CN106497864A - Ovarian follicle in vitro culture liquid and preparation method thereof before a kind of domestic animal chamber - Google Patents

Abstract

The invention discloses ovarian follicle in vitro culture liquid and preparation method thereof before a kind of domestic animal chamber, the nutrient solution is alpha linolenic acid, addition of the alpha linolenic acid in every 1mL cellar cultures liquid is 100～200 μ g, and the cellar culture liquid is by 100mL α MEM solution, 1.5mL transferrins, 150 μ L insulin, 100 μ L vitamin Cs, 0.5mL sodium selenites, 500 μ g bovine serum albumin(BSA)s, 100ng sodium alginates, 500IU FSH, 0.06g Benzylpenicillin sodium salt, 0.1g streptomycin sulphates composition.Compared with prior art, the present invention is first by the in vitro culture of ovarian follicle before alpha linolenic acid domestic animal ovarian cavity, effect reliability, it is easy to promote, it is adaptable to the in vitro culture of ovarian follicle before domestic animal chamber, is remarkably improved the Motility rate of ovarian follicle before in vitro culture domestic animal chamber.

Description

A kind of livestock preantral follicle in vitro culture medium and preparation method thereof

technical field

The invention relates to a biological preparation method, in particular to an in vitro culture solution of livestock preantral follicles and a preparation method thereof.

Background technique

Cell culture technology is the most basic technology in biological research. Its success provides an excellent research model for the study of cell proliferation and differentiation in vitro, cell survival and apoptosis, gene expression and regulation, etc. However, the current in vitro culture of cells And research, focusing more on a single cell type, has irreplaceable benefits for studying the function and mechanism of a single cell. However, in vitro culture of organism tissues composed of various types of cells, as well as the study of the interaction mechanism between different cells in vitro, has not yet achieved satisfactory progress. Ovarian follicles are composed of theca cells, granulosa cells and oocytes. The growth and development of follicles is the result of the interaction and coordination of the three cells. Synthesis and secretion, follicle growth mechanism and formation principle of follicle cavity, oocyte maturation and luteinization are all good models.

The development and maturation of ovarian follicles is a highly complex process, involving the coordination of animal hypothalamus, pituitary gland and ovary and the multi-directional communication between follicular cells. Although research has shown that many factors can regulate the development of follicles, however, The specific mechanisms by which these factors regulate follicular maturation are unclear. In addition, in the process of in vitro fertilization, since the number of healthy and high-quality eggs that scientists can directly obtain from the ovary is limited, how to cultivate follicles to obtain high-quality oocytes is the primary problem facing scientists. The success of follicle culture technology in vitro has broad application prospects both in basic research and in veterinary clinics. The in vitro culture of livestock follicles can provide reproductive guarantee for high-yield animals, and is of great significance to the establishment of "egg banks" of maternal genes, the study of the physiological and pathological processes of folliculogenesis and apoptosis, as well as conservation and commercial production.

α-linolenic acid is a kind of polyunsaturated fatty acid (n-3) and one of the essential nutrients for animals. As a regulator, polyunsaturated fatty acids may serve as precursors of steroid hormones and prostaglandins, and play an important role in multiple aspects of animal reproductive activities. Adding α-linolenic acid in the diet can increase the number of small and medium-sized follicles on the ovaries of cattle and sheep. At the same time, feeding different unsaturated fatty acids can increase the quality of bovine oocytes cultured in vitro and improve their fertilization rate. The cleavage rate and embryo quality of early embryos are significantly promoted. However, the effect of α-linolenic acid on the survival and development of follicles cultured in vitro has not been reported.

Contents of the invention

The object of the present invention is to provide a kind of livestock preantral follicle in vitro culture medium and its preparation method in order to solve the above problems.

The present invention achieves the above object through the following technical solutions:

The present invention is an in vitro culture solution of livestock preantral follicles, the culture solution is α-linolenic acid, and the amount of α-linolenic acid added to every 1 mL of conventional culture solution is 100-200 μg, and the conventional culture solution consists of 100 mL of α-MEM Solution, 1.5mL transferrin, 150μL insulin, 100μL vitamin C, 0.5mL sodium selenite, 500μg bovine serum albumin, 100ng sodium alginate, 500IU FSH, 0.06g penicillin sodium, 0.1g streptomycin sulfate.

A method for preparing livestock preantral follicle in vitro culture fluid, comprising the following steps:

1) Dissolve 1.5 mL of transferrin, 150 μL of insulin, 100 μL of vitamin C, 0.5 mL of sodium selenite, 0.06 g of penicillin sodium, and 0.1 g of streptomycin sulfate in 100 mL of α-MEM solution to prepare a basal culture medium;

2) Dissolve 500 μg of bovine serum albumin in the basal culture medium, mix slowly to avoid foaming, and prepare liquid I;

3) Dissolve 10 mg of sodium alginate in 10 ml of DMSO, mix slowly, absorb 100 μL of the mixed solution, add it to liquid I, mix slowly, and prepare liquid II;

4) Slowly dissolve 10-20mg of α-linolenic acid in 10ml of DMSO, mix slowly, absorb 100μL of the mixed solution, add it to solution II, mix slowly, and prepare a working solution;

5) Adjust the pH value of the working solution to 7.0-7.1, filter and sterilize, and store in a 4°C refrigerator for later use;

6) Take 500IU of FSH and add it into 100mL spare working solution for in vitro culture of follicles.

A method for in vitro culture of livestock preantral follicles, comprising the following steps:

(A) Obtain bovine ovaries from the slaughterhouse, put them in sterilized normal saline (temperature 30-35°C), and send them to the laboratory as soon as possible. After sterilizing with 70% alcohol, sterilize the ovaries with 30-35°C normal saline. Rinse off residual alcohol;

(B) Take out the follicle culture working solution stored in the 4°C refrigerator, preheat it in a 37°C water bath, and set aside;

(C) Cut the ovary in the step (A) into thin slices of 1 cm×0.5 cm×0.2 cm with a scalpel, then rinse with sterilized physiological saline for 1 to 2 times, and put it into a watch glass;

(D) Under a stereomicroscope, use a 25G needle to pick out the preantral follicles of livestock, and select the preantral follicles with a diameter of 160-220 μm for use;

(E) Put the selected preantral follicles into a 24-well culture dish, add preheated culture medium at 1ml/well for single follicle culture;

(F) Replace 70% of the culture medium at intervals of 24 hours;

(G) The diameter of follicles was measured daily with a graduated microscope, and changes in the follicle cavity were observed.

The beneficial effects of the present invention are:

The invention relates to an in vitro culture solution of preantral follicles of livestock and a preparation method thereof. Compared with the prior art, the present invention is the first in vitro culture of α-linolenic acid follicles of preantral follicles of livestock ovaries. The effect is reliable, easy to popularize, and is suitable for livestock The in vitro culture of preantral follicles can significantly improve the in vitro survival rate of livestock preantral follicles cultured in vitro.

Description of drawings

Fig. 1 is the bovine preantral follicle cultured in vitro of the present invention.

In the figure: A: the 1st day of culture; B: the 5th day of culture; C: the 12th day of culture; D the 18th day of culture.

detailed description

The present invention will be further described below in conjunction with accompanying drawing:

The present invention is an in vitro culture solution of livestock preantral follicles, the culture solution is α-linolenic acid, and the amount of α-linolenic acid added to every 1 mL of conventional culture solution is 100-200 μg, and the conventional culture solution consists of 100 mL of α-MEM Solution, 1.5mL transferrin, 150μL insulin, 100μL vitamin C, 0.5mL sodium selenite, 500μg bovine serum albumin, 100ng sodium alginate, 500IU FSH, 0.06g penicillin sodium, 0.1g streptomycin sulfate.

A method for preparing livestock preantral follicle in vitro culture fluid, comprising the following steps:

1) Dissolve 1.5 mL of transferrin, 150 μL of insulin, 100 μL of vitamin C, 0.5 mL of sodium selenite, 0.06 g of penicillin sodium, and 0.1 g of streptomycin sulfate in 100 mL of α-MEM solution to prepare a basal culture medium;

2) Dissolve 500 μg of bovine serum albumin in the basal culture medium, mix slowly to avoid foaming, and prepare liquid I;

3) Dissolve 10 mg of sodium alginate in 10 ml of DMSO, mix slowly, absorb 100 μL of the mixed solution, add it to liquid I, mix slowly, and prepare liquid II;

4) Slowly dissolve 10-20mg of α-linolenic acid in 10ml of DMSO, mix slowly, absorb 100μL of the mixed solution, add it to solution II, mix slowly, and prepare a working solution;

5) Adjust the pH value of the working solution to 7.0-7.1, filter and sterilize, and store in a 4°C refrigerator for later use;

6) Take 500IU of FSH and add it into 100mL spare working solution for in vitro culture of follicles.

A method for in vitro culture of livestock preantral follicles, comprising the following steps:

(A) Obtain bovine ovaries from the slaughterhouse, put them in sterilized normal saline (temperature 30-35°C), and send them to the laboratory as soon as possible. After sterilizing with 70% alcohol, sterilize the ovaries with 30-35°C normal saline. Rinse off residual alcohol;

(B) Take out the follicle culture working solution stored in the 4°C refrigerator, preheat it in a 37°C water bath, and set aside;

(C) Cut the ovary in the step (A) into thin slices of 1 cm×0.5 cm×0.2 cm with a scalpel, then rinse with sterilized physiological saline for 1 to 2 times, and put it into a watch glass;

(D) Under a stereomicroscope, use a 25G needle to pick out the preantral follicles of livestock, and select the preantral follicles with a diameter of 160-220 μm for use;

(E) Put the selected preantral follicles into a 24-well culture dish, add preheated culture medium at 1ml/well for single follicle culture;

(F) Replace 70% of the culture medium at intervals of 24 hours;

(G) The diameter of follicles was measured daily with a graduated microscope, and changes in the follicle cavity were observed.

Example 1

Accurately weigh 1.5mL transferrin, 150μL insulin, 100μL vitamin C, 0.5mL sodium selenite, 0.06g penicillin sodium, 0.1g streptomycin sulfate, dissolve in 100mL α-MEM solution, and prepare the basic culture medium; Dissolve 500 μg of bovine serum albumin in the basic culture medium, mix slowly to avoid foaming, and prepare solution I; measure 100 μL of 1 mg/ml sodium alginate solution, add it to solution I, mix slowly, and prepare solution II ; Take 120 μL of 1 mg/ml α-linolenic acid, add it to II solution, mix slowly, and prepare a working solution; adjust the pH value of the working solution to 7.0-7.1, filter and sterilize, and store it in a 4°C refrigerator for later use; take 500IU FSH Add 100mL spare working solution for in vitro culture of sheep follicles.

Example 2

Accurately weigh 1.5mL transferrin, 150μL insulin, 100μL vitamin C, 0.5mL sodium selenite, 0.06g penicillin sodium, 0.1g streptomycin sulfate, dissolve in 100mL α-MEM solution, and prepare the basic culture medium; Dissolve 500 μg of bovine serum albumin in the basic culture medium, mix slowly to avoid foaming, and prepare solution I; measure 100 μL of 1 mg/ml sodium alginate solution, add it to solution I, mix slowly, and prepare solution II ; Take 180 μL of 1 mg/ml α-linolenic acid, add it to the II solution, mix slowly, and prepare a working solution; adjust the pH value of the working solution to 7.0-7.1, filter and sterilize, and store it in a 4°C refrigerator for later use; take 500IU FSH Add 100mL spare working solution for in vitro culture of bovine follicles.

Comparative example

Accurately weigh 1.5mL transferrin, 150μL insulin, 100μL vitamin C, 0.5mL sodium selenite, 0.06g penicillin sodium, 0.1g streptomycin sulfate, dissolve in 100mL α-MEM solution, and prepare the basic culture medium; Dissolve 500 μg of bovine serum albumin in the basic culture medium, mix slowly to avoid foaming, and prepare solution I; measure 100 μL of 1 mg/ml sodium alginate solution, add it to solution I, mix slowly, and prepare solution II ; adjust the pH value of solution II to 7.0-7.1, filter and sterilize, and store in a refrigerator at 4°C for later use; take 500IU FSH and add it to 100mL spare working solution for in vitro culture of bovine follicles.

The results of the test example are shown in Figure 1 and Table 1:

Table 1 Effect of different concentrations of α-linolenic acid on the survival rate of ovarian preantral follicles cultured in vitro for 12 days

The basic principles and main features of the present invention and the advantages of the present invention have been shown and described above. Those skilled in the industry should understand that the present invention is not limited by the above-mentioned embodiments. What are described in the above-mentioned embodiments and the description only illustrate the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will also have Variations and improvements are possible, which fall within the scope of the claimed invention. The protection scope of the present invention is defined by the appended claims and their equivalents.

Claims (3)

1. An in vitro culture solution of livestock preantral follicles, characterized in that: the culture solution is α-linolenic acid, and the amount of α-linolenic acid in every 1 mL of conventional culture solution is 100 to 200 μg, and the conventional culture solution 100mL α-MEM solution, 1.5mL transferrin, 150μL insulin, 100μL vitamin C, 0.5mL sodium selenite, 500μg bovine serum albumin, 100ng sodium alginate, 500IU FSH, 0.06g penicillin sodium, 0.1g streptomycin sulfate prime composition. 2. a method for preparing livestock preantral follicle in vitro culture fluid, characterized in that, comprising the following steps: 1) Dissolve 1.5 mL of transferrin, 150 μL of insulin, 100 μL of vitamin C, 0.5 mL of sodium selenite, 0.06 g of penicillin sodium, and 0.1 g of streptomycin sulfate in 100 mL of α-MEM solution to prepare a basal culture medium; 2) Dissolve 500 μg of bovine serum albumin in the basal culture medium, mix slowly to avoid foaming, and prepare liquid I; 3) Dissolve 10 mg of sodium alginate in 10 ml of DMSO, mix slowly, absorb 100 μL of the mixed solution, add it to liquid I, mix slowly, and prepare liquid II; 4) Slowly dissolve 10-20mg of α-linolenic acid in 10ml of DMSO, mix slowly, absorb 100μL of the mixed solution, add it to solution II, mix slowly, and prepare a working solution; 5) Adjust the pH value of the working solution to 7.0-7.1, filter and sterilize, and store in a 4°C refrigerator for later use; 6) Take 500IU of FSH and add it into 100mL spare working solution for in vitro culture of follicles. 3. A method for in vitro cultivation of livestock preantral follicles, comprising the following steps: (A) Obtain bovine ovaries from the slaughterhouse, put them in sterilized normal saline (temperature 30-35°C), and send them to the laboratory as soon as possible. After sterilizing with 70% alcohol, sterilize the ovaries with 30-35°C normal saline. Rinse off residual alcohol; (B) Take out the follicle culture working solution stored in the 4°C refrigerator, preheat it in a 37°C water bath, and set aside; (C) Cut the ovary in the step (A) into thin slices of 1 cm×0.5 cm×0.2 cm with a scalpel, then rinse with sterilized physiological saline for 1 to 2 times, and put it into a watch glass; (D) Under a stereomicroscope, use a 25G needle to pick out the preantral follicles of livestock, and select the preantral follicles with a diameter of 160-220 μm for use; (E) Put the selected preantral follicles into a 24-well culture dish, add preheated culture medium at 1ml/well for single follicle culture; (F) Replace 70% of the culture medium at intervals of 24 hours; (G) The diameter of follicles was measured daily with a graduated microscope, and changes in the follicle cavity were observed.