• 대한수의학회지
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• 제38권2호
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• pp.394-400
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• 1998

Cryopreservation of embryos by vitrification is a simple method to preserve bovine embryos for subsequent embryo transfer, but embryonic viability after vitrification has been inconsistent and low compared with conventional slow freezing. The aim of the present study is to examine the effect of serum or serum albumin in a vitrification solution and epidermal growth factor(EGF) or fibroblast growth factor(FGF) on in vitro viability of bovine blastocysts frozen by vitrification. Bovine blastocysts were produced by in vitro maturation, fertilization of follicular oocytes and culture of embryos in a synthetic oviduct fluid medium(SOFM) containing BSA and 19 essential and nonessential amino acids. Blastocysts with excellent or good morphology were selected at 7 or 8 days after culture and utilized for vitrification. In experiment 1, blastocysts were vitrified in a solution containing semi-fetal calf serum(SFCS) or BSA(5 or 10mg/ml) and then their subsequent viabilities were examined by culturing thawed embryos in a SOFM containing BSA and 19 amino acids. Effect of EGF or FGF added to a SOFM containing polyvinyl alcohol(PVA) on the viability of vitrified-thawed blastocysts was investigated in experiment 2. BSA added at 5 or 10mg/ml to a vitrification solution showed significantly higher(p < 0.05) developmental rate to expanded and hatching blastocysts than SFCS, but there was no significant difference in the developmental rate to hatched blastocysts after thawing. Supplementation of a culture medium with EGF and/or FGF significantly increased(p < 0.05) embryo development to expanded blastocysts compared with control but showed no beneficial effect on the development to hatching or hatched blastocysts. Coculture of thawed embryos with granulosa cells in a TCM 199 containing 10% fetal calf serum(FCS) showed the highest developmental rate to expanded, hatching and hatched blastocysts among the groups tested. In conclusion, supplementation of a vitrification solution with BSA at 5mg/ml and culture of thawed blastocysts in a medium containing EGF and/or FGF can improve in vitro viability of bovine blastocysts frozen by vitrification.

• 한국가축번식학회지
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• 제15권1호
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• pp.49-57
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• 1991

생쥐 수정란의 동결보존법을 개선하고자 유리화 동결과정에서 제1보존액에서의 평형시간, 제2보존액에서의 전냉각 여부 및 straw loading 방법에 따른 동결보존 후 수정란의 생존율을 조사하고, 또한 유리화 동결보존 과정에서 수정란의 부분적인 손상 여부를 확인하기 위하여 동결보존 후 배양하여 수정란의 할구수를 Hoechst 33342로 염색하여 조사하였던 바 그 결과는 다음과 같다. 1. 동결융해 후 생존율은 Medium-1에서 10분간 평형시간을 준 경우(81.0%)는 5분간(40.0%) 및 15분간(74.1%)의 평형시간을 준 경우보다 유의적(P<0.05)으로 높았다. 2. Double Medium-2 column방법으로 얻은 생존율(81.0%)은 single Medium-2 column방법으로 얻은 생존율(62.8%)보다 유의적(P<0.01)으로 높았다. 그리고 Double Medium-2 column방법에서는 유리화 용액이 희석액에 오염되지 않아 순수하게 투명한 유리화를 이룰 수 있었다. 3. 전냉각을 않은 경우에 비하여 전냉각한 Medium-2로 동결한 수정란은 생존율에 있어서 다소 높은 성적을 보였으나 유의적인 효과는 없었다. 4. 상실배기의 수정란을 24~28시간동안 배양하여 얻은 후기 배반포를 Hoechst 33342로 염색하여 할구수를 조사하였던 바 신선란(53.3${\pm}$1.6)보다 동결란(41.4${\pm}$1.5)에서 유의적(P<0.05)으로할구수가 적었다. 이는 동결융해 과정에서의 부분적인 손상에 의하였던 것으로 사료된다.

• 한국자원식물학회지
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• 제29권6호
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• pp.635-641
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• 2016

Sweet potato (Ipomoea batatas L.) shoot tips grown in vitro were successfully cryopreserved by encapsulation-vitrification. Encapsulated explants are very easily manipulated, due to the relatively large size of the alginate beads, and a large number of samples can be treated simultaneously. In this study, the effects of sucrose preculture, cryoprotectant preculture, and post-warm recovery media on regrowth, following liquid nitrogen (LN) exposure, were investigated to establish an efficient encapsulation-vitrification protocol for sweet potato. Shoot tips of plants grown in vitro were precultured in 0.3 M sucrose for 2 d before encapsulation. Encapsulated shoot tips were pre-incubated in liquid MS (Murashige and Skoog) medium containing 0.5 M sucrose for 16 h, before preculturing in sucrose-enriched medium (0.7 M sucrose) for 8 h. Shoot tips were osmoprotected with 35% plant vitrification solution 3 (PVS3) for 3 h, before being dehydrated with PVS3 for 2 h at $25^{\circ}C$. The encapsulated and dehydrated shoot tips were transferred to 2 mL cryotubes, suspended in 0.5 mL PVS3, and plunged directly into liquid N. High levels of shoot formation were obtained for the cv. Yeulmi (65.7%) and Yeonwhangmi (80.3%). The regrowth rates of cryopreserved samples in Yeulmi (78.9%) and Yeonwhangmi (91.3%), following culture on ammonium-free MS medium for 5 d, were much higher than those cultured on standard MS medium (65.7% and 80.3%, respectively). This encapsulation-vitrification is a promising method for the long-term preservation of sweet potato.

• Reproductive and Developmental Biology
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• 제31권2호
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• pp.103-108
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• 2007

This study was carried out to investigate the effects of cryoprotectants, warming solution and removal of lipid on open pulled straw vitrification (OPS) method of porcine embryos produced by nuclear transfer (NT) of fetal fibroblasts. All solutions used during vitrification were prepared with holding medium consisting of 25 mM Hepes buffered TCM199 medium containing 20% fetal bovine serum (FBS) at $38.5^{\circ}C$. The blastocysts derived from NT with or without lipid were vitrified in each medium of different concentrations of dimethyl sulfoxide (DMSO) and ethylene glycol (EG). Also, blastocysts after cryopreservation were warmed into different concentrations of sucrose in warming solution. The optimal concentrations of cryoprotectants in vitrification solution were 10% DMSO + 10% EG in vitrification solution 1 (VS1) and 20% DMSO + 20% EG in vitrification solution 2 (VS2). The optimal concentrations of sucrose were 0.3 M sucrose in warming solution 1 (WS1) and 0.15 M sucrose in warming solution 2 (WS2). lipid removal from oocytes before NT enhanced the viability of NT embryos after vitrification. Our results show that use of the OPS method in conjunction with lipid removal provides effective cryopreservation of porcine nuclear transfer embryos.

• 한국자원식물학회지
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• 제19권3호
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• pp.385-391
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• 2006

In vitro-grown axillary buds of Melia aredarach were successfully cryopreserved by vitrification. On the MS medium supplemented with BA 1 mg/L, multiple shoots were developed within $4{\sim}5$ weeks. Plantlets of Melia azedarach were cold-hardened at $10^{\circ}C$ for a 16-hr photo-period for 6 weeks. Excised axillary shoot-tips from hardened plantlets were precultured on a solidified Murashige & Skoog agar medium (MS) supplemented with 0.7 M sucrose for 1 day at $25^{\circ}C$. Axillary shoot-tip meristems wert dehydrated using a highly concentrated vitrification solution (PVS2) for 60 min at $0^{\circ}C$ prior to a direct plunge into liquid nitrogen (LN). The PVS2 vitrification solution consisted of 30% glycerol (w/v), 15% ethylene glycol (w/v), 15% DMSO (w/v) in MS medium containing 0.4M sucrose. After short-term warming in a water bath at $40^{\circ}C$, the meristems were transferred into 2 ml of MS medium containing 1.2M sucrose for 15 min and then planted on solidified MS culture medium. Successfully vitrified and warmed meristems resumed growth within 2 weeks and directly developed shoots without intermediary callus formation. The survival rate of cold-hardened plantlets for 3 and 4 weeks was 90%. We did not find any difference in PCR-band patterns between control and cryopreserved plants. This method appears to be a promising technique for cryopreserving axillary shoot-tips from in vitro-grown plantlets of Medicinal plants.

• Clinical and Experimental Reproductive Medicine
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• 제32권2호
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• pp.177-185
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• 2005

Objective: This study was conducted to find an optimal condition for the vitrification of mouse morulae and expanded blastocysts. Materials and Methods: Mouse embryos were obtained at 2-cell stage and cultured to morula and expanded blastocyst stage in Human Tubal Fluid (HTF) medium supplemented with 10% Serum Substitute Supplement (SSS). The vitrification solutions used were EFS30, EFS35 and EFS40 that contains 30%, 35% and 40% ethylene glycol, respectively, with 18% ficoll and 0.5 M sucrose diluted in Dulbecco's phosphate-buffered saline (DPBS) medium supplemented with 10% SSS. The vitrification procedure was performed in EFS solution with three steps, followed by thawing in 6 steps with 0.5 M sucrose, and then survival and hatching-hatched rate per embryos recovered were compared among six groups. Results: After 24 h culture in different vitrification and thawing solution, the survival rate of morula embryos was 94.1%, 85.4% and 59.7% for EFS30, EFS35 and EFS40 group, respectively. Hatching rate of morula embryos after 72 h culture was 30.6%, 25% and 11.3% for EFS30, EFS35 and EFS40 group, respectively. The survival rate of expanded blastocyst embryos after 24 h culture was 90.4%, 98.5% and 100% for EFS30, EFS35 and EFS40 group, respectively. Hatching rate of expanded blastocyst embryos after 48 h culture was 46.2%, 57.6% and 64.3% for EFS30, EFS35 and EFS40 group, respectively. Conclusion: The EFS30 solution was the best for vitrification of mouse morulae. The EFS40 solution was the best for vitrification of mouse expanded blastocysts. The mouse expanded blastocyst was better than mouse morula for vitrification of mouse embryos.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.134-134
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• 2003

Bovine embryos produced by in vitro maturation, feretilization and development was examined for presevation and transfer. The fertilization medium used BO medium with 5 mM/$m\ell$ caffeine and 10$\mu\textrm{g}$/$m\ell$ heparin and adjusted to a pH of 7.2 to 7.4. The final concentration of spermatozoa was adjusted to 1$\times$$10^{6}$ cells/$m\ell$ motile sperm during fertilization in vitro. At 8~10 hrs after insemination, the oocytes were transferred into CR1aa medium and cultured for 7 days. Embryos were preserved by vitrification method for transfer. When the embryos of early, blastocyst and expanded blastocyst stages were frozen-thawed, the proportions of embryos with normal morphology 83.6, 88.1 and 85.2%. (중략)

• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2021년도 춘계학술대회
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• pp.36-36
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• 2021

Cryopreservation has been broadly used as an efficient method for a long-term conservation for many types of plants especially vegetatively propagated plants. Among several cryopreservation methods, a droplet-vitrification was the most widely applicable and efficient method. Studies have developed protocols for strawberry using droplet-vitrification method and suggested the practical use of the protocol for large number of germplasm with a little modification. In this study, the droplet vitrification method of shoot tip has been tested on 31 accessions provided around the world. Shoot tips were precultured on Murashige and Skoog (MS) liquid medium supplemented with 0.3~0.5M sucrose. Precultured explants were osmoprotected with loading solution, 35% of PVS3 (C4, 17.5% glycerol and 17.5% sucrose) for 40 min and exposed to dehydration solution, PVS3 (B1, 50% glycerol and 50% sucrose) for 60 min. Then, the explants were transferred onto droplets containing 2.5 uL PVS3 on sterilized aluminum foils prior to direct immersion in liquid nitrogen (LN) for 1hr. The cryopreserved shoot tips were rapidly warmed in a water bath at 40C and then unloaded in MS with 0.8M sucrose for 40 min. The shoot tips were cultured in NH₄NO₃-free MS post culture medium for 2 weeks. Subsequently, the explants were moved to the MS medium for 6 weeks and evaluated the regrowth rate. By this droplet-vitrification protocol, twenty-four accessions showed at least 40% regrowth rate. Out of 24 accessions, 'Nonsan1ho' had the highest regeneration rate of 85.8% and 'Jumbo pureberry' had the lowest with 42.1%.

• 한국자원식물학회지
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• 제36권6호
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• pp.571-579
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• 2023

In this study, cryopreservation by droplet-vitrification was applied to pear (Pyrus spp.) germplasm. We focused on the development and assessment of various strategies for the selection of suitable tissue, osmoprotection, and dehydration. We also evaluated post-thaw recovery of cryopreserved explants by droplet-vitrification. Preferentially, we tested the effects of preculture and loading treatments to determine which tissues were more suitable, either the apical shoot tips or the axillary buds. Apical shoot tips showed the better regrowth rate than in vitro axillary buds. The most effective techniques for cryopreservation were as follows. Shoots from in vitro seedlings which had been cultured for about 5-6 weeks were cold-hardened at 4℃ for one week, excised shoot tips were precultured on liquid MS medium including 0.3 M sucrose for 31 hours and 0.7 M sucrose for 17 hours, osmoprotected in loading solution (LS) for 40 min, and then cryoprotected in dehydration solution (PVS3) for 90 min. In addition, we found that regrowth rates of explants on regrowth medium after exposure to liquid nitrogen (LN) were higher than those on MS medium. Results indicated that the highest regrowth percentage was 95.6% for 'Bartlett' cultivar and 68.9% for 'BaeYun No.3' cultivar. Consequently, apical shoot tips of two pear cultivars, 'Bartlett' (P. communis) and 'BaeYun No.3' (P. pyrifolia), were successfully cryopreserved by droplet-vitrification. Results of this study show that the enhanced droplet-vitrification method described in the present study could be used as an effective means for long-term storage of pear genetic resources.

• 한국수정란이식학회지
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• 제23권4호
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• pp.269-274
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• 2008

The purpose of this study was to determine the effects of Taxol pre-treatment to in vitro matured bovine oocytes, and sucrose and trehalose added to vitrification solution on spindle morphology and embryonic development following cryopreservation. Bovine oocytes were collected from ovaries and matured in tissue culture medium 199 (TCM 199) supplemented with 10% Fetal Bovine Serum (FBS), 0.05ng/ml epidermal growth factor, 0.01 IU/ml luteinizing hormone and $1{\mu}g/ml$ estradiol for 22h in $39^{\circ}C$, 5% $CO_2$, TCM 199-HEPES containing 20% FBS was used as basic medium (BM) to prepare vitrification solution. Oocytes were pre-treated with $1\;{\mu}M$ Taxol in maturation medium for 15 min prior to vitrification. Oocytes were exposed to 1.6 M ethylene glycol (EG) and 1.3M dimethyl sulfoxide (DMSO) in BM and then were exposed to 3.2 M EG, 2.6 M DMSO and 0.5 M sucrose in BM or 3.2 M EG, 2.6 M DMSO and 0.5 M trehalose in BM. Oocytes with cumulus cells and oocytes without cumulus cells were considered as control 1 and control 2, respectively and held in TCM 199-HEPES at $39^{\circ}C$. Oocytes were frozen using modified solid surface vitrification and were stored in cryotubes in liquid nitrogen for more than 1 week. Frozen oocytes were thawed in TCM 199-HEPES containing 0.5 M, 0.25 M and 0.1 M sucrose in BM for 2 min, respectively or 0.5 M, 0.25 M and 0.1 M trehalose in BM for 2 min, respectively. Immunoflurorescence staining of oocytes was performed to assess spindle morphology and chromosome configuration of oocytes. The rates of cleavage and blastocyst were examined following in vitro fertilization. Normal spindle morphology rate of oocytes pre-treated with Taxol prior to vitrification was not higher than that of other vitrified groups. Taxol pre-treatment did not increase cleavage and blastocyst formation rates, although control groups showed significantly higher rates (p<0.05). Percentages of normal spindle and embryonic development were not significantly different among vitrified groups regardless of type of sugar. In conclusion, Taxol pre-treatment of oocytes before cryopreservation did not reduce the damage induced by vitrification and subsequently did not improve embryonic development following vitrification. Trehalose may be used as an alternative non-permeating cryoprotectant in vitrification solution.