玻璃化冷冻对小鼠GV期卵母细胞发育能力的影响

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。     

小鼠卵母细胞冷冻技术研究

采用PBS作为冷冻基础液,分别用甘油和二甲基亚砜(DMSO)作为冷冻保护液,在程序化冷冻保存和玻璃化冷冻保存条件下,研究小鼠生发泡期(GV期)卵母细胞的抗冻能力。结果表明,2种冷冻方法对小鼠GV期卵母细胞解冻后形态正常率和存活率无显著影响(P>0.05)。冷冻保护剂种类对小鼠GV期卵母细胞解冻后形态正常率无显著影响(P>0.05);但对存活率有显著影响,玻璃化冷冻采用二甲基亚砜作为冷冻保护液效果极显著优于甘油(P<0.01)。以冷冻效果较好的二甲基亚砜作为冷冻保护液,采用玻璃化冷冻不同发育阶段(GV期和MⅡ期)的小鼠卵母细胞,解冻后形态正常率无显著差异(P>0.05),但存活率GV期要显著优于MⅡ期卵母细胞(P<0.05)。     

开放式拉长细管冷冻法对牛卵母细胞体外受精后发育的影响

在常规牛体外受精(IVF)技术的基础上,分别采用开放式拉长细管 (OPS,open pulled straw)法和细管法对未经成熟培养的卵丘卵母细胞(COCs)进行玻璃化冷冻,解冻后再进行体外成熟(IVM)、IVF和早期胚胎的体外培养(IVC)。结果表明,细管组和 OPS组的解冻后 COCs正常率分别为 59.4%±4.3%和 77.9%±4.1%(P<0 01);成熟率分别为48.2%±5.3%和66.0%±5.8%(P<0 01);卵裂率分别为18.5%±2.0%和32.8%±1.4%(P<0 01);8 细胞阶段的成功率分别为14.8%±2.5%和 24.8%±1.5%(P<0 01);桑椹胚发育率分别为 0 和5 3%±1.1%,明显低于未经冷冻的鲜卵组(21.0%±3.8%;P<0 01);囊胚发育率分别为0和4.0%,明显低于鲜卵组(P<0 01)。说明OPS玻璃化冷冻法可以使未经成熟培养的牛 COCs冷冻后获得桑椹胚和囊胚,但桑囊胚发育率仍较低,方法有待改进。     

牛GV期卵母细胞冷冻保存后发育潜力的研究

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。     

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）。     

OPS玻璃化冷冻对小鼠四倍体胚胎体外发育的影响

为探究开放式拉长细管(OPS)玻璃化冷冻对四倍体胚胎发育的影响,本实验利用2-细胞胚胎电融合法制备四倍体胚胎,再对四倍体胚胎进行OPS玻璃化冷冻,分别观察记录二倍体胚胎、四倍体胚胎以及冷冻解冻后四倍体胚胎的发育情况。结果表明:2-细胞胚胎电融合效率为96.1%;二倍体胚胎组与电融合后四倍体胚胎组的囊胚率和孵化囊胚率差异不显著;冷冻解冻后四倍体胚胎的囊胚率(100%)与四倍体新鲜组(93.3%)差异不显著,其孵化囊胚率(72.3%)较新鲜组(64.9%)显著增高(P<0.05);四倍体冷冻解冻组的囊胚细胞数(31.96)与新鲜组(32.54)无显著差异;冷冻解冻后的四倍体早期囊胚进行体外培养时其发育速度比对照组更快。可见,冷冻对小鼠四倍体胚胎的囊胚率和囊胚细胞数均无显著影响,但孵化囊胚率显著提高,且OPS玻璃化冷冻后使四倍体胚胎的发育速度更快。     

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).     

玻璃化冻存对驴卵母细胞超微结构的影响

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。     

不同冷冻方法对水牛体外生产胚胎冷冻效果的研究

本研究采用不同来源的体外生产胚胎(屠宰场卵巢IVF胚胎,OPU-IVF胚胎,SCNT胚胎),系统研究了不同冷冻方法对水牛体外生产胚胎冷冻效果的影响,以完善水牛体外生产胚胎冷冻方法,进一步提高胚胎冷冻效果。试验选用6～7日龄囊胚分别用不同的冷冻液和不同冷冻方法进行胚胎冷冻。玻璃化冷冻液分别为40%EG、25%EG+25%DMSO和20%EG+20%DMSO+0.5 mol/L蔗糖;程序化冷冻液分别为10%甘油和0.05 mol/L海藻糖+1.8 mol/L EG+0.4%BSA。结果表明:(1)在玻璃化冷冻中,无论何种胚胎不同冷冻液的冷冻效果有明显的差异,但均以20%EG+20%DMSO+0.5 mol/L蔗糖作为冷冻液的冷冻效果最好,并且高于程序化冷冻的存活率;而对于程序化冷冻,用10%甘油作为冷冻液,屠宰场卵巢IVF胚胎的冷冻后存活率略高于用0.05 mol/L海藻糖+1.8 mol/L EG+0.4%BSA的冷冻后存活率,但差异不显著(P>0.05)。(2)VF胚胎利用程序化冷冻胚胎解冻后,在0～24 h内有76.5%胚胎复活,高于玻璃化冷冻的复苏率(48.9%)(P<0.05);而与此相反,在24～48 h内,玻璃化冷冻胚胎的复苏率(42.6%)则高于程序化冷冻(23.5%)(P<0.05)。综上所述,各种来源的水牛体外生产胚胎均可进行冷冻保存,应用玻璃化冷冻的效果好于程序化冷冻,且以20%EG+20%DMSO+0.5 mol/L蔗糖作为冷冻液进行玻璃化冷冻效果最好,但程序化冷冻后的胚胎复苏速度明显快于玻璃化冷冻的速度。     

添加HA纳米颗粒对牛GV期卵母细胞玻璃化冷冻后发育能力的影响

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...     

Effects of slow freezing and vitrification on embryo development in domestic cat

Cryopreservation of gametes and embryos is used to maintain genetic diversity of domestic and wild felids. However, felid oocytes and preimplantation embryos contain large amount of intracellular lipids, which affect their cryosensitivity. The objective was to compare the effects of slow freezing and vitrification and to study lipid phase transition (LPT) during cooling in cat embryos. In vitro-derived embryos were cultured 48 hr up to 4–8 cell stage, thereafter were either slow frozen or vitrified. Propylene glycol (PG) alone was used as a cryoprotective agent (CPA) for slow freezing, and a mixture of PG and dimethyl sulfoxide (DMSO) were used as CPAs for vitrification. After thawing/warming, embryos were in vitro cultured additionally for 72 hr. The total time of in vitro culture was 120 hr for all the groups including non-frozen controls. Effects of both cryopreservation procedures on the subsequent embryo development and nuclear fragmentation rate in embryonic cells were compared. There was no significant differences among the percentages of embryos achieved morula and early blastocyst stage in frozen-thawed group (36.4% and 20.0%), in vitrified-warmed group (34.3% and 28.6%) and in controls (55.6% and 25.9%). Cell numbers as well as nuclear fragmentation rate did not differ in these three groups. Average lipid phase transition (LPT) temperature (T*) was found to be relatively low (–2.2 ± 1.3°C) for the domestic cat embryos. It is supposed that the low LPT of LDs may provide a good background for successful application of slow freezing to domestic cat embryos. Generally, our study indicates that slow freezing and vitrification are both applicable for domestic cat embryo cryopreservation.     

Comparison of Different Cryopreservation Techniques: Higher Survival and Implantation Rate of Frozen–Thawed Mouse Pronuclear Embryos in the Presence of Beta‐Mercaptoethanol in Post‐Thaw Culture

The objective of this study was to investigate the effects of beta‐mercaptoethanol (β‐ME) on post‐thaw embryo developmental competence and implantation rate of mouse pronuclear (PN) embryos that were cryopreserved after slow freezing, solid surface vitrification (SSV) or open‐pulled straw (OPS) vitrification methods. Mouse PN embryos were cryopreserved by using slow freezing, SSV and OPS methods. After cryopreservation, freeze–thawed PN embryos were cultured up to blastocyst stage in a defined medium supplemented without or with 50 μm β‐ME. The blastocyst formation rate of embryos that were cryopreserved by slow freezing method (40.0%) or vitrified by OPS method (18.3%) were lower than those vitrified by SSV method (55.6%) and fresh embryos (61.9%) in the absence of 50 β‐ME in the culture media (p < 0.05). The blastocyst formation rate of embryos that were cryopreserved by slow freezing method (53.1%) or by OPS method (41.9%) were lower than those vitrified by SSV method (79.5%) and that of fresh (85.7%) in the presence of β‐ME in the culture media (p < 0.05). The embryos transfer results revealed that the implantation rate of blastocyst derived from mouse PN embryos vitrified by SSV method (31.9% vs 51.2%) was similar to that of the control (39.0% vs 52.5%), but higher than those cryopreserved by slow freezing (28.2% vs 52.0%) and by OPS method (0.0% vs 51.2%) (p < 0.05). In conclusion, supplementation of β‐ME in an in vitro culture medium was shown to increase survival of embryo development and implantation rate of frozen–thawed mouse PN embryos after different cryopreservation protocols.     

云岭山羊卵母细胞玻璃化冷冻的研究

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。     

Effect of vitrification at different meiotic stages on epigenetic characteristics of bovine oocytes and subsequently developing embryos

Vitrification by the Cryotop method is frequently used for bovine oocyte cryopreservation. Nevertheless, vitrified oocytes still have reduced developmental competency compared with fresh counterparts. The objective of this study was to compare the effect of vitrification either at the germinal vesicle (GV) stage or at the metaphase II (MII) stage on epigenetic characteristics of bovine oocytes and subsequently developing embryos. Our results demonstrated that vitrification of oocytes at each meiotic stage significantly reduced blastocyst development after in vitro fertilization (IVF). However, vitrification at the GV stage resulted in higher blastocyst development than did vitrification at the MII stage. Irrespective of the meiotic stage, oocyte vitrification did not affect 5-methylcytosine (5mC) immunostaining intensity in oocyte DNA. However, at both stages, it caused a similar reduction of 5mC levels in DNA of subsequently developing blastocysts. Oocyte vitrification had no effect on the intensity of H3K9me3 and acH3K9 immunostaining in oocytes and subsequent blastocysts. The results suggest that irrespective of meiotic stage, oocyte vitrification alters global methylation in resultant embryos although such alteration in the oocytes was not detected. Oocyte vitrification might not influence histone acetylation and methylation in oocytes and resultant embryos. Vitrification at the immature stage was more advantageous for blastocyst development than at the mature stage.     

Ultra‐Structural Alterations in In Vitro Produced Four‐Cell Bovine Embryos Following Controlled Slow Freezing or Vitrification

Cryopreservation is the process of freezing and preserving cells and tissues at low temperatures. Controlled slow freezing and vitrification have successfully been used for cryopreservation of mammalian embryos. We investigated the effect of these two cryopreservation methods on in vitro produced four‐cell stage bovine embryos which were classified according to their quality and separated into three groups. The first group was maintained as untreated controls (n = 350). Embryos of the second (n = 385) and the third (n = 385) groups were cryopreserved either by controlled slow freezing or by vitrification. Embryos in groups 2 and 3 were thawed after 1 day. Hundred embryos were randomly selected from the control group, and 100 morphologically intact embryos from the second and third group were thawed after 1 day and cultured to observe the development up to the blastocyst stage. The blastocyst development rate was 22% in the control group, 1% in the slow‐freezing group and 3% in the vitrification group. Remaining embryos of all three groups were examined by light microscopy, transmission electron microscopy and immunofluorescence confocal microscopy with subsequent histological staining procedures. Cryopreservation caused degenerative changes at the ultra‐structural level. Compared with vitrification, slow freezing caused an increased mitochondrial degeneration, cytoplasmic vacuolization, disruption of the nuclear and plasma membrane integrity, organelle disintegration, cytoskeletal damage, a reduced thickness of the zona pellucida and a formation of fractures in the zona pellucida. Further studies are required to understand and decrease the harmful effects of cryopreservation.     

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。     

Effect of vitrification procedures on the subsequent development of in vitro matured swamp buffalo oocytes following in vitro fertilization

Nowadays, the efficiency of buffalo oocytes cryopreservation is still low. The purpose of this study was to evaluate effects of two combinations of cryoprotectant agents (CPAs) and two vitrification devices for vitrification of swamp buffalo oocytes on their survival after vitrification warming, and subsequent developmental ability after in vitro fertilization. In vitro matured (IVM) oocytes were vitrified by either Cryotop (CT) or solid surface vitrification (SSV) interacting with vitrification solution A (VA) or B (VB). In the VA or VB solution exposed test, the oocytes showed similar survival rates, but decreased blastocyst rates after in vitro fertilization compared with that of untreated oocytes. After vitrification, the CT method combined with VA solution yielded a higher survival rate (91.3 ± 5.84%) of vitrified oocytes than that combined with VB solution (69.8 ± 4.19%–75.8 ± 4.55%); however, all the vitrification treatments showed lower blastocyst rates (1.1 ± 0.07%–5.2 ± 0.24%) compared with that of untreated oocytes (18.0 ± 1.09%). Our results indicated that combined vitrification treatments in this study did not improve the decreased ability of vitrified oocytes developing to the blastocyst stage.