Use of KRY-21i in preparing formula for promoting development of bovine embryos

Abstract

The application discloses a formula for promoting the development of bovine embryos by using KRY-21i and an application method thereof. By adding 10 muM of KRY-21i in the process of culturing bovine embryos after in vitro fertilization and combining the synergistic effect of LIF, FGF2 and IGF1, the formation rate of bovine blastocysts is significantly improved. The experimental results show that, compared with the control group, the blastocyst rate of the KRY-21i treatment group is significantly improved, proving that the addition of KRY-21i in the culture formula can effectively promote the normal development of bovine embryos. The formula and method provide a new technical approach for embryo engineering in the livestock industry, and can significantly improve the breeding efficiency and economic benefits.

Description

Technical Field

[0001] This invention relates to the fields of animal husbandry, veterinary medicine, and biotechnology, specifically to the application of KRY-21i in the preparation of formulations that promote bovine embryonic development. Background Technology

[0002] In vitro fertilization (IVF) of cattle, as an important technique for breeding and promoting superior cattle breeds in modern animal husbandry, has been widely applied and developed in recent years. IVF technology achieves the fusion of oocytes and sperm in a laboratory environment, forming embryos after successful fertilization. These embryos are then transferred back to the mother or used for further in vitro culture, thereby accelerating the dissemination of superior genes and improving cattle breeds. However, despite the significant theoretical advantages of IVF technology, it still faces many challenges in practical application, especially in the embryo culture stage, where the blastocyst formation rate is generally low. This phenomenon, to some extent, limits the widespread application and promotion of IVF technology in animal husbandry.

[0003] Many factors influence the blastocyst formation rate of bovine embryos, primarily including oocyte quality, sperm motility, optimization of fertilization conditions, and suitability of the in vitro culture environment. Specifically, oocyte quality directly affects the health and developmental potential of the fertilized egg; sperm motility and fertilization capacity determine the probability of successful fertilization and the early developmental stage of the embryo. Furthermore, optimizing conditions such as temperature, pH, and oxygen concentration during fertilization is also crucial for ensuring normal embryo development. Most critically, the in vitro culture environment, including the composition of the culture medium and the addition of cytokines and growth factors, plays a decisive role in embryo survival and developmental speed.

[0004] Currently, numerous studies and practices have demonstrated that adding specific growth factors and cytokines to the culture medium can significantly improve embryo survival and development speed. For example, the addition of factors such as interleukin-6 (LIF), fibroblast growth factor 2 (FGF2), and insulin-like growth factor 1 (IGF1) has been shown to promote normal embryo development and blastocyst formation to a certain extent. However, despite the positive effects of these additives in enhancing embryo development, how to further improve blastocyst formation rate and embryo quality remains a pressing challenge.

[0005] To address the aforementioned problems, this invention proposes an innovative method for introducing KRY-21i under standard in vitro culture conditions to promote bovine embryo development. KRY-21i is a novel bioactive molecule whose application in in vitro culture media can significantly improve the embryonic developmental environment, thereby increasing the blastocyst formation rate. By adding KRY-21i to a basal culture medium containing LIF, FGF2, and IGF1, the formulation of this invention not only retains the existing factors' promoting effects on embryonic development but also further enhances embryonic development efficiency and quality through the synergistic effect of KRY-21i.

[0006] The method proposed in this invention provides an effective technical approach to improve the overall efficiency of bovine in vitro fertilization (IVF). Specifically, the addition of KRY-21i optimizes the culture environment, enhances the division and expansion capabilities of embryonic cells, reduces developmental abnormalities, and increases the success rate of blastocyst formation. The application of this technology not only improves the success rate of IVF, reduces the number of ineffective fertilized eggs, and lowers production costs, but also enables large-scale breeding of superior cattle breeds in a short period, meeting the livestock industry's demand for high-quality cattle and thus significantly improving the production and economic benefits of the livestock industry.

[0007] In summary, this invention, by introducing KRY-21i into standard IVF culture medium, successfully solves the problems of low blastocyst formation rate and poor embryo quality in existing technologies, providing a highly efficient and reliable formula for promoting bovine embryo development. This not only provides new technical support for the breeding of superior livestock breeds but also opens up new directions for future research and applications in related fields, possessing significant production application value and broad market prospects. Summary of the Invention

[0008] The KRY-21i described in this invention is a small molecule compound, also known as LY2955303, with CAS number 1433497-19-8, and its SMILE formula is 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] This invention provides a method for applying KRY-21i in the preparation of a formula to promote bovine embryo development. This method is applicable to in vitro fertilization of bovine oocytes and subsequent in vitro culture (IVC) processes, and can significantly improve the blastocyst formation rate of bovine embryos.

[0010] Technical solution

[0011] 1.1. Application of the small molecule compound KRY-21i in the preparation of a formula to promote 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 basal conditions, 10 μM of KRY-21i is added to prepare a culture medium to promote bovine embryo development.

[0012] 1.2. Granulosa cells were removed from oocytes that had matured and been fertilized in vitro for 20 hours; the resulting cleavage embryos were added to the culture medium described in technical scheme 1.1; and cultured at 38.5°C and 5% CO2.

[0013] 1.3. The embryos described in technical solution 1.2 are continuously cultured and observed, and the blastocyst formation rate, embryo morphology and developmental characteristics are statistically analyzed. Beneficial effects

[0014] Formulas with added KRY-21i significantly improved blastocyst formation rates, promoted bovine embryo development, and thus increased IVF efficiency. This provides strong technical support for expanding high-quality cattle herds in the livestock industry. Detailed Implementation

[0015] Bovine ovarian samples: Fresh bovine ovaries were collected from local slaughterhouses, placed in physiological saline containing 100 IU / mL penicillin and streptomycin, and transported back to the laboratory within 2-4 hours at 37.0℃.

[0016] Reagents and solutions: Physiological saline (containing 100 IU / mL penicillin / streptomycin), 75% alcohol for surface disinfection; oocyte washing solution: M199 (Gibco, 12340) + 100 μg / mL heparin sodium (Sigma, H3393); IVM culture medium (IVFBioscience, 71001): 20 ng / mL LIF (Gibco, AF300-05), 40 ng / mL LGF2 (Thermo Fisher Scientific, PHG0266), and 20 ng / mL IGF1 (MCE, HY-P700093AF) added to the basal culture medium; IVF culture medium (IVFBioscience, 71004) for oocyte fertilization; IVC culture medium (IVF Bioscience, 71005) for embryo culture, with the same basic composition as IVM culture medium; KRY-21i: concentrated stock solution 50 μL. mM (stored in DMSO), dilute to 10 μM at a ratio of 1:5000 before use. DMSO (control solvent), dilution ratio 1:5000. 1% hyaluronidase is used to remove granular cells.

[0017] Instruments and equipment: Microscope for observing and selecting cumulus-oocyte complexes (COCs) and assessing embryo morphology; Incubator at a constant temperature of 38.5℃ and 5% CO2 for oocyte maturation and embryo culture; Aseptic workbench to ensure aseptic operation during the experiment; Water bath at 38.0℃ for sperm thawing; GraphPad Prism 10.0 software for data statistical analysis.

[0018] Bovine oocyte collection and maturation: Ovarian processing: The recovered ovaries were quickly washed with 75% alcohol and then thoroughly cleaned in saline containing antibiotics to remove excess tissue. Cumulus-oocyte complex (COC) collection: Follicles were incised with a sterile scalpel to allow COCs to flow out. Follicular fluid and its contents were collected in 15 mL centrifuge tubes and placed in a 37°C water bath for 10 minutes to settle. The supernatant was discarded, and the cells were gently resuspended in oocyte washing solution and transferred to a culture dish. COCs encapsulating three or more layers of cumulus cells were collected under a microscope. 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 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 allow the oocytes to complete maturation.

[0019] IVF (In Vitro Fertilization): 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. 6 Sperm / mL; Fertilization procedure: After washing mature oocytes 2-3 times with IVF culture medium, transfer them to a culture dish containing 400 μL of IVF culture medium, add sperm mixture, and culture at 38.5℃ and 5% CO2 for 20 hours to complete fertilization.

[0020] Embryo culture and grouping: Granulosa cells were removed. Twenty hours after fertilization, granulosa cells were removed using 1% hyaluronidase. The resulting cleavage embryos (2-cell or 4-cell stage) were then transferred to IVC medium.

[0021] 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.

[0022] Culture conditions and observations: The embryos were cultured in an incubator at 38.5℃ and 5% CO2. They were observed under a microscope on day 2 (D2), day 4 (D4), day 6 (D6), and day 8 (D8). The embryo development was recorded and the blastocyst formation rate was calculated.

[0023] Data Statistics and Analysis: Statistical analysis was performed using GraphPad Prism 10.0 software, and the experiment was repeated at least three times. One-way ANOVA was used for comparisons between groups. Data are expressed as mean ± standard error (Mean ± SEM). P < 0.05 was considered statistically significant.

[0024] Statistical analysis showed that the blastocyst rate of the LY group (10 μM KRY-21i) was significantly higher than that of the IVC and DMSO groups (P<0.05). This indicates that adding KRY-21i can effectively improve the blastocyst formation rate of bovine embryos.

[0025] During the culture process, at day 2, all embryos in the group were in the early cleavage stage with no obvious morphological differences. As the culture time increased (day 8), more embryos in the LY group formed blastocyst structures with good morphology, intact structure, high transparency, and more uniform cell division. Compared with the blastocysts formed in the DMSO and IVC groups, the quality was better.

[0026] Figure 1 The images show microscopic photographs of embryos in each group at D2 and D8, as well as a comparison of blastocyst rates. The LY group formed more high-quality blastocysts at D8, and statistical analysis also showed a significantly higher blastocyst rate.

[0027] One-way ANOVA and multiple comparisons yielded the following P-values: LY group vs. IVC group: P=0.0141; LY group vs. DMSO group: P=0.0003; IVC group vs. DMSO group: P=0.0069. All P-values ​​were <0.05, indicating significant differences among the groups, especially between the LY group and the IVC and DMSO groups.

[0028] In summary, the formulation proposed in this invention can significantly improve the blastocyst formation rate, which helps to 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.

[0029] The specific implementation methods described in this patent text are merely preferred embodiments of the present invention, and the scope of protection of the present invention is not limited thereto. Any obvious simple changes or equivalent substitutions made by those skilled in the art within the scope of the technology disclosed in this invention should be considered within the scope of protection of this invention. Attached Figure Description

[0030] Figure 1 This figure shows the experimental results data disclosed in the specific embodiments of the present invention, presented in micrographs and bar charts: Compared with the case without addition, the formulation with KRY-21i can significantly improve the blastocyst formation rate in cleavage embryo culture after in vitro fertilization of bovine oocytes.

Claims

1. The application of KRY-21i in the preparation of culture media that promote bovine embryonic development, characterized in that, The culture medium for promoting bovine embryo development consists of IVC basal medium, 20 ng / mL LIF, 40 ng / mL FGF2, 20 ng / mL IGF1, and 10 μM KRY-21i; the promotion of bovine embryo development is achieved by using the culture medium for promoting bovine embryo development in the culture of cleavage embryos after in vitro fertilization of bovine oocytes to improve the blastocyst formation rate; the CAS number of KRY-21i is 1433497-19-8.

2. The application according to claim 1, characterized by the following steps: Granulosa cells were removed from bovine oocytes that had matured and been fertilized in vitro for 20 hours. The resulting cleavage embryos were then given the culture medium for promoting bovine embryonic development as described in claim 1 and cultured at 38.5°C and 5% CO2.

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