• Animal Bioscience
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• v.35 no.9
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• pp.1360-1366
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• 2022

Objective: The present study analyzed the influence of co-transferring embryos with high and low cloning efficiencies produced via somatic cell nuclear transfer (SCNT) on pregnancy outcomes in dogs. Methods: Cloned dogs were produced by SCNT using donor cells derived from a Tibetan Mastiff (TM) and Toy Poodle (TP). The in vivo developmental capacity of cloned embryos was evaluated. The pregnancy and parturition rates were determined following single transfer of 284 fused oocytes into 21 surrogates and co-transfer of 47 fused oocytes into four surrogates. Results: When cloned embryos produced using a single type of donor cell were transferred into surrogates, the pregnancy and live birth rates were significantly higher following transfer of embryos produced using TP donor cells than following transfer of embryos produced using TM donor cells. Next, pregnancy and live birth rates were compared following single and co-transfer of these cloned embryos. The pregnancy and live birth rates were similar upon co-transfer of embryos and single transfer of embryos produced using TP donor cells but were significantly lower upon single transfer of embryos produced using TM donor cells. Furthermore, the parturition rate for TM dogs and the percentage of these dogs that remained alive until weaning was significantly higher upon co-transfer than upon single transfer of embryos. However, there was no difference between the two embryo transfer methods for TP dogs. The mean birth weight of cloned TM dogs was significantly higher upon single transfer than upon co-transfer of embryos. However, the body weight of TM dogs did not significantly differ between the two embryo transfer methods after day 5. Conclusion: For cloned embryos with a lower developmental competence, the parturition rate and percentage of dogs that remain alive until weaning are increased when they are co-transferred with cloned embryos with a greater developmental competence.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.11-11
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• 2001

In this study, we examined the developmental potential of reconstructed bovine embryos and the fate of donor mitochondria during their preimplantation development after nuclear transfer. Isolated cumulus cells were used as donor cells in nuclear transfer. Cumulus cells labelled with MitoTracker Green FM fluorochrome were injected into enucleated bovine MII oocytes and cultured in vitro. MitoTracker labelling on donor cells did not have a detrimental effect on blastocyst formation following nuclear transfer. Cleavage rate was about 69%(56/81) and blastocyst formation rate was 6.2% (5/81) at 7 days after nuclear transfer. The labelled mitochondria dispersed to the cytoplasm and became distributed among blastomeres and could be identified up to the 8- to 15-cell stages. Small patches of mitochondria were detected in some 8- to 15-cell stage embryos (5/20). However, donor mitochondria were not detected in embryos at the 16-cell stage and subsequent developmental stages. In the control group, mitochondria could be identified in arrested 1-cell embryos up to 7 days after nuclear transfer These results suggest that donor mitochondria disappear from recipient cytoplasm before 16-cell stage following nuclear transfer in reconstructed bovine embryos.

• Reproductive and Developmental Biology
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• v.28 no.1
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• pp.13-20
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• 2004

This study was conducted to investigate the developmental ability of caprine embryos after somatic cell interspecies nuclear transfer. Donor cells were obtained from an ear-skin biopsy of a caprine, digested with 0.25% trypsin-EDTA in PBS, and primary fibroblast cultures were established in TCM-199 with 10% FBS. After maturation, expanded cumulus cells were removed by vigorous pipetting in the presence of 0.3% hyaluronidase. The matured oocytes were dipped in D-PBS plus 10% FBS＋7.5 $\mu\textrm{g}$/ml cytochalasin B and 0.05 M sucrose. The reconstructed oocytes were electrically fused with donor cells in 0.3 M mannitol fusion medium. After the electofusion, embryos were activated by electric stimulation. Interspecies nuclear transfer embryos with bovine cytoplasts were cultured in TCM-199 medium supplemented with 10% FBS including bovine oviduct epithelial cells for 7∼9 day. On the other hand, the NT embryos with porcine cytoplasts were cultured in NCSU-23 medium supplemented with 10% FBS for 6∼8 day at $39^{\circ}C, 5% CO_2$ in air. In caprine-bovine NT embryos, the cleavage(2-cell) rate was 36.8% in confluence and 43.8% in serum starvation. The developmental rate of morula- and blastocyst-stage embryos was 0.0% in confluence and 18.8% in serum starvation. In caprine-porcine NT embryos, the cleavage(2-cell) rate was 76.7% in confluence and 66.7% in serum starvation. The developmental rate of morula and blastocyst stage embryos was 3.3% in confluence and 3.0% in serum starvation, and no significant difference was observed in synchronization treatment between donor cells. In caprine-bovine NT embryos, the cleavage(2-cell) rate of cultured donor cells was 30.8% and 17.6% in 5∼9 and 10∼14 passage(P＜0.05). The developmental rate of morula and blastocyst stage embryos were significantly higher(P＜0.05) in 5∼9 passage(23.1%) than in 10∼14 passage(0.0%) of cultured donor cells. In caprine-porcine NT embryos, the cleavage rate was significantly higher(P＜0.05) in 5∼9 passage(86.7%) than in 10∼14 passage(50.0%) of cultured donor cells. The developmental rate of morula and blastocyst stage embryos were 3.3 and 0.0% in 5∼9 and 10∼14와 passage of cultured donor cells. In caprine-bovine NT embryos, the developmental rate of morula and blastocyst stage embryos were 22.6% in interspecies nuclear transfer, 33.9% in in vitro fertilization and 28.1% in parthenotes, which was no significant differed. The developmental rate of morula and blastocyst stage embryos with caprine-porcine NT embryos were lower(P＜0.05) in interspecies nuclear transfer(5.1%) than in vitro fertiltzation(26.9%) and parthenotes(37.4%).

• Journal of Embryo Transfer
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• v.27 no.4
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• pp.193-203
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• 2012

The development of embryos reconstructed by nuclear transfer is dependent upon numerous factors including the type of recipient cell, method of enucleation, the type of donor cell, method of reconstruction, activation, the cell cycle stage of both the donor nucleus and the recipient cytoplasm and the method of culture of the reconstructed embryos. Many of these points which have been reviewed extensively elsewhere (Sun and Moor, 1995; Colman, 1999; Oback and Wells, 2002; Renard et al., 2002; Galli et al., 2003b), here we will concentrate on main area, the production of suitable cytoplast and nuclear donor, nuclear-cytoplasmic coordination, oocyte activation, culture of reconstructed embryos, and the effects that this may have on development.

• Journal of Embryo Transfer
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• v.30 no.3
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• pp.229-237
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• 2015

To improve the developmental potential of bovine somatic cell nuclear transfer (SCNT) embryos, this study compared the developmental rates to blastocyst stage in the SCNT embryos using donor fibroblasts treated with 5-azacytidine (5AC) and S-adenosylhomocysteine (SAH) at different concentrations. Their reprogramming efficiency level was investigated with level of telomerase activity. Donor fibroblasts isolated from adult ear skin of a cow were exposed to 5AC and SAH at different concentrations during 2 passages. After nuclear transfer into enucleated recipient oocytes, the cleavage and developmental rates were significantly (p<0.05) decreased in the SCNT embryos using 5AC-treated fibroblasts (5AC-SCNT embryos), compared with those of non-treated control (control-SCNT embryos) and SAH-treated fibroblasts (SAH-SCNT embryos). The developmental rates to blastocyst stage tended to be slightly increased in the SAH-SCNT embryos at each of the concentrations, and especially, the developmental rates in the SCNT embryos using 1.0 mM SAH-treated fibroblasts were significantly (p<0.05) higher than that of control SCNT embryos. The mean numbers of total and ICM cell in blastocysts were also significantly (p<0.05) decreased in the 5AC-SCNT embryos, compared with those of other SCNT blastocysts. Further, the level of telomerase activity was also significantly (p<0.05) decreased in the 5AC-SCNT embryos than those of control and SAH-SCNT embryos. Whereas, a significantly (p<0.05) up-regulated telomerase activity was observed in SAH-SCNT embryos, compare with that of control-SCNT embryos. In conclusion, SCNT embryos using hypomethylated donor cells with SAH, not 5AC, may improve the developmental potential and reprogramming efficiency.

• Journal of Embryo Transfer
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• v.10 no.1
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• pp.83-90
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• 1995

Production of calves after transfer of nuclear transplant embryos is the latest technology to be applied in commercial livestock breeding. The objective of this study was to establish an efficient procedure to produce offsprings from nuclear transplant embryos. The fusion rates (72.7% vs. 80.8%), cleavage rates (62.5% vs. 71.4%) and rates of development in vitro (12.0% vs. 15.2%) of nuclear transplant embryos were not significantly different between 30 and 40h maturation age of cytoplast. The in vivo and in vitro-derived embryos as nuclei donor were used in this system of bovine nuclear transplantation. Fusion rates of nuclear transplant embryos were not significantly different between in vivo and in vitro-derived embryos (73.0 and 79.2%, respectively). The percentage of embryos reaching the morulae or blastocysts were 21.8% for in vivo-derived embryos and 11.9% for in vitro-derived embryos (p<0.01). Pregnancy rates after embryo transfer of nuclear transplant embryos were not significantly different between in vivo and in vitro-derived embryos (45.9 and 40.5%, respectively). However, calving rates after embryo transfer of nuclear transplant embryos were significantly higher in the in vivo-derived embryos than in vitro (p<0.01). Further research for age of cytoplast and use of in vitro-derived embryos as nuclei donor is required in this system. In conclusion, these results clearly show that the use of in vitro-derived oocytes as recipient cytoplast can improve the nuclear transplant system for genetic progress in cattle.

• Journal of Embryo Transfer
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• v.10 no.3
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• pp.209-217
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• 1995

The present study was carried out to develop a cloning technology of mouse embryos by nuclear transplantation with electrofusion and to produce cloned offsprings by transfer of reconstituted embryos. A single nucleus from two- and eight-cell embryos was transplanted into the enucleated two-cell embryos by rnicromanipulation. The fusion of nucleus with recipient cytoplasm and the subsequent development of reconstituted embryos in vitro as well as in vivo to term were examined to determine the optimal electrofusion parameters for nuclear transplantation in mouse embryos. The successful enucleation of donor embryos was 84.9 and 83.3% in two- and eight-cell stage, respectively, and the successful injection of nucleus from two- and eight-cell donor embryos into the perivitelline space of enucleated two-cell embryos were 85.1 and 84.7%, respectively. No significant differences were found in enucleation or injection rate between the cell stages of donor embryos. When the blastomeres of intact two-cell mouse embryos were electrofused in 0.3 M mannitol medium(100 $\mu$sec., 3 pulses), the fusion rate was similarly 93.2, 92.2 and 92.0% in 1.0, 1.5 and 2.0 kV /crn, respectively, but in vitro development to blastocyst of the fused two-cell embryos was significantly(P<0.05) lower in 2.0 kV/cm (63.4%) than in 1.0 kV/cm (91.7%) or 1.5 kV/cm (82.4%). The development in vitro to eight-cell stage of the reconstituted embryos with nucleus from two-cell stage(45.5%) was significantly(P<0.05) higher than that from eight-cell stage blastomeres (16.7%). The number of blastomeres of the intact embryos at blastocyst stage was 50i0.6 and 55$\pm$2.4 in in vitro and in vivo cultured mouse embryos, respectively, but significantly(P<0.05) decreased to 35$\pm$0.7 in nuclear transplanted blastocyst embryos. The conception rate of mice following embryo transfer was 32.1% in the reconstituted two-cell embryos using two-cell donor nuclei, which was comparable to the fresh two-cell embryos(40.6%). However, the rate of development in vivo to term following embryo transfer of the reconstituted two-cell embryos using two-cell donor nuclei (23.5%) was significantly(P<0.05) lower compared with the percentage of two-cell fresh embryos(31.5%).

• Journal of Embryo Transfer
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• v.28 no.4
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• pp.307-314
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• 2013

Embryos formed in vivo were collected from 171 donors housed in Chung Cheong Buk-Do Institute of Livestock and Veterinary Research of the Chungbuk community during the years 2009~2012. We evaluated annual embryo collection, effect of follicle stimulating hormone (FSH), controlled internal drug release (CIDR) and prostaglandin (PG) administration to the donor for superovulation and controlling the estrus cycle, seasonal effects of embryo collection and compared the number of embryos recovered as per the collection days and pregnancy rate. In all, 1,243 embryos were collected from 118 donors with an average of $7.31{\pm}5.35$ embryos per donor, out of which 69.4% were transferable. Dosages of FSH required for inducing superovulation in various donors were compared. Average number of embryos collected from donors administered with 30 AU of FSH ($7.13{\pm}5.74$ per donor) was not significantly different from that of donors who were given an injection of 24 AU of FSH ($7.53{\pm}4.91$ per donor). However, the percentage of transferable embryos in the 30AU FSH-administered group (63.2 %, 449 of 711) was higher than that in the 24AU FSH-administered group (77.8%, 414 of 532). In the group of donors under a natural estrus cycle, the FSH dose administered did not influence the number of transferable embryos produced ($7.49{\pm}6.25$ per donor for 30 AU of FSH vs $7.49{\pm}4.92$ per donor for 24 AU of FSH). However, in donors administered with CIDR and PG for controlling the estrus cycle, the FSH dose affected the average number of transferable embryos collected ($4.25{\pm}2.87$ per donor for 30 AU of FSH vs $8.50{\pm}6.36$ per donor for 24 AU of FSH). We collected embryos from donors 6, 7 or 8 days after artificial insemination (AI). Results showed that the percentage of transferable embryos among those collected 8 days after AI was significantly higher than that among embryos collected 6 or 7 days after AI. Seasonal variations did not affect number of recovered embryos and pregnancy rates in natural estrus cycle and CIDR treatment groups (48.28% and 42.55%) but higher than pregnancy rate of frozen embryos (19.63%). These results indicated that administration of FSH beyond a threshold dose (at least 24 AU) has no beneficial effect on the production embryos and that collection of embryos 7~8 days after AI is optimal for embryo recovery. CIDR treatment induced superovulation in short term and had no influence on the natural estrus cycle. Finally, although good-quality embryos were transferred, freezing significantly reduced the pregnancy rates after transfer.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.2
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• pp.194-200
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• 2009

The aim of this study was to investigate the effects of donor cell passage, size and type on the development of nuclear transfer embryos. Porcine cumulus cells, fetal fibroblasts and oviductal epithelial cells from 1-2, 3-6 and 7-10 passages were used for the nuclear transfer. In the oocytes with the cumulus donor cells, fusion and cleavage rates of oocytes and cell numbers per blastocyst among the three different passage groups did not show any differences, but the rates of blastocyst formation from 1-2 and 3-6 passage groups were higher than those from 7-10 passage group. The rates of fusion, cleavage and blastocyst formation, and the cell numbers per blastocyst were higher in the embryos with the sizes of <20 and 20 ${\mu}m$ cumulus donor cells compared to the >20 ${\mu}m$ cumulus donor cell. In the oocytes with the fetal fibroblast donor cells, the rate of blastocyst formation from the 3-6 passage group was higher than from 1-2 and 7-10 passage groups. The embryos with the size of 20 $\mu{m}$ fetal fibroblast donor cell showed higher rate of blastocyst formation compared to those with <20 and >20 ${\mu}m$ donor cells. In the oocytes with the oviductal epithelial cells, the rates of blastocyst formation from 1-2 and 3-6 passage groups were higher compared to those from 7-10 passage group. The embryos with the sizes of <20 and 20 ${\mu}m$ oviductal epithelial donor cells had a higher rate of blastocyst formation compared to those with >20 ${\mu}m$ donor cell. Fusion and cleavage rates of oocytes, and cell numbers per blastocyst among the three different donor cell types from the 3-6 passage did not show any differences. However, the rate of blastocyst formation of somatic cell nuclear transfer (SCNT) embryos with the fetal fibroblast donor cell was higher than that of blastocyst formation of SCNT embryos with the cumulus and oviductal epithelial donor cells.

• Reproductive and Developmental Biology
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• v.34 no.2
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• pp.73-79
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• 2010

We investigated the microtubule dynamics, including the inheritance of donor centrosomes and the mitotic spindle assembly occurring during the first mitosis of somatic cell nuclear transfer (SCNT) embryos in pigs. SCNT embryos were fixed 15 min and 1 h after fusion in order to assess the inheritance pattern of the donor centrosome. The distribution and dynamic of the centrosome and microtubule during the first mitotic phase of SCNT embryos were also evaluated. The frequency of embryos evidencing $\gamma$-tubulin spots (centrosome) was 93.2% in the SCNT embryos 15 min after fusion. In the majority of the SCNT embryos (61.5%), however, no centrosome was observed 1 h after fusion. The frequency of the embryos with no or abnormal mitotic spindles 20 h after fusion was 19.6%. The $\gamma$-tubulin spots were detected near the nuclei of somatic cells regardless of cell cycle phase, whereas $\gamma$-tubulin spots in the SCNT embryos were observed only during the inter-anaphase transition. These results showed that the donor centrosome is inherited into the SCNT embryos, but failed to assemble the normal mitotic spindles during first mitotic phase in some SCNT embryos.