共查询到19条相似文献，搜索用时 359 毫秒
1.
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). 相似文献
2.
试验以屠宰场云岭黑山羊卵巢卵母细胞为材料,研究其玻璃化冷冻的效果。试验中选用20% EG+20% DMSO为冷冻液、冷冻环为载体,以20 s、40 s玻璃化时间冷冻GV和MⅡ期的卵母细胞。结果表明,GV期卵母细胞的形态正常率、成熟率和卵裂率都很低,且解冻成熟培养后冷冻组的成熟率和卵裂率极显著低于对照组(P<0.01)。而MⅡ期卵母细胞冷冻效果较好,毒性试验组和冷冻组形态正常率分别为91.1%和83.3%,明显高于GV期;孤雌激活后毒性组卵裂率与对照组无显著性差异(P>0.05),冷冻组的卵裂率显著低于对照组(P<0.05)。用20 s、40 s玻璃化时间冷冻的卵母细胞解冻后GV和MⅡ期各组均无显著差异。根据试验结果得出在冷冻保存中最好冷冻MⅡ期的卵母细胞,以便提高后期的卵裂率和囊胚率;卵母细胞玻璃化时间在40 s内均不影响卵母细胞的活力和发育潜力。 相似文献
3.
不同浓度玻璃化冷冻保护液及冷冻载体对驴卵母细胞成熟率的影响 《畜牧与饲料科学》2016,37(3):4-4
为了比较不同冷冻液及冷冻载体对驴卵母细胞成熟率的影响,试验设计4个浓度梯度（Ⅰ、Ⅱ、Ⅲ、Ⅳ）冷冻液和3种冷冻载体分别对生发泡（germinal vesicle,GV）期驴的卵母细胞进行玻璃化冷冻,对解冻后的卵母细胞进行体外成熟,通过比较卵母细胞的成熟率反映其冷冻效果。结果表明,冷冻液Ⅱ的卵母细胞成熟率（28.13%）显著（P〈0.05）高于其他3组,但显著（P〈0.05）低于正常组（51.77%）,在不同冷冻载体对驴卵母细胞成熟率的影响试验中,使用开放式拉长塑料细管（OPS）的卵母细胞成熟率（28.13%）显著（P〈0.05）高于普通玻璃细管组（11.67%）,但与GMP管组（23.53%）差异不显著（P〉0.05）。 相似文献
4.
为比较猪卵母细胞在GV期与MⅡ期的冷冻保存效果,试验在这两个成熟阶段对其进行玻璃化冷冻,GV期卵母细胞解冻后培养至成熟,MⅡ期卵母细胞解冻后恢复2 h,然后采用免疫荧光标记、Western blotting和链霉蛋白酶溶解方法分别检测它们的皮质颗粒分布、CD9蛋白表达水平和透明带消化时间上的差异。结果表明,GV期卵母细胞在解冻后2 h的存活率显著低于MⅡ期卵母细胞（P<0.05）,但极体排出率与对照卵母细胞无明显差异（P>0.05）;在冷冻MⅡ期卵母细胞中,皮质颗粒的皮质区分布比例和CD9的蛋白表达水平显著下降（P<0.05）,但冷冻GV期卵母细胞经体外成熟后则无明显变化（P>0.05）;冷冻GV期与MⅡ期卵母细胞均不会影响透明带的消化时间（P>0.05）。由此可见,猪卵母细胞在GV期的冷冻存活率虽然较MⅡ期低,但其体外成熟后极体排出率、皮质颗粒分布和CD9蛋白表达水平均未受到冷冻的影响。 相似文献
5.
试验旨在探究玻璃化冷冻对驴卵母细胞发育的影响，寻求驴卵母细胞冷冻的最佳条件。通过对不同发育时期的驴卵母细胞进行玻璃化冷冻，冷冻复苏后分别进行成熟培养和孤雌激活，并对GV期未冷冻组（对照组）、GV期冷冻组、IVM-M Ⅱ冷冻组卵母细胞微丝和线粒体超微结构进行免疫荧光标记，统计冷冻复苏后卵母细胞形态正常率、成熟率、孤雌激活卵裂率、超微结构正常率。结果表明，GV期冷冻组卵母细胞的形态正常率与GV期未冷冻组（对照组）间无显著差异（P>0.05），成熟率和卵裂率均显著低于对照组（P<0.05）；IVM-M Ⅱ冷冻组的卵裂率显著低于对照组（P<0.05），且卵裂后细胞发育受到阻滞。冷冻组微丝在皮质区分布明显减少的卵母细胞数目增多，冷冻组卵母细胞的线粒体数量明显低于对照组，由此可以说明冷冻对卵母细胞超微结构有损伤，从而导致复苏后成熟率下降，影响卵母细胞的受精和体外发育，且GV期冷冻组较IVM-M Ⅱ冷冻组在微丝与线粒体结构上有较小损伤，发育状态较好。 相似文献
6.
试验首次采用OPS法玻璃化冷冻小鼠GV期卵母细胞(不带卵丘细胞,下同),同时尝试用EDFS30对小鼠卵巢进行细管法玻璃化冷冻,以研究GV期卵母细胞冷冻后的发育潜力。首先,利用MEM培养和MEM-腔前卵泡培养新鲜GV期卵母细胞,并把较好的培养方式用于冷冻后培养试验。2种培养方式培养24h后新鲜GV期卵母细胞成熟率无显著性差异;OPS法冷冻的GV期卵母细胞解冻后成熟率及体外受精后卵裂率与对照组差异不显著(P>0.05)。细管法冷冻卵巢组织的GV期卵母细胞成熟率极显著低于对照组(P<0.01),其受精后未获得受精卵。结果表明:OPS法可有效地冷冻保存小鼠GV期卵母细胞,而细管法冷冻小鼠卵巢对GV期卵母细胞损伤较大。 相似文献
7.
二甲基亚砜(DMSO)、丙二醇(PROH)、乙二醇(EG)和甘油(GL)4种冷冻保护剂程序化冷冻牛GV期卵母细胞的结果表明,EG和PROH的保护效果比GL和DMSO好。4种不同冷冻方法冷冻保存牛GV期卵母细胞,比较解冻后卵母细胞的体外成熟率、受精后卵裂率。结果表明,在程序化冷冻法与细管玻璃化法(Straw)之间的差异不显著(P>0.05),在开放式拉管法(OPS)与毛细玻管法(GMP)之间的差异不显著(P>0.05);但OPS和GMP与程序化冷冻法和Straw之间的差异极显著(P<0.01)。玻璃化冷冻效果优于程序化冷冻。说明GMP和OPS玻璃化冷冻优于Straw玻璃化冷冻。说明可以采用GMP方法冷冻保存牛GV期卵母细胞。 相似文献
8.
为了探究羟基磷灰石(HA)纳米颗粒对牛GV期卵母细胞玻璃化冷冻效果的影响，试验以牛卵巢中GV期卵母细胞作为试验材料，将不同粒径的HA纳米颗粒添加到玻璃化冷冻液中，进行超声分散检测。首先，以卵母细胞存活率和成熟率为指标，将0、0.01%、0.05%、0.1%HA纳米颗粒分别添加到玻璃化冷冻液Ⅰ(VSⅠ)和玻璃化冷冻液Ⅱ(VSⅡ)中，不经冷冻直接进行毒性测试；然后，以形态正常率、成熟率、卵裂率和囊胚率为指标测定卵母细胞玻璃化冷冻后的发育能力；最后检测含有0.05%HA纳米颗粒的VSⅡ对冷冻后卵母细胞活性氧水平和线粒体膜电位水平的影响。结果表明：不同浓度HA纳米颗粒对牛GV期卵母细胞均无明显毒性；VSⅡ+0.05%HA纳米颗粒组卵母细胞冷冻后的形态正常率、成熟率、卵裂率、囊胚率显著高于VSⅠ+0.05%HA纳米颗粒组(P<0.05);VSⅡ+0.05%HA纳米颗粒组卵母细胞的活性氧水平显著低于VSⅡ组(P<0.05);VSⅡ+0.05%HA纳米颗粒组卵母细胞的线粒体膜电位水平显著高于VSⅡ组(P<0.05)。说明在VSⅡ中加入0.05%HA纳米颗粒能显著降低牛GV期卵母细胞... 相似文献
9.
玻璃化冻存对驴卵母细胞超微结构的影响 总被引：1，自引：1，他引：0
试验旨在探究玻璃化冷冻对驴卵母细胞发育的影响,寻求驴卵母细胞冷冻的最佳条件。通过对不同发育时期的驴卵母细胞进行玻璃化冷冻,冷冻复苏后分别进行成熟培养和孤雌激活,并对GV期未冷冻组(对照组)、GV期冷冻组、IVM-MⅡ冷冻组卵母细胞微丝和线粒体超微结构进行免疫荧光标记,统计冷冻复苏后卵母细胞形态正常率、成熟率、孤雌激活卵裂率、超微结构正常率。结果表明,GV期冷冻组卵母细胞的形态正常率与GV期未冷冻组(对照组)间无显著差异(P0.05),成熟率和卵裂率均显著低于对照组(P0.05);IVM-MⅡ冷冻组的卵裂率显著低于对照组(P0.05),且卵裂后细胞发育受到阻滞。冷冻组微丝在皮质区分布明显减少的卵母细胞数目增多,冷冻组卵母细胞的线粒体数量明显低于对照组,由此可以说明冷冻对卵母细胞超微结构有损伤,从而导致复苏后成熟率下降,影响卵母细胞的受精和体外发育,且GV期冷冻组较IVM-MⅡ冷冻组在微丝与线粒体结构上有较小损伤,发育状态较好。 相似文献
10.
试验旨在探究玻璃化冷冻及培养过程中添加甘氨酸（glycine，Gly）对水貂GV期卵母细胞冷冻解冻后存活率、核发育、线粒体和皮质颗粒分布的影响。试验分为3组：对照组（没有进行冷冻处理）、冷冻组和Gly添加处理组（1 mmol/L Gly）。对玻璃化冷冻解冻后的水貂GV期卵母细胞分别进行平衡恢复3 h和体外成熟培养，采用免疫荧光标记法检测各组GV期卵母细胞线粒体分布的差异及MⅡ期皮质颗粒分布的变化。结果显示，Gly添加处理组卵母细胞在解冻后3 h的存活率与冷冻组相比差异不显著（P>0.05），但显著低于对照组（P<0.05）；Gly添加处理组卵母细胞的减数分裂恢复率显著高于冷冻组（P<0.05），但与对照组相比差异不显著（P>0.05）。免疫荧光结果显示，Gly添加处理组的GV期卵母细胞线粒体正常分布率显著高于冷冻组（P<0.05），但Gly添加处理组和冷冻组的GV期卵母细胞线粒体正常分布率均显著低于对照组（P<0.05）。皮质颗粒分布结果显示，水貂GV期卵母细胞在冷冻后体外成熟培养至MⅡ期时，Gly添加处理组皮质颗粒的正常皮质区分布比例显著高于冷冻组（P<0.05），但Gly添加处理组与冷冻组的正常皮质区分布比例均显著低于对照组（P<0.05）。结果表明，添加Gly可以提高冻融后水貂卵母细胞的减数分裂恢复率，降低冷冻对其线粒体及皮质颗粒的损失。 相似文献
11.
探讨程序化冷冻与玻璃化冷冻对小鼠GV期卵母细胞及二细胞期胚胎的复苏率及其发育潜能的影响。通过小鼠的卵母细胞与早期胚胎的不同冷冻方法的比较,为后续阿旺绵羊的胚胎冷冻保存提供参考。采用程序化冷冻与玻璃化冷冻技术,分别冷冻小鼠GV期卵母细胞及二细胞期胚胎,复苏后培养,比较不同冷冻处理后的复苏率、成熟率与囊胚率。小鼠GV期卵母细胞程序化冷冻复苏率(48.00%±5.29%)显著低于玻璃化冷冻复苏率(65.00%±5.00%),有统计学差异(P=0.0147<0.05);而程序化冷冻后复苏卵母细胞的发育成熟率略高于玻璃化冷冻组,但无统计学意义。小鼠二细胞期胚胎程序化冷冻组复苏率(76.00%±2.00%)显著高于玻璃化冷冻组复苏率(70.00%±2.00%),有统计学差异(P=0.0213<0.05);冷冻后复苏胚胎发育的囊胚率程序化冷冻略低于玻璃化冷冻及对照组,但无统计学意义。 相似文献
12.
13.
Jiménez-Trigos E Naturil-Alfonso C Vicente JS Marco-Jiménez F 《Reproduction in domestic animals = Zuchthygiene》2012,47(3):472-478
Although much progress has been made in oocyte cryopreservation since 1971, live offspring have only been obtained in a few species and in rabbits. The aim of our study was to evaluate the effect of vitrification and slow freezing on the meiotic spindle, cortical granule (CG) distribution and their developmental competence. Oocytes were vitrified in 16.84% ethylene glycol, 12.86% formamide, 22.3% dimethyl sulphoxide, 7% PVP and 1% of synthetic ice blockers using Cryotop as device or slow freezing in 1.5 m PROH and 0.2 m sucrose in 0.25 ml sterile French mini straws. Meiotic spindle and CG distribution were assessed using a confocal laser-scanning microscope. To determine oocyte competence, in vitro development of oocytes from each cryopreservation procedure was assessed using parthenogenesis activation. Our data showed that oocytes were significantly affected by both cryopreservation procedures. In particular, meiotic spindle organization was dramatically altered after cryopreservation. Oocytes with peripheral CG distribution have a better chance of survival in cryopreservation after slow-freezing procedures compared to vitrification. In addition, slow freezing of oocytes led to higher cleavage and blastocyst rates compared to vitrification. Our data showed that, in rabbits, structural alterations are more evident in vitrified oocytes than in slow-frozen oocytes, probably as a consequence of sensitivity to high levels of cryoprotectants. Slow-freezing method is currently the recommended option for rabbit oocyte cryopreservation. 相似文献
14.
AM Luciano F Franciosi V Lodde F Perazzoli M Slezáková S Modina 《Reproduction in domestic animals》2009,44(3):480-488
Joining immature gamete cryopreservation and germinal vesicle transplantation (GVT) technique could greatly improve assisted reproductive technologies in animal breeding and human medicine. The present work was aimed to assess the most suitable cryopreservation protocol between slow freezing and vitrification for immature denuded bovine oocytes, able to preserve both nuclear and cytoplasmic competence after thawing. In addition, the outcome of germinal vesicle transfer procedure and gamete reconstruction was tested on the most effective cryopreservation system. Oocytes, isolated from slaughterhouse ovaries, were stored after cumulus cells removal either by slow freezing or by vitrification in open pulled straws. After thawing, oocytes were matured for 24 h in co-culture with an equal number of just isolated intact cumulus enclosed oocytes, and fixed in order to evaluate the stage of meiotic progression and cytoskeleton organization. Our results showed that after warming, vitrified oocytes reached metaphase II (MII) in a percentage significantly higher than oocytes cryopreserved by slow freezing (76.2% and 36.5% respectively, p < 0.05). Moreover, vitrification process preserved the organization of cytoskeleton elements in a higher proportion of oocytes than slow freezing procedure. Therefore vitrification has been identified as the elective method for denuded immature oocytes banking and it has been applied in the second part of the study. Our results showed that 38.3% of oocytes reconstructed from vitrified gametes reached the MII of meiotic division, with efficiency not different from oocytes reconstructed with fresh gametes. We conclude that vitrification represents a suitable method of GV stage denuded oocyte banking since both nuclear and cytoplasmic components derived from cryopreserved immature oocytes can be utilized for GVT. 相似文献
15.
16.
KGhM Mahmoud TH Scholkamy YF Ahmed GE Seidel Jr MF Nawito 《Reproduction in domestic animals》2010,45(4):565-571
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. 相似文献
17.
E Jiménez‐Trigos C Naturil‐Alfonso JS Vicente F Marco‐Jiménez 《Reproduction in domestic animals》2013,48(1):15-19
The aim of this study was to investigate the effect of Taxol and Cytochalasin B on the spindle, chromosome configuration and development to blastocyst stage after parthenogenesis activation of in vivo matured rabbit oocytes after vitrification. Oocytes were randomized into four groups: oocytes treated with Cytochalasin B or Taxol before vitrification, oocytes without treatment before vitrification and fresh oocytes. Oocytes were vitrified using Cryotop method, and meiotic spindle and chromosomal distribution were assessed with a confocal laser scanning microscopy. To determine oocyte competence, in vitro development of oocytes was assessed with parthenogenesis activation. There were no significant differences in the frequencies of normal spindle (33.0%, 31.0% and 32.6%, for non‐treated, Taxol‐treated and Cytochalasin B‐treated oocytes, respectively) and chromosome (48.3%, 46.6% and 34.8%, for non‐treated, Taxol‐treated oocytes and Cytochalasin B‐treated oocytes respectively) in vitrified groups, but significantly lower than those of fresh group (89.7% and 90.2%, for normal spindle and chromosome organization, respectively). No statistical differences were found in the cleavage and blastocyst development rates between non‐treated and Taxol‐treated oocytes (7.7% and 1.5% and 13.7% and 4.6%, for non‐treated and Taxol‐treated oocytes, respectively), although they were significantly lower than in the fresh group (42.3% and 32.1%, for cleavage and blastocyst development, respectively). Oocytes treated with Cytochalasin B failed to reach blastocyst stage. Normal spindle, chromosome configuration and blastocyst development of in vivo matured rabbit oocytes were damaged in vitrification, which was not improved by Taxol and Cytochalasin B pre‐treatment before vitrification. Moreover, a detrimental effect on blastocyst development of Cytochalasin B pre‐treatment before vitrification was observed. 相似文献
18.
Q-H Hong S-J Tian S-E Zhu J-Z Feng C-L Yan X-M Zhao G-S Liu S-M Zheng 《Reproduction in domestic animals》2007,42(1):34-38
The aim of this study was to investigate the effects of different vitrification solutions [EFS30 or EFS40 contains 30% (v/v) ethylene glycol (EG), 40% (v/v) EG; EDFS30 or EDFS40 contains 15% (v/v) EG and 15% (v/v) dimethyl sulfoxide (DMSO), 20% (v/v) EG and 20% (v/v) DMSO], equilibrium time during vitrification (0.5-2.5 min) and vitrification protocols [one-step straw, two-step straw and open-pulled straw (OPS)] on in vivo development of vitrified Boer goat morulae and blastocysts after embryo transfer. In the one-step straw method, the lambing rates of vitrified embryos in EFS30 (37.5%), EFS40 (40.5%) or EDFS30 (38.2%) group were similar to that of fresh embryos (57.5%) and conventional freezing method (46.7%) when the equilibrium time was 2 min. In the two-step straw method, the highest lambing rate was obtained when embryos were pretreated with 10% EG for 5 min and then exposed to EFS40 for 2 min (51.4%), showing similar lambing rates compared with fresh embryos (56.1%) or the embryos cryopreserved by conventional freezing method (45.2%). In the OPS method, the lambing rate in EFS40, EDFS30 or EDFS40 groups were similar to that (57.1%) of fresh embryos, or to that (46.0%) of embryos cryopreserved by conventional freezing method. The highest lambing rate (51.4%) of the group of OPS was obtained when the embryos were vitrified with EDFS30. In conclusion, either the two-step straw method in which embryos were pretreated in 10% EG for 5 min and then exposed to EFS40 for 2 min, or the OPS method in which embryos were pretreated in 10% EG + 10% DMSO for 30 s and then exposed to EDFS30 for 25 s was a simple and efficient method for the vitrification of Boer goat morulae and blastocysts. 相似文献
19.
【目的】研究内质网IP3受体（IP3R）抑制剂2-氨基乙基联苯基硼酸酯（2-APB）对玻璃化冷冻-解冻牛MⅡ期卵母细胞发育能力的影响。【方法】将体外成熟24 h的卵丘-卵母细胞复合体（COCs）用0.1%透明质酸酶消化去除卵丘颗粒细胞，获得的卵母细胞分为对照组和2-APB组，采用OPS法进行玻璃化冷冻，对照组直接进行玻璃化冷冻，2-APB组在玻璃化冷冻前先用10 μmol/L 2-APB预处理10 min。2 d后解冻卵母细胞，统计解冻后（0 h）及恢复培养2 h时卵母细胞的存活率，用FITC-PNA荧光探针检测恢复培养2 h时卵母细胞皮质颗粒（CG）的分布，用2'，7'-DCHFDA和Cell Tracker Blue CMF2HC分别检测胞内活性氧（ROS）和谷胱甘肽（GSH）含量。将成熟培养24 h未冷冻卵母细胞（Fresh组），与解冻后恢复2 h的对照组和2-APB组卵母细胞同时进行孤雌激活，于培养的第2、7天分别统计孤雌胚胎的卵裂率和囊胚率。【结果】与对照组相比，2-APB组冷冻-解冻卵母细胞0和2 h的存活率均显著增加（P＜0.05）；2-APB组皮质颗粒皮质区分布比例显著提高（P＜0.05），损伤型分布比例显著降低（P＜0.05）；2-APB组卵母细胞内GSH含量显著增加（P＜0.05），ROS含量显著降低（P＜0.05）；2-APB组卵母细胞孤雌激活胚胎的卵裂率、囊胚率均显著增加（P＜0.05），且与Fresh组无显著差异（P＞0.05）。【结论】2-APB预处理可通过增加卵母细胞内GSH含量，降低卵母细胞皮质颗粒损伤型分布比例和胞内ROS含量，提高玻璃化冷冻保存牛MⅡ期卵母细胞质量及早期胚胎发育能力。 相似文献