• Journal of Microbiology and Biotechnology
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• v.25 no.12
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• pp.1989-1996
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• 2015

Ice-binding proteins (IBPs) can inhibit ice recrystallization (IR), a major cause of cell death during cryopreservation. IBPs are hypothesized to improve cell viability after cryopreservation by alleviating the cryoinjury caused by IR. In our previous studies, we showed that supplementation of the freezing medium with the recombinant IBP of the Arctic yeast Glaciozyma sp. (designated as LeIBP) could reduce post-thaw hemolysis of human red blood cells and increase the survival of cryopreserved diatoms. Here, we showed that LeIBP could improve the viability of cryopreserved mammalian cells. Human cervical cancer cells (HeLa), mouse fibroblasts (NIH/3T3), human preosteoblasts (MC3T3-E1), Chinese hamster ovary cells (CHO-K1), and human keratinocytes (HaCaT) were evaluated. These mammalian cells were frozen in dimethyl sulfoxide (DMSO)/fetal bovine serum (FBS) solution with or without 0.1 mg/ml LeIBP at a cooling rate of -1℃/min in a -80℃ freezer overnight. The minimum effective concentration (0.1 mg/ml) of LeIBP was determined, based on the viability of HeLa cells after treatment with LeIBP during cryopreservation and the IR inhibition assay results. The post-thaw viability of mammalian cells was examined. In all cases, cell viability was significantly enhanced by more than 10% by LeIBP supplementation in 5% DMSO/5% FBS: viability increased by 20% for HeLa cells, 28% for NIH/3T3 cells, 21% for MC3T3-E1, 10% for CHO-K1, and 20% for HaCaT. Furthermore, addition of LeIBP reduced the concentrations of toxic DMSO and FBS down to 5%. Therefore, we demonstrated that LeIBP can increase the viability of cryopreserved mammalian cells by inhibiting IR.

• ALGAE
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• v.21 no.1
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• pp.125-131
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• 2006

Long-term preservation of bloom-forming cyanobacteria was evaluated using cryopreservation and freeze-drying of nine strains belonging to four genera and seven species. All test strains, except Aphanizomenon flos-aquae NIER- 10028, showed partial or complete survival following cryopreservation and freeze-drying. Frozen and freeze-dried strains were preserved for more than two years and were revived monthly. Most strains showed higher post-thaw viability after cryopreservation, especially without cryoprotectant compared to freeze-drying. Microcystis aeruginosa NIER-10010, M. viridis NIER-10020, M. ichthyoblabe NIER-10023, M. novacekii NIER-10029 and Oscillatoria sancta NIER-10027 were revived after 2.5 years of cryopreservation. These results suggest that cryopreservation may be an easy and timesaving long-term preservation method for bloom-forming cyanobacteria.

• Journal of Veterinary Clinics
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• v.4 no.2
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• pp.449-455
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• 1987

Quick freezing of bovine embryos was attempted after they were predehydrated at room temperature. Combined solutions of 2M glycerol or 2M ethylene glycol in the presence of either 0.5 or 1.0M sucrose in phosphated buffered saline+20% calf serum were compared. The quick freezing method in which embryos were directly transferred in liquid nitrogen vapor for 2 minutes at - l70$^{\circ}C$ before being plunged into liquid nitrogen was used. Post-thaw survival rates in 2M glycerol and 2M ethylene glycol were high with 0.5 M (55.6% and 53.3%) versus 1.0M(38.1% and 31.6%) sucrose(P < 0.05). But survival rates with 2M glycerol and 2M ethylene glycol were not significantly different. Transfer thawed embryos frozen with 2M glycerol and 2M ethylene glycol by 0.5M sucrose resulted in birthrates of 40.9% and 40.0%, respectively compared to 26.3% and 27.2%, respectively, for 1.0 M sucrose(p<0.05). This was 56.0% for fresh control.

• Korean Journal of Animal Reproduction
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• v.16 no.3
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• pp.217-224
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• 1992

These studies were carried out ot investigate on the transferred embryo development following ultrarapid frozen for 8-cell and morula of in vitro fertilization mouse embryos. The post-thaw embryo survival was evaluated and compared by cell stage of embryos and by equilibration time before ultrarapid freezing. The results obatined were summerized as follows: 1. The effects of equilibration time of 3 vs. 6 minutes before ultrarapid freezing and after thawing on the morphological survival and the viability of 8-cell and morulas embryos were not significant. 2. When the ultrarapid frozen-thawed 32 eight-cell and 33 morula embryos, and 30 fresh blastocysts were transferred to pseudopregnant recipient mice, the number of normal offsprings produced were 9(28.1%), 14(42.4%) and 18(60.0%), respectively. From the above resutls, it was concluded that the optimal conditions of pH osmolality of the media for mouse IVF and embryo culture, and the period of sperm preincubation might be 7.1, 310 mOsm and 120 min., respectively,a nd somewhat high conception rate might be resulted from transfer of frozen embryos of morula stage and fresh embryos of blastocyst stage.

• Journal of Embryo Transfer
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• v.9 no.2
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• pp.173-180
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• 1994

The present experirnents on cryopreservation were carried out to investigate effect of solution toxicity, equilibration time and cell stages on the post-thaw survival of mouse morulae and blastocyst embryos cryopreserved by vitrification in EFS solution. The mouse embryos were exposed to the EFS solution in one step at room temperature, kept in the EFS solution during different period for toxicity test, vitrified in liquid nitrogen and thawed rapidly. After the mouse morulae embryos were exposed to EFS solution for 2 and 5 ruin. at room temperature and then they were washed in 0.5 M sucrose solution and basal mediurn(D-PBS + 10% FCS), they were cultured to examined cryoprotectant toxicity induced injury during exposure, most of embryos developed to expanded blastocysts(100 and 90.0%). However, when the exposure time was extended to 10 and 20 min, these development rates dropped dramatically in 10 ruin. (75.0%) and 20 ruin. (4.5%), respectively. When the compacted morulae were vitrified in EFS solution after equilibration for 2 and 5 min, the embryos have developed to normal blastocyst following thawing, washing and culture processes was 89.3 and 89.6%. However, when the exposure time was expanded to 10 ruin, this survival rate dropped to 68.8%. When the blastocyst were vitrified in EFS solution after equilibration for 2, 5 and 10 minutes, the survival rate of embryos which developed to normal blastocyst following thawing and culture processing were 58.5, 46.7 and 22.4%, respectively. The optimal time of equilibration of mouse morula and blastocysts in EFS solution seemed o be 2 and 5 ruin.

• Journal of Embryo Transfer
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• v.24 no.4
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• pp.253-257
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• 2009

To enhance the embryo preservation technology and better application of embryo transfer technique to the field (dairy science or animal reproduction. etc.), we examined the viabilities of bovine embryos produced in vitro and in vivo after cryopreservation according to their developmental stage and thawing temperature. Bovine embryos from in vivo/vitro fertilization (Hanwoo) were examined at day 7, 8, and 9. Survival rates and total cell numbers of in vivo fertilized embryos were as follows: morulae 68.8% and $67\;{\pm}\;6.0$; blastocysts 80.5% and $120\;{\pm}\;10$; expanded blastocysts 77.4% and $138\;{\pm}\;9.7$, respectively. Rates of embryo development for blastocysts and expanded blastocysts after thawing were significantly higher than that of morula stage embryos (p<0.05). While survival rates of in vitro fertilized embryos according to developmental stage showed no significant difference among groups (morula 67.9%; blastocyst 74.3%; and expanded blastocyst 79.4%), total cell numbers were significantly lower than those of other groups (morula $64\;{\pm}\;5.9$; blastocyst $116\;{\pm}\;8.7$; and expanded blastocyst $135\;{\pm}\;9.1$) For the viability according to thawing temperature, survival rate was higher in $37^{\circ}C$.

• Journal of Embryo Transfer
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• v.17 no.2
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• pp.137-143
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• 2002

This study was conducted to evaluate the effect of sugar kinds and combination of various sugars, and straw size and pre-freezing time on motility of post-thaw spermatozoa in canine. In general, the extender was supplemented with fructose or glucose for semen freezing. The extender used in .this study was Tris-citric acid extender (Tris-buffer) supplemented with 20％ Egg-yolk, 8％ glycerol, 1％ Equex STM paste, and 70 mM sugars such as monosaccharide (fructose and xylose) and disaccharide(trehalose). To evaluate of sugar combination, the sugars supplemented in Tris-buffer. were combined such as control (fructose, xylose, trehalose), two combinations (Fru＋Tre, Fru＋Xy1, Tre＋Xy1) and three combinations (Fru＋Tre＋Xy1). The motility after CASA analysis in Fru＋Tre＋Xy1 was higher than those in fructose, trehalose, xylose, Fru＋Tre, Fru＋Xy1, Tre＋Xy1 (69 vs. 58, 61, 50, 65, 20, 54). However, the progressive motility after CASA analysis in Fru＋Tre group was higher than those in fructose, trehalose, xylose, Fru＋Xy1, Tre＋Xy1, Fru＋Tre＋Xy1 (59％ vs. 47, 55, 42, 13, 49, 44％). The survival rate of post-thaw spermatozoa in 0.25 $m\ell$ straw for 10 min pre-freezing was significantly higher than those in 0.25 $m\ell$ straw for 10 min, 0.25 and 0.5 $m\ell$ for 5 min (80＋0.0 vs. 65＋7, 68＋16, 58＋8％; P＜0.05). The results indicated that the progressive motility of post-thaw spermatozoa in 70 mM Fru ＋Tre (two combination) following CASA analysis and 0.25 ml straw for 10 min pre-freezing time could be better for freezing of semen in canine.

• Korean Journal of Animal Reproduction
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• v.23 no.4
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• pp.313-321
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• 1999

The purpose of these study was to investigate the use of a glass micropipette (GMP) as a vessel for vitrification of bovine IVP blastocysts, to compare the post-thaw survival rates of bovine blastocysts frozen in GMP with those frozen in OPS that have been previously investigated, and to improve the hatching rate following vitrification with GMP method. The GMP vessel permits higher freezing and warming rate than the OPS due to the higher heat conductivity of the glass and lower mass of the solution that contains the embryos. Groups of three bovine IVP blastocysts were sequentially placed into vitrification solution before being loaded into either the OPS or GMP vessels and immersed into L$N_2$within 20 to 25 sec. Post-thaw blastocysts were serially washed in 0.25 and 0.15 M sucrose in HM and TCM-199 for each 5 min, respectively, and then cultured in TCM 199 supplemented with 10% FCS for 24 h. The rate of blastocyst re-expanding did not significantly different for OPS (75.9%) and GMP (80.0%) methods (P＞0.05). The hatching rates in OPS (34.1%) and GMP (37.5%) methods were significantly lower than that in control group (54.3%) (P＞0.05). In addition, the rate of blastocyst re-expanding was significantly lower if blastocysts were vitrified in the wide portion of the micropipette rather than the narrow portion of the micropipette (83.3 vs 56.7%) (P＞0.05), even though three blastocysts were loaded per vessel. The hatching rate in 0.05% pronase solution treatment for 30, 60 and 90 see (45.9, 54.7 and 57.5%) were significantly higher than that in control (35.0%), even though there was not significantly different between 30 see and control. These results indicate that both vitrification vessels can provide high survival rates of bovine IVP blastocysts. However, the GMP vessel has the advantage over the OPS, in that the former does not need a cap to protect the vessel from floating after immersion in L$N_2$. The location of the embryos (narrow or wide portion of immersion) were considered to be limiting factors to the viability of bovine IVP embryos. The exposing in 0.05% pronase solution for 60 or 90 see can increase hatching rates of post-thaw bovine IVP blastocysts.

• Korean Journal of Animal Reproduction
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• v.16 no.1
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• pp.47-53
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• 1992

This study was carried out to investage the effect of cryoprotectant concentration and equilibration time on volume change and in vitro development of intact and bisected mouse embryos by rapid freezing. When compacted morulae were rapidly frozen in 3.0 to 4.0 glycerol or DMSO with 0.25M sucrose solution, the superior(P<0.05) post-thaw survival rate was obtained at the glycerol concentration of 4.0M(89.4%) than 3.0M(71.4%) or 5.0M(42.4%), but at the DMSO concentration of 3.0M(84.5%) than 4.0M(51.1%) or 5.0M(0.0%). The optimal equilibraton time for rapid freezing of ZP-free or bisected morulae in 4.0M glycerol with 0.25M sucrose was found tobe 3 minutes. The minimal volume of compacted morulaewhich corresponded with 61 to 62% of pre-equilibrated embryo volume was obtained from equilibration for 3 minutes in both 3.0 and 4.0M glycerol solutions with 0.25M sucrose.

• Korean Journal of Animal Reproduction
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• v.7 no.2
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• pp.57-71
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• 1983

1. Diluted chicken semen can be preserved at 2 to 5$^{\circ}C$ for 24 to 48 hr with resultant fertility of greater than 90% of that of fresh semen. Turkey semen can be preserved at 10 to 15$^{\circ}C$ for 6 to 24 hr and provide economical fertility. 2. Frozen chicken semen has given variable results; a 21 to 93% fertility ranges as compared to 92 to 94% expected with fresh semen. Highest fertility levels obtained with frozen turkey semen intravaginally inseminated have been 61 and 63% using DMSO and glycerol, respectively, as cryoprotectants. 3. The use of glycerol as a cryoprotectant reauires that its concentration in semen be reduced to less than 2% either by dialysis or centrifugation after thawing and before intravaginal insemination if optimal fertility is to be obtained. 4. The temperature at which cryoprotectants are added to semen and the time allowed for equilibration are important for subsequent fertility pre- and post-freezing. 5. The type of container used for packaging the semen, freeze or cooling rates, thaw rates and level of cryoprotectant all interact in affecting cell survival. 6. Plastic freeze straws as a packaging device for semen offers the following advantages: easy to handle, require minimal storage space, offer a wide range of freeze and thaw rates, and insemination can be made directly from them upon thawing. 7. Controlled slow cooling rates of 1 to 8$^{\circ}C$/min have thus far provided the best results for cooling chicken semen throught the transition phase change (liquid to solid) or critical temperature range of ＋5 to -20 or -35$^{\circ}C$. 8. Highest fertilities have been achieved with frozen chicken semen where a slow thaw rate (2。 to 5$^{\circ}C$) has been used regardless of the freeze rate. 9. To maintain a constant high level of fertility throughout a breeding season with frozen semen, a higher absolute number of spermatozoa must be inseminated (2 to 3 times as many) as compared to fresh semen since a, pp.oximately 50% are destroyed during processing and freezing. 10. The quality of semen may vary with season and age of the male. Such changes in sperm quality could be accentuated by storage effects. Thus, the correct number of spermatozoa may very well vary during the course of a breeding period. 11. As to time of insemination, it is best to avoid inseminating chicken hens within 1-2 hr after or 3-5 hr before oviposition; and turkey hens during or 7-10 hr before oviposition. 12. The physiological receptiveness of the oviduct at the time of insemination is a very important biological factor influencing fertility levels throughout the breeding season.