Application of metabolic small molecule additive in promotion of development process of bovine in-vitro embryos
By adding NAAG to bovine in vitro embryo culture medium, the problem of low blastocyst formation rate was solved, the blastocyst formation rate was improved and the developmental quality was maintained. It is applicable to existing culture systems and has industrialization potential.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHEJIANG UNIV
- Filing Date
- 2026-01-14
- Publication Date
- 2026-05-12
AI Technical Summary
In bovine in vitro embryo production, the blastocyst formation rate is low and the quality is unstable. Existing culture systems are unable to simulate the maternal uterine microenvironment, which affects the embryo's developmental potential.
The metabolic molecule N-acetylaspartate-glutamine (NAAG) was added to the in vitro maturation medium at a final concentration of 1 μM for use in bovine in vitro embryonic development.
It significantly improves blastocyst formation rate by nearly 15%, maintains embryo development stability, is compatible with existing culture systems, and is easy to operate.
Smart Images
Figure CN122012380A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of in vitro embryo production technology, and in particular relates to the application of a metabolic small molecule additive in promoting bovine in vitro embryo development. Background Technology
[0002] In recent years, with the increasing demand for high-quality germplasm resources in my country's dairy industry, in vitro embryo production (IVP) technology has gradually become an important auxiliary means for dairy cow breeding. Compared with traditional in vivo embryo transfer, IVP technology is not limited by the donor's estrous cycle, can be carried out year-round, and can significantly improve reproductive efficiency and shorten the breeding cycle. Therefore, it has important application value in the genetic improvement of dairy cows nationwide.
[0003] However, my country's bovine embryo IVP system still faces significant challenges. Although the in vitro fertilization rate is relatively stable, the blastocyst formation rate is generally low, significantly lower than the developmental efficiency of in vivo embryos. Unstable blastocyst quality and low survival rates after cryopreservation are also major factors restricting the large-scale promotion of this technology. The key to these problems lies in the fact that the in vitro culture environment cannot completely simulate the microenvironment of the maternal uterus and fallopian tubes. Embryos are easily restricted in terms of metabolism, oxidative stress regulation, and nutrient utilization, thus affecting their normal developmental potential.
[0004] Against this backdrop, how to improve blastocyst formation rate and blastocyst quality by optimizing the culture system and improving the composition of the culture medium has become a core research issue both domestically and internationally. Embryo development quality is closely related to the metabolite environment in the culture medium. To meet the needs of embryonic development, exogenous substances are often required to supplement the culture medium, and amino acids are a common additive.
[0005] N-acetylaspartate-glutamine (NAAG) is a neuropeptide with high abundance in the mammalian central nervous system. Currently, there are no publicly available reports on the application of NAAG in embryonic development or culture systems. Summary of the Invention
[0006] The purpose of this invention is to address the problems of low blastocyst formation rate and poor quality in existing bovine in vitro embryo production processes, and to provide a metabolic small molecule additive for promoting bovine in vitro embryo development. The metabolic small molecule additive, N-acetylaspartic acid-glutamine (NAAG), is safe, stable and easy to use.
[0007] This invention provides the application of a metabolic small molecule additive in promoting bovine in vitro embryonic development, wherein the metabolic small molecule additive is N-acetylaspartic acid-glutamine. The molecular structural formula of the additive is shown in (I): Furthermore, the application specifically involves adding metabolic small molecule additives in the form of droplets to an in vitro maturation culture medium used for culturing bovine in vitro embryos.
[0008] Furthermore, the final concentration of the metabolic small molecule additive in the in vitro maturation culture medium is 1 μM.
[0009] Furthermore, the metabolic small molecule additive is added at the time when bovine in vitro fertilized eggs begin in vitro culture.
[0010] Furthermore, the promotion of bovine in vitro embryonic development is manifested in increasing the blastocyst formation rate after bovine in vitro fertilization.
[0011] The present invention also provides a culture medium composition for promoting bovine in vitro embryonic development, comprising an in vitro maturation medium and N-acetylaspartic acid-glutamine at a final concentration of 1 μM in the culture medium.
[0012] This invention offers the following beneficial effects: The N-acetylaspartic acid-glutamine provided by this invention can significantly improve the blastocyst formation rate of bovine in vitro fertilized embryos, with a clearly defined effect. Experimental verification shows that adding NAGG to the bovine embryo in vitro culture system at a final concentration of 1 μM can significantly increase the blastocyst formation rate of bovine in vitro fertilized embryos from approximately 27% to approximately 43%, an increase of nearly 15 percentage points, indicating that NAAG has a significant and stable promoting effect on bovine embryo blastocyst formation. It exhibits strong compatibility with existing in vitro embryo culture systems, low application cost, and can be directly added to existing conventional bovine in vitro embryo culture systems without changing the basic culture medium formulation or culture process parameters. The operation is simple, demonstrating good system compatibility and promising prospects for industrialization. Attached Figure Description
[0013] The present invention will be further described below with reference to the accompanying drawings and embodiments; Figure 1 This is a graph showing the PCA cluster analysis results of samples from the three metabolomics groups: zygote, embryonic genome activation period (8-16 cells), and blastocyst.
[0014] Figure 2 A diagram showing the metabolic pathways enriched during the bovine blastocyst stage.
[0015] Figure 3 This is a graph showing the changes in α-ketoglutarate levels during three developmental stages: the zygote stage, the embryonic genome activation stage (8-16 cells), and the blastocyst stage, based on metabolomics data.
[0016] Figure 4The data are metabolomics data, showing the changes in NAAG content at three stages: bovine zygote, embryonic genome activation period (8-16 cells), and blastocyst.
[0017] Figure 5 Phenotypic diagrams of four concentration groups (NC, 500 μM, 300 μM, and 100 μM) developed to day 8.0.
[0018] Figure 6 Statistical charts showing blastocyst rates at four concentrations: NC, 500μM, 300μM, and 100μM.
[0019] Figure 7 Phenotypic diagrams of four concentration groups (NC, 100 μM, 10 μM, and 1 μM) developed to day 8.0.
[0020] Figure 8 Statistical chart of blastocyst rates for four concentration groups: NC, 100μM, 10μM, and 1μM.
[0021] Figure 9 Phenotypic diagrams of four concentration groups (NC, 1 μM, 100 nM, and 10 nM) developed to day 8.0.
[0022] Figure 10 Statistical chart of blastocyst rates for four concentration groups: NC, 1μM, 100nM, and 10nM.
[0023] Figure 11 Statistical charts showing the blastocyst rate in six replicates for the NC and 1μM groups. Detailed Implementation
[0024] The technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.
[0025] The NAAG used in this embodiment was purchased from SIGMA, part number A5930, CAS: 3106-85-2.
[0026] Example 1 The embryo reagents used in the following examples are shown in Table 1.
[0027] Table 1. Embryo Reagent List 1. In vitro maturation Preparation of mature plates: Add 200uL of mature culture medium IVM (specific formula shown in Table 2) to each well of a four-well plate, and place it in an embryo culture incubator with conditions of 38.5℃, 5% CO2, and 95% humidity two hours in advance for equilibration.
[0028] Maturation process: Collected cumulus-oocyte complexes (COCs), i.e. oocytes well-encapsulated by granulosa cells, are rinsed three times with 100uL of equilibrated maturation culture medium, and then placed in maturation culture medium in a four-well plate for maturation. The COCs are transferred and counted under a microscope using a pipette, with 50 COCs placed in each well. The maturation time is 22-24 hours.
[0029] Table 2 Maturation Culture Medium Formula 2. In vitro fertilization (1) Preparation of fertilization trays: Prepare fertilization trays 12 hours in advance. Place 200 μL of IVF (IVF bioscience) culture medium and 200 μL of SOF-FERT (Sperm Capacitation and Fertilization Media) (formulas shown in Tables 3 and 4) in each well of a four-well plate. Gently shake to evenly cover the entire bottom. Add 200 μL of paraffin oil and place the plate in an embryo incubator at 38.5°C and 5% CO2 for equilibration. At the same time, prepare 2 ml of Percoll solution (the composition of which is shown in Table 5), loosen the cap, and place the bottle in the incubator for equilibration.
[0030] Table 3 SOF-Base Formulation Table 4 SOF-Fert Formulation Table 5 Percoll Formulation (2) Preparation of oocytes: Rinse mature COCs three times with 100uL of SOF-FERT solution preheated to 37℃, and then add them to the fertilization dish prepared in step (1). Under a microscope, use a pipette to transfer and count 100 COCs added to each well.
[0031] (3) Sperm preparation: Add frozen sperm preheated to 37°C to equilibrated 90% Percoll solution, centrifuge at 800g for 8 minutes, remove the upper frozen sperm protectant, and retain the lower sperm layer (about 100uL); add 1mL of SOF-FERT solution preheated to 37°C, gently resuspend, centrifuge at 500g for 5 minutes, remove the supernatant, and retain the lower sperm layer (100uL).
[0032] (4) Fertilization process: Sperm density was calculated using a blood cell count method. The prepared sperm were slowly added to the vicinity of the mature COCs in the fertilization disc, so that the final sperm density in the fertilization disc was 5*10. 6 1 fertilization per mL, fertilization time 8-12 hours.
[0033] 3. In vitro culture Preparation of the culture dish: Prepare a 40uL IVC (IVF bioscience) droplet in a 35mm plastic culture dish and cover it with paraffin oil. The paraffin oil level should be higher than the IVC droplet. Place the dish in an embryo culture incubator with conditions of 38.5℃, 5% CO2 and 95% humidity in advance for equilibration.
[0034] Granulosa cells were removed and potential zygotes were selected: COCs were placed in T2 in vitro manipulation solution (TCM-199 + Hanks' Salts) and continuously rinsed with a pipette until the cumulus cells surrounding the zygote were completely removed. Cells containing polar bodies, with uniform, glossy black cytoplasm, intact shape, and a round, undamaged zona pellucida were selected as potential zygotes for subsequent experiments.
[0035] The animal material used in this embodiment (ovaries of Chinese Holstein dairy cows) came from a designated slaughterhouse in Ningbo, Zhejiang Province. The material was not subject to animal ethics review. The sperm used in this patent came from the dairy cow center of Beijing Shougang Livestock Development Co., Ltd. The sperm was conventional frozen Holstein dairy cow semen, with a grade of 0.4 or higher.
[0036] Example 2 Metabolomics analysis of bovine embryos at different stages In this embodiment, the internal metabolite composition of bovine in vitro fertilized embryos in the zygote, embryonic genome activation period (8-16 cells), and blastocyst stages was analyzed, and metabolomics results were combined to analyze metabolites that may be applied to promote embryo development in in vitro embryo culture systems.
[0037] (1) Metabolomics analysis of embryos at different developmental stages Bovine embryos obtained by in vitro fertilization as described in Example 1 were selected and sampled and analyzed at the following three developmental stages: The zygote stage is the starting point and first stage of embryonic development. The embryonic genome activation period (8–16 cell stage) is when the embryo's own genome begins to activate and the embryo begins to establish its own transcriptional regulatory network. The blastocyst stage is the period after the embryonic genome activation phase, during which the embryo undergoes cavitation and significant cell differentiation, forming a blastocyst with a well-defined structure.
[0038] For zygotic embryos, 9 hours after fertilization, embryos containing polar bodies were selected after granulosa cells were removed as zygotic samples. For 8–16 cell stage samples, embryos with 8–16 cells were collected on day 3.5 after fertilization. For blastocyst stage samples, embryos containing a blastocoel were collected on day 9 after fertilization. The collection method was as follows: after selecting embryos at the above stages, the zona pellucida was removed using hyaluronidase, the embryos were rinsed three times with 0.5% bovine serum albumin (BSA) solution, transferred to EP tubes, and flash-frozen in liquid nitrogen for preservation.
[0039] Fifty embryos from each stage were collected, with three replicates. All samples were then combined and subjected to metabolomics sequencing using LC-MS to obtain the internal metabolite composition. Based on the sequencing results, the small molecule metabolites inside embryos at different stages were compared and analyzed.
[0040] Figure 1 The results of PCA cluster analysis of metabolomics showed that there were significant differences in the metabolite composition of embryos at different developmental stages, and that significant metabolic reprogramming occurred during embryonic development.
[0041] Figure 2 The analysis focused on enrichment pathways during the blastocyst stage. The citrate cycle (TCA cycle) and the alanine, aspartate, and glutamate metabolism pathways showed the most significant enrichment during this period. The enrichment of the TCA cycle in the blastocyst stage indicates a shift in the embryo's metabolism from primarily relying on glycolysis and maternal reserves to a mode primarily driven by mitochondrial oxidative phosphorylation. The enrichment of alanine, aspartate, and glutamate metabolism pathways reflects the high degree of coupling between amino acid metabolism and energy metabolism during the blastocyst stage, suggesting that amino acids play a crucial role as substrates in metabolic regulation.
[0042] Figure 3This graph shows the changes in α-ketoglutarate (α-ketoglutarate) levels at different stages of bovine embryos. α-Ketoglutarate is an important intermediate in the citric acid cycle, and its levels are significantly upregulated during the blastocyst stage. This indicates that α-ketoglutarate plays a crucial role in embryonic energy metabolism during the blastocyst period, influencing blastocyst formation. Existing experiments have also demonstrated that adding α-ketoglutarate can improve the blastocyst formation rate in in vitro bovine embryo production. NAAG, as a small molecule polypeptide, is an important small molecule in the metabolic pathways of alanine, aspartic acid, and glutamate, and it also has metabolic linkages with the key intermediate of the citric acid cycle (α-ketoglutarate). It is highly correlated with the aforementioned enrichment metabolic pathways, and its levels also significantly increase during the blastocyst stage, exhibiting a more significant change compared to α-ketoglutarate.
[0043] Metabolomics results showed that the expression level of the small metabolic molecule NAAG was significantly increased during the blastocyst stage. Therefore, it is further speculated that by adding NAAG exogenously to the in vitro embryo culture system, the blastocyst stage embryos can be provided with metabolic support that matches their endogenous metabolic needs, thereby promoting the development of the embryo from the embryonic genome activation period to the blastocyst stage and improving the blastocyst formation rate.
[0044] Example 3 The fertilized eggs obtained according to the in vitro culture steps and screening criteria in Example 1 were grouped as follows: 100 fertilized eggs with the same diameter and uniform dark color were randomly divided into 4 groups of 25 eggs each.
[0045] NC group: Transfer to normal IVC droplets.
[0046] 500 μM NAAG group: Transferred into IVC droplets with a final concentration of 500 μM NAAG. (The concentrated NAAG stock solution was diluted with IVC solution to the corresponding final concentration and made into droplets. This method was used for subsequent groups of different concentrations in Examples 4 and 5).
[0047] 300 μM NAAG group: transferred into IVC droplets with a final concentration of 300 μM NAAG.
[0048] 100 μM NAAG group: transferred into IVC droplets with a final concentration of 100 μM NAAG.
[0049] The embryos were continuously cultured in the same culture medium at 38.5℃, 5% CO2, and 95% humidity until day 8.0, and the blastocyst formation rate was directly measured by photographing under a microscope. Blastocyst formation rate = (number of blastocysts observed on day 8 / total number of embryos that underwent cleavage) × 100%. The method for calculating the blastocyst formation rate is the same in the following examples.
[0050] Figure 5 This describes the formation of a bovine blastocyst as provided in the embodiments of this application. Figure 6 The figure shows a statistical chart of bovine blastocyst formation provided in the embodiments of this application. As shown in the figure, the concentrations of NAAG in these three groups are too high, and they have no significant promoting effect on bovine blastocyst formation.
[0051] Example 4 The fertilized eggs obtained according to the in vitro culture steps and screening criteria in Example 1 were grouped as follows: 100 fertilized eggs of uniform diameter and color were randomly divided into 4 groups of 25 eggs each.
[0052] NC group: Transfer to normal IVC droplets.
[0053] 100 μM NAAG group: transferred into IVC droplets with a final concentration of 100 μM NAAG.
[0054] 10 μM NAAG group: transferred into IVC droplets with a final concentration of 10 μM NAAG.
[0055] 1 μM NAAG group: transferred into IVC droplets with a final concentration of 1 μM NAAG.
[0056] When the embryos were continuously cultured in the same culture medium in an incubator at 38.5℃, 5% CO2, and 95% humidity until day 8.0, the blastocyst formation rate was statistically analyzed by directly photographing the embryos under a microscope.
[0057] Figure 7 This describes the formation of a bovine blastocyst. Figure 8 The figure shows the statistical graph of bovine blastocyst rate. As can be seen from the figure, the experimental groups with a final NAAG concentration of 1 μM and 10 μM in the IVC droplets both significantly promoted the formation of bovine blastocysts, and the experimental group with a final NAAG concentration of 1 μM in the IVC droplets showed a more significant promoting effect. Furthermore, combined with the results in Example 2, it is shown that high concentrations of NAAG inhibit blastocyst formation during the in vitro culture of bovine embryos.
[0058] Example 5 The fertilized eggs obtained according to the in vitro culture steps and screening criteria in Example 1 were grouped as follows: 100 fertilized eggs with the same diameter and uniform dark color were randomly divided into 4 groups of 25 eggs each.
[0059] NC group: Transfer to normal IVC droplets.
[0060] 1 μM NAAG group: transferred into IVC droplets with a final concentration of 1 μM NAAG.
[0061] 100 nM NAAG group: Transferred into IVC droplets with a final concentration of 100 nM NAAG.
[0062] 10 nM NAAG group: Transferred into IVC droplets with a final concentration of 10 nM NAAG.
[0063] The embryos were cultured continuously in the same culture medium in an incubator at 38.5℃, 5% CO2, and 95% humidity until day 8.0, and the blastocyst rate was statistically analyzed by taking pictures directly under a microscope.
[0064] Figure 9 This describes the formation of a bovine blastocyst as provided in the embodiments of this application. Figure 10 The figure shows a statistical chart of bovine blastocyst formation provided in the embodiments of this application. As can be seen from the figure, the promoting effect of lower final concentrations of NAAG on bovine blastocyst formation is not as great as that of NAAG with a final concentration of 1 μM in IVC droplets.
[0065] Figure 11 A summary of bovine blastocyst formation rates for all groups provided in the embodiments of this application is presented. It demonstrates that NAAG at a concentration of 1 μM best enhances the bovine blastocyst formation rate.
[0066] In summary, this invention addresses the key technical problem of low blastocyst formation rate in bovine in vitro embryo production. From the perspective of metabolic regulation during embryonic development, it systematically conducts technical research and application verification. Through metabolomics analysis of bovine in vitro fertilized embryos at three different developmental stages—zygotic, embryonic genome activation (8–16 cell stage), and blastocyst—significant stage-specific metabolic differences were identified, revealing the significantly active characteristics of energy and amino acid metabolism during the blastocyst stage. Based on this, this invention screened and identified the polypeptide N-acetylaspartate-glutamine (NAAG) as a functional small molecule metabolic additive in the in vitro embryo culture system, and systematically and comprehensively verified its application in bovine in vitro embryo culture.
[0067] This invention comprehensively evaluated the effect of NAAG in promoting blastocyst formation during in vitro embryo culture through multiple concentration gradient settings and repeated experiments. The experimental results consistently showed that adding NAAG at a concentration of 1 μM significantly improved the blastocyst formation rate of bovine in vitro fertilized embryos. Furthermore, this promoting effect was stable and reproducible, without adversely affecting the normal development of the embryo, demonstrating the safety and feasibility of NAAG as an embryo culture additive. Simultaneously, the application method of NAAG is highly compatible with existing in vitro embryo culture systems, requiring no changes to the basic culture medium formulation or culture process, making it simple to operate and easy to promote and apply in existing in vitro embryo production systems.
[0068] This invention combines metabolomics analysis results with in vitro culture applications, constructing a complete technical path from metabolic characteristic analysis to functional verification, providing a new technical approach and implementation scheme for optimizing in vitro embryo culture systems. The proposed NAAG application scheme can effectively improve the metabolic support environment for embryos under in vitro culture conditions, increasing embryo development efficiency and blastocyst quality. This has significant practical implications for solving problems such as low blastocyst formation rate and unstable development quality in current bovine in vitro embryo production. This invention can be widely applied to in vitro embryo production and breeding of cattle and other livestock, and has important application value and socio-economic significance for promoting the advancement of livestock genetics and breeding technology, improving the utilization efficiency of high-quality germplasm resources, and promoting the high-quality development of modern animal husbandry.
[0069] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.
Claims
1. The application of a small molecule metabolic additive in promoting bovine in vitro embryonic development, characterized in that, The metabolic small molecule additive is N-acetylaspartic acid-glutamine.
2. The application according to claim 1, characterized in that, The specific application involves adding metabolic small molecule additives in the form of droplets to an in vitro maturation culture medium used for culturing bovine embryos.
3. The application according to claim 2, characterized in that, The final concentration of the metabolic small molecule additive in the in vitro maturation culture medium is 1 μM.
4. The application according to claim 2, characterized in that, The metabolic small molecule additive was added when the bovine in vitro fertilized egg began in vitro culture.
5. The application according to any one of claims 1-4, characterized in that, The promotion of bovine in vitro embryonic development is manifested in increasing the blastocyst formation rate after bovine in vitro fertilization.
6. A culture medium composition for promoting bovine in vitro embryonic development, characterized in that, It includes an in vitro maturation medium and N-acetylaspartic acid-glutamine in the medium at a final concentration of 1 μM.