CN114403131B - A kind of semen cryopreservation liquid and its application - Google Patents

Abstract

The invention discloses a semen cryopreservation liquid and application thereof, and belongs to the technical field of cryopreservation. The semen cryopreservation liquid is prepared from Tris, citric acid, yolk, ethylene glycol, vitamin C, bioflavonoids and trehalose according to a specific ratio, so that the vitality and the survival rate of frozen and revived sperms can be maintained, the oxidative stress reaction caused by freezing is reduced, the acrosome and the membrane structure of the sperms are damaged, and a foundation is laid for a high-vitality sperm freezing method.

Description

A kind of semen cryopreservation liquid and its application

technical field

The invention belongs to the technical field of cryopreservation, and in particular relates to a semen cryopreservation solution and application thereof.

Background technique

The silver black fox is a seasonal animal with single estrus. The estrus is concentrated from mid-to-late January to mid-March every year. The estrus lasts for 5-10 days. During the entire estrus, it can mate 2-4 times continuously, and the rest period is long. Up to 10 months, the reproductive efficiency is extremely low, which has seriously affected the development of the fox industry.

At present, the normal temperature semen diluent and artificial insemination technology of silver black fox are relatively mature, but there are few studies on the cold storage diluent of silver black fox semen at home and abroad, and it has not been applied and promoted on a large scale. Carrying out artificial insemination with frozen semen can not only improve the breeding potential of fine male foxes, effectively preserve the semen of fine male foxes, speed up the breeding process, but also reduce the number of breeding male foxes in actual production and save feeding costs, and alleviate the frequent occurrence of large numbers of male foxes in the late breeding period. A situation where the male fox cannot be used.

There are many problems in the use of the existing silver black fox semen cryopreservation diluent for semen cryopreservation and artificial insemination, mainly: ① sperm can hardly resist the damage caused by liquid nitrogen rapid freezing, resulting in a large number of deaths; ② freezing is easy to cause oxidative stress 3) After thawing and resuscitating, the viability of sperm drops sharply, the survival time in vitro is shorter, and the reproductive rate after artificial insemination is very low, resulting in the waste of semen of fine male foxes; 4) Most of the current reports are Focused on the collection of silver black fox fresh semen, rapid liquid nitrogen freezing, and then thawing and insemination on the same day, there is a lack of research on semen quality identification and artificial insemination results evaluation during long-term cryopreservation.

Therefore, how to provide a fox semen cryopreservation solution and its application is an urgent problem to be solved in this field.

Contents of the invention

The invention discloses a fox semen cryopreservation solution and application thereof.

In order to achieve the above object, the present invention adopts the following technical solutions:

A semen cryopreservation solution, including: Tris 0.12g/L, citric acid 0.08g/L, egg yolk 2ml/L, ethylene glycol 0.6ml/L, vitamin C 6g/L, bioflavonoids 0.8g/L and seaweed Sugar 40-90mmol/L; preferably, the working concentration is 2/3 of the original concentration;

The original concentration is the concentration when not diluted into working concentration;

Preferably, it also includes: gentamicin with an original concentration of 1 million U/L;

Application of the above-mentioned semen cryopreservation solution in the preparation of semen preservation preparations;

Application of the above-mentioned semen cryopreservation solution in the preparation of fox semen preservation preparation;

A cold freezing method for semen, comprising the following steps:

Semen Collection:

Collect fox semen at intervals of 2-3 days and 1-2 days apart;

Frozen semen:

Mix the semen cryopreservation solution with fox semen at the working concentration to obtain pre-refrigerated semen, pre-cool the pre-refrigerated semen at 4°C; then pre-cool it to below -10°C with liquid nitrogen steam, and then quickly put it into liquid nitrogen save;

Preferably, pre-cooling is to put it in a refrigerator at 4°C until it is completely pre-cooled;

Preferably, place for 2h;

Preferably, the liquid nitrogen vapor is pre-cooled to below -20°C;

Preferably, the liquid nitrogen vapor is pre-cooled to below -30°C;

A cold thawing method for semen, the steps are as follows:

thaw:

Take out the frozen semen, place it at room temperature for 10 minutes, then move it to 70°C until the semen is completely dissolved, take it out, mix the semen and thawing solution at a ratio of 1:2-1:1, and thaw at 22-24°C thawing in liquid;

Preferably, the semen and the thawing solution are mixed at a ratio of 1:2;

Preferably, 0.5ml of semen is taken;

The semen is fox semen;

Preferably, the formula of the thawing liquid is: 100ml of distilled water, 1.7g of sodium citrate, 1.15g of sucrose, 0.09g of sodium bicarbonate, 0.35g of potassium dihydrogen phosphate, 0.3g of sulfonamide, and 5-5 g of penicillin and streptomycin. 100,000 units;

In summary, the present invention discloses a semen cryopreservation solution and its application. The present invention prepares a semen cryopreservation solution by Tris, citric acid, egg yolk, ethylene glycol, vitamin C, bioflavonoids and trehalose in a specific ratio. The preservation solution can maintain the vitality and survival rate of sperm after freezing and thawing, reduce the oxidative stress response caused by freezing and destroy the sperm acrosome and membrane structure, and lay the foundation for the high-viability freezing method of sperm.

Detailed ways

The following clearly and completely describes the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Embodiment 1 Materials and methods Main reagents and instruments

Main ingredients used to prepare the diluent: Tris, citric acid, egg, ethylene glycol, penicillin, streptomycin, trehalose, vitamin C, bioflavonoids, potassium dihydrogen phosphate, phosphoric acid disodium hydrogen. Sperm vital staining solution (eosin-aniline black method), Coomassie Brilliant Blue Kit. The instruments and consumables used include: fox cage, electric thermostatic water bath, liquid nitrogen tank, microscope (DM6B, LEIC A in Germany), ultra-clean bench, acidity meter, centrifuge, sperm collection cup, automatic sperm analyzer ( TOX I VO S, HamiltonThorne, USA), cell counting plate, fox stainless steel insemination device, fox insemination glass casing, frozen fine tube. The formula of thawing liquid for experimental animals: 100ml of distilled water, 1.7g of sodium citrate, 1.15g of sucrose, 0.09g of sodium bicarbonate, 0.35g of potassium dihydrogen phosphate, 0.3g of sulfonamide, and 50,000-100,000 units of penicillin and streptomycin. All experimental animals were raised in Matouying Town, Leting County, Tangshan City, Hebei Province. Select 5 male silver and black foxes during the healthy mating period, and feed them once a day at 08:00 am and 15:00 pm, with free access to food and water. Preparation of diluent for cryopreservation

Weigh 1.2g of Tris, 0.8g of citric acid, 20ml of egg yolk, 6ml of ethylene glycol, 100,000 U/100ml of gentamycin, 6mg/ml of vitamin C, 0.8mg/ml of bioflavonoids, trehalose (add 6 Different concentrations, respectively add 40mmol/L, 50mmol/L, 60mmol/L, 70mmol/L, 80mmol/L, 90mmol/L), distilled water to 100ml. The semen was collected by hand massage, and the semen of the fox was collected in a clean graduated semen collection cup (placed in a water bath at 37°C). The male fox collects semen 2-3 times a week, and should rest for 1-2 days after 2-3 consecutive days of semen collection. Do not increase the number of semen collection at will to prevent the quality of semen from decreasing, resulting in adverse consequences such as reduced utilization of male foxes. Thaw semen preparation in thin tubes and before thawing and freezing, dilute the semen at a ratio of 1:2, equilibrate the diluted semen in a refrigerator at 4°C for 2 hours, divide the balanced semen into frozen fine tubes, and seal with a sealing machine. Then place the thin tube horizontally on the pre-prepared thin tube holder (5 cm from the liquid nitrogen level), cover the container cover of the freezing tank, fumigate on the liquid nitrogen surface for 10 seconds, and quickly put the thin tube into liquid nitrogen for storage. When thawing, take a 500mL large beaker, pour warm water at 70°C, take out the thin tube stored in liquid nitrogen, place it at room temperature for 10 minutes, quickly place it in warm water and stir slowly, and when the semen is completely dissolved, quickly take out the thin tube and cut both ends , add semen to 0.5mL of thawed solution at 22-24°C, put the thawed solution containing semen into a CO2 incubator, and incubate at 37°C for subsequent semen quality testing. Semen Quality Testing

① Sperm motility detection: The main parameters of semen quality were measured by automatic sperm analyzer (Waite et al., 2008). The parameters of semen quality include: sperm density (× 10 ⁶ sperm/mL), motile sperm ratio (%), path velocity (VAP, μm/s), linear motion (VSL, μm/s), curved motion (VCL, μm /s), forwardness (STR=[VAP/VCL]100; %), linearity (LIN=[VSL/VCL]100; %).

②Plasma membrane integrity test: Take 1ml of hypotonic solution, preheat it in a 37°C water bath for 1 minute, add the diluted semen to the preheated hypotonic solution and incubate for 30 minutes, count 200 sperm under a high-power microscope, and count the sperm Percentage of tail swelling.

③Acrosome integrity detection (Coomassie Brilliant Blue staining method): Take 80ul semen dilution and add it to 1ml 0.9% normal saline, centrifuge at 10000 rpm for 15s, discard the supernatant; add 1ml 3.7% paraformaldehyde/PBS to suspend, Fix at room temperature for 30 min, centrifuge and discard supernatant; suspend with 1ml PBS, centrifuge and discard supernatant; suspend with 500-800u1 PBS and dry the smear; 0.22% Coomassie brilliant blue (prepared with an aqueous solution containing 50% methanol and 10% glacial acetic acid) Stain for 5 minutes; wash off the dye solution with dH ₂ O and dry, seal the slide with neutral gum, observe 200 sperm, the acrosome is blue, and the sperm with a smooth acrosome edge is the type of sperm with a complete acrosome.

Fresh Semen Analysis

After the semen was collected, the semen was diluted with fox semen diluent (Xinyuan, Jinzhou) at a ratio of 1:2, and the semen was quickly checked for routine quality. The results showed that the sperm concentration, motile sperm ratio, path velocity (VAP), linear motion (VSL), curved motion (VCL), forward sex (STR), linearity (LIN), plasma membrane integrity rate and There was no significant difference in the acrosome integrity rate (P>0.05) (Table 1).

Table 1 Determination results of fresh semen quality of different silver black fox individuals

Effects of Adding Different Concentrations of Trehalose on Semen Quality After Freezing-Thawing

It can be seen from Table 2 that there was no significant difference between the sperm concentration and LIN among the 6 groups (P>0.05); the ratio of motile sperm in the 50mmol/L and 60mmol/L groups was significantly higher than that in the 40mmol/L, 80mmol/L, and 90mmol/L groups Added group (P<0.05), no significant difference with 70mmol/L added group (P>0.05), 70mmol/L added group was significantly higher than 40mmol/L and 90mmol/L added group (P<0.05); VAP60mmol/L added VSL group was significantly higher than 40mmol/L, 70mmol/L, 80mmol/L, 90mmol/L addition group (P<0.05), and there was no significant difference with 50mmol/L addition group (P>0.05); VSL60mmol/L addition group was significantly higher than 40mmol/L, 50mmol/L, 80mmol/L, 90mmol/L added group (P<0.05), and 70mmol/L added group had no significant difference (P>0.05); VCL50mmol/L, 60mmol/L, 70mmol/L, The 80mmol/L addition group had no significant difference (P>0.05), but it was significantly higher than the 40mmol/L and 90mmol/L addition groups (P<0.05); STR and plasma membrane integrity were 50mmol/L, 60mmol/L, and 70mmol/L The difference in the addition group was not significant (P>0.05), but it was significantly higher than that in the 40mmol/L, 80mmol/L and 90mmol/L addition groups (P<0.05); the acrosome integrity rate in the 60mmol/L addition group was significantly higher than that in the 40mmol/L, 50mmol/L, 70mmol/L, 80mmol/L, 90mmol/L added groups (P<0.05).

Comparative example 1

In actual production, since there is no formula for the silver black fox cryopreservation solution, the blue fox cryopreservation solution is used to dilute the silver black fox semen for cryopreservation.

The existing formula of blue fox cryopreservation solution:

①Egg yolk 20ml, glycerin 4ml, fructose 3.8g, penicillin 1.2IU; as control group Ⅰ.

②Tris 240mmol/L, sodium citrate 63mmol/L, egg yolk 20ml, glycerol 4ml, trehalose 70mmol/L; as the control group II. All the other operations are the same as in Example 1, and the results are shown in Table 2.

It can be seen from Table 2 that there was no significant difference in LIN between the 6 experimental groups and 2 control groups (P>0.05); the sperm concentration, VSL and STR of the control group Ⅰ were significantly lower than those of the 50mmol/L supplemented group and 60mmol/L supplemented group. group (P<0.05), there was no significant difference (P>0.05) from the other four experimental groups, and the motile sperm ratio and plasma membrane integrity rate were significantly lower than those in the 50mmol/L supplemented group, 60mmol/L supplemented group and 70mmol/L supplemented group (P<0.05), not significantly different from the other 3 experimental groups (P>0.05), VAP, VCL and STR were not significantly different from the 90mmol/L supplemented group (P>0.05), but significantly lower than the other 5 experimental groups (P<0.05), the plasma membrane integrity rate was not significantly different from the 40mmol/L supplemented group and 90mmol/L supplemented group (P>0.05), but was significantly lower than the other 4 experimental groups (P<0.05); The sperm ratio, VAP, VCL, plasma membrane integrity rate, and acrosome integrity rate were significantly lower than those of the 6 experimental groups (P<0.05), and the sperm concentration, VSL, and STR of control group Ⅱ were not significantly different from those of the 90mmol/L addition group (P >0.05), but significantly lower than the other five experimental groups (P<0.05).

Table 2 Effect of adding different concentrations of trehalose on semen quality after freezing-thawing

Each embodiment in this specification is described in a progressive manner, each embodiment focuses on the difference from other embodiments, and the same and similar parts of each embodiment can be referred to each other.

The above description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the invention. Various modifications to the above-described embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention will not be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (8)

1. A semen cryopreservation liquid is characterized by comprising: tris0.12g/L, citric acid 0.08g/L, yolk 2ml/L, ethylene glycol 0.6ml/L, vitamin C6g/L, bioflavonoid 0.8g/L and trehalose 40-90mmol/L.

2. The semen cryopreservation solution of claim 1 wherein the working concentration is 2/3 of the original concentration.

3. The semen cryopreservation liquid as claimed in claim 1 or 2, further comprising: gentamicin with the original concentration of 100 ten thousand U/L.

4. Use of the semen cryopreservation solution as claimed in any one of claims 1 to 3 in the preparation of a semen storage preparation.

5. Use of a semen cryopreservation solution as claimed in any one of claims 1 to 3 in the preparation of a fox semen storage formulation.

6. A method for cryopreservation of semen, comprising the steps of:

semen collection:

collecting fox semen at a frequency of 2-3 days with interval of 1-2 days;

freezing semen:

mixing the semen cryopreservation solution of claim 1 with fox semen at a working concentration to obtain pre-refrigerated semen, and pre-cooling the pre-refrigerated semen at 4 ℃; then precooling to below-10 ℃ by using liquid nitrogen steam, and then quickly storing in liquid nitrogen.

7. A semen unfreezing method is characterized by comprising the following steps:

unfreezing:

taking out the semen cryopreservation solution of any one of claims 1 to 3, standing at room temperature for 10min, transferring to 70 ℃ environment until semen is completely dissolved, taking out, mixing semen and thawing solution uniformly according to 1:2-1:1, and thawing in 22-24 ℃ thawing solution.

8. The method for thawing semen according to claim 7, wherein said thawing solution has a formulation comprising: 100ml of distilled water, 1.7g of sodium citrate, 1.15g of cane sugar, 0.09g of sodium bicarbonate, 0.35g of monopotassium phosphate, 0.3g of sulfanilamide and 5-10 ten thousand units of streptomycin respectively.