Sheep semen diluent formula and freezing method

Abstract

The invention discloses a sheep semen diluent formula and a freezing method, and relates to the technical field of animal breeding, and every 100 mL of diluent is prepared from 0.28 g of functional amino acid, 0.07 g of bioactive peptide, 0.30 g of a buffering agent, 4.12 g of an osmotic pressure regulator and the balance ultrapure water. The preservation effect of the sheep semen is remarkably improved by accurately proportioning the functional amino acid, the bioactive peptide, the buffering agent and the osmotic pressure regulator, particularly, the specific concentration and proportion of the functional amino acid and the bioactive peptide provide necessary nutrition support for the semen, the activity and the survival rate of the semen are also promoted, and the survival rate of the semen is increased. By means of the innovative formula design, the sheep semen can still keep high quality after being diluted, a solid foundation is laid for follow-up cryopreservation, comprehensive protection of the biological activity of the sheep semen is achieved through careful component selection and concentration optimization, and the limitation of traditional diluents in component and effect is broken through.

Description

Technical Field

[0001] The invention relates to the technical field of animal reproduction, in particular to a formula of sheep semen diluent and a freezing method thereof. Background Art

[0002] In modern animal husbandry production, improving reproductive efficiency and optimizing genetic quality are key goals. Effective management and long-term preservation technology of semen from high-quality rams are important means to achieve this goal. The research and application of semen diluent formula and freezing methods can not only ensure the quality and vitality of semen, but also provide technical support for the long-term preservation and wide dissemination of genetic resources. In recent years, with the rapid development of reproductive biology, biochemistry and freezing technology, the optimization of semen diluent composition and the improvement of freezing preservation technology have become research hotspots.

[0003] There are many shortcomings in the traditional sheep semen diluent formula and freezing method. On the one hand, the traditional diluent composition is relatively simple and lacks targeted functional ingredients. It cannot meet the nutritional needs of sperm during the dilution and freezing process, which can easily lead to decreased sperm motility and survival rate. On the other hand, traditional freezing methods often lack precise temperature control and vibration regulation, which leads to the formation of ice crystals during the freezing process, damaging the sperm structure and affecting its fertilization ability. In addition, traditional technology also has a certain degree of blindness in the selection and use of cryoprotectants, which may cause adverse consequences such as sperm cell membrane rupture and DNA damage.

[0004] Therefore, in view of the defects of traditional sheep semen diluent formula and freezing method, a sheep semen diluent formula and freezing method are developed. Through these improvements, it is expected to significantly improve the cryopreservation effect of sheep semen, providing strong support for genetic improvement and reproductive efficiency improvement in animal husbandry. Summary of the invention

[0005] The purpose of the present invention is to make up for the shortcomings of the prior art and provide a sheep semen diluent formula and a freezing method thereof. The formula carefully proportions functional amino acids, bioactive peptides, buffers and osmotic pressure regulators, aiming to optimize the storage environment of semen. The combination of arginine and citrulline at specific concentrations, and the addition of lactoferrin peptide and GnRHa bioactive peptide effectively improve the stability and fertilization ability of semen. At the same time, the innovative freezing method combines the precise temperature control and vibration functions of the programmable freezer, as well as the use of osmotic cryoprotectants, which significantly improves the survival rate and fertilization rate of semen after cryopreservation, providing strong support for genetic improvement and reproductive efficiency in animal husbandry.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions: On the one hand, a formula of sheep semen diluent, the formula of each 100mL diluent is:

[0007] Functional amino acid 0.28g, bioactive peptide 0.07g, buffer 0.30g, osmotic pressure regulator 4.12g, the balance is ultrapure water

[0008] The functional amino acids consist of arginine, citrulline, cysteine ​​and methionine;

[0009] The bioactive peptides are composed of lactoferrin peptide, GnRHa, and insulin-like growth factor-1;

[0010] The buffer is Tris-HCl buffer;

[0011] The osmotic pressure regulator consists of sucrose and sodium chloride.

[0012] Furthermore, among the functional amino acids, arginine is a key participant in energy metabolism in sperm production and metabolism and can improve sperm motility, with a concentration of 5-10mmol / L. Citrulline is efficiently converted into arginine in vivo through the action of arginine succinate synthetase and arginine succinate lyase to provide an energy substrate for sperm, with a concentration of 3-8mmol / L, and the ratio of arginine to citrulline is about 2:1. Cysteine ​​participates in the synthesis and modification of sperm cell membrane proteins in the form of disulfide bonds, enhancing the stability of the cell membrane, with a concentration of 2-5mmol / L. Methionine participates in the construction of multiple important proteins in sperm, with a concentration of 3-6mmol / L.

[0013] Furthermore, the lactoferrin peptide in the bioactive peptide has a broad-spectrum antibacterial activity. It interacts with specific targets on the bacterial cell membrane, destroys the integrity of the cell membrane, causes the leakage of bacterial contents, and thus kills the bacteria at a concentration of 0.5-1 mg / mL. GnRHa can promote the maturation and development of sperm, improve sperm motility and fertilization ability at a concentration of 50-100 ng / mL. Insulin-like growth factor-1 can stimulate the proliferation and differentiation of sperm cells, improve the metabolic activity and motility of sperm at a concentration of 50-100 ng / mL.

[0014] Furthermore, the Tris-HCl buffer in the buffer can stabilize the pH of the diluent, resist pH changes caused by metabolites in semen and external factors, and provide a stable living environment for sperm. The concentration is 20-30mmol / L and the pH value is between 7.2-7.4.

[0015] Furthermore, the concentration of sucrose in the osmotic pressure regulator is 80-120 mmol / L, and the concentration of sodium chloride is 100-150 mmol / L, which jointly adjust the osmotic pressure of the diluent to make it close to the osmotic pressure of sheep semen, prevent sperm from losing water or absorbing water due to osmotic pressure differences, and maintain the normal morphology and function of sperm.

[0016] On the other hand, a method for freezing sheep semen is provided, wherein the specific steps of the freezing method are:

[0017] S100, semen collection and pretreatment: collect semen from a ram by using a semen collector, place the collected semen in a 32° C. insulated container, and perform a quality inspection on the semen. After the quality inspection is completed, centrifuge the semen with a centrifuge at a speed of 500-800 rpm for 10-15 minutes, and keep the temperature at 30-32° C. during the centrifugation process. After the centrifugation is completed, collect the precipitated sperm, add the semen to a diluent prepared by the formula of a sheep semen diluent according to claims 1-5, and use a rotary mixer to mix at a speed of 30-40 rpm for 5-8 minutes to evenly disperse the sperm in the diluent;

[0018] S200, preparation of freezing equipment: prepare a programmable freezer with a temperature control accuracy of ±0.05°C and a temperature control range of -200°C to 50°C. In terms of vibration function, the vibration frequency is between 1-1000Hz and can be adjusted with an accuracy of 1Hz. The amplitude is within the range of 0.1-5mm and can be adjusted with an accuracy of 0.1mm. A double-layer vacuum insulated liquid nitrogen tank with a capacity of 50-100L;

[0019] S300, semen freezing: the diluted semen is divided into 1.5-2.0mL plastic cryovials, 1.0-1.2mL per tube, and placed in the freezer to start the freezing program. At the same time, low-frequency vibration is turned on. The sucrose and amino acids pre-added in the diluent form a stable osmotic pressure environment around the sperm. The low-frequency vibration promotes the uniform diffusion of the cryoprotectant in the semen, so that the sperm can be more fully contacted, prevent ice crystals from excessively gathering locally, and reduce sperm dehydration damage. The freezer starts to cool down from 30-32℃ at a rate of 1-2℃ / min. When the temperature drops to -5 to -8℃, use a syringe pump to slowly add osmotic cryoprotectant solution to each cryovial. After adding, continue freezing. When the semen temperature is close to -0.5 to -1.5℃, start high-frequency vibration. High-frequency vibration interferes with the nucleation process of ice crystals, promotes the formation of more nuclei, and makes the ice crystals grow smaller. At the same time, glycerol gradually penetrates into the sperm to protect the intracellular structure.

[0020] S400, deep freezing and storage: after completing the high-frequency vibration, continue to lower the temperature at a rate of 2-5°C / min until the temperature drops to -80°C, transfer the cryovials to a liquid nitrogen tank, store them above the liquid nitrogen surface for 2-3 minutes, and then completely immerse the cryovials in liquid nitrogen for long-term storage;

[0021] S500, sperm quality test after freezing: frozen semen samples are taken out from liquid nitrogen regularly, thawed in a constant temperature water bath at 37-40℃, and the sperm acrosome integrity is tested by fluorescent staining. At the same time, in vitro fertilization experiments are performed to evaluate the fertilization ability of sperm.

[0022] Furthermore, in the S100, the ratio of semen to diluent in semen collection and pretreatment is 1:3-1:5.

[0023] Furthermore, in the S300, the vibration frequency of the low-frequency vibration in semen freezing is set to 3-7 Hz, and the amplitude is 0.5-1.5 mm.

[0024] Furthermore, in said S300, the osmotic cryoprotectant solution in the freezing of semen is a glycerol solution having a concentration of 5-8% v / v in semen.

[0025] Furthermore, in the S300, the frequency of high-frequency vibration in semen freezing is set to 300-700 Hz, the amplitude is 0.2-0.8 mm, and the duration is 3-5 minutes.

[0026] Compared with the prior art, the sheep semen diluent formula and freezing method thereof have the following beneficial effects:

[0027] 1. The present invention significantly improves the preservation effect of sheep semen by accurately matching functional amino acids, bioactive peptides, buffers and osmotic pressure regulators. In particular, the specific concentrations and ratios of functional amino acids and bioactive peptides not only provide necessary nutritional support for sperm, but also promote sperm vitality and survival rate. This innovative formula design enables sheep semen to maintain a high quality after dilution, laying a solid foundation for subsequent cryopreservation. Through careful ingredient selection and concentration optimization, comprehensive protection of the biological activity of sheep semen is achieved, breaking through the limitations of traditional diluents in terms of ingredients and effects.

[0028] 2. The present invention achieves fine regulation of the freezing process of sheep semen by accurately controlling the parameters of cooling rate, vibration frequency and amplitude, combined with the slow addition of osmotic cryoprotectant, which not only effectively avoids sperm damage during the freezing process, but also significantly improves the acrosome integrity and fertilization ability of sperm after freezing. In particular, the combined strategy of low-frequency and high-frequency vibrations is introduced during the freezing process to further optimize the freezing effect. Low-frequency vibration helps to evenly cool the semen during the cooling process, while high-frequency vibration helps to promote the stability of the internal structure of sperm at critical temperature points. This not only improves the cryopreservation efficiency of sheep semen, but also provides a useful reference for the cryopreservation of semen of other animals.

[0029] Other advantages, objectives and features of the present invention will be set forth in part in the following description and, in part, will be apparent to those skilled in the art based on an examination of the following or may be taught from the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0031] Figure 1 The present invention is a flow chart of a method for freezing sheep semen. DETAILED DESCRIPTION

[0032] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] Embodiment 1

[0034] Preparation of sheep semen diluent: prepare according to the formula of every 100mL diluent, take 0.28g of functional amino acids (including arginine concentration of 8mmol / L, citrulline concentration of 4mmol / L, cysteine ​​concentration of 3mmol / L, and methionine concentration of 4mmol / L), 0.07g of bioactive peptides (lactoferrin peptide concentration of 0.8mg / mL, GnRHa concentration of 80ng / mL, and insulin-like growth factor-1 concentration of 80ng / mL), 0.30g of buffer (Tris-HCl buffer concentration of 25mmol / L, pH value of 7.3), 4.12g of osmotic pressure regulator (sucrose concentration of 100mmol / L, sodium chloride concentration of 120mmol / L), and make up the rest with ultrapure water.

[0035] Semen collection and pretreatment: Use a semen collector to collect semen from the breeding ram, place the semen in a 32°C insulation container, and after quality inspection of the semen, use a centrifuge at 500 rpm for 12 minutes (the temperature is maintained at 31°C during the centrifugation process), collect the precipitated sperm, mix the semen with the above-prepared diluent in a ratio of 1:4, and use a rotary mixer to mix at 35 rpm for 6 minutes.

[0036] Preparation of freezing equipment: prepare a programmable freezer (temperature control accuracy of ±0.05°C, temperature control range of -200°C to 50°C, vibration frequency between 1-1000Hz with an accuracy of 1Hz, amplitude within the range of 0.1-5mm with an accuracy of 0.1mm) and a double-layer vacuum insulated liquid nitrogen tank with a capacity of 80L.

[0037] Semen freezing: The diluted semen was dispensed into 1.8 mL plastic cryovials, 1.1 mL per tube, placed in a freezer to start the freezing program, and low-frequency vibration was turned on at the same time (the vibration frequency was set to 5 Hz, and the amplitude was 1 mm). The freezer started to cool down from 31 °C at a rate of 1.5 °C / min. When the temperature dropped to -6 °C, a syringe pump was used to add a 6% v / v osmotic cryoprotectant solution of glycerol concentration to each cryovial. After the addition was completed, freezing was continued. When the semen temperature was close to -1 °C, high-frequency vibration was started (the frequency was set to 500 Hz, the amplitude was 0.5 mm, and the duration was 4 minutes).

[0038] Deep freezing and storage: After completing high-frequency vibration, continue to lower the temperature at a rate of 3°C / min until the temperature drops to -80°C. Transfer the cryovials to a liquid nitrogen tank and store them above the liquid nitrogen surface for 2.5 minutes. Then completely immerse the cryovials in liquid nitrogen for long-term storage.

[0039] Sperm quality testing after freezing: Frozen semen samples are taken out from liquid nitrogen regularly, thawed in a constant temperature water bath at 38°C, and the sperm acrosome integrity is tested by fluorescent staining. At the same time, in vitro fertilization experiments are performed to evaluate the fertilization ability of sperm.

[0040] Embodiment 2

[0041] Preparation of sheep semen diluent: for every 100 mL of diluent, take 0.28 g of functional amino acids (including arginine concentration of 7 mmol / L, citrulline concentration of 3.5 mmol / L, cysteine ​​concentration of 4 mmol / L, and methionine concentration of 5 mmol / L), 0.07 g of bioactive peptides (including lactoferrin peptide concentration of 0.7 mg / mL, GnRHa concentration of 70 ng / mL, and insulin-like growth factor-1 concentration of 70 ng / mL), 0.30 g of buffer (including Tris-HCl buffer concentration of 22 mmol / L, pH value of 7.2), 4.12 g of osmotic pressure regulator (including sucrose concentration of 90 mmol / L, sodium chloride concentration of 110 mmol / L), and make up the rest with ultrapure water.

[0042] Semen collection and pretreatment: Collect ram semen and place it in a 32°C insulated container. After checking the quality, use a centrifuge at 600 rpm for 10 minutes (temperature 30°C), collect the precipitated sperm, mix the semen with the diluent in a ratio of 1:3, and use a rotary mixer to mix at 30 rpm for 5 minutes.

[0043] Preparation of freezing equipment: prepare a programmable freezer (temperature control accuracy of ±0.05°C, temperature control range of -200°C to 50°C, vibration frequency between 1-1000Hz with an accuracy of 1Hz, amplitude within the range of 0.1-5mm with an accuracy of 0.1mm) and a double-layer vacuum insulated liquid nitrogen tank with a capacity of 70L.

[0044] Semen freezing: Diluted semen was divided into 1.6mL cryotubes, 1.0mL per tube, and placed in a freezer to start the freezing program and low-frequency vibration (vibration frequency 4Hz, amplitude 1.2mm). The freezer was cooled from 30℃ at a rate of 1.2℃ / min. When it reached -7℃, a 7% v / v osmotic cryoprotectant solution of glycerol concentration was added. When the semen temperature approached -1.2℃, high-frequency vibration was started (the frequency was set to 400Hz, the amplitude was 0.6mm, and it lasted for 3 minutes).

[0045] Deep freezing and storage: After completing high-frequency vibration, continue to cool down to -80°C at a rate of 2°C / min. Store the cryotube above the liquid nitrogen surface in the liquid nitrogen tank for 2 minutes and completely immerse the cryotube in liquid nitrogen for long-term storage.

[0046] Sperm quality testing after freezing: Frozen semen samples were taken from liquid nitrogen, thawed in a 39°C constant temperature water bath, and the sperm acrosome integrity was tested by fluorescent staining. At the same time, in vitro fertilization experiments were performed to evaluate the fertilization ability of the sperm.

[0047] Embodiment 3

[0048] Preparation of sheep semen diluent: For every 100mL of diluent, there are 0.28g of functional amino acids (including 6mmol / L arginine, 3mmol / L citrulline, 2.5mmol / L cysteine ​​and 3mmol / L methionine), 0.07g of bioactive peptides (including 0.6mg / mL lactoferrin peptide, 60ng / mL GnRHa and 60ng / mL insulin-like growth factor-1), 0.30g of buffer (Tris-HCl buffer with a concentration of 28mmol / L and a pH of 7.4), 4.12g of osmotic pressure regulator (including 110mmol / L sucrose and 140mmol / L sodium chloride), and the rest is made up with ultrapure water.

[0049] Semen collection and pretreatment: Collect ram semen in a 32°C insulated container. After quality inspection, use a centrifuge at 700 rpm for 13 minutes (the temperature is maintained at 32°C during centrifugation), collect the precipitated sperm, mix the semen with the diluent in a ratio of 1:5, and use a rotary mixer at 38 rpm for 7 minutes.

[0050] Preparation of freezing equipment: prepare a programmable freezer (with a temperature control accuracy of ±0.05°C, a temperature control range of -200°C to 50°C, a vibration frequency between 1-1000Hz with an accuracy of 1Hz, an amplitude within the range of 0.1-5mm with an accuracy of 0.1mm) and a double-layer vacuum insulated liquid nitrogen tank with a capacity of 90L.

[0051] Semen freezing: Diluted semen is divided into 2.0mL cryotubes, 1.2mL in each tube, and placed in a freezer to start freezing and low-frequency vibration (vibration frequency is set to 6Hz, amplitude is 0.8mm), cool down from 32℃ at 1.8℃ / min, add 5% v / v osmotic cryoprotectant solution of glycerol concentration at -5℃, and start high-frequency vibration (frequency is set to 600Hz, amplitude is 0.3mm, and last for 5 minutes) when the temperature approaches -0.8℃.

[0052] Deep freezing and storage: After completing high-frequency vibration, cool down to -80℃ at a rate of 4℃ / min. After storing above the liquid nitrogen surface in the liquid nitrogen tank for 3 minutes, completely immerse the cryovial in liquid nitrogen for long-term storage.

[0053] Sperm quality testing after freezing: Take the semen frozen in liquid nitrogen, use a 40℃ constant temperature water bath to thaw it, and use the fluorescent staining method to detect the integrity of the sperm acrosome. At the same time, perform in vitro fertilization experiments to evaluate the fertilization ability of the sperm.

[0054] Embodiment 4

[0055] Configuration of sheep semen diluent: per 100mL of diluent, there are 0.28g of functional amino acids (including 10mmol / L arginine, 5mmol / L citrulline, 5mmol / L cysteine ​​and 6mmol / L methionine), 0.07g of bioactive peptides (including 1mg / mL lactoferrin peptide, 100ng / mL GnRHa and 100ng / mL insulin-like growth factor-1), 0.30g of buffer (including 30mmol / L Tris-HCl buffer and 7.4 pH), 4.12g of osmotic pressure regulator (including 120mmol / L sucrose and 150mmol / L sodium chloride), and the rest is made up with ultrapure water.

[0056] Semen collection and pretreatment: Collect ram semen and place it in a 32℃ insulated container. After checking the quality, use a centrifuge at 800 rpm for 15 minutes (32℃), collect the precipitated sperm, mix the semen with the diluent in a ratio of 1:5, and use a rotary mixer at 40 rpm for 8 minutes.

[0057] Preparation of freezing equipment: prepare a programmable freezer (temperature control accuracy of ±0.05°C, temperature control range of -200°C to 50°C, vibration frequency between 1-1000Hz with an accuracy of 1Hz, amplitude within the range of 0.1-5mm with an accuracy of 0.1mm) and a double-layer vacuum insulation structure liquid nitrogen tank with a capacity of 100L.

[0058] Semen freezing: Divide the diluted semen into 2.0mL freezing tubes, 1.2mL in each tube, put them into the freezer and start freezing and low-frequency vibration (the vibration frequency is set to 7Hz, the amplitude is 1.5mm), cool down from 32℃ at 2℃ / min, add 8% v / v osmotic cryoprotectant solution of glycerol concentration at -8℃, and start high-frequency vibration (the frequency is set to 700Hz, the amplitude is 0.8mm, and it lasts for 5 minutes) when it is close to -1.5℃.

[0059] Deep freezing and storage: After completing high-frequency vibration, continue to cool down to -80℃ at a rate of 5℃ / min, transfer the cryovials to a liquid nitrogen tank, store them above the liquid nitrogen surface in the liquid nitrogen tank for 3 minutes, and then completely immerse the cryovials in liquid nitrogen for long-term storage.

[0060] Sperm quality testing after freezing: Take the semen frozen in liquid nitrogen, thaw it in a constant temperature water bath at 40℃, detect the integrity of the sperm acrosome by fluorescent staining, and conduct in vitro fertilization experiments to evaluate the fertilization ability of the sperm.

[0061] In different embodiments, by changing the concentration of each component in the functional amino acids, such as different concentrations of arginine and citrulline in Embodiments 2, 3, and 4 compared with Embodiment 1, and observing the quality of sperm after cryopreservation, it was found that a suitable concentration combination (such as arginine concentration of 8 mmol / L and citrulline concentration of 4 mmol / L in Embodiment 1) can make the sperm have better acrosome integrity after thawing and stronger in vitro fertilization ability, which reflects that a reasonable combination of functional amino acids provides a good metabolic and living environment for sperm, thereby improving quality.

[0062] The concentrations of the bioactive peptide components in each embodiment are different. For example, in Embodiment 2, the concentration of lactoferrin peptide is 0.7 mg / mL. Compared with other embodiments, when the concentration of the bioactive peptide is within an appropriate range, the sperm protection effect during the freezing process is better. From the sperm quality test results after freezing, it can be seen that the acrosome integrity and fertilization ability are improved, reflecting its function of protecting sperm and promoting development.

[0063] The frequency, amplitude and duration of low-frequency and high-frequency vibrations are different in different embodiments. For example, in Embodiment 3, the low-frequency vibration frequency is 6 Hz, the amplitude is 0.8 mm, and the high-frequency vibration frequency is 600 Hz, the amplitude is 0.3 mm. By comparison, it is found that appropriate vibration parameter settings can reduce the damage of ice crystals to sperm and improve the quality of sperm after thawing, which can be seen from the acrosome integrity and fertilization ability data.

[0064] In each embodiment, the osmotic cryoprotectant solution (with different glycerol concentrations) is combined with other freezing conditions. For example, in Example 4, the glycerol concentration is 8% v / v. Compared with other embodiments, different concentrations of glycerol work synergistically with other freezing parameters to further optimize the cryoprotection effect, which is reflected in the improvement of sperm quality after thawing.

[0065] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. A formula for sheep semen diluent, characterized in that: The formula for each 100mL dilution is: Functional amino acid 0.28g, bioactive peptide 0.07g, buffer 0.30g, osmotic pressure regulator 4.12g, the balance is ultrapure water; The functional amino acids consist of arginine, citrulline, cysteine ​​and methionine; The bioactive peptides are composed of lactoferrin peptide, GnRHa, and insulin-like growth factor-1; The buffer is Tris-HCl buffer; The osmotic pressure regulator consists of sucrose and sodium chloride.

2. A sheep semen diluent formulation according to claim 1, characterized in that: The concentration of arginine in the functional amino acids is 5-10 mmol / L, the concentration of citrulline is 3-8 mmol / L, and the ratio of arginine to citrulline is about 2:

1. The concentration of cysteine ​​is 2-5 mmol / L, and the concentration of methionine is 3-6 mmol / L.

3. A sheep semen diluent formula according to claim 1, characterized in that: The concentration of lactoferrin peptide in the bioactive peptides is 0.5-1 mg / mL, the concentration of GnRHa is 50-100 ng / mL, and the concentration of insulin-like growth factor-1 is 50-100 ng / mL.

4. A sheep semen diluent formulation according to claim 1, characterized in that: The concentration of the Tris-HCl buffer in the buffer is 20-30 mmol / L, and the pH value is between 7.2-7.

4.

5. A sheep semen diluent formula according to claim 1, characterized in that: The concentration of sucrose in the osmotic pressure regulator is 80-120 mmol / L, and the concentration of sodium chloride is 100-150 mmol / L.

6. A method for freezing sheep semen, characterized in that: The specific steps of the freezing method are: S100, semen collection and pretreatment: collect semen from a ram by using a semen collector, place the collected semen in a 32° C. insulated container, and perform a quality inspection on the semen. After the quality inspection is completed, centrifuge the semen with a centrifuge at a speed of 500-800 rpm for 10-15 minutes, and keep the temperature at 30-32° C. during the centrifugation process. After the centrifugation is completed, collect the precipitated sperm, add the semen to a diluent prepared by the formula of a sheep semen diluent according to claims 1-5, and use a rotary mixer to mix at a speed of 30-40 rpm for 5-8 minutes; S200, preparation of freezing equipment: prepare a programmable freezer with a temperature control accuracy of ±0.05°C and a temperature control range of -200°C to 50°C. In terms of vibration function, the vibration frequency is between 1-1000Hz and can be adjusted with an accuracy of 1Hz. The amplitude is within the range of 0.1-5mm and can be adjusted with an accuracy of 0.1mm. A double-layer vacuum insulated liquid nitrogen tank with a capacity of 50-100L; S300, semen freezing: the diluted semen is dispensed into 1.5-2.0mL plastic cryovials, 1.0-1.2mL per tube, and placed in a freezer to start the freezing program, while turning on low-frequency vibration. The freezer starts to cool down from 30-32℃ at a rate of 1-2℃ / min. When the temperature drops to -5 to -8℃, a syringe pump is used to add a permeable cryoprotectant solution to each cryovial. After the addition is completed, continue freezing. When the semen temperature approaches -0.5 to -1.5℃, start high-frequency vibration; S400, deep freezing and storage: after completing the high-frequency vibration, continue to lower the temperature at a rate of 2-5°C / min until the temperature drops to -80°C, transfer the cryovials to a liquid nitrogen tank, store them above the liquid nitrogen surface for 2-3 minutes, and completely immerse the cryovials in liquid nitrogen for long-term storage; S500, sperm quality test after freezing: frozen semen samples are taken out from liquid nitrogen regularly, thawed in a constant temperature water bath at 37-40℃, and the sperm acrosome integrity is tested by fluorescent staining. At the same time, in vitro fertilization experiments are performed to evaluate the fertilization ability of sperm.

7. A method for freezing sheep semen according to claim 6, characterized in that: In the S100, the ratio of semen to diluent during semen collection and pretreatment is 1:3-1:

5.

8. A method for freezing sheep semen according to claim 6, characterized in that: In the S300, the vibration frequency of the low-frequency vibration in semen freezing is set to 3-7 Hz, and the amplitude is 0.5-1.5 mm.

9. A method for freezing sheep semen according to claim 6, characterized in that: In the S300, the osmotic cryoprotectant solution in the freezing of semen is a glycerol solution with a concentration of 5-8% v / v in semen.

10. A method for freezing sheep semen according to claim 6, characterized in that: In the S300, the frequency of high-frequency vibration in semen freezing is set to 300-700 Hz, the amplitude is 0.2-0.8 mm, and the duration is 3-5 minutes.

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