Comparison of In Vitro Developmental Competence of Cloned Caprine Embryos Using Donor Karyoplasts from Adult Bone Marrow Mesenchymal Stem Cells vs Ear Fibroblast Cells

The aim of this study was to produce cloned caprine embryos using either caprine bone marrow‐derived mesenchymal stem cells (MSCs) or ear fibroblast cells (EFCs) as donor karyoplasts. Caprine MSCs were isolated from male Boer goats of an average age of 1.5 years. To determine the pluripotency of MSCs, the cells were induced to differentiate into osteocytes, chondrocytes and adipocytes. Subsequently, MSCs were characterized through cell surface antigen profiles using specific markers, prior to their use as donor karyoplasts for nuclear transfer. No significant difference (p > 0.05) in fusion rates was observed between MSCs (87.7%) and EFCs (91.3%) used as donor karyoplasts. The cleavage rate of cloned embryos derived with MSCs (87.0%) was similar (p > 0.05) to those cloned using EFCs (84.4%). However, the in vitro development of MSCs‐derived cloned embryos (25.3%) to the blastocyst stage was significantly higher (p < 0.05) than those derived with EFCs (20.6%). In conclusion, MSCs could be reprogrammed by caprine oocytes, and production of cloned caprine embryos with MSCs improved their in vitro developmental competence, but not in their fusion and cleavage rate as compared to cloning using somatic cells such as EFCs.     

Isolation and experimental chicken-embryo-inoculation studies with budgerigar papovavirus

Attempts to isolate and identify budgerigar papovavirus (BPV) were made during three separate outbreaks of disease diagnosed on pathological grounds. Direct electron microscopy was successful only when large areas of skin were extensively disrupted to release virus and then extracted with fluorocarbon to remove lipids. Direct inoculation of budgerigar tissue suspensions into chicken embryos or chicken cell cultures failed to produce detectable virus. However, when primary cultures of liver and kidney were prepared from affected budgerigars, BPV could be detected by electron microscopy and by the production of a cytopathic effect at the third or fourth passage in cell cultures. The isolated virus was pathogenic for 10-day-old but not 11- or 12-day-old chicken embryos. Inoculated 11- and 12-day-old embryos produced antibodies to BPV that were detectable 2 weeks after hatching by agar-gel-immunodiffusion tests.     

Migration and differentiation of gonadal germ cells under cross-sex germline chimeras condition in domestic chickens

A series of experiments was conducted to investigate migration, proliferation and differentiation of gonadal germ cells (GGCs) collected from the gonads of 7-day-old chick embryos under cross-sex germline chimera conditions. The migratory and proliferative abilities of exogenous GGCs were examined by transferring 50 fluorescently labeled GGCs collected from White Leghorn (WL) embryos into the blood of 2-day-old Rhode Island Red (RIR) embryos. No significant difference was observed in the number of fluorescently labeled GGCs in the gonads of recipient embryos among any of the four possible donor and recipient sex combinations. Cross-sex germline chimeras were produced to examine the differentiation of GGCs by transferring 100 GGCs from WL embryos into 2-day-old RIR embryos. Exogenous-GGC-derived progeny were obtained from both male and female recipients, except when female GGCs were transferred into male recipients. The migratory ability of GGCs recovered from the 7-day-old embryonic gonad was not influenced by cross-sex germ cell transfer conditions, whereas the differentiation of the GGCs was affected by the sex combinations of GGCs donors and recipients.     

Generation of Buffalo (Bubalus bubalis) Transgenic Chimeric and Nuclear Transfer Embryos Using Embryonic Germ-Like Cells Expressing Enhanced Green Fluorescent Protein

The possibility of producing transgenic buffalo embryos by chimera and nuclear transfer (NT) using buffalo embryonic germ (EG)‐like cells expressing enhanced green fluorescent protein (EGFP) has been explored in this study. Buffalo EG‐like cells and fibroblasts with two to eight passages were transfected with the lined plasmid (pCE‐EGFP‐IRES‐Neo‐dNdB) using Lipofectamine^TM 2000 and selected by culturing in 200 μg/ml G418 for 6–8 days. G418 resistant fibroblasts and EG‐like cells were used for embryo chimera and NT. To produce blastocysts by chimera, 8–16 cells embryos were injected with EG‐like and fibroblast cells. Then, to produce blastocysts by NT, in vitro maturated oocytes were enucleated and afterwards EG‐like/fibroblast cells transferred into the perivitelline space. No statistical differences were observed for the total blastocyst produced by the chimeric method, using EG‐like and fibroblasts as donor cells, resulting on an accomplishment of 35.6% vs 33.3%, respectively. Nevertheless, besides from the 37 blastocysts produced, 23 (62.2%) from EG‐like cells expressed EGFP, none of blastocysts from foetal fibroblasts expressed this protein. When the NT method was used, no statistical difference among different generations was observed in the percentage of oocytes fused, cleaved, and developed to blastocysts after NT for EG‐like cells. On average, the percentage of oocytes fused, cleaved, and developed to blastocysts after NT was respectively 81.8%, 67.7% and 10.7%. For the expression of EGFP, from the 12 blastocysts produced by NT, 7 of them were positive, while none of NT embryos from EGFP positive fibroblasts developed to blastocysts. Results of the present study clearly demonstrated that gene transfected buffalo EG‐like cells have the ability to form chimeric embryos after injecting into buffalo early embryos and reprogramming ability after NT, which can be employed to produce transgenic buffalos through either embryo chimera or NT.     

禽呼肠病毒琼扩抗原和血清的制备

以禽呼肠病毒S1133毒株尿囊腔接种10日龄无特定病原体(SPF)鸡胚,无菌收集24 h～120 h的死胚尿囊液,加1 mL/L的甲醛灭活,浓缩制得琼扩抗原;用该抗原制成灭活苗两次免疫SPF鸡,无菌采血分离制备阳性血清;选用SPF鸡无菌采血分离制备阴性血清.经过效价测定制备16单位琼扩抗原,8单位阳性血清;取免后的SPF鸡血样4个20～2-5稀释分别与1单位～8单位琼扩抗原做琼扩试验,确定了应该用4单位或8单位琼扩抗原做工作抗原;并以血清中和试验为参照证明琼扩试验在血清抗体监测中的可行性.本研究成功制备了禽呼肠病毒琼扩抗原与血清,为用琼扩试验定量检测禽呼肠病毒感染禽和疫苗免疫禽的抗体奠定了基础.     

生物频谱对家兔胚胎发育影响的初步研究

本文首次研究了周林生物频谱照射对家兔胚胎体外发育的影响，初步探讨了周林生物频谱在胚胎工程中应用的可能性。周林生物频谱分别照射家兔２－细胞胚胎３、５、７、１０、１５、２０、２５分钟，结果照射１０、１５、２０、２５分钟，可使７２小时（从注射ＨＣＧ后算起，即照射后４８小时）桑椹胚发育率显著增加，照射２０和２５分钟可提高７２、８４、９６和１０８小时囊胚发育率，照射１０分钟和１５分钟可提高１０８小时囊胚发育率，照射７、１０、１５、２０和２５分钟可提高９６小时扩张囊胚发育率及１０８小时孵化囊胚发育率。周林生物频谱照射家兔１６－细胞胚胎２０分钟，其６０小时（从注射ＨＣＧ后起，即照射后１２小时）致密桑椹胚发育率与对照比较差异极显著（Ｐ＜０．０１），囊胚、扩张囊胚和孵化囊胚发育率与对照比较无显著差异（Ｐ＞０．０５）     

Production of Wild Buffalo (Bubalus arnee) Embryos by Interspecies Somatic Cell Nuclear Transfer Using Domestic Buffalo (Bubalus bubalis) Oocytes

The objective of this study was to explore the possibility of producing wild buffalo embryos by interspecies somatic cell nuclear transfer (iSCNT) through handmade cloning using wild buffalo somatic cells and domestic buffalo (Bubalus bubalis) oocytes. Somatic cells derived from the ear skin of wild buffalo were found to express vimentin but not keratin and cytokeratin‐18, indicating that they were of fibroblast origin. The population doubling time of skin fibroblasts from wild buffalo was significantly (p < 0.05) higher, and the cell proliferation rate was significantly (p < 0.05) lower compared with that of skin fibroblasts from domestic buffalo. Neither the cleavage (92.6 ± 2.0% vs 92.8 ± 2.0%) nor the blastocyst rate (42.4 ± 2.4% vs 38.7 ± 2.8%) was significantly different between the intraspecies cloned embryos produced using skin fibroblasts from domestic buffalo and interspecies cloned embryos produced using skin fibroblasts from wild buffalo. However, the total cell number (TCN) was significantly (p < 0.05) lower (192.0 ± 25.6 vs 345.7 ± 42.2), and the apoptotic index was significantly (p < 0.05) higher (15.1 ± 3.1 vs 8.0 ± 1.4) for interspecies than that for intraspecies cloned embryos. Following vitrification in open‐pulled straws (OPS) and warming, although the cryosurvival rate of both types of cloned embryos, as indicated by their re‐expansion rate, was not significantly different (34.8 ± 1.5% vs 47.8 ± 7.8), the apoptotic index was significantly (p < 0.05) higher for vitrified–warmed interspecies than that for corresponding intraspecies cloned embryos (48.9 ± 7.2 vs 23.9 ± 2.8). The global level of H3K18ac was significantly (p < 0.05) lower in interspecies cloned embryos than that in intraspecies cloned embryos. The expression level of HDAC1, DNMT3a and CASPASE3 was significantly (p < 0.05) higher, that of P53 was significantly (p < 0.05) lower in interspecies than in intraspecies embryos, whereas that of DNMT1 was similar between the two types of embryos. In conclusion, these results demonstrate that wild buffalo embryos can be produced by iSCNT.     

Production of germ-line chimeras by the transfer of cryopreserved gonadal germ cells collected from 7- and 9-day-old chick embryos

Gonadal germ cells (GGC) were collected from the gonads of 7‐ or 9‐day‐old White Leghorn chick embryos and suspended in freezing medium containing 10% dimethylsulfoxide (DMSO). The cell suspension was frozen at ?1°C/min. until the temperature reached ?80°C. Then, the cells were immersed in liquid nitrogen at ?196°C and stored for 3–4 months. Approximately 50 frozen/thawed GGC were injected into the dorsal aorta of each 2‐day‐old Rhode Island Red (RIR) embryo, from which blood was drawn before germ‐cell injection. The injected embryos were incubated until they hatched and the chicks were raised until sexually mature. On reaching sexual maturity, a progeny test was performed by mating recipient chicks with normal RIR of the opposite sex. Progenies were obtained from male germ cell recipients that were injected with germ cells collected from 7‐ and 9‐day‐old embryos. The results demonstrated that frozen/thawed GGC collected from 7‐ or 9‐day‐old fertilized eggs can be used to produce male germ‐line chimeras.     

鸡胚大头开孔原壳体外培养方法的建立

通过原壳体外培养和抽蛋清的方法,就胚龄、蛋清抽取量对鸡胚发育的影响进行了研究。结果表明:①0日龄鸡胚抽取1mL、3mL和6mL蛋清后,其孵化率分别为38.6%,30.35%和8.6%。抽取蛋清1mL组(P<0.01)和3mL组种蛋(P<0.05)的孵化率明显优于抽取6mL组。②对3日龄鸡胚,抽取9mL蛋清后,鸡胚的发育受到的影响最大,其孵化率仅为33.3%,明显低于3mL和6mL组(P<0.01);而抽取3mL和6mL蛋清对3日龄鸡胚的影响较小,其孵化率仍高达74.3%和76.5%。③抽取6mL蛋清并在大头顶端开直径为1.0cm的圆孔进行鸡胚原壳体外培养获得成功,3日龄鸡胚的孵化率为18.5%。     

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.     

Effect of cryoprotectant composition on in vitro viability of in vitro fertilized and cloned bovine embryos following vitrification and in-straw dilution

In this study the efficacy of the combination of glycerol (GLY) and ethylene glycol (EG) as cryoprotectants in a vitrification method developed for direct embryo transfer was evaluated by in vitro development of in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos after vitrification. The IVF and SCNT blastocysts were vitrified in either 40% GLY, 30% GLY + 10% EG, or 20% GLY + 20% EG using French straws. After warming, the straws were held vertically for 1 min without shaking and were then placed horizontally for 5 min to dilute the cryoprotectants. After washing, the vitrified-warmed embryos were cultured in vitro for 72 h. There were no differences among the vitrification solutions with respect to the rates of vitrified-warmed IVF and SCNT embryos surviving and developing to the hatched blastocyst stage. However, the rates of development to the hatched blastocyst stage of the SCNT embryos vitrified with 40% GLY tended to be higher than those vitrified with 30% GLY + 10% EG or 20% GLY + 20% EG (26% vs. 7-8%, respectively). The development rates to the hatched blastocyst stage of the IVF and SCNT embryos vitrified with solution containing EG were significantly lower (P<0.05) than those of non-vitrified embryos. These results suggest that use of the combination of GLY and EG as cryoprotectants had no beneficial effect on the viability of embryos after in-straw dilution. However, this method is so simple that it can be used for practical direct transfer of vitrified embryos in the field.     

The use of embryonic stem cell derived bioactive material as a new protein supplement for the in vitro culture of bovine embryos

Embryonic stem (ES) cells are expanded versions of the inner cell mass cells that compose the early mammalian blastocyst. Components derived from ES cells may contain various bioactive materials (BM) helpful for early preimplantation embryo growth. In this study, we examined the effect of human ES cell derived BM (hES-BM) on in vitro culture of bovine embryos. When bovine parthenogenetic day 2 embryos were cultured in 10% hES-BM, a significantly higher embryo development rate (44.3%) and increased cell numbers were observed relative to control medium containing 3 mg/ml BSA (19.5%; P<0.01). Among the various concentrations (5, 10 and 15%) and days of treatment (2 or 4 days) tested, 10% hES-BM treatment for 4 days provided the best culture environment to support the growth of bovine embryos in vitro (P<0.05). Little difference was observed between 10% hES-BM and 10% FBS treatment in the examined parthenogenetic or in vitro fertilized embryos, although the hES-BM group developed at a slightly better rate. However, the ICM cell numbers were significantly higher in the hES-BM group in irrespective of embryo origin (P<0.05). In addition, the relative levels of pluripotency (Oct4, × 1.8 fold; Nanog. × 3.3 fold), embryogenesis (Stat3, × 2.8 fold; FGF4, × 18.8 fold; E-cad, × 2.0 fold) and growth (Glut5, × 2.6 fold) genes were significantly higher in the 10% hES-BM group than in the 10% FBS group (P<0.05), while the levels of other genes (Bax, Bcl2, MnSOD and Connexin43) were not different. This is the first report examining the positive effects of hES-BM on bovine embryo development in vitro. Based on our results, we conclude that hES-BM can be used as a new protein supplement for bovine preimplantation embryo development.     

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.     

Timing and uniformity of embryonic gene activation affect subsequent pre-implantation development of cloned bovine embryos

In the present study, we examined the timing of onset, intensity, and mosaicism of embryonic gene expression in bovine nuclear transfer (NT) embryos. The relationship between gene expression and early embryonic development was also examined. To monitor the gene expression of NT embryos, we produced NT embryos with bovine transfected fibroblasts carrying a firefly luciferase gene under the control of a chicken beta-actin promoter, an expression system that has previously been shown to be representative of embryonic gene expression in mice. Photon count imaging showed that luciferase luminescence began in NT embryos with fibroblasts 48 hours post fusion (hpf) and reached a plateau at the 4- to 8-cell stage at 60 hpf. Only 4- to 8-cell NT embryos luminescent by 60 hpf developed to the blastocyst stage. At 60 hpf, strongly luminescent embryos developed to the blastocyst stage at a higher rate (P<0.05) than embryos with weak or absent luminescence. However, embryos with mosaic luminescence developed at a much lower rate (P<0.05) than those with whole-embryo luminescence, even if the embryos exhibited strong luminescence. Our results indicate that precise and uniform embryonic gene expression at the 4- to 8-cell stage at 60 hpf may be closely related to development of bovine NT embryos to the blastocyst stage.     

Comparison of culturing Mycoplasma gallisepticum from fresh eggs and 18-day-old embryos

Twenty-four 70-week-old and sixteen 27-week-old white leghorn hens were challenged with R strain Mycoplasma gallisepticum (MG) by injection into the caudal thoracic air sac and infraorbital sinus. Eggs were collected daily and cultured within 7 days or incubated for 18 days. Vitelline membranes of eggs were cultured directly; in 18-day-old embryos, cultures were taken from the yolk sac, air sacs, and oral cavity. Culture of vitelline membrane of eggs within 2 days was compared with culture of eggs stored 10 days post oviposition. The first MG-positive egg was laid 2 days postinfection (PI). Hens continued to lay positive eggs to the end of the experiments. There was no significant difference in MG recovery between eggs cultured within 2 days and those cultured 10 days post oviposition. MG was isolated at a significantly higher rate from eggs than from 18-day-old embryos. MG was isolated at a higher rate from the yolk sac of 18-day-old embryos than from the air sacs or oral cavity of the same embryos.     

Production of quail-chick chimaeras by blastoderm cell transfer

1. Quail-chick chimaeras were produced by injecting dissociated quail blastoderm cells into chick embryos. 2. Quail blastoderms were removed from the yolk and the cells were dispersed by trypsin treatment or pipetting. The cell suspension (1 to 5 microliters) was injected into the subgerminal cavity of unincubated chick embryos. The chick embryos were then cultured in recipient eggshells. 3. Quail blastoderm cells injected into the chick embryos adhered to the chick embryonic cells. The rates of hatching were 8.6% (38 chicks from 441 eggs) and 40.3% (48 chicks from 119 eggs) when the volumes of the cell suspension injected were 3 to 5 microliters and 1 microliter, respectively. 4. Seven out of 86 hatched birds were clearly identified as being chimaeric because part of the feather colouring was of quail specificity. In addition to these chimaeric birds, there were 8 chimaeric embryos which died before hatching. The distribution patterns of the quail feathers were varied among the chimaeric birds and embryos. 5. This technique provides a basis for the investigation of chick embryo cryopreservation, genetic transformation and analysis of cell lineage of chickens.     

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.     

鸡胚中人工感染网状内皮增殖病病毒的检测

为了模仿禽网状内皮增生病毒(REV)垂直感染鸡胚,在1和3日龄的发育SPF鸡胚人工接种不同剂量的REV,并继续孵化至12日龄.在将发育鸡胚制成成纤维细胞单层后,再用抗REV单克隆抗体11B118作间接荧光抗体反应检测REV.结果表明,从所有人工感染REV的鸡胚12日龄制备的成纤维细胞中均能在培养48h检测出REV.该方法可用于检测生产弱毒疫苗的SPF鸡胚及其相应细胞有无REV的污染.