• 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 Veterinary Research
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• v.35 no.3
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• pp.635-641
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• 1995

The purpose of this study was to investigate the effects of developmental stage and equilibration time on survival of rabbit embryos following freezing by vitrification. Adult New Zealand White female rabbits were superovulated with PMSG and hCG. The 8-cell stage embryos were collected from 40 to 45 hours after hCG injection by flushing oviducts with Dulbecco's phosphated buffered saline and in vitro cultured in TCM-199 containing 10% fetal calf serum(FCS). Each embryos developed in vitro to 16-cell, compact morula and blastocyst was cryopreserved and cultured following thawing to examine their developmental potential to expanded blastocyst stage in vitro. The frozen-thawed-cultured embryos were stained with Hoechst 33342, and their nuclei were counted using a fluorescence microscope. On the toxicity test of EFS solution as cryopreservation, the survival rates of 8-cell stage embryos was decreased in reverse to increasing of exposure time over 5 minutes. The post-thaw survival rates of embryos on equilibration times was significantly(P<0.05) higher for 2 min. than for 5 or 10 minutes. From morula to blastocyst of rabbit embryos was more suitable than 8-cell stage for cryopreservation by vitrification. The higher post-thaw survival rate of embryos can be achieved by keeping the cryoprotectant at $4^{\circ}C$ than at $20^{\circ}C$. The mean number of nuclei per embryo following freezing by vitrification and in vitro culture to expanded blastocyst at compacted morula and blastcyst was not significantly differ from fresh blastocyst.

• Journal of Embryo Transfer
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• v.16 no.3
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• pp.239-243
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• 2001

Selection of oocyte cryopreservation method is a prerequisite factor for developing an effective bank system. Compared with slow freezing method, the vitrification has various advantages such as avoiding intracellular ice crustal formation. In our previous, we attempted to employ a vitrification method using ethylene glycol and an electron microscope grid for cryopreservation of mouse oocytes. However, A high incidence of spindle and chromosome abnormalities was detected in thawed oocytes after vitrification. We examined whether the addition of a cystoskeleton stabilizer Taxol $^{TM}$, to the vitrification solution could promote the post-thawed survival and subsequent development of stored oocytes. More oocytes developed to the 4-cell (44.7% vs. 69.7%), 8-cell (31.8% vs. 64.2%), morula (24.7% vs. 54.3%), and blastocyst (20.3% vs. 49.2%) stages after the addition of Taxol$^{TM}$ to the cryoprotectant than after no addition. 21 and 26 mouse pups were born after transfer of blastocyst derived from oocytes vitrified without and with Taxol. The addition of Taxol to vitrification solution greatly promoted post-thaw preimplantation development of ICR morose oocytes.tes.

• Korean Journal of Animal Reproduction
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• v.16 no.3
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• pp.269-276
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• 1992

Studies were carried out to find the freezing media which gives no ice crystals in single(glycerol, ethylene glycol, dimethyl sulfoxide(DMSO)) and mixture solutions(glycerol＋propylene glycol, glycerol＋ethylene glycol) of permeable cryoprotectants in vitrification solution and to study effects of VS on the survival of vitrified mouse morulae. The results are summarized as follows: 1. In toxicity test of permeable cryoprotectants, 30% glycerol of single solution showed the highest FDA-score(4.1) in mouse morulae frozen compared among other single solutions. The FDA-score(4.1) of 30% glycerol was higher than 30% ethylene glycol(3.6) and DMSO(1.4( (P<0.05). 2. 20, 30 or 40% single solution of permeable cryoprotectants containing m-PBS with 10% sucrose and 20% BSA was not crystallized during cooling, but crystallized during warming. However, the 30% mixture solution of the two permeable cryoprotectants was not crystallized both during cooling and warming.3. When mouse morulae were frozen in 30% mixture solutions of two permeable cryoprotectants(glycerol and propylene glycol, glycerol and ethylene glycol), highest FDA-score(4.5) was obtained in a mixture solution of 20% glycerol and 10% ethylene glycol(20G10E) than other 30% mixture solutions(10G20E, 15G15E, 20G10P, 15G15P, 10G20P) and there was significant difference between 20G10E and 10G20E(P<0.05).

• Clinical and Experimental Reproductive Medicine
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• v.37 no.4
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• pp.267-291
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• 2010

Life can be kept in suspended animation either before fertilization at oocyte stage or after fertilization at different stages of embryonic development for a variety of reasons. It not only has potential applications in fertility preservation and management in human but also has important roles in the preservation and management of animal genetic resources, low-cost international movement of selected genetics, and rapid dissemination of germplasm through assisted reproductive technologies (ART) and genetic engineering. Currently, slow-freezing and vitrification are the two approaches by which oocytes and embryos can be cryopreserved for long-term storage. Both of these methods have their own advantages and disadvantages but allow the cryopreservation of oocytes and embryos with comparable efficiency. Vitrification of oocyte and embryos, although proven successful just 13 years after slow-freezing, is generally considered an emerging technology and appears to slow gain acceptance in both animal and human ART despite having controversial storage and contamination issues. In this manuscript, we discuss the basic techniques of oocyt/embryo cryopreservation and review the current status and recent developments in vitrification.

• Journal of Embryo Transfer
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• v.11 no.2
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• pp.125-135
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• 1996

The present study was carried out to investigate the effects of cryoprotectants, equilibration step, freezing rate, culture condition following in vitro fertilization, and age and development stage of embryo by freezing with conventional slow freezing and vitrification on survival of frozen-thawed Korean native cattle(KNC) blastocysts produced in vitro. The KNC blastocysts produced in vitro were equilibrated in 1.8M ethylene glycol or 1.4M glycerol and cooled from -6$^{\circ}C$ to -35$^{\circ}C$ at -0.3$^{\circ}C$ or -O.6$^{\circ}C$ /minute. When equilibrated in 1.8M ethylene glycol, survival rate of fiozen4hawed blastocysts was sarne in both -0. 3$^{\circ}C$ /min and -0.6$^{\circ}C$ /min cooling rate(71.4%). With the equilibration in 1.4M glycerol, survival rate was higher in -0.3$^{\circ}C$ /min(63.6%) than in -0.6$^{\circ}C$ /min cooling rate(53.8%). For vitrification of the KNC blastocysts produced in vitro, they were equilibrated in 2-step or 3-step exposure to vitrification solution(25% ethylene glycol + 25% glycerol). Survival rate was sirilar in both 2-step(45.0%) and 3-step exposure(47.4%). According to culture condition following in vitro fertilization, higher survival rate was obtained for blastocysts co-cultured with bovine oviductal epithelial cell(BOEC, 77.3%) than for those cultured with epidermal growth factor(EGF, 65.7%) or for those co-cultured with BOEG + EGF (54.8%). According to embryo age and development stage, higher survival rate was obtained for 7-day ernbryos(70.0%) than 8-day(56.8%) or 9-day(20.0%) for blastocyst stage and obtained for 8-day embryos(74.3%) than 7-day(62.5%) or 9-day(42.9%) for exponded blastocyst. In surnmary, higher survival rate of frozen4hawed KNC blastocysts produced in vitro were obtained by using ethylene glycol for cryoprotectant and -0.3$^{\circ}C$ /min for cooling rate. And higher survival rate were obtained with co-culture with BOEC for culture condition following in vitro fertilization and with 7-day blastocyst or 8-day expanded blasto cyst for embryo age and development stage.

• Korean Journal of Veterinary Research
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• v.38 no.2
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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.

• Journal of Embryo Transfer
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• v.6 no.2
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• pp.30-36
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• 1991

The method of vitnilcation has various merits. It needs neither seeding nor slow freezing. It can freeze embryo by putting it directly into liquid nitrogen at the indoor temperature to $0^{\circ}C$. The operation process is quite easy. Moreover, higher promise of survival can be expected as there is no physical damage by any lumps of ice with the exception of cells. In Kasal's experiment (1990) using EFS liquid and Kang's experiment (1991) using GFS liquid the ratio of the damaged embryo was only 2-3%. But, the method of vitrification is now on the process of improvement, and the final or united method is not yet established. At the present time, most of the major institutes all over the world are using the traditional freezing method in the preservation of mouse embryo, but it is very likely that the vitrification will prevaIl in the near future considering the various merits of it. Calves can be begotten from the embryo by means of vitriilcated preservation in the cases of cow, rat, and rabbit as well as of mouse. In addition, recent experiments have shown that vitrificated preservation was successful in the case of drosophila embryo which was much bigger than mammalian embryo, which fact tells that this method is expected to be preferably used even in the preservation of living organs in the near future.

• 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.

• Journal of Embryo Transfer
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• v.4 no.1
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• pp.1-13
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• 1989

This paper reviews the most important steps that have generated consistent progress in principles and developmental progress of embryo cryopreservation, and also study on freezing procedure and its application by conventional method and current improved method for freezing procedure and its appilcation of embryo cryopreservation in farm animals. Four were of particular interest: 1.The transport of water across the ccli membrane (zona pellucida) during freezing and thawing accordinglyplays a role in determing whether the celi survives. This movement of water is controlied mainly by extracellular phase changes and by the nature and concentration of any cryoprotective agent present. Therates of cooling, freezing and warming, and the intervals over which they are applied are further decisi've factors in determining whether a cryopreservation procedure allows survival after thawing. 2.The first successful deep freezing experiments with sheep morula and blastocysts during the seventies were based on the early procedures used for mouse embryos.Current research during the eighties is developed with the aim of simplifying and improving current procedures such as one-step dilution and rapid or ultra-rapid cooling by using the model of laboratory animals. 3.The conventional method for the embryo cryopreservation is described. An alternative to this method which may result in high survival and also in reducing of the freezing and thawing time is done by combing a permeable cryoprotectant such as glycerol, DMSO or propanediol and a non-permeable compound such as sucrose, trehalose, raffinose or lactose. 4.Finally a different approach to the preservation of embryos, named vitrification, is introduced. This procedure depends upon the ability of concentrated solutions of cryoprotective agents such as glycerol and propanediol to supercool to very low temperature (-196$^{\circ}C$) during rapid cooling before solidifying without formation of ice. However, more complete data are necessary for successful vitrification of blastocysts.