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Effect of the Artificial Shrinkage on the Development of the Vitrified Bovine Embryos
하아나,Su-Jin Cho,Gautam-Kumar Deb,Jae-Il Bang,Tae-Hyeon Kwon,Byeong-Hyun Choi,공일근 사단법인 한국동물생명공학회 2010 한국동물생명공학회지 Vol.25 No.1
This study was conducted to find out the effects of artificial shrinkage (AS) on post-thaw development of bovine embryos. The blastocoelic cavity of blastocyst was punctured to remove its fluid contents and then incubated in the holding medium (HM) for 10 min. The punctured and non-punctured (control) blastocysts were equilibrated in vitrification solution 1 (VS1; TCM-199+20% FBS+10% EG) for 5 min and vitrification solution 2 (VS2; TCM199+20% FBS+35% EG+5% PVP+0.5 M Sucrose) for 1 min and vitrified by direct dropping into the liquid nitrogen. Vitrified blastocysts (punctured and control) were thawed and cultured in vitro (12 hr) for studying survival and hatching rates. The levels of shrinkage were measured by the volume of the blastocyst during equilibration in VS1 (at 1, 3 and 5 min of equilibration) and VS2 (at 30 and 60 sec of equilibration) that was considering the volume of non-punctured blastocyst in HM as 100%. The levels of shrinkage were higher in punctured group (62.4, 64.6, 64.3% at 1, 3 and 5 min in VS1; 50.6 and 52.7% at 30 and 60 sec in VS2) than control group (84.8, 86.6, 86.4% at 1, 3 and 5 min in VS1; 72.1 and 68.8% at 30 and 60 sec in VS2), but within each group the levels of shrinkage were similar. The survival (90.9%) and hatching (50.0%) rates of vitrified blastocysts at 12 hr post-thaw were higher in punctured group than that in control group (76.9% and 0.0% respectively). We confirmed that vitrification solutions (VS1 and VS2) have no toxic effect on the survival of blastocysts because the survival rates of blastocysts exposed to VS1 and VS2 for 24 hr were similar between punctured and control groups (94.3 vs. 96.0%; p>0.05). In conclusion, the preliminary data show that AS of blastocyst may improve survival and hatching rate after thawing.
Establishment of Cryopreservation of Leopard Cat Semen Collected by Electro-ejaculation Method
하아나,김유곤,윤진호,방재일,Gautam K. Deb,M. Fakruzzaman,임양묵,용환율,공일근 사단법인 한국동물생명공학회 2011 한국동물생명공학회지 Vol.26 No.4
The aim of this study was to evaluate the post-thawed characteristics of leopard cat semen. In this experiment,semen was collected from two leopard cats (A and B) at wild animal center in Seoul Grand Park in Korea. After collection, the sperms were washed with D-PBS and diluted by the freezing medium (Irvine science, USA) and stored in liquid nitrogen. The post-thawed concentration was 357 × 10^6sperms/ml for A and 97 × 10^6 sperms/ml for B. The viability of post-thawed sperm from A and B individual was 24.0% and 19.0%, respectively. Pre-freezing motility of A and B individual semen was 68.54% and 56.65. Leopard cat A had more normal sperm than that of B (69.5%vs. 54.5%). Acrosome integrity analysis detected live (14.5% vs. 9.0%), damage (39.0% vs. 44.0%) and dead (46.0%vs. 47.0%) in leopard cat A and B, respectively. The present results concluded that leopard cat semen can be collected successfully by electro-ejaculation method and cryopreserved successfullyfor future use in different assisted reproductive technologies. The cryopreservation protocol needs to be modified for increasing post-thawed viability of leopard cat spermatozoa
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.
방재일,하아나,진종인,정경일,이진권,류영실,민찬식,Gautam Kumar Deb,공일근 사단법인 한국동물생명공학회 2011 한국동물생명공학회지 Vol.26 No.4
The present research was carried out to evaluate the possibility of increasing female offspring production ratios using artificial insemination buffer (AIB) before artificial insemination (AI). In this experiment, we optimized AIB composition, made an AIB gun and analyze factors affecting AI non-return rate after AIB treatment. The AIB was made with the base of Tris-buffer supplemented with L-arginine and several other chemicals that might reduce the motility of male sperm compared to the female counterpart, therefore, increasing the possibility of fertilization by female sperm. AIB must be deposited into 2^nd to 4^th cervix by AIB gun. After 15 min of AIB deposition, frozen semen was deposited into the same place. A total of 348 cattle were inseminated with AIB insemination, and there were no significant differences between AIB and traditional AI non-return rates (56.8% vs. 55.7%). The AI non-return rate in AIB group, however, differed significantly among 7 Hanwoo farms. The parturition numbers (1^st to 7^th) of cows did not affect AIB AI rate. The proportion of AIB AI success rates was significantly higher in Hanwoo cows than in dairy cows (61.0% vs. 48.7%), but the average AI success rate did not differ significantly between AIB and conventional AI (56.8% vs. 55.7%). The female offspring production rate in 2^nd to 4^th cervix deposition place was significantly higher than that in the uterus body (77.7% vs. 59.6%, p<0.05). The injection volume of AIB in 5 and 10 ml was significantly higher than that in 2 ml (77.7%, 78.7% vs. 51.8%, p<0.05), but there were no differences in AIB injection volume between 5 and 10 ml. The best exposure time of AIB in the cervix was 10 to 15 min rather than 5 min (79.2%, 77.2% vs. 52.6%, p<0.05). AIB therefore needs to have an exposure time of at least over 10min for a higher production rate of female offspring. In conclusion, AIB could be used in AI industry to increase the female offspring ratio and AIB AI can increase the AI success rate.