Pig semen cryopreservation additive and cryopreservation agent

Abstract

The application provides a boar semen cryopreservation agent and an application method; the boar semen cryopreservation agent comprises alpha-amylase, and the content of the alpha-amylase is 10-1000 mu g / L. The application method of the cryopreservation agent is as follows: centrifuging the boar semen after collection and pretreatment, removing supernatant after centrifugation; adding a refrigerant, re-suspending the sperm, and then placing the sperm in a 4 DEG C refrigerator and standing for 3-4 hours; adding a freezing liquid and fully mixing; filling the semen into a thin tube at 4 DEG C, uniformly laying the thin tubes in a freezing rack, and then precooling on a liquid nitrogen surface and then placing in liquid nitrogen for freezing. The application has the advantages that: the overall components of the cryopreservation agent are simple, the cost is low, and the overall cost performance is high; due to the addition of alpha-amylase in the cryopreservation agent, the storage quality of the boar frozen semen can be significantly improved; and the activity, acrosome integrity, DNA integrity, mitochondrial activity and other physical and chemical properties of the boar frozen semen are effectively improved.

Description

Technical Field

[0001] This invention belongs to the field of porcine semen cryopreservation technology, and particularly relates to a porcine semen cryopreservation agent and its application method. Background Technology

[0002] Proteomics provides a macroscopic understanding of the composition and distribution of sperm proteins and helps uncover key factors in sperm cryotolerance based on phenotypic analysis. Sperm biological functions and their regulation are primarily achieved through post-translational modifications of proteins. After sperm are ejaculated from the testes, their protein synthesis capacity is largely lost. Proteomics will elucidate the differences in cryotolerance among individual boar semen.

[0003] Frozen semen technology in pigs refers to the process of collecting raw semen from boars, treating it specially, and then storing it in liquid nitrogen at -196°C to inhibit the physiological metabolic activities of the sperm, thereby achieving long-term storage. In the context of the African swine fever epidemic, frozen semen offers advantages such as ease of storage and transportation, and expanding the use of high-quality breeding stock.

[0004] However, sperm can suffer freezing damage during the cooling process, leading to poor sperm quality and low conception rates. Current technologies for improving sperm quality after cryopreservation of boar semen often involve controlling refrigeration and freezing conditions, adding cryoprotectants, or a combination of both. Research on cryoprotectants, besides conventional agents such as glycerol, amino acids, sugars, and buffers, focuses on additives that provide additional protection for boar sperm. However, most additives that produce the best beneficial effects are often produced in small quantities, are expensive, or require specific production processes. Therefore, their cost-effectiveness for boar semen preservation is not high, and their practicality is limited.

[0005] Therefore, it is essential to provide a readily available, inexpensive porcine semen cryopreservation additive that can provide excellent protection to reduce the physical and chemical damage to sperm during the freezing process and improve sperm motility after freezing. Summary of the Invention

[0006] To address the technical problem that existing additives that provide additional protection for boar sperm are not cost-effective for preserving boar semen, this invention provides a boar semen cryopreservation agent and its application method.

[0007] The technical solution provided by the present invention is as follows: a porcine semen cryopreservation agent, comprising α-amylase, wherein the content of α-amylase is 10-1000 μg / L.

[0008] Furthermore, the porcine semen cryopreservative includes a refrigeration solution and a freezing solution; the refrigeration solution includes: sterile distilled water, lactose, egg yolk, proline, and α-amylase; the freezing solution includes: a refrigeration solution (without α-amylase), glycerol, Equex.STM cryoprotectant, proline, and α-amylase.

[0009] Furthermore, the mass ratio of all components in the refrigeration solution except for α-amylase is 160:17-18:29-39:0.225-0.235; the amount of α-amylase added is 10-1000 μg / ml.

[0010] Furthermore, the mass ratio of all components in the cryogenic solution except for α-amylase is 92.5:5-7:29-39:0.225-0.235; the amount of α-amylase added is 10-1000 μg / ml.

[0011] Preferably, the amount of α-amylase added is 1000 μg / ml.

[0012] Furthermore, the preparation process of the refrigeration solution is as follows: Weigh the appropriate mass of lactose according to the proportion and add it to sterile distilled water, add egg yolk, mix thoroughly, and centrifuge at 2800 rpm for 30 min; take the supernatant, add proline to it, and then add α-amylase.

[0013] This invention also provides a method for applying the above-mentioned porcine semen cryopreservation agent, comprising the following steps:

[0014] S1. Add the collected and pre-processed boar semen to a centrifuge bottle, centrifuge the boar semen, and remove the supernatant after centrifugation.

[0015] S2. After removing the supernatant, add refrigeration solution to the pig semen to resuspend the semen and place it in a 4°C refrigerator for 3-4 hours.

[0016] S3. After the semen has been refrigerated and allowed to stand, add cryogenic liquid to achieve the effective target sperm concentration and mix thoroughly. In a 4°C environment, use a filling machine to draw the semen into thin tubes, then spread the thin tubes containing the semen evenly in batches on a freezing rack and pre-cool them on the surface of liquid nitrogen. Then, freeze them in liquid nitrogen.

[0017] Furthermore, step S1 also includes: recording the mass of the centrifuge bottle as M1, weighing the total weight of the centrifuge bottle and sperm after removing the supernatant as M2, and estimating the sperm mass as M3 = M2 - M1;

[0018] The volume of refrigeration solution added in step S2, V1 (mL), is equal to the total sperm count (hundred million) / [target (hundred million / mL) * 3] - M3;

[0019] In step S3, the volume of the cryosol added is V2 (mL) = 1 / 2V1 (mL).

[0020] Furthermore, the boar semen collection and pretreatment process in step S1 includes:

[0021] S11. Semen collection: 75-125 mL of semen from the mid-ejaculate portion of the pig was collected.

[0022] S12. Raw semen analysis: Raw semen is grayish-white or milky-white. The motility density of fresh semen is detected using a microscope. Pig semen with a motility ≥90% and a sperm density ≥200 million / mL is selected.

[0023] S13. Semen pre-dilution: Add 1 to 2 times the volume of BTS diluent to the collected boar semen. At an isothermal temperature of 32 to 35°C, slowly add the diluent to the original semen and gently shake well. After dilution, wrap the semen in cotton and place it in a constant temperature incubator at 17°C.

[0024] Furthermore, the application method of the porcine semen cryopreservation agent also includes step S4, semen thawing: thawing the frozen capillary tube at 50°C for 16 seconds, cutting off both ends of the capillary tube, and transferring the semen into a centrifuge tube preheated at 30°C for 1 minute.

[0025] The advantages of this invention are: the cryopreservative of this invention has a simple overall composition, low cost, and high overall cost-effectiveness; due to the addition of α-amylase to the cryopreservative, the preservation quality of frozen porcine semen can be significantly improved; it effectively improves multiple physicochemical properties of frozen porcine semen, including motility, acrosome integrity, DNA integrity, and mitochondrial activity. Attached Figure Description

[0026] Figure 1 This is a schematic diagram illustrating the classification of boar semen motility in the present invention.

[0027] Figure 2 Comparison of sperm motility after thawing in each experimental group and control group;

[0028] Figure 3 A comparison of sperm motility after thawing and incubation in each experimental group and the control group;

[0029] Figure 4 Microscopic fluorescence images showing the integrity of the sperm acrosome after thawing in each experimental group and control group;

[0030] Figure 5 Comparison of sperm acrosome integrity after thawing in each experimental group and control group;

[0031] Figure 6 Microscopic fluorescence images showing the integrity of sperm DNA after thawing in each experimental group and control group;

[0032] Figure 7 Comparison of sperm DNA integrity after thawing in each experimental group and control group;

[0033] Figure 8 Microscopic fluorescence images of sperm mitochondrial activity after thawing in each experimental group and control group;

[0034] Figure 9 A comparison of sperm mitochondrial activity after thawing in each experimental group and the control group. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] This invention, through GO enrichment analysis, discovered that α-amylase is highly expressed in frozen semen resistance. Based on this, research was conducted to design a cryopreservative with simple components, low cost, and significant improvement in the preservation quality of frozen porcine semen.

[0037] The experimental design of this invention first involves freezing boar semen using conventional methods, then measuring semen motility after thawing. While there were no significant differences in semen motility among boars of the same breed when collecting fresh semen, significant differences emerged after thawing. Based on this, 12 boars with high and low semen motility were selected as subjects for proteomic analysis. Specifically, 3 Large White boars had high motility (YT) and 3 had low motility (YS); 3 Landrace boars had high motility (LT) and 3 had low motility (LS). These were used to represent the average semen quality level of artificially raised boars. The classification results are as follows: Figure 1 As shown.

[0038] Proteomic analysis of semen from various experimental pigs revealed that GO enrichment analysis showed higher expression levels of α-amylase in cryopreservative-resistant pig sperm compared to cryopreservative-intolerant sperm. Based on this, the present invention designs a cryopreservative containing α-amylase and further designs experiments to enhance its specific effects on pig semen motility. Since α-amylase is readily available, relatively inexpensive, and easy to preserve, the cryopreservative with this as its main component also offers high overall cost-effectiveness.

[0039] The cryopreservative containing α-amylase used in this invention includes: a refrigeration solution and a freezing solution.

[0040] The cryogenic solution comprises sterile distilled water, lactose, egg yolk, proline, and α-amylase. The mass ratio of all components except α-amylase is 160:17-18:29-39:0.225-0.235; the amount of α-amylase added is 10–1000 μg / ml. Sugars are used to maintain osmotic pressure and provide energy; proline is used to stabilize cell structure and scavenge reactive oxygen species (ROS); egg yolk acts as a buffer, enabling sperm to adapt to diluents with significant osmotic pressure changes. This cryogenic solution is used in the pre-freezing stage of porcine semen to prevent cold shock and death caused by rapid cooling.

[0041] The preparation process of the refrigeration solution is as follows: Weigh the appropriate amount of lactose according to the proportion and add it to sterile distilled water. Add egg yolk, mix thoroughly, and centrifuge at 2800 rpm for 30 min. Take the supernatant, add proline to it, and then add α-amylase as needed.

[0042] The cryoprotectant comprises: a cryogenic fluid (excluding α-amylase), glycerol, Equex.STM cryoprotectant, proline, and α-amylase. The mass ratio of all components except α-amylase is 92.5:5-7:29-39:0.225-0.235; the α-amylase dosage is 10–1000 μg / ml. Glycerol is an osmotic protectant, while Equex.STM cryoprotectant is a non-osmotic protectant; both work together to protect the cell membranes of porcine semen.

[0043] The process of collecting fresh semen from experimental boars and the pretreatment procedure are as follows:

[0044] S1. Semen collection

[0045] Boar semen is primarily collected by hand. During the collection process, the collectors must ensure the hygiene and disinfection of themselves and the operating environment. When collecting fresh semen from boar ejaculation, select the midstream portion, typically approximately 75–125 mL. The preceding and following portions of ejaculation should be discarded, as these may contain higher levels of bacteria or impurities.

[0046] S2, Semen Analysis

[0047] The raw semen appears grayish-white or milky-white. The motility density of the collected fresh semen was tested on-site using a microscope, and sperm motility ≥90% and sperm density ≥200 million / mL were selected.

[0048] S3, Semen Pre-dilution

[0049] The collected raw semen is diluted isothermally. Boar semen is diluted with 1-2 times its volume of BTS diluent, and slowly added to the raw semen at an isothermal temperature (32-35℃), gently shaking to mix. After dilution, the solution is wrapped in cotton and placed in a 17℃ incubator. This step supplements the necessary nutrients for sperm, reduces sperm clumping, cushions against shocks during transport, and effectively maintains sperm motility.

[0050] The steps for using the cryopreservative of the present invention are as follows:

[0051] S4, Semen Centrifugation

[0052] The pretreated boar semen was added to a centrifuge bottle and centrifuged. The mass of the centrifuge bottle was recorded as M1 (g).

[0053] Because gelatinous substances or protoplasmic droplets in semen can easily cause sperm to clump together and reduce sperm motility, centrifugation is necessary to adjust sperm density and remove impurities.

[0054] Centrifuge at 17°C at 800 rpm for 15 minutes. Use an autoclaved needle to extract and discard the supernatant. After discarding the supernatant, weigh the total weight of the centrifuge bottle and sperm as M2 (g). (This process requires checking whether there are any sperm residues in the supernatant. If the standard is exceeded, centrifuge again.) The estimated sperm mass M3 (g) = M2 - M1.

[0055] S5, Semen Refrigeration

[0056] After centrifuging, add refrigeration solution I to the pig semen to resuspend the semen and place it in a 4°C refrigerator for 3-4 hours (sperm are in a dormant state at 0-5°C, and rapid cooling can easily cause cold shock and death).

[0057] The volume of refrigeration solution added, V1 (mL), is equal to the total number of sperm (hundred million) / [target (hundred million / mL) * 3] - M3.

[0058] S6, Semen Filling and Freezing

[0059] After refrigeration and settling, add cryogenic solution to the semen to achieve the target sperm count and mix thoroughly. In a 4°C environment, use a filling machine to draw the semen into 0.5 mL capillary tubes. Then, spread the capillary tubes containing semen evenly in batches on a freezing rack and pre-cool them for 8 minutes at a distance of 3 cm from the liquid nitrogen surface. Finally, freeze them in liquid nitrogen.

[0060] The volume of cryogenic fluid added, V2 (mL), is equal to 1 / 2 V1 (mL).

[0061] S7, Semen Thawing

[0062] After the semen capillary tubes were frozen for 24 hours, at least two capillary tubes from each experimental group were thawed for testing. The capillary tubes were thawed at 50°C for 16 seconds, the two ends of the capillary tubes were cut off, and the semen was transferred into centrifuge tubes preheated at 30°C and incubated for 1 minute.

[0063] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. These embodiments are merely illustrative and should not be construed as limiting the invention.

[0064] Seven additional experimental boars were selected, and their semen was collected and frozen according to the above-mentioned steps for collecting fresh semen from boars, pretreatment, and use of cryopreservatives. The effects were verified after thawing.

[0065] Specifically, the semen collected from all 7 experimental boars was mixed and then divided into 5 groups, including 4 experimental groups and 1 control group.

[0066] Experimental group: The concentrations of α-amylase in the refrigeration solution and the freezing solution were 10 μg / mL, 100 μg / mL, 1000 μg / mL, and 10000 μg / mL, respectively. Three samples were set for the same concentration in each group, and the average value of the measurement results was taken.

[0067] Control group: Neither the refrigeration solution nor the freezing solution contained α-amylase. Three samples were set up, and the average value of the measurement results was taken.

[0068] Example 1

[0069] Sperm motility testing:

[0070] After thawing the semen samples from each group using the method described above, drop them onto a glass slide. Analyze eight random fields of view at a time, and take the average of the eight field measurements. The test results are as follows: Figure 2 As shown, the cryopreservative containing α-amylase significantly improved sperm motility in boars compared to the cryopreservative without α-amylase. Even the group with the lowest concentration of α-amylase (10 μg / mL) showed a 10% improvement in sperm motility compared to the group without α-amylase; considering only the improvement in sperm motility, 1000 μg / mL was optimal.

[0071] Example 2

[0072] Sperm incubation motility test:

[0073] The thawed semen samples from each group were incubated in a 30°C water bath, and semen motility was tested every 30 minutes for a total of 60 minutes. The test results are as follows: Figure 3As shown, sperm motility decreased with prolonged incubation time, while the motility in the experimental groups was consistently higher than that in the control group. Within the experimental groups, sperm with added 1000 ug / ml of α-amylase exhibited higher motility in the first 50 minutes compared to other experimental groups.

[0074] Example 3

[0075] Sperm acrosome integrity testing:

[0076] ① Centrifuge the thawed semen at 800g for 5 minutes, remove the supernatant, add 1.5mL of 4% formaldehyde and fix for 30 minutes. After fixation, centrifuge at 800g for 2 minutes and discard the supernatant. After pipetting with PBS, centrifuge at 800 rpm for 2 minutes and resuspend in 1mL of PBS to obtain a sperm cell PBS suspension.

[0077] ② Spread 20 μL of sperm cell PBS suspension evenly onto a glass slide and allow it to air dry. Add 20 μL of FITC-PNA working solution (10 μg / mL) evenly to the sample and incubate at 37°C in the dark for 30 min. Then add 30 μL of Hoechst 33342 staining solution (10 μg / mL) and stain at room temperature for 5-10 min (the entire process should be performed in the dark). After staining, slowly rinse the slide with double-distilled water to remove excess dye, allow it to air dry, and quickly photograph it under a fluorescence microscope. Select at least three clear fields of view, ensuring that each field contains at least 200 sperm cells for observation and recording. Figure 4 , 5 The image shows the fluorescence effect of acrosome integrity in each group. It can be seen that except for the group with the highest concentration of α-amylase (10000 μg / mL), which has a negative effect on the sperm replacement integrity of boars, the other cryopreservatives with added α-amylase all have a certain effect on improving the sperm replacement integrity of boars.

[0078] Example 4

[0079] DNA integrity testing:

[0080] Following step ① of Example 2, a sperm cell PBS suspension was prepared. 300 μL of the sperm cell PBS suspension was taken, and 4.5 μL of Lacridine Orange Stain (15 μg / ml) was added. The mixture was stained at room temperature in the dark for 15–20 min, then dropped onto a glass slide and covered with a coverslip. The slide was then photographed and examined under a fluorescence microscope. Figure 6 , 7 As shown, the group with added α-amylase cryopreservatives significantly improved the DNA integrity of boars compared to the group without added α-amylase cryopreservatives.

[0081] Example 5

[0082] Mitochondrial activity assay:

[0083] Take 50 μL of thawed semen and place it in 0.5 mL of cell culture medium. Add 0.5 mL of JC-1 staining working solution, invert several times to mix, and incubate at 37°C for 20 minutes. After incubation, wash twice with JC-1 staining buffer (add 1 mL of JC-1 staining buffer, centrifuge at 600 rpm at 4°C for 3-4 minutes, discard the supernatant), and resuspend the cells in 300 μL of JC-1 staining buffer. Take 10 μL of the resuspended cell solution on a glass slide, cover with a coverslip, and photograph under a fluorescence microscope for analysis. Figure 8 , 9 As shown, the cryopreservation agent with added α-amylase significantly enhanced the mitochondrial activity of boars compared to the cryopreservation agent without added α-amylase; and the effect increased with increasing α-amylase concentration.

[0084] In summary, the addition of α-amylase to the cryopreservation agent significantly improved the motility, acrosome integrity, DNA integrity, and mitochondrial activity of frozen porcine semen. The improvement effect was closely related to the concentration of α-amylase; in particular, excessively high concentrations of α-amylase had a significant negative impact on the acrosome integrity of porcine sperm. The optimal concentration range for α-amylase to exert its effect was wide, from 10 to 1000 μg / mL. The preferred concentration of α-amylase was 1000 μg / mL.

[0085] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications or equivalent changes made to the above embodiments based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A cryopreservation agent for boar semen, characterized in that, Including refrigerant and freezing fluid; The refrigeration solution comprises: sterile distilled water, lactose, egg yolk, proline, and α-amylase; the freezing solution comprises: the refrigeration solution without α-amylase, glycerol, Equex.STM antifreeze, proline, and α-amylase; the mass ratio of the components in the refrigeration solution except for α-amylase is 160:17-18:29-39:0.225-0.235, and the amount of α-amylase added is 10~1000μg / ml; the mass ratio of the components in the freezing solution except for α-amylase is 92.5:5-7:29-39:0.225-0.235, and the amount of α-amylase added is 10~1000μg / ml.

2. The porcine semen cryopreservation agent according to claim 1, characterized in that, The amount of α-amylase added to both the refrigeration solution and the freezing solution was 1000 μg / ml.

3. The porcine semen cryopreservation agent according to claim 1, characterized in that, The preparation process of the refrigeration solution is as follows: Weigh the appropriate amount of lactose according to the proportion and add it to sterile distilled water. Add egg yolk, mix thoroughly, and centrifuge at 2800 rpm for 30 min. Take the supernatant, add proline to it, and then add α-amylase.

4. A method for applying a porcine semen cryopreservation agent, characterized in that, The application of the porcine semen cryopreservation agent as described in any one of claims 1-3 includes the following steps: S1. Add the collected and pre-processed boar semen to a centrifuge bottle, centrifuge the boar semen, and remove the supernatant after centrifugation. S2. After removing the supernatant, add refrigeration solution to the pig semen to resuspend the semen and place it in a 4°C refrigerator for 3-4 hours. S3. After the semen has been refrigerated and allowed to stand, add cryogenic liquid to achieve the effective target sperm concentration and mix thoroughly. In a 4°C environment, use a filling machine to draw the semen into thin tubes, then spread the thin tubes containing the semen evenly in batches on a freezing rack and pre-cool them on the surface of liquid nitrogen. Then, freeze them in liquid nitrogen.

5. The method of applying the porcine semen cryopreservation agent according to claim 4, characterized in that, Step S1, the process of collecting and preprocessing boar semen, includes: S11. Semen collection: 75-125 mL of semen from the mid-ejaculate portion of the pig was collected. S12. Raw semen analysis: Raw semen is grayish-white or milky-white. The motility density of fresh semen is detected using a microscope. Pig semen with a motility ≥90% and a sperm density ≥200 million / mL is selected. S13. Semen pre-dilution: Add 1 to 2 times the volume of BTS diluent to the collected boar semen. At an isothermal temperature of 32 to 35°C, slowly add the diluent to the raw semen and gently shake to mix. After dilution, wrap the mixture in cotton and place it in a constant temperature incubator at 17°C.

6. The method of applying the porcine semen cryopreservation agent according to claim 4, characterized in that, It also includes step S4, semen thawing: Thaw the frozen capillary tube at 50°C for 16 seconds, cut off both ends of the capillary tube, and transfer the semen into a centrifuge tube preheated at 30°C for 1 minute.

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