WO2021258423A1 - 一种无血清的牛卵母细胞体外成熟培养液及卵母细胞培养方法 - Google Patents

一种无血清的牛卵母细胞体外成熟培养液及卵母细胞培养方法 Download PDF

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WO2021258423A1
WO2021258423A1 PCT/CN2020/100828 CN2020100828W WO2021258423A1 WO 2021258423 A1 WO2021258423 A1 WO 2021258423A1 CN 2020100828 W CN2020100828 W CN 2020100828W WO 2021258423 A1 WO2021258423 A1 WO 2021258423A1
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serum
vitro maturation
bovine
oocytes
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French (fr)
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张景程
黄玥萌
张敏
王德宝
王永
张涌
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西北农林科技大学
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0608—Germ cells
    • C12N5/0609—Oocytes, oogonia
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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Definitions

  • the invention relates to the technical field of cell culture, and more specifically to a serum-free bovine oocyte in vitro maturation culture solution and an oocyte culture method.
  • In vitro maturation of oocytes is the basis and key link of embryo engineering technologies such as in vitro fertilization, somatic cell nuclear transfer, in vitro production of animal embryos, and transgenic cloning.
  • In vitro maturation of oocytes is not only affected by many factors such as ovarian cycle, animal species, follicle size, hormones, serum, etc., but also closely related to the procedures and methods of in vitro maturation of oocytes and the composition of the culture medium.
  • serum can lead to in vitro production Embryonic giant fetal syndrome.
  • the present invention provides a serum-free bovine oocyte in vitro maturation culture medium and an oocyte culture method, which improves the maturation rate of bovine oocyte in vitro culture and avoids the use of serum to reduce megafetal disease.
  • a serum-free bovine oocyte in vitro maturation culture medium including Hepes-free M199 basic culture medium, and the culture medium also includes the following additives: fatty acid-free BSA 2-8mg/ml, HMG 0.05-0.1IU/ml, 17 ⁇ -Estradiol 0.1-2 ⁇ g/ml, EGF 1-100ng/ml, L-cysteine 0.1-1mM, bFGF 1-100ng/ml, Glutamax (100x) 0.5-2 ⁇ L/ml, folic acid 1-100 ⁇ g/ml, cholic acid 1-10 ⁇ g/ml, CXCL12 1-100ng/ml.
  • a serum-free bovine oocyte in vitro maturation culture medium includes Hepes-free M199 basic culture medium, and the culture medium also includes the following additives: fatty acid-free BSA 6mg/ml, HMG 0.075IU/ml, 17 ⁇ -female Diol 1 ⁇ g/ml, EGF 60ng/ml, L-cysteine 0.57mM, bFGF 40ng/ml, Glutamax (100x) 1.6 ⁇ L/ml, folic acid 50 ⁇ g/ml, cholic acid 2 ⁇ g/ml, CXCL12 50ng/ml.
  • a method for in vitro maturation and culture of bovine oocytes uses the above-mentioned serum-free bovine oocyte in vitro maturation medium to culture bovine oocytes, and the specific steps are as follows: put the bovine oocytes into preheated serum-free cattle The oocytes are matured in the in vitro maturation culture medium and placed in an incubator at 38.5°C and 5% CO 2 saturated humidity for 18-22 hours to obtain mature bovine oocytes.
  • bovine oocyte is a GV stage oocyte.
  • serum-free bovine oocyte in vitro maturation culture medium is preheated for 1-3 hours.
  • serum-free bovine oocyte in vitro maturation culture solution is placed in an incubator at 38.5° C. and 5% CO 2 saturated humidity for preheating.
  • bovine oocytes are placed in each 500 ⁇ l serum-free bovine oocyte in vitro maturation culture medium.
  • the present disclosure provides a serum-free bovine oocyte in vitro maturation culture medium and an oocyte culture method.
  • the bovine oocyte in vitro maturation culture disclosed in the present invention Culturing bovine oocytes without using serum, improves the maturation rate of bovine oocytes, and effectively increases the early embryonic development rate of in vitro fertilized embryos and somatic cell nuclear transfer embryos.
  • Hepes-free M199 basic culture medium was purchased from Thermo Company, special grade fetal bovine serum (FBS) was purchased from Gibco Company, FGF, EGF, and CXCL12 were all purchased from R&D Company, and other unmarked products were purchased from sigma Company.
  • FBS fetal bovine serum
  • SOF solution preparation weighed 0.6294g of NaCl, 0.0534g of KCl, 0.162g of KH 2 PO 4 , 0.0172g of CaCl 2 ⁇ 2H 2 O, 0.00996g of MgCl 2 ⁇ 6H 2 O, 0.2106g of NaHCO 3.
  • SOFaa solution Use SOF solution as the basic culture medium, add 1% essential amino acids, 1% non-essential amino acids, 6mg/mL fatty acid-free bovine serum albumin, 12g/mL gentamicin and 12 ⁇ g/mL Cephalosporin.
  • mSOFaa solution Use SOFaa solution as the basic culture medium, add 1% ITS-G, 55 ⁇ M ⁇ -mercaptoethanol, 2.5mM hypotaurine, 0.4 ⁇ g/mL 1-oleoyl-SN-glycerol during preheating Sodium 3-phosphate, 0.1mM salidroside, 10ng/mL progesterone, 100 ⁇ M vitamin E, 1mM L-carnitine, 40ng/mL FGF, 10ng/mL corticosterone, 20ng/mL EGF, 25ng/mL IGF , 20ng/mL LIF, 2mg/mL hyaluronic acid.
  • the electrofusion solution includes: 0.3mol/L mannitol, 0.05mol/L calcium chloride, 0.1mol/L magnesium sulfate, 0.27mol/L histidine and 1mg/mL BSA.
  • Fertilization fluid use SOF solution without inositol as the basic culture medium, add 8mg/mL fatty acid-free bovine serum albumin, 12 ⁇ g/mL gentamicin, 12 ⁇ g/mL cephalosporin, 63 ⁇ g/mL L-arginine , 6 ⁇ g/mL L-aspartic acid, 50 ⁇ M L-carnitine, 2mM epinephrine, 20mM penicillamine, 10mM hypotaurine, 10 ⁇ g/mL heparin.
  • Serum-free bovine oocyte in vitro maturation medium including Hepes-free M199 basic medium, 6mg/mL fatty acid-free BSA, 0.075IU/ml HMG, 1 ⁇ g/ml 17 ⁇ -estradiol, 60ng/ml EGF, 0.57mM L-cysteine, 40ng/ml bFGF, 1.6 ⁇ L/ml Glutamax (100x), 50 ⁇ g/mL folic acid, 2 ⁇ g/mL cholic acid and 50ng/mL CXCL12.
  • Bovine oocytes have serum in vitro maturation medium, including Hepes-free M199 basic medium and 10% volume fraction of FBS, 0.075IU/ml HMG, 1 ⁇ g/ml 17 ⁇ -estradiol, 10ng/ml EGF, 1% volume fraction of ITSG, 10ng/ml bFGF, 50ng/mL CXCL12.
  • Bovine oocyte serum-free in vitro maturation medium including Hepes-free M199 basic medium and 6mg/mL fatty acid-free BSA, 0.075IU/ml HMG, 1 ⁇ g/ml 17 ⁇ -estradiol, 10ng/ml EGF , 1% volume fraction of ITSG, 10ng/ml bFGF, 50ng/mL CXCL12.
  • the bovine ovaries were collected from a designated slaughterhouse in Xi'an, Shaanxi province. The ovaries were placed in a thermos flask containing 100IU/ml penicillin and 100 ⁇ g/ml streptomycin in 20-25°C normal saline and transported back to the laboratory within 5h.
  • Treatment group Wash the selected COCs in serum-free bovine oocyte in vitro maturation culture medium (the in vitro maturation culture medium of Example 1, as the treatment group), and then transfer them to the in vitro maturation culture containing 3 mL of Example 1 Incubate in a 3 cm petri dish (equilibrate in an incubator for one hour in advance) at 38.5°C, 5% CO 2 , and saturated humidity for 18-22 hours.
  • the cultured and mature COCs were washed 3 times with PBS solution , and digested in Ca 2+ and Mg 2+ PBS containing 0.1% hyaluronidase for 1 to 2 minutes, and pipetted repeatedly with a 1000 mL pipette to remove oocytes Cumulus cells spread outside. After pipetting clean, wash 3 times in PBS, and then under the stereo microscope, use the foreign body needle to move the oocytes to select the oocytes with polar bodies for later use.
  • Control group 1 Wash the selected COCs twice in the in vitro maturation culture medium of bovine oocytes with serum (the in vitro maturation culture medium of Comparative Example 1, as the control group 1), and then transfer them into the body containing 3 mL of Comparative Example 1 In a 3cm petri dish of the mature culture medium (equilibrate in an incubator for one hour in advance), cultivate for 18-22 hours at 38.5°C, 5% CO 2 , and saturated humidity.
  • the cultured and mature COCs were washed 3 times with PBS solution , and digested in Ca 2+ and Mg 2+ PBS containing 0.1% hyaluronidase for 1 to 2 minutes, and pipetted repeatedly with a 1000 mL pipette to remove oocytes Cumulus cells spread outside. After pipetting clean, wash 3 times in PBS, and then under the stereo microscope, use the foreign body needle to move the oocytes to select the oocytes with polar bodies for later use.
  • Control group 2 Wash the selected COCs twice in a serum-free in vitro maturation culture medium of bovine oocytes (in vitro maturation culture medium of comparative example 2 as control group 2), and then transfer them into the in vitro culture medium containing 3 mL of comparative example 2 In a 3cm petri dish of the mature culture medium (equilibrate in an incubator for one hour in advance), cultivate for 18-22 hours at 38.5°C, 5% CO 2 , and saturated humidity.
  • the cultured and mature COCs were washed 3 times with PBS solution , and digested in Ca 2+ and Mg 2+ PBS containing 0.1% hyaluronidase for 1 to 2 minutes, and pipetted repeatedly with a 1000 mL pipette to remove oocytes Cumulus cells spread outside. After pipetting clean, wash 3 times in PBS, and then under the stereo microscope, use the foreign body needle to move the oocytes to select the oocytes with polar bodies for later use.
  • bovine fetal fibroblasts reach 80% confluence, aspirate the culture medium, wash the cells with Ca 2+ and Mg 2+-free PBS, add trypsin and EDTA mixed digestion solution to digest the cells. Observe the cells under an inverted microscope. When most of the cells have retracted, rounded, and the intercellular space expanded, stop the digestion with DMEM/F12 cell culture medium containing 10% fetal bovine serum, pipette and pipette, centrifuge to collect, and suspend , Inoculate a 24-well plate at a ratio of 1:3, and place it in a CO 2 incubator for cultivation. Two days before the preparation of somatic cloned embryos, the culture medium was changed to DMEM/F12 containing 0.5% fetal bovine serum.
  • the oocytes of the pre-enucleation treatment group and control group 1 and control group 2 were respectively incubated in M199 containing Hepes containing 7.5 ⁇ g/mL cytochalasin B and 4 mg/mL without fatty acid BSA for 15 minutes. Under the micromanipulator, the first polar body and the surrounding part of the protruding egg cytoplasm are sucked with a core removal tube with an inner diameter of 20 ⁇ m.
  • bovine fetal fibroblasts with a diameter of 15-20 ⁇ m were selected and injected under the zona pellucida of the enucleated treatment group and the control group 1, and control group 2.
  • the recombined embryos after nucleus injection are fused using the microelectrode method. Before fusion, the recombinant embryos were pre-equilibrated in the electrofusion solution for 3 minutes, and the electrofusion was performed under a 150-fold micromanipulator.
  • the top diameter of the two Z-shaped microelectrodes used for fusion operation is 15 ⁇ m, and the back end is connected to the micromanipulator, so that the contact surface of the donor and acceptor cell membranes of the recombinant embryo is perpendicular to the line connecting the two electrodes.
  • Fusion parameters Voltage 32V, pulse duration 20 ⁇ s, 2 pulse interval 10ms.
  • Half an hour after the fusion the fusion was observed under a microscope, and the fused embryos were put back into the oocyte in vitro maturation culture medium (corresponding Example 1, Comparative Example 1, and Comparative Example 2) and cultured for 3 hours.
  • the bovine somatic cell clone recombinant embryos of the treatment group and control group 1 and control group 2 were activated by ionomycin combined with 6-DMAP activation: Incubate in SOFaa solution of mycin for 4 min at room temperature, and then incubate in SOFaa solution containing 1-2 mmol/L 6-DMAP for 4 hours at 38.5°C, 5% CO 2 and saturated humidity.
  • blastocysts are transferable blastocysts.
  • Class C means that blastocyst transfer is not recommended.
  • the ABC classification of blastocysts is assessed according to the International Embryo Transfer Association's blastocyst quality scoring standards.
  • the serum-free bovine oocyte in vitro maturation culture fluid disclosed in the present invention is used after nuclear transplantation.
  • the transferable embryo ratio of the obtained embryos is significantly improved.
  • blastocysts are transferable blastocysts.
  • Class C means that blastocyst transfer is not recommended.
  • the ABC classification of blastocysts is assessed according to the International Embryo Transfer Association's blastocyst quality scoring standards.
  • the serum-free bovine oocyte in vitro maturation culture fluid disclosed in the present invention is used to obtain after fertilization
  • the transferable embryo ratio of embryos has increased significantly.
  • the serum-free bovine oocyte in vitro maturation culture medium disclosed in the present invention includes BSA, EGF, L-cysteine, bFGF, folic acid, cholic acid, CXCL12 and other components added to the basic culture medium;
  • the hot bovine oocytes are incubated in an in vitro maturation culture medium, and then placed in an environment with a CO 2 volume concentration of 5% for 18-22 hours to obtain mature oocytes.
  • the in vitro maturation culture medium and the in vitro maturation culture method of the present invention can improve the maturation efficiency of bovine oocytes in vitro culture.

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Abstract

一种无血清的牛卵母细胞体外成熟培养液及卵母细胞培养方法，属于细胞培养技术领域。所述无血清的牛卵母细胞体外成熟培养液包括无Hepes的M199基础培养液、无脂肪酸BSA、HMG、17β-雌二醇、EGF、L-cysteine、bFGF、100×Glutamax、叶酸、胆酸、CXCL12。该卵母细胞体外成熟培养液在不使用血清的情况下培养牛卵母细胞，提高了牛卵母细胞的成熟率，并有效提高体外受精胚胎和体细胞核移植胚胎的早期胚胎发育率。

Description

一种无血清的牛卵母细胞体外成熟培养液及卵母细胞培养方法 技术领域
本发明涉及细胞培养技术领域，更具体的说是涉及一种无血清的牛卵母细胞体外成熟培养液及卵母细胞培养方法。
背景技术
卵母细胞体外成熟培养是体外受精、体细胞核移植、动物胚胎体外生产和转基因克隆等胚胎工程技术的基础和关键环节。卵母细胞体外成熟不仅受到卵巢周期、动物品种、卵泡大小、激素、血清等多种因素影响，而且与卵母细胞体外成熟的程序和方法及培养液成分密切相关，特别是血清会导致体外生产胚胎的巨大胎儿综合征。虽然近几年卵母细胞体外成熟技术取得较大的进步，但是关于无血清培养体系的研究还并没有相关报道。
因此，提供一种无血清的牛卵母细胞体外成熟培养液及卵母细胞培养方法是本领域技术人员亟需解决的问题。
发明内容
有鉴于此，本发明提供了一种无血清的牛卵母细胞体外成熟培养液及卵母细胞培养方法，提高牛卵母细胞体外培养的成熟率，避免使用血清减少巨胎症。
为了实现上述目的，本发明采用如下技术方案：
一种无血清的牛卵母细胞体外成熟培养液，包括无Hepes的M199基础培养液，所述培养液还包括以下添加剂：无脂肪酸BSA 2-8mg/ml、HMG 0.05-0.1IU/ml、17β-雌二醇0.1-2μg/ml、EGF 1-100ng/ml、L-cysteine 0.1-1mM、bFGF 1-100ng/ml、Glutamax(100x)0.5-2μL/ml、叶酸1-100μg/ml、胆酸1-10μg/ml、CXCL12 1-100ng/ml。
进一步，一种无血清的牛卵母细胞体外成熟培养液，包括无Hepes的M199基础培养液，所述培养液还包括以下添加剂：无脂肪酸BSA 6mg/ml、HMG  0.075IU/ml、17β-雌二醇1μg/ml、EGF 60ng/ml、L-cysteine 0.57mM、bFGF 40ng/ml、Glutamax(100x)1.6μL/ml、叶酸50μg/ml、胆酸2μg/ml、CXCL12 50ng/ml。
进一步，一种牛卵母细胞体外成熟培养方法，利用上述无血清的牛卵母细胞体外成熟培养液培养牛卵母细胞，具体步骤如下：将牛卵母细胞放入预热的无血清的牛卵母细胞体外成熟培养液中，置于38.5℃、5％CO 2饱和湿度的培养箱中成熟培养18-22小时，获得成熟的牛卵母细胞。
进一步，所述牛卵母细胞为GV期卵母细胞。
进一步，所述无血清的牛卵母细胞体外成熟培养液预热1-3小时。
进一步，所述无血清的牛卵母细胞体外成熟培养液置于38.5℃、5％CO 2饱和湿度的培养箱中预热。
进一步，每500μl无血清的牛卵母细胞体外成熟培养液中放入40-50枚牛卵母细胞。
经由上述的技术方案可知，与现有技术相比，本发明公开提供了一种无血清的牛卵母细胞体外成熟培养液及卵母细胞培养方法，本发明公开的牛卵母细胞体外成熟培养液在不使用血清的情况下培养牛卵母细胞，提高了牛卵母细胞的成熟率，并有效提高体外受精胚胎和体细胞核移植胚胎的早期胚胎发育率。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
无Hepes的M199基础培养液购自Thermo公司，特级胎牛血清(FBS)购自Gibco公司，FGF、EGF、CXCL12均购自R&D公司，其他未标明的产品均购自sigma公司。
采卵液(PBS液)
26.168mg CaCl 2·2H 2O、37.218mg MgSO 4·7H 2O、0.0203g KCl、0.0200g KH 2PO 4、0.0036g丙酮酸钠、0.805g NaCl、0.1153g Na 2HPO 4、D-Glucose 0.1g、肝素钠6mg、胎牛血清3mL，去离子水定容到100mL，1mM NaOH调pH到7.0-7.2。
SOF溶液制备：将称量的0.6294g的NaCl、0.0534g的KCl、0.162g的KH 2PO 4、0.0172g的CaCl 2·2H 2O、0.00996g的MgCl 2·6H 2O、0.2106g的NaHCO 3、0.0033g的丙酮酸钠，及28.24L的乳酸钠加入98mL水中，1mM NaOH调pH到7.2～7.4，去离子水定容到100mL。
SOFaa溶液：以SOF溶液作为基础培养液，添加体积分数1％的必需氨基酸、体积分数1％的非必需氨基酸、6mg/mL无脂肪酸牛血清白蛋白、12g/mL庆大霉素和12μg/mL头孢霉素。
mSOFaa溶液：以SOFaa溶液作为基础培养液，预热时添加体积分数为1％的ITS-G、55μMβ-巯基乙醇、2.5mM亚牛磺酸、0.4μg/mL 1-油酰基-SN-甘油-3-磷酸钠盐、0.1mM红景天苷、10ng/mL的孕酮、100μM维生素E、1mM L-肉碱、40ng/mL FGF、10ng/mL皮质酮、20ng/mL EGF、25ng/mL IGF、20ng/mL LIF、2mg/mL透明质酸。
电融合液
电融合液包括：0.3mol/L甘露醇、0.05mol/L氯化钙、0.1mol/L硫酸镁、0.27mol/L组氨酸及1mg/mL的BSA。
受精液：以不含肌醇的SOF溶液作为基础培养液，添加8mg/mL无脂肪酸牛血清白蛋白，12μg/mL庆大霉素、12μg/mL头孢霉素、63μg/mL L-精氨酸、6μg/mL L-天冬氨酸、50μM L-肉碱、2mM肾上腺素、20mM青霉胺、10mM亚牛磺酸、10μg/mL肝素。
实施例1
无血清的牛卵母细胞体外成熟培养液，包括无Hepes的M199基础培养液，以及6mg/mL的无脂肪酸BSA、0.075IU/ml的HMG、1μg/ml的17β-雌二醇、60ng/ml的EGF、0.57mM的L-cysteine、40ng/ml的bFGF、1.6μL/ml的Glutamax(100x)、50μg/mL的叶酸、2μg/mL的胆酸和50ng/mL的CXCL12。
对比例1
牛卵母细胞有血清体外成熟培养液，包括无Hepes的M199基础培养液以及10％体积分数的FBS、0.075IU/ml的HMG、1μg/ml的17β-雌二醇、10ng/ml的EGF、1％体积分数的ITSG、10ng/ml的bFGF、50ng/mL的CXCL12。
对比例2
牛卵母细胞无血清体外成熟培养液，包括无Hepes的M199基础培养液以及6mg/mL无脂肪酸的BSA、0.075IU/ml的HMG、1μg/ml的17β-雌二醇、10ng/ml的EGF、1％体积分数的ITSG、10ng/ml的bFGF、50ng/mL的CXCL12。
实施例2牛卵母细胞的成熟培养
牛卵巢采自陕西省西安市定点屠宰场，将卵巢置于保温瓶内含100IU/ml青霉素和100μg/ml链霉素的20～25℃的生理盐水中，5h以内运回试验室。卵巢运回后，用灭菌剪刀剪除卵巢表面的结缔组织、脂肪和附着的输卵管，在无菌生理盐水中清洗三次，用装有21G针头的10mL注射器抽取卵巢表面2～8mm卵泡中的卵母细胞，放入6cm皿中，在实体显微镜下收集卵丘-卵母细胞复合体(cumulus-oocyte complexes，COCs)。收集后在PBS中清洗三遍。选择卵母细胞形态正常的COCs用于体外成熟培养。
处理组：将选择的COCs在无血清的牛卵母细胞体外成熟培养液(实施例1的体外成熟培养液，作为处理组)中洗两遍，然后移入装有3mL实施例1的体外成熟培养液的3cm平皿中(提前在培养箱平衡一小时)，在38.5℃、5％CO 2、饱和湿度条件下培养18～22h。将培养成熟的COCs用PBS液清洗3次，放入含0.1％透明质酸酶的无Ca 2+、Mg 2+PBS中消化1～2min，并用1000mL移液枪反复吹打，以除去卵母细胞外扩散的卵丘细胞。吹打干净后，在PBS中洗3次，然后在实体视显微镜下，以异物针拨动卵母细胞，挑选有极体的卵母细胞待用。
对照组1：将选择的COCs在牛卵母细胞有血清体外成熟培养液(对比例1的体外成熟培养液，作为对照组1)中洗两遍，然后移入装有3mL的对比例1的体外成熟培养液的3cm平皿中(提前在培养箱平衡一小时)，在38.5℃、5％CO 2、饱和湿度条件下培养18～22h。将培养成熟的COCs用PBS液清洗3次，放入含0.1％透明质酸酶的无Ca 2+、Mg 2+PBS中消化1～2min，并用1000mL移液枪反复吹打，以除去卵母细胞外扩散的卵丘细胞。吹打干净后，在PBS 中洗3次，然后在实体视显微镜下，以异物针拨动卵母细胞，挑选有极体的卵母细胞待用。
对照组2：将选择的COCs在牛卵母细胞无血清体外成熟培养液(对比例2的体外成熟培养液，作为对照组2)中洗两遍，然后移入装有3mL的对比例2的体外成熟培养液的3cm平皿中(提前在培养箱平衡一小时)，在38.5℃、5％CO 2、饱和湿度条件下培养18～22h。将培养成熟的COCs用PBS液清洗3次，放入含0.1％透明质酸酶的无Ca 2+、Mg 2+PBS中消化1～2min，并用1000mL移液枪反复吹打，以除去卵母细胞外扩散的卵丘细胞。吹打干净后，在PBS中洗3次，然后在实体视显微镜下，以异物针拨动卵母细胞，挑选有极体的卵母细胞待用。
观察并记录各组卵母细胞体外成熟情况，结果见表1。
表1卵母细胞体外成熟情况
Figure PCTCN2020100828-appb-000001
注：a、b上标不同表示差异显著，显著性使用卡方检验来检测。
由表1可以看出，与对照组1、对照组2的有血清和无血清体外成熟培养液体系相比，采用本发明公开的无血清的牛卵母细胞体外成熟培养液，牛卵母细胞的成熟率明显提高。
实施例3牛体细胞克隆胚胎的制备
(1)牛胎儿成纤维细胞的培养
从液氮中取一管荷斯坦奶牛的2～5代的牛胎儿成纤维细胞(采集自杨凌科元克隆股份有限公司牛场)于39℃解冻，加0.8mL的DMEM/F12细胞培养液，离心，弃上清，加细胞培养液重悬，取3mL细胞悬浮液接种于直径6cm的培养皿中，置于CO 2培养箱中38.5℃条件下培养。
待牛胎儿成纤维细胞达到80％汇合时，吸弃培养液，用无Ca 2+、Mg 2+的PBS冲洗细胞，加入胰蛋白酶与EDTA混合消化液，消化细胞。在倒置显微镜下观察细胞，待大多数细胞回缩、变圆、细胞间隙扩大时，用含10％胎牛血清的DMEM/F12细胞培养液终止消化，用移液器吹打后，离心收集，悬浮，按1:3的比例接种于24孔板，放入CO 2培养箱中培养。进行体细胞克隆胚制作的前两天，将培养液更换成含0.5％胎牛血清的DMEM/F12。
(2)卵母细胞的成熟培养
同实施例2。
(3)牛体细胞克隆胚的构建
去核：
去核前处理组和对照组1、对照组2的卵母细胞分别先在含7.5μg/mL细胞松弛素B、4mg/mL不含脂肪酸BSA的含有Hepes的M199中孵育15min。在显微操作仪下，用内径为20μm的去核管吸取第一极体及其周围的部分突出卵胞质。
注核和电融合：
注核时，挑选直径为15～20μm的牛胎儿成纤维细胞分别注入去核的处理组和对照组1、对照组2的卵母细胞透明带下。注核后的重组胚采用微电极的方法进行融合。融合前，重组胚在电融合液中预平衡3min，电融合在150倍的显微操作仪下进行。用于融合操作的两根“Z”字形微电极顶端直径为15μm，后端连于显微操作仪上，使重组胚的供体、受体细胞膜接触面与两电极的连线垂直，融合参数：电压32V、脉冲时长20μs、2次脉冲间隔10ms。融合后半小时在显微镜下观察融合情况，将融合好的胚胎分别放回卵母细胞体外成熟培养液(对应的实施例1、对比例1、对比例2)中培养3h。
(4)牛体细胞克隆重组胚的激活
在电融合之后3h，将处理组和对照组1、对照组2的牛体细胞克隆重组胚通过离子霉素(Ionomycin)联合6-DMAP的激活处理进行激活：首先在含2～5μmol/L离子霉素的SOFaa溶液中室温孵育4min，然后在含1～2mmol/L 6-DMAP的SOFaa溶液中，在38.5℃、5％CO 2、饱和湿度条件下培养4h。
(5)牛体细胞克隆胚胎的体外培养
四孔板每孔加入500μL的mSOFaa溶液，mSOFaa溶液上还覆盖有500μL石蜡油，石蜡油预先在CO 2培养箱中平衡至少2h，处理组和对照组1、对照组2的牛体细胞克隆重组胚在进行激活处理后，转移到上述四孔板中。每孔放置35～40枚牛体细胞克隆重组胚，在38.5℃、5％CO 2、7％O 2及饱和湿度条件下培养，记录发育情况，结果见表2。
表2克隆胚胎的发育情况
Figure PCTCN2020100828-appb-000002
注：a、b、c上标不同表示差异显著，显著性使用卡方检验来检测。AB级囊胚为可移植囊胚，C级为不推荐移植囊胚，囊胚ABC分级是根据国际胚胎移植协会的囊胚质量评分标准来评定。
由表2可以看出，与对照组1、对照组2的有血清和无血清体外成熟培养液体系相比，采用本发明公开的无血清的牛卵母细胞体外成熟培养液，在核移植后获得的胚胎的可移植胚胎比率显著提高。
实施例4牛体外受精胚胎的制备方法
(1)卵母细胞的成熟培养
同实施例2。
(2)精子的解冻，纯化，受精
将冻精(内蒙古赛科星公司)从液氮罐取出后，放置于37℃水浴解冻，离心管中做Percoll分层液。将2mL 90％Percoll液置于离心管底部，把2mL 45％Percoll液小心加到90％Percoll液面上，再将解冻后的精子置于45％Percoll液面上，1500rpm离心20min，小心吸取底部约200μL精液，分别加 入含有处理组和对照组1、对照组2卵母细胞的受精液中，放回培养箱内使精卵进行结合8-12h。
(3)受精胚胎的培养
受精完毕后，用口吸管或者枪头吹去卵子周围的残存卵丘细胞、精子。如果无法去除干净，可以在透明质酸酶溶液中进行去除。后移入mSOFaa溶液(液面上还覆盖有500μL石蜡油，并预先在CO 2培养箱中平衡至少2h)中培养，观察并记录发育情况，结果见表3。
表3体外受精胚胎的发育情况
Figure PCTCN2020100828-appb-000003
注：a、b、c上标不同表示差异显著，显著性使用卡方检验来检测。AB级囊胚为可移植囊胚，C级为不推荐移植囊胚，囊胚ABC分级是根据国际胚胎移植协会的囊胚质量评分标准来评定。
由表3可以看出，与对照组1、对照组2的有血清和无血清体外成熟培养液体系相比，采用本发明公开的无血清的牛卵母细胞体外成熟培养液，在受精后获得的胚胎的可移植胚胎比率显著提高。
本发明公开的无血清的牛卵母细胞体外成熟培养液包括添加于基础培养液的BSA、EGF、L-cysteine、bFGF、叶酸、胆酸和CXCL12等组分；将牛卵母细胞放入预热的牛卵母细胞体外成熟培养液中，然后置于CO 2体积浓度为5％的环境内成熟培养18～22小时，即获得成熟的卵母细胞。实验证实，本发明的体外成熟培养液及体外成熟培养方法，能够提高牛卵母细胞体外培养的成熟效率。
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。

Claims (7)

  1. 一种无血清的牛卵母细胞体外成熟培养液，包括无Hepes的M199基础培养液，其特征在于，所述培养液还包括以下添加剂：无脂肪酸BSA 2-8mg/ml、HMG 0.05-0.1IU/ml、17β-雌二醇0.1-2μg/ml、EGF 1-100ng/ml、L-cysteine 0.1-1mM、bFGF 1-100ng/ml、100xGlutamax 0.5-2μL/ml、叶酸1-100μg/ml、胆酸1-10μg/ml、CXCL12 1-100ng/ml。
  2. 根据权利要求1所述的一种无血清的牛卵母细胞体外成熟培养液，包括无Hepes的M199基础培养液，其特征在于，所述培养液还包括以下添加剂：无脂肪酸BSA 6mg/ml、HMG 0.075IU/ml、17β-雌二醇1μg/ml、EGF 60ng/ml、L-cysteine 0.57mM、bFGF 40ng/ml、100xGlutamax 1.6μL/ml、叶酸50μg/ml、胆酸2μg/ml、CXCL12 50ng/ml。
  3. 一种牛卵母细胞体外成熟培养方法，其特征在于，利用权利要求1或2所述的一种无血清的牛卵母细胞体外成熟培养液培养牛卵母细胞，具体步骤如下：将牛卵母细胞放入预热的无血清的牛卵母细胞体外成熟培养液中，置于38.5℃、5％CO 2饱和湿度的培养箱中成熟培养20-22小时，获得成熟的牛卵母细胞。
  4. 根据权利要求3所述的一种牛卵母细胞体外成熟培养方法，其特征在于，所述牛卵母细胞为GV期卵母细胞。
  5. 根据权利要求4所述的一种牛卵母细胞体外成熟培养方法，其特征在于，所述无血清的牛卵母细胞体外成熟培养液预热1-3小时。
  6. 根据权利要求5所述的一种牛卵母细胞体外成熟培养方法，其特征在于，所述无血清的牛卵母细胞体外成熟培养液置于38.5℃、5％CO 2饱和湿度的培养箱中预热。
  7. 根据权利要求6所述的一种牛卵母细胞体外成熟培养方法，其特征在于，每500μl无血清的牛卵母细胞体外成熟培养液中放入40-50枚牛卵母细胞。
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