• Journal of Embryo Transfer
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• v.30 no.3
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• pp.195-200
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• 2015

The objective of this study was to evaluate the toxicities of permeable cryoprotectants and finally to establish the cryopreservation method of surplus embryos obtained during assisted reproductive technology (ART). Toxicities of permeable cryoprotectants, dimethyl sulfoxide (DMSO), ethylene glycol (EG), Glycerol, and 1,2-PROH were investigated using a murine embryo model. Female $F-{_1}$ mice were stimulated with gonadotropin, induced ovulation with hCG and mated. Two cell embryos were collected and cultured after exposure to among DMSO, EG, Glycerol, and 1,2-PROH. Embryo development was evaluated up to the blastocyst stage. The total cell count of blastocysts that were treated with DMSO and Glycerol at the 2-cell stage was significantly lower than that were treated with EG ($81.1{\pm}15.1$), 1,2-PROH ($88.0{\pm}21.1$) or the control ($99.9{\pm}21.3$) (p<0.001). On comparison of four cryoprotectant treated groups, the DMSO and Glycerol treated group showed a decreased cell count compared with the EG and 1,2-PROH treated group (p<0.05). Both DMSO ($14.7{\pm}1.3$), EG ($12.1{\pm}1.1$), Glycerol ($15.2{\pm}1.8$), and 1,2-PROH ($11.5{\pm}1.3$) treated groups showed higher apoptosis rates of cells in the blastocyst compared with the control ($6.5{\pm}0.7$, p<0.0001). In addition, the DMSO or Glycerol treated group showed more apoptotic cells than the EG or 1,2-PROH treated group (p<0.001). The potential toxicity of cryoprotectants was uncovered by prolonged exposure of murine embryos to among DMSO, EG, Glycerol, and 1,2-PROH at room temperature. When comparing four permeable cryoprotective agents, EG and 1,2-PROH appeared to be less toxic than DMSO and Glycerol at least in a murine embryo model.

• 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.19 no.2
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• pp.127-132
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• 2004

Ninety-two heads of sire, ranging from 4 to 8 years old, and semen production records of 8,628 from those sire and the performance and carcass traits from 814 heads of those offspring were used to investigate the relationships of scrotal circumference(SC) of Hanwoo sire to semen characteristics and performance, carcass traits of those offspring. Average SC of sire were 38.7 cm. The semen volume, sperm concentration and total sperm number at first and second ejaculation were 5.63 mL and 5.32 mL, $17.9{\times}108/mL$ and 15.0${\times}10^8$/mL, and 100.3${\times}10^8$/ ejaculation and 79.4${\times}10^8$/ejaculation, respectively. SC is positively correlated with semen volume(1st : ${\gamma}$=0.27, P<0.05 ; 2nd : ${\gamma}$=0.19, P<0.10), sperm concentration(1st : ${\gamma}$=0.21, P<0.05) and total sperm number(1st $:{\gamma}=0.38$, 2nd : ${\gamma}$=0.28, P<0.01). The live weights of those offspring were 49.2, 281.1, 436.3 and 534.4 kg at 6, 12, 18, 22 months old, respectively, and average daily gain(ADG) were 0.81 kg/day. And, carcass weight, longissimus dorsi area, backfat thickness and marbling score were 313.8 kg, 77.9 cm, 0.62 cm and 2.47, respectively. There were tended to be positive relationships between SC of sire and live weight of 6 months old(${\gamma}$=0.08, P<0.10), 12(${\gamma}$=0.18, P=0.10), 18(${\gamma}$=0.21, P<0.10), 22(${\gamma}$=0.20, P<0.10), ADG(${\gamma}$=0.25, P<0.05), carcass weight(${\gamma}$=0.18, P<0.10) and longissimus dorsi area(${\gamma}$=0.18, P<0.10) of those offspring. However, SC and backfat thickness, marbling score have no significant relationship. This results indicate that SC of sire was related to semen production and the gain weight of those offspring, positively. However, further investigation are needed to confirm the results.

• Korean Journal of Animal Reproduction
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• v.27 no.1
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• pp.87-93
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• 2003

This study was conducted to investigate the effects of embryo development by fusion condition on the nuclear transfer with brindle coated cow's ear cells. Ear cells were transferred into an enucleated oocyte and fused with cytoplasm in the fusion condition with 1.9kv/cm, 2.0kv/cm, 2.1kv/cm each 10 and 20ug duration Nuclear transfer embryo were activeted with a combination of 5ug/ml and 1.9mM 6-DMAP (4min, 4h). Fusion rate was 51∼68% range among fusion condition (1.9, 2.0, 2.1kv/cm; 10, 20us). But, cytoplasm lysis rate was increased by higher electric condition (0∼51.8% range). Each parameter's cleavage and blastocyst formation rate were 1.9kv/cm for 10us (75.8 and 19.5%), 20us (69.8 and 48.6%), 2.0kv/cm for 10us (76.9 and 20.0%), 20 us (68.5 and 40.9%), 2.1kv/cm for 10us (70.5 and 44.2%), 20 us (68.5 and 27.0%). We compared the effectiveness of cloning for between brindle coated cow's ear cells and Hanwoo fetal fibroblast cells. There was no significant differences in the fusion rate and developmental rate to the blastocyst stage. After transfer of blastocysts derived from nuclear transfer embryos, pregnancy rates of the Hanwoo fetal fibroblast cells and brindle coated cow's ear cells were checked pregnant on day 60 as assessed by ultrasonography, 40% (2/5) and 15.8% (3/19), respectively. This studies conclude that brindle coated cow's ear cells have the developmental potentiality to term by nuclear transfer. These results demonstrate that the increased the field strength was to be profitable for development of blastocyst or reduce of cytoplasm's damage than increasing the pulse duration.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.9
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• pp.1260-1266
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• 2001

The effect of protein supplementation, $O_2$ concentration and co-culture on the development of embryos produced by nuclear transfer using cultured cumulus cell was investigated. Recipient oocytes and cumulus cells were obtained from the ovaries of the slaughtered Hanwoo cows. Donor cumulus cells were cultured in Dulbecco's modified Eagle medium containing 10% fetal bovine serum at 5% $CO_2$ in air at $38.5^{\circ}C$. The 1 to 6 passages of cumulus cells were isolated and used as donor cells. The in vitro matured oocytes were enucleated and then the isolated donor cells were introduced. One $15{\mu}s$ pulse of 180 volts was applied to induce the fusion between karyoplast and cytoplast. The fused embryos were activated with $10{\mu}M$ calcium ionophore for 5 min and 2 mM 6-dimethylaminopurine for 3 h. To examine the effect of protein supplementation, nuclear transfer (NT) embryos were cultured in one of the following 4 treatments : 1) CR1aa + 3 mg/ml BSA for 7 days ; 2) CR1aa + 10% FBS for 7 days ; 3) CR1aa + 1.5 mg/ml BSA + 5% FBS for 7 days ; and 4) CR1aa + 3 mg/ml BSA for first 3 days and then CR1aa + 1.5 mg/ml BSA + 5% FBS for 4 days. Culture took place at 5% $CO_2$, 5% $O_2$ and 90% $N_2$ at $38.5^{\circ}C$. Although there were no significant differences in cleavage rate among different protein supplements, the rates of blastocyst formation were significantly different. When NT embryos were cultured in the medium supplemented with only BSA, they could develop to only morula not to blastocyst. However, when FBS was supplemented, NT embryos developed to blastocyst stage. In order to investigate the effect of $O_2$ concentration and co-culture, NT embryos were cultured in CR1aa + 1.5 mg/ml BSA + 5% FBS with or without cumulus cell co-culture at an atmosphere of 5% $CO_2$ in air (20% $O_2$) or 5% $CO_2$, 5% $O_2$, 90% $N_2$ (5% $O_2$) at $38.5^{\circ}C$ for 7 days. The percentage of blastocyst development was significantly higher when the NT embryos were cultured at an atmosphere of 5% $O_2$ than that of 20% $O_2$ (p<0.05). However, there was no significant difference between with and without cumulus cell co-culture at an atmosphere of 5% $O_2$ or 20% $O_2$. Fifty embryos were transferred to 25 recipients and 5 recipients were pregnant at 100 days. From 5 pregnant cows, only one cow was delivered of female twin. In conclusion, the embryos reconstructed by enucleation of metaphase II oocytes and introduction of the cycling and quiescent cumulus donor cells in Hanwoo had developmental potential to term after embryo transfer to recipient cows.

• Journal of Embryo Transfer
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• v.19 no.2
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• pp.133-145
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• 2004

Serum-contain is commoly used for the production of in vitro-derived bovine embryos. However, were biological activity of serum varies from lot to lot, time consuming to choose better serum with good quality and risks of virus, bacteria and mycoplasma infection. This study established serum-free culture systems of in vitro embryo development to efficiently obtain a large number of blastocysts from ovaries of Hanwoo and oocytes maturation, cell number, tlerance of cryopreservation. Secondly, serum-contain medium is suspected of contributing to the large calf size, dystocia, cersarean sections, calf mortality and confirmed these blastocysts are high quality in terms of cyotolerance, high rates of pregancy and normal birth. For these reasons, Culture media (IVMD101 and IVD101) designed specifically for the preimplantation bovine embryo are rather simplistic, being based on salt solutions with additional energy substrates and growth factors. An improved serum-free medium (IVMD101) was developed for bovine oocytes maturation in vitro. Proportions of embryos developing to the blastocyst stage cultured in both IVD101(32.4%) and IVD101(34.5%) serum-free media were higher than in TCM199+10% FBS(12.4%) serumcontaining medium. Futhermore, the cell numbers per blastosyst obtained in the serum-free media were superior to those of blastocysts developed in serum-supplemented medium. Also, cell numbers of blastocysts obtained in the serum-free media were similar with blastocysts derived in vivo. Survival rate blastocysts after 24 hr incubation after thawing, the blastocysts cultured in both IVD101(94.5%) and IVD101(95.8%) serum-free media were higher than in TCM199+10% FBS (52.5%) serum-containing medium. After 72 hr incubation after thawing, hatching rates of blastocysts developed in IVD101(78.4%) and IVMD101(83.7%) were sighnificantly higher than that developed in the serum-supplemented medium(32.0%). The pregnancy rates almost not different between fresh blastocysts(38.2%) and frozen blastocysts(34.9%). The results suggested that the improved serum-free media(IVMD101 and IVD101) offer several advantages over culture in serum-cotaining medium, including increased rates of blastocyst formation and high cel numbers. Additionally, the survival and hatching rates of embryos product in serum-free media after post-thaw culture were superior to those of embryos produced in the serum-containg medium and useful for the production of high quality bovine embryos for cryo-preservation. These improved serum-free media are beneficial not only for the study of the mechanisms of early embryogenesis but also for mass production of good quality embryos for embryo transfer, cloning and transgenesis.

• Journal of Embryo Transfer
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• v.31 no.3
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• pp.243-247
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• 2016

Growth traits, such as body weight, directly influence productivity and economic efficiency in the swine industry. In this study, we estimate heritability for body weight traits usinginformation from pedigree and genome-wide single nucleotide polymorphism (SNP) chip data. Four body weight phenotypes were measured in 1,105 $F_2$ progeny from an intercross between Landrace and Jeju native black pigs. All experimental animals were subjected to genotypic analysis using PorcineSNP60K BeadChip platform, and 39,992 autosomal SNP markers filtered by quality control criteria were used to construct genomic relationship matrix for heritability estimation. Restricted maximum likelihood estimates of heritability were obtained using both genomic- and pedigree- relationship matrix in a linear mixed model. The heritability estimates using SNP information were smaller (0.36-0.55) than those which were estimated using pedigree information (0.62-0.97). To investigate effect of common environment, such as maternal effect, on heritability estimation, we included maternal effect as an additional random effect term in the linear mixed model analysis. We detected substantial proportions of phenotypic variance components were explained by maternal effect. And the heritability estimates using both pedigree and SNP information were decreased. Therefore, heritability estimates must be interpreted cautiously when there are obvious common environmental variance components.

• Journal of Embryo Transfer
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• v.31 no.3
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• pp.235-242
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• 2016

This study tested the association between genotypes of the single nucleotide polymorphism (SNP) marker, rs81437607 and capric acid (FA_C10_0) compositions in longissimus dorsi muscle in pigs. Eighteen fatty acid (FA) compositions were measured in a total of 974 $F_2$ animals among 1,106 $F_2$ progeny produced between Landrace and Jeju Black Pig (JBP). Among FA compositions tested, we identified a cluster of highly significant SNPs for capric acid compositions on 58 Mb position of Sus scrofa chromosome 12 (SSC12) using genome-wide association study (GWAS) with $F_2$ genotypes from SNP panel analysis. GWAS results showed that the rs81437607 was the highest trait-related SNP marker with capric acid levels. Three genotypes (C/C, C/T and T/T) of rs81437607 marker were found in $F_2$ population by further pyrosequencing. Association analysis results showed the significant differences between rs81437607 genotypes and capric acid compositions (P<0.05). The $F_2$ pigs harboring rs81437607 C/C ($0.119{\pm}0.002%$) and C/T ($0.116{\pm}0.002%$) genotypes showed additively higher levels of capric acid content than those of T/T homozygotes ($0.109{\pm}0.002%$) ($P=1.30{\times}10^{-12}$). These results suggested that the genetic variations of rs81437607 may be helpful to find causative variants and assist as molecular genetic markers for improving the capric acid contents in longissimus dorsi muscle in pigs.

• Journal of Embryo Transfer
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• v.24 no.1
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• pp.39-45
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• 2009

This study was carried out to evaluate the nuclear, cytoplasmic maturation and developmental potential of bovine oocytes selected by brilliant cresyl blue (BCB) as indirect measurement of oocytes growth phase. Cumulus-oocyte complexes (COCs) were collected from 2 to 8 mm follicles from slaughterhouse Hanwoo ovaries. The COCs were divided into stained cytoplasm to blue (BCB+) and unstained (BCB-) according to their ooplasm BCB coloration stained by $26{\mu}m$ of BCB after 90 min. Selected COCs were cultured in a TCM 199 for 18 to 26 h. Nuclear maturation and total cell number was evaluated after in vitro maturation (IVM) or in vitro culture (IVC) using $10{\mu}g/ml$ Hoechst 33342, and cytoplasmic maturation was evaluated by intracellular glutathione (GSH) assay before (0 h) and after (24 h) IVM. The oocyte diameters were not differed significantly between BCB+ ($157.4{\pm}5.8{\mu}m$) and BCB+ ($149.0{\pm}31.0{\mu}m$) groups (p>0.05). However, the proportion of metaphase II oocytes in BCB+ group was significantly higher than BCB- group after IVM (p<0.05). GSH content of BCB+ group oocytes was significantly higher than that of BCB- group just after collection ($7.3{\pm}0.6$ vs. $4.8{\pm}0.6\;pmol/oocyte$, p<0.05), but not varied after IVM($13.1{\pm}0.9$ and $12.6{\pm}2.5\;pmol/oocytes$ for BCB+ and BCB- respectively; p>0.05). The proportion of blastocyst formation and total cell number in BCB+ group (23.5% and $105.5{\pm}28.6$) was significantly higher than that in BCB- (9.8% and $72.4{\pm}26.1$; p<0.05). The results indicate that BCB+ group oocytes may provide a cellular and functional basis for the greater developmental competence in Korean Native Cow (KNC) oocytes.

• Journal of Embryo Transfer
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• v.30 no.3
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• pp.201-206
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• 2015

Selenium (Se) is an essential trace element for humans and animals, and several findings suggest that dietary Se intake may be necessary for bone health. Accumulating evidence indicates that Se compounds possess anticancer properties. Se is specifically incorporated into proteins in the form of selenocysteine and non-specifically incorporated as selenomethionine in place of methionine. This study evaluated protection by Se in the bone repair process in ovariectomized rats after irradiation. For such purpose, 80 ovariectomized female Sprague-Dawley rats were randomly divided into 4 experimental groups: ovariectomized (Ov), Ov/Se, Ov/irradiated (Irr) and Ov/ Se/Irr. A bone defect was created on the tibia of all animals 40 days after ovariectomy. Two days after surgery, only the Ov/Se and Ov/Se/Irr rats received 0.8 mg Se/kg. Three days after surgery, only the Ov/Irr and Ov/Se/Irr rats received 10 Gy of X-rays on the lower limb region. The animals were euthanized at 7, 15, 22 and 29 days after surgery to assess the repair process, which was evaluated by analysis of trabecular bone number (Masson Trichrome) and birefringence analysis (Picrosirius). It was possible to observe a delay in the bone repair process in the ovariectomized/irradiated group and similarity between the ovariectomized, Ov/ Se and Ov/Se/Irr groups. Our findings suggest that sodium selenite may influence a radioprotective effect in the bone repair of tibia of ovariectomized rats without toxicity.