哺乳动物卵母细胞玻璃化冷冻保存的研究进展

综述了近年来关于哺乳动物卵母细胞玻璃化冷冻保存的研究进展,并对这项技术的发展前景进行展望.重点讨论了影响哺乳动物卵母细胞玻璃化冷冻保存的几种因素,包括冷冻保护剂的种类和浓度、冷冻方法和卵母细胞所处的发育阶段等,以及冷冻过程中细胞膜、微丝、微管、皮质颗粒、纺锤体和线粒体等出现的损伤.目前,玻璃化冷冻法存在的最主要问题是冷冻过程中造成超微结构不可逆转的损伤影响胚胎发育,亟待解决.     

哺乳动物卵母细胞玻璃化冷冻保存的研究进展

综述了近年来关于哺乳动物卵母细胞玻璃化冷冻保存的研究进展,并对这项技术的发展前景进行展望。重点讨论了影响哺乳动物卵母细胞玻璃化冷冻保存的几种因素,包括冷冻保护剂的种类和浓度、冷冻方法和卵母细胞所处的发育阶段等,以及冷冻过程中细胞膜、微丝、微管、皮质颗粒、纺锤体和线粒体等出现的损伤。目前,玻璃化冷冻法存在的最主要问题是冷冻过程中造成超微结构不可逆转的损伤影响胚胎发育,亟待解决。     

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.     

猪卵母细胞玻璃化冷冻保存技术的研究进展

卵母细胞冷冻保存技术在动物种质资源保存、濒危动物保护等方面具有独特的作用,猪卵母细胞的冷冻保存仍是一项世界性难题,尚处于摸索阶段。现就猪卵母细胞冷冻保存的现状、冷冻原理与方法及冷冻损伤等问题进行综述。     

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.     

细胞松弛素B对绵羊GV期卵母细胞玻璃化冷冻效果的影响

本试验通过玻璃化冷冻前细胞松弛素B(CB)预处理来分析CB对绵羊GV期卵母细胞玻璃化冷冻/解冻后发育潜力的影响。分别从细胞毒性检测、冷冻检测和CB不同浓度（6.0、7.5和9.0 μg/ml）处理3个方面进行试验。试验结果表明毒性检验中CB处理组和未处理组卵母细胞的成熟率都显著低于对照组(P<0.05)，但相互之间差异不显著；玻璃化冷冻后，卵母细胞成熟率显著低于未冷冻组(P<0.05)，玻璃化冷冻CB处理组和未处理组卵母细胞体外成熟培养后成熟率之间差异不显著(P>0.05)；不同CB浓度处理后9.0 μg/ml组卵母细胞的成熟率显著高于对照组（P<0.05）。     

细胞松弛素B对牛GV期卵母细胞玻璃化冷冻效果的影响

本试验通过玻璃化冷冻前不同浓度细胞松弛素B(cytochalasin-B,CB)预处理来分析CB对牛GV期卵母细胞固体表面玻璃化(SSV)冷冻后发育潜力的影响。试验结果表明,玻璃化冷冻后,CB处理组和未处理组之间卵母细胞成熟率差异不显著(P>0.05),但均显著低于体外培养组。说明试验中所用CB浓度 (2.5、7.5、15、20和30 μg/mL)对牛GV期卵母细胞玻璃化冷冻效果的改善作用不明显。     

小鼠卵母细胞SSV法冷冻保存技术的研究

试验用2种前处理液(10%EG和10%EG 10%DMSO)和4种玻璃化冷冻液(EFS30、EFS40、EDFS30和EDFS40)对小鼠卵母细胞进行玻璃化(SSV)法冷冻保存,研究小鼠卵母细胞冷冻后的发育潜力。结果表明:小鼠未成熟卵母细胞形态正常率最高可达92.3%,成熟率达57.2%;成熟卵母细胞形态正常率最高可达91.5%。     

猪MII期卵母细胞的玻璃化冷冻的初步研究

试验采用3种冷冻载体(麦管、开放式拉长麦管和半麦管)对猪卵母细胞玻璃化冷冻后发育能力的影响进行研究,以探讨采用自制半麦管作为载体对猪MII期卵母细胞玻璃化冷冻后发育能力的影响.结果发现,采用半麦管法、OPS法玻璃化冷冻的猪卵母细胞的形态完整率、存活率、卵裂率分别为90.85%、60.07%、20.43%和84.15%、55.71%、14.71%,无显著差异(P＞0.05),但采用半麦管法的结果好于OPS法;与麦管法的形态完整率、存活率和卵裂率(分别为60.06%,39.64%和0)均有显著性差异(＜0.05).结论为采用半麦管法玻璃化冷冻猪卵母细胞可以稍微提高其冻后发育能力.     

Comparison of Cryotop and micro volume air cooling methods for cryopreservation of bovine matured oocytes and blastocysts

This study was designed to compare the efficiency of the Cryotop method and that of two methods that employ a micro volume air cooling (MVAC) device by analyzing the survival and development of bovine oocytes and blastocysts vitrified using each method. In experiment I, in vitro-matured (IVM) oocytes were vitrified using an MVAC device without direct contact with liquid nitrogen (LN₂; MVAC group) or directly plunged into LN₂ (MVAC in LN₂ group). A third group of IVM oocytes was vitrified using a Cryotop device (Cryotop group). After warming, vitrified oocytes were fertilized in vitro. There were no significant differences in cleavage and blastocyst formation rates among the three vitrified groups, with the rates ranging from 53.1% to 56.6% and 20.0% to 25.5%, respectively; however, the rates were significantly lower (P < 0.05) than those of the fresh control group (89.3% and 43.3%, respectively) and the solution control group (87.3% and 42.0%, respectively). In experiment II, in vitro-produced (IVP) expanded blastocysts were vitrified using the MVAC, MVAC in LN₂ and Cryotop methods, warmed and cultured for survival analysis and then compared with the solution control group. The rate of development of vitrified-warmed expanded blastocysts to the hatched blastocyst stage after 24 h of culture was lower in the MVAC in LN₂ group than in the solution control group; however, after 48–72 h of culture, the rates did not significantly differ between the groups. These results indicate that the MVAC method without direct LN₂ contact is as effective as the standard Cryotop method for vitrification of bovine IVM oocytes and IVP expanded blastocysts.     

Vitrification of in vitro matured oocytes of Mangalica and Large White pigs

The breeding of Mangalica, a native pig breed in Hungary, had been started in 1833, but this pig breed almost became extinct in Hungary in the past decades. In 1991, the number of sows was only 200. Although in these days the existing Mangalica population consists of more than 6000 animals representing different colour variations, the preservation of this traditional pig breed is still very important. Vitrification is a potential tool for the preservation of gametes and embryos of these animals. The aim of this study was to investigate the effects of vitrification on the developmental competence of Mangalica (M) and Large White (LW) oocytes following fertilisation. The oocytes were vitrified by the Open Pulled Straw (OPS) method using different concentrations of ethylene glycol and dimethyl sulphoxide as cryoprotectants. After rehydration the oocytes underwent in vitro fertilisation; the resultant zygotes were then cultured in vitro for four days to assess embryonic development. In the first experiment, in vitro maturation of M and LW oocytes was compared. No significant difference was observed in the nuclear maturation rate of LW and M oocytes. In the second experiment, the sensitivity of oocytes to vitrification was examined by evaluating oocyte morphology after thawing. A higher percentage of LW oocytes showed normal morphology compared to M oocytes, indicating that Mangalica oocytes are more sensitive to cryoprotectants than Large White oocytes. After warming and in vitro fertilisation, more than 50% of the oocytes started embryonic development and by the end of the incubation period morula stage embryos had developed in both groups. The results show that the OPS vitrification technique is well suited to preserve Mangalica oocytes and from these oocytes morula embryos can be produced.     

小鼠未成熟卵母细胞OPS法冷冻保存技术的研究

试验用OPS法对小鼠未成熟卵母细胞进行玻璃化冷冻保存研究,并对解冻后小鼠未成熟卵母细胞的成熟情况进行观察。结果表明:小鼠未成熟卵母细胞在10%EG、10%DMSO前处理后,在EFS30、EFS40、EDFS30和EDFS40中平衡15～45s进行冷冻保存,使卵母细胞解冻后的形态正常率最高达92.4%,与对照组无显著差异(P>0.05),成熟率达40.5%。     

Pregnancies from bovine oocytes matured and fertilized in vitro

Production of transgenic animals and embryo cloning are only a few examples of new biotechnological methods applied to animal embryos. All these techniques require large amounts of oocytes and early embryos. In many laboratory animals, embryos matured and fertilized in vivo are easily obtained, but with larger domestic species it requires laborious surgical procedures and the number of embryos obtained remains relatively small (Bracken et al. 1982). The in vitro maturation of follicular oocytes derived from slaughterhouse ovaries and their in vitro fertilization provides large numbers of oocytes and embryos with considerably less effort. The final proof of the success in the in vitro maturation and fertilization procedure is the birth of healthy progeny. Also the normal preimplantation development of the embryos gives useful information about the efficiency of the method employed.     

Meiosis in bovine oocytes matured in vitro and in vivo

Meiosis in bovine oocytes has; been studied after maturation in vitro or in vivo. Oocytes for in vitro maturation were collected from the ovaries of slaughtered cattle without regard to the phase of the estrous cycle while in vivo maturation was studied in oocytes from gonadotrophin-stimulated heifers at times varying between 6 and 36 h after the beginning of behavioural estrus. Oocytes from slaughtered cattle were classified according to their cumulus complex and ooplasm and were cultured for 6, 12, 18, 24, 36 or 48 h in modified Krebs-Ringer bicarbonate buffer before fixation) for cytogenetic analysis. Oocytes from stimulated heifers were aspirated from follicles or flushed from the oviducts, classified according to cumulus and ooplasm, and fixed within 6 h of collection. Nuclear maturation was more rapid in vitro than in vivo. The largest proportion of oocytes reached maturity (Mil) after 12 to 18 h in culture or 30 to 36 h after the onset of behavioural estrus. Oocytes devoid of cumulus cells or showing signs of vacuolation or degeneration had virtually no capacity for nuclear maturation.     

小鼠囊胚和扩张囊胚玻璃化冷冻保存的研究

以6M甘油+6.5%PVP(V1)和8MEG+7%PVP(V2)为玻璃化溶液,采用细管法和OPS一步法对小鼠囊胚进行冷冻。结果表明:胚胎在玻璃化溶液中平衡20S显著高于平衡60S后的存活率(P<0.05);蔗糖四步法解冻后的发育率与蔗糖三步法冷冻解冻后的发育率差异不显著(P>0.0 5);用OPS三步法冷冻后(V2)的小鼠囊胚的体外发育率显著高于细管法和OPS一步法冷冻后的发育率(P<0.01);在OPS三步法冷冻过程中,平衡时间对胚胎冷冻后的发育率有一定的影响。     

玻璃化冷冻对猪MⅡ卵母细胞皮质颗粒分布及其激活后胚胎发育能力的影响

本实验旨在探讨玻璃化冷冻保存对猪MⅡ期卵母细胞皮质颗粒分布和孤雌激活后早期胚胎发育能力的影响。实验将卵母细胞随机分为对照组、毒性实验组和冷冻组。采用EFS40和EDFS40两种玻璃化冷冻液处理,卵母细胞经恢复后对其进行染色,观察皮质颗粒的分布;并对另一部分卵母细胞实施孤雌激活,观察早期胚胎的发育。结果表明:毒性实验组和冷冻组卵母细胞皮质颗粒部分释放、完全释放的比例无显著差异,但均显著高于对照组(P<0.05)。不同毒性处理组和不同冷冻组间对皮质颗粒的分布无显著差异。与毒性实验组相比,冷冻处理组显著降低皮质颗粒在皮质区分布的比例(P<0.05)。EFS40毒性实验组孤雌激活后的存活率、卵裂率、囊胚发育率均显著高于EDFS40毒性实验组(86.6%vs.75.0%)、(61.8%vs.40.7%)、(30.2%vs.23.5%)(P<0.05)。EFS40冷冻组存活率显著高于EDFS40冷冻组,但均显著低于对照组。结果显示,抗冻保护剂处理和玻璃化冷冻均导致猪卵母细胞皮质颗粒释放,与EDFS40相比采用EFS40较适合猪MⅡ期卵母细胞冷冻保存。