Bovine oocyte in-vitro maturation culture solution and application thereof

Abstract

The invention relates to the technical field of in-vitro embryo culture, in particular to a bovine oocyte in-vitro maturation culture solution and application thereof. The bovine oocyte in-vitro maturation culture solution is prepared from Dac51, SAM and zinc sulfate, the ratio of Dac51 to SAM to zinc sulfate is (1-10) [mu] M to (40-200) [mu] M to (0.4-2) [mu] g / mL. According to the bovine oocyte in-vitro maturation culture solution capable of effectively promoting embryonic development, the Dac51, the SAM and the zinc sulfate cooperate to improve the nuclear maturation rate, the cleavage rate, the blastocyst rate, the hatching rate and the freezing survival rate of embryos, and the culture solution has important significance for improving the production efficiency and quality of the embryos.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro embryo culture, and particularly to an in vitro maturation culture solution for bovine oocytes and its application. Background Art

[0002] As an important means to improve the utilization efficiency of high-quality dairy and beef genetic resources, the technology of in vitro production (IVP) of bovine embryos has been widely applied in recent years. This technology system promotes the development of the industry through three core advantages: First, it significantly improves the embryo production and pregnancy numbers per unit time, especially the cultivation efficiency of calves with high genetic value genomes; Second, it greatly reduces the sperm consumption required for embryo production; Third, it precisely controls the sex ratio of offspring. Industry data shows that the IVP technology is gradually becoming the mainstream. At the commercial application level, the IVP technology has successfully helped livestock breeders achieve a double improvement in reproductive efficiency and genetic gain. Its core value is reflected in: by optimizing the donor oocyte collection (OPU) process and the utilization efficiency of recipients, a complete industrial chain covering domestic and cross-border trade has been constructed.

[0003] The innovation of cryopreservation technology has become the key support for the development of the IVP system. With the surge in demand for high-quality in vitro embryos, significant progress has been made in the cryopreservation technology of oocytes and embryos, which not only ensures the long-term preservation of genetic resources but also promotes the large-scale production of a large number of IVP embryos. In addition, IVP embryos also have important value in the field of basic research: as an ideal model for studying mammalian embryo development, its research results not only optimize livestock production traits but also provide important references for the study of the mechanism of human early embryogenesis.

[0004] Although the technology of in vitro maturation of bovine oocytes and embryo culture has continued to progress, the embryos produced in vitro are still difficult to reach the level of in vivo embryos in terms of vitality, quality, and developmental ability. Research shows that although in vitro embryos can develop to the blastocyst stage, their vitality is significantly lower than that of in vivo embryos, and adverse factors in the culture environment may lead to a decrease in blastocyst vitality, cryotolerance, and implantation potential. In addition, poor in vitro culture conditions may cause abnormal embryo metabolism, which not only reduces the blastocyst formation rate and embryo vitality but also affects pregnancy maintenance, fetal development, and offspring health. IVP embryos are different from in vivo embryos at the morphological, molecular, and metabolic levels. Problems such as their low developmental ability, decreased quality after oocyte maturation, and insufficient cryotolerance directly lead to a decrease in pregnancy rate and restrict the popularization of the technology. This limitation is further reflected in the application in the livestock industry, and the low utilization rate of IVP embryos significantly limits their development potential. Specifically, in vitro blastocysts often exhibit quality defect characteristics such as trophoblast vacuolization, sparse distribution of microvilli, reduced cell junctions, abnormal gene expression, and lipid metabolism disorders.

[0005] In the field of cryopreservation technology, currently, two methods, slow programmed freezing and vitrification, are mainly used for bovine IVP embryos. However, embryos are highly sensitive to freezing, and a stable and reliable technical solution has not been established yet. Therefore, improving embryo production and cryopreservation technologies to enhance post-thaw survival rate and pregnancy rate remains a key challenge that urgently needs to be overcome in the field of assisted reproduction. Summary of the Invention

[0006] To solve the problems existing in the prior art, the present invention provides an in vitro maturation medium for bovine oocytes and its application.

[0007] In a first aspect, the present invention provides a composition comprising: Dac51, SAM, and zinc sulfate; the ratio of Dac51, SAM, and zinc sulfate is (1 - 10) μM : (40 - 200) μM : (0.4 - 2) μg / mL.

[0008] Further, the concentration of Dac51 is (1 - 10) μM, the concentration of SAM is (50 - 200) μM, and the concentration of zinc sulfate is (0.5 - 2) μg / mL.

[0009] In a second aspect, the present invention provides an in vitro maturation medium for bovine oocytes, comprising the aforementioned composition.

[0010] Further, the in vitro maturation medium for bovine oocytes is based on TCM199 medium and further comprises: 0.005 - 0.015 IU / mL FSH, 0.005 - 0.015 IU / mL LH, 0.5 - 1.5 μg / mL E2, 25 - 75 ng / mL EGF, 20 - 60 ng / mL IGF, 5 - 15 μg / mL sodium heparin, and 50 - 150 IU / mL penicillin - streptomycin.

[0011] In a third aspect, the present invention provides a method for in vitro maturation of bovine oocytes, comprising: performing in vitro maturation of bovine oocytes using the aforementioned in vitro maturation medium for bovine oocytes and mineral oil.

[0012] Further, the conditions for in vitro maturation include: Culturing for 22 - 24 hours under the conditions of 4 - 10% CO2 and 37 - 40 °C.

[0013] In a fourth aspect, the present invention provides a method for improving the survival rate of cryopreserved bovine embryos, by using the aforementioned method for in vitro maturation of bovine oocytes during the in vitro maturation stage of bovine oocytes.

[0014] Further, vitrification is used during the freezing stage, and the thawing stage includes: The embryo to be thawed is incubated in a 0.2 - 0.4 M sucrose solution for 5 - 10 minutes, then transferred to a 0.2 - 0.4 M sucrose solution for another 5 - 10 minutes, and then cultured under the conditions of 4 - 10% CO2 and 37 - 40 °C until the morphology returns to normal.

[0015] In a fifth aspect, the present invention provides the use of the aforementioned composition, or the aforementioned in vitro maturation medium for bovine oocytes, in improving the production efficiency or quality of in vitro embryos.

[0016] Furthermore, the quality includes one or more of: nuclear maturation rate, cleavage rate, blastocyst rate, hatching rate, or cryosurvival rate.

[0017] The present invention has the following beneficial effects: The present invention provides an in vitro maturation medium for bovine oocytes comprising Dac51, SAM, and zinc sulfate. Through the synergistic effect of the three, the quality of bovine in vitro embryos is significantly improved, and its nuclear maturation rate, cleavage rate, blastocyst rate, hatching rate, and cryosurvival rate are enhanced. The in vitro maturation medium for bovine oocytes provided by the present invention can be used for cryopreservation of bovine in vitro embryos and can also be used to improve the production efficiency of bovine in vitro embryos, which has important application value in the field of bovine in vitro embryo production. Detailed Embodiments

[0018] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art without creative efforts based on the embodiments of the present invention fall within the scope of protection of the present invention.

[0019] The experimental methods involved in the following examples, unless otherwise specified, are all conventional methods in the art. For example, they can be referred to the experimental manuals in the art or carried out according to the conditions recommended by the manufacturer's instructions.

[0020] The experimental materials and reagents involved in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0021] Example 1 1. In vitro maturation of bovine oocytes Collect bovine ovaries from a local abattoir and keep the collected ovaries in a thermos cup containing physiological saline (containing 50 μg / mL streptomycin and 75 μg / mL penicillin) at 30 - 35 °C, and transport them back to the laboratory within 2 h. Then, repeatedly wash the collected ovaries 2 - 3 times with physiological saline, pick follicles with a diameter of 2 - 8 mm, and aspirate the follicular fluid into a 15 mL centrifuge tube with a needle through a vacuum pump. Use a Pasteur pipette to suck out the follicular fluid precipitate and place it in a 10 mm large dish, add egg washing solution, and select COCs tightly wrapped with 2 - 3 layers of cumulus cells under a microscope. Place the washed oocytes in a four-well plate containing 750 μL of IVM maturation medium and 250 μL of mineral oil (50 COCs per well), and culture them in an incubator with 5% CO2 at 38.5 °C for 22 - 24 h.

[0022] The basic components of the above IVM maturation medium include: TCM199 (1×) + 0.01 IU / mL FSH + 0.01 IU / mL LH + 1 μg / mL E2 + 50 ng / mL EGF + 40 ng / mL IGF + 10 μg / mL sodium heparin + 100 IU / mL penicillin-streptomycin. Store at 4 °C.

[0023] On this basis, the present invention respectively sets up multiple experimental groups: (1) Do not add respectively on the basis of the basic components (control group); add 1 μM Dac51; add 5 μM Dac51; 10 μM Dac51.

[0024] (2) Do not add respectively on the basis of the basic components (control group); add 50 μM SAM; add 100 μM SAM; 200 μM SAM.

[0025] (3) Do not add respectively on the basis of the basic components (control group); add 0.5 μg / mL zinc sulfate; add 1 μg / mL zinc sulfate; add 2 μg / mL zinc sulfate.

[0026] (4) Respectively on the basis of the basic components: do not add (control group); add 5 μM Dac51; add 100 μM SAM; add 1 μg / mL zinc sulfate; add 5 μM Dac51 + 1 μg / mL zinc sulfate; add 100 μM SAM + 1 μg / mL zinc sulfate; 5 μM Dac51 + 100 μM SAM; add 5 μM Dac51 + 100 μM SAM + 1 μg / mL zinc sulfate.

[0027] (5) On the basic components, respectively: without addition (control group); adding 5 μM Dac51; adding 100 μM SAM; adding 1 μg / mL zinc sulfate; adding 5 μM Dac51 + 1 μg / mL zinc sulfate; adding 100 μM SAM + 1 μg / mL zinc sulfate; adding 5 μM Dac51 + 100 μM SAM; adding 5 μM Dac51 + 100 μM SAM + 1 μg / mL zinc sulfate; adding 1 μM Dac51 + 50 μM SAM + 0.5 μg / mL zinc sulfate; adding 10 μM Dac51 + 200 μM SAM + 2 μg / mL zinc sulfate; adding 1 μM Dac51 + 50 μM SAM + 2 μg / mL zinc sulfate; adding 10 μM Dac51 + 200 μM SAM + 0.5 μg / mL zinc sulfate.

[0028] Maturation rate statistics: After in vitro maturation, select some COCs, use an oscillator to shake off the cumulus cells, observe under a microscope, and exclude the first polar body as nuclear maturation.

[0029] 2. In vitro fertilization of oocytes (1) Treat semen: Put the frozen semen taken out from liquid nitrogen into a 37 °C water bath for thawing for 40 s. Use sterilized scissors to cut off both ends of the thin tube, pour the semen into 7 mL of pre-warmed washing semen and mix well. Centrifuge at 1500 r / min for 5 min, discard the supernatant, and then add 7 mL of pre-warmed washing semen and centrifuge at 1500 r / min for 5 min. After centrifugation, discard the supernatant and add pre-warmed fertilization medium to resuspend, dilute the semen concentration to 1×10 7 / mL, and place it in an incubator for capacitation for 1.5 h.

[0030] (2) Treat oocytes: Use pre-warmed 1 mg / mL hyaluronidase to remove the expanded cumulus cells around the mature oocytes (only leaving 2 - 3 layers), wash the oocytes three times and then place them in an incubator to wait for fertilization.

[0031] (3) Fertilization: Put the treated oocytes into the fertilization drop, with 20 - 30 oocytes per drop, and culture them in an incubator with 5% CO2 at 38.5 °C. After culturing for 16 - 18 h, transfer the fertilized eggs to the pre-embryo culture medium for culturing for 48 h, and then transfer the embryos to the post-embryo culture medium for culturing for 5 d. Change half of the medium every 48 h until 2 and 7 d after fertilization, and count the cleavage rate and blastocyst rate.

[0032] 3. Vitrification and thawing of blastocysts (1)Vitrification cryopreservation: First, place the embryos in a cryoprotectant solution containing 10% EG and 10% DMSO for 30 s. Then, transfer the embryos to EDFSF40 for 25 s. EDFSF40 is composed of an FSF solution containing 20% (v / v) EG and 20% (v / v) DMSO. The FSF solution contains 300 g / L Ficoll, 0.5 M sucrose, and 20% (v / v) FBS in DPBS. Immediately plunge the embryos aspirated into OPS into liquid nitrogen.

[0033] (2)Thawing: Take out the OPS tube from liquid nitrogen, blow the embryos in the tube into a 0.25 M sucrose solution, incubate for 5 min, then transfer to a 0.25 M sucrose solution and incubate for 5 min. Then, place the embryos in an incubator with 5% CO2 at 38.5 °C for culture. If the morphology returns to normal within 30 minutes after thawing, it is considered viable. Calculate the cryopreservation survival rate.

[0034] 4. Blastocyst hatching experiment The blastocysts are cultured in vitro embryo culture medium for an additional 24 h to examine blastocyst hatching.

[0035] 5. Experimental results (1)As shown in Table 1, the nuclear maturation rate (78.10 ± 8.16%) with the addition of 5 μM Dac51 in bovine IVM maturation medium was significantly higher than that with 1 μM Dac51 (73.53 ± 7.06%), 10 μM Dac51 (69.90 ± 7.13%), and the control group (71.82 ± 6.84%, P <0.05).

[0036] Table 1 Effects of different concentrations of Dac51 on in vitro maturation of bovine COCs

[0037] (2)As shown in Table 2, the cleavage rate and blastocyst rate (84.76 ± 8.42%, 46.46 ± 4.15%) with the addition of 5 μM Dac51 in bovine IVF embryo culture medium were significantly higher than those with 1 μM Dac51 (76.47 ± 7.16%, 41.02 ± 4.03%), 10 μM Dac51 (72.82 ± 7.16%, 38.67 ± 3.58%), and the control group (71.30 ± 7.25%, 40.24 ± 3.98%, P <0.05).

[0038] Table 2 Effects of different concentrations of Dac51 on in vitro developmental ability of bovine IVF embryos

[0039] (3) As shown in Table 3, the nuclear maturation rate (80.51 ± 8.10%) of adding 100 μM SAM to the bovine IVM maturation medium was significantly higher than that of 50 μM SAM (75.65 ± 7.68%), 200 μM SAM (71.55 ± 7.43%), and the control group (72.48 ± 6.57%, P <0.05).

[0040] Table 3 Effects of different concentrations of SAM on in vitro maturation of bovine COCs

[0041] (4) As shown in Table 4, the cleavage rate and blastocyst rate (85.59 ± 8.18%, 47.52 ± 4.85%) of adding 100 μM SAM to the bovine IVF embryo culture medium were significantly higher than those of 50 μM SAM (77.39 ± 7.36%, 42.70 ± 4.16%), 200 μM SAM (75.86 ± 6.85%, 39.77 ± 3.94%), and the control group (72.73 ± 7.45%, 41.25 ± 4.06%, P <0.05).

[0042] Table 4 Effects of different concentrations of SAM on in vitro development ability of bovine IVF embryos

[0043] (5) As shown in Table 5, the nuclear maturation rate (80.53 ± 7.46%) of adding 1 μg / mL zinc sulfate to the bovine IVM maturation medium was significantly higher than that of 0.5 μg / mL zinc sulfate (75.68 ± 6.98%), 2 μg / mL zinc sulfate (71.19 ± 8.65%), and the control group (70.18 ± 6.44%, P <0.05).

[0044] Table 5 Effects of different concentrations of zinc sulfate on in vitro maturation of bovine COCs

[0045] (6) As shown in Table 6, the cleavage rate and blastocyst rate (82.64 ± 8.16%, 45.00 ± 4.62%) of adding 1 μg / mL zinc sulfate to the bovine IVF embryo culture medium were significantly higher than those of 0.5 μg / mL zinc sulfate (73.21 ± 6.85%, 40.24 ± 4.17%), 2 μg / mL zinc sulfate (71.43 ± 6.85%, 40.00 ± 3.98%), and the control group (70.40 ± 6.18%, 39.77 ± 4.06%, P <0.05).

[0046] Table 6 Effects of Different Concentrations of Zinc Sulfate on the in vitro Developmental Ability of Bovine IVF Embryos

[0047] (7) As shown in Table 7, the nuclear maturation rate (95.83 ± 9.15%) of adding 5 μM Dac51 + 100 μM SAM + 1 μg / mL zinc sulfate to the bovine IVM maturation medium was significantly higher than that of 5 μM Dac51 (79.25 ± 7.62%), 100 μM SAM (80.73 ± 9.05%), 1 μg / mL zinc sulfate (80.53 ± 7.56%), 5 μM Dac51 + 1 μg / mL zinc sulfate (87.60 ± 8.16%), 100 μM SAM + 1 μg / mL zinc sulfate (88.18 ± 9.32%), 5 μM Dac51 + 100 μM SAM (89.83 ± 8.45%) and the control group (71.77 ± 6.78%, P < 0.05).

[0048] Table 7 Effects of Combined Addition on the in vitro Maturation of Bovine COCs

[0049] (8) As shown in Table 8, the cleavage rate, blastocyst rate, cryosurvival rate and hatching rate (97.58 ± 8.48%, 63.64 ± 7.06%, 98.70 ± 9.14%, 97.37 ± 7.38%) of adding 5 μM Dac51 + 100 μM SAM + 1 μg / mL zinc sulfate to the bovine IVF embryo culture medium were significantly higher than those of 5 μM Dac51 (84.40 ± 7.65%, 45.65 ± 5.13%, 90.48 ± 8.09%, 89.47 ± 7.61%), 100 μM SAM (85.45 ± 9.12%, 45.74 ± 4.25%, 90.70 ± 9.12%, 89.74 ± 8.16%), 1 μg / mL zinc sulfate (83.33 ± 7.16%, 45.56 ± 5.16%, 90.24 ± 9.53%, 89.19 ± 9.23%).

[0050] In addition, in the previous experimental results, the present invention verified that it was difficult to significantly improve the cleavage rate and blastocyst rate by using 1 μM Dac51 alone; using 50 μM SAM alone slightly increased the cleavage rate, but it was difficult to significantly improve the blastocyst rate; using 0.5 μg / mL zinc sulfate alone was difficult to significantly improve the cleavage rate and blastocyst rate. However, the cleavage rate and blastocyst rate were significantly increased after the combination of the three, indicating that there was a synergistic effect among the three. In addition, the present invention also verified that multiple groups of synergistic combinations with different concentrations had similar effects on promoting the in vitro development ability of embryos.

[0051] Table 8 Effects of combined addition on the in vitro development ability of bovine IVF embryos

[0052] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composition, characterized in that, Comprising: Dac51, SAM and zinc sulfate; the ratio of Dac51, SAM and zinc sulfate is (1 - 10) μM : (40 - 200) μM : (0.4 - 2) μg / mL.

2. The composition according to claim 1, wherein The concentration of Dac51 is (1 - 10) μM, the concentration of SAM is (50 - 200) μM, and the concentration of zinc sulfate is (0.5 - 2) μg / mL.

3. An in vitro maturation culture medium for bovine oocytes, characterized in that, Comprising: The composition according to claim 1 or 2.

4. The in vitro maturation culture medium for bovine oocytes according to claim 3, characterized in that, The in vitro maturation culture medium for bovine oocytes is based on TCM199 medium and further comprises: 0.005 - 0.015 IU / mL FSH, 0.005 - 0.015 IU / mL LH, 0.5 - 1.5 μg / mL E2, 25 - 75 ng / mL EGF, 20 - 60 ng / mL IGF, 5 - 15 μg / mL sodium heparin and 50 - 150 IU / mL penicillin - streptomycin.

5. A method for in vitro maturation of bovine oocytes, characterized in that, Comprising: Using the in vitro maturation culture medium for bovine oocytes and mineral oil according to claim 3 or 4 for in vitro maturation of bovine oocytes.

6. The method for in vitro maturation of bovine oocytes according to claim 5, characterized in that, The conditions for in vitro maturation include: Culturing for 22 - 24 hours under the conditions of 4 - 10% CO2 and 37 - 40 °C.

7. A method for improving the survival rate of cryopreserved bovine embryos, characterized in that, Using the method for in vitro maturation of bovine oocytes according to claim 5 or 6 during the in vitro maturation stage of bovine oocytes.

8. The method according to claim 7, wherein During the freezing stage, vitrification freezing is used, and during the thawing stage, it includes: Placing the embryo to be thawed in a 0.2 - 0.4 M sucrose solution and incubating for 5 - 10 minutes, then transferring it to a 0.2 - 0.4 M sucrose solution and incubating for 5 - 10 minutes, and then culturing under the conditions of 4 - 10% CO2 and 37 - 40 °C until the morphology returns to normal.

9. Use of the composition according to claim 1 or 2, or the in vitro maturation culture medium for bovine oocytes according to claim 3 or 4 in improving the production efficiency or quality of in vitro embryos.

10. The application according to claim 9, wherein, The quality includes one or more of nuclear maturation rate, cleavage rate, blastocyst rate, hatching rate or cryosurvival rate.

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