应用奶牛性控精液生产体内性控胚胎及其移植试验初报

试验超排处理了3头供体牛,回收卵数19枚.可用胚胎11枚、退化胚6杖、未受精卵2枚.胚胎可用率为57.9%(11/19),头均可用胚3.7枚(11/3);同期发情处理了3头受体牛,妊娠率为66.6%(2/3),生下2头母犊,雌性胚胎率为100%.     

Ops法玻璃化冷冻兔胚胎的试验报告

采集中国大白兔的桑椹胚共545枚,采用OPS法,配制VSⅠ和VSⅡ两种冷冻液,分别冷冻兔的桑椹胚265枚和280枚,解冻后胚胎回收率分别为93.6%(248/265)和92.8%(260/280),两者差异不显著(P>0.05);解冻后胚胎形态良好率分别为95.2%(236/248)和94.2%(245/260),两者差异显著(P<0.05)。将解冻后形态良好的210枚桑椹胚分别移植到15只受体母兔体内,结果有8只母兔妊娠,妊娠率为53.3%(8/15),妊娠受体内移植胚胎数为110枚,产仔数占移植胚胎数的33.6%(37/110)。用VSⅠ冷冻的100枚桑椹胚移植到7只受体母兔体内,妊娠率为57.1%(4/7),产仔率为35.7%(20/56);用VSⅡ冷冻的110枚桑椹胚移植到8只受体母兔体内,妊娠率为50.0%(4/8),产仔率为31.5%(17/54)。无论妊娠率还是产仔率,VSⅠ都高于VSⅡ,但差异均不显著(P>0.05)。     

贵州胚胎工程技术研究及示范应用效果

21世纪以来,贵州开展了牛同期发情、超数排卵、胚胎分割、早期胚胎性别鉴定、胚胎移植等试验及其示范应用。研究表明:结合使用孕酮阴道栓和氯前列烯醇进行同期发情,受体合格率分别为50.12%(423/840)和37.82%(247/653),同期发情率分别为81.09%(343/423)和65.58%(162/247),受体妊娠率分别为51.52%(136/268)和18.57%(26/140),胚胎移植总效率分别为16.36%和3.98%,前者均极显著高于后者(P<0.01);促卵泡素法处理供体平均获得可用胚胎数4·8枚(43/9),促卵泡素 雌二醇法处理供体获可用胚9.8枚(117/12),差异极显著(P<0.01),说明促卵泡素和雌二醇结合使用进行超排,可改善牛的超排效果,显著增加可用胚胎数;8、10月份移植妊娠率分别为40.34%(48/119)、45.41%(84/185),均高于12月份的24.80%(26/105),且差异显著(P<0.05),可见,由于天气较寒冷且受体牛的饲草料不足,将严重影响胚胎移植妊娠率;8月份供体牛超数排卵的平均采胚数(8.40枚/头)及平均可用胚数均高于12月份的(5.81枚/头),其中平均可用胚数两者差异显著(P<0.05),在天气较寒冷的月份对供体牛进行超数排卵将会影响超排效果;移植双半胚和整胚的妊娠率分别为54.20%(13/24)和27.30%(6/22),前者为后者的2倍,表明在生产实践中通过胚胎分割不但可以成倍增加胚胎的数量,而且可以提高移植后的妊娠率;性别鉴定雌性胚胎冻胚的移植妊娠率40%(40/100),略低于常规冻胚的43.55%(27/62),但差异不显著(P>0.05);2003-2004年全省共移植牛胚胎1208枚,妊娠463头,移植妊娠率37.58%(454/1208)。初步建成了牛良种繁育体系,成效明显。     

蒙古绒山羊胚胎移植效果研究

通过评价双胚胎移植方法和单胚胎移植方法对蒙古绒山羊妊娠率及产羔率的影响,为优质种羊的快速扩繁提供技术支持。选取5只供体母羊进行同期发情与超数排卵处理,供体母羊发情后进行人工授精,收集胚胎;利用收集的可用胚胎对选取的39只受体母羊进行胚胎移植,其中10只受体母羊做双胚移植,29只受体母羊做单胚移植;比较双胚移植和单胚移植受体母羊的妊娠率及产羔率。结果表明:供体母羊经CIDR处理,发情率达100%,超数排卵、人工授精后共收集胚胎64枚,其中可用胚胎49枚,可用胚胎率为76.56%;双胚移植受体母羊的妊娠率为90.00%(9/10),产羔率为180.00%(18/10),单胚移植受体母羊的妊娠率为68.97%(20/29),产羔率为62.07%(18/29)。双胚移植方法比单胚移植方法可获得更高的妊娠率及产羔率,表明双胚移植方法适用性良好,具有极大的推广价值。     

常规冻精与性控冻精的授精效果分析

试验对常规冻精和性控冻精在荷斯坦牛超排后的授精效果作了分析.对29头做性控(X-精子)冻精的授精,获得111枚可用胚胎,头均可用胚为3.83枚;受精率:青年牛卵子受精率为90.60%,经产牛卵子受精率为86.15%.对34头做常规冻精的授精,获得212枚可用胚胎,头均可用胚为6.24枚;受精率:青年牛卵子受精率为92.17%,经产牛卵子受精率为88.50%.     

体内、外卵母细胞对猪体细胞克隆胚胎发育潜力的影响

为探讨猪体内、外成熟卵母细胞对核移植重组胚胎发育能力的影响,试验通过激素促排获得体内成熟卵母细胞和收集废弃卵巢获取体外成熟的卵母细胞,分别构建核移植重组胚,比较其卵裂率、囊胚率及胚胎移植受孕情况。结果显示,PGC+PMSG+HCG组的平均排卵数（27.8枚/头）显著高于PGC+HCG （12.5枚/头）、PMSG+HCG （13.7枚/头）及自然发情组（11.5枚/头）（P<0.05）,体内收集到的卵母细胞,可用于构建核移植重组胚的可用卵率均达到90%以上,与其他处理组差异不显著（P>0.05）,说明通过激素处理可获得更多的可用卵母细胞,而且卵母细胞的质量没有显著差异;以体内和体外成熟卵母细胞作为核移植受体构建的克隆胚胎,二者的胚胎融合率（80.31%和79.29%）和卵裂率（90.40%和86.51%）差异均不显著（P>0.05）,但来自体内成熟卵母细胞克隆的胚胎发育至囊胚期的比例显著升高（P<0.05）;将体内、外成熟卵母细胞构建的核移植重组胚分别移植代孕母猪,头平均移植30或60枚时,体内成熟卵母构建的克隆胚胎移植出生仔猪10头,而体外培养卵母细胞构建的克隆胚胎均未着床受孕,表明通过激素促排获得的卵母细胞质量更好,能显著提高克隆胚胎的囊胚率,减少胚胎移植数量,提高代孕母猪的怀孕率。     

转基因鲜胚和冻胚对黄牛受胎率的影响

本试验对转基因体细胞克隆胚胎经两种方式(鲜胚体温长途运送和冷冻胚胎复苏)移植到受体牛妊娠和产犊情况进行比较.结果为两组的妊娠率为33.08%和29.32%,出生率为5.70%和5.54%,差异均不显著(P>0.05),说明冻胚解冻后移植与鲜胚长途运输移植的效果无差异,转基因体细胞克隆胚胎低温冻存可应用于实际生产.     

胚胎因素对肉羊胚胎移植效果的影响

为提高肉羊胚胎移植的妊娠率,研究了胚胎发育阶段、胚胎体外保存时间和移植胚胎数对肉羊胚胎妊娠率的影响。结果表明:发育阶段对妊娠率有显著影响,在移植胚胎数量相同的情况下,囊胚的受胎率(58.7%)高于桑椹胚(56.4%)(P<0.05);胚胎体外保存0～10 min、10～30 min、30～60 min、60 min以上的妊娠率分别为66.7%、65.0%、60.0%、63.3%,四者间均没有显著差异(P>0.05);移植胚胎的数量对受胎率有显著影响,移植2枚囊胚的受胎率(67.7%)显著高于移植2枚桑椹胚(61.2%)、1枚囊胚(58.7%)、1枚桑椹胚的受胎率(56.4%)(P<0.05)。     

波尔山羊胚胎移植试验

应用FSH对8只供体波尔山羊进行了超排处理。在放栓的第9～12天,连续4d采用递减量肌肉注射FSH。结果有6只发情,同期发情率为75%。这6只羊配种后均采胚成功,共采胚83枚,只均13.8±7.36枚,其中可用胚75枚,只均12.5±5.99枚,可用胚胎率为90.36%(75/83)。将36枚可用胚移植受体奶山羊32只,妊娠18只,妊娠率56.25%(18/32)。     

波尔山羊腹腔内窥镜鲜胚移植试验

2006年10～11月在农五师兽医站种羊场使用2种超排药物对40只经产波尔山羊进行超排处理,供体羊有2只未发情,超排有效率为95%(38/40)。共回收胚胎388枚。其中,可用胚311枚,每只山羊收获胚胎10.21枚(388/38),平均可用胚8.18枚(311/38),可用胚率80.15%(311/388)。同期处理受体399只,有320只发情,同期率为80.20%,运用腹腔内窥镜技术移植受体267只,其中44只供体移植双胚。经试情150只受体未返情,受胎率为56.18%(150/267)。     

Developmental competence of HMC(TM) derived bovine cloned embryos obtained from somatic cell nuclear transfer of adult fibroblasts and granulosa cells

To enable us to handle a large number of oocytes at a given time and to have an increased throughput of cloned embryos, we attempted the Handmade cloning (HMC) technique, a zona-free method of bovine somatic cell nuclear transfer. Our objective was to study the developmental competence of the HMC derived embryos obtained using different types of somatic cells. A total of 6,874 cumulus-oocyte-complexes were used with either 7th or 11th passage fibroblasts (1st and 2nd groups, respectively), which were prepared from male animals, or granulosa cells (3rd group) as nuclei donors. The average cleavage rate was 65%, accompanied by a blastocyst rate of just 2% for the cleaved products and 5% for the >8-cell embryos, and there was no significant difference between the three groups. Out of 27 blastocysts recovered, 22 blastocysts were transferred to 22 recipients, resulting in two pregnancies. One pregnancy was lost after the fourth week while the other progressed to full term with the birth of a male calf. This first successful cloning of a male calf with the HMC technique in Europe indicates the successful adoption and establishment of this technique in our laboratory, and that this technique can be successful in producing viable embryos.     

Cryopreservation of bovine somatic cell nuclear-transferred blastocysts: effect of developmental stage

The effect of developmental stage on the survival of bovine somatic cell nuclear-transferred blastocysts after freezing and thawing was evaluated. We also investigated how freezing affects nuclear-transferred (NT) embryos and in vitro fertilized (IVF) bovine embryos. Advanced-stage bovine NT blastocysts survived freezing better than early-stage NT blastocysts (86 vs 14%). The trend was similar with IVF embryos (87 vs 30%). At the stages tested, there was no significant difference in the survivability of NT and IVF embryos from advanced (86 vs 87%) or early-stage blastocysts (14 vs 30%). The average survival rate did not differ between NT and IVF bovine embryos (50 vs 51%). The higher survival rate of advanced-stage blastocysts compared to early-stage blastocysts in NT and IVF bovine embryos might be due to their higher cell number. In NT (128 +/- 25 vs 53 +/- 20) and IVF (128 +/- 29 vs 75 +/- 22) groups, advanced-stage blastocysts contained a significantly higher total cell number than early-stage blastocysts. There was no difference in total cell number between advanced-stage NT and IVF blastocysts (128 +/- 25 vs 128 +/- 29), however, early-stage NT and IVF blastocysts (53 +/- 20 vs 75 +/- 22) differed significantly.     

序贯培养液G1/G2对山羊核移植胚胎发育和质量的影响

为了优化山羊核移植胚胎体外培养体系,提高核移植效率,本研究检测了山羊体细胞核移植(SCNT)胚胎在序贯培养液G1/G2中的发育率和囊胚细胞凋亡,以及核移植胚胎移植后的妊娠率,以传统mSOF-FBS培养液作为对照组,评估序贯培养液G1/G2支持山羊核移植胚胎的发育能力。结果显示,与对照组相比,G1/G2组的囊胚发育率差异不显著((27.7±3.1)%vs(25.3±1.0)%,P>0.05),囊胚细胞数和囊胚细胞凋亡率显著降低(分别为(93.2±4.5)vs(109.1±6.2)和(4.9±0.2)%vs(11.3±0.1)%,P<0.05),但移植后的妊娠率显著增高(21.4%vs 8.0%,P<0.05)。结果表明,与传统的培养液mSOF-FBS相比,序贯培养液G1/G2能更好地支持山羊核移植胚胎的发育。     

Effect of activation treatments of recipient oocytes on subsequent development of bovine nuclear transfer embryos

Effects of recipient oocyte activation methods on the development of nuclear transfer (NT) embryos were investigated. In Exp. 1, cell-cycle phase of serum-starved bovine cumulus cells was examined by flow cytometry. Majority (95.5%) of medium-sized (16-20 microm) cells that made up 56% of total cells was at the G0/G1 phase. NT embryos were constructed by electric fusion with the medium-sized serum-starved cumulus cells and bovine oocytes of 3 different preparations: enucleated oocytes treated with calcium ionophore A 23187 for 5 min and cycloheximide for 5 hr (A 23187/CHX), those treated with ethanol for 7 min and cycloheximide for 2 hr(ethanol/CHX) and those without treatment. In Exp. 2 and 3, developmental competence of NT embryos constructed with A 23187/CHX- and ethanol/CHX-treated oocytes was compared to that of NT embryos constructed with non-treated oocytes, respectively. Further, nuclear behavior in 3 different NT embryos was examined in Exp. 4. Within 1 hr after fusion, majority of the NT embryos constructed with non-treated oocytes showed condensed chromosome. Three hours after fusion, about 50% of NT embryos constructed with non-treated or ethanol/CHX-treated oocytes showed a single pronucleus-like structure. NT embryos constructed with ethanol/CHX-treated oocytes showed similar rates of fusion, cleavage and blastocyst formation to those of the non-treated oocytes. In contrast, NT embryos constructed with A 23187/CHX-treated oocytes did not show any pronucleus-like structure and showed lower cleavage rate and no development to blastocysts. The results indicate that ethanol/CHX-treated oocytes could support development of somatic cell NT embryos to the blastocyst stage at a similar rate to that of non-treated oocytes.     

In vitro development of canine somatic cell nuclear transfer embryos in different culture media

The objective of the present study was to investigate the effects of three different culture media on the development of canine somatic cell nuclear transfer (SCNT) embryos. Canine cloned embryos were cultured in modified synthetic oviductal fluid (mSOF), porcine zygote medium-3 (PZM-3), or G1/G2 sequential media. Our results showed that the G1/G2 media yielded significantly higher morula and blastocyst development in canine SCNT embryos (26.1% and 7.8%, respectively) compared to PZM-3 (8.5% and 0%) or mSOF (2.3% and 0%) media. In conclusion, this study suggests that blastocysts can be produced more efficiently using G1/G2 media to culture canine SCNT embryos.     

Effect of transfection and passage number of ear fibroblasts on in vitro development of bovine transgenic nuclear transfer embryos

The objective of this study was to determine if the transfection of human prourokinase (ProU) gene and passage number of transfected ear fibroblasts affected in vitro development of bovine transgenic nuclear transfer (NT) embryos. An expression plasmid for human ProU was constructed by inserting a bovine beta-casein promoter, a green fluorescent protein (GFP) marker and human ProU gene into a pcDNA3 plasmid and transfected into bovine ear fibroblasts using a lipid mediated method. Abattoir derived oocytes were enucleated at 18-20 hr post maturation and a single donor cell was transferred into the perivitelline space of a recipient oocyte. After fusion and activation, the couplets were cultured in modified synthetic oviductal fluid (mSOF) medium for 168 hr. In Experiment 1, significantly lower rate in blastocysts formation (10.3%) was observed in transfected donor cells at early passage than that in nontransfected counterparts (22.1%, P<0.05). In Experiment 2, development to blastocysts and GFP expression in blastocysts were not significantly different between early (3-7) and late (8-12) passage donor cells (10.3 vs. 11.3% and 54.5 vs. 41.7%, respectively). This study indicates that in vitro development of bovine transgenic NT embryos is negatively influenced by transfection of human ProU gene into donor fibroblasts. However, passage number of transfected ear fibroblasts does not affect in vitro development of bovine transgenic NT embryos.     

家兔胚胎细胞核移植与连续移植

将发育不同时期的兔胚和移核胚的卵裂球细胞核与去核的成熟卵母细胞共同组成移核胚,通过中间受体培养和移植实验检验胚胎的发育能力。结果表明,（１）兔囊胚之前各个时期的胚胎细胞核均可使移核胚发育到囊胚;（２）胚胎极化前后的卵裂球参与组成的移核胚发育到囊胚的比例无显著差异。但极化后 ６４细胞胚的卵裂球与去核卵母细胞的融合率低于极化前的 ８细胞胚胎卵裂球;（３）兔 １６细胞胚与去核的卵母细胞组成的移核胚可以发育到期,产仔率为 ３．１６％ ;（４）兔移核胚卵裂球用于连续核移植,其后 ２ 代均可发育成囊胚,其中第 １ 代移核胚与第 ２ 代移核胚发育率相似,但显著高于第 ３ 代移核胚;（５）兔移核胚和各代连续移核胚卵裂球与去核卵的融合率无显著差异。     

山羊胚胎分割及同卵双生试验

选择山羊晚期桑椹胚、囊胚、孵出囊胚和孵出增大胚泡,用简化分割法二分。将19对裸半胚移植于18只受体羊,结果有12只妊娠,其中两只胚胎消失,两只流产,其余8只足月分娩,共产半胚羔11只。晚期桑椹胚、囊胚、孵出囊胚和孵出增大胚泡各组的半胚发育为羔羊的发育率分别为12.5％(1/8)、20％(2/10)、25％(3/12)和62.5％(5/8)。前三组均未获得同卵双生羔羊。在第四组,将4对裸半胚移植于4只受体,有3只妊娠,足月分娩半胚羔5只,其中两对为同卵双生。本研究证明,对称分割山羊孵出增大胚泡,不仅其半胚在体内仍可继续发育形成正常胎儿,而且不装透明带移植其裸半胚,仍能获得较高的同卵双生率。山羊孵出增大胚泡更适宜用简化分割法分割。     

Effects of downregulating DNA methyltransferase 1 transcript by RNA interference on DNA methylation status of the satellite I region and in vitro development of bovine somatic cell nuclear transfer embryos

For the successful production of cloned animals by somatic cell nuclear transfer (NT), the epigenetic status of the differentiated donor cell is reversed to an embryonic totipotent status. However, in NT embryos, this process is aberrant, with genomic hypermethylation consistently observed. Here, we investigated the effects of silencing DNA methyltransferase 1 (DNMT1) mRNA by small interfering RNA (siRNA) on the DNA methylation status of the satellite I region and in vitro development of bovine NT embryos. First, the levels of DNMT1 expression were analyzed at 0, 24, 48, 72, 120 and 192 h after in vitro culture. Real-time PCR and western blotting analyses detected a significant decrease in DNMT1 mRNA in the siRNA-injected NT (siRNA-NT) group up to 72 h after in vitro culture. Next, the levels of DNA methylation of the satellite I region were analyzed at several time points after in vitro culture. The level of DNA methylation detected in siRNA-NT embryos was significantly less than those in NT embryos throughout in vitro development. Moreover, the developmental rate of embryos to blastocysts in the siRNA-NT group was significantly higher than that of NT embryos. Our data suggest that knockdown of DNMT1 mRNA in NT embryos can induce DNA demethylation, which may enhance reprogramming efficiency.