阅读说明：本技术 淫羊藿多糖用于制备山羊***冷冻保存稀释液的应用 (Application of epimedium polysaccharide in preparation of goat semen cryopreservation diluent ) 是由 胡建宏 李宇 张利坤 张星 任发 席华明 牛统娟 韩宁 王立强 李延华 于 2020-07-10 设计创作，主要内容包括：本发明提供了淫羊藿多糖用于制备山羊精液冷冻保存稀释液的应用。本发明利用淫羊藿多糖作为山羊精液的冷冻保存剂有效成分,能够在山羊精液冷冻保存过程中很好地保护精子的生理活性,包括一步稀释法和两步稀释法。尤其是,相对于现有山羊精液冷冻普遍采用两步稀释法的情况,本发明的含有淫羊藿多糖的冷冻保存剂配合一步稀释法冷冻程序即可达到冷冻保存的要求。(The invention provides application of epimedium polysaccharide in preparing goat semen cryopreservation diluent. The invention utilizes epimedium polysaccharide as the effective component of the cryopreservation agent of goat semen, can well protect the physiological activity of the sperm in the process of cryopreservation of the goat semen, and comprises a one-step dilution method and a two-step dilution method. Particularly, compared with the condition that the existing goat semen freezing generally adopts a two-step dilution method, the cryopreservation agent containing the epimedium polysaccharide can meet the requirement of cryopreservation by matching with a one-step dilution method freezing program.)

1. The epimedium polysaccharide is used for preparing dilution for freezing and preserving goat semen.

2. The goat semen cryopreservation diluent is characterized by comprising Tris, citric acid, fructose, epimedium polysaccharide, antibiotics and egg yolk.

3. The goat semen cryopreservation diluent as claimed in claim 2, wherein the cryopreservation diluent contains glycerol.

4. The goat semen cryopreservation diluent as claimed in claim 2, wherein the antibiotic is selected from one or a combination of two of penicillin and streptomycin at any ratio.

5. The goat semen cryopreservation diluent as claimed in claim 2, wherein the goat semen cryopreservation diluent formula is as follows: 200-300 mM Tris, 80-90 mM citric acid, 65-75 mM fructose, 15-25% (V/V) yolk, 1-4 mg/mL epimedium polysaccharide, 2000IU/mL penicillin, 0.4g/mL streptomycin and 100mL ultrapure water.

6. The goat semen cryopreservation diluent as claimed in claim 5, wherein the goat semen cryopreservation diluent formula contains 4% to 6% (V/V) of glycerol.

7. The goat semen cryopreservation diluent as claimed in claim 5, wherein the epimedium herb polysaccharide concentration is 2.5-3.5 mg/mL.

8. A goat semen cryopreservation method is characterized by comprising the following steps: isothermal mixing and dilution of goat semen with the cryopreservation diluent of any one of claims 3 or 6; and then gradually cooling the mixed diluent to 3-5 ℃, balancing at 3-5 ℃, and freezing for storage.

9. The method for cryopreservation of goat semen as claimed in claim 8, the method comprising: the mixed dilution adopts a multi-step dilution method, and the multi-step dilution method comprises dilution to a final concentration through one or more steps of intermediate concentrations.

10. A goat semen cryopreservation method is characterized by comprising the following steps: the goat semen and the cryopreservation diluent as claimed in any one of claims 2 or 5 are mixed and diluted isothermally, then the mixed diluent is gradually cooled to 3-5 ℃, after the mixed diluent is equilibrated at 3-5 ℃, the equilibrated mixed diluent and the cryopreservation diluent as claimed in any one of claims 3 or 6 are mixed and diluted isothermally, and then the goat semen is cryopreserved after the equilibration at 3-5 ℃.

Technical Field

The invention relates to the technical field of biology, in particular to application of epimedium polysaccharide in a diluent for freezing and storing goat semen and a freezing program by a one-step dilution method.

Background

Compared with a one-step dilution method, the two-step dilution method has ideal effect in the existing freezing preservation technology of goat semen, but the procedure of the two-step dilution method is relatively complex.

Disclosure of Invention

The invention provides the application of epimedium polysaccharide in preparing goat semen cryopreservation diluent in combination with the defects of the prior art.

Further, the invention provides a goat semen cryopreservation diluent. Therefore, the cryopreservation diluent provided by the invention comprises Tris, citric acid, fructose, epimedium polysaccharide, antibiotics and yolk.

Further, the cryopreservation diluent contains glycerol.

Optionally, the antibiotic is selected from one or two of penicillin and streptomycin in any ratio combination.

Optionally, the goat semen cryopreservation diluent formula is as follows: 200-300 mM Tris, 80-90 mM citric acid, 65-75 mM fructose, 15-25% (V/V) yolk, 1-4 mg/mL epimedium polysaccharide, 2000IU/mL penicillin, 0.4g/mL streptomycin and 100mL ultrapure water.

Furthermore, the goat semen cryopreservation diluent formula contains 4-6% (V/V) of glycerol.

Optionally, the concentration of the epimedium polysaccharide is 2.5-3.5 mg/mL.

Meanwhile, the invention provides a goat semen cryopreservation method. To this end, a method is provided comprising: isothermal mixing and diluting the goat semen and the cryopreservation diluent containing the glycerol; and then gradually cooling the mixed diluent to 3-5 ℃, balancing at 3-5 ℃, and freezing for storage.

Optionally, the mixed dilution is performed by a multi-step dilution method, which comprises dilution to a final concentration via one or more intermediate concentrations.

Another goat semen cryopreservation method comprises the following steps: and isothermally mixing and diluting the goat semen and the frozen and preserved diluent without the glycerol, then gradually cooling the mixed diluent to 3-5 ℃, balancing at 3-5 ℃, isothermally mixing and diluting the balanced mixed diluent and the frozen and preserved diluent with the glycerol, and continuously balancing at 3-5 ℃ and then freezing and preserving.

The invention adopts epimedium polysaccharide as the effective component of the cryopreservation agent of goat sperm, can well protect the physiological activity of the sperm in the process of cryopreservation of the goat sperm, and comprises a one-step dilution method and a two-step dilution method.

Particularly, compared with the condition that the conventional goat sperm freezing generally adopts a two-step dilution method, the cryopreservation diluent containing the epimedium polysaccharide can meet the requirement of cryopreservation by matching with a one-step dilution method freezing program.

Drawings

FIG. 1 is the effect of epimedium polysaccharide on the freeze-thaw rate of goat sperm in example 1;

FIG. 2SYBR-14/PI double staining method, A: live sperm fluorescence images; b: dead sperm fluorescence map; c: fluorescence map of sperm plasma membrane integrity;

FIG. 3 is the effect of epimedium polysaccharide on the integrity rate of the plasma membrane of the head of a goat after sperm freezing in example 1;

FIG. 4 is the effect of epimedium polysaccharide on the integrity rate of the plasma membrane of the tail part of a frozen goat sperm in example 1;

FIG. 5 is the detection of acrosome integrity after freezing of goat sperm in example 1, A: staining a fluorescence map of sperm acrosome; b: sperm nucleus staining fluorescence map; c: a Merge graph;

FIG. 6 is the effect of epimedium polysaccharide on the acrosome integrity of frozen goat sperm in example 1;

FIG. 7 is the effect of epimedium polysaccharide on the total antioxidant capacity (T-AOC) of goat after sperm freezing in example 1;

FIG. 8 is the effect of epimedium polysaccharide on the Malondialdehyde (MDA) content of a frozen goat sperm in example 1;

FIG. 9 is the effect of epimedium polysaccharide on the Reactive Oxygen Species (ROS) content of goat sperm after freezing in example 1;

FIG. 10 is the effect of epimedium polysaccharide on Catalase (CAT) activity after freezing of goat sperm in example 1;

FIG. 11 is the effect of epimedium herb polysaccharide on the activity of superoxide dismutase (SOD) after the freezing of goat sperm in example 1;

FIG. 12 is the effect of epimedium polysaccharide on ATP content of goat after sperm freezing in example 1;

FIG. 13 is the effect of epimedium polysaccharide on mitochondrial membrane potential of goat after sperm freezing in example 1;

FIG. 14 shows the cryopreservation ultrastructures (SEM) of goat sperm in example 1, wherein A-H are the cryopreservation ultrastructures of goat sperm obtained by randomly sampling and SEM;

FIG. 15 shows the cryopreservation ultrastructures (TEM) of goat sperm in example 1, wherein A to F are the random sampling and TEM samples of goat sperm obtained from the cryopreservation ultrastructures of goat sperm;

FIG. 16 is a graph showing the effect of the post-freezing survival rate of the goat sperm in examples 1 and 2;

FIG. 17 is a graph showing the effect of the percentage of intact head plasma membrane after freezing of goat sperm in examples 1 and 2;

FIG. 18 is a graph showing the effect of tail plasma membrane integrity after freezing of goat sperm in examples 1 and 2;

FIG. 19 is a graph showing the effect of frozen acrosome integrity of the goat sperm in examples 1 and 2.

Detailed Description

Unless otherwise indicated, the terms herein are to be understood in accordance with their ordinary knowledge in the art.

Herba Epimedii (Epimediumbrevicornu Maxim), also known as herba Epimedii, and Foliumet ramulus evodiae, is first recorded in Shen nong Ben Cao Jing, is a perennial herb belonging to berberidaceae, is a traditional Chinese medicine for invigorating,

has the functions of invigorating kidney, strengthening Yang, replenishing vital essence and invigorating vital energy. The epimedium polysaccharide is the main active ingredient in natural traditional Chinese medicine epimedium, and consists of galacturonic acid (GalA), galactose (Gal), rhamnose (Rha), xylose (Xyl), arabinose (Ara) and mannose (Man). The epimedium polysaccharide in the following examples is purchased from West Anwanfang Biotech limited and has a purity of more than or equal to 98%.

The one-step dilution method is characterized in that the semen is subjected to isothermal mixing and dilution to a certain concentration by using a freezing preservation diluent (containing glycerol), and is frozen after being cooled to 3-5 ℃; and a two-step dilution method, namely, diluting the frozen stock diluent without glycerol to a certain concentration at about 30 ℃, cooling to 3-5 ℃, diluting the frozen stock diluent with glycerol and then freezing.

The following is an embodiment provided by the inventor for carrying out cryopreservation on the goat semen by adopting epimedium polysaccharide cryopreservation diluent and a one-step dilution method and a two-step dilution method.