Application of KRY-21i in preparation of formula for promoting bovine embryonic development

Abstract

The invention discloses a formula for promoting bovine embryonic development by using KRY-21i and an application method of the formula. 10 [mu] M of KRY-21i is added in the embryo culture process after bovine in-vitro fertilization, and the synergistic effect of LIF, FGF2 and IGF1 is combined, so that the formation rate of the bovine blastocyst is remarkably improved. Experimental results show that compared with a control group, the blastocyst rate of a KRY-21i treatment group is remarkably increased, and it is proved that normal development of bovine embryos can be effectively promoted by adding KRY-21i into a culture formula. The formula and the method provided by the invention provide a new technical approach for embryo engineering in animal husbandry, and the breeding efficiency and the economic benefit can be remarkably improved.

Description

Technical Field

[0001] The invention relates to the fields of animal husbandry, veterinary medicine and biotechnology, and in particular to application of KRY-21i in preparing a formula for promoting bovine embryo development. Background Art

[0002] In vitro fertilization (IVF) of cattle has been widely used and developed in recent years as an important technical means to cultivate and breed excellent cattle in modern animal husbandry. IVF technology achieves in vitro combination of oocytes and sperm in a laboratory environment, forms embryos after successful fertilization, and then transplants these embryos back to the mother or uses them for further in vitro culture, thereby accelerating the spread of excellent genes and the improvement of cattle breeds. However, although IVF technology has significant advantages in theory, it still faces many challenges in its actual application, especially in the in vitro embryo culture stage, the blastocyst formation rate is generally low. This phenomenon has limited the widespread application and promotion of IVF technology in animal husbandry to a certain extent.

[0003] There are many factors that affect the blastocyst formation rate of bovine embryos, mainly including the quality of oocytes, sperm motility, optimization of fertilization conditions, and the suitability of the in vitro culture environment. Specifically, the quality of oocytes is directly related to the health of the fertilized egg and its subsequent developmental potential; the motility and fertilization ability of sperm determine the probability of successful fertilization and the initial development of the embryo. In addition, the optimization of temperature, pH value, oxygen concentration and other conditions during the fertilization process is also an important factor in ensuring the normal development of the fertilized egg. Most importantly, the in vitro culture environment, including the composition of the culture medium, the addition of cytokines and growth factors, etc., plays a decisive role in the survival rate and development rate of the embryo.

[0004] Currently, many studies and practices have shown that by adding specific growth factors and cytokines to the culture medium, the survival rate and development speed of embryos can be significantly improved. For example, adding factors such as interleukin-6 (LIF), fibroblast growth factor 2 (FGF2), and insulin-like growth factor 1 (IGF1) has been proven to promote the normal development of embryos and the formation of blastocysts to a certain extent. However, although these additives have played a positive role in improving embryo development, how to further improve the blastocyst formation rate and improve the quality of embryos on the existing basis is still a difficult problem that needs to be solved.

[0005] In view of the above problems, the present invention proposes an innovative method of introducing KRY-21i under standard in vitro culture conditions to promote the development of bovine embryos. KRY-21i is a new type of bioactive molecule. Its application in in vitro culture medium can significantly improve the development environment of embryos and thus increase the formation rate of blastocysts. By adding KRY-21i to the basic culture medium containing LIF, FGF2 and IGF1, the formula of the present invention not only retains the promoting effect of existing factors on embryo development, but also further improves the development efficiency and quality of embryos through the synergistic effect of KRY-21i.

[0006] The method proposed in the present invention provides an effective technical approach to improve the overall efficiency of in vitro fertilization of cattle. Specifically, the addition of KRY-21i can optimize the culture environment, enhance the ability of embryonic cells to divide and expand, reduce abnormalities in the development process, and improve the success rate of blastocyst formation. The application of this technology can not only improve the success rate of IVF technology, reduce the number of invalid fertilized eggs, and reduce production costs, but also can breed excellent cattle breeds on a large scale in a short period of time, meet the demand of animal husbandry for high-quality cattle herds, and thus significantly improve the production efficiency and economic benefits of animal husbandry.

[0007] In summary, the present invention successfully solves the problems of low blastocyst formation rate and low embryo quality in the prior art by introducing KRY-21i into the standard IVF culture medium, and provides an efficient and reliable formula for promoting bovine embryo development. This not only provides new technical support for the breeding of fine varieties in animal husbandry, but also opens up new directions for research and application in related fields in the future, and has important production application value and broad market prospects. Summary of the invention

[0008] The KRY-21i described in the present invention is a small molecule compound, also known as LY2955303, with a CAS number of 1433497-19-8, and a Smile formula of CC(C)(C)C1=CC(C2=CC(C3=CC=C(C(O)=O)C=C3)=NN2C4=CC=C(C=C4)C(N5CCN(CC5)C)=O)=CC(C(C)(C)C)=C1.

[0009] The present invention provides an application method of KRY-21i in preparing a formula for promoting bovine embryo development. The method is suitable for in vitro fertilization of bovine oocytes and the subsequent in vitro culture (IVC) process, and can significantly improve the blastocyst formation rate of bovine embryos. Technical Solution

[0010] 1.1. Application of the small molecule compound KRY-21i in the preparation of a formula for promoting bovine embryo development; characterized in that 20 ng / mL of LIF, 40 ng / mL of FGF2 and 20 ng / mL of IGF1 are added to the basal culture medium (IVC); under the above basic conditions, 10 μM KRY-21i is added to prepare a culture medium for promoting bovine embryo development.

[0011] 1.2. Remove granulosa cells from oocytes that have been matured in vitro and fertilized for 20 hours; add the culture medium described in Technical Solution 1.1 to the obtained cleavage embryos; and culture them under the conditions of 38.5°C and 5% CO2.

[0012] 1.3. The embryos described in Technical Solution 1.2 were continuously cultured and observed, and the blastocyst formation rate, embryo morphology and developmental characteristics were counted. Beneficial Effects

[0013] The blastocyst formation rate of the formula with KRY-21i was significantly improved, which promoted the development of bovine embryos and thus improved the efficiency of IVF. This provides strong technical support for the livestock industry to expand the high-quality cattle population. DETAILED DESCRIPTION

[0014] Bovine ovary samples: Fresh bovine ovaries were collected from a local slaughterhouse, placed in saline containing 100 IU / mL penicillin and streptomycin, and transported to the laboratory within 2-4 hours at 37.0°C.

[0015] Reagents and solutions: physiological saline (containing 100 IU / mL penicillin / streptomycin), 75% alcohol for surface disinfection, egg washing solution: M199 (Gibico, 12340) + 100 μg / mL heparin sodium (Sigma, H3393), IVM culture medium (IVFBioscience, 71001): add 20 ng / mL LIF (Gibco, AF300-05), 40 ng / mL FGF2 (Thermo Fisher, PHG0266), 20 ng / mL IGF1 (MCE, HY-P700093AF) to the basic culture medium, IVF culture medium (IVFBioscience, 71004) for oocyte fertilization, IVC culture medium (IVF Bioscience, 71005) for embryo culture, its basic composition is the same as IVM culture medium, KRY-21i: concentrated stock solution 50 mM (storage solvent is DMSO), diluted to 10 μM at a ratio of 1:5000 before use, DMSO (control solvent), dilution ratio 1:5000, 1% hyaluronidase was used to remove granular cells.

[0016] Instruments and equipment: Microscopes were used to observe and select cumulus-oocyte complexes (COCs) and evaluate embryo morphology; incubators were maintained at a constant temperature of 38.5°C and 5% CO2 for oocyte maturation and embryo culture; sterile operating tables ensured aseptic operation during the experiment; water baths at 38.0°C were used for sperm thawing; GraphPad Prism 10.0 software was used for data statistical analysis; Collection and maturation of bovine oocytes: Ovarian processing: The recovered ovaries were quickly washed with 75% alcohol, and then placed in saline containing antibiotics to be thoroughly washed to remove excess tissue; cumulus-oocyte complexes (COCs) were collected: the follicles were cut open with a sterile scalpel to allow the COCs to flow out, and the follicular fluid and its contents were collected in a 15 mL centrifuge tube, placed in a 37°C water bath for 10 minutes, and the supernatant was discarded. The tube was gently resuspended with egg washing solution and transferred to a culture dish. COCs wrapped in more than three layers of cumulus cells were collected under a microscope, and COCs with uniform cytoplasm and ≥3 layers of cumulus cells were selected for maturation culture; maturation culture: COCs were randomly divided into 4 groups, each group was placed in a culture dish containing 500 μL of maturation culture medium (IVM + LIF + FGF2 + IGF1), and cultured in an incubator at 38.5°C and 5% CO2 for 22.5 hours to complete the maturation of the oocytes.

[0017] Oocyte fertilization (IVF): Sperm preparation: thaw frozen bovine sperm in a 38.0°C water bath for 30 seconds and adjust the sperm concentration to 1×10 6 sperm / mL; fertilization step, the mature oocytes were washed 2-3 times with IVF culture medium, transferred to a culture dish containing 400 μL IVF culture medium, added with sperm mixture, and cultured at 38.5℃, 5% CO2 for 20 hours to complete fertilization.

[0018] Embryo culture and grouping: Granulosa cells were removed 20 hours after fertilization using 1% hyaluronidase, and the obtained cleavage embryos (2-cell or 4-cell stage) were transferred into IVC medium.

[0019] Grouping treatment: On the second day, the cleavage embryos were randomly divided into three groups: DMSO group: IVC basal culture medium + LIF (20 ng / mL) + FGF2 (40 ng / mL) + IGF1 (20 ng / mL) + 1 / 5000 DMSO; IVC group: IVC basal culture medium + LIF (20 ng / mL) + FGF2 (40 ng / mL) + IGF1 (20 ng / mL); LY group (10 μM KRY-21i group): IVC basal culture medium + LIF (20 ng / mL) + FGF2 (40 ng / mL) + IGF1 (20 ng / mL) + 10 μM KRY-21i.

[0020] Culture conditions and observation: The cells were cultured in an incubator at 38.5°C and 5% CO2. Observations were performed under a microscope on the second day (D2), the fourth day (D4), the sixth day (D6), and the eighth day (D8). The embryonic development was recorded and the blastocyst formation rate was calculated.

[0021] Data statistics and analysis: GraphPad Prism 10.0 software was used for statistical analysis, and the experiment was repeated at least 3 times. One-way analysis of variance was used for comparison between groups, and the data were expressed as mean ± standard error (Mean ± SEM). P < 0.05 indicated that the difference was statistically significant. The statistical analysis results showed that the blastocyst rate in the LY group (10 μM KRY-21i) was significantly higher than that in the IVC group and the DMSO group (P<0.05). This shows that the addition of KRY-21i can effectively improve the blastocyst formation rate of bovine embryos.

[0022] During the culture process, embryos in each group were in the early stage of cleavage at D2, with no obvious morphological differences. As the culture time increased (D8), more embryos in the LY group formed blastocyst structures with good morphology, complete structure, high transparency, more uniform cell division, and better quality than the blastocysts formed in the DMSO and IVC groups.

[0023] Figure 1 Microscope photos of each group of embryos at D2 and D8 and a comparison chart of the blastocyst rate were shown. The LY group formed more high-quality blastocysts at D8, and statistical analysis also showed that the blastocyst rate was significantly increased.

[0024] After one-way analysis of variance and multiple comparisons, the P values ​​were as follows: LY group vs IVC group: P=0.0141; LY group vs DMSO group: P=0.0003; IVC group vs DMSO group: P=0.0069; the above P values ​​were all <0.05, indicating that there were significant differences among the groups, especially the differences between the LY group and the IVC group and the DMSO group were particularly significant.

[0025] In summary, the formula proposed in the present invention can significantly improve the blastocyst formation rate, help expand the high-quality cattle population, and has positive significance for the modernization and efficient breeding of animal husbandry. It also provides theoretical and practical guidance for improving the efficiency of mammalian embryo culture.

[0026] The specific implementation method described in this patent text is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited thereto. Any obvious simple changes or equivalent substitutions made by any technician familiar with the technical field within the technical scope disclosed in the present invention shall be regarded as the protection scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This figure shows the experimental result data disclosed in the specific implementation mode of the present invention, which are respectively displayed by microphotographs and bar graphs: compared with the case without addition, the formula with KRY-21i added can significantly improve the blastocyst formation rate in the cleavage embryo culture after in vitro fertilization of bovine oocytes.

Claims

1. Application of KRY-21i in the preparation of formula for promoting bovine embryo development.

2. The formulation according to claim 1, wherein the preparation method comprises the following steps: The small molecule compound KRY-21i was added to the commercial IVC basal culture medium to make the final concentration of KRY-21i 10 μM; 20 ng / mL of LIF, 40 ng / mL of FGF2 and 20 ng / mL of IGF1 were further added to the culture medium.

3. The use according to claim 1, wherein the formula is used in the culture of cleavage embryos after in vitro fertilization of bovine oocytes to improve the blastocyst formation rate.

4. The method for culturing bovine embryos in vitro according to claim 3, characterized in that: The method comprises the following steps: removing granulosa cells from oocytes matured in vitro and fertilized 20 hours later; adding the culture medium of claim 2 to the obtained cleavage embryos; and culturing under the culture conditions of 38.5°C and 5% CO2.

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