CN113817668A - 一种体外受精优化方法及其在动物大量快速繁殖中的应用 - Google Patents
一种体外受精优化方法及其在动物大量快速繁殖中的应用 Download PDFInfo
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Abstract
本发明提供了一种体外受精(In vitrofertilization,IVF)优化方法及其在动物中大量快速繁殖中的应用,使用该方法能显著提高体外受精后的卵裂率和生仔率,本发明还提供了一种受精培养液(HTFG),包含该受精培养液的体外受精体系可以提高精子体外受精的卵裂率和生仔率。
Description
技术领域:
本发明属于辅助生殖技术领域,具体涉及一种体外受精优化方法及其在动物大量快速繁殖和保种中的应用。
背景技术:
基于科研和应用研究对所需C57BL/6小鼠(以下简称B6小鼠)品系的多种技术服务需求中,大量快速扩群繁殖尤为重要,其中冷冻精子品系的短时间大量获得活体小鼠更受关注。
目前针对小鼠快速扩群生产的方法主要包括交配扩繁和体外受精(In VitroFertilization,以下简称IVF)生产两种方式,其中基于IVF体系的辅助生殖技术具有快速、高效、节约成本、对小鼠的质量要求较低等优点。IVF是体外生产技术中的核心步骤,在大量快速繁殖中起着至关重要重要的作用。正常受精过程包括精卵识别、精卵结合、精卵融合和父母双方基因组的有序整合等等。IVF技术的重点内容是精子体外获能。目前,精子体外获能方法在许多物种已相当成熟。在一个稳定的获能体系中,精子质量和精子浓度常常是影响IVF技术的变量因素。IVF技术的实验操作时间虽然不长,但也可能产生一些重要问题,比如多精受精(Polyspermy),这一现象使得胚胎遗传物质的组成发生异常,导致胚胎发育率低。多精受精可能是由于精子浓度高造成,但也和卵细胞的成熟质量有关。还有研究表明微管微丝(Microfilaments)也影响着受精过程中纺锤体的旋转、精卵结合、皮质颗粒的分泌、第二极体的排出等一系列活动。在小鼠的精卵结合中,发现微丝微管的调节器JAS(Jasplakinolide)发挥着抑制作用。有研究报道在精卵结合中,与细胞骨架有关的ρ蛋白利于受精的发生。小鼠的IVF胚胎也存在一定的发育缺陷,特别是多精受精导致的胚胎异常发育对于小鼠IVF的效率影响很大,因此研究小鼠IVF胚胎发育过程中如何降低多精受精率,提高IVF胚胎卵裂率和发育率具有重要的实用价值。
传统的哺乳动物自然交配繁育技术受很多因素的影响,如动物性别、周龄和自身营养状态等。随着辅助生殖技术的发展,精子体外获能方法和胚胎体外培养体系在许多物种已相当成熟,但IVF技术的重点内容是提高精子体外获能和降低胚胎畸形率。在一个稳定的体外受精体系中,培养液常常是影响IVF技术的变量因素。
在体外受精过程中,一些物质被添加进受精液中以提高精子活力。GSH作为精子和卵细胞主要的非蛋白类抗氧化物质,具有提高精子活力,清除受精液中的ROS,调节精子中cAMP水平等作用。因此,在受精液中添加GHS能够提高受精后胚胎发育能力。
朱佳伟在其研究中发现,在小鼠体外受精中,通过在各个GSH添加组中受精后的卵裂率只有当GSH的浓度为300毫摩尔时才有较明显的提高,而其他各组之间差异不显著,从而认为低浓度的GSH并不能很高地保护精子使其免受ROS的伤害(“GSH对小鼠卵细胞体外受精成熟体外受精的影响”,硕士学位论文,东北农业大学,2008年)。
但是高浓度的GSH也会起到相反的作用。Boquest AC等(1999)的研究表面,在牛受精液中添加500微升的GSH会降低囊胚发育率。Jeong BS等(2001)也发现,在猪受精液中添加高浓度的GSH会降低卵裂率和囊胚率。因此降低对GSH的依赖也同时提高卵裂率和囊胚率。因此,寻找GSH浓度的最优范围是一个值得努力的方向。
并且,受精胚胎的卵裂依赖于培养体系的营养成分和PH值等,目前已成为亟待解决的问题。
发明内容:
本发明目的在于寻找一种更好的体外快速繁殖和保种的体系,提高受精和早期胚胎的质量,为动物的体外生产技术和辅助生殖技术提供优化的方案,提高繁育速度,缩短获得实验材料的时间,加速科研进程。
本发明的第一方面,提供了一种受精培养液(HTFG),所述的受精培养液包含受精液(HTF)和可选的包括还原型谷胱甘肽(GSH)。
优选的,所述GSH的浓度为0.006mg/ml~0.35mg/ml。更优选的,所述的GSH的浓度为0.006mg/ml~0.0307mg/ml,进一步优选的,所述的GSH的浓度为0.006mg/ml~0.0154mg/ml。优选的,所述GSH的浓度为上述范围内的任一值,例如0.007mg/ml、0.0077mg/ml、0.009mg/ml、0.01mg/ml、0.0154mg/ml、0.02mg/ml、0.03mg/ml、0.0307mg/ml、0.035mg/ml等等。
优选的,所述的HTFG中的受精液(HTF)包含电解质、碳源和氮源,例如钠盐、钾盐、镁盐、钙盐、葡萄糖和牛血清白蛋白。
优选的,所述的HTFG还包含氯化钠5.938mg/ml;氯化钾0.350mg/ml;七水硫酸镁0.049mg/ml;磷酸二氢钾0.054mg/ml;氯化钙0.570mg/ml;碳酸氢钠3 2.1mg/ml;葡萄糖0.500mg/ml;乳酸钠0.34ml/ml;丙酮酸钠0.037mg/ml;牛血清白蛋白4.0mg/ml。
本发明的第二方面,提供了一种体外受精体系,所述的体系包含如上所述的受精培养液(HTFG)。
优选的,所述的体系还包括精子获能液(TYH)和洗涤培养液。
优选的,所述的TYH包含钠盐、钾盐、钙盐、镁盐、葡萄糖、β-环糊精和聚乙烯醇。
优选的,所述的TYH包含氯化钠6.976mg/ml;氯化钾0.356mg/ml;二水氯化钙0.251mg/ml;葡萄糖1.0mg/ml;丙酮酸钠0.055mg/ml;七水硫酸镁0.293mg/ml;磷酸二氢钾0.162mg/ml;碳酸氢钠2.106mg/ml;β-环糊精0.983mg/ml;聚乙烯醇1.0mg/ml。
优选的,所述的洗涤培养液的成分为受精液。
优选的,所述的TYH、HTFG和洗涤培养液的形态为微滴。
优选的,所述的TYH微滴:HTFG微滴:洗涤培养液微滴个数比例为1:1:4。
优选的,所述的微滴还覆盖有矿物油。
更优选的,所述体外受精体系可以不包含GSH,使其适合新鲜精子的培养。
更优选的,所述体外受精体系中的GSH浓度为0.006mg/ml~0.35mg/ml,使其适合冷冻精子的培养。
本发明的第三方面,提供了一种如上所述的受精培养液或如上所述的体外受精体系在体外受精、体外繁殖和保种中的应用。
本发明的第四方面,提供了一种体外受精优化方法,其特征在于,所述的方法包括如下步骤:
(1)精子在精子获能液中培养;
(2)将(1)获能的精子与卵细胞在受精培养液中孵育;
(3)将(2)中受精的卵细胞在洗涤培养液中孵育;
其中,所述的方法采用如上所述的体外受精体系。
优选的,所述的精子为新鲜精子或冷冻精子。
更优选的,所述精子来源于小鼠的精子。
优选的,所述小鼠为C57BL/6(B6)、FVB、BALB/c、129等其他背景遗传修饰小鼠。
更优选的,所述的新鲜精子来源于10~32周龄雄鼠的新鲜精子,更优选的,所述雄鼠为12~18周龄;
更优选地,所述冷冻精子来源于使用麦管方式冻存的冷冻精子。
在一个具体实施方式中,对新鲜精子使用不含GSH的体外优化培养体系。
在一个具体实施方式中,对冷冻精子使用含GSH的如上所述的体外优化培养体系。
优选的,所述的受精培养液为受精培养液微滴。
优选的,所述的精子获能液为精子获能液微滴。
优选的,所述的洗涤培养液为洗涤培养液微滴。
优选的,所述的方法包含用移液器向培养皿中滴入精子获能液微滴,并覆盖矿物油,得到获能皿。
优选的,每个获能皿中有1滴如上所述的TYH微滴。
优选的,所述的TYH微滴的体积为90μl-100μl。
优选的,当所述精子为冷冻精子时,所述的TYH的体积为90μl;进一步优选的,当所述精子为新鲜精子时,所述的TYH微滴的体积为100μl。
优选的,所述的方法包含用移液器向培养皿中滴入受精培养液微滴,并覆盖矿物油,得到受精培养皿。
优选的,每个受精培养皿中有1滴如上所述的HTFG微滴。
优选的,所述的HTFG微滴的体积为90μl-200μl。
优选的,所述精子为冷冻精子时,所述的HTFG微滴的体积为90-150μl;当所述精子为新鲜精子时,所述的HTFG微滴的体积为150-200μl。
优选地,所述的方法包含用移液器向培养皿中滴入洗涤培养液微滴,并覆盖矿物油,得到洗涤培养皿;
优选的,每个培养皿中有4滴如上所述的洗涤培养液微滴,所述的皿称为洗涤培养皿。
优选的,所述的洗涤培养液微滴的体积为60μl-80μl。
优选的,所述的卵细胞通过超排的方法获得。优选的,所述的超排方法包含对受试个体施以一种或两种以上的激素,优选的,所述的激素为促性腺激素;优选的,所述激素为PMSG和HCG;优选的,所述的PMSG和HCG被先后施予受试个体;优选的,所述的PMSG的施与剂量为5-10IU/只;优选的,所述的HCG的施与剂量为5-10IU/只。
在一个具体实施方式中,所述的卵细胞通过以下方法获得:挑选3~4周龄(10~12g)B6雌鼠,超排,以10IU/只的剂量腹腔注射PMSG(Pregnant Mare SerumGonadotrophin),随后以10IU/只的剂量腹腔注射HCG(Human Chorionic Gonadotropin)。其中,HCG注射与PMSG的注射之间至少间隔47小时;优选的,HCG注射后16~18h取输卵管得到卵细胞。
优选地,所述的步骤(2)的受精时间是4-6h。
优选的,所述的步骤(3)的体外培养时间是12h以上。
优选的,对步骤(3)的胚胎数量和质量进行严格控制,每枚卵细胞数对应1微升洗涤培养液,并且根据极体的排出判断受精卵的质量,将质量好的受精卵放置一起进行培养。
本发明技术方案的优点在于:
通过本发明提供体外受精方法,打破传统认知,可以降低现有的体外受精体系对GSH的依赖,在更低的GSH浓度下,甚至可以在不包含GSH的情况下,获得相同的或者更高的2细胞发育率和生仔率。
本发明提供的受精培养液HTFG包含0.005mg/ml~0.035mg/ml的GSH,可以在比现有技术更低的GSH浓度的情况下,使冷冻精子体外受精更高的2细胞发育率和生仔率,都能够快速繁殖,具体地,体外受精率最高可达到80%以上,并且生仔率最高可达到40%以上。
优选的,本发明通过改进体外受精体系中的各个组分的含量,形态,以及比例,优化了体系的效果,适合多个物种,无论是对新鲜精子和冷冻精子都可以获得相同的或者更高的2细胞发育率和生仔率。
本发明采用了更为简便,高效的方式对常用小鼠品系的快速繁育IVF技术体系进行了确立,并改善了实验鼠选择、超排、受精孵育和洗涤培养环节的细节,提高了实验动物繁育的效率。
附图说明:
图1为采用本发明的IVF体系的精子的胚胎发育率和生仔率,其中图1A为新鲜精子采用本发明的IVF体系的胚胎发育率;图1B为冷冻精子采用本发明的IVF体系的胚胎发育率;图1C为新鲜精子采用本发明的IVF体系的生仔率;图1D为冷冻精子采用本发明的IVF体系的生仔率。
具体实施方式:
为了更好地说明本发明,以下结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,所描述的实施例仅是本发明的部分实施例,而不是全部。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。
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
以下实施例中,精子获能液采用简称TYH,受精液采用简称HTF,还原型谷胱甘肽采用简称GSH,受精培养液采用简称HTFG,体外受精技术采用简称IVF。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。所用试剂包括:激素(PMSG和HCG)购自宁波三生公司;试剂粉末GSH(G-4251),矿物油(M-8410),DPBS(D-8662),体外操作液M2(M-7167),麻醉剂Avertin购自Sigma公司;精子获能液、受精液和洗涤培养液均是实验室自行配制。洗涤培养液提供小鼠胚胎体外卵裂培养的营养。C57BL/6背景遗传修饰小鼠(以下简称B6小鼠)来自美国JAX的近交系小鼠。
实施例1激素对卵细胞超排的影响
选择4周龄B6雌鼠,按照表1的激素剂量进行超排操作,不同激素剂量对排卵数量影响如下:
表1不同激素剂量对排卵数量的影响
激素剂量(IU) | 小鼠数量(只) | 排卵数量(枚) | 平均排卵量(枚) |
0 | 10 | 0 | 0.00 |
5 | 42 | 1494 | 36.89 |
10 | 39 | 1635 | 41.62 |
数据显示5IU和10IU的激素注射剂量都可以使实验雌鼠达到超数排卵的效果。但是综合实验的稳定性和效率,优选10IU。
实施例2:建立小鼠IVF优化体系
1、配置TYH和制备获能皿
(1)配置TYH:每毫升TYH含有氯化钠6.976mg;氯化钾0.356mg;二水氯化钙0.251mg;葡萄糖1.0mg;丙酮酸钠0.055mg;七水硫酸镁0.293mg;磷酸二氢钾0.162mg;碳酸氢钠2.106mg;β-环糊精0.983mg;聚乙烯醇1.0mg;滤器过滤后得到TYH培养液;
(2)制备获能皿
用移液器向培养皿中滴入TYH微滴,所述获能液微滴的体积为100μl,并覆盖矿物油,得到获能皿A。
用移液器向培养皿中滴入TYH微滴,所述获能液微滴的体积为90μl,并覆盖矿物油,得到获能皿B。
2、配置实验组和对照组的受精培养液、制备受精培养皿
(1)配置不含GSH的HTFG:每毫升培养液含有氯化钠5.938mg;氯化钾0.350mg;七水硫酸镁0.049mg;磷酸二氢钾0.054mg;氯化钙0.570mg;碳酸氢钠3 2.1mg;葡萄糖0.500mg;乳酸钠0.34ml;丙酮酸钠0.037mg;1%双抗;牛血清白蛋白4.0mg;滤器过滤后得到受精液HTF即为对照组。
(2)配置含有不同浓度GSH的受精培养液(HTFG):称量0.0307gGSH粉末,溶解到1mlHTF内,取2.5μl到1mlHTF中混匀即0.25mM使用液实验例1(HTFG-1),取5μl到1mlHTF中混匀即0.5mM使用液实验组2(HTFG-2),再取10μl到1mlHTF中混匀即1.0mM使用液实验组3(HTFG-3)。于是获得不同浓度GSH的实验组HTFG(实验组1的GSH=0.0077mg/ml,实验组2的GSH=0.0154mg/ml,实验组3的GSH=0.0307mg/ml)。
(3)制备受精培养皿
以HTF作为对照组,以含有不同浓度GSH的HTFG为实验组,将这四组液体用移液器向不同的培养皿中滴入(对照组、HTFG-1、HTFG-2、HTFG-3)微滴,微滴的个数为1个,微滴的体积为200μl,并覆盖矿物油,得到四组受精培养皿A(对照组、HTFG-1、HTFG-2、HTFG-3)。
用移液器向不同培养皿中(对比例、HTFG-1、HTFG-2、HTFG-3)滴入微滴,微滴的个数为1个,微滴的体积为90μl,并覆盖矿物油,得到四组受精培养皿B(对照组、HTFG-1、HTFG-2、HTFG-3)。
3、制备洗涤培养皿
用移液器向培养皿中滴入HTF微滴,微滴的个数为4个,微滴的体积为80μl,并覆盖矿物油,得到洗涤培养皿。
实施例3:小鼠新鲜精子IVF优化方法
(1)新鲜精子IVF优化体系:选取成年12周龄雄鼠,最佳周龄是12~18周龄,安乐死取附睾尾于矿物油内,显微剪横切面剪开附睾尾顶端,将精子转移入实施例2的获能皿A中的TYH微滴内,置于37℃,二氧化碳5%的培养箱中孵育;选取3~4周龄(10~12g)B6雌鼠超排,PMSG腹腔注射10IU/只,47h后HCG腹腔注射10IU/只,HCG注射后16~18h,置于矿物油内,取得卵细胞转移至200μl实施例2制备的受精培养皿A的微滴内(对照组、HTFG-1、HTFG-2、HTFG-3);取获能后精子2μl或3μl加入HTF微滴;孵育受精4~6h后,将受精的卵细胞转移到实施例2制备的洗涤培养皿内的微滴内,清洗2~3次,按照每枚卵细胞数对应1毫升培养液,并且根据极体的排出判断受精卵的质量,将质量好的受精卵放置一起进行培养。第二天收集2细胞胚胎。
(2)统计B6小鼠新鲜精子的IVF及数据,结果如表2和图1A所示。
表2 B6小鼠新鲜精子的IVF及数据
受精卵数(枚) | 2细胞发育数(枚) | 2细胞发育率(%) | |
对照组 | 491 | 357 | 72.71% |
HTFG-1 | 607 | 444 | 73.15% |
HTFG-2 | 604 | 449 | 74.34% |
HTFG-3 | 612 | 502 | 82.03% |
根据上述结果可知:新鲜精子在本发明的体外受精体系下,添加不同浓度GSH,对于2细胞发育率提高均是不显著。这个数据说明,本发明的体外受精体系下,精子获能液和受精液本身给精子和卵子提供了足够的营养成分,不需要再增加GSH的辅助。而某商业化的试剂需要添加GSH提高鲜精的体外受精效率。因此,对于新鲜精子的体外受精体系添加GSH没有影响,可以采用不添加GSH的体外受精体系,减少实验步骤,降低成本。
实施例4:小鼠冷冻精子IVF优化方法
(1)冻存精子IVF优化体系:37℃水浴锅,从液氮内取出需要复苏的冻精麦管,室温10s,37℃水浴10min;眼科剪剪冻精两侧,用注射器将10μl冻精转移入实施例2制备的获能皿B中的TYH微滴内,置于37℃,5%二氧化碳的培养箱中孵育获能;选取3~4周龄(10~12g)B6雌鼠超排,PMSG腹腔注射10IU/只,47h后HCG腹腔注射10IU/只,HCG注射后16~18h,置于矿物油内,取得卵细胞转移至90μl受精培养皿A的微滴内(对照组、HTFG-1、HTFG-2、HTFG-3);取获能后精子10μl到20μl加入实施例2制备的受精皿B的HTF微滴中,孵育受精4~6h后,将卵细胞转移到实施例2的洗涤培养液微滴内,清洗2~3次,按照每枚卵细胞数对应1毫升培养液,并且根据极体的排出判断受精卵的质量,将质量好的受精卵放置一起进行培养。第二天收集2细胞胚胎。
(2)统计B6小鼠冷冻精子的IVF及数据,不同组间的数据差异性分析采用one-wayANOVA方法,利用Bonferroni’s post hoc检验组间差异是否显著,*表示P<0.05,结果如表3和图1B所示。
表3 B6小鼠冷冻精子的IVF及数据
受精卵数(枚) | 2细胞发育数(枚) | 2细胞发育率(%) | |
对照组 | 288 | 33 | 11.46% |
HTFG-1 | 342 | 44 | 12.87% |
HTFG-2 | 147 | 60 | 40.82% |
HTFG-3 | 379 | 132 | 34.83% |
根据上述结果可知:对于冷冻精子而言,随着GSH的浓度增加,2细胞发育率随之增加,在GSH的浓度达到0.0154mg/ml左右时,2细胞发育率达到最高,然后开始平缓下降,但GSH最高浓度为0.0307mg/ml时,其2细胞发育率依然高于对照组。因此,本发明发现,低浓度GSH可以增加冷冻精子的2细胞发育率,其浓度是某些商业化试剂添加浓度的一半。
实施例5:小鼠体外受精卵的繁殖
(1)准备代孕受体:选取7周龄以上ICR雄鼠进行结扎,试配成功后使用;将体重满足26~32g的ICR雌鼠做受体。每天下午3点将选取好的ICR雌鼠合笼结扎雄鼠,第二天早上见栓,见栓ICR雌鼠是代孕受体。
(2)用实施例2和实施例3获得的2细胞胚胎进行输卵管壁移植。
(3)统计生仔数,不同组间的数据差异性分析采用one-way ANOVA方法,利用Bonferroni’s post hoc检验组间差异是否显著,*表示P<0.05,结果如图1C和图1D所示。结果显示和2细胞发育率趋势一致,对于新鲜精子而言,增加GSH浓度并不会增加生仔率,而对于冷冻精子,低浓度GSH就可以大幅度增加生仔率。
以上详细描述了本发明的优选实施方式,但是,本发明并不限于上述实施方式中的具体细节,在本发明的技术构思范围内,可以对本发明的技术方案进行多种简单变型,这些简单变型均属于本发明的保护范围。
Claims (10)
1.一种受精培养液(HTFG),其特征在于,所述受精培养液包括受精液和可选的包括还原型谷胱甘肽(GSH)。
2.根据权利要求1所述的受精培养液,其特征在于,所述GSH的浓度为0.006mg/ml~0.35mg/ml。
3.一种体外受精体系,其特征在于,所述体系包括权利要求1-2任一所述的受精培养液。
4.根据权利要求3所述的体外受精体系,其特征在于,所述体系还包括精子获能液和洗涤培养液。
5.根据权利要求4所述的体外受精体系,其特征在于,受精培养液、精子获能液和洗涤培养液均为微滴形态。
6.权利要求1-2任一所述的受精培养液或权利要求3-5任一所述的体外受精体系在体外受精、体外繁殖和保种中的应用。
7.一种体外受精的方法,其特征在于,所述的方法包括如下步骤:
(1)精子在精子获能液中培养;
(2)将(1)获能的精子与卵细胞在受精培养液中孵育;
(3)将(2)中受精的卵细胞在洗涤培养液中孵育;
其中,所述的方法采用权利要求3-5任一所述的体外受精体系。
8.根据权利要求7所述的方法,其特征在于,所述的卵细胞通过超排方法获得,优选的,所述的超排方法包含对受试个体施以一种或两种以上的激素,优选的,所述的激素为促性腺激素。
9.根据权利要求7-8任一所述的方法,其特征在于,所述的精子来源于新鲜精子,优选的,所述的新鲜精子来源于10~32周龄雄鼠;或者,所述的精子来源于冷冻精子。
10.根据权利要求9所述的方法,其特征在于,当精子来源于冷冻精子时,所述的受精培养液包含还原型谷胱甘肽(GSH),所述的GSH的浓度为0.006mg/ml~0.35mg/ml。
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