CN114540283A - 用于牛体外胚胎生产的高效玻璃化冷冻方法 - Google Patents
用于牛体外胚胎生产的高效玻璃化冷冻方法 Download PDFInfo
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Abstract
本发明提供一种用于牛体外胚胎生产的高效玻璃化冷冻方法。本发明首次将β‑烟酰胺单核苷酸应用于牛体外胚胎生产中的牛IVF胚胎玻璃化冷冻技术中,通过在牛卵母细胞体外成熟液和牛体外受精胚胎培养液中添加一定浓度的β‑烟酰胺单核苷酸,然后将牛体外胚胎进行玻璃化冷冻,能够极大地提高牛IVF胚胎的玻璃化冷冻效率。进一步向牛卵母细胞体外成熟液和牛体外受精胚胎培养液中按比例添加生长因子和缝隙链接蛋白可进一步提高玻璃化冷冻效率。更进一步地,通过向玻璃化冷冻液中添加一定浓度的纳米颗粒可有助于进一步提高玻璃化冷冻效率。
Description
技术领域
本发明涉及牛体外胚胎生产技术领域,具体地说,涉及一种用于牛体外胚胎生产的高效玻璃化冷冻方法。
背景技术
牛体外胚胎生产具有重要的经济意义和社会价值,它可以用为克隆、核移植、转基因动物生产等提供胚胎来源,快速扩繁优良家畜群体,作为种质资源保存的手段保护濒危动物,具有重要的理论意义和社会意义。然而,牛体外胚胎生产冷冻后移植妊娠率(35.89+3.87%)显著低于新鲜体外胚胎生产(51.35+1.87%),这极大地限制了它们的应用(Niu etal.,2014)。据IETS统计数据显示,1997-2017年期间全球牛体外胚胎移植以鲜胚移植为主,冻胚移植比例仅为20%,与此同时,全球体内胚胎移植中冻胚移植比例超过60%(Ferréetal.,2020)。可见,牛体外胚胎生产冷冻技术已经成为制约牛体外胚胎产业化应用的关键瓶颈,亟需建立高效的用于牛体外胚胎生产的冷冻技术。
发明内容
本发明的目的是提供一种用于牛体外胚胎生产的高效玻璃化冷冻方法。
为了实现本发明目的,一方面,本发明提供β-烟酰胺单核苷酸在牛体外胚胎生产中提高牛IVF胚胎玻璃化冷冻效率中的应用。
研究表明,胚胎脂滴含量与其冷冻后发育能力呈负相关。通过对降低胚胎脂滴含量是否可促进其发育能力和改善冷冻效果进行研究。结果表明,通过离心去脂法和化学去脂法均可有效降低脂滴含量,并提高胚胎的发育能力与冷冻效果。离心去脂法主要通过CB处理增强细胞骨架稳定性,随后离心使脂滴极化,再利用显微打孔技术将脂滴吸出离心降脂法需要专业的显微操作设备、技术难度较大,且打孔会影响卵母细胞或胚胎的后续发育能力。因此,该方法的应用逐渐减少。相较于离心去脂法,添加降脂物质降脂法细胞毒性小、不存在机械损伤且去脂效果好。因此,添加降脂物质已成为当前胚胎去脂的主要方法。β-烟酰胺单核苷酸(Nicotinamide mononucleotide,NMN)又称为烟酰胺单核苷酸,是合成辅酶I--烟酰胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide,NAD+)的关键前体之一,广泛存在于各类生物细胞,参与众多代谢活动。在动物繁殖生理中,NMN可通过恢复线粒体功能、减少活性氧、维持正常的纺锤体与染色体结构提高衰老卵母细胞的减数分裂和受精能力,降低胚胎致死率。在促进脂代谢方面,NMN也具有巨大的应用潜能。Wang LF(2017)等报道烟酰胺磷酸核糖转移酶(Nicotinamide phosphoribosyltransferase,NAMPT)可通过激活Sirt1信号通路抑制FASN和ACC等成脂基因的表达进而减少脂质沉积;NMN作为NAMPT的酶产物,也可有效抑制FK866(NAMPT抑制剂)诱导的肝脏细胞脂质沉积。与之相似,UddinGM(2020)发现NMN可通过下调脂肪合成(脂肪酸合酶Fasn;乙酰辅酶a羧化酶1Acc1)、转运(脂肪酸转运蛋白CD36)相关基因的表达,上调脂肪分解代谢相关基因(肉碱脂酰转移酶1Cpt1)的表达抑制肝脏脂肪沉积。这些研究表明NMN有可能作为潜在的卵母细胞及胚胎去脂物质用以提高其耐冻性,促进牛体外冷冻胚胎的生产。
另一方面,本发明提供一种用于牛体外胚胎生产的高效玻璃化冷冻方法,包括以下步骤:
1)牛卵母细胞体外成熟;
2)牛体外受精胚胎的生产;
3)牛体外胚胎的冷冻。
其中,步骤1)将牛卵母细胞置于含有β-烟酰胺单核苷酸的体外成熟液中进行培养,且步骤2)将体外受精后的牛卵母细胞置于含有β-烟酰胺单核苷酸的牛体外受精胚胎培养液中进行培养,然后步骤3)将牛体外胚胎进行玻璃化冷冻。
进一步地,体外成熟液、体外受精胚胎培养液中β-烟酰胺单核苷酸的浓度分别为0.1-100μM,优选10μM。
进一步地,体外成熟液、体外受精胚胎培养液中还添加有生长因子和缝隙链接蛋白。
所述生长因子为胰岛素生长因子(如IGF1)和/或表皮生长因子,所述缝隙链接蛋白为CX37。
其中,胰岛素生长因子1(IGF1)是胰岛素生长因子家族中的重要成员,其可通过自分泌、内分泌和旁分泌的三种途径产生低分子多肽,IGF1调节细胞的增殖与胚胎发育(Adashi et al.,1985)。IGF1在卵母细胞功能中非常重要,由于它们的受体存在于卵丘-卵母细胞复合体(COCs)中,并且卵丘细胞特别产生大量这种因子,在牦牛COCs可测得IGF1及其受体IGFIR表达(Pan et al.,2015)。
表皮生长因子(EGF)是EGF配体家族成员,它的主要功能是促进卵母细胞成熟和卵丘细胞扩张。在体内,它能够协调LH信号通路,最终将LH从体细胞中传递进入卵母细胞中,进而促进卵母细胞成熟。缝隙链接蛋白37(Connexin37,Cx37)是一种链接蛋白,是组成卵母细胞与卵丘细胞之间缝隙链接(gap junction)的组成蛋白。而缝隙链接能够加强卵丘细胞和卵母细胞之间的物质、信息交流,进而加速卵母细胞的成熟进程。
上述三种物质对牛卵母细胞IVM效率及其IVF胚胎抗冻性的影响机制,目前还缺乏研究。
优选地,体外成熟液中胰岛素生长因子的浓度为200ng/mL,表皮生长因子的浓度为100ng/mL,CX37的浓度为25μg/mL。
体外受精胚胎培养液中胰岛素生长因子的浓度为200ng/mL,表皮生长因子的浓度为100ng/mL,CX37的浓度为25μg/mL。
进一步地,步骤3)将牛体外胚胎置于含有0.1%-1%纳米颗粒的玻璃化冷冻液中进行冷冻。
在本发明的一个具体实施方式中,用于牛体外胚胎生产的高效玻璃化冷冻方法,包括以下步骤:
(1)牛卵母细胞体外成熟
在牛卵巢上采集直径2-8mm卵泡中卵丘-卵母细胞复合体COCs,选择含有3层及以上颗粒细胞COCs用体外成熟液洗涤3-4遍后,按30-50枚COCs/每孔/500μL体外成熟液放入预平衡(2h)的四孔板培养液中,于38.5℃、5%CO2及饱和湿度二氧化碳培养箱中培养;其中,所述体外成熟液为TCM199+10μg/mL FSH+10μg/mL LH+10μg/mL E2+10%FBS,且体外成熟液中添加有0.1-100μM的β-烟酰胺单核苷酸;
(2)牛体外受精胚胎的生产
COCs体外培养22-24h后,用0.1%透明质酸酶消化2-3min去除颗粒细胞,挑选具有第一极体且胞质均匀的牛卵母细胞进行体外受精;牛冷冻细管精液于37-38℃水浴解冻后用洗精液(BO液+2.5mM Caffeine),经400-500rpm离心5-7min,共离心两次;精子沉淀用受精液(BO液+20mg/mL BSA+20mg/mL肝素钠+100IU/mL青霉素钾+100μg/mL链霉素)调整精子密度为4×106-5×106个/mL。吸取20μL精液加入含有20枚卵母细胞的80μL受精液小滴中,精子终密度为1×106/mL,于38.5℃、5%CO2及饱和湿度二氧化碳培养箱中受精18-20h;
将受精后的牛卵母细胞移入牛体外受精胚胎培养液小滴(100μL)中培养,间隔46-48h半量换液,受精卵体外培养7d时统计发育到囊胚阶段的胚胎数;
其中,所述牛体外受精胚胎培养液为含10%FBS的CR1aa培养液,且牛体外受精胚胎培养液中添加有0.1-100μM的β-烟酰胺单核苷酸;
(3)牛体外胚胎的冷冻
室温下将囊胚移入预处理液(含10%乙二醇和10%二甲基亚砜的DPBS溶液)中平衡30s,然后移入玻璃化冷冻液中平衡25s后吸入OPS管,直接投进液氮中保存;其中,所述玻璃化冷冻液为乙二醇、DMSO和FS液按体积比(1.5-2):(1.5-2):(6-7)的混合液(优选1.5:1.5:7),且玻璃化冷冻液中添加有0.1%-1%的纳米颗粒。
FS液:含30%(w/v)聚蔗糖Ficoll 70和0.5M蔗糖的DPBS溶液。
优选地,步骤(1)的体外成熟液中添加有10μM的β-烟酰胺单核苷酸。
优选地,步骤(2)的牛体外受精胚胎培养液中添加有10μM的β-烟酰胺单核苷酸。
优选地,步骤(3)的玻璃化冷冻液中添加有0.5%的纳米颗粒。
所述纳米颗粒为平均粒径5nm左右的金纳米粒子。
借由上述技术方案,本发明至少具有下列优点及有益效果:
本发明首次将β-烟酰胺单核苷酸应用于牛体外胚胎生产中的牛IVF胚胎玻璃化冷冻技术中,通过在牛卵母细胞体外成熟液和牛体外受精胚胎培养液中添加一定浓度的β-烟酰胺单核苷酸,然后将牛体外胚胎进行玻璃化冷冻,能够极大地提高牛IVF胚胎的玻璃化冷冻效率。进一步向牛卵母细胞体外成熟液和牛体外受精胚胎培养液中按比例添加生长因子和缝隙链接蛋白可进一步提高玻璃化冷冻效率。更进一步地,通过向玻璃化冷冻液中添加一定浓度的纳米颗粒可有助于进一步提高玻璃化冷冻效率。
附图说明
图1为本发明较佳实施例中去脂、生长因子和链接蛋白、纳米粒子联合处理对玻璃化牛IVF胚胎基因表达的影响。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。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。
实施例1用于牛体外胚胎生产的高效玻璃化冷冻方法
一、实验方法
1、卵母细胞体外成熟
在牛卵巢(采自河北大厂)上采集直径2-8mm卵泡中卵丘-卵母细胞复合体(cumulus-oocytecomplexes,COCs),选择含有3层及以上颗粒细胞COCs用体外成熟液(IVM液:TCM199+10μg/mL FSH+10μg/mL LH+10μg/mL E2+10%FBS)洗涤3遍后,按50枚COCs/每孔/500μL成熟培养液放入预平衡2h的四孔板培养液中,于38.5℃、5%CO2及饱和湿度二氧化碳培养箱中培养。
2、体外受精胚胎的生产
牛体外受精生产胚胎参照Nedambale等的方法,略有改动。
COCs体外培养22-24h后,用0.1%透明质酸酶消化2-3min去除颗粒细胞,挑选具有第一极体且胞质均匀的卵母细胞进行体外受精。奶牛冷冻细管精液(牛体编号:11101930,北京市奶牛中心)于37℃水浴解冻后用7mL洗精液(BO液+2.5mM Caffeine),经500rpm离心5min,离心两次;精子沉淀用受精液(BO液+20mg/mL BSA+20mg/mL肝素钠+100IU/mL青霉素钾+100μg/mL链霉素)调整精子密度为5×106个/mL。吸取20μL精液加入含有20枚卵母细胞的80μL受精液小滴中(精子终密度为1×106/mL),于38.5℃、5%CO2及饱和湿度二氧化碳培养箱中受精18-20h。
将受精后的卵母细胞移入牛体外受精胚胎培养液(IVC液:含10%FBS的CR1aa培养液)培养小滴100μL中培养,间隔48h半量换液,受精卵体外培养7d时统计发育到囊胚阶段的胚胎数。
3、体外胚胎冷冻
冷冻:室温下将囊胚移入平衡液(10%ED+10%DMSO)中平衡30s,然后移入玻璃化溶液EDFS30平衡25s后吸入OPS管,直接投进液氮中保存。
解冻:室温下从液氮灌中取出OPS管,迅速浸入解冻液液滴中,体视显微镜下将胚胎移入0.5M蔗糖溶液小滴中平衡5min,CR1aa中洗涤3次。
预处理液:含10%乙二醇(EG)和10%二甲基亚砜(DMSO)的DPBS溶液。
FS液:含30%(w/v)聚蔗糖Ficoll 70和0.5M蔗糖的DPBS溶液。
玻璃化冷冻液EDFS30:EG、DMSO和FS液按照体积比(v/v)1.5:1.5:7混匀。
解冻液:含0.5M蔗糖的DPBS溶液。
4、荧光定量PCR
采用荧光实时定量PCR进行检测,具体流程如下:
采用BIO-RAD(美国)CFX96TM实时荧光定量PCR仪进行定量分析。采用oligo7软件进行引物设计,引物序列如表1所示。实验采用15μL反应体系,包括:10μM上游引物、下游引物各0.5μL,cDNA模板2μL,TB Green Premix Ex Taq II(2I)7.5μL,RNase free ddH2O 4.5μL。反应程序:95℃预变性30s;95℃5s,60℃30s,39个循环。每个样品重复三次,以牛GAPDH为内参基因,采用2-△△Ct法计算基因的相对表达量。
表1实时荧光定量PCR
5、囊胚细胞数
囊胚采用0.5%链霉蛋白酶处理1-2分钟去除透明带,而后采用10ug/mlHochest33342染色10min,压片后置于荧光显微镜下观察并拍照,进而统计囊胚细胞数。
6、脂滴染色
牛卵母细包或胚胎采用4%多聚甲醛固定;含0.1%PVA的PBS洗涤3遍,置于尼罗红染色液中,37℃避光孵育10min;0.1%PVA的PBS清洗2遍,置于荧光显微镜下观察脂滴含量变化并拍照。采用Nikon EZ-C1 Free Viewr软件分析尼罗红荧光强度。
7、实验设计
(1)去脂对牛卵母细胞IVM效率、IVF胚胎发育和冷冻效率得影响
在卵母细胞IVM液、IVF胚胎IVC液中添加不同浓度NMN,获得的囊胚直接进行玻璃化冷冻,进而研究NMN对牛卵母细胞IVM效率、IVF胚胎发育和冷冻效率得影响,筛选最佳NMN浓度。
(2)生长因子和链接蛋白对牛卵母细胞IVM效率、IVF胚胎发育和冷冻效率的影响
在卵母细胞IVM液、IVF胚胎IVC液中分别或同时添加IGF+EGF+CX37,获得的囊胚直接进行玻璃化冷冻,进而研究生长因子和链接蛋白对牛卵母细胞IVM效率、IVF胚胎发育和冷冻效率得影响,筛选最佳组合。
(3)纳米粒子对牛IVF胚胎冷冻效率的影响
在玻璃化冷冻液中添加不同浓度纳米粒子,而后对牛IVF囊胚进行玻璃化冷冻,进而研究纳米粒子对牛IVF囊胚冷冻效率的影响,筛选最佳纳米粒子浓度。
所述纳米粒子为平均粒径5nm左右的金纳米粒子。
(4)联合处理组
在卵母细胞IVM液、IVF胚胎IVC液中添加NMN,获得的囊胚采用添加纳米粒子的玻璃化冷冻液进行玻璃化冷冻。
①IGF+EGF+CX37+10μM NMN组:在卵母细胞IVM液中同时添加200ng/mL IGF、100ng/mL EGF、25μg/mL CX37、10μM NMN,进而研究该处理对牛卵母细胞IVF胚胎冷冻效率的影响。
②IGF+EGF+CX37+0.5%纳米组:在卵母细胞IVM液中添加200ng/mL IGF、100ng/mLEGF、25μg/mL CX37、在玻璃化冷冻液中添加0.5%纳米粒子,进而研究该处理对牛卵母细胞IVF胚胎冷冻效率的影响。
③10μM NMN+0.5%纳米组:在卵母细胞IVM液中添加10μM NMN、玻璃化冷冻液中添加0.5%纳米粒子,进而研究该处理对牛卵母细胞IVF胚胎冷冻效率的影响。
④IGF+EGF+CX37+10μM NMN+0.5%纳米组:在卵母细胞IVM液中同时添加200ng/mLIGF、100ng/mL EGF、25μg/mL CX37、10μM NMN,玻璃化冷冻液中添加0.5%纳米粒子,进而研究该处理对牛卵母细胞IVF胚胎冷冻效率的影响。
8、结果
(1)去脂对牛卵母细胞IVM效率、IVF胚胎发育和冷冻效率的影响
如表2所示,在牛卵母细胞IVM、IVF胚胎体外培养过程中添加NMN,结果表明,10μMNMN组的核成熟率(95.14±8.32%)显著高于对照组(85.08±7.05%)和对照组(85.08±7.05%),10μM NMN组IVF卵裂率和囊胚率(90.09±8.47%、50.00±4.87%)显著高于其他处理组(41.70±3.98%-42.26±3.74%)和对照组(35.22±3.25%)。同时,10μM NMN组的荧光强度显著低于对照组和0.1μM NMN组、1μM NMN组、对照组,10μM NMN组玻璃化冷冻后存活率(92.37%±7.94)显著高于对照组(84.31%±7.04)。
表2去脂对牛卵母细胞IVM效率、IVF胚胎发育和冷冻效率得影响
注:不同小写字母表示差异显著。下同。
(2)生长因子和链接蛋白对牛卵母细胞IVM效率、IVF胚胎发育和冷冻效率的影响
如表3所示,在牛卵母细胞IVM、IVF胚胎体外培养过程中添加生长因子和CX37,结果表明,生长因子+CX37组的核成熟率(93.28±7.27%)显著高于对照组(85.17±7.93%),生长因子+CX37组IVF卵裂率和囊胚率(93.22±8.03%、45.45±4.13%)显著高于对照组(85.07±8.14%、35.09±3.29%)。同时,生长因子+CX37组玻璃化冷冻后存活率(92.00±7.84%)显著高于对照组(85.00±6.93%)。
表3生长因子和链接蛋白对牛卵母细胞IVM效率、IVF胚胎发育和冷冻效率的影响
(3)纳米粒子对牛IVF胚胎玻璃化冷冻效率的影响
在玻璃化冷冻液中添加不同浓度的纳米粒子,而后对牛IVF囊胚进行玻璃化冷冻,进而研究纳米粒子添加对IVF胚胎玻璃化冷冻效率的影响。如表4所示,在牛IVF胚胎玻璃化冷冻液中添加纳米颗粒,结果表明,0.5%纳米颗粒组冷冻存活率(90.43±8.35%)显著高于对照组(85.60±6.37%)。
表4纳米粒子对牛IVF胚胎玻璃化冷冻效率的影响
组别 | 囊胚数 | 冷冻存活率 |
0.1%纳米颗粒 | 90 | 80(88.89±7.94%)ab |
0.5%纳米颗粒 | 94 | 85(90.43±8.35%)a |
1%纳米颗粒 | 93 | 81(87.10±7.52%)ab |
冷冻对照组 | 125 | 107(85.60±6.37%)b |
(4)去脂、生长因子和链接蛋白、纳米粒子联合处理对牛IVF胚胎玻璃化冷冻效率的影响
为了进一步提高牛卵母细胞IVF胚胎玻璃化冷冻效率,我们对比较了不同处理组合的冷冻效率。结果如表5所示,IGF+EGF+CX37+10μM NMN+0.5%纳米组的冷冻存活率、囊胚孵化率、囊胚细胞数(99.30±8.68%)显著高于其他处理组(91.53±8.38%-93.55±6.03%)、冷冻对照组(84.40±7.95%),IGF+EGF+CX37+10μMNMN+0.5%纳米组的囊胚孵化率、囊胚细胞数(98.58±8.78%,129.76±10.87)显著高于其他处理组(82.52±6.38-86.21±6.92%,93.41±8.24-99.65±9.04)、冷冻对照组(80.00±7.93%,90.65±8.93)、新鲜对照组(90.08±8.54%,118.93±7.76b)。
表5去脂、生长因子和链接蛋白、纳米粒子联合处理对牛IVF胚胎玻璃化冷冻效率的影响
(5)冷冻对囊胚基因表达的影响
为了进一步提高牛卵母细胞IVF胚胎玻璃化冷冻效率,比较了不同处理组合对囊胚冷冻后基因表达的影响。结果如图1所示,生长因子+CX37+去脂+纳米颗粒组IFN-tau表达量、抗凋亡基因显著高于其他处理组、冷冻对照组、新鲜对照组,而该组促凋亡基因的表达量高于其他处理组、冷冻对照组、新鲜对照组。
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,可以对之做一些修改或改进,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.β-烟酰胺单核苷酸在牛体外胚胎生产中提高牛IVF胚胎玻璃化冷冻效率中的应用。
2.用于牛体外胚胎生产的高效玻璃化冷冻方法,其特征在于,包括以下步骤:
1)牛卵母细胞体外成熟;
2)牛体外受精胚胎的生产;
3)牛体外胚胎的冷冻;
其中,步骤1)将牛卵母细胞置于含有β-烟酰胺单核苷酸的体外成熟液中进行培养,且步骤2)将体外受精后的牛卵母细胞置于含有β-烟酰胺单核苷酸的牛体外受精胚胎培养液中进行培养,然后步骤3)将牛体外胚胎进行玻璃化冷冻。
3.根据权利要求2所述的方法,其特征在于,体外成熟液、体外受精胚胎培养液中β-烟酰胺单核苷酸的浓度分别为0.1-100μM,优选10μM。
4.根据权利要求2所述的方法,其特征在于,体外成熟液、体外受精胚胎培养液中还添加有生长因子和缝隙链接蛋白。
5.根据权利要求4所述的方法,其特征在于,所述生长因子为胰岛素生长因子和/或表皮生长因子,所述缝隙链接蛋白为CX37。
6.根据权利要求5所述的方法,其特征在于,体外成熟液中胰岛素生长因子的浓度为200ng/mL,表皮生长因子的浓度为100ng/mL,CX37的浓度为25μg/mL;
体外受精胚胎培养液中胰岛素生长因子的浓度为200ng/mL,表皮生长因子的浓度为100ng/mL,CX37的浓度为25μg/mL。
7.根据权利要求2-6任一项所述的方法,其特征在于,步骤3)将牛体外胚胎置于含有0.1%-1%纳米颗粒的玻璃化冷冻液中进行冷冻。
8.根据权利要求2所述的方法,其特征在于,包括以下步骤:
(1)牛卵母细胞体外成熟
在牛卵巢上采集直径2-8mm卵泡中卵丘-卵母细胞复合体COCs,选择含有3层及以上颗粒细胞COCs用体外成熟液洗涤3-4遍后,按30-50枚COCs/每孔/500μL体外成熟液放入预平衡的四孔板培养液中,于38.5℃、5%CO2及饱和湿度二氧化碳培养箱中培养;其中,所述体外成熟液为TCM199+10μg/mL FSH+10μg/mL LH+10μg/mL E2+10%FBS,且体外成熟液中添加有0.1-100μM的β-烟酰胺单核苷酸;
(2)牛体外受精胚胎的生产
COCs体外培养22-24h后,用0.1%透明质酸酶消化2-3min去除颗粒细胞,挑选具有第一极体且胞质均匀的牛卵母细胞进行体外受精;牛冷冻细管精液于37-38℃水浴解冻后用洗精液,经400-500rpm离心5-7min,共离心两次;精子沉淀用受精液调整精子密度为4×106-5×106个/mL;吸取20μL精液加入含有20枚卵母细胞的80μL受精液小滴中,精子终密度为1×106/mL,于38.5℃、5%CO2及饱和湿度二氧化碳培养箱中受精18-20h;
将受精后的牛卵母细胞移入牛体外受精胚胎培养液小滴中培养,间隔46-48h半量换液,受精卵体外培养7d时统计发育到囊胚阶段的胚胎数;
其中,所述牛体外受精胚胎培养液为含10%FBS的CR1aa培养液,且牛体外受精胚胎培养液中添加有0.1-100μM的β-烟酰胺单核苷酸;
(3)牛体外胚胎的冷冻
室温下将囊胚移入预处理液中平衡30s,然后移入玻璃化冷冻液中平衡25s后吸入OPS管,直接投进液氮中保存;其中,所述玻璃化冷冻液为乙二醇、DMSO和FS液按体积比(1.5-2):(1.5-2):(6-7)的混合液,且玻璃化冷冻液中添加有0.1%-1%的纳米颗粒。
9.根据权利要求8所述的方法,其特征在于,步骤(1)的体外成熟液中添加有10μM的β-烟酰胺单核苷酸;
步骤(2)的牛体外受精胚胎培养液中添加有10μM的β-烟酰胺单核苷酸;
步骤(3)的玻璃化冷冻液中添加有0.5%的纳米颗粒。
10.根据权利要求8或9所述的方法,其特征在于,所述纳米颗粒为平均粒径5nm的金纳米粒子。
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