牛胚胎玻璃化冷冻技术研究进展

胚胎冷冻保存是保存和繁殖遗传优势动物的主要工具,是胚胎移植产业的重要组成部分。目前广泛使用的冷冻方法主要有慢速冷冻和玻璃化冷冻2种。由于玻璃化冷冻具有成本低、效率高、操作简单等优点,玻璃化冷冻法越来越受到人们的重视。经过多年研究,研究人员已经在玻璃化冷冻方法、冷冻液等方面取得了大量进展,玻璃化冷冻法已经开始进入商业化应用。本文综述了牛胚胎冷冻保存技术的研究进展,旨在为相关从业人员提供一定借鉴和参考。     

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。     

哺乳动物胚胎玻璃化冷冻保存的研究进展

玻璃化(vitrification)是指利用物理学原理将高浓度的抗冻保护剂急速降温后，由液态转化为外形类似玻璃状的稳定而透明的非晶体化固体状态的过程。玻璃化的固体保留了液体正常的分子和离子分布，可以视为一种无序黏稠的超冷液体，因而避免了细胞内形成冰晶而对细胞产生伤害。1985年，Rall等率先将玻璃化冷冻方法用于小鼠胚胎的冷冻保存，推动了该方法在哺乳动物胚胎冷冻保存的方面的应用。之后，玻璃化冷冻保存不断改进，     

牛胚胎玻璃化冷冻保存技术及国内近期研究进展

本文就胚胎玻璃化冷冻保存的原理、冷冻保护剂的作用和冷冻方法及近期国内牛胚胎玻璃化冷冻的研究进展情况进行了详细的综述.     

牛胚胎冷冻保存的研究进展

目前,牛的胚胎冷冻保存巳成为一种较成熟的常规技术,广泛应用于牛的繁殖科研与生产。本文回顾了牛胚冷冻保存技术的发展概况,对牛胚胎的常规冷冻技术、直接冷冻法及玻璃化冷冻技术的进展作了简要论述。     

哺乳动物胚胎玻璃化冷冻研究进展

近40年来,利用冷冻保存技术将哺乳动物胚胎长期保存起来,建立"胚胎库"是保护物种资源和拯救濒危动物的有效手段,同时也是加快家畜品种改良、建立动物基因库和实施胚胎移植产业化的重要组成部分,也可以为克隆、转基因等现代生物技术提供丰富的试验材料,使胚胎的供给不受时间和空间的限制。胚胎冷冻保存技术在人类辅助生殖方面也具有广阔的应用前景。本文主要介绍了玻璃化冷冻过程中胚胎冷冻主体承载工具特点和应用,并展望未来哺乳动物胚胎冷冻发展的方向。     

哺乳动物胚胎玻璃化冷冻保存的研究进展

自1985年胚胎玻璃化冷冻(v itrification)保存技术发明以来,玻璃化法先后在小鼠、兔、绵羊、牛、猪、山羊等动物胚胎上获得成功,近年来有关哺乳动物胚胎玻璃化冷冻保存的研究主要集中在冷冻和解冻方法上,相继发明了一些新的玻璃化冷冻方法:冷环玻璃化法(cryo loop)和开放式细管法(open pu lled straw,OPS),并且对解冻后细管内直接脱除防冻剂进行了广泛深入的研究,使得冷冻胚胎移植更易于在生产上推广应用。现将胚胎玻璃化冷冻的原理、冷冻保护剂、冷冻方法、解冻后保护剂脱除方法的最新研究进展作一综述。     

哺乳动物胚胎冷冻保存研究进展

哺乳动物胚胎冷冻保存效果受冷冻保护剂、冷冻方法、解冻方法等多种因素影响,其中冷冻方法是一个关键性因素.目前胚胎冷冻方法主要有常规慢速冷冻法和玻璃化冷冻法两种.常规慢速冷冻法是指利用甘油、乙二醇等做冷冻保护剂通过缓慢降温的方式进行胚胎冷冻;玻璃化冷冻法是指利用高浓度的冷冻保护剂通过快速降温的方式进行胚胎冷冻.与常规慢速冷冻法相比,玻璃化冷冻法简化了操作过程,大大缩短了操作时间,不需昂贵的程序控制冷冻仪.     

山羊胚胎冷冻保存技术研究

本研究探讨了不同抗冻保护剂、不同冷冻方法、玻璃化液(EFS40)中添加FCS和BSA,以及冷冻前细胞松驰素B处理对山羊胚胎冷冻保存效果的影响。结果表明,山羊胚胎常规冷冻时以1.5 mol/L EG为抗冻保护剂的保护效果最好,解冻后胚胎发育率为70.59%,孵化率为58.82%;玻璃化冷冻细管法和OPS法以EFS40为保护液的冷冻效果较好,其解冻后胚胎的发育率分别为67.57%和52.94%;EFS40中添加BSA的冷冻保护效果显著地高于不添加其他成分的EFS40;山羊胚胎冷冻前用细胞松驰素B处理,能提高冷冻保存的效果。     

哺乳动物胚胎冷冻保存的研究进展

哺乳动物胚胎冷冻保存技术按其进展分为缓慢冷冻法、快速冷冻法和玻璃冷冻法。玻璃化冷冻法又分为常规细管法和OPS法。本文综述了缓慢冷冻法、快速冷冻法以及玻璃化冷冻法的操作过程及所需的抗冻保护剂,并对各种方法的优缺点进行了初步分析。     

奶牛冷冻胚胎分割和分割后半胚冷冻技术

用简易分割方法,分割奶牛冷冻胚胎16枚,获得半胚32枚,分割成功率为100％(16/16),可移植半胚29枚,移植于16头受体牛,到第3个情期未返情者经直肠检查有6头妊娠,妊娠率为37.5％(6/16)。其中,在分割前或者分割后经恢复培养0.5～2h再移植的10头受体牛,5头妊娠;而未经恢复培养,分割后直接移植的6头受体牛中,只有1头妊娠。移植后3个月,直肠检查确定2头流产。已有1头受体黄牛生出1头奶牛牛犊(母)和1头受体奶牛产1头奶牛牛犊。其余的2头妊娠受体牛将于9月份产犊。此外,用简易分割法分割奶牛胚胎5枚,得到半胚10枚,裸半胚直接冷冻,解冻后回收可移植半胚5枚,移植于4头受体牛,无一头妊娠。结果表明,冷冻胚胎的分割半胚优于分割后冷冻半胚移植效果;冷冻胚胎分割前或者分割后恢复培养移植优于未经恢复培养而直接移植;简易分割法可应用于冷冻胚胎的分割。     

胚胎冻融技术及其在我国肉羊产业中的应用

胚胎冻融技术可使胚胎移植不受时间、地点的限制，从而达到提高胚胎移植效率的目的，研究和优化胚胎冻融技术对胚胎移植工程的发展具有重要的意义。主要对胚胎冻融技术的原理、特点及其在肉羊产业中的应用情况进行了综述，旨在阐明胚胎冻融技术对我国肉羊产业发展的重要作用。     

牛胚胎冷冻与移植受胎技术研究

本文进行了牛胚胎冷冻与移植受胎技术研究。摸索出了提高牛冷冻胚胎成活率和移植受胎率的综合配套技术。牛冷冻胚胎解冻成活率达８４．４％（５７／１２２）。其中１９９６年移植受胎率达到５５．６％（２０／３６）。Ａ级胚胎达以７７．８％（２１／２７），并在我国首次获得中国荷斯坦牛冷冻胚胎的二分胚的同孵孪生牛犊。且达到４２．９％（３／７）移植受胎率。     

小鼠卵母细胞和胚胎不同冷冻方法的比较研究

探讨程序化冷冻与玻璃化冷冻对小鼠GV期卵母细胞及二细胞期胚胎的复苏率及其发育潜能的影响。通过小鼠的卵母细胞与早期胚胎的不同冷冻方法的比较,为后续阿旺绵羊的胚胎冷冻保存提供参考。采用程序化冷冻与玻璃化冷冻技术,分别冷冻小鼠GV期卵母细胞及二细胞期胚胎,复苏后培养,比较不同冷冻处理后的复苏率、成熟率与囊胚率。小鼠GV期卵母细胞程序化冷冻复苏率(48.00%±5.29%)显著低于玻璃化冷冻复苏率(65.00%±5.00%),有统计学差异(P=0.0147<0.05);而程序化冷冻后复苏卵母细胞的发育成熟率略高于玻璃化冷冻组,但无统计学意义。小鼠二细胞期胚胎程序化冷冻组复苏率(76.00%±2.00%)显著高于玻璃化冷冻组复苏率(70.00%±2.00%),有统计学差异(P=0.0213<0.05);冷冻后复苏胚胎发育的囊胚率程序化冷冻略低于玻璃化冷冻及对照组,但无统计学意义。     

牛体外胚胎冷冻保存的研究进展

体外胚胎冷冻保存技术是胚胎移植技术的重要组成部分,在辅助生殖技术中发挥重要作用,同时对种质资源保存、加强遗传改良和促进优质种源国际交流等方面具有重要意义。然而,体外胚胎冷冻过程中存在脂质含量过高、活性氧水平升高及机械损伤等问题,导致体外胚胎冷冻效率低,这极大地限制了体外胚胎冷冻保存技术的广泛应用。大量研究表明,通过去脂质、优化体外胚胎培养液、人工塌陷囊胚腔和优化冷冻程序等手段,可以有效提高冷冻后胚胎的存活率和发育能力。因此,本文概述了体外胚胎冷冻保存技术的研究进展和胚胎冷冻过程中存在的问题,总结了提高体外胚胎冷冻效率的方法措施,旨在为提高体外胚胎冷冻保存效率提供一定参考。     

Influence of Embryonic Size and Manipulation on Pregnancy Rates of Mares After Transfer of Cryopreserved Equine Embryos

Many years of poor results of equine embryo cryopreservation has produced a lack of confidence in this technique. Embryo cryopreservation has been successfully used for more than 20 years in other species like bovine and human. The large size of the embryos and the presence of a capsule impermeable to cryoprotectants have been the two main reasons for the failure. In the last few years, a mayor breakthrough for this technique was obtained when large equine embryos could be successfully cryopreserved after breaching the capsule and collapsing the blastocoel cavity. In the present study, we compared the pregnancy rates obtained by vitrification or cryopreservation by slow freezing of embryos smaller than 300 μm. No difference was found between vitrification and slow freezing of embryos <180 μm (pregnancy rate on day 16: 34/61, 55.7%; 6/8, 75%) but produced very low results for embryos between 180 and 300 μm in diameter (0/11, 0%; 1/7, 14.3%). Embryos larger than 300 μm were collapsed before cryopreservation, and two different types of carriers, hemi-straw or Stripper-Tip, were used for vitrification. High pregnancy rates were obtained when the hemi-straw was used as a carrier (7/10, 70% vs. 0/5, 0%), demonstrating that a minimum vitrification volume was essential to preserve the embryo viability. These findings establish that, due to the large range in diameter, equine embryos need to be cryopreserved using different protocols depending on their size.     

Recent progress in bovine in vitro-derived embryo cryotolerance: Impact of in vitro culture systems,advances in cryopreservation and future considerations

Cryopreservation of in vitro-derived bovine embryos is a crucial step for the widespread reproduction and conservation of valuable high-merit animals. Given the current popularity of bovine in vitro embryo production (IVP), there is a demand for a highly efficient ultra-low temperature storage method in order to maximize donor ovum pickup (OPU) turn-over, recipient availability/utilization and domestic/overseas commercial trading opportunities. However, IVP bovine embryos are still very sensitive to chilling and cryopreservation, and despite recent progress, a convenient (simple and robust) protocol has not yet been developed. At the moment, there are two methods for bovine IVP embryo cryopreservation: slow programmable freezing and vitrification. Both of the aforementioned techniques have pros and cons. While controlled-rate slow cooling can easily be adapted for direct transfer (DT), ice crystal formation remains an issue. On the other hand, vitrification solved this problem but the possibility of successful DT commercial incorporation remains to be determined. Moreover, simplification of the vitrification protocol (including warming) through the use of an in-straw dilution without the use of a microscope is a prerequisite for its use under farm conditions. This review summarizes the bovine IVP embryo cryopreservation achievements, strengths and limitations of both freezing systems and prospective improvements to enhance cryosurvival, as well as perspectives on future directions of this assisted reproductive technology.     

不同冷冻和解冻方法对小鼠桑椹胚发育的影响

本试验以2种程序化冷冻液和2种玻璃化冷冻液对昆明白系小鼠的桑椹胚进行细管法冷冻保存,比较程序化冷冻-管外解冻和玻璃化冷冻-管内解冻对胚胎体内、外发育的影响。胚胎体外培养结果表明:玻璃化冷冻组及程序化冷冻组胚胎发育率(95.3%￣95.8%,98.9%)无显著(P>0.05)差异。将程序化冷冻、EFS30玻璃化冷冻以及新鲜的胚胎各168枚移植给假孕受体鼠,妊娠受体产活仔率各组间相比(50.8%,58.3%,54.9%)无显著性(P>0.05)差异。结果证明,玻璃化冷冻保存的胚胎管内解冻效果好,为生产中家畜的胚胎移植提供了理论和技术参考。     

Effects of reduced glutathione supplementation in semen freezing extender on frozen-thawed bull semen and in vitro fertilization

During cryopreservation, spermatozoa may suffer cold and cryo-induced injuries ―associated with alterations in cell defense systems― that are detrimental to their function and subsequent fertility. This study aimed to determine the efficacy of supplementing the semen freezing extender with the antioxidant reduced glutathione (GSH) in cattle. Semen was collected from four bulls and diluted in a freezing extender supplemented with or without GSH (0, 1, 5, and 10 mM) before the cooling step of the cryopreservation process. After thawing, the quality of the frozen-thawed semen was investigated for motility, viability, acrosomal and DNA integrity, and subsequent embryo development after in vitro fertilization of bovine oocytes. Additionally, semen from one of the bulls was used to analyze semen antioxidative potential, sperm penetration into oocytes, male pronucleus formation rate, and embryo DNA integrity. The sperm quality varied among bulls after GSH supplementation. One bull had decreased sperm total motility, and two bulls had decreased sperm DNA integrity. GSH supplementation had positive effects on embryo development for three bulls. Two of them showed both improved cleavage and blastocyst formation rates, while the other one only showed an improved cleavage rate. We observed positive effects on early male pronucleus formation and no negative effects on DNA integrity and cell number in blastocyst stage embryos. Although the effect varies depending on individual bulls and GSH concentration, GSH supplementation in semen may improve in vitro embryo production from frozen semen.