• Journal of Embryo Transfer
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• v.17 no.2
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• pp.129-136
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• 2002

The purpose of this study was to investigate the comparison of viability of bovine blastocysts following glass micropipette (GMP) vitrification and the possibility of production of Korean Native Cow ("Hanwoo,"Bos taurus coreanae) following embryo transfer into Mongolia cows (Bos taurus mongolian). The embryos of Korean Native Cow were produced by IVMFC or superovulation methods in Korea, cryopreserved by GMP vitrification, and subsequently trans-ported to Mongolia. The recipient cows were synchronized using a CIDR plus and prostaglandin $F_2\alpha$($PGF_2\alpha$) treatment. To produce in vivo embryos, seven cows were superovulated using FSH and PGF$_2$／sub $\alpha$／ treatment. A total of 64 blastocysts ( $9.1\pm2.94$ per cow) were collected. In vitro embryos were produced using a defined culture system which cleaved in 80.1％ ova (174／217), and developed to blastocyst stage embryos of 40.8％ (71／174). The post-thaw survival rate of in vivo blastocysts (93.7％; 45／48) was significantly higher than that of in vitro blastocysts (82.5％; 52／63, P＜0.05). Embryo transfer was carried out using 8 Mongolian recipient cows and 2 post-thaw blastocysts per recipient. Five of 8 recipients were found pregnant at Day 60 but one abortion occurred by Day 240. Two of offspring were produced from the Mongolian cows at 275 days after embryo transfer. These results indicated that a GMP vitrification method could be used as a cryopreservation technique for in vivo or in vitro bovine blastocysts and produced effectively a Korean Native Cow following embryo transfer into a Mongolian recipient cow.

• Korean Journal of Animal Reproduction
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• v.17 no.3
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• pp.233-242
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• 1993

The effects of in vitro maturation and sperm treatment condition on the in vitro fertilization (IVF) and developmental capacity of bovine oocytes were investigated and the development of embryos was compared under the 2 different co-culture system, with GC or BOEC. The cultured embryo to 16 cell or morula wre transferred into recipients or frozen by 2 different freezing method. The results obtained were summarized as follows; 1. In vitro maturation rates of vovine follicular oocytes cultrued in TCM199 with 10% FCS or ECS were 64.0% and 72.7%, but the case of addition of 10% FCS or ECS to TCM199 co-cultured with granulosa cells were 81.3% and 84.0%, respectively. IVM rate of three TCM199 added to granulosa cells was higher than that of media without granulosa cells. 2. When bovine follicular oocytes were matured in TCM199 with 10% FCS and GC and then fertilized in vitro by sperm treated with caffeine, embryo developments of bovine oocytes co-cultured with BOEC were 38.4% and 51.4%, respectively. But those of bovine oocytes co-cultured with GC were 52.2% by sperm treated with caffeine-heparin. 3. Cleavage rates of bovine oocytes cultured with 10% FCS alone and fertilized in vitro by sperm treated with caffeine-heparin was 33.0%. 4. When bovine follicular oocytes were matured in TCM199 with 10% FCS and GC, embryo developments of bovine ooctyes co-cultured with BOEC of GC were 46.0% and 50.2%, respectively. 5. When bovine follicular oocytes were matured in TCM199 with 10% ECS and GC, embryo developments co-cultured with BOEC or GC were 45.2% and 51.4%, respectively. 6. When Korean Native cow's follicular oocytes matured in TCM199 with 10% FCS and GC, embryo developed co-cultured with BOEC or GC were 45.2% and 51.4%, respectively. 6. When Korean Native cow's follicular oocytes matured in TCM199 with 10% FCS and GC, embryo developments of the bovine oocyte co-cultured with BOEC and GC were 41.8% and 60.1%. But with FCS 10% those of the bovine oocytes co-cultured with BOEC and GC were 42.0% and 48.4%, respectively. 7. When Holstein's follicular oocytes were matured in TCM199 with 10% ECS and GC, embryo developments fo the bovine oocytes co-cultured with BOEC and GC were 50.0% and 57.7%, but with ECS 10% those of the bovine oocytes co-cultured with BOEC and GC were 52.2% and 56.5%, respectively. 8. The viability of frozen-thawed embryos ranged from 60~80% and those of frozen-thawed embryos from vitrification was lower than that from conventional metiod. 9. The selected fresh embryos were transferred nonsurgically to 7 recipients but did not result in pregnancy.

• Fisheries and Aquatic Sciences
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• v.21 no.9
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• pp.23.1-23.8
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• 2018

Background: Russian sturgeon (Acipenser gueldenstaedtii) is an emerging candidate species in the Korean aquaculture domain owing to its highly valued caviar. Although the embryonic development of this species was previously described, the complete image data on the morphological differentiation of developing embryos have not been yet fully available. Further, with the viewpoint of larval production in hatchery, the effects of temperature on embryonic viability and the temporal window of hatching event have not been extensively studied. Hence, the objective of this study was to provide a complete set of photographic image data on the embryogenesis and also to examine the effects of incubation temperatures on embryonic viability and hatching event in farm-bred Russian sturgeon. Results: Typical characteristics of embryonic development including uneven, holoblastic cleavages with unequal blastomeres, followed by the formation of germ layer, neurulation, and organogenesis until hatching, were documented. Under different temperature conditions (12, 16, or $20^{\circ}C$), viability of embryos incubated at $12^{\circ}C$ was significantly lower as relative to those of 16 and $20^{\circ}C$ incubated embryos. Hatchability of embryos was higher, and the timing of hatching event was more synchronized at $20^{\circ}C$ than at 12 and $16^{\circ}C$. Conclusion: Data from this study suggest that the incubation of Russian sturgeon embryos at $20^{\circ}C$ would be desirable in the hatchery practice with respect to the good hatchability of embryos and the synchronization of hatching events. Additionally, the updated image data for complete embryonic development could be a useful reference guide for not only developmental researches but also artificial propagation of Russian sturgeon in farms.

• Forest Science and Technology
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• v.14 no.4
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• pp.160-168
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• 2018

The seeds of Elaeocarpus serratus, a tropical underutilized fruit tree are characterized by hard seed coat and consequent poor water uptake and low germination. To improve the regeneration through seeds, various parameters such as viability of seeds, water uptake, and effect of seed mass on germination and pretreatments were performed using a completely randomized design (CRD). Tetrazolium (TZ) test was conducted using fresh, mature seeds revealed $50{\pm}2.56%$ mean viability. Seeds of different weight classes showed similar pattern of water uptake and the saturation level was achieved at 60 hrs of soaking. Seeds belong to weight class 2.6-3.5g were germinated ($12.5{\pm}1.26%$) with $175{\pm}1.75days$ (d) of mean time taken for germination (MTG). Germination capacity of seeds varied significantly among different populations and Varkala population gave $12.5{\pm}1.1%$ germination with $174.6{\pm}2.5d$ MTG. Among various seed treatments, mechanical scarification was superior in germination and significant reduction in MTG ($p{\leq}0.05$). The mechanical scarification by complete removal of seed coat resulted in $49.2{\pm}1.52%$ germination within a short period of time ($9.52{\pm}0.89d$ MTG). However, the complete removal of seed coat without damaging to embryo is a difficult task. An alternate treatment (Mechanical scarification II) by making cracks on nut faces vertically followed by soaking in distilled water for 24 hrs gave $48.4{\pm}1.73%$ germination with significantly reduced MTG ($12.14{\pm}0.56d$) over unsoaked, untreated control ($6.5{\pm}1.84%$ germination and $197.18{\pm}1.79d$ MTG; $p{\leq}0.05$). This treatment (Mechanical scarification II) is therefore recommended for E. serratus seeds as it can adopt easily and can achieve 7 fold increases in germination over control. The recorded germination through mechanical scarification is in tune with realized viability percentage of the seeds.

• Journal of Embryo Transfer
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• v.30 no.1
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• pp.7-15
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• 2015

The objective of this study was to investigate the efficiency of nicotinic acid on sperm cryosurvival and fertilization ability in frozen-thawed boar semen. Boar semen was collected by glove-hand method and was frozen using freezing solution treated to 0, 5, 10 and 20 mM of nicotinic acid. The frozen sperm for sperm characteristic analysis was thawed such as viability, acrosome reaction, and mitochondrial integrity. The frozen-thawed sperm was estimated by SYBR14/PI double staining for viability, FITC-PNA/PI double staining for acrosome reaction and Rhodamine123/PI double staining for mitochondrial integrity using a flow cytometry. The embryo was estimated in vitro development and DCFDA staining for reactive oxygen species assessment. As results, frozen-thawed sperm viability was significantly higher in 5 and 10 mM ($61.1{\pm}1.5%$,$64.7{\pm}2.0%$) of nicotinic acid than other groups (0 mM, $52.1{\pm}2.3%$; 20 mM, $47.8{\pm}5.1%$, P<0.05). The live sperm with acrosome reaction was significantly higher in 5 and 10 mM of nicotinic acid ($26.1{\pm}1.8%$, $24.9{\pm}1.5%$) than other groups (0 mM, $35.3{\pm}0.8%$; 20 mM, $36.5{\pm}1.9%$, P<0.05). The live sperm with mitochondrial integrity was significantly higher in 5 and 10 mM ($84.2{\pm}3.6%$, $88.4{\pm}2.3%$) of nicotinic acid than other groups (0 mM, $77.3{\pm}4.4%$; 20 mM, $73.3{\pm}3.6%$, P<0.05). Blastocyst rate of in vitro development was significantly higher in 10 mM ($17.0{\pm}1.3%$) of nicotinic acid than other groups (0 mM, $9.4{\pm}0.5%$; 5mM, $12.6{\pm}0.8%$; 20 mM, $5.0{\pm}1.0%$, P<0.05). Moreover, total cell number was higher in 5 and 10 mM ($53.6{\pm}2.9%$, $57.9{\pm}2.8%$) of nicotinic acid than other groups (0 mM, $41.0{\pm}1.4%$; 20 mM, $23.2{\pm}2.8%$, P<0.05). Hydrogen peroxide in embryos was lower in 5 mM nicotinic acid ($0.7{\pm}0.1%$) than other groups (0 mM, $1.0{\pm}0.1%$; 10mM, $0.9{\pm}0.0%$; 20 mM, $1.4{\pm}1.0%$, P<0.05). In conclusion, nicotinic acid-treated semen improves cryosurvival and quality of spermatozoa. Also, the fertilized oocytes with nicotinic acid improve quality of embryo and blastocyst formation.

• Journal of Embryo Transfer
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• v.30 no.4
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• pp.277-282
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• 2015

Cryopreservation affects osmotic tolerance and intracellular ion concentration through changes in expression levels of water and ion channels. Control of these changes is important for cell survival after cryopreservation. Relatively little is known about changes in $K^+$ channel expression compared to water channel expression. This study was performed to investigate changes in TASK-2 channel (KCNK5: potassium channel, subfamily K, member 5), a member of two-pore domain $K^+$ channel family, in cryopreserved mouse ovaries. Cryopreservation increased TASK-2 mRNA expression in mouse ovaries. In addition, TASK-2 protein expression was upregulated in vitrified and slowly frozen ovaries. TASK-2 protein was expressed in all area of granulosa cells that surround the oocyte within the follicle, except nucleus. Viability of cells overexpressed with TASK-2 was higher than that of vector-transfected cells. Our results found that TASK-2 expression was increased by cryopreservation and overexpression of TASK-2 decreased cryopreservation-induced cell death. These results suggest that TASK-2 upregulation might reduce cryodamage.

• Korean Journal of Animal Reproduction
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• v.12 no.1
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• pp.31-36
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• 1988

There studies were conducted using inbred ICR mice to examine the sex of preimplantation mouse embryo. The morphological normality of mice embryos treated with the culture medium containing rat H-Y antiserum(10%, v/v) plus complement(20%,v/v) was observed and also the sexing of embryos was investigated by chromosomal analysis. The results obtained were summarized as follows: 1. The viability of preimplantation mouse embryos, which were incubated in vitro with different media condition, was scored 68.9-85.5% in control group. However, 151 embryos normally developed up to blastocyst and 160 embryos were retarded growth or destroyed out of total 311 embryos treated in the medium containing H-Y antiserum(10%, v/v) plus complement(20%,v/v). 2. H-Y antiserum was prepared from inb red rats (Wistar and Donryu strain) with different immunization times (4, 5 and 6th) to examine the specific titer of embryos by the number of immunization. Precentage of normally developed embryos incubated either in the medium containing the antiserum of Wistar plus complement or Donryu plus complement was revealed 50.9, 47.4 and 50.0% (4, 5 and 6th immunization and 47.8, 41.2 and 48.7%, respectively. 3. Twenty two females and five males were identified out of fourty-eight normally developed embryos incubated in the medium containing H-Y antiserum plus complement by chromosomal analysis.

• Journal of Embryo Transfer
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• v.21 no.1
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• pp.1-5
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• 2006

본 연구는 개 채취 정액의 동결후의 생존성과 신선 및 동결 정자의 capacitation, acrosome reaction과 생존성을 조사하고, 아울러 신선 및 동결 정액을 자연 발정 또는 발정 유기 암캐 에게 인공수정 후 임신율을 조사하였다. 개 채취 정액의 동결 융해 후의 생존성은 $64.5{\pm}2.30%$로서 신선 정액의 생존성에 비해 유의하게 낮게 나타났다(p<0.05). 신선 및 동결 정액의 capacitation, acrosome reaction 및 생존성은 각각 $52.5{\pm}4.5%,\;9.5{\pm}0.6%,\;68.8{\pm}4.5%$ 및 $16.2{\pm}3.2%,\;3.2{\pm}0.5%,\;24.5{\pm}2.5%$로 나타났다. 신선 및 동결 정액을 자연 발정 또는 발정을 유기한 암캐에 인공수정했을 때 임신율은 각각 50.0% 및 33.3%로서 동결 정액을 이용했을 때 임신율이 신선 정액에 비해 낮은 임신율을 나타냈다.

• Journal of Embryo Transfer
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• v.33 no.1
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• pp.41-48
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• 2018

In the last several decades, cell therapy research has increased worldwide. Many studies have been conducted on cell therapy, and have revealed that transplanted cells did not survive for long, and implanted cells remained inactive causing immune rejection depending on the patient's condition. Therefore, studies on cell-free therapy need to be conducted. To overcome these limitations, an alternative is the use of supernatant from cells, called "conditioned media (CM)." During in vitro cell culture, culture media supply nutrients to maintain cell characteristics and viability. In the culture, cells not only consume nutrients but also release beneficial proteins and substances, which are called "secretome." CM from cells can be stored for a long time and is easy to handle. Moreover, secretome in CM can also be measured; exact amount of secretome is important to set the standard value for disease treatment. Here, we reviewed studies on CM and confirmed that various secretomes from CM were identified in these studies. Moreover, these findings could benefit cell and animal studies in future. In conclusion, CM could be a potential candidate for an alternative to cell therapy.

• Journal of Embryo Transfer
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• v.31 no.1
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• pp.9-12
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• 2016

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are oocyte-specific growth factors that regulate many critical processes involved in early folliculogenesis and oocyte maturation. In this study, effects of GDF9 and BMP15 treatment during in vitro maturation of porcine oocytes upon development after parthenogenetic activation were investigated. Neither GDF, BMP15 alone nor in combination affects the number and viability of cumulus cells or the rates of oocyte maturation and blastocyst development. However, the treatment of GDF9 on porcine oocytes increased the number of trophectodermal (TE) cells of blastocysts derived from activated oocytes (P<0.05). The treatment of BMP15 increased the cell numbers of both inner cell mass (ICM) and TE cells (P<0.05). The treatment with the combination of GDF9 and BMP15 further increased the numbers of ICM and TE cells, compared with GDF9 or BMP15 treatment alone (P<0.05). In conclusion, the treatment of GDF9 or BMP15 (or both) enhanced the quality of blastocysts via the increased number of ICM and/or TE cells.