This study was conducted to examine the effects of human follicular fluid and gonadotropin (FSH+HCG+rhEGF) on in vitro maturation, fertilization and development of human immature oocytes. Cumulus-oocyte complexes (COCs) were collected following for in vitro fertilization and embryo transfer (IVF-ET) cycles of the patients. At the time of oocytes collection, oocytes were classified into MII, MI and GV in accordance with their appearance (MII: Fully mature oocyte at metaphase II of meiosis; MI: Nearly mature oocytes at metaphase I of meiosis; GV: Immature oocytes at prophase I of meiosis). After controlled ovarian stimulation using gonadotropin(FSH) and human chorionic gonadotropin (HCG) in 70 ICSI cycles, 158 MI to MII matured oocytes were intracytoplasmic sperm injection (ICSI) h after in vitro culture and 553 MII oocytes were ICSI after denudation. The aspirated MI and GV oocytes were cultured in culture medium containing 10% (v/v) serum protein substitute (SPS), 10% (v/v) human follicular fluid (hFF) and 10% (v/v) serum protein substitute (SPS)+1 IU/ml FSH+10 IU/ml HCG+10 ng/ml recombinant human epidermal growth factor (rhEGF). The maturation rate of immature oocytes was similar among the three group. When maturation medium was supplemented with 10% SPS, 10% hFF or gonadotropins, the fertilization rate of in vitro matured oocytes was higher in 10% SPS (80.0%), but there was no statistical significance (78.2%; hFF, 76.9%; gonadotropin, p>0.05). The development rate of human embryos developed to cells were not significant difference in the medium containing SPS, hFF and gonadotropins (65.6%, 65.9% and 66.7%). The results of these study suggest that human follicular fluid and gonadotropins supplemented in the culture medium was not effected on the in vitro maturation, fertilization and development of human immature oocytes.

The objective of this study was to examine the effect of caffeine and sodium bicarbonate in a fertilization medium on the fertilizability of boar spermatozoa that were frozen in straws. Boar spermatozoa were extended with Beltsville F5 extender and frozen in 0.25‐ml straws. In vitro matured porcine oocytes were fertilized in vitro (IVF) with frozen‐thawed boar spermatozoa for 6 h in a modified tris‐buffered medium (mTBM) or in its modified medium by substituting the tris with 25 mM sodium bicarbonate (modified bicarbonate‐buffered medium; mBBM). Some of inseminated oocytes were fixed and stained for examination of sperm penetration. IVF embryos were cultured in a North Carolina State University‐23 medium for embryo development. The percentage of live sperm was 47±4% and morphological abnormality of acrosome was found in 14±3% of spermatozoa. Optimal sperm concentration for IVF was 0.75～1.0×106 sperms/ml when mTBM containing 5 mM caffeine was used as the fertilization medium. Sperm penetration was significantly (p<0.05) stimulated by increasing caffeine concentration in the IVF medium. In addition, mBBM significantly (p<0.05) increased sperm penetration (92%) compared to mTBM (65%). More (p<0.05) blastocysts (22% vs. 32%) developed from the oocytes that were fertilized in mBBM containing 1 mM caffeine than from those fertilized in mTBM with 5 mM caffeine. Our results indicate that boar spermatozoa can be frozen successfully in straws with holding their normal fertilizability and that caffeine and sodium bicarbonate stimulates sperm penetration in vitro.

본 연구는 돼지 수정란의 체외 성숙 및 체외 배양액의 retinol 첨가 효과를 규명하기 위하여 체외 성숙 및 체외 배양액에 retinol을 첨가하여 수정란의 체외 발달에 미치는 영향을 구명하고자 수행되었다. 체외 성숙 배양액에 retinol을 첨가한 결과 성숙율은 으로 각 처리구 간의 유의적인 차이가 없었다(p>0.05). 체외 수정 후 배반포로의 발달율은 첨가구에서 의 발달율을 나타내어 타 처리구에 비하여 유의적으로(p<0.05) 높게 나타났으며,

본 연구는 체외성숙된 돼지난포란을 액상정액으로 수정시 수정시간과 배양배지가 난포란의 발달에 미치는 영향을 조사하기 위하여 실시하였다. 정자농후정액 (30∼60 ml)을 채취하여 실온에서 2시간 정도 서서히 냉각시킨 후, 정액을 15 ml 튜브에 담아 800×g로 10분간 원심분리하였다. 상층액은 버리고 하부의 정자는 5 ml LEN 희석액으로 1×10/sup 9/ 전자/ml가 되도록 재희석하였다. 희석된 정액은 4℃ 냉장고에 보존하였다. 미성숙 난모세포의 성숙에 사용된 배지는 26.19 mM sodium bicarbonate, 0.9 mM sodium pyruvate, 10 ㎍/ml insulin, 2 ㎍/ml vitamin B/sub 12/, 25 mM HEPES, 10 ㎍/ml bovine apotransferrin, 150 μM cysteamine, 10 IU/ml PMSG, 10 IU/ml PMSG, 10 IU/ml EGF, 0.4% BSA, 75 ㎍/ml sodium penicillin G, 50 ㎍/ml streptomycin sulfate그리고 10% pFF를 첨가한 TCM-199 배지였다. 22시간 성숙 배양한 후 난모세포는 cysteamine과 hormone들을 배제한 후 38.5℃, 5% CO₂ incubator에서 22시간 더 성숙시켰다. 성숙된 난모세포는 채취 후 2일간 4℃에 보존된 액상정액으로 수정되었다. 난모세포는 500 μl mTBM 수정 배지에서 1×10/sup 6/ 정자/ml의 농도로 1, 3, 6 그리고 9시간 동안 수정시켰다. 그 후 난모세포는 500 μl NCSU-23, Hopes buffered NCSU-23, PZM-3 그리고 PZM-4 배양배지에 옮겨서 6, 48 그리고 144시간을 더 배양하였다. 정자침투율, 웅성전핵형성율 그리고 난모세포의 난할율은 6 및 9시간 수정시간에서 1 및 3시간 수정시간 보다 높았다. 6시간 수정시 배반포형성율 (33.6%)은 1, 3 그리고 9시간 수정시 배반포형성율 (11.4, 23.0 그리고 29.6%) 보다 높았다. 배반포의 평균세포수는 6, 9, 3 그리고 1시간 수정시 각각 32.9, 27.6, 26.3 그리고 24.4개 였다. 분할된 난모세포의 배반포형성율 그리고 배반포의 평균세포수는 NCSU-23, PZM-3 그리고 PZM-4 배양배지보다 HEPES buffered NCSU-23 배양배지가 우수하였다. 결론적으로 4℃ 보존 돼지액상정액은 체외성숙된 돼지 난모세포의 체외수정에 사용될 수 있음이 입증되었다. 또한 체외성숙된 돼지 난모세포는 500 μl mTBM 수정배지에서 1×10/sup 6/ 정자/ml로 6시간 공배양시키는 것이 바람직하며, HEPES buffered NCSU-23 배양배지에서 배양하는 것이 좋다는 결과를 얻었다.

Essential and non-essential amino acids supplemented to culture medium stimulate mammalian embryo development in vitro. Amino acids such as glycine, taurine and alanine are concentrated in the lumen of oviduct and uterus and it can he thought that these amino acids may have physiological role on fertilization and embryo development. Our aim of this experiment was to investigate the effects of essential and non-essential amino acids, taurine or glycine supplemented to fertilization medium on the cleavage and subsequent in vitro development of bovine oocytes matured and fertilized in vitro. Immature oocytes were obtained from slaughtered Holstein cows and heifers and matured in TCM199 containing 10% fetal calf serum, 2.5 g /mL of FSH and LH and 1 g / mL of estradiol with granulosa cells in vitro. After maturation, oocytes were coincubated with sperm in fertilization medium supplemented with Minimum Essential Medium (MEM) essential and non-essential amino acids, taurine (3.75 mM) or glycine (10 mM) for 30 hours in vitro. Inseminated oocytes were cultured in synthetic oviduct fluid medium (SOEM) containing MEM essential, non-essential amino acids and 1 mM glutarnine up to 8 days after fertilization.Supplementation of fertilization medium with MEM essential and non-essential amino acids lowered significantly (p<0.05 and p<0.001) the cleavage rate after 30 hours of IVF (53.3%) and at Day 3 (62.7%: Day 0: the day of I VF) compared to control (64.3% and 77.3%, respectively). Subsequent developmental rates to morulae (Mo) and expanding blastocysts (ExBL) also significantly decreased (p<0.001 and p<0.05 for Mo and ExBL) when oocytes were coincubated with sperm in the medium containing MEM amino acids. Taurine added to fertilization medium have not increased the cleavage rate over the control, whereas glycine showed significantly lower (p<0.01) cleavage rate at Day 3 than that of taurine, but there was no significant difference in the developmental rates to Mo and ExBL of bovine embryos irrespective of the supplementation of taurine or glycine to fertilization medium. In conclusion, supplementation of fertilization medium with essential and non-essential amino acids, taurine or glycine has no beneficial effect on in vitro cleavage and development of bovine oocytes matured and fertilization in vitro.

The study was conducted to determine the optimal hormone and glucose levels during the in vitro culture of bovine oocytes matured and fertilized in vitro for blastocyst development. Oocytes matured in TCM 199 + 10% FCS + hormones and glucose were fertilized in vitro in a TALP medium with swim up separated and heparin-treated epididymal cauda spermatozoa. Oocytes were cultured for 2~5 days in synthetic oviduct fluid medium (SOFM) supplemented with 10% FGS and with different hormone and glucose levels, and further cultured 5 days same medium in SOFM. The results are summarized as follows : The in vitro maturation and penetration rates of porcine oocytes cultured in TCM 199 media containing PMSG, hCG, PMSG + hCG, hCG + estradiol, PMSG + estradiol 0 to20 hours after insemination were 88.0% and 81.8%, 82.6% and 68.4%, 80.0% and 75.0%, 80.0% and 65.0%, 77.3% and 64.7%, respectively. The in vitro maturation and penetration rates of porcine oocytes cultured in TCM 199 media containing PMSG, hCG, PMSG + hCG, hCG + estradiol, PMSG + estradiol 20 to 40 hours after insemination were 92.0% and 87.0%, 92.0% and 82.6%, 91.3% and 81.0%, 85.2% and 73.9%, 87.5% and 81.0%, respectively. The cleavage and in vitro developmental rates to blastocyst of porcine oocytes cultured in TCM 199 media containing 0.05 mM, 0.10 mM, 0.30 mM, 0.50 mM, 1.00 mM, and 3.00 mM glucose lelvels 0~3 days after insemination were 31.5~48.1% and 10.0~16.7%, respectively. The cleavage and in vitro developmental rates to blastocyst of porcine oocytes cultured in TCM 199 media containing 0.05 mM, 0.10 mM, 0.30 mM, 0.50 mM, 1.00 mM, and 3.00 mM glucose levels 4~8 days after insemination were 30.0~53.8% and 8.7~19.2%, respectively. The cleavage and in vitro developmental rates to blastocyst were higher in TCM 199 media containing various glucose levels 0~3 days after insemination than 4~8 days.

본 실험은 성선(性腺)자극호르몬과 우태아혈청(牛胎兒血淸) 첨가가 돼지 난포란(卵胞卵)의 체외성숙(體外成熟) 및 체외수정(體外受精)에 미치는 영향을 조사하기 위하여 실시하였다. 미경산돈(未經産豚)(체중 80~90kg)의 난소(卵巢)를 도살된 직후에 절취하여 의 보온병에 담아 실험실로 운반하여 난포(卵胞)직경이 3~5mm되는 것만을 골라 난포(卵胞)를 찔러서 난포란(卵胞卵)을 채취하였다. 성숙모돈(成熟牡豚)(체중 130~150kg)의 정소상체미부정자(精巢上體尾部精子)를 농도로 희석하여 체외수정(體外受精)에 이용하였다. 본 실험의 경과를 요약하면 다음과 같다. 1. m-KRB와 10% FCS를 m-KRB에 첨가한 경우 성숙율(成熟率)은 82.37%이며, 10% FCS가 첨가된 배양액에 PMSG, hCG 그리고 PMSG와 hCG를 각각 첨가한 경우 66, 58, 68%로서 성숙율(成熟率)이 향상되었다. 2. 난구세포(卵丘細胞)의 팽화(膨化)는 m-KRB와 10% FCS가 첨가된 배양액에서 일어나지 않았으나 10% FCS가 첨가된 배양액에 PMSG, hCG 그리고 PMSG와 hCG를 각각 첨가시 92, 13, 91%로서 팽화율(膨化率)이 향상되었다. 3. m-KRB에서 성숙된 난포란(卵胞卵)의 체외수정(體外受精)에서 정자침입율과 웅성전핵(雄性前核) 형성율은 각각 93.7%였으나 FCS와 성선(性腺)자극호르몬을 첨가한 경우 각각 100, 80%로서 웅성전핵(雄性前核) 형성율이 향상되었다.

This study was carried out to investigate the optimum application time of basal fertilization in direct-seeded rice on flooded paddy surface at medium saline soil in southwestern reclaimed saline area which is Munpo series and have 0.3 or more soil salinity. The nitrogen fertilizer level were 14, 17, 20kg per 10a, and the basal fertilization were applied before sowing, 10, 20, 30 days after sowing along with topdressing at tillering stage, respectively. The summarized experimental results are as follows. Seedling stand was proper when basal fertilization was applied before sowing, but lower than appropriate seedling stand when basal fertilization were applied 10, 20, 30 days after sowing along with topdressing at tillering stage. The rice yield were 509-541kg/10a when 14-20kg/10a basal fertilization was applied before sowing, and 368-481kg/10a when basal fertilization were applied 10, 20, 30 days after sowing along with topdressing at tillering stage. Considering the yield and salt injury at medium saline soil in southwestern reclaimed saline area, the application of basal fertilization before sowing was superior for improving seedling stand, and 14-20kg/10a fertilization rate was allowable.