Sheep semen cryopreservation solution and semen preservation method

Abstract

The present invention belongs to the technical field of semen cryopreservation, and more specifically, relates to sheep semen freezing solution and semen preservation method. The sheep semen freezing solution components of the present invention include Tris, citric acid, fructose, lactose, trehalose, penicillin, streptomycin, soy lecithin, nobiletin, glycerol and water. By using a specific content of nobiletin as an effective antioxidant component of a sheep sperm cryopreservative, it plays an antioxidant role when the sperm is frozen, making the sperm more stable during the cryopreservation process. After adding nobiletin, the vitality and motility of sheep sperm after freezing and thawing can be significantly improved. It effectively reduces the oxidative stress damage of sperm during the freezing process and improves the antioxidant capacity of sperm.

Description

Technical Field

[0001] The present invention belongs to the technical field of semen cryopreservation, and more specifically, relates to sheep semen cryopreservation liquid and a semen preservation method. Background Art

[0002] Sheep breeding is a key pillar of the livestock industry. Breeding high-quality sheep is crucial for increasing mutton production, improving wool quality, and preserving genetic resources. Artificial insemination, a core breeding method, relies heavily on the effectiveness of semen cryopreservation. Cryopreserved semen allows for efficient and multi-temporal utilization of high-quality breeding rams, reduces introduction costs, and avoids the risk of disease during live transport, making it crucial for the industrialization of sheep breeding.

[0003] Although semen cryopreservation technology is relatively mature in livestock such as cattle and pigs, sheep semen faces significant challenges due to its unique biological characteristics. Sheep sperm is highly sensitive to freezing damage, and the proportion of polyunsaturated fatty acids in its cell membrane phospholipids is high. During the freezing process, sheep sperm will be damaged by oxidative stress, leading to problems such as decreased sperm motility and damaged membrane structure integrity.

[0004] Therefore, there is an urgent need for a sheep semen freezing solution to solve the above problems. Summary of the Invention

[0005] The present invention aims to provide a sheep semen freezing solution and a semen preservation method.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] The present invention provides a sheep semen freezing solution. The freezing solution comprises the following components per 100 mL: 2.71 g of Tris, 1.4 g of citric acid, 1.12 g of fructose, 6.31 g of lactose, 1.50 g of trehalose, 60 mg of penicillin, 100 mg of streptomycin, 5.5 g of soybean lecithin, 0.568 mg of nobiletin, 7 mL of glycerol, and the remainder being water.

[0008] The sheep semen freezing solution provided by the present invention has an antioxidant effect on sperm freezing by adding a specific proportion of nobiletin, making the sperm more stable during the cryopreservation process. After adding the specific proportion of nobiletin, the vitality and motility of sheep sperm after freezing and thawing can be significantly improved. The oxidative stress damage to sperm during the freezing process is effectively reduced, and the antioxidant capacity of sperm is improved.

[0009] Furthermore, the dilution ratio of the working concentration of the freezing solution is 4 / 5 of the original concentration.

[0010] The present invention also provides application of the sheep semen freezing solution in preparing semen preservation preparations.

[0011] The present invention also provides application of the sheep semen freezing solution in freezing and preserving sheep semen.

[0012] The present invention also provides a method for freezing and preserving sheep semen using the sheep semen freezing solution, comprising the following steps: collecting sheep semen, preheating the semen and mixing it with the freezing solution to obtain a mixed solution, and precooling the mixed solution and then freezing it.

[0013] Furthermore, the sperm motility of the semen is greater than 0.8.

[0014] Furthermore, the preheating temperature is 32°C.

[0015] Furthermore, the volume ratio of the semen to the semen freezing solution is 1:4.

[0016] Furthermore, the pre-cooling condition is 4°C for 2h.

[0017] The present invention has the following beneficial effects:

[0018] The sheep semen freezing solution provided by the present invention is formulated by combining a specific ratio of nobiletin with the pH buffer Tris, the energy-supplying components fructose and lactose, and the antibacterial component penicillin-streptomycin. This solution can significantly improve the vitality, motility, and membrane integrity of sheep sperm after freezing and thawing. It effectively reduces oxidative stress damage to sperm during the freezing process and improves the sperm's antioxidant capacity. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The statistical graphs show the effects of different concentrations of nobiletin on the motility of thawed sheep sperm, where A is the statistical graph of overall sperm motility, B is the statistical graph of sperm progressive motility, and C is the statistical graph of sperm rapid progressive motility.

[0020] Figure 2 Statistical graphs showing the effects of different concentrations of nobiletin on the antioxidant capacity of sperm during freezing, where A is the statistical graph for catalase content, B is the statistical graph for superoxide dismutase content, C is the statistical graph for glutathione peroxidase content, and D is the statistical graph for malondialdehyde content. * indicates p < 0.05, and ** indicates p < 0.01. DETAILED DESCRIPTION

[0021] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0022] Example 1

[0023] 1. Experimental setup.

[0024] 0 mg, 0.284 mg, 0.568 mg, 0.852 mg, and 1.136 mg of nobiletin were weighed into groups, and 2.71 g of Tris, 1.4 g of citric acid, 1.12 g of fructose, 6.31 g of lactose, 1.50 g of trehalose, 60 mg of penicillin, 100 mg of streptomycin, 5.5 g of soy lecithin, and 7 mL of glycerol were added to each group. The volume was made up to 100 ml with ultrapure water, and semen freezing solutions with nobiletin concentrations of 0 μmol / L, 4 μmol / L, 8 μmol / L, 12 μmol / L, and 16 μmol / L were prepared as experimental groups.

[0025] 2. Semen collection and freezing.

[0026] 1. Preparations for semen collection: Thermos, collection tube, collection cup, cloth cover, scissors, basin, kitchen paper, and towel. Place 40°C water in the thermos and collection tube. Install the collection tube, collection cup, and cloth cover in order.

[0027] 2. Semen Collection: Place the ewe and ram into a small pen at the same time and secure the ewe to stimulate the ram's sexual desire. The semen collector is ready to collect the semen. When the ram begins to mount, the ram's penis is lowered into the semen collection tube to complete the collection. The semen is then transported to the laboratory in a dark place.

[0028] 3. Observation of sperm motility and equipment preparation: microscope, slide, cover slip, pipette, pipette tip, blood cell counting chamber, fully automatic semen filling machine, frozen sperm tubes, marker pen, frozen semen diluent, constant temperature water bath, test tube rack, 1mL syringe, 50mL centrifuge tube, 3% NaCl solution.

[0029] 4. Semen motility determination: Place the freshly collected semen together with the semen collection cup into a pre-prepared constant temperature water bath at 32°C. Observe the original sperm motility under a microscope. Semen with motility greater than 0.8 is used for frozen semen preparation. Use a 1mL syringe to measure 5 tubes of 1mL semen, and add 4mL of semen freezing solution with concentrations of nobiletin of 0μmol / L, 4μmol / L, 8μmol / L, 12μmol / L, and 16μmol / L to make 5-fold dilutions.

[0030] 5. Sample Aliquoting and Pre-cooling: Place five tubes of diluted semen into 50mL centrifuge tubes. Start the filling machine, insert the frozen sperm tubes, insert the semen aspiration tube into the bottom of the 50mL centrifuge tubes, and run the machine to dispense. Wrap the sealed frozen sperm tubes with kitchen paper, label them, lay them flat, and protect them from light in at least three layers. Place them in a 4°C refrigerator, away from light, and cool for 2 hours. Once cooled, remove them and place them on a pre-cooled frozen sperm rack.

[0031] 6. Preparation of semen freezing equipment: small program freezing instrument, liquid nitrogen, tweezers, gauze bag, thumb tube, matching foam box, and semen freezing rack.

[0032] 7. Semen Freezing: Turn on the equipment and purge the tubes. After purging, pour liquid nitrogen into the foam box to pre-cool the instrument. When the instrument temperature registers 4°C, immediately cross-place the pre-arranged semen tubes, close the lid, and begin freezing. Freezing is complete when the display reads -140°C. Once freezing is complete, quickly place the frozen semen into the liquid nitrogen. Then, slowly place the thumb tube into the liquid nitrogen, transfer it to a gauze bag, and place it in a liquid nitrogen tank for storage.

[0033] 8. Thaw the semen and check its motility: Prepare a water bath in advance. Once the temperature reaches 37°C, remove the frozen semen from the liquid nitrogen tank and place it in the water bath. After thawing for 30 seconds, check the semen's motility under an optical microscope. If the motility is above 0.35, it can be stored for future use.

[0034] 3. Experimental testing of the effect.

[0035] 1. Determination of sperm motility: Quickly remove the frozen sperm tube from liquid nitrogen and place it in a constant temperature water bath at 37°C for 30 seconds. After thawing, transfer the semen to a 1.5mL centrifuge tube, take 4uL and add it to a special slide for sperm analysis. Use a sperm analyzer to detect the overall sperm motility, progressive motility, and rapid progressive motility after thawing.

[0036] 2. Superoxide dismutase detection: The superoxide dismutase content in semen was detected using a superoxide dismutase detection kit according to the kit instructions. The kit was purchased from Yinova Biotechnology Co., Ltd., catalog number E-NWOO57Sh.

[0037] 3. Catalase Detection: The catalase content in semen was determined using a catalase detection kit according to the kit instructions. The kit was purchased from Yinova Biotechnology Co., Ltd., catalog number E-NW0006Sh.

[0038] 4. Glutathione peroxidase activity detection: Glutathione peroxidase detection kit was purchased from Yinova Biotechnology Co., Ltd., catalog number E-NW0062Wh.

[0039] 5. Malondialdehyde content determination: The malondialdehyde content detection kit was purchased from Yinova Biotechnology Co., Ltd., with the product number E-NW00865Sh.

[0040] 4. Experimental results.

[0041] 1. Experimental results are as follows Figure 1As shown in the results, adding nobiletin to sheep semen cryo-diluent can improve the overall motility, progressive motility and rapid progressive motility of sperm compared with the 0 μmol / L group, and the nobiletin 8 μmol / L group was better.

[0042] 2. Experimental results are as follows Figure 2 As shown, the addition of nobiletin to sheep semen cryo-diluent significantly increased the levels of superoxide dismutase, catalase, and glutathione peroxidase, and reduced malondialdehyde content, compared to 0 μmol / L. Nobiletin and glutathione peroxidase synergistically enhanced sperm antioxidant capacity and reduced lipid peroxidation, with the 8 μmol / L nobiletin group showing the best results.

[0043] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

Claims

1. Sheep semen cryopreservation solution, characterized in that: The freezing solution includes the following components per 100 mL: 2.71 g of Tris, 1.4 g of citric acid, 1.12 g of fructose, 6.31 g of lactose, 1.50 g of trehalose, 60 mg of penicillin, 100 mg of streptomycin, 5.5 g of soy lecithin, 8 μmol / L of nobiletin, 7 mL of glycerol, and the remainder is water.

2. The sheep semen cryopreservation solution according to claim 1, characterized in that: The dilution ratio of the working concentration of the freezing solution is 4 / 5 of the original concentration.

3. Use of the sheep semen freezing solution according to claim 1 in preparing semen preservation preparations.

4. Use of the sheep semen freezing solution according to claim 1 in cryopreservation of sheep semen.

5. The method for preserving sheep semen using the sheep semen freezing solution according to claim 1, characterized in that: The following steps are involved: Sheep semen is collected, preheated, and mixed with the semen freezing solution of claim 1 to obtain a mixed solution, which is then precooled and frozen.

6. The method for preserving sheep semen using the sheep semen freezing solution according to claim 5, wherein: The sperm motility of the semen is greater than 0.

8.

7. The method for preserving sheep semen using the sheep semen freezing solution according to claim 5, wherein: The preheating temperature is 32°C.

8. The method for preserving sheep semen using the sheep semen freezing solution according to claim 5, wherein: The volume ratio of the semen to the semen freezing solution is 1:

4.

9. The method for preserving sheep semen using the sheep semen freezing solution according to claim 5, wherein: The pre-cooling condition is 4° C., 2 h.

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