• 한국수정란이식학회지
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• 제30권4호
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• pp.289-298
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• 2015

Edaravone (Eda) is a potent scavenger of inhibiting free radicals including hydroxyl radicals ($H_2O_2$). Reactive oxygen species (ROS) such as $H_2O_2$ can alter most kinds of cellular molecules such as lipids, proteins and nucleic acids, cellular apoptosis. In addition, oxidative stress from over-production of ROS is involved in the defective embryo development of porcine. Previous study reported that Eda has protective effects against oxidative stress-like cellular damage. However, the effect of Eda on the preimplantation porcine embryos development under oxidative stress is unclear. Therefore, in this study, the effects of Eda on blastocyst development, expression levels of ROS, and apoptotic index were first investigated in preimplantation porcine embryos. After in vitro fertilization, porcine embryos were cultured for 6 days in PZM medium with Eda ($10{\mu}M$), $H_2O_2$ ($200{\mu}M$), and Eda+$H_2O_2$ treated group, respectively. Rate of blastocyst development was significantly increased (P<0.05) in the Eda treated group compared with only $H_2O_2$ treated group. And, we measured intracellular levels of ROS by DCF-DA staining methods and investigated numbers of apoptotic nuclei by TUNEL assay analysis is in porcine blastocyst, respectively. Both intracellular ROS levels and the numbers of apoptotic nucleic were significantly decreased (P<0.05) in porcine blastocysts cultured with Eda ($10{\mu}M$). More over, the total cell number of blastocysts were significantly increased (P<0.05) in the Eda-treated group compared with untreated group and the only $H_2O_2$ treated group. Based on the results, Eda was related to regulate as antioxidant-like function according to the reducing ROS levels during preimplantation periods. Also, Eda is beneficial for developmental competence and preimplantation quality of porcine embryos. Therefore, we concluded that Eda has protective effect to ROS derived apoptotic stress in preimplantation porcine embryos.

• Reproductive and Developmental Biology
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• 제35권3호
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• pp.287-294
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• 2011

Autophagy is a process of intracellular bulk protein degradation, in which the accumulated proteins and cytoplasmic organelles are degraded. It plays important roles in cellular homeostasis, apoptosis, and development, but its role during early embryo development remains contentious. Therefore, in the present study, we investigated the effects of 3-methyladenine (3-MA) on early embryonic development in pigs, we also investigated several indicators of developmental potential, including mitochondrial distribution, genes expressions (autophagy-, apoptosis- related genes), apoptosis and ER-stress, which are affected by 3-MA. After in vitro maturation and fertilization, presumptive pig embryos were cultured in PZM-3 medium supplemented with 3-MA for 2 days at $39^{\circ}C$ 5% $CO_2$ in air. Developmental competence to the blastocyst stage in the presence of 3-MA was gradually decreased according to increasing concentration. Thus, all further experiments were performed using 2 mM 3-MA. Blastocysts that developed in the 3-MA treated group decreased LC3-II intensity and expressions of autophagy related genes than those of the untreated control, resulting in down-regulates the autophagy. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that the number of containing fragmented DNA at the blastocyst stage increased in the 3-MA treated group compared with control ($6.0{\pm}1.0$ vs $3.3{\pm}0.6$, p<0.05). Also, the expression of the pro-apoptotic gene Bax increased in 3-MA treated group, whereas expression of the anti-apoptotic gene Bcl-XL decreased. Mito Tracker Green FM staining showed that blastocysts derived from the 3-MA treated group had lower mitochondrial integrity than that of the untreated control, resulting in decrease the embryonic qualities of preimplantation porcine blastocysts. Then, the expression of the spliced form of pXBP-1 product (pXBP-1s) increased in 3-MA treated group, resulting increase of ER-stress. Taken together, these results indicate that inhibition of autophagy by 3-MA is closely associated with apoptosis and ER-stress during preimplantation periods of porcine embryos.

• 농업과학연구
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• 제11권1호
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• pp.68-76
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• 1984

수도상자육묘(水稻箱子育苗)에 있어서 이상적(理想的)인 치묘(稚苗)와 중묘(中苗) 그리고 성묘(成苗)의 육성(育成)에 알맞은 재배조건(栽培條件)을 구명(究明)하고져 파종밀도(播種密度)와 비료용량(肥料用量), 육묘(育苗)의 시기(時期)와 일수(日數)의 영향(影響)을 분석(分析)한바, 파종량(播種量)은 적을수록, 시비량(施肥量)은 많을수록, 만파(晩播)일수록, 육묘기간(育苗期間)이 연장(延長)될수록 초장(草長), 엽수(葉數) 및 건물중(乾物重)은 증대(增大)하였는데 그 영향(影響)은 파종기(播種期)>육묘일수(育苗日數)>파종밀도(播種密度)>시비량(施肥量)의 순(順)으로 컸으며 조기파종시(早期播種時)에는 상자당(箱子當) 200g의 파종량(播種量)과 4g까지의 시비량하(施肥量下)에서 알맞은 치묘육성(稚苗育成)이 가능(可能)하였고 적당(適當)한 중묘(中苗)는 25일간(日間)의 육묘시(育苗時)에는 50g의 박파(薄播)에서, 그리고 35일묘(日苗)에서는 100~125g 범위(範圍)의 파종밀도하(播種密度下)에서 육성가능(育成可能)하나 45일묘(日苗)는 지나치게 웃자라 기계이앙용(機械移秧用)으로는 부적당(不適當)하였다. 만파시(晩播時)에도 같은 경향(傾向)이나 처리간차이(處理間差異)가 확대(擴大)되었으며 치묘(稚苗)는 상자당(箱子當) 100g 이하(以下)의 박파조건(薄播條件)에서 육성가능(育成可能)하였고 100내지(乃至) 150g의 파종밀도(播種密度)에 25일간(日間) 육묘(育苗)하므로서 알맞은 중묘(中苗)를 얻을 수 있었으나 35일(日) 이상(以上) 육묘일수(育苗日數)를 연장(延長)하면 지나치게 생장(生長)하거나 도장(徒長)하였고 성묘비율(成苗比率)도 현저(顯著)히 저하(低下)하였다.

• Asian-Australasian Journal of Animal Sciences
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• 제31권9호
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• pp.1420-1430
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• 2018

Objective: Epigallocatechin-3-gallate (EGCG) is a major ingredient of catechin polyphenols and is considered one of the most promising bioactive compounds in green tea because of its strong antioxidant properties. However, the protective role of EGCG in bovine oocyte in vitro maturation (IVM) has not been investigated. Therefore, we aimed to study the effects of EGCG on IVM of bovine oocytes. Methods: Bovine oocytes were treated with different concentrations of EGCG (0, 25, 50, 100, and $200{\mu}M$), and the nuclear and cytoplasmic maturation, cumulus cell expansion, intracellular reactive oxygen species (ROS) levels, total antioxidant capacity, the early apoptosis and the developmental competence of in vitro fertilized embryos were measured. The mRNA abundances of antioxidant genes (nuclear factor erythriod-2 related factor 2 [NRF2], superoxide dismutase 1 [SOD1], catalase [CAT], and glutathione peroxidase 4 [GPX4]) in matured bovine oocytes were also quantified. Results: Nuclear maturation which is characterized by first polar body extrusion, and cytoplasmic maturation characterized by peripheral and cortical distribution of cortical granules and homogeneous mitochondrial distribution were significantly improved in the $50{\mu}M$ EGCG-treated group compared with the control group. Adding $50{\mu}M$ EGCG to the maturation medium significantly increased the cumulus cell expansion index and upregulated the mRNA levels of cumulus cell expansion-related genes (hyaluronan synthase 2, tumor necrosis factor alpha induced protein 6, pentraxin 3, and prostaglandin 2). Both the intracellular ROS level and the early apoptotic rate of matured oocytes were significantly decreased in the $50{\mu}M$ EGCG group, and the total antioxidant ability was markedly enhanced. Additionally, both the cleavage and blastocyst rates were significantly higher in the $50{\mu}M$ EGCG-treated oocytes after in vitro fertilization than in the control oocytes. The mRNA abundance of NRF2, SOD1, CAT, and GPX4 were significantly increased in the $50{\mu}M$ EGCG-treated oocytes. Conclusion: In conclusion, $50{\mu}M$ EGCG can improve the bovine oocyte maturation, and the protective role of EGCG may be correlated with its antioxidative property.

• Weed & Turfgrass Science
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• 제6권2호
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• pp.124-129
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• 2017

사료용 피 직파재배시 적정 파종량 시험에서 입모수는 파종량이 증가할수록 많았으며, 입모율은 41-42% 정도였다. 경수도 파종량이 증가할수록 많았고, 초장은 파종량이 많을수록 증가하였으나 $50kg\;ha^{-1}$에서는 감소하였다. 파종량이 많았던 $50kg\;ha^{-1}$에서 도복지수가 3이었다. 건물중은 파종량 $40kg\;ha^{-1}$에서 가장 높았고, TDN 생산량은 파종량이 적을수록 높았다. 수량반응 곡선상 $36kg\;ha^{-1}$에서 최대수량 건물수량이 추정되었다. 변화 상태의 회귀식은 $Y=0.0098X^2+0.7030X+2.6267$으로 표시되었다. 출수기는 파종량이 많을수록 늦어지는 경향이었다. 사료용 피 직파 재배시 질소질 비료 $150-250kg\;ha^{-1}$ 시비에서 건초 수량의 차이는 없었으나, 무시용 대비 건초 수량은 200 및 $250kg\;ha^{-1}$ 시비에서 16.6톤 $ha^{-1}$ 및 16.9톤 $ha^{-1}$로 43% 및 45% 정도 높았다. 조단백질 수량(CP)은 건물수량과 비슷한 경향으로 무질소구에서 0.28톤 $ha^{-1}$이었던 것이 질소 $250kg\;ha^{-1}$ 시비구에서 0.54톤 $ha^{-1}$ 이었다.

• 한국수정란이식학회지
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• 제15권1호
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• pp.67-75
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• 2000

This study was conducted to establish the optimal conditions for in vitro embryo production using oocytes derived from follicles of slaughter-house ovaries. The ovaries of Hanwoo were obtained from a local slaughter-house. The oocytes were aspirated from visible follicles of 2~7mm in diameter. The recovered oocytes which were completely surrounded by at least 2 layers of cumulus cells and a homogeneous cytoplasmic pigmentation were used. The selected oocytes were washed 3 or 4 times with D-PBS containing 10% bovine calf serum (BCS) and matured in vitor (IVM) in Ham's F-10 supplemented with 10% BCS or 0.01 $\mu\textrm{g}$/ml epidermal growth factor(EGF) at 39$^{\circ}C$ under 5% CO2 in air for 24 hours. They were fertilizqed in vitro (IVF) with fresh sperm separated by Percoll density gradient or swim-up in TALP media. The zygotes were cultrued with or without bovine oviductal epitherial cells(BOEC) in media(HECM-6 supplemented with 11 amino acid and / or TCM-199 supplemented with 10% BCS) for 7 to 10 days. The results obtained were as follow: The cleavage rate and developmental rate to blastocyst after maturation and IVF were not significantly different between Ham's F-10 with EGF(76.0% vs. 44.0%) and BCS(75.9% vs. 43.6%)(P<0.05). The cleavage rate and development rate to blastocyst after fertilizing by swim-up or Percoll method were not signifciantly(P<0.05) different between swim-up (80.2% vs. 29.2%) and Percoll(81.9% vs. 26.5%) (P<0.05). The cleavage rate in TCM 199(80.5) was signficiantly higher than that in HECM-6 (72.0%) (P<0.05). However, developmental rate to blastocyst using TCM 199 following HECM-6 for 3 or 4 days (42.2%) was significantly higher than that in TCM-199 alone(26.7%)(P<0.05). The cleavage rate and development rate of embryos produced in vitro by exchange timing for HECM-6 media were not significantly different between in day 3(78.6% vs. 45.5%) and day 4(75.0% vs. 43.2%)(P<0.05). The cleavage rate and developmental rate to blastocysts according to co-culture system were not significantly different between with (74.2% vs. 41.4%) and without BOEC(73.95 vs. 43.5%) (P<0.05). The number of blastomere in blastocyst stage after co-culture with or without BOEC was not significantly different (106.7$\pm$5.1 and 96.6$\pm$4.0). In conclusion, the most transferable IVP embryos could be produced from Ham's F-10 medium for IVM, Percoll density gradient method for IVF sperm separation and in vitro culture in HECM-6 until day 3 or day 4, and then transferred into TCM-199 until day 9 within adequate embryo density in culture droplets after insemination.

• 한국가축번식학회지
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• 제27권2호
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• pp.115-123
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• 2003

본 연구는 돼지난포란의 체외배양액에 황화합물인 Catalase 첨가 및 저분압산소조건 하에서 첨가배양함으로서 돼지난포란의 체외성숙과 체외배발달에 미치는 영향을 구명하고자 실시하였다. 본 연구에서 얻어진 결과를 요약하면 다음과 같다. 1. 돼지난포란을 체외성숙배양액인 0.9mM cy-steine이 함유된 NCSU-23 배양액에 CAT를 각각 0, 100, 500 및 1000uni1s/m1 첨가하여 체외성숙 및 수정을 실시한 결과, 모든 평가요소에서 처리구가 대조구에 비하여 유의적으로 낮은 결과를 나타내어 체외수정에 좋지 않은 영향을 미치는 것으로 판단된다(P>0.05). 2. 체외수정을 실시한 후, 배발달배양액인 NCSU-23에 7일 동안 배양한 결과, 배반포형성률과 총세포수에 있어서 처리구가 대조구에 비하여 유의적으로 낮은 결과를 나타내어 체외성숙시 CAT 첨가배양은 배발달에 좋지 않은 것으로 판단된 다(P>0.05). 3. 배발달배양액인 NCSU-23에 CAT를 각각 0, 100, 500 및 1000units/m1을 첨가하고 5 및 20% 산소농도하에서 7일간 배양을 실시한 결과, 산소농도에 따른 차이는 인정되지 않았으며, CAT 첨가에 따른 배반포형성률과 총세포수에 있어서도 처리구가 대조구보다 유의적으로 낮은 결과를 나타냈다(P>0.05). 결론적으로 돼지난포란을 이용하여 체외수정란을 생산할 때, NCSU-23 배양액에 catalase의 첨가 및 산소조건은 영향을 미치지 않는 것으로 조사되었다.

• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2018년도 춘계학술발표회
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• pp.45-45
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• 2018

We had already reported the successful germination for green pods of purple lady's slipper orchid (Cypripedium macranthos Sw.). The green pod methods is to take immature seeds in green capsules, sterilize the capsule, and take out the sterile seeds. This method, however, needs very critical time of harvest. The critical time of seed harvest changes depending upon the species, condition of the specimen, and climatic influence, and the right time lies between 5 and 12 weeks after fertilization. In this study, the mature seeds were collected after 120-130 days with hand-polination of lady's slipper orchids. Mature seeds are usually dormant and it has to be overcome, either with hormone or storing the seeds near freezing for two or three months to break dormancy. The seeds were first surface sterilized with 70% ethanol and then transferred 1% NaOCl for 10-15 minutes, followed by rinses 3 times with sterilized distilled water. The cypripedium seeds consists of an embryo within a seed coat known as a testa. The testa is water repellent and the seed has a large air space between the embryo and testa so the seed tends to float on water. We had resolved the problems with vacuum pump to soak water into the testa before sterilization. The seeds were placed on liquid or agar solidified germination media. Cultures were incubated at $24{\pm}1^{\circ}C$ in dark. The seeds were germinated in 6-8 weeks in liquid suspension culture (germination percentage over 18%); however, the seeds on agar solidified media took more than 5 months to germinate and the germination percentage less than 5%. The most effective media for liquid culture was 1/4 strength Murashige and Skoog (MS) medium with 50 ml/l coconut water ($4brix^{\circ}$) at pH 5.8.

• Clinical and Experimental Reproductive Medicine
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• 제25권1호
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• pp.43-50
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• 1998

This study was performed to determine the effects of nitric oxide on human sperm cell function. Semen samples were obtained from normal healthy volunteers. Motile spermatozoas collected by swim-up method were incubated up to 24 hours in Ham's F-10 medium supplemented with a various concentration of sodium nitroprusside (nitric oxide releasing agent). Sperm motility, hyperactivation, acrosome reaction rate, and acrosin activity were determined. The results are as follows; 1. 1mM of SNP resulted in a significant decrease in sperm motility ($44.8%{\pm}8.9%:78.1%{\pm}6.3%$, and hyperactivation $(10.4%{\pm}6.4%:47.7%:{\pm}9.5%)$ after incubation for 3 hours compared with the control group (Ham's F-10 alone), but had no effect on acrosome reaction. 2. At $100{\mu}M$ SNP, sperm motility was reduced after incubation for 6 hours $(54.8%{\pm}3.2%)$ compared with that of the control group $(82.7%{\pm}8.9%)$, but hyperactivation and acrosome reaction were not affected. 3. However, a lower concentration (less than $10{\mu}M$) of SNP had no effect on sperm motility and hyperactivation for 8 hours of incubation but significantly decreased them when incubation periods were increased up to 24 hours compared with the control group. On the other hand, $1{\mu}M$ and $10{\mu}M$ SNP significantly increased the acrosome reaction rate in both acrosomal status ($17.3%{\pm}5.2%$, $23.5%{\pm}4.7%$, respectively) and acrosin activity ($34.3{\mu}IU{\pm}10.5{\mu}IU,\;45.6{\mu}IU{\pm}5.6{\mu}IU$, respectively) as compared with the control group $(7.0%{\pm}4.0%,\;9.5{\mu}IU{\pm}3.4{\mu}IU)$. These results indicate that SNP, NO releasing agent, has a dose-dependent effects on the sperm cell function. Therefore it may positively affect the fertilization by promoting acrosomal reaction at a lower concentration (less than $10{\mu}M$).

• 한국수정란이식학회지
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• 제18권3호
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• pp.237-242
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• 2003

본 연구에서는 소에 있어서 수정란 이식을 다각적으로 분석하고 개선하기 위해 이식한 결과를 비교 검토하였고 그 결과는 다음과 같다. 1. 채란된 한우 난포란을 이용하여 생산된 체외수정란을 서로 다른 지역별로 이식한 바 B지역(경주)에서는 106의 수란우에 이식하여 51두(48.1％)가 수태하였고, A지역(김천) 수태율(33.8％)과 D 지역(경산) 수태율(35.3％)보다 유의하게 높았지만, C 지역(탑리)에서는 유의차가 존재하지 않았다. 따라서, 수정란이식에 있어서 이식 지역과 사육 환경, 시술자의 숙련도에 따라 수태율이 달라질 수 있다는 것을 알 수 있었다. 2. 수란우의 산차에 따른 수태율은, 미경산우 42.9％로서 경산우의 36.6％에 비해 다소 높은 수태율을 보였으나 유의적인 차이는 인정되지 않았다(P<0.05). 3. 이식 수정란의 발육단계는 중기 배반포 또는 후기 배반포로서 구분하여 수란우에 이식하였다. 중기 배반포가 45.5％, 후기 배반포 41.0％로서 중기 배반포가 후기 배반포보다 높은 수태율을 나타내었지만 유의한 차이는 인정되지 않았다. 배반포기가 생성된 날짜에 따른 수란우의 수태율은 7일째 생성된 배반포 이식시 수태율은 43.8％, 8일째 생성된 배반포 이식시 수태율은 32.9％, 9일째 생성된 배반포 이식시 수태율은 20.0％로서 7일째 배반포의 수태율 이 다른 두 처리군보다 수태율은 높았지만, 유의차는 인정되지 않았다.