A method for improving the effect of cryopreservation of semen

Abstract

The application provides a method for improving the effect of sperm freezing preservation, and particularly relates to adding sperm into a freezing preservation solution containing carnosine to perform freezing preservation. The freezing preservation solution added with carnosine can effectively improve the activity of the sperm after freezing. The addition of carnosine into the sperm freezing preservation solution can effectively improve the quality of the sperm after freezing preservation, and provides certain technical support for sperm preservation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of animal reproduction technology, and particularly relates to a method for cryopreservation of semen. BACKGROUND

[0002] Sperm ultra-low temperature freezing technology has been widely used in artificial insemination of cattle, sheep and chicken. It can not only realize the deployment of high-quality semen across time and space, but also has important significance in the protection and full use of excellent local genetic resources. However, due to the low membrane cholesterol-phospholipid ratio and high content of unsaturated fatty acids of pig sperm, it is more sensitive to temperature change and is easily damaged by freezing. Therefore, the current pig frozen semen technology is still immature, and the application effect in actual production is not ideal.

[0003] Cyclodextrin is a small cylindrical, non-toxic and harmless oligosaccharide with a hydrophobic center and an external hydrophilic. It is often used for coating and carrier of drugs and food. Cyclodextrin can bind cholesterol and fatty acids and transport them in and out of the cell membrane through a concentration gradient, which can increase the cholesterol content of the plasma membrane and improve the ability of sperm of sheep, cattle, horses and rabbits to resist low-temperature freezing damage. Carnosine (β-alanyl-L-histidine) is a dipeptide composed of β-alanine and L-histidine, which is widely present in excitable tissues and has the ability of pH buffering, metal ion chelation, antioxidant capacity and preventing the formation of lipid oxidation end products. There is no application of carnosine or carnosine combined with cyclodextrin loaded cholesterol (CLC) in pig semen cryopreservation medium. SUMMARY

[0004] The purpose of the present application is to provide a method for improving the effect of semen cryopreservation, in order to solve the problem of low sperm cholesterol content and poor freezing activity caused by oxidative damage.

[0005] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0006] A method for improving the effect of semen cryopreservation, characterized in that the semen is added to a cryopreservation solution containing carnosine for cryopreservation.

[0007] As described above, preferably, the concentration of carnosine in the cryopreservation solution is 1.2-10 mM. Further preferably, the concentration of carnosine in the cryopreservation solution is 1.33 mM.

[0008] As described above, preferably, the semen is first pretreated by adding cyclodextrin loaded cholesterol to the semen, incubating, centrifuging, discarding the supernatant, and then adding the cryopreservation solution to the semen. Further preferably, the concentration of cyclodextrin loaded cholesterol added is 0.3-1.5 mg / 1.2x10 8Sperm, the temperature of incubation is 17℃, the time of incubation is 15-30min.

[0009] The method as described above, preferably, the cryopreservation solution further comprises 10-15% egg yolk and 3-8% glycerol.

[0010] Further, the semen is pig semen, cow semen, mouse semen, sheep semen, human semen.

[0011] Application of carnosine and cyclodextrin loaded cholesterol in improving the quality of semen cryopreservation. Carnosine and cyclodextrin loaded cholesterol are used to treat semen, so as to improve the quality of semen after cryopreservation.

[0012] Specifically, cyclodextrin loaded cholesterol is first added to the semen, and after incubation, centrifugation is performed, and the supernatant is discarded, and then a cryopreservation solution containing carnosine is added for cryopreservation.

[0013] Further, the concentration of the added cyclodextrin loaded cholesterol is 0.3-1.5mg / 1.2x10 8 Sperm, the temperature of incubation is 17℃, the time of incubation is 15-30min; the concentration of carnosine in the cryopreservation solution is 1.2-10mM.

[0014] The application provides a method for improving the effect of semen cryopreservation, which can effectively improve the integrity of the plasma membrane, sperm motility, cholesterol content and ATP content of semen after cryopreservation, and reduce the content of active oxygen. The application also provides a new use of carnosine, i.e., adding carnosine to the semen cryopreservation solution, which can effectively improve the quality of cryopreserved semen, and provides certain technical support for semen preservation. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 It is a schematic diagram of the experimental process in Example 1.

[0016] Figure 2 It is a schematic diagram of the experimental process in Example 2. DETAILED DESCRIPTION

[0017] The following examples are used to further illustrate the application, but should not be construed as limiting the application. Modifications or replacements made to the application without departing from the spirit and essence of the application shall all belong to the scope of the application.

[0018] Unless otherwise specified, the technical means used in the examples is the conventional means familiar to those skilled in the art, and unless otherwise specified, the reagents used in the application are all analytical pure or above.

[0019] Example 1

[0020] Normal temperature diluent: 45g Premium powder (Germany, Minitube, item number 13533 / 3001) was added to 1000 mL of autoclaved water and mixed well. After the powder was dissolved, the solution was left to stand for 30 minutes to stabilize the osmotic pressure and pH. The solution was filtered through a 0.22 μm filter using a syringe and was ready for use.

[0021] Freezing base solution: 84.9 g Cryo Plus powder (Germany, Minitube, item number 13531 / 3001) was placed in a glass container that had been autoclaved, and was dissolved in 770 mL of autoclaved water. After the powder was completely dissolved, the solution was left to stand for 30 minutes to stabilize the osmotic pressure and pH. The solution was filtered through a 0.22 μm filter using a syringe and was ready for use.

[0022] Carnosine stock solution: 90.5 mg of L-carnosine was dissolved in 1 mL of the freezing base solution to obtain a 400 mM carnosine stock solution.

[0023] Glutathione stock solution: 153.66 mg of glutathione was dissolved in 10 mL of the freezing base solution to obtain a 50 mM glutathione stock solution.

[0024] Semen cryopreservation diluent A (referred to as A solution): the control group was 8 mL of the freezing base solution, to which 2 mL of fresh chicken egg yolk was added; the carnosine 1 group was 7.9 mL of the freezing base solution, to which 0.1 mL of the carnosine stock solution and 2 mL of fresh chicken egg yolk were added; the carnosine 2 group was 7.8 mL of the freezing base solution, to which 0.2 mL of the carnosine stock solution and 2 mL of fresh chicken egg yolk were added; the glutathione group was 6 mL of the freezing base solution, to which 2 mL of the glutathione stock solution and 2 mL of fresh chicken egg yolk were added, and the mixture was mixed well and cooled to 17°C.

[0025] Semen cryopreservation diluent B (referred to as B solution): 6.5 mL of the freezing base solution, to which 2 mL of fresh chicken egg yolk and 1.5 mL of glycerol were added, and the mixture was cooled to 5°C.

[0026] The test variety in this example was a Large White pig, and the following tests were carried out using the semen cryopreservation diluent prepared according to the present application. A flowchart of the test is shown in Figure 1

[0027] (1) Semen collection

[0028] Fresh semen was collected from a boar farm (hand grip method). The color of the boar semen was light cream or grayish white, and had a specific slight fishy smell, no rotten odor, and the sperm motility was greater than 85% and the sperm abnormality rate was less than 10%. The semen was mixed with the normal temperature diluent at a ratio of 1:1, and the bottle was placed in a 17°C constant temperature box for 1 hour before being sent to the laboratory.

[0029] (2) Semen treatment and freezing

[0030] ​Semen was concentrated by centrifugation at 700xg for 9 minutes, and the supernatant was removed. The concentrated sperm was divided into four groups and mixed with four kinds of A solution at a volume ratio of 1:1. After mixing, the concentrations of the additives in each group were as follows: control group: no carnosine and glutathione; carnosine group 1: 2 mM carnosine; carnosine group 2: 4 mM carnosine; glutathione group: 5 mM glutathione. The mixture was wrapped with toilet paper and placed in a 4°C refrigerator for 2 hours to slowly cool to 4°C. According to the volume ratio of sperm + A solution to B solution (2:1), B solution was added to the cooled sample to make the final concentrations of egg yolk and glycerol 13.3% and 5%, respectively. The mixture was mixed well and loaded into a 0.5 mL tube, which was sealed with sealing powder after being moistened with water.

[0031] Freezing method: The sample tube was placed horizontally at a distance of 4 cm from the liquid nitrogen surface for fumigation freezing. During the process, the tube was rolled every two minutes, and the fumigation freezing was performed for a total of 15 minutes. After freezing, the sample tube was moved into liquid nitrogen for storage.

[0032] (3) Thawing of the sperm in the tube and evaluation of sperm quality

[0033] After one week of freezing, the sperm freezing tube was quickly removed from the liquid nitrogen and placed in a 50°C water bath for 16 seconds. The thawed semen was then flowed into a 1:10 volume ratio of Premium diluent in a 38°C water bath. After the mixture was well mixed and equilibrated for 15 minutes, the semen sample was measured.

[0034] 1) Measurement of pig sperm motility

[0035] 4 μL of the semen sample was spotted on a disposable sperm counting slide preheated to 38°C. The computer-aided sperm analysis system (Tsinghua Tongfang Sperm Analysis System) with a constant temperature hot plate was used to analyze and evaluate the sample in four fields of view in the up, down, left, and right directions.

[0036] 2) Measurement of pig sperm reactive oxygen species (ROS)

[0037] The ROS activity of the semen sample was detected using a reactive oxygen species detection kit (China, Biyun Tian Company). The DCFH-DA probe was added at a final concentration of 10 μM, and the sample was gently mixed and then incubated in a 38°C water bath for 25 minutes (the negative control of reactive oxygen species did not add the probe, and the positive control added the probe and a positive control stimulant). PBS solution was used to wash away the impurities in the sample after the reaction was completed. The sperm sample was diluted to the original volume and then added to a 96-well plate. The fluorescence value was detected using an enzyme-labeled instrument.

[0038] 3) Measurement of pig sperm lipid oxidation (MDA)

[0039] ​The sample of sperm washed by PBS was prepared for MDA detection using RIPA (strong) lysis solution. The sample was broken by ultrasonic in ice bath environment, with power 270W, ultrasonic on for 1 second and off for 3 seconds alternately for 3 minutes, and then centrifuged at 2000xg for 10 minutes. The upper solution obtained by centrifugation was detected using a lipid oxidation (MDA) detection kit.

[0040] 4) ATP determination of pig sperm

[0041] The sample of sperm washed by PBS was prepared for MDA detection using RIPA (strong) lysis solution. The sample was broken by ultrasonic in ice bath environment, with power 270W, ultrasonic on for 1 second and off for 3 seconds alternately for 3 minutes, and then centrifuged at 2000xg for 10 minutes. The upper solution obtained by centrifugation was detected using a lipid oxidation (MDA) detection kit.

[0042] (4) Evaluation results of pig sperm quality

[0043] The measured results are shown in Table 1.

[0044] Table 1 Effect of carnosine on sperm freezing

[0045]

[0046] According to the detection results of the activity before freezing, it is shown that the antioxidant carnosine and glutathione group have played a certain role in the cooling process from 17℃ to 4℃.

[0047] After freezing and thawing, the activity and ATP content of the carnosine group after thawing are significantly higher than those of the control group and the glutathione group, which shows that the antioxidant effect of carnosine is better than that of glutathione in the freezing process of pig sperm, and can effectively reduce the production of reactive oxygen species and malondialdehyde.

[0048] Example 2 Combined application of carnosine and CLC in cryopreservation agent

[0049] In this example, carnosine and cyclodextrin loaded cholesterol (CLC) are combined. The normal temperature dilution solution, carnosine stock solution, cryopreservation agent A solution and cryopreservation agent B solution are the same as in Example 1.

[0050] CLC preparation: cyclodextrin solution (1g methyl-β-cyclodextrin and 2mL methanol) and cholesterol solution (200mg cholesterol and 1mL chloroform) were prepared respectively. After mixing the cholesterol solution with the cyclodextrin solution, the solution was evaporated by nitrogen flow to obtain white CLC powder.

[0051] CLC stock solution: CLC powder was dissolved in Tris-citric acid-glucose (TCG) buffer (300 mM Tris base, 95 mM citric acid monohydrate, 28 mM D-(+)-glucose, pH = 7.0) to prepare a CLC solution at a concentration of 50 mg / mL. The solution was placed in a 38°C water bath for 10 minutes, then vortexed and ultrasonicated for 30 minutes, and finally added with bovine serum albumin (BSA) at a concentration of 3 mg / mL.

[0052] The test object of this example was a Large White pig, and the following tests were performed using the semen cryopreservation diluent prepared by the application. The flowchart is shown in Figure 2

[0053] (1) Semen collection: same as Example 1

[0054] (2) Pig semen processing and freezing

[0055] After determining the sperm density, the semen was evenly divided into two groups. The CLC group was added with 0.5 mg of CLC stock solution per 1.2 x 10 8 Sperm was added with CLC stock solution, and the control group was added with the same volume of normal temperature diluent as the CLC stock solution, and incubated at 17°C for 20 min. Then, it was concentrated by centrifugation at 700 x g for 9 min, and the supernatant containing CLC was removed. The concentrated sperm in each group was then evenly divided into two groups, and control group A liquid and carnosine group A liquid were added in a volume ratio of 1:1, and mixed uniformly at the same temperature (17°C). It was wrapped with toilet paper and placed in a 4°C refrigerator for 2 hours to slowly cool down. B liquid was added to the cooled sample at the same temperature according to the volume ratio of sperm + A liquid to B liquid of 2:1, so that the final concentrations of egg yolk and glycerol were 13.3% and 5%, respectively. After mixing and detecting the activity, the sample was loaded into a 0.5 mL tube and sealed with sealing powder after being moistened with water, and then frozen.

[0056] Freezing method: the sample tube was placed horizontally at a distance of 4 cm from the liquid nitrogen surface for fumigation freezing, and the tube was rolled every two minutes during the process, for a total of 15 minutes of fumigation freezing. After freezing, the sample tube was moved into liquid nitrogen for storage.

[0057] (3) Thawing of the tube and evaluation of sperm quality

[0058] 1) Sperm thawing method same as Example 1.

[0059] 2) Sperm motility, reactive oxygen species, lipid oxidation (MDA), and ATP determination methods same as Example 1.

[0060] 3) Determination of cholesterol content in pig sperm

[0061] ​The sperm sample was added with PBS solution, and the impurities in the sample were washed away by centrifugation. Isopropyl alcohol was added to the sperm precipitate, and after ultrasonic crushing in an ice bath environment, the sample was placed at 4°C or on ice for 5-10 min, and then centrifuged at 12000 rpm for 10 min to obtain the detection sample. The total cholesterol (TC) detection kit (China, Solabio Company) was used for detection.

[0062] 4) Membrane integrity assay

[0063] The sperm sample was treated by PI and H33342 mixed staining method. 50 μL of sperm sample was mixed with 10 μL of PI and 5 μL of H33342 staining agent, and then incubated in a 37°C water bath for 15 min. After incubation, the staining solution was washed away with PBS, and then fixed with 4% paraformaldehyde solution for 10 min. The sample was observed under a fluorescence inverted microscope. Three fields of view were randomly selected for each sample, and about 400 sperm cells were recorded for their plasma membrane integrity. The percentage of sperm membrane integrity was calculated based on the coloration state of sperm membrane.

[0064] (4) Evaluation results of pig sperm quality

[0065] The results of the determination are shown in Table 2.

[0066] Table 2 Effect of CLC addition on sperm motility

[0067]

[0068] The above results show that the addition of CLC effectively increases the cholesterol content of frozen sperm. The combination of CLC and carnosine can further improve the motility, cholesterol content and ATP content of frozen sperm, and reduce the content of reactive oxygen species, and the antioxidant effect is better than that of carnosine alone. It is indicated that the two have a synergistic effect.

Claims

1. A method for improving the cryopreservation effect of semen, characterized in that, The method involves adding semen to a cryopreservation solution containing carnosine for cryopreservation, wherein the concentration of carnosine in the cryopreservation solution is 1.2~10mM; Furthermore, the semen undergoes pretreatment by adding cyclodextrin-loaded cholesterol to the semen at a concentration of 0.3~1.5 mg / 1.2×10⁻⁶. 8 After incubation, the sperm is centrifuged, and the supernatant is discarded. Cryopreservation solution is then added to the semen.

2. The method according to claim 1, characterized in that, The incubation temperature is 17℃, and the incubation time is 15~30min.

3. The method according to claim 1, characterized in that, The cryopreservation solution also includes 10-15% egg yolk and 3-8% glycerol.

4. The method according to claim 1, characterized in that, The semen is pig semen, cow semen, rat semen, sheep semen, or human semen.

5. The application of the method for improving the effect of semen cryopreservation as described in claim 1 in improving the quality of semen cryopreservation.

6. The application according to claim 5, characterized in that, Cyclodextrin-loaded cholesterol was first added to the semen, incubated, centrifuged, and the supernatant was discarded. Then, a cryopreservation solution containing carnosine was added to the semen for cryopreservation.

7. The application according to claim 5, characterized in that, The concentration of the cyclodextrin loaded with cholesterol added is 0.3~1.5 mg / 1.2×10⁻⁶. 8 Sperm were incubated at 17°C for 15-30 minutes; carnosine concentration in cryopreservation solution was 1.2-10 mM.

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