• Asian-Australasian Journal of Animal Sciences
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• v.26 no.12
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• pp.1680-1688
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• 2013

Many different approaches have been developed to improve the efficiency of animal cloning by somatic cell nuclear transfer (SCNT), one of which is to modify histone acetylation levels using histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA). In the present study, we examined the effect of TSA on in vitro development of porcine embryos derived from SCNT. We found that TSA treatment (50 nM) for 24 h following oocyte activation improved blastocyst formation rates (to 22.0%) compared with 8.9% in the non-treatment group and total cell number of the blastocysts for determining embryo quality also increased significantly ($88.9{\rightarrow}114.4$). Changes in histone acetylation levels as a result of TSA treatment were examined using indirect immunofluorescence and confocal microscopy scanning. Results showed that the histone acetylation level in TSA-treated embryos was higher than that in controls at both acetylated histone H3 lysine 9 (AcH3K9) and acetylated histone H4 lysine 12 (AcH4K12). Next, we compared the expression patterns of seven genes (OCT4, ID1; the pluripotent genes, H19, NNAT, PEG1; the imprinting genes, cytokeratin 8 and 18; the trophoblast marker genes). The SCNT blastocysts both with and without TSA treatment showed lower levels of OCT4, ID1, cytokeratin 8 and 18 than those of the in vivo blastocysts. In the case of the imprinting genes H19 and NNAT, except PEG1, the SCNT blastocysts both with and without TSA treatment showed higher levels than those of the in vivo blastocysts. Although the gene expression patterns between cloned blastocysts and their in vivo counterparts were different regardless of TSA treatment, it appears that several genes in NT blastocysts after TSA treatment showed a slight tendency toward expression patterns of in vivo blastocysts. Our results suggest that TSA treatment may improve preimplantation porcine embryo development following SCNT.

• Clinical and Experimental Reproductive Medicine
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• v.26 no.2
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• pp.231-238
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• 1999

The objective of this study was to examine the effect of developmental stage and embryo age of in vitro produced bovine blastocysts after vitrification and thawing. In vitro cultured day 8 blastocysts after IVF were equilibrated 20% ethylene glycol (EG) for 3 min. and were vitrified using EFS40, which is consisted of 40% EG, 18% ficoll, 0.3M sucrose and 10% FBS added in mDPBS for 30 sec. before being plunged into $LN_2$. Also, survival in vitro was assessed by re-expansion and hatching or hatched at 24 hand 48 h postwarming, respectively. The results obtained in these experiments were summarized as follows; 1) When the embryos were cultured for 8 day after IVF, 41.0% of the cleaved embryos developed to the blastocysts (early; 7.6%, expanded; 22.9%, hatching; 4.6% and hatched; 5.9%). 2) When the embryos were exposed or vitrified to the freezing solution, the re-expansion of vitrified embryos (73.3%) was significantly lower than that of control and exposed embryos (100, 97.0%) (p<0.05). But the formation rate of hatching or hatched blastocysts of vitrified embryos (66.7, 46.7%) at 48h after thawing was similar to that of exposed embryos (66.7, 39.4%) but not control (100, 100%) (p<0.01). However, in the total cell numbers of those developed hatched blastocysts, there were not significantly different among the treatment groups. 3) When the embryo survival rates by different developmental stage were examined, the re-expansion was not different among the groups $(64.5{\sim}75.6%)$. After warming 48 h, the hatching and hatched formation of early blastocysts (25.8, 9.7%) was significantly lower than those of expanded (69.7, 39.4%) and hatching blastocysts (53.3, 43.3%) (p<0.05). 4) In addition, when the expanded blastocysts at day 7, 8 and 9 were vitrified, the re-expansion of day 8 and 9 embryos was significantly lower than that of day 7 (day 7; 93.9%, day 8; 75.8% and day 9; 87.5%) (p<0.05). However, the rates of development to hatched blastocysts were no difference among the groups (day 7; 36.4%, day 8; 36.4% and day 9; 31.3%). These results suggested that in vitro produced expanded or hatching blastocysts can be efficiently cryopreserved by the two-step vitrification method using EFS40.

• Journal of Embryo Transfer
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• v.29 no.1
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• pp.59-66
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• 2014

Transcription activator like effector nucleases (TALENs) are artificial restriction enzymes generated by fusing a TALE DNA binding domain to a DNA cleavage domain which remove and introduce specific genes to produce transgenic animals. To investigate the efficient laboratory techniques for the injection of TALEN mRNA, pEGFP-N1 commercial plasmid were microinjected into porcine parthenogenetic and in vitro fertilization (IVF). In Experiment 1, to investigate injection time, compared 4 different time durations (2 hr, 4 hrs, 6 hrs & 8 hrs) after post activation of parthenogenetic embryos and after 6 hrs of co-incubation with sperms in IVF embryos. There were significant difference (P<0.05) in development to the blastocysts (4.4, 8.9, 3.9, 0.6%), GFP expression in blastocysts (1.3, 5.7, 2.3, 0.0%) which injected after post activation of 4 hrs compared with other 3 groups. IVF embryos after 2 hrs and 4 hrs injected were expressed GFP significantly higher than rest of two groups (P<0.05). In Experiment 2, compared development of 2 different concentrations ($20ng/{\mu}l$ and $50ng/{\mu}l$) of EGFP injection. There were significant difference (P<0.05) between two treatments which has higher cleavage (58.8 vs 41.9%), blastocysts development rate (13.0 vs 11.1%) and GFP expressed blastocysts (5.7 vs 0.0%) in $20ng/{\mu}l$ than the $50ng/{\mu}l$ in parthenogenetic embryos. In IVF embryos, only $20ng/{\mu}l$ injected embryos were expressed GFP (4.2%) after 7 days of incubation and 77.3 vs 64.7% of cleavage, 26.4 vs 23.5% development to blastocysts. In Experiment 3, three different volumes (5, 10 and 20 pl) were microinjected into porcine embryos to determine the most appropriate volume. Out of 3 groups, significantly higher development rates of cleavage (68.3, 58.0, 29.3%), blastocysts (11.7, 12.7, 0.5%) and GFP expressed blastocysts (2.9, 7.8, 0.0%) were shown in the 10 pl group (P<0.05). In conclusion, these results imply that $20ng/{\mu}l$ concentration, 10 pl of volume and injection at 4 hrs after post activation for parthenogenetic and 2~4 hrs after IVF, $20ng/{\mu}l$ concentration and 10 pl volume for IVF embryos were more effective microinjection conditions.

• Korean Journal of Animal Reproduction
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• v.21 no.2
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• pp.131-137
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• 1997

This study was carried out to investigate the in vivo development rates of vitrified-thawed mouse expanding, hatching and hatched blastoc ysts(BL). In vitro fertilization produced blastocysts were vitrified in EFS40(40% ethylene glycol, 30% Ficoll and 0.3 M sucrose in phosphate buffer saline containing 10% FBS). Expanding a and hatching blastocysts were equilibrated in 20% ethylene glyco](EG) for 5 min. before exposure to EFS40 at 25°C for 1 min., they were then vitrified in liquid nitrogen. Hatched blastocysts which cultured in m-CR1 medium supple mented 0.4% bovine serum albumin on day 5. were equilibrated in 10% EG for 5 min. and then vitrified in EFS40 for 30 sec. After thawing, re-expanding blastocysts were transferred to recipients(3 day of pseudopregnant) on one or both uterine horns(6-8 embryos per a horn). Preg¬n nancy rates of recipients and implantation were a assccessed by autopsy on 15 gestation. The res¬u ults obtained in these experiments were summar¬1 ized as follows; 1) The pregnancy and live fetus rates, for vitrified expanding BL(77.8 and 25.0%) and hatching BL(77.8 and 26.4%)n vitro were not significantly difference in those of control BL (66.7 and 42.9%: 83.3 and 40.4%), respectively, 2) in vitro development of vitrified hatched BL was 34.0%. and 3) in vivo developmental rate of vitrified hatched BL was only 33.3%. These results suggested that proposed rapid vitrification p procedures used EFS40 cryoprotectant can be effectively performed in mouse expanding Ihatching blastocysts and that mouse blastocysts a after being hatched from zona pellucida can be successfully cryopreserved.

• Journal of Embryo Transfer
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• v.19 no.3
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• pp.239-244
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• 2004

The present study was to investigate the pregnancy rate, gestation length and abortion rate of the recipients which transferred blastocysts produced by in-vivo collection, in-vitro fertilization (IVF) and nuclear transfer (NT). In addition, we investigate the birth weight and survival rate of the calves derived from the same methods. The pregnancy rate was 56.3％ in-vivo blastocysts, significantly higher than 19.4％ in NT blastocysts (p<0.05) but not significantly different from 30.0％ in IVF blastocysts. The abortion rate and the gestation length did not differ among the treatment groups (abortion rate: 0, 22.2 and 16.7％ respectively; gestation length: 278.8, 289.4 and 281.4 days respectively). The mean birth weight was significantly higher in NT calves (39.9kg) than in-vivo calves (25.5kg p<0.05). Recipients of in-vivo blastocysts (n=9) had all normal delivery and all of their calves survived on the 60$^{th}$ day from the birth. Recipients of IVF blastocysts (n=7) had all normal delivery but one of their calves died on the 48$^{th}$ day from the birth. Among recipients of NT blastocysts (n=5), three had normal delivery and two had Caesarean section. Among calves born through normal delivery (n=3) two died just after delivery but those born through Caesarean section all survived on the 60th day from the birth.

• 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.

• Asian-Australasian Journal of Animal Sciences
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• v.2 no.4
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• pp.591-593
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• 1989

Bovine blastocysts were obtained by totally in-vitro technique and then transferred to recipient cows. Total of 15 calves (including 4 premature calves) were obtained from 11 recipients. Four calves were obtained from the transfers of fresh blastocysts and 9 calves were obtained from the transfers of frozen-thawed blastocysts. Two calves were obtained from the bisected fresh blastocyst. The males and 5 females were delivered. Birth weight of calves was within normal range except one female, and all calves appeared to be completely normal.

• Asian-Australasian Journal of Animal Sciences
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• v.11 no.3
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• pp.307-310
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• 1998

The present studies were undertaken to evaluate the effects of a low concentration of dimethyl-sulfoxide (DMSO) on in vitro maturation and development of bovine oocytes fertilized in vitro. Significantly more oocytes reached the metaphase stage of the second meiotic division in TCM-199 supplemented with $50{\mu}M$ DMSO than in the control medium (p < 0.05), and the highest rates of development up to the blastocyst stage were obtained when $50{\mu}M$ DMSO was added to the maturation and culture media (p < 0.05). The avarage of cell numbers of the blastocysts, expanded and hatched blastocysts cultured with $50{\mu}M$ DMSO were 81.7, 125.7 and 129.9 cells, respectively. The proportion of blastocysts with normal chromosome numbers was 90.5%. These results suggest that the addition of $50{\mu}M$ DMSO is beneficial for the maturation of bovine oocytes and production of the blastocysts with high quality.

• Asian-Australasian Journal of Animal Sciences
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• v.12 no.2
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• pp.169-173
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• 1999

Studies were made to evaluate the specific and combined effects of different fatty acids on the in vitro development of 8-cell rat embryo in culture media with and without carbohydrate substrate. Palmitic, oleic, linoleic and arachidonic acids were added singly and in combination to media which contained fatty acid-free BSA. Cell numbers in blastocysts cultured in the media were counted and compared with cell numbers in blastocysts at the corresponding stage collected from the uterus. Oleic, linoleic and arachidonic acids promoted the rat embryo development from 8-cell to the blastocysts. especially in the absence of carbohydrate substrates. Among these three, oleic acid was the most effective but embryo development was not accelerated by the addition of palmitic acid in either the presence or the absence of carbohydrate substrates. Addition of the mixture of four fatty acids was more effective for rat embryo development than single treatment with any of fatty acids tested. Cell numbers per blastocyst in the presence and absence of carbohydrate substrate were similar, and did not differ from those for blastocysts obtained from the uterus.

• Journal of Embryo Transfer
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• v.23 no.1
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• pp.1-4
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• 2008

This study was performed to investigate the effect of laser-assisted hole in the zona pellucida (ZP) of frozen-thawed ICR mouse embryos on the process of hatching that is critical for expanded blastocysts to implant into endometrium, Vitrification medium, composed of ethylene glycol and sucrose supplemented with 7.5% (w/v) PVP, was used to freeze $2{\sim}4$ cell stage embryos recovered from oviducts of superovulated and mated female mice before storing them in $LN_2$. Right after thawing them, a laser beam was shot to make a hole in ZP followed by culturing in KSOM for $96{\sim}120\;hr$ and examining development to blastocyst and hatching every 12 hr. Laser-treated embryos showed significantly higher hatching rate compared to control (92.9% vs. 22.1%, p<0.05). From around Day 4, blastocysts developed from laser-treated embryos started hatching while the blastocysts of control group failed to hatch showing a lot of shrinkage. This study shows that a laser-assisted hole in ZP improves the hatching rate of blastocysts developed from frozen-thawed, in vitro cultured ICR mouse embryos.