Composition, method and application thereof for improving egg in vitro maturation quality and embryo pregnancy rate after in vitro fertilization

Abstract

The present invention discloses a composition, method and application thereof for improving the quality of in vitro maturation of eggs and the pregnancy rate of embryos after in vitro fertilization. The present invention aims at the problems of low in vitro maturation rate and poor subsequent embryo development potential caused by metabolic disorders during in vitro maturation of oocytes, and provides a composition including two or more regulators in vitamin C, L-carnitine and thrombopoietin to regulate the metabolic mode of oocytes, thereby greatly improving the in vitro maturation rate of oocytes and the subsequent embryo development potential. The present invention not only improves the efficiency of in vitro embryo production of livestock, but also provides a reference for improving the quality of in vitro maturation of human assisted reproductive eggs. The method is simple, safe and efficient, and has great application value and broad application prospects.

Description

[0001] This application is a divisional application with the application date of July 17, 2024, application number CN202410955964.5, and invention name "A composition, method and application for improving the quality of in vitro maturation of eggs and the pregnancy rate of embryos after in vitro fertilization", which is incorporated herein by reference in its entirety. Technical Field

[0002] The invention belongs to the field of biotechnology, and in particular relates to a composition, method and application thereof for improving the quality of in vitro maturation of eggs and the pregnancy rate of embryos after in vitro fertilization by the combined use of vitamin C, L-carnitine and THPO. Background Art

[0003] In the past few decades, mammalian in vitro embryo production technology has been widely used in the fields of livestock genetic improvement, breeding and human assisted reproduction. However, in vitro embryos have technical bottlenecks such as low development efficiency and poor embryo quality, which have been difficult to break through. It is generally believed that the in vitro culture environment is different from the in vivo development environment, especially the imperfect composition of the in vitro maturation medium for eggs, which leads to insufficient in vitro maturation of eggs. This is an important reason for the low development potential of in vitro embryos, especially the low pregnancy rate of embryos after transplantation, which greatly limits the development of in vitro embryo production technology. Therefore, optimizing the composition of the in vitro maturation medium for eggs, improving the quality of egg maturation, and then improving the subsequent embryo development potential is currently the most direct and effective strategy to improve the efficiency and quality of in vitro embryo development and improve in vitro embryo production technology.

[0004] Oocyte maturation is strictly regulated by multiple signals. Oocytes gradually develop to mature ovulation in vivo as follicles develop, providing good material and environmental guarantees for high-quality oocyte maturation. However, the in vitro oocyte maturation environment is an artificial culture medium, and the oxygen concentration in the culture environment is higher than that in the follicles in vivo, which can easily cause oxidative stress in the oocytes and cannot meet the high-quality maturation requirements of the oocytes. In addition, the metabolism in the oocyte, especially lipid metabolism, provides sufficient energy and regulators for oocyte maturation. Under the in vivo environment, a complex and dynamic lipid metabolism regulatory network is formed in the oocyte, but the key factors or pathways that play a core role are still unknown. Although lipid metabolites are indispensable, long-term exposure to a high-fat environment can also cause irreversible damage to oocytes.

[0005] At present, many strategies have been reported to improve the quality of in vitro maturation of eggs by adding antioxidants to in vitro maturation culture medium or promoting lipid metabolism. For example, adding cysteamine to in vitro maturation fluid of eggs can increase the glutathione content in oocytes and reduce oxidative stress damage; resveratrol, quercetin, and melatonin can reduce the level of active oxygen in eggs and the level of cell apoptosis in early fertilized embryos, and increase the number of mitochondria in eggs and the amount of ATP produced. CN100432219A discloses a bovine egg in vitro maturation culture medium, which mainly adds antioxidant tea polyphenols to remove free radicals such as hydroxyl radicals and superoxide anions, thereby improving the in vitro maturation rate of bovine eggs. CN110846272A discloses a method for reducing the content of active oxygen in eggs, which is obtained by adding 5-20% mature follicular fluid to the basic culture medium to improve the in vitro maturation rate of pig eggs.

[0006] However, due to the complexity of oxidative stress and lipid metabolism regulation, current correction technologies for oocyte maturation in vitro still have problems of low developmental efficiency and poor quality.

[0007] The information in the background technology is only for illustrating the general background of the present invention and should not be regarded as admitting or suggesting in any form that such information constitutes the prior art known to a person skilled in the art. Summary of the invention

[0008] The present invention is based on the results of previous studies, namely, there is a large amount of expression of the thrombopoietin (THPO) gene in the parietal granulosa cells of cattle and sheep. The main functional protein encoded by the THPO gene is a humoral growth factor, which is necessary for megakaryocyte proliferation, maturation and platelet production, and can affect the mitochondrial metabolism of hematopoietic stem cells. Based on the above research, the present invention creatively developed a composition for in vitro maturation culture of eggs and established a new in vitro maturation method of eggs. Specifically, the present invention provides a composition and application for improving the quality of in vitro maturation of eggs and the pregnancy rate of embryos after in vitro fertilization.

[0009] In a first aspect of the present invention, a composition is provided for improving the quality of in vitro maturation of eggs and the pregnancy rate of embryos after in vitro fertilization, which comprises two or more regulators selected from vitamin C or its precursor, L-carnitine and THPO.

[0010] In certain embodiments, in the composition for improving the quality of in vitro maturation of eggs and the pregnancy rate of embryos after in vitro fertilization according to the present invention, the composition comprises any combination of (a)-(d):

[0011] (a) vitamin C or its precursor and L-carnitine, wherein the weight ratio of the vitamin C or its precursor to the L-carnitine is 1:2-50;

[0012] (b) vitamin C or its precursor and THPO, wherein the weight ratio of the vitamin C or its precursor to the THPO is 200-1000:1;

[0013] (c) L-carnitine and THPO, wherein the weight ratio of L-carnitine to THPO is 1000-10000:1;

[0014] (d) vitamin C or its precursor, L-carnitine and THPO, and the weight ratio of the vitamin C or its precursor, the L-carnitine and the THPO is (200-1000):(1000-10000):1.

[0015] In certain embodiments, in the composition for improving the quality of in vitro maturation of eggs and the pregnancy rate of embryos after in vitro fertilization according to the present invention, the amount of vitamin C or its precursor can make the working concentration of vitamin C reach 0.5-1000 μg / ml during in vitro culture; the amount of L-carnitine can make the working concentration of L-carnitine reach 10-1000 μg / ml during in vitro culture; or the amount of THPO can make the working concentration of THPO reach 0.001-5 μg / ml during in vitro culture.

[0016] In certain embodiments, the composition for improving the quality of oocyte maturation in vitro and the pregnancy rate of embryos after in vitro fertilization according to the present invention further comprises a basal culture medium.

[0017] The second aspect of the present invention provides a method for improving the quality of in vitro maturation of eggs and the pregnancy rate of embryos after in vitro fertilization, which comprises the step of contacting the eggs with the composition of the first aspect in vitro.

[0018] In certain embodiments, in the method for improving the quality of in vitro maturation of eggs and the pregnancy rate of embryos after in vitro fertilization according to the present invention, the improvement includes improving the efficiency and / or quality of embryo development.

[0019] The third aspect of the present invention provides a method for improving in vitro maturation of an egg, comprising the step of contacting the egg with the composition of the first aspect in vitro.

[0020] In certain embodiments, according to the method for improving in vitro maturation of eggs described in the present invention, the improvement includes increasing the blastocyst development rate, the number of blastocyst cells, reducing the number of blastocyst apoptotic cells, and increasing the pregnancy rate after embryo transfer.

[0021] The fourth aspect of the present invention provides use of the composition described in the first aspect in animal genetic improvement, breeding, in vitro embryo production or culture.

[0022] According to data released by the International Embryo Tissue Society (IETS), the number of in vitro embryos of cattle has exceeded the number of in vivo embryos since 2016, and it has shown a trend of increasing year by year. Therefore, in vitro embryo production technology has become an effective way to efficiently utilize improved resources. The present invention provides effective correction approaches and methods for the problems of low embryo development rate and low subsequent development potential caused by poor quality of egg in vitro maturation, greatly improves the development rate and quality of embryos after in vitro fertilization, and provides an effective strategy for the upgrading of the animal husbandry industry. At the same time, it has important reference value in the field of assisted reproduction, especially in the acquisition of high-quality embryos. Therefore, the present invention will generate huge economic value and social value for my country to promote the production of in vitro embryos for livestock. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments or methods of the present invention, the drawings used in the embodiments are briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0024] Figure 1 The following is a dynamic analysis of THPO gene expression in the parietal granulosa cells and oocytes of follicles at different developmental stages in cattle and sheep. Among them, small (diameter <5mm), medium (5mm<diameter<12mm), and large (diameter>12mm) follicles of cattle were taken from ovaries in slaughterhouses; sheep follicles were taken before the first injection of FSH (FSH 0h), 24h after the first injection of FSH (FSH 24h), 24h after the second injection of FSH (FSH 48h), before LH injection (LH 0h), 8h after LH injection (LH 8h), and 26h after LH injection (LH26h). Single-cell transcriptome sequencing was performed on the parietal granulosa cells and oocytes at each stage, and TPM values ​​were obtained by analysis.

[0025] Figure 2 This is a schematic diagram of the process of in vitro maturation of cattle and sheep eggs, in vitro fertilization, in vitro culture and embryo transplantation.

[0026] Figure 3 This is a statistical analysis of the total number of blastocyst cells after different treatments of sheep oocyte maturation in vitro.

[0027] Among them, the control group was a conventional in vitro maturation group, and treatment groups 1-7 were: VitC alone group, L-carnitine alone group, THPO alone group, VitC and L-carnitine combined group, VitC and THPO combined group, L-carnitine and THPO combined group, VitC, L-carnitine and THPO combined group. The total number of blastocyst cells was obtained by counting the number of cells after DAPI immunofluorescence staining. DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0030] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0031] As used herein, the term "egg" refers to a cumulus-oocyte complex extracted from an in vivo oocyte collection or an isolated ovarian follicle.

[0032] Herein, the term "embryo" refers to an early stage larvae developed from a fertilized egg. Since development is a continuous process, the embryo herein includes larvae of different forms generated in any developmental process or stage, generally referring to pre-implantation embryos or pre-implantation embryos, such as 2-cell embryos, 4-cell embryos, 8-cell embryos, morulae, blastocysts, etc.

[0033] As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, in a cell culture, in a petri dish, etc., rather than in an organism (eg, an animal, plant, or microorganism).

[0034] Herein, the term "regulation", sometimes also referred to as "regulation", refers to any behavior that changes the level of oxidative stress or lipid metabolism.

[0035] Herein, the term "vitamin C" or "ascorbic acid", abbreviated as "VitC", is associated with a variety of diseases and is identified as an essential medicine in the list of essential medicines by the World Health Organization.

[0036] Herein, the term "precursor" refers to any substance, molecule or entity that can act as or produce vitamin C after chemical or physical changes. The precursor can be covalently bound or chelated in some manner and released or converted into an active ingredient, particularly vitamin C, before, simultaneously or after administration to a subject or target cell or tissue. Precursors can be prepared by modifying the functional groups present in the compound in a manner that allows the modification to be cleaved into the parent compound in routine manipulation or in vivo. Precursors include compounds in which hydroxyl, amino, sulfhydryl or carboxyl groups are bound to any group that is cleaved to form free hydroxyl, amino, sulfhydryl or carboxyl groups, respectively, when administered to a mammalian subject.

[0037] In this article, the term "L-carnitine", also known as L-carnitine, L-carnitine, vitamin BT, chemical formula is C 7 H 15 NO 3 , is an amino acid that helps convert fat into energy.

[0038] In this article, the term "THPO", which stands for Thrombopoietin in full and is expressed in Chinese as thrombopoietin, refers to thrombopoietin protein, which is closely related to the metabolic regulation of hematopoietic stem cells.

[0039] Herein, the term "working concentration" is also referred to as "use concentration", which refers to the initial concentration when the regulator is brought into contact with the embryo and effectively treated. The concentration unit is not limited, and can be, for example, ng / ml, μg / ml, mg / ml, etc.

[0040] [Composition]

[0041] The first aspect of the present invention provides a composition for improving the quality of in vitro maturation of eggs and the pregnancy rate of embryos after in vitro fertilization, which is sometimes referred to herein as the "composition of the present invention", and includes a combination of different types of regulators, wherein the regulators are selected from two or more of vitamin C or its precursor, L-carnitine and THPO. The regulators in the regulator combination of the present invention interact with each other to produce a synergistic effect, and compared with a single regulator, the combination greatly improves the regulatory effect. The form of the composition of the present invention is not limited, and can be a solid such as a dry powder, or a liquid such as a solution.

[0042] In certain embodiments, the composition of the present invention may be a combination of the above-mentioned conditioning agents, i.e., it does not contain any component other than the conditioning agents except for the inevitable impurities. In the composition of the present invention, each conditioning agent may exist in a mixed form, or two or more conditioning agents may exist in a form of being present alone. When used, the conditioning agents present alone may be pre-mixed and then used, or each conditioning agent may be used alone simultaneously or separately in sequence.

[0043] In certain embodiments, the composition of the present invention comprises the above-mentioned combination of regulators, and further comprises other ingredients in addition. The composition or type of other ingredients is not limited and can be freely selected as needed. Such other ingredients can select any known ingredients, particularly reagents and compositions related to embryo culture, such as culture medium, culture fluid or its additives, etc.

[0044] In certain embodiments, the regulator combination of the present invention is a combination of vitamin C or its precursor and L-carnitine. Preferably, the weight ratio of vitamin C to L-carnitine is 1:2-50, such as 1:2, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, etc. Within the above range, effective synergy between vitamin C and L-carnitine can be achieved, greatly improving the regulatory effect.

[0045] In certain embodiments, the regulator combination of the present invention is a combination of vitamin C or its precursor and THPO. Preferably, the weight ratio of vitamin C to THPO is 200-1000:1, such as 200:1, 250:1, 300:1, 350:1, 400:1, 450:1, 500:1, 550:1, 600:1, 650:1, 700:1, 750:1, 800:1, 850:1, 900:1, 950:1, 1000:1, etc. Within the above range, effective synergy between vitamin C and THPO can be achieved, greatly improving the regulatory effect.

[0046] In certain embodiments, the regulator combination of the present invention is a combination of L-carnitine and THPO. Preferably, the weight ratio of L-carnitine to THPO is 1000-10000:1, such as 1000:1, 1200:1, 1400:1, 1600:1, 1800:1, 2000:1, 2200:1, 2400:1, 2500:1, 2750:1, 3000:1, 3500:1, 4000:1, 4500:1, 5000:1, 5500:1, 6000:1, 6500:1, 7000:1, 7500:1, 8000:1, 8500:1, 9000:1, 9500:1, 10000:1, etc. Within the above range, effective synergy of L-carnitine and THPO can be achieved, greatly improving the regulatory effect.

[0047] In certain embodiments, the amount of vitamin C or its precursor of the present invention can make the working concentration of vitamin C reach 0.5-500 μg / ml when used. As long as vitamin C can reach the above range when used, the concentration or amount in the composition is not limited. In the case where the composition is a solution and is used as an added component, the concentration of vitamin C or its precursor in the composition can be higher than the working concentration, so that when the composition is added to the embryo culture medium, it can be diluted to reach the above working concentration. In the case where the composition is a solution and is directly used as a culture medium, the concentration of vitamin C or its precursor in the composition is usually substantially equal to the working concentration. The working concentration of vitamin C is preferably 1-400 μg / ml, such as 1.5 μg / ml, 2 μg / ml, 2.5 μg / ml, 3 μg / ml, 3.5 μg / ml, 4 μg / ml, 4.5 μg / ml, 5 μg / ml, 5.5 μg / ml, 6 μg / ml, 6.5 μg / ml, 7 μg / ml, 7.5μg / ml, 8μg / ml, 8.5μg / ml, 9μg / ml, 9.5μg / ml, 10μg / ml, 11μg / ml, 12μg / ml, 13μg / ml, 14μg / ml, 15μg / ml, 16μg / ml, 17μg / ml, 18 μg / ml, 19μg / ml, 20μg / ml, 21μg / ml, 22μg / ml, 25μg / ml, 30μg / ml, 35μg / ml, 40μg / ml, 50μg / ml, 60μg / ml, 70μg / ml, 80μg / ml, 90μg / ml, 100 μg / ml, 120μg / ml, 140μg / ml, 160μg / ml, 180μg / ml, 200μg / ml, 220μg / ml, 240μg / ml, 260μg / ml, 280μg / ml, 300μg / ml, 350μg / ml, 400μg / ml.

[0048] In certain embodiments, the amount of L-carnitine of the present invention can make the working concentration of L-carnitine reach 10-1000 μg / ml. As long as L-carnitine can reach the above range when used, the concentration or amount of L-carnitine in the composition is not limited. In the case where the composition is a solution and is used as an added component, the concentration of L-carnitine in the composition can be a working concentration, so that when the composition is added to the embryo culture medium, it can be diluted to reach the above working concentration. In the case where the composition is a solution and is directly used as a culture medium, the concentration of L-carnitine in the composition is usually substantially equal to the working concentration. The working concentration of L-carnitine is preferably 50-500 μg / ml, such as 55 μg / ml, 60 μg / ml, 65 μg / ml, 70 μg / ml, 75 μg / ml, 80 μg / ml, 85 μg / ml, 90 μg / ml, 95 μg / ml, 100 μg / ml, 125 μg / ml, 150 μg / ml, 1 75μg / ml, 200μg / ml, 225μg / ml, 250μg / ml, 275μg / ml, 300μg / ml, 325μg / ml, 350μg / ml, 375μg / ml, 400μg / ml, 425μg / ml, 450μg / ml, 475μg / ml, 500μg / ml.

[0049] In certain embodiments, the amount of THPO of the present invention is such that the working concentration of THPO is 0.001-5 μg / ml. The working concentration of THPO is preferably 0.05-3μg / ml, such as 0.06μg / ml, 0.07μg / ml, 0.08μg / ml, 0.09μg / ml, 0.1μ g / ml, 0.15μg / ml, 0.20μg / ml, 0.25μg / ml, 0.3μg / ml, 0.35μg / ml, 0.4μg / ml, 0.45μg / ml, 0.5μg / ml, 0.55μg / ml, 0.6μg / ml, 0.65μg / ml, 0.7μg / ml, 0.75μg / ml, 0.8μg / ml , 0.85μg / ml, 0.9μg / ml, 0.95μg / ml, 1μg / ml, 1.5μg / ml, 2μg / ml, 2.5μg / ml, 3μg / ml.

[0050] [Methods for improving in vitro maturation of oocytes]

[0051] The second aspect of the present invention provides a method for improving in vitro maturation of an egg, comprising the step of contacting an egg with the composition of the first aspect in vitro.

[0052] The improvements of the present invention include improving the efficiency and / or quality of embryo development, including improving the blastocyst development rate, the number of blastocyst cells, and the implantation rate after transplantation.

[0053] [use]

[0054] The third aspect of the present invention provides the use of the composition of the first aspect in animal genetic improvement, breeding and in vitro embryo production. The composition and its regulator have been described in detail in the first aspect and will not be repeated here.

[0055] In certain embodiments, the animal of the present invention refers to a non-human animal. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, a cow, a primate, or a pig). In some embodiments, animals include but are not limited to mammals, birds, reptiles, amphibians, fish, and worms. In some embodiments, animals are transgenic animals, genetically engineered animals, or clones. In certain embodiments, animals of the present invention are livestock, including pigs, cows, and sheep.

[0056] In certain embodiments, the genetic improvement and enrichment of the present invention include genetic improvement or enrichment relying on in vitro embryo production technology.

[0057] Example 1

[0058] This example shows the application of combined administration of VitC, L-carnitine and THPO in improving the in vitro maturation quality and developmental potential of sheep eggs.

[0059] 1. Experimental Materials

[0060] Collection of ovine ova (cumulus-oocyte complexes)

[0061] Ovary was collected from slaughterhouse, placed in 35℃ saline solution, and transported back to the laboratory within 2 hours. The ovary was washed three times with saline solution, and the follicle was punctured with a 20G needle disposable syringe containing 5mL of egg aspiration solution to release the cumulus-oocyte complex. The oocyte cumulus-oocyte complex with uniform cytoplasm and more than 3 layers of cumulus cells was collected under a stereoscope, i.e., oocyte. The subsequent experimental procedures were as follows: Figure 2 shown.

[0062] 2. Experimental Methods

[0063] (1) Experimental groups:

[0064] Control group: conventional in vitro maturation group;

[0065] Treatment group 1: VitC (100 μg / ml) alone administration group;

[0066] Treatment group 2: L-carnitine (1 mg / ml) alone administration group;

[0067] Treatment group 3: THPO (200 ng / ml) alone administration group;

[0068] Treatment group 4: VitC (50 μg / ml) and L-carnitine (500 μg / ml) combined administration group;

[0069] Treatment group 5: VitC (50 μg / ml) and THPO (100 ng / ml) co-administration group;

[0070] Treatment group 6: L-carnitine (500 μg / ml) and THPO (100 ng / ml) co-administration group;

[0071] Treatment group 7: the group to which VitC (33.3 μg / ml), L-carnitine (333.3 μg / ml) and THPO (66.7 ng / ml) were administered in combination.

[0072] The control group was the basic in vitro maturation solution, and the treatment groups (1-7) were the basic maturation solution with different components or combined components added thereto.

[0073] (2) In vitro maturation of sheep eggs

[0074] The collected sheep eggs were washed three times with egg washing solution, and then washed three times with oocyte in vitro maturation basal culture medium pre-equilibrated for 3 hours in advance, and transferred to the control group and experimental group in vitro maturation culture medium, and placed in 5% CO 2 , 38.5℃ saturated humidity incubator for 22-24 hours. Four-well plate culture, each well contains 600μL in vitro maturation culture medium, 300μL mineral oil cover, 30-35 sheep cumulus oocyte complexes per well. Sheep oocytes were matured in vitro according to different groups.

[0075] The control group used sheep oocyte in vitro maturation basal medium, and the configuration of the oocyte in vitro maturation basal medium was as follows: TCM199 + 10% estrus sheep serum + 1 μg / mL follicle-stimulating hormone + 1 μg / mL luteinizing hormone + 1 μg / mL β-estradiol + 12 μg / mL DL-cysteine ​​+ 100 μg / mL streptomycin + 100 IU / mL penicillin.

[0076] (3) In vitro fertilization of sheep eggs

[0077] Sheep eggs matured in vitro for 22-24 hours were placed in 0.5% hyaluronidase, and most of the cumulus cells on the surface of the mature eggs were removed by gentle and repeated blowing. The eggs were washed three times with fertilization solution, and transferred into 400 μL fertilization solution that had been pre-equilibrated in an incubator for more than 2 hours. The eggs were covered with 300 μL mineral oil, with 30 mature eggs in each well for in vitro fertilization.

[0078] Take the frozen sperm out of the liquid nitrogen with tweezers, thaw quickly in a 38°C water bath, and then transfer the thawed sperm to 600 μL sperm upstream solution and incubate in an incubator for 30 minutes to allow the sperm to fully float. After 30 minutes, take 100 μL of supernatant and add it to the four-well plate with mature eggs, and incubate the sperm and eggs for 20 hours. The fertilization conditions are 100% saturated humidity, 38.5°C, 5% CO 2 , 95% air.

[0079] The fertilization solution is configured as follows: mSOFaa + 2% estrus sheep serum by volume. The formula of mSOFaa solution is shown in Table 1, and all reagents were purchased from Sigma-Aldrich.

[0080] (4) Sheep embryo culture in vitro

[0081] After 20 hours of co-incubation, the fertilized eggs were aspirated and gently blown to remove the cumulus cells and sperm remaining on the surface of the fertilized eggs. The eggs were washed three times with embryo culture medium and transferred to embryo culture medium that had been equilibrated in an incubator for more than 2 hours and placed at 38.5°C and 5% O 2 +5% CO 2 +90%N 2 The embryos were cultured in a 100% saturated humidity incubator. Each well of a four-well plate contained 600 μL embryo culture medium and covered with 300 μL mineral oil.

[0082] The above embryo in vitro culture medium was configured as follows: mSOFaa+8 mg / mL BSA (A8806, Sigma).

[0083] After 48 h of culture, the cleavage rate was counted, and culture was continued until the 7th day, and the blastocyst rate was counted.

[0084] (5) Total cell number of blastocyst

[0085] The blastocysts on the 7th day were collected, washed three times with PBS containing 0.1% PVA by volume, and then DAPI staining was used to detect the total number of cells in the blastocysts. The specific steps are as follows: remove the zona pellucida with benchtop solution, then fix in 4% paraformaldehyde solution for 30 minutes, and permeabilize with PBS solution containing 0.5% TritonX-100 by volume for 20 minutes at room temperature. Use 1μg / mL DAPI to stain the nuclei of the blastocysts, incubate in the dark for 5 minutes, and then seal the slides. Observe and count the number of DAPI cells under an upright fluorescence microscope.

[0086] (6) Sheep embryo transfer and pregnancy testing

[0087] Secure the recipient sheep on the operating frame, fix the tip of the uterine horn on the side of the ovary with the corpus luteum or the side with a good corpus luteum, pierce the uterine horn wall at the 1 / 3 of the uterine horn without blood vessels with a paper clip, then insert the transplant gun tip into the uterine cavity through the needle hole, swing the gun tip, and when it is confirmed that the gun tip is in the uterine cavity, push the syringe piston connected to the embryo transfer tube to inject the embryo that was fertilized in vitro and developed into a blastocyst, then withdraw the embryo transfer tube, and finally return the uterine horn to the abdominal cavity and disinfect it.

[0088] B-ultrasound was used to detect pregnancy on the 40th to 45th day after pregnancy, and the embryo transfer pregnancy rate was calculated. The pregnancy rate calculation formula is:

[0089] Pregnancy rate = (number of pregnant recipient sheep / total number of transplanted recipient sheep) × 100%.

[0090] 3. Test results

[0091] The statistical results of in vitro embryo development rate are shown in Table 1, and the total number of blastocyst cells is shown in Table 1. Figure 2 shown.

[0092] The data in Table 1 show that compared with the control group (32.39±1.69%), the cleavage rate of the experimental group 7 supplemented with vitamin C, L-carnitine, and THPO (84.44±3.56%) was significantly higher than that of the control group (p<0.05). The combined treatments of VitC+L-carnitine, VitC+THPO, L-carnitine+THPO, and VitC+L-carnitine+THPO produced a synergistic effect, which was significantly higher than the blastocyst development efficiency of VitC, L-carnitine, and THPO added alone; and the combination of VitC+L-carnitine+THPO had the best effect. It can be seen that the combined use of VitC, L-carnitine, and THPO helps to improve the development efficiency of sheep IVF embryos.

[0093] Table 1 Effect of combined administration of VitC, L-carnitine and THPO on embryonic development rate after in vitro fertilization during in vitro maturation of sheep eggs

[0094]

[0095] Note: The data of development rate were obtained through one-way analysis of variance and expressed as mean ± standard error. Superscripts a, b, c with different letters in the same column indicate significant differences (p<0.05).

[0096] The total cell number of blastocyst is an important indicator for evaluating the quality of blastocyst. Figure 2The data showed that the combined treatments of VitC+L-carnitine (91.0±3.0), VitC+THPO (91.4±2.6), L-carnitine+THPO (91.9±2.5), and VitC+L-carnitine+THPO (97.5±2.6) produced a synergistic effect, which was significantly higher than the total number of cells added separately by VitC (80.6±3.2), L-carnitine (82.6±2.7), and THPO (79.4±2.3), and the combination of VitC+L-carnitine+THPO had the best effect. It can be seen that the combined use of VitC, L-carnitine, and THPO can help increase the number of embryonic cells in sheep in vitro fertilization.

[0097] The in vitro matured eggs of each group were subjected to in vitro fertilization and embryo culture, and the pregnancy rate was statistically calculated after the embryos were transplanted. The results are shown in Table 3. The results showed that compared with the control group (51.33±3.36%), the combination of VitC+L-carnitine, VitC+THPO, L-carnitine+THPO, and VitC+L-carnitine+THPO produced a synergistic effect, and were significantly higher than the transplantation efficiency of VitC, L-carnitine, and THPO added alone; and the combination of VitC+L-carnitine+THPO (66.7%) was the best, and significantly better than the effect of VitC, L-carnitine, and THPO added in combination. It can be seen that the combined use of VitC, L-carnitine, and THPO is the most effective solution to improve the pregnancy rate of sheep in vitro embryo transplantation.

[0098] Table 2 Effect of combined administration of VitC, L-carnitine and THPO on pregnancy rate of embryo transfer after in vitro fertilization during in vitro maturation of sheep oocytes

[0099]

[0100] Note: Pregnancy rate data were obtained through chi-square test analysis. Different letters in the same column, a, b, c, indicate significant differences among different groups (p<0.05).

[0101] Example 2

[0102] This example shows the application of combined administration of VitC, L-carnitine and THPO in improving the in vitro maturation quality and developmental potential of bovine oocytes.

[0103] 1. Experimental Design and Methods

[0104] (1) Experimental groups

[0105] Control group: conventional in vitro maturation group;

[0106] Treatment group 1: VitC (100 μg / ml) alone administration group;

[0107] Treatment group 2: L-carnitine (1 mg / ml) alone administration group;

[0108] Treatment group 3: THPO (200 ng / ml) alone administration group;

[0109] Treatment group 4: VitC (50 μg / ml) and L-carnitine (500 μg / ml) combined administration group;

[0110] Treatment group 5: VitC (50 μg / ml) and THPO (100 ng / ml) co-administration group;

[0111] Treatment group 6: L-carnitine (500 μg / ml) and THPO (100 ng / ml) co-administration group;

[0112] Treatment group 7: the group to which VitC (33.3 μg / ml), L-carnitine (333.3 μg / ml) and THPO (66.7 ng / ml) were administered in combination.

[0113] The control group was the basic in vitro maturation solution, and the treatment groups (1-7) were the basic maturation solution with different components or combined components added thereto.

[0114] (2) Bovine oocyte in vitro maturation and embryonic development

[0115] The experimental procedure is as follows Figure 2 shown.

[0116] ① Oocyte collection, in vitro oocyte maturation, and in vitro fertilization

[0117] The ovaries of dairy cows were kept warm in 32℃-35℃ physiological saline and sent to the laboratory. The follicles visible on the surface of the ovaries were extracted with a 20ml syringe. The cumulus-oocyte complex (i.e., egg) containing more than 3 layers of cumulus cells was washed twice with oocyte washing solution, and then washed twice with basic egg in vitro maturation solution, and then placed in a pre-CO 2 40-50 eggs were placed in a four-well plate maturation medium (500 μl volume of maturation medium) that had been equilibrated for more than 2 hours in an incubator with 5% CO 2 air, temperature 39℃, saturated humidity, and incubation time is 24h.

[0118] Further, the semen in the capillary tube was thawed in a 38°C water bath, placed in a 15 mL centrifuge tube containing semen washing solution, centrifuged at 1800 r / min for 5 min, and the supernatant was discarded, and repeated twice. Then 50 μl of sperm suspension was added to the 50 μl fertilization drop that had been balanced for more than 2 h to form a 100 μl fertilization drop, which was balanced in an incubator for 1.5 h to allow the sperm to fully capacitate.

[0119] After 22-24 hours of in vitro culture, COCs were digested with 0.1% hyaluronidase for about 1 minute to remove the outer layer of expanded granulosa cells, washed three times with pre-equilibrated fertilization solution, and mature oocytes with uniform cytoplasm were aspirated and placed in fertilization drops (15 oocytes / 100 μl). 38.5°C, 5% CO 2 After the sperm and egg are incubated together for 8-10 hours in an incubator, the eggs are placed in embryo culture medium and washed thoroughly to remove the sperm adhering to the surrounding area, and then cultured in microdroplets of embryo culture medium that have been pre-equilibrated for more than 2 hours.

[0120] ② Embryo in vitro culture

[0121] The fertilized zygotes were transferred to four-well plates for culture, and cultured in 500 μl of early development medium for 2 days. After counting the cleavage embryos, they were transferred to 500 μl of late development medium for culture and continued to be cultured for 5 days. Thereafter, half of the medium was replaced every other day. After fertilization, the number of in vitro fertilized blastocysts was counted and the blastocyst rate was calculated after 7-8 days of in vitro culture.

[0122] ③Embryo uterus transplantation and pregnancy testing.

[0123] The recipient cow was restrained and anesthetized according to the requirements of artificial insemination. After the vulva was cleaned and disinfected, the cervix was opened and the fallopian tube was flushed. The embryo transfer tube was inserted deep into the uterine horn on one side by rectal grasping method. The syringe piston connected to the embryo transfer tube was pushed to inject the embryo that developed into blastocyst after in vitro fertilization, and then the embryo transfer tube was withdrawn. B-ultrasound was used to detect pregnancy on the 45th to 50th day of pregnancy, and the pregnancy rate was calculated.

[0124] 2. Experimental results

[0125] As can be seen from Table 3, compared with the control group (26.8±2.2%), treatment groups 1 / 2 / 4 / 5 / 6 / 7 did not affect the 48h cleavage rate of in vitro fertilized embryos, but all promoted the blastocyst development efficiency; among them, the combination treatments of VitC+L-carnitine, VitC+THPO, L-carnitine+THPO, and VitC+L-carnitine+THPO produced a synergistic effect, which was significantly higher than the blastocyst development rate of VitC, L-carnitine and THPO added alone.

[0126] As can be seen from Table 4, compared with the control group (40.0%), L-carnitine alone can improve the pregnancy rate after embryo transfer, but the combination of VitC + L-carnitine, VitC + THPO, L-carnitine + THPO, and VitC + L-carnitine + THPO treatments are significantly higher than the pregnancy rate after embryo transfer treated with VitC, L-carnitine, and THPO alone.

[0127] Therefore, the combined administration of VitC, L-carnitine, and THPO or the combined administration of the three can significantly improve the development efficiency and quality of in vitro fertilization embryos in dairy cows.

[0128] Table 3 Effect of combined administration of VitC, L-carnitine and THPO on embryo development rate after in vitro fertilization during in vitro maturation of bovine oocytes

[0129]

[0130] Note: The superscripts a, b, and c indicate significant differences, and the significance was tested using one-way analysis of variance. Cleavage rate = number of two cells / number of eggs × 100%, blastocyst rate = number of blastocysts / number of eggs × 100%.

[0131] Table 4 Effect of combined administration of VitC, L-carnitine and THPO on pregnancy rate of in vitro fertilization embryo transfer during in vitro maturation of bovine oocytes

[0132]

[0133] Note: The superscripts a, b, and c indicate significant differences, and the significance was analyzed using the chi-square test. Pregnancy rate = number of pregnant females / number of recipient females × 100%.

[0134] Although the present invention has been described with reference to exemplary embodiments, it should be understood that the present invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments of the present specification without departing from the scope or spirit of the present invention. The scope of the claims should be based on the broadest interpretation to cover all modifications and equivalent structures and functions.

Claims

1. A composition for improving the quality of in vitro maturation of eggs and the pregnancy rate of embryos after in vitro fertilization, characterized in that: The method comprises a basic in vitro culture medium and any combination of the following (a)-(c) added to the basic in vitro culture medium: (a) vitamin C and thrombopoietin, wherein the weight ratio of the vitamin C to the thrombopoietin is 200-1000:1; (b) L-carnitine and thrombopoietin, wherein the weight ratio of the L-carnitine to the thrombopoietin is 500-3000:1; (c) vitamin C, L-carnitine and thrombopoietin, wherein the weight ratio of the vitamin C, the L-carnitine and the thrombopoietin is (200-1000):(500-3000):1; The amount of vitamin C can make the working concentration of vitamin C reach 0.5-500 μg / ml during in vitro culture; the amount of L-carnitine can make the working concentration of L-carnitine reach 10-1000 μg / ml during in vitro culture; the amount of thrombopoietin can make the working concentration of thrombopoietin reach 0.001-5 μg / ml during in vitro culture.

2. A method for improving the quality of in vitro maturation of eggs and the pregnancy rate of embryos after in vitro fertilization, characterized in that: The method comprises the step of contacting an egg with a composition according to claim 1 in vitro.

3. The method for improving the quality of in vitro maturation of eggs and the pregnancy rate of embryos after in vitro fertilization according to claim 2, characterized in that: The improvement includes improving the efficiency and / or quality of embryo development.

4. A method for improving in vitro maturation of eggs, characterized in that: The method comprises the step of contacting an egg with a composition according to claim 1 in vitro.

5. The method for improving in vitro maturation of oocytes according to claim 4, characterized in that: The improvement includes increasing the blastocyst development rate, the number of blastocyst cells, reducing the number of blastocyst apoptotic cells, and increasing the pregnancy rate after embryo transplantation.

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