CN112322579A - 牛体外受精使用的培养液和提高牛体外受精的方法 - Google Patents
牛体外受精使用的培养液和提高牛体外受精的方法 Download PDFInfo
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- CN112322579A CN112322579A CN202110010657.6A CN202110010657A CN112322579A CN 112322579 A CN112322579 A CN 112322579A CN 202110010657 A CN202110010657 A CN 202110010657A CN 112322579 A CN112322579 A CN 112322579A
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Abstract
本发明涉及牛体外受精使用的培养液和提高牛体外受精的方法。牛体外受精的方法包括如下步骤:将成熟的卵丘‑卵母细胞复合体在受精培养液中洗涤,再转移到受精培养液中,放入培养箱中,备用;从液氮中取冻精细管,37℃水浴解冻,使精液注入盛有精液制备培养液的离心管中,离心后弃上清;将精液制备培养液加入上述离心管,重悬精子沉淀,取适当的精子悬液进行精子计数;将计算好体积的精子悬液加入盛有卵母细胞的受精培养液滴中,并将培养盘放入培养箱,使精卵共孵育,完成体外受精操作,以备用于胚胎体外培养。涉及的受精培养液和精液制备培养液包含丙酮酸钠和肝素等成分。本发明方法能够显著提高牛体外受精的效率。
Description
技术领域
本发明属于动物繁殖技术领域,涉及一种用于农业-畜牧兽医繁殖的技术,具体涉及一种牛体外受精的方法,另外,本发明还涉及牛体外受精中使用的相关工作试液在牛体外受精中的应用。本发明牛体外受精的方法具有优异的技术效果。
背景技术
体外受精(In Vitro Fertilization)或(external fertilization)是指哺乳动物的精子和卵子在体外人工控制的环境中完成受精过程的技术,英文简称为IVF。由于它与胚胎移植技术(ET)密不可分,又简称为IVF-ET。在生物学中,把体外受精胚胎移植到母体后获得的动物称试管动物(test-tube animal)。这项技术成功于20世纪50年代,在最近20年发展迅速,现已日趋成熟而成为一项重要而常规的动物繁殖生物技术。
体外受精技术对动物生殖机理研究、畜牧生产、医学和濒危动物保护等具有重要意义。如用小鼠、大鼠或家兔等作实验材料,体外受精技术可用于研究哺乳动物配子发生、受精和胚胎早期发育机理。在家畜品种改良中,体外受精技术为胚胎生产提供了廉价而高效的手段,对充分利用优良品种资源,缩短家畜繁殖周期,加快品种改良速度等有重要价值。在人类,IVF-ET技术是治疗某些不孕症和克服性连锁病的重要措施之一。体外受精技术还是哺乳动物胚胎移植、克隆、转基因和性别控制等现代生物技术不可缺少的组成部分。
随着现代农业科技的发展,为了更充分利用良种母牛的繁殖潜力,加速遗传育种进程,在生产实践中应用高效的繁殖新技术成为必然。活体采卵(Ovum pick UP,OPU)和体外受精技术(In Vitro Fertilization,IVF)是二十世纪八十年代快速发展起来的胚胎工程新技术,二者相结合可获得大量遗传系谱明确的胚胎,从而缩短世代间隔。目前,这两种技术已经成为欧美和大洋洲等畜牧业发达国家的农场主为扩大良种母牛群而采用的重要繁殖技术。然而,采用常规的牛胚胎培养体系(CR1aa和SOF液),牛体外受精的囊胚发育率较低,而且胚胎质量也远不及体内胚胎,导致胚胎移植受体后的妊娠率低,因此如何提高囊胚发育率及胚胎质量成为体外受精胚胎生产的重点和研究的焦点。
早在1878年,德国人Scnenk就以家兔和豚鼠为材料,开始探索哺乳动物的体外受精技术。但直到1951年,美籍华人张民觉和Austin分别发现精子体外获能现象后,体外受精技术才获得了突破性进展。牛体外受精技术受到卵母细胞的体外成熟、精子的体外获能、受精卵的体外培养环境等多个方面的影响。
胚胎的体外培养是IVF技术的一个关键环节,亦是卵母细胞体外成熟和体外受精技术最终效果的体现和检验。在体外受精后,受精卵在向囊胚发育过程中将需经历一系列重要的变化,包括合子的形成、第一次卵裂、胚胎基因组的激活、致密化以及形成囊胚。这一过程中,外界环境的变化会导致基因表达发生改变,从而影响胚胎的正常发育及质量。目前,哺乳动物早期胚胎的体外培养研究主要集中在改善培养液成分以满足不同发育阶段的胚胎营养需求。基于Rosenkrans等(Rosenkrans, C.F.,Jr. and N.L. First, Effect offree amino acids and vitamins on cleavage and developmental rate of bovinezygotes in vitro. J Anim Sci, 1994.72(2): p.434-7)开发的Charles Rosenkrans 1(CR1)培养液和Tervit等(Tervit, H.R., D.G. Whittingham, and L.E. Rowson,Successful culture in vitro of sheep and cattle ova. J Reprod Fertil, 1972.30(3): p.493-7)开发的合成输卵管液(Synthetic Oviductal Fluid,SOF),经多年不断改进逐步形成了两种培养体系。据Hakan Sagirkaya等(Sagirkaya, H., et al.,Developmental potential of bovine oocytes cultured in different maturationand culture conditions. Anim Reprod Sci, 2007.101(3-4): p.225-40)和Somfai等(Somfai,T., et al., Development of bovine embryos cultured in CR1aa andIVD101 media using different oxygen tensions and culture systems. Acta VetHung, 2010.58(4): p.465-74)研究成果表明,CR1aa培养液用于牛胚胎培养有较好的效果,可以广泛应用于牛的胚胎培养;Thompson,J.G.等(Thompson,J.G., et al., Effectof inhibitors and uncouplers of oxidative phosphorylation during compactionand blastulation of bovine embryos cultured in vitro. J Reprod Fertil,2000.118(1): p.47-55)和Jean M.Feugang等(Feugang, J.M., O. Camargo-Rodriguez,and E. Memili, Culture systems for bovine embryos. Livestock Science,2009.121(2-3): p.141-149)的研究结果显示,SOF培养液也是一种适合于牛胚胎培养的培养体系。张志平等(张志平, 安志兴, 张锈, 张涌, 牛胚胎培养体系的优化. 西北农林科技大学学报, 2006. 34)和桑国俊等(桑国俊, 牛卵母细胞及体外胚培养技术研究. 2008)研究结果也显示,经过优化的CR1aa和SOF培养液均适合于牛的体外胚胎培养,均取得良好的培养效果。哺乳动物早期胚胎发育是一个高度协调且精确调节的过程。在进化过程中,配子细胞逐步形成了一系列的分子级联网络,以保证胚胎发育周期系统地进行。在发育过程中,胚胎的内外活性氧自由基(Reactive Oxygen Species,ROS)与抗氧化剂的平衡对早期胚胎发育起着决定性作用。
大多生化反应均产生ROS,其在细胞内、外均有着重要的作用,一部分ROS起着信号分子的作用,但是大多数ROS对机体是有害的。Brooker, R.J.等(Brooker, R.J.,Genetics: analysis and principles (4th ed.). McGraw-Hill Science, 2011)报道,ROS可以引起细胞DNA损伤、不饱和脂肪酸的氧化、蛋白质中氨基酸的氧化甚至可以导致某些酶的失活。一般来说,ROS以四种形式存在,其中H2O2氧化作用较强,是引起氧化伤害的最主要因素。
大量研究表明,谷胱甘肽(GSH)是以非蛋白质形式存在的一种抗氧化剂,能够清除多种自由基:超氧阴离子自由基、羟基自由基、过氧化氢、次氯酸和脂氧自由基,并且能够维持细胞内外氧化还原平衡。细胞内外环境GSH和ROS水平是影响受精卵发育过程中的两个重要因素。早在2000年,de Matos等(de Matos, D.G. and C.C. Furnus, The importanceof having high glutathione(GSH) level after bovine in vitro maturation onembryo development effect of beta-mercaptoethanol, cysteine and cystine.Theriogenology, 2000.53(3): p.761-71)曾通过在体外胚胎培养过程中添加β-巯基乙醇、半胱氨酸和胱氨酸来提高囊胚率。
尽管体外受精技术能成功应用于许多哺乳动物,但由于体外受精的囊胚率低而导致体外受精胚胎的生产成本高、效率低,限制了该技术在牛快速扩繁实践中的广泛应用。因此,如何能够降低成本并提高牛IVF胚胎生产效率和胚胎质量成为亟待解决的问题。
目前,牛体外受精的技术体系中主要以CR1aa和SOF液为胚胎体外培养液,并在此基础上进行改进,囊胚发育率均有不同程度的提高,囊胚发育率平均为30%-40%。对于囊胚质量,可以通过囊胚细胞总数、ICM细胞数/总细胞数比例、细胞凋亡率来评估。囊胚细胞总数根据囊胚所处阶段的不同而不同,S. Iwasaki等(Iwasaki,S. and T.Nakahara, Cellnumber and incidence of chromosomal anomalies in bovine blastocystsfertilized in vitro followed by culture in vitro or in vivo in rabbitoviducts. Theriogenology, 1990.33(3): p.669-75)获得的牛早期囊胚总细胞数平均为44,ICM细胞数/囊胚总细胞数比例为15.8%左右;Andrew J. Watson等(Watson, A.J., etal., Impact of bovine oocyte maturation media on oocyte transcript levels,blastocyst development, cell number, and apoptosis. Biol Reprod, 2000.62(2):p.355-64)统计牛囊胚细胞凋亡率约为7.7%-13%。
CN103898046B(中国专利申请号201410073635.4)公开了一种专用于牛体外受精胚胎的培养液,所述培养液配方为:NaCl 109.5mM、KCl 3.1mM、NaHCO3 26.2mM、MgCl2·6H2O0.8mM、KH2PO3 1.19mM、丙酮酸钠0.4mM、葡萄糖1.5mM、半乳糖酸钙5mM、10v/v%胎牛血清、L-谷氨酰胺1mM、2v/v%必需氨基酸、1v/v%非必须氨基酸和谷胱甘肽3mM,以水配制;所述必需氨基酸为以下氨基酸按比例混合后配制的水溶液,其中,各氨基酸含量为:L-盐酸精氨酸6.32g/L、L-胱氨酸二盐酸盐1.564g/L、L-盐酸组氨酸一水物2.1g/L、L-异亮氨酸2.625g/L、L-亮氨酸2.62g/L、L-赖氨酸盐酸盐3.625g/L、L-蛋氨酸0.755g/L、L-苯丙氨酸1.65g/L、L-苏氨酸2.38g/L、L-色氨酸0.51g/L、L-酪氨酸1.8g/L和L-缬氨酸2.34g/L;所述非必须氨基酸为以下氨基酸按比例混合后配制的水溶液,其中,各氨基酸含量为:L-丙氨酸0.89g/L、L-天门冬酰胺一水物1.5g/L、L-天冬氨酸1.33g/L、L-谷氨酸1.47g/L、甘氨酸0.75g/L、L-脯氨酸1.15g/L和L-丝氨酸1.05g/L。将牛体外受精胚胎置于上述培养液中进行体外培养,据信结果显示明显优于未添加GSH的对照组,提高了囊胚发育率及胚胎质量,降低体外生产胚胎的成本,为牛IVF技术应用于实践提供实验基础,可大大加速遗传育种进程。
在先申请案CN108728404A(申请号:201810576806.3)公开了一种牛体外受精胚胎培养的方法,其包括如下步骤:(1)卵母细胞的采集和体外成熟离体采集:取屠宰场卵巢盛放于加双抗生理盐水的保温桶中,31-33℃条件下,3h内运回实验室;抽取表面2-8mm的卵泡,收集沉淀,在体视显微镜下捡出至少含有3层卵丘细胞包裹的卵母细胞COCs(即,卵丘-卵母细胞复合体),在洗卵液中洗涤2遍,去除多余杂质;活体采集:对牛进行活体采卵,将所得卵泡液在体视显微镜下捡出至少含3层卵丘细胞包裹的卵丘-卵母细胞复合体(COCs),放入包含HEPES的成熟培养液中38.8℃、3h内运回实验室;将以上离体采集或者活体采集所得COCs在卵母细胞成熟培养液中洗涤1次,再转移到新的成熟培养液中培养22-24h,培养条件为38.8℃、5.5-6.5% CO2、饱和湿度;(2)体外受精将成熟的COCs在受精培养液中洗涤1次,再转移到受精培养液中,放入培养箱中,备用;从液氮中取一支冻精细管,37℃水浴解冻;无菌操作剪开细管两端,使精液注入盛有精液制备培养液的15mL离心管中,328×g离心2次,每次5min,离心后弃上清;将300μL精液制备培养液加入上述离心管,重悬精子沉淀,取适当的精子悬液进行精子计数;将计算好体积的精子悬液加入盛有卵母细胞的受精培养液滴中,并将培养盘放入培养箱,使精卵共孵育16-20h,培养条件为38.8℃、5.5-6.5% CO2、饱和湿度;(3)胚胎体外培养及保存体外受精操作完毕后,将胚胎周围的颗粒细胞用剥卵针去除干净,放入胚胎培养液中培养,此时记为胚胎培养的第1天,培养条件为38.8℃、6% O2、88%N2、饱和湿度,第3天记录卵裂率;第7天记录囊胚率,至第9天时统计囊胚孵化率(其为孵化囊胚数除以囊胚数所得百分数),并进行质量鉴定;将可用胚胎在保存液中洗涤3次,在平衡液中平衡10min后转移入冷冻液,按5段装液法装入胚胎,做好标记,再放入程序降温仪中按照0.5℃/min的速度降至-35℃后,将胚胎细管迅速取出,置入液氮中冷冻保存。
众所周知,胚胎生物技术是良种牛培育及快速扩繁的有效技术手段,其中活体采卵-体外胚胎生产(OPU-IVP)技术通过活体采集优秀个体卵母细胞,经体外成熟(IVM)、体外受精(IVF)、胚胎体外培养(IVC)等,在体外条件下可快速获得大量优质胚胎。相比体内胚胎,其生产效率高达4~8倍,能有效提升优良母畜繁殖潜力,但一般条件下牛卵母细胞供体牛场与胚胎生产实验室距离较远,需要数小时的运输,而运输时间和运输条件对维持卵母细胞活力和质量至关重要。传统的解决方式有两种,一是采集的卵母细胞先放在预成熟液中抑制卵母细胞成熟,到达实验室后再转移到成熟液中置于培养箱中进行体外成熟培养;二是采集的卵母细胞放在成熟液中,置于微型培养箱内运输,期间一直不间断供应CO2,但是运输状态下维系二氧化碳的供应是比较困难的。
在先申请案CN111254109A(中国专利申请号2020100820171)为克服上述运输困难的问题,提供了一种运输培养液,其中包含甘氨酸、丙氨酸、精氨酸盐酸盐、天冬氨酸、胱氨酸二盐酸盐、谷氨酸、L-谷氨酰胺、组氨酸、苏氨酸、色氨酸、酪氨酸、缬氨酸、抗坏血酸、生物素、氯化胆碱、泛酸钙、叶酸、甲萘醌等,使用该运输培养液进行牛卵母细胞的体外受精和胚胎培养的方法包括如下步骤:卵母细胞的采集和体外成熟、体外受精、胚胎体外培养及保存。该发明方法和运输培养液取得了优异的技术效果。上述CN108728404A和CN111254109A通过引用以其全部内容并入本文。
然而,牛冷冻精液在与卵母细胞进行体外受精的过程中,其体外受精的效率仍然是有限的。因此,有必要开发一种方法,以提高牛冷冻精液在与卵母细胞进行体外受精的过程中的体外受精的效率。
发明内容
本发明的目的在于提供一种提高牛冷冻精液在与卵母细胞进行体外受精的过程中的体外受精的效率的方法。更具体的,本发明为达成上述目的而提供一种专用于牛体外受精时冷冻精子的精液制备培养液和受精培养液,使用这些精液制备培养液和受精培养液预先处理冷冻精子以提高其在后续的体外受精过程中的效率。本发明人已经出人意料的发现,本发明方法和相关工作试液呈现优异的技术效果,本发明因此发现而得以完成。
为此,本发明第一方面提供了一种牛体外受精胚胎培养的方法,其包括如下步骤:
(1)卵母细胞的采集和体外成熟
a)离体采集:取屠宰场所得牛卵巢,抽取表面2-8mm的卵泡,收集沉淀,在体视显微镜下捡出至少含有3层卵丘细胞包裹的卵母细胞COCs(即,卵丘-卵母细胞复合体),放入包含HEPES的运输培养液中38.8℃、无二氧化碳供应、24h内运回实验室;
b)将以上离体采集或者活体采集所得COCs在卵母细胞成熟培养液中洗涤1次,再转移到新的成熟培养液中培养22-24h,培养条件为38.8℃、5.5-6.5% CO2、饱和湿度;
(2)体外受精
将成熟的COCs在受精培养液中洗涤1次,再转移到受精培养液中,放入培养箱中,备用;
从液氮中取一支冻精细管,37℃水浴解冻;无菌操作剪开细管两端,使精液注入盛有精液制备培养液的15mL离心管中,328×g离心2次,每次5min,离心后弃上清;将300µL精液制备培养液加入上述离心管,重悬精子沉淀,取适当的精子悬液进行精子计数;
将计算好体积的精子悬液加入盛有卵母细胞的受精培养液滴中,并将培养盘放入培养箱,使精卵共孵育16-20h,培养条件为38.8℃、5.5-6.5% CO2、饱和湿度;
(3)胚胎体外培养及保存
体外受精操作完毕后,将胚胎周围的颗粒细胞用剥卵针去除干净,放入胚胎培养液中培养,此时记为胚胎培养的第1天,培养条件为38.8℃、6% O2、88% N2、饱和湿度,第3天记录卵裂率;第7天记录囊胚率,至第9天时统计囊胚孵化率(其为孵化囊胚数除以囊胚数所得百分数),并进行质量鉴定;
将可用胚胎在保存液中洗涤3次,在平衡液中平衡10min后转移入冷冻液,按5段装液法装入胚胎,做好标记,再放入程序降温仪中按照0.5℃/min的速度降至-35℃后,将胚胎细管迅速取出,置入液氮中冷冻保存。
根据本发明第一方面的方法,其中步骤(1)之(b)中,所述成熟培养液是添加了100mL/L FBS、10μg/mL FSH、10μg/mL LH、1μg/mL E2、20ng/mL EGF的BY基础培养液。
根据本发明第一方面的方法,其中步骤(1)之(a)中,所述运输培养液中包含:甘氨酸50.0mg/L、L-丙氨酸25.0mg/L、L-精氨酸盐酸盐70.0mg/L、L-天冬氨酸30.0mg/L、L-胱氨酸二盐酸盐26.0mg/L、L-谷氨酸75.0mg/L、L-谷氨酰胺100.0mg/L、L-组氨酸盐酸盐一水合物21.88mg/L、L-羟基脯氨酸10.0mg/L、L-异亮氨酸40.0mg/L、L-亮氨酸60.0mg/L、L-赖氨酸盐酸盐70.0mg/L、L-甲硫氨酸15.0mg/L、L-苯丙氨酸25.0mg/L、L-脯氨酸40.0mg/L、L-丝氨酸25.0mg/L、L-苏氨酸30.0mg/L、L-色氨酸10.0mg/L、L-酪氨酸二钠盐二水合物58.0mg/L、L-缬氨酸25.0mg/L、抗坏血酸0.05mg/L、生物素0.01mg/L、氯化胆碱0.5mg/L、D-泛酸钙0.01mg/L、叶酸0.01mg/L、甲萘醌0.01mg/L、烟酰胺0.025mg/L、烟酸0.025mg/L、对氨基苯甲酸0.05mg/L、盐酸吡哆醛0.025mg/L、盐酸吡哆醇0.025mg/L、核黄素0.01mg/L、盐酸硫胺素0.01mg/L、维生素A醋酸酯0.1mg/L、维生素D2即骨化醇0.1mg/L、α-生育酚磷酸钠盐0.01mg/L、肌醇0.05mg/L、无水氯化钙200.0mg/L、硝酸铁九水合物0.7mg/L、无水硫酸镁97.67mg/L、氯化钾400.0mg/L、氯化钠6800.0mg/L、磷酸二氢钠一水合物140.0mg/L、硫酸腺嘌呤10.0mg/L、5'-磷酸腺苷0.2mg/L、三磷酸腺苷1.0mg/L、胆固醇0.2mg/L、葡萄糖1000.0mg/L、脱氧核糖0.5mg/L、还原型谷胱甘肽0.05mg/L、盐酸鸟嘌呤0.3mg/L、次黄嘌呤钠0.354mg/L、酚红20.0mg/L、核糖0.5mg/L、醋酸钠50.0mg/L、胸腺嘧啶0.3mg/L、吐温80为20.0mg/L、尿嘧啶0.3mg/L、黄嘌呤钠0.3mg/L,以及0.01IU/mL的FSH、0.01IU/mL的LH、1μg/mL的E2,50ng/mL的EGF、100ng/mL的IGF、10%庆大霉素、55µg/mL丙酮酸钠、1.2mM/L半胱氨酸、3mg/mL的BSA、10mM/L的HEPES。
根据本发明第一方面的方法,其中HEPES即4-羟乙基哌嗪乙磺酸在所述成熟培养液中的浓度为5~15mmol/L,例如10 mmol/L。在本发明中,EGF—— 表皮生长因子,FSH——卵泡刺激素,FBS——胎牛血清,E2——雌二醇,LH——促黄体生成素,IGF——胰岛素样生长因子,BSA——牛血清白蛋白。
根据本发明第一方面的方法,其中步骤(1)中,所述运输培养液中还包含牛磺酸和葡萄糖酸锌。
根据本发明第一方面的方法,其中步骤(1)中,所述运输培养液中牛磺酸的浓度为30~50mg/L,例如40mg/L或45mg/L。
根据本发明第一方面的方法,其中步骤(1)中,所述运输培养液中葡萄糖酸锌的浓度为1~3mg/L,例如2mg/L。
根据本发明第一方面的方法,其中步骤(2)中,所述受精培养液是包含112.0mM 氯化钠、4.02mM 氯化钾、2.25mM的氯化钙二水合物、0.52mM六水氯化镁、0.83mM磷酸二氢钾、37.0mM碳酸氢钠、1.25mM丙酮酸钠、10μg/ml肝素、4mg/ml牛血清白蛋白(BSA)、100U/ml青霉素、100μg/ml链霉素、10umoL/L α-硫辛酸、25μg/ml叶酸、3mg/ml谷氨酸钠的水溶液。
根据本发明第一方面的方法,其中步骤(2)中,所述精液制备培养液是包含112.0mM 氯化钠、4.02mM 氯化钾、2.25mM的 氯化钙二水合物、0.52mM六水氯化镁、0.83mM磷酸二氢钾、37.0mM碳酸氢钠、1.25mM丙酮酸钠、10μg/ml肝素、4mg/ml牛血清白蛋白(BSA)、10mM咖啡因、100U/ml青霉素、100μg/ml链霉素、10umoL/L α-硫辛酸、25μg/ml叶酸、3mg/ml谷氨酸钠的水溶液。
根据本发明第一方面的方法,其中步骤(3)中,所述胚胎培养液包含:109.5mM氯化钠、3.1mM氯化钾、26.2mM碳酸氢钠、0.8mM六水氯化镁、1.19mM 磷酸二氢钾、0.4mM丙酮酸钠、1.5mM葡萄糖、5mM半乳糖酸钙、2.5v/v%胎牛血清(FBS)、L-谷氨酰胺1mM、2v/v%必需氨基酸、1v/v%非必须氨基酸、3mM的谷胱甘肽、枸橼酸钠0.04w/v%、麦芽糖0.02w/v%的水溶液;所述必需氨基酸为以下氨基酸按重量比例添加:L-盐酸精氨酸6.32g、L-胱氨酸二盐酸盐1.564g、L-盐酸组氨酸一水物2.1g、L-异亮氨酸2.625g、L-亮氨酸2.62g、L-赖氨酸盐酸盐3.625g、L-蛋氨酸0.755g、L-苯丙氨酸1.65g、L-苏氨酸2.38g、L-色氨酸0.51g、L-酪氨酸1.8g和L-缬氨酸2.34g,所述非必须氨基酸为以下氨基酸按重量比例添加:L-丙氨酸0.89g、L-天门冬酰胺一水物1.5g、L-天冬氨酸1.33g、L-谷氨酸1.47g、甘氨酸0.75g、L-脯氨酸1.15g和L-丝氨酸1.05g。
根据本发明第一方面的方法,其中步骤(1)中,所述BY基础培养液是包含如下组分的水溶液:氯化钙180~220mg/L、九水硝酸铁0.70~0.75mg/L、氯化钾380~420mg/L、硫酸镁90~100mg/L、氯化钠6500~7000mg/L、一水磷酸二氢钠130~150mg/L、碳酸氢钠2000~2500mg/L、醋酸钠40~60mg/L、L-丙氨酸20~30mg/L、L-精氨酸盐酸盐60~80mg/L、L-天冬氨酸25~35mg/L、L-半胱氨酸盐酸盐一水合物0.10~0.12mg/L、L-胱氨酸二盐酸盐20~30mg/L、L-谷氨酸70~80mg/L、甘氨酸40~60mg/L、L-组氨酸盐酸盐一水合物20~25mg/L、L-羟脯氨酸8~12mg/L、L-异亮氨酸15~25mg/L、L-亮氨酸50~70mg/L、L-赖氨酸盐酸盐60~80mg/L、L-蛋氨酸10~20mg/L、L-苯丙氨酸20~30mg/L、L-脯氨酸30~50mg/L、L-丝氨酸20~30mg/L、L-苏氨酸25~35mg/L、L-色氨酸8~12mg/L、L-酪氨酸二钠二水合物55~60mg/L、L-缬氨酸20~30mg/L、抗坏血酸0.04~0.06mg/L、α-D-生育酚磷酸酯0.008~0.012mg/L、生物素0.008~0.012mg/L、骨化醇0.08~0.12mg/L、D-泛酸钙0.008~0.012mg/L、氯化胆碱0.4~0.6mg/L、叶酸0.008~0.012mg/L、肌醇0.04~0.06mg/L、三水合甲萘醌亚硫酸氢钠0.015~0.025mg/L、烟酸0.02~0.03mg/L、烟酰胺0.02~0.03mg/L、p-氨基苯甲酸0.04~0.06mg/L、盐酸吡哆辛0.04~0.06mg/L、核黄素0.008~0.012mg/L、盐酸硫胺素0.008~0.012mg/L、维生素A乙酸酯0.1~0.2mg/L、硫酸腺嘌呤8~12mg/L、腺嘌呤0.15~0.25mg/L、腺苷三磷酸二钠0.8~1.2mg/L、胆固醇0.15~0.25mg/L、2-脱氧-D-核糖0.4~0.6mg/L、D-葡萄糖800~1200mg/L、谷胱甘肽0.04~0.06mg/L、盐酸鸟嘌呤0.25~0.35mg/L、次黄嘌呤钠0.3~0.4mg/L、核糖0.4~0.6mg/L、胸腺素0.25~0.35mg/L、吐温80为4~6mg/L、尿嘧啶0.25~0.35mg/L、黄嘌呤钠0.3~0.4mg/L、酚红8~12mg/L。
根据本发明第一方面的方法,其中步骤(1)中,所述BY基础培养液是包含如下组分的水溶液:氯化钙200mg/L、九水硝酸铁0.72mg/L、氯化钾400mg/L、硫酸镁97.7mg/L、氯化钠6800mg/L、一水磷酸二氢钠140mg/L、碳酸氢钠2200mg/L、醋酸钠50mg/L、L-丙氨酸25mg/L、L-精氨酸盐酸盐70mg/L、L-天冬氨酸30mg/L、L-半胱氨酸盐酸盐一水合物0.11mg/L、L-胱氨酸二盐酸盐26mg/L、L-谷氨酸75mg/L、甘氨酸50mg/L、L-组氨酸盐酸盐一水合物21.88mg/L、L-羟脯氨酸10mg/L、L-异亮氨酸20mg/L、L-亮氨酸60mg/L、L-赖氨酸盐酸盐70mg/L、L-蛋氨酸15mg/L、L-苯丙氨酸25mg/L、L-脯氨酸40mg/L、L-丝氨酸25mg/L、L-苏氨酸30mg/L、L-色氨酸10mg/L、L-酪氨酸二钠二水合物57.66mg/L、L-缬氨酸25mg/L、抗坏血酸0.05mg/L、α-D-生育酚磷酸酯0.01mg/L、生物素0.01mg/L、骨化醇0.1mg/L、D-泛酸钙0.01mg/L、氯化胆碱0.5mg/L、叶酸0.01mg/L、肌醇0.05mg/L、三水合甲萘醌亚硫酸氢钠0.019mg/L、烟酸0.025mg/L、烟酰胺0.025mg/L、p-氨基苯甲酸0.05mg/L、盐酸吡哆辛0.05mg/L、核黄素0.01mg/L、盐酸硫胺素0.01mg/L、维生素A乙酸酯0.14mg/L、硫酸腺嘌呤10mg/L、腺嘌呤0.2mg/L、腺苷三磷酸二钠1mg/L、胆固醇0.2mg/L、2-脱氧-D-核糖0.5mg/L、D-葡萄糖1000mg/L、谷胱甘肽0.05mg/L、盐酸鸟嘌呤0.3mg/L、次黄嘌呤钠0.354mg/L、核糖0.5mg/L、胸腺素0.3mg/L、吐温80为5mg/L、尿嘧啶0.3mg/L、黄嘌呤钠0.34mg/L、酚红10mg/L。
根据本发明第一方面的方法,其中步骤(1)中,所述BY基础培养液中还包括亚硒酸钠,其浓度为0.2~0.3mg/L,例如0.25mg/L;和/或,所述BY基础培养液中还包括硫酸铜,其浓度以无水物计为0.05~0.1mg/L,例如0.075mg/L。
在本发明中,例如在步骤(3)中,胚胎所用的保存液、平衡液、冷冻液等均是本领域公知的并且是可以容易从市售途径获得的,例如冷冻液可以是瑞典Vitrolife公司在国内销售的FreezeKitTM Cleave(本发明如未特别说明,实例中所用冷冻液为FreezeKitTMCleave,胚胎保存液为含有20%牛血清和500IU/ml青霉素G钾的Whittingham改良杜氏磷酸缓冲液,平衡液为日本Kitazato Biopharma公司ES液)。
进一步的,本发明第二方面提供了一种卵母细胞的运输培养液,其如本发明第一方面所述。
进一步的,本发明第三方面提供了牛体外受精的方法,其包括如下步骤:
将成熟的卵丘-卵母细胞复合体(COCs)在受精培养液中洗涤1次,再转移到受精培养液中,放入培养箱中,备用;
从液氮中取一支冻精细管,37℃水浴解冻;无菌操作剪开细管两端,使精液注入盛有精液制备培养液的15mL离心管中,328×g离心2次,每次5min,离心后弃上清;将300µL精液制备培养液加入上述离心管,重悬精子沉淀,取适当的精子悬液进行精子计数;
将计算好体积的精子悬液加入盛有卵母细胞的受精培养液滴中,并将培养盘放入培养箱,使精卵共孵育16-20h,培养条件为38.8℃、5.5-6.5% CO2、饱和湿度,完成体外受精操作,以备用于胚胎体外培养。
根据本发明第三方面的方法,其中所述受精培养液是包含112.0mM 氯化钠、4.02mM 氯化钾、2.25mM的氯化钙二水合物、0.52mM六水氯化镁、0.83mM磷酸二氢钾、37.0mM碳酸氢钠、1.25mM丙酮酸钠、10μg/ml肝素、4mg/ml牛血清白蛋白(BSA)、100U/ml青霉素、100μg/ml链霉素、10umoL/L α-硫辛酸、25μg/ml叶酸、3mg/ml谷氨酸钠的水溶液。
根据本发明第三方面的方法,其中所述精液制备培养液是包含112.0mM 氯化钠、4.02mM 氯化钾、2.25mM的 氯化钙二水合物、0.52mM六水氯化镁、0.83mM磷酸二氢钾、37.0mM碳酸氢钠、1.25mM丙酮酸钠、10μg/ml肝素、4mg/ml牛血清白蛋白(BSA)、10mM咖啡因、100U/ml青霉素、100μg/ml链霉素、10umoL/L α-硫辛酸、25μg/ml叶酸、3mg/ml谷氨酸钠的水溶液。
根据本发明第三方面的方法,其中所述卵丘-卵母细胞复合体是通过如下方法采集和体外成熟的:
a)离体采集:取屠宰场所得牛卵巢,抽取表面2-8mm的卵泡,收集沉淀,在体视显微镜下捡出至少含有3层卵丘细胞包裹的卵母细胞COCs(即,卵丘-卵母细胞复合体),放入包含HEPES的运输培养液中38.8℃、无二氧化碳供应、24h内运回实验室;
b)将以上离体采集或者活体采集所得COCs在卵母细胞成熟培养液中洗涤1次,再转移到新的成熟培养液中培养22-24h,培养条件为38.8℃、5.5-6.5% CO2、饱和湿度,得到成熟的卵丘-卵母细胞复合体。
根据本发明第三方面的方法,其具有本发明第一方面或者第二方面所述的特征。
进一步的,本发明第四方面提供了用于牛体外受精的受精培养液,其是包含112.0mM 氯化钠、4.02mM 氯化钾、2.25mM的氯化钙二水合物、0.52mM六水氯化镁、0.83mM磷酸二氢钾、37.0mM碳酸氢钠、1.25mM丙酮酸钠、10μg/ml肝素、4mg/ml牛血清白蛋白(BSA)、100U/ml青霉素、100μg/ml链霉素、10umoL/L α-硫辛酸、25μg/ml叶酸、3mg/ml谷氨酸钠的水溶液。
进一步的,本发明第五方面提供了用于牛体外受精的精液制备培养液,其是包含112.0mM 氯化钠、4.02mM 氯化钾、2.25mM的 氯化钙二水合物、0.52mM六水氯化镁、0.83mM磷酸二氢钾、37.0mM碳酸氢钠、1.25mM丙酮酸钠、10μg/ml肝素、4mg/ml牛血清白蛋白(BSA)、10mM咖啡因、100U/ml青霉素、100μg/ml链霉素、10umoL/L α-硫辛酸、25μg/ml叶酸、3mg/ml谷氨酸钠的水溶液。
本发明任一方面或该任一方面的任一实施方案所具有的任一技术特征同样适用其它任一实施方案或其它任一方面的任一实施方案,只要它们不会相互矛盾,当然在相互之间适用时,必要的话可对相应特征作适当修饰。下面对本发明的各个方面和特点作进一步的描述。
本发明所引述的所有文献,它们的全部内容通过引用并入本文,并且如果这些文献所表达的含义与本发明不一致时,以本发明的表述为准。此外,本发明使用的各种术语和短语具有本领域技术人员公知的一般含义,即便如此,本发明仍然希望在此对这些术语和短语作更详尽的说明和解释,提及的术语和短语如有与公知含义不一致的,以本发明所表述的含义为准。
本发明使用的胎牛血清可以容易的从市场购得其标准化商品形式,例如可以从各种代理商处购得Gibco公司的澳洲胎牛血清(货号:10099141)、新西兰胎牛血清(货号:10091148)、北美胎牛血清(货号:16000044)、墨西哥胎牛血清(货号:10437028)等。在本发明上下文的试验中,如未特别说明,所用的胎牛血清是Gibco公司的澳洲胎牛血清(货号:10099141)。
具体实施方式
通过下面的实施例可以对本发明进行进一步的描述,然而,本发明的范围并不限于下述实施例。本领域的专业人员能够理解,在不背离本发明的精神和范围的前提下,可以对本发明进行各种变化和修饰。本发明对试验中所使用到的材料以及试验方法进行一般性和/或具体的描述。虽然为实现本发明目的所使用的许多材料和操作方法是本领域公知的,但是本发明仍然在此作尽可能详细描述。若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段,所用原料均为市售商品。在本发明中,如未特别说明,提及谷氨酸或者谷氨酸钠时,均是指L-谷氨酸或者L-谷氨酸钠。
实施例1:牛体外受精胚胎的培养方法(离体采集)
本实施例在步骤(1)中,将采集的卵母细胞置于包含HEPES的运输培养液中38.8℃、无二氧化碳供应、24h内运回实验室。
一、试剂
本发明的具体试验中,如未另外说明,使用的相关试剂详述如下:
本实施例所用的包含HEPES的运输培养液,其中包含:甘氨酸50.0mg/L、L-丙氨酸25.0mg/L、L-精氨酸盐酸盐70.0mg/L、L-天冬氨酸30.0mg/L、L-胱氨酸二盐酸盐26.0mg/L、L-谷氨酸75.0mg/L、L-谷氨酰胺100.0mg/L、L-组氨酸盐酸盐一水合物21.88mg/L、L-羟基脯氨酸10.0mg/L、L-异亮氨酸40.0mg/L、L-亮氨酸60.0mg/L、L-赖氨酸盐酸盐70.0mg/L、L-甲硫氨酸15.0mg/L、L-苯丙氨酸25.0mg/L、L-脯氨酸40.0mg/L、L-丝氨酸25.0mg/L、L-苏氨酸30.0mg/L、L-色氨酸10.0mg/L、L-酪氨酸二钠盐二水合物58.0mg/L、L-缬氨酸25.0mg/L、抗坏血酸0.05mg/L、生物素0.01mg/L、氯化胆碱0.5mg/L、D-泛酸钙0.01mg/L、叶酸0.01mg/L、甲萘醌0.01mg/L、烟酰胺0.025mg/L、烟酸0.025mg/L、对氨基苯甲酸0.05mg/L、盐酸吡哆醛0.025mg/L、盐酸吡哆醇0.025mg/L、核黄素0.01mg/L、盐酸硫胺素0.01mg/L、维生素A醋酸酯0.1mg/L、维生素D2即骨化醇0.1mg/L、α-生育酚磷酸钠盐0.01mg/L、肌醇0.05mg/L、无水氯化钙200.0mg/L、硝酸铁九水合物0.7mg/L、无水硫酸镁97.67mg/L、氯化钾400.0mg/L、氯化钠6800.0mg/L、磷酸二氢钠一水合物140.0mg/L、硫酸腺嘌呤10.0mg/L、5'-磷酸腺苷0.2mg/L、三磷酸腺苷1.0mg/L、胆固醇0.2mg/L、葡萄糖1000.0mg/L、脱氧核糖0.5mg/L、还原型谷胱甘肽0.05mg/L、盐酸鸟嘌呤0.3mg/L、次黄嘌呤钠0.354mg/L、酚红20.0mg/L、核糖0.5mg/L、醋酸钠50.0mg/L、胸腺嘧啶0.3mg/L、吐温80为20.0mg/L、尿嘧啶0.3mg/L、黄嘌呤钠0.3mg/L,以及0.01IU/mL的FSH、0.01IU/mL的LH、1μg/mL的E2,50ng/mL的EGF、100ng/mL的IGF、10%庆大霉素、55µg/mL丙酮酸钠、1.2mM/L半胱氨酸、3mg/mL的BSA、10mM/L的HEPES,以及牛磺酸40mg/L、葡萄糖酸锌2mg/L。
加双抗生理盐水:包含青霉素400IU/mL、链霉素400μg/mL的生理盐水。
洗卵液:添加了3mg/mL牛血清白蛋白的BY基础培养液。
成熟培养液:添加了100mL/L FBS、10μg/mL FSH、10μg/mL LH、1μg/mL E2、20ng/mLEGF的BY基础培养液。
包含HEPES的成熟培养液:添加了15 mmol/L HEPES、100mL/L FBS、10μg/mL FSH、10μg/mL LH、1μg/mL E2、20ng/mL EGF的BY基础培养液。
其中,EGF—— 表皮生长因子,FSH——卵泡刺激素,FBS——胎牛血清,E2——雌二醇,LH——促黄体生成素。
受精培养液:包含112.0mM 氯化钠、4.02mM 氯化钾、2.25mM的 氯化钙二水合物、0.52mM六水氯化镁、0.83mM磷酸二氢钾、37.0mM碳酸氢钠、1.25mM丙酮酸钠、10μg/ml肝素、4mg/ml牛血清白蛋白(BSA)、100U/ml青霉素、100μg/ml链霉素的水溶液。
精液制备培养液:包含112.0mM 氯化钠、4.02mM 氯化钾、2.25mM的 氯化钙二水合物、0.52mM六水氯化镁、0.83mM磷酸二氢钾、37.0mM碳酸氢钠、1.25mM丙酮酸钠、10μg/ml肝素、4mg/ml牛血清白蛋白(BSA)、10mM咖啡因、100U/ml青霉素、100μg/ml链霉素的水溶液。
胚胎培养液:包含:109.5mM氯化钠、3.1mM氯化钾、26.2mM碳酸氢钠、0.8mM六水氯化镁、1.19mM 磷酸二氢钾、0.4mM丙酮酸钠、1.5mM葡萄糖、5mM半乳糖酸钙、10v/v%胎牛血清(FBS)、L-谷氨酰胺1mM、2v/v%必需氨基酸、1v/v%非必须氨基酸、3mM的谷胱甘肽、枸橼酸钠0.04w/v%、麦芽糖0.02w/v%的水溶液;所述必需氨基酸为以下氨基酸按重量比例添加:L-盐酸精氨酸6.32g、L-胱氨酸二盐酸盐1.564g、L-盐酸组氨酸一水物2.1g、L-异亮氨酸2.625g、L-亮氨酸2.62g、L-赖氨酸盐酸盐3.625g、L-蛋氨酸0.755g、L-苯丙氨酸1.65g、L-苏氨酸2.38g、L-色氨酸0.51g、L-酪氨酸1.8g和L-缬氨酸2.34g,所述非必须氨基酸为以下氨基酸按重量比例添加:L-丙氨酸0.89g、L-天门冬酰胺一水物1.5g、L-天冬氨酸1.33g、L-谷氨酸1.47g、甘氨酸0.75g、L-脯氨酸1.15g和L-丝氨酸1.05g。
BY基础培养液是包含如下组分的水溶液:氯化钙200mg/L、九水硝酸铁0.72mg/L、氯化钾400mg/L、硫酸镁97.7mg/L、氯化钠6800mg/L、一水磷酸二氢钠140mg/L、碳酸氢钠2200mg/L、醋酸钠50mg/L、L-丙氨酸25mg/L、L-精氨酸盐酸盐70mg/L、L-天冬氨酸30mg/L、L-半胱氨酸盐酸盐一水合物0.11mg/L、L-胱氨酸二盐酸盐26mg/L、L-谷氨酸75mg/L、甘氨酸50mg/L、L-组氨酸盐酸盐一水合物21.88mg/L、L-羟脯氨酸10mg/L、L-异亮氨酸20mg/L、L-亮氨酸60mg/L、L-赖氨酸盐酸盐70mg/L、L-蛋氨酸15mg/L、L-苯丙氨酸25mg/L、L-脯氨酸40mg/L、L-丝氨酸25mg/L、L-苏氨酸30mg/L、L-色氨酸10mg/L、L-酪氨酸二钠二水合物57.66mg/L、L-缬氨酸25mg/L、抗坏血酸0.05mg/L、α-D-生育酚磷酸酯0.01mg/L、生物素0.01mg/L、骨化醇0.1mg/L、D-泛酸钙0.01mg/L、氯化胆碱0.5mg/L、叶酸0.01mg/L、肌醇0.05mg/L、三水合甲萘醌亚硫酸氢钠0.019mg/L、烟酸0.025mg/L、烟酰胺0.025mg/L、p-氨基苯甲酸0.05mg/L、盐酸吡哆辛0.05mg/L、核黄素0.01mg/L、盐酸硫胺素0.01mg/L、维生素A乙酸酯0.14mg/L、硫酸腺嘌呤10mg/L、腺嘌呤0.2mg/L、腺苷三磷酸二钠1mg/L、胆固醇0.2mg/L、2-脱氧-D-核糖0.5mg/L、D-葡萄糖1000mg/L、谷胱甘肽0.05mg/L、盐酸鸟嘌呤0.3mg/L、次黄嘌呤钠0.354mg/L、核糖0.5mg/L、胸腺素0.3mg/L、吐温80为5mg/L、尿嘧啶0.3mg/L、黄嘌呤钠0.34mg/L、酚红10mg/L、0.25mg/L亚硒酸钠和0.075mg/L无水硫酸铜。
二、牛体外受精和胚胎培养:
步骤(1)、卵母细胞的采集和体外成熟
(a)取屠宰场所得牛卵巢,抽取表面2-8mm的卵泡,收集沉淀,在体视显微镜下捡出至少含有3层卵丘细胞包裹的卵母细胞COCs(即,卵丘-卵母细胞复合体),放入包含HEPES的运输培养液中38.8℃、无二氧化碳供应、24h内运回实验室(本例是于15小时运回实验室进行下一步骤处理);
(b)将所得COCs在卵母细胞成熟培养液中洗涤1次,再转移到新的成熟培养液中培养22-24h(实际操作24小时),培养条件为38.8℃、5.5-6.5% CO2、饱和湿度;
步骤(2)、体外受精
将成熟的COCs在受精培养液中洗涤1次,再转移到受精培养液中,放入培养箱中,备用;
从液氮中取一支冻精细管,37℃水浴解冻;无菌操作剪开细管两端,使精液注入盛有精液制备培养液的15mL离心管中,328×g离心2次,每次5min,离心后弃上清;将300µL精液制备培养液加入上述离心管,重悬精子沉淀,取适当的精子悬液进行精子计数;
将计算好体积的精子悬液加入盛有卵母细胞的受精培养液滴中,并将培养盘放入培养箱,使精卵共孵育16-20h(实际操作18小时),培养条件为38.8℃、5.5-6.5% CO2、饱和湿度;
步骤(3)、胚胎体外培养及保存
体外受精操作完毕后,将胚胎周围的颗粒细胞用剥卵针去除干净,放入胚胎培养液中培养,此时记为胚胎培养的第1天,培养条件为38.8℃、6% O2、88% N2、饱和湿度,第3天记录卵裂率;第7天记录囊胚率,至第9天时统计囊胚孵化率(其为孵化囊胚数除以囊胚数所得百分数),并进行质量鉴定;
将可用胚胎在保存液中洗涤3次,在平衡液中平衡10min后转移入冷冻液,按5段装液法装入胚胎,做好标记,再放入程序降温仪中按照0.5℃/min的速度降至-35℃后,将胚胎细管迅速取出,置入液氮中冷冻保存。
三、胚胎差异染色的方法
1.选取体外培养第7天的囊胚,使用2%多聚甲醛固定20min。
2.使用含0.5% BSA的磷酸盐缓冲液(PBS-BSA)洗两次,放入透化液(50μl Triton、5μl 吐温80和9.945ml PBS)中,室温放置30min。
3.使用2M盐酸室温处理20min,然后使用100mM的Tris-HCl室温处理10min,使CDX2蛋白能够与一抗结合。
4.使用PBS-BSA清洗三次,将囊胚放入封闭液(1ml山羊血清、5μl 吐温80和8.995ml PBS)中,室温封闭1h,然后转入4℃冰箱封闭过夜。
5.弃去封闭液,CDX2一抗用封闭液按1:200稀释,室温孵育2h,弃去一抗稀释液,用PBS-BSA清洗3次,每次5min。
6.caspase-3一抗(购自Cell Signaling Technology公司)用封闭液按1:200稀释,室温孵育2h,弃去一抗稀释液,用PBS-BSA清洗3次,每次5min。
7.在避光条件下用封闭液按1:200稀释CDX2特异性二抗(购自Sigma公司),室温下避光放置1h。避光条件下弃去二抗稀释液,用PBS清洗3次,每次5min。
8.在避光条件下用封闭液按1:200稀释caspase-3特异性二抗(购自LifeTechnologies公司),室温下避光放置1h。避光条件下弃去二抗稀释液,用PBS清洗3次,每次5min。
9.加入10μg/mL的Hochest 33342染液染细胞核,室温作用5min,在荧光显微镜下观察并拍照。
10.实验重复三次,每次随机选取10个囊胚,计算凋亡率、ICM细胞数/总细胞数来评估囊胚质量。
数据统计方法:实验数据采用统计软件SAS V8中的ANOVA程序进行分析,Duncan’smultiple-range检验方法判定处理间的差异显著性,当p<0.05时认为差异显著。
在本发明中,卵裂率=受精卵裂数/受精卵数。在本发明中,囊胚率=囊胚数/卵裂胚胎数。
四、卵母细胞的体外成熟(IVM)效果——成熟率
在本实施例的步骤(1)中,经体外成熟培养完毕后,在倒置显微镜下观察,将卵母细胞有第一极体释放、保持卵丘细胞间分泌粘稠的基质、细胞层显著膨大,细胞以卵子为中心,大体呈放射状向四周扩散者判定为已成熟,记录成熟的卵母细胞数,计算成熟率。
五、结果
本实施例对中国黄牛(南阳牛,役用品种)进行试验。结果,卵裂率86.4%、桑葚胚率为66.3%、囊胚率为51.6%、凋亡率5.3%;另外,在第9天时囊胚孵化率达75.1%。卵母细胞体外成熟的成熟率达87.5%。
实施例1A:牛体外受精胚胎的培养方法(离体采集)
参照实施例1的方法,分别针对荷斯坦牛(乳牛品种)、西门塔尔牛(肉牛品种)、中国水牛(役用品种)三种牛进行试验,结果:卵裂率均在85~90%范围内、桑葚胚率均在62~67%范围内、囊胚率均在50~55%范围内、凋亡率均在4~6%范围内、第9天囊胚孵化率均在72~77%范围内;荷斯坦牛、西门塔尔牛、中国水牛三种牛的卵母细胞体外成熟的成熟率分别为83.3%、80.6%、75.6%。
实施例1B:牛体外受精胚胎的培养方法(离体采集)
参照实施例1的方法,分别针对中国黄牛(南阳牛,役用品种)、荷斯坦牛(乳牛品种)、西门塔尔牛(肉牛品种)、中国水牛(役用品种)四种牛进行试验,但是将受精培养液和精液制备培养液的配方分别作修改,其余所用试液和操作不变。受精培养液配方改为:包含112.0mM 氯化钠、4.02mM 氯化钾、2.25mM的 氯化钙二水合物、0.52mM六水氯化镁、0.83mM磷酸二氢钾、37.0mM碳酸氢钠、1.25mM丙酮酸钠、10μg/ml肝素、4mg/ml牛血清白蛋白(BSA)、100U/ml青霉素、100μg/ml链霉素、10umoL/L α-硫辛酸、25μg/ml叶酸、3mg/ml谷氨酸钠的水溶液;精液制备培养液配方改为:包含112.0mM 氯化钠、4.02mM 氯化钾、2.25mM的 氯化钙二水合物、0.52mM六水氯化镁、0.83mM磷酸二氢钾、37.0mM碳酸氢钠、1.25mM丙酮酸钠、10μg/ml肝素、4mg/ml牛血清白蛋白(BSA)、10mM咖啡因、100U/ml青霉素、100μg/ml链霉素、10umoL/L α-硫辛酸、25μg/ml叶酸、3mg/ml谷氨酸钠的水溶液。结果:四种牛的卵裂率均在88~91%范围内、桑葚胚率均在64~67%范围内、囊胚率均在77~82%范围内、凋亡率均在3~5%范围内、第9天囊胚孵化率均在74~78%范围内,例如中国黄牛的卵裂率为89.7%、桑葚胚率为66.1%、囊胚率为79.4%、凋亡率为3.8%、第9天囊胚孵化率75.3%;中国黄牛、荷斯坦牛、西门塔尔牛、中国水牛四种牛的卵母细胞体外成熟的成熟率分别为85.3%、86.4%、81.8%、75.3%。
实施例1C:牛体外受精胚胎的培养方法(离体采集)
参照实施例1B进行,不同的仅是其中的受精培养液和精液制备培养液中不添加叶酸。结果:四种牛的卵裂率均在85~89%范围内、桑葚胚率均在62~65%范围内、囊胚率均在53~56%范围内、凋亡率均在3~5%范围内、第9天囊胚孵化率均在72~76%范围内,例如中国黄牛的卵裂率为87.7%、桑葚胚率为64.8%、囊胚率为53.4%、凋亡率为4.4%、第9天囊胚孵化率75.3%;中国黄牛、荷斯坦牛、西门塔尔牛、中国水牛四种牛的卵母细胞体外成熟的成熟率分别为85.7%、83.4%、82.1%、76.8%。
实施例1D:牛体外受精胚胎的培养方法(离体采集)
参照实施例1B进行,不同的仅是其中的受精培养液和精液制备培养液中不添加谷氨酸钠。结果:四种牛的卵裂率均在86~90%范围内、桑葚胚率均在63~65%范围内、囊胚率均在53~56%范围内、凋亡率均在3~6%范围内、第9天囊胚孵化率均在73~76%范围内,例如中国黄牛的卵裂率为85.8%、桑葚胚率为63.4%、囊胚率为54.2%、凋亡率为4.7%、第9天囊胚孵化率74.2%;中国黄牛、荷斯坦牛、西门塔尔牛、中国水牛四种牛的卵母细胞体外成熟的成熟率分别为86.1%、84.3%、81.5%、75.8%。
实施例1E:牛体外受精胚胎的培养方法(离体采集)
参照实施例1B进行,不同的仅是其中的受精培养液和精液制备培养液中不添加叶酸也不添加谷氨酸钠。结果:四种牛的卵裂率均在86~90%范围内、桑葚胚率均在61~64%范围内、囊胚率均在52~55%范围内、凋亡率均在4~6%范围内、第9天囊胚孵化率均在71~75%范围内,例如中国黄牛的卵裂率为88.2%、桑葚胚率为63.6%、囊胚率为53.9%、凋亡率为5.1%、第9天囊胚孵化率74.4%;中国黄牛、荷斯坦牛、西门塔尔牛、中国水牛四种牛的卵母细胞体外成熟的成熟率分别为85.6%、82.4%、82.1%、76.8%。
文献报道活性氧(ROS)引起的氧化应激是动物生殖细胞和早期胚胎发生凋亡或永久性发育阻滞的主要原因之一,在体外培养系统中添加抗氧化剂成为降低氧化应激对动物生殖细胞损伤和提高早期胚胎发育能力的方法之一。α-硫辛酸作为一种抗氧化剂,据信可以通过直接清除自由基、螯合金属离子并促进其他抗氧化物质形成从而发挥抗氧化作用。在生殖细胞和早期胚胎中,硫辛酸可以通过降低ROS水平、促进抗氧化酶活性和线粒体活性来提高生殖细胞质量和早期胚胎发育能力。本发明人尝试在受精培养液和精液制备培养液中添加α-硫辛酸以期待提高牛体外受精的效率,然而,如上述试验结果所示的,在添加了α-硫辛酸的受精培养液和精液制备培养液中,只有在同时增补添加叶酸和谷氨酸钠二者后,才能显著地提高囊胚率,进而表明使用上述受精培养液和精液制备培养液预先处理冷冻精子,能够提高精子在后续的体外受精过程中的效率。
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
Claims (10)
1.用于牛体外受精的受精培养液,其是包含112.0mM 氯化钠、4.02mM 氯化钾、2.25mM的氯化钙二水合物、0.52mM六水氯化镁、0.83mM磷酸二氢钾、37.0mM碳酸氢钠、1.25mM丙酮酸钠、10μg/ml肝素、4mg/ml牛血清白蛋白、100U/ml青霉素、100μg/ml链霉素、10umoL/L α-硫辛酸、25μg/ml叶酸、3mg/ml谷氨酸钠的水溶液。
2.用于牛体外受精的精液制备培养液,其是包含112.0mM 氯化钠、4.02mM 氯化钾、2.25mM的 氯化钙二水合物、0.52mM六水氯化镁、0.83mM磷酸二氢钾、37.0mM碳酸氢钠、1.25mM丙酮酸钠、10μg/ml肝素、4mg/ml牛血清白蛋白、10mM咖啡因、100U/ml青霉素、100μg/ml链霉素、10umoL/L α-硫辛酸、25μg/ml叶酸、3mg/ml谷氨酸钠的水溶液。
3.牛体外受精的方法,其包括如下步骤:
将成熟的卵丘-卵母细胞复合体在受精培养液中洗涤1次,再转移到受精培养液中,放入培养箱中,备用;
从液氮中取一支冻精细管,37℃水浴解冻;无菌操作剪开细管两端,使精液注入盛有精液制备培养液的15mL离心管中,328×g离心2次,每次5min,离心后弃上清;将300µL精液制备培养液加入上述离心管,重悬精子沉淀,取适当的精子悬液进行精子计数;
将计算好体积的精子悬液加入盛有卵母细胞的受精培养液滴中,并将培养盘放入培养箱,使精卵共孵育16-20h,培养条件为38.8℃、5.5-6.5% CO2、饱和湿度,完成体外受精操作,以备用于胚胎体外培养,
其中,
所述受精培养液是包含112.0mM 氯化钠、4.02mM 氯化钾、2.25mM的氯化钙二水合物、0.52mM六水氯化镁、0.83mM磷酸二氢钾、37.0mM碳酸氢钠、1.25mM丙酮酸钠、10μg/ml肝素、4mg/ml牛血清白蛋白、100U/ml青霉素、100μg/ml链霉素、10umoL/L α-硫辛酸、25μg/ml叶酸、3mg/ml谷氨酸钠的水溶液。
4.根据权利要求3的方法,其中所述精液制备培养液是包含112.0mM 氯化钠、4.02mM氯化钾、2.25mM的 氯化钙二水合物、0.52mM六水氯化镁、0.83mM磷酸二氢钾、37.0mM碳酸氢钠、1.25mM丙酮酸钠、10μg/ml肝素、4mg/ml牛血清白蛋白、10mM咖啡因、100U/ml青霉素、100μg/ml链霉素、10umoL/L α-硫辛酸、25μg/ml叶酸、3mg/ml谷氨酸钠的水溶液。
5.根据权利要求3的方法,其中所述卵丘-卵母细胞复合体是通过如下方法采集和体外成熟的:
a)离体采集:取屠宰场所得牛卵巢,抽取表面2-8mm的卵泡,收集沉淀,在体视显微镜下捡出至少含有3层卵丘细胞包裹的卵母细胞COCs即卵丘-卵母细胞复合体,放入包含HEPES的运输培养液中38.8℃、无二氧化碳供应、24h内运回实验室;
b)将以上离体采集所得COCs在卵母细胞成熟培养液中洗涤1次,再转移到新的成熟培养液中培养22-24h,培养条件为38.8℃、5.5-6.5% CO2、饱和湿度,得到成熟的卵丘-卵母细胞复合体。
6.根据权利要求3的方法,其还包括如下操作:
(3)胚胎体外培养及保存
体外受精操作完毕后,将胚胎周围的颗粒细胞用剥卵针去除干净,放入胚胎培养液中培养,此时记为胚胎培养的第1天,培养条件为38.8℃、6% O2、88% N2、饱和湿度,第3天记录卵裂率;第7天记录囊胚率,至第9天时统计囊胚孵化率,并进行质量鉴定;
将可用胚胎在保存液中洗涤3次,在平衡液中平衡10min后转移入冷冻液,按5段装液法装入胚胎,做好标记,再放入程序降温仪中按照0.5℃/min的速度降至-35℃后,将胚胎细管迅速取出,置入液氮中冷冻保存。
7.根据权利要求5的方法,所述成熟培养液是添加了100mL/L FBS、10μg/mL FSH、10μg/mL LH、1μg/mL E2、20ng/mL EGF的BY基础培养液;
所述运输培养液中包含:甘氨酸50.0mg/L、L-丙氨酸25.0mg/L、L-精氨酸盐酸盐70.0mg/L、L-天冬氨酸30.0mg/L、L-胱氨酸二盐酸盐26.0mg/L、L-谷氨酸75.0mg/L、L-谷氨酰胺100.0mg/L、L-组氨酸盐酸盐一水合物21.88mg/L、L-羟基脯氨酸10.0mg/L、L-异亮氨酸40.0mg/L、L-亮氨酸60.0mg/L、L-赖氨酸盐酸盐70.0mg/L、L-甲硫氨酸15.0mg/L、L-苯丙氨酸25.0mg/L、L-脯氨酸40.0mg/L、L-丝氨酸25.0mg/L、L-苏氨酸30.0mg/L、L-色氨酸10.0mg/L、L-酪氨酸二钠盐二水合物58.0mg/L、L-缬氨酸25.0mg/L、抗坏血酸0.05mg/L、生物素0.01mg/L、氯化胆碱0.5mg/L、D-泛酸钙0.01mg/L、叶酸0.01mg/L、甲萘醌0.01mg/L、烟酰胺0.025mg/L、烟酸0.025mg/L、对氨基苯甲酸0.05mg/L、盐酸吡哆醛0.025mg/L、盐酸吡哆醇0.025mg/L、核黄素0.01mg/L、盐酸硫胺素0.01mg/L、维生素A醋酸酯0.1mg/L、维生素D2即骨化醇0.1mg/L、α-生育酚磷酸钠盐0.01mg/L、肌醇0.05mg/L、无水氯化钙200.0mg/L、硝酸铁九水合物0.7mg/L、无水硫酸镁97.67mg/L、氯化钾400.0mg/L、氯化钠6800.0mg/L、磷酸二氢钠一水合物140.0mg/L、硫酸腺嘌呤10.0mg/L、5'-磷酸腺苷0.2mg/L、三磷酸腺苷1.0mg/L、胆固醇0.2mg/L、葡萄糖1000.0mg/L、脱氧核糖0.5mg/L、还原型谷胱甘肽0.05mg/L、盐酸鸟嘌呤0.3mg/L、次黄嘌呤钠0.354mg/L、酚红20.0mg/L、核糖0.5mg/L、醋酸钠50.0mg/L、胸腺嘧啶0.3mg/L、吐温80为20.0mg/L、尿嘧啶0.3mg/L、黄嘌呤钠0.3mg/L,以及0.01IU/mL的FSH、0.01IU/mL的LH、1μg/mL的E2,50ng/mL的EGF、100ng/mL的IGF、10%庆大霉素、55µg/mL丙酮酸钠、1.2mM/L半胱氨酸、3mg/mL的BSA、10mM/L的HEPES。
8.根据权利要求7的方法,所述运输培养液中还包含牛磺酸和葡萄糖酸锌,牛磺酸的浓度为30~50mg/L,葡萄糖酸锌的浓度为1~3mg/L。
9.根据权利要求6的方法,所述胚胎培养液包含:109.5mM氯化钠、3.1mM氯化钾、26.2mM碳酸氢钠、0.8mM六水氯化镁、1.19mM 磷酸二氢钾、0.4mM丙酮酸钠、1.5mM葡萄糖、5mM半乳糖酸钙、2.5v/v%胎牛血清(FBS)、L-谷氨酰胺1mM、2v/v%必需氨基酸、1v/v%非必须氨基酸、3mM的谷胱甘肽、枸橼酸钠0.04w/v%、麦芽糖0.02w/v%的水溶液;所述必需氨基酸为以下氨基酸按重量比例添加:L-盐酸精氨酸6.32g、L-胱氨酸二盐酸盐1.564g、L-盐酸组氨酸一水物2.1g、L-异亮氨酸2.625g、L-亮氨酸2.62g、L-赖氨酸盐酸盐3.625g、L-蛋氨酸0.755g、L-苯丙氨酸1.65g、L-苏氨酸2.38g、L-色氨酸0.51g、L-酪氨酸1.8g和L-缬氨酸2.34g,所述非必须氨基酸为以下氨基酸按重量比例添加:L-丙氨酸0.89g、L-天门冬酰胺一水物1.5g、L-天冬氨酸1.33g、L-谷氨酸1.47g、甘氨酸0.75g、L-脯氨酸1.15g和L-丝氨酸1.05g。
10.根据权利要求7的方法,所述BY基础培养液是包含如下组分的水溶液:氯化钙180~220mg/L、九水硝酸铁0.70~0.75mg/L、氯化钾380~420mg/L、硫酸镁90~100mg/L、氯化钠6500~7000mg/L、一水磷酸二氢钠130~150mg/L、碳酸氢钠2000~2500mg/L、醋酸钠40~60mg/L、L-丙氨酸20~30mg/L、L-精氨酸盐酸盐60~80mg/L、L-天冬氨酸25~35mg/L、L-半胱氨酸盐酸盐一水合物0.10~0.12mg/L、L-胱氨酸二盐酸盐20~30mg/L、L-谷氨酸70~80mg/L、甘氨酸40~60mg/L、L-组氨酸盐酸盐一水合物20~25mg/L、L-羟脯氨酸8~12mg/L、L-异亮氨酸15~25mg/L、L-亮氨酸50~70mg/L、L-赖氨酸盐酸盐60~80mg/L、L-蛋氨酸10~20mg/L、L-苯丙氨酸20~30mg/L、L-脯氨酸30~50mg/L、L-丝氨酸20~30mg/L、L-苏氨酸25~35mg/L、L-色氨酸8~12mg/L、L-酪氨酸二钠二水合物55~60mg/L、L-缬氨酸20~30mg/L、抗坏血酸0.04~0.06mg/L、α-D-生育酚磷酸酯0.008~0.012mg/L、生物素0.008~0.012mg/L、骨化醇0.08~0.12mg/L、D-泛酸钙0.008~0.012mg/L、氯化胆碱0.4~0.6mg/L、叶酸0.008~0.012mg/L、肌醇0.04~0.06mg/L、三水合甲萘醌亚硫酸氢钠0.015~0.025mg/L、烟酸0.02~0.03mg/L、烟酰胺0.02~0.03mg/L、p-氨基苯甲酸0.04~0.06mg/L、盐酸吡哆辛0.04~0.06mg/L、核黄素0.008~0.012mg/L、盐酸硫胺素0.008~0.012mg/L、维生素A乙酸酯0.1~0.2mg/L、硫酸腺嘌呤8~12mg/L、腺嘌呤0.15~0.25mg/L、腺苷三磷酸二钠0.8~1.2mg/L、胆固醇0.15~0.25mg/L、2-脱氧-D-核糖0.4~0.6mg/L、D-葡萄糖800~1200mg/L、谷胱甘肽0.04~0.06mg/L、盐酸鸟嘌呤0.25~0.35mg/L、次黄嘌呤钠0.3~0.4mg/L、核糖0.4~0.6mg/L、胸腺素0.25~0.35mg/L、吐温80为4~6mg/L、尿嘧啶0.25~0.35mg/L、黄嘌呤钠0.3~0.4mg/L、酚红8~12mg/L、亚硒酸钠0.2~0.3mg/L、硫酸铜0.05~0.1mg/L。
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