Inducer for expression of endogenous heat shock protein 70 in goat sperm, method for increasing expression level of endogenous heat shock protein 70 in goat sperm and application

Abstract

The application discloses a goat sperm endogenous heat shock protein 70 expression inducer, a method for improving the expression level of goat sperm endogenous heat shock protein 70 and application thereof. The goat sperm endogenous heat shock protein 70 expression inducer comprises the following components: Tris, citric acid, glucose, glutamine, glycerol, a penicillin-streptomycin mixed solution, egg yolk liquid and ultrapure water. The application further provides a method for improving the expression level of goat sperm endogenous heat shock protein 70, which comprises the following steps: preparing a goat sperm membrane protective agent; collecting semen; diluting the semen with the goat sperm endogenous heat shock protein 70 expression inducer; and freezing the semen. The application further provides application of glutamine in improving the expression level of goat sperm endogenous heat shock protein 70. The application can improve the expression level of heat shock protein 70 in sperm in the freezing recovery process, effectively reduces the membrane damage of sperm, guarantees the sperm activity and the intact rate of biological membrane structure, and guarantees the normal physiological function of sperm.

Description

Technical Field

[0001] This invention belongs to the field of sheep semen preservation technology, specifically relating to a sheep sperm endogenous heat shock protein 70 expression inducer and a method and application for improving the expression level of sheep sperm endogenous heat shock protein 70. Background Technology

[0002] Modern sheep farming is moving towards greater intensification and scale, but the scarcity of superior breeding sires has become a key bottleneck restricting the sustainable development of the sheep industry. Semen cryopreservation technology, as an efficient means of utilizing superior germplasm resources, can preserve sperm for long periods, bringing significant economic value enhancement to the sheep industry.

[0003] Heat shock protein 70 plays a crucial role in sperm cryopreservation. It not only protects sperm cells under high-temperature conditions, maintaining protein conformational stability and transmembrane transport, but also plays a key role in protecting sperm from cryopreservation damage. It participates in regulating protein folding and refolding, recognizing and processing damaged or misfolded proteins, thereby ensuring normal sperm function after freezing and thawing.

[0004] Currently, research on sperm diluents and cryopreservatives mainly focuses on conventional aspects such as ensuring sperm motility, meeting nutritional needs, improving antibacterial capabilities, and regulating osmotic pressure. However, these studies have several limitations: First, most studies only focus on these conventional aspects and fail to fully consider the important physiological changes in sperm during cryopreservation, especially the regulation of heat shock protein 70 expression levels, which is crucial for sperm cryopreservation. There are currently no reports on this, yet heat shock protein 70 plays a vital role in the function and quality of sperm after cryopreservation, and its expression level regulation can significantly impact subsequent sperm development and fertilization capacity. Existing sperm protection and cryopreservation technologies face technical bottlenecks. Second, existing technologies often use recombinant proteins and cell transfection to improve the expression levels of related intracellular proteins. However, recombinant proteins are mostly human-derived, leading to high costs and increasing the economic burden of research and application. Cell transfection technology is not suitable for sperm.

[0005] Therefore, existing technologies urgently need to overcome the aforementioned technical problems. Innovative research on sperm diluents and preservatives should not only meet the conventional technical requirements of ensuring sperm motility and providing nutrition, but also focus on improving the expression level of heat shock protein 70 during sperm cryopreservation and recovery, thereby improving the cryopreservation effect and subsequent performance of sperm. Summary of the Invention

[0006] The primary objective of this invention is to overcome the shortcomings of the existing technology and provide an inducer for the expression of endogenous heat shock protein 70 in sheep sperm.

[0007] The first objective of this invention is achieved through the following technical solution: a sheep sperm heat shock protein 70 expression inducer, comprising the following components: 300 mmol / L Tris, 95 mmol / L citric acid, 56 mmol / L glucose, 5 mmol / L glutamine, 6% glycerol (v / v), 1% penicillin-streptomycin mixture (v / v), 15% egg yolk fluid (v / v), and ultrapure water, wherein the above substances are dissolved in the ultrapure water.

[0008] The second objective of this 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 objective of this 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:

[0010] 1) Preparation of sheep sperm membrane protectant: Dissolve 300 mmol / L Tris, 95 mmol / L citric acid, 56 mmol / L glucose, 5 mmol / L glutamine, 0.02%-0.1% DMSO, 6% glycerol, 1% penicillin-streptomycin mixture, and 15% egg yolk in ultrapure water and bring to a final volume to prepare a sheep sperm heat shock protein 70 expression inducer; transfer to a reagent bottle through a disposable syringe filter, seal, and store at 4°C;

[0011] 2) Semen collection;

[0012] 3) The semen was diluted with a sheep sperm heat shock protein 70 expression inducer;

[0013] 4) Semen freezing.

[0014] Furthermore, in step 2), the semen collection is performed using a simulated vagina method under sterile conditions. After collection, the simulated vagina is immediately erected to prevent backflow of semen, and the collected fresh semen is kept warm at 37°C.

[0015] Furthermore, in step 3), the dilution of semen with the sheep sperm heat shock protein 70 expression inducer is as follows: take fresh semen with a motility of 75% or higher and a rapid linear motility of 50% or higher; take the sheep sperm heat shock protein 70 expression inducer, heat the sheep sperm heat shock protein 70 expression inducer to 37°C, and at 37°C, add the semen to the sheep sperm heat shock protein 70 expression inducer for dilution, with a volume ratio of semen to sheep sperm membrane protectant of 1:10, and mix well after dilution.

[0016] Furthermore, in step 4), the semen freezing is performed by cooling in a constant temperature refrigerator at 4°C for 4 hours; after cooling, the semen is placed into a 0.25ml capillary tube, equilibrated for another 2 hours, then moved to a distance of 4cm from the liquid nitrogen surface, fumigated for 7 minutes, and then quickly immersed in liquid nitrogen for cryopreservation.

[0017] Furthermore, it also includes step 5) thawing of semen, wherein the semen is thawed by removing the frozen capillary from liquid nitrogen and then immersing it in a 37°C water bath for 30 seconds.

[0018] The third objective of this invention is to overcome the shortcomings of the prior art and provide the application of glutamine in increasing the expression level of heat shock protein 70 in sheep sperm.

[0019] The third objective of this invention is achieved through the following technical solution: the application of glutamine in increasing the expression level of heat shock protein 70 in sheep sperm, characterized by: taking 300 mmol / L Tris, 95 mmol / L citric acid, 56 mmol / L glucose, 5 mmol / L glutamine, 6% glycerol (v / v), 1% penicillin-streptomycin mixture (v / v), and 15% egg yolk solution (v / v), dissolving and adjusting the volume of the above substances in ultrapure water to prepare a heat shock protein 70 expression inducer for sheep sperm; transferring the solution to a reagent bottle through a disposable syringe filter and sealing it, storing it at 4°C; raising the temperature of the heat shock protein 70 expression inducer to 37°C, and adding semen to the heat shock protein 70 expression inducer at 37°C, diluting the semen and the heat shock protein 70 expression inducer at a volume ratio of 1:10, mixing well after dilution; and then freezing for preservation.

[0020] The beneficial effects of this invention are: it can increase the expression level of heat shock protein 70 in sperm during cryopreservation, effectively reducing membrane damage to sperm and ensuring sperm motility and the integrity of biological membrane structure. This invention can greatly improve the resistance of sheep sperm to cryopreservation damage, ensuring that sperm can perform normal physiological functions, significantly increasing the utilization rate of breeding rams, and facilitating livestock improvement and breeding work.

[0021] Compared with existing conventional sheep sperm diluents, using the sheep sperm endogenous heat shock protein 70 expression inducer of this invention to dilute and preserve semen resulted in the following improvements under the same conditions: sheep sperm showed a 7.0% increase in heat shock protein expression level, a 50.8% increase in motility, a 7.0% increase in plasma membrane integrity, and a 5.7% increase in acrosome membrane integrity; goat sperm showed a 17.2% increase in heat shock protein expression level, a 25.0% increase in motility, an 18.2% increase in plasma membrane integrity, and a 12.8% increase in acrosome membrane integrity. Attached Figure Description

[0022] Figure 1 This is a bar chart showing the expression level of heat shock protein 70 in sheep sperm in Example 1.

[0023] Figure 2 This is a bar chart of sheep sperm motility (TM) from Example 1;

[0024] Figure 3 This is a bar chart of sheep sperm forward motility (PM) from Example 1;

[0025] Figure 4 This is a bar chart showing the integrity of sheep sperm plasma membranes in Example 1;

[0026] Figure 5 This is a bar chart showing the integrity of the acrosome of sheep sperm in Example 1;

[0027] Figure 6 This is a bar chart showing the expression level of heat shock protein 70 in goat sperm in Example 2.

[0028] Figure 7 This is a bar chart of goat sperm motility (TM) from Example 2;

[0029] Figure 8 This is a bar chart of forward motility (PM) of goat sperm in Example 2;

[0030] Figure 9 This is a bar chart showing the integrity of the goat sperm plasma membrane in Example 2;

[0031] Figure 10 This is a bar chart showing the integrity of the acrosome of goat sperm in Example 2. Detailed Implementation

[0032] The present invention will now be described in detail with reference to the accompanying drawings.

[0033] Example 1: Effect of an endogenous heat shock protein 70 expression inducer in sheep sperm on sperm quality during cryopreservation revival.

[0034] I. Laboratory Animals

[0035] The sheep 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 under uniform feeding and management.

[0036] II. Test Methods

[0037] (1) Preparation of protective agent

[0038] Control group diluent: Dissolve 1.8g Tris (tris(hydroxymethyl)aminomethane), 1g citric acid, 0.5g glucose, 0.5ml penicillin-streptomycin mixture (15140122, Gibco), 3ml glycerol, and 15ml egg yolk solution in ultrapure water and bring the volume to 50ml. Transfer the solution through a 0.22 μm disposable syringe filter to a reagent bottle, seal, and store at 4℃. Use within one week.

[0039] Experimental group sheep sperm membrane protectant: Dissolve 1.8g Tris, 1g citric acid, 0.5g glucose, 0.5ml penicillin-streptomycin mixture, 3ml glycerol, 15ml egg yolk fluid, and 0.033g glutamine in ultrapure water and bring the volume to 50ml. Transfer the solution through a 0.22 μm disposable syringe filter to a reagent bottle, seal, and store at 4℃. Use within one week.

[0040] (2) Semen collection: Semen was collected every two days using a prosthetic vagina. A cotton ball soaked in 75% alcohol was used to disinfect the inner tube of the prosthetic vagina. Then, sterile saline solution was applied 2-3 times. The inner tube was rinsed with sterile Tris-citric acid-glucose basal solution (Tris 36 g / L, citric acid 20 g / L, glucose 10 g / L) and allowed to air dry. Hot water, filling 2 / 3 of the cavity, was injected into the water inlet of the prosthetic vagina shell at a temperature of 45-50℃. The inlet was then sealed with a rubber stopper. The temperature of the inner tube was measured with a thermometer and maintained at 38-40℃. A sterile glass rod was used to apply sterile lubricant (medical petroleum jelly) to the anterior 1 / 3-1 / 2 of the prosthetic vagina to lubricate its cavity. The collection cup, sterilized and dried under high pressure, was inserted into the un-applied end. The pressure inside the prosthetic vagina was adjusted according to the insertion depth of the collection cup. During semen collection, the collector stands to the right rear of the collection table. As the male animal mounts, the penis is quickly inserted into the artificial vagina. After collection, the artificial vagina is immediately erected to prevent backflow of semen. The collected fresh semen is then incubated at 37°C and sent to the laboratory for processing.

[0041] Fresh semen quality testing and processing. 5 μl of fresh semen was diluted with Tris-citric acid-glucose basal solution. Another 5 μl of the diluted semen was placed on a glass slide, covered with a coverslip, and analyzed using a computer-aided semen analysis system. At least five fields of view were examined, and the system captured at least 1000 sperm cells. Semen samples with a fresh semen motility of 75% or higher and more than 50% of sperm exhibiting rapid linear motility were used for subsequent testing.

[0042] (3) Freezing and thawing of sheep semen: Fresh sheep semen was diluted with the control group diluent and the experimental group sheep semen membrane protectant at 37°C to adjust the sperm density to 2×10⁻⁶. 8The volume / ml of diluted semen was placed in a 37°C water bath and then placed in a 4°C freezer for 4 hours to cool down. After the water bath temperature dropped to 4°C, 200μl was pipetted into 250μl of frozen semen tubes, which were then sealed with sealing powder and equilibrated at 4°C for 2 hours. These frozen semen tubes were placed at equal intervals on a freezing rack and then fumigated with liquid nitrogen at a distance of 4cm above the liquid nitrogen surface for 7 minutes before being placed in liquid nitrogen for storage. The frozen semen was thawed in a water bath at 37°C for 30 seconds after three days.

[0043] (4) Flow cytometry was used to detect the expression of heat shock protein 70 in frozen semen. 400 μL of phosphate-buffered saline (DPBS) was added to the semen sample, and the sample was centrifuged at 1350 r / min for 3 min, repeated twice, and the supernatant was completely removed. 250 μL of fixative was added to the semen sample and mixed well. The sample was incubated at room temperature for 30 min to fix the cell structure. Then, 200 μL of permeabilization buffer was added, and the sample was centrifuged for 3 min, repeated three times to remove the fixative. 300 μL of permeabilization buffer was added to treat the cells for 15 min, followed by centrifugation and washing. 400 μL of blocking buffer was added to the sample and the cells were blocked at room temperature for 30 min. After blocking, the permeabilization buffer was centrifuged to remove excess blocking agent. Then, diluted mouse monoclonal antibody against heat shock protein 70 (ab2787, abacm) was added to the cells and incubated for 1 h to label the target protein. Mouse monoclonal IgG2a was used as an isotype control to evaluate non-specific binding. After incubation, the cells were washed again by centrifugation with the membrane-breaking solution to remove unbound primary antibody. Then, diluted donkey anti-mouse FITC fluorescent secondary antibody (1:200) was added, and the cells were incubated for 30 min in the dark to detect primary antibody binding. Finally, the sperm cells were thoroughly washed and resuspended with DPBS for flow cytometry analysis. During flow cytometry, at least 10,000 cells were collected and analyzed to assess cellular fluorescence intensity. The geometric mean fluorescence intensity accurately reflects the expression level of the target protein by subtracting the background fluorescence intensity of isotype control cells from the fluorescence intensity of cells stained with the specific antibody.

[0044] The results of the detection of sperm heat shock protein 70 expression level are as follows: Figure 1 As shown, Control represents the control group and Gln represents 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%, while that in the experimental group was 75.7%.

[0045] (5) Sperm TM and PM detection: Transfer the frozen and thawed semen to a 1.5ml centrifuge tube, place it on a constant temperature stage at 37℃, take 3μl of the thawed semen and drop it onto a glass slide, cover it with a glass slide, and use a computer-aided semen analysis system to detect sperm TM and PM. At least 5 fields of view with a total of more than 1000 sperm should be detected and the data should be recorded.

[0046] Sperm™ results as follows Figure 2 As shown, the sperm PM results are as follows: Figure 3 As shown; Figure 2 and Figure 3 The horizontal axis represents the grouping situation, with Control being the control group and Gln being the experimental group. The vertical axis represents the proportion of TM and PM. The TM of the control group was 59.23% and PM was 40.20%, while the TM of the experimental group was 72.9% and PM was 48.3%. Both TM and PM in the experimental group were significantly higher than those in the control group (P < 0.05).

[0047] (6) Plasma membrane integrity detection: The plasma membrane integrity was detected by flow cytometry using PI dye. After thawing, the sperm were centrifuged at 300×g, the supernatant was removed, and the sperm were resuspended in 499ul of phosphate buffered saline (PBS) and 1ul of PI dye. The mixture was incubated in the dark for 10min, and finally detected by flow cytometry.

[0048] The results of sperm plasma membrane integrity testing are as follows: Figure 4 As shown, Control represents the control group and Gln represents 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%, while that of the experimental group was 59.8%.

[0049] (7) Acrosome membrane integrity detection: The acrosome membrane integrity was detected by flow cytometry using a peanut lectin fluorescent labeling (PNA-FITC) kit. Thawed semen was incubated at 37°C for 30 min, centrifuged at 300g for 5 min, the supernatant was discarded, and GENMED preservation solution was added to adjust the sperm concentration to 2 × 10⁻⁶. 7 Sperm count / ml was measured, and 100 μl of suspension was extracted. 500 μl of GENMED cleaning solution was added to mix the sperm. The supernatant was collected by centrifugation (using the same centrifugation method as described above). Then, 200 μl of GENMED staining solution B was added, and the sperm population was mixed. After incubation at room temperature in the dark for 20 min, the supernatant was removed by centrifugation (using the same centrifugation method as described above). 200 μl of self-prepared propidium iodide (0.4 μl / mg) was added, and the mixture was incubated at room temperature in the dark for 5 min. After centrifugation, the supernatant was collected (using the same centrifugation method as described above), and 1 ml of GENMED cleaning solution was added. Finally, the sample was analyzed by flow cytometry.

[0050] The results of the sperm acrosome integrity test are as follows: Figure 5As shown, Control is the control group and 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 that of the experimental group was 79.1%.

[0051] Example 2: Effects of an inducing agent for endogenous heat shock protein 70 expression in goat sperm on sperm quality during cryopreservation revival.

[0052] I. Laboratory Animals

[0053] 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 under uniform feeding and management.

[0054] II. Test Methods

[0055] (1) Preparation of protective agent

[0056] Control group 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 bring the volume to 50ml. Transfer the solution through a 0.22 μm disposable syringe filter to a reagent bottle, seal, and store at 4℃. Use within one week.

[0057] Experimental group sheep sperm membrane protectant: Dissolve 1.8g Tris, 1g citric acid, 0.5g glucose, 0.5ml penicillin-streptomycin mixture, 3ml glycerol, 15ml egg yolk fluid, and 0.033g glutamine in ultrapure water and bring the volume to 50ml. Transfer the solution through a 0.22 μm disposable syringe filter to a reagent bottle, seal, and store at 4℃. Use within one week.

[0058] (2) Semen collection: Semen was collected every two days using a prosthetic vagina. A cotton ball soaked in 75% alcohol was used to disinfect the inner tube of the prosthetic vagina. Then, sterile saline solution was applied 2-3 times. The inner tube was rinsed with sterile Tris-citric acid-glucose basal solution (Tris 36 g / L, citric acid 20 g / L, glucose 10 g / L) and allowed to air dry. Hot water, filling 2 / 3 of the cavity, was injected into the water inlet of the prosthetic vagina shell at a temperature of 45-50℃. The inlet was then sealed with a rubber stopper. The temperature of the inner tube was measured with a thermometer and maintained at 38-40℃. A sterile glass rod was used to apply sterile lubricant (medical petroleum jelly) to the anterior 1 / 3-1 / 2 of the prosthetic vagina to lubricate its cavity. The collection cup, sterilized and dried under high pressure, was inserted into the un-applied end. The pressure inside the prosthetic vagina was adjusted according to the insertion depth of the collection cup. During semen collection, the collector stands to the right rear of the collection table. As the male animal mounts, the penis is quickly inserted into the artificial vagina. After collection, the artificial vagina is immediately erected to prevent backflow of semen. The collected fresh semen is then incubated at 37°C and sent to the laboratory for processing.

[0059] Fresh semen quality testing and processing. 5 μl of fresh semen was diluted with Tris-citric acid-glucose basal solution. Another 5 μl of the diluted semen was placed on a glass slide, covered with a coverslip, and analyzed using a computer-aided semen analysis system. At least five fields of view were examined, and the system captured at least 1000 sperm cells. Semen samples with a fresh semen motility of 75% or higher and more than 50% of sperm exhibiting rapid linear motility were used for subsequent testing.

[0060] (3) Freezing and thawing of goat sperm: Fresh goat semen was diluted with the control group diluent and the experimental group goat sperm membrane protectant at 37°C to adjust the sperm density to 2×10⁻⁶. 8 The volume / ml of diluted semen was placed in a 30°C water bath and then placed in a 4°C freezer for 2 hours to cool down. After the water bath temperature dropped to 4°C, 200μl was pipetted into 250μl frozen semen tubes, sealed with sealing powder, and equilibrated at 4°C for 1 hour. The tubes were then placed at equal intervals on a freezing rack and fumigated with liquid nitrogen at a distance of 4cm above the liquid nitrogen surface for 7 minutes before being placed in liquid nitrogen for storage. The frozen semen was thawed in a water bath at 37°C for 30 seconds after three days.

[0061] (4) Flow cytometry was used to detect the expression of heat shock protein 70 in frozen semen. 400 μL of DPBS was added to the semen sample, and the mixture was centrifuged at 1350 r / min for 3 min, repeated twice, and the supernatant was completely removed. 250 μL of fixative was added to the semen sample and mixed well. The sample was incubated at room temperature for 30 min to fix the cell structure. Then, 200 μL of permeabilization buffer was added, and the mixture was centrifuged for 3 min, repeated three times to remove the fixative. 300 μL of permeabilization buffer was added to treat the cells for 15 min, followed by centrifugation and washing. 5% BSA was added to the sample for blocking at room temperature for 30 min. After blocking, the permeabilization buffer was centrifuged to remove excess blocking agent. Then, diluted mouse monoclonal antibody against heat shock protein 70 was added to the cells and incubated for 1 h to label the target protein. Mouse monoclonal IgG2a was used as an isotype control to assess non-specific binding. After incubation, the cells were washed again with the permeabilization buffer to remove unbound primary antibody. Subsequently, diluted donkey anti-mouse FITC fluorescent secondary antibody (1:200) was added, and the cells were incubated in the dark for 30 min to detect primary antibody binding. Finally, sperm cells were thoroughly washed and resuspended with DPBS in preparation for flow cytometry analysis. During flow cytometry, at least 10,000 cells were collected and analyzed to assess cellular fluorescence intensity. Geometric mean fluorescence intensity was calculated by subtracting the background fluorescence intensity of isotype control cells from the fluorescence intensity of cells stained with the specific antibody, thus accurately reflecting the expression level of the target protein.

[0062] The results of sperm heat shock protein 70 expression level detection are as follows: Figure 6 As shown, Control represents the control group and Gln represents 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 level in the control group was 51.4%, while that in the experimental group was 60.3%.

[0063] (5) Sperm TM and PM detection: Transfer the frozen and thawed semen to a 1.5ml centrifuge tube, place it on a constant temperature stage at 37℃, take 3μl of the thawed semen and drop it onto a glass slide, cover it with a glass slide, and use a computer-aided semen analysis system to detect sperm TM and PM. At least 5 fields of view with a total of more than 1000 sperm should be detected and the data should be recorded.

[0064] Sperm™ results as follows Figure 7 As shown, the sperm PM results are as follows: Figure 8 As shown; Figure 7 and Figure 8The horizontal axis represents the grouping situation, with Control being the control group and Gln being the experimental group. The vertical axis represents the proportion of TM and PM. The TM of the control group was 46.6% and PM was 30.7%, while the TM of the experimental group was 58.3% and PM was 36.4%. Both TM and PM in the experimental group were significantly higher than those in the control group (P < 0.05).

[0065] (6) Plasma membrane integrity detection: The plasma membrane integrity was detected by flow cytometry using PI dye. After thawing, the sperm were centrifuged at 300×g, the supernatant was removed, and the sperm were resuspended in 499ul of PBS and 1ul of PI dye. The mixture was incubated in the dark for 10min, and finally detected by flow cytometry.

[0066] The results of sperm plasma membrane integrity testing are as follows: Figure 9 As shown, Control is the control group and 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 that of the experimental group was 49.8%.

[0067] (7) Detection of acrosome membrane integrity: The acrosome membrane integrity was detected by flow cytometry using a peanut lectin fluorescent labeling (PNA-FITC) kit. The thawed semen was incubated at 37°C for 30 min, centrifuged at 300g for 5 min to remove the supernatant, and GENMED preservation solution was added to adjust the sperm concentration to 2×107 sperm / ml. 100 μl of the suspension was extracted, and 500 μl of GENMED cleaning solution was added to mix the sperm. The supernatant was collected by centrifugation (using the same centrifugation method as above). Then, 200 μl of GENMED staining solution B was added, and the sperm were mixed. The mixture was incubated at room temperature in the dark for 20 min, and the supernatant was removed by centrifugation (using the same centrifugation method as above). 200 μl of self-prepared propidium iodide (0.4 μl / mg) was added, and the mixture was incubated at room temperature in the dark for 5 min. The supernatant was collected by centrifugation (using the same centrifugation method as above), and 1 ml of GENMED cleaning solution was added. Finally, the mixture was detected by flow cytometry.

[0068] The results of the sperm acrosome integrity test are as follows: Figure 10 As shown, Control is the control group and 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 that of the experimental group was 70.5%.

[0069] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A sheep sperm heat shock protein 70 expression inducer, characterized in that... It comprises the following components: 300 mmol / L Tris, 95 mmol / L Citric Acid, 56 mmol / L Glucose, 5 mmol / L Glutamine, 6% glycerol (v / v), 1% penicillin-streptomycin mixture (v / v), 15% egg yolk solution (v / v), and ultrapure water, all of which 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... Includes the following steps: 1) Preparation of sheep sperm membrane protectant: Dissolve 300 mmol / L Tris, 95 mmol / L citric acid, 56 mmol / L glucose, 5 mmol / L glutamine, 0.02%-0.1% DMSO, 6% glycerol, 1% penicillin-streptomycin mixture, and 15% egg yolk in ultrapure water and bring to a final volume to prepare a sheep sperm heat shock protein 70 expression inducer; transfer the solution to a reagent bottle through a disposable syringe filter, seal, and store at 4°C. 2) Semen collection; 3) The semen was diluted with a sheep sperm heat shock protein 70 expression inducer; 4) Semen 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 collection is performed using a simulated vagina, with aseptic operation throughout. After the semen collection is completed, the simulated vagina is immediately erected to prevent semen backflow, 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 dilution of semen with the sheep sperm heat shock protein 70 expression inducer is as follows: Semen with a fresh sperm motility of 75% or higher and a rapid linear motility of 50% or higher is collected; sheep sperm heat shock protein 70 expression inducer is taken and heated to 37°C; under the condition of 37°C, the semen is added to the sheep sperm heat shock protein 70 expression inducer for dilution, with a volume ratio of semen to sheep sperm membrane protectant of 1:10, and then mixed well 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 freezing is performed by cooling in a constant temperature refrigerator at 4°C for 4 hours; after cooling, the semen is placed into a 0.25ml capillary tube, equilibrated for another 2 hours, then moved to a distance of 4cm from the liquid nitrogen surface, fumigated for 7 minutes, and then quickly immersed in 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: It also includes step 5) thawing of semen, wherein the semen is thawed by taking out the frozen capillary from liquid nitrogen and then immersing it in a 37°C water bath for 30 seconds.

7. The application of glutamine in increasing the expression level of heat shock protein 70 in sheep sperm, characterized by: Dissolve 300 mmol / L Tris, 95 mmol / L Citric Acid, 56 mmol / L Glucose, 5 mmol / L Glutamine, 6% glycerol (v / v), 1% penicillin-streptomycin mixture (v / v), and 15% egg yolk solution in ultrapure water and bring the volume to a final volume to prepare a sheep sperm heat shock protein 70 expression inducer. Transfer the solution to a reagent bottle through a disposable syringe filter, seal, and store at 4°C. Heat the sheep sperm heat shock protein 70 expression inducer to 37°C. At 37°C, add semen to the sheep sperm heat shock protein 70 expression inducer at a volume ratio of 1:10, mix well, and then freeze.

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