Expression inducer for endogenous heat shock protein 70 of sheep sperms, method for improving expression level of endogenous heat shock protein 70 of sheep sperms and application of expression inducer

Abstract

The invention relates to a sheep sperm endogenous heat shock protein 70 expression inducer, a method for improving the expression level of sheep sperm endogenous heat shock protein 70 and application of the sheep sperm endogenous heat shock protein 70 expression inducer. The sheep sperm internal heat shock protein 70 expression inducer comprises the following components: Tris, citric acid, glucose, glutamine, glycerol, a penicillin-streptomycin mixed solution, a yolk solution and ultrapure water. The invention further provides a method for improving the expression level of the endogenous heat shock protein 70 of the sheep sperms. The method comprises the following steps: preparing a sheep sperm membrane protective agent; collecting semen; a sheep sperm internal heat shock protein 70 expression inducer is adopted to dilute the sperm; freezing the semen. The invention also provides an application of glutamine in improving the expression level of heat shock protein 70 in sheep sperms. The method can improve the expression level of the heat shock protein 70 in the sperm in the freezing recovery process, effectively reduces membrane damage to the sperm, ensures the sperm motility and the integrity rate of a biological membrane structure, and ensures that the sperm exerts normal physiological functions.

Description

Technical Field

[0001] The present invention belongs to the technical field of sheep semen preservation, and in particular relates to an inducer for the expression of endogenous heat shock protein 70 in sheep sperm and a method for improving the expression level of endogenous heat shock protein 70 in sheep sperm and application thereof. 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] Heat shock protein 70 plays a vital role in the process of sperm cryopreservation. Heat shock protein 70 can not only protect sperm cells in a high temperature environment, maintain protein conformational stability and transmembrane transport, but also play a key role in resisting freezing damage during sperm freezing. It is involved in regulating protein folding and refolding, identifying and processing damaged or misfolded proteins, 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 regulation of heat shock protein 70 expression level, which is crucial in the sperm cryopreservation process. There is no relevant report at present, and heat shock protein 70 plays an important role in the function and quality of sperm after cryopreservation. The regulation of its expression level can have an important impact on the subsequent development and fertilization ability of sperm. The existing technology of sperm protection and sperm cryopreservation technology has technical bottlenecks; secondly, in order to improve the expression level of related proteins in cells, the existing technology often uses recombinant proteins and cell transfection methods, but 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 overcome the above-mentioned technical problems. By conducting innovative research on sperm diluents and protective agents, it is necessary not only to meet the conventional technical requirements of ensuring sperm vitality and providing nutrition, but also to focus on increasing the expression level of heat shock protein 70 during the sperm freezing and recovery process, so as to improve the sperm freezing preservation 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 an inducer for the expression of endogenous heat shock protein 70 in sheep sperm.

[0007] The first object of the present invention is achieved through the following technical scheme: an inducer for the expression of heat shock protein 70 in sheep sperm, comprising the following components: 300mmol / L Tris, 95mmol / L citric acid, 56mmol / L glucose, 5mmol / L glutamine, 6% by volume glycerol, 1% by volume penicillin-streptomycin mixture, 15% by volume yolk liquid and ultrapure water, wherein the above substances are dissolved in the ultrapure water.

[0008] The second purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for increasing the expression level of endogenous heat shock protein 70 in sheep sperm.

[0009] The second object of the present invention is achieved through the following technical solution: a method for increasing the expression level of endogenous heat shock protein 70 in sheep sperm, comprising the following steps: 1) Prepare sheep sperm membrane protective agent: take 300mmol / L Tris, 95mmol / L citric acid, 56mmol / L glucose, 5mmol / L glutamine, 0.02%-0.1% DMSO, 6% glycerol, 1% penicillin-streptomycin mixture, 15% egg yolk solution, dissolve the above substances in the ultrapure water and make up to volume to prepare the inducer of heat shock protein 70 expression in sheep sperm; transfer to a reagent bottle through a disposable syringe filter, seal it, and store it at 4°C; 2) Semen collection; 3) Diluting semen with an inducer of heat shock protein 70 expression in sheep sperm; 4) Sperm freezing.

[0010] 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.

[0011] Furthermore, in step 3), the sheep sperm heat shock protein 70 expression inducer dilutes semen by: collecting semen with a fresh sperm motility of more than 75% and a rapid linear motion of more than 50%; taking the sheep sperm heat shock protein 70 expression inducer, heating the sheep sperm heat shock protein 70 expression inducer to 37°C, and adding the semen to the sheep sperm heat shock protein 70 expression inducer at 37°C for dilution, with the volume ratio of semen to sheep sperm membrane protective agent being 1:10, and mixing after dilution.

[0012] 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.

[0013] 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.

[0014] The third purpose of the present invention is to overcome the shortcomings of the prior art and provide the use of glutamine in improving the expression level of heat shock protein 70 in sheep sperm.

[0015] The third object of the present invention is achieved through the following technical scheme: the application of glutamine in improving the expression level of heat shock protein 70 in sheep sperm, characterized in that: 300mmol / L Tris, 95mmol / L citric acid, 56mmol / L glucose, 5mmol / L glutamine, 6% glycerol by volume, 1% penicillin-streptomycin mixed solution by volume and 15% egg yolk solution by volume are taken, and the above substances are added into ultrapure water in turn to dissolve and fix the volume to prepare an inducer for the expression of heat shock protein 70 in sheep sperm; the mixture is transferred to a reagent bottle through a disposable syringe filter, sealed, and stored at 4°C; the inducer for the expression of heat shock protein 70 in sheep sperm is heated to 37°C, and under the condition of 37°C, semen is added to the inducer for the expression of heat shock protein 70 in sheep sperm, and the volume ratio of semen to the inducer for the expression of heat shock protein 70 in sheep sperm is 1:10, and the mixture is mixed after dilution; and then frozen and stored.

[0016] The beneficial effects of the present invention are as follows: the present invention can increase the expression level of heat shock protein 70 in sperm during the freezing recovery process, effectively reduce the membrane damage of sperm, and ensure the sperm vitality and the integrity of the biological membrane 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.

[0017] Compared with the existing conventional sheep sperm diluent, the sheep sperm endogenous heat shock protein 70 expression inducer of the present invention is used to dilute and preserve semen. Under the same conditions, the expression level of heat shock protein in sheep sperm is increased by 7.0%, the vitality is increased by 50.8%, the plasma membrane integrity rate is increased by 7.0%, and the acrosome membrane integrity rate is increased by 5.7%; the expression level of heat shock protein in goat sperm is increased by 17.2%, the vitality is increased by 25.0%, the plasma membrane integrity rate is increased by 18.2%, and the acrosome membrane integrity rate is increased by 12.8%. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1This is a bar graph of the expression level of heat shock protein 70 in sheep sperm in Example 1; Figure 2 is a bar graph of sheep sperm motility (TM) in Example 1; Figure 3 is a histogram of sheep sperm progressive motility (PM) in Example 1; Figure 4 is a bar graph of the plasma membrane integrity of sheep sperm in Example 1; Figure 5 The histogram of the acrosome integrity of sheep sperm in Example 1; Figure 6 The bar graph of the expression level of heat shock protein 70 in goat sperm of Example 2; Figure 7 is a bar graph of goat sperm motility (TM) in Example 2; Figure 8 is a bar graph of goat sperm forward movement (PM) in Example 2; Fig. 9 is a bar graph of goat sperm plasma membrane integrity in Example 2; Fig.10 This is a bar graph of the acrosome integrity of goat sperm in Example 2. DETAILED DESCRIPTION

[0019] The present invention is described in detail below with reference to the accompanying drawings.

[0020] Example 1 Effect of endogenous heat shock protein 70 expression inducer in sheep sperm on sperm quality during cryopreservation of sheep semen 1. Experimental Animals 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.

[0021] 2. Test methods (1) Preparation of protective agent 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.

[0022] Experimental group sheep sperm membrane protectant: Take Tris 1.8g, citric acid 1g, glucose 0.5g, penicillin-streptomycin mixture 0.5ml, glycerol 3ml, egg yolk liquid 15ml and glutamine 0.033g, 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.

[0023] (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, and glucose is 10 g / L). 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 sterilized glass rod to dip in sterilized lubricant (medical vaseline) and apply it to 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.

[0024] Fresh semen quality testing and processing. 5 μl of fresh semen was diluted with Tris-citrate-glucose basic solution, and 5 μl of the diluted semen was placed on a 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. Semen samples with a motility of more than 75% and more than 50% of sperm with rapid linear motion were used for subsequent tests.

[0025] (3) Freezing and thawing of sheep sperm: Fresh sheep semen was diluted with the diluent of the control group and the sheep sperm membrane protective agent of the experimental group at 37°C to adjust the sperm density to 2×10 8 / ml; place the diluted semen in a 37℃ water bath and place it in a 4℃ freezer for 4h; after the water bath temperature drops to 4℃, use a pipette to transfer 200μl to a 250μl frozen sperm tube, seal it with sealing powder, and balance it at 4℃ for 2h; place these frozen sperm tubes equidistantly on a 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 it for 30s at a water bath temperature of 37℃.

[0026] (4) Detect the expression of heat shock protein 70 in frozen semen by flow cytometry. Add an appropriate amount of 400 μL phosphate buffered saline (DPBS) to the semen, 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 it 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 the permeabilization solution again to centrifuge and wash. 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 heat shock protein 70 mouse monoclonal primary antibody (ab2787, abacm) to the cells and incubate for 1 h to label the target protein. At the same time, set up mouse monoclonal IgG2a as an isotype control to evaluate nonspecific binding. After the incubation, the membrane was centrifuged and washed again to remove the unbound primary antibody. Subsequently, the diluted donkey anti-mouse 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 and resuspended with DPBS in preparation for flow cytometry analysis. During the flow cytometry test, 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.

[0027] The results of sperm heat shock protein 70 expression level were as follows Figure 1 As shown in the figure, Control is the control group, Gln is the experimental group, the expression level of heat shock protein 70 in the experimental group was significantly higher than that in the control group (P < 0.05), the expression level of heat shock protein 70 in the control group was 70.9%, and the expression level of heat shock protein 70 in the experimental group was 75.7%.

[0028] (5) Detection of sperm TM and PM: Transfer the frozen and 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 and PM. Detect at least 1,000 sperm in at least 5 fields of view and record the data.

[0029] Sperm TM results Figure 2 As shown, sperm PM results are as follows Figure 3 As shown; Figure 2 and Figure 3The horizontal axis represents the grouping, Control is the control group and Gln is the experimental group, and the vertical axis represents the proportion of TM and PM; the TM of the control group is 59.23%, and the PM is 40.20%, while the TM of the experimental group is 72.9%, and the PM is 48.3%. The TM and PM of the experimental group are significantly higher than those of the control group (P < 0.05).

[0030] (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.

[0031] The results of sperm plasma membrane integrity test are as follows Figure 4 As shown in the figure, Control is the control group, Gln 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 55.9%, and the plasma membrane integrity rate of the experimental group was 59.8%.

[0032] (7) Detection of acrosome membrane integrity: Peanut agglutinin fluorescence labeling (PNA-FITC) detection kit was used for detection by flow cytometry. Thawed semen was incubated at 37°C for 30 min, centrifuged at 300 g for 5 min, and the supernatant was removed. GENMED preservation solution was added to adjust the sperm concentration to 2×10 7 Sperm / ml, extract 100μl suspension, add 500μl GENMED cleaning solution to mix the sperm, centrifuge to get the supernatant (same as the above centrifugation method), then add 200μl GENMED staining solution B, mix the sperm group, incubate at room temperature in the dark for 20min, centrifuge to get the supernatant (same as the above centrifugation method), then add 200μl of self-prepared propidium iodide (0.4μl / mg), incubate at room temperature in the dark for 5min, centrifuge to get the supernatant (same as the above centrifugation method), add 1ml of GENMED cleaning solution, and finally detect by flow cytometer.

[0033] The results of sperm acrosome integrity test are as follows Figure 5 As shown, Control is the control group, Gln is the experimental group, the acrosome integrity rate of the experimental group was significantly higher than that of the control group (P < 0.05), the acrosome integrity rate of the control group was 74.9%, and the acrosome integrity rate of the experimental group was 79.1%.

[0034] Example 2 Effect of endogenous heat shock protein 70 expression inducer in goat sperm on sperm quality during cryopreservation of goat semen 1. Experimental Animals 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.

[0035] 2. Test methods (1) Preparation of protective agent 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 the volume to 50ml. Transfer to a reagent bottle through a 0.22 μm disposable syringe filter, seal it, store it in a 4°C refrigerator, and use it within one week.

[0036] Experimental group sheep sperm membrane protectant: Take Tris 1.8g, citric acid 1g, glucose 0.5g, penicillin-streptomycin mixture 0.5ml, glycerol 3ml, egg yolk liquid 15ml, glutamine 0.033g, 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.

[0037] (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, and glucose is 10 g / L). 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 sterilized glass rod to dip in sterilized lubricant (medical vaseline) and apply it to 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.

[0038] Fresh semen quality testing and processing. 5 μl of fresh semen was diluted with Tris-citrate-glucose basic solution, and 5 μl of the diluted semen was placed on a 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. Semen samples with a motility of more than 75% and more than 50% of sperm with rapid linear motion were used for subsequent tests.

[0039] (3) Freezing and thawing of goat sperm: Fresh goat semen was diluted with the diluent of the control group and the goat sperm membrane protective agent 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 for 2h; After the water bath temperature drops to 4℃, use a pipette to transfer 200μl to a 250μl 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 with liquid nitrogen for 7min at a distance of 4cm above the liquid nitrogen surface, and then store it in liquid nitrogen; Thaw the frozen semen in a water bath three days later, and thaw it for 30s at a water bath temperature of 37℃.

[0040] (4) Detect the expression of heat shock protein 70 protein in frozen semen by flow cytometry. Add an appropriate amount of 400 μL of DPBS to the semen, 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 it 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 mouse monoclonal primary antibody against heat shock protein 70 to the cells and incubate for 1 h to label the target protein. At the same time, set mouse monoclonal IgG2a 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 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 flow cytometry analysis. During the flow cytometry test, 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.

[0041] The results of sperm heat shock protein 70 expression level detection are as follows Figure 6 As shown in the figure, Control is the control group, Gln is the experimental group, the expression level of heat shock protein 70 in the experimental group was significantly higher than that in the control group (P < 0.05), the heat shock protein 70 in the control group was 51.4%, and the heat shock protein 70 in the experimental group was 60.3%.

[0042] (5) Detection of sperm TM and PM: Transfer the frozen and 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 and PM. Detect at least 1,000 sperm in at least 5 fields of view and record the data.

[0043] Sperm TM results Figure 7 As shown, sperm PM results are as follows Figure 8 As shown; Figure 7 and Figure 8 The horizontal axis represents the grouping, Control is the control group and Gln is the experimental group, and the vertical axis represents the proportion of TM and PM; the TM of the control group is 46.6% and the PM is 30.7%, while the TM of the experimental group is 58.3% and the PM is 36.4%. The TM and PM of the experimental group are significantly higher than those of the control group (P < 0.05).

[0044] (6) Plasma membrane integrity detection: Use PI dye to detect the plasma membrane integrity using flow cytometry. 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.

[0045] The results of sperm plasma membrane integrity test are as follows Fig. 9 As shown, Control is the control group, Gln 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 42.2%, and the plasma membrane integrity rate of the experimental group was 49.8%.

[0046] (7) Detection of acrosomal membrane integrity: Peanut agglutinin fluorescent labeling (PNA-FITC) detection kit was used for flow cytometry. Thawed semen was incubated at 37°C for 30 min, centrifuged at 300g for 5 min to remove the supernatant, GENMED preservation solution was added to adjust the sperm concentration to 2×107 sperm / ml, 100 μl of suspension was extracted, 500 μl of GENMED cleaning solution was added to mix the sperm, and the supernatant was obtained by centrifugation (same as the above centrifugation method), then 200 μl of GENMED staining solution B was added to mix the sperm group, incubated at room temperature in the dark for 20 min, and the supernatant was removed by centrifugation (same as the above centrifugation method), and 200 μl of self-prepared propidium iodide (0.4 μl / mg) was added, and the supernatant was removed by centrifugation at room temperature in the dark for 5 min. After that, 1 ml of GENMED cleaning solution was added, and finally the flow cytometer was used for detection.

[0047] The results of sperm acrosome integrity test are as follows Fig.10As shown, Control is the control group, Gln is the experimental group, the acrosome integrity rate of the experimental group was significantly higher than that of the control group (P < 0.05), the acrosome integrity rate of the control group was 76.16%, and the acrosome integrity rate of the experimental group was 70.5%.

[0048] 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. An inducer for the expression of heat shock protein 70 in sheep sperm, characterized in that The composition comprises the following components: 300mmol / L Tris, 95mmol / L citric acid, 56mmol / L glucose, 5mmol / L glutamine, 6% by volume glycerol, 1% by volume penicillin-streptomycin mixture, 15% by volume egg yolk liquid and ultrapure water, wherein the above substances are dissolved in the ultrapure water.

2. A method for increasing the expression level of endogenous heat shock protein 70 in sheep sperm, characterized in that The steps include: 1) Prepare sheep sperm membrane protective agent: take 300mmol / L Tris, 95mmol / L citric acid, 56mmol / L glucose, 5mmol / L glutamine, 0.02%-0.1% DMSO, 6% glycerol, 1% penicillin-streptomycin mixture, 15% egg yolk solution, dissolve the above substances in the ultrapure water and make up to volume to prepare the inducer of heat shock protein 70 expression in sheep sperm; transfer to a reagent bottle through a disposable syringe filter, seal it, and store it at 4°C; 2) Semen collection; 3) Diluting semen with an inducer of heat shock protein 70 expression in sheep sperm; 4) Sperm freezing.

3. The method for increasing the expression level of endogenous heat shock protein 70 in sheep 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 increasing the expression level of endogenous heat shock protein 70 in sheep sperm according to claim 2, characterized in that: In step 3), the sheep sperm heat shock protein 70 expression inducer dilutes semen by: collecting semen with a fresh sperm motility of more than 75% and a rapid linear motion of more than 50%; taking the sheep sperm heat shock protein 70 expression inducer, heating the sheep sperm heat shock protein 70 expression inducer to 37° C., adding the semen to the sheep sperm heat shock protein 70 expression inducer at 37° C. for dilution, the volume ratio of semen to sheep sperm membrane protective agent being 1:10, and mixing after dilution.

5. The method for increasing the expression level of endogenous heat shock protein 70 in sheep 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 increasing the expression level of endogenous heat shock protein 70 in sheep 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. The use of glutamine in increasing the expression level of heat shock protein 70 in sheep sperm, characterized in that: Take 300mmol / LTris, 95mmol / L citric acid, 56mmol / L glucose, 5mmol / L glutamine, 6% glycerol, 1% penicillin-streptomycin mixture and 15% egg yolk solution, add the above substances into ultrapure water in turn to dissolve and make up to volume, and prepare the inducer of heat shock protein 70 expression in sheep sperm; transfer it to a reagent bottle through a disposable syringe filter, seal it, and store it at 4℃; heat the inducer of heat shock protein 70 expression in sheep sperm to 37℃, add semen to the inducer of heat shock protein 70 expression in sheep sperm at 37℃, dilute the semen and the inducer of heat shock protein 70 expression in sheep sperm in a volume ratio of 1:10, mix after dilution; then freeze and store it.

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