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Study on In Vitro Aggregation and Culture of Mouse Embryos by Phytohemagglutinin-P (Phytohemagglutinin-P 첨가(添加)에 따른 생쥐배(胚)의 시험관내(試驗管內) 응집(凝集)과 배양(培養)에 관하여)

  • Park, Hang Kyun;Ryou, Zae Yoong
    • Current Research on Agriculture and Life Sciences
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    • v.7
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    • pp.83-97
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    • 1989
  • This study was carried out to obtain basic information necessary for aggregation and in-vitro culture of mouse embryos by treating phytohemagglutinin-p (PHA-P). The 4-, 8-cell and morula embryos were obtained from female mice of albino BALE/C, CBA and C57BL strains, those were injected 5 i.u pregenant mare serum gonadotrophin and 5 i.u human chorionic gonadotrophin to superovulation. The zona pellucidia was removed by placing the embryos in Acidic Tyrode solution containing 1.0% protease or/and 5 ug/ml PHA-P. The pairs of zona free embryos were subjected to aggregation by glassneedle in BMOC-3 containing 5 ug/ml PHA-P. The aggregation embryos were cultured in Brinster's mouse ova culture-3(BMOC-3) medium under the gas phase of 5% $CO_2$ in air $37^{\circ}C$ for 13 to 50 hours. The results obtained in this study are summarised as follows : 1. When 4-, 8-cell and morula embryos were zona-freed in acidic Tyrode solution containing 1.0% protease or/and 5 ug/ml PHA-P, and cultured in vitro to blastocysts, the 4- and 8-cell embryos showed slightly less development rates than the morula one did, and solution of 5 ug/ml PHA-P brought some higher development rate than negative control. 2. As 2, 5 or 10 ug/ml PHA-P was added to the solution to aggregate 4-, 8-cell or morula embryos, 2 ug/ml solution represented slightly lower aggregation rate than the higher levels solutions, and 4- and 8-cell embryos showed higher rates than morula one did (P<.05). 3. In respect to the development rates of aggregated embryos to morula no significant difference was found among PHA-P levels and between 4-and 8-cell embryos. With respect to those of aggregated embryos to blastocysts the different levels of PHA-P showed similar results, however, the 4- and 8-cell embryos represented higher rates than the morula one did (P<.05). 4. The mean time necessary for development of aggregated 4-, 8-cell and morula embryos to blastocysts were 38.5-40, 26-27 and 19-20hrs. Respectively in solution for aggregation. 5. The aggregation rates of embryos were 34-94%, when treated protease or/and PHA-P. Supplementation of 5 ug/ml PHA-P to the solution for aggregation showed a trend demonstrating higher aggregation rate compared to negative control, although no significance was found. However, 4- and 8-cell embryos represented significantly higher aggregation rates than the morula one did (P<.05). 6. The development rates of 4- and 8-cell embryos to morula were 52.7-84.7 and 73.8-87.2%, respectively, showing no significant difference between two cell stages. However, the aggregation rates of embryos treated with solution containing PHA-P were higher than negative control (P<.05). 7. The development rates of 4- and 8-cell and morula embryos to blastocysts were 41.7-77.7 78.7-83.0 and 0-19.2%, respectively. The rates of 4-cell embryos treated with PHA-P were significant higher than the negative control (P<.05). The 8-cell and morula embryos also showed more rates when treated PHA-P.

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The Role of Camera-Based Coincidence Positron Emission Tomography in Nodal Staging of Non-Small Cell Lung Cancer (비소세포폐암의 림프절 병기 결정에서 Coincidence PET의 역할)

  • Lee, Sun-Min;Choi, Young-Hwa;Oh, Yoon-Jung;Cheong, Seong-Cheoll;Park, Kwang-Joo;Hwang, Sung-Chul;Lee, Yi-Hyeong;Park, Chan-H;Hahn, Myung-Ho
    • Tuberculosis and Respiratory Diseases
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    • v.47 no.5
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    • pp.642-649
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    • 1999
  • Background: It is very important to determine an accurate staging of the non-small cell lung cancer(NSCLC) for an assessment of operability and it's prognosis. However, it is difficult to evaluate tumor involvement of mediastinal lymph nodes accurately utilizing noninvasive imaging modalities. PET is one of the sensitive and specific imaging modality. Unfortunately PET is limited use because of prohibitive cost involved with it's operation. Recently hybrid SPECT/PET(single photon emission computed tomography/positron emission tomography) camera based PET imaging was introduced with relatively low cost. We evaluated the usefulness of coincidence detection(CoDe) PET in the detection of metastasis to the mediastinal lymph nodes in patients with NSCLC. Methods: Twenty one patients with NSCLC were evaluated by CT or MRI and they were considered operable. CoDe PET was performed in all 21 patients prior to surgery. Tomographic slices of axial, coronal and sagittal planes were visually analysed. At surgery, mediastinal lymph nodes were removed and histological diagnosis was performed. CoDe PET findings were correlated with histological findings. Results: Twenty of 21 primary tumor masses were detected by the CoDe PET. Thirteen of 21 patients was correctly diagnosed mediastinal lymph node metastasis by the CoDe PET. Pathological N0 was 14 cases and the specificity of N0 of CoDe PET was 64.3%. Sensitivity, specificity, positive predictive value, negative predictive value and accuracy of N1 node was 83.3%, 73.3%, 55.6%, 91.7%, and 76.2% respectively. Sensitivity, specificity, positive predictive value, negative predictive value and accuracy of N2 node was 60.0%, 87.5%, 60.0%,87.5%, and 90.0% respectively. There were 3 false negative cases but the size of the 3 nodes were less than 1cm. The size of true positive nodes were 1.1cm, 1.0cm, 0.5cm respectively. There were 1 false positive among the 12 lymph nodes which were larger than 1cm. False positive cases consisted of 1 tuberculosis case, 1 pneumoconiosis case and 1 anthracosis case. Conclusion: CoDe PET has relatively high negative predictive value in the enlarged lymph node in staging of mediastinal nodes in patients with NSCLC. Therefore CoDe PET is useful in ruling out metastasis of enlarged N3 nodes. However, further study is needed including more number of patients in the future.

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Effects of Light Intensity, Nutrient Solution Compositions before Harvest and the Time of Nutrient Solution Removal on Nitrate Contents in Hydroponically-Grown Leaf Lettuces in Closed Plant Production System (폐쇄형 식물생산시스템에서 광도, 수확 전 양액조성 및 양액결제시기가 잎상추의 체내 질산염 함량에 미치는 영향)

  • Yeo, Kyung-Hwan;Choi, Gyeong-Lee;Lee, Jung-Sup;Lee, Jae-Han;Park, Kyoung-Sub;Kim, Jin-Hyun
    • Journal of Bio-Environment Control
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    • v.26 no.4
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    • pp.393-401
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    • 2017
  • The nitrate ($NO_3{^-}$) accumulation of hydroponically grown leafy vegetables may increase in the condition of a closed-type plant production system with low light intensity due to low activity of enzymes involved in nitrogen assimilation and the use of $NO_3-N$ as major nitrogen source. The objective of this study is to investigate the effects of light intensities, nutrient solution compositions and the time of nutrient solution removal before harvest on nitrate contents of hydroponically-grown lettuces in a closed plant production system. The reduction of nitrate contents in leafy lettuces 'Cheongchima' was higher in the treatments of 'TW' (nutrient solution removal) and '$(NH_4)_2CO_3$' (use of ammonium carbonate as nitrogen source) than those in other treatments, which significantly lowered fresh weight and leaf area of the plants. In the light intensity of $100{\mu}mol{\cdot}m^{-2}{\cdot}s^{-1}$, the nitrate content was effectively reduced without causing any growth retardation, by substitution of the nutrient solution composition that $NO_3-N$ was removed ('$NO_3-N$ removal' treatment) or the half strength of standard nutrient solution was applied ('1/2 S' treatment), for 7days before harvest. The effects of light intensity and the time of nutrient solution removal before harvest on growth and nitrate contents in leafy lettuces were investigated. The nitrate contents in leaves under the light condition of $300{\mu}mol{\cdot}m^{-2}{\cdot}s^{-1}$ before nutrient solution removal were lower than those of 100 or $200{\mu}mol{\cdot}m^{-2}{\cdot}s^{-1}$. The removal of nutrient solution for 7 days before harvest quickly reduced the amount of nitrates in leaves in all the light intensities with a greater degree under the $300{\mu}mol{\cdot}m^{-2}{\cdot}s^{-1}$ of light condition, while the 7 days-removal with both 200 and $300{\mu}mol{\cdot}m^{-2}{\cdot}s^{-1}$ of light conditions caused decrease in 16~31% of leaf area and 20~35% of fresh weight, compared to the 3 days-removal treatment. The nitrate contents were greatly reduced from 3,018 to 1,035 in $200{\mu}mol{\cdot}m^{-2}{\cdot}s^{-1}$, and 2,021 to 480 ppm in the light condition of $300{\mu}mol{\cdot}m^{-2}{\cdot}s^{-1}$, with the nutrient solution removal for 3 days before harvest, without causing any deterioration in growth and product quality. The vitamin C contents in leaves were higher in the treatment of nutrient solution removal for both 3 and 5 days before harvest with the light condition of $300{\mu}mol{\cdot}m^{-2}{\cdot}s^{-1}$ than those in the light condition of 100 or $200{\mu}mol{\cdot}m^{-2}{\cdot}s^{-1}$.

In the Treatment I-131, the Significance of the Research that the Patient's Discharge Dose and Treatment Ward can Affect a Patient's Kidney Function on the Significance of Various Factors (I-131 치료시 환자의 신장기능과 다양한 요인으로 의한 퇴원선량 및 치료병실 오염도의 유의성에 관한 연구)

  • Im, Kwang Seok;Choi, Hak Gi;Lee, Gi Hyun
    • The Korean Journal of Nuclear Medicine Technology
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    • v.17 no.1
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    • pp.62-66
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    • 2013
  • Purpose: I-131 is a radioisotope widely used for thyroid gland treatments. The physical half life is 8.01 and characterized by emitting beta and gamma rays which is used in clinical practice for the purpose of acquiring treatment and images. In order to reduce the recurrence rate after surgery in high-risk thyroid cancer patients, the remaining thyroid tissue is either removed or the I-131 is used for treatment during relapse. In cases of using a high dosage of radioactive iodine requiring hospitalization, the patient is administered dosage in the hospital isolation ward over a certain period of time preventing I-131 exposure to others. By checking the radiation amount emitted from patients before discharge, the patients are discharged after checking whether they meet the legal standards (50 uSv/h). After patients are discharged from the hospital, the contamination level is checked in many parts of the ward before the next patients are hospitalized and when necessary, decontamination operations are performed. It is expected that there is exposure to radiation when measuring the ward contamination level and dose check emitted from patients at the time of discharge whereby the radiation exposure by health workers that come from the patients in this process is the main factor. This study analyzed the correlation between discharge dose of patients and ward contamination level through a variety of factors such as renal functions, gender, age, dosage, etc.). Materials and Method: The study was conducted on 151 patients who received high-dosage radioactive iodine treatment at Soon Chun Hyang University Hospital during the period between 8/1/2011~5/31/2012 (Male: Female: 31:120, $47.5{\pm}11.9$, average dosage of $138{\pm}22.4$ mCi). As various factors expected to influence the patient discharge dose & ward contamination such as the beds, floors, bathroom floors, and washbasins, the patient renal function (GFR), age, gender, dosage, and the correlation between the expected Tg & Tg-Tb expected to reflect the remaining tissue in patients were analyzed. Results: In terms of the discharge dose and GFR, a low correlation was shown in the patient discharge dose as the GFR was higher (p < 0.0001). When comparing the group with a dosage of over 150mCi and the group with a lower dosage, the lower dosage group showed a significantly lower discharge dose ($24{\pm}10.4uSv/h$ vs $28.7{\pm}11.8uSv/h$, p<0.05). Age, gender, Tg, Tg-Tb did not show a significant relationship with discharge dose (p> 0.05). The contamination level in each spot of the treatment ward showed no significant relationship with GFR, Tg, Tg-Tb, age, gender, and dosage (p>0.05 ). Conclusion: This study says that discharge of the dose in the patient's body is low in GFR higher and Dosage 150mCi under lower. There was no case of contamination of the treatment ward, depending on the dose and renal association. This suggests that patients' lifestyles or be affected by a variety of other factors.

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Effects of Ovarian Function on the Hypophyseal Gonadotropin Secretion in Rats (흰쥐의 난소기능(卵巢機能)이 하수체(下垂體)의 성선(性腺) 날극(剌戟)호르몬 분비(分泌)에 미치는 영향(影響))

  • Seo, Kil Woong;Kim, Chong Sup;Park, Chang Sik;Lee, Kyu Seung
    • Korean Journal of Agricultural Science
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    • v.16 no.2
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    • pp.169-178
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    • 1989
  • The study was carried out to elucidate the feedback mechanism on the hypothalamo-hypophyseal system from the functional changes of ovary in female rats. One hundred and forty-four mature female rats were lloted into the three groups; ovariectoimzed group, estradiol treated group and intact control group. The varies of 48 heads of rat were completely removed. Forty eight heads of rat were administered with $200{\mu}g$ of estradiol benzoate every 48 hours. Serum FSH, LH and prolactin levels were determined with radioimmunoassay method at 3,6,12,24 ours, and 5,10, and 15days after treatment. The rats were necropsied to measure the weights of hypophysis and to examin the histological changes in the organs. The results obtained were as follows: The weights of hypophysis were increased after ovariectomy and decreased after estradiol injection. The differences in hypophysis weights were significant between the group from 5 days after treatment. The histological changes in hypophysis were appeared from 5th day after ovariectomy. Proliferation and hypertrophy began to occur in basophilic from 10th day after ovariectomy, chromophobes were slightly hypertrophied and acidophilic cells were atrophied. In estradiol injected rats the histological findings were appeard to be contrary to those of ovariectomized rats. Serum FSH levels significantly changed after ovariectomy and estradiol injection and were higher in both the treated groups than in the intact control group. Within 18 hours after treatment the level was the highest in ovariectomized group, and thereafter the highest level was found in estradiol treated gorup. In ovariectomized rats the levels were rapidly increased 3 hours after treatment and maximum levels were found 18 hours after treatment. In estradiol treated rats the levels started to increase 18 hours after treatment and reached maximum levels 24 hours treatment. 4. Serum LH levels started to increase 3 hours after ovariectomy and estradiol injection and reached maximum levels 12 hours after ovariectomy and 24 hours after estradiol injection. There were significant differences in LH levels between the groups in each observation time. Up to 18 hours after treatment levels were higher in ovariectomized rats than in estradiol treated rats. but thereafter the levels were higher in estradiol treated rats than in ovariectomized rats. The multiple range test showed that a significant difference in LH levels was not found between ovariectomized group and estradiol treated group 18 hours and 5 days after treatment. 5. Serum prolactin levels were significantly changed after ovariectomy and estradiol injection. The levels were lower in ovariectomized rats than in intact control rats.

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Utility of Wide Beam Reconstruction in Whole Body Bone Scan (전신 뼈 검사에서 Wide Beam Reconstruction 기법의 유용성)

  • Kim, Jung-Yul;Kang, Chung-Koo;Park, Min-Soo;Park, Hoon-Hee;Lim, Han-Sang;Kim, Jae-Sam;Lee, Chang-Ho
    • The Korean Journal of Nuclear Medicine Technology
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    • v.14 no.1
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    • pp.83-89
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    • 2010
  • Purpose: The Wide Beam Reconstruction (WBR) algorithms that UltraSPECT, Ltd. (U.S) has provides solutions which improved image resolution by eliminating the effect of the line spread function by collimator and suppression of the noise. It controls the resolution and noise level automatically and yields unsurpassed image quality. The aim of this study is WBR of whole body bone scan in usefulness of clinical application. Materials and Methods: The standard line source and single photon emission computed tomography (SPECT) reconstructed spatial resolution measurements were performed on an INFINA (GE, Milwaukee, WI) gamma camera, equipped with low energy high resolution (LEHR) collimators. The total counts of line source measurements with 200 kcps and 300 kcps. The SPECT phantoms analyzed spatial resolution by the changing matrix size. Also a clinical evaluation study was performed with forty three patients, referred for bone scans. First group altered scan speed with 20 and 30 cm/min and dosage of 740 MBq (20 mCi) of $^{99m}Tc$-HDP administered but second group altered dosage of $^{99m}Tc$-HDP with 740 and 1,110 MBq (20 mCi and 30 mCi) in same scan speed. The acquired data was reconstructed using the typical clinical protocol in use and the WBR protocol. The patient's information was removed and a blind reading was done on each reconstruction method. For each reading, a questionnaire was completed in which the reader was asked to evaluate, on a scale of 1-5 point. Results: The result of planar WBR data improved resolution more than 10%. The Full-Width at Half-Maximum (FWHM) of WBR data improved about 16% (Standard: 8.45, WBR: 7.09). SPECT WBR data improved resolution more than about 50% and evaluate FWHM of WBR data (Standard: 3.52, WBR: 1.65). A clinical evaluation study, there was no statistically significant difference between the two method, which includes improvement of the bone to soft tissue ratio and the image resolution (first group p=0.07, second group p=0.458). Conclusion: The WBR method allows to shorten the acquisition time of bone scans while simultaneously providing improved image quality and to reduce the dosage of radiopharmaceuticals reducing radiation dose. Therefore, the WBR method can be applied to a wide range of clinical applications to provide clinical values as well as image quality.

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The Experimental Study for Myocardial Preservation Effect of Ischemic Preconditioning (허혈성 전조건화 유발이 심근보호에 미치는 영향에 관한 실험적 연구)

  • 이종국;박일환;이상헌
    • Journal of Chest Surgery
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    • v.37 no.2
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    • pp.119-130
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    • 2004
  • Decrease in cardiac function after open heart surgery is due to an ischemia induced myocardial damage during surgery, and ischemic preconditioning, a condition in which the myocardial damage does not accumulate after repeated episodes of ischemia but protects itself from damage after prolonged ischemia due to myocytes tolerating the ischemia, is known to diminish myocardial damage, which also helps the recovery of myocardium after reperfusion, and decreases incidences of arrythmia. Our study is performed to display the ischemic preconditioning and show the myocardial protective effect by applying cardioplegic solution to the heart removed from rat. Material and Method: Sprague-Dawley male rats were used, They were fixed on a modified isolated working heart model after cannulation. The reperfusion process was according to non-working and working heart methods and the working method was executed for 20 minutes in which the heart rate, aortic pressure, aortic flow and coronary flow were measured and recorded. The control group is the group which the extracted heart was fixed on the isolated working heart model, recovered by reperfusion 60 minutes after infusion and preserved in the cardioplegic solution 20 minutes after the working heart perfusion and aortic cross clamp, The thesis groups were divided into group I, which ischemic hearts that were hypoxia induced were perfused by cardioplegic solution and preserved for 60 minutes; group II, the cardioplegic solution was infused 45 seconds (II-1), 1 minutes (II-2), 3 minutes (II-3), after the ischemia induction, 20 minutes after working heart perfusion and aortic cross clamp; and group III, hearts were executed on working heart perfusion for 20 minutes and aortic cross clamp was performed for 45 seconds (III-1), 1minute (III-2), 3 minutes (III-3), reperfused for 2 minutes to recover the heart, and then aortic cross clamping was repeated for reperfusion, all the groups were compared based on hemodynamic performance after reperfusion of the heart after preservation for 60 minutes. Result: The recovery time until spontaneous heart beat was longer in groups I, II-3, III-2 and III-3 to control group (p<0.01). Group III-1 (p<0.05) had better results in terms of recovery in number of heart rates compared to control group, and recovered better compared to II-1 (p<0.05). The recovery of aortic blood pressure favored group III-1 (p<0.05) and had better outcomes compared with II-1 (p<0.01). Group III-1 also showed best results in terms of cardiac output (p<0.05) and group III-2 was better compared to II-2 (p<0.05). Group I (p<0.01) and II-3 (p<0.05) showed more cardiac edema than control group. Conclusion: When the effects of other organs are dismissed, protecting the heart by infusion of cardioplegic solution after enforcing ischemia for a short period of time before the onset of abnormal heart beats for preconditioning has a better recovery effect in the cardioplegic group with preconditioning compared to the cardioplegic solution itself. we believe that further study is needed to find a more effective method of preconditioning.

Studies on the Viability of Frozen Removed Seminal Plasma by Saline(RSP-S) and Tris-buffer(RSP-T) Semen of Small Spcies Dogs (소형 개 RSP-S와 RSP-T 정액의 동결 융해후의 생존성에 관한 연구)

  • 김상근
    • Korean Journal of Animal Reproduction
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    • v.25 no.3
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    • pp.269-275
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    • 2001
  • This study was carried out to investigate the general characteristics such as volume, sperm concentration, sperm motility, sperm abnormality on whole semen, RSP-S and RSP-T semen and fractional semen of small size dogs, and the effect of temperature and preservation time and cryoproservation on motility of whole and RSP-S and RSP-T semen. Multiple ejaculates were collected from small dogs by the digital manipulation of penis. 1. The volume per ejaculate semen, sperm of concentration and motility and abnormal sperm rate of 1st fractional semen were 0.65$\pm$0.09 $m\ell$, 4.52$\pm$0.35$\times$10$^{6}$ cells/$m\ell$, 15.64$\pm$3.85% and 5.50$\pm$0.62%. Also, 2nd fractional semen were 1.25$\pm$0.20$m\ell$, 3.35$\pm$0.48$\times$10$^{6}$ cells/$m\ell$, 96.25$\pm$4.65% and 4.24$\pm$0.46%. And 3rd fractional semen were 1.45$\pm$0.21$m\ell$, 3.55$\pm$0.52$\times$10$^{6}$ cells/$m\ell$, 92.82$\pm$4.24% and 4.66 $\pm$0.58%, respectively. 2. The sperm of concentration and motility and abnormal sperm rates of whole, RSP-S and RSP-T semen were 5.45$\pm$0.82$\times$10$^{6}$ cells/$m\ell$, 95.55 $\pm$4.65%, 4.58$\pm$0.45% and 4.82$\pm$0.36$\times$10$^{6}$ cells/$m\ell$, 90.10$\pm$3.42%, 6.48$\pm$0.68% and 4.55$\pm$0.45$\times$10$^{6}$ cells/$m\ell$, 93.25$\pm$3.85%, 4.82$\pm$0.58%, respectively. 3. The motility of whole, RSP-S and RSP-T semen were higher at 4$^{\circ}C$ than at 38$^{\circ}C$. When preservation temperature was at 4$^{\circ}C$, survival rates of RSP-S and RSP-T sperm were 97.54~6.25% at 1~72 hrs, 97.40~5.62% at 1~100 hrs, respectively. 4. The survival rates of slow and rapid frozen 2nd fraction, RSP-S and RSP-T semen were 67.3$\pm$4.45%, 88.8$\pm$4.46% and 46.4$\pm$3.84%, 74.4$\pm$4.20%, respectively. Survival rates was significantly higher in frozen RSP-S and RSP-T semen than that in control group(8.5$\pm$2.12%).

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Effect on In­Vitro Fertilization of Pig Oocytes Matured in Different In­Vitro Maturation Media according to Sperm Concentration of Liquid Boar semen (돼지 액상정액의 정자 주입농도가 서로 다른 체외성숙배지에서 배양된 난포란의 체외수정에 미치는 영향)

  • 박창식;이영주;고현진;양창범;손동수;서길웅;이규승
    • Korean Journal of Animal Reproduction
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    • v.26 no.1
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    • pp.1-7
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    • 2002
  • The present study was carried out to investigate the effects of maturation media on penetrability of pig oocytes by liquid boar sperm coincubated with different sperm concentrations in a modified Tris­buffered medium (mTBM). Follicular oocytes collected from ovaries of prepubertal gilts were matured in a modified TCM-199 (mTCM-199) medium, modified Waymouth MB 752/1 (mWaymouth MB 752/1) medium or NCSU-23 medium. Oocytes (30~40) were transferred into each well of a Nunc 4-well multidish containing 0.5 $m\ell$ maturation medium. The sperm­ich portion of ejaculates with greater than 90% motile sperm were used in the experiment. The semen was cooled 22 to 24$^{\circ}C$ over 2 h period. The semen was diluted with Beltsville Thawing Solution (BTS) extender at room temperature to give 2$\times$10$^{8}$ sperm/$m\ell$ in 100 $m\ell$ plastic bottle. Liquid boar semen of 30 $m\ell$ in 100 $m\ell$ plastic bottle was kept at 17$^{\circ}C$ far 5 days. The sperm with greater than 70% motility after day 5 of storage were used for in-vitro fertilization (IVF). After 44 h maturation of immature oocytes in 5% $CO_2$in air at 38.5$^{\circ}C$, cumulus cells were removed and oocytes (30~40) were coincubated for 6 h in 0.5 $m\ell$ mTBM fertilization medium with five different (1$\times$10$^{6}$ , 2$\times$10$^{6}$ , 4$\times$10$^{6}$ , 6$\times$10$^{6}$, 10$\times$10$^{6}$ $m\ell$) sperm concentrations. At 6 h after IVF, oocytes were transferred into 0.5 $m\ell$ NCSU-23 culture medium fur further culture of 6 h. At 12 h after IVF, sperm penetration, polyspermy and male pronuclear formation of oocytes were evaluated. Oocytes of NCSU-23 maturation medium decreased polyspermy and increased male pronuclear formation compared to those of mTCM­199 and mWaymouth MB 752/1 maturation media. Of oocytes matured in NCSU-23 medium and inseminated in mTBM medium with 2~4$\times$10$^{6}$ $m\ell$ sperm concentrations, 50.8~50.9% showed sperm penetration, 13.3~19.5% polyspermy and 43.9~45.4% male pronuclear formation. In conclusion, we found out that oocytes matured in NCSU­23 medium and inseminated in mTBM medium showed superior in­vitro fertilization compared to those matured in mTCM­199 and mWaymouth MB 752/1 maturation media and inseminated in mTBM medium. The optimum sperm concentrations for in-vitro fertilization of oocytes matured in NCSU-23 medium by liquid boar semen stored at 17$^{\circ}C$ for 5 days were 2~4$\times$10$^{6}$ $m\ell$.

In Vitro Fertilization of Pig Oocytes Matured In­Vitro by liquid Boar Spermatozoa (체외성숙 돼지 난포란의 액상정액을 이용한 체외수정)

  • 박창식;이영주
    • Korean Journal of Animal Reproduction
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    • v.26 no.1
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    • pp.17-23
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    • 2002
  • The present study was carried out to investigate the effects of the maturation media such as a modified TCM-199 (mTCM-199) medium, modified Waymouth MB 752/1 (mWaymouth MB 752/1) medium or NCSU-23 medium on penetrability of pig oocytes by liquid boar sperm. Oocytes (30~40) were transferred into each well of a Nunc 4-well multidish containing 0.5 $m\ell$ maturation medium. When immature pig oocytes were cultured in mTCM-199, mWaymouth MB 752/1 and NCSU-23 maturation media for 44 h in 5% $CO_2$, in air at 38.5$^{\circ}C$, the germinal vesicle breakdown (CVBD) rates of the oocytes were 95.6, 94.1 and 94.9%, respectively, and the maturation rates (metaphase II) of oocytes were 92.5, 90.1 and 91.1%, respectively. No differences were observed among the maturation media. The spermrich portion of ejaculates with greater than 90% motile sperm were used in the experiment. The semen was cooled 22 to 24$^{\circ}C$ over 2 h period. The semen was diluted with Beltsville Thawing Solution (BTS) extender at room temperature to give 2$\times$10$^{8}$ sperm/$m\ell$ in 100 $m\ell$ plastic bottle. Liquid boar semen of 30 $m\ell$ in 100 $m\ell$ plastic bottle was kept at 17$^{\circ}C$ for 5 days. The sperm with greater than 70% motility after day 5 of storage were used for in-vitro fertilization (IVF). After 44 h maturation of immature oocytes, cumulus cells were removed and oocytes (30~40) coincubated far 6 h in 0.5 $m\ell$ mTCM-199 and mTBM fertilization media with 2$\times$1061$m\ell$ sperm concentration. At 6 h after IVF, oocytes were transferred into 0.5 $m\ell$ mTCM-199 and NCSU-23 culture media for further culture 6 or 42 h. Sperm penetration, polyspermy and male pronuclear formation of oocytes at 12 h after IVF, and developmental ability of oocytes at 48 h after IVF were evaluated. The oocytes in combination with NCSU-23 medium for maturation and mTBM medium for IVF increased male pronuclear formation (48.0%) compared to those in combination with mTCM-199 media for maturation and IVF, and mWaymouth MB 752il medium for maturation and mTCM-199 medium far IVF. The rates of cleaved embryos (2~4 cell stage) at 48 h after IVF were 24.1% in combination with mTCM-199 media for maturation, IVF and culture, 43.6% in combination with mWaymouth MB 75211 medium fur maturation and mTCM-199 media for IVF and culture, and 71.2% in combination with NCSU-23 medium for maturation, mTBM medium for IVF and NCSU-23 medium for culture. In conclusion, we found out the oocytes matured in vitro were fertilized by liquid boar sperm stored in BTS extender at 17$^{\circ}C$ for 5 days. We recommend the simple defined NCSU-23 medium for nuclear maturation, mTBM medium and liquid boar sperm for IVF, and NCSU-23 medium for embryo culture.