钌红对玻璃化冷冻牛卵母细胞线粒体Ca~(2+)的调控研究

玻璃化冷冻会严重损伤哺乳动物卵母细胞的线粒体功能,进而极大地限制了其解冻后的发育能力。为此,本试验设置3个钌红(RR)处理组,即牛卵母细胞用含0.5、1、2μmol/L RR的玻璃化冷冻液进行冷冻,解冻后放入含0.5、1、2μmol/L RR的体外成熟液中继续培养0.5h,同时,新鲜卵母细胞一部分不进行冷冻,一部分用不含RR的冷冻液进行玻璃化冷冻,分别作为新鲜对照组和玻璃化冷冻对照组,然后共检测5组牛卵母细胞线粒体Ca~(2+)水平、ATP含量及孤雌激活后胚胎的发育能力,进而研究RR对玻璃化冷冻牛卵母细胞线粒体Ca~(2+)水平的调控作用。结果显示:(1)玻璃化冷冻显著提高了牛卵母细胞中线粒体Ca~(2+)水平(P0.05),而2μmol/L RR处理组线粒体Ca~(2+)水平显著低于冷冻对照组(P0.05),但与新鲜组相比无显著差异(P0.05);(2)玻璃化冷冻显著降低了牛卵母细胞中ATP含量(P0.05),2μmol/L RR处理组卵母细胞中ATP含量显著高于冷冻对照组及0.5、1μmol/L RR处理组(P0.05);(3)玻璃化冷冻对照组卵裂率、囊胚率显著低于新鲜对照组(P0.05),1μmol/L处理组卵裂率、囊胚率与新鲜对照组相比无显著差异(P0.05)。综上所述,RR处理能显著抑制解冻后牛卵母细胞线粒体Ca~(2+)流入,保护线粒体功能,提高其发育能力。本试验结果为正向调控玻璃化冷冻卵母细胞线粒体Ca~(2+)水平,进而提高其发育能力,促进玻璃化冷冻卵母细胞的广泛应用提供了参考依据。     

钌红对玻璃化冷冻牛卵母细胞线粒体Ca2+的调控研究

玻璃化冷冻会严重损伤哺乳动物卵母细胞的线粒体功能,进而极大地限制了其解冻后的发育能力。为此,本试验设置3个钌红（RR）处理组,即牛卵母细胞用含0.5、1、2 μmol／L RR的玻璃化冷冻液进行冷冻,解冻后放入含0.5、1、2 μmol／L RR的体外成熟液中继续培养0.5 h,同时,新鲜卵母细胞一部分不进行冷冻,一部分用不含RR的冷冻液进行玻璃化冷冻,分别作为新鲜对照组和玻璃化冷冻对照组,然后共检测5组牛卵母细胞线粒体Ca²⁺水平、ATP含量及孤雌激活后胚胎的发育能力,进而研究RR对玻璃化冷冻牛卵母细胞线粒体Ca²⁺水平的调控作用。结果显示：①玻璃化冷冻显著提高了牛卵母细胞中线粒体Ca²⁺水平（P＜0.05）,而2 μmol／L RR处理组线粒体Ca²⁺水平显著低于冷冻对照组（P＜0.05）,但与新鲜组相比无显著差异（P＞0.05）;②玻璃化冷冻显著降低了牛卵母细胞中ATP含量（P＜0.05）,2 μmol／L RR处理组卵母细胞中ATP含量显著高于冷冻对照组及0.5、1 μmol/L RR处理组（P＜0.05）;③玻璃化冷冻对照组卵裂率、囊胚率显著低于新鲜对照组（P＜0.05）,1 μmol／L处理组卵裂率、囊胚率与新鲜对照组相比无显著差异（P＞0.05）。综上所述,RR处理能显著抑制解冻后牛卵母细胞线粒体Ca²⁺流入,保护线粒体功能,提高其发育能力。本试验结果为正向调控玻璃化冷冻卵母细胞线粒体Ca²⁺水平,进而提高其发育能力,促进玻璃化冷冻卵母细胞的广泛应用提供了参考依据。     

不同浓度紫杉醇预处理对绵羊卵母细胞玻璃化冷冻保存效果的影响

细胞骨架系统的稳定对于提高卵母细胞或胚胎冷冻后的存活率及其发育能力具有重要作用。紫杉醇(Taxol)作为细胞骨架稳定剂,可增强α、β-微管蛋白二聚体的紧密联系,增大微管交联,促进微管聚合和稳定。本实验以绵羊体外成熟卵母细胞为材料,旨在探讨紫杉醇预处理对玻璃化冷冻绵羊卵母细胞存活率、形态正常率及体外受精后胚胎发育率的影响。采用0、0.5、1.0、1.5μmol/L的紫杉醇预处理绵羊卵母细胞并进行玻璃化冷冻保存,未经处理的新鲜卵母细胞为对照组。结果表明:采用浓度为0.5μmol/L紫杉醇预处理绵羊卵母细胞冷冻保存效果最佳,其解冻后的存活率(82.38%)与其他3个试验组相比有显著差异性(P0.05);体外受精卵裂率(51.17%)和桑囊胚率(20.30%)也显著高于其他3个试验组(P0.05),但低于对照组(69.42%,34.77%)(P0.05)。由此可见,以0.5μmol/L浓度的紫杉醇预处理组绵羊体外成熟卵母细胞冷冻后能获得较好的效果。     

牛卵泡卵母细胞的体外成熟和冷冻保存

在简化牛卵母细胞体外成熟的基础上,观察了保护剂、平衡温度、预平衡方法、解冻方法以及冷冻方法对牛卵泡卵母细胞冷冻的影响。结果表明:在体外成熟培养液中添加26.2 mmol/L的NaHCO3和对屠宰场卵巢进行选择更能促进牛卵母细胞的体外成熟;无论是程序冷冻还是玻璃化冷冻,乙二醇(EG)与甘油(GLY)相比更能促进牛卵泡卵母细胞的冷冻效果;在程序冷冻中,冷冻液中添加0.1 mol/L蔗糖比添加0.3mol/L的蔗糖更能促进牛卵泡卵母细胞的冷冻;在玻璃化冷冻中,37℃和25℃的平衡温度比4℃更适合牛卵泡卵母细胞的冷冻;在10%、5%、1%的预平衡浓度之间,5%的预平衡浓度即可达到预平衡的效果;在解冻时,多步脱除保护剂更能保护冷冻的卵母细胞;比较了几种最小样本量(minimum size sample,MSS)玻璃化冷冻方法,解冻成熟培养后的成熟率,拉细毛细玻璃管冷冻法为(41.67±3.19)%,极显著高于未拉细毛细玻璃管冷冻法(glassmicropipette,GMP)的(30.19±1.93)%和固体表面冷冻法(solid surface vitrification,SSV)的(28.33±2.89)%(P<0.01)。     

甘氨酸对水貂GV期卵母细胞冷冻保存效率的影响

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(P0.05),但显著低于对照组(P0.05);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(P0.05),但与对照组相比差异不显著(P0.05)。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(P0.05),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(P0.05)。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(P0.05),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(P0.05)。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。     

甘氨酸对水貂GV期卵母细胞冷冻保存效率的影响

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。     

玻璃化冷冻牛卵母细胞单精子注射后体外发育能力的研究

实验研究了不同成熟培养时间的牛卵母细胞玻璃化冷冻及胞质内单精子注射(ICSI)后的受精效果。结果表明:成熟后的新鲜牛卵母细胞按照ICSI注射方法穿刺而不注射精子组与未经穿刺的对照组相比,孤雌激活后的卵裂率、囊胚发育率及囊胚细胞数无显著差异(P>0.05);成熟培养16h(MⅠ)和23h(MⅡ)卵母细胞冷冻解冻后形态正常率均显著低于新鲜对照组(76.66%、87.33%vs100.0%)(P<0.05),冷冻解冻后二者分别成熟培养至24h,ICSI后胚胎的囊胚发育率(5.29%、14.41%)显著低于新鲜对照组(24.40%)(P<0.05);成熟培养23h与成熟培养16h的卵母细胞冷冻解冻后形态正常率及ICSI后囊胚发育率(14.41%vs5.29%)均有显著性差异(P<0.05)。实验证明,ICSI操作不会影响卵母细胞发育潜力;玻璃化冷冻影响卵母细胞解冻后形态正常率以及ICSI后胚胎的发育能力;成熟培养23h比16h的卵母细胞冷冻保存后经ICSI的胚胎发育潜力高。     

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。     

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).     

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%。     

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.     

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.     

Vitrification with Glutamine Improves Maturation Rate of Vitrified / Warmed Immature Bovine Oocytes

The current study examined the protective effects of l ‐glutamine and cytochalasin B during vitrification of immature bovine oocytes. Oocyte vitrification solution (PBS supplemented with 10% FCS, 25% EG, 25% DMSO and 0.5 m trehalose) was the vitrification control. Treatments were the addition of 7 μg/ml cytochalasin B, 80 mm glutamine or both cytochalasin and glutaminine for 30 s. After warming, oocytes were matured in vitro for 24 h, fixed and stained with Hoechst (33342) for nuclear maturation evaluation. l ‐glutamine improved the vitrified/warmed immature bovine oocytes viability (32.8%), increasing the nuclear maturation rates compared to other treatments and the no treatment vitrified control (17.4%). There was, however, no effect of cytochalasin B on in vitro maturation (14.4%).     

Effect of Meiotic Stages During <Emphasis Type="Italic">In Vitro</Emphasis> Maturation on the Survival of Vitrified-Warmed Buffalo Oocytes

The present study was conducted to investigate the effect of meiotic stages during in vitro maturation (IVM) on the survival of vitrified-warmed buffalo oocytes, vitrified at different stages of IVM. Cumulus oocyte complexes obtained from slaughterhouse ovaries were randomly divided into 6 groups: control (non-vitrified, matured for 24 h at 38 ± 1^°C, 5% CO₂ in humidified air), and those matured for 0 h (vitrified before IVM) or 6, 12, 18 and 24 h before vitrification. Cumulus oocyte complexes were vitrified in solution consisting of 40% w/v propylene glycol and 0.25 mol/L trehalose in phosphate-buffered saline supplemented with 4% w/v bovine serum albumin. Vitrified cumulus oocyte complexes were stored at −196^∘C (liquid nitrogen) for at least 7 days and then thawed at 37^°C; cryoprotectant was removed with 1 mol/L sucrose solution. Cumulus oocyte complexes in the 0, 6, 12, 18 and 24 h groups were then matured for an additional 24, 18, 12, 6 and 0 h, respectively, to complete 24 h of IVM. Among the five vitrification groups, 89–92% of cumulus oocyte complexes were recovered, after warming, of which 84–91% were morphologically normal. Overall survivability of vitrified cumulus oocyte complexes was lower (p < 0.05) than that of non-vitrified cumulus oocyte complexes (94.5%). Survival rates of cumulus oocyte complexes matured 24 h prior to vitrification (61.3%) were higher (p < 0.05) than those matured for 12 h (46.7%), 6 h (40.6%) and 0 h (37.6%). Nuclear status following 24 h IVM was assessed. A higher proportion of non-vitrified (control) oocytes (72.7%) reached metaphase II (M-II) stage in control than oocytes vitrified for 24 h (60.0%), 18 h (54.4), 12 h (42.3%), 6 h (33.3%) and 0 h (31.6%) (p < 0.05). The results suggest that length of time in maturation medium prior to vitrification influences post-thaw survivability of buffalo oocytes; longer intervals resulted in higher survival rates.     

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%。     

The Effect of Different Vitrification and Staining Protocols on the Visibility of the Nuclear Maturation Stage of Equine Oocytes

In this study, we compared two staining protocols assessing the nuclear chromatin stage of equine oocytes after vitrification using permeable and nonpermeable cryoprotectants. Slaughterhouse-derived oocytes (n = 155) were obtained from a total of 32 mares and in vitro matured in M199 medium for 42 hours at 38.5°C in 5% CO_2. In the first experiment, two concentrations of Hoechst 33342 (HO) were tested (10 μg/mL; P1 and 2.5 μg/mL; P2) combined with 50 μg/mL of propidium iodide as staining protocols to evaluate the visibility of matured oocytes (n = 44). In the second experiment, 111 oocytes were evaluated using the staining protocol P2, before (C, control) and after vitrification following a two-step conventional protocol with (15% dimethyl sulfoxide, 15% ethylene glycol, and 0.5 M sucrose; V1) or without (1 M sucrose; V2) using permeable cryoprotectants. Our results showed that P2 provided a higher percentage of oocytes with outstanding visibility of the nuclear chromatin stage (52.17%; P < .05) in comparison with P1 (19.04%). In the second experiment, no cryoprotectant-free vitrified oocytes reached the metaphase II maturation stage. This result was significantly lower (P < .05) than conventional vitrification (15.38%) and both lower in comparison with the nonvitrified control group (42.11%). In conclusion, permeable cryoprotectant-free vitrification of equine oocytes obtained poor results and therefore cannot be considered an alternative to vitrification using permeable cryoprotectants. In addition, a staining protocol with a low concentration of HO is recommended to evaluate the nuclear chromatin stage of equine oocytes after in vitro maturation.     

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。     

High survival rate of bovine oocytes matured in vitro following vitrification

Improving pregnancy rates associated with the use of cryopreserved human oocytes would be an important advance in human assisted reproductive technology (ART). Vitrification allows glasslike solidification of a solution without ice crystal formation in the living cells. We have attempted to improve the survival rates of oocytes by a vitrification technique using bovine models. In vitro matured oocytes with or without cumulus cells were vitrified with either 15.0% (v/v) ethylene glycol (EG) + 15% (v/v) dimethylsulfoxide (DMSO) + 0.5 M sucrose or 15% (v/v) EG + 15% (v/v) 1,2-propanediol (PROH) + 0.5 M sucrose, using 'Cryotop' or 'thin plastic sticker', respectively. The oocyte survival rates after vitrifying-warming, and the capacity for fertilization and embryonic development were examined in vitro. The rate of embryonic development to blastocyst was significantly higher (P<0.05) in the oocytes vitrified with 15% (v/v) EG + 15% (v/v) PROH + 0.5 M sucrose than in the oocytes vitrified with 15% (v/v) EG + 15% (v/v) DMSO + 0.5 M sucrose (7.4% +/- 4.1 vs. 1.7% +/- 3.0, respectively). Oocytes vitrified without cumulus cells had a higher survival rate after thawing and a superior embryonic developmental capacity compared with oocytes vitrified with cumulus cells. Prolonged pre-incubation time after thawing adversely affected the rates of embryonic cleavage and development. These results indicate that in vitro matured bovine oocytes can be vitrified successfully with the mixture of the cryoprotectants, EG + PROH, the absence of cumulus cells for vitrification does not affect oocyte survival rate after warming, and vitrified and warmed oocytes do not require pre-incubation before in vitro fertilization.