A kind of goat semen preservation liquid and preservation method thereof

Abstract

The present invention belongs to the technical field of semen preservation solution, in particular to a goat semen preservation solution and a preservation method thereof. The preservation solution comprises the following components per 100 mL: component A: 1.2-1.3 g of fructose, 1.7-1.8 g of citric acid monohydrate, 3.5-3.6 g of tris(hydroxymethyl)aminomethane; component B: 19-21 mL of fresh egg yolk, 3.5-4.5 mL of vitamin E, 0.4-0.6 g of bovine serum albumin, 0.4-0.6 g of vitamin C, 3.5-4.5 mL of glycerol, 100 00-15000IU of penicillin sodium, 10000-15000IU of streptomycin sulfate, 0.5-0.7mg of Y-27632·2HCl, 5-6mg of ectoine, 0.2-0.3mg of bifendate, 0.3-0.4mg of tiopronin, 0.5-0.6mg of amifostine, 2-2.5mg of azithromycin, 0.1-5mg of dexamethasone; the rest is double distilled water. The present invention can effectively solve the problem of yolk aggregation of goat semen in yolk-containing preservation solution, prolong the storage time of goat semen under low temperature conditions, and achieve the best quality of sperm.

Description

Technical Field

[0001] The invention belongs to the technical field of semen preservation solution, and in particular relates to a goat semen preservation solution and a preservation method thereof. Background Art

[0002] Sheep farming is an important part of animal husbandry. Since the 1950s, with the increase in market demand for mutton and the decrease in demand for wool and sheepskin products, the world's sheep farming industry has gradually developed from wool-oriented to meat-oriented. Mutton is high in protein, low in fat, low in cholesterol, contains many amino acids needed by the human body, is nutritionally comprehensive, and has health benefits. It is a green and safe food that conforms to modern consumer concepts. With the improvement of people's living standards, the demand for mutton has increased significantly, and large-scale and intensive mutton farms have developed rapidly. As a modern biological rapid propagation technology, artificial insemination technology for mutton sheep has accelerated the large-scale and intensive development of mutton sheep.

[0003] The combination of semen preservation and artificial insemination technology can break through the time and geographical restrictions of semen breeding of excellent breeding animals, and has the effect of accelerating the process of herd improvement and breeding, protecting livestock germplasm resources and genetic diversity. The establishment of semen preservation technology has greatly promoted the industrialization process of artificial insemination of livestock, and has great commercial value and application prospects. Semen preservation is a major innovation in artificial insemination technology. At present, the semen preservation of cattle, horses and other livestock has achieved satisfactory results.

[0004] Since Philips first discovered in 1939 that egg yolk has a protective effect on bull sperm, egg yolk has become an indispensable and important component of the preservation solution as a sperm protective agent. It has been widely used in the preservation of sperm of different animals. However, progress in the preservation of goat semen has been slow. The main reason is that in 1957, ROY first discovered that goat seminal plasma can cause yolk aggregation and eventually cause sperm death. This yolk aggregation effect is caused by a protease derived from the goat's bulbourethral gland, and this protease was named EYCE. In the 1960s, Iritani et al. defined this enzyme as phospholipase A. Phospholipase A can hydrolyze phosphatidylcholine in yolk into fatty acids and lysophosphatidylcholine, resulting in increased plasma membrane permeability, and ultimately inducing sperm acrosome reaction and chromosome depolymerization. These structural changes are not conducive to sperm survival. In order to solve the problem that goat seminal plasma causes yolk aggregation and eventually causes sperm death, some researchers remove goat seminal plasma containing phospholipase A by centrifugation, and then resuspend the sperm precipitated by centrifugation with yolk preservation solution, so that yolk aggregation no longer occurs in the preservation solution. However, in the process of removing goat seminal plasma, low-speed centrifugation will cause incomplete sperm precipitation, nearly half of the sperm are still in the goat seminal plasma, and the sperm vitality of the precipitated sperm is significantly reduced, affecting the preservation effect. Although high-speed centrifugation can completely precipitate the sperm in the semen, the precipitated sperm will lose vitality and utilization value. Therefore, a goat semen preservation solution and a preservation method thereof are urgently needed to solve the above problems. Summary of the invention

[0005] In order to overcome the defects in the prior art, the present invention provides a goat semen preservation solution and a preservation method thereof. The present invention can solve the problem of aggregation of phospholipase A in goat semen due to interaction with egg yolk, and at the same time improve the preservation time and quality of goat semen.

[0006] To achieve one of the above purposes, the present invention adopts the following technical solution:

[0007] A goat semen preservation solution, comprising the following components per 100 mL of the preservation solution:

[0008] Component A: 1.2-1.3 g of fructose, 1.7-1.8 g of citric acid monohydrate, 3.5-3.6 g of tris(hydroxymethyl)aminomethane;

[0009] Component B: 19-21 mL of fresh egg yolk, 3.5-4.5 mL of vitamin E, 0.4-0.6 g of bovine serum albumin, 0.4-0.6 g of vitamin C, 3.5-4.5 mL of glycerol, 10,000-15,000 IU of penicillin sodium, 10,000-15,000 IU of streptomycin sulfate, 0.5-0.7 mg of Y-27632·2HCl, 5-6 mg of ectoine, 0.2-0.3 mg of bifendate, 0.3-0.4 mg of tiopronin, 0.5-0.6 mg of amifostine, 2-2.5 mg of azithromycin, and 0.1-5 mg of dexamethasone;

[0010] The rest was double distilled water.

[0011] Preferably, the concentration of dexamethasone is 20 μg / mL.

[0012] Preferably, the preservation solution comprises the following components per 100 mL:

[0013] Component A: 1.26 g fructose, 1.72 g citric acid monohydrate, 3.53 g tris(hydroxymethyl)aminomethane;

[0014] Component B: 20 ​​mL of fresh egg yolk, 4 mL of vitamin E, 0.5 g of bovine serum albumin, 0.5 g of vitamin C, 4 mL of glycerol, 15,000 IU of penicillin sodium, 15,000 IU of streptomycin sulfate, 0.64 mg of Y-27632·2HCl, 5.68 mg of ectoine, 0.21 mg of bifenthrin, 0.33 mg of tiopronin, 0.5 mg of amifostine, 2 mg of azithromycin, and 2 mg of dexamethasone;

[0015] The rest was double distilled water.

[0016] The second object of the present invention is to provide a method for preserving goat semen preservative solution, comprising the following steps:

[0017] The collected qualified goat semen was diluted 8 times with preservation solution, and then the diluted semen was divided into centrifuge tubes, wrapped with absorbent cotton, and stored in a 4°C refrigerator.

[0018] The third object of the present invention is to provide a method for preserving goat semen preservative solution, comprising the following steps:

[0019] The collected qualified sheep semen is diluted 8 times with preservation solution, and then the diluted semen is divided into straw tubes. The straw tubes are sealed with polyvinyl alcohol powder, and then the straw tubes are placed in pre-prepared liquid nitrogen for fumigation, and finally the straw tubes are placed in liquid nitrogen for preservation.

[0020] The advantages of the present invention are:

[0021] (1) The functions of the components in the preservation solution of the present invention are as follows:

[0022] Dexamethasone: Like other glucocorticoids, it has pharmacological effects such as anti-inflammatory, anti-endotoxin, immunosuppressive, anti-shock and enhanced stress response, so it is widely used to treat a variety of diseases. Dexamethasone mainly exerts anti-inflammatory and analgesic effects by inhibiting the activity of phospholipase A and the synthesis of its metabolites. Dexamethasone binds to the glucocorticoid receptors of target cells, induces the target cells to produce lipocortin, inhibits the production of phospholipase A by blocking the synthesis of mRNA, reduces the production of arachidonic acid metabolites, and achieves a protective effect on target cells and organs. Dexamethasone can also activate adenylate cyclase, inhibit phosphodiesterase, increase cAMP levels and the sensitivity of bronchial β receptors to adrenergic drugs and theophylline, and indirectly exert a bronchial antispasmodic effect.

[0023] Y-27632·2HCl: As an inhibitor of ROCK-II, it has the ability to selectively inhibit Ca 2+ Sensitization, inhibiting smooth muscle contraction induced by a variety of stimulants. Y-27632·2HCl acts on cultured cells to inhibit the formation of Rho-induced p160 ROCK-regulated stress fibers. 10μM Y-27632·2HCl treatment of human embryonic stem cells in serum-free medium can significantly reduce cell apoptosis and increase cell cloning efficiency (from 1% to 27%).

[0024] Fructose: Carbohydrates are important energy substances in sheep semen preservation fluid or diluent. Their main function is to provide nutrition and energy for sheep sperm. In addition, carbohydrates can stabilize sperm membrane protein-lipid complexes and play a role in protecting sperm plasma membrane.

[0025] Citric acid monohydrate and tris(hydroxymethylaminomethane): both are buffer substances, which are essential components in goat semen preservation solution or diluent. The purpose of adding buffer substances to the diluent is to adjust and maintain the normal pH of semen to facilitate the survival of sperm.

[0026] Vitamin E and Vitamin C: Adding a certain concentration of vitamin E and vitamin C to the semen preservation solution can reduce the excessive superoxide produced during sperm preservation, reduce the oxidative stress damage to the sperm plasma membrane, and improve the quality of sperm preservation.

[0027] Fresh egg yolk: As a protective and energy substance, it is widely used in semen preservation or diluent. In addition, the low-density lipoprotein in the egg yolk directly penetrates the cell plasma membrane and can play a role in stabilizing and protecting the sperm plasma membrane.

[0028] Sodium penicillin and streptomycin sulfate: During the collection and storage process, semen is inevitably contaminated by certain microorganisms. Adding appropriate doses of antibacterial substances to the preservation solution or diluent can inhibit the growth of bacterial microorganisms, improve the ability to kill harmful bacteria, and prolong the storage time of sperm.

[0029] Glycerol: As an osmotic protective agent, it can lower the freezing point of cells, reduce the formation of ice crystals, alleviate the damage of free radicals to cells, and change the permeability of the cell membrane to electrolytes, drugs, toxins or metabolites.

[0030] Ectohydropyrimidine: It is an osmotic pressure compensating solute that can balance the osmotic pressure of cells. Ectohydropyrimidine will bind the surrounding water molecules to form a complex, wrap around cells, enzymes, proteins and other organic molecules, and form a protective layer with protective, nourishing and hydration-stabilizing functions. It stabilizes the enzyme molecular structure of enzyme preparations under adverse conditions such as high temperature, freezing and drying, maintains enzyme activity under extreme conditions such as freezing and thawing, and high temperature, and provides protection for enzymes, DNA, cell membranes and the entire cell.

[0031] Tiopronin: It is a new type of glycine derivative containing free thiol groups. It can remove free radicals in cells through the reversible binding of thiol groups with free radicals, activate metabolic enzymes, and chelate with toxic substances that hinder the activity of HS-enzymes to play a detoxifying role.

[0032] Bifendate: It has the function of stabilizing the surface structure of cell membranes, can enhance the cell's ability to resist damage, inhibit the damage to the cell plasma membrane structure and function caused by toxic metabolites, inhibit microsomal lipid peroxidation, inhibit the covalent binding of toxic substances to microsomes and lipids, and inhibit the consumption of reduced coenzyme II and oxygen during metabolism. In addition, bifendate has a significant induction effect on the terminal oxidase cytochrome P450.

[0033] Azithromycin: It is a new generation of macrolide antibiotics. Compared with erythromycin, it has stronger antibacterial activity, a wider antibacterial spectrum, and fewer adverse reactions. In addition to its antimicrobial effect, it also inhibits the expression and secretion of secretory phospholipase A, induces phospholipid denaturation to prevent the binding of phospholipase A to phospholipids, reduces the production and release of cell membrane arachidonic acid and eicosanoids, and inhibits inflammatory responses. Azithromycin accumulates in lysosomes, leading to increased extracellular secretion of lysosomal enzymes, promoting phagocytosis and digestion. By inhibiting the ERK1 / 2MAP and NF-KB signaling pathways, it inhibits the production of mucin and cytokines, and inhibits inflammatory responses.

[0034] (2) In order to solve the problem of aggregation of goat semen and egg yolk, the preservation solution prepared by the present invention is used to preserve goat semen, which can solve the problem of aggregation of goat semen and egg yolk, leading to sperm death, prolong the storage time of goat sperm under low temperature conditions, and achieve the best sperm quality.

[0035] (3) The storage method of the present invention achieves the maintenance of goat sperm for a long time and high quality under low temperature conditions by studying the composition of the storage solution and the ratio of each component. Low temperature storage effectively prolongs the utilization time of goat semen and reduces the metabolic efficiency of sperm. The components in the sperm storage solution can protect the sperm plasma membrane and provide the nutritional requirements of sperm low temperature metabolism. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail in conjunction with the embodiments below. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the scope of protection of the present invention. The raw materials involved in the following embodiments are all from common commercially available products:

[0037] A goat semen preservation solution and a preservation method thereof, wherein each 100 mL of the preservation solution comprises the following components:

[0038]

[0039]

[0040] Specifically, component A is a basic preservative solution, which can be stored for a long time after preparation; component B is added on the basis of component A, and the egg yolk in component B needs to be fresh and prepared before use. Other ingredients in component B are added to component A when needed, so it needs to be divided into component A and component B.

[0041] Example 1

[0042] A goat semen preservation solution, comprising the following components per 100 mL of the preservation solution:

[0043] Component A: 1.26 g fructose, 1.72 g citric acid monohydrate, 3.53 g tris(hydroxymethyl)aminomethane;

[0044] Component B: 20 ​​mL of fresh egg yolk, 4 mL of vitamin E, 0.5 g of bovine serum albumin, 0.5 g of vitamin C, 4 mL of glycerol, 15000 IU of penicillin sodium, 15000 IU of streptomycin sulfate, 0.64 mg of Y-27632·2HCl, 5.68 mg of ectoine, 0.21 mg of bifenthrin, 0.33 mg of tiopronin, 0.5 mg of amifostine, 2 mg of azithromycin, 0.1 mg of dexamethasone; the rest is double distilled water.

[0045] The concentration of dexamethasone was 1 μg / mL.

[0046] Example 2

[0047] A goat semen preservation solution, comprising the following components per 100 mL of the preservation solution:

[0048] Component A: 1.26 g fructose, 1.72 g citric acid monohydrate, 3.53 g tris(hydroxymethyl)aminomethane;

[0049] Component B: 20 ​​mL of fresh egg yolk, 4 mL of vitamin E, 0.5 g of bovine serum albumin, 0.5 g of vitamin C, 4 mL of glycerol, 15000 IU of penicillin sodium, 15000 IU of streptomycin sulfate, 0.64 mg of Y-27632·2HCl, 5.68 mg of ectoine, 0.21 mg of bifenthrin, 0.33 mg of tiopronin, 0.5 mg of amifostine, 2 mg of azithromycin, and 0.5 mg of dexamethasone; the rest is double distilled water.

[0050] The concentration of dexamethasone was 5 μg / mL.

[0051] Example 3

[0052] A goat semen preservation solution, comprising the following components per 100 mL of the preservation solution:

[0053] Component A: 1.26 g fructose, 1.72 g citric acid monohydrate, 3.53 g tris(hydroxymethyl)aminomethane;

[0054] Component B: 20 ​​mL of fresh egg yolk, 4 mL of vitamin E, 0.5 g of bovine serum albumin, 0.5 g of vitamin C, 4 mL of glycerol, 15000 IU of penicillin sodium, 15000 IU of streptomycin sulfate, 0.64 mg of Y-27632·2HCl, 5.68 mg of ectoine, 0.21 mg of bifenthrin, 0.33 mg of tiopronin, 0.5 mg of amifostine, 2 mg of azithromycin, and 2 mg of dexamethasone; the rest is double distilled water.

[0055] The concentration of dexamethasone was 20 μg / mL.

[0056] Example 4

[0057] A goat semen preservation solution, comprising the following components per 100 mL of the preservation solution:

[0058] Component A: 1.26 g fructose, 1.72 g citric acid monohydrate, 3.53 g tris(hydroxymethyl)aminomethane;

[0059] Component B: 20 ​​mL of fresh egg yolk, 4 mL of vitamin E, 0.5 g of bovine serum albumin, 0.5 g of vitamin C, 4 mL of glycerol, 15000 IU of penicillin sodium, 15000 IU of streptomycin sulfate, 0.64 mg of Y-27632·2HCl, 5.68 mg of ectoine, 0.21 mg of bifenthrin, 0.33 mg of tiopronin, 0.5 mg of amifostine, 2 mg of azithromycin, and 5 mg of dexamethasone; the rest is double distilled water.

[0060] The concentration of dexamethasone is 50 μg / mL.

[0061] Comparative Example 1

[0062] A goat semen preservation solution, comprising the following components per 100 mL of the preservation solution:

[0063] Component A: 1.26 g fructose, 1.72 g citric acid monohydrate, 3.53 g tris(hydroxymethyl)aminomethane;

[0064] Component B: 20 ​​mL of fresh egg yolk, 4 mL of vitamin E, 0.5 g of bovine serum albumin, 0.5 g of vitamin C, 4 mL of glycerol, 15,000 IU of penicillin sodium, 15,000 IU of streptomycin sulfate, 0.64 mg of Y-27632·2HCl, 5.68 mg of ectoine, 0.21 mg of bifendate, 0.33 mg of tiopronin, 0.5 mg of amifostine, 2 mg of azithromycin; the rest is double distilled water;

[0065] In the above Examples 1-4 and Comparative Example 1,

[0066] 1. The steps for collecting semen are:

[0067] In the breeding sheep farm, a semen collection site is set up. Before semen collection, the ram is penned into the shed, and a fixing frame for fixing ewes is placed in the shed. Before semen collection, the shed is cleaned. In the ewe shed, a healthy, disease-free, strong, and docile ewe is found and fixed on the fixing frame to ensure that the female animal is within the male animal's field of vision in order to arouse the ram's sexual desire. Clean the false vagina, the semen collection cup, and the glass rod, and install the prepared semen collection cup at one end of the false vagina. Then pour 40℃ hot water into the interlayer of the false vagina and install the air nozzle with a piston; use the glass rod to take a small amount of vaseline and evenly apply a layer on the inner tube. After blowing and pressurizing, the end with the vaseline applied should be triangular.

[0068] 2. The cryopreservation method of sheep semen is:

[0069] The collected qualified sheep semen was diluted 8 times with the prepared preservation solution, and the diluted semen was divided into centrifuge tubes or capillary tubes, wrapped with absorbent cotton, and stored in a 4°C refrigerator.

[0070] 3. Semen index detection is:

[0071] Sperm motility and movement parameters were tested using a sperm quality analyzer (CASA). Before testing, the diluted semen was shaken evenly, 10 μL of the middle layer of semen was taken on a slide, covered with a cover slip, and placed under a microscope (400×) equipped with a 37°C constant temperature stage for testing and analysis. More than 5 fields of view were randomly selected under the microscope for measurement, with at least 200 sperm in each field of view, and the sperm motility and movement parameter values ​​of each field of view were measured.

[0072] Method for determining the plasma membrane integrity rate: using the osmotic swelling test (HOST) test, take 200 μL of hypotonic solution (composition: 4.9g trisodium citrate and 9.0g fructose in 1000mL double distilled water) and add it to 20 μL of semen, mix and incubate at 37°C for 35 minutes, then smear and air dry, fix with 2% glutaraldehyde for 15 minutes and then wash with water. After drying, test under a microscope, count 200 sperms, and divide the number of sperms that meet the test criteria by the total number of sperms. Sperm plasma membrane integrity rate = (number of sperms with intact plasma membranes / total number of sperms) × 100%.

[0073] Method for determining the acrosome integrity rate: using the Giemsa staining method, take 20 μL of diluted semen smear and air-dry it, then fix it with 4% formaldehyde for 15 minutes, rinse it and air-dry it, and use a pipette to absorb the newly prepared Giemsa dye solution for staining, ensuring that the dye solution is evenly spread on the smear for 2 hours. Finally, wash and air-dry it, and detect it under a microscope according to the detection standards, and count 200 sperm. Sperm acrosome integrity rate = (number of sperm with normal acrosomes / total number of sperm) × 100%.

[0074] 4. The results are analyzed as follows:

[0075] The effects of the preservation solutions prepared in Examples 1-4 and Comparative Example 1 on goat sperm under low temperature storage are shown in Tables 1-24 below:

[0076] Note: In the following table, a, b, c, and d represent the significant differences between different groups at each time point. Different letters indicate significant differences (p<0.05), and the same letters indicate no significant differences (p>0.05).

[0077] Table 1 Effect of dexamethasone concentration on goat sperm motility (%) stored at 4°C

[0078]

[0079] As shown in Table 1, after the goat semen was stored at 4°C for 1 day, the treatment groups of Examples 1-4 can effectively improve sperm motility, and the difference between the Examples and Comparative Example 1 with different concentrations of dexamethasone added reached a significant level (p<0.05). After the 6th day, the sperm motility of the treatment groups of Examples 1-4 was significantly higher than that of Comparative Example 1 (p<0.05), and the sperm motility of the 20μg / mL dexamethasone treatment group (Example 3) was significantly higher than that of the 1μg / mL, 5μg / mL and 50μg / mL treatment groups (Example 1, Example 2 and Example 4) (p<0.05). In addition, the sperm motility of the 20μg / mL dexamethasone treatment on the 7th day was still 64.99%, while the sperm motility of Comparative Example 1 on the 7th day was only 38.52%.

[0080] The above results show that when goat sperm is stored at 4°C, adding dexamethasone to the storage solution can effectively improve the sperm survival rate. At the same time, using 20μg / mL dexamethasone can more effectively prolong the storage time of semen at 4°C. In addition, from the analysis of the results, sperm motility does not increase with the increase of dexamethasone concentration, indicating that dexamethasone is a non-dose-dependent semen preservation additive.

[0081] Table 2 Effect of dexamethasone concentration on plasma membrane integrity of goat sperm stored at 4°C (%)

[0082]

[0083] As shown in Table 2, the plasma membrane integrity rate decreases with the increase of storage time, but the addition of dexamethasone helps to improve the plasma membrane integrity rate of goat sperm during storage. Among them, when stored for 1 day, the sperm plasma membrane integrity rate of the treatment group of Examples 1-4 was significantly higher than that of the sperm plasma membrane integrity rate of Comparative Example 1 (p<0.05); when stored for 7 days, the plasma membrane integrity rate of the treatment group of Examples 3-4 was significantly higher than that of the treatment group of Examples 1-2 and Comparative Example 1 (p<0.05). The results confirmed that the sperm plasma membrane integrity rate of the 20μg / mL treatment group remained the highest during the storage process and was significantly higher than that of Comparative Example 1 (p<0.05).

[0084] Table 3 Effect of dexamethasone concentration on acrosome integrity rate (%) of goat sperm stored at 4°C

[0085]

[0086]

[0087] As can be seen from Table 3, the acrosome integrity rate decreases with the increase of storage time, but the addition of dexamethasone helps to improve the acrosome integrity rate of goat sperm during storage. Among them, when stored for 1 day, the sperm acrosome integrity rate of the treatment group of Example 1-4 was significantly higher than that of the sperm acrosome integrity rate of Comparative Example 1 (p<0.05); when stored for 7 days, the sperm acrosome integrity rate of the treatment group of Example 3-4 was significantly higher than that of the treatment group of Example 1-2 and Comparative Example 1 (p<0.05). The results confirmed that the sperm acrosome integrity rate of the 20μg / mL treatment group remained the highest during the storage process, and was significantly higher than that of Comparative Example 1 (p<0.05).

[0088] Table 4 Effect of dexamethasone concentration on the linear velocity (μm / s) of goat sperm stored at 4℃

[0089] Time / d Comparative Example 1 Example 1 Example 2 Example 3 Example 4 1 <![CDATA[23.55±2.83 a ]]> <![CDATA[24.87±0.86 a ]]> <![CDATA[22.88±1.07 a ]]> <![CDATA[24.76±2.15 a ]]> <![CDATA[23.82±1.11 a ]]> 2 <![CDATA[21.07±1.92 b ]]> <![CDATA[22.14±2.3 ab ]]> <![CDATA[22.41±1.86 ab ]]> <![CDATA[23.94±1.69 ab ]]> <![CDATA[24.89±1.49 a ]]> 3 <![CDATA[21.44±1.33 a ]]> <![CDATA[21.62±1.09 a ]]> <![CDATA[22.86±2.07 a ]]> <![CDATA[22.50±1.74 a ]]> <![CDATA[23.37±1.74 a ]]> 4 <![CDATA[21.00±0.97 a ]]> <![CDATA[21.78±2.36 a ]]> <![CDATA[21.68±1.05 a ]]> <![CDATA[22.63±1.31 a ]]> <![CDATA[22.84±1.17 a ]]> 5 <![CDATA[18.28±0.93 a ]]> <![CDATA[19.56±0.78 a ]]> <![CDATA[19.98±1.83 a ]]> <![CDATA[20.05±1.91 a ]]> <![CDATA[19.17±1.35 a ]]> 6 <![CDATA[19.64±0.99 ab ]]> <![CDATA[18.23±0.95 b ]]> <![CDATA[19.34±2.05 ab ]]> <![CDATA[20.93±0.88 a ]]> <![CDATA[18.68±0.89 b ]]> 7 <![CDATA[18.18±1.24 a ]]> <![CDATA[16.29±1.16 b ]]> <![CDATA[16.04±0.84 b ]]> <![CDATA[18.11±0.29 a ]]> <![CDATA[17.35±1.07 ab ]]>

[0090] As shown in Table 4, on the 7th day, the linear sperm velocity of the treatment group of Example 3 was not significantly different from that of the control group 1 (p>0.05), and the linear sperm velocity of the treatment group of Example 3 was significantly higher than that of the treatment groups of other examples (p<0.05).

[0091] Table 5 Effect of dexamethasone concentration on the curve velocity (μm / s) of goat sperm stored at 4℃

[0092]

[0093]

[0094] As shown in Table 5, the sperm curve velocity of the treatment groups of Examples 1-4 was significantly lower than that of Comparative Example 1 (p<0.05) at 7 days, and the sperm curve velocity of the treatment groups of Examples 3-4 was significantly higher than that of the treatment groups of Examples 1-2 (p<0.05).

[0095] Table 6 Effect of dexamethasone concentration on path velocity (μm / s) of goat sperm stored at 4°C

[0096]

[0097] As shown in Table 6, on the 7th day, the sperm path speed of the Example 1-4 treatment group was significantly lower than that of the Comparative Example 1 (p<0.05), and the Example 3-4 treatment group was significantly higher than that of the Example 1-2 treatment group (p<0.05).

[0098] Table 7 Effect of dexamethasone concentration on the lateral swing amplitude (μm) of goat sperm stored at 4℃

[0099]

[0100]

[0101] As shown in Table 7, the sperm lateral swing amplitude of Examples 1-4 is significantly lower than that of Comparative Example 1 (p<0.05), and the Example 3-4 treatment group is significantly higher than that of the Example 1-2 treatment group (p<0.05).

[0102] Table 8 Effect of dexamethasone concentration on the motility of goat sperm stored at 4°C

[0103] Time / d Comparative Example 1 Example 1 Example 2 Example 3 Example 4 1 <![CDATA[0.93±0.05 a ]]> <![CDATA[0.95±0.01 a ]]> <![CDATA[0.94±0.03 a ]]> <![CDATA[0.91±0.05 a ]]> <![CDATA[0.94±0.04 a ]]> 2 <![CDATA[0.95±0.05 a ]]> <![CDATA[0.92±0.03 a ]]> <![CDATA[0.93±0.04 a ]]> <![CDATA[0.95±0.04 a ]]> <![CDATA[0.97±0.02 a ]]> 3 <![CDATA[0.95±0.04 a ]]> <![CDATA[0.94±0.06 a ]]> <![CDATA[0.96±0.03 a ]]> <![CDATA[0.93±0.04 a ]]> <![CDATA[0.92±0.02 a ]]> 4 <![CDATA[0.88±0.1 a ]]> <![CDATA[0.89±0.11 a ]]> <![CDATA[0.94±0.06 a ]]> <![CDATA[0.95±0.04 a ]]> <![CDATA[0.96±0.03 a ]]> 5 <![CDATA[0.88±0.1 a ]]> <![CDATA[0.89±0.11 a ]]> <![CDATA[0.94±0.06 a ]]> <![CDATA[0.95±0.04 a ]]> <![CDATA[0.96±0.03 a ]]> 6 <![CDATA[0.89±0.07 a ]]> <![CDATA[0.95±0.03 a ]]> <![CDATA[0.89±0.04 a ]]> <![CDATA[0.93±0.03 a ]]> <![CDATA[0.91±0.01 a ]]> 7 <![CDATA[0.96±0.05 a ]]> <![CDATA[0.73±0.06 c ]]> <![CDATA[0.82±0.09 b ]]> <![CDATA[0.91±0.06 ab ]]> <![CDATA[0.92±0.04 ab ]]>

[0104] As shown in Table 8, on the 7th day, the sperm motility of the treatment group of Example 3-4 was not significantly different from that of Comparative Example 1 (p>0.05), the sperm motility of the treatment group of Example 1-2 was significantly lower than that of Comparative Example 1 (p<0.05), and the sperm motility of the treatment group of Example 2 was not significantly different from that of the treatment group of Example 3-4 (p>0.05).

[0105] Table 9 Effect of dexamethasone concentration on the whipping frequency of goat sperm stored at 4℃

[0106] Time / d Comparative Example 1 Example 1 Example 2 Example 3 Example 4 1 <![CDATA[0.68±0.03 a ]]> <![CDATA[0.76±0.05 a ]]> <![CDATA[0.71±0.07 a ]]> <![CDATA[0.71±0.04 a ]]> <![CDATA[0.74±0.06 a ]]> 2 <![CDATA[0.72±0.09 a ]]> <![CDATA[0.73±0.07 a ]]> <![CDATA[0.74±0.02 a ]]> <![CDATA[0.74±0.05 a ]]> <![CDATA[0.73±0.07 a ]]> 3 <![CDATA[0.72±0.05 a ]]> <![CDATA[0.73±0.05 a ]]> <![CDATA[0.74±0.07 a ]]> <![CDATA[0.72±0.05 a ]]> <![CDATA[0.72±0.06 a ]]> 4 <![CDATA[0.67±0.02 b ]]> <![CDATA[0.68±0.03 ab ]]> <![CDATA[0.72±0.04 a ]]> <![CDATA[0.71±0.04 ab ]]> <![CDATA[0.71±0.02 ab ]]> 5 <![CDATA[0.66±0.02 a ]]> <![CDATA[0.67±0.06 a ]]> <![CDATA[0.67±0.07 a ]]> <![CDATA[0.73±0.04 a ]]> <![CDATA[0.72±0.05 a ]]> 6 <![CDATA[0.71±0.05 a ]]> <![CDATA[0.77±0.07 a ]]> <![CDATA[0.71±0.04 a ]]> <![CDATA[0.75±0.03 a ]]> <![CDATA[0.71±0.04 a ]]> 7 <![CDATA[0.82±0.05 a ]]> <![CDATA[0.72±0.07 b ]]> <![CDATA[0.75±0.04 ab ]]> <![CDATA[0.72±0.03 b ]]> <![CDATA[0.75±0.04 ab ]]>

[0107] As shown in Table 9, on the 7th day, the sperm whipping frequency of the treatment groups of Example 1 and Example 3 was significantly lower than that of Comparative Example 1 (p<0.05), and there was no significant difference between the treatment groups of Example 2 and Example 4 and Comparative Example 1 (p>0.05).

[0108] Table 10 Effect of dexamethasone concentration on the linearity of goat sperm stored at 4°C

[0109] Time / d Comparative Example 1 Example 1 Example 2 Example 3 Example 4 1 <![CDATA[0.43±0.04 a ]]> <![CDATA[0.41±0.01 a ]]> <![CDATA[0.43±0.04 a ]]> <![CDATA[0.44±0.02 a ]]> <![CDATA[0.42±0.03 a ]]> 2 <![CDATA[0.41±0.06 a ]]> <![CDATA[0.42±0.05 a ]]> <![CDATA[0.42±0.04 a ]]> <![CDATA[0.41±0.03 a ]]> <![CDATA[0.40±0.04 a ]]> 3 <![CDATA[0.42±0.03 a ]]> <![CDATA[0.40±0.06 a ]]> <![CDATA[0.41±0.04 a ]]> <![CDATA[0.41±0.03 a ]]> <![CDATA[0.43±0.04 a ]]> 4 <![CDATA[0.46±0.05 a ]]> <![CDATA[0.43±0.06 ab ]]> <![CDATA[0.43±0.03 ab ]]> <![CDATA[0.39±0.04 b ]]> <![CDATA[0.41±0.02 ab ]]> 5 <![CDATA[0.46±0.01 a ]]> <![CDATA[0.46±0.07 a ]]> <![CDATA[0.43±0.03 a ]]> <![CDATA[0.43±0.03 a ]]> <![CDATA[0.46±0.02 a ]]> 6 <![CDATA[0.44±0.05 a ]]> <![CDATA[0.38±0.06 b ]]> <![CDATA[0.46±0.03 a ]]> <![CDATA[0.44±0.01 a ]]> <![CDATA[0.45±0.02 a ]]> 7 <![CDATA[0.34±0.03 c ]]> <![CDATA[0.52±0.05 a ]]> <![CDATA[0.50±0.02 a ]]> <![CDATA[0.43±0.02 b ]]> <![CDATA[0.45±0.01 b ]]>

[0110] As shown in Table 10, on the 7th day, the sperm linearity of Examples 1-4 was significantly higher than that of Comparative Example 1 (p<0.05), and the Example 1-2 treatment group was significantly higher than that of the Example 3-4 treatment group (p<0.05).

[0111] Table 11 Effect of dexamethasone concentration on the movement angle of goat sperm stored at 4℃

[0112] Time / d Comparative Example 1 Example 1 Example 2 Example 3 Example 4 1 <![CDATA[92.35±10.94 b ]]> <![CDATA[158.18±12.35 a ]]> <![CDATA[140.34±34.03 a ]]> <![CDATA[94.04±15.93 b ]]> <![CDATA[96.60±30.43 b ]]> 2 <![CDATA[103.45±25.11 b ]]> <![CDATA[121.04±37.74 b ]]> <![CDATA[166.06±23.97 a ]]> <![CDATA[177.63±23.7 a ]]> <![CDATA[138.00±24.58 ab ]]> 3 <![CDATA[108.40±16.66 ab ]]> <![CDATA[100.72±7.81 b ]]> <![CDATA[124.14±31.59 ab ]]> <![CDATA[138.34±26.26 a ]]> <![CDATA[122.18±6.66 ab ]]> 4 <![CDATA[102.47±11.96 bc ]]> <![CDATA[99.19±14.9 c ]]> <![CDATA[127.94±26.56 b ]]> <![CDATA[176.67±16.59 a ]]> <![CDATA[90.58±4.96 c ]]> 5 <![CDATA[89.07±5.18 b ]]> <![CDATA[80.53±23.27 b ]]> <![CDATA[89.77±24.87 b ]]> <![CDATA[124.18±15.08 a ]]> <![CDATA[102.15±14.09 ab ]]> 6 <![CDATA[129.16±17.99 ab ]]> <![CDATA[130.82±29.63 ab ]]> <![CDATA[110.76±11.42 b ]]> <![CDATA[157.74±4.34 a ]]> <![CDATA[146.76±28.04 a ]]> 7 <![CDATA[55.15±7.72 b ]]> <![CDATA[49.58±5.2 b ]]> <![CDATA[61.48±10.75 b ]]> <![CDATA[122.31±16.98 a ]]> <![CDATA[126.43±21.65 a ]]>

[0113] As shown in Table 11, on the 7th day, the sperm movement angle of the Example 3-4 treatment group was significantly higher than that of the Example 1-2 treatment group (p<0.05), and there was no significant difference between the Example 1-2 treatment group and the comparative example 1 (p>0.05).

[0114] Table 12 Effect of dexamethasone concentration on the forward mobility of goat sperm stored at 4°C

[0115]

[0116]

[0117] As shown in Table 12, on the 7th day, the sperm forwardness of Examples 1-4 was significantly higher than that of Comparative Example 1 (p<0.05), and the Example 1-2 treatment group was significantly higher than that of the Example 3-4 treatment group (p<0.05).

[0118] In summary, it can be seen from Tables 4 to 12 that when goat sperm is stored at 4°C, adding dexamethasone to the storage solution can effectively slow down sperm motility. At the same time, using 20 μg / mL dexamethasone can more effectively prolong the storage time of semen at 4°C.

[0119] Ultra-low temperature cryopreservation method of goat semen

[0120] Dexamethasone of different concentrations, 0 μg / mL and 20 μg / mL, was added to the goat sperm preservation solution for ultra-low temperature cryopreservation in liquid nitrogen. The collected qualified goat semen was diluted 8 times with the prepared preservation solution, and the diluted semen was divided into capillaries, sealed with polyvinyl alcohol powder, wrapped with absorbent cotton, placed in a 4°C refrigerator for slow cooling and equilibrium, and then the capillaries were placed in the pre-prepared liquid nitrogen for fumigation, and finally stored in liquid nitrogen.

[0121] Semen index test:

[0122] The goat semen stored in the ultra-low temperature cryopreservation of liquid nitrogen is placed in a water bath at about 40°C for thawing, and the sperm motility, plasma membrane integrity rate and acrosome integrity rate of the thawed goat semen are respectively measured. The specific steps include:

[0123] Method for determination of motility and motility parameters: Use sperm quality analyzer (CASA) to detect sperm motility index and motility parameters. Shake the diluted semen before testing, take 10μL of the middle layer of semen on a slide, cover it with a cover slip, and place it under a microscope (400×) equipped with a 37°C constant temperature stage for detection and analysis. Randomly select more than 5 fields of view under the microscope for measurement, with at least 200 sperm in each field of view, and measure the sperm motility and motility parameter values ​​of each field of view.

[0124] Method for determining the plasma membrane integrity rate: using the osmotic swelling test (HOST) test, take 200 μL of hypotonic solution (composition: 4.9g trisodium citrate and 9.0g fructose in 1000mL double distilled water) and add it to 20 μL of semen, mix and incubate at 37°C for 35 minutes, then smear and air dry, fix with 2% glutaraldehyde for 15 minutes and then wash with water. After drying, test under a microscope, count 200 sperms, and divide the number of sperms that meet the test criteria by the total number of sperms. Sperm plasma membrane integrity rate = (number of sperms with intact plasma membranes / total number of sperms) × 100%.

[0125] Method for determining the acrosome integrity rate: using the Giemsa staining method, take 20 μL of diluted semen smear and air-dry it, then fix it with 4% formaldehyde for 15 minutes, rinse it and air-dry it, and use a pipette to absorb the newly prepared Giemsa dye solution for staining, ensuring that the dye solution is evenly spread on the smear for 2 hours. Finally, wash and air-dry it, and detect it under a microscope according to the detection standards, and count 200 sperm. Sperm acrosome integrity rate = (number of sperm with normal acrosomes / total number of sperm) × 100%.

[0126] result

[0127] Table 13 Effect of dexamethasone on goat cryopreserved sperm motility (%)

[0128] Dexamethasone (μg / mL) vitality(%) 0 <![CDATA[64.04±1.70 a ]]> 20 <![CDATA[73.44±1.62 b ]]>

[0129] Note: a, b indicate significant differences between different groups. Different letters indicate significant differences (p<0.05), and the same letters indicate no significant differences (p>0.05).

[0130] As shown in Table 13, the sperm motility of the cells supplemented with 20 μg / mL dexamethasone after freezing was significantly higher than that of the control group (p<0.05).

[0131] Table 14 Effect of dexamethasone on plasma membrane of goat cryopreserved sperm (%)

[0132] Dexamethasone (μg / mL) Plasma membrane (%) 0 <![CDATA[0.65±0.02 a ]]> 20 <![CDATA[0.73±0.01 b ]]>

[0133] Note: a, b indicate significant differences between different groups. Different letters indicate significant differences (p<0.05), and the same letters indicate no significant differences (p>0.05).

[0134] As shown in Table 14, the sperm plasma membrane integrity rate of the sperm added with 20 μg / mL dexamethasone after freezing was significantly higher than that of the control group (p<0.05).

[0135] Table 15 Effect of dexamethasone on goat cryopreserved sperm acrosome (%)

[0136] Dexamethasone (μg / mL) Acrosome (%) 0 <![CDATA[0.65±0.01 a ]]> 20 <![CDATA[0.73±0.02 b ]]>

[0137] Note: a, b indicate significant differences between different groups. Different letters indicate significant differences (p<0.05), and the same letters indicate no significant differences (p>0.05).

[0138] As shown in Table 15, the acrosome integrity rate of sperm added with 20 μg / mL dexamethasone after freezing was significantly higher than that of the control group (p<0.05).

[0139] Table 16 Effect of dexamethasone on linear velocity (μm / s) of cryopreserved sperm in goats

[0140] Dexamethasone (μg / mL) Linear velocity (μm / s) 0 <![CDATA[19.01±1.49 a ]]> 20 <![CDATA[21.54±0.85 a ]]>

[0141] Note: a, b indicate significant differences between different groups. Different letters indicate significant differences (p<0.05), and the same letters indicate no significant differences (p>0.05).

[0142] As shown in Table 16, there was no significant difference in the linear velocity of sperm added with 20 μg / mL dexamethasone after freezing compared with the control group (p>0.05).

[0143] Table 17 Effect of dexamethasone on the curve velocity (μm / s) of goat cryopreserved sperm

[0144] Dexamethasone (μg / mL) Curve speed (μm / s) 0 <![CDATA[39.58±6.65 a ]]> 20 <![CDATA[52.84±2.71 b ]]>

[0145] Note: a, b indicate significant differences between different groups. Different letters indicate significant differences (p<0.05), and the same letters indicate no significant differences (p>0.05).

[0146] As shown in Table 17, the sperm curve velocity of the sperm with 20 μg / mL dexamethasone added after freezing was significantly higher than that of the control group (p<0.05).

[0147] Table 18 Effect of dexamethasone on path velocity (μm / s) of cryopreserved sperm in goats

[0148] Dexamethasone (μg / mL) Path speed (μm / s) 0 <![CDATA[27.99±4.70 a ]]> 20 <![CDATA[37.37±1.92 b ]]>

[0149] Note: a, b indicate significant differences between different groups. Different letters indicate significant differences (p<0.05), and the same letters indicate no significant differences (p>0.05).

[0150] As shown in Table 18, the sperm path velocity (μm) of the cells supplemented with 20 μg / mL dexamethasone after freezing was significantly higher than that of the control group (p<0.05).

[0151] Table 19 Effect of dexamethasone on the lateral swing amplitude (μm) of goat cryopreserved sperm

[0152] Dexamethasone (μg / mL) Side swing amplitude (μm) 0 <![CDATA[11.59±1.95 a ]]> 20 <![CDATA[15.48±0.80 b ]]>

[0153] Note: a, b indicate significant differences between different groups. Different letters indicate significant differences (p<0.05), and the same letters indicate no significant differences (p>0.05).

[0154] As shown in Table 19, the lateral swing amplitude of sperm added with 20 μg / mL dexamethasone after freezing was significantly higher than that of the control group (p<0.05).

[0155] Table 20 Effect of dexamethasone on goat cryopreserved sperm motility

[0156] Dexamethasone (μg / mL) Swing 0 <![CDATA[0.85±0.05 a ]]> 20 <![CDATA[0.95±0.03 a ]]>

[0157] Note: a, b indicate significant differences between different groups. Different letters indicate significant differences (p<0.05), and the same letters indicate no significant differences (p>0.05).

[0158] As shown in Table 20, there was no significant difference in sperm motility between the group with 20 μg / mL dexamethasone added after freezing and the control group (p>0.05).

[0159] Table 21 Effect of dexamethasone on whipping frequency of cryopreserved sperm in goats

[0160] Dexamethasone (μg / mL) Whipping frequency 0 <![CDATA[0.7±0.1 a ]]> 20 <![CDATA[0.74±0.01 a ]]>

[0161] Note: a, b indicate significant differences between different groups. Different letters indicate significant differences (p<0.05), and the same letters indicate no significant differences (p>0.05).

[0162] As shown in Table 21, there was no significant difference in the sperm whipping frequency between the group with 20 μg / mL dexamethasone added after freezing and the control group (p>0.05).

[0163] Table 22 Effect of dexamethasone on the linearity of goat cryopreserved sperm

[0164] Dexamethasone (μg / mL) Linearity 0 <![CDATA[0.49±0.04 a ]]> 20 <![CDATA[0.41±0.01 b ]]>

[0165] Note: a, b indicate significant differences between different groups. Different letters indicate significant differences (p<0.05), and the same letters indicate no significant differences (p>0.05).

[0166] As shown in Table 22, the linearity of sperm added with 20 μg / mL dexamethasone after freezing was significantly higher than that of the control group (p<0.05).

[0167] Table 23 Effect of dexamethasone on the movement angle of goat cryopreserved sperm

[0168] Dexamethasone (μg / mL) Moving Angle 0 <![CDATA[108.66±19.66 a ]]> 20 <![CDATA[122.56±3.45 a ]]>

[0169] Note: a, b indicate significant differences between different groups. Different letters indicate significant differences (p<0.05), and the same letters indicate no significant differences (p>0.05).

[0170] As shown in Table 23, there was no significant difference in the sperm movement angle between the group with 20 μg / mL dexamethasone added after freezing and the control group (p>0.05).

[0171] Table 24 Effect of dexamethasone on the forward tendency of goat cryopreserved sperm

[0172] Dexamethasone (μg / mL) Forward 0 <![CDATA[0.69±0.06 a ]]> 20 <![CDATA[0.58±0.01 b ]]>

[0173] Note: a, b indicate significant differences between different groups. Different letters indicate significant differences (p<0.05), and the same letters indicate no significant differences (p>0.05).

[0174] As shown in Table 24, the forward tropism of sperm added with 20 μg / mL dexamethasone after freezing was significantly higher than that of the control group (p<0.05).

[0175] It can be seen from Tables 13 to 24 that when cryopreserving goat sperm, adding 20 μg / mL dexamethasone to the preservation solution can effectively improve the quality of semen in liquid nitrogen ultra-low temperature cryopreservation.

[0176] The goat semen preservation solution and the preservation method thereof of the present invention are also applicable to the preservation of sheep semen.

[0177] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A goat semen preservation solution, characterized in that: The preservation solution contains the following components per 100 mL: Component A: 1.2-1.3 g of fructose, 1.7-1.8 g of citric acid monohydrate, 3.5-3.6 g of tris(hydroxymethyl)aminomethane; Component B: 19-21 mL of fresh egg yolk, 3.5-4.5 mL of vitamin E, 0.4-0.6 g of bovine serum albumin, 0.4-0.6 g of vitamin C, 3.5-4.5 mL of glycerol, 10,000-15,000 IU of penicillin sodium, 10,000-15,000 IU of streptomycin sulfate, 0.5-0.7 mg of Y-27632·2HCl, 5-6 mg of ectoine, 0.2-0.3 mg of bifendate, 0.3-0.4 mg of tiopronin, 0.5-0.6 mg of amifostine, 2-2.5 mg of azithromycin, and 0.1-5 mg of dexamethasone; The rest was double distilled water.

2. The goat semen preservation solution according to claim 1, characterized in that: The concentration of dexamethasone is 20 μg / mL.

3. The goat semen preservation solution according to claim 1, characterized in that: The preservation solution contains the following components per 100 mL: Component A: 1.26 g fructose, 1.72 g citric acid monohydrate, 3.53 g tris(hydroxymethyl)aminomethane; Component B: 20 ​​mL of fresh egg yolk, 4 mL of vitamin E, 0.5 g of bovine serum albumin, 0.5 g of vitamin C, 4 mL of glycerol, 15,000 IU of penicillin sodium, 15,000 IU of streptomycin sulfate, 0.64 mg of Y-27632·2HCl, 5.68 mg of ectoine, 0.21 mg of bifenthrin, 0.33 mg of tiopronin, 0.5 mg of amifostine, 2 mg of azithromycin, and 2 mg of dexamethasone; The rest was double distilled water.

4. A method for preserving goat semen using the preservative solution according to any one of claims 1 to 3, characterized in that: The following steps are involved: The collected qualified goat semen is diluted 8 times with the preservation solution, and the diluted semen is then dispensed into centrifuge tubes or capillaries, wrapped with absorbent cotton, and stored in a refrigerator at 4°C.

5. A method for preserving goat semen using the preservative solution according to any one of claims 1 to 3, characterized in that: The following steps are involved: The collected qualified sheep semen is diluted 8 times with the preservation solution, and the diluted semen is then packaged into straw tubes, the straw tubes are sealed with polyvinyl alcohol powder, and then the straw tubes are placed in pre-prepared liquid nitrogen for fumigation, and finally the straw tubes are placed in liquid nitrogen for preservation.

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