Goat Somatic Cell Nuclear Transfer Research Articles

Effects of Crotonylation on Reprogramming of Cashmere Goat Somatic Cells with Different Differentiation Degrees.

Simple SummaryCurrently, not enough is known about the effect of histone modification on the epigenetic reprogramming of somatic cells, and the lack of basic study limits the development of somatic cell nuclear transfer technology. The aim of this study was to explore the influence of lysine crotonylation, a newly discovered histone post-translational modification, on the reprogramming of somatic cells from Cashmere goats. The results showed that the crotonylation level was increased in somatic cells with sodium crotonate treatment. At the same time, the treatment of somatic cells improved the cloned embryo cleavage rate. In conclusion, an increasing crotonylation level could promote the reprogramming of somatic cells and cloned embryo development. This finding provides an important reference for future improvements in the efficiency of in vitro Cashmere goat somatic cell nuclear transfer embryo production.Failure in the epigenetic reprogramming of somatic cells is considered the main reason for lower cloned embryo development efficiency. Lysine crotonylation (Kcr) occupies an important position in epigenetic modification, while its effects on somatic cell reprogramming have not been reported. In this study, we detected the influence of sodium crotonate (NaCr) on the Kcr levels in three types of somatic cells (muscle-derived satellite cells, MDSCs; fetal fibroblast cells, FFCs; and ear tip fibroblast cells, EFCs). The three types of somatic cells were treated with NaCr for cloned embryo construction, and the cleavage rates and Kcr, H3K9cr, and H3K18cr levels in the cloned embryos were analyzed. The results showed that the abnormal levels of Kcr, H3K9cr, and H3K18cr were corrected in the treatment groups. Although there was no significant difference in the cloned embryo cleavage rate in the FFC treatment group, the cleavage rates of the cloned embryos in the MDSCs and EFCs treatment groups were increased. These findings demonstrated that the Kcr level was increased with NaCr treatment in somatic cells from Cashmere goat, which contributed to proper reprogramming. The reprogramming of somatic cells can be promoted and cloned embryo development can be improved through the treatment of somatic cells with NaCr.

Long non-coding RNA lnc_3712 impedes nuclear reprogramming via repressing Kdm5b

Long non-coding RNAs (lncRNAs) are involved in shaping chromosome conformation and regulation of preimplantation development. However, the role of lncRNA during somatic cell nuclear transfer (SCNT) reprogramming remains largely unknown. In the present study, we identified 114 upregulated lncRNAs in the 8-cell SCNT embryos as candidate key molecules involved in nuclear reprogramming in goat. We found that H3K4me3 was an epigenetic barrier in goat nuclear reprogramming that and injection of Kdm5b mRNA greatly improved SCNT embryos development through removal of H3K4me3. We further reported that knockdown of lnc_3712 increased the expression of Kdm5b, which led to H3K4me3 demethylation. Of note, the development of goat SCNT embryos was improved when lnc_3712 was knocked down, whereas the blastocyst rate showed no difference in lnc_3712 and Kdm5b double knockdown SCNT embryos compared with the negative control SCNT embryos. Specifically, in lnc_3712 knockdown SCNT embryos, partial of the transcriptional activity and the expression of critical embryonic genes (Wee1, Ctsb, and Ybx1) were similar with that of in vitro fertilization embryos. Therefore, our results elucidate the critical role of lnc_3712 in regulating the development of goat SCNT embryos via repressing Kdm5b, which advances our current understanding of the role of lncRNAs during nuclear reprogramming.

Aberrant DNA and histone methylation during zygotic genome activation in goat cloned embryos

In somatic cell nuclear transfer (SCNT) embryos, developmental defects first appear at the time of zygotic genome activation (ZGA), a process that is under the control of DNA and histone methylation. However, dynamics of 5-mC and 5-hmC during ZGA differ between porcine and bovine SCNT embryos, and histone methylation during ZGA in goat SCNT embryos remains poorly understood. Therefore, in the present study, we investigated the dynamic changes of 5-mC, 5-hmC, H3K4me2/3, and H3K9me3, as well as the expression of key genes related to these epigenetic modifications, during ZGA in goat cloned embryos. Compared with the IVF embryos, the 5-mC signal intensity was significantly increased at the 2- and 4-cell stage SCNT embryos, and the H3K4me3 and H3K9me3 signal intensity was significantly increased at 2- to 8-cell stage SCNT embryos, while the 5-hmC and H3K4me2 signal intensity was significantly lower at the 4- and 8-cell stage SCNT embryos. Of note, the H3K9me3 level was also significantly higher, whereas H3K4me3 signal intensity showed no statistical difference in the pronuclear stage SCNT embryos. Moreover, the expression of TET2, DNMT3B, KDM4A, SUV39H1, G9A, and SETDB1 was significantly increased, while the expression of UHRF1, PCNA, KDM4B, KDM4D, KDM5A, KDM5B, and KDM5C was significantly decreased at the 8-cell stage SCNT embryos. Our data revealed aberrant DNA and histone methylation during ZGA in goat cloned embryos. We further inferred that the abnormally higher level of 5-mC, H3K4me3, and H3K9me3 might serve as epigenetic barriers of the reprogramming and modifying these aberrant modifications might be a promising strategy to improve cloning efficiency in goat.

Dynamic changes in chromatin and microtubules at the first cell cycle in SCNT or IVF goat embryos.

We investigated the dynamic changes in chromatin and microtubules at the first cell cycle in goat somatic cell nuclear transfer (SCNT)-derived and in vitro fertilization (IVF)-derived embryos. Stage-dependent and characteristic changes to chromatin and microtubules occurred in SCNT-derived embryos at different times after activation. About half donor nuclei underwent premature chromosome condensation (PCC) at 1 h post activation, and furtherly reached telophase at 2 h after activation. However, we discovered that the separated chromosomes reaggregated, not keeping two independent nuclei; and formed one pronucleus at 2.5 h after activation. One pronucleus was found in all reconstructed oocytes except other no nucleus oocytes from 3 to 22 h after activation. Reconstructed oocytes reached the first mitotic metaphase at 23 h post activation, which was later than that of IVF-derived embryos at 16 h after insemination. SCNT-derived embryos showed significantly higher abnormalities in the first mitotic metaphase spindle, compared with IVF-derived embryos. Abnormal spindles included multi polar and half spindles. SCNT-derived embryos began to cleave at 24 h after activation, which was later than that of IVF-derived embryos at 21 h after insemination. SCNT-derived embryos showed delayed conversion from telophase to interphase than IVF-derived embryos during cleavage. These might lead to poor development in SCNT-derived embryos.

Overexpression of Tet3 in donor cells enhances goat somatic cell nuclear transfer efficiency.

Ten-eleven translocation 3 (TET3) mediates active DNA demethylation of paternal genomes during mouse embryonic development. However, the mechanism of DNA demethylation in goat embryos remains unknown. In addition, aberrant DNA methylation reprogramming prevalently occurs in embryos cloned by somatic cell nuclear transfer (SCNT). In this study, we reported that TET3 is a key factor in DNA demethylation in goat pre-implantation embryos. Knockdown of Tet3 hindered DNA demethylation at the two- to four-cell stage in goat embryos and decreased Nanog expression in blastocysts. Overexpression of Tet3 in somatic cells can initiate DNA demethylation, reduce 5-methylcytosine level, increase 5-hydroxymethylcytosine level and promote the expression of key pluripotency genes. After SCNT, overexpression of Tet3 in donor cells corrected abnormal DNA hypermethylation of cloned embryos and significantly enhanced invitro and invivo developmental rate (P<0.05). We conclude that overexpression of Tet3 in donor cells significantly improves goat SCNT efficiency.

Treating donor cells with 2-PCPA corrects aberrant histone H3K4 dimethylation and improves cloned goat embryo development

ABSTRACTEpigenetic modifications extensively occur in mammalian embryonic development and cell differentiation process. They play an essential role in the reprogramming of nuclei during somatic cell nuclear transfer (SCNT) and subsequent in vitro embryonic development. Recently, SCNT embryos have been verified to contain a subnormal level of histone H3K4 dimethylation (H3K4me2) in contrast to in vitro fertilized embryos. This finding suggested that increasing H3K4me2 levels may ameliorate the aberrant development of cloned embryos. In this study, we investigated the influence of treating donor cells with trans-2-Phenylcyclopropylamine (2-PCPA), a specific inhibitor of lysine-specific demethylase 1 (LSD1), on embryogenesis, H3K4me2 level, and gene expression in cloned goat embryos. Treated goat fetal fibroblast cells (GFFs) with 2-PCPA served as donor cells for subsequent SCNT. Results showed that H3K4me2 levels in treated GFFs increased gradually with the increasing 2-PCPA concentration (p < 0.05) and had no obvious influence in cell viability. The 2-PCPA-induced up-regulation of H3K4me2 levels led to G0/G1 cell cycle arrest and the difference was significant at 2μM compared with the control group (p < 0.05). Interestingly, the development rate of goat SCNT embryos in vitro was significantly improved and aberrant H3K4me2 levels were effectively corrected in 2-PCPA-treated SCNT embryos in contrast to that in SCNT control embryos. Moreover, 2-PCPA treatment promoted the mRNA expression of key developmental genes Oct4 and Sox2 (p < 0.05) without affecting the expression levels of imprinted genes IGF2R and H19 in goat SCNT embryos. These results indicated that abnormal H3K4me2 status can be corrected and SCNT embryo development can be promoted through treatment of donor cells with 2-PCPA.Abbreviations: SCNT: somatic cell nuclear transfer; H3K4me2: H3K4 dimethylation; 2-PCPA: trans-2-Phenylcyclopropylamine; LSD1: lysine-specific demethylase 1; GFFs: goat fetal fibroblast cells; IVF: in vitro fertilization; iPS: induced pluripotent stem; PBS: phosphate-buffered saline; IVM: in vitro maturation; RNAPII: RNA polymerase II; HMTs: histone methyltransferase

Treatment donor cells with UNC0638 modify the abnormal histone H3K9 dimethylation and gene expression in cloned goat embryos

Epigenetic modifications are considered crucial to the reprogramming of somatic cell nuclear transfer (SCNT) embryos and subsequent in vitro development. The abnormal histone H3K9 dimethylation (H3K9me2) status has recently been reported in SCNT embryos. The present study was designed to evaluate the effect of treatment donor cells with UNC0638, a specific inhibitor of H3K9 methyltransferase G9A, on in vitro development, H3K9me2 levels and gene expression in goat SCNT embryos. Different concentration of UNC0638 was applied to treat goat fetal fibroblasts (GFFs), and the treated GFFs were used as donor cells for SCNT. The results showed that 0.2μM UNC0638 decreased significantly the level of H3K9me2 in treated GFFs (P<0.05), and did not influence the cell viability (P>0.05). Compared with intracytoplasmic sperm injection (ICSI) embryos, SCNT embryos derived from untreated GFFs (C-SCNT) presented higher level of H3K9me2 (P<0.05). Although the in vitro developmental rate was not improved, the abnormal H3K9me2 level was corrected in SCNT embryos from UNC0638-treated GFFs (T-SCNT) compared with C-SCNT embryos. Furthermore, UNC0638 treatment could promote the mRNA expression of key developmental genes Nanog and Oct4 (P<0.05), but did not affect the imprinted genes H19 and IGF2R expression in goat SCNT embryos (P>0.05). In conclusion, these results indicated that treatment donor cells with UNC0638 may have beneficial effects in terms of modifying the abnormal H3K9me2 status and gene expression in cloned goat embryos.

36 SERIAL SOMATIC CELL NUCLEAR TRANSFER INCREASES PREGNANCY LOSSES IN GOATS

Serial cloning by somatic cell nuclear transfer (SCNT) has been successful in several mammalian species. This method can be beneficial for transgenic line expansion or resetting the lifespan of transgenic cells. Previous studies in bovine and porcine have shown a decrease in efficiency over multiple iterations of serial cloning. However, the contradictory data has been reported in mice where no decrease in cloning efficiency was observed after 25 generations of recloning. To our knowledge, no data have been reported investigating the efficiency of serial cloning in goats. The aim of this study was to evaluate whether there is an effect of recloning on goat SCNT efficiency. αMHC-TGF-β1 fetal fibroblast cells (containing transforming growth factor-β under control of a cardiac-specific promoter) were produced by electroporation and used for the first round of SCNT. For serial cloning, we used neonatal fibroblast cells obtained from skin biopsies used as nuclear donors. These cells were collected from the transgenic cloned goats generated by the first round of SCNT. Cumulus-oocyte complexes recovered from abattoir-derived ovaries using slicing technique were matured in vitro for 20 to 24 h. The first polar body and metaphase plate were removed from a cumulus cell-free oocyte, and a donor fibroblast cell was subsequently transferred into the enucleated oocyte. Fused embryos were then activated for 5 min in 5 mM ionomycin followed by 4 h in 2 mM DMAP with 5 mg mL−1 cycloheximide. Activated embryos were cultured in G1 medium with 5 mg mL−1 BSA for 12 h, followed by surgical transfer into the oviducts of recipients synchronized to show oestrus within 12 h of SCNT. In total, 592 and 395 embryos were transferred to 37 and 25 recipient goats, respectively, for the first and second round of SCNT. Pregnancy rate, rate of pregnancy loss, and term rate were analysed by Chi-squared with a 2-tailed P-value. No significant difference was observed in Day 40 pregnancy rates (32.4 v. 36%) and term rates (32.4 v. 20%) between the first round of cloning and the successive recloning. However, the rate of pregnancy losses was significantly greater in recloning group (P &lt; 0.05), with 4 out of 9 pregnancies lost between Day 40 of gestation and term, whereas no pregnancy losses were observed after Day 40 of gestation in the first-round cloning group. The greater pregnancy loss in the recloning procedure might be caused by accumulation of epigenetic errors resulting from incomplete reprogramming. We are assessing the DNA methylation pattern of differentially methylated regions (DMR) of 2 paternally imprinted genes (H19 and IGF2R) in the cloned and recloned goats and expect to see a difference in their imprinted gene DNA methylation pattern, which could explain the greater rate of pregnancy loss in recloned goats.

Oocytes from small and large follicles exhibit similar development competence following goat cloning despite their differences in meiotic and cytoplasmic maturation

Reduced developmental competence after IVF has been reported using oocyte derived from small follicles in several species including cattle, sheep, and goats. No information is currently available about the effect of follicle size of the cytoplast donor on in vivo development after somatic cell nuclear transfer (SCNT) in goats. Oocytes collected from large (≥3 mm) and small follicles (<3 mm) were examined for maturation and in vivo developmental competence after SCNT. Significantly greater maturation rate was observed in oocytes derived from large follicles compared with that of small follicles (51.6% and 33.7%, P < 0.05). Greater percent of large follicle oocytes exhibited a low glucose-6-phosphate dehydrogenase activity at germinal vesicle stage compared with small follicle oocytes (54.9% and 38.7%, P < 0.05). Relative mRNA expression analysis of 48 genes associated with embryonic and fetal development revealed that three genes (MATER, IGF2R, and GRB10) had higher level of expression in metaphase II oocytes from large follicles compared with oocytes from small follicles. Nevertheless, no difference was observed in pregnancy rates (33.3% vs. 47.1%) and birth rates (22.2% vs. 16.7%) after SCNT between the large and small follicle groups). These results indicate that metaphase II cytoplasts from small and large follicles have similar developmental competence when used in goat SCNT.

Cytokine gene expression at the maternal-fetal interface after somatic cell nuclear transfer pregnancies in small ruminants.

The present retrospective study investigated pregnancy rates, the incidence of pregnancy loss and large offspring syndrome (LOS) and immune-related gene expression of sheep and goat somatic cell nuclear transfer (SCNT) pregnancies. We hypothesised that significantly higher pregnancy losses observed in sheep compared with goat SCNT pregnancies are due to the increased amounts of T-helper 1 cytokines and proinflammatory mediators at the maternal-fetal interface. Sheep and goat SCNT pregnancies were generated using the same procedure. Control pregnancies were established by natural breeding. Although SCNT pregnancy rates at 45 days were similar in both species, pregnancy losses between 45 and 60 days of gestation and the incidence of LOS were significantly greater in sheep than in goats. At term, the expression of proinflammatory genes in sheep SCNT placentas was increased, whereas that in goats was similar to that in control animals. Genes with altered expression in sheep SCNT placentas included cytotoxic T-lymphocyte-associated protein 4 (CTLA4), interleukin 2 receptor alpha (IL2RA), cluster of differentiation 28 (CD28), interferon gamma (IFNG), interleukin 6 (IL6), interleukin 10 (IL10), transforming growth factor beta 1 (TGFB1), tumor necrosis factor alpha (TNF-α), interleukin 1 alpha (IL1A) and chemokine (C-X-C motif) ligand 8 (CXCL8). Major histocompatibility complex-I protein expression was greater in sheep and goat SCNT placentas at term than in control pregnancies. An unfavourable immune environment is present at the maternal-fetal interface in sheep SCNT pregnancies.

31 EFFECT OF ACTIVATION METHOD ON IN VIVO DEVELOPMENT FOLLOWING SOMATIC CELL NUCLEAR TRANSFER IN GOATS

The effects of activation method and timing between fusion and activation in goat somatic cell nuclear transfer (NT) were investigated. In vivo-ovulated oocytes were surgically flushed from donors 54 to 62 h after CIDR withdrawal in the breeding season and enucleated after brief ultraviolet exposure. Transfected fibroblasts and epithelial cells from 4 clonal strains were serum-starved for 4 days before NT. Two direct current electric pulses (2 kV cm–1 each for 10 μs) were used to induce fusion and simultaneous activation. Forty-five minutes after successful fusion, reconstructs received a second activation stimulus delivered either electrically as above (group 1) or by exposure to 2.5 μM ionomycin for 1 min (group 2). Non-fused couplets received another electrical stimulus in a second fusion attempt (group 3). Fused reconstructs from all three groups were cultured in 5 μg mL–1 of cycloheximide and 5 μg mL–1 of cytochalasin B for 3 h before culture overnight in AgResearch SOF media. Embryos at the 1- and 2-cell stages were transferred to the oviducts of synchronized recipients 2 days after oestrus. Each recipient received on average 10 to 12 embryos. Pregnancy and fetal development was monitored regularly by ultrasound. Parturition was induced up to 5 days before expected full term. Kids were reared on the recipients until weaning, with supplemental feeding as required. Embryo survival data were analysed by Fisher's exact test. There were no significant differences between groups 1 and 2 in terms of pregnancy and embryo survival rates throughout development. In group 1, 110 embryos were transferred to 11 recipients. Four does (36%) were diagnosed pregnant on Day 30 of gestation, carrying a total of 8 fetuses (7.3%). All 8 were delivered at term; however, one died at birth and another before weaning. In group 2, 202 embryos were transferred to 20 recipients. Thirteen does (65%) were pregnant on Day 30 of gestation, with a total of 23 fetuses (11.4%). One pregnancy was lost by Day 50 and another by Day 100. The remaining 11 pregnancies (55%) were maintained to term, with 18 kids delivered (8.9%). Four died within 1 day of birth, with the other 14 surviving to weaning. In group 3, a total of 63 embryos were transferred to five recipients. However, no fetuses were detected at Day 30; significantly less than for either group 1 (P &lt; 0.05) or 2 (P &lt; 0.003). Overall embryo survival, in terms of live kids at weaning from embryos transferred, was 5.5, 6.9 and 0% for groups 1, 2 and 3, respectively. Using in vivo-ovulated oocytes, activation with either ionomycin or electrical stimulation, combined with cycloheximide and cytochalasin B, are similar in promoting successful development following NT. However, with re-fusion (group 3) the first electrical stimulus delivered 45 min before fusion may cause a partial pre-activation of the oocyte cytoplasm sufficient to preclude development. Supported by GTC and the NZ Ministry of Science and Innovation.

Development of an Optimized Zona-Free Method of Somatic Cell Nuclear Transfer in the Goat

The purpose of this study was to develop an improved zona-free method of goat somatic cell nuclear transfer (SCNT) that has both ease of operation and efficiency. The main steps involved were: (1) optimization of in vitro oocyte maturation, (2) parthenogenetic activation of zona-free oocytes, (3) SCNT of zona-free anaphase II-telophase II (AII-TII) oocytes that subverted the need for long term UV-exposure of the oocytes, and (4) in vitro culture of groups of cloned embryos in wells in a highly efficient continuous serum-free embryo medium to the blastocyst stage before transfer to the recipients. Percentages of transgenic blastocyst production were 22.3 and 33.1% for adult and fetal cell lines, respectively. After transfer of cloned and transgenic blastocysts, 28.6 and 36.4% of the recipients were confirmed pregnant and 75 and 33.3% of the pregnancies resulted in the delivery of viable offspring, respectively. To our knowledge, this is the first report of successful live and survived birth of cloned and transgenic offspring through a whole procedure of in vitro oocyte maturation and embryo development to the blastocyst stage, and in this study the in vitro efficiencies of cloned and transgenic embryo production were higher than the available reports.

Optimization of electrofusion protocols for somatic cell nuclear transfer

Electrofusion is one of the critical steps used in somatic cell nuclear transfer (SCNT). This low effectiveness of electrofusion of the somatic donor cell into the recipient oocyte limits the cloning success in certain mammals. In this study, chamber fusion (CF) and micro-electrode fusion were compared in goat SCNT. A 15 μm tip-end, 100 μm frustum-end and 200 μm parallel micro-electrodes were employed to perform micro-electrofusion with the aid of a micromanipulator. Different combinations of micro-electrodes, tip-end plus tip-end (TT), tip-end plus frustum-end (TF), frustum-end plus frustum-end (FF) and parallel micro-electrodes (PM) were evaluated. To improve fusion efficiency a couple of fibroblast karyoplast and oocyte cytoplast cells were pressurized or unpressurized during fusion in each group. No significant differences in the fusion rate of the unpressurized groups and chamber fusion (73.1% in TT, 75.3% in TF, 73.3% in FF, 74.2% in PM and 74.8% in CF) were recorded. The fusion rates in the TT (94.9%) and TF (92.2%) were significantly higher than in the FF (83.0%) and PM (83.9%) groups. The highest fusion rate and the lowest degeneration rate were obtained in the TT group. Compared with chamber fusion, the fusion rate was increased from 72.2 to 89.0% for the granulosa cells, 77.1 to 94.6% for fetal fibroblast cells and 51.2 to 78.0% for mammary gland epithelial cells in goat SCNT. These results showed that the pressurized fusion protocol carried out by a pair of tip-end micro-electrodes is optimal to improve the fusion efficiency of SCNT.

Cloning of Asian yellow goat (C. hircus) by somatic cell nuclear transfer: Telophase enucleation combined with whole cell intracytoplasmic injection

Our and other previous studies have shown that telophase enucleation is an efficient method for preparing recipient cytoplasts in nuclear transfer. Conventional methods of somatic cell nuclear transfer either by electro-fusion or direct nucleus injection have very low efficiency in animal somatic cell cloning. To simplify the manipulation procedure and increase the efficiency of somatic cell nuclear transfer, this study was designed to study in vitro and in vivo development of Asian yellow goat cloned embryos reconstructed by direct whole cell intracytoplasmic injection (WCICI) into in vitro matured oocytes enucleated at telophase II stage. Our results demonstrated that the rates of cleavage and blastocyst development of embryos reconstructed by WCICI were slightly higher than in conventional subzonal injection (SUZI) group, but no statistic difference (P > 0.05) existed between these two methods. However, the percentage of successful embryonic reconstruction in WCICI group was significantly higher than that in SUZI group (P < 0.05). After embryo transfer at 4-cell stage, the foster in both groups gave birth to offspring. Therefore, the present study suggests that the telophase ooplasm could properly reprogram the genome of somatic cells, produce Asian yellow goat cloned embryos and viable kids, and whole cell intracytoplasmic injection is an efficient protocol for goat somatic cell nuclear transfer.

Nuclear transplantation in the early porcine embryo

Electrofusion is one of the critical steps used in somatic cell nuclear transfer (SCNT). This low effectiveness of electrofusion of the somatic donor cell into the recipient oocyte limits the cloning success in certain mammals. In this study, chamber fusion (CF) and micro-electrode fusion were compared in goat SCNT. A 15 μm tip-end, 100 μm frustum-end and 200 μm parallel micro-electrodes were employed to perform micro-electrofusion with the aid of a micromanipulator. Different combinations of micro-electrodes, tip-end plus tip-end (TT), tip-end plus frustum-end (TF), frustum-end plus frustum-end (FF) and parallel micro-electrodes (PM) were evaluated. To improve fusion efficiency a couple of fibroblast karyoplast and oocyte cytoplast cells were pressurized or unpressurized during fusion in each group. No significant differences in the fusion rate of the unpressurized groups and chamber fusion (73.1% in TT, 75.3% in TF, 73.3% in FF, 74.2% in PM and 74.8% in CF) were recorded. The fusion rates in the TT (94.9%) and TF (92.2%) were significantly higher than in the FF (83.0%) and PM (83.9%) groups. The highest fusion rate and the lowest degeneration rate were obtained in the TT group. Compared with chamber fusion, the fusion rate was increased from 72.2 to 89.0% for the granulosa cells, 77.1 to 94.6% for fetal fibroblast cells and 51.2 to 78.0% for mammary gland epithelial cells in goat SCNT. These results showed that the pressurized fusion protocol carried out by a pair of tip-end micro-electrodes is optimal to improve the fusion efficiency of SCNT.