A preparation for preserving pig semen at room temperature and its use method

Abstract

The present invention discloses the application of puerarin in the preparation of a product for the room temperature preservation of pig semen. The present invention also discloses a room temperature preservation preparation for pig semen, the raw materials of which include: puerarin with a concentration of 0.1-100 μmol / L and a diluent with a pH value of 6.5-7.3; the components of the diluent include: glucose, ethylenediaminetetraacetic acid, sodium citrate, sodium bicarbonate, trishydroxymethylaminomethane, citric acid, cysteine, bovine serum albumin, kanamycin sulfate and water. The present invention also discloses a method for using the above-mentioned room temperature preservation preparation for pig semen, comprising the following steps: placing a boar semen stock solution in a constant temperature environment of 17°C, adding the above-mentioned room temperature preservation preparation for pig semen to dilute it at a volume ratio of 1:4-9, and the temperature difference between the boar semen stock solution and the pig semen room temperature preservation preparation during the dilution process is less than or equal to 1°C; mixing once every 12 hours during the preservation process.

Description

Technical Field

[0001] The invention relates to the technical field of artificial insemination, and in particular to a pig semen room temperature storage preparation and a use method thereof. Background Art

[0002] Artificial insemination technology (AI) has been widely adopted in the production process of pigs worldwide, mainly due to the appropriate reduction of the cost of boar breeding. In the selection of excellent offspring, excellent boars can also be selected to mate with sows. The offspring inherit the excellent genes of their parents, while reducing the production cycle and improving productivity. In the process of artificial insemination of pigs, it is necessary to preserve the semen of the breeding pigs, but due to the particularity between species, the sperm of pigs is extremely sensitive to temperature compared with other species. For example, cryopreservation will cause crystallization of pig sperm due to the sudden drop in temperature, which will cause serious damage to the plasma membrane of pig sperm, especially freezing stress, which is more sensitive to peroxidation damage; while the temperature damage to sperm can be reduced during storage at room temperature. The main reason for the decline in sperm motility is that sperm will metabolize and produce highly active ROS in the in vitro environment. Excessive ROS will affect sperm capacitation, superactivation, and acrosome reaction in the external environment, thereby affecting the health of sperm. Pig semen stored at room temperature can meet the daily needs of production, and the storage time is generally about seven days, which is convenient to use.

[0003] Boar semen is generally stored at about 17°C for about five days. The physiological activities of sperm are inhibited, and their movement and metabolism are inefficient. Once the temperature is restored to 37°C, sperm metabolism and movement reach their most active state. A large number of practices have proved that when boar semen is stored at a constant temperature of 17°C, sperm survival time is longer and sperm motility decreases more slowly.

[0004] Antioxidants are a class of chemical substances that, when present in small amounts in a polymer system, can delay or inhibit the oxidation process of the polymer, thereby preventing the aging of the polymer and extending its service life. They are also called "antioxidants". There are many different types of antioxidants based on their different antioxidant reaction mechanisms and functional groups. They remove oxidizing substances from sperm, fight ROS, and inhibit lipid oxidation in sperm cell membranes, thereby allowing sperm to maintain their shape and function. Among them, the most widely used antioxidants in recent years include: non-enzymatic antioxidants and enzymatic antioxidants.

[0005] Non-enzymatic antioxidants include: vitamins (vitamins A, C, E, α-lipoic acid, β-carotene, L-carnitine), trace elements (Se, Cu, Zn, Mn), Chinese herbal medicines and plant extracts (polysaccharides, flavonoids, alkaloids), etc. Enzymatic antioxidants include: superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT). This type of antioxidant exists in the cell membrane and can catalyze the dismutation reaction of active oxygen free radicals, convert lipid peroxides into alcohols and other specific chemical reactions, and block the side chain cycle reaction of lipid peroxidation, convert H2O2 into H2O and O2, and promptly clean up a large number of free radicals and intermediates produced in the reaction.

[0006] Puerarin, also known as puerarin, is a white and slightly yellow crystalline powder separated and purified from Chinese herbal medicine plants. It belongs to the isoflavone class. Its chemical name is 8-beta-D-glucopyranose-4',7-dihydroxyisoflavone (8-(β-D-Glucopyranosyl)-4',7-dihydroxyisoflav one), and its molecular formula is C 21 H 20 O9, relative molecular weight 416.37. In terms of antioxidant, in vitro cell test results show that puerarin (0.1, 1, 10 μmol / L) pretreatment can reduce the production of reactive oxygen species in PC12 cells induced by β-amyloid protein 25-35, and the effect is most obvious with low concentration of puerarin. Puerarin can remove superoxide anions and -OH, inhibit H2O2-induced hemolysis of erythrocytes and production of lipid peroxides, and increase the activity of superoxide dismutase in blood and brain cells. A large number of experiments have shown that puerarin has the effects of improving immunity, enhancing myocardial contractility, protecting myocardial cells, lowering blood pressure, and resisting platelet aggregation, but it has not yet been found that puerarin can be used in preparations for the storage of pig semen at room temperature. Summary of the invention

[0007] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a pig semen room temperature storage preparation and a method of using the same.

[0008] Application of puerarin in the preparation of products for the storage of pig semen at room temperature.

[0009] A preparation for storing pig semen at room temperature, the raw materials of which include: puerarin with a concentration of 0.1-100 μmol / L and a diluent with a pH value of 6.5-7.3; the components of the diluent include: glucose, ethylenediaminetetraacetic acid, sodium citrate, sodium bicarbonate, tris(hydroxymethyl)aminomethane, citric acid, cysteine, bovine serum albumin, kanamycin sulfate and water; the mass fraction of glucose is 5-7 g / 100 mL, the mass fraction of ethylenediaminetetraacetic acid is 0.4-0.5 g / 100 mL, and the mass fraction of sodium citrate is The mass fraction of the aqueous phase is 1.2-1.6 g / 100 mL, the mass fraction of sodium bicarbonate is 0.1-0.3 g / 100 mL, the mass fraction of tris(hydroxymethyl)aminomethane is 0.8-1.2 g / 100 mL, the mass fraction of citric acid is 0.4-0.6 g / 100 mL, the mass fraction of cysteine ​​is 0.005-0.015 g / 100 mL, the mass fraction of bovine serum albumin is 0.6-1.0 g / 100 mL, and the mass fraction of kanamycin sulfate is 0.04-0.08 g / 100 mL.

[0010] Puerarin is an isoflavone derivative extracted, separated and purified from Pueraria root, and exists in the root of Pueraria lobata, a leguminous plant. The applicant has confirmed through experiments that the use of puerarin for pig sperm preservation can not only reduce the production of ROS to a certain extent to alleviate oxidative stress, which is related to the mechanism of scavenging free radicals and reducing the production of ROS; it can also promote mitochondrial function by regulating the PARP1 / PI3K / AKT signaling pathway, and provide ATP for sperm energy metabolism through the protection of mitochondrial functional integrity and the glycolysis pathway, thereby maintaining the normal function of sperm cells.

[0011] During room temperature storage, bacteria may be present in the semen concentrate due to factors such as operator errors, bacterial infection of the boar's reproductive system, and bacterial contamination of instruments. These bacteria are mainly Gram-positive bacteria, Gram-negative bacteria, and fungi. Boar semen is rich in nutrients, which can be easily utilized by bacteria, resulting in a reduction in nutrients available to sperm and a reduction in sperm survival time. Puerarin can inhibit the growth of bacteria in semen and prolong its storage time.

[0012] Preferably, the mass ratio of glucose, ethylenediaminetetraacetic acid, sodium citrate, sodium bicarbonate, tris(hydroxymethyl)aminomethane, citric acid, cysteine, bovine serum albumin and kanamycin sulfate is 6:0.45:1.38:0.5:1:0.5:0.01:0.8:0.06.

[0013] Preferably, the concentration of puerarin is 50 μmol / L.

[0014] Preferably, the raw materials also include: formononetin and aurantin, the concentration of formononetin is 4-6 μmol / L, and the concentration of aurantin is 13-17 μmol / L.

[0015] Formononetin, derived from Astragalus and Licorice, is an isoflavone with anti-tumor and anti-cancer effects and estrogenic effects, and is often used as a diuretic in clinical practice. Auricularia cassiae is an anthraquinone monomer compound isolated from the anti-lipidemic effective part of Cassia seed.

[0016] The applicant has found through experiments that further compounding puerarin with formononetin and aurantia cassiae can further reduce the damage of oxidative stress to sperm during room temperature storage, improve the quality of semen storage, and extend the storage time of semen. The applicant controls the molar ratio of puerarin, formononetin, and aurantia cassiae to 0.1-100: 1-10: 0.5-20; the sperm motility can be maintained at more than 70% after 7 days of room temperature preservation, which can meet the production needs and improve the utilization efficiency of excellent boars.

[0017] The method for using the above-mentioned pig semen room temperature preservation preparation comprises the following steps: placing the boar semen stock solution in a constant temperature environment of 17°C, adding the above-mentioned pig semen room temperature preservation preparation to dilute it at a volume ratio of 1:4-9, wherein the temperature difference between the boar semen stock solution and the pig semen room temperature preservation preparation during the dilution process is less than or equal to 1°C; and mixing once every 12 hours during the preservation process.

[0018] The invention has simple components and low cost, can effectively prolong the survival time of pig sperm stored at room temperature, has good preservation effect, and is expected to improve the efficiency of pig farms. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a comparison chart of the total antioxidant capacity test results of the pig semen room temperature storage preparation obtained in Example 3 and the blank group.

[0020] Figure 2 This is a comparison chart of the results of active oxygen determination of the pig semen room temperature storage preparation obtained in Example 3 and the blank group.

[0021] Figure 3 This is a comparison chart of the ATP measurement results of the pig semen room temperature storage preparation obtained in Example 3 and the blank group.

[0022] Figure 4 The figure is a comparison chart of the sperm motility and ATP test results of the pig semen room temperature storage preparations obtained in Example 3, Example 6 and the comparative example.

[0023] Figure 5 The figure is a comparison chart of the results of active oxygen and total antioxidant capacity determination of the pig semen room temperature storage preparations obtained in Example 3, Example 6 and the comparative example. DETAILED DESCRIPTION

[0024] The present invention will be further explained below in conjunction with specific embodiments.

[0025] Semen stock solution: Huoshou black pig. The Huoshou black pig used in this application adopts artificial semen collection, and the original sperm motility is above 90%.

[0026] Puerarin: purchased from Shengqing Biological Company, the purity of puerarin is above 98.1%.

[0027] Formononetin: purchased from Hubei Jusheng Technology Co., Ltd., with a purity of 99%.

[0028] Auricularia cassiae: purchased from Shanghai Yuanye Biotechnology Co., Ltd., the purity of auricularia cassiae is above 98%.

[0029] Chemicals: The reagents used in the experiment were from Nanjing Jiancheng Company.

[0030] The diluent (pH=7) is prepared by adding 6 g glucose, 0.45 g ethylenediaminetetraacetic acid, 1.38 g sodium citrate, 0.2 g sodium bicarbonate, 1 g tris(hydroxymethyl)aminomethane, 0.5 g citric acid, 0.01 g cysteine, 0.8 g bovine serum albumin, and 0.06 g kanamycin sulfate, and distilled water is added to 100 mL.

[0031] Preparation of pig semen: Transport the black pig semen to the laboratory and place it in a constant temperature refrigerator at 17°C. Dilute it with the following pig semen room temperature storage preparation at a volume ratio of 1:4 or 1:9. During dilution, the temperature difference between the black pig semen and the pig semen room temperature storage preparation should not exceed 1°C. Mix it every 12 hours during the storage process to prevent sperm precipitation and death.

[0032] Example 1

[0033] The preparation for storing pig semen at room temperature is composed of puerarin and the above diluent, and the concentration of puerarin is 10 μmol / L.

[0034] Example 2

[0035] The preparation for storing pig semen at room temperature is composed of puerarin and the above diluent, and the concentration of puerarin is 25 μmol / L.

[0036] Example 3

[0037] The preparation for storing pig semen at room temperature is composed of puerarin and the above diluent, and the concentration of puerarin is 50 μmol / L.

[0038] Example 4

[0039] The preparation for storing pig semen at room temperature is composed of puerarin and the above diluent, and the concentration of puerarin is 75 μmol / L.

[0040] Example 5

[0041] The preparation for storing pig semen at room temperature is composed of puerarin and the above diluent, and the concentration of puerarin is 100 μmol / L.

[0042] The porcine semen room temperature storage preparation obtained in Examples 1-5 was compared with a blank control group (diluent, without puerarin), and 3 replicates were set for each group. Each group was subjected to a sperm motility test: sperm motility was measured by computer-assisted sperm analysis (CASA). The detection method referred to the test operation steps provided by the "Hong Shi Ye" porcine sperm computer-assisted analysis system CASA, and optimized operations were performed on this basis.

[0043] The results are as follows:

[0044] Table 1: Effect of puerarin on the motility of pig sperm stored at room temperature

[0045]

[0046]

[0047] Note: a, b, c, d, e represent differences. Data in the same column with different letters on their shoulders indicate significant differences (P<0.05), while data with the same letters or no letters indicate no significant differences (P>0.05).

[0048] From Table 1, we can see that:

[0049] i. On the first day of storage, there was no significant difference in sperm motility between each group and the blank control group (P>0.05).

[0050] ii. On the third day of storage, there was no significant difference in sperm motility between the Example 4-5 groups and the blank control group (P>0.05). However, there was a significant difference in sperm motility between the blank control group and the Example 1-3 groups (P<0.05), indicating that adding a certain concentration of puerarin has a protective effect on sperm motility; there was a significant difference in sperm motility between the Example 1 group and the Example 3 group (P<0.05), indicating that adding different concentrations of puerarin also has a certain difference in sperm motility.

[0051] iii. On the fifth day of storage, there was a significant difference in sperm motility between the blank control group and the Example 2-4 groups (P < 0.05). At the same time, the blank control group was compared with the Example 1-4 groups, which proved that different concentrations of puerarin had an effect on sperm motility, and the effect of low concentrations of puerarin gradually weakened over time.

[0052] iv. On the seventh day of storage, there was a significant difference in sperm motility between the blank control group and the Example 3-5 groups (P < 0.05), and there was no significant difference in sperm motility between Example 2 and the blank control group and the other Example groups (P > 0.05), indicating that the puerarin concentration of 25 μmol / L after seven days of storage was the dividing point between the blank group and the puerarin group.

[0053] The applicant found that when the concentration of puerarin was greater than 25 μmol / L, sperm motility was significantly higher than that of the blank control group and other example groups from the third day to the seventh day (P < 0.05). Therefore, the optimal concentration of puerarin was 50 μmol / L.

[0054] Based on the above conclusions, the applicant used the room temperature storage preparation of pig semen obtained in Example 3 as the test group, and compared it with the blank group (diluent, without puerarin) by the following measurements, with 3 replicates for each group.

[0055] a. Determination of total antioxidant capacity (T-AOC): add 200 μL phosphate buffered saline (PBS) to the sperm collected at 3, 5, and 7 days old (>1 million), and fully break the homogenate to break the antioxidants in the sperm. Centrifuge at 12,000 r / min for 5 minutes, and take the supernatant for determination. The prepared ABTS is oxidized into green ABTS under the action of oxidants. When antioxidants in sperm exist, the production of ABTS will be inhibited. The absorbance of ABTS at 405 nm can be measured and calculated to determine the total antioxidant capacity of sperm.

[0056] like Figure 1 As shown in the figure, with the extension of storage time, the T-AOC capacity of semen continued to decrease. On the third day, the total antioxidant capacity of the blank group was slightly higher than that of the test group, which may be due to the strong total antioxidant capacity of the original semen. After five days of storage, the T-AOC capacity of the test group was slightly higher than that of the blank group, and the difference was still not significant (P>0.05). However, on the seventh day, the T-AOC capacity of the test group was higher than that of the blank group, and the difference was significant (P<0.05).

[0057] b. Determination of reactive oxygen species (ROS): The sperm collected on days 3, 5, and 7 (>1 million) were washed repeatedly 2-3 times with 0.01 mol / L phosphate buffered saline (PBS), centrifuged at 1000 r / min for 5 min, the supernatant was aspirated, and the cell pellet was retained for determination. The probe (DCFH-DA) was added according to the sample concentration determined in the pre-experiment, and the sperm was incubated at 37°C for 0.5-3 h, mixed every 3-5 min, centrifuged again at 1000 r / min for 5 min, the supernatant was aspirated, and the precipitate was collected for fluorescence determination. When there is reactive oxygen in sperm, DCFH is oxidized to a strong green fluorescent substance DCF, with a maximum peak at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Its fluorescence intensity is proportional to the reactive oxygen in the sperm.

[0058] like Figure 2 As shown in the figure, with the extension of storage time, the content of ROS shows an upward trend, which is in stark contrast to the change in T-AOC capacity in semen. As the storage time of semen increases, oxidative metabolism in sperm produces more and more ROS. Excessive ROS has a damaging effect on sperm capacitation, fertility and cell membrane. Therefore, reducing the production of ROS is one of the important factors to prolong sperm motility.

[0059] As time went on, the ROS content in sperm stored for three and five days gradually increased. The ROS level in the experimental group was lower than that in the blank group (P>0.05). On the seventh day, the ROS content in the experimental group was significantly reduced compared with the blank group (P<0.05), indicating that the growth rate of ROS in the added experimental group slowed down on the seventh day of storage, confirming that puerarin (50μmol / L) has a significant scavenging effect on ROS production in sperm.

[0060] c. Determination of ATP: The sperm collected at 3, 5, and 7 days (>1 million) were centrifuged at 1000r / min for 5 minutes, the supernatant was aspirated, and the cell pellet was added with 300-500μL double distilled water, and the pellet was homogenized in a 90℃ water bath. The cell suspension was heated in boiling water for 10 minutes, taken out, mixed, and extracted for 1 minute for determination. Creatine kinase catalyzes adenosine triphosphate and creatine to generate phosphocreatine, which was determined by the phosphomolybdic acid colorimetric method.

[0061] like Figure 3 As shown in the figure, the ATP level in semen decreases with the extension of semen storage time. On the third and fifth days of semen storage, the ATP level in the experimental group was always higher than that in the blank group. On the seventh day of storage, the ATP level in the experimental group was significantly higher than that in the blank group, and the difference was significant (P < 0.05). This shows that puerarin (50 μmol / L) has a significant effect on improving the ATP level in semen.

[0062] Example 6

[0063] The preparation for storing pig semen at room temperature is composed of puerarin, formononetin, aurantin and the above diluent, wherein the concentration of puerarin is 50 μmol / L, the concentration of formononetin is 5 μmol / L, and the concentration of aurantin is 10 μmol / L.

[0064] Comparative Example

[0065] The preparation for storing pig semen at room temperature is composed of formononetin, aurantin and the above diluent, the concentration of formononetin is 5 μmol / L, and the concentration of aurantin is 10 μmol / L.

[0066] According to the above measurement results, as time goes by, the difference between the groups will be more significant. Therefore, based on the previous results, the applicant used the pig semen room temperature storage preparations obtained in Example 3, Example 6 and the comparative example to store them under the same conditions for 7 days, and each group was set up with 3 replicates. After 7 days, sperm motility test, total antioxidant capacity (T-AOC) determination, reactive oxygen species (ROS) determination, and ATP determination were carried out, and the results were as follows: Figure 4 and Figure 5 shown.

[0067] pass Figure 4 and Figure 5 It can be seen that after 7 days of storage, the antioxidant performance of pig sperm in Example 3 group is better than that of the control group, and the levels of Example 6 are all better than the other two groups. It shows that the use of puerarin, formononetin and cassia bark in combination for semen storage at room temperature can synergistically enhance the antioxidant capacity of pig sperm, have a significant scavenging effect on the ROS production of sperm, and increase semen ATP, thereby prolonging the vitality of pig sperm during storage at room temperature.

[0068] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A preparation for storing pig semen at room temperature, characterized in that: The raw materials include: puerarin with a concentration of 0.1-100 μmol / L and a diluent with a pH value of 6.5-7.3; Its raw materials also include: formononetin and aurantin, the concentration of formononetin is 4-6 μmol / L, and the concentration of aurantin is 13-17 μmol / L; The components of the diluent include: glucose, ethylenediaminetetraacetic acid, sodium citrate, sodium bicarbonate, tris(hydroxymethylaminomethane), citric acid, cysteine, bovine serum albumin, kanamycin sulfate and water; wherein the mass fraction of glucose is 5-7 g / 100 mL, the mass fraction of ethylenediaminetetraacetic acid is 0.4-0.5 g / 100 mL, the mass fraction of sodium citrate is 1.2-1.6 g / 100 mL, the mass fraction of sodium bicarbonate is 0.1-0.3 g / 100 mL, the mass fraction of tris(hydroxymethylaminomethane) is 0.8-1.2 g / 100 mL, the mass fraction of citric acid is 0.4-0.6 g / 100 mL, the mass fraction of cysteine ​​is 0.005-0.015 g / 100 mL, the mass fraction of bovine serum albumin is 0.6-1.0 g / 100 mL, and the mass fraction of kanamycin sulfate is 0.04-0.08 g / 100 mL.

2. The pig semen room temperature storage preparation according to claim 1, characterized in that: The mass ratio of glucose, ethylenediaminetetraacetic acid, sodium citrate, sodium bicarbonate, tris(hydroxymethyl)aminomethane), citric acid, cysteine, bovine serum albumin and kanamycin sulfate is 6:0.45:1.38:0.5:1:0.5:0.01:0.8:0.

06.

3. The pig semen room temperature storage preparation according to claim 1, characterized in that: The concentration of puerarin was 50 μmol / L.

4. A method for using the pig semen room temperature storage preparation according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: placing a boar semen stock solution in a constant temperature environment of 17°C, adding the boar semen room temperature preservation preparation described in any one of claims 1 to 3 to dilute the solution at a volume ratio of 1:4-9, wherein the temperature difference between the boar semen stock solution and the boar semen room temperature preservation preparation during the dilution process is less than or equal to 1°C; and mixing once every 12 hours during the preservation process.

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