• Korean Journal of Plant Resources
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• v.32 no.6
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• pp.689-697
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• 2019

This study describes an efficient and widely applicable droplet-vitrification following cryopreservation for shoot tips of strawberry (Fragaria × ananassa Duch.) cvs. 'Wonkyo3114' and 'Gurumi40'. The shoot tips were precultured in Murashige and Skoog (MS) liquid medium supplemented with sucrose (0.3-0.5M). Precultured explants were osmoprotected with loading solution (LS, C4) containing 20% glycerol and 20% sucrose for 40 min and exposed to dehydration solution (B5) containing 40% glycerol and 40% sucrose for 40 min at 25℃, Subsequently, the explants were transferred onto droplets containing 2.5 μL PVS3 on sterilized aluminum foils (4 cm × 0.5 cm) prior to direct immersion in liquid nitrogen (LN) for 1 h. The highest regrowth rate (%) in both the cultivars was obtained when the shoot tips were precultured with MS + 0.3M sucrose for 40 h at 25℃. The cryopreserved shoots tips exhibited 55% regrowth rate by culturing in NH₄NO₃-free MS medium supplemented with 3% sucrose, 1.0 g/L casein, 1.0 mg/L GA₃, and 0.5 mg/L BA for 5 weeks and in MS medium supplemented with 0.5 mg/L GA₃ for 8 weeks. This result shows that droplet-vitrification could be employed as a promising method for cryostorage of strawberry germplasm.

• Journal of Embryo Transfer
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• v.26 no.3
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• pp.201-207
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• 2011

The present study was performed to investigate the survival and subsequent embryonic developmental rate of immature and mature oocytes after vitrification and pronuclear stage embryos after slow-freezing and vitrification. We have also tried to examine the dependency of concentrations (7.5, 15%) and exposure time (5, 10, 20 min) of ED cryoprotectant on developmental rate of pronuclear stage embryos. The developmental rates of 2-ce1l and blastocyst embryos at mature oocytes were significantly (p<0.05) higher than immature oocytes. After slow freezing, vitrification and thawing of pronuclear stage embryo, the survival and developmental rates of blastocysts and hatched blastocysts were significantly (p<0.05) higher after vitrification than after slow-freezing. On contrary, the developmental rates of 2-cell embryos were significantly (p<0.05) higher after slow freezing than after vitrification. The cryopreservation methods of pronuclear stage embryos vitrified by exposed to 7.5% ED solution for 5 minutes was significantly (p<0.05) higher than other experimental group. The results of our study suggest 1hat the developmental rates of mature oocytes have been more successful than immature oocytes during vitrification. Vitrification was more efficient than slow freezing in case of pronuclear stage embryos. The effective cryopreservation method of pronuclear stage embryos was vitrified by exposed to 7.5% ED solution for 5 minutes.

• 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.

• Clinical and Experimental Reproductive Medicine
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• v.28 no.4
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• pp.295-300
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• 2001

Objective : To assess the effects of progesterone and acetyl-L-carnitine used after treated with Isolate�� gradient before semen cryopreservation-thawing on sperm parameters and membrane integrity. Material and Methods : From April 2001 to July 2001, ten normal male partner of couples who were visited in vitro fertilization (IVF) clinics. the semens were treated with $Isolate^{(R)}$ gradient before cryopreservation, spermatozoa was incubated with progesterone (1, 5 and $10{\mu}M$), acetyl-L-carnitine (2.5, 5 and $10{\mu}M$), or both (progesterone, $1{\mu}M$; and acetyl-L-carnitine, $5{\mu}M$) for 30 min. Results: There were no differences in sperm parameters and vital stain among isolate only treated group, progesterone (1, 5 and $10{\mu}M$), acetyl-L-carnitine (2.5, 5 and $10{\mu}M$) and both (progesterone, $1{\mu}M$; and acetyl-L-carnitine, $5{\mu}M$). But, in high concentration of acetyl-L-carnitine ($10{\mu}M$) treated group, sperm parameters and vital stain were decreased. The statistical method was used ANOVA (Kruskal-Wallis test) and p value was <0.01. Conclusions : Neither progesterone nor acetyl-L-carnitine show to be protective effect on the cryodamage assessed by sperm parameters and vital stain (eosin-Y stain) in normal sperm. High concentration of acetyl-L-carnitine ($10{\mu}M$), however, was harmful effect on cryoprevention.

• 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.

• Animal Bioscience
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• v.37 no.7
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• pp.1177-1184
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• 2024

Objective: Semen cryopreservation is an effective method of preserving genetic material, particularly in native chicken breeds facing a substantial decline. In this study, we evaluated the quality of frozen/thawed rooster semen treated with different concentrations of oral administrations of black ginger (Kaempferia parviflora: KP) extract and determined its fertility. Methods: Thirty-two Thai native roosters (Pradu Hang Dum, 42 weeks old) were used in this study. The treatments were classified into four groups according to the concentration of KP extract administered to the roosters: 0, 100, 150, and 200 mg/kg body weight. The quality of fresh semen was analyzed before cryopreservation. Post-thaw sperm quality and fertility potential were determined. Also, lipid peroxidation was determined. Results: The results showed that sperm concentration and movement increased in roosters treated with 200 mg/kg of KP extract (p<0.05). The malondialdehyde (MDA) in the roosters receiving 200 mg/kg KP extract was lower than that in the other but had an insignificant difference within the KP treatment groups (p>0.05). The highest MDA levels were observed in the control group (p<0.05). The percentage of motile sperm (total motility and progressive motility) after semen thawing was higher in roosters that received 150 and 200 mg/kg KP extract than in those that received 100 mg/kg KP extract and the control (p<0.05). MDA levels decreased significantly in roosters that received 150 and 200 mg/kg KP extract than in those that received 100 mg/kg KP extract and the control (p<0.05). Fertility and hatchability were greater in the KP150 and KP200 groups than in the KP100 and control groups (p<0.05). Conclusion: The optimal amount of KP extract influencing initial sperm quality was determined to be 200 mg/kg. However, 150 mg/kg was the optimal low dosage of KP extract administration that maintained sperm quality and fertility following semen cryopreservation.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.44 no.6
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• pp.665-670
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• 2011

The rotifer Brachionus plicatilis is one of the most important food organisms in aquaculture. The resting eggs produced by mictic female rotifers are easily stored and hatched, making them useful as the starter for the mass culture of rotifers in marine larval culture. This study examined the optimum preservation method for resting eggs to ensure a high hatching rate. To produce resting eggs, the marine rotifer B. plicatilis was cultured with Nannochloris oculata (KMMCC 16). The resting eggs were harvested and cryopreserved using 5% and 10% methanol (MeOH), dimethylsulfoxide (DMSO), and glycerol as cryoprotectant agents (CPAs). The cryopreservation comprised slow or rapid freezing and the resting eggs were stored for one month in liquid nitrogen ($-196^{\circ}C$). The resting eggs were also dried at different temperatures (30, 40, and $50^{\circ}C$) and for different times (1, 2, and 3 h). In general, the hatching rates of the resting eggs preserved with CPA were higher than those without CPA and the slow freezing method was better than the rapid freezing method. However, the optimum CPA concentration for the hatching rate of the resting eggs varied with the freezing method and kind of CPA, and the CPA also affected the viability of the resting eggs. Dried resting eggs had a high, rapid hatching rate over 80%. The moisture content of the resting eggs cryopreserved in liquid nitrogen affected the hatching rate. Drying at $30^{\circ}C$ for 1 hour resulted in a high hatching rate of the resting eggs. In conclusion, drying at $30^{\circ}C$ for 1 hour and preservation in liquid nitrogen with the slow freezing method, without CPA, is recommended for a high hatching rate (ca. 95%) of rotifer resting eggs.

• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.9-9
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• 2002

This study was to evaluate the in vitro survival of vitrified-thawed bovine MII enucleated (MIIe) oocytes according to activation timing and minimun volume cooling (MVC) method and their in vitro development after somatic cell nuclear transfer (SONT). Bovine oocytes were recovered from slaughtered bovine ovary and matured in TCM-199 supplemented with 10% FBS. (omitted)

• Journal of Chest Surgery
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• v.42 no.3
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• pp.283-291
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• 2009

Background: Tracheal reconstruction after extended tracheal resection still remains as a major surgical challenge because good clinical outcomes are usually correlated with limited tracheal resection. Recent investigations with a using cryopreserved trachea for the reconstruction of a trachea have been carried out to overcome this problem. In this study, we analyzed viability of tracheas, which is an important determining factor for the success of transplanting a cryopreserved trachea and the development of post-transplantation tracheal stenosis, according to three different experimental factors: 1) the warm-ischemic time, 2) the cryopreservation solution and 3) the preserving temperature, to determine a better cryopreservation protocol and a better composition of the cryopreservation solution. Material and Method: Rats tracheas were harvested for different warm-ischemic times (0 hr, 12 hrs, 24 hrs). The tracheas were treated with recombinant insulin growth factor-1 (IGF) and they were stored at three different temperatures $(4^{\circ}C,\;-80^{\circ}C,\;-196^{\circ}C)$ for two weeks. After two weeks, we thawed the stored trachea and isolated the cells of the tracheas with using type II collagenase. We cultured the cells for seven days and then we compared the cellular viability by the MTT reduction assay. Result: Though cryopreservation is required to preserve a trachea for a longer time period, the viability of the tracheas stored at $-80^{\circ}C$ and $-196^{\circ}C$ was significantly reduced compared to that of the tracheas stored at $4^{\circ}C$. The viability of the tracheas with warm-ischemic times of 12 hrs and 24 hrs was also reduced in comparison to the tracheas with a warm-ischemic time of 0 hrs. Our data showed that the warm ischemic time and the parameters of crypreservation negatively affect on trachea viability. However, a cryopresrvation solution containing IGF-1 improved the cellular viability better than the existing cryopreservation solution. For the warm ischemic time group of a 0 hr, the addition of IGF-1 improved the viability of trachea at all the preserving temperatures. Conclusion: These experiments demonstrate that the viability of cryopreserved trachea can improved by modifying the components of the crypreservation solution with the addition of IGF-1 and reducing the warm-ischemic time.

• Microbiology and Biotechnology Letters
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• v.48 no.2
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• pp.205-214
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• 2020

Cyanobacteria are microorganisms which have important roles in the nitrogen cycle due to their ability to fix nitrogen in water and soil ecosystems. They also produce valuable materials that may be used in various industries. However, some species of cyanobacteria may limit the use of water resources by causing harmful algal blooms in water ecosystems. Many culture collection depositories provide cyanobacterial strains for research, but their systematic preservation is not well-developed in Korea. In this study, we developed a method for the cryopreservation of the cyanobacteria Trichormus variabilis (syn. Anabaena variabilis), using alginate microcapsules. Two approaches were used for the experiments and their outputs were compared. One of the methods involved the cryopreservation of cells using only a cryoprotectant and the other used the cryoprotectant within microcapsules. After cryopreservation for 35 days, cells preserved with both methods were successfully regenerated from the initial 1.0 × 10⁵ cells/ml to a final concentration of 6.7 × 10⁶ cells/ml and 1.1 × 10⁷ cells/ml. Irregular T. variabilis shapes were found after 14 days of regeneration. T. variabilis internal structures were observed by transmission electron microscopy (TEM), revealing that lipid droplets were reduced after cryopreservation. The expression of the mreB gene, known to be related to cell morphology, was downregulated (54.7%) after cryopreservation. Cryopreservation using cryoprotectant alone or with microcapsules is expected to be applicable to other filamentous cyanobacteria in the future.