• 한국수정란이식학회지
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• 제14권2호
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• pp.83-88
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• 1999

This study was undertaken with three objectives : to determine the optimal time of blood collection for plasma urea nitrogen(PUN) analysis, to examine the frequency distribution of PUN levels in recipient herd and to relate concentration of PUN to conception rate in Hanwoo recipients. The relationship between PUN level and time postfeeding was examined for 5 individual cows. Mean concentration of PUN rose for 4hrs postfeeding and decreased to PUN level before feeding at 10hrs postfeeding. And then, the blood for PUN analysis was collected at the time before feeding in next experiments. The ratio of cows with PUN concentration of < 12, 12∼18 and 18mg/dl were 50.6, 39.9 and 9.5% in 163 recipients, individually. The pregnancy rate of cows with PUN concentration 12∼16 mg/dl (63.3%) was higher than that of cows with PUN concnetration < 12 mg/dl (46.7%) or > 16mg/dl (42.9%). There results suggest that the PUN test may be beneficial for management of recipient herd in effects to maintain or improve reproductive efficiency.

• 한국수정란이식학회지
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• 제32권3호
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• pp.105-109
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• 2017

Autophagy is an intracellular degradation and recycling system. Oocyte maturation is dynamic process, in which various proteins should be synthesized and degraded. In our previous study, we reported the loci of autophagosome and dynamics of autophagic activity in porcine oocytes during in vitro maturation. In this study, we verified loci of autophagosome in porcine follicular cumulus-oocyte complex by detection of microtubule-associated protein 1A/1B-light chain 3 (LC3) which is the reliable marker of autophagosome. Porcine ovary including various sizes of follicles was fixed within 1 hour after collection from slaughterhouse. After fixation, immunohistochemistry was conducted on sliced ovary tissue containing various sizes of follicles by using LC3 antibody. As a result, LC3 signal was clearly detected in both cumulus and oocytes of various sizes of follicles. We also found ring shaped signal which represent autophagosome near oocyte membrane. Most of the signals in oocytes were localized nearby cellular membrane while evenly dispersed in cumulus cells. Therefore, this result suggests that autophagy occurs in porcine COCs (cumulus-oocyte complexes) at follicular stage.

• 한국수정란이식학회지
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• 제24권3호
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• pp.145-150
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• 2009

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) ${\sim}4$ 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 $6{\sim}8$ 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.

• 한국수정란이식학회지
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• 제13권2호
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• pp.107-115
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• 1998

The present study was carried out for the comparative study on the collection of bovine follicular oocytes by ultrasound-guided ovum pick-up(OPU) and slaughterhouse-derived (SHD) ovary aspiration and in vitro production of bovine embryos with the follicular oncytes in Korean native cows. Bovine follicular nocytes were observed with a 6.5 MHz convex-array ultrasound transducer designed for intravaginal use and the oocytes were collected with the aspiration equipment attached to the ultrasonograph. Bovine ovaries were collected and transported in phosphate buffered saline from the local slaughterhouse, the follicular oocytes were collected by the aspiration method. The collected follicular oocytes in good quality were matured, fertilized and cultured in the media. The total number of the visible follicles and the recovery rate of follicular oocytes were increased in ultrasonography following follicle stimulating hormone(FSH) treatment in Korean native cows. The mean recovery rate of oocytes was 66.2, 52.8 and 41.7% in the FSH-OPU, non-treatment-OPU and SHD ovaries, respectively. The mean number of recorved oocytes per cow were not significantly(P<0.05) different between the FSH-OPU(14.0$\pm$11.54) and SHD(17.1i6.21) groups, but the numbers in both groups were significantly(P<0.05) higher than the number in the non-treatment-OPU(3.7$\pm$1.57) group. The mean number of usable nocytes in Grade T /11 per ovary was 6.3, 4.8 and 1.3 in the cows of the SHD, FSH-OPU and non-treatment-OPU groups, respectively. The in vitro developmental rate to the blastocyst was not significantly different between the oocytes obtained via OPU(37.1%) and SHD(29.3%). Therefore, the ultrasound-guided OPU technique can be applied to the production of excellent embryos from the high-quality cows, and for the large scale production of in vitro bovine oocytes and embryos, the SHD ovary aspiration method is valuable.

• 한국수정란이식학회지
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• 제12권3호
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• pp.293-300
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• 1997

Supplementation of maturation medium with additional granulosa cells has beneficial effect on in vitro maturation of bovine follicular oocytes and their subsequent cleavage and development in vitro. However, maturation system using granulosa cells have some disadvantages that collection of granulosa cells is cumbersome and metabolic activity of the cells is variable according to ovarian cycle or follicular size. We hypothesized that bovine immsture oocytes matured without granulosa cell coculture can fertilize and develop normally if the medium volume per oocyte is reduced during in vitro maturation. Immature oocytes were matured for 24 hours in a TCM199 containing 10% fetal calf serum, anterior pitultary hormone (0.02 AU /ml Antrinⓡ) and estradiol with or without granulosa cells in vitro. In Group 1, 35 to 40 oocytes were matured in a well of 4-well plastic dish containing 500 $\mu$l of maturation medium and granulosa cells, and 9 to 10 oocytes were matured in a 50-$\mu$l drop of maturation medium without granulosa cells in Group 2. After maturation, oocytes were coincubated with sperm for 30 hours in a modified Tyrode's medium (IVF). Inseminated oocytes were cultured in a microdrop (30 $\mu$l) of a synthetic oviduct fluld medium (SOFM) containing BSA, Minimum Essential Medium essential and non-essential amino acids for 9 days. As a preliminary experiment, we investigated the beneficial effect of granulosa cells during maturation on subsequent cleavage and development using the same type of culturedishes (4-well dish). Granulosa cells could not increase embryo cleavage after fertilization but significantly improved (p<0.05) embryo development to expanding blastocyst (Table1 and 2). In Group 1, 68 and 80% of inseminated oocytes have cleaved at 30 hours and 2 days after IVF, respectively, which is similar (p>0.05) to the result of Group 2 (69% at 30 hours and 78% at 2 days after IVF). The oocytes in Group 2 showed 21 and 11% of developmental rates to expanding and hatching blastocysts, respectively, which was not significantly different (p>0.05) from those (20 and 10%, respectively) of oocytes in Group 1. In conclusion, it has been clarified that a microdrop culture system without granulosa cells for in vitro maturation can support bovine embryonic development to blastocyst in vitro as readily as a granulosa cell coculture system.

• 한국수정란이식학회지
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• 제29권3호
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• pp.265-271
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• 2014

본 연구에서 한우를 공란우로 사용하여 OPU 방법으로 가장 더운 계절의 hot season과 선선한 cool season의 두 계절의 차이에 따른 생성된 난포의 수, 난자 회수율, 난자 등급율, 수정율 및 배반포 발달 능력을 분석하여, 두 계절이 공란우의 번식 능력에 미치는 영향에 관하여 조사하였다. 1. 계절의 영향이 OPU 공란우의 난포 생성 수에 미치는 결과는 난포 생성 개수는 1154개($18.32{\pm}2.26$), 971개($15.41{\pm}3.34$)로 hot season 그룹이 유의적으로 높은 것을 알 수 있었다(p<0.05). 2. 계절에 따른 난자 수 및 난자 회수율은 hot season 그룹의 475개($7.54{\pm}3.14$), 41.16%로 cool season 그룹 448개($7.11{\pm}3.42$), 46.14%와 비교하여 유의적인 차이가 없었다(p<0.05). 3. OPU를 통하여 회수된 두 계절별 난자 등급은 Grade A는 Hot season 그룹 110개($1.75{\pm}1.86$), Cool season 그룹 63개($1.00{\pm}1.46$)로 hot season 그룹이 cool season 그룹과 비교하여 유의적으로 높았다(p<0.05). 하지만 다른 등급인 Grade B는 87개($1.38{\pm}1.60$) vs. 97개($1.54{\pm}1.39$), Grade C는 166개($2.63{\pm}2.43$) vs. 170개($2.70{\pm}2.04$), Grade D는 112개($1.78{\pm}2.65$) vs. 118개($1.87{\pm}1.86$)로 hot season과 cool season 간의 유의적인 차이를 보이지 않았다(p<0.05). 4. 계절에 따른 체외 수정 후의 수정률은 hot season과 cool season 각각 242(66.67%)와 209(63.3%), 배반포 발달율 214(58.95%) vs. 188(56.97%)로 수정률과 배반포 발달율은 유의적인 차이가 없었다(p<0.05). 본 연구의 결과로 계절에 따른 영향에 의해 공란우의 난포생성수와 A등급의 난자 출현율에서 유의적인 차이를 보였다. 하지만 나머지 등급의 난자 출현율, 수정률 및 배반포 발달율은 차이가 없는 것으로 보아, 계절의 차이로 인한 한우 공란우의 번식 능력에 미치는 영향은 미비하다고 판단된다.

• 한국수정란이식학회지
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• 제31권1호
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• pp.47-52
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• 2016

본 연구는 희소한우의 증식을 위해 성 감별 처리된 정자를 체내수정란 생산 및 이식 기술에 적용하고자 정자의 농도별 수정란 회수 결과를 조사하고, 생산된 수정란은 이식을 통해 후대 생산 기술을 정립하고자 실시하였다. 가축유전자원센터에서 보유 중인 칡소 암소를 선발하여 공란우로 공시하였으며, 과배란처리는 발정주기에 관계없이 CIDR-Plus(Intravaginal Progesterone Releasing Device)를 질 내에 삽입하고, 4일 후부터 FSH(Antorin)를 12시간 간격으로 4일간 총 28AU(Amour unit) 절감 주사하고, FSH 주사 3일째 CIDR-Plus을 제거함과 동시에 $PGF_{2{\alpha}}$(Lutalyse) 3.0 ml를 주사해 과배란을 유기하였다. 인공수정은 발정이 확인된 후 12시간 간격으로 3회 실시하였다. 대조구의 경우, 보편적으로 사용하는 인공수정용 스트로우인 2,000만 농도의 칡소 동결정액 스트로우를 이용하였으며, 성감별 처리구의 경우, 스트로우 당 400만과 1,000만의 두 실험군으로 인공수정을 실시하였다. 수정란의 회수는 인공수정 후 7일째 되는 날 실시하였으며, 이식가능 수정란은 IETS의 기준에 따라 판정하였다. 대조구에서 체내수정란 회수 결과는 이식가능 수정란이 두 당 $6.20{\pm}2.28$개로 전체 회수 수정란의 67.39%로 나타났으나, 성감별 정자처리구에서는 $4{\times}10^6$ 농도에서 $4.33{\pm}5.39$개(26.53%), $10{\times}10^6$ 농도에서 $1.57{\pm}1.72$개(24.44%)로, 대조구에 비해 유의적으로 낮은 이식가능 수정란 결과를 나타냈다. 미수정란의 비율은 성감별 처리구가 각각 52.04%와 57.78%로 대조구의 8.70%에 비해 유의적으로 매우 높았다(p<0.05). 이식가능 수정란의 발달 단계에 대한 조사 결과, 대조구의 경우 초기배반포배 단계의 수정란이 30.43%로 가장 높은 비율을 차지했으며, 그 다음이 확장배반포배(19.57%), 상실배(17.39%) 단계였다. 반면에 처리구의 경우, 두 처리구 모두 상실배 단계의 수정란이 15.31%와 24.44%로 가장 높은 비율을 나타냈다. 생산된 체내수정란을 한우 수란우에 이식한 결과, 대조구의 경우 35.00%, 처리구의 경우 12.50%의 수태율을 나타내었다. 성감별 정자 유래 암송아지의 염색체 핵형분석 결과, 2n=60으로 일반 한우와 다르지 않은 염색체 분포 양상을 나타내었으며, 29개의 상동염색체와 XX 성 염색체를 가진 정상 암컷 개체임을 확인하였다.

• 한국수정란이식학회지
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• 제11권3호
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• pp.249-258
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• 1996

To improve the efficiency of in vitro production of embryos with follicular oocytes in Korean Native cows, the recovery rates, in vitro maturation, fertilization and development, and the time required for collecting and processing oocytes by aspiration with or without slicing were evaluated comparatively. The ovaries were obtained from a local abattoir and placed in physiological saline at 25~28$^{\circ}C$ and brought to the laboratory within 3 hrs. The oocytes were collected by aspiration of follicles(2~6mm) with or without slicing ovaries after aspiration, and classified into Grade I, Grade II, Denuded, Expanded oocytes by the morphology of cumulus cells attached and the homogeneity of cytoplasmic granules. Also the time required for each step of collecting and processing oocytes were measured. The cumulus cells were removed in some Grade I oocytes to measure their size and nuclear configuration before and after in vitro maturation. The Grade I oocytes were matured in vitro(IVM) for 24 hrs. in TGM-199 supplemented with 35$\mu$g /ml FSH, 10$\mu$g /ml LH, 1 $\mu$g /ml at 39$^{\circ}C$ under 5% C02 in air. They were fertilized in vitro(IVF) by epididymal spermatozoa treated with heparin for 24hrs. and then the zygotes were cocultured in vitro (IVC) with bovine oviductal epithelial cells for 10 days. The results obtained were as follows: The number of oocytes recovered per ovary was averaged 6.6 by aspiration and 11.2 by slicing post aspiration, which summed to 17.8. The number of Grade I oocytes recovered per ovary was averaged 3.1 by aspiration and 3.6 by slicing, which summed to 6.7. The percentage of Grade I to total oocytes recovered was significantly(P<0.05) higher as 48.0 % in aspiration than 31.6% in slicing post aspiration. The time requlred for recovering a Grade I oocyte by aspiration and slicing was 1.1 and 2.5 min, respectively. The mean diameter of Grade I oocytes by aspiration and slicing was similar as 148.7 and 151.5$\mu$m, respectively. The percentage of Metaphase II stage oocytes after IVM for 24 hours was significantly (P

• 한국수정란이식학회지
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• 제25권4호
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• pp.263-266
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• 2010

Prediction of semen's fertilizing ability used in artificial insemination (AI) is one of very important factors on pig reproductive performance. In vitro fertilization (IVF) has been used for indirect evaluation of sperm's fertilizing ability and it has been showed as highly correlated index. In swine industry, increasing interest in preservation of boar semen raises questions on the sperm motility from semen used in commercial AI centers. Mitochondria in sperm mid-piece generate the energy to support motility and could be an explanation of impaired fertility. Objective of this study was to suggest usable sperm motility to farms in measuring the effect of sperm motility and sperm abnormality on in vitro production of embryo in which sperm's fertilizing ability can be determined indirectly. Semen samples were provided from local AI center and used within 3 days after collection. Semen samples were divided by 4 different motile groups (>70%; 61~70%; 51~60%; <50%) using CASA (computer-assisted sperm analysis) on the days of IVF. Developmental rate to the blastocyst stage from over 61% motile sperm group showed significantly higher rate than below 60% motile sperm group ($16.5{\pm}0.7{\sim}18.4{\pm}0.8%$ vs $6.3{\pm}0.8{\sim}11.5{\pm}0.7%$, p<0.05). In experiment to determine the relationship between sperm motility and viability and abnormality, over 61% motile sperm groups showed significantly higher viability rate compared to below 60% motile sperm groups ($84.8{\pm}4.0{\sim}88.1{\pm}4.0%$ vs $69.1{\pm}4.0{\sim}74.2{\pm}4.0%$, p<0.05). On the other hand, morphological sperm abnormality showed significantly higher in over 70% motile sperm group ($10.2{\pm}2.2$ vs $16.0{\pm}2.2{\sim}21.0{\pm}2.2%$, p<0.05). In experiment to find the correlation between sperm motility of 4 different motile groups and amount of mitochondria, lower motility group also showed lower level of mitochondria (p<0.05). The mitochondria parameter used in this study showed another possibility to differentiate the sperm motility. Taken together, because below 60% motile semen used in AI reduce the fertility, AI centers should provide the over 60% motile sperm to the farms at the time of AI.

• 한국산림과학회지
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• 제38권1호
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• pp.1-12
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• 1978

휴면기간(休眠期間)이 긴 은행나무 종자(種子)의 냉습적처리(冷濕積處理)에 따른 생장촉진물질(生長促進物質)과 생장억제물질(生長抑制物質)의 증감(增減)을 구명(究明)하기 위하여 본(本) 연구(硏究)를 수행(遂行)하고 얻어진 결과(結果)를 요약(要約)하면 다음과 같다. 1. 무처리종자(無處理種子)는 배(胚), 배유(胚乳), 종피(種皮)의 각추출물(各抽出物)에서 생장억제물질(生長抑制物質)을 볼 수 있었으며, 특(特)히 종피(種皮)에서 가장 현저(顯著)하게 나타났다. 2. 냉습적처리종자(冷濕積處理種子)의 배(胚)와 종피(種皮)에서 생장억제물질(生長抑制物質)의 현저(顯著)한 감소(減少)가 있었으나, 생장촉진물질(生長促進物質)은 배부분(胚部分)에서 증가(增加)가 약간 나타났다. 3. 이상(以上)의 결과(結果)에서 은행나무 종자(種子)의 휴면(休眠)에는 종피(種皮), 배(胚), 배유(胚乳)의 각부분(各部分)이 생장억제물질(生長抑制物質)이 존재(存在)하며 발아(發芽)에 관계하는 것으로 추측(推測)된다. 4. 은행나무 종자(種子)와 발아촉진(發芽促進)과의 관계는 생장촉진물질(生長促進物質)의 증가(增加)보다는, 배(胚)와 종피(種皮)의 생장억제물질(生長抑制物質)의 감소(減少)에 영향된다고 추측(推測)된다. 5. 생장촉진물질(生長促進物質)인 Gibberellin은 자외선형광검사등검정(紫外線螢光檢査燈檢定)에서 Rf 0.26, 에서 나타났고, 생장억제물질(生長抑制物質)인 Abscisic acid는 Rf 0.62, Rf 0.70, Rf 0.78에서 각각(各各) purple, gray, blue의 형광색(螢光色)으로 나타났다.