秋水仙胺对电融合方法体外制备牛四倍体胚胎的影响

通过添加秋水仙胺从而改善电融合方法体外制备牛四倍体胚胎的过程。首先检测牛早期胚胎的分裂时间,并由此确定于体外受精后26~30h时间段内挑选既同期化且优质的2细胞期胚胎用于电融合。电融合参数选取2次直流电压为0.75kV/Cm且脉冲时间60μs。并通过免疫荧光染色监测电融合后细胞核的重组过程。设立电融合对照组和电融合秋水仙胺处理组,处理组在电融合后随即处理0.05mg/L秋水仙胺6h,后彻底洗涤继续体外培养至囊胚期。处理组的融合率（84.4%vs 84.8%）、分裂率（74.3%vs 77.6%）和囊胚率（36.1%vs 39.4%）皆低于对照组,但差异均不显著。获取的囊胚期胚胎的核型分析结果显示,处理组的四倍体制备率显著高于对照组（59.4%vs 26.9%）。综上所述,对电融合后的胚胎处理0.05mg/L秋水仙胺6h显著提高了牛四倍体电融合方法体外制的备率。     

不同电融合参数及融合液对昆明小鼠2-细胞胚胎融合及囊胚率的影响

四倍体胚胎在研究哺乳动物配子发生、发育、四倍体的发生、遗传、免疫机制等方面具有重要作用,因此提高融合及发育效率至关重要。为找出适合小鼠2-细胞胚胎融合的最佳方案,试验采用两种融合液0.3mol/L甘露醇溶液和0.3mol/L蔗糖溶液,两种融合液均采用了120V/mm,20μs×2、100V/mm,40μs×2、80V/mm,80μs×2三种电融合参数。其中采用0.3mol/L甘露醇溶液作为融合液且融合参数采用80V/mm,80μs×2获得了92.5%的融合率和90.6%的囊胚率,是小鼠2-细胞胚胎融合的最佳方案。     

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。     

猪2-细胞胚胎细胞的电融合

为了探索猪胚胎细胞核移植技术中细胞融合参数，进行了猪２－细胞胚胎细胞的电融合。电融合时，直流电场强度为２ｋＶ／ｃｍ，脉冲时程选４０μｓ时，可获得７０．４％的融合率和６８．４％的发育率。非电解质融合液（甘露醇）对于融合率和融合胚的发育均优于电解质融合液（ＤＰＢＳ）。在直流电脉冲前的交流脉冲是必要的，可以显著提高融合率（Ｐ＜０．０５）。     

提高牛胚胎体外生产效率的研究进展

作者从采卵率、卵母细胞成熟率、受精率、胚胎发育率、胚胎冷冻解冻后生存率等方面综述了提高牛胚胎体外生产效率的研究进展。     

影响牛胚胎采集效果的因素

本文在全面分析胚胎采集过程中的每个技术环节基础上,指出影响采胚效果的影响因素,并提出了相应的解决办法及应对措施.     

体外培养体系对牛体外受精胚胎性比的影响

研究了不同体外培养体系对牛体外受精胚胎性比的影响。结果表明，添加血清的2组培养系统中所得雄性囊胚与雌性囊胚的性比虽偏离了1：1，但差异不显著（P〉0．05），而总的体外胚胎的性比则显著偏离了1：1（P〈0．05）。这说明在体外培养系统中雄性胚胎比雌性胚胎发育能力更强，并且血清的存在对于雄性胚胎的发育有明显的促进作用。     

牛早期胚胎体外培养的研究进展

本文就目前牛早期胚胎体外培养的常用基础培养液、牛早期胚胎的体外培养方法以及影响牛早期胚胎发育的一些常规添加成分的发展现状做了较全面系统的概括,并指出了体外培养体系的研究价值和意义。     

体细胞核移植生产转基因牛胚胎的研究

为了优化利用体细胞核移植生产转基因牛早期胚胎的体系,以携带绿色荧光蛋白-新霉抗性双标记基因的pMSCV质粒转染的胎牛耳成纤维细胞为供体,以体外成熟的牛卵母细胞为受体构建克隆胚,研究供体细胞的传代次数对转基因胚的影响和重构胚在不同电场条件下（电场强度、直流电脉冲次数）融合效率及其在不同体外培养系统中的发育效果。结果表明：传代5次的细胞做核供体较有利于转基因胚的发育;在电场条件为电场强度1.6 kV/cm、直流脉冲1次的融合率最高;负压单层细胞共培养体系中的转基因囊胚发育率较好,与未转基因体细胞核移植胚的发育相比无显著差异（P〉0.05）。     

牛体细胞核移植胚胎的批量化生产

利用2枚显微去核后的半卵与1枚供体细胞同步融合,并结合微穴法(WOWs)培养体系批量化生产成年牛克隆胚胎(日产40～80枚).结果,重构胚电融合率95.7%(3059/3197)、卵裂率87.1%(2637/3027)、囊胚率41.1%(1244/3027)和可冻胚率(72.5%,933/1244)均达到较高水平.克隆胚胎采用玻璃微管玻璃化冷冻保存数月后移植,30日龄时妊娠率为28.1%(48/171),已经产下5头足月克隆牛续.结果表明,该方法具有产业开发潜力.     

Increase in calf production by the transfer of bisected bovine embryos

A total of 132 embryos were recovered from 17 superovulated donor cows 7 d after estrus. Seventy-four embryos were selected and assigned to 2 treatment groups. The number of whole embryos that were directly transferred (Group A) and bisected (Group B) were 44 and 30 embryos, respectively. Sixty demi-embryos were produced from 30 morulae to blastocyst-stage embryos that were bisected. One hundred-three embryos, including whole and demi-embryos without zonae pellucidae, were nonsurgically transferred. Only one whole or demi-embryo was transferred to each recipients. The pregnancy rate for whole embryos (A) was 63.6% (28/44), while for demi-embryos (B) it was 74.6% (44/59). There was no significant difference between the pregnancy rates of whole embryos (A) and bisected embryos (B) transferred 7 d after estrus. Forty-three calves including the 14 sets of identical twins were obtained from 30 original embryos (143.3%) using the embryo bisection technique.     

输卵管和颗粒细胞单层对牛体外受精胚胎发育的影响

以屠宰场牛卵巢为试验材料,研究输卵管细胞单层(OCM)和颗粒细胞单层(GCM)对牛卵母细胞体外成熟(IVM)、体外受精(IVF)和体外培养(IVC)后胚胎发育能力的影响。(1)从卵泡抽取卵丘卵母细胞复合体(COCs),并根据卵母细胞外面卵丘细胞的层数将其分为3类:1级(≥4层);2级(2～3层);3级(0～1层)。作分别在IVM和IVC培养液中添加GCM(1×106个/mL)与不添加的对比试验。结果显示:添加GCM对1级卵母细胞的卵裂率、6～8细胞发育率和囊胚率无明显影响(P>0.05);但添加GCM的2级、3级卵母细胞,受精后的卵裂率、6～8细胞发育率和囊胚率分别高于未添加组(P<0.05)。(2)所有卵母细胞(包括COCs和裸卵)被随机分为3个组,在其IVM和IVC培养液中分别添加OCM、GCM或不添加体细胞(对照组)。结果显示:OCM和GCM组的卵裂率、6～8细胞发育率和囊胚率均高于对照组(P<0.05),而两试验组之间差异不显著。     

Male and female effects on the in vitro production of bovine embryos

A 3-year study was carried out to evaluate male and female effects on the efficiency of an in vitro fertilization (IVF) programme. The semen of different bulls used for artificial insemination was tested for the in vitro production of transferable blastocysts. The fertilization capacity was recorded for each bull. Bovine oocytes were matured in vitro, fertilized with frozen/thawed semen of 63 individual bulls and cultured during 8 days. The semen of one bull was used as control. The percentage of cleavage (36.3-93.4%) and blastocysts on day 7 (6.9-51.2%) varied from bull to bull. Despite high variability, blastocysts were produced with the semen of all bulls in the first trial. Moreover, oocytes fertilized with 85% of tested bulls reached a blastocyst rate not different to the control bull. The correlation coefficients of six bulls showed no significant male effect but an influence of oocytes on the cleavage rate (F-value 0.38, P > 0.05, and 12.4, P < 0.001, respectively). The development to blastocysts on day 7 was significantly influenced by sperms and also oocytes and session (P < 0.01), but no combined interaction was observed between female and male. It is concluded that transferable embryos can be produced in vitro in the first trial with frozen/thawed semen of 63 tested bulls. The results show different capacities of bulls to produce embryos and high male and female effects on the efficiency of an IVF programme.     

牛卵泡液对牛卵母细胞体外成熟及受精胚发育力的影响

研究了牛卵母细胞体外成熟液和胚胎培养液中添加不同浓度的牛卵泡液对其体外成熟率和受精胚发育力的影响。结果表明:添加10%牛卵泡液的实验组,卵母细胞的成熟率与血清对照组没有显著差异(P>0.05);添加10%卵泡液的实验组与血清对照组相比,卵裂率和囊胚率没有显著差异,却显著高于添加5%和20%牛卵泡液的实验组(P<0.01),且各实验组囊胚内细胞数差异不大(P>0.05)。因此,用10%的牛卵泡液可以取代成熟液和胚胎培养液中的血清,并可降低实验成本。     

Blastocyst production from in vitro-produced day-2 bovine embryos classified by cleavage stage, and cytogenetical evaluation of the resultant day-8 blastocysts

The present study was conducted to determine the criteria for selecting good quality embryos on Day-2 post-insemination and at the blastocyst stage. Bovine oocytes were matured, fertilized and cultured in vitro. First, Day-2 embryos were classified based on the number of blastomeres into 2-cell, 3- to 4-cell, 5- to 8-cell and >8-cell stage embryos; chromosome samples were then prepared. In the second experiment, the Day-2 embryos classified according to the number of blastomeres were cultured separately for an additional 6 days (Day 8). The resultant Day-8 blastocysts from each group of Day-2 embryos were classified into the following 3 grades based on morphology and diameter: Grade A, hatched and hatching blastocysts; Grade B, expanded blastocysts; and Grade C, unexpanded blastocysts. Chromosome samples were then prepared. The 5- to 8-cell stage Day-2 embryos had the lowest incidence of chromosomal abnormalities (13.5%, P<0.05) and the highest development rate to blastocysts (59.2%, P<0.05). Furthermore, the blastocysts derived from the 5- to 8-cell stage embryos had the largest mean number of cells (102.8+/-42.4, P<0.05), largest number of metaphases per blastocyst (9.5+/-4.8, P<0.05) and lowest incidence of chromosomal abnormalities (24.6%, P<0.05). The Grade A blastocysts had the largest mean number of cells (136.6+/-33.4, P<0.05), a large number of metaphases per blastocyst (11.9+/-5.5, P<0.05) and a low incidence of severe chromosomal abnormalities (17.3%). The results showed that, at Day 2, the 5- to 8-cell stage embryos were of better quality since they had the lowest incidence of chromosomal abnormalities and the highest blastocyst rate and the resultant blastocysts had the largest number of cells and lowest incidence of chromosomal abnormalities. In particular, selection of Grade A blastocysts can improve the development rate to term.     

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.