Bovine embryo refrigerated preservation liquid and application thereof

Abstract

The invention provides a bovine embryo refrigerated preservation solution. The bovine embryo refrigerated preservation solution is prepared from hydroxyethyl starch, dimethyl sulfoxide, fetal calf serum, cane sugar, cepharanthine and deionized water. The ratio of the components is further optimized, and the components comprise 5% of hydroxyethyl starch, 5% of dimethyl sulfoxide, 55% of fetal calf serum, 37% of deionized water, 2% of cane sugar and 1% of cepharanthine. Under the optimal raw material ratio condition that the concentration of cepharanthine is 600 [mu] mol / L and the concentration of cane sugar is 2 mol / L, cepharanthine is added into the bovine embryo preserving fluid, through a triple synergistic mechanism of oxidation resistance, apoptosis resistance and immunoregulation, the defect of biochemical damage protection in the prior art is overcome, and full-period protection is provided for embryos.

Description

Technical Field

[0001] The present invention belongs to the technical field of embryo preservation, specifically relates to the technical field of bovine embryo cold storage, and more specifically relates to the technical field of a bovine embryo cold storage solution and application thereof. Background Art

[0002] Superovulation technology can yield a larger number of embryos, but it also requires more synchronized estrus females for embryo transfer, which is time-consuming, labor-intensive, and costly. Cryopreserving embryos and waiting for estrus before transplanting them to a suitable recipient not only frees embryo transfer from time and space constraints but also allows for rigorous recipient screening, ensuring recipient quality and improving conception rates. This will significantly reduce costs and promote the application and widespread adoption of embryo transfer technology in production, becoming a crucial technical support for the industrialization of embryo transfer. Embryo cryopreservation is crucial for preserving genetic diversity and protecting superior breeds.

[0003] Cepharanthine (Ceph) is a natural product extracted from the Menispermaceae plant Stephania japonica. Ceph has become a research hotspot in recent years, but its impact in the field of reproduction has rarely been reported.

[0004] How to add cephalanx to bovine embryo preservation fluid and screen suitable preservation fluid components, through the triple synergistic mechanism of anti-oxidation, anti-apoptosis and immune regulation, to fill the deficiencies of existing technologies in protecting against biochemical damage and provide "full-cycle protection" for embryos. Summary of the Invention

[0005] In view of the technical problem that there are currently no research reports on adding cephalanthrin to bovine embryo cold storage solution and screening suitable storage solution components in the existing technology, a bovine embryo cold storage solution and its application are provided. This method combines cephalanthrin with bovine embryo cold storage solution in a specific component ratio and screens suitable storage solution components. Through the triple synergistic mechanism of anti-oxidation, anti-apoptosis and immune regulation, it fills the deficiencies of the existing technology in protecting against biochemical damage and provides "full-cycle protection" for the embryo.

[0006] To achieve this technical purpose, the present invention adopts the following technical solutions: The present application provides a bovine embryo cold storage solution, which comprises: hydroxyethyl starch, dimethyl sulfoxide, fetal bovine serum, sucrose, cepharanthin, and deionized water.

[0007] Preferably, the bovine embryo cold storage solution comprises, by mass percentage, 7%-3% hydroxyethyl starch, 7%-3% dimethyl sulfoxide, 40%-60% fetal bovine serum, 21.6%-52.4% deionized water, 1%-3% sucrose, and 1.4%-0.6% cepharanthin.

[0008] More preferably, the bovine embryo cold storage solution comprises, by mass percentage, 5% hydroxyethyl starch, 5% dimethyl sulfoxide, 50% fetal bovine serum, 37% deionized water, 2% sucrose, and 1% cepharanthin.

[0009] Preferably, the concentration of cepharanthin used in the bovine embryo cold storage solution is 100-1000 μmol / L.

[0010] More preferably, the concentration of cepharanthin used in the bovine embryo cold storage solution is 600 μmol / L.

[0011] Preferably, the sucrose concentration selected in the bovine embryo cold storage solution is 0.5 mol / L-4 mol / L.

[0012] More preferably, the sucrose concentration selected in the bovine embryo cold storage solution is 2 mol / L.

[0013] Furthermore, the present application also provides the use of the bovine embryo cold storage solution in the cold storage of bovine embryos.

[0014] Regarding the issue of new inventions and creations using existing common knowledge as the basis of existing technology. In fact, except for groundbreaking inventions, any invention and creation is inseparable from the existing technical means and technical elements as the basis, and is the result of further innovation on the basis of existing technical means. Technologies such as embryo cold preservation solution are all based on existing technology, but how to combine cephalothin with bovine embryo preservation solution, and screen suitable preservation solution components, through the triple synergistic mechanism of anti-oxidation, anti-apoptosis, and immune regulation, how to coordinate the ratio between raw materials and auxiliary materials, and how to make the bovine embryo cold preservation solution obtain the characteristics of significantly improved recovery culture survival rate and hatching rate after a series of processes at 4°C for 24h, 36h, and 48h, all need to be repeatedly verified through a series of unforeseen scientific experiments. It is precisely for these reasons that, although this application utilizes conventional technical means as a basis, and on this basis provides a bovine embryo cold preservation solution and its application through scientific experiments, the various technical steps in the provided bovine embryo cold preservation solution preparation method form a whole, which is integral and cannot be arbitrarily dismembered. These preparation methods as a whole can achieve the effect of significantly improving the survival rate and hatching rate of resuscitation culture after low-temperature storage at 4°C for 24h, 36h, and 48h, and have broad practical value.

[0015] By implementing the technical solution of the present invention, the following beneficial effects can be achieved: The present application provides a method for preparing a bovine embryo cold storage solution, which uses hydroxyethyl starch, dimethyl sulfoxide, fetal bovine serum, sucrose, cepharanthin, and deionized water as raw material ratios. The ratios between the components are further optimized, with 5% hydroxyethyl starch, 5% dimethyl sulfoxide, 55% fetal bovine serum, 37% deionized water, 2% sucrose, and 1% cepharanthin. Under the optimal raw material ratio conditions of 600 μmol / L cepharanthin concentration and 2 mol / L sucrose concentration, a specific component ratio is compounded and added to the bovine embryo preservation solution in combination with cepharanthin, and suitable preservation solution components are screened. Through the triple synergistic mechanism of anti-oxidation, anti-apoptosis, and immunomodulation, the method fills the deficiencies of the existing technology in protecting against biochemical damage and provides "full cycle protection" for the embryo. DETAILED DESCRIPTION

[0016] The technical solutions of the present invention are described clearly and completely below with reference to the embodiments. It is obvious that the embodiments described are only a portion of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are also within the scope of protection of the present invention.

[0017] In this example, the reagents used were: Cephalaenopsis indole purchased from MedChemExpress; fetal bovine serum (FBS) purchased from TrinityTek; dimethyl sulfoxide, hydroxyethyl starch, and sucrose purchased from Hunan Yunbang Biotechnology Co., Ltd., and all of the above reagents were of analytical grade.

[0018] The instruments used in this application are: constant temperature and humidity incubator LHS-100CH from Shanghai Yiheng Scientific Instrument Co., Ltd.; digital display constant temperature water bath HH-S4 from Jiangsu Jinyi Instrument Technology Co., Ltd.; high temperature and high pressure sterilizer ALPCL-40L from Shanghai Simo Biotechnology Co., Ltd.; ultra-clean workbench VD-850 from Suzhou Purification Equipment Co., Ltd., etc.

[0019] Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.

[0020] Example 1: A bovine embryo cold storage solution The present application provides a bovine embryo cryopreservation solution, comprising: 7% hydroxyethyl starch, 7% dimethyl sulfoxide, 60% fetal bovine serum, 21.60% deionized water, 3% sucrose, and 1.40% cepharanthin, wherein the cepharanthin concentration is 1000 μmol / L and the sucrose concentration is 4 mol / L.

[0021] Example 2: A bovine embryo cold storage solution The present application provides a bovine embryo cryopreservation solution, comprising: 6% hydroxyethyl starch, 6% dimethyl sulfoxide, 55% fetal bovine serum, 29.30% deionized water, 2.50% sucrose, and 1.20% cepharanthin, wherein the cepharanthin concentration is 800 μmol / L and the sucrose concentration is 3 mol / L.

[0022] Example 3: A bovine embryo cold storage solution The present application provides a bovine embryo cryopreservation solution, comprising: 5% hydroxyethyl starch, 5% dimethyl sulfoxide, 55% fetal bovine serum, 37% deionized water, 2% sucrose, and 1% cepharanthin, wherein the cepharanthin concentration is 600 μmol / L and the sucrose concentration is 2 mol / L.

[0023] Example 4: A bovine embryo cold storage solution The present application provides a bovine embryo cryopreservation solution, comprising: 4% hydroxyethyl starch, 4% dimethyl sulfoxide, 45% fetal bovine serum, 44.70% deionized water, 1.50% sucrose, and 0.80% cepharanthin, wherein the cepharanthin concentration is 100 μmol / L and the sucrose concentration is 1 mol / L.

[0024] Example 5: A bovine embryo cold storage solution The present application provides a bovine embryo cryopreservation solution, comprising: 3% hydroxyethyl starch, 3% dimethyl sulfoxide, 40% fetal bovine serum, 52.40% deionized water, 1% sucrose, and 0.60% cepharanthin, wherein the cepharanthin concentration is 50 μmol / L and the sucrose concentration is 0.5 mol / L.

[0025] Example 6: Comparison of the effects of bovine embryo cold storage solutions Based on the description in Examples 1 to 5 above, the effects of the obtained bovine embryo cold storage solutions numbered M1, M2, M3, M4, and M5 were compared.

[0026] Production of bovine in vitro embryos (1) Oocyte acquisition: Ovaries were collected from the slaughterhouse and placed in a thermos containing saline solution. The samples were then transported to the laboratory within 3 hours. The ovaries were then washed 3-5 times in 35°C saline solution supplemented with penicillin and streptomycin. The ovaries were then placed in a beaker and saline solution containing double-antibody antibodies was added until the ovaries were covered. The beaker was placed in a 35°C water bath. Follicular fluid from the upper 2-8 mm of the ovary was aspirated using an 18-gauge syringe and placed in a 15 mL centrifuge tube. After complete sedimentation, the precipitate was aspirated using a Pasteur pipette and placed in a 90 mm culture dish. The oocytes were then diluted with egg washing solution. Oocytes were then isolated under a stereomicroscope and placed in a 35 mm culture dish.

[0027] (2) Oocyte maturation: Place a four-well culture plate with 750 μL LIV and 250 μL mineral oil per well in an incubator at 38.5°C, 5% CO2, and saturated humidity for equilibration for at least 2 hours. From the eggs, select cumulus-oocyte complexes (COCs) with uniform cytoplasm and completely surrounded by 3-4 layers of dense cumulus cells and wash them 2-3 times in egg washing solution. Transfer them to the equilibrated four-well plate, placing approximately 50 COCs per well. Place the four-well plate with COCs in an incubator at 38.5°C, 5% CO2, and saturated humidity for maturation for 22-24 hours.

[0028] (3) In vitro fertilization: Add 7 drops of 50 μL of fertilization solution to a 35 mm culture dish, cover with mineral oil, and place in an incubator to equilibrate for more than 2 hours. Thaw the semen in a 37.5°C water bath, mix the semen with 6 mL of semen washing solution, and centrifuge at 1800 rpm for 8 minutes to wash the sperm. After centrifugation, remove the supernatant, repeat the washing twice, and then resuspend the sperm to keep the sperm density at 2×10 6 After washing the mature oocytes three times in fertilization solution, transfer them into 50μL droplets of fertilization solution, placing about 15 oocytes in each droplet. Add 50μL of semen and place in an incubator for 8-18 hours to complete in vitro fertilization.

[0029] (4) In vitro development: 2-3 hours in advance, use 100μL M1-M5 droplets to make development culture plates and wash plates (35mm culture dishes) and place them in the incubator for equilibrium. After fertilization, transfer the oocytes from the droplets into 1.5mL centrifuge tubes, with about 200 oocytes per tube. Vortex on a vortex machine for about 3 minutes to remove the granulosa cells. After washing the fertilized eggs three times in M1-M5, place them in M1-M5 culture plates, with about 25 oocytes per drop. After 48 hours of culture, count the cleavage status of the eggs and transfer them to M1-M5 for continued culture. Change half the medium every 48 hours. After 7-8 days, count the number of blastocysts and use the resulting embryos for subsequent experiments.

[0030] (5) Select the above-mentioned cryopreservation solution according to different tests. After washing the embryos three times in the cryopreservation solution, 3-5 embryos are loaded into 0.25mL wheat tubes according to the five-stage method and stored in a 4℃ refrigerator. The cryopreservation time of bovine embryos is 24h, 36h and 48h. After the storage is completed, the embryos are taken out of the refrigerator, the contents are pushed out into a 90mm culture dish at room temperature, washed three times with holding solution and then transferred to a balanced culture dish. After continuing to culture with the preservation solution in a constant temperature incubator at 37℃, 5% CO2 and saturated humidity for 24h-48h, the survival and hatching conditions of the embryos in different groups are identified by morphological methods under a stereo microscope and corresponding records are made.

[0031] The above experiment was repeated three times per group. After 24 and 48 hours of culture, the number of embryos that survived and hatched from cryopreserved embryos was counted, and the survival rate (number of embryos that survived / number of embryos that were revived) and hatching rate (number of embryos that hatched / number of embryos that were revived) were calculated. The results are shown in Table 1.

[0032] Table 1: Effects of cryopreservation for different periods of time using different preservation solutions on embryo survival and hatching rates

[0033] Bovine embryos were cryopreserved at 4°C using M1-M5 for 24, 36, and 48 hours, respectively. Survival and hatching rates were then calculated after 24-48 hours of thawing and culture. The results are shown in Table 1. After 24 and 36 hours of cryopreservation, the survival and hatching rates of the M1-M5 groups initially increased and then decreased. The M5 group exhibited the highest survival and hatching rates. The results indicate that a combination of 5% hydroxyethyl starch, 5% dimethyl sulfoxide, 55% fetal bovine serum, 37% deionized water, 2% sucrose, and 1% cepharanthin was added to the bovine embryo preservation solution at a concentration of 600 μmol / L and 2 mol / L sucrose. The addition of cepharanthin to the solution, along with screening for appropriate preservation solution components, addresses the shortcomings of existing technologies in protecting against biochemical damage through its synergistic antioxidant, anti-apoptotic, and immunomodulatory mechanisms, providing "full-cycle protection" for embryos.

[0034] The above embodiments are only for illustrating the technical concept and features of the present invention in a specific scenario. Its purpose is to enable people who need this technology to understand the content of the present invention and implement it. It does not limit the scope of protection of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. A bovine embryo cold storage solution, characterized in that: The bovine embryo cold storage solution comprises: hydroxyethyl starch, dimethyl sulfoxide, fetal bovine serum, sucrose, cepharanthin and deionized water.

2. The bovine embryo cold storage solution according to claim 1, wherein The bovine embryo cold storage solution comprises, by mass percentage, 7%-3% hydroxyethyl starch, 7%-3% dimethyl sulfoxide, 40%-60% fetal bovine serum, 21.6%-52.4% deionized water, 1%-3% sucrose, and 1.4%-0.6% cepharanthin.

3. The bovine embryo cold storage solution according to claim 1, wherein The bovine embryo cold storage solution comprises, by mass percentage, 5% hydroxyethyl starch, 5% dimethyl sulfoxide, 50% fetal bovine serum, 37% deionized water, 2% sucrose, and 1% cepharanthin.

4. The bovine embryo cold storage solution according to claim 1, wherein The concentration of cepharanthin selected in the bovine embryo cold storage solution is 100-1000 μmol / L.

5. The bovine embryo cold storage solution according to claim 1, wherein The concentration of cepharanthin used in the bovine embryo cold storage solution is 600 μmol / L.

6. The bovine embryo cold storage solution according to claim 1, wherein The sucrose concentration selected in the bovine embryo cold storage solution is 0.5 mol / L-4 mol / L.

7. The bovine embryo cold storage solution according to claim 1, wherein The sucrose concentration selected in the bovine embryo cold storage solution is 2 mol / L.

8. Use of the bovine embryo cold storage solution as described in any one of claims 1-7 in the cold storage of bovine embryos.

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