난자생검(Ovum pick up, 이하 OPU) 기법은 유효축군 조기확보 및 우수한우 유전자 원의 조기증식, 희소유전자원 다량확보를 위해 매우 중요한 기법으로 주목받고 있다. 이 러한 OPU기술에는 체외배양기술 외에도 현장의 환경과 시기에 따라 회수되는 난자의 수 역시 중요하다. 현재, 가축센터에서는 한우의 유전자 다양성 확보를 위한 일반 한우 외에 도 희소한우 증식을 위한 연구를 진행 중에 있다. 본 연구는 OPU기법을 활용하여 가축 유전자원센터에서 보유중인 한우 또는 칡소를 대상으로 계절별 및 모색별로 난포란의 개 수와 난자회수율의 차이를 비교 · 검토하고자 실험을 진행하였다. 실험시기는 3월에서 5 월(봄), 6월에서 8월(여름), 9월부터 11월(가을)까지 구분하여 실시하였으며, 계절별 및 모 색별로 채취되는 난자의 개수와 회수율을 분석을 하였다. 봄에는 8.20±6.57, 여름에는 5.47±4.01, 가을에는 5.88±3.65개가 회수되었으며, 또한 계통별로는 가을에 실시한 결과 한 우는 6.40±6.15개, 칡소는 2.75±0.55개가 회수되었다. 그리고, 계통별로 수정란발달률은 한 우는 난할률이 25.0±44.4%, 배반포발달률이 6.3±50.0%, 칡소의 경우 난할률이 27.3±21.0%, 배반포발달률이 9.1±16.7%로 나타났다. 계절별로 OPU를 실시한 결과 여름, 가을경에 실 시하는 것에 비해서 봄에 실시를 하였을 경우 회수되는 난자의 수가 많은 것으로 확인을 하였다. 그리고, 현재 일반한우가 칡소에 비해서 회수되는 난자가 더 많은 것으로 확인되 었다. 이에 반하여 난할률 및 배반포발달률은 차이가 없는 것으로 확인되었으나, 향후 실 험진행에 따라 정확한 결과를 도출할 것으로 사료된다.

Laparoscopic ovum pick-up (LOPU) is a convenient method for collecting oocytes in small ruminants. LOPU has the advantage of being a less invasive means of oocyte collection, thereby allowing for a repeated usage of the oocyte donor animals. A total of 25 Korean black goats were used in the winter season (December to February) and LOPU was applied to the goats which had been treated for superovulation more than two times during the last twelve months. Estrus was synchronized with an intravaginal insert containing 0.3 g progesterone for 10 to 12 days. Ovaries were hyperstimulated with eCG 1,000 IU oneshot, FSH with eCG (50 mg / 1,000 IU; 70 mg / 500 IU; 70 mg / 1,000 IU) oneshot or FSH multiple-shot with eCG oneshot (20 mg × 6 / 300 IU) given intramuscularly 72 h prior to LOPU. For these groups, the number of follicles (mean ± SEM) observed which developed to larger than 2 mm in diameter were 1.6 ± 2.5, 4.3 ± 3.1, 5.5 ± 4.2, 6.6 ± 2.1 and 8.8 ± 7.8, respectively. Oocytes were aspirated by using OPU needles and a vacuum pump. The overall oocyte retrieval rates were 41.4%. Oocytes were matured in TCM-199 supplemented with 10% (w/v) bovine serum albumin + 10 μg/ml FSH + 1 μg/ml 17β-estradiol for 27 h at 39℃ in 5% CO2 in air. Oocytes were parthenogenetically activated by ionomycin combined with 6-diethylaminopurine (6- DMAP). Total oocyte maturation and cleavage rate were 67.3% and 78.8%, respectively. In summary, LOPU is a useful oocyte collection method in Korean black goats that can provide immature oocytes for transgenesis or nuclear transfer.

The brilliant cresyl blue (BCB) has been used to select the developmental competent oocytes in pigs, goats and cows. Growing oocytes have a higher level of active glucose-6-phosphate dehydrogenase(G6PDH) compare to mature oocytes and are rarely stained compared to mature oocytes, because G6PDH converts BCB to colorless. First polar body extrusion regard as a guideline of meoisis completion. Selection of polar body extrude oocyte is more developmental competent to blastocyst than unselected. This study was conducted to compare the BCB test to the polar body extrusion on selection of developmental competent porcine oocytes for the production of blastocyst. Cumulus-Oocytes complex were exposed to 26uM BCB stain diluted in NCSU-23 for 90 min. There was no significant difference embryo development to blastocysts between BCB treated and not treated(19.58±1.99 vs 18.75±2.27 %), which means there was no detrimental effect of BCB exposure to oocytes. Normal fertilization is not differed among treatment groups from 70.0 to 78.4% development to blastocyst, beside polyspermy did not. To compare two different selection methods, BCB test and polar body extrusion, evaluate the developmental competent of IVP embryos. BCB+PB+(blue stained and polar body extruded, 20.71±0.45%) and BCB－PB+(colorless and polar body extruded, 20.04±1.29%) groups are significantly (p<0.05) higher developed than those of BCB+PB－(blue stained and no polar body, 13.24±0.73%) and BCB－PB－(colorless and no poladbody, 7.25±0.77%). These results showed that selection of polar body extruded oocytes method is more efficient than that of BCB test.

본 연구는 체외성숙/체외수정 유래의 돼지 난자를 이용하여 체외발달시 배양액의 종류나 교체에 따른 영향을 구명하고자 수행하였다. mNCSU-23에서 체외성숙시킨 다음 mTBM에서 체외수정시킨 난자를 목적에 따라 두 가지로 나누어 실험한 결과는 다음과 같다. 1. 체외성숙/체외수정란을 NCSU-23에서 배양액 교체없이 7일 동안 배양하거나 CZB에서 4일 배양한 다음 Pig-MEM으로 옮겨서 나머지 3일간 배양한 결과, 난분할율은 배양액간 차이를 보이지 않

본 연구는 mNCSU-23에서 체외성숙시킨 난자를 이용하여 돼지 체외수정시 적절한 배양액과 정자농도를 구명하고자 수행하였다. 정액은 액상정액을 이용하였고 체외수정배양액으로 mTBM과 mTLP-PVA, 정자농도는 운동정자수 5, 1 , 5 sperm/로 체외수정 한 다음 NCSU-23에서 체외발달을 유도한 결과는 다음과 같다. 1. 돼지 체외성숙란을 mTBM 또는 mTLP-PVA에서 운동정자수 1 sperm/로 체외수정시킨 다음 체외발달시킨 결과, 정자침

본 연구는 pFF, cysteine, -mercaptoethanol, 성선자극호르몬 등 여러 가지 체외성숙 촉진 물질이 첨가딘 성숙배양액에 EGF 첨가가 돼지 미성숙 난포란의 체외성숙에 효과적인지 또한 그 효가는 배양소적당 COC의 수에 영향을 받는지을 구명하고자 EGF의 첨가 유무와 배양소적당 COC수(50개 또는 15개)를 조합한 요인시험을 실시했다. 도축돼지의 난소에서 채취한 COCs를 각 처리별로 mNCSU-23 에서 성숙배양하고 mTBM에서 운

본 연구는 보다 안정된 돼지 체외수정란 생산시스템 확립을 목적으로 체외성숙배양액 mNCSU-37, mNCSU-23 및 TCM-199 가 각각 미성숙 난포란의 체외성숙, 체외수정, 체외발달에 미치는 영향을 구명하기 위하여 수행하였다. 도축돼지의 난소에서 채취한 COCs를 10% pFF가 포함된 각각의 성숙배양액에서 최종동도가 의 농도로 체외수정 시킨 다음, NCSU-23 에서 체외발달을 유도한 결과는 다음과 같다. 1. TCM-199에서 성숙시킨 난자가

This study was carried out to investigate the effect of biological factors on the in vitro production(IVP) of bovine oocytes for development of simple culture methods and medium. Oocytes from the slaughterhouse ovaries were matured and fertilized using general protocol and this study was examined if there were necessary to co-culture, media change, media type and embryo density. This results were as follows: 1. The development rate according to co-culture with cumulus cells and non co-culture as drop culture was not significantly different in cleavage (68.9 vs 71.7%), 8-cell stage (41.2 vs 44.1%) and blastocyst stage (12.2 vs 13.8%), respectively (p<0.05) 2. The blastocyst development rates in YS and CRIaa were higher than that in TCM199 (12.4, 10.4 vs 3.7%), but the cleavage (69.0, 77.8 and 61.0%) and 8-cell stage (31.7, 37.0 and 35.7%) development accoring to YS, TCM199 and CRIaa ws not significantly different, respectively (p<0.05). 3. There was no significantly different in cleavage (62.6, 59.5 and 61.2%), 8-cell(34.7, 37.9 and 34.0%) and blastocyst (9.5, 11.6 and 12.8%) development among medium change time as control, Group I and Group II, respectively (p<0.05). 4. Blastocyst formation of 8-cell stage according to embryo density was not significantly different in 1, 10 and 25 embryos (27.3, 22.5 and 34.0%), respectively (p<0.05). These results indicated that simple culture system could reduce bovine IVP embryos as drop culture as non co-culture system, high density embryo (25 embryos/50 1 drop). YS defined medium and no medium change for whole culture period, although other biological factors need to examine in order to produce efficient IVP bovine embryos.

The objective of this study was to produce calves by transfer of embryos derived from slaughter house(SHD) and ultrasound-guided ovum pick-up (OPU). At 60 hrs after injection of 400 mg FSH dissolved in 25% polyvinylpyrrolidone(PVP) by single dose, ultrasound-guided follicular oocyte aspiration was ferformed. Day-7 and day-8 blastocysts produced by in vitro maturation (IVM), fertilization (IVF) and culture(IVC) of the oocytes derived from SHD and OPU were nonsurgically transferred into recipients. The results obtained were as follows. The cleavage rate and the development rate to blastocysts were not significantly (P<0.05) different between the oocytes obtained by SHD (72.9% vs. 34.1%) and OPU (75.9% vs. 38.4%). The oocyte recovery rate from the number of follicles by ultrasound-guided aspiration were not significantly (P<0.05) different between Holstein (61.7%) and Hanwoo (60.1%), but the rate of oocytes useful for IVF was significantly (P<0.05) higher in Hanwoo (69.3%) than Holstein (59.6%). The cleavage rate and the development rate to blastocysts was not significantly (P<0.05) different between Holstein (74.9% vs. 39.2%) and recipients on day 8 of estrus cycle resulted in 13 pregnancies (34.2%). One of them was sacrificed during gestation period due to mastitis and another was aborted spontaneous. The resulting 14 calves were morphologically normal at birth. Seventy eight fresh OPU-IVF embryos were transferred into 21 recipients on day 8 of estrus cycles, resulting in pregnancy of 12 recipients (41.4%). Two of them were sacrificed during gestation period due to mastitis and the other two were aborted. Nevertheless, the 11 OPU-calves have been born normally.

This experiment was carried out to develop the anesthetic methods for ultrasonography and a new simplified disposable needle guidance device for ovum pick-up(OPU) in cows. Three different anesthetic methods were applied as. 1) epidural analgesia only with 2% lidocaine(20~30 ml), 2) epidural analgesia with 2% under general sedation with xylazine, 3) epidural analgesia with 2% lidocaine under general sedation with detomidine. We evaluated the anesthetic effects with items such as relaxation of anal sphincter, tail movement and rectal wall, retractability of both ovaries, additional anesthesia and possibility of OPU. Through this experiment, the above three anesthetic methods were applicable to OPU, but the epidural anlagesia under general sedation with detomldine was most effective for OPU. We developed a new disposable needle guidance device with stainless steel tube. With this, disposable needles can be easily attatchable to any other intravaginal probes. And also, it was found to he practical, economic and effective for OPU with the recovery rate of 51.2%.

This study was carried out to compare the actual size(length and height) of ovaries, follicles and corpora lutea of Korean native cow with those on sonograms. We used 3 different probes(3.5 MHz abdominal probe, 6.5 MHz transvaginal probe and 5.0 MHz transrectal probe) and a calipher for measurements of ovaries, follicles and corpora lutea on sonograms and actual size. Under water immersion, 157 ovaries were scanned with 3 probes and measured in actual size and compared each other. The average height and width of ovaries of Korean native cows were 17.403.99 and 34.236.02mm, respectively. In comparison of height, length of ovaries and preovulation follicles, we found that image with a transvaginal probe was nearly the same as the actual size(p<0.01), but with an abdominal probe the image was appeared larger than the actual size. In measurement(diameter) of preovulation follicles the transvaginal probe was proven to be more accurate to the actual size than other probes and in corpus luteum measurement all probes were accurate. In the comparison of number of follicles by different size ranges, there was no statistical difference in the count of follicles over 10 mm in diameter between the transvaginal probe and naked eyes.

In order to determine the optimum condition and timing for in vitro maturation of oocytes to metaphase of meiosis II (M II), the immatured follicular oocytes were recovered by puncturing the large(1.0~1.5 mm in diameter) and small(<1.0 mm in diameter) follicles in the ovaries of rabbits treated intramuscularly with a single dose of 100 TU PMSG 68 hours previously. The follicular oocytes were classified into three grades by the attachment of cumulus cells. The Grade I and II follicular oocytes from large follicles were cultured in BO-DM medium with 10% FCS, 35 g /nl of FSH, 10 g /ml of LH and 1 g /ml of estradiol-17 at 39t in a 5% incubator for 11 to 23 hours. In 3 hours interval during the culture period, the oocytes were harvested and their cumulus cells were removed with hyaluronidase. The denuded oocytes were stained with Hoechst 33342 dye and their meiotic status and extrusion of the first polar body (PB) were examined under a fluorescence microscope. Also the fragmentation of the first PB and the distance between the first PB and nucleus were examined. The results obtained were as follows: 1. The mean recovery rate of follicular oocytes from the large and small follicles was 59. 9 and 31.3%, respectively. The mean number of oocytes recovered per rabbit and the Grade I percentage were 14.6 and 94.4% in large follicles, but 2.1 and 61.1% in small follicles, respectively. All the parameters examined were different significantly (p<0.05) between both the folliclular size. 2. Most of the follicular oocytes(86.8%) were matured in vitro to M II phase in 14 hours in Grade I oocytes, but the significantly(p<0.05) less oocytes(45.5%) were matured in Grade II oocytes. 3. The first PB was extruded in most of the oocytes(94.7%) in 14 hours of culture with the fragmentation rate of 29.6%, but the fragmentation rate of the first PB increased significantly (p<0.05) as the culture period for maturation was longer to 20 hours(63.5%). 4. The distance between the first PB and nucleus was increased linearly (p<0.05) as the maturation time passed from 14(7.1rn) to 23 hours(58.4m). 5. From the above results it was concluded that the optimum time for in vitro maturation culture might be 14 hours in the follicular oocytes from rabbit primed with PMSG for 68 hours, expecially when these follicular oocytes were used for recipient cytoplasms in embryo cloning.

본 연구는 종모우의 선발방법으로 난포란을 이용하여 실험실내 정자의 수정능력을 직접 검정하여 평가코자 시도되었다. 즉 본 실험은 후대검정중에 있는 한우 후보종모우 15두의 동결융해정자의 수정능력을 평가하기 위하여 정액을 고장액(HIS)에 처리한 후 DM에서 6시간 그리고 소 난포액이 20% 첨가된 DM에서 4시간 전배양하여 수정능을 획득시켜 정자의 활력과 첨체 반응율을 조사하였고 전배양된 정자의 체내(토끼 난관) 또는 체외수정능력을 조사하기 위하여 FCS