A bovine embryonic development formula containing hydroxytyrosol and a method of making the same

Abstract

This invention discloses a bovine embryo development formula containing hydroxytyrosol. The formula is used for in vitro bovine embryo development and includes the following ingredients: hydroxytyrosol stock solution, tocopherol acetate stock solution, and L-carnitine stock solution. The invention also discloses its preparation method, including the following steps: S1 Stock solution preparation: Hydroxytyrosol powder is dissolved in dimethyl sulfoxide to prepare a hydroxytyrosol stock solution; tocopherol acetate is dissolved in propylene glycol to prepare a tocopherol acetate stock solution; L-carnitine is weighed and dissolved in deionized water to prepare an L-carnitine stock solution; S2 Working solution preparation: The stock solutions prepared in step S1 are added to the IVC basal culture medium, mixed, and filtered to obtain the bovine embryo development formula. This invention significantly improves the developmental ability of bovine embryos during the maternal-zygotic transition period, effectively promotes the embryo's overcoming of developmental bottlenecks, and improves subsequent blastocyst formation and hatching ability.

Description

Technical Field

[0001] This invention relates to the field of bovine in vitro embryo culture technology, and in particular to a bovine embryo development formula containing hydroxytyrosol and its preparation method. Background Technology

[0002] In vitro embryo production (IVF) technology in cattle is a core method in modern livestock breeding, mainly comprising three stages: in vitro maturation of oocytes (IVM), in vitro fertilization (IVF), and in vitro embryo culture (IVC). The IVC stage is crucial for the development of fertilized eggs into blastocysts. However, the in vitro culture environment presents significant oxidative stress compared to the in vivo uterine environment. Excessive accumulation of reactive oxygen species (ROS) can easily lead to lipid peroxidation, protein oxidation, and DNA damage, resulting in high rates of embryonic developmental arrest, low blastocyst formation rates, and poor embryo quality.

[0003] In the in vitro development of bovine embryos, the major embryonic gene activation (EGA) period, specifically the 8-cell to 16-cell stage, is the main bottleneck period. During this stage, the embryo transitions from maternal gene regulation to zygotic gene regulation, resulting in a dramatic shift in metabolic patterns and making it extremely sensitive to oxidative stress. However, current techniques that directly add antioxidants such as β-mercaptoethanol to the IVC culture medium primarily promote glutathione synthesis through indirect pathways, targeting only a single site and exhibiting some cytotoxicity. Furthermore, strategies involving adding antioxidants to the IVM culture medium or semen diluent, due to their premature intervention or targeting a non-embryo stage, cannot guarantee a direct and sustained protective effect on embryonic development during the EGA window in the IVC stage.

[0004] Hydroxytyrosol (HT) is a natural polyphenolic compound extracted from olives. It has a strong ability to scavenge reactive oxygen species (ROS) and exerts its antioxidant effect by directly scavenging free radicals and activating the Nrf2 signaling pathway. However, the current application of HT in bovine in vitro fertilization (IVP) is limited to the semen preservation stage, where it is added to semen diluents to improve sperm quality. HT is diluted or metabolized before fertilization and cannot provide direct protection for embryonic development during the IVC stage.

[0005] How to provide direct, safe, and multi-dimensional synergistic antioxidant protection for bovine embryos during the IVC stage to improve blastocyst rate and embryo quality has become a pressing technical problem to be solved in this field. Summary of the Invention

[0006] The purpose of this invention is to provide a bovine embryo development formula containing hydroxytyrosol and its preparation method, so as to solve the technical problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A bovine embryo development formula containing hydroxytyrosol, the formula being used for in vitro bovine embryo development, the formula comprising the following ingredients: hydroxytyrosol stock solution, tocopherol acetate stock solution, and L-carnitine stock solution.

[0008] Furthermore, the concentration of the hydroxytyrosol mother liquor is 10 mM; the concentration of the tocopherol acetate is 100 mM; and the concentration of the L-carnitine is 2 M.

[0009] Furthermore, the concentration of hydroxytyrosol in the formulation is 0.1-10 μM; the concentration of tocopherol acetate in the formulation is 100 μM; and the concentration of L-carnitine in the formulation is 1.5 mM.

[0010] Furthermore, the hydroxytyrosol mother liquor is prepared by reacting hydroxytyrosol with dimethyl sulfoxide solvent; the tocopherol acetate mother liquor is prepared by reacting tocopherol acetate with propylene glycol solvent; and the L-carnitine mother liquor is prepared by reacting L-carnitine with deionized water solvent.

[0011] The preparation method of the bovine embryo development formula containing hydroxytyrosol includes the following steps: S1 stock solution preparation: Dissolve hydroxytyrosol powder in dimethyl sulfoxide to prepare hydroxytyrosol stock solution; dissolve tocopherol acetate in propylene glycol to prepare tocopherol acetate stock solution; weigh L-carnitine and dissolve it in deionized water to prepare L-carnitine stock solution; Preparation of working solution S2: Add the stock solution prepared in step S1 to the IVC basic culture medium, mix well, filter and obtain the bovine embryo development formula.

[0012] Furthermore, the IVC basic culture medium is mCR1aa culture medium; the mCR1aa culture medium is divided into early stage medium and late stage medium.

[0013] Furthermore, the preparation method of the mCR1aa culture medium is as follows: weigh 640 mg NaCl, 23 mg KCl, 220 mg NaHCO3, 16.26 mg MgCl2·6H2O, 4.4 mg sodium pyruvate, 27 mg glucose, 200 μL phenol red, 357.46 mg Hepes, and 55 mg calcium galactonate, add the above raw materials to deionized water to dissolve, and then filter to prepare the solution.

[0014] Furthermore, the preparation method of the pre-treatment solution is as follows: take 9.6 mL of mCR1aa base solution, take 60 mg of BSA, 200 μL of EAA (50×), 100 μL of Non-EAA (100×), and 100 μL of glutamine, mix the above raw materials with 9.6 mL of mCR1aa base solution and filter to prepare the solution; the preparation method of the post-treatment solution is as follows: add 10% FBS to the pre-treatment solution.

[0015] The principle of this invention is as follows: The formula is a ternary compound antioxidant system, composed of three active ingredients: hydroxytyrosol (HT), tocopheryl acetate, and L-carnitine.

[0016] HT is a natural polyphenol compound with ROS scavenging activity. This invention demonstrates that adding 1 μM HT to IVC culture medium can reduce the ROS level in 8-cell embryos by 42.1% compared to the control group, suggesting that HT reduces the level of oxidative stress in embryonic cells by directly scavenging excess ROS, thereby reducing the inhibitory effect of oxidative damage on embryonic development.

[0017] Tocopheryl acetate is a stable derivative of vitamin E and a lipid-soluble antioxidant. Its hydrolysis products can be distributed in cell membranes and organelle membranes, maintaining membrane structural integrity by inhibiting lipid peroxidation. In the formulation of this invention, tocopheryl acetate mainly plays a membrane protective role, complementing the water-soluble ROS scavenging function of HT.

[0018] L-carnitine is an important carrier of fatty acid metabolism. It can provide the necessary energy support for embryonic division and differentiation during major EGA by ensuring mitochondrial function.

[0019] Among hydroxytyrosol (HT), tocopherol acetate, and L-carnitine, HT first reduces intracellular ROS levels, weakening the initial damage to membrane structures caused by oxidative stress; tocopherol acetate further protects the integrity of the cell membrane and mitochondrial membrane; and L-carnitine maintains mitochondrial function by stabilizing mitochondrial membrane potential. Together, these three substances improve the developmental microenvironment of the embryo during the maternal-zygotic transition period, promoting the embryo's progress through the 8-cell to 16-cell developmental bottleneck.

[0020] The advantages of this invention compared to the prior art are as follows: This invention significantly improves the developmental capacity of bovine embryos during the maternal-zygotic transition period by directly adding hydroxytyrosol, tocopheryl acetate, and L-carnitine during the IVC stage. It can effectively promote the embryo to overcome the 8-cell to 16-cell developmental bottleneck and improve the subsequent blastocyst formation and hatching capacity. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, preferred embodiments are described below to further illustrate the invention in detail. However, it should be noted that many details listed in the specification are merely to provide the reader with a thorough understanding of one or more aspects of the invention, and these aspects can be achieved even without these specific details.

[0022] 1. Screening for hydroxytyrosol concentration Test method: 1.1 Experimental Animals and Live Oocyte Collection (OPU) The ovarian tissue surrounding healthy bovine ovaries collected from slaughterhouses was removed, and follicular fluid from follicles measuring 2-8 mm was collected using a syringe. Under a microscope, the oocytes were classified into four grades: A, B, C, and D, based on their morphological characteristics. Grade A and B oocytes were selected for experiments.

[0023] The grading criteria are as follows: Grade A oocytes contain 4 or more layers of cumulus cells, which are dense, have uniform cytoplasm, and are dark and shiny; Grade B oocytes contain 1 to 3 layers of cumulus cells, with uniform cytoplasm and dark and shiny cytoplasm; Grade C oocytes are called degenerate oocytes, which include naked oocytes, with loose cumulus cells or heterogeneous cytoplasm.

[0024] 1.2 Oocyte selection and in vitro maturation (IVM) The IVM culture medium consisted of: TCM199 (1×), 10% FBS (v / v), 0.01 IU / mL FSH, 0.01 IU / mL LHL, 1 μg / mL 17β-E2, 0.2 mM sodium pyruvate, 50 μg / mL gentamicin, and 1% penicillin-streptomycin (v / v).

[0025] Prepare 100 μL droplets of IVM culture medium in a 35 mm culture dish, cover with mineral oil, and equilibrate for 4 hours in a 38.5°C, 5% CO2, saturated humidity incubator. Add 15-20 oocytes to each droplet and incubate for 24 hours.

[0026] 1.3 In Vitro Fertilization (IVF) Commercially available frozen semen was used and thawed in a 38°C water bath for 45 seconds. After thawing, the semen was washed twice with BO buffer, centrifuged at 1500 rpm for 8 minutes each time, the supernatant was discarded, and BO capacitation solution was added to adjust the sperm density to 1 × 10⁻⁶. 6 Cells / mL were placed in a 38.5℃, 5% CO2 incubator for 2 hours to acquire energy.

[0027] After IVM, mature oocytes are digested with 0.1% hyaluronidase to remove cumulus cells, and MII stage oocytes that have expelled the first polar body are selected for fertilization.

[0028] Prepare 50 μL fertilization microdroplets in a 35 mm culture dish, cover with mineral oil, add 15-20 oocytes to each droplet, and add 50 μL of capacitated sperm suspension to achieve a final sperm concentration of 1 × 10⁻⁶. 6 The number of fertilized eggs per mL was determined. 16-18 hours after fertilization, pronucleus formation was observed under a stereomicroscope to confirm that the fertilized eggs were used for subsequent embryo culture.

[0029] 1.4 Preparation of Mother Liquor Weigh 1.54 mg of HT powder, dissolve it in 1 mL of chromatographic grade DMSO, vortex for 30 s, and sonicate for 5 min to prepare a 10 mM stock solution.

[0030] 1.5 Embryo in vitro culture (IVC) and grouping treatment IVC culture medium uses the mCR1aa culture system, which is divided into early stage medium and late stage medium.

[0031] mCR1aa basic solution (100mL): Weigh 640 mg NaCl, 23 mg KCl, 220 mg NaHCO3, 16.26 mg MgCl2·6H2O, 4.4 mg sodium pyruvate, 27 mg glucose, 200 μL phenol red (1% stock solution), 357.46 mg Hepes, and 55 mg calcium galactonate. Dissolve the above raw materials in deionized water and bring the volume to 100 mL. Filter the solution through a 0.22 μm filter for sterilization.

[0032] Early stage solution (Day 1~2): Take 9.6 mL of mCR1aa base solution, add 60 mg of BSA, 200 μL of EAA (50×), 100 μL of Non-EAA (100×), and 100 μL of glutamine. Mix and dissolve the above raw materials with the mCR1aa base solution, and then filter and sterilize using a 0.22 μm filter to prepare the solution.

[0033] Later stage solution (Day 3~7): FBS is added to the initial solution to make the final FBS concentration 10% (v / v), mixed well and then filtered through a 0.22μm filter to sterilize.

[0034] Fertilized eggs were randomly divided into 5 groups, with 60 fertilized eggs in each group. Each replicate consisted of 60 fertilized eggs, for a total of 3 independent replicates, totaling 180 fertilized eggs. The grouping details are as follows: Control group (Con): Add 10 μL DMSO and 10 μL propylene glycol to 10 mL of basal IVC culture medium, mix well and filter. Final solvent concentration: DMSO 0.1%, propylene glycol 0.1%.

[0035] HT-1 group: Take 0.1 μL of HT stock solution and add it to 10 mL of basic IVC culture medium, then add 9.9 μL of DMSO and 10 μL of propylene glycol, mix well and filter to make the HT concentration 0.1 μM.

[0036] HT-2 group: Take 1 μL of HT stock solution and add it to 10 mL of basic IVC culture medium, then add 9 μL of DMSO and 10 μL of propylene glycol, mix well and filter to make the HT concentration 1 μM.

[0037] HT-3 group: Take 5 μL of HT stock solution and add it to 10 mL of basic IVC culture medium, then add 5 μL of DMSO and 10 μL of propylene glycol, mix well and filter to make the HT concentration 5 μM.

[0038] HT-4 group: Take 10 μL of HT stock solution and add it to 10 mL of basic IVC culture medium, add 10 μL of propylene glycol, mix well and filter to make the HT concentration 10 μM.

[0039] The final concentrations of DMSO and propylene glycol solvents were strictly kept consistent across all treatment groups to rule out any potential effects of the solvents themselves on embryonic development.

[0040] On the first day after fertilization, i.e., the day the fertilized eggs are transferred into the IVC medium, the process begins. The fertilized eggs are transferred into the pre-equilibrated IVC medium for 4 hours. 50 μL droplets of pre-IVC medium are prepared in 35 mm culture dishes, covered with mineral oil, and 15-20 fertilized eggs are placed in each droplet. The dishes are then incubated in a tri-gas incubator at 38.5℃, 5% CO2, 5% O2, 90% N2, and saturated humidity. On the third day of culture (48-72 hours after fertilization), the embryos are transferred into the corresponding late-stage medium for each group, and thereafter, half the medium is changed every 48 hours.

[0041] 1.6 Embryonic Development On the second day of culture, the cleavage was observed and recorded under an inverted microscope, and the cleavage rate was calculated as (number of embryos ≥2-cell / total number of fertilized eggs cultured × 100%).

[0042] On the seventh day of culture, the embryonic development stage was assessed under a stereomicroscope according to the International Embryo Transfer Society (IETS) standards, and the blastocyst rate was calculated.

[0043] The criteria for blastocyst identification are: the formation of a distinct blastocoel, and the identifiable inner cell mass and trophoblast cells. The ratio of expanded blastocysts (blastocoel significantly enlarged, larger in volume than the unexpanded stage) and hatching blastocysts (trophoblast cells beginning to emerge from the zona pellucida) is also recorded.

[0044] Statistical analysis was performed using SPSS 20.0 software. Data are expressed as mean ± standard deviation. One-way ANOVA was used for comparisons between groups, and Duncan's method was used for multiple comparisons. P < 0.05 was considered statistically significant. The results are shown in Table 1.

[0045] Table 1. Effects of different concentrations of hydroxytyrosol on embryonic development Note: Different lowercase letters in the same column indicate significant differences (P<<0.05), while the same or no letters indicate no significant differences (P>0.05).

[0046] As shown in Table 1, the cleavage rate in the control group was 73.3±4.5%, while the cleavage rates in the HT-1, HT-2, HT-3, and HT-4 groups were 74.5±5.0%, 76.2±4.3%, 73.8±5.2%, and 71.5±5.8%, respectively, with no significant difference between the groups (P>0.05). This indicates that the intervention target of HT is the maternal-zygotic transition period (major EGA). The cleavage rate was not significantly affected, ruling out the direct toxicity or mitotic-promoting effect of HT on early mitosis of fertilized eggs, demonstrating that its effect is stage-specific.

[0047] Regarding blastocyst rates, the control group had a blastocyst rate of 28.3±4.2%, while the HT-1 group had a blastocyst rate of 31.7±4.8%, slightly higher than the control group but not statistically significant (P>0.05). The HT-2 group had a blastocyst rate of 40.5±3.9%, significantly higher than the other groups (P<0.05). The HT-3 group had a blastocyst rate of 34.2±4.5%, lower than the HT-2 group, but still significantly higher than the control group (P<0.05). The HT-4 group had a blastocyst rate of 25.8±5.0%, slightly lower than the control group, but the difference was not significant (P>0.05).

[0048] Based on the experimental data above, the optimal concentration of hydroxytyrosol is 1 μM, and this concentration will be used in subsequent experiments.

[0049] 2. Effects of formulation components on bovine embryonic development 2.1 Preparation of Mother Liquor HT stock solution: Weigh 1.54 mg of HT powder, dissolve it in 1 mL of DMSO, vortex for 30 seconds, and sonicate for 5 minutes to prepare a 10 mM stock solution.

[0050] Tocopherol acetate stock solution: Weigh 47.5 mg of tocopherol acetate, dissolve it in 1 mL of propylene glycol, vortex to mix, and prepare a 100 mM stock solution.

[0051] L-Carnitine stock solution: Weigh 322.4 mg of L-carnitine, dissolve it in 1 mL of ultrapure water, vortex to dissolve, filter through a 0.22 μm filter to remove bacteria, and prepare a 2 M stock solution.

[0052] 2.2 Experimental Grouping Fertilized eggs were obtained according to methods 1.1-1.3 and divided into 8 groups of 60 fertilized eggs each. Each replicate consisted of 60 fertilized eggs and was performed three times independently. The grouping is as follows: CON group: Add 10 μL DMSO and 10 μL propylene glycol to both the early and late phases of 10 mL IVC culture medium, vortex to mix, filter through a 0.22 μm filter, and equilibrate at 4 °C for 2 h.

[0053] HT group: Add 1 μL of HT stock solution + 9 μL of LDMSO + 10 μL of propylene glycol to both the early and late stages of 10 mL of IVC culture medium, vortex mix, filter through a 0.22 μm filter, and equilibrate at 4℃ for 2 h to make the final HT concentration 1 μM.

[0054] VitE group: 10 μL of tocopherol acetate stock solution, 10 μL of LDMSO and 2.5 μL of propylene glycol were added to the early and late phases of 10 mL IVC culture medium. The mixture was filtered through a 0.22 μm filter and equilibrated at 4 °C for 2 h to achieve a final concentration of 100 μM for tocopherol acetate.

[0055] Carn group: 7.5 μL L-carnitine stock solution, 10 μL LDMSO, and 10 μL propylene glycol were added to both the early and late phases of 10 mL LVC culture medium. The mixture was filtered through a 0.22 μm filter and equilibrated at 4 °C for 2 h to achieve a final L-carnitine concentration of 1.5 mM.

[0056] HT+VitE group: 1 μL of HT stock solution, 10 μL of tocopherol acetate stock solution, 9 μL of LDMSO, and 2.5 μL of propylene glycol were added to the early and late stages of 10 mL of IVC culture medium. The mixture was filtered through a 0.22 μm filter and equilibrated at 4 °C for 2 h to achieve a final HT concentration of 1 μM and a final tocopherol acetate concentration of 100 μM.

[0057] HT+Carn group: 1 μL HT stock solution + 7.5 μL L-carnitine stock solution + 9 μL LDMSO + 10 μL propylene glycol were added to the early and late phases of 10 mL IVC culture medium. The mixture was filtered through a 0.22 μm filter and equilibrated at 4℃ for 2 h to achieve a final HT concentration of 1 μM and a final L-carnitine concentration of 1.5 mM.

[0058] The VitE+Carn group: 10 μL of tocopherol acetate stock solution + 7.5 μL of L-carnitine stock solution + 10 μL of LDMSO + 2.5 μL of propylene glycol were added to both the early and late stages of 10 mL IVC culture medium. The mixture was filtered through a 0.22 μm filter and equilibrated at 4℃ for 2 h to achieve a final concentration of 100 μM for tocopherol acetate and 1.5 mM for L-carnitine.

[0059] HTC group: 1 μL of HT stock solution, 10 μL of tocopheryl acetate stock solution, 7.5 μL of L-carnitine stock solution, 9 μL of LDMSO, and 2.5 μL of propylene glycol were added to both the early and late stages of 10 mL IVC culture medium. The mixture was filtered through a 0.22 μm filter and equilibrated at 4℃ for 2 h to achieve a final concentration of 1 μM for HT, 100 μM for tocopheryl acetate, and 1.5 mM for L-carnitine.

[0060] By adding the appropriate solvents, the final concentrations of DMSO and propylene glycol in all groups were made to be 0.1% (v / v), thus eliminating the influence of the solvents.

[0061] The IVC procedure is the same as in 1.5. Fertilized eggs are transferred to the pre-equilibrated early-stage medium for each group (equilibrated for 4 hours). 50 μL droplets of early-stage IVC medium are prepared in 35 mm culture dishes, covered with mineral oil. Each droplet contains 15-20 fertilized eggs. The dishes are then incubated in a tri-gas incubator at 38.5℃, 5% CO2, 5% O2, 90% N2, and saturated humidity. On day 3 of culture, the embryos are transferred to the corresponding late-stage medium for each group. The medium is then changed at half volume every 48 hours.

[0062] Day 2 of culture: Observe under an inverted microscope and count the cleavage rate.

[0063] Day 3 of culture: Observe each embryo individually under a 200x inverted microscope, and determine the developmental stage based on the number and morphology of blastomeres. Statistically determine the proportion of embryos that have developed to the 8-cell stage and the 9- to 16-cell stage. The 8-cell-16-cell success rate is defined as the percentage of embryos that have developed to the ≥8-cell stage out of the total number of fertilized eggs.

[0064] On day 7 of culture, the following indicators were assessed and recorded under a stereomicroscope: Blastocyst rate: The percentage of embryos that have developed to the blastocyst stage (including early blastocysts, expanded blastocysts, and hatching blastocysts) out of the total number of fertilized eggs.

[0065] Expanded blastocyst rate: The percentage of blastocysts whose blastocoel volume exceeds 50% of the total embryo volume out of the total number of fertilized eggs.

[0066] Blastocyst hatching rate: The percentage of blastocysts that have ruptured the zona pellucida and begun to emerge from the trophoblast cells out of the total number of fertilized eggs.

[0067] Blastocyst total cell count detection: On day 7 of culture, blastocysts from each group were washed three times in PBS for 5 minutes each time. They were then transferred to PBS containing 10 μg / mL Hoechst 33342 and incubated at room temperature in the dark for 15 minutes. Subsequently, they were washed three times with PBS for 5 minutes each time. The blastocysts were placed on a glass slide, covered with a coverslip, and observed under a fluorescence microscope using a DAPI filter (excitation wavelength 350–370 nm, emission wavelength 460–490 nm). Images of each blastocyst were taken at least three times from each focal plane, and cell nucleus counting was performed using ImageJ software.

[0068] Statistical analysis was performed using SPSS 20.0 software, and data are expressed as mean ± standard deviation. One-way ANOVA was used to compare cleavage rate, 8-cell-16-cell success rate, blastocyst rate, expanded blastocyst rate, and hatched blastocyst rate among groups, followed by Duncan's method for multiple comparisons. One-way ANOVA was also used to compare the total number of cells in blastocysts among groups, followed by Duncan's method for multiple comparisons. P < 0.05 indicated statistical significance, and P < 0.01 indicated highly statistical significance. The results are shown in Table 2.

[0069] Table 2 Effects of formulation components on bovine embryonic development Note: Different lowercase letters in the same column indicate significant differences (P<<0.05), while the same or no letters indicate no significant differences (P>0.05).

[0070] As can be seen from Table 2: Regarding cleavage rate, the cleavage rate ranged from 73.0±4.5% to 76.8±3.5%, and the difference between groups was not statistically significant, indicating that neither single component nor compound system had a significant effect on early embryonic cleavage events.

[0071] Regarding the 8-cell to 16-cell pass rates, significant differences in the effect gradient were observed among the treatment groups. The pass rate in the Con group was 47.5±5.5%; the pass rates in the HT group, VitE group, and Carn group were 58.0±4.8%, 55.0±5.0%, and 56.5±4.6%, respectively, all showing some improvement compared to the Con group. The HT group showed the largest improvement, indicating that hydroxytyrosol played a core driving role. Among the two-component groups, the pass rates in the HT+VitE group were 65.0±4.2%, the HT+Carn group was 67.0±4.0%, and the VitE+Carn group was 63.0±4.3%, all significantly higher than the single-component groups. The 8-cell to 16-cell pass rate in the HTC group was as high as 76.5±3.5%, an improvement of 61.1% compared to the Con group, and also higher than other single-component and two-component groups, with significant differences (p<0.005).

[0072] Regarding blastocyst rates, the Con group had a blastocyst rate of 27.5±4.0%, while the HT, Vitamin E, and Carn groups had rates of 39.0±3.5%, 33.0±3.8%, and 34.0±3.6%, respectively. The blastocyst rates of the two-component groups ranged from 40.0±3.4% to 46.0±3.0%. Notably, the HT+VitE and HT+Carn groups, which contained hydroxytyrosol, had higher blastocyst rates than the Vitamin E+Carn groups, demonstrating the crucial role of hydroxytyrosol. The HTC group achieved a blastocyst rate of 54.0±2.8%, significantly higher than the other single-group and two-group groups (p<0.005).

[0073] In terms of blastocyst expansion rate and hatching blastocyst rate, the HTC group had an expansion blastocyst rate of 40.0±2.5% and a hatching blastocyst rate of 24.0±2.0%, both significantly higher than all experimental groups. Furthermore, the total number of cells in the blastocysts of the HTC group reached 145±10, which was 64.8% higher than the Con group and 34.3% higher than the HT group, demonstrating that the formula can improve the probability of blastocyst formation while also significantly optimizing the intrinsic quality of the blastocysts.

[0074] Based on the above data analysis, hydroxytyrosol, tocopheryl acetate, and L-carnitine all have significant positive regulatory effects on indicators of bovine embryonic development at various stages, and this effect shows a clear component-dependent synergistic enhancement characteristic.

[0075] 3. Effects of the formulation on embryonic antioxidant capacity and mitochondrial function 3.1 Experimental Grouping The HTC group and the Con group were compared using the methods described in 2.2. Fertilized eggs were cultured according to the methods in 1.1-1.3, and the IVC procedure was the same as in 1.5.

[0076] 3.2 Detection indicators and methods.

[0077] On day 3 of culture, embryos developed to the 8-cell to 16-cell stage were carefully aspirated using a micropipette under an inverted microscope. The embryos were transferred to PBS preheated to 38.5°C and washed three times for 5 minutes each time to remove residual culture medium. The Con group and HTC group from Experiment 2.2 were selected for comparison. At least 20 embryos at the target stage were randomly selected from each group, with at least 20 embryos per replicate, for a total of three independent replicates.

[0078] Intracellular ROS level detection: Day 3 (8-cell stage) embryos were sampled. Embryos were placed in PBS-PVA solution containing 10 μM DCFH-DA and incubated at 38.5℃ in the dark for 20 min. After washing three times with PBS-PVA, the embryos were placed on slides and observed for green fluorescence (excitation wavelength 488 nm, emission wavelength 525 nm) under a fluorescence microscope. All images were acquired using fixed exposure time and gain parameters, and background fluorescence subtraction was performed. At least three focal plane images were captured for each embryo, and relative fluorescence intensity was analyzed using Image-ProPlus software.

[0079] Mitochondrial membrane potential (MMP) detection: JC-1 probe method. Embryos were incubated with JC-1 working solution (5 μg / mL) at 38.5℃ in the dark for 20 min. After washing three times with PBS-PVA, the embryos were observed under a fluorescence microscope: red fluorescence represented J-aggregates (highly polarized mitochondria, normal membrane potential, excitation wavelength 540 nm, emission wavelength 590 nm), and green fluorescence represented JC-1 monomers (lowly polarized mitochondria, depolarized membrane potential, excitation wavelength 485 nm, emission wavelength 535 nm). All images were acquired using fixed exposure time and gain parameters, and background fluorescence was subtracted. At least three focal plane images were taken for each embryo, and the red / green fluorescence intensity ratio was analyzed using Image-ProPlus software. A decreased ratio indicates mitochondrial dysfunction.

[0080] Statistical analysis was performed using SPSS 20.0 software. Data are expressed as mean ± standard deviation. Independent samples t-tests were used to compare two groups. P < 0.05 was considered statistically significant, and P < 0.01 was considered extremely significant. The results are shown in Table 3.

[0081] Table 3. Effects of the formulation on embryonic antioxidant capacity and mitochondrial function As shown in Table 3, in the ROS detection, the average fluorescence intensity of embryos in the Con group was 45.8±7.2 AU, while the average fluorescence intensity of embryos in the HTC group decreased to 26.5±4.8 AU, which was 42.1% lower than that of the control group. The difference was extremely significant (P<0.01), indicating that the amount of reactive oxygen species accumulated in the cells of embryos in the HTC group was significantly reduced, and the level of oxidative stress was effectively alleviated.

[0082] In the mitochondrial membrane potential detection, the JC-1 red-green fluorescence ratio of embryos in the Con group was 1.78±0.24, while that in the HTC group was 3.45±0.32, which was significantly higher than that in the control group (P<0.01). The mitochondrial membrane potential stability of embryos in the HTC group was significantly better than that in the control group, and the mitochondrial functional integrity was significantly improved.

[0083] The above results indicate that hydroxytyrosol, tocopheryl acetate, and L-carnitine can simultaneously reduce the level of reactive oxygen species in embryonic cells and significantly improve the stability of mitochondrial membrane potential during the 8-cell to 16-cell stages. This improvement in cellular levels is consistent with the developmental outcomes observed in Experiment 2, where the HTC group showed significantly higher 8-cell to 16-cell stage success rate, blastocyst rate, and total number of blastocyst cells compared to the control group. This suggests that the relief of oxidative stress and the maintenance of mitochondrial function are important cellular biological bases for this formula to promote embryonic development over the 8-cell to 16-cell stage bottleneck.

[0084] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A bovine embryo development formula containing hydroxytyrosol, characterized in that, The formula is used for in vitro development of bovine embryos and includes the following ingredients: hydroxytyrosol mother liquor, tocopherol acetate mother liquor, and L-carnitine mother liquor.

2. The bovine embryo development formula containing hydroxytyrosol according to claim 1, characterized in that: The concentration of the hydroxytyrosol mother liquor is 10 mM; the concentration of the tocopherol acetate is 100 mM; and the concentration of the L-carnitine is 2 M.

3. The bovine embryonic development formula containing hydroxytyrosol according to claim 1, characterized in that: The concentration of hydroxytyrosol in the formulation is 0.1-10 μM; the concentration of tocopherol acetate in the formulation is 100 μM; and the concentration of L-carnitine in the formulation is 1.5 mM.

4. The bovine embryonic development formula containing hydroxytyrosol according to claim 1, characterized in that: The hydroxytyrosol mother liquor is prepared by mixing hydroxytyrosol with dimethyl sulfoxide solvent; the tocopherol acetate mother liquor is prepared by mixing tocopherol acetate with propylene glycol solvent; and the L-carnitine mother liquor is prepared by mixing L-carnitine with deionized water solvent.

5. The method for preparing the bovine embryo development formulation containing hydroxytyrosol according to any one of claims 1-4, characterized in that, Includes the following steps: S1 stock solution preparation: Dissolve hydroxytyrosol powder in dimethyl sulfoxide to prepare hydroxytyrosol stock solution; dissolve tocopherol acetate in propylene glycol to prepare tocopherol acetate stock solution; weigh L-carnitine and dissolve it in deionized water to prepare L-carnitine stock solution; Preparation of working solution S2: Add the stock solution prepared in step S1 to the IVC basic culture medium, mix well, filter and obtain the bovine embryo development formula.

6. The method for preparing the bovine embryo development formulation containing hydroxytyrosol according to claim 5, characterized in that: The IVC basic culture medium is mCR1aa culture medium; the mCR1aa culture medium is divided into early stage medium and late stage medium.

7. The method for preparing the bovine embryonic development formula containing hydroxytyrosol according to claim 6, characterized in that: The preparation method of the mCR1aa culture medium is as follows: Weigh 640mg NaCl, 23mg KCl, 220mg NaHCO3, 16.26mg MgCl2·6H2O, 4.4mg sodium pyruvate, 27mg glucose, 200μL phenol red, 357.46mg Hepes, and 55mg calcium galactonate. Dissolve the above raw materials in deionized water and filter to prepare the solution.

8. The method for preparing the bovine embryonic development formula containing hydroxytyrosol according to claim 7, characterized in that: The preparation method of the pre-treatment solution is as follows: take 9.6 mL of mCR1aa base solution, take 60 mg of BSA, 200 μL of EAA (50×), 100 μL of Non-EAA (100×), and 100 μL of glutamine, mix the above raw materials with 9.6 mL of mCR1aa base solution and filter to prepare the solution; the preparation method of the post-treatment solution is as follows: add 10% FBS to the pre-treatment solution.

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