CN106834216B - In-vitro culture solution and culture method for swine parthenogenetic activation embryos - Google Patents
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Abstract
The invention discloses an in-vitro culture solution (mPZM-3) for a swine parthenogenetic activated embryo and a culture method, belonging to the field of in-vitro culture of animal embryos, wherein the raw materials of the in-vitro culture solution (mPZM-3) comprise mPZM-3 basic culture solution, calcium lactate pentahydrate and sodium pyruvate, the concentration of the calcium lactate pentahydrate in the in-vitro culture solution is 0.606g/L, and the concentration of the sodium pyruvate is 0.022 g/L; the solvent used in the in vitro culture solution is hydrogen-rich water, and H is2The content of (A) is 1.2-1.6 ppm; the method for culturing the in-vitro culture solution for the swine parthenogenetic activation embryo comprises the following steps: washing a swine parthenogenetic activated embryo with an in-vitro culture solution containing 5 mu mol/L cytochalasin B, treating for 3-5 h, washing with an in-vitro culture solution without cytochalasin B, and culturing for 168h until the embryo develops into a blastocyst. The invention can ensure that energy and a development environment are better provided for the parthenogenetic activation of the pig embryo, thereby improving the embryo culture quality and effect.
Description
Technical Field
The invention relates to the field of in-vitro culture of animal embryos, in particular to an in-vitro culture solution and a culture method for swine parthenogenetic activated embryos.
Background
In vitro culture of animal embryos is an important link in embryo biotechnology. Under the condition of in vitro culture, the early embryo often cannot complete the whole process of development from fertilized eggs to blastula, but stops at a certain specific development period, which is called as embryo in vitro development blocking (in vitro block of embryo development), so that the in vitro development quality of the early embryo is seriously influenced, for example, the in vitro blastula development rate of the pig is only about 20 percent generally, the number of inner cell masses is about 30, the birth rate of offspring is reduced, and the research and application of the pig in agriculture, bioengineering and medicine are greatly hindered.
The in vitro culture of animal embryos is related to three factors, namely embryo types, culture conditions and components of culture solution, wherein the improvement of the components of the culture solution is the basis and key link of the in vitro culture technology, and directly influences the in vitro culture effect and the quality of in vitro embryo production.
Parthenogenetically activated embryos are embryos that are activated so that they complete a mature division and thereby transition to mitosis, starting a new ontogenesis. The research on parthenogenetic activation of mammals deepens the understanding of a plurality of important theoretical problems of embryogenesis, nuclear transplantation and the like. The success of parthenogenetic activation also has important theoretical and practical significance for wild animal protection, transgenic animal manufacture, deep research on biological development mechanism and the like.
At present, the existing in-vitro culture solution and culture method for the swine parthenogenetic activation embryo cannot ensure the stable activation of the oocyte, cannot provide required energy for cell metabolism well, and are not enough to obtain higher embryo culture quality and effect.
Disclosure of Invention
1. Technical problem to be solved
The invention aims to provide an in-vitro culture solution for a swine parthenogenetic activated embryo and a culture method, which are used for promoting the in-vitro development of the embryo.
2. Technical scheme
In order to solve the problems, the invention adopts the following technical scheme:
an in vitro culture solution for culturing swine parthenogenetic activation embryos, wherein raw materials of the in vitro culture solution comprise mPZM-3 embryo culture solution, calcium lactate pentahydrate and sodium pyruvate, the concentration of the calcium lactate pentahydrate in the in vitro culture solution is 0.606g/L, and the concentration of the sodium pyruvate is 0.022 g/L; the solvent used in the in vitro culture solution is hydrogen-rich water, and H is2The content of (B) is 1.2 to 1.6 ppm.
Further, the raw materials of the mPZM-3 embryo culture solution comprise mPZM-3 basic culture solution, L-glutamine, hypotaurine, L-cysteine, penicillin, streptomycin sulfate and bovine serum albumin, and the proportion of the raw materials is 1L: 0.146 g: 0.546 g: 0.069 g: 100000 IU: 70000 IU: 3g of the total weight.
Still further, the mPZM-3 basal medium includes solutes at the following concentrations: 6.312g/L of sodium chloride, 2.119g/L of sodium bicarbonate, 0.746g/L of potassium chloride, 0.048g/L of monopotassium phosphate and 0.048g/L of magnesium sulfate; also included are 2% essential amino acids and 1% nonessential amino acids.
For swine parthenogenetic activation of embryos, H in hydrogen-rich water in the in vitro culture solution2The concentration is preferably 1.4 ppm.
The invention also provides a method for culturing the swine parthenogenetic activation embryo by using the in-vitro culture solution, which comprises the following steps:
(1) washing the swine parthenogenetic activation embryo in an in-vitro culture solution containing 5 mu mol/L cytochalasin B for three times, and transferring the swine parthenogenetic activation embryo into the in-vitro culture solution containing 5 mu mol/L cytochalasin B for culturing for 3-5 h;
(2) the treated embryos are washed three times with a cytochalasin B-free culture solution, transferred into an in vitro culture solution without cytochalasin B, covered with 500 mu L of mineral oil at the same time, and cultured for 168 hours until the embryos develop into blastula.
Preferably, the porcine parthenogenetically activated embryo in step (1) above is cultured in an in vitro culture medium containing 5. mu. mol/L cytochalasin B for 4 h.
Specifically, the culture density of the swine parthenogenetic activation embryo in the step (1) is 40-60 embryos cultured in 500 mu L of in vitro culture solution under the conditions of 38.5 ℃ and 5% CO2And saturation humidity.
3. Advantageous effects
(1) Pyruvate is an energy substance necessary for cell metabolism, is a substrate generated by sugar metabolism, plays a role of replacing a carbon source in a culture medium, and participates in cell nutrition metabolism. Glucose, sodium lactate, etc. also function similarly in cell culture, but the mechanism of action varies from animal to animal. Especially the effect of glucose on the early embryo culture, the glucose has proved to have obvious inhibiting effect on mouse and hamster zygotes, and is a barrier for embryo to overcome development block, and pigs also have similar phenomena. Therefore, the invention adopts pyruvic acid as an energy substance for cell culture.
(2) The calcium ions play an important role in maintaining the stability and permeability of cell membranes and connection between cells and play an important role in the contraction of morula and the formation and expansion of blastocysts; in addition, an increase in calcium ion concentration is an important induction signal for oocyte activation. Calcium lactate pentahydrate has high solubility and can provide high-concentration calcium ions.
(3) The solvent used in the invention is hydrogen-rich water, and hydrogen in the solution can directly react with oxygen free radicals in cells to generate nontoxic and harmless water which can be utilized by the cells, thereby having obvious protective effect on oxidative damage of in vitro blastula.
(4) According to the method for culturing the swine parthenogenetic activation embryo, cytochalasin B is added into an in-vitro culture solution, and can inhibit the formation of microtubules and microfilaments in cells, so that cytoskeletons in oocytes are relaxed, the flexibility of the cells is increased, and cell division is facilitated; meanwhile, cytochalasin B destroys the formation of a spindle, inhibits the formation of polar bodies and the discharge of chromosomes, ensures the chromosome multiple, can improve the blastula development rate of parthenogenetic activated oocytes when being used with other activators, increases the number of cells in blastula, reduces the active oxygen level in blastula cells, and thus improves the number and quality of blastula.
The in-vitro culture solution for culturing the swine parthenogenetic activation embryo and the application method thereof can ensure the stable activation of the oocyte, better provide required energy for cell metabolism and are beneficial to improving the embryo culture quality and effect.
Drawings
FIG. 1 shows the result of nuclear staining of blastocysts in control and hydrogen-rich porcine parthenogenetically activated embryos cultured up to 168 h;
FIG. 2 shows the results of the potential staining of the blastocyst mitochondrial membrane in the control group and the hydrogen-rich group after culturing the swine parthenogenetically activated embryos for 168 h;
FIG. 3 shows the results of in situ fluorescent staining of blastocysts Pan-caspase in control and hydrogen-rich pigs cultured to 168 h.
Detailed Description
In order that the invention may be more readily understood, specific embodiments thereof will be described further below. The following examples illustrate standard laboratory practice of the inventors for illustrating the mode of the invention, and the invention should not be construed as being limited in scope to these examples. This example is provided in light of the present disclosure and the general level of skill in the art, and it will be understood by those skilled in the art that the following is illustrative only and that various changes, modifications and alterations can be made without departing from the scope of the invention. The techniques referred to therein, unless otherwise indicated, are conventional laboratory techniques well known to those skilled in the art; unless otherwise specified, reagents and materials used in the following examples are commercially available.
Examples
Preparation of mature culture solution of porcine oocyte
The in vitro maturation medium was prepared by mixing TCM-199 with 10% (V/V) fetal bovine serum (Gibco, USA), 10% (V/V) porcine follicular fluid (self-made in the laboratory), 10IU/mL pregnant mare serum gonadotropin (Ningbo, third hormone products Co., Ltd., China), 10IU/mL human chorionic gonadotropin (Ningbo, third hormone products Co., Ltd., China), 10IU/mL double antibody (Sigma, USA), 0.1mg/mL L-cysteine (Sigma, USA), and 10ng/mL epidermal growth factor (Sigma, USA).
Second, obtaining and in vitro maturation of porcine oocytes
Pig ovaries were harvested from slaughterhouses and placed in saline at 37 ℃ (containing 1000IU/mL penicillin and streptomycin) and transported back to the laboratory within 2 hours. Cumulus Oocyte Complexes (COCs) with the follicle diameter of 2-8 mm are extracted by a No. 18 needle syringe and injected into a 15mL centrifuge tube, supernatant is discarded after 30min of precipitation, COCs with more than 3 layers of granular cells and uniform cytoplasm are selected under a microscope, washed three times by Taiwan liquid and TCM-199(Gibco, USA) in sequence, and placed into in-vitro maturation culture solution with pre-temperature balance for culture for 44 h. The culture conditions were 38.5 ℃ and 5% CO2And saturation humidity.
Preparation of parthenogenetic activating solution and embryo culture solution
Parthenogenetic activating solution: 0.3mol/L mannitol, 0.05mmol/L calcium chloride and 0.1mmol/L magnesium chloride.
In vitro culture solution: 6.312g/L of sodium chloride, 2.119g/L of sodium bicarbonate, 0.746g/L of potassium chloride, 0.048g/L of potassium dihydrogen phosphate, 0.048g/L of magnesium sulfate, 0.606g/L of calcium lactate pentahydrate, 0.022g/L, L g/L of sodium pyruvate-glutamine 0.146g/L, 0.546g/L of hypotaurine, 0.069g/L of cysteine, 10000IU/L of penicillin, 70000IU/L of streptomycin, 2% of essential amino acid (50X), 1% of non-essential amino acid (100X) and 3g/L of bovine serum albumin. The solvent used in the experimental group was hydrogen rich water, wherein H2The content was 1.4ppm (experimental group may also be referred to as hydrogen-rich group), and the solvent used in the control group was distilled water.
Parthenogenetic activation of porcine MII oocytes
The COCs cultured for 44h are transferred into 0.1% hyaluronidase (Sigma, USA) and blown by a pipette gun for 2min, oocytes which are uniform in cytoplasm, normal in shape and discharged from the first polar body are picked out under a microscope, transferred into TCM-199 and washed for 3 times, and placed on a constant temperature table at 37 ℃ for standby.
The selected oocytes are balanced in the activating solution for 5min, transferred into a 0.2mm activating groove paved with the activating solution and discharged in parallel with the electrodes for electric activation, and the activating parameters are 1.2kV/cm, 30 mus and one-time pulse.
In vitro culture of swine parthenogenetic activation embryo
The activated oocytes were washed three times in a culture solution containing 5. mu. mol/L cytochalasin B (Sigma, USA), cultured therein for 3-5 h, and then washed three times with a culture solution containing no cytochalasin B.
In vitro culture solutions of the hydrogen-rich group and the control group were prepared in advance, and each of the prepared in vitro culture solutions was filled in two four-well plates (NUNC, Denmark) of 500. mu.L.
The activated and washed oocytes were divided into two portions and transferred to prepared four-well plates, each covered with 500. mu.L of mineral oil. The culture conditions were 38.5 ℃ and 5% CO2And saturation humidity.
And sixthly, counting the cleavage rate, the blastula rate and the blastula cell number of the parthenogenetic activated embryo.
And (4) counting the cleavage rate of the embryo after parthenogenetic activation at the 44 th hour, and counting the blastocyst rate at the 168 th hour. After incubation with 5. mu.g/mL Hoechst33342 staining solution for 10min in the dark, nuclei were stained, then washed 3 times with TCM-199, and the stained nuclei were placed on a slide glass and pressed into a tablet, and observed under an inverted fluorescence microscope (IX71, OLYMPUS, Japan), as shown in FIG. 1, nuclei of all cells in the blastocyst were stained with Hoechst33342 to show blue fluorescence, and the number of blastocysts was counted.
Seventhly, detecting the level of mitochondrial membrane potential (delta Ψ m) in the blastocyst
The above three groups of blastocysts were subjected to mitochondrial membrane potential detection using PZM-3 solution containing 10. mu.g/mL JC-1(5, 5 ', 6, 6' -tetrachloro-1, 1 ', 3, 3' -tetraethylene-imidocynanine iodide) (Byunnan, China) under staining conditions of 37 ℃ for 20min in the absence of light, followed by 3 washes with TCM-199 and detection with laser confocal microscopy (TCS SP2, Leica, Germany). At the maximum cross-section of the blastocyst cells, the red and green fluorescence signal intensities were observed and recorded, respectively. As a result, as shown in FIG. 2, the ratio of the fluorescence intensity of Red (RITC) to that of green (FITC) was Δ Ψ m. Each set of experiments was repeated 3 times, approximately 20 blastocysts each time.
Eighth, detection of Pan-caspase Activity in blastocysts
caspases are responsible for selectively cleaving certain proteins, thereby causing apoptosis. I.e., the more active they are, the faster the cells will die. Staining was performed using the Pan-caspase in situ fluorescent staining kit manufactured by Promega (USA). FITC-VAD-FMK is diluted to 10 mu mol/L by TCM-199, blastocysts are incubated in staining solution at 37 ℃ for 15min in the dark, washed 3 times by TCM-199 and then photographed under a fluorescence microscope. The gel was washed 3 times with TCM-199 and photographed under a fluorescent microscope. The results are shown in FIG. 3, and the fluorescence photographs were quantified using Image-Pro Plus 6.0 software and the fluorescence intensity values were recorded, and the results are expressed as the fluorescence average relative density. Each set of experiments was repeated 3 times, approximately 20 blastocysts each time.
Nine results
1. And (4) statistics of cleavage rate, blastocyst rate and blastocyst cell number.
Results of calculation of cleavage rate, blastocyst rate and blastocyst cell number of the control group and the hydrogen-rich group are shown in table 1.
TABLE 1 Effect of Hydrogen-rich Water on porcine parthenogenetic activation of embryo in vitro development
Note: data in the same column are marked with different letters to indicate significant difference (P < 0.05).
As can be seen from table 1, the cleavage rate of the hydrogen-rich group was significantly higher than that of the control group when parthenogenetically activated embryos developed in vitro to 44 h. Parthenogenetic activation embryos developed in vitro to 168h, the blastula rate of the hydrogen-rich group was significantly higher than that of the control group, and the number of cells in blastula was also significantly higher than that of the control group (P < 0.05).
In FIG. 1, the number of cells in hydrogen-rich blastocysts was significantly higher than that of the control group.
2. Mitochondrial membrane potential (. DELTA.. PSI.m) levels and Pan-caspase Activity assays for blastocysts
The control and hydrogen-rich groups showed Δ Ψ m and Pan-caspase activity statistics as shown in Table 2.
TABLE 2 Effect of hydrogen-rich water on porcine parthenogenetically activated blastocyst mitochondrial membrane potential and Pan-caspase activity
| Group of | ΔΨm | Pan-caspase Activity |
| Control group | 0.91±0.07(58)b | 10.65±1.26(69)a |
| Hydrogen rich component | 1.12±0.12(56)a | 7.41±2.24(63)b |
As can be seen from Table 2, the mitochondrial membrane potential (. DELTA.. PSI.m) of the hydrogen-rich blastocysts was significantly higher than that of the control group. While the Pan-caspase activity was significantly lower than that of the control group (P < 0.05).
In FIG. 2, the hydrogen-rich blastocysts exhibited a significantly higher yellow or orange fluorescence intensity than the control.
In FIG. 3, the fluorescence intensity of the blastocysts of the control group was significantly higher than that of the hydrogen-rich group.
As is clear from fig. 1, fig. 2, fig. 3 and the above results, the effect of hydrogen-rich water on the in vitro culture of swine parthenogenetically activated embryos was as follows: the hydrogen-rich water can improve the cell cleavage rate, improve the blastocyst development rate and increase the number of cells in the blastocyst; it also enhances mitochondrial membrane potential in the blastocyst; and the activity of the Pan-caspase in the blastula is obviously reduced, which is beneficial to maintaining the activity of the cells.
It should be understood by those skilled in the art that the above embodiments are only for illustrating the present invention and are not to be used as a limitation of the present invention, and that changes and modifications to the above embodiments are within the scope of the claims of the present invention as long as they are within the spirit and scope of the present invention.
Claims (7)
1. An in vitro culture solution for swine parthenogenetic activation embryos, which is characterized in that: the raw materials of the in vitro culture solution comprise mPZM-3 embryo culture solution, calcium lactate pentahydrate and sodium pyruvate, wherein the concentration of the calcium lactate pentahydrate in the in vitro culture solution is 0.606g/L, and the concentration of the sodium pyruvate is 0.022 g/L; the solvent used in the in vitro culture solution is hydrogen-rich water, and H is2The content of (B) is 1.2 to 1.6 ppm.
2. The in vitro culture solution for swine parthenogenetically activated embryos of claim 1, wherein the culture solution is prepared by culturing a swine parthenogenetically activated embryo in a culture medium comprising a culture medium containing a culture medium and a culture medium containing a culture mediumThe raw materials of the mPZM-3 embryo culture solution comprise mPZM-3 basic culture solution, L-glutamine, hypotaurine, L-cysteine, penicillin, streptomycin sulfate and bovine serum albumin, and the mixture ratio of the mPZM-3 embryo culture solution to the bovine serum albumin is 1L: 0.146 g: 0.546 g: 0.069 g: 100000 IU: 70000 IU: 3g of the total weight of the mixture; the solvent used by the mPZM-3 embryo culture solution is hydrogen-rich water, and H in the solvent2The content of (B) was 1.4 ppm.
3. The in vitro culture solution for the swine parthenogenetically activated embryo according to claim 2, wherein the mPZM-3 basal culture solution comprises solutes at the following concentrations: 6.312g/L of sodium chloride, 2.119g/L of sodium bicarbonate, 0.746g/L of potassium chloride, 0.048g/L of monopotassium phosphate and 0.048g/L of magnesium sulfate; also included are 2% essential amino acids and 1% nonessential amino acids.
4. A method for culturing a swine parthenogenetically activated embryo by using the in vitro culture solution according to any one of claims 1 to 3, comprising the steps of:
(1) washing the swine parthenogenetic activation embryo in an in-vitro culture solution containing 5 mu mol/L cytochalasin B for three times, and transferring the swine parthenogenetic activation embryo into the in-vitro culture solution containing 5 mu mol/L cytochalasin B for culturing for 3-5 h;
(2) and washing the processed embryo with a culture solution without cytochalasin B for three times, transferring the washed embryo into an in-vitro culture solution without cytochalasin B, and culturing for 168 hours until the embryo develops into a blastocyst.
5. The method for culturing the swine parthenogenetically activated embryo according to claim 4, wherein the swine parthenogenetically activated embryo in step (1) is cultured in the in vitro culture solution containing 5 μmol/L cytochalasin B for 4 h.
6. The method for culturing the swine parthenogenetically activated embryo according to claim 4, wherein the culturing density of the swine parthenogenetically activated embryo in step (1) is 40-60 embryos per 500 μ L of the in vitro culture solution.
7. The method for culturing swine parthenogenetically activated embryos in vitro according to claim 4, wherein the culturing conditions in step (2) are 38.5 ℃ and 5% CO2And saturation humidity.
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| CN110184233B (en) * | 2019-05-15 | 2023-07-25 | 湖北省农业科学院畜牧兽医研究所 | Oocyte in-vitro maturation culture solution additive and application thereof |
| CN110205284B (en) * | 2019-05-27 | 2021-11-19 | 中国农业大学 | Application of L-proline in improving early embryo development and oxidation resistance of oocyte |
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