Regulation method for improving superovulation efficiency of huaxi cattle by creatine treatment and application

Abstract

This invention discloses a method and application for regulating and improving superovulation efficiency in West China cattle using creatine treatment, belonging to the field of animal reproductive technology in animal husbandry. During the superovulation treatment of West China cattle, exogenous creatine (500-1000 mg / head) is administered to the donor cow via intramuscular or subcutaneous administration, simultaneously with follicle-stimulating hormone (FSH) administered twice daily, morning and evening. Creatine, as a natural energy reserve and buffer substance, reaches the ovarian tissue through blood circulation and is actively taken up by follicular cells, converted into phosphocreatine, maintaining a stable intracellular ATP / ADP ratio, improving the local energy metabolism microenvironment of the follicles, and achieving a comprehensive improvement in the number and quality of ovulation, estrus rate, and embryo formation rate after superovulation. This invention opens up a new technical path for improving the efficiency of superovulation in cattle based on energy metabolism regulation, and is simple to operate and highly compatible with existing superovulation processes.

Description

Technical Field

[0001] This invention belongs to the field of animal reproduction technology in animal husbandry, and in particular relates to a method and application of creatine treatment to improve the superovulation efficiency of West China cattle. Background Technology

[0002] As an important economic livestock species, the large-scale and efficient breeding of bovines is of great significance for ensuring meat supply and promoting the development of animal husbandry. However, the natural reproductive system of cattle has significant biological limitations: firstly, the singleton pregnancy rate is as high as 99%, while the twin pregnancy rate is less than 1%; secondly, the gestation period is as long as 280-290 days; and thirdly, the postpartum recovery period for cows is 40-60 days. These biological characteristics severely restrict the efficiency of breeding and propagation of superior breeds, and traditional breeding models can no longer meet the urgent needs of modern animal husbandry for high-quality breeding stock.

[0003] Superovulation, a cutting-edge technology in modern animal reproduction, provides an innovative solution for cattle breeding improvement. Superovulation involves artificially administering exogenous gonadotropins during specific stages of the cow's estrous cycle to stimulate ovarian development and the release of more follicles than naturally occurring. The resulting oocytes can be used for mating, artificial insemination, early embryo culture, or embryo transfer. This technology breaks down seasonal and geographical limitations, fully tapping into the reproductive potential of cows and significantly improving reproductive efficiency.

[0004] Currently, the mainstream approach to superovulation technology involves first stimulating follicle growth and development with follicle-stimulating hormone (FSH), followed by inducing ovulation with human chorionic gonadotropin-releasing hormone (hCG), luteinizing hormone (LH), or gonadotropin-releasing hormone analogs to obtain more eggs. Although this technology has been widely applied in livestock production and scientific research, several technical bottlenecks remain to be addressed: the number of high-quality embryos available for transfer is insufficient in practice; the use of gonadotropins may damage the maternal uterus or embryos; and while the number of ovulations increases, embryo viability is low, severely affecting embryo transfer success rates and subsequent pregnancy outcomes, thus limiting the further promotion and application of superovulation technology in bovine reproduction. Summary of the Invention

[0005] To overcome the above shortcomings, this invention provides a method and application for regulating and improving the efficiency of superovulation in West China cattle using creatine treatment. By simultaneously supplementing with exogenous creatine during the superovulation hormone administration cycle, the ovarian energy metabolism is effectively improved, thereby increasing the quantity and quality of oocytes, estrus rate, and embryo formation rate after superovulation in West China cattle. This opens up a new path for improving the efficiency of bovine superovulation technology, as detailed below: A method for regulating creatine treatment to improve superovulation efficiency in West China cattle involves administering exogenous creatine to donor cows during superovulation treatment to improve at least one of the following indicators: oocyte count, ovulation quality, estrus rate, and embryo formation rate after superovulation.

[0006] Preferably, the superovulation treatment is as follows: vaginal progesterone suppositories are inserted on any day of the donor cow's estrous cycle, and follicle-stimulating hormone is administered daily from day 9 to day 12 after suppository insertion.

[0007] Preferably, the creatine is administered via intramuscular or subcutaneous injection.

[0008] Preferably, the creatine administration regimen is as follows: creatine is administered continuously from day 9 to day 12 after vaginal progesterone suppositories are placed in the donor cow (starting from day 9 and continuing until day 12), twice daily, with each administration of creatine at a dose of 500-1000 mg / head (preferably 1000 mg / head).

[0009] Preferably, from day 9 to day 12, creatine and follicle-stimulating hormone are administered once in the morning and once in the evening, with a 12-hour interval between the two administrations, and creatine and follicle-stimulating hormone are administered simultaneously with each administration.

[0010] Preferably, the total dose of follicle-stimulating hormone used is 500-700 IU / head (specifically: Day 1: 90-125 IU / time / head, Day 2: 70-100 IU / time / head, Day 3: 60-75 IU / time / head, Day 4: 30-50 IU / time / head), and the creatine and follicle-stimulating hormone are administered twice a day, morning and evening, at 12-hour intervals.

[0011] Preferably, the regulation method further includes: after the last administration of creatine, administering prostaglandin to the donor cow to induce estrus, and performing artificial insemination after estrus, wherein the prostaglandin is cloprostenol, and the dosage is 0.5-0.7 mg / head.

[0012] Preferably, the Huaxi cattle are multiparous cows with 2-4 parities.

[0013] On the other hand, the present invention discloses the use of creatine in the preparation of a drug for improving the superovulation efficiency of West China cattle, wherein the drug is used in combination with follicle-stimulating hormone, vaginal progesterone suppositories and / or prostaglandins.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention effectively improves the energy metabolism environment for follicle development by simultaneously applying exogenous creatine during superovulation. Experimental data show that the proportion of high-quality embryos in the control group is only 4.48%, while the CR8 group of this invention significantly increases the rate of high-quality embryos to 14.13%, overcoming the technical bottleneck of increasing the number of ovulations but having low embryo viability in the prior art.

[0015] 2. In existing superovulation techniques, a high percentage of medium / poor quality embryos are formed after the development of a large number of follicles, which seriously affects the success rate of implantation. This invention supplements energy metabolism substrates with creatine, which significantly reduces the percentage of medium / poor quality embryos, with the CR8 group reduced to 64.13%, effectively reducing the number of embryos with poor developmental potential.

[0016] 3. Excessive use of gonadotropins in the prior art may damage oocytes or early embryos, leading to an increased proportion of degenerated or unfertilized eggs. The proportion of invalid embryos in the CR8 group of this invention is only 21.73%, avoiding the risk of embryo degeneration caused by overstimulation, and demonstrating the technical characteristics of balance and high efficiency.

[0017] 4. While focusing on increasing the number of ovulations, this invention also emphasizes the comprehensive improvement of ovulation quality and subsequent embryo formation rate. Experimental data show that the total number of embryos in the creatine treatment group increased from 67 in the control group to 92 in the CR8 group, while the rate of high-quality embryos also increased, achieving dual optimization of quantity and quality.

[0018] 5. Existing superovulation techniques mainly rely on the dosage and timing regulation of exogenous gonadotropins. This invention introduces creatine regulation into the field of superovulation for the first time. Creatine is a naturally occurring nitrogenous organic acid in organisms, serving as an energy reserve and buffer. Under the catalysis of creatine kinase, it interconverts with phosphocreatine. When cellular ATP is sufficient, it stores phosphate groups; when ATP is rapidly consumed, it quickly regenerates ATP, thereby maintaining a stable intracellular ATP / ADP ratio. During the superovulation treatment of West China cattle, exogenous creatine is simultaneously applied, allowing it to reach the ovarian tissue through blood circulation. After being actively taken up by follicular cells, it is converted into phosphocreatine, increasing the local energy reserve level of the follicles. Specifically... Its effects include: promoting high-quality follicle development, enhancing the sensitivity of granulosa cells to follicle-stimulating hormone (FSH), and increasing the number of effective ovulations; improving oocyte quality, preventing sudden ATP depletion during meiosis, reducing the risk of chromosomal abnormalities, and increasing the embryo formation rate and high-quality embryo rate after fertilization; mitigating oxidative stress damage, maintaining mitochondrial ATP synthesis efficiency, and protecting oocytes and early embryos; supporting the developmental potential of early embryos, providing sufficient energy substrates for fertilized eggs, and promoting blastocyst formation; and synergistic effects with FSH to address the problems of insufficient energy supply for follicle development, poor oocyte maturation quality, and low early embryo viability in existing superovulation techniques through energy metabolism regulation.

[0019] 6. This invention, through serum non-targeted metabolomics analysis, confirms that serum creatinine levels significantly increased after creatine intervention, indicating that the creatine-phosphocreatine system was active and ATP regeneration was accelerated. Differential metabolites between the mating period and the embryo flushing period were significantly enriched in glycerophospholipid metabolism, linoleic acid metabolism, arachidonic acid metabolism, and cAMP signaling pathway, verifying the molecular basis for creatine to improve the follicular development microenvironment and enhance embryo quality, and providing clear metabolic mechanism support for the method of this invention. Attached Figure Description

[0020] Figure 1 To illustrate the effects of different creatine treatment groups on estrus rate and ovarian response in West China cattle, A represents a comparison of estrus rates among different treatment groups, B represents a comparison of the average number of corpora lutea among different treatment groups, and C represents a comparison of the average number of follicles among different treatment groups. Figure 2 The effect of different creatine treatment groups on the number of donor embryos from West China cattle is shown in Figure 1. A represents the average total number of embryos, B represents the number of untransferable embryos, C represents the number of transferable embryos, D represents the transferable embryo rate, and E represents the untransferable embryo rate. Figure 3 The effect of different creatine treatment groups on the distribution of embryo grades in West China cattle donors: A represents the number distribution of embryos of different quality grades, B represents the rate of high-quality embryos, and C represents the rate of medium / low-quality embryos. Figure 4 For the comparison of serum reproductive hormone concentrations in West China cattle under different treatment groups, A represents the change in anti-Müllerian hormone concentration, and B represents the change in progesterone concentration. Figure 5 A comparison of the morphology of some embryos in the control group and the CR8 group; Figure 6 A complete data processing workflow for metabolite identification, differential screening, and functional analysis; Figure 7 The results of multivariate statistical analysis of serum metabolic profiles after creatine intervention are shown. In this figure, A is the PLS-DA score plot, B is the result of the permutation test, and C is the result of the quality control analysis. Figure 8 The graphs show the statistical distribution of differential metabolites, where A is a bar chart of differential metabolites and B is a Venn diagram of differential metabolites. Figure 9 This refers to the dynamic changes in creatinine levels during superovulation. Figure 10 These are heatmaps and volcano maps of differential metabolites at different stages. Among them, A is a heatmap of differential metabolites during the mating period, B is a heatmap of differential metabolites during the embryonic flushing period, C is a volcano map of differential metabolites during the mating period, and D is a volcano map of differential metabolites during the embryonic flushing period. Figure 11 Results of KEGG metabolic pathway enrichment analysis during the mating season; Figure 12 The results are from the KEGG metabolic pathway enrichment analysis during the embryo flushing period. Detailed Implementation

[0021] The donor cows used in the following examples meet the following conditions: Variety and Appearance: Excellent variety, meets the evaluation criteria for variety appearance, and has good production performance.

[0022] Standard weight: 450-650 kg. A suitable weight range helps ensure that the cow is in good physical condition and can better cope with superovulation and subsequent pregnancy.

[0023] Genetic background: Stable genetic performance and clear pedigree. A clear genetic background helps ensure the superior traits of offspring, meeting the needs of propagating superior varieties.

[0024] Health and reproductive status: No history of miscarriage, no pregnancy for more than 90 days postpartum, at least two normal estrous cycles, normal reproductive organs and reproductive function, and no genetic or infectious diseases. Good health and reproductive status are the foundation for ensuring successful superovulation and subsequent successful pregnancy.

[0025] Example 1 Vaginal progesterone suppositories were inserted on any day of the donor cow's estrous cycle. Creatine and follicle-stimulating hormone (FSH) were injected daily from day 9 to 12 after suppository insertion to promote superovulation. The dosage of creatine was 750 mg / dose / head, and the total dose of FSH was 500 IU / head (Day 1: 90 IU / dose / head, Day 2: 70 IU / dose / head, Day 3: 60 IU / dose / head, Day 4: 30 IU / dose / head). Creatine and FSH were injected twice daily, morning and evening, 12 hours apart, with both creatine and FSH administered simultaneously with each injection. By precisely controlling the timing, dosage, and injection interval of creatine and FSH, their synergistic effect promoted the full development and maturation of follicles, increased the number of ovulations, and improved the quality of the released eggs.

[0026] Example 2 Vaginal progesterone suppositories were inserted on any day of the donor cow's estrous cycle. Creatine and follicle-stimulating hormone (FSH) were injected daily from day 9 to 12 after suppository insertion to promote superovulation. The dosage of creatine was 850 mg / dose / head, and the total dose of FSH was 700 IU / head (Day 1: 125 IU / dose / head, Day 2: 100 IU / dose / head, Day 3: 75 IU / dose / head, Day 4: 50 IU / dose / head). Creatine and FSH were injected twice daily, morning and evening, 12 hours apart, with both creatine and FSH administered simultaneously with each injection. By precisely controlling the timing, dosage, and injection interval of creatine and FSH, their synergistic effect promoted the full development and maturation of follicles, increased the number of ovulations, and improved the quality of the released eggs.

[0027] Example 3 Vaginal progesterone suppositories were inserted on any day of the donor cow's estrous cycle. Creatine and follicle-stimulating hormone (FSH) were injected daily from day 9 to 12 after suppository insertion to promote superovulation. The dosage of creatine was 1000 mg / dose / head, and the total dose of FSH was 700 IU / head (Day 1: 125 IU / dose / head, Day 2: 100 IU / dose / head, Day 3: 75 IU / dose / head, Day 4: 50 IU / dose / head). Creatine and FSH were injected twice daily, morning and evening, 12 hours apart, with both creatine and FSH administered simultaneously with each injection. By precisely controlling the timing, dosage, and injection interval of creatine and FSH, their synergistic effect promoted the full development and maturation of follicles, increased the number of ovulations, and improved the quality of the released eggs.

[0028] Example 4 Vaginal progesterone suppositories were inserted on any day of the donor cow's estrous cycle. Creatine and follicle-stimulating hormone (FSH) were injected daily from day 9 to 12 after suppository insertion to promote superovulation. The dosage of creatine was 500 mg / dose / head, and the total dose of FSH was 600 IU / head (Day 1: 110 IU / dose / head, Day 2: 90 IU / dose / head, Day 3: 60 IU / dose / head, Day 4: 40 IU / dose / head). Creatine and FSH were injected twice daily, morning and evening, 12 hours apart, with both creatine and FSH administered simultaneously with each injection. By precisely controlling the timing, dosage, and injection interval of creatine and FSH, their synergistic effect promoted the full development and maturation of follicles, increased the number of ovulations, and improved the quality of the released eggs.

[0029] Example 5 Superovulation in Huaxi cattle was promoted using the regulation method described in Example 3. Following the injection of creatine and follicle-stimulating hormone on the morning of day 11, the number of follicles was counted using ultrasound before insemination, and the number of corpora lutea was counted on day 7 after the last artificial insemination, before the embryo flushing procedure. Creatine supplementation helps regulate the levels of reproductive hormones in the cows. After thrombolysis, the combined effect of creatine and hormone level changes enhances ovarian response in Huaxi cattle.

[0030] After the final injection of creatine and follicle-stimulating hormone, 0.6 mg / head of cloprostenol is injected into the donor cow. Insemination is then performed after the donor cow comes into estrus. Cloprostenol dissolves the corpus luteum, regulates the estrous cycle of the cow, and concentrates the estrus time, facilitating insemination and increasing the success rate of insemination, thereby improving the embryo formation rate after superovulation.

[0031] Experimental Section 1. Test materials Reagents: Creatine was purchased from Beijing Penglai Pharmaceutical Co., Ltd., with a specification of 0.5g / vial, and each vial was dissolved in 2.5mL of physiological saline.

[0032] Follicle-stimulating hormone (FSH) was supplied by Ningbo No. 2 Hormone Factory, with a specification of 500 IU / vial, each vial dissolved in 10 mL of physiological saline.

[0033] Cloprostenol (PG) was purchased from Ningbo No. 2 Hormone Factory, with a specification of 0.2 mg / 2 mL.

[0034] The vaginal progesterone extended-release suppositories (CIDR) are from Zoetis Animal Health Products Co., Ltd., with a specification of 1.38g / suppository and 20 suppositories / pack.

[0035] Donor cattle: Huaxi cattle were selected as donor cattle, with an average weight between 450-650 kg, an age between 24-72 months, good body condition and body condition, and no reproductive diseases. All donor cattle had 2-4 parities. Donor cattle had free access to grazing and water. Their daily diet consisted of a premix of silage, concentrate, and hay. The temperature in the cattle shed was controlled between 10-30℃. After tethering, each experimental cattle was fed sufficient high-quality forage daily, supplemented with adequate vitamins and trace elements.

[0036] Experimental grouping and creatine dosage regimen: Donor cows were randomly divided into 5 groups: a control group (CON group) and 4 creatine treatment groups (CR4 group, CR6 group, CR8 group, and CR10 group), with 10 cows in each group. The creatine injection dosage for each group is as follows: CON group: No creatine injection; CR4 group: 500mg creatine per head per injection; CR6 group: 750mg creatine per head per injection; CR8 group: 1000mg creatine per head per injection; CR10 group: 1250mg creatine per head per injection.

[0037] 2. Experimental Methods A vaginal progesterone suppository is inserted on any day of the cow's estrous cycle, and the day of insertion is recorded as day 0. After restraining the donor cow with a restraint frame or neck clamp, the cow's vulva is disinfected with a 0.1-0.2% solution of benzalkonium bromide (the main component of which is benzalkonium bromide), and then dried. The sterile CIDR suppository is inserted into the suppository applicator according to aseptic technique, leaving the silicone thread end protruding from the rear. The cow's rectum is examined with the left hand to further confirm the absence of reproductive diseases and to determine the location of the cervix. The suppository applicator is inserted into the cow's vulva with the right hand, and after reaching the bottom, the piston of the applicator is pushed with the right hand to insert the CIDR suppository into the cow's vagina. Finally, the applicator is removed, completing the suppository insertion procedure.

[0038] From day 9 to day 12, each cow was injected with follicle-stimulating hormone (FSH) and creatine twice a day, morning and evening (creatine was administered according to the regimen for each group; the CON group received only FSH). The FSH dosage is shown in Table 1.

[0039] Table 1. Follicle-stimulating hormone dosage (IU / head)

[0040] Note: x = (weight - 450kg) × 0.125.

[0041] On the evening of the 12th day, after the injection of follicle-stimulating hormone and creatine, cloprostenol was injected at a dose of 0.6 mg / head, and the progesterone suppository in the vagina was removed.

[0042] Twenty-four hours after unblocking, the cow's estrus status is closely monitored. The onset of estrus is determined by the cow's ability to consistently accept mounting behavior, and the time of estrus is recorded. Eight hours after recording estrus, the first artificial insemination is performed, using frozen semen with a sperm motility of at least 40%. Thereafter, insemination is performed every 12 hours for a total of three inseminations. Seven days after insemination, a non-surgical embryo flushing procedure is performed from the uterine horn to retrieve the embryo. The donor cow is then separated into different pens post-surgery, with ample water and feed provided, and the pens kept dry and clean to prevent infection.

[0043] 3. Experimental Results Experiment 1: Effects of different creatine treatment groups on estrus rate and ovarian response in West China cattle. To evaluate the effect of creatine dosage on superovulation in West China cattle, a gradient dosage experiment was set up (CR4, CR6, CR8, CR10 groups), with the group without creatine injection serving as the control (CON group). The sample size for each group was 10 cattle.

[0044] The test results are shown in Table 2 and Figure 1 , Figure 1 In the figure, A represents the comparison of estrus rate among different treatment groups, B represents the comparison of average corpus luteum number among different treatment groups, and C represents the comparison of average follicle number among different treatment groups. From the figure, we can see that: Estrus rate: All creatine treatment groups reached over 90%, with no significant difference from the control group, confirming that creatine supplementation does not affect normal estrus. Ovarian response: The average number of corpora lutea and follicles increased significantly with increasing creatine dosage. The CR8 and CR10 groups showed the best results. The number of corpus luteum was 10.6±1.35 in the CR8 group and 11.3±2.67 in the CR10 group, which was significantly higher than that in the control group (7.3±3.47). Follicle count: CR8 group was 13.3±1.88 (P<0.05), CR10 group was 14.6±3.63 (P<0.01), both significantly better than the control group's 10.5±2.27.

[0045] In conclusion, creatine dosages of CR8 and CR10 can significantly improve the superovulation efficiency of West China cattle, with the CR10 group showing the best results.

[0046] Table 2. Effects of different creatine treatment groups on estrus rate and ovarian response in West China cattle.

[0047] Experiment 2: Effects of different creatine treatment groups on the number of embryos from West China bovine donors To further verify the actual effect of creatine on improving superovulation efficiency in West China cattle and to systematically evaluate the application value of creatine in embryo production, this study statistically analyzed the embryo production data of different treatment groups. Some cattle did not obtain embryos due to individual reasons. The actual effective data are shown in Table 3 below. Figure 2 , Figure 2 In the graph, A represents the average total number of embryos, B represents the number of transferable embryos, C represents the number of non-transferable embryos, D represents the transferable embryo rate, and E represents the non-transferable embryo rate. From the graph, we can see that: Embryo yield: The total number of embryos in the CR8 group and the CR10 group were 10.22±7.06 and 10.8±7.20, respectively, both significantly higher than the 6.7±2.16 in the CON group (P<0.01), confirming that creatine can significantly increase the ovulation base.

[0048] Number of usable embryos: The number of usable embryos in the CR8 group (8.0±6.10) and the CR10 group (7.2±6.61) was significantly higher than that in the CON group (P<0.01). The CR8 group showed the best performance in increasing the absolute number, indicating that creatine not only increases the number of ovulations, but also effectively increases the proportion of high-quality embryos with transfer value.

[0049] Appropriate dose (CR8): In terms of embryo utilization, the CR8 group was 78.26%, which was not significantly different from the CON group (80.59%) (P>0.05), indicating that the 1000mg dose maintained high quality while ensuring quantity.

[0050] Excessive dose (CR10): The unusable embryo rate in the CR10 group increased significantly to 33.33%, and the embryo utilization rate decreased significantly to 66.66% (P<0.01), indicating that although the 1250mg dose further increased the total number of embryos, it may have caused some embryos to develop abnormally, resulting in a decrease in the overall utilization rate.

[0051] In conclusion, the CR8 group represents the optimal dosage for enhancing superovulation efficiency in West China cattle, maximizing the number of embryos produced while ensuring embryo quality and utilization.

[0052] Table 3. Effects of different creatine treatment groups on the number of embryos from West China bovine donors.

[0053] Experiment 3: Effects of different creatine treatment groups on the grade distribution of West China bovine donor embryos To further evaluate the regulatory effect of creatine on embryonic developmental potential, this application analyzed the embryo quality grades of different treatment groups. The results are shown in Table 4 and... Figure 3 , Figure 3 In the figures, A represents the number distribution of embryos of different quality grades, B represents the rate of high-quality embryos, and C represents the rate of medium / low-quality embryos. The results showed that the proportion of high-quality embryos (EB+B grade) in each creatine treatment group was significantly higher than that in the CON group. Specifically, the high-quality embryo rate in the CR8 group reached 14.13%, which was significantly different from that in the CON group (P<0.001), indicating that this dose was most effective in promoting embryo differentiation to the blastocyst stage and significantly increasing the proportion of top-quality embryos. Regarding the proportion of medium / low-quality embryos (CM1+CM2 grade), except for the CR6 group, all other creatine treatment groups showed a significant decrease (P<0.01), with the CR8 group decreasing to 64.13%, confirming that creatine can effectively inhibit embryonic developmental arrest and reduce the proportion of low-quality embryos with developmental delays.

[0054] Comprehensive benefit analysis shows that the CR8 group performed well in terms of total number of embryos (92), number of usable embryos (72), rate of high-quality embryos (14.13%), and rate of medium / poor quality embryos (64.13%), demonstrating the optimal comprehensive benefit that balances yield and quality. It is the best implementation plan to improve the efficiency of superovulation in West China cattle.

[0055] Table 4. Effects of different creatine treatment groups on the grade distribution of donor embryos from West China cattle.

[0056] Experiment 4: Effects of Creatine Injection on Reproductive Hormone Levels in Western Chinese Bovines To further verify the physiological regulatory mechanism of creatine during superovulation, this application analyzed bovine serum samples during superovulation, focusing on the concentration changes of anti-Müllerian hormone (AMH) and progesterone (P4). 10 mL of blood was collected from the jugular vein at three time points: before superovulation treatment (before FSH injection), before artificial insemination, and before embryo collection. Serum was separated and stored at -20°C. AMH and P4 concentrations were detected using a bovine-specific ELISA kit. Each group consisted of 10 bovines, with the CR8 group as representative and the CON group as the control group. Data are expressed as mean ± standard deviation. Independent samples t-tests were used for intergroup comparisons. The results showed: AMH secretion characteristics: Before follicle-stimulating hormone (FSH) injection, there was no significant difference in basal AMH levels between the two groups; however, before artificial insemination, the AMH concentration in the CR group reached 4.35 ng / mL, which was significantly higher than the 1.87 ng / mL in the CON group (P<0.01), indicating that creatine pretreatment significantly enhanced the sensitivity and responsiveness of follicular granulosa cells to FSH stimulation. (See attached figures). Figure 4 A represents the change in anti-Müllerian hormone (AMH) concentration, and B represents the change in progesterone (P4) concentration. The graph shows that: P4 secretion characteristics: Before embryo collection, the P4 concentration in the CR group reached 23.22 ng / mL, which was significantly higher than that in the CON group (16.94 ng / mL), demonstrating that creatine treatment effectively promoted luteinization and maintained corpus luteum function.

[0057] The above analysis shows that creatine plays a stage-specific synergistic regulatory role in superovulation cycles: in the mid-superovulation stage, it promotes follicle recruitment and development by increasing AMH levels, and in the late stage, it optimizes luteal function by increasing P4 levels. This demonstrates the effectiveness of this protocol in improving superovulation efficiency from an endocrine perspective. Figure 5 The figure shows a comparison of the morphology of some embryos in the control group and the CR8 group. As shown in the figure, the embryos in the control group show partial fragmentation, developmental delay and irregular morphology; while the embryos in the CR8 group have complete morphology, clear and compact cell clusters, and present typical blastocyst morphology, which further verifies the significant effect of the method of the present invention in improving the quality of embryos after superovulation in West China cattle.

[0058] Experiment 5 Effects of creatine on serum metabolites in West China bovines To systematically analyze the specific effects of creatine intervention on metabolism during superovulation in West China cattle, this study selected serum samples at key time points for untargeted metabolomics analysis. The specific procedure is as follows: (1) Extraction of metabolites Take 100 μL of thawed serum sample into a centrifuge tube, add 400 μL of pre-chilled extraction solution (methanol and acetonitrile mixed in a 1:1 volume ratio, containing an isotopic internal standard), and vortex for 30 seconds to mix. Then, sonicate in an ice-water bath for 10 minutes and incubate at -40°C for 1 hour to fully precipitate proteins. After centrifuging at 12,000 rpm for 15 minutes at 4°C, transfer the supernatant to a vial for subsequent instrument analysis.

[0059] (2) Liquid chromatography-mass spectrometry (LC-MS) analysis Data acquisition was performed using a Vanquish ultra-high performance liquid chromatograph (Thermo Fisher Scientific, USA) combined with an Orbitrap Exploris 120 mass spectrometer (Thermo Fisher Scientific, USA). Chromatographic separation was performed using a Waters ACQUITY UPLC BEH Amide column (2.1 × 100 mm, 1.7 μm); mobile phase A consisted of 25 mmol / L ammonium acetate + 25 mmol / L ammonia solution, and mobile phase B consisted of acetonitrile; the sample pan temperature was 4 °C, and the injection volume was 2 μL. Mass spectrometry acquisition was performed using a high-resolution Orbitrap detector, with primary and secondary mass spectrometry scans performed in positive and negative ion modes, respectively, to ensure the accuracy of metabolite identification.

[0060] (3) Data processing and metabolite identification The raw mass spectrometry data were first converted to .mzXML format using ProteoWizard software, and then chromatographic peak extraction, baseline correction, and normalization were performed using a self-developed R package. Metabolite identification was achieved by comparing the data with a self-built secondary mass spectrometry database. An algorithm scoring threshold of 0.3 was set to screen for highly reliable metabolite identification results.

[0061] (4) Differential metabolites and functional analysis XCMS software was used for peak alignment, deconvolution, and quality control (QC), while MetaX software was used for metabolite identification and relative quantification. The criteria for screening differentially expressed metabolites were: ① Fold Change (FC) ≥ 1.5 or ≤ 0.67; ② P-value < 0.05. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis and metabolite correlation network construction were performed on the identified differentially expressed metabolites to illustrate the changes in key metabolic pathways induced by creatine intervention. The complete data processing workflow for the above metabolite identification, differential screening, and functional analysis is as follows: Figure 6 As shown.

[0062] All experiments were independently repeated at least three times. Count data, including the number of embryos recovered, the number of usable embryos, and the percentage of embryos at each stage, are expressed as mean ± standard deviation (Mean ± SD). Data were analyzed using GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA). To analyze the differences between the different creatine dosage groups (CR4 / 6 / 8 / 10) and the control group (CON), the Brown-Forsythe test was first used to test for homogeneity of variance. If homogeneity of variance was met, one-way ANOVA was used, followed by multiple comparisons between groups using Tukey's test; if variances were unequal, Welch-corrected one-way ANOVA was used, followed by pairwise comparisons between groups using Dunnett's T3 test.

[0063] To further elucidate the molecular mechanism by which creatine (CR) enhances superovulation efficiency in West China cattle, this study conducted non-targeted metabolomics analysis on serum samples from different time points within the superovulation cycle. Based on the blood collection time, the data were divided into two comparison periods: the mating period (Day 13), with the control group designated as D and the creatine group as C; and the flushing period (Day 20), with the control group designated as F and the creatine group as E. A systematic statistical analysis was then performed on these groups.

[0064] 1. Multivariate statistical analysis of the effects of creatine intervention on serum metabolic profile The results of multivariate statistical analysis of the effects of creatine intervention on serum metabolic profiles are as follows: Figure 7 As shown, the PLS-DA model analysis results reveal the distribution characteristics within and between groups: samples within the same group tend to cluster in the score space, showing good intra-batch repeatability; while between groups, a clear separation trend is observed. P <0.05, revealing a significant metabolic difference between the two groups ( Figure 7 A). The permutation test results confirmed the robustness and reliability of the model (R²=0.93, Q²=0.82), indicating that creatine intervention induced significant metabolic remodeling. Figure 7 B). Furthermore, the QC samples (Quality Control) are densely and centrally distributed in the PCA score chart, verifying the stability of the instrument's condition and the high quality and reproducibility of the data throughout the testing process. Figure 7 C).

[0065] 2. Screening of differentially expressed metabolites Based on the annotation results of secondary ion metabolites, this study screened and quantitatively analyzed the differentially expressed metabolites among the groups. (See attached data.) Figure 8Data showed that 69 differentially expressed metabolites were identified during the mating period (day 13), of which 45 were upregulated and 24 were downregulated; in contrast, 48 differentially expressed metabolites were identified during the embryo flushing period (day 20), with 17 upregulated and 31 downregulated. Figure 8 A). To further explore the correlation of metabolic regulation between the two periods, an intersection analysis of common differential metabolites was performed using Venn diagrams. The results showed that there were 6 overlapping differential metabolites between the two periods ( Figure 8 (B) The six common differential metabolites are: 5-methyl-2(3H)-furanone, cis-8,11,14-eicosatetrienoic acid, bergamot, eicosenoic acid, ethyl octadecanoate-9,12,15-trienoic acid, and urolithin A-8-O-glucuronide. Preliminary functional analysis suggests that these metabolites may participate in the regulation of energy metabolism through different mechanisms. Cis-8,11,14-eicosatetrienoic acid and eicosenoic acid can serve as direct substrates for energy metabolism, while bergamot and urolithin A-8-O-glucuronide play important roles in maintaining mitochondrial function and homeostasis. 5-methyl-2(3H)-furanone and ethyl octadecanoate-9,12,15-trienoic acid may indirectly participate in energy-related biosynthesis and signal transduction processes.

[0066] 3. Changes in creatinine, a secondary metabolite of creatine Creatinine is a metabolic product of creatine; elevated levels indicate an active creatine-phosphocreatine system and accelerated ATP regeneration. (Test results) Figure 9 The results showed that the relative creatinine level in the CR group before artificial insemination was significantly higher than that in the control group (CON). The trend in the graph shows that creatinine levels exhibited a dynamic change over time, first increasing and then decreasing: the baseline levels of both groups were similar before FSH (follicle-stimulating hormone) injection; however, before artificial insemination, the creatinine level in the CR group reached its peak (approximately 2.38 × 10⁻⁶). 8 The levels were significantly higher than those in the control group at the same time point (approximately 2.23 × 10⁻⁶). 8 Before embryo collection, creatinine levels in both groups decreased, and the difference between the groups narrowed.

[0067] 4. Differential metabolite heatmaps and their volcano maps We categorized and summarized the differentially metabolites, and the treatment group and the control group showed significant differences in their metabolic profiles. Figure 10 ).

[0068] Among the differentially regulated metabolites during mating, the upregulated metabolites mainly include arachidonic acid and serotonin, which are involved in biological processes such as fatty acid metabolism, energy regulation, and neurotransmitter signal transduction, respectively; the downregulated metabolites are mainly concentrated in lipid molecules such as sphingomyelin, ceramide, and phosphatidylcholine, which are closely related to cell membrane energy metabolism, signal transduction, and structural integrity. Figure 10 A, C).

[0069] Among the differentially regulated metabolites during the embryo flushing stage, the most upregulated metabolite was lysophosphatidylcholine glycerol-1-palmitate, which is associated with triglyceride catabolism and lipid energy supply. The most downregulated metabolites included phosphatidylcholine and 1,3-dihydroxyacetone, an intermediate product of glycolysis. The former affects the physicochemical properties of the cell membrane and the metabolic microenvironment, while the latter is closely related to the energy supply of glucose metabolism. Figure 10 B, D).

[0070] 5. KEGG metabolic pathway enrichment analysis Pathway enrichment analysis based on the KEGG database showed that ( Figure 11 , 12 Mating period ( Figure 11 ) and embryo flushing period ( Figure 12 The enrichment patterns of differential metabolites exhibit significant stage specificity. During the mating period, differential metabolites are mainly enriched in arachidonic acid metabolism (ARA). P <0.0001), linoleic acid metabolism, cAMP signaling pathway, and glycerophospholipid metabolism pathways (all P <0.05); while during the embryonic flushing stage, there was a significant enrichment in glycerophospholipid metabolism ( P <0.0001), linoleic acid metabolism and unsaturated fatty acid biosynthesis pathways, etc. (all P <0.05). Both groups showed significant enrichment in the glycerophospholipid metabolism and linoleic acid metabolism pathways, indicating that creatine has a certain regulatory effect on lipid metabolism in donor cattle. Creatine supplementation may improve energy supply by regulating metabolic pathways: on the one hand, by optimizing the energy regulation network through influencing the membrane lipid environment and signal transduction pathways (mating period); on the other hand, by promoting fatty acid oxidation and coenzyme system efficiency to enhance energy supply capacity (embryo flushing period). This different regulation of the lipid metabolism network helps to improve the energy microenvironment for follicle and embryo development, thereby improving oocyte quality and embryonic developmental potential, ultimately reflected in an increase in the number of usable embryos and an increase in the proportion of high-quality embryos. Figure 11 , 12 ).

Claims

1. The use of creatine combined with follicle stimulating hormone in the preparation of a drug for improving the efficiency of superovulation in Huaxi cattle, characterized in that, The improvement of superovulation efficiency in Huaxi cattle is to increase at least one of the following indicators: number of eggs, ovulation quality, estrus rate, and embryo formation rate after superovulation.

2. The application according to claim 1, characterized in that, The drug is used to administer to donor cows during superovulation treatment in West China cattle.

3. The application according to claim 2, characterized in that, The superovulation treatment is as follows: vaginal progesterone suppositories are inserted on any day of the donor cow's estrous cycle, and the drug is administered daily from day 9 to day 12 after suppository insertion.

4. The application according to claim 2, characterized in that, The drug is administered via intramuscular or subcutaneous injection.

5. The application according to claim 3, characterized in that, The drug was administered to the donor cows for 9-12 days after vaginal progesterone suppositories were inserted, twice daily, with each dose being 500-1000 mg per cow.

6. The application according to claim 5, characterized in that, On days 9-12 after vaginal progesterone suppositories were inserted into donor cows, creatine and follicle-stimulating hormone were administered twice daily, morning and evening, with a 12-hour interval between the two administrations, and creatine and follicle-stimulating hormone were administered simultaneously with each administration; the dose of creatine administered each time was 1000 mg / head.

7. The application according to claim 6, characterized in that, The total dose of follicle-stimulating hormone used is 500-700 IU / head.

8. The application according to any one of claims 1-7, characterized in that, The Huaxi cattle mentioned are multiparous cows, with parity of 2-4.

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