Optimization of parthenogenetic activation of rabbit oocytes and development of rabbit embryo by somatic cell nuclear transfer

The present study explored a suitable parthenogenetic activation (PA) procedure for rabbit oocytes and investigated the developmental potential of somatic cell nuclear transfer (SCNT) embryos using rabbit foetal fibroblasts (RFFs). The electrical activation had the optimal rate of blastocyst (14.06%) when oocytes were activated by three direct current (DC) pulses (40 V/mm, 20 μs each) followed by 6‐dimethylaminopurine (6‐DMAP) and cycloheximide (CHX) treatment; the blastocyst rate of ionomycin (ION) + 6‐DMAP + CHX (12.07%) activation was higher than that of ION + 6‐DMAP (8.6%) activation or ION + CHX (1.24%) activation; there was no significant difference in blastocyst rate between ION + 6‐DMAP + CHX and DC + 6‐DMAP + CHX groups. The blastocyst rate of ION + 6‐DMAP + CHX‐activated oocytes in the basic rabbit culture medium (M‐199) + 10% foetal bovine serum (FBS; 14.28%) was higher than that in buffalo conditioned medium (5.75%) or G1/G2 medium (0), and the blastocyst rate was increased when M‐199 + 10% FBS was supplemented with amino acids. Refreshing culture medium every day or every other day significantly increased the blastocyst rate. Treatment of donor cells with 0.5% FBS for 3–5 days increased blastocyst rate of SCNT embryos (33.33%) than no serum starvation (22.47%) or 0.5% FBS treatment for 6–9 days (23.61%); the blastocyst rate of SCNT embryos derived from nontransgenic RFFs was higher than that derived from transgenic RFFs by electroporation. The blastocyst development ability of SCNT embryos derived from RFFs by electroporation (32.22%) was higher than that of liposome (19.11%) or calcium phosphate (20.00%) transfection, and only the embryos from electroporation group have the EGFP expression (24.44%). In conclusion, this study for the first time systematically optimized the conditions for yield of rabbit embryo by SCNT.     

Chemical Activation with a Combination of Ionomycin and Dehydroleucodine for Production of Parthenogenetic,ICSI and Cloned Bovine Embryos

The aim of this study was to evaluate the potential of dehydroleucodine (DhL), a new drug isolated from a medicinal herb used in Argentina, for activation of bovine oocyte. Several DhL concentrations and exposure times after ionomycin (Io) treatment were tested. The optimal DhL treatment, found for parthenogenetic development, was employed to produce bovine embryos by intracytoplasmic sperm injection (ICSI) and somatic cell nuclear transfer (SCNT). The best parthenogenic embryo developments were observed with 5 μm Io for 4 min followed by 5 μm DhL concentration and after 3‐h exposure time (52.3% cleavage; 17.4% morulae; 7.3% blastocyst; n = 109). This treatment generated no significant differences with standard Io plus 6‐dimethylaminopurine (DMAP) treatment in preimplantation embryo development. In our conditions, the embryo development reached after ICSI and SCNT assisted by the DhL treatment did not differ in terms of cleavage and blastocyst development from activation with standard Io plus DMAP treatment (p > 0.05). In conclusion, DhL utilization to activate oocytes and induce development of parthenogenotes, ICSI‐embryos or SCNT‐embryos is reported here for first time.     

Activation of Zona‐Free Buffalo (Bubalus bubalis) Oocytes by Chemical or Electrical stimulation,and Subsequent Parthenogenetic Embryo Development

Parthenogenetic activation using zona‐free oocytes offers an alternative model that could be applied to develop protocols for the activation of reconstructed embryos for cloning. The aim of this study was to compare the efficacy of different methods for the activation of zona‐free buffalo oocytes in terms of their effects on the developmental competence of parthenogenetic embryos. The effects of zona removal on parthenogenetic activation and in vitro developmental competence of metaphase II oocytes were also examined. All activation methods were followed by incubation of 2 mm 6‐dimethylaminopurine (6‐DMAP) for 4 h. Out of three different pulse strengths (1.2, 2.1 or 3.3 kV/cm) used, 2.1 kV/cm resulted in the highest blastocyst rate (25.3%). On comparing different chemical agents and electric pulse, highest blastocyst rate was observed for calcium ionophore (CaI) (28.6%) followed by ethanol (25.0%), electric pulse (22.5%) and combined CaI and ethanol treatment (16.7%) although differences among them were not significant. Furthermore, a significantly reduced developmental potential was observed in zona‐free oocytes when compared to zona‐intact ones up to the blastocyst stage (44.3% vs 27.1%). In conclusion, zona‐free buffalo oocytes can be successfully activated for parthenogenetic development using chemical or electrical stimulation. Out of different agents examined, CaI followed by 6‐DMAP resulted in the highest blastocyst rate.     

Effect of Pre‐Treatment of Ovine Sperm on Male Pronuclear Formation and Subsequent Embryo Development Following Intracytoplasmic Sperm Injection

The objective of this study was to determine the effects of various methods of sperm pre‐treatment on male pronuclear (MPN) formation and subsequent development of ovine embryos derived from in vitro‐matured oocytes and intracytoplasmic sperm injection (ICSI). The effect of treatment of injected oocytes with dithiothreitol (DTT) on embryo development was also assessed. In Exp. 1, the injected oocytes with non‐treated sperm were activated with three different procedures. The cleavage and blastocyst rates in those activated with DTT was lower (p < 0.05) than those activated with either ionomycin (Io) + 6‐dimethylaminopurine (6‐DMAP) or DTT + I + 6‐DMAP. In Exp. 2, the effects of sperm pre‐incubated with DTT, sodium dodecyl sulphate (SDS) or DTT + SDS as well as two‐time frozen/thawed sperm (without cryoprotectant) on MPN formation and oocyte activation were examined. The non‐treated sperm served as controls. The MPN formation in DTT + SDS group was higher (p < 0.05) than other groups except for freeze–thaw group. No difference in the rate of activated ICSI oocytes was observed among groups. In Exp. 3, the effect of pre‐treatment of sperm on subsequent development of ICSI embryos and blastocyst cell numbers were examined. The rates of cleavage and blastocyst formation as well as the blastocyst cell numbers were similar among the pre‐treated and control groups. In conclusion, pre‐treatment of sperm with DTT + SDS positively affected MPN formation, although the subsequent development capacity of the resulting embryos remained limited. Moreover, DTT was not effective on oocyte activation compared with Io + 6‐DMAP after ICSI.     

An intracytoplasmic injection of deionized bovine serum albumin immediately after somatic cell nuclear transfer enhances full-term development of cloned mouse embryos

In mouse somatic cell nuclear transfer (SCNT), polyvinylpyrrolidone (PVP) is typically included in the nuclear donor injection medium. However, the cytotoxicity of PVP, which is injected into the cytoplasm of oocytes, has recently become a cause of concern. In the present study, we determined whether bovine serum albumin deionized with an ion-exchange resin treatment (d-BSA) was applicable to the nuclear donor injection medium in SCNT as an alternative to PVP. The results obtained showed that d-BSA introduced into the cytoplasm of an enucleated oocyte together with a donor nucleus significantly enhanced the rate of in vitro development of cloned embryos to the blastocyst stage compared with that of a conventional nuclear injection with PVP in SCNT. We also defined the enhancing effects of d-BSA on the blastocyst formation rate when d-BSA was injected into the cytoplasm of oocytes reconstructed using the fusion method with a hemagglutinating virus of Japan envelope before oocyte activation. Furthermore, immunofluorescence experiments revealed that the injected d-BSA increased the acetylation levels of histone H3 lysine 9 and histone H4 lysine 12 in cloned pronuclear (PN) and 2-cell embryos. The injection of d-BSA before oocyte activation also increased the production of cloned mouse offspring. These results suggested that intracytoplasmic injection of d-BSA into SCNT oocytes before oocyte activation was beneficial for enhancing the in vitro and in vivo development of mouse cloned embryos through epigenetic modifications to nuclear reprogramming.     

Effect of synchronization of donor cells in early G1‐phase using shake‐off method on developmental potential of somatic cell nuclear transfer embryos in cattle

In this study, we compared the developmental ability of somatic cell nuclear transfer (SCNT) embryos reconstructed with three bovine somatic cells that had been synchronized in G0‐phase (G0‐SCNT group) or early G1‐phase (eG1‐SCNT group). Furthermore, we investigated the production efficiency of cloned offspring for NT embryos derived from these donor cells. The G0‐phase and eG1‐phase cells were synchronized, respectively, using serum starvation and antimitotic reagent treatment combined with shaking of the plate containing the cells (shake‐off method). The fusion rate in the G0‐SCNT groups (64.2 ± 1.8%) was significantly higher than that of eG1‐SCNT groups (39.2 ± 1.9%) (P < 0.05), but the developmental rates to the blastocyst stage of SCNT embryos per fused oocytes were similar for all groups. The overall production efficiency of the clone offspring in eG1‐SCNT groups (12.7%) per recipient cow was higher than that in G0‐SCNT groups (3%) (P < 0.05). The mean birth weight of cloned calves and the average calving score in the G0‐SCNT groups (48.1 ± 3.4 kg and 3.3 ± 0.3, respectively) was significantly higher (P < 0.05) than those of eG1‐SCNT groups (37.2 ± 2.1 kg and 2.3 ± 0.2, respectively). Results of this study indicate that synchronization of donor cells in eG1‐phase using the shake‐off method improved the overall production efficiency of the clone offspring per transferred embryo.     

Comparative study of the developmental competence of cloned pig embryos derived from spermatogonial stem cells and fetal fibroblasts

Spermatogonial stem cells (SSC) are promising resources for genetic preservation and restoration of male germ cells in humans and animals. However, no studies have used SSC as donor nuclei in pig somatic cell nuclear transfer (SCNT). This study investigated the potential for use of porcine SSC as a nuclei donor for SCNT and developmental competence of SSC‐derived cloned embryos. In addition, demecolcine was investigated to determine whether it could prevent rupture of SSC during SCNT. When the potential of SSC to support embryonic development after SCNT was compared with that of foetal fibroblasts (FF), SSC‐derived SCNT embryos showed a higher (p < .05) developmental competence to the blastocyst stage (47.8%) than FF‐derived embryos (25.6%). However, when SSC were used as donor nuclei in the SCNT process, cell fusion rates were lower (p < .05) than when FF were used (61.9% vs. 75.8%). Treatment of SSC with demecolcine significantly (p < .05) decreased rupture of SSC during the SCNT procedure (7.5% vs. 18.8%) and increased fusion of cell‐oocyte couplets compared with no treatment (74.6% vs. 61.6%). In addition, SSC‐derived SCNT embryos showed higher blastocyst formation (48.4%) than FF‐derived embryos without (28.4%) and with demecolcine treatment (17.4%), even after demecolcine treatment. Our results demonstrate that porcine SSC are a desirable donor cell type for production of SCNT pig embryos and that demecolcine increases production efficiency of cloned embryos by inhibiting rupture of nuclei donor SSC during SCNT.     

Propagation of elite rescue dogs by somatic cell nuclear transfer

The objective of the present study was to compare the efficiency of two oocyte activation culture media to produce cloned dogs from an elite rescue dog and to analyze their behavioral tendencies. In somatic cell nuclear transfer procedure, fused couplets were activated by calcium ionophore treatment for 4 min, cultured in two media: modified synthetic oviduct fluid (mSOF) with 1.9 mmol/L 6‐dimethylaminopyridine (DMAP) (SOF‐DMAP) or porcine zygote medium (PZM‐5) with 1.9 mmol/L DMAP (PZM‐DMAP) for 4 h, and then were transferred into recipients. After embryo transfer, pregnancy was detected in one out of three surrogate mothers that received cloned embryos from the PZM‐DMAP group (33.3%), and one pregnancy (25%) was detected in four surrogate mothers receiving cloned embryos from the SOF‐DMAP group. Each pregnant dog gave birth to one healthy cloned puppy by cesarean section. We conducted the puppy aptitude test with two cloned puppies; the two cloned puppies were classified as the same type, accepting humans and leaders easily. The present study indicated that the type of medium used in 6‐DMAP culture did not increase in cloning efficiency and dogs cloned using donor cells derived from one elite dog have similar behavioral tendencies.     

Effect of Transgene Introduction and Recloning on Efficiency of Porcine Transgenic Cloned Embryo Production In Vitro

Retrovirus-mediated exogenous gene transfection of somatic cells is an efficient method to produce transgenic embryos by somatic cell nuclear transfer (SCNT). This study evaluated whether efficiency of transgenic embryos production, by SCNT using fibroblast cells transfected by retrovirus vector, is influenced by the introduced transgene and whether recloning could further improve its efficiency. Transgenic cloned embryos were produced by SCNT of porcine foetal fibroblast cells transfected by either LNβ-Z or LNβ-enhanced green fluorescent protein (EGFP) retrovirus vector and evaluated for their developmental ability in vitro . Blastomeres from four-cell stage porcine embryos, produced by SCNT of foetal fibroblast cells transfected with LNβ-EGFP retroviral vector, were subsequently recloned into enucleated metaphase II oocytes and evaluated for changes in chromatin configuration, in vitro embryo development and gene expression. Analysis of results showed that cleavage and blastocyst rates of porcine SCNT embryos, using LacZ (53.6 ± 6.4%; 12.0 ± 5.7%) or EGFP (57.5 ± 6.3%; 10.1 ± 4.1%) transfected fibroblasts, did not differ (p > 0.05) from those of non-transfected controls (60.9 ± 8.2%; 12.3 ± 4.0%). Recloning of blastomeres did not further improve the in vitro development rate. Interestingly, the nuclei of blastomere underwent slower remodelling process than somatic cell nuclei. Both cloned and recloned embryos showed 100% transgene expression and there were no evidence of mosaicism. In conclusion, our data shows that the efficiency of transgenic cloned embryos production by SCNT of somatic cells transfected with replication-defective retrovirus vector is not influenced by the transgene introduction into donor cells and recloning of four-cell stage blastomere could not further improve its efficiency.     

Stage‐specific effects of osmolarity of a culture medium on development of pig oocytes and miniature pig somatic cell nuclear transfer embryos activated by ultrasound treatment

Whether high osmolarity of a culture medium at the early culture stage affects the development of pig oocytes and miniature pig somatic cell nuclear transfer (SCNT) embryos activated by ultrasound was examined. When oocytes were cultured in modified porcine zygote medium‐3 (mPZM‐3) with increased NaCl to 138 mmol/L (mPZM‐3+NaCl; 326 mOsm) or 50 mmol/L sucrose (mPZM‐3+sucrose; 318 mOsm) for the first 2 days and then cultured in normal mPZM‐3 (273 mOsm) for 5 days, the cleavage and blastocyst formation rates were significantly (P < 0.05) higher than those of oocytes cultured in mPZM‐3 for 7 days. The cleavage and blastocyst formation rates of SCNT embryos cultured in mPZM‐3+NaCl for the first 2 days and then cultured in mPZM‐3 for 5 days were also significantly (P < 0.05) higher than those of embryos cultured in mPZM‐3 for 7 days. These results showed that the high osmolarity of a culture medium induced by increasing NaCl concentration during the first 2 days improves the development of pig oocytes and miniature pig SCNT embryos activated by ultrasound.     

Effects of Chemical Activation Treatment on Development of Swamp Buffalo (Bubalus bubalis) Oocytes Matured In Vitro and Fertilized by Intracytoplasmic Sperm Injection

The objective of this study was to optimize the activation protocol for buffalo oocytes after intracytoplasmic sperm injection (ICSI). The release of the second polar body (PB) at 3, 6 and 9 h after ICSI of in‐vitro matured oocytes activated either with 5 μm ionomycin (Io) or with 7% ethanol (EtOH) was preliminary examined. The highest rate of second PB extrusion occurred at 3 h of activation, and the second PB extrusion in EtOH group was significantly higher than that in Io group. Oocytes that extruded the second PB were selected and cultured either with 1.9 mm 6‐dimethylaminopurine (6‐DMAP) for 3 h or with 10 μg/ml cycloheximide (CHX) for 5 h. Significantly higher rate of oocytes formed 2 pronuclei in EtOH combined with CHX (EtOH + CHX) (62%) group compared to those of Io + CHX (42%) and EtOH + 6‐DMAP (48%) groups (p < 0.01) whereas Io + 6‐DMAP group showed intermediate value (58%). Significantly higher blastocyst formation rates were obtained in Io + 6‐DMAP (29%) and EtOH + CHX (24%) groups than in Io + CHX (6%) and EtOH + 6‐DMAP (17%) groups. Our results indicate that buffalo ICSI oocytes are effectively activated by combination treatment of Io with 6‐DMAP and EtOH with CHX resulting in the highest cleavage and blastocyst formation rates.     

Effects of cumulus cells on in vitro maturation of oocytes and development of cloned embryos in the pig

The purpose of this study was to investigate the role of porcine cumulus cells (CC) in oocyte maturation and somatic cell nuclear transfer (SCNT) embryo development in vitro. Denuded pig oocytes were co-cultured with CC or routinely cultured in maturation medium without a feeder layer. Porcine CC inactivated with mitomycin C or non-inactivated were used for the feeder layer in co-culture with porcine SCNT embryos to investigate comparatively the developmental competence of cloned embryos. The DNA damage aspects of apoptosis and expression pattern of genes implicated in apoptosis (Fas/FasL) as well as the mRNA expression of DNA methyltransferase (Dnmt1, Dnmt3a) of porcine SCNT embryos were also evaluated by comet assay or real-time RT-PCR, respectively. The results showed that co-culture with CC improved the extrusion rate of pbI (49.3% vs 31.5%, p<0.05) and survival rate (75.7% vs 53.3%, p<0.05) of denuded oocytes, but had no effects on blastocyst developmental rate or 2-cell-stage survival rate of in vitro fertilization embryos. Co-culture with CC inactivated by mitomycin C improved the blastocyst developmental rate (26.6% vs 13.0%, p<0.05) and decreased the apoptotic incidence (27.6% vs 46.2%, p<0.05) of porcine cloned embryos. Co-culture with inactivated CC reduced Fas and FasL mRNA expression of cloned embryos at the blastocyst stage compared with NT controls (p<0.05), but there were no differences in Dnmt1 and Dnmt3a mRNA expression among groups. Co-culture with inactivated cumulus cell monolayer significantly increased blastocyst formation and decreased the apoptotic incidence in porcine cloned embryos during in vitro development.     

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.     

DNA methylation analysis on satellite I region in blastocysts obtained from somatic cell cloned cattle

Many observations have been made on cloned embryos and on adult clones by somatic cell nuclear transfer (SCNT), but it is still unclear whether the progeny of cloned animals is presenting normal epigenetic status. Here, in order to accumulate the information for evaluating the normality of cloned cattle, we analyzed the DNA methylation status on satellite I region in blastocysts obtained from cloned cattle. Embryos were produced by artificial insemination (AI) to non‐cloned or cloned dams using semen from non‐cloned or cloned sires. After 7 days of AI, embryos at blastocyst stage were collected by uterine flushing. The DNA methylation levels in embryos obtained by using semen and/or oocytes from cloned cattle were similar to those in in vivo embryos from non‐cloned cattle. In contrast, the DNA methylation levels in SCNT embryos were significantly higher (P < 0.01) than those in in vivo embryos from non‐cloned and cloned cattle, approximately similar to those in somatic cells used as donor cells. Thus, this study provides useful information that epigenetic status may be normal in the progeny of cloned cattle, suggesting the normality of germline cells in cloned cattle.     

影响猪ICSI转基因技术效率的主要因素研究

以猪体外成熟卵子和冷冻解冻的死精子为材料,以pEGFP-N1为模式基因,探讨注射台温度、激活后6-DMAP的处理和精子与PEGFP-N1孵育液添加BSA(牛血清白蛋白)对精子胞质内注射(ICSI)转基因效率的影响。结果表明:注射台温度为30℃时的阳性率为40.07%,而38.5℃时为20.97%,差异极显著(P<0.01)。添加BSA的囊胚转基因率为55.56%,对照组为33.33%,差异极显著(P<0.01)。6-DMAP处理组与对照组的转基因率分别为52.53%和26.25%,差异极显著(P<0.01);而且6-DMAP处理组的囊胚率(9.96%)显著高于(P<0.05)对照组(2.30%)。研究表明注射台温度对转基因效率有明显影响,温度高转基因率低;精子与PEGFP-N1孵育液添加BSA对转基因胚胎发育有一定促进和保护作用,有利于提高囊胚转基因率;激活后用6-DMAP处理能提高转基因率和囊胚率。     

Cloned porcine embryos can maintain developmental ability after cryopreservation at the morula stage

The aim of the present study was to clarify the overall efficiency of porcine somatic cell nuclear transfer (SCNT) by incorporating cryopreservation of the cloned embryos before transfer. The SCNT embryos reconstructed with preadipocytes and in vitro-matured (IVM) oocytes were cultured to harvest morula stage embryos; they were then subjected to delipation (removal of cytoplasmic lipid droplets) and vitrification. After warming and culture, the embryos developing to blastocysts were transferred to recipients to obtain cloned piglets. From 372 reconstructed embryos, 188 (50.5%) reached the morula stage and 117 (31.5%) developed to blastocysts after vitrification. Transfer of 98 (26.3%) morphologically normal blastocysts gave rise to 6 (1.6%) piglets, including 1 stillborn. The efficiency of the cloned piglet production was comparable with that obtained using SCNT embryos without cryopreservation (2.7%, 17/635). Here, we demonstrate that porcine somatic cell cloning can be performed without a significant reduction in efficiency even when the SCNT embryos are cryopreserved before transfer.     

Effects of trichostatin A on in vitro development and transgene function in somatic cell nuclear transfer embryos derived from transgenic Clawn miniature pig cells

The present study was carried out to examine the effects of post‐activation treatment of trichostatin A (TSA), a histone deacetylase inhibitor, on in vitro development and transgene function of somatic cell nuclear transfer (SCNT) embryos derived from Clawn miniature pig embryonic fibroblast (PEF) transfected with a bacterial endo‐β‐galactosidase C gene (removal of the α‐galactosyl (Gal) epitope). SCNT embryos were incubated with or without TSA (50 or 100 nmol/L) after activation, cultured in vitro and assessed for cleavage, blastocyst formation and transgene function. The rate of blastocyst formation was significantly higher in SCNT embryos treated with 50 nmol/L TSA than that in control (P < 0.05), whereas the rate of cleavage and cell number of blastocyst did not differ. Following labelling with fluorescein isothiocyanate‐labelled BS‐I‐B₄ isolectin, the intensity of fluorescence observed on cell‐surface was dramatically reduced in transgenic SCNT blastocyst in comparison with non‐transgenic SCNT blastocyst. However, the reduction of α‐Gal epitope expression in transgenic SCNT blastocyst was not affected by TSA treatment. The results of this study showed that post‐activation treatment with 50 nmol/L TSA is effective to improve in vitro developmental capacity of transgenic SCNT miniature pig embryos without the modification of transgene function.     

Efficiency of Two Enucleation Methods Connected to Handmade Cloning to Produce Transgenic Porcine Embryos

The purpose of our work was to establish an efficient-oriented enucleation method to produce transgenic embryos with handmade cloning (HMC). After 41–42 h oocytes maturation, the oocytes were further cultured with or without 0.4 μg/ml demecolcine for 45 min [chemically assisted handmade enucleation (CAHE) group vs polar body (PB) oriented handmade enucleation (OHE) group respectively]. After removal of the cumulus cells and partial digestion of the zona pellucida, oocytes with visible extrusion cones and/or polar bodies attached to the surface were subjected to oriented bisection. Putative cytoplasts without extrusion cones or PB were selected as recipients. Two cytoplasts were electrofused with one transgenic fibroblasts expressing green fluorescent protein (GFP), while non-transgenic fibroblasts were used as controls. Reconstructed embryos were cultured in Well of Wells (WOWs) with porcine zygote medium 3 (PZM-3) after activation. Cleavage and blastocyst rates were registered on day 2 and day 7 of in vitro culture respectively. Meanwhile, the total blastocyst cell number was counted on day 7. We found that the difference was only observed between blastocyst rates (38.6 ± 2% vs 48.1 ± 3%) of cloned embryos with GFP transgenic fibroblast cells after CAHE vs OHE. With adjusted time-lapse for zonae-free cloned embryos cultured in WOWs with PZM-3, it was obvious that in vitro developmental competence after CAHE was compromised when compared with the OHE method. OHE enucleation method seems to be a potential superior alternative method used for somatic cell nuclear transfer (SCNT) with transgenic fibroblast cells.     

Effect of mSOF and G1.1/G2.2 Media on the Developmental Competence of SCNT‐Derived Bovine Embryos

The objective of this study was to compare the effect of two culture media: modified synthetic oviductal fluid (mSOF) and G1.2/G2.2, on the developmental competence of bovine somatic cell–cloned embryos. Cloned embryos were produced by transferring adult skin fibroblasts into enucleated MII oocytes. After activation, the reconstructed embryos were randomly allotted to either mSOF or G1.2/G2.2 for culture (the embryos were transferred from G1.2 to G2.2 on days 3 of culture). The development competence of cloned embryos in these two culture systems was compared in terms of cleavage rate, blastocyst formation rate and apoptosis cell number in day 7 blastocyts. To investigate the in vivo developmental competence of cloned embryos in the two culture systems, a total of 87 and 104 blastocysts derived from mSOF and G1.2/G2.2 medium groups were transferred individually to recipient Angus cows, respectively. No differences were observed in terms of cleavage rate, day 7 blastocyst rate and blastocyst cell number between these two culture systems. However, the day 6 blastocyst formation rate was significantly higher in G1.2/G2.2 than that in mSOF. In addition, blastocysts cultured in mSOF have a higher percentage of apoptotic blastomeres compared to those in G1.2/G2.2 (8.5 ± 1.2 vs 16.8 ± 1.5, p < 0.05). Although difference in pregnancy rate was not observed 40 days after embryo transfer, significantly higher pregnancy rate was observed in G1.2/G2.2 group after 90 days of embryo transfer (12.4% vs 37.5%, p < 0.05). Moreover, calving rate was significantly improved in G1.2/G2.2 group compared to mSOF group (27.9% vs 6.7%, p < 0.05). In conclusion, our results indicate that G1.2/G2.2 can improve developmental competence of bovine SCNT embryos both in vitro and in vivo, which is more suitable for culture of bovine SCNT embryos than mSOF medium.     

Effect of activation methods for bovine oocytes after intracytoplasmic injection

A principal nuclear transfer procedure is to inject a donor cell into the perivitelline space in an enucleated oocyte and then electric fusion is performed (cell fusion method). The effects of activation methods in reconstructed oocytes for the serum-starved somatic cell cloning procedure were investigated in this study by means of intracytoplasmic injection (i.c.i.). Bovine oocytes were enucleated at 18-22 h for in vitro maturation, and subsequently the nucleus of cumulus cell collected from Japanese Black Bulls (JBCC) after 5-7 days of starved culture was injected into the recipient cytoplast with a piezo-micromanipulator. At 1 h after i.c.i., reconstructed oocytes were stimulated with ethanol (ET) or calcium ionophore (CaI) as the first activation treatment, followed by cycloheximide (CHX) or 6-dimethylaminopurin (DMAP) treatment as the second activation. In the experiment on the first activation method, the proportion of reconstructed oocytes developing to the blastocyst stage was significantly (p<0.01) higher in the ET activation method than that with CaI (10.5% and 4.7%, respectively). And the experiment on the second activation method after ET treatment showed similar proportions of blastocyst development in both CHX and DMAP treatments (5.9% and 2.8%, respectively). The present results indicated that combined activation treatment with ET and CHX was efficient for reconstructed bovine oocytes by i.c.i.