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This experiment was carried out to clarify the physical and chemical characteristics of whole semen, seminal plasma and spermatozoa in Korean native bull. The results obtained were summarized as follows: 1. Semen volume, sperm density and total number of sperm were on the average 5.9±0.23 ㎖, 10.74±0.71 10^8/㎖ and 63.43±2.13 10^8 /ejaculate respectively. All of them were higher in Spring and Winter than in Summer and Autumn. 2. The pH value, viscosity, specific gravity and dry weight of whole semen were 6.5±0.03, 5.5±0.43, 1.036±0.002 and 9.9±0.20% respectively. The pH value was lowest in Summer while the viscosity and specific gravity were highest in winter and the dry weight was heaviest in Spring. 3. The mean values of sodium in whole semen, seminal plasma and spermatozoa were 195.87±76.8 ㎎/100㎖, 197.36±34.94㎎/100㎖ and 56.46±24.75γ/10^8 and those of potassium were 183.75±42.32 ㎎/100m1, 191.59±43.23 ㎎/100 ㎖ and 56.91±18.85 γ/10^8 respectively. The mean values of chloride were 353.23±44.82 ㎎/100 ㎖, 225.43±30.68 ㎎/100㎖ and 136.08±13.02γ/10^8 and those of total phosphorus were 60.37±6.21㎎/100㎖. 33.19±2.77 ㎎/100㎖ and 25.03±2.14 γ/10^8 respectively. 4. Concentrations of fructose and citric acid in whole semen were 393.60±77.55 ㎎/100 ㎖ and 552.58±67.12 ㎎/100 ㎖, in seminal plasma 722.40±181.14 ㎎/100 ㎖ and 649.06±61.82 ㎎/100 ㎖, and in spermatozoa 46.19±8.81 γ/10^8 arid 110.64±8.32γ/10^8. 5. Average total nitrogen values in whole semen, seminal plasma and spermatozoa were 1577.95±86.92 ㎎/100㎖, 1266.1±198.92 ㎎/100 ㎖ and 536.84±119.34γ/10^8, non-protein nitrogen in these were 351.8±99.21 ㎎/100 ㎖, 374.9±73.88 ㎎/100 ㎖ and 159.20±29.29 γ/10^8, and urea were 26.89±8.29 ㎎/100 ㎖, 38.24±10.25 ㎎/100 ㎖ and 5.94±1.45γ/10^8 respectively. 6. Total amino acid in whole semen, seminal plasma and spermatozoa were 8474.32 ㎎/100m1, 6901.73 ㎎/100 ㎖ and 3220.72γ/10^8 respectively. 7. Average values of the total lipid in whole semen, seminal plasma and spermatozoa were 702.4±53.9 ㎎/100㎖, 492.5±6.1 ㎎/100 ㎖ and 322.2±56.5 γ/10^8, phospho lipid 130.9±8.8 ㎎/100 ㎖, 83.7±6.8 ㎎/100 ㎖ and 70.5±9.9 γ/10^8, and total. cholesterol 282.4±41.3 ㎎/100 ㎖, 189.8±7.1 ㎎/100 ㎖ and 81.6±13.4γ/10^8 respectively.
To establish the industrial utilization of frozen bovine embryo, bovine embryos were frozen to -196℃, storaged, thawed and transferred. The results obtained from these experiments are summarized as follows : 1. Superovulation was induced from 27 Holstein donor cows. The average numbers of corpus luteum per donor cow was 10.3. The egg recovery rate was 56.3%, and the average number of recovered embryo was 4.6 per donor cow, 2. The pregnancy rates of frozen embryos by embryonic stages such as late morula, early blastocyst and blastocyst were 37.5%, 50.0%o and 42.9%, respectively, and those of fresh embryos were 55.0℃, 64.3% and 69.2%, respectively. 3. When fresh and frozen embryos were transferred to recipients whose estrus were naturally induced, the pregnancy rates were 53.3% and 36.8%, respectively. However, the pregnancy rates were 65.7% and 66.0%, respectively when fresh and frozen embryos were transferred to recipients whose estrus were artificially induced by PGF₂α. 4. When fresh and frozen embryos were transferred into tip of uterine horn, the pregnancy rates were 77.4% and 72.4%, respectively. On the other hand, pregnancy rates were 31.3% and 21.4%, respectively when fresh and frozen embryos were transferred into the bottom of uterine horn.
本 硏究에서는 卵巢內에 존재하는 다수의 未成熟 卵胞卵을 회수한 다음 이를 in vitro system에서 成熟, 受精 및 胚發達에 있어서 卵丘細胞(cumulus cell)가 미치는 영향과 그 기전을 조사하였다. 卵丘細胞에 대하여 좀더 구체적인 검토를 수행할 목적으로 牛卵胞卵에 대한 抗 卵丘細胞 抗體(Rabbit anti-bovine cumulus cell antibody)를 제조하여 Ammonium sulfate precipitation과 Protein A-sepharose CL-4B affinity chromatography의 의해 IgG만을 분리 정제한 다음, indirect ELISA와 Immunoprecipitation法에 의해 확인된 이들 각각의 抗體(intact cumulus cell antibody, solubilized cumulus cell antibody)를 牛卵胞卵의 體外成熟과 受精時 첨가하는 卵丘細胞가 體外成熟과 受精 및 갑發達에 미치는 영향을 조사하여 다음과 같은 결과를 얻었다. 1) 無處理 卵丘細胞와 溶解 卵丘細胞를 抗原으로 처리한 개체로부터 血淸을 채취하여 間接酸素免疫分析法으로 抗體 형성여부를 확인한 결과 1:12,800의 稀釋倍率에서도 두 抗血淸 處理區가 對照 血淸區에 비해 높은 O.D.價를 나타냄으로써 두 가지 형태의 抗原이 다같이 抗卵丘細胞 抗體를 생산하는 것으로 확인되었다. 2) Iodogn과 Immunoprecipitation를 이용하여 항체의 생성을 재차 確認하였는데 Iodogen method를 이용한 牛卵丘細胞의 ?? Iabelling은 25,000/10㎕ cmp이었다. 또한 ??-bovine cumulus cell과 각각의 抗血淸 反應 결과 생성된 Immunoprecipitates의 radioactivity와 O.D價를 조사하였던바, 無處理 혹은 溶解化된 卵丘細胞에서 공히 높은 抗體의 생성을 확인했으며 두 處理群을 비교해 볼 때 溶解化된 卵丘細胞 抗體가 無處理 卵丘細胞 抗體보다 더 높은 反應性과 O.D價를 가지고 있음을 알 수 있었다. 3) 無處理 卵丘細胞에 대한 抗血淸과 溶解化된 卵丘細胞에 대한 抗血淸으로부터 抗體를 분리해 냈을 때 정제된 Ig G의 O.D.價를 각각 1.19mg/ml 이상이었다. 4) 卵丘細胞가 부착된 卵胞卵과 제거된 卵胞卵에 無處理 卵丘細胞에 의해 생성된 抗體를 처리한 결과, Metaphase Ⅱ 段階에까지 발달한 卵胞卵은 卵丘細胞가 제거된 卵胞卵의 對照區를 포함한 모든 抗體 處理區의 경우 47.6∼59.1%로써 卵丘細胞가 부착된 卵胞卵의 對照區 78.8%에 비해 유의하게 낮았다. (P<0.01). 5) 卵丘細胞가 부착된 卵胞卵과 제거된 卵胞卵의 溶解 卵丘細胞에 의해 생성된 抗體를 처리한 결과, M Ⅱ 단계에까지 발달한 卵胞卵은 卵丘細胞가 제거된 卵胞卵의 對照區를 포함한 모든 抗體 處理區의 경우 47.6∼59.1%로써 卵丘細胞가 附着된 卵胞卵의 對照區 82.1%에 비해 有意하게 낮았다.(P<0.01). 抗卵丘細胞 抗體의 작용으로 인하여 이러한 卵丘細胞의 효과는 억제되는 결과로 미루어 볼 때 卵丘細胞는 體外成熟시 卵胞卵의 核成熟을 증진시키는 것으로 사료된다. 6) 卵丘細胞가 부착되었거나 또는 제거된 成熟 牛卵胞卵에 武處理 卵丘細胞에 대한 抗體를 처리한 다음 體外受精을 실시한 결과 卵丘細胞가 제거된 卵胞卵에 있어서 對照區를 포함한 모든 抗體 處理區의 受精率 45.0∼53.7%는 卵丘細胞가 부착된 卵胞卵에 있어서 對照區의 受精率 64.3%에 비해 다소 낮았다. 한편 雌性 및 雄性 前核이 동시에 형성된 牛卵胞卵의 비율은 卵丘細胞를 제거한 卵胞卵의 對照毆를 포함한 모든 抗體 處理區가 18.2∼38.9%로써 卵丘細胞가 부착된 卵胞卵에 있어서의 對照區 51.9%에 비해 有意하게 낮았다.(P<0.05). 7) 卵丘細胞가 부착되었거나 또는 제거된 成熟 卵胞卵에 溶解 卵丘細胞에 대한 抗體를 처리한 다음 體外受精을 실시한 결과 卵丘細胞가 제거된 卵胞卵에 있어서 對照區를 포함한 모든 抗體 處理區의 受精率 45.0∼52.5%는 卵丘細胞가 부착된 卵胞卵에 있어서 對照區의 受精率 62.8%에 비해 다소 낮았다. 또한 雌性 및 雄性 前核이 동시에 형성된 卵子의 비율은 卵丘細胞가 제거된 卵胞卵에 있어서의 對照區를 포함한 모든 부착된 卵胞卵가 26.3∼38.3%로써 卵丘細胞가 부착된 卵胞卵에 있어서의 對照區 55.6%에 비해 有意하게 낮았다.(P<0.05). 8) 抗體處理를 받은 牛卵胞卵의 체외발생을 조사하였던 바, 卵丘細胞가 제거된 卵胞卵에 있어서 對照區를 포함한 모든 抗體 處理區의 受精率이 8-細胞期, 16-細胞期 및 桑實胚期 혹은 胚盤胞期까지 발달한 비율은 7.1∼14.5, 2.9∼5.9 및 1.5%∼2.9%로써 卵丘細胞가 부착된 난자에 있어서 對照區의 18.6, 10.0 및 8.6%에 비해 낮은 胚發生率을 나타냈다. 抗卵丘細胞 抗體를 처리함으로써 卵丘細胞 작용이 억제된다는 本 硏究 결과는 卵丘細胞가 卵胞卵의 體外受精率과 前核形成率 및 胚發生能에 유의한 효과가 있다는 것을 시사한다. These experiments were carried out to investigate the effect of cumulus cells on in vitro maturation, fertilization, and subsequent development of follicular oocytes recovered from the bovine ovaries as well as to study the mechanisms involved in these processes. To examine the specific role of cumulus cell, antisera to bovine cumulus cell were produced Japanese Giant rabbit by repeated immunization of intact or solubilized bovine cumulus cell and purified IgG by the Ammonium sulfate precipitation and Protein A-Sepharose affinity chromatography. Then, these IgG of the bovine cumulus cell-specific antibodies were confirmed by indirect ELISA and Immunoprecipitation, and the effects of theses antibodies were investigated in vitro maturation, fertilizaiton, and development of bovine follicular oocytes. The results obtained in these experiment were summarized as follows: 1. The titer of the antibodies to cumulus cell determined by indirect ELISA using intact or solubilized bovine cumulus cell coasted plated was very high in both intact and solubilized cumulus cell. Especially, the optical density(O.D.) at 1: 12,800 dilution of antibodies was significantly higher than those of non-immunized control sera. 2. To reexamine the immunity of antibody, cumulus cells were labelled with I using the Iodogen method and the radioactivity of I into cumulus cell was shown approximately 25,000cpm/10㎕. The high radioactivity and O.D. value in immunoprecipitating reaction of I-bovine cumulus cell with anti-cumulus cell antisera were reconfirmed the existence of immuno-reactivity of antibody to cumulus cell. In addition, reactivities and O.D. values of solubilized cumulus cell antibodies were significantly higher than those of intact cumulus cell antibody. 3. O.D. values of the purified intact and solubilized cumulus cell antibodies were more than 1.19mg/ml. 4. When the follicular oocytes were treated with antibody to intact cumulus cell, the maturation rates of cumulus-enclosed and removed oocytes were ranged from 47.6 to 59.1%. These values are significantly lower than those(78.8%) fo follicular oocytes cultured without the antibody (p<0.01). 5. The maturation rates of cumulus-enclosed and removed oocytes treated with antibody against solubilized cumulus cell were ranged from 46.7 to 59.1%, showing significantly lower than those(82.1%) of oocytes cultured in antibody free medium(P<0.01). These results that the beneficial effect of cumulus cell to the oocyte maturation was inhibited by the action of antibody to cumulus cell suggest that cumulus cell enhances nuclear maturation of follicular oocytes. 6. When the follicular oocytes matured in vitro were treated with antibody to intact cumulus cell, the fertilization rates of cumulus intact and removed oocytes were ranged from 45.0 to 53.7%. These values are slightly lower than those(64.3%) of follicular oocytes not treated with the antibody, and increased frequencies of both male and female pronucler formations were detected in cumulus intact oocytes cultured in medium without antibody (P<0.05). 7. The fertilizaiton rates of cumulus intact and removed oocytes treated with antibody to solubilized cumulus cell were ranged from 45.0 to 52.5%, significantly lower than those(62.8%) of oocytes cultured in antibody free medium, and increased frequencies of ova with male and female pronuclei formations were found in the presence of cumulus cells(P<0.05). 8. The development rates of cumulus intact and removed oocytes to 8-, 16-cell, and morula or blastocyst stage after treatment of intact and solubilized cumulus cell antibody were ranged from 7.1 to 14.5 from 2.9, and from 1.5 to 2.9T, respectively. These rates were slightly lower than 18.6, 10.0 and 8.6% of cumulus intact oocytes cultured in medium without the antibody. The results that beneficial effects of cumulus cell to the pronuclear formation and embryo development were blocked by the action of antibody to cumulus cell suggest that cumulus cell significantly promotes normal fertilizaiton with proper pronuclear formation and embryo development.
In vascular smooth muscle cells (VSMCs), inductionof the heme oxygenase-1 (HO-1) confers vascularprotection against cellular proliferation mainly viaits up-regulation of the cyclin-dependent kinaseinhibitor p21WAF1/CIP1 that is involved in negativeregulation of cellular proliferation. In the presentstudy, we investigated whether the phytochemicalcurcumin and its metabolite tetrahydrocurcumincould induce HO-1 expression and growth inhibitionin rat VSMCs and, if so, whether their antiproliferativeeffect could be mediated via HO-1 expression. Atnon-toxic concentrations, curcumin possessingtwo Michael-reaction acceptors induced HO-1 expressionby activating antioxidant response element(ARE) through translocation of the nuclear transcriptionfactor E2-related factor-2 (Nrf2) into thenucleus and also inhibited VSMC growth triggeredby 5% FBS in a dose-dependent manner. In contrast,tetrahydrocurcumin lacking Michael-reaction accep -tor showed no effect on HO-1 expression, AREactivation and VSMC growth inhibition. The antiproliferativeeffect of curcumin in VSMCs was accompaniedby the increased expression of p21WAF1/CIP1. Inhibition of VSMC growth and expression ofp21WAF1/CIP1by curcumin were partially, but notcompletely, abolished when the cells were coincubatedwith the HO inhibitor tin protoporphyrin. In human aortic smooth muscle cells (HASMCs),curcumin also inhibited growth triggered by TNF-αand increased p21WAF1/CIP1expression via HO-1-dependent manner. Our findings suggest that curcuminhas an ability to induce HO-1 expression,presumably through Nrf2-dependent ARE activation,in rat VSMCs and HASMCs, and provide evidence thatthe antiproliferative effect of curcumin is considerablylinked to its ability to induce HO-1 expression.
These experiments were carried out to obtain the basic informations and to establish the best conditions needed for the activation of mouse eggs by ethanol treatment. The influence of ethanol concentration and the age of eggs, time from hCG injection to recovery, on activation frequency of eggs was investigated, To activate the mouse eggs parthenogenetically, cumulus masses including eggs were released into a fresh solution of ethanol for 5min and then rinsed out 5-6 times with ethanol-free medium. After incubation of cumulus masses in the drops of medium for 5 hrs at 37℃ in the humidified atmosphere of 5% CO₂ in air, the adherent cumulus cells were removed with hyaluronidase (100unit/㎖). The types of parthenogenetic eggs were morphologically classified into haploid (H), immediate cleavage (IC) and diploid (D) under a inverted microscope. These parthenogenetic eggs were then incubated into the drops of mKRB medium for 96 hrs under the same conditions as above. The results obtained in these experiments were summarized as follows : 1. ICR female mice (1) When the eggs were treated with a variety of ethanol concentrations, 0 to 11%, the highest parthenogenetic activation rate (94.4%) was observed at 7% ethanol concentration. (2) Activation frequency was comparatively high when the eggs were recovered from the oviducts in more than 18 hrs after hCG injection. 2. (C₃H × C57BL) F₁ hybrid female mice (1) Of 341 F₁ eggs treated with 7% ethanol for 5 min at 18 hrs after hCG injection, 317 eggs (93.0%) were activated and a majority of parthenogenetic eggs were haploids (71.9%). (2) After 96 hrs of in vitro culture, the developmental rate of haploid, immediate cleavage and diploid eggs to morphologically normal morula or blastocyst stage was 16.7, 66.7 and 72.3%, respectively. (3) Of 18 haploid eggs recovered from the uteri on the 4th day of pregnancy after transfer to the oviducts of recipients, 12 eggs (60.0%) were normally developed to morula or blastocyst stage.
Animal industry in Korea urgently needs the domestic introduction no the industrial application of embryo transfer technique. Namely, this technique can be utilized effectively. as means of the improvement of livestocks, as means of the increase of meat production, as means of substitute for the livestock import, and dissemination of new breed. However, as this technique available in our country is remaining initial stage, we can not make use of the technique industrially unless we make much improvement as follows; induction of superovulation, non-surgical recovery of embryos, synchronization between the estrus cycles of donor and recipient, non-surgical transfer of embryos, etc. Simultaneously, the basic studies such as harvesting oocytes from ovary, in vitro culture of oocytes. in vitro capacitation of spermatozoa, cloning by culture of blastomeres and transfer of nuclei, sexing embryo, etc. should not be neglected in order to make the technique of embryo transfer more simple and convenient. For the success of these studies, universities, national and public institutes, large scale cattle farms, and small scale cattle farms should cooperate each other. For instance, universities undertake basic researches, and then national and public institutes apply the results of the researches to animal industry along with cooperation by large scale cattle farms. By the help of the cooperative organizations, the technique relevant to our environment and farm condition may be able to be finalized, and to be applied to small scale cattle farm. Consequently, being served to stimulate animal productivity, this technique can be contributed to the development of livestock industry in Korea.
This experiment was performed to clarify the effect of lipid decreasing factor included in the seminal plasma of bull semen on the content of various phospholipids and neutral lipids in boar spermatozoa. Total phospholipids and total neutral lipids extracted from boar spermatozoa after incubation and freezing with the solution of bull plasma protein were fractionated quantitatively on the column of Dowex-I and silica gel, respectively. The results obtained in this experiment were as follows: 1. Total phospholipids extracted from boar spermatozoa were fractionated into 12 fractions and the content of each fraction in boar spermatozoa were decreased significantly(P$lt;0.02) by the action of lipid decreasing factor included in bull plasma protein. Decreasing rates were ranged from 38.24 to 47.36%. 2. The content of neutral lipids such as cholesterol-ester, free cholesterol, mono-, di-, and tri-glyceride in boar spermatozoa were also decreased significantly (P$gt;0.01) by the action of lipid decreasing factor included in bull plasma protein. Decreasing rates of cholesterol-ester, free cholesterol, mono-, di-, and tri-glyceride were 28.62, 30.85, 59.65, 55.49, and 54.32, respectively. 3. Possibility that lipid decreasing factor included in bull seminal plasma is not a lipid hydrolyzing enzyme but a specific factor which extracts all sort of lipids from spermatozoa was increased by the results of present experiment.
This experiment was carried out to develop a new luteolytic reagent and its effective administration technique which could be used for the synchronization of estrus cycle in dairy cattle. Five miligram of prostaglandin F₂x and 2 ㎎ of nor-adrenalin were administrated by way of intravulvosubmucous, respectively, and the induction of estrus and the rates of synchronization were observed. The changes in the concentration of steroid hormones such as progesterone, estradiol-17β and testosterone in blood plasma of dairy cattle prior to and after administration of the reagents were also estimated. The results obtained in this experiment were summarized as follows; 1) Standing estrus was induced in 8 cows out of 10 cows within 2 to 4 days after administration of prostaglandin F 2α injected by way of intravulvosubmucous at 10 days after natural ovulation. 2) Six cows out of 10 cows were resulted in standing estrus within 5 to 6 days after administration of nor-adrenalin via intravulvosubmucous at 10 days after natural ovulation. 3) The concentrations of steroid hormens in blood plasma of dairy cattle responded to the administration of prostaglandin F2α and nor-adrenalin were determined. The Plasma progesterone levels had fallen from 7.615±0.81ng/㎖ and 6.242±182ng/㎖ to 2.839±0.341ng/㎖ and 1.583±0.520 ng/㎖, respectively, within 6 hoots after administration of prostaglandin F2α and noradrenalin and then declined to a baseline level within 24 hours. 4) The concentration of plasma estradiol-17β rose to a peak level, 55.7±13.19 pg/㎖, within 3 days after injection of prostaglandin F2α and 60.3±8.15 pg/㎖ within 5 days after injection of nor-adrenalin, respectively, and the estrus were induced at the time when estradiol-17β levels pose to the peak, respectively. 5) The peak levels of estradiol-17β during the estrus induced by the administration of prostaglandin F2α or nor-adrenalin were lower significantly than those of estradiol-17β during the natural estrus, respectively. 6) Testosterone concentration increased during the 3 days preceding estrus and attained peak values, 59.4±9.73pg/㎖ and 55.8±6.14pg/㎖, respectively, 48 hour before the onset of estrus induced by prostaglandin F2X or nor-adrenalin. 7j Progesterone concentrations in blood plasma of dairy cattle not responded to the administration of prostaglandin or nor-adrenalin were also fallen sharply within 6 hours after administration of the reagents but no levels below 1.0 ng/㎖ were observed. 8) From the results above mentioned it could be concluded that nor-adrenalin was as effective reagent as prostaglandin F2α for the synchronization of estrus cycle in dairy cattle and that intravulvosubmucous injection method was the most effective technique for the administration of the reagents.