Cryopreservation agent for goat semen as well as preparation method and application of cryopreservation agent

Abstract

The invention belongs to the technical field of artificial insemination of milk goats, and particularly relates to a cryopreservation agent for goat semen, a preparation method and application. According to the cryopreservation agent for the goat semen, the egg yolk, the glycerinum and the idebenone are all cryoprotectants, the egg yolk is crucial for preventing sperms from being damaged by freezing, the glycerinum serves as a permeable protective agent, formation of ice crystals can be reduced, damage of free radicals to cells can be relieved, the permeability of cell membranes can be changed, and the effect of preventing the sperms from being damaged by freezing is achieved. The sperm activity is improved. As an antioxidant, idebenone effectively improves semen activity, plasma membrane integrity, acrosome integrity and fertilization ability after freeze thawing, significantly improves oxidation resistance, reduces ROS level, further reduces lipid peroxide generation, reduces cell membrane damage, obviously improves milk goat sperm cryopreservation effect, and improves sperm activity. The preparation method of the cryopreservation agent is simple, and the cryopreservation operation method is simple.

Description

Technical Field

[0001] The present invention belongs to the technical field of artificial insemination of dairy goats, and specifically relates to a cryopreservative for goat semen, a preparation method and an application thereof. Background Art

[0002] The popularization of artificial insemination technology for dairy goats can give full play to the genetic potential of male animals. Semen cryopreservation is an important part of artificial insemination. Semen cryopreservation can break through the limitations of time and space for the exchange of high-quality germplasm resources, and at the same time avoid the death loss caused by the long-distance transportation of animals, as well as the input of human and material resources and epidemic prevention costs, greatly reducing the cost of germplasm exchange in the breeding process. At the same time, semen cryopreservation combined with technologies such as estrus synchronization, artificial insemination, and embryo transfer can more reasonably arrange breeding plans and improve the efficiency of animal reproduction and breed improvement. Semen cryopreservation technology has important practical significance for the sustainable development of the livestock industry and the supply of livestock products.

[0003] Cryopreservation will cause a significant decrease in sperm motility, viability, plasma membrane integrity rate, acrosome integrity rate, mitochondrial activity and DNA integrity rate in goat semen, resulting in the inability to maintain fertilization ability, and then reducing the pregnancy rate and birth rate. Therefore, it is very necessary to improve the basic cryopreservative to improve the quality of frozen-thawed sperm and fertilization ability. Summary of the Invention

[0004] In view of the problems existing in the above-mentioned prior art, the present invention provides a cryopreservative for goat semen, which improves the quality and fertilization ability of frozen-preserved dairy goat semen.

[0005] In order to solve the above technical problems, the following technical solutions are proposed:

[0006] The present invention provides a cryopreservative for goat semen, comprising: a normal temperature diluent and a cryoprotective substance;

[0007] The cryoprotective substance includes egg yolk, glycerol and idebenone; the composition of the normal temperature diluent includes: D-fructose, tris(hydroxymethyl)aminomethane, citric acid, streptomycin sulfate and sodium ampicillin.

[0008] Preferably, the normal temperature diluent uses ultrapure water as a solvent, and the composition includes: 8-12 g / L of D-fructose, 25-30 g / L of tris(hydroxymethyl)aminomethane, 12-16 g / L of citric acid, 0.1-0.3 g / L of streptomycin sulfate and 0.1-0.3 g / L of sodium ampicillin.

[0009] Preferably, the cryopreservative is divided into cryopreservative Ⅰ and cryopreservative Ⅱ;

[0010] The cryopreservative Ⅰ includes a first room-temperature diluent and a first cryoprotective substance; the first cryoprotective substance includes egg yolk and idebenone; based on the volume of the first room-temperature diluent, the added amount of egg yolk in the cryopreservative Ⅰ accounts for 10% - 20% of its volume, and the final concentration of idebenone is greater than 0 and less than 10 μM;

[0011] The cryopreservative Ⅱ includes a second room-temperature diluent and a second cryoprotective substance; the second cryoprotective substance includes egg yolk, glycerol and idebenone; based on the volume of the second room-temperature diluent, the added amount of egg yolk in the cryopreservative Ⅱ accounts for 10% - 20% of its volume, the added amount of glycerol accounts for 4% - 6% of its volume, and the final concentration of idebenone is greater than 0 and less than 10 μM.

[0012] The present invention provides a preparation method of the cryopreservative described in the above technical solution, including: mixing the room-temperature diluent and the cryoprotective substance to obtain the cryopreservative.

[0013] The present invention provides the application of the cryopreservative described in the above technical solution or the cryopreservative prepared by the preparation method described in the above technical solution in improving the quality and / or antioxidant capacity of goat semen.

[0014] Preferably, the goat includes a dairy goat.

[0015] The present invention provides a method for preserving goat semen, including the following steps:

[0016] Mixing and equilibrating the cryopreservative Ⅰ with goat semen to obtain an equilibration solution;

[0017] Mixing and performing a second equilibration of the cryopreservative Ⅱ with the equilibration solution to obtain a second equilibration solution;

[0018] Performing cryopreservation on the second equilibration solution; the cryopreservative Ⅰ is the cryopreservative Ⅰ described in the above technical solution; the cryopreservative Ⅱ is the cryopreservative Ⅱ described in the above technical solution.

[0019] Preferably, the mixing volume ratio of the cryopreservative Ⅰ to goat semen is 1:3 - 5;

[0020] The temperature of the equilibration is 4°C, and the time of the equilibration is 2 - 3 h;

[0021] The mixing method of the cryopreservative Ⅱ and the equilibration solution includes equal-volume and isothermal mixing; the temperature of the second equilibration is 4°C, and the time of the second equilibration is 2 - 3 h;

[0022] Preferably, the cryopreservation method includes liquid nitrogen preservation.

[0023] The present invention provides a thawing method for frozen semen obtained by the preservation method described in the above technical solution, including:

[0024] After taking out the frozen semen, warm water thawing and water bath incubation are carried out in sequence. The temperature of the warm water thawing is 37-40 °C and the time is 25 s; the temperature of the water bath incubation is 37 °C and the time is 3-5 min.

[0025] Beneficial effects:

[0026] The present invention provides a cryopreservative for goat semen. The cryopreservative can play a role in protecting sperm during the freezing process. D-fructose in the normal temperature diluent can supplement the energy consumed by physiological activities such as sperm glycolysis and oxidative phosphorylation, and extend the sperm preservation time; tris(hydroxymethyl)aminomethane (Tris) can maintain the pH value of the diluent stable and help sperm maintain morphological function; citric acid is non-toxic and has good pH regulation, can regulate and stabilize the pH value of the diluent, and maintain sperm motility; streptomycin sulfate and sodium ampicillin are antibiotics, which can effectively kill the germs harmful to sperm cells in the diluent; the egg yolk, glycerol and idebenone are all cryoprotective substances. The combination of the three can not only achieve cryopreservation but also inhibit peroxidative damage during cryopreservation. The three are crucial for sperm to avoid cryodamage: lecithin and unsaturated fatty acids in the egg yolk can reduce the action of free radicals, stabilize the cell membrane, and have the effects of promoting lipoprotein synthesis and improving sperm metabolism. Glycerol, as a permeating cryoprotectant, can reduce ice crystal formation, reduce the damage of free radicals to cells, change the cell membrane permeability, and provide sperm activity. Idebenone, as an antioxidant, can effectively improve the semen motility, plasma membrane integrity, acrosome integrity and fertilization ability after freezing and thawing, significantly improve the antioxidant performance, reduce the ROS level, thereby reducing the generation of lipid peroxides, reducing cell membrane damage, significantly improving the cryopreservation effect of dairy goat sperm, and improving sperm activity.

[0027] During the freezing process, sperm are prone to intracellular metabolic disorders, DNA damage, protein structure changes, mitochondrial function impairment, cytoplasmic instability and excessive generation of reactive oxygen species (ROS) free radicals caused by factors such as ice crystal formation and oxidative stress, reducing the conception rate of semen. However, the cryopreservative of the present invention can inhibit peroxidative damage during cryopreservation, is applicable to the field of long-term cryopreservation of dairy goat semen in liquid nitrogen and artificial insemination. The preparation method of the cryopreservative is simple, the cryopreservation operation method is simple, and it is easy to promote, significantly improving the quality and fertilization ability of dairy goat semen after freezing and thawing. Description of the drawings

[0028] Figure 1 Effects of different concentrations of Ide on the tail plasma membrane integrity rate (MembraneIntegrity%) of dairy goat semen after freezing and thawing;

[0029] Figure 2 Effect of Ide at different concentrations on the acrosome integrity rate (Acrosome Integrity%) of frozen-thawed semen of dairy goats;

[0030] Figure 3 Effect of Ide at different concentrations on the total antioxidant capacity (T-AOC) of frozen-thawed semen of dairy goats;

[0031] Figure 4 Effect of Ide at different concentrations on the activity of total superoxide dismutase (T-SOD) in frozen-thawed semen of dairy goats;

[0032] Figure 5 Effect of Ide at different concentrations on the activity of glutathione peroxidase (GSH-Px) in frozen-thawed semen of dairy goats;

[0033] Figure 6 Effect of Ide at different concentrations on the content of malondialdehyde (MDA) in frozen-thawed semen of dairy goats;

[0034] Figure 7 Detection diagram of sperm plasma membrane integrity in thawed semen;

[0035] Figure 8 Fluorescent staining diagram of sperm nucleus in thawed semen;

[0036] Figure 9 Fluorescent staining diagram of sperm acrosome in thawed semen;

[0037] Figure 10 For Figure 8 and Figure 9 Combined diagram. Specific implementation mode

[0038] The present invention provides a cryopreservative for goat semen, comprising: a room temperature diluent and a cryoprotective substance;

[0039] The cryoprotective substance includes egg yolk, glycerol and idebenone; the composition of the room temperature diluent includes: D-fructose, Tris, citric acid, streptomycin sulfate and sodium ampicillin.

[0040] As an optional implementation mode, the cryopreservative of the present invention is divided into cryopreservative I and cryopreservative II.

[0041] The cryopreservative I of the present invention includes a first room temperature diluent and a first cryoprotective substance; the first cryoprotective substance includes egg yolk and idebenone; as an optional implementation mode, based on the volume of the first room temperature diluent, the addition amount of egg yolk in the cryopreservative I accounts for 10% - 20% of its volume, more preferably 15%, and the final concentration of idebenone is greater than 0 and less than 10 μM, and can also be 2 - 6 μM.

[0042] The cryopreservative II of the present invention comprises a second room-temperature diluent and a second cryoprotective substance; the second cryoprotective substance comprises egg yolk, glycerol and idebenone. As an alternative embodiment, based on the volume of the second room-temperature diluent, the addition amount of egg yolk in the cryopreservative II accounts for 10% to 20% of its volume, more preferably 15%; the addition amount of glycerol accounts for 4% to 6% of its volume, more preferably 5%; the final concentration of idebenone is greater than 0 and less than 10 μM, and may also be 2 to 6 μM. As an alternative embodiment, the idebenone is dissolved in DMSO and added after obtaining an idebenone solution.

[0043] As an alternative embodiment, the room-temperature diluent of the present invention uses ultrapure water as a solvent, and its composition includes: 8 to 12 g / L of D-fructose, 25 to 30 g / L of Tris, 12 to 16 g / L of citric acid, 0.1 to 0.3 g / L of streptomycin sulfate, and 0.1 to 0.3 g / L of sodium ampicillin; the concentration of D-fructose in the room-temperature diluent of the present invention is 8 to 12 g / L, more preferably 10 g / L; the concentration of Tris in the room-temperature diluent is 25 to 30 g / L, more preferably 27 g / L; the concentration of citric acid in the room-temperature diluent is 12 to 16 g / L, more preferably 14 g / L; the concentration of streptomycin sulfate in the room-temperature diluent is 0.1 to 0.3 g / L, more preferably 0.2 g / L; the concentration of sodium ampicillin in the room-temperature diluent is 0.1 to 0.3 g / L, more preferably 0.2 g / L. D-fructose in the room-temperature diluent provides an energy source as a semen cryopreservative, which is essential, and can supplement the consumption caused by physiological activities such as sperm glycolysis and oxidative phosphorylation, and extend the sperm preservation time; Tris can maintain the stability of the diluent pH, and citric acid can adjust the diluent pH to improve sperm motility; streptomycin sulfate can inhibit bacterial protein synthesis, especially having a strong antibacterial effect on Mycobacterium tuberculosis; sodium ampicillin has a broad antibacterial spectrum and has a good antibacterial effect on Gram-positive bacilli, Gram-negative bacteria, and Gram-positive cocci. With the specific concentration ratio of D-fructose, Tris, citric acid, streptomycin sulfate, and sodium ampicillin, an appropriate environment with sufficient energy substances, stable osmotic pressure, moderate pH value, and low harmful pathogen concentration in the diluent is achieved to provide a suitable environment for sperm cell survival.

[0044] The cryopreservative prepared by the present invention can play a role in protecting sperm during the freezing process. Among them, egg yolk can protect sperm from freezing damage. Lecithin and unsaturated fatty acids in egg yolk can also reduce the damage of free radicals during cryopreservation, achieving the effects of stabilizing cell membranes, promoting lipoprotein synthesis, and improving sperm metabolism. Glycerol can reduce the amount of ice crystal formation during the freezing process, reduce the damage of free radicals to cells, and reduce cell damage at an appropriate concentration. At the same time, glycerol is also a permeating protective agent that can change the permeability of cell membranes, enter cells, and maintain the osmotic balance inside and outside cells, thereby improving the activity of sperm. Idebenone can effectively improve semen quality, especially sperm motility, viability, and acrosome integrity rate, and maintain a high conception rate. Egg yolk, glycerol, and idebenone cooperate with each other to reduce oxidative damage during the cryopreservation of goat sperm, improve the preservation quality of semen, and significantly improve the semen quality and fertilization ability of dairy goats after thawing under the combined action of D-fructose, Tris, citric acid, streptomycin sulfate, sodium ampicillin, egg yolk, glycerol, and idebenone.

[0045] The present invention has no special limitation on the sources of the egg yolk, glycerol, idebenone, D-fructose, Tris, citric acid, streptomycin sulfate, and sodium ampicillin, and conventional commercially available products can be used.

[0046] The present invention provides a preparation method of the cryopreservative described in the above technical solution, including: mixing the normal-temperature diluent and cryoprotective substances to obtain a cryopreservative. The present invention has no special limitation on the mixing method, and conventional methods can be used.

[0047] The present invention provides the application of the cryopreservative described in the above technical solution or the cryopreservative prepared by the preparation method described in the above technical solution in improving the quality and antioxidant capacity of goat semen. As an optional implementation manner, the goats in the present invention include dairy goats. In a specific embodiment of the present invention, the semen of Saanen dairy goats is used for effect verification. As an optional implementation manner, the application method includes: mixing and equilibrating the cryopreservative Ⅰ described in the above technical solution with goat semen to obtain an equilibration solution; mixing and performing a second equilibration on the cryopreservative Ⅱ described in the above technical solution with the equilibration solution to obtain a second equilibration solution; encapsulating the second equilibration solution in a freezing straw for cryopreservation. The equilibration and the second equilibration are achieved by static placement. After the cryopreservative Ⅰ of the present invention is prepared, it is preferably equilibrated at 37 °C to mix and dilute the cryopreservative Ⅰ and goat semen at the same temperature, reducing the stimulation received by sperm during the dilution process to obtain a first equilibration solution; the cryopreservative Ⅱ of the present invention is prepared and equilibrated at 4 °C to isothermally and isovolumetrically mix and dilute the cryopreservative Ⅱ and the first equilibration solution at 4 °C, reducing the damage received by sperm due to excessive temperature change during the dilution process and improving sperm motility.

[0048] As an alternative embodiment, improving the quality of goat semen according to the present invention includes one or more of the following 1) to 7): 1) increasing the sperm motility rate and vitality of goat semen; 2) increasing the average curvilinear velocity of sperm in goat semen; 3) increasing the average straight-line velocity of sperm in goat semen; 4) increasing the average path velocity of sperm in goat semen; 5) increasing the average moving angle of sperm in goat semen; 6) increasing the integrity rate of the sperm tail plasma membrane of dairy goats; 7) the integrity rate of sperm acrosomes after freeze-thawing of goat semen. The sperm vitality described in the present invention is the percentage of sperm moving linearly under a microscope among the sperm in the field of view. The sperm motility rate described in the present invention is the percentage of sperm with motility ability in the semen.

[0049] As an alternative embodiment, improving the antioxidant capacity of goat semen according to the present invention includes one or more of the following 1) to 4): 1) increasing the total antioxidant capacity of goat semen; 2) increasing the total superoxide dismutase activity of goat semen; 3) increasing the glutathione peroxidase activity of goat semen; 4) reducing the content of malondialdehyde in goat semen.

[0050] The cryopreservative of the present invention can increase the sperm motility rate and vitality, average curvilinear velocity, average straight-line velocity, average path velocity and average moving angle of sperm in goat semen, increase the integrity rate of the sperm tail plasma membrane of dairy goats, the integrity rate of sperm acrosomes after freeze-thawing of goat semen, and increase the conception rate; the cryopreservative of the present invention can also increase the total antioxidant capacity, total superoxide dismutase activity, glutathione peroxidase activity of goat semen and reduce the malondialdehyde content; the conception rate of goat semen preserved by the cryopreservative of the present invention is high.

[0051] The present invention provides a method for preserving goat semen, which includes the following steps: mixing and equilibrating the cryopreservative Ⅰ described in the above technical solution with goat semen to obtain an equilibrium solution; mixing and second-equilibrating the cryopreservative Ⅱ described in the above technical solution with the equilibrium solution to obtain a second equilibrium solution; and cryopreserving the second equilibrium solution.

[0052] As an optional embodiment, the present invention mixes the cryopreservative I with goat semen in a volume ratio of 1:3-5, more preferably 1:4; the mixing temperature is 37°C, and the mixing temperature and volume ratio can reduce the stress on sperm cells during the mixing of the diluent and semen, and reduce the damage to sperm in this process. The equilibrium temperature of the present invention is 4°C, and the equilibrium time is 2-3h, or 2h; the equilibrium temperature and time can slowly cool the sperm cells to 4°C, gradually adapt to the low temperature environment, reduce the damage to sperm during the cooling process, and provide the freezing process after early adaptation to the low temperature environment. After the cryopreservative I of the present invention is prepared, it is preferably placed at 37°C for equilibrium, so that the cryopreservative I and goat semen are mixed and diluted at the same temperature, reducing the stimulation to the sperm during the dilution process, and obtaining a first equilibrium solution; the cryopreservative II of the present invention is prepared and balanced at 4°C, so that the cryopreservative II and the first equilibrium solution are isothermally and equal-volume mixed and diluted at 4°C, reducing the damage to the sperm due to excessive temperature changes during the dilution process, and improving the sperm motility.

[0053] As an optional embodiment, the mixing method of the cryopreservative II and the balancing solution of the present invention includes equal volume and isothermal mixing; the temperature of the second balancing solution is 4°C, and the time of the second balancing solution is 2 to 3 hours; the temperature and time of the second balancing solution can gradually adapt the sperm to the low temperature environment. The reason why cryopreservative I and cryopreservative II are divided into cryopreservative I and cryopreservative II for gradual dilution with goat semen is that the damage to the goat semen during the dilution process can be reduced and the sperm motility can be improved.

[0054] As an optional embodiment, the present invention encapsulates the second balance liquid in a frozen sperm tube for cryopreservation. In a specific embodiment of the present invention, the specification of the frozen sperm tube used is 0.25 mL. As an optional embodiment, the method of cryopreservation described in the present invention includes liquid nitrogen preservation. In the present invention, the temperature of the cryopreservation is preferably -196°C. As an optional embodiment, before liquid nitrogen preservation, the present invention fumigates the frozen sperm tube containing semen 4 cm above the liquid nitrogen surface for 15 minutes, and then puts it into a canvas bag. Liquid nitrogen fumigation is simple to operate and easy to use. Putting it into a canvas bag can effectively distinguish and package semen samples from different rams.

[0055] The present invention provides a method for thawing frozen semen obtained by the above-described cryopreservation method, including: taking out the frozen semen and sequentially performing warm water thawing and water bath incubation. The temperature of the warm water thawing in the present invention is 37-40°C, or can be 38-39°C. The time of the warm water thawing in the present invention is 25 s; the temperature of the water bath incubation in the present invention is 37°C; the time of the water bath incubation is 3-5 min, or can be 4 min. The warm water thawing and the water bath incubation can better resuscitate the sperm in the semen sample to be thawed. The thawing method of the present invention is a better choice explored through multiple practices, rather than a limitation on the method for thawing semen samples.

[0056] Cryopreservation can inhibit sperm motility, reduce energy consumption and metabolic activities, and make life temporarily in a static state to achieve the purpose of extending the sperm preservation time. The present invention provides a cryopreservative that can simultaneously improve the quality and fertilization ability of cryopreserved dairy goat semen. Compared with the prior art, the present invention adds idebenone, an antioxidant with a concentration of 6 μM, to the semen cryopreservative, effectively reducing the generation of lipid peroxides, reducing cell membrane damage, improving the motility, plasma membrane integrity, acrosome integrity and fertilization ability of frozen-thawed semen, significantly improving the antioxidant performance, reducing the ROS level, and increasing sperm activity; combined with the protective effects of egg yolk and glycerol, the three together can significantly improve the cryopreservation effect of dairy goat sperm. The technical solution of the present invention is applicable to the long-term cryopreservation of dairy goat semen in liquid nitrogen and the field of artificial insemination. The preparation method of the cryopreservative of the present invention is simple, the cryopreservation operation method is simple, and it is easy to promote, and can significantly improve the quality and fertilization ability of dairy goat semen after freezing and thawing.

[0057] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0058] The data analysis method for the following examples:

[0059] Graphpad Prism 10.1.2 was used to plot and analyze the data. SPSS 26.0 was used to perform one-way analysis of variance (ANOVA) on all data according to LSD (Least Significance Difference) to detect the significance of the results, and multiple t-tests were used to evaluate the differences between groups. Different letters in the subscripts indicate significant differences (P<0.05), and the same letters indicate no significant differences (P>0.05). The data in Table 1 are expressed as mean ± standard deviation.

[0060] In the examples, the cryodiluents are the cryopreservatives. In Example 1, the cryodiluent I is the cryopreservative I. In Example 2, the cryodiluent II given is the cryopreservative II.

[0061] Example 1

[0062] 1 Test materials

[0063] 1.1 Test animals: The test rams were provided by the Longxian (Heshuǐ) Dairy Goat Test and Demonstration Base. Ten healthy, strong, good in physical appearance and reproductive function Saanen dairy goats aged 3 to 5 years were selected.

[0064] 2 Test methods

[0065] 2.1 Preparation of freezing diluent

[0066] 2.1.1 Formulation of freezing diluent Ⅰ:

[0067] The preparation method of the normal temperature diluent is as follows: 10 g of D-fructose, 27 g of Tris, 14 g of citric acid, 0.2 g of streptomycin sulfate and 0.2 g of sodium ampicillin were dissolved in 1 L of pure water.

[0068] Idebenone was dissolved with DMSO to obtain an idebenone solution.

[0069] Freezing diluent Ⅰa: Add 15% of the volume of egg yolk to the normal temperature diluent, and do not add the idebenone (ldebenone, abbreviated as Ide) solution, and mix well for use.

[0070] Freezing diluent Ⅰb: Add 15% of the volume fraction of egg yolk to the normal temperature diluent, and the final concentration of idebenone is 2 μM, and mix well for use.

[0071] Freezing diluent Ⅰc: Add 15% of the volume fraction of egg yolk to the normal temperature diluent, and the final concentration of idebenone is 4 μM, and mix well for use.

[0072] Freezing diluent Ⅰd: Add 15% of the volume fraction of egg yolk to the normal temperature diluent, and the final concentration of idebenone is 6 μM, and mix well for use.

[0073] Freezing diluent Ⅰe: Add 15% of the volume fraction of egg yolk to the normal temperature diluent, and the final concentration of idebenone is 8 μM, and mix well for use.

[0074] Freezing diluent Ⅰf: Add 15% of the volume fraction of egg yolk to the normal temperature diluent, and the final concentration of idebenone is 10 μM, and mix well for use.

[0075] 2.1.2 Formulation of freezing diluent Ⅱ:

[0076] Cryodiluents Ⅱa: Add 15% (by volume) of egg yolk and 5% (by volume) of glycerol to the room-temperature diluent, without adding idebenone solution, and mix well for use.

[0077] Cryodiluents Ⅱb: Add 15% (by volume) of egg yolk, 5% (by volume) of glycerol, and idebenone with a final concentration of 2 μM to the room-temperature diluent, and mix well for use.

[0078] Cryodiluents Ⅱc: Add 15% (by volume) of egg yolk, 5% (by volume) of glycerol, and idebenone with a final concentration of 4 μM to the room-temperature diluent, and mix well for use.

[0079] Cryodiluents Ⅱd: Add 15% (by volume) of egg yolk, 5% (by volume) of glycerol, and idebenone with a final concentration of 6 μM to the room-temperature diluent, and mix well for use.

[0080] Cryodiluents Ⅱe: Add 15% (by volume) of egg yolk, 5% (by volume) of glycerol, and idebenone with a final concentration of 8 μM to the room-temperature diluent, and mix well for use.

[0081] Cryodiluents Ⅱf: Add 15% (by volume) of egg yolk, 5% (by volume) of glycerol, and idebenone with a final concentration of 10 μM to the room-temperature diluent, and mix well for use.

[0082] 2.1.3 Before collecting semen, pre-equilibrate the prepared Cryodiluents Ⅰ in a 37 °C water bath and Cryodiluents Ⅱ in a 4 °C refrigerator for use.

[0083] 2.2 Semen Collection and Processing

[0084] Method for determining sperm motility (visual observation and counting): Adjust the thermostatic heating microscope to 37 °C, use a pipette to take 10 μL of semen and drop it on a glass slide. After covering the glass slide, observe five fields of view at the four corners and the center of the glass slide under the microscope. The proportion of sperm moving straight forward in the field of view to all sperm in the field of view is the sperm motility.

[0085] Collect semen from Saanen dairy goats using the artificial vagina method, and keep the collected semen in a 37 °C thermostatic cup and bring it to the laboratory for testing within 1 h. Samples with sperm motility above 0.80 (i.e., 80%) and normal color and odor are used for the experiment.

[0086] 2.3 Dilution and Cryopreservation of Dairy Goat Semen

[0087] 0 μM group (denoted as A): Mixing and dilution were completed using a freezing capillary tube with a specification of 0.25 mL. The semen of dairy goats was isothermally diluted to 1:4 at 37 °C with freezing diluent Ⅰa, wrapped with multiple layers of gauze and absorbent cotton, and equilibrated in a 4 °C refrigerator for 2 - 3 h, then slowly cooled to 4 °C; it was isothermally and equally volumetrically diluted with freezing diluent Ⅱa in a 4 °C environment and the semen after the previous equilibration. After thoroughly mixing the diluted semen, it was equilibrated again in a 4 °C refrigerator for 2 h; after the equilibration ended, it was aspirated into the freezing capillary tube and sealed; the freezing capillary tube containing the semen of dairy goats was placed 4 cm above the liquid nitrogen surface for fumigation for 15 min, and then put into a canvas bag and immersed in liquid nitrogen for storage.

[0088] 2 μM group (denoted as B): The same as the 0 μM group, the only difference is that freezing diluent Ⅰa was replaced with freezing diluent Ⅰb, and freezing diluent Ⅱa was replaced with freezing diluent Ⅱb.

[0089] 4 μM group (denoted as C): The same as the 0 μM group, the only difference is that freezing diluent Ⅰa was replaced with freezing diluent Ⅰc, and freezing diluent Ⅱa was replaced with freezing diluent Ⅱc.

[0090] 6 μM group (denoted as D): The same as the 0 μM group, the only difference is that freezing diluent Ⅰa was replaced with freezing diluent Ⅰd, and freezing diluent Ⅱa was replaced with freezing diluent Ⅱd.

[0091] 8 μM group (denoted as E): The same as the 0 μM group, the only difference is that freezing diluent Ⅰa was replaced with freezing diluent Ⅰe, and freezing diluent Ⅱa was replaced with freezing diluent Ⅱe.

[0092] 10 μM group (denoted as F): The same as the 0 μM group, the only difference is that freezing diluent Ⅰa was replaced with freezing diluent Ⅰf, and freezing diluent Ⅱa was replaced with freezing diluent Ⅱf.

[0093] 2.4 Quality evaluation of frozen semen of dairy goats

[0094] 2.4.1 Thawing of semen

[0095] After 2 months of cryopreservation, the semen of Saanen dairy goats in groups A, B, C, D, E, and F was thawed. The specific thawing steps were as follows: Place a sufficient number of 2 mL sterilized centrifuge tubes in a 37 °C water bath. Then, take out the canvas bags containing the frozen capillary tubes of groups A, B, C, D, E, and F from the liquid nitrogen tank, and immerse the canvas bags in a foam box filled with liquid nitrogen. Perform the operation inside the foam box: Use forceps to take out the frozen capillary tubes from the canvas bag, quickly put the frozen capillary tubes into a 37 °C constant temperature cup for thawing for 25 s, take them out, dry the moisture on the surface of the frozen capillary tubes, cut off one side of the sealed part of the frozen capillary tube, and slowly transfer the semen into the centrifuge tube placed in the water bath in advance, and incubate for 3 - 5 min for use.

[0096] 2.4.2 Detection of sperm motility performance

[0097] The sperm computer - aided analysis system (CASA) was used for analysis. First, the temperature of the microscope was set at 37 °C. Then, the glass slide was pre - heated for a few seconds. Next, 10 μL of thawed semen sample was dropped onto it, and a coverslip was quickly covered to prepare for microscopic examination. Through the CASA system, sperm motility parameters such as sperm motility rate (TM, %), sperm motility (PM, %), average curvilinear velocity (VCL, μm / s), average straight - line velocity (VSL, μm / s), average path velocity (VAP, μm / s), average amplitude of lateral head displacement (ALH, μm), and average angle of sperm movement (MAD, °) could be measured. The effects of cryodiluents Ⅰ with different concentrations of idebenone (Ide) on the motility rate, motility, and motility performance of frozen - thawed semen of dairy goats are shown in Table 1.

[0098] Table 1 Effects of idebenone on the motility rate, motility, and motility performance of thawed sperm

[0099]

[0100]

[0101] Note: When presenting data, the superscript letters were used to indicate the statistical differences between different groups. If there are no identical letters in the superscript letters of the data in the same column for different groups, it means that the statistical differences between them are significant (P < 0.05). On the contrary, if there are identical letters in the superscript letters of the data, it indicates that the statistical differences between these groups are not significant (P > 0.05).

[0102] According to Table 1, when 2 μM, 4 μM, 6 μM, 8 μM, and 10 μM of Ide were added to cryodiluent Ⅰ, the motility performance such as the motility rate and motility of dairy goat sperm after thawing was improved. Among them, except for ALH, all sperm attributes in the 6 μM treatment group were significantly higher than those in the control group (P < 0.05); there were no significant differences in TM and MAD when 2 μM, 4 μM, 6 μM, 8 μM, and 10 μM of Ide were added (P > 0.05), but they were all significantly higher than those in the control group (P < 0.05); there were no significant differences in PM when 2 μM, 4 μM, and 6 μM of Ide were added (P > 0.05), and they were all significantly higher than those in the control group (P < 0.05).

[0103] 2.4.3 Detection of sperm plasma membrane integrity

[0104] Plasma membrane integrity was detected by the hypoosmotic swelling test (HOST). Sperm with intact cell membranes with complete biological functions would absorb water and swell in a hypoosmotic environment, causing the sperm tails to bend, while sperm that had lost plasma membrane integrity would not show tail bending. Specific operation: The fructose in the HOST solution was 0.9 g / mL, and the sodium citrate was 0.49 g / mL; 10 μL of thawed semen (semen after freezing and thawing) was added to 1 mL of HOST solution, incubated at 37 °C for 20 min, then 10 μL of the mixture was taken out, and the total number of sperm and the number of sperm with bent tails were counted under a microscope. Each group was repeated three times, and three fields of view were taken each time. After counting, the plasma membrane integrity rate was calculated: Plasma membrane integrity rate = number of sperm with bent tails / total number of sperm × 100%. The results of sperm plasma membrane integrity detection are shown in Figure 7 , Figure 7 . Among them, the sperm indicated by the arrow has an intact plasma membrane at the tail, and the sperm indicated by the triangle has damaged plasma membrane.

[0105] The effects of adding different concentrations of Ide on the plasma membrane integrity rate of the tails of thawed dairy goat semen are shown in Figure 1 and Table 2. When 4 μM, 6 μM, 8 μM, and 10 μM of Ide were added to the freezing diluent, the plasma membrane integrity rate of the tails of dairy goat sperm measured after thawing was significantly higher than that of the control group and the 2 μM group (P < 0.05).

[0106] Table 2 Plasma membrane integrity rate of the tail (%)

[0107]

[0108] 2.4.4 Detection of sperm acrosome integrity

[0109] The fluorescein isothiocyanate-labeled peanut agglutinin (FITC-PNA) staining method was used and the operation was carried out in the dark throughout the process: 50 μL of frozen-thawed semen was evenly smeared on a clean glass slide and air-dried naturally; after the sample was air-dried, it was fixed with absolute methanol for 10 min and then air-dried; 30 μL of FITC-PNA staining solution was taken to cover the sample, placed at 37 °C, and incubated in the dark for 30 min; after the incubation, the sample was rinsed 2 - 3 times with PBS, air-dried naturally, sealed with colorless nail polish, and then observed under a fluorescence microscope. The detection diagram of sperm acrosome integrity is shown in Figures 8 - 10 , Figure 8 is the fluorescence staining diagram of the sperm nucleus; Figure 9 is the fluorescence staining diagram of the sperm acrosome; Figure 10 is Figure 8 and 8 Figure 9 combined diagram. Figure 10 . Among them, the sperm indicated by the arrow has an intact acrosome, and the sperm indicated by the triangle has a damaged acrosome. Figure 8The blue color in it is the fluorescence map of sperm nucleus staining. Blue indicates sperm with intact sperm nuclei, and sperm with damaged sperm nuclei cannot be stained by DAPI; Figure 9 The green color in it is the acrosome staining map of sperm. The acrosomes of sperm are shown by the green fluorescence in the figure, and sperm with damaged acrosomes cannot be stained by FITC-PNA.

[0110] The effects of adding different concentrations of Ide on the acrosome integrity rate of frozen-thawed semen of dairy goats are as Figure 2 shown in and Table 3. When 6 μM of Ide is added to the freezing diluent, the acrosome integrity rate of sperm in the frozen-thawed semen of dairy goats is significantly higher than that of the control group (P < 0.05), and there is no significant difference between other treatment groups and the control group (P > 0.05).

[0111] Table 3 Acrosome integrity rate of sperm (%)

[0112]

[0113] 2.4.5 Fertility assessment

[0114] At the dairy goat experimental demonstration base in Longxian (Heshui), 215 healthy ewes with good body conditions and similar ages were selected for estrus synchronization treatment. On the 11th day after inserting the estrus sponge, 200 IU of pregnant mare serum gonadotropin PMSG was injected intramuscularly. On the 12th day, the sponge was removed and 1 mL of sodium cloprostenol injection PG was injected intramuscularly at the same time. The component content of the sodium cloprostenol injection was 0.1 mg / mL. The ewes came into heat 12 - 14 hours after sponge removal. 150 estrus ewes were selected and randomly divided into 3 groups (fresh sperm group, frozen sperm control group, and frozen sperm experimental group), with 50 ewes in each group. Different colored ear tags were put on as markers to distinguish the groups, and artificial insemination was carried out. The fresh sperm group used freshly collected semen that was not frozen-thawed and did not add Ide, the control group used the frozen semen with an idebenone concentration of 0 μM in 2.3 of Example 1, and the experimental group used the frozen semen with an idebenone concentration of 6 μM in 2.3 of Example 1. The first insemination was carried out 8 - 12 hours after estrus, and the insemination volume for each ewe each time was 0.2 mL. Insemination was carried out twice a day, morning and evening, during the estrus period until the estrus ended. On the 50th day after the insemination of this batch of ewes was completed, B-ultrasound detection was carried out on them, and the conception rates of each group were counted, as shown in Table 4.

[0115] Table 4 Comparison of artificial insemination conception rate results

[0116] Item Number of inseminated ewes / head Number of pregnant ewes / head Conception rate % Fresh semen group (0 μM) 50 41 82 Experimental group (6 μM) 50 37 74 Control group (0 μM) 50 31 62

[0117] Note: The fresh sperm group used freshly collected semen, which was not the same batch of semen as the experimental group and the control group; the experimental group and the control group were the same batch of frozen semen samples.

[0118] The results of the effect of adding 6 μM Ide on the conception rate of artificial insemination of dairy goat semen are shown in Table 4. Under the same conditions, the conception rate was 82% when using fresh semen for artificial insemination, 74% when using frozen-thawed dairy goat semen added with 6 μM Ide for artificial insemination, and 62% when using frozen-thawed dairy goat semen without adding Ide for artificial insemination. It can be seen that adding 6 μM Ide can effectively improve the sperm motility of frozen-thawed semen, thereby increasing the conception rate when using frozen semen in the field of artificial insemination.

[0119] 2.5 Evaluation of the antioxidant performance of frozen-thawed semen

[0120] The semen obtained after thawing in Step 2.4.1 was used for the following tests:

[0121] (1) Detection of total antioxidant capacity (T-AOC)

[0122] Detection of T-AOC enzyme content: It was processed according to the instructions of the total antioxidant capacity (T-AOC) detection kit (product number A015-2, batch number 20240402, Nanjing Jiancheng Bioengineering Institute). According to the standard curve y = -1.1214x + 1.1262, R 2 = 0.9957, where x is the OD value and y is the T-AOC enzyme concentration, the T-AOC enzyme activity was calculated. Finally, the absorbance at 593 nm was detected by an enzyme-labeling instrument. Four repeated experiments were carried out. The detection results of the total antioxidant capacity are as Figure 3 shown in Table 5. When adding 2 μM, 4 μM, 6 μM, 8 μM, and 10 μM Ide to the freezing diluent, the T-AOC of the frozen-thawed dairy goat semen was significantly higher than that of the control group (P < 0.05). Among them, the 4 μM and 8 μM treatment groups had the best effects and there was no significant difference between them (P > 0.05).

[0123] Table 5 Detection results of semen T-AOC in different treatment groups

[0124]

[0125]

[0126] (2) Detection of the activity of total superoxide dismutase (T-SOD)

[0127] Detection of T-SOD enzyme content: It was processed according to the instructions of the T-SOD kit (total superoxide dismutase test kit, product number A001-1, batch number 20240308, Nanjing Jiancheng Bioengineering Institute). Finally, the absorbance at 550 nm was detected by an enzyme-labeling instrument. Four repeated experiments were carried out. The detection results of the T-SOD enzyme activity are as Figure 4As shown in Table 6, when 2 μM, 4 μM, 6 μM, 8 μM, and 10 μM of Ide were added to the freezing diluent, the T-SOD of thawed dairy goat semen was significantly higher than that of the control group (P < 0.05). Among them, the 6 μM treatment group had the best effect and there was no significant difference between it and the 2 μM treatment group (P > 0.05).

[0128] Definition of T-SOD enzyme activity: The amount of SOD corresponding to a 50% SOD control rate in 1 mL of reaction solution per milligram of tissue protein is one SOD activity unit (U). SOD activity (U / mgprot) = (control OD value - measured OD value) / control OD value / 50% × total volume of reaction solution / sampling volume / protein concentration of test sample.

[0129] Table 6 Detection results of semen T-SOD in different treatment groups

[0130]

[0131]

[0132] (3) Detection of glutathione peroxidase (GSH-Px) activity

[0133] Detection of GSH-Px enzyme content: The GSH-Px enzyme content in sperm was processed according to the instructions of the glutathione peroxidase (GSH-Px) test kit (product number A005, batch number 20240118, manufacturer Nanjing Jiancheng Bioengineering Research Institute). Finally, after standing at room temperature for 5 min, the absorbance at 412 nm was measured. Four repeated experiments were carried out. The GSH-Px enzyme activity in tissues was calculated according to the instructions of the test kit. The formula is as follows:

[0134]

[0135] The detection results of GSH-Px enzyme activity are as Figure 5 As shown in Table 7, when 6 μM of Ide was added to the freezing diluent, the GSH-Px activity of thawed dairy goat semen was significantly higher than that of the control group and other treatment groups (P < 0.05).

[0136] Table 7 Detection results of semen GSH-Px in different treatment groups

[0137]

[0138]

[0139] (4) Detection of malondialdehyde (MDA) content

[0140] The content of MDA in sperm was processed according to the instructions of the malondialdehyde (MDA) assay kit (product number G4300-96T, batch number MPC2311056, manufacturer Servicebio). Finally, 0.01 mL was taken and the absorbance at 532 nm was detected by an enzyme-linked immunosorbent assay (ELISA) reader. Four replicate experiments were carried out.

[0141] Content of MDA (nmol / mgprot) = (measured OD value - blank OD value) / (standard OD value - blank OD value) * standard concentration (10 nmol / ml) / protein concentration of the sample to be measured (mgprot / ml).

[0142] The detection results of MDA content are as Figure 6 shown in Table 8. When 8 μM of Ide was added to the freezing diluent, the content of MDA in the thawed semen of dairy goats was significantly lower than that of the control group (P < 0.05), and there was no significant difference among the treatment groups of 2 μM, 6 μM, 8 μM, and 10 μM (P > 0.05).

[0143] Table 8 Detection results of semen MDA in different treatment groups

[0144]

[0145]

[0146] In summary, adding 6 μM of Ide to the freezing diluent of dairy goat semen can improve the quality and fertilization ability of thawed semen of dairy goats, and enhance the application value of frozen semen in artificial insemination.

[0147] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A cryopreservative for goat semen, characterized in that, Comprising: Normal temperature diluent and cryoprotective substances; The cryoprotective substances include egg yolk, glycerol and idebenone; The composition of the normal temperature diluent includes: D-fructose, tris(hydroxymethyl)aminomethane, citric acid, streptomycin sulfate and sodium ampicillin.

2. The cryopreservative according to claim 1, wherein The normal temperature diluent uses ultrapure water as a solvent, and its composition includes: 8-12 g / L of D-fructose, 25-30 g / L of tris(hydroxymethyl)aminomethane, 12-16 g / L of citric acid, 0.1-0.3 g / L of streptomycin sulfate and 0.1-0.3 g / L of sodium ampicillin.

3. The cryopreservative according to claim 1 or 2, characterized in that, The cryopreservative is divided into cryopreservative I and cryopreservative II; The cryopreservative I includes a first normal temperature diluent and a first cryoprotective substance; the first cryoprotective substance includes egg yolk and idebenone; calculated based on the volume of the first normal temperature diluent, the added amount of egg yolk in the cryopreservative I accounts for 10%-20% of its volume, and the final concentration of idebenone is greater than 0 and less than 10 μM; The cryopreservative II includes a second normal temperature diluent and a second cryoprotective substance; the second cryoprotective substance includes egg yolk, glycerol and idebenone; calculated based on the volume of the second normal temperature diluent, the added amount of egg yolk in the cryopreservative II accounts for 10%-20% of its volume, the added amount of glycerol accounts for 4%-6% of its volume, and the final concentration of idebenone is greater than 0 and less than 10 μM.

4. The preparation method of the cryopreservative according to any one of claims 1 to 3, characterized in that, Comprising: After mixing the normal temperature diluent and the cryoprotective substances, a cryopreservative is obtained.

5. Use of the cryopreservative according to any one of claims 1-3 or the cryopreservative prepared by the preparation method according to claim 4 in improving the quality and / or antioxidant capacity of goat semen.

6. The application according to claim 5, characterized in that, The goats include dairy goats.

7. A method for preserving goat semen, characterized in that, Comprising the following steps: After mixing and equilibrating the cryopreservative I with goat semen, an equilibration solution is obtained; After mixing and second equilibration of the cryopreservative II with the equilibration solution, a second equilibration solution is obtained; The second equilibration solution is cryopreserved; the cryopreservative I is the cryopreservative I according to claim 3; the cryopreservative II is the cryopreservative II according to claim 3.

8. The preservation method according to claim 7, characterized in that, The mixing volume ratio of the cryopreservative I to goat semen is 1:3-5; The temperature of the equilibration is 4°C, and the time of the equilibration is 2-3 h; The mixing method of the cryopreservative II and the equilibration solution includes equal-volume and isothermal mixing; the temperature of the second equilibration is 4°C, and the time of the second equilibration is 2-3 h.

9. The preservation method according to claim 7, wherein The cryopreservation method includes liquid nitrogen preservation.

10. A thawing method for the frozen semen obtained by the preservation method according to any one of claims 7 to 9, characterized in that, Comprising: After taking out the frozen semen, it is thawed in warm water and incubated in a water bath in sequence. The temperature of the warm water thawing is 37-40°C, and the time is 25 s; the temperature of the water bath incubation is 37°C, and the time is 3-5 min.

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