CN110074098A - A kind of mouse sperm frozen stock solution and its preparation method and application - Google Patents

Abstract

The present invention provides a kind of mouse sperm frozen stock solutions and its preparation method and application, belong to technical field in vitro fertilization.A kind of mouse sperm frozen stock solution, including the Rosmarinic acid mother liquor independently dispensed and basal liquid；The basal liquid is the aqueous solution comprising 2~4% skimmed milk power of 16~20% gossypose of mass concentration and mass concentration；The solvent of the Rosmarinic acid mother liquor is the basal liquid.The mouse sperm frozen stock solution is hybridly prepared into the working solution of the Rosmarinic acid containing 20~50mmol/L when use.Contain Rosmarinic acid in spermatozoa cryopreservation liquid; for resist freeze-resuscitation process in the excessive active oxygen that generates, optimize the metabolic process of life of sperm, enhance immune function and anti-oxidation function; have the function that the ability for protecting sperm fertilization, to improve mouse sperm rate in vitro fertilization.

Description

A kind of mouse sperm cryopreservation liquid and its preparation method and application

technical field

The invention belongs to the technical field of in vitro fertilization, and in particular relates to a mouse sperm cryopreservation solution and a preparation method and application thereof.

Background technique

Due to the needs of biomedical research, in the experimental process, it is often necessary to prepare or import genetically modified mice from abroad. However, due to the technical difficulty and high price of the prepared or imported mice, the number of mice obtained is limited, and they are used for scientific research. The experimental mice need to reach a certain statistically significant number. Due to the long cycle of natural reproduction, sperm cryopreservation and multiple in vitro fertilization methods can quickly obtain a sufficient number of mice for experiments; scientific research work When it's over, the mice are often euthanized, leaving the resources gone. At present, resources can be preserved by adopting the method of sperm cryopreservation; at the same time, in the case of genital trauma or near death, mice can also adopt the method of sperm cryopreservation to preserve the resources; preservation of cryopreserved sperm can also be carried out repeatedly. The second in vitro fertilization operation completed the microbiological purification of the mice. Glutamine solution has the functions of enhancing immunity and anti-oxidation, and is often used as a traditional mouse sperm cryopreservation solution for inbred mice or genetically engineered mice, but its ability to protect mouse sperm needs to be further improved.

SUMMARY OF THE INVENTION

In view of this, the object of the present invention is to provide a mouse sperm cryopreservation solution for improving the in vitro fertilization rate of mice, its preparation method and application.

The invention provides a mouse sperm cryopreservation liquid, which comprises an independently packaged rosmarinic acid mother liquid and a base liquid; the base liquid contains 16-20% raffinose and 2-4% degreased An aqueous solution of milk powder; the solvent of the rosmarinic acid mother liquor is the base liquid.

Preferably, the base liquid is an aqueous solution comprising a mass concentration of 18% raffinose and a mass concentration of 3% skimmed milk powder.

Preferably, the concentration of the rosmarinic acid mother liquor is 100mmol/L.

Preferably, the working solution of the mouse sperm cryopreservation solution is a base solution containing 20-50 mmol/L rosmarinic acid.

Preferably, the working solution of the mouse sperm cryopreservation solution is a base solution containing 30-40 mmol/L rosmarinic acid.

The invention provides a kind of preparation method of described mouse sperm cryopreservation liquid, comprises the following steps:

1) Mix raffinose, skimmed milk powder and water, sterilize, and dispense into a tube of 990 μL each to obtain the base solution;

2) Prepare the rosmarinic acid mother solution with a concentration of 100 mmol/L by using the base solution, and divide into one tube per 10 μL to obtain the rosmarinic acid mother solution.

Preferably, when preparing the working solution of the mouse sperm cryopreservation solution, discard 10×(n-1) μL of the base solution from each tube and mix it with n tubes of rosmarinic acid mother solution to prepare rosemary A working solution of mouse sperm cryopreservation solution with a concentration of balmic acid of 10×nmmol/L; wherein n=2～5.

Preferably, the sterilization described in step 1) is sterilization by filter membrane; the pore size of the filter membrane is 0.22 μm.

The invention provides the application of the mouse sperm cryopreservation solution or the mouse sperm cryopreservation solution prepared by the preparation method in the cryopreservation of mouse sperm.

Preferably, the method for cryopreserving mouse sperm comprises the following steps:

A. Prepare the mouse sperm cryopreservation solution as the working solution of the mouse sperm cryopreservation solution, and equilibrate to 37-39°C to obtain the preheated working solution;

B. After the mice to be frozen for sperm were killed, the tail of the epididymis was taken out, cut into pieces, and fragments of the tail of the epididymis were obtained;

C. Mix the fragments of the tail of the epididymis with the preheated working solution, separate the sperm, pre-cool the working solution containing the sperm, and store it in liquid nitrogen.

The mouse sperm cryopreservation solution provided by the present invention provides an appropriate amount of rosmarinic acid in the base solution, which is used to fight against excessive active oxygen generated during the cryopreservation-resuscitation process, optimize the life metabolism process of sperm, enhance immune function and Antioxidant function, so as to achieve the effect of protecting sperm fertilization ability. Experiments have shown that using the traditional mouse sperm cryopreservation solution (basic solution) for inbred mice or genetically engineered mice, the in vitro fertilization rate after recovery is low, and it is often impossible to obtain a sufficient number of embryos for embryo transfer. The conservation work of inbred mice brings certain difficulties. When a certain amount of rosmarinic acid is added to the mouse sperm cryopreservation solution, it can supplement the energy in the process of sperm cryopreservation and recovery, restore the active oxygen produced in the process of sperm cryopreservation and recovery, and relieve the toxic effect of ROS on sperm. Positive meaning, it is conducive to the maintenance of sperm motility, and a higher in vitro fertilization rate is obtained, and the fertilization rate reaches 73-85%, which has significant advantages compared with low concentration (10mmol/L) or traditional mouse sperm cryopreservation solution.

Further, the present invention further limits the content of rosmarinic acid in the working liquid of the mouse sperm cryopreservation liquid to be 30-40 mmol/L. Experiments have shown that: after adopting the working solution of 30mmol/L rosemary to preserve mouse sperm, the in vitro fertilization rate reaches 87%, which is higher than the 100mmol/L glutamine mouse sperm cryopreservation solution reported by the prior art (78% in vitro fertilization rate). ) increased by 11.54%, which shows that the mouse sperm cryopreservation solution provided by the invention has strong immune-enhancing and anti-oxidative functions, and significantly improves the in vitro fertilization rate of mouse sperm.

Detailed ways

The invention provides a mouse sperm cryopreservation liquid, which comprises an independently packaged rosmarinic acid mother liquid and a base liquid; the base liquid contains 16-20% raffinose and 2-4% degreased An aqueous solution of milk powder; the solvent of the rosmarinic acid mother liquor is the base liquid.

The mouse sperm cryopreservation solution provided by the present invention includes separately packaged rosmarinic acid mother solution and base solution. The base liquid is preferably an aqueous solution comprising a mass concentration of 18% raffinose and a mass concentration of 3% skimmed milk powder. The present invention has no special limitation on the concentration of the rosmarinic acid mother liquor, which can be prepared to be 10 to 100 times the working concentration of rosmarinic acid. In order to illustrate the preparation method of the working solution of the mouse sperm cryopreservation solution of the present invention, the preparation of 100 mmol/L of rosmarinic acid mother solution is taken as an example for specific illustration. The present invention has no special restrictions on the sources of the raffinose, skimmed milk powder and rosmarinic acid, and they can be obtained by commercial purchase methods well known in the art. The water solvent used in the base liquid is preferably double distilled water.

In the present invention, the mouse sperm cryopreservation solution is formulated with a working solution of the mouse sperm cryopreservation solution before use, so as to prevent the immune function and anti-oxidation function from being reduced, thereby reducing the ability to preserve the vitality of mouse sperm. The working solution of the mouse sperm cryopreservation solution is preferably a base solution containing 20-50 mmol/L rosmarinic acid, more preferably a base solution containing 30-40 mmol/L rosmarinic acid.

The present invention provides a kind of preparation method of described mouse sperm cryopreservation liquid, preferably comprises the following steps:

1) Mix raffinose, skimmed milk powder and water, sterilize, and dispense into a tube of 990 μL each to obtain the base solution;

2) Prepare the rosmarinic acid mother solution with a concentration of 100 mmol/L by using the base solution, and divide into one tube per 10 μL to obtain the rosmarinic acid mother solution.

In the present invention, the sterilization is preferably filter sterilization; the filter membrane preferably has a pore size of 0.22 μm. Prior to sterilization, the resulting mixture is preferably centrifuged in order to remove insoluble impurities. The rotational speed of the centrifugation is preferably 10000-14000 rpm, more preferably 12000 rpm, and the centrifugation time is preferably 10-30 min, more preferably 20-30 min.

In the present invention, the base liquid and the mother liquid of rosmarinic acid prepared by the above preparation method are used for cryopreservation.

In the present invention, when preparing the working solution of the mouse sperm cryopreservation solution, it is preferred to discard 10×(n-1) μL of the base solution from each tube and mix it with n tubes of rosmarinic acid mother solution to prepare A working solution of mouse sperm cryopreservation solution with a rosmarinic acid concentration of 10×n mmol/L was obtained; wherein n=2˜5.

The invention provides the application of the mouse sperm cryopreservation solution or the mouse sperm cryopreservation solution prepared by the preparation method in the cryopreservation of mouse sperm.

In the present invention, the method for cryopreserving mouse sperm preferably comprises the following steps:

A. Prepare the mouse sperm cryopreservation solution as the working solution of the mouse sperm cryopreservation solution, and equilibrate to 37°C to obtain the preheated working solution;

B. After the mice to be frozen for sperm were killed, the tail of the epididymis was taken out, cut into pieces, and fragments of the tail of the epididymis were obtained;

C. Mix the fragments of the tail of the epididymis with the preheated working solution, separate the sperm, pre-cool the working solution containing the sperm, and store it in liquid nitrogen.

In the present invention, the mouse sperm cryopreservation solution is formulated into the working solution of the mouse sperm cryopreservation solution, and the temperature is balanced to 37-39° C. to obtain the preheated working solution. The method for balancing the temperature of the working fluid is preferably a water bath at 37-39°C. Balance the working solution of the mouse sperm cryopreservation solution to 37-39°C to make the subsequent sperm separation as close as possible to body temperature and avoid cold stress on the sperm.

In the present invention, after the mice whose sperm are to be frozen are killed, the cauda epididymis is taken out and shredded to obtain fragments of the cauda epididymis. The mouse whose sperm is to be frozen is preferably a 12-14 week old mouse. The present invention has no special limitation on the type of the mouse, and the target mouse to be preserved in the field can be used. In summary of the embodiments of the present invention, the mouse is an inbred mouse C57BL/6 mouse. The method of killing is not particularly limited, and the conventional method of killing by cervical vertebra dislocation in the art can be used.

After obtaining the fragments of the cauda epididymis and the preheated working solution, the present invention mixes the fragments of the cauda epididymis with the preheated working solution, separates the sperm, pre-cools the working solution containing the sperm, and stores it in liquid nitrogen.

In the present invention, the tail epididymis fragments of each mouse are preferably mixed with 1 mL of preheated working solution. The time for separating the sperm is preferably 5-7 minutes, which is conducive to allowing the sperm to fully swim out and enter the working fluid. Before pre-cooling, the working solution containing sperm is preferably subpackaged. The dispensing container is preferably a 250 μL straw, and each tube is preferably filled with 50-80 μL of working solution containing sperm, and heat-sealed. The pre-cooling method is preferably to place the divided straws in liquid nitrogen vapor for 3-5 minutes, and then put them into liquid nitrogen for long-term storage.

In the present invention, in order to verify the cryopreservation effect of the cryopreservation solution provided by the present invention, the cryopreserved sperm were recovered and fertilized in vitro, and the fertilization rate was selected and counted.

A mouse sperm cryopreservation solution provided by the present invention and its preparation method and application are described in detail below in conjunction with the examples, but they should not be interpreted as limiting the protection scope of the present invention.

Example 1

Preparation method of mouse sperm cryopreservation solution

Dissolve 18g of raffinose and 3g of skimmed milk powder in 100mL of double distilled water and mix. Centrifuge the resulting mixture at 12,000rpm for 30min. Take the supernatant and filter it with a 0.22μm filter to sterilize it. Pack in 990μL and mark it as liquid A. Store in a -20°C refrigerator; use Solution A to prepare a solution of rosmarinic acid at a concentration of 100mM, aliquot 10μL, label it as Solution B, and store in a -20°C refrigerator. Liquids A and B are mouse sperm cryopreservation liquids.

Embodiment 2 to Embodiment 5

Preparation method of working solution of mouse sperm cryopreservation solution

When in use, discard 10 μL of the solution A prepared in Example 1 and then mix it with 2 tubes of solution B to obtain a 20 mM rosmarinic acid freezing solution.

Discard 20 μL of solution A prepared in Example 1 and mix it with 3 tubes of solution B to obtain 30 mM rosmarinic acid freezing solution.

Discard 30 μL of solution A prepared in Example 1 and mix it with 4 tubes of solution B to obtain 40 mM rosmarinic acid freezing solution.

Discard 40 mL of the liquid A prepared in Example 1 and then mix it with 5 liquids B to obtain a 50 mM rosmarinic acid freezing solution.

Comparative Example 1

Liquid A and liquid B prepared in Example 1 were directly mixed to obtain 10 mM rosmarinic acid freezing solution.

Comparative Example 2

The liquid A prepared in Example 1 was used as the traditional mouse sperm preservation liquid.

Embodiment 6-10

Comparative Examples 1 to 2 and Examples 2 to 5 are sperm cryopreservation solutions containing different concentrations of rosmarinic acid, which are respectively marked as groups 1, 2, 3, 4, 5, and 6, of which glutamine in group 1 The concentration is zero, and the content of the components corresponding to each number is shown in Table 1.

The preparation situation of the mouse sperm cryopreservation solution of different components in table 1

components 1 2 3 4 5 6 Raffinose (w/v) 18% 18% 18% 18% 18% 18% Skimmed milk powder (w/v) 3% 3% 3% 3% 3% 3% Rosmarinic acid (mM) 0 10 20 30 40 50

Sperm cryopreservation: 12-14 weeks-old inbred mouse C57BL/6 mice were killed by cervical dislocation, the cauda epididymis was aseptically removed, cut into pieces with ophthalmic scissors, and immersed in 1 mL of frozen sperm equilibrated to 37°C In the storage solution, let the sperm swim out fully. After 5-7 minutes, divide into 250 μL straws, each tube contains 50-80 μL, and heat seal. Place in liquid nitrogen vapor for 3 to 5 minutes to pre-cool, then put into liquid nitrogen for storage.

Sperm resuscitation: Take out the sperm freezing straw from the liquid nitrogen, shake it in the air for 15-30 seconds, put it into a 37°C water bath for 30-60 seconds to completely thaw, cut off the straw seal, and blow the sperm into 1mL of in vitro fertilization solution After centrifugation at 2500-3000rpm for 3-5min, discard the supernatant, retain 50 μL of liquid, blow the sperm away, and check the motility of the sperm.

Ovum acquisition: SPF-grade female mice of the same strain as male mice were taken at 5-6 weeks, injected with PMSG (pregnant mother serum hormone) 30-50IU 3 days before the experiment, and injected with HCG (human chorionic gonadotropin) 25 hours after 48 hours ~40IU, 14~16 hours later, sacrificed by cervical dislocation, opened the abdominal cavity, separated the fallopian tubes, found the enlarged part under a stereomicroscope, released the egg mass, picked the egg mass into 100~200 μL of preheated 37°C in vitro fertilization fluid, and Wash 2-3 times and cover with mineral oil. Place in a 37°C, 5% carbon dioxide incubator.

Fertilization process: Take 10 μL of the recovered sperm fluid and place it in the egg mass fluid, and place it in an incubator at 37°C and 5% carbon dioxide to continue culturing to complete the fertilization process.

Selection of fertilized eggs: After 6 to 7 hours, place the fertilized liquid droplet under a microscope to select the scattered eggs, transfer them to 100 μL of KSOM culture medium preheated to 37°C covered with mineral oil, and continue to cultivate for 20 to 22 hours Afterwards, the embryos developed to the 2-4 cell stage were selected for transplantation, and the fertilization rate was calculated.

Table 2 In vitro fertilization rate after resuscitation of different mouse sperm cryopreservation solutions

From the above results, it can be seen that using the traditional mouse sperm cryopreservation solution for inbred mice or genetically engineered mice has a low in vitro fertilization rate after recovery, and often cannot obtain a sufficient number of embryos for embryo transfer. The breeding work of inbred mice brings certain difficulties. A certain amount of rosmarinic acid is added to the mouse sperm cryopreservation solution to supplement and restore energy during sperm cryopreservation and recovery. The active oxygen produced has a positive significance in removing the toxic effect of ROS on sperm, which is conducive to the maintenance of sperm vitality and a higher in vitro fertilization rate. Comparative experiments show that the addition of rosmarinic acid in the mouse sperm cryopreservation solution A better fertilization effect can be obtained in the range of 20-50mM, and rosmarinic acid at a concentration of 30mM has the best effect.

Comparative Example 3

According to literature reports, 100mM glutamine basal solution also has the functions of enhancing immunity and anti-oxidation, and is used as a mouse sperm cryopreservation solution for mouse sperm cryopreservation.

Example 11

According to the 30mM rosmarinic acid mouse sperm cryopreservation liquid working solution with the best effect obtained in the above-mentioned examples and the 100mM glutamine prepared in comparative example 3 respectively as the sperm cryopreservation liquid according to the method of embodiment 5 to measure mouse In vitro fertilization rate of sperm, the obtained experimental results are shown in Table 3.

The preparation situation of the mouse sperm cryopreservation solution of table 3 different components

components 30mM rosmarinic acid 100mM Glutamine In vitro fertilization rate (%) 87 78

Experiments have shown that: after adopting the working solution of 30mmol/L rosemary to preserve mouse sperm, the in vitro fertilization rate reaches 87%, which is higher than the 100mmol/L glutamine mouse sperm cryopreservation solution reported by the prior art (78% in vitro fertilization rate). ) increased by 11.54%, which shows that the mouse sperm cryopreservation solution provided by the invention has strong immune-enhancing and anti-oxidation functions, and significantly improves the in vitro fertilization rate of mouse sperm.

The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

Claims (10)

1. a kind of mouse sperm frozen stock solution, which is characterized in that including the Rosmarinic acid mother liquor independently dispensed and basal liquid；The base Plinth liquid is the aqueous solution comprising 2~4% skimmed milk power of 16~20% gossypose of mass concentration and mass concentration；The Rosmarinic acid The solvent of mother liquor is the basal liquid.

2. mouse sperm frozen stock solution according to claim 1, which is characterized in that the basal liquid is to include mass concentration 18% The aqueous solution of 3% skimmed milk power of gossypose and mass concentration.

3. mouse sperm frozen stock solution according to claim 1, which is characterized in that the concentration of the Rosmarinic acid mother liquor is 100mmol/L。

4. mouse sperm frozen stock solution described in any one according to claim 1~3, which is characterized in that the mouse sperm freezes The working solution of liquid is the basal liquid of the Rosmarinic acid containing 20~50mmol/L.

5. mouse sperm frozen stock solution according to claim 4, which is characterized in that the working solution of the mouse sperm frozen stock solution is The basal liquid of the Rosmarinic acid containing 30~40mmol/L.

6. a kind of preparation method of mouse sperm frozen stock solution described in Claims 1 to 5 any one, which is characterized in that including with Lower step:

1) gossypose, skimmed milk power and water mixing, degerming, every 990 μ L packing, one pipe obtain basal liquid；

2) Rosmarinic acid is made into the Rosmarinic acid mother liquor of 100mmol/L concentration with the basal liquid, every 10 μ L packing, one pipe obtains To Rosmarinic acid mother liquor.

7. preparation method according to claim 6, which is characterized in that when preparing the working solution of mouse sperm frozen stock solution, Basal liquid described in every pipe is discarded and is mixed with n pipe Rosmarinic acid mother liquor after 10 × (n-1) μ L basal liquids, Rosmarinic acid is prepared Concentration is the working solution of the mouse sperm frozen stock solution of 10 × n mmol/L；Wherein n=2~5.

8. preparation method according to claim 6, which is characterized in that degerming described in step 1) is membrane filtration degerming； The aperture of the filter membrane is 0.22 μm.

9. preparation side described in mouse sperm frozen stock solution or claim 6~8 any one described in Claims 1 to 5 any one Application of the mouse sperm frozen stock solution of method preparation in mouse sperm freezing preservation.

10. application according to claim 9, which is characterized in that the method that the mouse sperm freezing saves includes following Step:

A., mouse sperm frozen stock solution is configured to the working solution of mouse sperm frozen stock solution, balances to 37~39 DEG C, is preheated Working solution；

B. by after the mouse of sperm freezing is put to death, cauda epididymidis is taken out, shreds, obtains cauda epididymidis fragment；

C. the cauda epididymidis fragment and the working solution of the preheating are mixed, separated sperm, the working solution containing sperm is pre-chilled Afterwards, it saves into liquid nitrogen.