Ovum Pick-Up 기술을 이용한 난자 채취 시 회수압력에 따른 엘크 암사슴의 수정란 생산 효율조사

이은도,김동교,최봉환,김관우 한국산학기술학회 2022 한국산학기술학회논문지 Vol.23 No.8

본 연구는 엘크 암사슴에서 초음파 유래 OPU(Ovum Pick-Up) 기술을 이용하여 난자를 채취할 시 회수압력을 다르게 하여 회수효율과 수정란발달 효율을 비교하기 위하여 실시하였다. 난자는 초음파 진단기를 이용하여 사슴 난소의 난포를 흡입하였고, 회수 시 압력은 50-80 mmHg 범위 내에서 회수하였다. 회수 압력은 4그룹으로(50 mmHg, 60 mmHg, 70 mmHg, 80 mmHg) 나누어 회수율과 수정율 그리고 배반포 발달율을 조사하였다. 회수율은 66.03%, 57.22%, 7 62.33%, 81.17%의 회수율을 보였다. A+B의 난자 등급 비율은 55.71%, 36.31%로 50 mmHg 그룹과 60 mmHg 그룹에서 유의적으로 높은 결과를 보였다. 수정율에 있어서는 81.25%, 66.67%로 50 mmHg 그룹과 60 mmHg 그룹에서 유의적으로 높은 결과를 보였다. 배반포 발달율은 36.94%, 41.67%, 16.67%, 10.37%의 발달율을 보였다. 50 mmHg 그룹과 60 mmHg 그룹에서 유의적으로 높은 결과를 보였다. 본 연구 결과로, 난자 채취 시 회수압력이 높을수록 회수율이 높지만 난자등급, 수정율 및 배반포 발달율에 있어서는 효율이 떨어지는 결과를 확인하였다. 추가적인 연구를 통해 엘크 체외수정란 이식을 성공시키기 위한 난자 회수조건 최적화 및 체외배양 효율을 향상시키는 연구를 진행해야 한다. This study was undertaken to determine the oocyte recovery and embryo development efficiency in elk using an ultrasonic-derived OPU (Ovum Pick-Up) technology. The oocytes were collected after retrieving the follicles of the elk ovary using an ultrasonic diagnostic device under recovery pressures ranging from 50 to 80 mmHg. Based on the recovery pressure used, the oocytes were segregated into four groups: 50 mmHg, 60 mmHg, 70 mmHg, and 80 mmHg. The influence of increasing recovery pressure was evaluated on parameters such as recovery rate, fertilization rate, and embryonic development. We obtained a recovery rate of 66.03%, 57.22%, 62.33%, and 81.17% for the 50, 60, 70, and 80 mmHg groups, respectively. The ratios of grade A and B oocytes retrieved were significantly higher in the 50 mmHg and 60 mmHg groups (55.71% and 36.31%, respectively). The 50 and 60 mmHg groups also showed significantly higher fertilization rate percentages of 81.25% and 66.67%, respectively and significantly higher blastocyst development rates of 36.94% and 41.67%, respectively. Our results indicate that although a higher recovery pressure improved the recovery rate, it negatively influenced the oocyte grade, fertilization rate, and blastocyst development rate. Future research should focus on optimizing the recovery conditions and in vitro culture efficiency for successful Elk embryo transplantation.

초음파 유래 Ovum Pick-Up 기술을 이용한 엘크 암사슴의 수정란 생산

이은도,이상훈,김동교,이진욱,이성수,김관우 한국산학기술학회 2020 한국산학기술학회논문지 Vol.21 No.10

This study, which was designed to improve and increase the utilization of female elk, examined the possibility of collecting their embryos during the non-reproductive period using ultrasound-guided Ovum Pick-Up (OPU) technology. Once follicles in the ovaries of an elk were observed, the embryos were retrieved through the absorption of the follicles using an OPU ultrasound probe. Fifty-seven embryos were retrieved from 85 follicles, giving a retrieval rate of 67.1%. Morphological evaluation of the recovered embryos revealed 14.0% embryos to be in grade A, 19.2% in grade B, 15.7% in grade C, and 50.8% in grade D. The developmental efficiency of the retrieved embryos was also investigated using in vitro maturation, in vitro fertilization, and in vitro culture. After cultivating 28 embryos through in vitro fertilization, 19 embryos were found to be fertilized; the fertilization rate was 67.9%. Four embryos developed to the blastocyst stage, indicating a 14.3% development rate. This study confirmed that the production of fertilized embryos from a seasonal breeder, such as elk, is possible via the ultrasound-guided OPU method. If the efficiency of in vitro fertilization and in vitro culture can be improved through further research, it will help improve the efficiency of elk embryo production through the transplantation of their fertilized embryos. 본 연구는 초음파 유래 OPU (Ovum Pick-Up) 기술을 이용하여 엘크 암사슴의 개량 및 활용성 증진을 위해 엘크 암사슴 난자를 채취하였다. 비번식 계절시기에도 체외 수정란이 생산 가능한지 확인하기 위하여 OPU 초음파 진단기를 이용하여 사슴 난소에서 난포를 흡입한 후 난자를 회수 하였다. 총 85개의 난포를 흡입 한 결과 57개의 난자가 회수되어 67.1%의 회수율을 나타내었다. 난자의 등급을 평가한 결과 A 등급 14.0%, B 등급 19.2%, C 등급 15.7% 및 D 등급 50.8%로 조사되었다. 본 연구에서는 A~C 등급의 난자를 이용하여 체외성숙, 체외수정 및 체외배양을 실시하였다. 총 28개의 난자를 이용하여 67.9% (19/28)의 수정율을 보였으며 배반포 발달율은 14.3% (4/28)로 나타났다. 본 연구 결과를 토대로 하여 볼 때, 계절번식을 하는 것으로 알려져 있는 엘크에 초음파 유래 OPU 방법이 적용 가능한 것으로 확인하였다. 따라서, 향후 추가적인 연구를 통해 체외수정과 체외배양 효율을 보다 향상시킨다면 엘크 수정란 이식을 통해 사슴의 개량기반 구축과 암사슴의 활용성 향상에 기여 할 수 있을 것으로 사료된다.

초음파유도에 의한 소 난포란의 채취에 관한 연구 I. 발정주기, 계절 및 bST처치 영향에 관하여

이병천,윤기영,김현일,노상호,이강남,황우석,Lee, Byeong-chun,Yoon, Ki-young,Kim, Hyun-il,Roh, Sang-ho,Lee, Kang-nam,Hwang, Woo-suk 대한수의학회 1997 大韓獸醫學會誌 Vol.37 No.4

Estrus cycle of cow, season and follicldar stimulating treatment, having an effect on the number of follicle, are investigated for the oocyte recovery rate in ovum pick-up(OPU). The number of follicle aspirated and oocyte collected on the different days of estrus cycle(D 4~5, D 9~10 and D 14~15) were not significantly different among the groups. The higher number of viable oocytes were produced on Jan-May(79.0%) than Jun-Aug(33.3%) by OPU in cow. The number of follicle and aspirated oocyte in cows treated FSH or PMSG combined with bovine somatotropin(bST) were 1.2~1.5 times higher than in cows treated alone follicular stimulating hormone(FSH) or pregnant mare serum gonadotropin(PMSG). In conclusion, OPU can be repeatedly practiced 2 or 3 times in an estrus cycle. In addition, the high environmental temperature is not good for ovarian function of cow and the bST co-treatment with FSH or PMSG is increasing the number of aspiratable follicle.

한우에서 Ovum Pick-Up 유래 수정란의 임신 시기별 임신율 비교

고응규(Yeoung-Gyu Ko),이재영(Jae-Yeong Lee),김남태(Nam-Tea Kim),김찬란(Chan-Lan Kim),이세영(Se Young Lee),최창용(Changyong Choe) 한국산학기술학회 2023 한국산학기술학회논문지 Vol.24 No.7

본 연구는 한우에서 난소 내 난자 채취(Ovum Pick-Up, OPU) 기술을 적용하여 수정란이식을 실시하였을 경우 임신기간 동안 임신율을 어떻게 유지하면서 분만에 이르는지 확인하고자 수행하였다. 수정란이식 후 시기별 임신율은 45일령 50%, 2개월령 47.5%, 6.5개월령 35.0%였다. 신선 수정란과 동결 수정란의 임신율은 45일령, 2개월령, 4개월령, 6.5개월령에 신선 수정란 64.3%, 64.3%, 57.1%, 50.0%였으며 동결 수정란은 임신 전체 기간동안 16.7%를 나타내었다. 상실배, 초기배반포, 배반포기 수정란을 이식하였을 때 임신기간이 경과할수록 임신율이 떨어졌으며, 수정란의 발달 단계가 성숙한 것을 이식할수록 임신율이 높아지는 것을 확인하였다. 공란우별로 7∼8개의 수정란을 이식하고 이들의 임신율을 조사한 결과 이식 후 6.5개월령에서 임신율이 12.5%∼71.4%를 나타내어 공란우별로 임신율에 큰 차이를 나타내었다. 수란우의 임신율은 6.5개월령에서 미경산우 16.7%, 경산우 57.9%로 나타났다. 본 연구 결과 한우에서 OPU 유래 수정란을 이식하였을 경우 신선·동결 수정란, 수정란 발달 단계, 공란우 개체, 수란우 분만 경력에 따라 수정란이식 후 임신율에 차이가 나는 것을 확인하였는데, 이는 수정란이식 후 임신율 및 분만율에 다양한 요인이 작용하는 것을 확인하는 계기가 되었으므로, 본 연구를 바탕으로 한우의 수정란이식 임신율을 향상 시킬 수 있는 다양한 연구를 지속적으로 추진하여야 할 것으로 사료된다. This study investigates how to maintain the pregnancy rate and reach parturition subsequent to embryo transfer achieved by applying the Ovum Pick-Up (OPU) technology in Korean native cattle (Hanwoo). The overall pregnancy rates after embryo transfer were determined to be 50% at 45 days, 47.5% at 2 months, and 35.0% at 6.5 months after embryo transfer. The pregnancy rates obtained after AI for fresh and frozen embryos were 64.3%, 64.3%, 57.1%, and 50.0% for fresh embryos at 45 days, 2 months, 4 months, and 6.5 months, respectively, and 16.7% for frozen embryos during the entire period. Transferring morula, early blastocyst, and blastocyst stage embryos resulted in decreased pregnancy rate during the pregnancy period, whereas transferring embryos at a mature developmental stage resulted in increased pregnancies. In all, 7 to 8 embryos of each donor cow were cultured to the blastocyst stage and transferred to recipient cows. This resulted in increasing their pregnancy rates from 12.5% to 71.4%. At 6.5 months of gestation, the pregnancy rates obtained were 16.7% in heifers and 57.9% in multiparous cows. Results of this study indicate the need for continuous promotion of further research that will help improve the pregnancy rate after embryo transfer of Hanwoo cows.

핵이식 기법을 이용한 한우 쌍태생산에 관한 연구 I. Ovum pick-up(OPU), 전기적 세포융합 및 체외배양 기법을 이용한 복제수정란 생산

황우석,신태영,노상호,이병천,Hwang, Woo-suk,Shin, Tae-young,Roh, Sang-ho,Lee, Byeong-chun 대한수의학회 1998 大韓獸醫學會誌 Vol.38 No.1

The purpose of present study is to improve the efficiency of fusion and the developmental rates of nuclear transplanted embryos to produce genetically identical twins from Korean native cattle. The diameter of aspirated follicles had no significant effect on the recovery rates of oocytes collected by ovum pick-up technique. The fusion rates of nuclear transplanted embryos were significantly higher in 50 and $100{\mu}s$ DC duration groups(73.3 and 72.0% ; respectively) than that in $30{\mu}s$ group(55.6% ; p<0.05). The cleavage rates of nuclear transplanted embryos appeared to be significantly higher in donor nuclei derived from in vivo (65.0%) than in those from in vitro (50.5% ; p<0.01), but the developmental rates to morulae and blastocysts were not significantly different between them(13.7 vs 10.9%, respectively).

OPU 채란계절이 한우의 난자 품질 및 발달 능력에 미치는 영향

김성수,최병현,조현태,진종인,하아나,민찬식,조규완,공일근 사단법인 한국동물생명공학회 2014 한국동물생명공학회지 Vol.29 No.3

Implementation of smart embryo technologies in cattle e.g. ovum pick-up followed by in vitro embryo production(OPU-IVP). Seasonal variation is important factor for follicular growth, oocytes quality, quantity and developmentalcompetence. Therefore the aim of present study was carried out to investigated whether the seasons (hot and cool) effecton follicular development, oocyte recovery and subsequent embryo development. Follicular oocytes were aspirated fromKorean native cows (Hanwoo) by the ovum pick-up (OPU) method, which was performed 24 times during two differentseasons, the hot (July to September) and cool (October to December), from OPU donors. The recovered oocytes wereclassified according to morphological categories and used for in vitro embryo production (IVEP). The mean numberof total follicles was significantly higher (p<0.05) during the hot season (18.32 ± 2.26) compared to cool season (15.41± 3.34). Furthermore, seasons did not significantly effect on the number of oocytes recovered (hot season: 41.16%vs. cool season: 46.14%). However, the average number of Grade A oocytes was significantly greater during hot (1.75± 1.86) season compared to the cool season (1.00 ± 1.46), but there was no significant difference of other gradesoocytes. The cleavage rate (hot: 66.67% vs. cool: 63.3%) and embryo development (hot: 58.95% vs. cool: 56.97%)did not differ significantly between the seasons. In conclusion, the results of present study suggest that the season(hot and cool) does not have effects on the oocyte recovery and embryo developmental competence of in vitro culturedembryos.

한우 공란우 및 생체내 난자 회수(ovum pick-up) 조건이 체외수 정란의 발달에 미치는 효과

박용수 ( Yong Soo Park ),공준호 ( Jun Ho Kong ),이준구 ( Jun Koo Yi ),오동엽 ( Dong Yep Oh ),정기화 ( Ki Hwa Chung ) 한국동물위생학회 2021 한국동물위생학회지 (KOJVS) Vol.44 No.4

Artificial insemination of Korean native cattle (KNC) is the predominant method for breed improvement. However, industrialization of embryo production and transfer is necessary to utilize the genetic potential of KNC. The aim of this study was to examine associations between KNC donor cows and ovum pick-up (OPU) conditions, in-vivo oocyte recovery, and embryo development. Oocyte recovery and blastocyst development rates were higher at 50 and 60 mmHg OPU vacuum pressure than at 40 mmHg, which was, however, not significant. Regarding follicle growth, injection of 500 mg GnRH 36 hours before OPU significantly increased the number of OPU oocytes from an average of 4.6 to 7.6 (P<0.05); no significant difference in embryo development rates was observed. Significant differences were observed in the numbers of OPU oocytes, embryo development rates, and transplantable blastocysts per individual among nine KNC donors (P<0.05). Furthermore, although there was no difference in OPU oocyte recovery intervals in approximately 2∼8 weeks, the number of recovered oocytes significantly decreased at the 12-week interval (P<0.05); there was no difference in embryo development rates. The number of oocytes and embryonic development rates only tended to decrease until the seventh OPU session, but decreased significantly until the eighth session (P<0.05). The average pregnancy rate after transfer of OPU-derived in-vitro embryos into recipient cows was 41.8%. To improve the efficiency of OPU egg recovery and in-vitro embryo production, considering KNC donor characteristics, vacuum pressure of 60 mmHg, GnRH pretreatment to induce follicle growth, and effective OPU egg recovery up to seven times at intervals of 2∼4 weeks appears to be most suitable. This study may facilitate the industrialization of KNC embryo production and transfer using high-quality cows.

핵이식 기법을 이용한 한우 쌍태생산에 관한 연구 II. Ovum pick-uo(OPU) 유래 공여핵 및 활성화 유도 수핵난자의 핵이식

황우석,신태영,노상호,박종임,이병천,Hwang, Woo-suk,Shin, Tae-young,Roh, Sang-ho,Park, Jong-im,Lee, Byeong-chun 대한수의학회 1998 大韓獸醫學會誌 Vol.38 No.3

The efficiency of nuclear transfer using donor embryos originated from ovum pickup(OPU) and activated recipient cytoplasts were examined for induction of twinning in Korean native cattle(KNC). After aspiration of follicle by OPU, regardless of the vacuum applied, we obtained same result in proportions of recovered cumulus-oocyte complex (COCs) with compact cumulus. Under electric stimulation(1.0kV/cm DC for $40{\mu}s$), most of activated oocytes proceed to anaphase II/telophase II within 3h(84.7%). In the treatment of oocyte activation, the preactivation which was performed before fusion had significant effect on the developmental rates to morula/blastocyst stage(9.4 vs 4.0%). In embryo transfer of nuclear transferred embryos, we obtained 2 twins from KNC recipients and 1 twin from a Holstein recipient. Our results showed that it is possible to obtain twins using nuclear transfer technique in KNC.

초음파유도 난포채란기의 개발과 이의 검증

최민철,이효종,조성근,강태영,원현희,Choi, Min-cheol,Lee, Hyo-jong,Cho, Seong-keun,Kang, Tae-yung,Won, Hyun-hui 대한수의학회 1999 大韓獸醫學會誌 Vol.39 No.6

This study was carried out to develop a newly designed ovum pick-up(OPU) instrument for ultrasound-guided transvaginal follicular aspiration in cows. This new instrument consists of out- & inner-layer stainless pipes and a grip with a trigger(hand) switch. Some gauge types of disposable needles and tubes can be attached to this inner pipe. With this instrument, while grasping an ovary with one hand, the other hand can handle in apiration and vacuum on/off with the least assitant's help. With this instrument the mean recovery rate of bovine follicular oocytes was 45.2%. In recovered oocytes, usable oocytes(Grade I & II) were 30.4% and this rate meant 1.4 oocytes per ovary. For 30 days after initial aspiration with this instrument, some adverse effects such as adhesion, hemorrhage, hematoma and other mass formation in/with ovaries were also examined by rectal examination, ultrasonographic and endoscopic images. Adhesion was found in one ovary 1 week after aspiration, and hemorrhagic lesion was found 1-2 days and petechia were found 3-5 days after aspiration and there was no remarkable adverse effects. It was found that this instrument could be applicable and safe for ovum pick-up in cows.

OPU 채란계절이 한우의 난자 품질 및 발달 능력에 미치는 영향

김성수,최병현,조현태,진종인,하아나,민찬식,조규완,공일근 한국수정란이식학회 2014 한국동물생명공학회지 Vol.29 No.3

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±2.26), 971개(15.41±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±3.14), 41.16%로 cool season 그룹 448개(7.11±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±1.86), Cool season 그룹 63개(1.00±1.46)로 hot season 그룹이 cool season 그룹과 비교하여 유의적으로 높았다(p<0.05). 하지만 다른 등급인 Grade B는 87개(1.38±1.60) vs. 97개(1.54±1.39), Grade C는 166개(2.63±2.43) vs. 170개(2.70±2.04), Grade D는 112개(1.78±2.65) vs. 118개(1.87±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. Implementation of smart embryo technologies in cattle e.g. ovum pick-up followed by in vitro embryo production (OPU-IVP). Seasonal variation is important factor for follicular growth, oocytes quality, quantity and developmental competence. Therefore the aim of present study was carried out to investigated whether the seasons (hot and cool) effect on follicular development, oocyte recovery and subsequent embryo development. Follicular oocytes were aspirated from Korean native cows (Hanwoo) by the ovum pick-up (OPU) method, which was performed 24 times during two different seasons, the hot (July to September) and cool (October to December), from OPU donors. The recovered oocytes were classified according to morphological categories and used for in vitro embryo production (IVEP). The mean number of total follicles was significantly higher (p<0.05) during the hot season (18.32±2.26) compared to cool season (15.41±3.34). Furthermore, seasons did not significantly effect on the number of oocytes recovered (hot season: 41.16% vs. cool season: 46.14%). However, the average number of Grade A oocytes was significantly greater during hot (1.75±1.86) season compared to the cool season (1.00±1.46), but there was no significant difference of other grades oocytes. The cleavage rate (hot: 66.67% vs. cool: 63.3%) and embryo development (hot: 58.95% vs. cool: 56.97%) did not differ significantly between the seasons. In conclusion, the results of present study suggest that the season (hot and cool) does not have effects on the oocyte recovery and embryo developmental competence of in vitro cultured embryos.