Application of tributyrin as an additive for embryo in vitro development culture medium

Abstract

The invention discloses the use of tributyrin as an additive for embryo in vitro development culture medium, and belongs to the technical field of animal reproductive biology. The invention applies tributyrin as an additive to mouse embryo in vitro development culture medium for the first time, and the tributyrin can significantly improve the blastocyst rate, reduce the ROS content in the embryo, improve the mitochondrial membrane potential in the embryo, improve the ATP level in the embryo and the expression of antioxidant genes, improve the DNA methylation and histone modification level in the embryo, and promote the embryo in vitro development; and tributyrin, as a natural antioxidant and epidrug, is safe and has no toxic side effects, is cheap and easy to obtain, does not need to replace the culture medium during the entire development and culture process, and is simple to operate; the tributyrin provides strong support for in vitro embryo engineering technologies such as the efficient development of embryos in human assisted reproductive technology, mammalian fertilized embryos, parthenogenetic embryos and somatic cell cloned embryos, and has good application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of animal reproductive biology, and in particular relates to the use of tributyrin as an additive for embryo in vitro development culture medium. Background Art

[0002] During the in vitro development of embryos (IVC), one of the important issues is the generation and accumulation of reactive oxygen species (ROS) in the environment, culture medium, mineral oil and embryos themselves. Insufficient ROS removal causes cellular oxidative stress, which seriously reduces the embryo's developmental capacity by oxidizing RNA, DNA and protein. Unlike in vitro culture, somatic cells in the in vivo environment can consume oxygen and provide a low-oxygen environment for gamete and early embryo development; moreover, the body's efficient antioxidant system can promptly remove freshly generated ROS and protect cells from ROS damage. Compared with the in vivo environment, the antioxidant capacity of in vitro embryos is reduced, making them more susceptible to oxidative stress and leading to increased ROS levels. Therefore, in order to improve the developmental capacity of in vitro embryos, a variety of antioxidants have been applied to IVC.

[0003] In the early stages of embryonic development, the transcription level of the zygotic genome is in a silent state, and development depends on maternal substances stored in the oocyte. As embryonic development proceeds, maternal substances will gradually degrade, and the zygotic genome will gradually activate transcription, marking the transition of embryonic development from maternal regulation to zygotic genome regulation. During the zygotic genome activation (ZGA) period, epigenetic modifications on the genome undergo large-scale reprogramming, and the developmental arrest of in vitro embryos first appears at the ZGA stage, largely due to the failure of some key epigenetics to be completely reprogrammed by oocytes, among which DNA and histone epigenetic modifications are more important. In vitro embryos of various animals have been found to have abnormal levels of DNA methylation and histone acetylation at the ZGA stage, resulting in insufficient ZGA initiation and delayed embryonic development. Therefore, epigenetic barriers are an important barrier to in vitro embryonic development. Therefore, in order to improve the developmental capacity of in vitro embryos, a variety of epigenetic modification drugs have been tried in IVC.

[0004] Tributyrin (TB) is an ester compound formed by glycerol and butyric acid. It has a variety of biological activities and pharmacological properties, which has attracted people's attention and research. However, there are no reports on the use of TB for embryonic development in vitro. Summary of the invention

[0005] The purpose of the present invention is to provide the use of tributyrin as an additive for embryo in vitro development culture medium. In the present invention, tributyrin is used as an additive for the first time in mouse embryo in vitro development culture medium, and the tributyrin can significantly increase the blastocyst rate, reduce the ROS content in the embryo, improve the mitochondrial membrane potential in the embryo, increase the ATP level in the embryo and the expression of antioxidant genes, improve the DNA methylation and histone modification levels in the embryo, and promote the in vitro development of the embryo.

[0006] In a first aspect, the present invention provides the use of tributyrin as an additive for embryo in vitro development culture medium, and the use includes promoting embryo development.

[0007] In some embodiments, promoting embryonic development includes any of the following: A1) reducing ROS content in the embryo; A2) improving mitochondrial membrane potential in the embryo; A3) increasing ATP level and expression of antioxidant genes in the embryo; A4) improving DNA methylation and histone modification levels in the embryo.

[0008] In some embodiments, the concentration of tributyrin is 50-200 μmol / L, for example, 50 μmol / L, 80 μmol / L, 100 μmol / L, 120 μmol / L, 150 μmol / L, 160 μmol / L, 180 μmol / L, 200 μmol / L or other values ​​within this range.

[0009] In some embodiments, the concentration of tributyrin is 100 μmol / L.

[0010] In some embodiments, the embryo in vitro development medium comprises KMSO basal medium.

[0011] In a second aspect, the present invention provides an embryo in vitro development culture medium additive for promoting embryo development, the additive comprising tributyrin.

[0012] In a third aspect, the present invention provides an embryo in vitro development culture medium for promoting embryo development, the culture medium comprising the above-mentioned additive.

[0013] In some embodiments, the concentration of the additive is 50-200 μmol / L, for example, 50 μmol / L, 80 μmol / L, 100 μmol / L, 120 μmol / L, 150 μmol / L, 160 μmol / L, 180 μmol / L, 200 μmol / L or other values ​​within this range.

[0014] In some embodiments, the concentration of the additive is 100 μmol / L.

[0015] In some embodiments, the culture medium further comprises KMSO basal culture medium.

[0016] The beneficial effects of the present invention are as follows: Different from the prior art, the present invention applies tributyrin as an additive to the culture medium for in vitro development of mouse embryos for the first time, and the tributyrin can significantly improve the blastocyst rate, reduce the ROS content in the embryo, improve the mitochondrial membrane potential in the embryo, increase the ATP level in the embryo and the expression of antioxidant genes, improve the DNA methylation and histone modification levels in the embryo, and promote the in vitro development of the embryo; and tributyrin, as a natural antioxidant and epigenetic drug, is safe, has no toxic side effects, is cheap and easy to obtain, does not need to replace the culture medium during the entire development and culture process, and is simple to operate; the tributyrin provides strong support for in vitro embryo engineering technologies such as the efficient development of embryos in human assisted reproductive technology, mammalian fertilized embryos, parthenogenetic embryos and somatic cell cloned embryos, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The results of the effect of tributyrin on the blastocyst formation rate of mice in different treatment groups in Example 2 of the present invention are shown in Figure (A) is a result of microscopic observation of mouse embryos, and Figure (B) is a result of blastocyst formation rate of mice;

[0018] Figure 2 The results of the effect of tributyrin on ROS levels in mouse embryos in different treatment groups in Example 3 of the present invention are shown in Figure (A) and Figure (B) respectively show the results of ROS fluorescence in mouse embryos and ROS levels in mouse embryos;

[0019] Figure 3 The results of the effect of tributyrin on mitochondrial membrane potential in mouse embryos in different treatment groups in Example 3 of the present invention are shown in Figure 1, wherein Figure (A) is a fluorescence result of JC-1 staining in mouse embryos, and Figure (B) is a result of JC-1 red / JC-1 green levels in mouse embryos;

[0020] Figure 4 This is a graph showing the effect of tributyrin on ATP levels in mouse embryos in different treatment groups in Example 3 of the present invention;

[0021] Figure 5 This is a graph showing the effect of tributyrin on the expression level of antioxidant-related genes in mouse embryos in different treatment groups in Example 3 of the present invention;

[0022] Figure 6 The results of the effect of tributyrin on the 5hmC level in the mouse embryo genome in different treatment groups in Example 3 of the present invention are shown in Figure (A) and Figure (B) respectively show the results of 5hmC fluorescence in the mouse embryo and the results of 5hmC fluorescence intensity in the mouse embryo;

[0023] Figure 7 These are the results of the effect of tributyrin on the histone H3K27ac modification level in the mouse embryo genome in different treatment groups in Example 3 of the present invention, wherein Figure (A) is a graph of the H3K27ac fluorescence results in mouse embryos, and Figure (B) is a graph of the H3K27ac fluorescence intensity results in mouse embryos. DETAILED DESCRIPTION

[0024] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] The experimental methods without specific conditions in the examples are usually carried out according to conventional conditions and conditions described in the manual, or according to conditions recommended by the manufacturer. The general equipment, materials, reagents, etc. used are all available from commercial channels unless otherwise specified.

[0026] Example 1 Preparation of Mouse Embryo In Vitro Development Culture Medium

[0027] The preparation method of the mouse embryo in vitro development culture medium is as follows:

[0028] (1) Preparation of tributyrin stock solution: Dissolve 87.8 μL tributyrin (purchased from Sigma, catalog number 91010) in 1 mL DMSO to obtain a 300 mmol / L tributyrin stock solution, which was aliquoted into 5 μL tubes for later use and stored at -20°C for later use.

[0029] (2) Add the tributyrin storage solution prepared in step (1) to KSOM basal embryo culture medium (purchased from Sigma, catalog number MR-101-D) to obtain mouse embryo in vitro development culture medium.

[0030] Among them, the culture medium was divided into the following two groups for subsequent experimental tests:

[0031] Basic development culture medium (control group): KSOM basic embryo culture medium;

[0032] Developmental culture medium supplemented with tributyrin (treatment group): Based on KSOM basal culture medium, supplemented with 50 μmol / L, 100 μmol / L or 200 μmol / L tributyrin (prepared and used immediately).

[0033] Example 2 Effect of tributyrin on the in vitro development of mouse embryos

[0034] Specifically, at 4:00 p.m. on the first day, 10 IU of pregnant mare serum gonadotropin was intraperitoneally injected into 6- to 9-week-old SPF-grade Kunming female mice, and 8 IU of chorionic gonadotropin was injected 48 h later. At 9:00 a.m. the next day, 9- to 12-week-old male mice were killed by cervical dislocation, and the tail of the epididymis next to the vas deferens was taken. After cutting with ophthalmic scissors in a 200 μL insemination droplet (Sigma, MR-070-D), sperm was released for 60 min. The insemination droplet needed to be pre-balanced the night before. At the same time, 4 female mice were killed by cervical dislocation, and the enlarged oviduct was taken and placed in the insemination solution. An insulin needle was used to open one side of the ampulla under the microscope. Under the action of squeezing or its internal pressure, the cumulus-oocyte complex (COCs) of the oocytes would be released from the ampulla. 8 to 10 μL of sperm and COCs were taken in the insemination droplet for insemination. 6 hours after insemination, the fertilized zygotes (with male and female pronuclei) were taken, cleaned and transferred to KSOM culture drops with different treatments for culture. The specific groups are as follows:

[0035] Control group: Add the basic mouse embryo in vitro development culture medium to a small dish dedicated for embryo culture, make 2 drops, each drop has 50 μL culture medium, cover with paraffin oil, pre-equilibrate in an incubator at 37°C, 5% CO2, saturated humidity for more than 6 hours, place the embryos in the well-equilibrated culture drops, 25 to 35 embryos in each drop;

[0036] Treatment group 1: Add mouse embryo in vitro development culture medium containing 50 μmol / L tributyrin to a small dish dedicated for embryo culture, make 2 drops, each drop is 50 μL culture medium, covered with paraffin oil, pre-equilibrated in an incubator at 37°C, 5% CO2, saturated humidity for more than 6 hours, and place the embryos in the well-equilibrated culture drops, 25 to 35 embryos in each drop;

[0037] Treatment group 2: Add mouse embryo in vitro development culture medium containing 100 μmol / L tributyrin to a small dish dedicated for embryo culture, make 2 drops, each drop is 50 μL culture medium, covered with paraffin oil, pre-equilibrated in an incubator at 37°C, 5% CO2, and saturated humidity for more than 6 hours, and place the embryos in the well-equilibrated culture drops, 25 to 35 embryos in each drop;

[0038] Treatment group 3: Add mouse embryo in vitro development culture medium containing 200 μmol / L tributyrin to a small dish dedicated for embryo culture, make 2 drops, each drop is 50 μL culture medium, covered with paraffin oil, pre-equilibrated in an incubator at 37°C, 5% CO2, and saturated humidity for more than 6 hours, and place the embryos in the well-equilibrated culture drops, 25 to 35 embryos in each drop;

[0039] The above different treatment groups were cultured in an incubator at 37°C, 5% CO2, and saturated humidity for 24h, 48h, 72h, and 96h, corresponding to the 2-cell, 4-cell, morula, and blastocyst stages of embryonic development, and the embryonic development was observed. The results are shown in Tables 1 and Figure 1 shown.

[0040] Table 1 Effects of tributyrin on in vitro development of mouse embryos

[0041]

[0042] Note: Data in the same column with different letters on their shoulders represent significant differences (P<0.05), while data without letters or with the same letters represent no significant differences (P>0.05).

[0043] from Figure 1 As can be seen from the results in Table 1, the number of cells in the treatment group increased significantly compared with the control group ( Figure 1 A), compared with the control group, the blastocyst rate of treatment group 1 was increased, but the effect was not significant (78.37±8.33% vs 69.53±4.08%, P>0.05); compared with the control group, the blastocyst rate of treatment group 2 was significantly increased (86.07±16.53% vs 69.53±4.08%, P<0.05); compared with the control group, the blastocyst rate of treatment group 3 was increased, but the effect was not significant (80.23±10.70% vs 69.53±4.08%, P>0.05). The above results showed that tributyrin at a concentration of 50-200μmol / L can promote the in vitro development of mouse embryos and increase the blastocyst rate; further, tributyrin at a concentration of 100μmol / L can significantly promote the in vitro development of mouse embryos and significantly increase the blastocyst rate.

[0044] Among them, the data statistics and processing methods are as follows:

[0045] Cleavage rate (%) = number of cleavage embryos / number of fertilized eggs with male and female pronuclei × 100%

[0046] Blastocyst rate (%) = number of blastocysts / number of cleavage embryos × 100%

[0047] Example 3 Effect of tributyrin on improving the quality of mouse embryo development in vitro

[0048] According to the experimental results in the above Example 2, 100 μmol / L tributyrin can significantly increase the mouse blastocyst rate. Therefore, illustratively, in the experimental group of this example, the culture medium with 100 μmol / L tributyrin was used for the experiment. Specifically, the experiment was divided into the following two groups:

[0049] Control group: Add the basic mouse embryo in vitro development culture medium to a small dish dedicated for embryo culture, make 2 drops, each drop has 50 μL culture medium, cover with paraffin oil, pre-equilibrate in an incubator at 37°C, 5% CO2, saturated humidity for more than 6 hours, place the embryos in the well-equilibrated culture drops, 25 to 35 embryos in each drop;

[0050] Experimental group: Add mouse embryo in vitro development culture medium containing 100 μmol / L tributyrin into a special small dish for embryo culture, make 2 drops, each drop is 50 μL culture medium, covered with paraffin oil, pre-equilibrated in an incubator at 37°C, 5% CO2, and saturated humidity for more than 6 hours, and place the embryos in the balanced culture drops, 25 to 35 embryos in each drop.

[0051] (1) Detection of reactive oxygen species (ROS) in mouse embryos

[0052] Referring to the steps of in vitro development and culture in Example 2, the above two treatment groups were subjected to in vitro development and culture; according to the instructions of the reactive oxygen detection kit (Biyuntian, S0033S), DCFH-DA was diluted with KSOM culture medium to a working solution concentration of 5 μM. After collecting the 2-cell stage embryos from the control group and the experimental group, they were placed in preheated culture medium and washed twice, then transferred to droplets containing DCFH-DA working solution, incubated in a light-proof incubator at 37°C and 5% CO2 for 30 minutes, and then washed 2 to 3 times with clean KSOM culture medium and observed and photographed under an inverted fluorescence microscope. ImageJ software was used to analyze the average fluorescence intensity of ROS in the embryos. The results are shown in the figure. Figure 2 shown.

[0053] from Figure 2 It can be seen that after treatment with 100 μmol / L tributyrin, the average fluorescence intensity of ROS in mouse embryos decreased. The results showed that 100 μmol / L tributyrin can significantly reduce the ROS level in mouse embryos.

[0054] (2) Mitochondrial membrane potential detection

[0055] Referring to the steps of in vitro development and culture in Example 2, the above two treatment groups were subjected to in vitro development and culture; according to the instructions of the mitochondrial membrane potential JC-1 detection kit (Biyuntian, C2006), the 2-cell embryos of the control group and the experimental group were transferred into 1×JC-1 staining working solution, incubated in a 37°C, 5% CO2 incubator in the dark for 30 minutes, and then the embryos were fully washed with 1×JC-1 staining buffer, and placed under an inverted fluorescence microscope for observation and photography. ImageJ software was used to analyze the average fluorescence intensity of JC-1 red and JC-1green of the embryos. The results are shown in Figure 3 shown.

[0056] from Figure 3It can be seen that after treatment with 100 μmol / L tributyrin, the relative fluorescence intensity of JC-1 red / green in mouse embryos increased. The results showed that 100 μmol / L tributyrin can significantly improve the mitochondrial membrane potential level.

[0057] (3) Detection of ATP levels in mouse embryos

[0058] Referring to the steps of in vitro development and culture in Example 2, the above two treatment groups were subjected to in vitro development and culture; according to the instructions of the ATP detection kit (Biyuntian, S0027), the 2-cell embryos of the control group and the experimental group were subjected to ATP detection. Add 16 μL of ATP detection lysate to a 200 μL centrifuge tube, and transfer 40 2-cell embryos. Use tweezers to grab the centrifuge tube, place it in liquid nitrogen for freezing, and after the liquid at the bottom of the centrifuge tube solidifies, place it in room temperature H2O for thawing, and repeat this twice. Quickly vortex at high speed, centrifuge after vortexing, so that the liquid reaches the bottom of the tube, and no bubbles appear. After centrifugation, place the sample on ice. Take 64 μL of ATP working solution (ATP detection reagent diluent: ATP detection reagent = 4:1) into the tube, vortex and centrifuge to the bottom of the tube, and quickly transfer to a black 96-well plate for detection on a multifunctional microplate reader. The results are as follows Figure 4 shown.

[0059] from Figure 4 It can be seen that after treatment with 100 μmol / L tributyrin, the level of ATP in mouse embryos increased significantly. The results show that 100 μmol / L tributyrin can significantly increase the ATP content of embryos.

[0060] (4) Detection of antioxidant gene expression in mouse embryos

[0061] Referring to the steps of in vitro development and culture in Example 2, the above two treatment groups were subjected to in vitro development and culture; 2-cell embryos of the control group and the experimental group were obtained, and high-purity RNA was extracted according to the instructions of the RNAprep Pure micro-sample total RNA extraction kit (Tiangen, DP420). Reverse transcription was performed using the HiScript Ⅱ Q RT SuperMix for Qrcr reverse transcription kit (Nuoweizan, R223) under the conditions of 50°C, 15min, and 85°C, 5s. PCR amplification was performed according to the system of 1.5μL cDNA, 10μL 2×ChamQUniversal SYBR Qpcr Master Mix (Nuoweizan, Q711), 0.5μL of each forward and reverse primer, and 7.5μL of H2O. Reaction conditions: 95°C, (pre-denaturation) 3min; 95°C (denaturation) 10s; 55°C (annealing) 30s; 40 cycles (PCR amplification); fluorescence signals were collected during extension at 55°C, RPLP0 gene was used as an internal reference, and 2 -ΔΔct The expression level was calculated by the formula. The primer sequences of each gene are shown in Table 2 below:

[0062] Table 2 Primer sequence list

[0063]

[0064] The results are as follows Figure 5 shown.

[0065] from Figure 5 It can be seen that after treatment with 100 μmol / L tributyrin, the antioxidant genes in mouse embryos ( SOD2 , GPX4 ) expression level was significantly increased. The results showed that 100 μmol / L tributyrin could significantly promote the expression of embryonic antioxidant genes.

[0066] (5) DNA methylation level detection

[0067] Referring to the steps of in vitro development and culture in Example 2, the above two treatment groups were subjected to in vitro development and culture; 2-cell embryos of the control group and the experimental group were obtained. In order to detect the DNA methylation level in mouse embryos, the cells were first fixed with paraformaldehyde, and Triton X-100 enhanced the permeability of the cell membrane to facilitate the binding of antibodies and antigens. After acidification and alkali neutralization of the cells, 2% BSA blocked nonspecific proteins, and then incubated with the corresponding primary antibody and fluorescently labeled secondary antibody. The specific method and steps are as follows:

[0068] ① Fixation and permeabilization of mouse embryos: Place the mouse embryos in a cleaning solution (PBS containing 0.1% Tween-20 and 0.01% Triton X-100) and quickly wash once, then transfer to a fixation and permeabilization solution (PBS containing 4% paraformaldehyde and 0.5% Triton) and treat at room temperature for 50 minutes. After fixation and permeabilization, transfer the embryos to the cleaning solution and wash them three times, 2 minutes each time.

[0069] ② Acidification: Acidify with 4N hydrochloric acid for 15 minutes.

[0070] ③ Neutralization: Neutralize with 100 mM Tris-HCl, pH 8.0, for 20 min.

[0071] ④ Blocking: Transfer the cleaned embryos into blocking solution (cleaning solution containing 2% BSA, prepared immediately before use) and block at room temperature for 1 hour.

[0072] ⑤ Primary antibody incubation: After blocking, transfer the embryos directly to 5hmC primary antibody (ActiveMotif, 91309, 1:200) diluted in blocking solution and incubate overnight at 4°C. Wash with washing solution three times the next day, 8 minutes each time.

[0073] ⑥ Secondary antibody incubation: After washing, transfer the embryos to the secondary antibody diluted in blocking solution and incubate in a 37℃ wet box in the dark for 1 hour. After incubation, wash with cleaning solution 3 times, 8 minutes each time.

[0074] ⑦DNA staining: Incubate the embryos in DAPI at room temperature in the dark for 5 minutes. After incubation, wash with cleaning solution 3 times, 3 minutes each time.

[0075] ⑧ Sealing: Select a square area the size of a coverslip on a clean glass slide, add a small amount of supporting agent around it, then drop about 5μL of quenching sealing agent in the center, quickly transfer the washed embryos to evenly spread them in the liquid, quickly cover them with a coverslip, and gently press the coverslip under a stereoscope until the embryo becomes 1.2 times larger.

[0076] The results are as follows Figure 6 shown.

[0077] from Figure 6 It can be seen that after treatment with 100 μmol / L tributyrin, the 5hmC level in mouse embryos was significantly increased. The results show that 100 μmol / L tributyrin can increase the 5hmC level in mouse embryos.

[0078] (6) Detection of histone acetylation levels

[0079] Referring to the in vitro development and culture steps in Example 2, the above two treatment groups were subjected to in vitro development and culture; 2-cell embryos of the control group and the experimental group were obtained, and the histone H3K27ac level was detected by immunofluorescence. The specific method and steps are as follows:

[0080] ① Fixation and permeabilization of mouse embryos: Place the mouse embryos in a cleaning solution (PBS containing 0.1% Tween-20 and 0.01% Triton X-100) and quickly wash once, then transfer to a fixation and permeabilization solution (PBS containing 4% paraformaldehyde and 0.5% Triton) and treat at room temperature for 50 minutes. After fixation and permeabilization, transfer the embryos to the cleaning solution and wash them three times, 2 minutes each time.

[0081] ② Blocking: Transfer the cleaned embryos into blocking solution (cleaning solution containing 2% BSA, prepared immediately before use) and block at room temperature for 1 hour.

[0082] ③ Primary antibody incubation: After blocking, embryos were directly transferred to H3K27ac primary antibody (Cell Signaling Technology, A7253, 1:200) diluted in blocking solution and incubated overnight at 4°C. The next day, the cells were washed three times with washing solution, each time for 8 minutes.

[0083] ④ Secondary antibody incubation: After washing, transfer the embryos to the secondary antibody diluted in blocking solution and incubate in a 37℃ wet box in the dark for 1 hour. After incubation, wash with cleaning solution 3 times, 8 minutes each time.

[0084] ⑤DNA staining: Incubate the embryos in DAPI at room temperature in the dark for 5 minutes. After incubation, wash with cleaning solution 3 times, 3 minutes each time.

[0085] ⑥ Sealing: Select a square area the size of a coverslip on a clean glass slide, add a small amount of supporting agent around it, then drop about 5μL of quenching sealing agent in the center, quickly transfer the cleaned embryos to evenly spread them in the liquid, quickly cover them with a coverslip, and gently press the coverslip under a stereoscope until the embryo becomes 1.2 times larger.

[0086] The results are as follows Figure 7 shown.

[0087] from Figure 7 It can be seen that after treatment with 100 μmol / L tributyrin, the histone H3K27ac level in mouse embryos was significantly increased. The results show that 100 μmol / L tributyrin can increase the histone H3K27ac level in mouse embryos.

[0088] In summary, the present invention applies tributyrin as an additive to the culture medium for in vitro development of mouse embryos for the first time. The tributyrin can significantly increase the blastocyst rate, reduce the ROS content in the embryo, improve the mitochondrial membrane potential in the embryo, increase the ATP level in the embryo and the expression of antioxidant genes, improve the DNA methylation and histone modification levels in the embryo, and promote the in vitro development of the embryo.

[0089] It should be noted that the above embodiments all belong to the same inventive concept, and the description of each embodiment has its own focus. For matters that are not described in detail in some embodiments, reference may be made to the description in other embodiments.

[0090] The above-mentioned embodiments only express the implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Use of tributyrin as an additive for embryo in vitro development culture medium, characterized in that: The uses include promoting embryonic development; The embryo is a mouse embryo.

2. The use according to claim 1, characterized in that The concentration of the tributyrin is 50-200 μmol / L.

3. The use according to claim 2, characterized in that: The concentration of tributyrin is 100 μmol / L.

4. An embryo in vitro development culture medium for promoting embryo development, characterized in that: The culture medium includes tributyrin.

5. The embryo in vitro development culture medium for promoting embryo development according to claim 4, characterized in that: The concentration of the tributyrin is 50-200 μmol / L.

6. The embryo in vitro development culture medium for promoting embryo development according to claim 5, characterized in that: The concentration of tributyrin is 100 μmol / L.

7. The embryo in vitro development culture medium for promoting embryo development according to any one of claims 4 to 6, characterized in that: The culture solution also includes KMSO basic culture solution.

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