牛卵母细胞的玻璃化冷冻研究

本试验通过牛卵母细胞玻璃化冷冻过程中采用载体、冷冻时卵母细胞所带颗粒细胞层数和其成熟培养时间这3个方面对牛卵母细胞的玻璃化冷冻进行了探讨。(1)不同载体的对比:玻璃OPS管和塑料OPS管冷冻效果较好。玻璃OPS管冷冻卵母细胞,解冻后形态正常率、体外受精后卵裂率和囊胚率分别为91.2%、41.3%和14.52%。塑料OPS管冷冻卵母细胞,解冻后形态正常率、体外受精后卵裂率和囊胚率分别为89.09%、40.90%和14.54%。(2)保留2￣3层颗粒细胞的卵母细胞玻璃化冷冻的冷冻效果较好,其冷冻解冻后形态正常率、体外受精后卵裂率和囊胚率分别为90.79%、40.13%和15.13%。(3)体外成熟培养22h的卵母细胞冷冻效果较好,其冷冻解冻后形态正常率、体外受精后卵裂率和囊胚率分别为94.14%、41.80%和15.23%。     

不同冷冻载体对牛未成熟卵母细胞玻璃化冷冻效果的影响

为了研究不同冷冻载体制备方法及其对牛未成熟卵母细胞发育能力的影响,以牛未成熟卵母细胞为实验材料,分别探究开放式拉长细管(OPS,open pulled straw)和玻璃微管(GMP,glass micropipette)的制备方法,并以OPS、GMP和冷冻叶片为冷冻载体玻璃化冷冻牛未成熟卵母细胞,解冻后经体外成熟培养和体外受精,统计形态正常率、成熟率、卵裂率及囊胚率。结果显示:以简易小酒精灯为热源,以细管为原材料可以制备出适用的OPS冷冻载体;以酒精喷灯为热源,以细玻璃管为原材料可以制备出适用的GMP冷冻载体。OPS和GMP组形态正常率分别为(74.3%±1.8)%和(72.5%±2.6)%;无显著差异(P0.05),上述二组均显著低于冷冻叶片组(82.1%±1.3)%,P0.05,但三组间成熟率、卵裂率和囊胚率均无显著差异(P0.05)。研究表明,分别以简易小酒精灯和酒精喷灯为热源,以细管和细玻璃管为原材料可以成功制备出OPS和GMP载体;以OPS、GMP和冷冻叶片为冷冻载体均可成功地玻璃化冷冻牛未成熟卵母细胞。     

不同冷冻方法对牛卵母细胞及孤雌胚发育的影响

研究了麦管和OPS管法冷冻以及OPS法中保护剂种类对牛卵母细胞体外成熟及孤雌胚发育的影响。结果发现,OPS管冷冻牛卵母细胞形态正常率、卵裂率、囊胚率极显著高于麦管法(P<0.05)。在OPS法中,应用两种保护剂冷冻后,卵母细胞形态正常率、卵裂率、囊胚率差异极显著(P<0.01);采取38℃与25℃温度平衡,冷冻后卵母细胞各发育指标差异不显著(P>0.05);采用4℃平衡,冷冻后的卵母细胞激活后没有出现囊胚,各发育指标极显著降低(P<0.01)。结果表明,OPS法可以有效地保护卵母细胞,保证其后孤雌激活;采用低温平衡会对孤雌发育的囊胚阶段有较大影响。     

绵羊体外成熟卵母细胞OPS法玻璃化冷冻保存试验

研究以EDFS30为玻璃化冷冻液,以卵母细胞解冻后孤雌激活和体外受精后的卵裂率、囊胚发育率作为评价指标,探讨了以OPS法玻璃化冷冻保存体外成熟绵羊卵母细胞的效果。结果表明:卵母细胞孤雌激活后的卵裂率,冷冻组(64.2%)显著(P<0.05)低于毒性组(76.7%)和对照组(79.1%),而毒性组和对照组无显著(P>0.05)差异;卵母细胞孤雌激活后的囊胚发育率,冷冻组(4.2%)和毒性组(5.8%)均显著(P<0.05)低于对照组(20.2%),毒性组和冷冻组无显著(P>0.05)差异;冷冻组和毒性试验组卵母细胞体外受精后的卵裂率和囊胚发育率(67.6%和7.1%;62.3%和9.1%)均显著低于对照组(78.4%和28.4%)(P<0.05),而毒性组和冷冻组无显著(P>0.05)差异。可见以EDFS30为玻璃化冷冻液,采用OPS法冷冻保存绵羊体外成熟卵母细胞会在一定程度上降低其受精能力和胚胎发育能力。     

玻璃化冷冻对猪GV期卵母细胞成熟及发育能力的影响

为了获得最适的冷冻猪GV期卵母细胞的冷冻保护液配方和最适冷冻载体,本研究就目前应用最多的7种冷冻保护液配方进行筛选,并且比较3种不同冷冻载体冷冻卵母细胞的效果差异。结果表明:7组冷冻保护液对GV期卵母细胞有低毒性作用,各组分裂率、囊胚率、囊胚细胞数均低于对照组,综合各组指标选取3组对猪MII期卵母细胞的发育能力损伤最小的冷冻保护剂,应用GMP管对其冷冻保护效果进行比较,发现第6组的极体率29.47%,第5组的极体率23.54%与第3组的极体率8.28%相比差异显著(P0.05),且第5组的存活率、极体率与第3组相比差异显著(P0.05)。综合考虑,选取第5组HM+7.5%(DMSO+EG),HM+15%(DMSO+EG)+0.5 mol/L Su组作为冷冻保护液来比较3种不同冷冻裁体冷冻卵母细胞的效果差异,发现半麦管冻融后的卵母细胞存活率高达54.11%与OPS管、GMP管的33.11%和26.79%差异显著(P0.05),半麦管法更适合GV期猪卵母细胞的玻璃化冷冻。     

水牛卵母细胞玻璃化冷冻保存

以水牛MII期的卵母细胞为材料,利用玻璃化冷冻液EDS33(16.5%EG+16.5%DMSO+sucrose)对水牛MII期的卵母细胞进行两步法(玻璃毛细管(GMP)和拉细的开口塑料细管(OPS))玻璃化冷冻保存,即卵母细胞首先放入预平衡液(7.5%EG+7.5%DMSO+sucrose)中平衡3 min,再移入玻璃化冷冻液中30 s后装管直接投入液氮.解冻是在蔗糖浓度逐渐降低的解冻液中进行的.解冻后存活的卵母细胞孤雌激活,通过囊胚发育率作为评定卵母细胞冷冻效果的指标.结果发现,GMP和OPS法冷冻保存的水牛卵母细胞解冻后的存活率(分别为96.80%和97.41%)与对照组卵母细胞的存活率(100%)3者之间差异均不显著(P>0.05).GMP法和OPS法冷冻的水牛卵母细胞激活后的胚胎分裂率和囊胚发育率2者均明显低于对照组(分别为30.58%和28.32% vs50.94%,10.81%和9.38% vs 29.63%,P<0.05),而这2种方法冷冻的水牛卵母细胞激活后的分裂率和囊胚发育率差异均不显著(P>0.05).这表明GMP和OPS玻璃化冷冻方法可以用于水牛卵母细胞的冷冻,并且玻璃化冷冻的卵母细胞能继续分裂并发育到囊胚.     

玻璃化冷冻对猪MII期卵母细胞发育能力的影响

以MII期卵母细胞作为试验材料,应用玻璃化冷冻方法,就目前应用最多的7组冷冻保护液配方进行筛选,并且比较3种不同冷冻载体冷冻卵母细胞的效果。结果发现:7组冷冻保护液对MII期卵母细胞有低毒性作用,各组分裂率、囊胚率、囊胚细胞数均低于对照组;综合各组指标,选取3组对猪MII期卵母细胞的发育能力损伤最小的冷冻保护剂,应用GMP管对其冷冻保护效果进行比较,发现第7组冷冻保护液配方EFS不适于MII期卵母细胞的冷冻,其形态正常率、分裂率分别为1.19%、0,与第3组、第6组形态正常率、分裂率(59.48%、53.55%;24.19%、35.02%)差异显著(P0.05)。综合考虑,选取HM+7.5%(DMSO+EG),HM+17%(DMSO+EG)+0.4 mol/L Su作为冷冻保护剂来比较3种不同冷冻载体冷冻卵母细胞的效果差异,发现半麦管冻融后MII期卵母细胞的分裂率高达53.67%,与OPS法的29.33%及GMP法的35.00%相比差异显著(P0.05),半麦管法更适合MII期猪卵母细胞的玻璃化冷冻。     

3种冷冻方法对猪MⅡ期卵母细胞线粒体分布和损伤的影响

本研究旨在探讨3种冷冻方法对猪MⅡ期卵母细胞玻璃化冷冻后线粒体的分布和超微结构变化的影响.通过透射电镜、Rhodamine-123 (R-123)荧光染色和体外发育观察,结果显示,(1)无论是FDA-DAPI复染存活率,还是孤雌激活卵裂率,CLV (Cryoloop vitrification)法(72.00%,7.22％)效果最好,OPS(Open Pulled Straw)法(60.00％,4.85％)次之,straw法(42.22％,0％)最低；(2)CLV法经R-123染色后,卵母细胞线粒体的正常分布率(52.24％)要比其它2组要高(OPS,48.65％；straw,37.68%),但三者之间没有显著差异(P＞0.05)；(3)透射电镜超微结构表明,冻后卵母细胞线粒体变得粗糙和模糊,有的线粒体嵴减少甚至消失.结果表明,冷冻过程中卵母细胞线粒体的分布和形态损伤严重,CLV法可提高冷冻速率,增强冻后卵母细胞的发育能力,降低冷冻造成的卵母细胞线粒体异常分布比例.     

水牛卵母细胞冷冻保存初步研究

①用EFS30、EFS40、EDFS30、EDFS40四种玻璃化冷冻液对MⅡ期水牛卵母细胞进行毒性试验,结果表明:试验组卵母细胞形态正常率与对照组均无显著性差异(P>0.05);对卵母细胞孤雌激活后EDFS30、EDFS40组的卵裂率与对照组(75.28%)及EFS30、EFS40组差异显著(P<0.05);利用4种冷冻保护剂采用OPS法冷冻保存MⅡ期水牛卵母细胞,其中以EDFS40作为冷冻液时,卵母细胞冷冻解冻后孤雌激活卵裂率最高,达31.60%;以EDFS40作为冷冻液,比较了GMP法和OPS法的冷冻效果,结果表明GMP法冷冻效果好于OPS法。②采用不同预处理时间和平衡时间使用细管法常规冷冻G V期卵母细胞,结果表明预处理5 min、平衡15min组的形态正常率和极体排出率相对较高,分别为72.73%、27.27%。     

不同冷冻和解冻方法对绵羊卵母细胞发育效果的影响

卵母细胞冷冻保存具有广泛而潜在的应用价值.试验主要分析了不同冷冻及解冻方法对绵羊GV期和MII卵母细胞发育效果的影响.GV期卵母细胞程序化冷冻解冻后形态正常率(54.8%)以及体外培养成熟率(14.7%)均显著低于细管玻璃化(71.8%,29.5%;P＜0.05)和OPS玻璃化(78.3%、35.4%;P＜0.05),卵裂率OPS玻璃化高于细管玻璃化,但差异不显著(26.1%,16.7%;P＞0.05);MII期卵母细胞形态正常率程序化冷冻显著低于细管玻璃化和OPS玻璃化冷冻(67.2%,77.6%,84.8%;P＜0.05),卵裂率OPS玻璃化显著高于程序化冷冻法(31.3%,10.3%;P＜0.05);3种不同方法冷冻不同发育时期卵母细胞成熟率和卵裂率与对照组相比较均差异极显著(P＜0.01).解冻后卵母细胞形态正常率三步与五步解冻法极显著高于一步解冻法(85.9%,82.7%,63.1%; P＜0.01);成熟率为三步法和五步法显著高于一步法(10.8%,26.9%,25.3%;P＜0.05);卵裂率三步法和五步法高于一步法,但没有差异显著性(14.3%,23.9%,21.1%; P＞0.05).     

Piezo-actuated zona-drilling improves the fertilisation of OPS vitrified mouse oocytes

The present study was designed to investigate fertilisation of open pulled straw (OPS) vitrified mouse oocytes drilled with piezo-micromanipulation method and their subsequent in vitro and in vivo developmental capacity. Ovulated mouse oocytes were vitrified using the OPS method. After warming, the zona pellucida of a group of vitrified-warmed oocytes was drilled by piezo-micromanipulation. Groups of (a) vitrified, (b) vitrified/drilled and (c) fresh control oocytes were fertilised in vitro. The fertilisation rate of vitrified-warmed oocytes was significantly lower than that of fresh oocytes (45.0 +/- 12.6% vs. 85.2 +/- 6.8%, P < 0.05), and was significantly improved by zona-drilling (85.4 +/- 7.3%). However, blastocyst formation rates of the vitrified and vitrified/drilled groups were significantly lower than those of the fresh controls (65.7 +/- 7.0% and 66.4 +/- 2.5% vs. 86.6 +/- 4.3%, respectively, P < 0.05). The cell number of blastocysts from the vitrified/drilled or the vitrified group was not different from that of the controls. Embryo transfer resulted in pregnancy in all three groups, but the rate of development to term was lower in the vitrified/drilled or vitrified groups than in the controls (16.6 +/- 0.7% or 36.0 +/- 2.4% vs. 51.3 +/- 2.9%, respectively). Our results demonstrated that zona-drilling with piezo-micromanipulation could improve fertilisation in OPS vitrified mouse oocytes but did not increase the overall number of vitrified oocytes developing to term.     

Effect of Different Combinations of Cryoprotectants on In Vitro Maturation of Immature Buffalo (Bubalus bubalis) Oocytes Vitrified by Straw and Open‐Pulled Straw Methods

This study was designed to evaluate effects of different combinations of cryoprotectants on the ability of vitrified immature buffalo oocytes to undergo in vitro maturation. Straw and open‐pulled straw (OPS) methods for vitrification of oocytes at the germinal vesicle stage also were compared. The immature oocytes were harvested from ovaries of slaughtered animals and were divided into three groups: (i) untreated (control); (ii) exposed to cryoprotectant agents (CPAs); or (iii) cryopreserved by straw and OPS vitrification methods. The vitrification solution (VS) consisted of 6 m ethylene glycol (EG) as the standard, control vitrification treatment, and this was compared with 3 m EG + 3 m dimethyl sulfoxide (DMSO), 3 m EG + 3 m glycerol, and 3 m DMSO + 3 m glycerol. Cryoprotectants were added in two steps, with the first step concentration half that of the second (and final) step concentration. After warming, oocyte samples were matured by standard methods and then fixed and stained for nuclear evaluation. Rates of MII oocytes exposed to CPAs without vitrification were lower (54.3 ± 1.9% in EG, 47.5 ± 3.4% in EG + DMSO, 36.8 ± 1.2% in EG + glycerol and 29.9 ± 1.0% in DMSO + glycerol; p < 0.05) than for the control group (79.8 ± 1.3%). For all treatments in each vitrification experiment, results were nearly identical for straws and OPS, so all results presented are the average of these two containers. The percentages of oocytes reaching telophase‐I or metaphase‐II stages were lower in oocytes cryopreserved using all treatments when compared with control. However, among the vitrified oocytes, the highest maturation rate was seen in oocytes vitrified in EG + DMSO (41.5 ± 0.6%). Oocytes cryopreserved in all groups with glycerol had an overall low maturation rate 19.0 ± 0.6% for EG + glycerol and 17.0 ± 1.1% for DMSO + glycerol. We conclude that the function of oocytes was severely affected by both vitrification and exposure to cryoprotectants without vitrification; the best combination of cryoprotectants was EG + DMSO for vitrification of immature buffalo oocytes using either straw or OPS methods.     

小鼠2-细胞胚胎细管法和OPS法玻璃化冷冻保存技术的研究

本试验在室温 (2 0℃和 2 5℃ )条件下 ,利用不同浓度的玻璃化溶液 (EFS和EDFS) ,对小鼠 2 细胞胚胎进行细管法和OPS法玻璃化冷冻保存。在 2 0℃室温条件下 ,用EFS4 0平衡 1min细管一步法冷冻 ,解冻后囊胚发育率仅为35 .0 % ,和新鲜 2 细胞体外培养的对照组 (6 5 .0 % )的差异极显著 (P <0 .0 1)。当 2 细胞胚胎在 10 %EG +10 %D溶液中预处理 5min ,再移入EDFS中平衡 30s二步法冷冻保存 ,解冻后囊胚发育率达 4 7.8%～ 4 8.8% ;当室温升至2 5℃时 ,二步法冷冻保存后 2 细胞的囊胚发育率达到 5 2 .2 % ,与对照组无显著差异 (P >0 .0 5 )。改用OPS二步法EFS30冷冻组保存后的 2 细胞胚胎的囊胚发育率高达 6 2 .2 % ,为试验中的最佳组。用最佳细管法和OPS法冷冻组解冻后培养至囊胚移植给受体母鼠均获得产仔     

小鼠胚胎的玻璃化冷冻研究

用不同冷冻载体(玻璃管、塑料管和0.25 mL细管)及不同冷冻方法(程序化冷冻和玻璃化冷冻)对小鼠3.5 d~4 d桑椹胚和囊胚进行冷冻保存,并与不做任何冷冻保存处理直接培养进行对比。结果表明,使用玻璃管、塑料管和0.25 mL细管作为胚胎的承载材料进行玻璃化冷冻,效果差异不显著;采用程序化冷冻与OPS玻璃化冷冻法,对小鼠胚胎进行冷冻保存可以取得较好的结果。从而得出,用不同材质的冷冻载体进行玻璃化冷冻,可以获得与程序化冷冻相同的良好效果。     

不同冷冻方法对GV期绵羊卵母细胞发育效果的影响

卵母细胞冷冻保存具有广泛而潜在的应用价值。文章主要分析了不同冷冻方法对绵羊GV期卵母细胞发育效果的影响。GV期卵母细胞程序化冷冻解冻后形态正常率（54.8%）和体外培养成熟率（14.7%）均显著或极显著低于细管玻璃化冷冻（71.8%,29.5%;P〈0.05）和OPS玻璃化冷冻（78.3%,P〈0.01;35.4%,P〈0.05）;3种不同方法冷冻GV期卵母细胞成熟率与对照组相比均差异极显著（P〈0.01）。     

Effect of open pulled straw (OPS) vitrification on the fertilisation rate and developmental competence of porcine oocytes

Freezing technologies are very important to preserve gametes and embryos of animals with a good pedigree or those having high genetic value. The aim of this work was to compare immature and in vitro matured porcine oocytes regarding their morphology and ability to be fertilised after vitrification by the open pulled straw (OPS) method. In four experiments 830 oocytes were examined. To investigate the effect of cumulus cells on oocyte survival after OPS vitrification, both denuded and cumulus-enclosed oocytes were vitrified at the germinal vesicle (GV) stage, then after vitrification they were matured in vitro. Besides, in vitro matured oocytes surrounded with a cumulus and those without a cumulus were also vitrified. The survival of oocytes was evaluated by their morphology. After in vitro fertilisation the rates of oocytes penetrated by spermatozoa were compared. Our results suggest that the vitrification/warming procedure is the most effective in cumulus-enclosed oocytes (22.35 +/- 1.75%). There was no difference between the order of maturation and vitrification in cumulus-enclosed oocytes, which suggests the importance of cumulus cells in protecting the viability of oocytes during cryopreservation.     

培养介质、卵丘细胞和卵泡直径对猪卵母细胞体外成熟的影响

A类卵母细胞在mTCM 199、NCSU2 3和NCSU37体系中培养 4 4～ 5 2小时后 ,成熟率分别为 76 .1%、78.1%和 6 5 .2 %。前两者差异不显著 (P >0 .0 5 ) ,但显著高于后者 (P <0 .0 5 )。卵母细胞在添加eCG和hCG的NCSU2 3体系中的成熟率 (75 .6 % )明显高于添加FSH的LH和成熟率 (6 5 .2 % ) (P <0 .0 5 )。A、B、C三类卵母细胞在NCSU2 3的成熟率分别为 73.3%、6 0 .4 %和 11.0 % ,三者间差异显著 (P <0 .0 5 )。大 (ф >6mm)、中 (ф =3～ 6mm)和小 (ф <3mm)三种卵泡中的卵母细胞在NCSU2 3中培养后 ,成熟率分别为 5 6 .2 % ,78.1%和 5 1.9% ,中等卵泡中卵母胞的体外成熟率显著高于其他两组 (P <0 .0 5 )。