应用体细胞克隆技术和胚胎移植技术生产克隆水牛

为了解决克隆水牛技术在供核细胞、受体细胞中的选用问题,探索不同条件对移植受孕的影响,完善克隆技术的理论体系。试验以摩拉奶水牛的耳成纤维细胞作为供核细胞,本地水牛卵母细胞为受体胞质进行细胞克隆构建重组胚胎;将发育5～10 d的重组胚胎移植于本地水牛、杂交水牛的子宫内,观察不同发情方式、胚胎类型、胚龄及季节对移植受孕的影响。结果表明:水牛的克隆胚胎妊娠率较低,分别为12.05%、12.04%、13.95%。自然发情和同期发情对克隆胚胎移植受胎率的影响差异不显著;鲜胚胎移植186头,受胎22头,受胎率为11.83%(22/186);冻胚胎移植174头,受胎23头,受胎率为13.22%(23/174)。克隆水牛从出生至12月龄体尺增长明显高于本地水牛;水牛克隆胚胎移植受胎在秋季最好,秋季受胎数占全年的53.33%,显著高于其他季节。     

受体母猪排卵状况对体细胞克隆胚胎移植效率的影响

为了确定猪体细胞克隆胚胎的受体母猪最佳发情阶段,本研究比较了供受体同期化方案对猪体细胞克隆胚胎输卵管内移植后30 d受胎率和妊娠足月率的影响。将体外培养2 d的猪体细胞克隆胚胎,移植到发情第1~2天的受体母猪。根据受体母猪卵泡发育与排卵状况将受体猪群分为2个类别:卵泡发育处于即将排卵或正在排卵阶段(组一);卵泡发育处于排卵前阶段或完全排卵阶段(组二)。结果发现:(1)组一6头受体母猪胚胎移植后30d受胎率为100%;而组二10头受体母猪受胎率为40%,二者差异显著(P0.05)。(2)组一6头受体母猪全部产仔,妊娠足月率为100%;组二10头受胎母猪2头产仔,妊娠足月率为20%,二者差异极显著(P0.01)。以上结果表明:体外培养2 d的猪体细胞克隆胚胎发育阶段与即将排卵或正在排卵的受体母猪的子宫环境同期性最好,得到了理想的胚胎移植效率。     

世界首例体细胞克隆水牛在广西诞生

11月19日上午，世界首例体细胞克隆水牛在广西大学诞生。这项技术的成功，将使我国具有培养更多优质水牛的能力。     

我国第一个体细胞克隆牛胚胎移植中心成立

本刊讯 :中国农业大学和芦台经济技术开发区日前签署协议 ,中国第一个体细胞克隆牛胚胎移植中心在唐山正式成立。据了解 ,早在2001年 ,中国农业大学就和芦台经济技术开发区合作 ,建立了生物技术实验基地。第二年4月 ,中国第一头地方优质奶牛在芦台经济技术开发区诞生。此后 ,在这个开发区又陆续培育出5头体细胞克隆优质奶牛。今年又培育出具有世界最新药物蛋白基因的2头克隆奶牛。目前 ,芦台经济技术开发区的克隆牛成活率处于我国领先水平。此次成立的我国第一个体细胞克隆牛胚胎移植中心将成为华北地区克隆奶牛繁育基地和生物工程服务中心…     

水牛克隆胚胎和超数排卵胚胎移植情况报告

对温州水牛体细胞克隆胚胎和超数排卵胚胎的移植情况进行总结。从高产温州母水牛采集耳部组织制备成纤维细胞作为供体细胞进行核移植,buffao培养获得体细胞克隆胚胎后,非手术移植到同期发情处理的温州水牛子宫内。将98枚体细胞克隆胚胎移植到49头受体水牛子宫内,8头受体水牛妊娠,最后产下克隆水牛2头;将8枚超数排卵胚胎移植到8头受体水牛子宫内,2头水牛妊娠,产下胚胎移植水牛2头。由此表明,在本地条件下,进行水牛体细胞克隆和胚胎移植是可行的。     

影响牛胚胎移植妊娠因素的研究

牛妊娠的确立与胚胎、子宫环境和黄体之间存在相当复杂的关系。据Sreenan报道,乳牛在输精后胚胎的死亡率为30—40％,且主要发生于妊娠后的18日内。因胚胎损失的机理很复杂,故将影响牛胚胎移植妊娠的主要因素报道如下: 一、供受体发情同期化程度因素的影响受精后,胚胎是在一个因内分泌的变化而引起不     

影响牛胚胎移植妊娠率的因素

牛的胚胎移植自70年代中期国际上已从实验转入应用阶段,商业性机构纷纷建立。80年代以来,发展更为迅速,并在胚胎显微切割、基因导入和胚胎性别鉴定等技术方面取得了突破性进展。胚胎移植由许多复杂的环节组成,无论是那一个环节的疏忽,都会影响移植的效果。目前国际上非手术鲜胚移植妊娠率为60—70％,我国为50—60％,冻胚妊娠率则比鲜胚低15—20％。因此,研究影响牛胚移妊娠率的因素,对提高胚胎移植效果,降低成本,具有重要的现实意义。一,胚胎质量胚胎质量对移植能否成功关系颇大。目前胚胎质量的形态学评价及质量级别的划分是主观的,常因评定人员及实验室的不同而有差别。但一般来     

世界首例成活的体细胞克隆水牛在广西自然诞生

2005年3月17日,世界首例成活的体细胞克隆水牛在广西大学自然诞生。该项技术使我国具有了培养更多优质水牛的能力。     

奶水牛活体采卵胚胎移植的应用

主要探讨利用胚胎移植技术加快优良奶水牛的繁育。本研究共进行175头次胚胎移植,受胎44头,流产8头,产下36头健康犊牛,受胎率为25.14%(44/175),流产率为18.18%(8/44)。数据分析,受胎率方面,后备牛>干奶牛>挤奶牛;鲜胚移植受胎率优于冻胚;自然发情移植受胎率与同期发情移植受胎率相差不显著;两种同期发情（CIDR+PG和GnRH+PG+GnRH）方法处理移植受胎率相当。流产率方面,挤奶牛>后备牛>干奶牛;鲜胚移植流产率与冻胚移植流产率基本一致;自然发情流产率小于同期发情流产率;CIDR+PG移植流产率高于GnRH+PG+GnRH移植流产率。结论：OPU胚胎移植技术可用于育种扩群。胚胎移植前后,加强饲养管理,减少应激,纠正内分泌平衡,有利于提高受胎率和降低流产率,获得更好的经济效益。     

Comparison of efficiency of in vitro cloned sheep embryo production by conventional somatic cell nuclear transfer and handmade cloning technique

Conventional somatic cell nuclear transfer (SCNT) technique of in vitro production of cloned embryos involves use of costly and complicated micromanipulators. Handmade cloning (HMC) technique has been applied as efficient and cost‐effective alternative in many livestock species. The aim of the present study was to compare the efficiency of in vitro production and in vitro development of cloned sheep embryos by the two techniques. Cloned embryos were produced by conventional SCNT using micromanipulator apparatus and by HMC technique. Enucleation efficiency and efficiency of fusion with somatic cell (nucleus donor) were compared. Cleavage percentage was observed on day 2 of in vitro culture (IVC), and morula and blastocyst percentages were calculated on day 7 of IVC. Higher enucleation efficiency (96.98 ± 1.01 vs. 93.62 ± 1.03; p > .05) as well as fusion efficiency was obtained with HMC technique than with conventional SCNT (96.26 ± 1.34 vs. 92.63 ± 0.70, p < .05); 181 cloned sheep embryos were produced in vitro by conventional SCNT and 92 by HMC. Cleavage percentage observed on day 2 of in vitro culture was higher in HMC than SCNT (66.92 ± 3.72 vs. 55.97 ± 2.5, respectively, p < .05). Morula percentage obtained was higher in SCNT than HMC (44.12 ± 2.93 vs. 30.43 ± 6.79, respectively, p < .05), whereas blastocyst percentage obtained by HMC was higher (12.46 ± 4.96) than SCNT (5.31 ± 2.25; p > .05). It was inferred that HMC technique provides a cost‐effective and efficient method of in vitro production of cloned sheep embryos with a comparatively simpler technique with a possibility of automation. Efficiency of cloned embryo production could be improved further by propagating and standardizing this technique.     

水牛体细胞连续核移植的效果

为探讨水牛体细胞连续核移植的效果,以水牛胎儿成纤维细胞为核供体,进行了水牛体细胞连续核移植。结果显示,连续核移植的融合率显著高于原代核移植(87.9%vs76.2%,P<0.05),但两者之间的分裂率和囊胚率没有显著差异(P>0.05);这说明水牛体细胞核移植胚胎可被再次克隆而不降低其发育能力,水牛体细胞连续核移植是可行的。     

延边黄牛卵母细胞与体细胞克隆胚胎内脂滴变化规律的研究

用苏丹Ⅳ和苏丹黑B 2种染色方法,对体外培养的延边黄牛各发育阶段卵母细胞和体细胞克隆胚胎内脂滴变化进行研究,并对2种染色方法进行了比较。结果表明,随着细胞发育阶段的不同,脂滴的含量也随之变化,从未经成熟培养的卵母细胞到8细胞期胚胎内脂滴不断增多,脂滴直径不断增大;而从8细胞期到囊胚期胚胎内脂滴不断减少,脂滴直径也不断减小。也显示苏丹IV染色效果较好,可作为测定活体细胞中脂滴的检测方法。     

Survival of embryos and calves derived from somatic cell nuclear transfer in cattle: a nationwide survey in Japan

To obtain data concerning the survival of embryos and calves derived from somatic cell nuclear transfer (SCNT) in Japan, a nationwide survey was carried out in April, 2009. As a result, data concerning 3264 embryo transfers (ETs) with SCNT embryos which produced 301 calves were accumulated and their survival was analyzed. The present survey revealed that survival rates of transferred bovine embryos and produced calves derived from SCNT had not improved over a decade (1998–2007). A remarkable feature of the pregnancies with SCNT embryos was a high incidence of spontaneous abortions. When the decade was divided by the occurrence of bovine spongiform encephalopathy (BSE) in 2001, significant decreases in the ‘after BSE’ period (2002–2007) were observed in the percentages of calves born (P < 0.01), calves living at birth (P < 0.05), calves living for 24 h (P < 0.05) and 6 months (P < 0.01). Abortions that occurred during 61–99 days after ETs were significantly increased (P < 0.01) in the ‘after BSE’ period. Certain kinds of regeneration that occurred in oocytes during the 15–20 h of storage of bovine ovaries at 10–15°C as a part of BSE inspection might have had some negative effects on SCNT embryos when these oocytes were used as recipients of SCNT.     

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.     

Supplement of autologous ooplasm into porcine somatic cell nuclear transfer embryos does not alter embryo development

Somatic cell nuclear transfer (SCNT) is considered as the technique in which a somatic cell is introduced into an enucleated oocyte to make a cloned animal. However, it is unavoidable to lose a small amount of the ooplasm during enucleation step during SCNT procedure. The present study was aimed to uncover whether the supplement of autologous ooplasm could ameliorate the oocyte competence so as to improve low efficiency of embryo development in porcine SCNT. Autologous ooplasm‐transferred (AOT) embryos were generated by the supplementation with autologous ooplasm into SCNT embryos. They were comparatively evaluated with respect to embryo developmental potential, the number of apoptotic body formation and gene expression including embryonic lineage differentiation, apoptosis, epigenetics and mitochondrial activity in comparison with parthenogenetic, in vitro‐fertilized (IVF) and SCNT embryos. Although AOT embryos showed perfect fusion of autologous donor ooplasm with recipient SCNT embryos, the supplement of autologous ooplasm could not ameliorate embryo developmental potential in regard to the rate of blastocyst formation, total cell number and the number of apoptotic body. Furthermore, overall gene expression of AOT embryos was presented with no significant alterations in comparison with that of SCNT embryos. Taken together, the results of AOT demonstrated inability to make relevant values improved from the level of SCNT embryos to their IVF counterparts.     

Constant transmission of mitochondrial DNA in intergeneric cloned embryos reconstructed from swamp buffalo fibroblasts and bovine ooplasm

Although interspecies/intergeneric somatic cell nuclear transfer (iSCNT) has been proposed as a tool to produce offspring of endangered species, conflict between donor nucleus and recipient cytoplasm in iSCNT embryos has been identified as an impediment to implementation for agricultural production. To investigate the nuclear–mitochondrial interactions on the developmental potential of iSCNT embryos, we analyzed the mtDNA copy numbers in iSCNT embryos reconstructed with water buffalo (swamp type) fibroblasts and bovine enucleated oocytes (buffalo iSCNT). As controls, SCNT embryos were derived from bovine fibroblasts (bovine SCNT). Buffalo iSCNT and bovine SCNT embryos showed similar rates of cleavage and development to the 8‐cell stage (P > 0.05). However, buffalo iSCNT embryos did not develop beyond the 16‐cell stage. Both bovine and buffalo mtDNA content in buffalo iSCNT embryos was stable throughout the nuclear transfer process, and arrested at the 8‐ to 16‐cell stage (P > 0.05). In bovine SCNT embryos that developed to the blastocyst stage, mtDNA copy number was increased (P < 0.05). In conclusion, both the donor cell and recipient cytoplast mtDNAs of buffalo iSCNT embryos were identified and maintained through the iSCNT process until the 8–16‐cell stage. In addition, the copy number of mtDNA per embryo was a useful monitor to investigate nuclear–mitochondrial interactions.     

Cloned calves derived from somatic cell nuclear transfer embryos cultured in chemically defined medium or modified synthetic oviduct fluid

Somatic cell nuclear transfer (SCNT) is considered to be a critical tool for propagating valuable animals. To determine the productivity calves resulting from embryos derived with different culture media, enucleated oocytes matured in vitro were reconstructed with fetal fibroblasts, fused, and activated. The cloned embryos were cultured in modified synthetic oviduct fluid (mSOF) or a chemically defined medium (CDM) and developmental competence was monitored. After 7 days of culturing, the blastocysts were transferred into the uterine horn of estrus-synchronized recipients. SCNT embryos that were cultured in mSOF or CDM developed to the blastocysts stages at similar rates (26.6% vs. 22.5%, respectively). A total of 67 preimplantational stage embryos were transferred into 34 recipients and six cloned calves were born by caesarean section, or assisted or natural delivery. Survival of transferred blastocysts to live cloned calves in the mSOF and the CDM was 18.5% (to recipients), 9.6% (to blastocysts) and 42.9% (to recipients), 20.0% (to blastocysts), respectively. DNA analysis showed that all cloned calves were genetically identical to the donor cells. These results demonstrate that SCNT embryos cultured in CDM showed higher viability as judged by survival of the calves that came to term compared to blastocysts derived from mSOF cultures.