체외수정 돼지 배아의 초기 분할 양상 분석에 의한 발달능 예측

전유별,비샤스 디비엔두,윤기영,현상환 韓國受精卵移植學會 2009 한국동물생명공학회지 Vol.24 No.1

The aim of the present study was to investigate the cleavage pattern, its developmental ability and apoptosis of porcine embryo in vitro. Morphology data on a total of 919 embryos were analyzed retrospectively. Forty-eight hours after insemination, embryos were classified into five groups based on the cleavage state as follows; 1 cell, 2 cell, 4 cell, 5 to 8 cell and fragmentation. These groups were cultured another 120 hours and then evaluated for blastocyst formation. Blastocyst formation rates were significantly higher in 4 cell (42.5%) and 5 to 8 cell (48.6%) cleaving groups than in other groups (p<0.05). On the other hand, 2 cell and fragmentation groups produced 4.9% and 3,9% blastocysts, respectively. And we could verify that in the event of 2 cell block and fragmentation of embryo. To analyze the apoptotic frequency in preimplantation development of porcine IVF embryos, all cells of each blastocyst were performed by TUNEL assay. There were no significantly differences in the total cell numbers of embryos and apoptotic cell rate in blastocysts among the each classified groups. Data suggest that 4 cell and 5 to 8 cell cleaving embryos at 48 hour after insemination have high developmental competence, and may be an useful parameter to predict the development of preimplantation embryos and to study using preimplanation embryonic research.

액상 보존액 내 동결 보호제가 4℃에 보관된 액상 돼지 정액의 운동성과 생존성에 미치는 효과

전유별,박광우,정승아,윤준철,현상환 사단법인 한국동물생명공학회 2012 한국동물생명공학회지 Vol.27 No.1

The objective of this study was to investigate the motility and kinematics of boar sperm that while stored at 4C. The samples of fresh boar semen were place into an extender, Androhep, and stored at 4℃. In three of these samples, cryoprotectants were added. The sperm's motilities and kinematics were evaluated by using microscope (×400) and the viability status was evaluated by using with eosin staining method. The 5 sample groups are; Goup A:Androhep (extender), stored at 17℃. Group B:Androhep (extender), stored at 4℃. Group C:Androhep (extender), + 3% glycerol (cryoprotectant), stored at 4℃. Group D:Androhep (extender), + 3% DMSO (cryoprotectant), stored at 4℃. Group E: Androhep (extender), + 3% ethylene glycol (cryoprotectant), stored at 4℃. In group A, the sperm's motility was reduced. On day one the sperm's motility was (85.7 ± 2.3) and day 5 the motility was (43.9 ± 3.3). In group B, C and D the sperm's motility were reduced to 0 on day 5. In group E the sperm's percentage of motility decreased. On day one the sperm's motility was (42.0 ± 0.5) and day 5 the motility was (2.3 ± 0.3). When comparing cryoprotectant in samples of boar sperm there is a slight improvement in the results when the use of Androhep Lite (extender), + 3% ethylene glycol (cryoprotectant), stored at 4℃ are used. Based on these results, ethylene glycol can protect sperm from heat shock at 4℃, but not satisfactory level. However, it showed the possibilities of liquid semen preservation at 4℃ by using cryoprotectant.

액상 보존액 내 동결 보호제가 에 보관된 액상 돼지 정액의 운동성과 생존성에 미치는 효과

전유별,박광우,곽성성,정승아,윤준철,현상환 韓國受精卵移植學會 2012 한국동물생명공학회지 Vol.27 No.1

The objective of this study was to investigate the motility and kinematics of boar sperm that while stored at 4C. The samples of fresh boar semen were place into an extender, Androhep, and stored at . In three of these samples, cryoprotectants were added. The sperm's motilities and kinematics were evaluated by using microscope () and the viability status was evaluated by using with eosin staining method. The 5 sample groups are; Goup A:Androhep (extender), stored at . Group B:Androhep (extender), stored at . Group C:Androhep (extender), + 3% glycerol (cryoprotectant), stored at . Group D:Androhep (extender), + 3% DMSO (cryoprotectant), stored at . Group E:Androhep (extender), + 3% ethylene glycol (cryoprotectant), stored at . In group A, the sperm's motility was reduced. On day one the sperm's motility was () and day 5 the motility was (). In group B, C and D the sperm's motility were reduced to 0 on day 5. In group E the sperm's percentage of motility decreased. On day one the sperm's motility was () and day 5 the motility was (). When comparing cryoprotectant in samples of boar sperm there is a slight improvement in the results when the use of Androhep Lite (extender), + 3% ethylene glycol (cryoprotectant), stored at are used. Based on these results, ethylene glycol can protect sperm from heat shock at , but not satisfactory level. However, it showed the possibilities of liquid semen preservation at by using cryoprotectant.

착상 전 돼지 체외수정 배아 발달 단계에서의 세포 자멸사 현상

홍성민,전유별,현상환 韓國受精卵移植學會 2009 한국동물생명공학회지 Vol.24 No.3

In this study, we aimed to determine whether the evaluated markers of cell death could be found at particular developmental stages of normal porcine in vitro fertilization (IVF) embryos. We investigated the characteristics of spontaneous and induced apoptosis during preimplantation development stages of porcine IVF embryos. In experiment 1, to induce apoptosis of porcine IVF embryos, porcine IVF embryos at 22h post insemination were treated at different concentration of actinomycin D (0, 5, 50 and 500 ng/ml in NCSU medium). Treated embryos were incubated at in 5% , 5% for 8h, and then washed to NCSU medium and incubated until blastocyst (BL) stage. We examined cleavage rate at 2days and BL development rate at 7days after in vitro culture. A significantly lower rate of cleavage was found in the 500 ng/ml group compared to others (500 ng/ml vs. 0, 5, 50 ng/ml; 27.8 % vs. 50.0%, 41.2%, 35.9%), and BL formation rate in 500 ng/ml was lower than that of others (500 ng/ml vs. 0, 5, 50 ng/ml; 8.0% vs. 12.6%, 11.2%, 12.6%). In experiment 2, to evaluate apoptotic cells, we conducted TUNEL assay based on morphological assessment of nuclei and on detection of specific DNA degradation under fluorescence microscope. This result showed that apoptosis is a normal event during preimplantation development in control group (0 ng/ml actinomycin D). A high number of BL derived control group contained at least one apoptotic cell. Actinomycin D treated BLs responded to the presence of apoptotic inductor by significant decrease in the average number of blastomeres and increase in the incidence of apoptotic cell death. In 500 ng/ml group, the incidence of apoptosis increased at 4-cell stage and later. This result suggested that apoptosis is a process of normal embryonic development and actinomycin D is useful tool for the apoptosis study of porcine preimplantation embryos.

Effects of Green Tea (Camellia sinensis) Extract Supplementation at Different Dilution Steps on Boar Sperm Cryopreservation and in vitro Fertilization

박상현,전유별,유일정 한국임상수의학회 2018 한국임상수의학회지 Vol.35 No.2

We evaluated the effects of green tea extract (GTE) supplementation at different dilution steps on boar spermfreezing and in vitro fertilization. Sperm intracellular hydrogen peroxide (H2O2), motility, viability, acrosome integrityand morphology were determined. In addition, sperm IVF parameters (penetration and monospermy) and glutathione(GSH) levels of presumptive zygotes (PZs) were evaluated. Semen was diluted in lactose egg yolk (LEY) and cooledat 5oC for 3 h (first dilution step) and then diluted in LEY with 9% glycerol and maintained at 5oC for 30 min (seconddilution step). Four experimental groups were compared: first and second dilution steps without GTE (control), firstdilution step with GTE (Step 1), second dilution step with GTE (Step 2) and first and second dilution step with GTE(Step 1+2). The spermatozoa were frozen in nitrogen vapor. Higher sperm motility, viability and acrosome integrityafter thawing were observed in Step 1, Step 2 and Step 1+2 groups compared with the control (P < 0.05). LowerH2O2 level was observed in Step 1+2 compared with control and Step 1 (P < 0.05). For IVF, matured oocytes wereco-cultured with spermatozoa frozen according to the experimental groups. GSH levels of PZs were significantly higherin Step 2 and Step 1+2 than in control and Step 1 (P < 0.05) without a significant difference in IVF parameters. Inconclusion, supplementation with GTE in both first and second dilution steps during the freezing process resulted inbetter boar sperm cryopreservation and might be beneficial for further embryo development.

개 정액의 냉동보존 시 κ-Carrageenan이 정자 성상에 미치는 영향

김은지,전유별,유일정,Kim, Eun-Ji,Talha, Nabeel A.H.,Jeon, Yu-Byeol,Yu, Il-Jeoung 한국동물생명공학회 2019 한국동물생명공학회지 Vol.34 No.1

This study was conducted to find out the effect that ${\kappa}-Carrageenan$ has on the properties of dog sperm when it was added to the cryoprotectant. Extender basically was contained 1.21 g Trizma base, 0.67 g citric acid, 0.4 g glucose, 0.03 g penicillin G, 0.05 g streptomycin sulfate. Extender1 was added with 0.1%, 0.2%, 0.3%, and 0.5% carrageenan, while extender2 was supplemented with glycerol. After freezing-thawing, the motility, viability, acrosome integrity, apoptosis, and ROS (reactive oxygen specifications) of sperm were measured to analyze the effects of the supplementation of carrageenan. Total Motile (TM), Rapid Progressive Motile (RPM), Medium Progressive Motile (MPM), and Immotile were measured through the CASA system after thawing in 37 degree water. Extender with 0.2% ${\kappa}-carrageenan$ ($64.26{\pm}0.49$) was significantly higher than control ($40.24{\pm}8.27$) (p < 0.05). RPMs of extender with 0.1%, 0.2% ${\kappa}-carrageenan$ ($57.64{\pm}6.34$, $56.47{\pm}1.35$) were significantly higher than the other groups (p < 0.05). Acrosome integrity was measured by dyeing to PSA-FITC with an epifluorescence microscope. Normal acrosome ratio of extender with 0.5% ${\kappa}-carrageenan$ ($61{\pm}8.03$) was higher than the other groups (p < 0.05). Apoptosis was measured with a FACSCalibur flow cytometer using FITC (FITC Annexin V Apoptosis Detection Kit). Treated groups of ${\kappa}-carrageenan$ of 0.1% ($0.81{\pm}0.05$), 0.2% ($0.85{\pm}0.05$) were significantly higer (p < 0.05) than control. Modified SYBR/PI staining was used for determination of viability and DCF staining was used for evaluation of ROS. Viability and ROS were not significantly different from other groups. In conclusion, adding a certain concentration of carrageenan to the extender of cryopreservation, carrageenan contributes to the improvement of the sperm motility, acrosome integrity and prevention of apoptosis.

바이오장기 연구를 위한 이동식 아이솔레이터 내 복제 미니 돼지의 생리 활성 평가

김해성,전유별,곽성성,정승아,정의만,현상환,정의배 韓國受精卵移植學會 2011 한국동물생명공학회지 Vol.26 No.3

The present study investigated the physiological evaluation of cloned mini-pigs in a transportable isolator. Transportable isolator was designed and manufactured by our research team for transporting gnotobiotic pig. Until now, no previous reports are available regarding the physiological activities and harmful effects when pigs were transported in this isolator. Five cloned mini-pigs of 1~2 year (s) old female with a body weight between 80~90 kg were used. The effects of transportable isolator on stress-related hormone, adrenocorticotrophic hormone (ACTH) and cortisol levels, and heart rate were evaluated. In addition, it was also examined the effects of transportable isolator on blood chemistry factors (alanine aminotransferase: ALT, aspartate aminotransferase: AST, blood urea nitrogen: BUN, glucose, and creatinine). Blood was sampled just before the beginning of transport (T0), at the end of transport (30min after the transport; T1), and 30 min after the end of transport (T2). At the same time, heart rate was also evaluated. As a result, heart rate had no significant (p>0.05) differences at the various-time points of study (T0, T1, T2). However, heart rate was slightly higher than normal range in T1 and T2. The ACTH level was higher than normal range. Whereas, the cortisol level was lower than normal range. There were no statistical significant differences both ACTH and cortisol level between different time groups. Also, there were no significant differences in blood chemistry factors. Therefore, our present study shows that transportable isolator has no harmful effect on stress and physiological condition in cloned mini-pigs.

Histological Features of the Testis in the Long-beaked Common Dolphin from Korean Waters

김주환,장성웅,전유별,안두해,김두남,현상환,문창종 사단법인 한국동물생명공학회 2013 한국동물생명공학회지 Vol.28 No.1

The present study examined the histological characteristics of adult testis in the long-beaked common dolphin (Delphinus capensis) from Korean waters and the localization of DEAD-box polypeptide 4 (DDX4; a germ cell marker)and vimentin (a Sertoli cell marker) expression in the dolphin testis compared with that in terrestrial mammals, including dogs and rats. The seminiferous tubules of dolphin testis have very small or completely closed lumens, and spermatogenic cells and Sertoli cells within the tubules cannot be differentiated. Immunohistochemical analysis showed that, in the dolphin testis, DDX4- and vimentin-positive cells were scattered extensively within the tubule, whereas in the dog and rat testis, DDX4 immunoreactivity was localized in spermatogenic cells of the adluminal compartment,and vimentin immunoreactivity was localized in Sertoli cells of the basal compartment in the seminiferous epithelium. These results suggest that the histological characteristics of the seminiferous tubules in the dolphin testis differ from those of terrestrial species.

Effects of Antioxidants Supplement in Porcine Sperm Freezing on in vitro Fertilization and the Glutathione and Reactive Oxygen Species Level of Presumptive Zygotes

박상현,유일정,전유별 사단법인 한국동물생명공학회 2017 한국동물생명공학회지 Vol.32 No.4

The present study was aimed to determine the effects of green tea extract (GTE) and beta-mercaptoethanol (β-ME) supplementation in boar sperm freezing extender on in vitro fertilization (IVF) and reactive oxygen species (ROS) and glutathione (GSH) levels of presumptive zygotes (PZs). Experimental groups were allocated into lactose egg yolk (LEY) without antioxidant (control), GTE (1,000 mg/l in LEY) and β-ME (50 μM in LEY). In freezing, spermatozoa extended with LEY were cooled to 5°C for 3 h and then kept at 5°C for 30 min following dilution with LEY containing 9% glycerol and 1.5% Equex STM. The final sperm concentration was 1 × 108/ml. Spermatozoa were loaded into straws and frozen in nitrogen vapor for 20 min. For IVF, oocytes were matured in NCSU-23 medium and co-cultured with spermatozoa following thawing at 37°C for 25 sec. At 12 h following IVF, IVF parameters (sperm penetration and monospermy) were evaluated. In addition, GSH and ROS levels of PZs were determined by Cell Tracker Blue CMF2HC and DCHFDA, respectively. IVF parameters did not show any significant difference among the experimental groups. GSH and ROS levels of PZs were not significantly different between groups. In conclusion, antioxidant supplementation in boar sperm freezing could not influence IVF parameters, ROS and GSH levels of PZs.

The Effects of 3-Isobutyl-1-methylxanthine (IBMX) on Nuclear and Cytoplasmic Maturation of Porcine Oocytes In Vitro

곽성성,장성훈,정세헌,전유별,Dibyendu Biswas,현상환 사단법인 한국동물생명공학회 2012 한국동물생명공학회지 Vol.27 No.3

The 3-isobutyl-1-methylxanthine (IBMX) is non-selective phosphodiesterase and is able to prevent resumption of meiosis by maintaining elevated cyclic AMP (cAMP) concentrations in the oocyte. The present study was conducted to analyze: (1) nuclear maturation (examined by the Hoechst staining), (2) whether cytoplasmic maturation (examined by the intracellular glutathione (GSH) concentration) of porcine oocytes is improved during meiotic arrest after prematuration (22 h) with IBMX. Before in vitro maturation (IVM), oocytes were treated with 1 mM IBMX for 22 h. After 22 h of pre-maturation, the higher rate of IBMX treated group oocytes were arrested at the germinal vesicle (GV) stage (42.3%) than control IVM oocytes (10.1%). It appears that the effect of IBMX on the resumption of meiosis has shown clearly. In the end of IVM, the reversibility of the IBMX effect on the nuclear maturation has been corroborated in this study by the high proportions of MII stage oocytes (72.5%) reached after 44 h of IVM following the 22 h of inhibition. However, intracellular GSH concentrations were lower in the oocytes treated with IBMX than the control oocytes (6.78 and 12.94 pmol/oocyte, respectively). These results demonstrate that cytoplasmic maturation in porcine oocytes pre-treated with IBMX for 22 h did not equal that of control oocytes in the current IVM system. These results indicate that pre-maturation with IBMX for 22 h may not be beneficial in porcine IVM system.