolk liquid and ultrapure water, and the above substances are dissolved in the ultrapure water. The present invention also provides a method for protecting endogenous iron death inhibitory protein in sheep sperm, comprising the following steps: preparing an endogenous iron death inhibitory protein protective agent for sheep sperm; semen collection; diluting semen with an endogenous iron death inhibitory protein protective agent for sheep sperm; and freezing semen. The present invention also provides an application of an endogenous iron death inhibitory protein protective agent for sheep sperm. The present invention can protect the iron death inhibitory protein during the freezing recovery process, ensure the expression level of the iron death inhibitory protein, ensure the sperm motility and the integrity of the biofilm structure, and greatly improve the resistance of sheep sperm to freezing damage.

Description

Technical Field

[0001] The invention belongs to the technical field of sheep semen preservation, and specifically relates to a sheep sperm endogenous ferroptosis inhibitory protein protective agent and a protection method and application. Background Art

[0002] Modern sheep farming is moving towards a more intensive and large-scale direction, and the scarcity of excellent breeding stock has become a key bottleneck restricting the sustainable development of the sheep industry. As a means of efficiently utilizing excellent germplasm resources, semen cryopreservation technology can preserve sperm for a long time, bringing significant economic value to the sheep industry.

[0003] Ferroptosis is the main type of cell death during sperm cryopreservation, which is associated with the decline of ferroptosis inhibitory proteins. Ferroptosis inhibitory proteins include GPX4 and FTH1, which are essential for maintaining the normal physiological function of sperm and play a key role in anti-ferroptosis during sperm freezing, inhibiting the occurrence of ferroptosis through redox regulation and iron regulation, thereby ensuring the normal function of sperm after freezing and thawing.

[0004] At present, the research on sperm diluents and protective agents mainly focuses on how to ensure sperm vitality, how to meet nutrient requirements, how to improve antibacterial ability, and how to regulate osmotic pressure. However, these studies have many limitations: First, most studies only focus on the above conventional aspects and fail to fully consider the important physiological changes of sperm during the cryopreservation process, especially the regulatory role of ferroptosis inhibitory proteins in the sperm cryopreservation process. There are few relevant reports, and ferroptosis inhibitory proteins play an important role in the function and quality of sperm after cryopreservation. The regulation of their expression levels can have an important impact on the subsequent development and fertilization ability of sperm; secondly, in order to increase the expression level of related proteins in cells, the existing technology often uses recombinant proteins and cell transfection methods. However, recombinant proteins are mostly human-derived, which will bring high costs and increase the economic burden of research and application. Cell transfection technology is not suitable for sperm.

[0005] Therefore, the existing technology urgently needs to conduct research on sperm diluents and protective agents to protect ferroptosis inhibitory proteins during the sperm cryopreservation process, thereby improving the sperm cryopreservation effect and subsequent use performance. Summary of the invention

[0006] The first purpose of the present invention is to overcome the shortcomings of the prior art and provide a goat sperm endogenous ferroptosis inhibitory protein protector.

[0007] The first objective of the present invention is achieved through the following technical scheme: a sheep sperm endogenous iron death inhibition protein protector, comprising the following components: 300mmol / L Tris (trishydroxymethylaminomethane), 95mmol / L citric acid, 56mmol / L glucose, 5μmol / L chloroquine, 6% volume fraction glycerol, 1% volume fraction penicillin-streptomycin mixture and 15% volume fraction egg yolk liquid, the above substances are dissolved in ultrapure water to form the sheep sperm endogenous iron death inhibition protein protector.

[0008] The second purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for protecting endogenous ferroptosis inhibitory protein in sheep sperm.

[0009] The second object of the present invention is achieved through the following technical solution: a method for protecting endogenous ferroptosis inhibitory protein in sheep sperm, comprising the following steps:

[0010] 1) Prepare the sheep sperm endogenous ferroptosis inhibitory protein protective agent, transfer the prepared sheep sperm endogenous ferroptosis inhibitory protein protective agent into a reagent bottle through a disposable syringe filter, seal it, and store it at 4°C;

[0011] 2) Semen collection;

[0012] 3) Using sheep sperm endogenous iron death inhibitor protein protector to dilute semen;

[0013] 4) Sperm freezing.

[0014] Furthermore, in step 2), the semen is collected by using a false vagina collection method, and the whole process is aseptically operated; after the semen collection is completed, the false vagina is immediately erected to prevent the semen from flowing back, and the collected fresh semen is kept warm at 37°C.

[0015] Furthermore, in step 3), the method of diluting semen with endogenous ferroptosis inhibitory protein protective agent of sheep sperm is as follows: semen with fresh sperm vitality of more than 75% and rapid linear motion of more than 50% is collected; the endogenous ferroptosis inhibitory protein protective agent of sheep sperm is taken, and the temperature of the endogenous ferroptosis inhibitory protein protective agent of sheep sperm is raised to 37° C., and at 37° C., the semen is added to the endogenous ferroptosis inhibitory protein protective agent of sheep sperm for dilution, the volume ratio of semen to endogenous ferroptosis inhibitory protein protective agent of sheep sperm is 1:10, and the mixture is mixed after dilution.

[0016] Furthermore, in step 4), the semen is frozen by cooling in a 4°C constant temperature refrigerator for 4 hours; after cooling, the semen is placed in a 0.25 ml capillary tube, and after balancing for 2 hours, it is moved to a place 4 cm away from the liquid nitrogen surface, fumigated for 7 minutes, and then quickly put into liquid nitrogen for cryopreservation.

[0017] Furthermore, the method further comprises step 5) thawing of semen, wherein the semen is thawed by taking out the frozen straw from liquid nitrogen and placing it in a 37° C. water bath for thawing for 30 seconds.

[0018] The third purpose of the present invention is to overcome the shortcomings of the prior art and provide an application of an endogenous ferroptosis inhibitory protein protective agent for sheep sperm.

[0019] The third objective of the present invention is achieved through the following technical scheme: the application of endogenous ferroptosis inhibitory protein protective agent of sheep sperm, including the following contents: preparing endogenous ferroptosis inhibitory protein protective agent of sheep sperm; transferring to a reagent bottle through a disposable syringe filter, sealing, and storing at 4°C; heating the endogenous ferroptosis inhibitory protein protective agent of sheep sperm to 37°C, adding semen to the endogenous ferroptosis inhibitory protein protective agent of sheep sperm at 37°C, diluting the semen and the endogenous ferroptosis inhibitory protein protective agent of sheep sperm in a volume ratio of 1:10, mixing after dilution; and then freezing and storing.

[0020] The beneficial effects of the present invention are: the present invention can protect the ferroptosis inhibitory protein during the freezing recovery process, ensure the expression level of the ferroptosis inhibitory protein, effectively reduce the membrane damage of sperm, and ensure the sperm motility and the integrity of the biomembrane structure. The present invention can greatly improve the resistance of sheep sperm to freezing damage, ensure that sperm can play a normal physiological function, can fully improve the utilization rate of breeding rams, and is conducive to the development of livestock breeding work.

[0021] Compared with the existing conventional sheep sperm diluent, the sheep sperm endogenous iron death inhibitor protein protectant of the present invention is used to dilute and preserve semen. Under the same conditions, the expression level of iron death inhibitor protein GPX4 in sheep sperm is increased by 17.0%, the vitality is increased by 22.4%, the plasma membrane integrity rate is increased by 7.5%, the lipid peroxidation level is reduced by 26.0%, and the Fe 2+ concentration decreased by 80.4%; the expression level of ferroptosis inhibitory protein FTH1 in goat sperm increased by 408.2%, the plasma membrane integrity rate increased by 38.3%, the lipid peroxidation level decreased by 47.9%, and Fe 2+ The level dropped by 65.8%. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a bar graph of the expression level of GPX4 in sheep sperm in Example 1;

[0023] Figure 2 is a bar graph of sheep sperm motility (TM) in Example 1;

[0024] Figure 3 is a bar graph of the plasma membrane integrity of sheep sperm in Example 1;

[0025] Figure 4is a bar graph of lipid peroxidation levels in sheep sperm of Example 1;

[0026] Figure 5 The sheep Fe of Example 1 2+ Horizontal bar chart;

[0027] Figure 6 The bar graph of FTH1 expression level of goat sperm in Example 2;

[0028] Figure 7 is a bar graph of goat sperm plasma membrane integrity in Example 2;

[0029] Figure 8 is a bar graph of lipid peroxidation levels of goat sperm in Example 2;

[0030] Fig. 9 The goat sperm Fe of Example 2 2+ Horizontal bar chart. DETAILED DESCRIPTION

[0031] The present invention is described in detail below in conjunction with the accompanying drawings.

[0032] Example 1 Effect of endogenous ferroptosis inhibitor protein on sperm quality during cryopreservation of sheep semen

[0033] 1. Experimental Animals

[0034] The sheep semen used in this experiment came from Inner Mongolia Jinlai Animal Husbandry Technology Co., Ltd., and four 2-year-old adult rams were selected, which were required to be healthy, disease-free, and uniformly raised and managed.

[0035] 2. Test methods

[0036] (1) Preparation of protective agent

[0037] Control dilution: Dissolve 1.8g Tris (trishydroxymethylaminomethane), 1g citric acid, 0.5g glucose, 0.5ml penicillin-streptomycin mixture (15140122, Gibco), 3ml glycerol and 15ml egg yolk solution in ultrapure water and adjust the volume to 50ml. Transfer to a reagent bottle through a 0.22 μm disposable syringe filter, seal it, store it in a 4℃ refrigerator, and use it within one week.

[0038] Experimental group sheep sperm endogenous ferroptosis inhibitor protein protectant: Take Tris 1.8g, citric acid 1g, glucose 0.5g, penicillin-streptomycin mixture 0.5ml, glycerol 3ml, egg yolk liquid 15ml and chloroquine 0.0799mg, dissolve the above substances in ultrapure water, and make up to 50ml. Transfer to a reagent bottle through a 0.22 μm disposable syringe filter, seal it, store it in a refrigerator at 4℃, and use it within one week.

[0039] (2) Semen collection: Use the pseudo vagina method to collect semen once every two days. Use long-handled tweezers to grab a 75% alcohol cotton ball and rub it on the pseudo vagina inner tube for disinfection. Then rub it with sterile saline 2-3 times. Rinse the pseudo vagina inner tube with sterile Tris-citrate-glucose basic solution (Tris is 36 g / L, citric acid is 20 g / L, glucose is 10 g / L and water). Let the residual liquid in the pseudo vagina dry naturally. Inject hot water that accounts for 2 / 3 of the volume of the pseudo vagina interlayer cavity into the water injection hole of the pseudo vagina shell. The water temperature is 45-50℃, and the water injection port is plugged with a rubber plug. Use a thermometer to measure the inner tube temperature and keep it at 38-40℃. Use a sterile glass rod to dip in sterile lubricant (medical vaseline) and apply it to the 1 / 3-1 / 2 of the front section of the pseudo vagina to lubricate its inner cavity. Insert the semen collection cup that has been sterilized and dried by high pressure into the other end that is not coated with vaseline. Adjust the pressure in the pseudo vagina cavity according to the insertion depth of the semen collection cup. During semen collection, the semen collector is at the right rear of the semen collection platform. When the male animal mounts, the penis is quickly introduced into the false vagina. After the semen collection is completed, the false vagina is immediately erected to prevent the semen from flowing back. The collected fresh semen is kept warm at 37℃ and sent to the laboratory for processing.

[0040] Fresh semen quality testing and processing. Take 5μl of fresh semen and dilute it with Tris-citrate-glucose basic solution (200μL), take 5μl of the diluted semen on a slide, cover the slide with a glass slide, and use a computer-assisted semen analysis system to detect at least 5 fields of view. The detection system captures at least 1000 sperm cells. The fresh semen motility reaches more than 75%, and the semen samples with more than 50% fast linear motion sperm are used for subsequent tests.

[0041] (3) Freezing and thawing of sheep sperm: Fresh sheep semen was diluted with the diluent of the control group and the endogenous iron death inhibitor protein protectant of the experimental group at 37°C until the sperm density reached 2×10 8 / ml; place the diluted semen in a 37℃ water bath and place it in a 4℃ freezer to cool for 4h; after the water bath temperature drops to 4℃, use a pipette to pipette 200μl to 250μl into frozen sperm tubes, seal with sealing powder, and balance at 4℃ for 2h; place these frozen sperm tubes equidistantly on the freezing rack, fumigate with liquid nitrogen for 7min at a distance of 4cm above the liquid nitrogen surface, and then store them in liquid nitrogen; thaw the frozen semen in a water bath three days later, and thaw for 30s at a water bath temperature of 37℃.

[0042] (4) Detection of GPX4 expression in frozen semen by flow cytometry. Add 400 μL of phosphate-buffered saline (DPBS) to the semen and centrifuge at 1350 r / min for 3 min, repeat twice and completely remove the supernatant. Add 250 μL of fixative to the semen sample and mix well. Let stand at room temperature for 30 min to fix the cell structure. Then add 200 μL of permeabilization solution and centrifuge for 3 min and repeat 3 times to remove the fixative. Add 300 μL of permeabilization solution to treat the cells for 15 min, and then add permeabilization solution again for centrifugation and washing. Add 400 μL of blocking solution to the sample and block at room temperature for 30 min. After blocking, centrifuge the permeabilization solution to remove excess blocking agent. Then add diluted GPX4 rabbit polyclonal primary antibody (30388-1-AP) to the cells and incubate for 1 h to label the target protein. At the same time, set rabbit polyclonal IgG as an isotype control to evaluate nonspecific binding. After incubation, centrifuge the permeabilization solution again to remove unbound primary antibody. Subsequently, diluted donkey anti-rabbit FITC fluorescent secondary antibody (1:200) was added and incubated for 30 min in the dark to detect the binding of the primary antibody. Finally, the sperm cells were thoroughly washed with DPBS and resuspended in preparation for analysis by flow cytometry. During the flow cytometric detection process, at least 10,000 cells were collected and analyzed to evaluate the cell fluorescence intensity. The geometric mean fluorescence intensity is obtained by subtracting the fluorescence intensity background of the isotype control stained cells from the fluorescence intensity of the specific antibody stained cells, thereby accurately reflecting the expression level of the target protein.

[0043] The results of sperm GPX4 expression level detection are as follows Figure 1 As shown in the figure, Control is the control group, CQ is the experimental group, the ordinate is the GPX4 expression level, the GPX4 expression level of the experimental group was significantly higher than that of the control group (P < 0.05), the GPX4 expression level of the control group was 51.3%, and the GPX4 expression level of the experimental group was 60.0%.

[0044] (5) Sperm TM detection: transfer the frozen thawed semen into a 1.5 ml centrifuge tube and place it on a constant temperature platform at 37°C. Take 3 μl of the thawed semen and drop it on a glass slide. After pressing the slide with a coverslip, use a computer-assisted semen analysis system to detect sperm TM. Detect at least 1,000 sperm in at least 5 fields of view and record the data.

[0045] Sperm TM results Figure 2 As shown, the horizontal axis represents the grouping, Control is the control group, CQ is the experimental group, and the vertical axis represents the proportion of TM; the TM of the control group is 59.4%, and the TM of the experimental group is 72.7%. The TM of the experimental group is significantly higher than that of the control group (P < 0.05).

[0046] (6) Plasma membrane integrity detection: PI dye was used to detect the plasma membrane integrity using flow cytometry. Thawed sperm was centrifuged at 300×g, the supernatant was removed, 499ul of phosphate buffered saline (PBS) and 1ul of PI dye were added to resuspend the sperm, incubated in the dark for 10min, and finally detected by flow cytometry.

[0047] The results of sperm plasma membrane integrity test are as follows Figure 3 As shown in the figure, Control is the control group, CQ is the experimental group, the plasma membrane integrity rate of the experimental group was significantly higher than that of the control group (P < 0.05), the plasma membrane integrity rate of the control group was 60.89%, and the plasma membrane integrity rate of the experimental group was 65.45%.

[0048] (7) Lipid peroxidation detection: The lipid peroxidation level was detected using BODIPY™ 581 / 591 C11 lipid peroxidation probe. 100 μL of thawed semen was taken from each group and centrifuged at 300 × g for 5 minutes. The supernatant was discarded and the precipitate was washed with PBS. The sperm concentration was adjusted to 2 × 10 using 499.5 μL PBS and 0.5 μL BODIPY C11 (10 mM). 6 sperm / mL, and incubate the mixture at 37°C in the dark for 10 minutes. Wash the cells as above and resuspend in 200 μL of PBS before flow cytometric analysis. The flow cytometer uses 488 nm excitation light as the light source for oxidized C11-BODIPY (oxC11-BODIPY). The signal intensity of oxC11-BODIPY is collected using a 525 / 40 BP filter. The sample flow rate is maintained at 200-400 cells per second; at least 10,000 cells are analyzed for each sample.

[0049] Sperm lipid peroxidation test results Figure 4 As shown, C is the control group, CQ is the experimental group, the lipid peroxide level of the control group was significantly higher than that of the experimental group (P < 0.05), the lipid peroxide level of the control group was 4084.93, and the lipid peroxide level of the experimental group was 3034.

[0050] (8) Fe 2+ Detection: Fe²⁺ levels were detected using FerrOrange. 100 μL of thawed semen was taken from each group and centrifuged at 300×g for 5 minutes. The supernatant was discarded and the pellet was washed with PBS. The sperm concentration was adjusted to 2×10 using 495 μL PBS and 5 μL FerrOrange (1 mM). 6sperm / mL and incubate the mixture at 37°C in the dark for 10 minutes. Wash the cells as above, resuspend in 200 μL of PBS, and then perform flow cytometric analysis. The flow cytometer uses 488 nm excitation light as the light source for Fe²⁺. The signal intensity of Fe²⁺ is collected using a 585 / 42 BP filter. The sample flow rate is maintained at 200-400 cells per second; at least 10,000 cells are analyzed for each sample.

[0051] Sperm Fe 2+ The level test results are as follows Figure 5 As shown, Control is the control group, CQ is the experimental group, and the Fe 2+ The level of Fe 2+ The level was 7737.7, and the Fe 2+ The level is 1517.7.

[0052] Example 2 Effect of endogenous ferroptosis inhibitor protein on sperm quality during cryopreservation of goat semen

[0053] 1. Experimental Animals

[0054] The goat semen used in this experiment came from Inner Mongolia Jinlai Animal Husbandry Technology Co., Ltd. Four 2-year-old adult rams were selected, and they were required to be healthy, disease-free, and uniformly raised and managed.

[0055] 2. Test methods

[0056] (1) Preparation of protective agent

[0057] Control dilution: Dissolve 1.8g Tris, 1g citric acid, 0.5g glucose, 0.5ml penicillin-streptomycin mixture, 3ml glycerol and 15ml egg yolk solution in ultrapure water and adjust to 50ml. Transfer to a reagent bottle through a 0.22μm disposable syringe filter, seal it, store it in a refrigerator at 4℃, and use it within one week.

[0058] Experimental group sheep sperm endogenous ferroptosis inhibitor protein protectant: Take Tris 1.8g, citric acid 1g, glucose 0.5g, penicillin-streptomycin mixture 0.5ml, glycerol 3ml, egg yolk liquid 15ml and chloroquine 0.0799mg, dissolve the above substances in ultrapure water, and make up to 50ml. Transfer to a reagent bottle through a 0.22 μm disposable syringe filter, seal it, store it in a refrigerator at 4℃, and use it within one week.

[0059] (2) Semen collection: Use the pseudo vagina method to collect semen once every two days. Use long-handled tweezers to grab a 75% alcohol cotton ball and rub it on the pseudo vagina inner tube for disinfection. Then rub it with sterile saline 2-3 times. Rinse the pseudo vagina inner tube with sterile Tris-citrate-glucose basic solution (Tris is 36 g / L, citric acid is 20 g / L, glucose is 10 g / L and water). Let the residual liquid in the pseudo vagina dry naturally. Inject hot water that accounts for 2 / 3 of the volume of the pseudo vagina interlayer cavity into the water injection hole of the pseudo vagina shell. The water temperature is 45-50℃, and the water injection port is plugged with a rubber plug. Use a thermometer to measure the inner tube temperature and keep it at 38-40℃. Use a sterile glass rod to dip in sterile lubricant (medical vaseline) and apply it to the 1 / 3-1 / 2 of the front section of the pseudo vagina to lubricate its inner cavity. Insert the semen collection cup that has been sterilized and dried by high pressure into the other end that is not coated with vaseline. Adjust the pressure in the pseudo vagina cavity according to the insertion depth of the semen collection cup. During semen collection, the semen collector is at the right rear of the semen collection platform. When the male animal mounts, the penis is quickly introduced into the false vagina. After the semen collection is completed, the false vagina is immediately erected to prevent the semen from flowing back. The collected fresh semen is kept warm at 37℃ and sent to the laboratory for processing.

[0060] Fresh semen quality testing and processing. 5 μl of fresh semen was diluted with Tris-citrate-glucose basic solution (200 μL), and 5 μl of diluted semen was placed on a glass slide, covered with a cover glass, and tested at least 5 fields of view using a computer-assisted semen analysis system. The detection system captured at least 1,000 sperm cells. The fresh semen motility reached more than 75%, and the semen samples with more than 50% fast linear motion sperm were used for subsequent tests.

[0061] (3) Freezing and thawing of goat sperm: Fresh goat semen was diluted with the diluent of the control group and the endogenous iron death inhibitor protein protective agent of the goat sperm of the experimental group at 37°C, and the sperm density was adjusted to 2×10 8 / ml; place the diluted semen in a 30℃ water bath and place it in a 4℃ freezer to cool for 2h; after the water bath temperature drops to 4℃, use a pipette to draw 200μl to 250μl into a frozen sperm tube, seal it with sealing powder, and balance it at 4℃ for 1h; place it on the freezing rack at equal distances, fumigate it with liquid nitrogen for 7min at a distance of 4cm above the liquid nitrogen surface, and then put it into liquid nitrogen for storage; the frozen semen is thawed in a water bath after three days, and the water bath temperature is 37℃ for 30s.

[0062] (4) Detect the expression of FTH1 in frozen semen by flow cytometry. Add 400 μL of DPBS to the semen and centrifuge at 1350 r / min for 3 min, repeat twice and completely remove the supernatant. Add 250 μL of fixative to the semen sample and mix well. Let stand at room temperature for 30 min to fix the cell structure. Then add 200 μL of permeabilization solution and centrifuge for 3 min and repeat 3 times to remove the fixative. Add 300 μL of permeabilization solution to treat the cells for 15 min, and add permeabilization solution again for centrifugation and washing. Add 5% BSA to the sample and block at room temperature for 30 min. After blocking, centrifuge the permeabilization solution to remove excess blocking agent. Then add diluted rabbit monoclonal primary antibody against FTH1 to the cells and incubate for 1 h to label the target protein. At the same time, set rabbit monoclonal IgG as an isotype control to evaluate nonspecific binding. After the incubation is completed, centrifuge the permeabilization solution again to remove unbound primary antibody. Subsequently, diluted donkey anti-mouse FITC fluorescent secondary antibody (1:200) was added and incubated for 30 minutes in the dark to detect the binding of the primary antibody. Finally, the sperm cells were thoroughly washed with DPBS and resuspended in preparation for analysis by flow cytometry. During the flow cytometry detection process, at least 10,000 cells were collected and analyzed to evaluate the cell fluorescence intensity. The geometric mean fluorescence intensity is obtained by subtracting the fluorescence intensity background of the isotype control stained cells from the fluorescence intensity of the specific antibody stained cells, thereby accurately reflecting the expression level of the target protein.

[0063] The results of sperm FTH1 expression level test are as follows Figure 6 As shown in the figure, Control is the control group, CQ is the experimental group, and the FTH1 expression level in the experimental group is significantly higher than that in the control group (P<0.05). The FTH1 expression level in the control group is 17.0%, and the FTH1 expression level in the experimental group is 86.4%.

[0064] (5) Plasma membrane integrity detection: Use PI dye and flow cytometry to detect the plasma membrane integrity. Thawed spermatozoa were centrifuged at 300×g, the supernatant was removed, 499ul of PBS and 1ul of PI dye were added to resuspend the spermatozoa, incubated in the dark for 10min, and finally detected by flow cytometry.

[0065] The results of sperm plasma membrane integrity test are as follows Figure 7 As shown in the figure, Control is the control group, CQ is the experimental group, the plasma membrane integrity rate of the experimental group was significantly higher than that of the control group (P < 0.05), the plasma membrane integrity rate of the control group was 48.1%, and the plasma membrane integrity rate of the experimental group was 66.5%.

[0066] (6) Lipid peroxidation detection: The lipid peroxidation level was detected using BODIPY™ 581 / 591 C11 lipid peroxidation probe. 100 μL of thawed semen was taken from each group and centrifuged at 300 × g for 5 minutes. The supernatant was discarded and the precipitate was washed with PBS. The sperm concentration was adjusted to 2 × 10 using 499.5 μL PBS and 0.5 μL BODIPY C11 (10 mM). 6 sperm / mL, and incubate the mixture at 37°C in the dark for 10 minutes. Wash the cells as above and resuspend in 200 μL of PBS before flow cytometric analysis. The flow cytometer uses 488 nm excitation light as the light source for oxidized C11-BODIPY (oxC11-BODIPY). The signal intensity of oxC11-BODIPY is collected using a 525 / 40 BP filter. The sample flow rate is maintained at 200-400 cells per second; at least 10,000 cells are analyzed for each sample.

[0067] Sperm lipid peroxidation test results Figure 8 As shown, Control is the control group, CQ is the experimental group, the lipid peroxide level of the experimental group was significantly lower than that of the control group (P < 0.05), the lipid peroxide level of the control group was 2993.7, and the lipid peroxide level of the experimental group was 1559.

[0068] (8) Fe 2+ Detection: Fe²⁺ levels were detected using FerrOrange. 100 μL of thawed semen was taken from each group and centrifuged at 300×g for 5 minutes. The supernatant was discarded and the pellet was washed with PBS. The sperm concentration was adjusted to 2×10 using 495 μL PBS and 5 μL FerrOrange (1 mM). 6 sperm / mL and incubate the mixture at 37°C in the dark for 10 minutes. Wash the cells as above, resuspend in 200 μL of PBS, and then perform flow cytometric analysis. The flow cytometer uses 488 nm excitation light as the light source for Fe²⁺. The signal intensity of Fe²⁺ is collected using a 585 / 42 BP filter. The sample flow rate is maintained at 200-400 cells per second; at least 10,000 cells are analyzed for each sample.

[0069] Sperm Fe 2+ The level test results are as follows Fig. 9 As shown, Control is the control group, CQ is the experimental group, and Fe 2+ The level of Fe 2+ The level was 16050.3, and the Fe 2+ The level is 5493.

[0070] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A sheep sperm endogenous ferroptosis inhibitor protein protective agent, characterized in that The invention comprises the following components: 300mmol / LTris, 95mmol / L citric acid, 56mmol / L glucose, 5μmol / L chloroquine, 6% glycerol by volume, 1% penicillin-streptomycin mixed solution by volume and 15% egg yolk solution by volume. The above substances are dissolved in ultrapure water to prepare the sheep sperm endogenous ferroptosis inhibitory protein protective agent.

2. A method for protecting endogenous ferroptosis inhibitory protein in goat sperm, characterized in that The steps include: 1) preparing the sheep sperm endogenous ferroptosis inhibitory protein protective agent according to claim 1, transferring the prepared sheep sperm endogenous ferroptosis inhibitory protein protective agent into a reagent bottle through a disposable syringe filter, sealing it, and storing it at 4°C; 2) Semen collection; 3) Using sheep sperm endogenous iron death inhibitor protein protector to dilute semen; 4) Sperm freezing.

3. The method for protecting endogenous ferroptosis-inhibiting protein in goat sperm according to claim 2, characterized in that: In step 2), the semen is collected by using a false vagina collection method, and the whole process is sterile. After the semen is collected, the false vagina is immediately erected to prevent the semen from flowing back, and the collected fresh semen is kept warm at 37°C.

4. The method for protecting endogenous ferroptosis-inhibiting protein of goat sperm according to claim 2, characterized in that: In step 3), the use of endogenous ferroptosis inhibitory protein protective agent for sheep sperm to dilute semen is as follows: collect semen with fresh semen vitality of more than 75% and rapid linear motion of more than 50%; take endogenous ferroptosis inhibitory protein protective agent for sheep sperm, heat the endogenous ferroptosis inhibitory protein protective agent for sheep sperm to 37°C, add semen to endogenous ferroptosis inhibitory protein protective agent for sheep sperm at 37°C for dilution, the volume ratio of semen to endogenous ferroptosis inhibitory protein protective agent for sheep sperm is 1:10, and mix after dilution.

5. The method for protecting endogenous ferroptosis-inhibiting protein of goat sperm according to claim 2, characterized in that: In step 4), the semen is frozen by cooling in a 4°C constant temperature refrigerator for 4 hours; after cooling, the semen is placed in a 0.25 ml capillary tube, equilibrated for 2 hours, moved to a place 4 cm away from the liquid nitrogen surface, fumigated for 7 minutes, and then quickly put into liquid nitrogen for cryopreservation.

6. The method for protecting endogenous ferroptosis-inhibiting protein in goat sperm according to claim 2, characterized in that: The method further comprises step 5) thawing of semen, wherein the semen is thawed by taking out the frozen straw from liquid nitrogen and then putting it into a 37° C. water bath to thaw for 30 seconds.

7. Application of endogenous ferroptosis inhibitor protein protective agent for goat sperm, characterized in that The method comprises the following contents: preparing the endogenous ferroptosis inhibitory protein protective agent for sheep sperm as described in claim 1; transferring the protective agent to a reagent bottle through a disposable syringe filter, sealing the bottle, and storing the bottle at 4°C; heating the endogenous ferroptosis inhibitory protein protective agent for sheep sperm to 37°C, adding semen to the endogenous ferroptosis inhibitory protein protective agent for sheep sperm at 37°C, diluting the semen to the endogenous ferroptosis inhibitory protein protective agent for sheep sperm in a volume ratio of 1:10, and mixing the diluted products evenly; and then freezing and preserving the products.

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