牛胚胎玻璃化冷冻技术的发展和应用概况

胚胎冷冻保存是动物胚胎工程的重要组成部分,它是胚胎移植技术在生产中推广应用的重要基础。和现在通用的常规冷冻法相比,玻璃化冷冻具有用时短,所需设备少和操作简单等优点,成为目前国内外胚胎冷冻保存领域发展新方向。本文就牛胚胎玻璃化冷冻的概念、方法和国内外发展状况做一简要综述。     

影响玻璃化冷冻兔胚胎效果的一些因素

试验对影响玻璃化冷冻兔胚胎效果的一些因素进行探讨，以找出理想的玻璃化冷冻方法。在测试的５种玻璃化溶液中，含３５％乙二醇（ＥＧ）和１．０ｍｏｌ／Ｌ蔗糖的溶液（ＶＳ１）对胚胎的毒性最小。用ＶＳ１冷冻桑椹胚和囊胚的理想程序是：在室温下使胚胎分别在２０％ＥＧ和３５％ＥＧ中平衡２、３分钟后，移入ＶＳ１中，０．５分钟内（囊胚也可在２分钟后）投入液氮中冷冻。桑椹胚的存活率为９１．７％（３３／３６），囊胚的存活率为９７．１％（３３／３４）～９７．３％（３６／３７）。８～１６细胞胚胎的理想冷冻程序为：在室温下使胚胎在２０％ＥＧ、３５％ＥＧ中平衡２、３分钟，移入４℃的３７％ＥＧ＋１．０ｍｏｌ／Ｌ蔗糖溶液中平衡２分或１０分钟后冷冻，胚胎存活率分别为１００％（３７／３７）、８６．１％（３１／３６）。     

A History of Equine Embryo Transfer and Related Technologies

The first successful equine embryo transfer was reported in 1972, 21 years after the first reported embryo transfer in cattle. Adaptations of embryo transfer and the various related technologies have generally been more rapid in the equine than in cattle, with the exceptions of superovulation, in vitro fertilization and cryopreservation. Recent progress has been achieved in all three of these areas. This paper presents a time line of various events in the history of equine embryo transfer and related technologies. Approximately 25,000 equine embryo transfers are being conducted per year, worldwide.     

Studies on Survival of Horse Oocytes After Rapid-i Method Vitrification

The aim of the present study was to determine the vitality and developmental competence of equine oocytes after in vitro maturation (IVM) and vitrified by Rapid-i method. In experiment 1, oocytes after IVM were vitrified using media: EquiPro VitKit (group 1) or medium containing 18% Ficoll, 40% ethylene glycol, and 0.3 M sucrose (group 2). For evaluation of toxicity effect, oocytes were exposed to media without a plug to liquid nitrogen. To evaluate viability, oocytes were stained with fluorescein diacetate and ethidium bromide. In experiment 2, oocytes after IVM and vitrification were activated by 7.5 μM ionomicin in TCM 199 (5 minutes) combined with 2 mM 6-DMAP in TCM 199 with 10% fetal bovine serum (4.5 hours). Survival rate was: 63% in group 1 (n = 54), 55% in group 2 (n = 69), and 73.2% (n = 56) in the control group. After parthenogenetic activation, 10.2% (n = 49) of 2–4 blastomeres were observed. This percentage was lower than in the nonvitrified group: 38.5% (n = 53).     

小鼠的胚胎冷冻保存研究

随着生物工程技术的进步，出现了许多生物工程小鼠品系，促使小鼠胚胎冷冻保存技术得到了广泛的应用，采用胚胎冷冻的方法来长期的保存这些品系，可以节省大量的人力和物力，目前，在小鼠胚胎冷冻方面的研究主要是围绕提高存活率和简化操作过程，本文综述了在小鼠胚胎冷冻保存方面的国内外研究进展。     

Effect of eFSH on Ovarian Cyclicity and Embryo Production of Mares in Spring Transitional Phase

The use of equine FSH (eFSH) for inducing follicular development and ovulation in transitional mares was evaluated. Twenty-seven mares, from 3 to 15 years of age, were examined during the months of August and September 2004, in Brazil. Ultrasound evaluations were performed during 2 weeks before the start of the experiment to confirm transitional characteristics (no follicles larger than 25 mm and no corpus luteum [CL] present). After this period, as the mares obtained a follicle of at least 25 mm, they were assigned to one of two groups: (1) control group, untreated; (2) treated with 12.5 mg eFSH, 2 times per day, until at least half of all follicles larger than 30 mm had reached 35 mm. Follicular activity of all mares was monitored. When most of the follicles from treated mares and a single follicle from control mares acquired a preovulatory size (≥35 mm), 2,500 IU human chorionic gonadotropin (hCG) was administered IV to induce ovulation. After hCG administration, the mares were inseminated with fresh semen every other day until ovulation. Ultrasound examinations continued until detection of the last ovulation, and embryo recovery was performed 7 to 8 days after ovulation. The mares of the treated group reached the first preovulatory follicle (4.1 ± 1.0 vs 14.9 ± 10.8 days) and ovulated before untreated mares (6.6 ± 1.2 vs 18.0 ± 11.1 days; P < .05). All mares were treated with prostaglandin F_2α (PGF_2α), on the day of embryo flushing. Three superovulated mares did not cycle immediately after PGF_2α treatment, and consequently had a longer interovulatory interval (22.4 vs 10.9 days, P < 0.05). The mean period of treatment was 4.79 ± 1.07 days and 85.71% of mares had multiple ovulations. The number of ovulations (5.6 vs 1.0) and embryos (2.0 vs 0.7) per mare were higher (P < 0.05) for treated mares than control mares. In conclusion, treatment with eFSH was effective in hastening the onset of the breeding season, inducing multiple ovulations, and increasing embryo production in transitional mares. This is the first report showing the use of FSH treatment to recover embryos from the first cycle of the year.     

OPS玻璃化冷冻对小鼠四倍体胚胎体外发育的影响

为探究开放式拉长细管(OPS)玻璃化冷冻对四倍体胚胎发育的影响,本实验利用2-细胞胚胎电融合法制备四倍体胚胎,再对四倍体胚胎进行OPS玻璃化冷冻,分别观察记录二倍体胚胎、四倍体胚胎以及冷冻解冻后四倍体胚胎的发育情况。结果表明:2-细胞胚胎电融合效率为96.1%;二倍体胚胎组与电融合后四倍体胚胎组的囊胚率和孵化囊胚率差异不显著;冷冻解冻后四倍体胚胎的囊胚率(100%)与四倍体新鲜组(93.3%)差异不显著,其孵化囊胚率(72.3%)较新鲜组(64.9%)显著增高(P<0.05);四倍体冷冻解冻组的囊胚细胞数(31.96)与新鲜组(32.54)无显著差异;冷冻解冻后的四倍体早期囊胚进行体外培养时其发育速度比对照组更快。可见,冷冻对小鼠四倍体胚胎的囊胚率和囊胚细胞数均无显著影响,但孵化囊胚率显著提高,且OPS玻璃化冷冻后使四倍体胚胎的发育速度更快。     

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。     

几种冷冻保存方法对奶牛胚胎保护效果的研究

选取奶牛体内生产胚胎93枚(其中囊胚54枚,桑椹胚39枚),比较研究了不同冷冻方法和保护剂对其的保护效果。结果表明:乙二醇处理组对奶牛囊胚的保护效果优于One-Step-Freezing组、甘油组和玻璃化组(回收率、形体正常率和继续发育率四组分别为91.0%、82.0%、66.6%;91.0%、91.0%、88.8%;86.6%、85.0%、83.3%和80.0%、66.7%、50.0%),其中继续发育率在乙二醇组、甘油组和玻璃化组间差异显著(P<0.05);而桑椹胚的各项指标以乙二醇处理组最高,玻璃化组最低,但各组间差异不显著。结果说明乙二醇处理组对胚胎的保护效果优于其它几个处理组。     

鲜整胚及分割双半胚移植试验研究

本试验结合我省胚胎移植的现状,用7头纯种西门塔尔牛作供体,46头杂交牛作受体,进行同期发情和超数排卵,获得鲜整胚59枚,双半胚32枚;用鲜整胚22枚,双半胚24枚进行移植,研究鲜整胚与分割双半胚的移植效果。结果表明:鲜整胚单枚移植妊娠率为27.3%(6/22),分割双半胚移植妊娠率为54.2%(13/24),鲜整胚单枚移植妊娠率比分割双半胚移植妊娠率低26.9个百分点,经t检验差异显著(P<0.05)。     

哺乳动物胚胎冷冻技术的研究进展

胚胎冷冻保存已广泛用于胚胎移植、动物克隆以及动物资源保护.此技术的应用需保证胚胎在冷冻-解冻后具有较高的成活率.自从1972年小鼠胚胎冷冻保存获得成功以来,许多学者在简化冷冻程序,缩短冷冻时间,方面进行了深入的研究.本文就胚胎冷冻的原理、保护剂、冷冻方法以及解冻方法等方面进行了综述.     

哺乳动物胚胎冷冻技术的研究进展

胚胎冷冻保存已广泛用于胚胎移植、动物克隆以及动物资源保护。此技术的应用需保证胚胎在冷冻—解冻后具有较高的成活率。自从1972年小鼠胚胎冷冻保存获得成功以来,许多学者在简化冷冻程序、缩短冷冻时间等方面进行了深入的研究。文章就胚胎冷冻的原理、保护剂、冷冻方法以及解冻方法等方面进行了综述。     

In Vitro Production of Equine Embryos

The development of methods to produce embryos in vitro in the horse has been delayed compared with other domestic species. Oocytes can be collected from excised ovaries or from the small or preovulatory follicles of live mares. Intracytoplasmic sperm injection is the only reliable method to fertilize equine oocytes in vitro. Intracytoplasmic sperm injection-produced embryos can be transferred into the oviducts of recipient mares or cultured to the morula or blastocyst stage of development for nonsurgical embryo transfers into recipients' uteri. Embryos cultured in vitro have some morphological differences compared with embryos collected from the mares' uteri. Most notably, the embryonic capsule does not form in culture, and the zona pellucida fails to expand completely. However, embryo produced in vitro can result in viable pregnancies and healthy offspring.     

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。