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).     

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。     

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）。     

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。     

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。     

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)。     

毛细玻管玻璃化冷冻法冷冻保存牛卵母细胞的研究

为了探讨毛细玻管玻璃化冷冻法(GMP)对牛卵母细胞冷冻效果的影响,试验采用毛细玻管玻璃化冷冻法冷冻不同发育阶段的牛卵母细胞,并且对不同预处理方式和玻璃化时间对玻璃化冷冻牛卵母细胞解冻后形态及体外受精卵裂率的影响进行了比较.结果表明:采用毛细玻管玻璃化冷冻法冷冻GV期和体外培养6 h、18 h、22 h各发育阶段的卵母细胞,解冻后体外受精后卵裂率分别是36.67%、40.98%、41.27%、52.38%,前三者之间差异不显著(P>0.05),前三者与培养22 h卵母细胞(52.38%)和对照细胞(68.42%)差异极显著(P<0.01);采用不同浓度的稀溶液预处理卵母细胞,3%乙二醇稀溶液预处理玻璃化前牛卵母细胞可分别获得较高的卵裂率(36.96%);卵母细胞在玻璃化溶液中暴露时间越长受到损伤越严重,暴露30 s能获得最好的玻璃化冷冻效果.     

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-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。     

母源基因Gdf9、Zar1、Mater和Dnmt1 mRNA在绵羊卵母细胞和早期胚胎中的表达

为探讨母源基因在绵羊卵母细胞和胚胎发育过程中的表达模式,运用Real-time PCR技术研究4种母源基因:Gdf9(生长分化因子9)、Zar1(合子阻泄因子)、Mater(胚胎必要的母体抗原)及Dnmt1(DNA甲基化转移酶1)的mRNAs在绵羊GV期卵母细胞,24h成熟卵母细胞,2-细胞、4-细胞、8-细胞、16-细胞胚胎以及囊胚中的含量变化情况。结果表明:在GV期卵中Zar1、Mater、Gdf9以及Dnmt1m RNA相对含量最高(P0.05);从2-细胞胚胎开始,4种基因的mRNA相对含量显著降低(P0.05),各基因mRNA的含量在不同发育阶段的卵母细胞和胚胎中存在动态变化,这对卵子生长和胚胎发育有重要意义。     

C型钠肽预处理对绵羊卵母细胞体外成熟及相关基因表达的影响

本实验旨在研究C型钠肽(CNP)预处理对绵羊卵母细胞体外成熟(IVM)及相关基因(Dnmt1、Zar1、Mater、HAS2、PTX3和PTGS2)表达的影响。以体外成熟24 h为对照组,研究CNP预处理组(200nmol/L CNP预处理4 h然后体外成熟24 h)中卵母细胞经孤雌激活(PA)后的发育情况,并通过RT-q PCR检测CNP预处理对成熟相关基因表达的影响。结果表明:200 nmol/L CNP可在4 h内有效抑制卵母细胞减数分裂进程。CNP预处理组中孤雌胚胎囊胚率显著高于对照组(P0.05);绵羊卵母细胞成熟培养前,CNP预处理组中卵丘细胞扩展相关基因HAS2、PTGS2和PTX3表达量较对照组极显著上调(P0.01);CNP预处理组中母源基因Dnmt1、Zar1和Mater表达量与对照组均无显著差异(P0.05)。绵羊卵母细胞成熟培养后,CNP预处理组中PTX3表达量极显著高于对照组(P0.01),而CNP预处理组中HAS2表达量较对照组极显著下调(P0.01);CNP预处理组中母源基因Dnmt1、Zar1和Mater表达量均显著高于对照组(P0.05)。综上表明,CNP预处理可影响绵羊卵丘细胞扩展相关基因HAS2、PTGS2、PTX3和母源基因Dnmt1、Zar1、Mater的表达,并提高绵羊卵母细胞成熟质量及早期胚胎发育能力。     

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。     

Antioxidants and glycine can improve the developmental competence of vitrified/warmed ovine immature oocytes

Despite the numerous potential applications of oocyte cryopreservation, the poor success rate has limited its practical applications. In livestock, particularly in ovine, the oocytes have low developmental competence following vitrification/warming process. Considering the occurrence of osmotic and oxidative stresses during the vitrification/warming process, the application of antioxidants and osmolytes may improve the developmental competence of vitrified/warmed oocytes. In the present study, we aimed to evaluate the effects of the addition of ascorbic acid (AA) and N‐acetyl cysteine (NAC) as antioxidants and glycine as an organic osmolyte either to the vitrification/warming solutions (VWS) or to the IVM medium on the developmental competence of vitrified/warmed ovine germinal vesicle stage oocytes. The survival rate in the vitrified groups was significantly lower than fresh ones. In vitrified/warmed oocytes, there was no significant difference in survival rate between supplemented and non‐supplemented groups. The addition of AA and/or NAC to the VWS or IVM medium and adding glycine to the IVM medium reduced the proportion of apoptotic oocytes and fragmented embryos, which was reflected as an increase in the proportions of metaphase II stage oocytes and blastocyst production. The best result was achieved by supplementing the IVM medium with NAC. In our study condition, antioxidants and glycine could improve the developmental competence of vitrified/warmed ovine immature oocytes, especially when added during IVM.     

猪卵母细胞在GV期与MⅡ期的冷冻保存效果比较研究

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。     

Comparative Effects of Porcine Oocytes Vitrified at the GV and MⅡ Stages

This study was designed to compare the efficiency of porcine oocytes vitrified at the GV and MⅡ stages. The vitrified GV oocytes were matured in vitro and then evaluated their cortical granule distribution with immunofluorescence, CD9 protein level with Western blotting and zona pellucida dissolution time with pronse digestion method, as compared to vitrified MⅡ oocytes.The results showed that the survival percentages of oocytes vitrified at the GV stage were significantly lower than those vitrified at the MⅡ stage after 2 h of warming (P<0.05);However, there was no difference in maturation rate between vitrified and fresh oocytes (P>0.05). The oocytes vitrified at MⅡ stage resulted in significantly decreased normal cortical granule distribution and CD9 protein level (P<0.05), but no such results were found in vitrified GV oocytes after maturation (P>0.05). In addition, vitrification did not affect the dissolution time of zona pellucida in GV and MⅡ oocytes (P>0.05). The data demonstrated that despite of lower survival rate compared with MⅡ oocytes, vitrified porcine GV oocytes after maturation showed normal polar body extrusion, cortical granule distribution and CD9 protein level.     

Effect of vitrification of feline ovarian cortex on follicular and oocyte quality and competence

Cryopreservation of ovarian cortex has important implications in the preservation of fertility and biodiversity in animal species. Slow freezing of cat ovarian tissue resulted in the preservation of follicular morphology and in the follicular development after xenografting. Vitrification has been recently applied to ovarian tissues of different species, but no information is available on the effect of this method on feline ovarian cortex. Moreover, meiotic competence of fully grown oocytes isolated from cryopreserved tissue has not been reported. The aim of this study was to evaluate the effect of vitrification of feline ovarian cortex on follicular morphology and oocyte integrity, as well as meiotic competence. A total of 352 fragments (1.5-2 mm(3) ) were obtained from ovarian cortical tissues: 176 were vitrified and 176 were used fresh as control. Histological evaluation of fresh and vitrified fragments showed intact follicles after cryopreservation procedures with no statistically significant destructive effect from primordial to antral follicles. After IVM, oocytes collected from vitrified ovarian fragment showed a higher proportion of gametes arrested at germinal vesicle (GV) stage compared to those isolated from fresh control tissue (33.8% vs 2.9%; p < 0.001). However, oocytes isolated from vitrified tissues were able to resume meiosis, albeit at lower rate than those collected from fresh tissues (39.8% vs 85.9%; p < 0.00001). Vitrification induced changes in the organization of cytoskeletal elements (actin microfilaments and microtubules) of oocytes, but significantly only for actin network (p < 0.001). Finally, chromatin configuration within the GV was not affected by the cryopreservation procedure. Our study demonstrated that vitrification preserves the integrity of ovarian follicles and that oocytes retrieved from cryopreserved tissue maintain the capability of resuming meiosis. To our knowledge, this has not previously been reported in the cat.     

牛体外成熟卵母细胞冷冻保存的研究

本实验探讨了用６各不同方法冷冻保存的牛体外成熟卵母细胞体受精后的发育潜力及其冷冻损伤，尽管用不同方法冷冻的牛卵母细胞多数（８６．１％）形态正常，但体外受精后的受精率（３０．０％）和卵裂率（７．４％）明显低于对照组（６９．０％和５２．９％）。卵裂后继续发育的能力严重受损，在６种冷冻方法中，１．６Ｍ丙二醇分步平衡、程序冷冻效果最好，受精率和卵裂率分别达３６．４％和１３．５％，冷冻后卵母细胞发育潜力下降     

Vitrification of fully grown and growing porcine oocytes using germinal vesicle transfer

The survival rate of vitrified germinal vesicle (GV) stage porcine oocytes is very low, and it is not known if the vitrification damages the nucleus, cytoplasm or both. We have evaluated the eventual GV or cytoplasmic damage in fully grown (FG) and growing vitrified oocytes. Fifty-five percent of nonvitrified FG cumulus-denuded oocytes reached the metaphase II (MII) stage in culture. When growing oocytes from preantral (PA) and early antral (EA) follicles were matured in vitro, almost all oocytes were arrested at the GV stage (GV stage: PA 88.9 and EA 79.5%, respectively). When fresh GVs from FG, PA and EA oocytes were transferred into fresh enucleated FG oocytes and matured in vitro, some of them reached the MII stage (MII stage: FG/FG 57.5%, PA/FG 9.3% and EA/FG 35.3%, respectively). The maturation rate of vitrified FG oocytes was only 6.1% but increased dramatically when vitrified GVs from FG, PA and EA oocytes were transferred into fresh enucleated FG oocytes (MII stage: VitFG/FG 43.9%, VitPA/FG 7.1% and VitEA/FG 26.3%, respectively). These results were not significantly different from those for the nonvitrified groups (MII stage: FG/FG 57.5%, PA/FG 9.3% and EA/FG 35.3%, respectively). We activated the reconstructed oocytes that received fresh or vitrified GVs (FG/FG, EA/FG, VitFG/FG and VitEA/FG) and examined their embryonic development. Cleaved embryos (nonvitrified groups 13.0-61.8%, vitrified groups 33.3-40.0%) and blastocysts (nonvitrified groups 0.0-18.2%, vitrified groups 0.0-2.9%) were obtained after activation. These results demonstrate that vitrified porcine GVs maintain maturational and developmental competence and that vitrification predominantly damages the cytoplasm.     

Developmental competence of in vitro matured ovine oocytes vitrified in solutions with different concentrations of trehalose

This study aimed to determine the optimum concentration of trehalose in solutions used for vitrification of in vitro matured (IVM) ovine oocytes. IVM oocytes were randomly divided into four experimental (vitrified) and one control (fresh) groups. Experimental groups were treated with different concentrations (0.0, 0.25, 0.5 and 1.0 M) of trehalose. After warming, some viable oocytes were exposed to 0.25% pronase to test zona pellucida hardening, whereas the others were fertilized and cultured in vitro for 8 days to evaluate their developmental competence. Blastocysts quality was assessed by differential staining and TUNEL test. Survival and developmental rates of oocytes vitrified in the presence of 0.5 M trehalose were significantly higher than those of the other vitrified groups. Furthermore, there was a significant difference between fresh and vitrified groups in total blastocyst rate. Analysis of blastocysts quality also revealed a significant difference between the group treated with 0.5 M trehalose and other groups in terms of apoptotic index. Furthermore,zona pellucida digestion time period was longer in trehalose‐free (0.0 M) group compared to other groups. In conclusion, we found that IVM ovine oocytes vitrified in solutions containing 0.5 M trehalose are fertilization‐competent and are able to produce good‐quality blastocysts with an apoptotic index comparable to that of the fresh oocytes. Therefore, 0.5 M may be considered the optimum concentration of trehalose to be used in solutions prepared for vitrification of oocytes.     

Cryopreservation of immature buffalo oocytes: Effects of cytochalasin B pretreatment on the efficiency of cryotop and solid surface vitrification methods

The aim of the present study was to compare the efficiency of the solid surface (SSV), cryotop (CT) vitrification methods and cytochalasin B (CB) pretreatment for cryopreservation of immature buffalo oocytes. Cumulus‐oocyte complexes (COCs) were placed for 1 min in TCM199 containing 10% dimethylsulfoxide (DMSO), 10% ethylene glycol (EG), and 20% fetal bovine serum, and then transferred for 30 s to base medium containing 20% DMSO, 20% EG and 0.5 mol/L sucrose. CB pretreated ((+)CB) or non‐pretreated ((?)CB) COCs were vitrified either by SSV or CT. Surviving vitrified COCs were selected for in vitro maturation (IVM) and in vitro fertilization (IVF). The rate of viable oocytes after vitrification in CT groups (82%) was significantly lower (P < 0.05) than that in a fresh control group (100%), but significantly higher (P < 0.05) than those in SSV groups (71–72%). Among vitrified groups, the highest maturation rate was obtained in the CT (?)CB group (32%). After IVF, the cleavage and blastocyst formation rates were similar among vitrified groups but significantly lower than those of the control group. In conclusion, a higher survival rate of oocytes after vitrification and IVM was obtained in the CT group compared with that in the SSV group, indicating the superiority of the CT method. Pretreatment with CB did not increase the viability, maturation or embryo development of vitrified oocytes.     

Improvement of cumulus-free oocyte maturation in vitro and its application to microinsemination with primary spermatocytes in mice

In micromanipulation experiments using immature oocytes, final ooplasmic maturation is often compromised because the oocytes are usually first freed from their nurturing cumulus cells. This study was undertaken to determine whether cumulus-free in vitro maturation (IVM) in mice could be improved by modifying IVM medium having defined components. Cumulus-free germinal vesicle (GV) stage oocytes were subjected to IVM in either alphaMEM medium, TYH medium, or a 1:1 mixture of the two (termed TaM). TYH medium produced a better maturation rate (181/196; 92.3%) than alphaMEM (184/257; 71.6%). However, alphaMEM supported better embryo development to the morula/blastocyst stage than TYH following in vitro fertilization (93.3% vs. 76.5%) or parthenogenetic activation (82.4% vs. 60.4%). Mitochondrial distribution in MII oocytes was diffuse following IVM in alphaMEM, but was aggregated with TYH. The maturation promoting factor (MPF) activity in MII oocytes was significantly higher in TYH than in alphaMEM (P<0.05). Oocytes cultured in TaM had intermediate characteristics and essentially resembled in vivo matured oocytes, with the mitochondrial distribution pattern being most typical of that condition. The highest rate of development from GV oocytes to full-term fetuses following in vitro fertilization and embryo transfer to foster mothers (23.8%) was obtained using TaM. When this IVM system was applied to MI oocytes injected with spermatocytes, offspring were first obtained without cytoplasmic replacement at MII. Thus, optimization of the culture medium can considerably improve the quality of cumulus-free oocyte IVM in mice.