Methods for promoting in vitro development of mammalian embryos and uses thereof

Abstract

The present disclosure provides a method for promoting in vitro development of mammalian embryos, comprising the step of adding alpha-ketoglutarate in the embryo development fluid at 90-180h after in vitro fertilization. Compared with the method of adding throughout the whole embryo development, the present disclosure finds that the promotion of embryo development efficiency is more significant when alpha-ketoglutarate is added after 90 hours after fertilization (90 hours to 180 hours after fertilization).

Description

Technical Field

[0001] This invention belongs to the field of animal genetics and breeding, specifically relating to a method for promoting in vitro development of mammalian embryos and its application. Background Technology

[0002] In vitro embryo production technology has significantly improved the fertility of female animals, with the number of embryos produced annually increasing in both humans and farm animals. However, due to the stark differences between the in vitro and in vivo environments, in vitro embryos experience greater oxidative stress and the accumulation of reactive oxygen species (ROS), impacting subsequent embryo development and implantation. Improving the efficiency and quality of in vitro embryo development, and consequently, the transplantation efficiency of in vitro cultured embryos, has remained a hot research topic for scientists and engineers. Summary of the Invention

[0003] This disclosure provides a method for promoting in vitro development of mammalian embryos. Specifically, in bovine in vitro embryo development, α-ketoglutarate is added from 90 hours post-fertilization until 180 hours after blastocyst formation to promote in vitro embryo development efficiency and blastocyst quality. Compared to methods that add α-ketoglutarate throughout the entire embryonic development process (0 to 180 hours post-fertilization) to improve embryonic development efficiency, this disclosure finds that adding α-ketoglutarate from 90 hours post-fertilization (from 90 to 180 hours post-fertilization) significantly improves embryonic development efficiency.

[0004] According to a first aspect of this disclosure, a method for promoting in vitro development of mammalian embryos is provided, comprising the step of culturing an embryo development medium with α-ketoglutarate added 90-180 h after in vitro fertilization of a cumulus-oocyte complex.

[0005] In some embodiments, the concentration of the α-ketoglutaric acid is 100-300 μM, preferably 120-180 μM, and more preferably 150 μM.

[0006] In some embodiments, the embryonic development fluid includes monosodium glutamate, sodium pyruvate, sodium citrate, inositol, sodium lactate, and BSA.

[0007] In some embodiments, the embryonic development fluid further includes NaCl, KCl, NaHCO3, KH2PO4, and gentamicin.

[0008] In some embodiments, the embryonic development fluid comprises: 80-120 mM NaCl, 4-8 mM KCl, 20-30 mM NaHCO3, 0.5-2 mM KH2PO4, 0.5-2 mM sodium glutamate, 0.1-0.5 mM sodium pyruvate, 0.2-0.8 mM sodium citrate, 2-4 mM inositol, 4-6 mM sodium lactate, 3-5 mg / mL BSA, and 20-25 μg / mL gentamicin.

[0009] In some embodiments, the embryonic development fluid comprises: 90-110 mM NaCl, 6.5-6.8 mM KCl, 23-24 mM NaHCO3, 1-1.2 mM KH2PO4, 0.9-1 mM sodium glutamate, 0.3-0.5 mM sodium pyruvate, 0.4-0.6 mM sodium citrate, 2.4-2.6 mM inositol, 4.8-5.2 mM sodium lactate, 3.6-3.8 mg / mL BSA, and 23-25 ​​μg / mL gentamicin.

[0010] In some specific embodiments, the embryonic development fluid comprises: 100 mM NaCl, 6.64 mM KCl, 23.27 mM NaHCO3, 1.10 mM KH2PO4, 0.93 mM sodium glutamate, 0.37 mM sodium pyruvate, 0.46 mM sodium citrate, 2.56 mM inositol, 4.92 mM sodium lactate, 3.7 mg / mL BSA, and 23.3 μg / mL gentamicin.

[0011] In some embodiments, the embryo development fluid further includes essential and non-essential amino acids. Preferably, in some embodiments, the embryo development fluid further includes 18-20 μL / mL of essential amino acids (50x concentrate, purchased from Sigma, M5550) and 8-10 μL / mL of non-essential amino acids (100x concentrate, purchased from Sigma, M7145).

[0012] The key aspect of the method for promoting animal embryonic development using α-ketoglutarate disclosed in this invention lies in the timing of α-ketoglutarate addition. Furthermore, α-ketoglutarate, when used in conjunction with specific culture media or substances, can further promote in vitro embryonic development. The culture media used can be any existing culture media suitable for various animal embryos, or other undisclosed culture media, including but not limited to commonly used media such as KSOM, M16, and PZM-3.

[0013] In some embodiments, the method includes the following steps:

[0014] (1) In vitro fertilization of mammalian cumulus-oocyte complexes after in vitro maturation and culture;

[0015] (2) 90-180 hours after in vitro fertilization, α-ketoglutarate is added to the embryo development medium for embryo culture.

[0016] In some embodiments, the in vitro maturation culture time in step (1) is 10-30 hours, preferably 20-25 hours. In some specific embodiments, the in vitro maturation culture time in step (1) is 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, or any value between them.

[0017] In some embodiments, the maturation culture medium used in the in vitro maturation culture includes: M199 culture medium, FBS (fetal bovine serum), FSH (follicle-stimulating hormone), LH (luteinizing hormone), E2 (estradiol), sodium pyruvate, cysteine, EGF (epidermal growth factor), penicillin and streptomycin.

[0018] In some embodiments, the maturation culture medium comprises: M199 culture medium supplemented with 8-20% FBS (fetal bovine serum), 0.01-0.05 IU / mL FSH (follicle-stimulating hormone), 0.01-0.05 IU / mL LH (luteinizing hormone), 0.5-5 μg / mLE2 (estradiol), 10-30 μg / mL sodium pyruvate, 0.05-0.2 mg / mL cysteine, 10-30 ng / mL EGF (epidermal growth factor), 80-120 μg / mL penicillin, and 80-120 μg / mL streptomycin.

[0019] In some embodiments, the maturation culture medium comprises: M199 culture medium supplemented with 8-12% FBS (fetal bovine serum), 0.01-0.03 IU / mL FSH (follicle-stimulating hormone), 0.01-0.03 IU / mL LH (luteinizing hormone), 1-3 μg / mL LE2 (estradiol), 20-25 μg / mL sodium pyruvate, 0.05-0.2 mg / mL cysteine, 18-22 ng / mL EGF (epidermal growth factor), 90-110 μg / mL penicillin, and 90-110 μg / mL streptomycin.

[0020] In some specific embodiments, the maturation culture medium comprises: M199 culture medium supplemented with 10% FBS (fetal bovine serum), 0.02 IU / mL FSH (follicle-stimulating hormone), 0.02 IU / mL LH (luteinizing hormone), 2 μg / mL E2 (estradiol), 22 μg / mL sodium pyruvate, 0.1 mg / mL cysteine, 20 ng / mL EGF (epidermal growth factor), 100 μg / mL penicillin, and 100 μg / mL streptomycin.

[0021] In some embodiments, the method further includes step (3) calculating the embryo cleavage rate 2 days after embryo culture, and calculating the blastocyst development rate and inner cell mass cell number 7 days and / or 8 days after embryo culture.

[0022] In some embodiments, the mammal includes livestock. Preferably, the mammal includes pigs, cattle, sheep, horses, donkeys, or rabbits, with cattle being the most preferred.

[0023] According to a second aspect of this disclosure, the application of the method described in the first aspect in promoting in vitro development of mammalian embryos is provided.

[0024] In some implementations, the method is used to improve blastocyst development efficiency.

[0025] In some embodiments, the method is used to improve blastocyst quality, preferably to increase the number of cells in the inner cell mass, the total number of cells in the blastocyst, and the ratio of inner cell mass cells to total number of cells in the blastocyst.

[0026] The method disclosed herein is primarily applicable to improving in vitro embryonic development technology for mammals, overcoming the first critical hurdle in the development from morula to blastocyst, thus enhancing in vitro embryo production efficiency. Further application in production practice can effectively improve embryonic development efficiency, increase blastocyst quality, and subsequently improve pregnancy success rates. Attached Figure Description

[0027] Figure 1 The effects of adding α-ketoglutarate during in vitro embryonic development are shown, including (A) blastocyst morphology after 8 days of in vitro culture following fertilization; (B) cleavage rate after 2 days of in vitro culture following fertilization; (C) blastocyst development rate after 7 days of in vitro culture following fertilization; and (D) blastocyst development rate after 8 days of in vitro culture following fertilization.

[0028] Figure 2 The effects of α-ketoglutarate addition on blastocyst quality during in vitro embryonic development are shown, including (A) representative immunofluorescence images of SOX2-labeled blastocysts in each group; (B) number of cells in the inner cell mass of the blastocyst; (C) total number of cells in the blastocyst; and (D) the ratio of the number of cells in the inner cell mass of the blastocyst to the total number of cells.

[0029] Figure 3 The effects of adding α-ketoglutarate at different time points in in vitro embryonic development were shown, including (A) cleavage rate after 2 days of in vitro culture following fertilization; and (B) blastocyst development rate after 8 days of in vitro culture following fertilization.

[0030] Figure 4The effects of adding α-ketoglutarate at different stages of in vitro embryonic development on blastocyst quality are shown. (A) Representative immunofluorescence images of SOX2-labeled blastocysts in each group; (B) Number of cells in the inner cell mass of the blastocyst; (C) Total number of cells in the blastocyst; (D) Ratio of inner cell mass cells to total number of cells in the blastocyst. Detailed Implementation

[0031] Alpha-ketoglutarate (α-ketoglutarate) is an important metabolite in the tricarboxylic acid cycle, and recent studies have found that it can effectively delay aging in mammals. Adding α-ketoglutarate during embryonic development can promote embryonic development in pigs and mice, improve in vitro blastocyst quality, and increase the number of inner cell mass cells and implantation success rate. However, previous studies have all added α-ketoglutarate to the embryonic development medium throughout the entire process, without exploring which stage of addition yields the optimal effect on improving embryonic development. This publication, through extensive experiments, unexpectedly discovered that in bovine in vitro embryonic development, adding α-ketoglutarate 90-180 hours after in vitro fertilization significantly improves developmental efficiency compared to the group adding α-ketoglutarate throughout the entire process or at other time points.

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0033] The reagents and materials used in the following examples are all commercially available products.

[0034] Example

[0035] Example 1

[0036] (1) Bovine ovaries obtained from slaughterhouses were used. After aspirating follicular fluid, cumulus-oocyte complexes were separated and transferred to maturation culture medium for in vitro maturation. The maturation culture medium consisted of M199 medium supplemented with 10% FBS (fetal bovine serum), 0.02 IU / mL FSH (follicle-stimulating hormone), 0.02 IU / mL LH (luteinizing hormone), 2 μg / mL E2 (estradiol), 22 μg / mL sodium pyruvate, 0.1 mg / mL cysteine, 20 ng / mL EGF (epidermal growth factor), 100 μg / mL penicillin, and 100 μg / mL streptomycin.

[0037] (2) In vitro fertilization was performed 22 hours after maturation, and embryos were transferred to embryo development medium for culture 16 hours after fertilization. The embryo development medium was supplemented with 30 μM, 150 μM, and 750 μM α-ketoglutarate, respectively. The embryo development medium contained: 100 mM NaCl, 6.64 mM KCl, 23.27 mM NaHCO3, 1.10 mM KH2PO4, 0.93 mM sodium glutamate, 0.37 mM sodium pyruvate, 0.46 mM sodium citrate, 2.56 mM inositol, 4.92 mM sodium lactate, 3.7 mg / mL BSA, 18.6 μL / mL essential amino acids (50x concentrate, purchased from Sigma, M5550), 9.3 μL / mL non-essential amino acids (100x concentrate, purchased from Sigma, M7145), and 23.3 μg / mL gentamicin, with the remainder being water.

[0038] (3) Two days after embryo culture (i.e. 48 hours after fertilization), the embryo cleavage rate was observed and counted by the naked eye. Seven and eight days later, the blastocyst development rate was observed and counted by the naked eye, and the number of inner cell mass cells was counted by immunofluorescence staining.

[0039] The results are as follows:

[0040] 1. Adding 150 μM α-ketoglutarate during bovine in vitro embryonic development can significantly improve blastocyst development efficiency.

[0041] Compared with the control group without α-ketoglutarate, the addition of different concentrations of α-ketoglutarate to the embryo development medium during in vitro embryonic development had no significant effect on the cleavage rate. Figure 1 B); however, the addition of 150 μM α-ketoglutarate significantly improved the blastocyst development rate on day 7 (B). Figure 1 C) and blastocyst development rate on day 8 ( Figure 1 D) The morphology of the blastocyst after 8 days of in vitro culture following fertilization is as follows: Figure 1 As shown in Figure A.

[0042] 2. Adding 150 μM α-ketoglutarate during bovine in vitro embryonic development can significantly improve blastocyst quality.

[0043] Seven days after fertilization and in vitro culture, the inner cell mass cells of the blastocyst were labeled with SOX2 and counted. Figure 2 A) The results showed that adding 150 μM α-ketoglutarate to the embryo development fluid significantly increased the number of cells in the inner cell mass of the blastocyst. Figure 2 B) and the ratio of inner cell mass cells to total cells ( Figure 2 D), but different concentrations of α-ketoglutarate did not affect the total number of cells in the blastocyst. Figure 2 C).

[0044] Example 2

[0045] (1) Bovine ovaries obtained from slaughterhouses were used. After aspirating follicular fluid, cumulus-oocyte complexes were separated and transferred to maturation culture medium for in vitro maturation. The maturation culture medium consisted of M199 medium supplemented with 10% FBS (fetal bovine serum), 0.02 IU / mL FSH (follicle-stimulating hormone), 0.02 IU / mL LH (luteinizing hormone), 2 μg / mL E2 (estradiol), 22 μg / mL sodium pyruvate, 0.1 mg / mL cysteine, 20 ng / mL EGF (epidermal growth factor), 100 μg / mL penicillin, and 100 μg / mL streptomycin.

[0046] (2) In vitro fertilization was performed 22 hours after maturation, and the embryos were transferred to embryo development medium for embryo culture. Specifically, 150 μM α-ketoglutarate was added to the embryo development medium at 0-48h, 48-90h, 90-180h and 0-180h after fertilization, respectively, and the embryos were cultured in embryo development medium without α-ketoglutarate for the remaining time periods. The embryonic development fluid formula is as follows: 100mM NaCl, 6.64mM KCl, 23.27mM NaHCO3, 1.10mM KH2PO4, 0.93mM sodium glutamate, 0.37mM sodium pyruvate, 0.46mM sodium citrate, 2.56mM inositol, 4.92mM sodium lactate, 3.7mg / mL BSA, 18.6μL / mL essential amino acids (50x concentration), 9.3μL / mL non-essential amino acids (100x concentration), and 23.3μg / mL gentamicin.

[0047] Two days after embryo culture (i.e. 48 hours after fertilization), the cleavage rate of the embryos was visually observed and counted. Eight days later, the blastocyst development rate was visually observed and counted, and the number of inner cell mass cells was counted by immunofluorescence staining.

[0048] The results are as follows:

[0049] 1. Adding α-ketoglutarate 90-180 hours after fertilization significantly improves blastocyst development efficiency.

[0050] Adding 150 μM α-ketoglutarate to the embryonic development fluid at 0-48 h, 48-90 h, 90-180 h post-fertilization, and throughout the entire 0-180 h period, did not significantly change the cleavage rate of the embryos in any of the groups. Figure 3 A). The blastocyst development rates in the 90-180h, 0-48h, 48-90h, and 0-180h full-process supplementation groups were all higher than those in the control group without α-ketoglutarate supplementation. Among them, the blastocyst development rate in the 90-180h supplementation group was the highest, significantly higher than that in the other supplementation groups. Figure 3 B).

[0051] 2. Adding α-ketoglutarate 90-180 hours after fertilization significantly improved blastocyst quality.

[0052] The inner cell mass cells of the blastocyst were labeled with SOX2 and counted. Figure 4 A) Compared with the control group, the groups that received 150 μM α-ketoglutarate supplementation throughout the 90-180h and 0-180h embryonic growth cycles had significantly higher numbers of inner cell mass cells and an increased ratio of inner cell mass cells to total cells. Figure 4 B, 4D). However, the addition of 150 μM α-ketoglutarate during the 90-180h stage not only significantly increased the number of inner cell mass cells and the ratio of inner cell mass cells to total cells in the blastocyst, but also significantly increased the total number of cells in the blastocyst (B, 4D). Figure 4 B-4D).

[0053] The above description is merely a general illustration and specific embodiment of the present invention, and is not intended to limit the present invention in any other way. Any person skilled in the art can make modifications or variations based on the disclosed technical content of the present invention to create equivalent embodiments. Any modifications or variations made to the present invention without departing from the concept and spirit of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for promoting in vitro development of mammalian embryos, comprising the steps of adding α-ketoglutarate to an embryo development medium and culturing the embryo 90-180 hours after in vitro fertilization of the cumulus-oocyte complex.

2. The method according to claim 1, characterized in that, The concentration of the α-ketoglutaric acid is 100-300 μM, preferably 120-180 μM, and more preferably 150 μM.

3. The method according to claim 1 or 2, characterized in that, The embryonic development fluid includes monosodium glutamate, sodium pyruvate, sodium citrate, inositol, sodium lactate, and BSA; Preferably, the embryo development fluid further includes NaCl, KCl, NaHCO3, KH2PO4, and gentamicin; Preferably, the embryo development fluid comprises: 80-120 mM NaCl, 4-8 mM KCl, 20-30 mM NaHCO3, 0.5-2 mM KH2PO4, 0.5-2 mM sodium glutamate, 0.1-0.5 mM sodium pyruvate, 0.2-0.8 mM sodium citrate, 2-4 mM inositol, 4-6 mM sodium lactate, 3-5 mg / mL BSA, and 20-25 μg / mL gentamicin; More preferably, the embryo development fluid comprises: 90-110 mM NaCl, 6.5-6.8 mM KCl, 23-24 mM NaHCO3, 1-1.2 mM KH2PO4, 0.9-1 mM sodium glutamate, 0.3-0.5 mM sodium pyruvate, 0.4-0.6 mM sodium citrate, 2.4-2.6 mM inositol, 4.8-5.2 mM sodium lactate, 3.6-3.8 mg / mL BSA, and 23-25 ​​μg / mL gentamicin.

4. The method according to any one of claims 1-3, characterized in that, The method includes the following steps: (1) In vitro fertilization of mammalian cumulus-oocyte complexes after in vitro maturation and culture; (2) 90-180 hours after in vitro fertilization, α-ketoglutarate is added to the embryo development medium for embryo culture.

5. The method according to claim 4, characterized in that, In step (1), the in vitro maturation culture time is 10-30 hours, preferably 20-25 hours; and / or The maturation culture medium used in the in vitro maturation culture includes: M199 culture medium, fetal bovine serum, follicle-stimulating hormone, luteinizing hormone, estradiol, sodium pyruvate, cysteine, epidermal growth factor, penicillin and streptomycin. Preferably, the maturation culture medium comprises: M199 culture medium supplemented with 8-20% fetal bovine serum, 0.01-0.05 IU / mL follicle-stimulating hormone, 0.01-0.05 IU / mL luteinizing hormone, 0.5-5 μg / mL estradiol, 10-30 μg / mL sodium pyruvate, 0.05-0.2 mg / mL cysteine, 10-30 ng / mL epidermal growth factor, 80-120 μg / mL penicillin, and 80-120 μg / mL streptomycin; More preferably, the maturation culture medium comprises: M199 culture medium supplemented with 8-12% fetal bovine serum, 0.01-0.03 IU / mL follicle-stimulating hormone, 0.01-0.03 IU / mL luteinizing hormone, 1-3 μg / mL estradiol, 20-25 μg / mL sodium pyruvate, 0.05-0.2 mg / mL cysteine, 18-22 ng / mL epidermal growth factor, 90-110 μg / mL penicillin, and 90-110 μg / mL streptomycin.

6. The method according to claim 4, characterized in that, The method further includes step (3): after 2 days of embryo culture, the embryo cleavage rate is counted, and after 7 days and / or 8 days, the blastocyst development rate and the number of inner cell mass cells are counted.

7. The method according to any one of claims 1-6, characterized in that, The mammals include livestock. Preferably, the mammal includes a pig, a cow, a sheep, a horse, a donkey, or a rabbit, with a cow being the most preferred.

8. The use of the method according to any one of claims 1-7 in promoting the in vitro development of mammalian embryos.

9. The application according to claim 8, characterized in that, The method described is used to improve blastocyst development efficiency.

10. The application according to claim 8, characterized in that, The method is used to improve blastocyst quality, and is preferably used to increase the number of cells in the inner cell mass, the total number of cells in the blastocyst, and the ratio of the number of cells in the inner cell mass to the total number of cells in the blastocyst.

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