• Asian-Australasian Journal of Animal Sciences
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• 제22권3호
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• pp.343-349
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• 2009

This study was designed to simplify a cryopreservation program for embryonic stem cells (ESCs) by selection of cooling method and cryoprotectant. Commercially available mouse E14 embryonic stem cells (ESCs) were cryopreserved with various protocols, and morphology and viability of the frozen-thawed ESCs and their reactive oxygen species (ROS) production were subsequently monitored. Post-thaw colony-formation of ESCs was detected only after a slow freezing using dimethyl sulfoxide (DMSO) by stepwise placement of a freezing container into a $-80^{\circ}C$ deep freezer and subsequently into -$196^{\circ}C$ liquid nitrogen, while no proliferation was detected after vitrification. When the simplified protocol was employed, the replacement of DMSO with a mixture of DMSO and ethylene glycol (EG) further improved the post-thaw survival. ROS generation in ESCs frozen-thawed with the optimized protocol was not increased compared with non-frozen ESCs. The use of fresh mouse embryonic fibroblasts as feeder cells for post-thaw subculture did not further increase post-thaw cell viability. In conclusion, a simplified slow-freezing program without employing programmable freezer but using DMSO and EG was developed which maintains cell viability and colony-forming activity of ESCs during post-thaw subculture.

• 한국산림과학회지
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• 제98권1호
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• pp.115-124
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• 2009

새로운 균주가 육종되었을 때 그 균주가 장기간 고유특성을 유지하기란 쉽지 않으며, 시간이 경과 할수록 균주는 퇴화를 거듭한다. 이에 따라 현재 많이 이용되고 있는 초저온에서의 균주보존 후 균사의 활력과 온도가 생존에 미치는 영향과 표고균주가 동결하는 동안 나타나는 상태변화를 이해하고자 하였다. 저온 처리별 균주보존결과 표고균주의 생존은 $-20^{\circ}C$에서 50일간 보존하면 사멸했으며, $-80^{\circ}C$와 $-196^{\circ}C$에서 보존한 균주들은 온도에 영향을 받지 않고 생존하였다. 저온 처리별 균사생장속도는 $-80^{\circ}C$에서 보존한 균주들의 재생속도가 가장 빨랐다. 균사의 정단부분과 말단부분에 대한 저온 처리별 균주보존 결과 부분별 위치는 균사의 활력과 생존에 영향을 끼치지 못했다. 프로그램이 가능한 동결장치를 이용하여 표고균주의 동결과정에서 나타나는 현상을 조사한 결과 균주별 각기 다른 어는점을 가지고 있었다. 또한 표고의 자실체 발생 온도형과 균주별 어는점과의 관계는 관련이 없었다. 프로그램이 가능한 동결장치를 이용하여 표고균주의 동결과정에서 나타나는 상태변화를 최소화하기 위해 일시적으로 낮은 온도로 동결을 시도하였지만 균주들은 온도에 영향을 받지 않았다.

• 한국임상수의학회지
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• 제19권1호
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• pp.73-79
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• 2002

To investigate the usability of frozen canine embryos for embryo transfer in the dog, 19 donors, 3 recipients, and 6 male dogs were used for the experiment. Natural mating or artificial insemination was performed for breeding the bitches in natural estrus. Vaginal smear test along with progesterone titre test were performed to detect the appropriate mating time and the bitches were bred twice during 3-6days following LH surge. Embryo collection was done on 8, 9-11, 12-13 days after the second mating to collect morula and blastocyst. Embryos were frozen using a programmable freezer and preseued in LNE tank. Embryos were thawed in 37$^{\circ}C$ water for 15 seconds and transferred into each uterine horn within 30 minutes. Embryos were collected from 13 bitches of 19 donors(68.4%) and the collected embryos were from between 9 and 13 days after 2nd mating. Embryos were produced both by natural mating(60.0%, 9115) and AI with frozen semen(100.0%, 4/4). Embryos were collected from the donors weighed between 2.5 and 30 kg and their age was from 1.5 to 3 years. 52 embryos were collected from 13 donors and the mean number of embryos was four. The stage of embryos was from 2-cell to gastrula and morulae were colledted mostly from 10 to 11 days after 2nd mating. Embryos were collected evenly from each uterine horn and the rate of embryo collection for the number of corpus luteum was 83.9%. Embryos were transferred to 3 recipients(morula 8, blastocyst 1, gastrula 8), however, no offspring was produced.

• 한국발생생물학회지:발생과생식
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• 제2권1호
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• pp.63-68
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• 1998

본 연구에서는 보다 효율적인 동결 보존법을 수립하기 위하여 현재 사용되는 동결 보존액과 동결방법을 정자의 운동성 측면에서 비교해 보았다. 즉, 세 종류의 조성이 다른 동결보존액인 TYB, dithiothreitol을 첨가한 TYB+DTT, KS II 등이 동결보존 전후에 있어 운동성에 미치는 영향을 조사하였으며, 또한 vapor freezing 방법과 computerized freezer를 사용한 동결방법이 정자 운동성에 미치는 영향을 알아보았다. 정자의 분석은 현미경적 방법과 두 종류의 컴퓨터 정자 자동측정기인 SAIS와 Hamilton Thorn을 사용하여 동결 전 후의 정자 운동성과 VCL, VSL, VAP, ALH, LIN 등의 sub-motility 패턴을 측정하였다. 정액성상이 정상인 군에서 동결보존액을 비교한 실험결과는 TYB군과 TYB+DTT군, 그리고 KS II군의 융해 후 운동성이 각각 28.3%, 23.0%, 34.8%로 KS II군이 우수하였고, 동결방법을 비교한 실험에서는 vapor freezing 군과 computerized freezing 군의 융해 후 정자 운동성이 각각 27.8%, 33.2%로 유의차는 없었다. 또한 무력정자증을 보인 정액군에서는 TYB군과 TYB+DTT군, 그리고 KS II군에서 융해후 정자 운동성이 각각 13.6%, 10.0%, 18.5%로 여기 KS II군이 우수하였으며, vapor freezing군과 computerized freezing군의 융해 후 정자 운동성은 12.8%, 12.9%로 유의차가 없었다. 이상의 결과로 보아 운동성이 정상인 정액군과 무력정자증을 보이는 정액군에서 KS II를 사용해 동결하는 것이 TYB나 TYB+DTT를 사용하는 것보다 운동성 있는 정자를 회수하는데 더 효율적이며, 동결방법 측면에서는 vapor freezing 방법과 computerized freezing방법간에 큰 차이가 없음을 볼 수 있었다.

• 한국임상수의학회지
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• 제17권1호
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• pp.234-237
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• 2000

The dornor, 2 years old, 20kg and mixed breed, was bred naturally on day 1 and day 3 of estrus and eight gastrulae were collected by flushing the uterus of the donor after laparatomy on day 13 after the second mating. The embryos were frozen by programmable freezer and preserved for about 3 months in liquid nitrogen. Another bitch in natural estrus, 2 years old, 30kg, mixed breed, was selected as the recipient and the frozen embryos(8 gastrulae) were thawed and each 4 embryos were transferred into upper partr of left and right uterine horn, respectively, on day 13 after the proper mating day determined by vaginal smear. The ecipient delivered 6 offspring 48 days after embryo transfer. Of 6 puppies, one was still birth and two puppies died one month after birth. Parentage test was performed by DNA analysis using microsatellite sequences for 3 puppiers, the recipient, the donor, the male dog bred with the donor, and the male dog raised near to the recipient. The markers selected for the test were CXX 873(133-157 base pair) and CXX 894(141-165 base pair). Using primers manufactured according to the markers, the blood samples were processed for polymerase chain reaction and the PCR products were treated for electrophoresis. The three puppies showed identical band to that of recipient, consequently, it was concluded that the puppies were offspring of the recipient mated naturally by the male dog, not the offspring by embryo transfer.

• 식물조직배양학회지
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• 제26권4호
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• pp.229-233
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• 1999

본 연구에서는 낙엽송 배발생조직의 장기저장을 위한 가능성에 대하여 조사하였다. 0.4M혹은 20% PEG로 24시간 전 처리하여 -0.33$^{\circ}C$/min의 동결온도 하강율로 초저온시켰을 때 높은 조직생중량을 보인 반면 -0.5 혹은 -1.$0^{\circ}C$/min의 경우 초저온 후 조직의 생중량이 저조한 것으로 나타났다. 1, 7, 및 28일간 초저온보존된 조직을 회수하여 재생장시킨 다음 PCR을 이용하여 변이분석을 한 결과 초저온보존 기간에 관계없이 DNA변이는 전혀 나타나지 않았다. 초저온보존된 배발생 조직으로부터 체세포배 유도 및 식물체 재분화를 유도할 수 `있었다.

• 한국수정란이식학회지
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• 제23권3호
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• pp.223-227
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• 2008

The cryopreservation of Hanwoo embryos has become an integral part of assisted reproduction in animal. The objective of this study was to assess the effect of The objectives of this study were: (1) to evaluate the influence of bovine embryo developmental stage on in vitro embryo development after freezing, (2) to study the efficiency compared with conventional freezed embryos at different embryo source. For conventional slow-freezing, day 7 or 8 expanded blastocysts were collected. The standard freezing medium was 1.8 M ethylene glycol (EG). Embryos were equilibrated in 1.8 Methylene glycol(EG) with 0.1 M sucrose in Dulbecco's phosphate-buffered saline (D-PBS) supplemented with 0.5% bovine serum albumin. Embryos were then loaded individually into 0.25 ml-straw and placed directly into cooling chamber of programmable freezer precooled to $-7^{\circ}C$, after 2 min, the straw was seeded, maintained at $-7^{\circ}C$ for 8 min, and then cooled to $-35^{\circ}C$ at $0.3^{\circ}C$/min, plunged and stored in liquid nitrogen for at least 3 days. For thawing, the straw containing embryos were warmed in air for 10 see and exposed to $37^{\circ}C$ water for 20 sec. Straws were then removed from $37^{\circ}C$ water. Rates of blastocyst survive and hatched were evaluated at 12 to 48h post-warming. The re-expansion and hatched rates of morula embryos were significantly lower than those obtained for blastocysts and expansion blastocysts (31.6%, 10.5% vs, 68.9%, 22.2% vs, 73.7%, 53.6%, respectively). No differences in re-expansion rates were found between in vivo and in vitro blastocysts. whereas hatched rates was significantly higher (51.2%) in vivo compared with in vitro embryos (18.6%). in conclusion, demonstrate that conventional freezing can be used successfully in cryopreservation of in vitro and in vivo bovine embryos, and that it might be considered for use in commercial programs and embryo preservation.

• 한국수정란이식학회지
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• 제29권1호
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• pp.13-19
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• 2014

This study was carried out to study the survival rate of thawed Hanwoo embryos frozen by the slow-rate freezing or the cryotop vitrification method. Hanwoo cumulus-oocyte complexes were recovered from ovaries at a slaughter house, matured for 20~22 hours, fertilized with Hanwoo semen for 5~6 hours, and cultured for 7~9 days in $38.5^{\circ}C$, 5% $CO_2$ incubator. For freezing, Day 7~9 blastocysts were collected. Embryos for the slow-rate freezing were equilibrated in 1.8 M ethylene glycol (EG) with Dulbecco's phosphate-buffered saline (D-PBS). Programmable cell freezer was precooled down to $-7^{\circ}C$, and the straw was seeded during 8 minutes-holding time, and was cooled to $-35^{\circ}C$ at the cooling rate of $0.3^{\circ}C/min$, and then was plunged and stored in liquid nitrogen. Embryos for the cryotop vitrification were treated in TCM199 with 0.5 M sucrose, 16% EG, 16% dimethylsulfoxide (DMSO). Embryos were then loaded individually onto cryotop and plunged directly into liquid nitrogen. The survival rates of embryos frozen by these two freezing methods were evaluated at 12 to 24h post-thawing. The survival rates of frozen/thawed Hanwoo embryos by the cryotop vitrification method ($56.86{\pm}26.53%$) were slightly higher than those by the slow-rate freezing method ($55.07{\pm}26.43%$) with no significant difference. Using the cryotop vitrification and the slow-rate freezing of Hanwoo blastocysts on Day 7 following in-vitro fertilization (IVF) treatment, the survival rates of frozen/thawed Hanwoo embryos were $72.65{\pm}18.3%$ and $79.06{\pm}17.8%$, respectively. The survival rates by the cryotop vitrification were higher than those by the slow-rate freezing on both Day 8 and 9 with significantly higher survival rate on Day 9 (p<0.05). Using the cryotop vitrification and the slow-rate freezing of Hanwoo embryos to compare between three different blastocyst stages, the survival rates of the blastocyst stage embryos were $66.22{\pm}18.8%$ and $45.76{\pm}12.8%$, respectively with higher survival rate by the vitrification method (p<0.05). And the survival rate of expanded blastocysts was higher than those of early blastocysts and blastocysts in two freezing methods with significantly higher survival rate by the slow-rate freezing method (p<0.05).

• Journal of Animal Science and Technology
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• 제48권6호
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• pp.797-804
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• 2006

본 연구에서는 동결 보호제 EG l에 sucrose 첨가 농도에 따른 생존성의 실험의 결과를 요약하면 다음과 같다. 1.5 M EG와 1.8 M EG 만을 이용하여 동결융해 후 생존서의 조사한 결과 71.1%와 70.2%로 각각 나타났다. 총세포수에 있어서도 127±1.3개와 124±1.6개로 생존율과 총세포수에 있어서도 두 그룹간에는 유의적인 차이를 보이지 않았다. 1.5 M EG와 1.8 M EG에 0.1 M sucrose를 각각 첨가한 후 동결 보존하여 융해 하였을 때 생존율과 총세포수 조사 결과는 1.5 M EG에 0.1 M sucrose 처리구가 73.6% 그리고 1.8 M EG 에 0.1 M sucrose 첨가군은 76.9%의 결과를 보였으며 총세포수 에 있어서도 118±1.2 와 112±1.2 개의 결과를 보여 생존성과 총세포수에 있어서도 두 처리군 모두 유의적인 차이를 나타내지 않았으나 1.5 M EG 처리구에서 총세포수는 다소 높은 경향을 보였다. 1.5 M EG 와 1.8 M EG에 0.3 M sucrose를 첨가하여 각각 생존성과 총세포수 조사 결과는 70.8%와 88.7%의 생존율을 나타내어 1.8 M EG 에 0.3 M sucrose 처리구가 유의적으로(P<0.05) 높은 결과를 보였다. 따라서 소 체외수정란을 conventional slow-freezing 방법으로 동결 보존할 경우는 1.8 M EG 동결보호제에 0.3 M sucrose를 병행하여 사용하는 방법이 수정란을 최상의 상태로 유지할 수가 있어 수정란이식에 적용할 경우 효과적일 것으로 사료된다.

• 한국수정란이식학회지
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• 제18권1호
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• pp.43-50
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• 2003

정자의 동결보존을 위한 새로운 기술개발 목적은 동결과정에서 최소한의 손상으로, 응해 후 최대한 높은 활력도의 정자를 얻는 것이다 정자가 난자와 수정하기 위해서는 적당한 생존성과 운동성을 유지해야 하는데, 가장 일반적인 방법으로는 정자의 진진 운동성과 첨체의 정상 여부 및 형태 검사방법 등이 있다 본 연구는 사람 정액을 동결보존 할 때 semi-programmable freezer를 이용한 완만동결 방법과, 액체질소의 vapor를 이용한 급속동결 방법이 응해 후 정자의 운동양상과 생존율 및 형태에 미치는 영향을 알아보기 위해 실시하였다. 동결-응해 후 정자의 MOT, VCL, VSL, VAP는 각각 급속 동결방법에서 47.40$\pm$20.06%, 38.12$\pm$15.58 $\mu$m/s, 28.19$\pm$14.10 $\mu$m/s, 33.64$\pm$15.15 $\mu$m/s로 완만 동결방법인 43.39$\pm$18.79%, 33.91$\pm$13.50 $\mu$m/s, 19.98$\pm$10.88 $\mu$m/s, 24.60$\pm$11.72 $\mu$m/s보다 유의적으로 높았으나(p<0.05), LIN은 완만동결 방법이 34.64$\pm$11.36으로 급속동결 방법인 28.83$\pm$10.35보다 더 좋은 성적을 나타내었다(p<0.05). 고 활력정자를 나타내는 HYP 역시 급속동결 방법에서 2.77$\pm$2.71%로 완만동결 방법의 1.33$\pm$1.57%보다 유의적으로 높게 나타났다(p<0.05). 그러나 정자의 운동양태를 나타내는 MAD, WOB, DNC, DNM에 있어서는 완만동결 방법이 급속동결 방법에 비해 조금 더 좋은 성적을 보였으나 유의적인 차이는 없었다. 동결-응해 후 정자의 생존율 및 정상형태의 정자는 완만동결 방법이 각각 62$\pm$2.1%, 44$\pm$8.3%, 급속동결 방법은 60$\pm$2.2%, 46$\pm$7.7%로 유의적인 차이가 없이 비슷한 결과를 보였다. 따라서 사람 정액의 동결 보존 시 많은 시간과 고가의 장비가 필요한 완만동결 방법보다는 짧을시간동안 액체 질소만으로 간단히 시행할 수 있는 급속동결 방법이 더 효과적이라고 사료된다.