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     

Stepwise in-straw dilution and direct transfer using open pulled straws (OPS) in the mouse: a potential model for field manipulation of vitrified embryos

In the present study, mouse blastocysts were employed to investigate the feasibility and efficiency of stepwise in-straw dilution and direct transfer using the open pulled straw (OPS) method. In experiment I, the effects of various vitrification solutions (VS) on embryo survival were examined. After thawing, the expanded blastocyst rates (97.59 and 95.05%) and hatching rates (80.48 and 78.95%) achieved in the EDFS30 [15% ethylene glycol (EG), 15% dimethyl sulfoxide (DMSO), Ficoll, and sucrose] and EFS40 [40% EG, Ficoll, and sucrose] groups were no different from those (96.15% and 83.33%) of the control group. However, the rates in the EFS30 [30% EG, Ficoll, and sucrose] (87.80 and 55.43%) and EDFS40 [20% EG, 20% DMSO, Ficoll, and sucrose] (95.69 and 70.97%) groups were significantly lower than those (96.15 and 83.33%) of the control group (P<0.05). In the experiment II, the effects of the volume of VS in the OPS on the survival of embryos after in-straw thawing were investigated. When the length of the VS in the column was less than 1 cm, the in vitro viability of embryos thawed by stepwise in-straw dilution was no different among the experimental and control groups. The embryos could be successfully thawed by immersing the OPS in 0.5 M sucrose for 3 min and then 0.25 M sucrose for 2 min. In experiment III, the effect of immersion time of the OPS in diluent (PBS) on the viability of vitrified embryos was investigated. After in-straw thawing, OPSs were immersed immediately in 1 ml PBS for 0 to 30 min. When the immersion time of the OPSs in PBS was less than 12 min, in vitro development of the in-straw thawed embryos was no different from that of the controls. In experiment IV, in-straw thawed blastocysts were directly transferred to pseudopregnant mice to examine their in vivo developmental viability. The pregnancy (91.67%) and birth rates (42.42%) of embryos in-straw thawed and directly transferred were no different from those of the unvitrified controls (90.90 and 40%) and embryos thawed by the conventional method (84.61 and 46.94%). These results demonstrate that mouse embryos vitrified with OPS could be successfully thawed by stepwise in-straw dilution and transferred directly to a recipient and that this method might be a model for field manipulation of vitrified embryos in farm animals.     

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%。     

Viability of OPS Vitrified Sheep Embryos After Direct Transfer

The aim of this study was to evaluate the viability in the effect of open pulled straw (OPS) vitrification procedure of sheep embryos after direct transference. Embryos were produced in vivo and cryopreserved in slow freezing or OPS vitrification. The survival rates of cryopreserved embryos were compared to non-frozen standard pattern. In a first set of experiments, embryos at morula and blastocyst stages were dived in ethylene glycol (1.5 M) and frozen in an automatic freezer. After being thawed, they were directly or indirectly transferred to ewes recipient. A second group of embryos were drawn into OPS and plunged into liquid nitrogen after being exposed at room temperature for 1 min and 45 s in 10% EG plus 10% dimethyl sulphoxide (DMSO), then again for 30 s in 20% EG + 20% DMSO + 0.5 M sucrose. After being warmed, embryos were also directly transferred using a French mini straw as the catheter for the transplantation process or after in vitro dilution of cryoprotectants (two-step-process). No significant difference was observed among fresh, frozen or vitrified embryos on pregnancy rate (50.0%, 38.6% and 55.8%). However, when we evaluated only the direct transference, the pregnancy rate of OPS vitrified embryos was higher than that of frozen embryos (57.1% vs 34.8%) (p = 0.07). In addition, vitrified morulae had a higher pregnancy rate than the one with frozen embryos (64.0% vs 38.9%) (p = 0.07). Finally, our results indicate that OPS vitrification technique in association with direct transference improves the viability of sheep embryos with potential applications to field conditions.     

Post-thaw development of in vitro produced buffalo embryos cryopreserved by cytoskeletal stabilization and vitrification

The present study was conducted to examine post-thaw in vitro developmental competence of buffalo embryos cryopreserved by cytoskeletal stabilization and vitrification. In vitro produced embryos were incubated with a medium containing cytochalasin-b (cyto-b) in a CO₂ incubator for 40 min for microfilament stabilization and were cryopreserved by a two-step vitrification method at 24℃ in the presence of cyto-b. Initially, the embryos were exposed to 10% ethylene glycol (EG) and 10% dimethylsulfoxide (DMSO) in a base medium for 4 min. After the initial exposure, the embryos were transferred to a 7 µl drop of 25% EG and 25% DMSO in base medium and 0.3 M sucrose for 45 sec. After warming, the embryos were cultured in vitro for 72 h. The post-thaw in vitro developmental competence of the cyto-b-treated embryos did not differ significantly from those vitrified without cyto-b treatment. The hatching rates of morulae vitrified without cyto-b treatment was significantly lower than the non-vitrified control. However, the hatching rate of cyto-b-treated vitrified morulae did not differ significantly from the non-vitrified control. This study demonstrates that freezing of buffalo embryos by cytoskeletal stabilization and vitrification is a reliable method for long-term preservation.     

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 medium composition and exposure time on post-thaw development of buffalo embryos produced in vitro

This study examined the effects of different vitrification medium compositions and exposure times (2, 4 and 6min) on the post-thaw development of buffalo embryos produced in vitro (IVP). The compositions were (1) 40% ethylene glycol (EG); (2) 25% glycerol (G)+25% EG, and (3) 25% EG+25% dimethylsulfoxide (DMSO). The base medium was 25mM Hepes-buffered TCM-199+10% steer serum +50microg/mL gentamycin. The IVP embryos were cryopreserved by a two-step vitrification method at 24 degrees C. After warming, the embryos were cultured in vitro for 72h. The vitrification of morulae and blastocysts in 25% EG+25% DMSO with an exposure time of 2 and 4min, respectively, resulted in a better hatching rate than other combinations. The hatching rate of morulae vitrified in 25% EG+25% G, 25% EG+25% DMSO, and blastocysts vitrified in 40% EG, 25% EG+25% DMSO were negatively correlated with exposure time. However, the hatching rate of blastocysts vitrified in 25% EG+25% G was positively correlated with exposure time. The study demonstrated that the post-thaw in vitro development of IVP buffalo embryos was affected by the vitrification medium composition and exposure time.     

奶牛GV期卵母细胞OPS法和GMP法超低温冷冻效果

采用OPS管和GMP管对GV期的牛的卵母细胞进行玻璃化冷冻.在不同的前处理液中平衡5 min,然后在冷冻液(EFS30,EFS40,EDFS30或EDFS40)中平衡30 s,进行OPS法和GMP法玻璃化冷冻保存.结果显示,OPS法用EFS40液和EDFS40液冷冻后形态正常卵率为69.6%和76.1%,2组差异显著(P<0.05),成熟率最高达19.2%和33.3%,2组差异显著(P<0.05);GMP法用EFS40液和EDFS40液冷冻后形态正常卵率最高达75.6%和80.8%,2组差异显著(P<0.05),成熟率最高达15.6%和34.9%,2组差异显著(P<0.05).而采用EDFS40液,OPS法和GMP法对GV期卵母细胞体外发育的影响差异均不显著,但GMP法的冷冻效率较高.表明采用EDFS40液GMP法对GV期卵母细胞的冷冻效率优于OPS法.     

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.     

Equilibrium vitrification of mouse embryos at various developmental stages using low concentrations of cryoprotectants

We previously developed a new vitrification method (equilibrium vitrification) by which two-cell mouse embryos can be vitrified in liquid nitrogen in a highly dehydrated/concentrated state using low concentrations of cryoprotectants. In the present study, we examined whether this method is effective for mouse embryos at multiple developmental stages. Four-cell embryos, eight-cell embryos, morulae, and blastocysts were vitrified with EDFS10/10a, 10% (v/v) ethylene glycol and 10% (v/v) DMSO in FSa solution. The FSa solution was PB1 medium containing 30% (w/v) Ficoll PM-70 plus 0.5 M sucrose. The state of dehydration/concentration was assessed by examining the survival of vitrified embryos after storage at –80°C. When four-cell embryos and eight-cell embryos were vitrified with EDFS10/10a in liquid nitrogen and then stored at –80°C, the survival rate was high, even after 28 days, with relatively high developmental ability. On the other hand, the survival of morulae and blastocysts vitrified in liquid nitrogen and stored at –80°C for four days was low. Therefore, morulae and blastocysts cannot be vitrified in a highly dehydrated/concentrated state using the same method as with two-cell embryos. However, when blastocysts were shrunken artificially before vitrification, survival was high after storage at –80°C for four days with high developmental ability. In conclusion, the equilibrium vitrification method using low concentrations of cryoprotectants, which is effective for two-cell mouse embryos, is also useful for embryos at multiple stages. This method enables the convenient transportation of vitrified embryos using dry ice.     

影响山羊胚胎冷冻效果因素的研究

分别用浓度为1.5 mol/L的乙二醇(EG),1.5 mol/L 1,2-丙二醇(PROH)和1.5 mol/L甘油为冷冻保护液对山羊胚胎进行常规冷冻保存,结果三者对山羊胚胎的冷冻保护效果无显著差异,其中以1.5 mol/L EG的冷冻保护效果为佳。以EFS40为玻璃化液对山羊胚胎进行细管法和OPS法玻璃化冷冻,其结果与常规冷冻间差异不显著,表明常规冷冻法、玻璃化细管法和OPS法均可用于山羊胚胎的冷冻保存。采用25℃和37℃水浴对常规冷冻和玻璃化冷冻后的山羊胚胎进行解冻,从解冻后的发育效果看,二者间无显著差异,但37℃水浴解冻后的胚胎发育效果略好于25℃。还比较了玻璃化液EFS40中添加FCS和BSA后与不添加其他成分的EFS40对胚胎冷冻保护效果的影响,结果表明添加BSA的EFS40的冷冻保护效果显著地高于不添加其他成分的EFS40,但与添加FCS的EFS40间不存在统计学上的差异。     

Vitrification of biopsied mouse embryos

Solid surface vitrification (SSV) was compared with in-straw vitrification for cryopreservation of biopsied mouse embryos. Eight-cell stage embryos were zona drilled and one blastomere was removed. Developed morulae or blastocysts were vitrified in microdrop (35% EG + 5% PVP + 0.4 M trehalose) or in straw (7.0 M EG + 0.5 M sucrose). Following recovery, embryos were cultivated in vitro or transferred into recipients. Cryopreservation had an effect not only on the survival of biopsied embryos but also on their subsequent development in vitro. Cryosurvival of biopsied morulae vitrified in straw was significantly inferior to SSV. The post-warm development of biopsied and non-biopsied morulae was delayed on Day 3.5 and 4.5 in both vitrification groups. A delay in development was observed on Day 5.5 among vitrified non-biopsied blastocysts. The percentage of pups born from biopsied morulae or blastocysts following cryopreservation did not differ from that of the control. No significant differences could be detected between methods within and between embryonic stages in terms of birth rate. The birth rate of biopsied embryos vitrified in straw was significantly lower compared to the non-biopsied embryos. The novel cryopreservation protocol of SSV proved to be effective for cryopreservation of morula- and blastocyst-stage biopsied embryos.     

冷冻方法和冷冻保护液对山羊卵母细胞发育的影响

比较了不同冷冻方法和冷冻保护液对山羊卵母细胞冷冻-解冻后体外受精和激活后的发育效果.结果表明,开放式拉长塑料细管(OPS)法的效果好于玻璃化细管法和常规冷冻法;在OPS法中,20%EG 20%DMSO对卵母细胞的冷冻保护效果好于EDFS40,而在玻璃化细管法冷冻中,则是EDFS40好于20%EG 20%DMSO.对于常规冷冻法而言,则是1.5mol/L EG的冷冻效果好于1.5mol/L PROH.     

冷冻后小鼠卵母细胞的透明带和质膜对体外受精的影响

以冷冻保存后的小鼠卵母细胞为实验材料,研究其膜对体外受精效果的影响。在室温(25±0.5)℃条件下,以10%乙二醇(EG)+10%二甲基亚砜(DMSO)为预处理液,EDFS30为玻璃化溶液。将卵母细胞于10%EG+10%DMSO溶液中预处理30s,然后再移入EDFS30中处理25s,以开放式拉长细管(OPS)为承载器投入液氮中,即两步法玻璃化冷冻保存。结果表明:冷冻卵母细胞受精后的卵裂率(46.67%)显著低于新鲜组的(86.06%)(P<0.05)。冷冻卵母细胞去透明带后质膜上的平均精子结合数(10.70)与对照组(10.81)无显著性差异(P>0.05),形成雌雄原核数也无显著性差异(2.49vs.2.59)(P>0.05)。冷冻卵母细胞透明带打孔后,其体外受精后卵裂率与新鲜组差异不显著(84.73%vs.91.19%)(P>0.05)。因此玻璃化冷冻保存小鼠卵母细胞透明带的变化是影响体外受精效果的主要因素之一。     

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.     

Effect of cryoprotectant composition on in vitro viability of in vitro fertilized and cloned bovine embryos following vitrification and in-straw dilution

In this study the efficacy of the combination of glycerol (GLY) and ethylene glycol (EG) as cryoprotectants in a vitrification method developed for direct embryo transfer was evaluated by in vitro development of in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos after vitrification. The IVF and SCNT blastocysts were vitrified in either 40% GLY, 30% GLY + 10% EG, or 20% GLY + 20% EG using French straws. After warming, the straws were held vertically for 1 min without shaking and were then placed horizontally for 5 min to dilute the cryoprotectants. After washing, the vitrified-warmed embryos were cultured in vitro for 72 h. There were no differences among the vitrification solutions with respect to the rates of vitrified-warmed IVF and SCNT embryos surviving and developing to the hatched blastocyst stage. However, the rates of development to the hatched blastocyst stage of the SCNT embryos vitrified with 40% GLY tended to be higher than those vitrified with 30% GLY + 10% EG or 20% GLY + 20% EG (26% vs. 7-8%, respectively). The development rates to the hatched blastocyst stage of the IVF and SCNT embryos vitrified with solution containing EG were significantly lower (P<0.05) than those of non-vitrified embryos. These results suggest that use of the combination of GLY and EG as cryoprotectants had no beneficial effect on the viability of embryos after in-straw dilution. However, this method is so simple that it can be used for practical direct transfer of vitrified embryos in the field.     

转基因兔胚胎玻璃化冷冻保存的研究

在２５℃条件下,将兔体外受精精子载体转基因兔桑椹胚置于含有４０％乙二醇、１８％Ｆｉｃｏｌ、０．３ｍｏｌ蔗糖的ｍＰＢＳ溶液（ＥＦＳ４０）中平衡２分钟,然后直接投入液氮,成功地进行了玻璃化冷冻保存。解冻后桑椹胚发育至囊胚和孵化囊胚的比例分别为６５．８１％和３９．２４％,与未经冷冻的鲜胚发育比例（７１．０５％和４３．４２％）相比,没有明显的差异。７８枚经玻璃化冷冻和解冻的桑椹胚移植给５只受体,其中２只妊娠,共产下８只活仔兔。     

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。     

Vitrification of mouse two-cell and blastocyst stage embryos in simplified closed system using either a hemi-straw or a hollow fiber device

Two-cell stage and blastocyst stage mouse embryos were equilibrated in a medium containing 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for 8–15 min. Vitrification was performed in a medium containing 0.5 M sucrose and either 15% EG + 15% DMSO, 17.5% EG + 17.5% DMSO, or 20% EG + 20% DMSO for 30 s. They were then placed either on a hemi-straw (HS) or a hollow fiber vitrification (HFV) device and vitrified by cooled air inside a 0.5-ml straw. In two-cell embryos, a 100% survival rate was obtained from all groups except the 20% HS group (P > .05). All vitrified two-cell groups showed similar rates of blastocyst development to that of fresh control group (P > .05), except 17.5% and 20% HFV groups, which were significantly lower than the other groups (P < .05). In the blastocyst embryos, the HFV groups were divided into two subgroups (non-collapsed; HFV-NC and collapsed; HFV-C blastocyst). Re-expansion rate in 15% HFV-NC, 17.5% HFV-NC, and 15% HFV-C groups was reduced (P < .05), whereas the rest were similar to control. In conclusion, we established a simplified, reliable, and closed system for HFV vitrification applying hemi-straw, which does not require skilled practitioners.     

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。