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Effect of Cytochalasin B Treatment on the Improvement of Survival Rate in Vitrified Pig Oocyte
황인설,박미령,곽태욱,박상현,임지현,김성우,황성수 한국발생생물학회 2018 발생과 생식 Vol.22 No.3
To improve survival rates of vitrified pig oocytes, the treatment of cytoskeletal stabilizer on an appropriate time is one of the possible approaches. However, the exact treatment timing and effect of cytoskeletal stabilizer such as cyto-chalasin B (CB) is not well known during oocyte vitrification procedures. Thus, the present study was conducted to determine optimal treatment timing of CB during vitrification and warming procedures. In experiment 1, the survival rates of the post-warming pig oocytes were analyzed by fluorescein diacetate (FDA) assays with 4 classifications. In results, post-warming oo-cytes showed significantly (p<0.05) decreased number of alive oocytes (31.8% vs. 86.4%) compared to fresh control. In detail, the significant difference (p<0.05) was found only in strong fluorescence (18.2% vs. 70.5%) not in intermediate fluorescence groups (13.6% vs. 15.9%). In experiment 2, CB was treated before (CB-Vitri) and after (Vitri-CB) vitrification. In results, group of Vitri-CB showed significantly (p<0.05) higher (91.6%) survival rates compared to group of CB-Vitri (83.7%), sig-nificantly (p<0.05) and comparable with group of Vitri Control (88.7%) by morphological inspection. In FDA assay results, group of Vitri-CB showed significantly (p<0.05) higher (44.2%) survival rates compared to groups of CB-Vitri (36.7%) and Vitri Control (35.1%). In conclusion, the increased survival rates of post-warming pig oocyte treated with Vitri-CB method are firstly described here. The main finding of present study is that the CB treatment during recovery could be helpful to re-fresh the post-warming pig oocyte resulting its improved survival rates.
Fertility preservation in pig using ovarian tissues by vitrification method
황인설 사단법인 한국동물생명공학회 2022 한국동물생명공학회지 Vol.37 No.2
Cryopreservation of porcine ovarian tissue by vitrification method is a promising approach to preserve genetic materials for future use. However, information is not enough and technology still remains in a challenge stage in pig. Therefore, the objective of present study was to determine possibility of vitrification method to cryopreserve porcine ovarian tissue and to confirm an occurrence of cryoinjuries. Briefly, cryoinjuries and apoptosis patterns in vitrified-warmed ovarian tissue were examined by histological evaluation and TUNEL assay respectively. In results, a damaged morphology of oocytes was detected among groups and the rate was significantly (p < 0.05) lower in vitrification group (25.8%) than freezing control group (67.7%), while fresh control group (6.6%) showed significantly (p < 0.05) lower than both groups. In addition, cryoinjury that form a wave pattern of tissues around follicles was found in the frozen control group, but not in the fresh control group as well as in the vitrification group. Apoptotic cells in follicle was observed only in freezing control group while no apoptotic cell was found in both fresh control and vitrification. Similarly, apoptotic patterns of tissues not in follicle were comparable between fresh control and vitrification groups while freezing control group showed increased tendency. Conclusively, it was confirmed that vitrification method has a prevention effect against cryoinjury and this method could be an alternative approach for cryopreservation of genetic material in pigs. Further study is needed to examine the viability of oocytes derived from vitrified-warmed ovarian tissue.
Effect of a short-term in vitro exposure time on the production of in vitro produced piglets
황인설,권대진,곽태욱,이주영,형남웅,양현,오건봉,옥선아,박응우,임기순,황성수 사단법인 한국동물생명공학회 2016 한국동물생명공학회지 Vol.31 No.2
Although piglets have been delivered by embryo transfer (ET) with in vitro produced (IVP) embryos and blastocysts, a success rate has still remained lower level. Unlike mouse, human, and bovine, it is difficult to a production of piglets by in vitro fertilization (IVF) because of an inappropriate in vitro culture (IVC) system in pig. Therefore, the present study was conducted to investigate whether minimized exposure time in IVC can improve the pregnancy and delivery rates of piglets. Immediately after IVM, the oocytes were denuded and co-incubated with freshly ejaculated boar semen for 3.5 to 4 hours at 38.5 ˚C under 5% CO2 in air. To avoid long-term exposure to in vitro state, we emitted IVC step after IVF. After that the presumptive zygotes were transferred into both oviducts of the surrogate on the same day or 1 day after the onset of estrus. Pregnancy was diagnosed on day 28 after ET and then was checked regularly every month by ultrasound examination. The 3 out of 4 surrogates were determined as pregnant (75%) and a total of 5 piglets (2 females and 3 males) were delivered at 118.3 ± 2.5 days of pregnancy period. In conclusion, a short-term exposure time may be an important factor in the production of IVP-derived piglets. It can be apply to the in vitro production system of transgenic pig by IVF, cloning, and pronuclear microinjection methods.
황인설,박미령 한국산학기술학회 2022 한국산학기술학회논문지 Vol.23 No.7
This study endeavored to develop a vitrification device and examine the survival rates of pig oocytes following vitrification and warming. General lab equipment, such as nylon mesh and inoculation loop, were used to develop the vitrification device. The survival rate was examined using in vitro matured pig oocytes. Our results reveal that 40 and 50 μm nylon mesh showed significantly (P<0.05) higher survival rates (71.7 and 75.5%, respectively) compared to other mesh sizes of 30, 60, and 70 μm (58.3, 65.0, and 56.7%, respectively), and were comparable to survival obtained with Cryotop (71.7%). Cryoinjuries by dislocation of oocytes (with 60 and 70 μm mesh) and higher volume of vitrification solution (30 μm) resulted in reduced survival rates. However, the oocytes could be safely located and excessive vitrification solution could be removed easily in the 40 and 50 μm groups. In conclusion, we confirm that 40 and 50 μm of nylon mesh is very useful for preserving a high amount of pig oocytes with survival rates comparable to a commercialized vitrification device. 본 연구에서는 돼지 난자용 유리화동결 기구를 고안하고 그 생존율을 분석하고자 하였다. 일반적인 실험실에 구비되어 있는 연구 기자재인 nylon mesh와 inoculation loop를 활용하여 유리화동결용 기구를 제작하였고, 체외성숙 돼지 난자를 이용하여 유리화동결 과정을 진행하였다. 그 결과, 40과 50㎛의 nylon mesh를 사용하여 유리화동결 과정을 진행하였을 때 각각 71.7과 75.5%의 생존율을 보였으며, 이미 상용화된 동결기구인 Cryotop의 생존율 71.7%와 유사한 결과를 획득하였다. 하지만, 30, 60, 70㎛의 nylon mesh를 사용한 경우, 각각 58.3, 65.0, 56.7%의 생존율로 Cryotop과 비교하여 유의하게 (p<0.05) 감소되는 결과를 보였으며, 유리화동결과정 중 발생하는 동결상해로 인한 것으로 사료되었다. 또한 본 연구에서 제작된 nylon mesh 동결기구를 사용할 경우, 유리화동결과정 중 동결액 부피의 최소화를 용이하게 할 수 있었다. 결론적으로, 본 연구에서 고안된 간이 동결기구를 사용할 시 기존의 상용 기구인 Cryotop과 비교하여 유리화동결의 과정이 간소화됨은 물론 다량의 난자를 일시에 처리할 수 있었다. 특히 40과 50㎛의 nylon mesh 동결기구를 사용할 경우 높은 생존율을 확보할 수 있고, 이를 활용하여 다량의 난자를 처리해야하는 가축이나 동물의 유전자원보존에 있어 활용성이 매우 높을 것으로 사료된다.