Effect of Oocytes in vivo and in vitro on the Developmental Potential of Porcine Somatic Cell Cloned Embryos

To investigate the impact of porcine oocytes in vivo and in vitro maturation (IVM) on the development of porcine somatic cell cloned embryos,the somatic cell cloned embryos cultured in vitro and the sows were treated with hormones to collect mature oocytes in vivo,and the cleavage rate, blastocyst rate and embryo implantation were compared. The results showed that the average number of ovulation in PGC+PMSG+HCG group was significantly higher than that of PGC+HCG,PMSG+HCG and the natural estrus groups (P<0.05). The oocytes collected in vivo could be used for the construction, and the available oocytes rate reached more than 90%,and there was no significant difference among the four groups (P>0.05),which indicated that groups treated by hormone could obtain more available oocytes and the quality of oocytes was not significant different. In vivo and in vitro matured oocytes were used as nuclear transfer embryos of recombinant receptor,the fusion efficiency (80.31% and 79.29%) and cleavage rate (90.40% and 86.51%) were not significant different (P>0.05), but the proportion of in vivo matured oocytes cloned embryos developed into the blastocyst stage was significantly higher (P<0.05). The reconstructed embryos made from in vivo and in vitro matured oocytes were transplanted into surrogate sows (transferred 30 or 60 embryos),10 piglets were born in in vivo maturation of cloned embryo transfer group,while there was no implantation in in vitro maturation of cloned embryo transfer group. The results showed that high quality oocytes obtained by superovulation could significantly increase the blastocyst rate of embryos,reduce the number of embryos transferred and improve the pregnancy rate of surrogate sows.     

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。     

不同共培养体细胞和胎牛血清浓度对牛体外受精后早期胚胎的影响

利用屠宰黄牛的卵母细胞经体外成熟(IVM)、体外受精(IVF)后的早期胚胎,与单层颗粒细胞(GC)、输卵管上皮细胞(BOEC)等体细胞共培养及在胎牛血清的胚胎培养液中的后续发育进行了研究,并探讨了其影响因素,以期筛选出最佳的体外培养条件。结果表明:使用GC和BOEC体外共培养牛体外受精后胚胎,均取得了较好的囊胚发育率;且牛体外受精后早期胚胎体外培养体系中,添加10%血清能有效地促进牛体外受精后胚胎的囊胚率。     

转GFP基因核移植牛胚胎的研究

试验采用脂质体转染法与电穿孔法,以携带绿色荧光蛋白（GFP）-新霉抗性（neo-）双标记基因的pMSCV质粒转染胎牛耳成纤维细胞为供体与体外成熟的牛卵母细胞为受体构建克隆胚。研究了体外成熟培养液中添加EGF（表皮生长因子）对转基因胚的影响,不同转染方法构建供体细胞对重构胚发育的影响和在不同体外培养系统中的发育效果。结果显示,体外成熟培养液中添加EGF 30 ng/mL组的卵母细胞成熟率最高,但对后期转基因重构胚的囊胚发育率的影响,以添加EGF 20 ng/mL组的最高;以胎牛耳成纤维细胞为供体细胞,不同转染方法转染供体细胞构建重构胚,其囊胚发育率差异不显著（P>〖JP2〗0.05）;mSOFaa+颗粒细胞单层细胞共培养体系中的转基因囊胚发育率最好,该体系更适合体细胞核移植法生产转基因牛胚胎。     

Development of interspecies cloned monkey embryos reconstructed with bovine enucleated oocytes

This study was carried out to determine whether culture media reconstructed with bovine enucleated oocytes and the expression pattern of Oct-4 could support dedifferentiaton of monkey fibroblasts in interspecies cloned monkey embryos. In this study, monkey and bovine skin fibroblasts were used as donor cells for reconstruction with bovine enucleated oocytes. The reconstructed monkey interspecies somatic cell nuclear transfer (iSCNT) embryos were then cultured under six different culture conditions with modifications of the embryo culture media and normal bovine and monkey specifications. The Oct-4 expression patterns of the embryos were examined at the two-cell to blastocyst stages using immunocytochemistry. The monkey iSCNT embryos showed similar cleavage rates to those of bovine SCNT and bovine parthenogenetic activation (PA). However, the monkey iSCNT embryos were not able to develop beyond the 16-cell stage under any of the culture conditions. In monkey and bovine SCNT embryos, Oct-4 could be detected from the two-cell to blastocyst stage, and in bovine PA embryos, Oct-4 was detectable from the morula to blastocyst stage. These results suggested that bovine ooplasm could support dedifferentiation of monkey somatic cell nuclei but could not support embryo development to either the compact morula or blastocyst stage. In conclusion, we found that the culture conditions that tend to enhance monkey iSCNT embryo development and the expression pattern of Oct-4 in cloned embryos (monkey iSCNT and bovine SCNT) are different than in bovine PA embryos.     

Development and Status of Cattle Embryo Cloning in Germany

Contents: A review about experiments in bovine embryo cloning performed by different working groups in Germany is given. The procedure is shortly described and the achieved results are specified. Average enucleation rates in the experiments were 58–74%, electrofusion rates were 31 to 85%. Between 3 and 17% of the in vitro cultured embryos cleaved to transferable embryos. The first calf emerging from nuclear transfer in Germany was born in August, 1992. A clone of three identical calves was given birth in April, 1993. Four months later a calf was born, which exclusively emerged from in vitro techniques (in vitro maturation of recipient oocytes, in vitro production of blastomere donor embryo, in vitro culture of cloned embryos). Finally some future aspects of bovine embryo cloning in Germany are illustrated.     

Different Culture Media Requirements of IVF and Nuclear Transfer Bovine Embryos

Important differences exist between in vitro fertilized (IVF) and nuclear transfer (NT) bovine embryos. Studies have shown that although in vitro development is comparable, post-implantation survival is greatly reduced in NT embryos. In this study, we compare serum and bovine serum albumin (BSA) supplementation during oocyte maturation and embryo culture of IVF and NT embryos. In experiment 1, oocytes and embryos were randomly distributed into different treatment groups consisting of synthetic oviductal fluid (SOF) medium supplemented with either serum, fatty acid-free BSA (FAF) or fraction V BSA during maturation and/or culture to assess IVF embryo development. In experiment 2, oocytes were matured in SOF + serum or SOF + FAF and reconstructed embryos were cultured in SOF + FAF to assess NT embryo development. Among the IVF treatment groups, a greater number of blastocysts were observed in the steer serum (SER) group (IVM and IVC in SOF + serum) on day 6; however, no significant differences were seen in blastocyst development from day 8 onwards. Hatching frequencies on days 8 and 9 were significantly greater in groups with serum, with the exception of FAF (IVM and IVC in SOF + FAF) on day 9. For the NT treatment groups, the presence of serum during IVM resulted in a higher proportion of MII oocytes and increased blastocyst development and hatching rates were compared with supplementation of FAF. These results indicate that both serum and FAF provide comparable embryo development for IVF but not for NT bovine embryos.     

Japanese Society for Animal Reproduction: award for outstanding research 2002. The development and prevalence of the transfer technique for frozen-thawed embryos of Japanese black beef cattle in Tochigi Prefecture

The conditions of embryo transfer by the stepwise method, in which frozen-thawed embryos are transferred on day 7 (day 0=onset of estrus), were investigated with the aim of increasing pregnancy rates in frozen-thawed embryo transfer. The use of a vaginal speculum to prevent bacterial infection when passing an embryo transfer gun through the vagina yielded a pregnancy rate equal to or higher than that with application of a sheath cover to the transfer gun. Administration of a sedative, xylazine, to recipient cattle for preventing movement at the time of embryo transfer improved the pregnancy rate. The influence of the time from thawing of frozen embryos to transfer and of the transportation of the recipient by truck upon pregnancy rate was investigated. Embryo transfer within 60 minutes after aspiration into a straw or transportation of the bovine recipient, 1.5 hours each way before and after transfer, had no influence on pregnancy rate. Relations of the embryonic developmental stage and morphological quality after thawing of frozen embryos to pregnancy rate were investigated in recipients of nulliparous Holstein heifers. The pregnancy rate increased as the embryonic developmental stage advanced from compacted morula, early blastocyst, and blastocyst in that order. The pregnancy rate obtained with blastocyst stage embryos was significantly (P<0.05) higher than that with compacted morula stage embryos, and there was no significant difference in pregnancy rates between excellent morphological quality and good morphological quality for compacted morula stage embryos. When correlation of luteal function and pregnancy rate was investigated in bovine recipients, pregnancy rate showed a tendency to increase with increasing blood progesterone (P) concentration on the day before (on day 6 after estrus) and the day of embryo transfer. The pregnancy rate in bovine recipients, which showed a blood P concentration of > or =2.5 ng/ml on the day before embryo transfer, was significantly (P<0.05) higher than that in those with a blood P concentration of <2.5 ng/ml. Pregnancy rate showed a tendency to increase with decreasing blood estradiol-17beta (E2) concentration on the day of embryo transfer. Activation of luteal function by administration of human chorionic gonadotropin (hCG) in cycling cattle was investigated for its effect on increasing pregnancy rate in bovine recipients. A follicle coexisting with cyclic CL ovulated and induced CL formed after injection of hCG 1,500 IU 5 days after ovulation. The blood P concentration was significantly (P<0.05) higher in the administration group than in the control group, and the blood E2 concentration rapidly decreased, showing a lower concentration than in the control group. These results suggest the possibility that the pregnancy rate could be improved by administration of hCG. Pregnancy rate following intramuscular injection hCG 1,500 IU was comparatively investigated in parous Japanese Black beef cattle receiving frozen-thawed embryos 7 days after estrus. Pregnancy rate was 67.5% in the group in which hCG was administered on day 6 after estrus, and was significantly (P<0.05) higher than that in the control group (45.0%) and the group in which hCG was administered on day 1 after estrus (42.5%), revealing that hCG administration facilitated pregnancy. Transfer of frozen-thawed embryos in the blastocyst stage within 60 minutes after the aspiration into a straw, with a vaginal speculum after administration of xylazine is suggested as a way of improving pregnancy rate in bovine recipients with favorable luteal function and in those with luteal function activated by administration of hCG on the day before embryo transfer.     

添加不同发情时期山羊血清对牛胚胎体外发育的影响

为了提高牛体外胚胎囊胚发育率,添加不同发情时期山羊血清对牛胚胎进行体外培养。分别采集发情周期D0、D2、D4和D7山羊血清（发情当天为D0）,灭活除菌备用。结果表明不同发情时期羊血清对孤紫激活胚胎孵裂率影响差异不显著,囊胚发育率分别为29.5%、32.4%、33.9%和41.3%,D7血清能显著提高囊胚发育率（P＜0.05）,且扩张和孵化胚出现较早。添加D0血清体外受精胚胎卵裂率较高,但对囊胚发育率影响差异不显著。说明孤紫激活和体外受精胚胎发育有差异,D7血清对胚胎后期发育有利。受精胚无血清培养3d后添加D7血清,囊胚发育率为42.9%,是较为理想的培养方法。     

Importance of oil overlay for production of porcine embryos in vitro

Technologies to edit the zygote genome have revolutionized biomedical research not only for the creation of animal models for the study of human disease but also for the generation of functional human cells and tissues through interspecies blastocyst complementation technology. The pig is the ideal species for these purposes due to its great similarity in anatomy and physiology to humans. Emerging biotechnologies require the use of oocytes and/or embryos of good quality, which might be obtained using in vitro production (IVP) techniques. However, the current porcine embryo IVP systems are still suboptimal and result in low monospermic fertilization and blastocyst formation rates and poor embryo quality. During recent years, intensive investigations have been performed to evaluate the influence of specific compounds on gametes and embryos and to avoid the use of undefined supplements (serum and serum derivate) in the incubation media. However, little consideration has been given to the use of the mineral oil (MO) to overlay incubation droplets, which, albeit being a routine component of the IVP systems, is a totally undefined and thus problematic product for the safety of gametes and embryos. In this review, we provide an overview on the advantages and disadvantages of using MO to cover the incubation media. We also review one important concern in IVP laboratories: the use of oils containing undetected contamination. Finally, we discuss the effects of different types of oils on the in vitro embryo production outcomes and the transfer of compounds from oil into the culture media.     

Developmental competence of bovine embryos reconstructed by the transfer of somatic cells derived from frozen tissues.

The ability of frozen-thawed fetal skin was examined to generate viable cell lines for nuclear transfer. Fetal skin frozen at -35 degrees C or -80 degrees C in the presence of 5% DMSO were used as tissue explants to generate somatic cells. The resultant confluent cells were then used as donors for nuclear transfer (NT). Of the bovine NT embryos reconstructed from the somatic cells, 78% to 81% showed cleavage, 43% to 48% reached the stage of morula formation and 34% to 35% reached blastocyst formation. There were no significant differences in development (P>0.05) when the NT embryos were compared with those reconstructed from fresh somatic-cell-derived skin tissues (75%, 45%, and 38%, for cleavage, and development to morula and blastocyst stages, respectively). The results indicated that cell lines derived from bovine fetal skin cryopreserved by a simple method could be used as donors in nuclear transfer. The resulting embryos showed similar development in vitro to those reconstructed from unfrozen fetal-skin-derived somatic cells.     

紫外线照射时间对孤雌激活和体外受精胚胎发育的影响

用核荧光染料 Hoechst3 3 3 42对 5组牛成熟卵母细胞染色后 ,以紫外线 ( UV)分别照射0 ,10 ,2 0 ,3 0 s和 40 s。观察和分析对孤雌激活胚胎和体外受精胚胎卵裂及体外发育的影响。结果表明 ,经 UV照射 0 ,10 ,2 0 ,3 0 s和 40 s的卵母细胞孤雌激活后的卵裂率分别为 80 .8% ,77.9% ,72 % ,61.4%和 45 % ,囊胚发育率分别为 3 9.2 % ,3 6.4% ,19.4% ,14 .5 %和 11.1% ;UV照射 2 0 s以上的囊胚发育率都极显著低于UV未照射和照射 10 s的卵母细胞 ( P <0 .0 1) ,UV照射卵母细胞超过 2 0 s降低了孤雌激活胚胎的体外发育潜力 ;体外受精胚胎中 ,UV照射2 0 s以上与未照射和照射 10 s组相比 ,卵裂率和囊胚发育率显著差异 ( P >0 .0 5 ) ,U V照射卵母细胞超过 2 0 s时 ,显著降低了体外受精胚胎的发育潜力。由此可知 ,卵母细胞经染色后 ,UV照射时间应控制在 2 0 s以内 ,并以照射 10 s时 ,对孤雌激活胚和体外受精胚的体外发育影响最小     

Expression of Interferon-tau mRNA in Bovine Embryos Derived from Different Procedures

Interferon-tau (IFN-τ) is a secreted conceptus protein which plays a critical role in the establishment of ruminant pregnancy by its antiluteolytic and antiviral effects. In the present study, we hypothesized that IFN-τ expression was temporally and spatially regulated in different pre-implantation embryos and the levels of IFN-τ expression were different among bovine embryos derived from parthenogenetic activation (PA), in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). By using in situ hybridization with Digoxingenin (DIG)-labelled IFN-τ cDNA as a probe, we detected IFN-τ mRNA in bovine embryos from days 3 to 9 in culture. However, the timing of the initiation of IFN-τ mRNA expression was different among PA, IVF and SCNT embryos. Interferon-τ mRNA was first expressed in 16-cell stage IVF embryos on day 4, in SCNT morula on day 5 and early PA blastocyst on day 6. Semi-quantitative RT-PCR analysis showed that the expression levels of IFN-τ mRNA did not differ significantly among IVF, SCNT and PA embryos on day 7. In addition, freezing and thawing did not have a major impact either on IFN-τ mRNA expression in IVF or in vivo -produced bovine blastocysts.     

牛、羊卵母细胞孤雌激活的研究

本试验主要比较了离子霉素、电脉冲两种方法激活牛、羊体外成熟卵母细胞的效率。两种激活方法中。牛胚胎卵裂率无显著差异（90．61％对94．40％，P〉0．05），而离子霉素激活胚胎的囊胚发育率极显著高于电激活方法（12．3％对2．4％，P〈0．01）。两种方法对羊胚胎的研究中，羊胚胎卵裂率无显著差异（72．4％对77．4％，P〉0．05）。但是离子霉素激活胚胎的囊胚发育率显著高于电激活方法（3．67％对10．40％，P〈0．05）。本试验中还比较了用化学激活法（离子霉素）激活牛体外成熟卵母细胞后，用SOFaa体系培养，换液与不换液对孤雌激活胚胎体外发育的影响。结果表明：在第4天不换液的胚胎卵裂率和囊胚率极显著高于换液的胚胎（11．64％对3．49％。P〈0．01）。     

In vitro development of bovine embryos encapsulated in sodium alginate

The objective of this study was to evaluate the in vitro development of bovine embryos encapsulated in alginate. Day-4 embryos produced in vitro (n = 110) were encapsulated with 1.5% sodium alginate and co-cultured with oviduct cells. Unencapsulated embryos (n = 106) were used as controls. In vitro development rate to the blastocyst stage at Day 7 was similar between encapsulated, 42.7%, (47/110) and control. 34% (36/106). embryos. Although encapsulated embryos were able to hatch on Day 9, they did so in a lower proportion than controls (P < 0.05). In conclusion, alginate encapsulation of bovine embryos does not disturb the in vitro development up to the blastocyst stage but significantly reduces the hatching process.     

In-straw cryoprotectant dilution for bovine embryos vitrified using Cryotop

The aim of this study was to develop an in-straw dilution method suitable for 1-step bovine embryo transfer of vitrified embryos using the Cryotop vitrification-straw dilution (CVSD) method. The development of embryos vitrified using the CVSD method was compared with those of embryos cryopreserved using in-straw vitrification-dilution (ISVD) and conventional slow freezing, outside dilution of straw (SFODS) methods. In Experiment 1, in vitro-produced (IVP) embryos cryopreserved using the CVSD method were diluted, warmed and exposed to the dilution solution at various times. When vitrified IVP embryos were exposed to the dilution solution for 30 min after warming, the rates of embryos developing to the hatched blastocyst stage after 72 h of culture (62.0-72.5%) were significantly lower (P<0.05) than those of embryos exposed to the solution for 5 and 10 min (82.4-94.3%), irrespective of supplementation with 0.3 M sucrose in the dilution solution. In Experiment 2, the rate of embryos developing to the hatching blastocyst stage after 48 h of culture in IVP embryos cryopreserved using the SFODS method (75.0%) was significantly (P<0.05) lower than those of embryos cryopreserved using the CVSD and ISVD methods (93.2 and 97.3%, respectively). In Experiment 3, when in vivo-produced embryos that had been cryopreserved using the CVSD, ISVD and SFODS methods and fresh embryos were transferred to recipient animals, no significant differences were observed in the conception and delivery rates among groups. In Experiment 4, when IVP embryos derived from oocytes collected by ovum pick-up that had been cryopreserved using the CVSD and ISVD methods and fresh embryos were transferred to recipient animals, no significant differences were observed in the conception rates among groups. Our results indicate that this simplified regimen of warming and diluting Cryotop-vitrified embryos may enable 1-step bovine embryo transfer without the requirement of a microscope or other laboratory equipment.     

牛体外受精早期胚胎与小鼠胎仔成纤维细胞共培养的研究

探讨了人工合成培养液ＣＲ１ａａ和小鼠胎仔成纤维细胞对牛体外受精早期胚胎体外发育的影响。结果表明，牛体外受精卵在ＣＲ１ａａ液中的卵裂率达７６．２％，８细胞胚的比率达４４．８％。小鼠胎仔成纤维细胞能够显著促进牛体外受精的早期囊胚以上胚胎的发育。牛体外受精后第５、６天的早期胚胎分别与小鼠胎仔成纤维细胞共培养，在受精后第７天发育至囊胚以上的比率分别达１９．８％和２４．６％；受精后第８天，孵化的囊胚比例分别达５．２％和７．５％。实验表明，受精后第５、６天的牛体外受精早期胚胎与小鼠胎仔成纤维细胞共培养，可显著提高扩张囊胚和孵化囊胚数量。小鼠成纤维细胞对胚胎发育的支持作用取决于胚胎发育阶段     

DNA methylation status of bovine blastocyst embryos obtained from various procedures

DNA methylation is an important factor for the regulation of gene expression in early embryos. It is well known that the satellite I sequence is more heavily methylated in bovine somatic cell nuclear transfer (NT-SC) embryos than in embryos derived from in vitro fertilization (IVF). However, the methylation status of bovine embryos obtained by other procedures is not well known. To clarify DNA methylation levels of bovine embryos obtained from various procedures, we examined satellite I sequences in bovine blastocyst (BC) embryos derived from NT-SC, NT using embryonic blastomeres (NT-EM), in vivo (Vivo), IVF and parthenogenetic treatment (PA). Furthermore, in order to evaluate the efficacy of DNA demethylation by the NT procedure, we determined the DNA methylation levels in bovine embryos in which NT was recapitulated (Re-NT). Although the DNA methylation levels in the NT-SC embryos were higher than those in the other embryos, the NT-EM embryos exhibited lower DNA methylation levels. The satellite I sequence in the NT-SC embryos was more demethylated than that in the donor cells. Although the DNA methylation level in the individual NT-SC embryos showed variation, the full-term developmental efficacy of these embryos were not different. These findings suggest that the methylation level of the satellite I sequence at the BC stage is not related to the abnormalities of bovine embryos produced by NT-SC. There was no difference in methylation levels between Re-NT and NT-SC embryos. Our results indicated that the DNA methylation status differed among embryos produced by various methods and that at least some of the demethylation of the donor cell genome occurred in the recipient cytoplast after NT-SC, but the demethylation ability of the NT procedure was noted in the first NT but not in the second NT.