• Korean Journal of Animal Reproduction
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• v.20 no.2
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• pp.155-161
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• 1996

For a large sclase production of genetically identical or cloned animals, the effect of cryopreservation by vitrification on the post-thaw viability of nuclear transplant rabbit embryos were investigated. The embryos of 16-cell stage were collected from the mated does at 48 hours post-hCG injection, and they were synchronized to G1 phase of 32-cell stage were injected into enucleated recipient cytoplasms by micromanipulation. After culture until 20h post-hCG injection, the nuclear transplant oocytes were electrofused and activated by electrical stimulation. After in vitro culture for 48h, the nuclear transplant embryos developed to morula stage were cryoperserved with EFS solution by vitrification method. The forzen nuclear transplant embryos were thawed and cultured for 72h and the nuclear transplant of blastomeres under a fluorescence microscopy. The in vitro development to blastocyst of intact-fresh and intact-frozen 16-cell embryos was found to be 96.9 and 63.9%, respectively. The in vitro development to blastocyst of nuclear transplant and frozen-thawed nuclear transplant embryos was found to be 74.5 and 42.9%, respectively. Also, their mean blastomere numbers and mean cell cycles/day was 153 and 105, 145 and 1.34, respectively. From the above results it was concluded that the present cryopreservation by vitrification of nuclear transplant rabbit embryos might be useful though was decreased significantly.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.7-7
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• 2001

Cryopreservation by vitrification of Hanwoo blastocysts derived from nuclear transfer with Hanwoo adult ear cell was compared with that of in vitro fertilized blastocysts. For vitrification, day 7 or day 8 blastocysts were serially exposed in glycerol (G) or/and ethylene glycol (EG) mixtures (10% G for 5 min, 10% G plus 20% EG for 5 min, and 25% G plus 25% EG for 30 sec) which was diluted in 10% FBS added D-PBS. Thawing of straw was carried out in air for 10 sec and then in water bath of $25^{\circ}C$ for 20 sec. The contents of the straw (0.2 ml) was expelled into a culture dish contained with 0.5M sucrose and 10% FBS(S-DPBS) by cutting the cotton plug and then the recovered embryos were put into fresh 0.25M and 0.125M S-DPBS for 30 sec, respectively, The embryos were transferred into D-PBS with 10% FBS for 5 min. and were cultured in a 10u1 droplet of co-culture environment (cumulus cell monolayer + 10% added CRl medium) for 24 h. In the result, survival rates were 88.9% and 85.4% for nuclear transfer embryos and in vitro fertilized embryos, respectively. After transfer of vitrified-thawed blastocysts produced from nuclear transfer, 4 of 5 total recipients did not return to the subsequent estrus cycle at 30 days. It is concluded that the Hanwoo blastocysts derived from nuclear transfer can be successfully cryopreserved using simple and efficient vitrification method.

• Journal of Embryo Transfer
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• v.16 no.3
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• pp.245-250
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• 2001

This study was performed to evaluate whether vitrification method using ethyle glycol and eletron microscopic (EM) grid could be used far the cryopreservation of human oocytes in ART program. Surplus oocytes were obtained from consented IVF patients. These surplus human oocytes were frozen with our vitrification method, Oocytes were exposed to 1.5M ethylene glycol (EG) in DPBS far 2,5 minutes, followed by 5.5M EG plus 1.0M Sucrose in DPBS for 20 seconds. Then oocytes were transferred onto the EM grid and the grid was plunged into LN2 for storage. For thawing, oocytes containing EM grid were sequentially transferred in 1.0M, 0.5M, 0.25M, 0.125M and 0 M sucrose in DPBS solution at the intervals of 2.5 minutes. Thawed and survived oocytes were provided for ICSI. Embryos from vitrified oocytes were transferred to uterus of the patient on 4 to 5 days after ovulation in natural cycles of on 15 to 17 day of hormone replacement cycles. A total of 370 oocytes from 26 patients were thawed and 159 (43.0%) of them survived. One hundred thirty four oocytes (84.3%) were fertilized normally and 126 pre-embryos were transferred to 26 patients, resulting in 5 clinical pregnancies. The pregnancy rate per transfer was 19.2% and implantation rate was 4.0%. Among the five pregnant, 4 patients delivered 4 healthy babies and the one patient was 32-week ongoing pregnancy. From this results, vitrification using ethylene glycol as cryoprotectant and EM grid is a rapid and simple method that can be effectively applied for the cryopreservation of human oocytes in ART program.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2019.04a
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• pp.81-81
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• 2019

Ginseng seeds are one of short-lived seeds species which loose their viability easily in the condition of conventional storage. Cryopreservation using liquid nitrogen (LN) has been recommended as a alternative storage for this kind of germplasm short lived or dessiccation-sensitive. This study was performed to find out whether cryopreservation could affect physiological change such as enzyme activity induced by reactive oxygen species. In this work, the redox ratio of ascorbate and glutathione were examined onto ginseng seedlings before and after LN storage of seeds for 1 day using spectrophotometer method. Reduced ascorbate (ASA) was increased while oxidized ascorbate (DHA) was decreased slightly for both after 1d-LN storage. And for glutathione also, reduced form (GSH) was increased while oxidized form (GSSG) was decreased slightly for both after 1d-LN storage. Consequently total phenol compound and ion leakage after LN storage showed no significant differences. Additionally root growth from the seeds after LN storage was not affected by ultra low temperature. From the above results, we may suggest that cryopreservation could be recommended for storage tool of ginseng seeds even with low viability also and expected to make slower seed aging process during preservation period through further study.

• Journal of Embryo Transfer
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• v.14 no.3
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• pp.163-169
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• 1999

This study was carried out to investigate the effect of co-culture system(bovine oviduct epithelial cells; BOEC) and defined culture system(modified TALP ; mTALP) on the development of IVM-IVF embryos, and survival of in vitro produced blastocysts after freezing and thawing. Occytes from the slaugheterhous ovaries were matured and fertilized using general protocol. The results obtained were as the following: 1. Survival rates of frozen-thawed blastocysts using 10% glycerol as cryoprotectant was higher in day 7 blastocysts than in Day 8 and 9 blastocysts from co-cultrue system, but survival rate of frozen-thawed blastocysts was higher in Day 10 blastocysts than in day 8 and 9 blastocysts from defined culture system. Regardless of their age, survival rate of frozen-thawed blastocysts was significantly higher (p<0.05) in co-culture system than in defined culture system. 2. The cell number of blastocysts was significanlty higher (p<0.05) in Day 7 blasotcysts than in Day 8 and 9 blastocysts from co-cultures, but the cell number of blsstocysts was significantly higher (p<0.05) in Day 10 blastocysts than in Day 8 and 9 blastocysts from defined culture system. Regardless of the culture system, blastocysts with higher cell number showed higher survival rates after freezing and thawing.

• Korean Journal of Plant Tissue Culture
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• v.26 no.4
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• pp.229-233
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• 1999

The possibility for long-term preservation of Larix leptolepis embryogenic tissue was tested in this study. Higher relative increase of the tissue fresh weight was observed when embryogenic tissue was pretreated for 24 hrs in a medium containing 0.4 M sorbitol or 20% polyethyleneglycol with cooling rate of -0.33$^{\circ}C$/min. The fast cooling rate of -0.5$^{\circ}C$ and -1.$0^{\circ}C$/min appeared to be less effective in regrowth of tissues from cryopreservation. No DNA variants have been observed by PCR analysis among the embryogenic tissues recovered after 1-, 7-, and 28-day-cryopreservation. The post-thaw embryogenic tissue gave rise to mature somatic embryos which developed into plants.

• Reproductive and Developmental Biology
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• v.32 no.2
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• pp.111-115
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• 2008

The aim of this study was to examine more effective cryoprotectant for the cryopreservation of mouse preantral follicles. Enzymetically isolated preantral follicles from 12-day-old mice were cryopreserved by a slow freezing protocol with 1.5 M propanediol (PROH), dimethyl sulphoxide (DMSO) or glycerol (GLY) and then grown and matured in vitro for 11 days after thawing. The survival of preantral follicles immediately after freezing and thawing was not different among the PROH (68.2%), DMSO (72.4%) and GLY (72.1%). After grown and matured in vitro, the rates of survival and metaphase II oocytes were 54.9% and 36.6% for PROH which was significantly higher rates (p<0.05) compared with the rates obtained from DMSO (16.9% and 9.0%) and GLY (16.3% and 7.5%). The diameter of metaphase II oocytes from pre antral follicles frozen in PROH ($67.4{\pm}1.8\;{\mu}m$) was significantly (p<0.05) smaller than that of the fresh preantral follicles ($69.1{\pm}2.3\;{\mu}m$). The results from the present study revealed that PROH is more suitable cryoprotectant for the cryopreservation of mouse preantral follicles.

• Korean Journal of Animal Reproduction
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• v.19 no.1
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• pp.43-48
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• 1995

The purpose of this study was to examine the suvival rates of cryopreserved porcine embryos collected on Day 6 (Day 0=onset of estrus) with various cryprotectants. Eighty two embryos at different stages, ranged from expanded blastocyst to hatched blastocyst, were allocated to 6 experimental groups in different combinations of cryoprotectants glycerol, egg yolk and/or trehalose. Porcine embryos were cryopreserved using conventinal slow freezing precedures. The embryos were equilibrated with one of the freezing solutions, cooled from 25 to -7$^{\circ}C$ at 1$^{\circ}C$/min, seeded at -7$^{\circ}C$ frozen to -36$^{\circ}C$ at 0.5$^{\circ}C$/min, and then plunged into liquid nitrogen. The frozen embryos were thawed by immersion in 37$^{\circ}C$ water and the cryoprotectants were removed by dilution with 0.5M sucrose solution. Embryonic survival was estimated from the normal development of embryos for 12, 24 and 48hrs culture. Then the embryos were stained and their cell nuclei was counted. The survival rates of morphological embryos were significantly higher in group I (10% glycerol) or group IV (10% glycerol＋10% egg yolk＋0.5M trehalose) than those in other groups, although the nuclei number was quite higher in embryos treated with 10% glycerol and 0.25M trehalose at expended blastocyst. However, hatched blastocysts showed higher viability and nuclei number treated with either egg york or trehalose, but the survival rates after 48hrs of culture were quite low. These results indicate that egg yolk and trehalos as a supplement to freezing solutions can be useful to the cryopreservation ofn porcine embryos.

• Journal of Plant Biotechnology
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• v.44 no.4
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• pp.478-483
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• 2017

The expression profiles of low-temperature-related genes were examined in in vitro potatoes exposed to a cold condition for 1-3 days. The expression levels of PPI1 and CI21B genes were lineraly elevated from day 1 to day 3, while the opposite trend was observed for CBF4 and CI21A. In addition, the expression of the genes CI21A and CI21B varied, along with specific tissues (leaf, stem, and tuber) and the treatment periods. Therefore, potato shoot tips were cryopreserved with LS and PVS2 containing different oncentrations of AFP. It can thus be inferred that the presence of AFP in LS and PVS2 was likely to elevate expression pattern of the genes. Furthermore, the concentration of AFP (1,500 ng/ml for LS and 500 ng/mg for PVS2) was the best for the cryopreservation of potatoes.

• Journal of Embryo Transfer
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• v.23 no.3
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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.