• Korean Journal of Animal Reproduction
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• v.24 no.3
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• pp.225-230
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• 2000

This study was designed to investigate effect of cryoprotectant kinds and cell stages on the viability of bovine embryos cryopreserved by vitrification. The oocytes were collected from ovarian follicles of Korean native cows. The follicular oocytes were cultured in TCM-199 medium containing hormone and 10%(v/v) FCS for 24~48hrs in a incubator with 5% $CO_2$, in air at 38.5$^{\circ}C$ and then matured oocytes were again cultured for 7~10 hrs with spermatozoa capacitated by preincubation. The vitrification solutions of EFS and EDS were consisted of 40%(v/v) ethylene glycol, 18%(v/v) Ficoll and 0.3M sucrose, and 20%(v/v) ethylene glycol, 16.5%(v/v) DMSO and 0.5M sucrose in TCM-199 medium supplemented with 10% FCS, respectively. The embryos were exposed to EFS or EDS at $25^{\circ}C$ and loaded into OPP straw for 30 sec. The plug end of each straws was heat-sealed and straws was slowly immersed into liquid nitrogen(L$N_2$). The results obtained were summarized as follows : 1 . The rates of cleavage and hatching of embryos frozen with vitrification, rapid and slow freezing methods were 67.5%, 27.5% and 42.5%, 20.0% and 52.5%, 25.0%, respectively And rates of cleavage and hatching of embryos frozen with vitrification method were significantly(p<0.05) higher than those in other methods, and the rates were lower than those in control group(82.5% and 37.5%). 2. The rates of cleavage and hatching of embryos were significantly(p<0.05) different between EFS(47.5% and 22.5%) and EPS(52.5% and 27.5%), and the rates were lower than those in control group(82.5% and 37.5%). 3. After vitrification freezing of bovine embryos at zygote, 2 cell, 8 cell, morulae and blastocyst stage, the rate of cleavage and hatching were 25.0% and 15.0%, 32.5% and 20.0%, 37.5% and 20.0%, 52.5%, 27.5%, 47.5% and 25.0%, respectively. And developmental rates to the expended blastocyst stage of embryos frozen at zygote stage was significantly(p<0.05) lower rather than those in 2, 8-cell and morulae stage.

• Korean Journal of Animal Reproduction
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• v.18 no.4
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• pp.251-256
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• 1995

Two experiments were conducted to study the production of in vitro fertilized bovine embryos and the viability of blastocysts cryopreserved by vitrification. In experiment 1, production rate of in vitro matured bovine oocytes after fertilization in medium containing bovine oviduct epithelial cells (BOEC), cumulus cells and granulosa cells to blastocysts were 18.4, 14.6 and 13.1%, respectively. Developmental percentages of blastocysts produced at day 6, 7 and 8 were 8.5, 10.6 and 15.2% respectively. Hatching rate of bovine embryos produced was 60.0%. In experiment 2, post-thawed surviving embryos in a vitrification solution consisting of 7.15M ethylene glycol, 2.5 mM ficoll and 0.3 M sucrose were 36.4% (56/154). Also, survival rate of bovine embryos after exposed to vitrification solution at 1, 2, 3, 4 and 5 min were 84.0, 88.0, 71.0, 48.0 and 24.0% respectively.

• Korean Journal of Plant Resources
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• v.31 no.6
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• pp.675-683
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• 2018

This study describes an efficient and widely applicable droplet-vitrification following cryopreservation for shoot tips of Chrysanthemum morifolium (Ramat.) cvs. 'Borami' and 'Yes morning'. The shoot tips of Chrysanthemum were precultured in Murashige and Skoog (MS) liquid medium supplemented with sucrose (0.3-0.7 M). Precultured explants were treated with loading solution (LS, C6) containing glycerol 20% and sucrose 20% for 30 min and exposed to dehydration solution (B5) containing 40% of glycerol and 40% of sucrose for 60 min at $25^{\circ}C$, and then transferred onto droplets containing $2.5{\mu}l$ PVS3 on sterilized aluminum foils ($4cm{\times}0.5cm$) prior to direct immersion in liquid nitrogen (LN) for 1 h. The highest regeneration rate (%) was obtained when shoot tips were precultured with treatment-2 (exposing of shoot tips to MS + 0.3M sucrose for 30 h and then treated with MS+0.5 M sucrose for 16 h) at $25^{\circ}C$ in both the cultivars. The viability of cooled samples, followed by culturing on $NH_4NO_3$-free MS medium for first 5 days was increased to two-fold (80.7%) regrowth rate over those cultured on normal MS medium or MS medium containing plant growth regulators. This result shows droplet-vitrification would be a promising method for cryobanking chrysanthemum germplasm.

• Journal of Embryo Transfer
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• v.17 no.2
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• pp.117-121
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• 2002

This study was carried out to investigate the in vitro fertilization rate of canine immature oocytes cryopreserved by vitrification freezing. The vitrification solutions of EPS and EDS were consisted of 40％ ethylene glycol 18％ Ficoll and 0.3M sucrose, and 20％ ethylene glycol, 16.5％ DMSO and 0.5M sucrose in TCM-199 medium supplemented with 10％ FCS, respectively. The oocytes were exposed The developmental rate of in vitro cultured oocytes recovered from ovaries collected at different stages of the reproductive cycle were 3.8％, 10.7％, 46％, respectively. to EFS or EDS at $25^{\circ}C$ and loaded into straw fer 30 sec. The straws was slowly immersed into L$N_2$. Fertilization and survival rate was defined as development rate on in vitro culture or FDA-test. 1. The fertilization rate after vitrification freezing of immature oocytes at 1, 6, 12 and 24 hrs after collection from ovaries was very low(5.3％～31.4％) than the unfrozen oocyte(60.0％). And the fertilization rate after vitrification freezing of immature oocytes was very higher than that of mature oocytes. 2. The survival rate after vitrification freezing of immature oocytes at 1, 6, 12 and 24 hrs after collection from ovaries was 55.0％, 40.0％, 28.6％ and 17.1％, respectively. And the survival rate after vitrification freezing of immature oocytes was slightly higher than that of mature oocytes.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2010.05a
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• pp.12-12
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• 2010

We developed droplet-vitrification protocol, a combination of droplet-freezing and solution-based vitrification, and applied to germplasm collections of garlic, potato, lily as well as cell lines, including hairy roots, somatic embryos. To establish a garlic cryobank, four Korean garlic field collections at Danyang, Suwon, Mokpo and Namhae were cryopreserved last five years. The protocol applied consisted of preculture for 3-4 days at $10^{\circ}C$ on solid MS medium with 0.3M sucrose, loading for 40 min in liquid medium with 35% PVS3, dehydration with PVS3 for 150 min, cooling in $5{\mu}l$ droplets of PVS3 placed on aluminum foil strips by dipping these strips in liquid nitrogen, warming them by plunging the foil strips into pre-heated($40^{\circ}C$) 0.8M sucrose solution for 30s. A total of over 900 accessions of garlic were stored in liquid nitrogen for long-term conservation using unripe inflorescences, cloves or bulbils. Twelve alternative plant vitrification solutions were designed by modifying cryoprotectant concentrations from the original PVS2 and PVS3. The results suggest that PVS2-based vitrification solutions with increased glycerol and sucrose and/or decreased DMSO and EG concentrations can be applied for medium size explants which are tolerant to chemical toxicity and moderately sensitive to osmotic stress. PVS3 and variants can be used widely when samples are heterogeneous, of large size and/or very sensitive to chemical toxicity and tolerant to osmotic stress.

• Korean Journal of Agricultural Science
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• v.38 no.2
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• pp.227-233
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• 2011

This study aimed at developing cryopreservation protocol for chrysanthemum (Dendranthema grandiflora Tzelevcv. peak) shoot apices based on droplet-vitrification procedure, which is a combination of droplet-freezing and solution based vitrification. Progressive preculture of shoot apices in liquid MS medium supplemented with 0.3 and 0.7 M sucrose for 31 and 17 hours, respectively, was found optimum among preculture treatments tested. The composition of both loading and vitrification solutions significantly affected recovery growth of shoot tips before and after cryopreservation. Balancing glycerol and sucrose concentrations in the solutions was beneficial for recovery growth. The highest recovery after cryopreservation was observed when apical shoot tips were extracted from 4-week-old in vitro plantlets, progressively precultured with 0.3-0.5-0.7 M sucrose for 32-16-6 hours, respectively, then treated with loading solution comprising of 1.9 M glycerol + 0.5 M sucrose (35% PVS3 solution). Apices were then dehydrated with the vitrification solution consisted of 50% glycerol + 50% sucrose for 90 minutes then directly immersed in liquid nitrogen.

• Korean Journal of Plant Resources
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• v.19 no.6
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• pp.665-669
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• 2006

Somatic embryos do not survive at exposure to liquid nitrogen temperatures without cryoprotective treatments. A simplified technique which simultaneously induces and cryoprotects embryogenic calli using plant vitrification solution 2 (PVS2) followed by dehydration was developed for the cryopreservation of Soap berry genetic resources. Vitrification is a way of removing the moisture in vegetation through PVS2. The PVS2 vitrification solution consisted of 30% glycerol (w/v), 15% ethylene glycol (w/v), 15% Dimethylsulfoxide (w/v) in B5 medium containing 0.4M sucrose. Two tests were done. The one was to eliminate moisture at $0^{\circ}C$ and the other at $25^{\circ}C$. In both cases the best results came out at a vitrification time of $10{\sim}20$ minutes. It was also found that the survival rate was higher at $0^{\circ}C$ than at $25^{\circ}C$. In particular, the survival rate reached more than 80%. Water-damaged embryos turned brown and stoped growth, but energetic embryos took on a milky hue and show a very vigorous growth rate. Successful cryopreservation of somatic embryos of soapberry can be used to establish in vitro genebanks for long-term conservation of Soapberry genetic resources to complement field genebanks and other in vitro methods already being used.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.84-84
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• 2002

The advantages of the OPS techniques(Vajta G et al, Mol Reprod Dev 51: 53-58,1998) give 1) high survival rates of various types of eggs, 2) quick and simple process, 3) inexpensive equipment and reduced chilling injury. The efficiency of IVM/IVF technique in the porcine species is relatively lower than that obtained in other species such as ruminants. Two experiments were designed to investigate the effects of in-vitro fertilization of porcine oocytes matures using different OPS protocol for chilling and warming of vitrification. Porcine oocytes from ovaries collected at abattoir were matured for 44 hours in TCM199 Earle's salt supplemental with pyruvate, pff, L-cysteine, hormones and gentamycin. Oocytes were denuded and fertilized with frozen boar semen by common method. Porcine embryos produced routinely by in-vitro culture system of NCSU23 medium. The vitrification and the warming were conducted by OPS method with the glass micropipette instead of straw vessels and modified the protocol of G.Vajta(1999). In Exp 1, Chilling/Warming:Holding Medium(HM)+EG+DMSO/HM +sucrose Medium(SM) at 39$^{\circ}C$ warm stage. In Exp 2, : PBS+CS+EG+Ficoll+ Trehalose/PBS+Trehalose at 25$^{\circ}C$ stage. Filling, freezing, packing, thawing out and further culturing were performed to follow the basic protocol of G Vajta. During IVM-lVC and post-warming, fertilization parameter and developmental potential were compared to and statistically analysed. It was not significantly different from Exp 1 and Exp 2 but 25$^{\circ}C$ of stage was slightly higher on the morula/blastocyst forming rate and better atmosphere for worker than that at 39$^{\circ}C$ stage.

• Korean Journal of Plant Resources
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• v.31 no.6
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• pp.684-694
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• 2018

This study describes the successful establishment of a cryopreservation protocol for Citrus limon cultivars: 'Frost Eureka limon' and 'Cook Eureka limon', using a droplet-vitrification method. The shoot tips that were excised from in vitro grown seedlings of the two cultivars were preserved in liquid nitrogen (LN) and successfully regenerated into whole plants. Excised shoot tips were pre-cultured for 1 or 2 days in 0.3 M and 0.5 M sucrose solutions at $25^{\circ}C$ and incubated in a loading solution (LS) composed of 17.5% glycerol + 17.5% sucrose in Murashige and Skoog (MS) medium for 40 min at $25^{\circ}C$. Prior to direct immersion in LN for 1 h, the shoot tips were dehydrated with plant vitrification solution 2 (PVS2) at $0^{\circ}C$ or PVS3 at $25^{\circ}C$. The frozen shoot tips were re-warmed and unloaded with 1.2 M sucrose in $\text\tiny{^1/_2}$ MS for 30 min at $25^{\circ}C$. Shoot tips were post-cultured overnight on survival medium and then micrografted onto 'trifoliate orange' (Poncirus trifoliate (L.) Raf. seedling rootstocks for recovery and to produce whole plants. The highest regrowth rates were 53.5% and 50.3% for cryopreserved shoot tips of 'Frost Eureka limon' and 'Cook Eureka limon', respectively, when pre-cultured in 0.3 M and 0.5 M sucrose concentrations in a sequencing manner, with LS and treated with PVS2 for 60 min at $0^{\circ}C$. We also investigated whether the ammonium ion concentration on post-culture medium affected the viability of the cryopreserved Citrus shoot tips. The viability of cooled samples, following culturing on woody plant media (WPM) containing $\text\tiny{^1/_4}$ ammonium nitrate overnight before micrografting, was the highest (70.3%) in 'Frost Eureka limon'. The study described here is a cost-effective and safe method to conserve Citrus fruit cultivars, for the improvement and large-scale multiplication of fruit plants and for breeding disease resistance.

• Journal of Animal Reproduction and Biotechnology
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• v.36 no.4
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• pp.299-306
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• 2021

Vitrification methods are commonly used for mammalian reproduction through the long-term storage of blastocyst produced in vitro. However, the survival and quality of embryos following vitrification are significantly low compared with blastocyst from in vitro production (IVP). This study evaluates that the survival of frozen-thawed bovine embryos was relevant to mitochondrial superoxide derived mitochondrial activity. Here we present supplementation of the cryopreservation medium with Mito-TEMPO (0.1 µM) induced a significant (p < 0.001; non-treated group: 56.8 ± 8.7%, reexpanded at 24 h vs Mito-TEMPO treated group: 77.5 ± 8.9%, re-expanded at 24 h) improvement in survival rate of cryopreserved-thawed bovine blastocyst. To confirm the quality of vitrified blastocyst after thawing, DNA fragmentation of survived embryos was examined by TUNEL assay. As a result, TUNEL positive cells rates of frozen-thawed embryos were lower in the Mito-TEMPO treated group (4.2 ± 1.4%) than the non-treated group (7.1 ± 3.5%). In addition, we investigated the intracellular ROS and mitochondrial specific superoxide production using DCF-DA and Mito-SOX staining in survived bovine embryos following vitrification depending on Mito-TEMPO treatment. As expected, intracellular ROS levels and superoxide production of vitrified blastocysts after cryopreservation were significantly reduced (p < 0.05) according to Mito-TEMPO supplement in freezing medium. Also, mitochondrial activity measured by MitoTracker Orange staining increased in the frozen-thawed embryos with Mito-TEMPO compared with non-treated group. These results indicate that the treatment of Mito-TEMPO during cryopreservation might induce reduction in DNA fragmentation and apoptosis-related ROS production, consequently increasing mitochondrial activation for developmental capacity of frozen-thawed embryos.