CN107668026A - Application of the rutin as livestock semen Cryoprotectant - Google Patents

Abstract

The invention discloses the application of rutin as a cryopreservation agent for livestock semen. The invention also provides a cryopreservation solution for livestock semen, including frozen base fluid, egg yolk, rutin and glycerin. The invention uses rutin as an antifreeze agent for the first time Used for cryopreservation of pig, sheep and cattle semen, suitable for artificial insemination of pig, sheep and cattle semen frozen in thin tubes, long-term storage of semen after ultra-low temperature freezing and insemination after long-distance transportation, can significantly improve the sperm viability of pigs, sheep and cattle after freezing-thawing rate, acrosome integrity rate, and plasma membrane integrity rate.

Description

Application of rutin as cryopreservative for domestic animal semen

technical field

The invention belongs to the technical field of ultra-low temperature cryopreservation of mammalian semen, and in particular relates to the application of rutin as an antifreeze agent in the cryopreservation of livestock semen.

Background technique

Sperm freezing technology is of great significance to the development of livestock. It not only solves the problem of long-term preservation of male animal semen, but also facilitates the preservation of genetic resources. It is also convenient to apply and is not limited by time and space. It plays an important role in establishing a global gene bank of excellent species. The wide application of sperm freezing technology is conducive to improving the utilization rate of excellent breeding males, reducing the number of breeding males, reducing economic costs, and improving the efficiency of animal husbandry, especially in the field of artificial insemination. Play an important role. Semen freezing technology has important practical significance for the sustainable development of domestic and international animal husbandry and the satisfactory supply of animal products.

Sperm are sensitive to temperature changes. In the process of freezing and cooling, sperm are susceptible to freezing damage, mainly including physical damage, chemical damage and oxidative damage. Compared with fresh semen, the motility, acrosome integrity, mitochondrial activity, DNA integrity, and membrane integrity were significantly decreased in frozen-thawed spermatozoa. The quality of sperm is closely related to pregnancy rate, delivery rate and litter size. So far, frozen semen artificial insemination technology has been applied to cattle production, and it is most widely used in the breeding and improvement of dairy cattle. But after freezing and thawing, there are still 40%-50% sperm inactivation, which seriously reduces the utilization rate of the fine-bred bull. However, pig sperm and sheep sperm are more sensitive to freezing stimulation. After thawing, the sperm recovery rate is low, there are many abnormal sperm, and the conception rate is lower than the normal conception rate. Therefore, the freezing of pig sperm and sheep sperm is still in the experimental stage in the world.

During the freezing process, due to the drop in temperature, some ice crystals will first form in the extracellular solution. When the extracellular ice crystals are formed, a hypertonic solution environment will be formed outside the cells, resulting in the loss of intracellular water. As the temperature continues to drop, ice crystals also form inside the cells. When a large number of extracellular and intracellular ice crystals are produced, they will cause severe mechanical damage to the sperm cell membrane, internal structure of the cell and organelles, destroy cell function, cell membrane integrity, and acrosome damage, and can also cause sperm DNA structure integrity Sex changes, causing DNA damage, resulting in the death of sperm and reducing the survival rate. The formation of ice crystals is inevitable during the freezing process, and the degree of damage is closely related to the size of the ice crystals. How to reduce the harm of ice crystals in the freezing process, the frozen ice crystals should be in the state of microcrystals as much as possible, so as to reduce the damage of cold shock.

Contents of the invention

In view of the deficiencies in the prior art, the present invention provides a livestock semen cryopreservation agent and its application in pig, sheep and cattle semen cryopreservation, which solves the problem of low sperm viability after freezing-thawing of livestock in the prior art.

In order to solve the above-mentioned deficiencies, the technical scheme adopted in the present invention is as follows:

The invention provides the application of rutin as a livestock semen cryopreservation agent.

The present invention also provides livestock semen cryopreservation agent, which comprises freezing base liquid, egg yolk, glycerin and rutin.

The formula of the freezing base liquid of the present invention is glucose 1.1g, citric acid 1.48g, Tris 2.42g, penicillin sodium 0.06g, streptomycin sulfate 0.1g, double distilled water 100ml; wherein the added amount of glycerin is livestock semen cryopreservative 6%-12% by volume; the volume ratio of frozen base liquid to egg yolk is 4:1.

In one embodiment, the amount of rutin added to 100 ml of the livestock semen cryopreservative of the present invention is 0.010 mmol-0.100 mmol.

In another embodiment, the added amount of rutin in 100ml of the livestock semen cryopreservative of the present invention is 0.030mmol-0.080mmol.

In yet another embodiment, the amount of rutin added in 100ml of the livestock semen cryopreservative of the present invention is 0.050mmol-0.100mmol.

The beneficial effects of the present invention are:

The present invention uses rutin for the first time in pig, sheep and cattle semen cryopreservation, the effect is reliable, and it is easy to popularize. Significantly improve the sperm viability, acrosome integrity and plasma membrane integrity of pigs, sheep and cattle after freezing and thawing, and maintain the fertilization ability of sperm.

Description of drawings

Fig. 1 is a comparison diagram of sperm acrosome integrity and plasma membrane integrity after adding rutin antifreeze, and Fig. 1 (a) is a schematic diagram of sperm acrosome integrity, Indicated is the sperm with intact acrosome, △ indicates the sperm with damaged acrosome, Figure 1(b) is a schematic diagram of sperm plasma membrane integrity, ↑ is sperm with intact plasma membrane, △ is damaged sperm.

Figure 2 is a comparison chart of sperm acrosome integrity and plasma membrane integrity after adding resveratrol antifreeze agent, Figure 2 (a) is a schematic diagram of sperm acrosome integrity, Indicated is the sperm with intact acrosome, △ indicates the sperm with damaged acrosome, Figure 2(b) is a schematic diagram of sperm plasma membrane integrity, ↑ is sperm with intact plasma membrane, and △ is damaged sperm.

Figure 3 is a comparison diagram of sperm acrosome integrity and plasma membrane integrity after adding phytic acid antifreeze, Figure 3 (a) is a schematic diagram of sperm acrosome integrity, Indicated is the sperm with intact acrosome, △ indicates the sperm with damaged acrosome, Figure 3(b) is a schematic diagram of sperm plasma membrane integrity, ↑ is sperm with intact plasma membrane, △ is damaged sperm.

detailed description

Rutin, also known as rutin, vitamin P, and rutin, belongs to flavonoids and widely exists in plants in nature, mainly in roots, stems, and leaves. It also exists in some traditional Chinese medicines after research, such as Hawthorn, ginkgo, wolfberry, etc. all contain a certain amount of rutin. Rutin is a yellow crystalline powder with a molecular formula of C ₂₇ H ₃₀ O ₁₆ , and a relative molecular mass of 610.51. It is easily soluble in organic solvents such as ethanol, dimethyl sulfoxide, and ether. Rutin is antibacterial, anti-inflammatory, anti-radiation, and regulates the permeability of capillaries. It is clinically used for hypertension, cardiovascular and cerebrovascular diseases, and has a good therapeutic effect. The structure of rutin contains multiple hydroxyl groups. We speculate that hydroxyl groups can replace water molecules, weaken the crystallization process of water during freezing, reduce the damage of ice crystals to sperm, improve the survival rate of sperm, and may play a very good role in freezing cells. It can also play a good role in complexing metal ions.

We have proved through many experiments that adding rutin antifreeze can play a certain protective role in the process of livestock semen freezing. At present, there are no related reports at home and abroad about the use of rutin as a cryoprotectant for the cryopreservation of livestock such as pigs, sheep and cattle, or even any mammalian semen.

In the following examples, the normal temperature diluent (Betsville Thawing Solution, BTS) formula involved is: glucose 3.7g, trisodium citrate dihydrate 0.6g, EDTA 0.125g, sodium bicarbonate 0.125g, potassium chloride 0.075 g, 0.06 g of penicillin sodium, 0.1 g of streptomycin sulfate, mixed with double distilled water, and the volume was adjusted to 100 ml, and the pH was adjusted to 7.2, and the osmotic pressure was 310 mosm/L.

The livestock semen cryopreservation agent of the present invention comprises freezing base liquid, egg yolk and rutin.

The volume ratio of the amount of glycerin added to the above-mentioned livestock semen cryopreservation agent is 6%-12%, that is, to obtain another kind of livestock semen cryopreservation agent of the present invention, for example, adding glycerol to the above-mentioned 94ml livestock semen cryopreservation agent Quantity is 6ml, promptly obtains another kind of livestock semen cryopreservation agent of the present invention and is used for preserving pig semen.

The formula of described freezing base liquid is glucose 1.1g, citric acid 1.48g, Tris2.42g, sodium penicillin 0.06g, streptomycin sulfate 0.1g, double distilled water 100ml.

Example 1:

The preparation method of pig semen cryopreservation agent of the present invention, comprises the steps:

Step 1, prepare the frozen base solution: accurately measure 1.1g of glucose, 1.48g of citric acid, 2.42g of Tris, 0.06g of penicillin sodium, and 0.1g of streptomycin sulfate, dissolve them in 100ml double distilled water, adjust the pH to 6.21, and adjust the osmotic pressure 286mosm/L, prepared as frozen base fluid;

Step 2, accurately measure 80ml of freezing base liquid, add fresh egg yolk 20ml, rutin 0.01mmol-0.10mmol, and mix to obtain a kind of livestock semen cryopreservative (I liquid) of the present invention.

Take 94ml of the above-mentioned livestock semen cryopreservative and add 6ml of glycerin, filter and sterilize, and cool to room temperature to obtain another livestock semen cryopreservative (II solution) of the present invention for storing pig semen, and put it in a 4°C refrigerator Reserved for the following test tests.

The inventor has carried out following test on the use effect of the pig semen cryopreservation diluent of this embodiment:

(1) Semen collection

Collect semen by hand holding, filter through 4 layers of sterile gauze to remove jelly, and conduct routine quality inspection with an optical microscope at 34-37°C. Choose semen with no peculiar smell, milky white color, normal sperm morphology, motility rate above 0.75, and "dense" density, and keep it warm at 30°C for the test.

(2) Semen processing and freezing

Add the fresh semen to the isothermal diluent at room temperature (30°C, 1:1 dilution), wrap it in multiple layers of gauze, and equilibrate in a 17°C incubator for 0.5h-1h. Centrifuge the equilibrated semen at 17°C (1200rpm, 10min), discard the supernatant, add isothermal solution I with twice the volume, wrap it with multiple layers of gauze, and place it in a refrigerator at 4°C for 1h-1.5h. Afterwards, add the equilibrated semen to an equal volume of II solution at 4°C, wrap it with gauze and place it in a refrigerator at 4°C for another 1.5-2.5 hours to balance. Inhale the semen into a 0.25ml thin tube with a special syringe, quickly seal the tube, place it in a programmed freezer at a rate of 1°C/min and slowly drop it from 4°C to -5°C, and then quickly move it to 2.5-3.5cm above the liquid nitrogen for fumigation After 10-15 minutes, put the thin tube into liquid nitrogen for storage.

(3) Thawing of frozen semen in thin tubes and evaluation of sperm quality

1. Sperm motility

Take out the frozen semen in the thin tube and put it into a 37°C water bath to thaw for 45 seconds, collect it in a small test tube, then add an equal volume of diluent at room temperature preheated at 37°C to dilute, incubate for 10 minutes, and take 10 μL of semen on a glass slide Cover the slices with a cover glass, and evaluate sperm motility (percentage of linearly moving sperm) under a 400× inverted microscope. At least 200 spermatozoa were checked each time and repeated 5 times.

2. Integrity rate of sperm acrosome

After staining with FITC-PNA staining solution, the morphology of sperm acrosome was observed by fluorescence microscope.

After the frozen fine tube is thawed, add the semen to the surface of 2ml 3% PVP liquid, centrifuge at 1500rpm for 6min, discard the supernatant; wash twice with PBS, centrifuge at 1500rpm for 6min, discard the supernatant; resuspend the sperm in PBS (37°C), Adjust the density to 1-2×10 ⁶ spe/ml; draw 30 μl of sperm suspension, smear, air-dry, and fix with methanol for 10 minutes; then add 30 μl FITC-PNA staining solution, and incubate in a dark and humid environment at 37°C for 30 minutes; wash with PBS, Air dry, drop a small amount of mounting medium, cover the slide, seal the slide with colorless nail polish, and observe under a 400× fluorescent microscope as soon as possible. At least 200 spermatozoa were checked each time and repeated 5 times.

3. Integrity rate of sperm plasma membrane

Hypotonic swelling test (HOST) was performed using fructose-sodium citrate hypotonic solution. Dilute the thawed semen with hypotonic solution, adjust the sperm density to 1×l0 ⁶ sperm/ml, incubate at 37°C for 30 minutes, take 20 μl of the semen suspension and drop it on a blood cell counting plate, observe under a 400× microscope, and count the bent-tailed sperm Percentage, check at least 200 sperm each time, repeat 5 times.

(4) Sperm quality assessment results

Application pig semen cryopreservation antifreeze of the present invention and seminal fluid freezing-thawing method, evaluation result is as follows:

When 0.010mmol-0.100mmol rutin is added to the freezing solution for pigs, the sperm viability rate after freezing-thawing reaches 65%, the acrosome integrity rate reaches 70%, and the plasma membrane integrity rate reaches 68%.

The results of the test example are shown in Table 1 below:

Table 1

deal with sperm motility acrosome integrity plasma membrane integrity control group 30% 45% 35% Example 1 65% 70% 68%

Comparative example 1:

In this comparative example, the preparation method of the refrigerant for pig semen and the quality detection of pig sperm are the same as those in Example 1, except that the amount of phytic acid added as an antifreeze agent is 0.02-0.10 mmol.

The test results showed that the motility rate of pig sperm was 43%, the acrosome integrity rate was 50%, and the plasma membrane integrity rate was 48%.

Comparative example 2:

In this comparative example, the preparation method of the refrigerant for pig semen and the quality detection of pig sperm are the same as those in Example 1, except that resveratrol is added as an antifreezing agent in an amount of 0.1-0.3 mmol. The test results showed that the porcine sperm motility rate was 47%, the acrosome integrity rate was 50%, and the plasma membrane integrity rate was 56%.

Both comparative example 1 and comparative example 2 are tests done by the inventor adding phytic acid or resveratrol to the antifreeze agent, and the addition amount of phytic acid or resveratrol is the optimum addition obtained from multiple tests , but the experimental results were not satisfactory. When rutin is used as an antifreeze agent, its activity rate, acrosome integrity rate and plasma membrane integrity rate are significantly better than those of phytic acid or resveratrol, which may be due to the obvious effect of rutin as a strong antifreeze agent. Higher than phytic acid and resveratrol.

Fig. 1-Fig. 3 are comparison diagrams of sperm acrosome integrity and plasma membrane integrity after adding three different antifreeze agents. As shown in the figure:

In the acrosome integrity of rutin, most of them are cap-shaped and hemispherical, and they are sperm with complete acrosome. In the detection of plasma membrane integrity, most sperm have obvious bending tail phenomenon, and some sperm have tail bending Less obvious but more intact plasma membrane and less damaged spermatozoa.

In the acrosome integrity detection of resveratrol, the number of uneven fluorescent coloring was significantly more than that in rutin, and the acrosome integrity was significantly lower than that after adding rutin. In the plasma membrane integrity assay, the number of inconspicuous tail bends increased more than the inconspicuous tail bends after adding rutin.

In the acrosome integrity test of phytic acid, there were cap-shaped hemispherical green fluorescence and uneven fluorescence coloring, which were lower than the acrosome integrity of rutin. In the detection of plasma membrane integrity, both obvious and inconspicuous bent tails existed, but the number of bent tails was lower than that after adding rutin.

Comparing the three, it can be concluded that the integrity of the acrosome of rutin is higher than that of phytic acid and resveratrol, and the integrity of the plasma membrane is also higher than that of the two. Adding rutin as an antifreeze can significantly improve the activity of sperm.

Example 2:

The preparation method of goat semen cryopreservation agent of the present invention, comprises the steps:

Step 1, preparing frozen base liquid, the method is the same as in Example 1;

Step 2: Accurately measure 85ml of frozen base solution, add 15ml of fresh egg yolk and 0.030mmol-0.080mmol of rutin, and mix evenly to prepare a cryopreservative for livestock semen (I liquid) of the present invention.

Get the above-mentioned livestock semen cryopreservation agent of 88ml and add 12ml glycerin to be mixed with the mixed solution of 100ml, filter sterilize, cool to room temperature, namely obtain another kind of livestock semen cryopreservation agent (II liquid) of the present invention and be used for preserving sheep semen, Put it in a refrigerator at 4°C for the following tests.

(1) Semen collection

The semen is collected by the hand holding method, and the routine quality inspection is carried out at 34-37°C with a microscope. Select semen with no peculiar smell, milky white color, normal sperm morphology, motility rate above 0.7, and "dense" density, and keep it warm at 30°C for the test.

(2) Semen processing and freezing

Add isothermal and equal volume of solution I to the fresh semen, wrap it with multi-layer gauze, and equilibrate it in a refrigerator at 4°C for 2 hours. Add isothermal and equal volume II solution to the equilibrated seminal fluid, wrap it with multiple layers of gauze, and place it in a refrigerator at 4°C for 1-1.5 hours to equilibrate. Inhale the semen into a 0.25ml thin tube with a special syringe, quickly seal the tube, place it in a programmed freezer at a rate of 1°C/min and slowly drop it from 4°C to -5°C, and quickly move it to 2.5-3.5cm above the liquid nitrogen for fumigation 6 -8min, put the thin tube into liquid nitrogen for storage.

(3) Thawing of frozen semen in thin tubes and evaluation of sperm quality

1. Sperm motility

Take out the frozen semen in the thin tube and put it into a 37°C water bath to thaw for 25 seconds, collect it in a small test tube, then dilute it with an equal volume of diluent at room temperature preheated at 37°C, incubate for 10 minutes, and take 10 μL of semen on a glass slide Cover the slices with a cover glass, and evaluate sperm motility (percentage of linearly moving sperm) under a 400× inverted microscope. At least 200 spermatozoa were checked each time and repeated 5 times.

2. Integrity rate of sperm acrosome

After staining with FITC-PNA staining solution, the morphology of sperm acrosome was observed by fluorescence microscope.

After the frozen fine tube is thawed, add the semen to the surface of 2ml 3% PVP liquid, centrifuge at 1500rpm for 6min, discard the supernatant; wash twice with PBS, centrifuge at 1500rpm for 6min, discard the supernatant; resuspend the sperm in PBS (37°C), Adjust the density to 1-2×10 ⁶ sperm/ml; draw 30 μl of sperm suspension, smear, air-dry, and fix with methanol for 10 minutes; then add 30 μl FITC-PNA staining solution, and incubate in a dark and humid environment at 37°C for 30 minutes; wash with PBS, Air dry, drop a small amount of mounting medium, cover the slide, seal the slide with colorless nail polish, and observe under a 400× fluorescent microscope as soon as possible. At least 200 spermatozoa were checked each time and repeated 5 times.

3. Integrity rate of sperm plasma membrane

Hypotonic swelling test (HOST) was performed using fructose-sodium citrate hypotonic solution. Dilute the thawed semen with a regular diluent, adjust the sperm density to 1×l0 ⁶ sperm/ml, incubate at 37°C for 30 minutes, take 20 μl of the semen suspension and drop it on a blood cell counting plate, observe under a 400× microscope, and count the bent-tailed sperm Percentage, check at least 200 sperm each time, repeat 5 times.

(4) Sperm quality assessment results

Application sheep semen cryoprotectant of the present invention and semen freezing-thawing method, evaluation result is as follows:

When 0.030mmol-0.080mmol of rutin is added to the freezing liquid for sheep, the sperm viability rate after freezing-thawing reaches 60%, the acrosome integrity rate reaches 65%, and the plasma membrane integrity rate reaches 56%. The results are shown in Table 2 below Table 2

deal with sperm motility acrosome integrity plasma membrane integrity control group 43% 51% 40% Example 2 60% 65% 56%

Comparative example 1:

In this comparative example, the preparation method of the refrigerant used for sheep semen and the quality detection of pig sperm are the same as those in Example 1, except that the amount of phytic acid added as an antifreeze agent is 0.02-0.05 mmol.

The test results showed that the porcine sperm motility rate was 44%, the acrosome integrity rate was 53%, and the plasma membrane integrity rate was 46%.

Comparative example 2:

In this comparative example, the preparation method of the refrigerant for sheep semen and the quality detection of pig sperm are the same as those in Example 1, except that 0.4 mmol of resveratrol is added as an antifreeze agent. The test results showed that the sheep sperm motility rate was 49%, the acrosome integrity rate was 54%, and the plasma membrane integrity rate was 51%.

Both comparative example 1 and comparative example 2 are tests done by the inventor adding phytic acid or resveratrol to the antifreeze agent, and the addition amount of phytic acid or resveratrol is the optimum addition obtained from multiple tests amount, but the test results are not satisfactory, when rutin is used as antifreeze, its activity rate, acrosome integrity rate and plasma membrane integrity rate are significantly better than those of phytic acid or resveratrol, which may be due to the strong antifreeze The rutin in the freezer has a significantly higher effect than phytic acid and resveratrol.

Example 3:

The preparation method of bovine semen cryopreservation agent of the present invention, comprises the steps:

Step 1, preparing frozen base liquid, the method is the same as in Example 1;

Step 2, accurately measure freezing base liquid 80ml, add fresh egg yolk 20ml and rutin 0.050mmol-0.100mmol, promptly obtain a kind of livestock semen cryopreservation agent (I liquid) of the present invention.

Add 6ml of glycerin to the above-mentioned livestock semen cryopreservation agent, mix well, filter and sterilize, and cool to room temperature to obtain another livestock semen cryopreservation agent (II liquid) of the present invention for preserving bovine semen, and put it in 4°C Store in the refrigerator for the following experiments.

(1) Semen collection

Semen was collected by hand holding method, and routine quality inspection was carried out at 37°C with a microscope. Select semen with no peculiar smell, milky white color, normal sperm morphology, motility rate above 0.7, and "dense" density, and keep it warm at 37°C for the test.

(2) Semen processing and freezing

Add the fresh semen to the isothermal II solution, adjust the sperm density to 1.5×10 ⁶ /ml, wrap it in gauze, and equilibrate it in a refrigerator at 4°C for 4 hours.

Inhale the balanced semen into a 0.25ml thin tube with a special syringe, quickly seal the tube, place it in a programmed freezer at a rate of 1°C/min and slowly drop it from 4°C to -5°C, and quickly transfer it to liquid nitrogen Fumigate 2-3cm above for 5-10min, put the thin tube into liquid nitrogen for storage.

(3) Thawing of frozen semen in thin tubes and evaluation of sperm quality

1. Sperm motility

After taking out the thin tube of frozen semen, quickly put it into a 37°C water bath to thaw for 45 seconds, collect it in a small test tube, then add an equal volume of room temperature diluent to dilute, and incubate for 10 minutes, take 10 μL of semen on a glass slide, and cover with a cover glass , Evaluate sperm motility (percentage of linearly moving sperm) under a 400× inverted microscope. At least 200 spermatozoa were checked each time, repeated 5 times.

2. Integrity rate of sperm acrosome

After staining with FITC-PNA staining solution, the morphology of sperm acrosome was observed by fluorescence microscope.

After the frozen fine tube is thawed, add the semen to the surface of 2ml 3% PVP liquid, centrifuge at 1500rpm for 6min, discard the supernatant; wash twice with PBS, centrifuge at 1500rpm for 6min, discard the supernatant; resuspend the sperm in PBS (37°C), Adjust the density to 1-2×10 ⁶ spe/ml; draw 30 μl of sperm suspension, smear, air-dry, and fix with methanol for 10 minutes; then add 30 μl FITC-PNA staining solution, and incubate in a dark and humid environment at 37°C for 30 minutes; wash with PBS, Air dry, drop a small amount of mounting medium, cover the slide, seal the slide with colorless nail polish, and observe under a 400× fluorescent microscope as soon as possible. At least 200 spermatozoa were checked each time and repeated 5 times.

3. Integrity rate of sperm plasma membrane

Hypotonic swelling test (HOST) was performed using fructose-sodium citrate hypotonic solution. Dilute the thawed semen with a regular diluent, adjust the sperm density to ¹ ×106/ml, incubate at 37°C for 30 minutes, take 20 μl of the semen suspension and drop it on a hemocytometer, observe under a 400× microscope, and count the bent-tailed sperm Percentage, check at least 200 sperm each time, repeat 5 times.

(4) Sperm quality assessment results

Apply bovine semen cryopreservation antifreeze of the present invention and seminal fluid freezing-thawing method, evaluation result is as follows:

When 0.050mmol-0.100mmol of rutin is added, the sperm viability after freezing-thawing reaches 68%, the acrosome integrity rate reaches 75%, and the plasma membrane integrity rate reaches 62%. The results are shown in Table 3.

table 3

deal with sperm motility acrosome integrity plasma membrane integrity control group 44% 58% 52% Example 3 68% 75% 62%

Comparative example 1:

In this comparative example, the preparation method of the refrigerant used for bovine semen and the quality detection of pig sperm are the same as those in Example 1, except that the amount of phytic acid added as an antifreeze agent is 0.03-0.09 mmol.

The test results showed that the porcine sperm motility rate was 46%, the acrosome integrity rate was 59%, and the plasma membrane integrity rate was 53%.

Comparative example 2:

In this comparative example, the preparation method of the refrigerant for bovine semen and the quality detection of pig sperm are the same as those in Example 1, except that 0.03-0.08 mmol of resveratrol is added as an antifreezing agent. The test results showed that the bovine sperm motility rate was 48%, the acrosome integrity rate was 61%, and the plasma membrane integrity rate was 56%.

Both comparative example 1 and comparative example 2 are tests done by the inventor adding phytic acid or resveratrol to the antifreeze agent, and the addition amount of phytic acid or resveratrol is the optimum addition obtained from multiple tests amount, but the test results are not satisfactory, when rutin is used as antifreeze, its activity rate, acrosome integrity rate and plasma membrane integrity rate are significantly better than those of phytic acid or resveratrol, which may be due to the strong antifreeze The rutin in the freezer has a significantly higher effect than phytic acid and resveratrol.

It should be understood that those skilled in the art can make improvements or changes based on the above description, and all these improvements and changes should belong to the protection scope of the appended claims of the present invention.

Claims (6)

1. The application of rutin as a cryopreservative for livestock semen. 2. the livestock semen cryopreservation agent described in claim 1, comprises freezing base liquid, egg yolk, glycerin and rutin. 3. livestock semen cryopreservation agent as claimed in claim 2, is characterized in that, the formula of described freezing base liquid is glucose 1.1g, citric acid 1.48g, Tris2.42g, penicillin sodium 0.06g, streptomycin sulfate 0.1g , double distilled water 100ml; the added amount of the glycerin is 6%-12% of the volume of the livestock semen cryopreservative; the volume ratio of the frozen base liquid to egg yolk is 4:1. 4. The livestock semen cryopreservation agent as claimed in claim 3, characterized in that, the added amount of rutin in 100ml of the livestock semen cryopreservation agent is 0.010mmol-0.100mmol. 5. The livestock semen cryopreservation agent according to claim 3, wherein the rutin addition amount is 0.030mmol-0.080mmol in 100ml of the livestock semen cryopreservation agent. 6. The livestock semen cryopreservation agent according to claim 3, wherein the rutin addition amount is 0.050mmol-0.100mmol in 100ml of the livestock semen cryopreservation agent.