• Journal of Embryo Transfer
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• v.19 no.3
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• pp.307-313
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• 2004

본 연구는 고양이의 신선 및 동결 정소상체 정액과 정소상체 정액 성상과 및 동결보존시의 생존성 및 난포란과 정소상체 정자의 체외수정 후 체외수정율과 분할율에 대해 조사하였다. 고양이 정소상체 정액의 정자농도는 3.25$\pm$0.75${\times}$$10^{6}$ cells/$m\ell$, 정자의 활력은 70.85$\pm$4.20%, 기형정자 수는 8.55$\pm$1.85%로서 대조군인 사출정액의 정자농도는 5.05$\pm$0.40${\times}$$10^{6}$ cells/$m\ell$, 정자의 활력은 90.24$\pm$455%, 기형정자 수는 4.20$\pm$0.50%와 비교할 때 정자농도와 활력은 낮았으며, 기형정자 수는 많았다. 고양이 정액과 tris-buffer로 희석한 정액을 20분간 배양했을 때 정자농도는 3.50$\pm$0.40${\times}$$10^{6}$ cells/$m\ell$, 정자활력 은 75.50$\pm$2.55%, 기 형정자 수는 6.75$\pm$0.58%로서 희석하지 않은 정소상체 정액의 성상에 비해 약간 높게 나타났다. Tris-buffer로 희석한 고양이 정소상체 정액을 동결 융해했을 때 생존율은 54.50$\pm$4.45, 활력은 47.50$\pm$6.40%로서 희석하지 .않은 대조군의 생존을 74.50$\pm$6.25%와 활력 78.50$\pm$5.20%에 비해 현저히 높게 나타났다. 고양이의 난포란과 신선 및 동결 정소상체 정자를 수정시켰을 때 체외수정율과 분할율은 68.30$\pm$5.35%, 57.25$\pm$4.35% 및 48.65$\pm$4.95%, 35.65 $\pm$4.75%로서 신선 정자에 비해 동결 정소상체 정자로 수정시킨 군의 분할율이 유의하게 낮았다.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.1
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• pp.47-57
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• 2000

Objective: The effects of cryopreservation with or without coculture on the in vitro development of blastomere-biopsied 8-cell mouse embryos were investigated. This experimental study was originally designed for the setup of a preclinical mouse model for the preimplantation genetic diagnosis (PGD) in human. Methods: Eight-cell embryos were obtained after in vitro fertilization (IVF) from F1 hybrid mice (C57BL(표현불가)/CBA(표현불가)). Using micromanipulation, one to four blastomeres were aspirated through a hole made in the zona pellucida by zona drilling (ZD) with acid Tyrode's solution (ATS). A slow-freezing and rapid-thawing protocol with 1.5M dimethyl sulfoxide (DMSO) and 0.1M sucrose as cryoprotectant was used for the cryopreservation of blastomere- biopsied 8-cell mouse embryos. After thawing, embryos were cultured for 110 hours in Ham's F-10 supplemented with 0.4% bovine serum albumin (BSA). In the coculture group, embryos were cultured for 110 hours on the monolayer of Vero cells in the same medium. The blastocyst formation was recorded, and the embryos developed beyond blastocyst stage were stained with 10% Giemsa to count the total number of nuclei in each embryo. Results: The survival rate of embryos after cryopreservation was significantly lower in the blastomere-biopsied (7/8, 6/8, 5/8, and 4/8 embryos) groups than in the non-biopsied, zona intact (ZI) group. Without the coculture, the blastocyst formation rate of embryos after cryopreservation was not significantly different among ZI, the zona drilling only (ZD), and the balstomere-biopsied groups, but it was significantly lower than in the non-cryopreserved control group. The mean number of cells in embryos beyond blastocyst stage was significantly higher in the control group ($50.2{\pm}14.0$) than in 6/8 ($26.5{\pm}6.2$), 5/8 ($25.0{\pm}5.5$), and 4/8 ($17.8{\pm}7.8$) groups. With the coculture using Vero cells, the blastocyst formation rate of embryos after cryopreservation was significantly lower in 5/8 and 4/8 groups, compared with the control, 7/8, and 6/8 groups. The mean number of cells in embryos beyond blastocyst stage was also significantly lower in 4/8 group ($25.9{\pm}10.2$), compared with the control ($50.2{\pm}14.0$), 7/8 ($56.0{\pm}22.2$), and 6/8 ($55.3{\pm}25.5$) groups. Conclusion: After cryopreservation, blastomere-biopsied mouse embryos have a significantly impaired developmental competence in vitro, but this detrimental effect might be prevented by the coculture with Vero cells in 8-cell mouse embryos biopsied one or two blastomeres. Biopsy of mouse embryos after ZD with ATS is a safe and highly efficient preclinical model for PGD of human embryos.

• Reproductive and Developmental Biology
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• v.28 no.2
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• pp.83-87
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• 2004

The objective of this study was performed to establish the in vitro culture system of porcine in vitro maturation and in vitro fertilization(IVM/IVF) embryo. These studies was to determine the effects of melatonin, nitric oxide donor(SNP), and the combination effects of SNP and melatonin in porcine IVM/IVF embryos. In routine porcine IVM/IVF procedure, oocytes were cultured for 40∼44h incubation, and the zygotes were cultured for 40∼44h in NCSU 23 medium. Then 2 to 8 cell embryos were removed cumulus cell and were allotted randomly to NCSU 23 containing different concentration of melatonin, SNP and SNP plus melatonin in 5% $O_2$, 5% $CO_2$ and 90% $N_2$ at 38.5$^{\circ}C$. Cell numbers of blastocyst were also counted using double fluorescence stain method. In NCSU 23 medium treated with melatonin 0, 1, 5 and 10 nM, the developmental rate of morula plus blastocysts were 33.3%, 39.1%, 33.3% and 27.9%, respectivly. This result show that the developmental rate of morula and blascytocys treated with 1 nM melatonin was higher than in any other groups(P<0.05). The developmental rates of morula plus blastocysts were 41.9% in 0 uM SNP, 25.6% in 50 uM and 28.4% in 100 uM, respectively. The developmental rate of morula plus blastocysts were decreased treated with SNP in NCSU 23. In combined effects of SNP plus melatonin (0, SNP 50 uM, SNP 50 uM plus melatonin 1 nM, SNP 50 uM plus melatonin 5 nM and SNP 50 uM plus melatonin 10 nM), the developmental rates beyond morula stage of porcine embryos were 31.3%, 34.1%, 39.5%, 29.4% and 39.5%, respectively. The addition of SNP 50 uM plus maltonin 1 nM, developmental rates of blastocyst was higher rate than in any other groups. Cell numbers of blastocyst in NCSU 23 treated with melatonin 0, 1, 5 and 10 nM were 41.0, 42.6, 39.6 and 33.0, respectively. In combined effects of SNP plus melatonin (0, SNP 50 uM, SNP 50 uM plus melatonin 1 nM , SNP 50 uM plus melatonin 5 nM and SNP 50 uM plus melatonin 10 nM), cell numbers of developed blastocyst were 36.3, 34.6, 39.0, 39.9 and 39.0, respectively. These result show that the cell numbers of blastocyst treated with 0, 1 and 5 nM melatonin were higher than in 10 nM group(P<0.05), but cell numbers of blatocyst produced by SNP plus melatonin were not significantly difference in all experimental groups.

• Journal of Embryo Transfer
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• v.30 no.2
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• pp.73-82
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• 2015

This study was designed to evaluate the possibility of increase through dairy female offspring's ratio by transfer of pre-selected transferrable blastocyst that was produced by pre-selected X-bearing semen with OPU derived oocytes. Elite dairy female cow is demanded strongly compared with male, the so called, farmer wants to produce only an elite female dairy offspring as a candidate female dairy cow for producing milk. In our study, we selected 2 elite dairy bull semen from National Agricultural Cooperative Federation to pre-select X-bearing semen and 5 elite dairy female cows as donor for collecting of OPU derived oocytes. OPU derived embryo production system was carried out an aspiration of immature oocytes from 5 donor cows 2 times per week, total 200 times for 2 to 7 months by an ultrasonographic guided follicular aspiration system and then produced in vitro-produced blastocysts by in vitro maturation, fertilization and culture. Dairy donor semen selected H-319, 320 bull in National Agricultural Cooperative federation was sorted X-bearing semen by flow-cytometer and frozen for using IVF with OPU derived oocytes. Donor cows were selected 5 elite dairy cows from Gyeongju Dairy Cow Community and then disease tests such as 4 kinds of disease before selecting was checked. Oocyte proportion of grade 1 to 3 from total collected oocytes was significantly lower in donor A and B than those in donor C, D and E (82.16 and 70.03% vs. 90.0, 91.78 and 93.57%), respectively (p<0.05). However, number of oocytes per session in donor A, C and E was significantly higher than those in donor B and D ($7.77{\pm}3.26$, $5.85{\pm}2.10$ and $7.03{\pm}2.14$ vs. $4.68{\pm}2.61$ and $5.21{\pm}1.97$ oocytes), but donor A was significantly higher than donor C (p<0.05). Development to blastocyst in donor B, C and E was significantly higher than those in donor A and D (31.0, 25.0 and 25.0% vs. 14.3 and 4.5%), but donor A was not different in donor C and E (p<0.05). Nine out of 10 blastocysts (90.0%) derived from OPU blastocysts were confirmed male embryos that was induced with Y-bearing semen to confirm sex ratio only. Total 96 blastocysts derived from female bearing semen were transferred into synchronized recipients and then confirmed 42 recipients (43.8%) pregnancy rate, 36 offspring (37.5%) and 91.7% female sex ratio (33 female vs. 3 male offspring). Taken together all data, elite dairy female offspring could be produced effectively by in vitro production system between pre-selected x-bearing semen and OPU derived oocytes that would be influential breeder in the breeding of dairy farm to increase effectively elite dairy offspring ratio as well as net income in the dairy farmer.

• Reproductive and Developmental Biology
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• v.28 no.2
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• pp.77-82
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• 2004

The purpose of this study was performed to establish the in vitro culture system of porcine in vitro maturation and in vitro fertilization(IVM/IVF) embryo. These studies was to determine the effects of antioxidants(aesculetin, taurine and melatonin) in porcine IVM/IVF embryos. In routine porcine IVM/IVF procedure, oocytes were cultured for 40∼44h incubation in NCSU 23 mediumand matured oocytes were inseminated with frozen semen. Then 2 to 8 cell embryos were removed cumulus cell and were allotted randomly to NCSU 23 containing different concentration of antioxidants in 5% $O_2$ and 5% $CO_2$ at 38.5$^{\circ}C$. Cell numbers of blastocyst were also counted using double fluorescence stain method. Aesculetin were added to NCSU 23 medium at concentration of 1 ug, 5 ug, and 10 ug, when treated with 10 ug(35.7%) of aesucletin at the rate of embryos of the morula plus blatocsyts were higher than those of any other groups (30.2%, 29.5% and 29.2%)(P<0.05). The developmental rates beyond morula stage of porcine embryos in NCSU 23 medium supplemented with taurine 0, 2.5 and 5.0 mM were 26.1%, 26.9% and 31.7%, respectively. The addition of 5.0 mM taurine was higher the developmental rate beyond morula stage than in any other groups. In NCSU 23 medium treated with melatonin 0, 1, 5 and 10 nM, the developmental rate of morula plus blastocysts were 33.3%, 39.1%, 33.3% and 27.9%, respectively. The developmental rate of morula and blascytocys treated with 1nM melatonin was higher than in any other groups(P<0.05). Cell numbers of blastocyst in NCSU 23 treated with melatonin 0, 1, 5 and 10nM were 41.0, 42.6, 39.6 and 33.0, respectively. These results indicate that aesculetin, taurine and melation can increase the developmental rate beyond the morulae and blastocysts in porcine embryos.

• Horticultural Science & Technology
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• v.19 no.3
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• pp.378-383
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• 2001

Embryo culture, ovule culture and ovary slice culture were tested to find optimum method for overcoming post fertilization barrier in interspecific crosses between L. longiflorum 'Gelria' and L. cernuum. Although reciprocal crosses between the species were carried out by cut-style pollination method, fruits developed only in crosses of L. longiflorum${\times}$L. cernuum. On the 40 days after pollination, ovaries were sliced into 2-4mm thickness and cultured on a hormone-free Murashige-Skoog (MS) medium, supplemented with 2%, 4%, 6%, 8% and 10% sucrose. For the L. longiflorum Gelria'${\times}$L. cernuum cross, ovule development was found to be best at 6% sucrose and a lot of hybrid plant lets established directly from the ovary slice culture and subsequent ovule culture. High concentration of sucrose above 8% made ovules abort or vitrificate from 40 days after culture. In contrast, ovules from the L. cernuum${\times}$L. longiflorum 'Gelria' cross swelled well in ovary slice culture, however, they did not germinated in subsequent ovule culture. On the 60 days after pollination, ovules thicker than 0.6mm was interpreted as one containing embryo. The embryo size ranged from 1.2 mm to 1.7 mm, and in vitro development of the excised embryos was found to be best with the MS medium (pH 5.8), supplemented with $0.1-1 mg{\cdot}L^{-1}$ NAA and 6% sucrose. Thick ovules excised 60 days after pollination germinated about 60% as normal seeds in MS medium supplemented with 6% sucrose and free hormone. The ovule culture 60 days after pollination was concluded to be most recommendable to produce interspecific hybrids in large scale crosses between L. longiflorum 'Gelria' and L. cernuum by the reason of easy procedure.

• Korean Journal of Animal Reproduction
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• v.26 no.1
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• pp.41-51
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• 2002

The present study was carried out to examine the effect of cysteamine in vitro maturation (IVM) of porcine oocytes and development of porcine IVM/IVF Embryos. The results were summarized as follows : 1. The rates of nuclear maturation, penetrated oocytes, pronuclear formation, polyspermic oocytes and mean numbers of the penetrated sperm were not different in NCSU23 maturation medium with 0, 25, 50 and 100 $\mu$M cysteamine (P〉0.05). 2. The rates of blastocyst formation at day 7 after in vitro fertilization in 0, 25, 50 and 100 $\mu$M cysteamine were 17.9$\pm$6.1, 17.4$\pm$6.3, 24.2$\pm$1.9 and 16.9$\pm$2.0%, respectively. And the total cells were 30.7$\pm$2.4, 34.9$\pm$2.8, 39.6$\pm$2.3 and 36.8$\pm$3.6, respectively. Fifty $\mu$M cystealnine group was significantly higher than those of any other treatment groups (P＜0.05). 3. The ratios of ICM/total cells in 20~40% category were 20.5, 41.6, 19.5 and 31.5%, respectively. Twenty five $\mu$M cysteamine group was higher than those of other groups. 4. The rates of blastocyst formation at day 7 in the NCSU-23 culture medium of porcine IVF-produced embryos with 0, 25, 50, and 100 $\mu$M cysteamine were 16.0$\pm$0.2, 13.6$\pm$1.7, 25.0$\pm$0.8 and 15.7$\pm$4.5%, respectively. And the total cells were 27.0$\pm$3.7, 36.1$\pm$4.8, 34.0$\pm$3.8 and 25.2$\pm$4.4, respectively. Fifty $\mu$M cysteamine group was significantly higher than those of any other treatment groups (P＜0.05). 5. The ratios of ICM/total cells in 20~40% category were 53.8, 30.0, 16.6 and 11.1%, respectively. The addition groups of cysteamine were lower than those of control group. In conclusion, these results suggested that the addition of 50 $\mu$M cysteamine in the IVM medium and 25~50 $\mu$M cysteamine in IVC medium were effective on the blastocyst formation and total cells of blastocysts.

• Korean Journal of Animal Reproduction
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• v.26 no.3
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• pp.215-221
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• 2002

Antioxidants(N-acetyl-1-cysteine, ebselen and glutathione) and growth factors(EGF, PDGF) were studied as a mean of increasing the development of porcine embryos produced by in vitro maturation (IVM) and in vitro fertilization(IVF). Porcine embryos developed to the 2~8 cell stage after IVF were cultured fer 6 to 7 days at 38.5$^{\circ}C$ in NCSU 23 medium containing antioxidants plus growth factors. Cell numbers of blastocysts were counted by fluorescence staining method. The developmental rate beyond morula stages in NCSU 23 containing NAC(1nm) or NAC(1nm) plus EGF(100ng/$m\ell$) or PDGF(5ng/$m\ell$) were 28.1, 32.3 and 35.3%, respectively. NAC plus PDGF group was slightly higher than control group(P>0.05). The developmental capacity in NCSU 23 containing ebselen(10 $\mu$M) or ebselen(10 $\mu$M) plus EGF(100ng/$m\ell$) or PDGF(5ng/$m\ell$) were 17.8, 36.9 and 40.3%, respectively Ebselen plus growth factor groups were significantly higher than control group(P<0.05). The developmental capacity in NCSU 23 containing glutathione(100 $\mu$M) or glutathione(100 $\mu$M) plus EGF (100ng/$m\ell$) or PDGF(5ng/$m\ell$) were 24.1, 30.5 and 27.7%, respectively. There were not difference in all experimental groups(P>0.05). In all experimental groups, there was no significantly differences on the cell number of blastocysts, but ebselen plus growth factor groups were significantly higher than control group. These studies indicate that antioxidants plus growth factors can increase the proportion of embryos that developed beyond morulae stage.

• Journal of Embryo Transfer
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• v.20 no.3
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• pp.289-295
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• 2005

This study was performed to investigate the relationship between Heat shock protein 70 (HSP70) gene polymorphism and in vitro fertilization(IVF) embryo development in the pigs. The single strand conformation polymorphism(SSCP) genotypes from HSP70 K1, K3 and K4 PCR products were detected different patterns. In cleavage rate of oocyte fertilized in vitro, HSP70 K1-AA genotype($73.1\%$) and K1-AB genotype($62.3\%$) showed significantly higher oocyte cleavage rate than HSP70 K1-BB genotype($49.3\%$)(p<0.05). And HSP70 K3-AA genotype ($72.4\%$) and K3-AB($62.2\%$) also showed significantly higher oocyte cleavage rate than HSP70 K3-BB genotype($49.1\%$)(p<0.05). The IVF embryo development of 2-cell stage according to HSP70 genotypes of sperm and pig breeds also showed a significant difference. The number of embryos developed to 2-cell stage in Landrace(28.8) and Duroc(29.8) were significantly higher than in Yorkshire(10.9)(p<0.05). And also HSP70 K4-AB genotype group(29.6) higher than HSP70 K4-AA genotype group(10.6)(p<0.05). However, the number of embryos developed to blastocyst stage did not showed significant differences among breeds as well as HSP70 genotypes. These resrults suggest that in vitro development in porcine early embryos may be affected by HSP70 genotypes and breeds.

• Journal of Embryo Transfer
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• v.19 no.2
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• pp.155-163
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• 2004

The aims of this study are 1) to test oocytes and embryos collected from in-vitro to achieving the valuable protocol by culturing, vitrifying and thawing of oocytes/embryos, and 2) to transfer them to recipient, and finally have resulted in pregnancies from recipient females after surgical or nonsurgical transfer. In vitro maturation and fertilization were performed according to Funahashi et al (1994). Glucose-free NCSU 23 supplemented with 5 mM sodium pyruvate, 0.5 mM sodium lactate and 4 mg/ml bovine serum albumin for 2 days at $39^{\circ}C$, and 10% fetal bovine serum albumin was added to the culture medium thereafter. Embryos were treated with 7.5 ${\mu}g/ml$ cytochalasin-B for 30 min, centrifuged at 13,000 rpm for 13 min and then exposed sequentially to an ethylene glycol(EG) vitrification solution, aspirated into OPS, and plunged/thawed into/from liquid nitrogen. In vivo embryos were surgically collected from three dornors after AI for control group. Forty-nine embryos were washed 3 times in mPBS + 10% FBS, followed treatments : cultured, centrifuged, vitrified, recovered and transferred to recipients as in vitro prepared embryos. Three recipients were transferred individually with 100, 100 frozen embryos derived from abattoir and 34 fresh embryos by surgically, and another three recipients were transferred individually with 150, 150 frozen embryos and 100 fresh embryos by nonsurgically, respectively. all recipient sows exhibited delayed returns to estrus. To our knowledge, theses results suggest that required an improved techniques, more vigorous embryos preparation and substitute to gilt with cleaner uterous condition.