• Journal of Microbiology and Biotechnology
• /
• 제10권3호
• /
• pp.349-354
• /
• 2000

Abstract A facultative alkalophilic gram-negative Vibrio metschnikovii strain RH530, isolated from the wastewater, produced several alkaline proteases (VAP) including six alkaline serine proteases and a metalloprotease. From this strain, high yielding YAP mutants were isolated by NTG treatment. The isolated mutant KS1 showed nine times more activity than the wild-type after optimization of the culture media. The production was regulated by catabolite repression when glucose was added to the medium. The effects of several organic nitrogen sources on the production of the YAP were investigated to avoid catabolite repression. The combination of 4% wheat gluten meal (WGM), 1.5% cotton seed flour (eSF), and 5% soybean meal (SBM) resulted in the best production when supplemented with 1% NaCl. The YAP showed a resistance to surfactants such as $sodium-{\alpha}-olefin$ sulfonate (AOS), polyoxy ethylene oxide (POE), and sodium dodecyl sulfate (SDS), yet not to linear alkylbenzene sulfonate (LAS). However, the activity of the YAP was restored completely when incubated with LAS in the presence of POE or $Na_2SO_4$. The YAP was stable in a liquid laundry detergent containing 6.6% SLES (sodium lauryl ether sulfate), 6.6% LAS, 19.8% POE, and stabilizing agents for more than two weeks at $40^{\circ}C$, but the stability was sharply decreased even after 1 day when incubated at $60^{\circ}C$. A washing performance test with the YAP exhibited it to be a good washing power by showing 51 % and 60% activity at $25^{\circ}C{\;}and{\;}40^{\circ}C$, respectively, thereby indicating that the YAP also has a good detergency at a low temperature. All the results suggest that the YAP produced from the mutant strain KSI has suitable properties for use in laundry detergents.rgents.

• Clinical and Experimental Reproductive Medicine
• /
• 제27권4호
• /
• pp.367-372
• /
• 2000

Objective: This study was performed to evaluate whether vitrification method could be used for the cryopreservation of human blastocysts derived from IVF program. Methods: Surplus embryos were obtained from consented IVF patients. Controlled ovarian hyperstimulation was done with midluteal GnRH agonist, gonadotropin and hCG. After oocyte retrieval and insemination, fresh embryo transfer was done at $4{\sim}8$ cell stage. The surplus embryos after ET were cultured in blastocyst medium up to 6 days after oocyte retrieval. Obtained blastocysts were cryopreserved with our vitrification method. Blastocysts were exposed to 1.5 Methylene glycol (EG) in phosphate buffered saline (PBS) for 2.5 minutes, followed by 5.5 M EG plus 1 M sucrose for 20 seconds. Then 1 to 3 blastocysts were mounted on electron microscope (EM) grid and the grid was plunged into liquid nitrogen for storage. For thawing, blastocyst-containing EM grids were sequentially transferred in 1.0 M, 0.5 M, 0.25 M, 0.125 M and 0 M sucrose solution at the intervals of2.5 minutes. And blastocysts were cultured for about 6 hours and only re-expanded blastocysts were transferred to uterus of the patients on 4 to 5 days after ovulation in natural cycle or on 18 to 19 day of artificial cycle. Results: From Oct. 1998 to Jul. 1999, 34 patients were agreed to participate in this study. The mean age and duration of infertility of the patients were 31.6 years and 4.1 years, respectively. Among 34 cycles. replacements could be done in 20 cycles (58.8%). A total 93 blastocysts were thawed and 48 (51.6%) of them survived. Thirty-eight blastocysts, mean 1.9 embryos per patient, were transferred, resulting in 5 clinical pregnancies which consisted of 1 triplet, 2 sets of twins and 2 singleton pregnancies. The pregnancy rate per transfer was 25% and implantation rate was 23.6%. Five patients delivered 7 healthy babies including 2 sets of twins at term. Conclusion: Successful pregnancies and deliveries were established after transfer of vitrified human blastocysts. Vitrification using ethylene glycol as cryoprotectant and electron microscope grid is a rapid and simple method that can be effectively applied for the cryopreservation of human blastocysts.

• 한국수정란이식학회:학술대회논문집
• /
• 한국수정란이식학회 1997년도 국제학술대회 및 Workshop
• /
• pp.27-34
• /
• 1997

Bovine oocytes surrounded with compact cumulus cells were cultured for 20 to 22 hours($38^{\circ}C$, 5% $CO_2$) in modified TCM-199 medium supplemented with 5% superovulated cow serum(SCS) and inseminated by in vitro capacitated spermatozoa. Day 7 to 8 embryos were equilibrated for 10 minutes in 1.3M methyl cellosolve(MC) <1.1M diethylene glycol(DEG), 1.8M ethylene glycol(EG), 1.6M propylene glycol(PG) and 1.1 M 1,3-butylene glycol(BG) solutions. They were then loaded into 0.25ml straws, placed into an alcohol bath freezer at $0^{\circ}C$, cooled from $0^{\circ}C$ to $-6^{\circ}C$ at $-1^{\circ}C$/minute, seeded, held for 10 minutes, and stored in liquid nitrogen. After thawing in $30^{\circ}C$ water, the embryos wee rehydrated in TCM-199 medium and then cultured for 48 hours in TCM-199 plus 5% SCS. Embryos were considered viable if they progressed to later developmental stages with a good morphology. Some of the embryos frozen in each cryoprotectant were thawed and transferred non-surgically without removing the cryoprotectant. Hatched embryos survived freezing and one-step dilution as follows : EG(50.0%), MC(53.6%), DEG(56.9%), PG(58.0%) and BG(11.5%). The survival rate of embryos cooled at -0.3^{\circ}C$ vs. $-0.5^{\circ}C$/minute was not significantly different(P<0.05), however, blastocysts hatched most often (P<0.01) in vitro when cooled at a rate of $0.3^{\circ}C$/minute(64.6%), 31/48) than at $-0.5^{\circ}C$/minute(22.6%, 12/53). Pregnancy rates resulting from embryos frozen in the different cryoprotectants were as follows : MC(48%, 10/21); DEG(30%, 3/10); EG(74%, 20/27); and PG(40%, 4/10). These results indicate that MC, DEG, EG and PG have utility as cryoprotectants for the freezing and thawing of IVF Bovine embryos.

• 한국수정란이식학회지
• /
• 제24권3호
• /
• pp.207-212
• /
• 2009

The aim of present experiment was to examine commercial synthetic extender(AndroMed) for semen cryopreservation of Korean Black Bull. Semen was collected from a Korean Black Bull using an artificial vagina and transported to the laboratory. The semen was diluted 1:1 by AndroMed. The pellect was diluted to final sperm concentration of $5{\times}10^5/ml$ by doubling in every 10 minutes at $4^{\circ}C$ cold chamber. The semen was equilibrated for 1 hr at cold chamber and packed to 0.5 ml straw. The semen straws were located above 5 cm of liquid nitrogen for 5 minutes, above 5 cm for 10 minutes and above 10 cm for 10 min. And then the frozen straw was plunged to $LN_2$. The presented straws were examined the viability and motility after thawed at $37^{\circ}C$ water bath. Hanwoo semen was used as KPN (Korea Proven Bull Number) in this experiment. The survival rates was significantly higher in fresh semen than frozen semen ($80{\pm}14%\;and\;43{\pm}11%$). However, the motility rates was similar (80.7% and 66.4%). The survival and motility rates were higher in 5cm, 10 min treatment group than the other two groups in straw-located height and duration above $LN_2$ ($50{\pm}14%$ and 70.7% vs, 33.18% and $65{\pm}7%$ vs, 30.14% and 65.7%, respectively). The development rates to cleavage was higher in Black Cow than Hanwoo semen (62.2%, 64.4%), However, The development rates to blastocyst was higher in Hanwoo than Black cow semen (25.9%, 23.0%). In conclusion. The present results that acceptable fertilization and cryopreservation could be obtained by in vitro fertilization with frozen-thawed semen using a synthetic semen extender (AndroMed).

• Journal of Animal Science and Technology
• /
• 제49권5호
• /
• pp.585-592
• /
• 2007

본 연구는 풍산개 동결정액 제조기술을 정립하기 위하여 정액성상과 정액 동결 시 희석액에 첨가되는 Glycerol 농도, 동결속도, 융해온도와 시간에 따라 정액의 운동성과 생존율 및 CASA를 이용한 운동성 등에 대하여 조사하여 최적의 동결조건을 확립하기 위해 실시하였다.1.풍산개의 평균 정액량 5.9ml, 정액농도 116.3 ×106sperm/ml 총정자수 789.3×106sperm, 운동성 88.7±1.77% 및 생존율 87.6±1.85% 였다. 2.1차 희석액을 상온에서 희석 후 상온에서 4°C까지 하강시킨 후 glycerol 농도가 3%, 5% 및 7% 첨가된 2차 희석액을 희석 후 6일간 운동성을 측정한 결과 5일째 3%일 때 46.2±9.3%, 5% glycerol에서는 48.1±8.5% 및 7%일 때 52.7±8.2%로 glycerol 7%가 유의적으로 높은 운동성을 나타냈다.3. 각기 다른 glycerol 농도를 함유한 동결보존액에서 동결보존 후 융해하였을 때 7%의 glycerol 농도에서 각각 52.7%와 57.7±10.3%로서 다른 처리구에 비해 유의적인(P<0.01) 운동성 및 생존율을 나타냈다.4.정액을 동결함에 있어 예비동결시 57 및 10cm의 높이에서 정치시킨 후 동결을 실시하였을 때 액체질소의 표면 7cm의 높이에서 동결을 실시한 처리구에서 전체적으로 유의한 운동성과 생존율을 나타냈다.

• 한국수정란이식학회지
• /
• 제23권3호
• /
• pp.223-227
• /
• 2008

The cryopreservation of Hanwoo embryos has become an integral part of assisted reproduction in animal. The objective of this study was to assess the effect of The objectives of this study were: (1) to evaluate the influence of bovine embryo developmental stage on in vitro embryo development after freezing, (2) to study the efficiency compared with conventional freezed embryos at different embryo source. For conventional slow-freezing, day 7 or 8 expanded blastocysts were collected. The standard freezing medium was 1.8 M ethylene glycol (EG). Embryos were equilibrated in 1.8 Methylene glycol(EG) with 0.1 M sucrose in Dulbecco's phosphate-buffered saline (D-PBS) supplemented with 0.5% bovine serum albumin. Embryos were then loaded individually into 0.25 ml-straw and placed directly into cooling chamber of programmable freezer precooled to $-7^{\circ}C$, after 2 min, the straw was seeded, maintained at $-7^{\circ}C$ for 8 min, and then cooled to $-35^{\circ}C$ at $0.3^{\circ}C$/min, plunged and stored in liquid nitrogen for at least 3 days. For thawing, the straw containing embryos were warmed in air for 10 see and exposed to $37^{\circ}C$ water for 20 sec. Straws were then removed from $37^{\circ}C$ water. Rates of blastocyst survive and hatched were evaluated at 12 to 48h post-warming. The re-expansion and hatched rates of morula embryos were significantly lower than those obtained for blastocysts and expansion blastocysts (31.6%, 10.5% vs, 68.9%, 22.2% vs, 73.7%, 53.6%, respectively). No differences in re-expansion rates were found between in vivo and in vitro blastocysts. whereas hatched rates was significantly higher (51.2%) in vivo compared with in vitro embryos (18.6%). in conclusion, demonstrate that conventional freezing can be used successfully in cryopreservation of in vitro and in vivo bovine embryos, and that it might be considered for use in commercial programs and embryo preservation.

• 한국식품위생안전성학회지
• /
• 제28권1호
• /
• pp.13-18
• /
• 2013

약용식물 중 잔류농약의 안전성을 평가하고자 2012년 전국 9개 도시에서 유통되고 있는 인삼과 도라지에 대하여 전체 112점의 시료를 수거하여 잔류농약을 분석하였다. 122종의 농약에 대해 GC-ECD, GC-NPD 및 HPLC-UVD를 이용한 다종농약 다성분 분석법으로 잔류농약을 분석하였고, 분석 결과 12점의 시료에서 7종의 농약이 검출되어 10.7% 검출률을 보였다. 농약 성분별 검출 빈도는 procymidone, kresoxim-methyl, endosulfan, cypermethrin, tralomethrin, tetraconazole, chlorfluazuron 순이었다. 농약이 검출된 시료 중 잔류허용기준을 초과한 시료는 2점으로 1.8% 검출률을 보였으며, 도라지 1점에서 tetraconazole, 인삼 1점에서 cypermethrin이 검출되었다. 해당 작물에 대한 잔류허용기준이 설정되어 있지 않거나 품목고시 되어 있지 않은 시료는 10점에서 5종의 농약이 검출되어 8.9% 검출률을 보였다. 본 연구에서 검출된 농약이 해당 약용 식물의 섭취로 인체에 유입될 일일섭취허용량 대비 일일 섭취추정량은 최저 0.006%에서 최고 0.333%로 낮은 %ADI 값을 보여 인체 위해도는 낮은 것으로 판단되었다.

• 한국안광학회지
• /
• 제11권3호
• /
• pp.165-172
• /
• 2006

Cr, Cu, Fe 등을 고의적으로 첨가한 MgO 단결정에 자외선 및 X선을 조사하여 여기하고 액체질소온도로부터 500K까지의 범위에서 열자극 발광 그로우곡선과 발광 스펙트럼을 측정 분석하였다. 이 시료에서 얻어진 TSL 그로우곡선은 136.5K, 223.5K, 360K, 390K, 440K, 5개의 봉우리가 얻어졌다. 그들 봉우리의 활성화 에너지 값은 각각 0.27eV와 0.63eV, 1.08eV, 1.19eV, 1.33eV이었다. 우리는 200nm에서 650nm의 파장 범위 내에서 MgO 단결정의 TSL 스펙트럼을 측정하였다. 이 TSL 스펙트럼 측정으로부터 얻어진 345nm, 375nm, 410nm 파장에서 봉우리를 가진 스펙트럼을 분석하였으며, 이들의 발광 기구를 기술하였다. MgO:Cr, MgO:Cu, and MgO:Fe에서 방출된 TSL 스펙트럼의 봉우리는 346nm, 360nm, 375nm의 파장에서 나타났다.

• 한국물환경학회지
• /
• 제34권1호
• /
• pp.46-56
• /
• 2018

A mcyB-specific Ultra-Rapid quantitative PCR was developed for the quantitative detection of Microcystis aeruginosa, which is often a dominant species in green tide. McyB-specific UR-qPCR was optimized under extremely short times of each step in thermal cycles, based on the specific primers deduced from the mcyB in microcystin synthetase of M. aeruginosa. The M. aeruginosa strain KG07 was used as a standard for quantification, after the microscopic counting and calculation by mcyB-specific UR-qPCR. The water samples from the river water with the Microcystis outbreak were also measured by using both methods. The $1.0{\times}10^8$ molecules of mcyB-specific DNA was recognized inner 4 minutes after beginning of UR-qPCR, while $1.0{\times}10^4$ molecules of mcyB-specific templates was detected inner 7 minutes with quantitative manner. From the range of $1.0{\times}10^2$ to $1.0{\times}10^8$ initial molecules, quantification was well established based on $C_T$ using mcyB-specific UR-qPCR (Regression coefficiency, $R^2=0.9977$). Between the numbers of M. aeruginosa cell counting under microscope and calculated numbers using mcyB-specific UR-qPCR, some differences were often found. The reasons for these differences were discussed; therefore, easy compensation method was proposed that was dependent on the numbers of the cell counting. Additionally, to easily extract the genomic DNA (gDNA) from the samples, a freeze-fracturing of water-sample using liquid nitrogen was tested, by excluding the conventional gDNA extraction method. It was also verified that there were no significant differences using the UR-qPCR with both gDNAs. In conclusion, the mcyB-specific UR-qPCR that we proposed would be expected to be a useful tool for rapid quantification and easy monitoring of M. aeruginosa in environmental water.

• 한국발생생물학회지:발생과생식
• /
• 제20권3호
• /
• pp.219-225
• /
• 2016

Most of the commercial devices for vitrification are directly immersed into the warming solution (WS) for increasing of warming rate. However, the previous modified cut standard straw (MCS) which has reported is difficult to immerse into the WS. The aim of this study was to investigate whether the long cut straw (LCS) could be useful as a stable tool for vitrified-warmed human blastocysts. A total of 138 vitrified-warmed cycles were performed between November 2013 and November 2014 (exclusion criteria: women ${\geq}38$ years old, poor responder, surgical retrieval sperm, and severe male factor). The artificial shrinkage was conducted using 29-gauge needles. Ethylene glycol and dimethyl sulfoxide (7.5% and 15% (v/v)) were used as cryoprotectants. Freezing and warming were conducted using the LCS tool. The cap of LCS was removed using the forceps in the liquid nitrogen ($LN_2$) and then directly immersed into the first WS for 1 min at $37^{\circ}C$ (1 M sucrose). Only re-expanded blastocysts were transferred after it was cultured in sequential media for 18-20 h. A total of 294 blastocysts were warmed, and all were recovered (100%). Two hundred eighty-five embryos were survived (96.9%). The vitrified-warmed blastocysts of all patients were transferred without any cancellation. We were able to achieve a reasonable implantation (24.2%), following by clinical pregnancy (36.2%), which then continued to ongoing pregnancy (36.2%), and live birth (31.2%). Using LCS is achieved the acceptable rates of survival, pregnancy and live birth. Therefore, the LCS could be considered as a stable and simple tool for human embryo vitrificaton.