• Journal of Embryo Transfer
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• v.26 no.1
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• pp.65-70
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• 2011

This study was conducted to investigate the survival rate of AndroMed and Tris-egg yolk extender for cryopreservation of Korean Native Bull Semen (Chick Cow). Semen was collected from a Korean Native Bull Semen over 3 year's old. The semen was diluted 1:1 by AndroMed and Tris-egg yolk extender. The pellet was diluted to final sperm concentration of $5{\times}10^7$ cell/ml by doubling in every 10 minutes at $4^{\circ}C$ cold chamber. The semen was equilibrated for 1 hrs at cold chamber and packed to 0.5 ml straw. The semen straws were located above 5 cm of liquid nitrogen for 5 minutes. And then the frozen straw was plunged to LN2. The presented straws were examined the viability and motility after thawed at $37^{\circ}C$ water bath. The survival rates was significantly higher (p<0.05) in Tris-egg yolk extender than AndroMed extender ($89.7{\pm}19.8$ vs. $73.4{\pm}11.2$). However, motility was no significant differences ($78.4{\pm}18.7$ vs. $67.9{\pm}14.6$). Survival rate in time of equilibration between visual and CASA program had higher in 2 h ($86.33{\pm}9.4$ vs. $92.32{\pm}12.4$) than in 5 h ($78.20{\pm}7.8$ vs. $88.28{\pm}13.1$) 15 h ($65.24{\pm}6.6$ vs. $76.48{\pm}17.3$) 20 h ($56.26{\pm}4.6$ vs. $67.73{\pm}18.4$). The development rates to cleavage was higher in Tris-egg yolk extender than AndroMed extender (82.2% vs. 81.7%). Similarly, the development rates to blastocyst was significantly higher (p<0.05) in Tris-egg yolk extender than AndroMed extender (42.3% vs. 29.6%). In conclusion, the obvious impact of this study will be its practical application to improve viability and fertilizing ability of cryopreserved spermatozoa used for in vitro fertilization (IVF) and AI, Which in turn will be beneficial to animal genetic resources conservation.

• Journal of Embryo Transfer
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• v.16 no.3
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• pp.193-201
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• 2001

This study was designed to examine the factors affecting in fertilization and development of embryos in vitro, and to examine whether zone drilling by laser irradiation can improve the hatching rate of IVF embryos from DNA marker-proved Hanwoo. DNA markers related to marbling score were identified using DNA fingerprinting with Ml3 probe and restriction enzyme Hae III. Oocytes were aspirated from immature ovarian follicles using a combined method of rectal ovarian-palpation and transvaginal ultrasound-guidance(6.5MHz) under local anesthesia. The aspirated oocytes were washed twice with fresh D-PBS containing 5% FBS and were rewashed 4 to 5 times with TCM-199 containing 5% FBS. A morphological grade of I to IV was assigned to each oocyte. Data were analyzed using the GLM procedure of SAS. Sperm separation methods did not have any significant effect on cleavage or developmental abilities of IVF embryos. Significantly(P<0.05) higher cleavage rate was observed in embryos from GI(60.0%, 3/5), GII(69.2%, 18/26) and GIII(62.1%, 59/95) compared to embryos from GIV oocytes(36.2%, 25/69). And the developmental rate to blastocyst stage was higher(P<0.05) in embryos from GI(33.3%, 1/3) and GII oocytes(38.9%, 7/18) than those from GIII(16.9%,10/59) and GIV oocytes(4.0%, 1/25). There was no significant difference in development of IVF embryos to blastocyst by media for in vitro culture. Proportion of hatched blastocyst was significantly(P<0.05) higher in embryos received zona drilling by laser than those of non-drilled.

• Journal of Embryo Transfer
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• v.17 no.1
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• pp.45-53
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• 2002

The main goal of this study was to produce transgenic porcine embryos by direct injection of sperm-mediated exogenous DNA. Spermatozoa (6$\times$10$^{6}$ sperms of final concentration) were mixed with pcDNA LAC Z (20 ng/$\mu$l) and subjected into electroporation (300~750 volts, 25 $\mu$F, 0.4 cm electrode). After sperm injection, the oocytes were activated electrically (1.7 KV/cm, 30$\mu$sec, single pulse) in 0.3 M mannitol solution or not. The sperm injected eggs were cultured in NCSU 23 medium (0.4% BSA) at 39$^{\circ}C$, 5% $CO_2$ in air fur 144 h. The rates of cleavage and development into blastocyst stage in activation group were significantly higher than those of non-activation group (79.6% and 24.1% vs. 46.3% and 14.4%, respectively, p<0.05). Control oocytes and shame injection were developed to blastocysts low (2.5%). Sixty five (27.1%) out of 240 embryos observed in activation and non-activation groups were showed positive by X-gal staining. However, all embryos in both groups were expressed partial or mosaic pattern. These results suggested that electrical stimulation far oocytes activation after sperm injection enhances the incidence of both fertilization and development fellowing sperm injection in the pig. Our study also suggested that sperm-mediated transfer of exogenous DNA by ICSI would be used as a valuable tool for the production of transgenic porcine embryos.

• Korean Journal of Soil Science and Fertilizer
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• v.45 no.4
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• pp.544-550
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• 2012

This study was conducted to investigate the characteristic factors which have been influenced on nitrous oxide ($N_2O$) emissions related to the environment change of nitrogen application level, rainfall and temperature during the potato cultivation at black volcanic ash soil from 2010 to 2011. During the potato cultivation, the more amount of nitrogen fertilizer applied, $N_2O$ emissions amounts were released much. $N_2O$ emissions with the cultivation time were released much at the first and middle of cultivation with heavy rainfall, but it was released very low until the end of cultivation and drought season. $N_2O$ emissions mainly were influenced by the rainfall and soil water content. The correlation (r) with $N_2O$ emissions, soil wate, soil temperature in 2010 were very significant at $0.6251^{**}$ and $0.6082^{**}$ respectively, but soil EC was not significant to 0.10824. In 2011, soil temperature was very significant at $0.4879^{**}$, but soil water and soil EC were not significant at 0.0468 and 0.0400 respectively. Also, $NH_4$-N was very significant at $0.7476^{**}$, but $NO_3$-N and soil nitrogen ($NO_3-N+NH_4-N$) were not significant at 0.0843 and 0.1797, respectively. During the potato cultivation period, the average emissions factor of 2 years released by the nitrogen fertilizer application was presumed to be 0.0040 ($N_2O-N\;kg\;N^{-1}\;kg^{-1}$). This factor was lower about 2.5 times than the IPCC guideline default value (0.0100 $N_2O-N\;kg\;N^{-1}\;kg^{-1}$).

• Korean Journal of Soil Science and Fertilizer
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• v.45 no.6
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• pp.1230-1236
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• 2012

To verify that the electrokinetic remediation is effective for decreasing salinity of fields of the plastic-film house, field tests for physical property, chemical property, and crop productivity of soils have been conducted. The abridged result of those tests is as follows. In the EK treatment, the electrokinetic remediation has been treated at the constant voltage (about 0.8 V $cm^{-1}$) for fields of the farm household. At this time, an alternating current (AC) 220 V of the farm household was transformed a direct current. The HSCI (High Silicon Cast Iron) that the length of the stick for a cation is 20cm, and the Fe Plate for an anion have been spread out on the ground. As the PVC pipe that is 10 cm in diameter was laid in the bottom of soils, cations descend on the cathode were discharged together. For soil physical properties according to the EK treatment, the destruction effect of soil aggregate was large, and the infiltration rate of water was increased. However, variations of bulk density and porosity were not considerable. Meanwhile, in chemical properties of soils, principal ions of such as EC, $NO_3{^-}$-N, $K^+$, and $Na^+$ were better rapidly reduced in the EK treated control plot than in the untreated control plot. And properties such as pH, $P_2O_5$ and $Ca^{2+}$ had a small impact on the EK. For cropping season of crop cultivation according to the EK treatment, decreasing rates of chemical properties of soils were as follows; $NO_3{^-}$-N 78.3% > $K^+$ 72.3% > EC 71.6% $$\geq_-$$ $Na^+$ 71.5% > $Mg^{2+}$ 36.8%. As results of comparing the experimental plot that EK was treated before crop cultivation with it that EK was treated during crop cultivation, the decreasing effect of chemical properties was higher in the case that EK was treated during crop cultivation. After the EK treatment, treatment effects were distinct for $NO_3{^-}$-N and EC that a decrease of nutrients is clear. However, because the lasting effect of decreasing salinity were not distinct for the single EK treatment, fertilization for soil testing was desirable carrying on testing for chemical properties of soils after EK treatments more than two times. In the growth of cabbages according to the EK treatment, the rate of yield increase was 225.5% for the primary treatment, 181.0% for the secondary treatment, and 124.2% for third treatment compared with the untreated control plot. The yield was increased by a factor of 130.0% for the hot pepper at the primary treatment (Apr. 2011), 248.1% for the lettuce at the secondary treatment (Nov.2011), and 125.4% for the young radish at the third treatment (Jul. 2012). In conclusion, the effect of yield increase was accepted officially for all announced crops.

• Clinical and Experimental Reproductive Medicine
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• v.28 no.2
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• pp.95-103
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• 2001

연구목적: 본 연구는 생쥐 preantral follicles의 체외 배양 조건을 확립하고 이를 기초로 높은 체외 발달률 그리고 산자 생산률을 얻고자 하였다. 연구재료 및 방법 : Preantral follicles의 oocyte-granulosa cell complexes (OGCs)는 생후 12일된 FI ($C57BL{\times}CBA$)으로부터 난소를 적출하여 효소를 이용한 방법을 통해 획득하였다. 회수된 complexes는 10일 또는 12일 동안 체외 성장을 위해 Transwell-COL membrane insert로 옮겨졌고 5% FBS, 100 mIU/ml FSH, 100 mIU/ml hMC가 첨가된 ${\alpha}MEM$에서 배양되었다. 체외 성숙을 위해 1.5 IU/ml hCG가 첨가된 ${\alpha}MEM$에서 18 hrs 배양을 실시하였다. 그 후 M16에서 수정능력이 획득된 정자와 수정을 하여 4 hrs, 7 hts, 9 hrs 후에 10% FBS가 첨가된 modified M16 배양액에서 4일간 배양하거나 또는 bovine cumulus cell과 co-culture를 실시하였다. 그리고 형태적으로 정상적인 22개의 상실배와 포배를 2마리의 위임신 대리모 (ICR)의 자궁에 이식하여 산자 생산을 유도하였다. 결과: 1) OGCs 크기가 mouse preantral follicles의 핵 및 세포질 성숙에 미치는 영향을 조사하였을 때 $120{\sim}150{\mu}m$의 preantral follicles (MII: 33.0%, 난할률: 36.7%, 상실배 이상; 20.9%)은 핵 및 세포질 성숙에 있어서 $70{\sim}110{\mu}m$ (MII: 12.2%, 난할률: 10.2%, 상실배 이상: 4.8%)보다 더 높았다(p<0.001). 2)체외 성장기간의 연장이 mouse preantral follicles의 핵 및 세포질 성숙에 미치는 영향을 조사하였을 때 10일 (난할률: 38.2%)은 12일 (난할률: 20.0%)보다 난할률에서만 더 높았다 (p<0.01). 3) 체외 수정 시간의 연장이 mouse preantral follicle의 세포질 성숙에 미치는 영향을 조사하였을 때 9 hrs (난할룰 31.5%, 상실배 이상: 14.3%)은 4 hrs (난할률: 17.5%, 상실배 이상: 4.8%), 7 hrs (난할률: 20.4%, 상실배 이상: 6.1%) 보다 세포질성숙에 있어서 유의하게 높은 발달률을 나타냈다 (p<0.01). 4) 공배양이 mouse preantral follicle의 세포질성숙에 미치는 영향을 조사하였을 때 공배양 (상실배 이상: 17.4%)을 실시했을 때와 M16 (상실배 이상17.4%)에서 배양되었을 때는 차이가 없었다. 5)preantral follicle의 크기 ($120{\sim}150{\mu}m$), 체외 성장기간 (10일), 체외 수정 기간 (9시간), 배양 환경 (단지 medium만 존재)의 적절한 결과들을 종합하여 수행하였을 때 MII 성숙률, 난할률, 상실배 이상의 발달률은 30.2%, 39.3%, 22.5%이었고 총 22개의 상실배 및 포배를 2마리의 대리모에 이식했을 때 1마리가 임신했고 1마리의 산자를 생산했다. 결론: 따라서, 본 실험은 preantral follicle을 이용한 체외 배양 시스템이 생쥐 oocyte를 공급하는 또 다른 방법으로 효과적으로 이용될 수 있다는 것을 시사한다.

• Clinical and Experimental Reproductive Medicine
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• v.31 no.2
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• pp.105-110
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• 2004

Objectives: Despite severe oligospermia, males with Y chromosome microdeletion can achieve conception through ICSI (Intracytoplasmic Sperm Injection). However, ICSI may not only result in the transmission of microdeletions but also the expansion of deletion to the offspring. The purpose of this study was to screen vertical transmission, expansion of microdeletions and de novo deletion in male fetuses conceived by ICSI. Materials and Methods: A total of 32 ICSI treated patients with their 33 (a case of twin) male fetuses conceived by ICSI were used to make this study group. Sequence-tagged sites (STSs)-based PCR analyses were performed on genomic DNA isolated from peripheral blood of fathers and from the amniocytes of male fetuses. Ten primer pairs namely, sY134, sY138, MK5, sY152, sY147, sY254, sY255, SPGY1, sY269 and sY158 were used. The samples with deletions were verified at least three times. Results: We detected a frequency of 12.5% (4 of the 32 patients) of microdeletions in ICSI patients. In 4 patients with detected deletions, two patients have proven deletions on single STS marker and their male fetuses have the identical deletion in this region. Another two patients have two and three deletions, but their male fetuses have more than 3 deletions which include deletions to their father's. Meanwhile, seven male fetuses, whose fathers were analyzed to have all 10 STS markers present, have deletions present in at least one or more of the markers. Conclusions: Although the majority of deletions on the Y chromosome are believed to arise de novo, in some cases a deletion has been transmitted from the fertile father to the infertile patient. In other cases the deletion was transmitted through ICSI treatment, it is likely that one sperm cell is injected through the oocyte's cytoplasm and fertilization can be obtained from spermatozoa. Our tests for deletion were determined by PCR and our results show that the ICSI treatment may lead to vertical transmission, expansion and de novo Y chromosome microdeletions in male fetuses. Because the sample group was relatively small, one should be cautious in analyzing these data. However, it is important to counsel infertile couples contemplating ICSI if the male carries Y chromosomal microdeletions.

• Clinical and Experimental Reproductive Medicine
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• v.29 no.1
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• pp.1-12
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• 2002

Objective: In order to acquire the technique for the establishment of human embryonic stem cells (ESe) derived from the human frozen-thawed embryos produced in IVF-ET program, this study was performed to establish mouse ESC derived from the in vitro fertilized embryos. Materials and Methods: After Fl hybrid (C57BL female $\times$ CBA mael) female mice were superovulated with PMSG and hCG treatment, their oocytes were retrieved and inseminated, and the fertilized embryos were cultured for 96-120 hours until the expected stages of blastocysts were obtained. To isolate the inner cell mass (ICM), either the blastocysts were treated with immunosurgery, or the whole embryos were cultured for 4 days. Isolated ICMs were then cultured onto STO feeder cell layer, and the resultant ICM colonies were subcultured with trypsin-EDTA treatment. During the subculture process, ESC-like cell colonies were observed with phase contrast microscopy. To identify ESC in the subcultured ESC-like cell colonies, alkaline phosphatase activity and Oct-4 (octamer-binding transcription factor-4) expression were examined by immunohistochemistry and RT-PCR, respectively. To examine the spontaneous differentiation, ESC-like cell colonies were cultured without STO feeder cell layer and leukemia inhibitory factor (LIF). Results: Seven ESC-like cell lines were established from ICMs isolated from the in vitro fertilized embryos. According to the developmental stage, the growth of ICMs isolated from the expanded blastocysts was significantly better than that of ICMs isolated from the hatched blastocysts (80.3% vs. 58.7%, p<0.05). ESC-like cell colonies were only obtained from ICMs of expanded blastocysts. However, the ICMs isolated from the embryos treated with immunosurgery were poorly grown and frequently differentiated during the culture process. The established ESC-like cell colonies were positively stained with alkaline phosphatase and expressed Oct-4, and their morphology resembled that observed in the previously reported mouse ESC. In addition, following the extended in vitro culture process, they maintained their expression of cell surface markers characteristic of the pluripotent stem cells such as alkaline phosphatase and Oct-4. When cultured without STO feeder cell layer and LIF, they were spontaneously differentiated into the various types of cells. Conclusion: The findings of this study suggest that the establishment of mouse ESC can be successfully derived from the in vitro fertilized embryos. The established ESC-like cells expressed the cell surface markers characteristic of the pluripotent stem cells and spontaneously differentiated into the various types of cells.

• Development and Reproduction
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• v.16 no.2
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• pp.137-143
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• 2012

The main objective of this study is to investigate the effects of ovulation induction by treating HCG, LHRHa, GnRHa, ovaprim and pimozidein in long snout bullhead, L. longriostris. All hormons were injected into the muscles of back. Concentration of LHRHa to injection were 20, 50, 100, 150 and 200 ${\mu}g/kg$ and same concentration of LHRHa was injected after 24 hour. The ovulation induction rate was 100% and fertilization and hatching rates were 68.4, 45.2, 58.4 and 33.6% in 50 and 100 ${\mu}g/kg$. The times to ovulation were between 28 and 44 h. HCG was injected in long snout bullhead at 5,000, 10,000, 15,000, 20,000 and 25,000 IU/kg. The ovulation induction rate was 50% in 15,000 and 20,000 IU/kg. Fertlilzation and hatching rates were 55.2, 45.6, 52.8 and 44.8%. Ovulation time was between 72~80 h. HCG concentration of 500, 1,000, 2,000 and 4,000 IU/kg were injected with $50{\mu}g$ of LHRHa. Ovulation and hatching rates in 2,000 IU/kg were 75 and 35%. Ovulation time was 28~48 h. Ovaprim of 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mL/kg were injected to the abdominal cavity. The ovulation induction rate was highest at 2.5 mL/kg to 50% and ovulation time was between 66~86 h. LHRHa concentration of 50, 100, 200, 300 and 400 ${\mu}g/kg$ was injected with pimozide (1,000 ${\mu}g/kg$). Ovulation induction rate was 87% at 100 and 200 IU/kg with pimozide. Ovulation time was between 66~86 h.

• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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• pp.361-361
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• 2017

The NPK are known as macro elements that affect crop growth and yield. In 1989, Cambodia Agricultural Research and Development Institute (CARDI) gave a recommendation rate of fertilizer on rice production based on soil types. This recommended rate of NPK seems however relatively low as compared to farmers' practices nowadays and the amount in the neighboring countries. The CARDI recommended rate for Prateah Lang soil type is 50kg N, $25kg\;P_2O_5$, $25kg\;K_2O\;ha^{-1}$ while recent farmers' practice rates are 55 - 64kg N, 24 - 46kg $P_2O_5$, $30kg\;K_2O\;ha^{-1}$. However, the overuse of chemical fertilizer will lead to un-preferable plant growth, insect pest, disease and economic yield. Thus, we examined the effect of different NPK application rates on the growth and yield components in Prateah Lang soil type in Takeo province to investigate appropriate rates for improving rice productivity with economic efficiency. This study was conducted from July to November during wet season in 2013. A multi-locational trial with 6 treatments (T0 - T5) of NPK rates in 5 locations (trial 1 - 5) with 3 replications was conducted. The different combinations of NPK application were employed from 0, 50, 60, 80, 100, $120kg\;N\;ha^{-1}$, 0, 25, 30 45, $60kg\;P_2O_5\;ha^{-1}$ and 0, 15, 25, 30, $45kg\;K_2O\;ha^{-1}$. Urea, DAP and KCl were used for fertilization. Split application was employed [basal: 20% of N, 100% of P and K, top dressing-1st: 40% of N (30DAT), 2nd: 40% of N (PI stage)]. Three-week-old seedlings of var. Phka Rumdoul were transplanted with 2 - 3 seedlings $hill^{-1}$ with $20cm{\times}20cm$ spacing. Plant length, tiller number at the maximum tillering stage and yield components were measured. The different rates of NPK application affected some yield components. The panicle number per hill was the most important key component followed by the spikelet number per panicle. However, the other parameters such as the filled grain percentage and 1000 grains weight had small effect or weak relation with the yield. Although the panicle number per hill had a significantly positive correlation with the stem number per hill, it was not correlated with the percentage of productive culms. The variation in the grain yield among the 5 trials was small and the difference was not significant. Although the yield tended to be higher at higher N and P application, there was no significant difference above 60kg N and $30kg\;P_2O_5$. The yield was the highest at 15, 30 and $45kg\;K_2O$ followed by $25kg\;K_2O$. The relationships between N, P and the stem number per hill were significantly linear positive, though it was not linear between K and the stem number. From these results, to increase rice productivity in the target area, farmers' effort to increase N and P input rather than CARDI recommendation up to 60kg N and $30kg\;P_2O_5$ will be sufficient considering economic efficiency. Besides, the amount of K application should be reconsidered.