• Journal of Embryo Transfer
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• v.26 no.3
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• pp.171-180
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• 2011

In this study I report that in vitro development rates of bovine nuclear transfer embryos activated either with boar sperm cytosolic factor (SCF) or with ionomycin followed by cycloheximide (CHX) and subsequent in vivo developmental rates after embryo transfer are related to blastocyst quality as evaluated by apoptosis analysis. SCF was extracted from porcine semen then purified for post-activation injection after nuclear transfer. The optimal timing for SCF injection was determined to be at least 22 h post-IVM for parthenogenetic activation of bovine oocyies. A total of 364 oocytes were successfully enucleated and 268 (73.6%) fused and were injected with SCF. The survival rate of fused and injected embryos was 109/113 (96.5%) after 2 h. The cleavage rates of nuclear transfer embryos after 3 d of culture in the ionomycin/CHX treated group were significantly higher than those of the SCF-activated group (93.3% vs 81.7%, p<0.01, respectively). However, at 7 d and 9 d there was no significant difference between the total developmental rates to blastocyst for either treatment group. Total blastocyst cell numbers were also not significantly different between the two activation treatments (ionomycin/CHX: 149.5${\pm}$7.7 vs. SCF: 139.3${\pm}$4.4 cells). In contrast, the apoptotic levels in the SCF blastocysts were higher than those produced after the chemical treatment (28.2${\pm}$5.1% vs. 8.8${\pm}$0.6%, respectively). A total of 18 expanded or hatching blastocysts was transferred to nine synchronized recipients in each activation group; 5/9 (55.5%) and 2/9 (22.2%) were pregnant at 40 d in the ionomycin/CHX treatment and SCF activated group, respectively. However, only one went to term in the ionomycin/CHX treatment while none of the pregnancies from the SCF group were maintained by 90 d. In conclusion, these results suggest that SCF derived from different species is a limited activator to be used for activation after bovine nuclear transfer in lieu of a chemical activation protocol.

• Reproductive and Developmental Biology
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• v.28 no.1
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• pp.21-27
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• 2004

This study was conducted to investigate the developmental ability of caprine embryos after somatic cell interspecies nuclear transfer. Recipient bovine and porcine oocytes were obtained from slaughterhouse and were matured in vitro according to established protocols. 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. The matured oocytes were dipped in D-PBS plus 10% FBS ＋ 7.5 $\mu$ g/ml cytochalasin B and 0.05M sucrose. Enucleation were accomplished by aspirating the first polar body and partial cytoplasm which containing metaphase II chromosomes using a micropipette with an out diameter of 20∼30 $\mu$m. A Single donor cell was individually transferred into the perivitelline space of each enucleated oocyte. The reconstructed oocytes were electric fusion with 0.3M mannitol fusion medium. After the electrofusion, 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. And 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. Interspecies nuclear transfer by recipient bovine oocytes were fused with electric length 1.95 kv/cm and 2.10 kv/cm. There was no significant difference between two electric length in fusion rate(47.7 and 44.6%) and in cleavage rate(41.9 and 54.5%). Using electric length 1.95 kv/cm and 2.10 kv/cm in caprine-porcine NT oocytes, there was also no significant difference between two treatments in fusion rate(51.3 and 46.1%) and in cleavage rate(75.0 and 84.9%). The caprine-bovine NT oocytes fusion rate was lower(P＜0.05) in 1 pulse for 60 $\mu$sec(19.3%), than those from 1 pulse for 30 $\mu$sec(50.8%) and 2 pulse for 30 $\mu$sec(31.0%). The cleavage rate was higher(P＜0.05) in 1 pulse for 30 $\mu$sec(53.3%) and 2 pulse for 30 $\mu$sec(50.0%), than in 1 pulse for 60 $\mu$sec(18.2%). The caprine-porcine NT oocytes fusion rate was 48.1% in 1 pulse for 30 $\mu$sec, 45.2% in 2 pulse for 30 $\mu$sec and 48.6% in 1 pulse for 60 $\mu$sec. The cleavage rate was higher(P＜0.05) in 1 pulse for 30 $\mu$sec(78.4%) and 1 pulse for 60 $\mu$sec(79.4%), than in 2 pulse for 30 $\mu$sec(53.6%). In caprine-bovine NT embryos, the developmental rate of morula and blastocyst stage embryos were 22.6% in interspecies nuclear transfer and 30.6% 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 parthenotes(37.4%).

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.68-68
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• 2001

Blastocyst formation, consisting of the inner cell mass (ICM) and trophectoderm (TE), is the first differentiation process during embryonic development in mammals. It has been hypothesized that the proportion of ICM to TE in the blastocyst may be crucial for subsequent developmental competence of early embryos, which it may be expressed as a sensitive indicator for evaluating in vitro systems. In this study ICM/total cell ratio of nuclear transfer (NT) embryos was compared with IVF-derived and in vivo embryos. Somatic cell nuclei obtained from a fetus at Day 40 of gestation were transferred into the enucleated oocyte and then cultured in NCSU 23 medium for 6 days as previously described (Koo et al., Biol. Reprod. 2000; 63:986-992). ICM and TE cells of blastocysts were determined by using a differential staining method (Han et al., Biol. Reprod. 1999; 60:1110-1113). Development rate (9.8$\pm$2.5%, 23/225) to the blastocyst stage of NT embryos was lower than IVF embryos (23.8$\pm$2.7%, 53/223). Thus, a difference was detected in the in vitro developmental rate to blastocyst stage between NT and IVF-derived embryos (P<0.05). In the next experiment, we investigated ICM and TE nuclei to assess the quality of blastocysts that produced by NT, IVF and in vivo, respectively. NT blastocysts (27.6$\pm$8.3) showed a smaller total cell number than IVF-derived (42.6$\pm$17.4) and in vivo embryos (283.9$\pm$103.5) (P<0.05). Ratios of ICM/total cells in NT, IVF and in vivo blastocysts were 15.1$\pm$ 18.6% (n=56), 12.3$\pm$9.2% (n=57) and 30.4$\pm$6.8% (n=40), respectively. Individual blastocysts for the ratio of ICM/total cells were assigned to 3 groups (I; <20%, II; 20 to 40% and III;>40%). As the results, most in vivo blastocysts (97.5%, 39/40) were distributed into group II while most NT (78.6%, 44/56) and IVF-derived blastocysts (82.5%, 47/57) were allocated to group I. Thus, our data show that NT or IVF-derived embryos have aberrant morphology during early development in vitro systems, suggesting that these anomalies may result in developmental failures of the NT embryos to term.

• Journal of Embryo Transfer
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• v.23 no.2
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• pp.101-108
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• 2008

This study investigated the developmental ability and gene expression of somatic cell nuclear transfer embryos using ear skin fibroblast cells derived from miniature pig. When miniature pig (m) and landrace pig (p) were used as donor cells, there were no differences in cleavage (79.2 vs. 78.2%) and blastocyst rates (27.4 vs. 29.7%). However, mNT blastocysts showed significantly higher apoptosis rate than that of pNT blastocysts (6.1 vs. 1.7%) (p<0.05). The number of nuclei in pNT blastosysts was significantly higher than that of mNT (35.8 vs. 29.3) (p<0.05). Blastocysts were analyzed using Realtime RT-PCR to determine the expression of Bax-${\alpha}$, Bcl-xl, H19, IGF2, IGF2r and Xist. Bax-${\alpha}$ was higher in mNT blastocyst than pNT blastocyst (p<0.05). There was no difference in Bcl-xl between two NT groups. Bax-${\alpha}$/Bcl-xl was, however, significantly higher in mNT blastocyst compared to pNT. The expression of imprinting genes were aberrant in blastocysts derived from NT compared to in vivo blastocysts. H19 and IGF2r were significantly lower in mNT blastocysts (p<0.05). The expression of IGF2 and Xist was similar in two NT groups. However, imprinting genes were expressed aberrantly in mNT compared to pNT blastocysts. The present results suggest that the NT between donor cells derived from miniature pig and recipient oocytes derived from crossbred pig might affect reprogramming of donor cell, resulting in high apoptosis and aberrant expression patterns of imprinting genes.

• Journal of Embryo Transfer
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• v.27 no.1
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• pp.1-7
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• 2012

Somatic cell nuclear transfer (SCNT) for miniature pig has been developed for xenotransplantation and many other biomedical experiments. However, the efficiency of SCNT is still very low due to many factors. To optimize the surrogate mother condition for improvement of cloned miniature pigs efficiency, we investigated the effect of the status of surrogate mother on pregnancy, farrowed rate in SCNT pigs. After SCNT with mesenchymal stem cells as donor cells, the SCNT embryos were surgically transferred into the oviduct of surrogated pigs. To compare the effects of status of surrogate pigs on pregnancy, surrogate pigs were prepared by artificial abortion at day 20~29 (Group 1), 30~39 (Group 2), and 40~45 (Group 3) of gestation. After SCNT embryos transfer in three different status of surrogate pigs, Group 2 (56.3%) and 3 (55.6%) had significantly ($p$ <0.05) higher the pregnancy rate than group 1 (0%) at day 30 of gestation. The status of ovulation in surrogate pig also was investigated. Post-ovulation status (54.8%) had higher proportion than pre-ovulation status (38.7%) and ovulation status (6.5%). We obtained 19 cloned miniature piglets from seven surrogate gilts and five piglets are living healthy but fourteen piglets died soon after birth or stillbirth. The weights of piglets greatly differ from 254 to 1,296 g. Microsatellite analysis showed that cloned piglets were genetically different from the surrogate mother and cloned piglets were genetically equal to the donor cell. In conclusion, the present result indicates that artificially abortion method can improve the efficiency of pregnancy after SCNT in pigs. This study will provide available method for the further study and application in the field of xenotransplantation.