Culture solution for reducing lipid of animal embryo before freezing and preparation method and application thereof

Abstract

The invention discloses a culture solution for reducing lipid of animal embryos before freezing as well as a preparation method and application of the culture solution. The culture solution is prepared by adding bezafibrate and cholic acid into an NCSU23 culture solution. The invention also discloses a preparation method of the culture solution, which comprises the following steps: preparing an NCSU23 culture solution, and adding bezafibrate and cholic acid into the NCSU23 culture solution. Compared with a conventional NCSU23 culture solution, the culture solution provided by the invention has the advantages that the added bezafibrate and cholic acid have a synergistic lipid-lowering effect, so that the content of lipid in an embryo can be remarkably reduced, and the survival rate and the blastocyst hatching rate after the embryo is frozen and unfrozen are finally effectively improved. The culture solution provided by the invention has application prospects in the aspects of animal embryo lipid reduction, frozen embryo survival and development and the like.

Description

Technical Field

[0001] The invention relates to an animal embryo culture medium and application thereof, in particular to a lipid-reducing culture medium before embryo freezing and a preparation method thereof and application thereof in improving the survival rate of embryos after freezing and thawing or the hatching blastocyst rate, and belongs to the field of animal embryo culture medium. Background Art

[0002] In vivo embryos refer to embryos formed by fertilization of oocytes in the body of female animals after mating. On the 6th day after the first mating, the uterine horns are flushed with embryo flushing fluid, and the embryo flushing fluid is examined under a stereomicroscope to obtain embryos of the 6th day of embryonic age, with morula embryos being the main developmental stage.

[0003] The high lipid content in pig embryos is a species-related cell characteristic. However, high lipid content is also considered to be an important factor restricting the improvement of pig embryo freezing efficiency. Embryo cryopreservation technology mainly preserves early embryos under extremely low temperature conditions to maintain their vitality and developmental potential for a long time. At present, the transplantation of frozen embryos has obtained surviving offspring in more than 20 species; the freezing of cattle and sheep embryos has been maturely applied to commercial introduction. During embryo cryopreservation, a large amount of intracellular lipids hinders the penetration of cryoprotectants and limits the effect of embryo vitrification. It is one of the main reasons for the loss of cell activity and developmental potential after pig embryo freezing, resulting in the current situation that the embryo freezing efficiency of pigs is lower than that of cattle, sheep and other livestock. It is a difficult problem that urgently needs to be broken through in pig breeding and germplasm resource protection. Under the premise of ensuring that the embryonic developmental potential is not damaged, achieving embryo lipid reduction and improving the embryo freezing effect after lipid reduction are issues that need to be urgently addressed in pig embryo cryopreservation.

[0004] Embryo lipid reduction is mainly divided into invasive micromanipulation and non-invasive lipid regulation. The early invasive micromanipulation method is to polarize lipids through high-speed centrifugation and extract them through a microinjection needle to achieve the purpose of fat removal. Although this method has a significant fat removal effect, high-speed centrifugation leads to embryonic dysplasia, and the damage to the zona pellucida caused by microinjection will damage the cell development potential and increase the risk of pathogen contamination. Therefore, using the biological activity of the embryo to achieve non-invasive lipid reduction is the best solution to take into account both pig embryo lipid reduction and the survival and development of frozen embryos. Summary of the invention

[0005] One of the purposes of the present invention is to provide a culture medium for reducing lipids before embryo freezing.

[0006] The second object of the present invention is to provide a method for preparing a lipid-lowering culture medium before embryo freezing.

[0007] The third purpose of the present invention is to apply the culture solution to reduce the lipid content in the embryo or to improve the survival rate and hatching blastocyst rate of the embryo after freezing and thawing.

[0008] In order to achieve the above object, the main technical solutions adopted by the present invention include:

[0009] On one hand, the present invention provides a culture medium for reducing lipids before freezing of animal embryos.

[0010] In a preferred embodiment, the culture medium is prepared by adding bezafibrate and bile acid to animal embryo culture medium.

[0011] A preferred specific embodiment, the animal embryo culture medium is NCSU23 culture medium, which is specifically composed of the following ingredients per 100 mL of culture medium: 0.6356 g sodium chloride, 0.2106 g sodium bicarbonate, 0.0356 g potassium chloride, 0.0162 g potassium dihydrogen phosphate, 0.0294 g magnesium sulfate heptahydrate, 0.0250 g calcium chloride dihydrate, 0.1000 g D-glucose, 0.0876 g taurine, 0.0546 g hypotaurine, 1.0000 mL penicillin-streptomycin solution, 0.4000 g bovine serum albumin, and the balance is water, and the pH value is 7.2; wherein the concentration of the penicillin-streptomycin solution is 10000 IU / mL.

[0012] In a preferred embodiment, the content of bezafibrate in the culture solution is 40-60 μM per 100 mL of culture solution; preferably, the content of bezafibrate in the culture solution is 50 μM per 100 mL of culture solution.

[0013] In a preferred specific embodiment, the content of bile acid in the culture medium is 40-60 μM per 100 mL of culture medium; preferably, the content of bile acid in the culture medium is 50 μM per 100 mL of culture medium.

[0014] Another aspect of the present invention provides a method for preparing a lipid-lowering culture medium for animal embryos before freezing, comprising: preparing an animal embryo culture medium, and adding bezafibrate and bile acid to the animal embryo culture medium to obtain the culture medium.

[0015] Another aspect of the present invention is to apply the culture solution to reduce the lipid content in embryos, or to increase the survival rate of embryos after freezing and thawing and the hatching blastocyst rate.

[0016] In a preferred specific implementation scheme, the specific application steps of the culture medium include: (1) preheating the culture medium in an environment of 37-39°C and 4-6% carbon dioxide for 0.5-2 hours; (2) transferring animal embryos to the preheated culture medium and incubating them in an environment of 37-39°C and 4-6% carbon dioxide for 5-7 hours.

[0017] In a preferred embodiment, in step (1), the culture medium is preheated at 38.5°C and 5% carbon dioxide for 1 hour; in step (2), the animal embryos are transferred to the preheated culture medium and incubated at 38.5°C and 5% carbon dioxide for 6 hours.

[0018] In a preferred embodiment, the incubated animal embryos are washed 2-4 times with embryo culture medium before being frozen.

[0019] In a preferred embodiment, the animal embryo is a pig embryo, preferably an in vivo pig embryo at the morula stage of development.

[0020] The present invention adds bezafibrate and cholic acid to an animal embryo culture medium to obtain a lipid-lowering culture medium for animal embryos before freezing. Because bezafibrate and cholic acid have a synergistic lipid-lowering effect, compared with the original animal embryo culture medium, the culture medium can significantly reduce the lipid content in the embryo and ultimately effectively improve the survival rate and hatching blastocyst rate of the embryo after freezing-thawing. The present invention has the advantages of simple method, low technical requirements for operators, and high repeatability in practical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The lipid-lowering effect of incubating pig embryos with the culture medium provided by the present invention for 6 hours, the lipid-lowering effect when only one lipid-lowering agent is contained, and the lipid-lowering effect when any one of the two agents is replaced by the conventional lipid-lowering agent L-carnitine or niacin; wherein, Figure 1 -A is a fluorescence quantitative detection diagram of lipid droplets in embryos;

[0022] Figure 1 -B is a statistical analysis of the relative fluorescence intensity of lipid droplets when embryos were incubated with bezafibrate and bile acid alone or in combination; Figure 1 -C is a statistical analysis of the relative fluorescence intensity of lipid droplets in embryos when bezafibrate and bile acid were replaced by L-carnitine or niacin, respectively; LDs: lipid droplets, embryos were incubated with Nile red fluorescent dye to quantitatively detect lipid content; *: P < 0.05;

[0023] Figure 2 The lipid content and embryonic cell division level of pig embryos incubated with the culture solution provided by the present invention before and after freezing are measured; wherein, Figure 2 -A is the fluorescence quantitative detection of lipid droplets and the fluorescence localization detection of cell nuclei; Figure 2 -B is a statistical analysis of the relative fluorescence intensity of lipid droplets; Figure 2-C is a statistical analysis of the number of cell nuclei; LDs: lipid droplets, embryos were incubated with Nile red fluorescent dye to quantitatively detect lipid content; Hoechset33342: a nuclear fluorescent dye that reflects the level of embryonic cell division through the number of cell nuclei; Merge: a composite image of lipid droplets and cell nuclei; NCSU23: embryos incubated with NCSU23 culture medium (without adding any other components); Embryo culture medium 1: lipid-lowering embryos incubated with embryo culture medium 1 prepared in Example 1; NCSU23+freezing: embryos incubated with NCSU23 culture medium (without adding any other components), frozen-thawed, and then cultured in NCSU23 culture medium for 12 hours; Embryo culture medium 1+freezing: embryos incubated with embryo culture medium 1 prepared in Example 1, frozen-thawed, and then cultured in NCSU23 culture medium for 12 hours; *: P<0.05; ***: P<0.001; ****: P<0.0001; ns: P>0.05;

[0024] Figure 3 The graph shows the survival results of frozen-thawed pig embryos after incubation with NCSU23 culture medium and embryo culture medium 1 prepared in Example 1, and the results of continued development to form hatching blastocysts. DETAILED DESCRIPTION

[0025] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, it should be understood that the embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall into the protection scope of the present invention. Although many materials and operating methods used to achieve the purpose of the present invention are well known in the art, the present invention is still described in as much detail as possible here. If not specifically stated, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the raw materials used are all commercially available commodities.

[0026] Example 1 Preparation of lipid-lowering culture medium before embryo freezing

[0027] (1) Take 60 mL of ultrapure water, add 0.6356 g of sodium chloride, 0.2106 g of sodium bicarbonate, 0.0356 g of potassium chloride, 0.0162 g of potassium dihydrogen phosphate, 0.0294 g of magnesium sulfate heptahydrate, 0.0250 g of calcium chloride dihydrate, 0.1000 g of D-glucose, 0.0876 g of taurine, 0.0546 g of hypotaurine, and 1.0000 mL of 10000 IU / mL penicillin-streptomycin solution, adjust the pH to 7.2, and dilute to 100 mL with ultrapure water;

[0028] (2) Filter through a 0.22 μm filter and add 0.4000 g of bovine serum albumin;

[0029] (3) 0.0018 g of bezafibrate was added to 500 μL of anhydrous bile acid (10 mM in DMSO), dissolved, and then added to 100 mL of NCSU23 culture medium to obtain;

[0030] (4) The culture medium should not be stored at 4°C for more than 1 month.

[0031] Experimental Example 1 Experiment on reducing lipid content in embryos, improving embryo survival rate after freezing and thawing, and hatching blastocyst rate using animal embryo culture medium

[0032] 1 Test method

[0033] 1.1 Embryo culture medium for experiment:

[0034] (1) Embryo culture medium 1: embryo culture medium 1 prepared in Example 1; (2) Embryo culture medium 2: except for the absence of bile acid, the other components and preparation method are exactly the same as those of embryo culture medium 1 prepared in Example 1; (3) Embryo culture medium 3: except for the absence of bezafibrate, the other components and preparation method are exactly the same as those of embryo culture medium 1 prepared in Example 1; (4) Embryo culture medium 4: embryo culture medium obtained by replacing bezafibrate in embryo culture medium 1 prepared in Example 1 with L-carnitine of the same concentration; (5) Embryo culture medium 5: embryo culture medium obtained by replacing bezafibrate in embryo culture medium 1 prepared in Example 1 with niacin of the same concentration; (6) Embryo culture medium 6: embryo culture medium obtained by replacing bile acid in embryo culture medium 1 prepared in Example 1 with L-carnitine of the same concentration; (7) Embryo culture medium 7: embryo culture medium obtained by replacing bile acid in embryo culture medium 1 prepared in Example 1 with niacin of the same concentration; (8) NCSU23 culture medium (as a control).

[0035] 1.2 Test methods

[0036] Obtaining, incubating and freezing pig embryos:

[0037] The sow is impregnated by mating or artificial insemination. On the 6th day after mating or artificial insemination, the uterine horns of the sow are flushed with embryo flushing fluid using conventional surgical methods, or the uterus and ovaries are completely removed immediately after the sow is killed, and the uterine horns are flushed with embryo flushing fluid; the flushing fluid is collected and examined under a stereomicroscope to obtain pig embryos in the morula stage.

[0038] Preheat the culture medium with reduced lipids before freezing for 1 hour at 38.5°C and 5% carbon dioxide. Transfer the morula embryos from the embryo flushing solution to the culture medium with reduced lipids before freezing, incubate for 6 hours at 38.5°C and 5% carbon dioxide, wash 3 times with conventional embryo culture medium, and then use for freezing.

[0039] Freezing: The embryos were transferred to vitrification medium I (NCSU23 culture medium supplemented with 20% fetal bovine serum, 7.5% dimethyl sulfoxide and 7.5% ethylene glycol) for 3 min, transferred to vitrification medium II (NCSU23 culture medium supplemented with 20% fetal bovine serum, 15% dimethyl sulfoxide, 15% ethylene glycol and 397 mM sucrose) for 10 s, loaded onto freezing rods and immersed in liquid nitrogen.

[0040] Thawing: Take the cryostat out of liquid nitrogen, and immediately immerse the embryo loading end in thawing solution I to separate the embryo from the cryostat. Maintain the embryo in thawing solution I (NCSU23 culture medium supplemented with 20% fetal bovine serum and 300mM sucrose) for 5 minutes, transfer to thawing solution II (NCSU23 culture medium supplemented with 20% fetal bovine serum and 150mM sucrose) and maintain it for 5 minutes. Wash it three times with regular embryo culture medium and incubate it in regular embryo culture medium for 6 to 12 hours.

[0041] Nile red fluorescent dye was used to stain the lipid droplets in the embryos, and Hoechst33342 fluorescent dye was used to stain the cell nuclei in the embryos. Laser scanning confocal microscopy was used to photograph and quantify the lipid droplets and cell nuclei in the embryos. ImageJ software was used to digitize the images. IBM SPSS Statistics software was used to perform one-way ANOVA analysis. The results were expressed as mean ± standard error and statistical graphs were drawn.

[0042] 2 Test results

[0043] 2.1 Results of experiments on reducing lipid content in embryos

[0044] Depend on Figure 1 -A, Figure 1 -B It can be seen that incubating embryos with culture medium containing only bezafibrate or only bile acid can slightly reduce the lipid content of the embryos, but there is no significant statistical difference compared with the embryo control group incubated with NCSU23 culture medium (without adding any other ingredients); however, the embryo culture medium 1 prepared in Example 1 contains both bezafibrate and bile acid, and incubating pig embryos for 6 hours can significantly reduce the lipid content in the embryo; therefore, the combined use of bezafibrate and bile acid in the embryo culture medium 1 prepared in Example 1 has a synergistic effect on lipid-lowering in pig embryos.

[0045] Depend on Figure 1 -A, Figure 1-C It can be seen that when the drug combination that exerts a lipid-lowering effect is bezafibrate and cholic acid, the lipid content of the embryo after incubation is the lowest among the results of various drug combinations, and the lipid-lowering effect is the highest; when either bezafibrate or cholic acid is replaced, the lipid content of the embryo after incubation is not significantly different from that of the fresh control, and the lipid-lowering effect is not as good as the embryo culture medium 1 prepared in Example 1.

[0046] The results of the test on lipid content of embryos after freezing and thawing are as follows Figure 2 -A, Figure 2 As shown in -B, after the embryos incubated with the culture medium prepared in Example 1 were frozen and thawed, the lipid content in the embryos was significantly lower than that in the frozen embryo control.

[0047] 2.2 Experimental results of embryo survival rate and blastocyst hatching rate after freezing and thawing

[0048] The results of the test for the ability of embryonic cells to divide are as follows Figure 2 -A, Figure 2 -C, the embryos incubated with the embryo culture medium 1 prepared in Example 1 were cultured in NCSU23 culture medium for 12 hours after freezing and thawing, and the embryo cell division ability was significantly higher than that of the embryos incubated with NCSU23 culture medium (without adding any other ingredients), then frozen and thawed, and then cultured in NCSU23 culture medium for 12 hours.

[0049] The frozen and thawed embryos were observed and identified by morphology. If the embryonic cytoplasm recovered from the condensed glassy state and expanded to form a blastocyst cavity, and the embryonic morphology was complete, the outline was clear, the color tone and cell density were good, then it was considered alive. Figure 3 As shown, after the embryos were incubated with the embryo culture solution 1 prepared in Example 1 for 6 hours, 91.30% of the morulae developed into early blastocysts or blastocysts. The survival rate of the embryos incubated with the embryo culture solution 1 prepared in Example 1 after freezing and thawing reached 75.76% after 6 hours, which was significantly higher than the survival rate of 60.61% of the embryos incubated with the NCSU23 culture solution after freezing and thawing. After 12 hours of freezing and thawing, 66.67% of the embryos incubated with the embryo culture solution 1 prepared in Example 1 developed into expanded blastocysts and hatched blastocysts, which was significantly higher than the survival rate of 42.42% of the embryos incubated with the NCSU23 culture solution after 12 hours of freezing and thawing.

Claims

1. A culture medium for reducing lipids before freezing animal embryos, characterized in that: The culture fluid is prepared by adding bezafibrate and bile acid into animal embryo culture fluid.

2. The culture solution according to claim 1, characterized in that The animal embryo culture medium is NCSU23 culture medium; the composition of the NCSU23 culture medium is: every 100 mL of culture medium contains the following ingredients: 0.6356 g sodium chloride, 0.2106 g sodium bicarbonate, 0.0356 g potassium chloride, 0.0162 g potassium dihydrogen phosphate, 0.0294 g magnesium sulfate heptahydrate, 0.0250 g calcium chloride dihydrate, 0.1000 g D-glucose, 0.0876 g taurine, 0.0546 g hypotaurine, 1.0000 mL penicillin-streptomycin solution, 0.4000 g bovine serum albumin, the balance is water, and the pH value is 7.2; wherein the concentration of the penicillin-streptomycin solution is 10000 IU / mL.

3. The culture solution according to claim 1, characterized in that Each 100 mL of culture medium contains 40-60 μM bezafibrate and 40-60 μM bile acid.

4. The culture solution according to claim 3, characterized in that Each 100 mL of culture medium contained 50 μM bezafibrate and 50 μM cholic acid.

5. A method for preparing the culture solution according to any one of claims 1 to 4, characterized in that: include: An animal embryo culture medium is prepared; and bezafibrate and bile acid are added to the animal embryo culture medium to obtain the product.

6. Use of the culture solution described in any one of claims 1 to 4 in reducing the fat content of animal embryos, or in improving the survival rate of animal embryos after freezing and thawing or the hatching blastocyst rate.

7. The use according to claim 6, characterized in that: include: (1) preheating the culture medium at 37-39° C. and 4-6% carbon dioxide for 0.5-2 h; (2) Transfer the animal embryos into preheated culture medium and incubate at 37-39°C and 4-6% carbon dioxide for 5-7 hours.

8. The use according to claim 7, characterized in that: In step (1), the culture medium is preheated at 38.5° C. and 5% carbon dioxide for 1 hour; in step (2), the animal embryos are transferred to the preheated culture medium and incubated at 38.5° C. and 5% carbon dioxide for 6 hours.

9. The use according to claim 7, characterized in that: The incubated animal embryos are washed 2-4 times with embryo culture medium and then used for freezing.

10. The use according to claim 6, characterized in that: The animal embryo is a pig embryo, preferably an in vivo pig embryo at the morula stage of development.

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