• Clinical and Experimental Reproductive Medicine
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• 제27권3호
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• pp.283-289
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• 2000

Objective: The study was performed to compare the survival rate and the development of day 2 mouse embryos which had freezing procedures done. Methods: We used three different vitrification solutions (EFS, VS14, DPS) and a ultrarapid freezing solution (UFS) for cryopreservation of day 2 mouse embryo. Results: We tested toxicity by exposing embryos to vitirification solutions and a ultrarapid freezing solution. The survival rates are 100%, 97.8%, 95.6% and 100% (EFS, VS14, DPS and UFS). After cultured for 96 hours, hatching rates of each group are 93.5% (no freezing), 95.6% (EFS), 86.4% (VS14), 93.0% (DPS), and 93.0% (UFS). There is no significant differences among groups. The survival rates after thawing cryopreserved embryos are 80.2%, 91.7%, 69.5%, 0% and 91.8% (slow freezing, EFS, VS14, DPS and UFS). Also cultured for 96 hours, the hatching rates are 93.5% (no freezing), 84.1% (slow freezing), 93.9% (EFS), 48.5% (VS14) and 70.1% (UFS). Conclusion: The survival rates of vitrification in EFS solution and ultrarapid freezing are higher than slow freezing (p<0.05). The hatching rate of vitrification in EFS solution cultured for 96 hours is highest, so vitrification of day 2 mouse embryos in EFS solution considered as more effective for cryopreservation.

• 한국축산식품학회지
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• 제41권6호
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• pp.1012-1021
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• 2021

This study evaluated the effect of freezing rate on the quality characteristics of pork loin to establish an objective standard for rapid freezing. To generate various freezing rates, three air flow rates (0, 1.5, and 3.0 m/s) were applied under three freezing temperatures (-20℃, -30℃, and -40℃). Based on the results, freezing rates ranged from 0.26-1.42 cm/h and were graded by three categories, i.e, slow (category I, >0.4 cm/h), intermediate (category II, 0.6-0.7 cm/h) and rapid freezing (category III, >0.96 cm/h). Both temperature and the air flow rate influenced the freezing rate, and the freezing rate affected the ice crystal size and shear force in pork loin. However, the air flow rate did not affect thawing loss, drip loss or the color of pork loins. In the comparison of freezing rates, pork belonging to category II did not show a clear difference in quality parameters from pork in category I. Furthermore, pork in category III showed fresh meat-like qualities, and the quality characteristics were clearly distinct from those of category I. Although the current standard for rapid freezing rate is 0.5 cm/h, this study suggested that 0.96 cm/h is the lowest freezing rate for achieving meat quality distinguishable from that achieved with conventional freezing, and further increasing the freezing rate did not provide advantages from an energy consumption perspective.

• 한국가축번식학회지
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• 제21권3호
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• pp.287-292
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• 1997

This study was carried out to investigate the effects of concentration and kinds of cryoprotectants, equilibraction time, thawing temperature and time, sucrose concentration on the survival rates of frozen bovine demi-embryos. The bovine demi-embryos following dehydration by cryoprotectants a various concentration of sucrose were freezed by cell freezer and thawed in 3$0^{\circ}C$ water bath. Survival and in vitro developmental rates was defined as development rates on in vitro culture or FDA-test. The results are summarized as follows : 1. The high survival rates of demi-embryos after frozen-thawing in freezing medium was attained 2.0M glycerol. The high survival rates of demi-embryos after frozen-thawing in freezing medium was obtained using single cryoprotectant(25.0~30.0%) than mixed cryoprotectants(16.7~19.0%). 2. The survival rates of demi-embryos after frozen-thawing in freezing medium added 1.5M, 2.0M glycerol＋0.25M sucrose(37.5~33.3%) were higher survival rates than those of sucrose concentration of 0.50, 0.75M(12.5~26.7%). 3. The equilibration time on the survival rates of demi-embryos was attained after short period of time(30.0~35.0%) in the freezing medium higher than long period of time(21.1%). 4. The thawing temperature on the survival rates of demi-embryos was attained at 3$0^{\circ}C$ of thawing temperature(26.7~40.0%) higher than $25^{\circ}C$ or 37$^{\circ}C$ of thawing temperature(13.3~20.0%). 5. The thawing time on the survival rates of demi-embryos was attained at 1~5 minutes of thawing time(26.7~33.3%) in the freezing medium higher than 10 minutes of thawing time(13.3~18.8%).

• 한국식품위생안전성학회지
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• 제3권1호
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• pp.19-26
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• 1988

Most of meat spoilage bacteria area Gram negative, which are very sensitive to freezing ; for instance , 90% of E. coli cells are killed or sub-lethally injured by freezing at -3$0^{\circ}C$, and the freeze-injury rate is dependent upon freezing rate. Since the injured bacterial cells are sensitive to selective agents, they fail to multiply in selective media. Injured bacterial cells are, however, capable of spontaneous repair at appropriate environmental and nutritional conditions . Enumeration of injured bacterial cells involves artificial induction of repair at these conditions. Cubic beef samples(3$\times$3$\times$3cm) were frozen at -6$0^{\circ}C$, -4$0^{\circ}C$, or -18$^{\circ}C$. The samples frozen at each temperature were thawed at 4$^{\circ}C$, 2$0^{\circ}C$, or by microwave . After these respective freezing an thawing treatments, the percentage of sub-lethally injured total coliforms out of total surviving ones was measured and compared. The results were as follows: 1. The interaction between freezing and thawing on injury rate was not significant. 2. The injury rates(as means of all three thawing treatments post-freezing) by freezing at -6$0^{\circ}C$, -4$0^{\circ}C$, or -18$^{\circ}C$ were 32.2$^{\circ}C$ and 19.2$^{\circ}C$ respectively . 3. The injury rates(as means of all three freezing treatments)by thawing at 4$^{\circ}C$, 2$0^{\circ}C$, or by microwave were 49.3%, 11.7% and 21.0% respectively. The highest injury rate was caused by freezing at -6$0^{\circ}C$ and subsequent thawing at 4$^{\circ}C$. However since the injury rates by freezing treatment were not significantly different, freezing at -18$^{\circ}C$ and subsequent thawing at 4$^{\circ}C$ can also be recommended , from an economic perspective.

• 한국가축번식학회지
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• 제21권2호
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• pp.95-102
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• 1997

The studies on the carried out to investigate the effective concentration of cryoprotectant agents and sucrose by one-step straw method of bovine embryos. The follicular oocytes were cultured in TCM-199 medium containing 10 IU/ml PMSG(Sigma, USA), 10 IU/ml hCG(Sigma, USA), 1$\mu\textrm{g}$/ml $\beta$-estradiol(Sigma, USA) and 10% FCS for 24~48 hrs in incubator with 5% CO2 in air at 38.5$^{\circ}C$ and then matured oocytes were again cultured for 12~18 hrs with motile capacitated sperm by preincubation of heparin. The bovine embryos following dehydration by cryoprotective agents and various concentrations of sucrose were directly plunged into liquid nitrogen and thawed in 3$0^{\circ}C$ water. Survival and in vitro developmental rate was defined as devellpmental rate on in vitro culture or FDA-test. The results are smmarized as followes : 1. The high in vitro developmental rates of bovine frozen embryos after rapidly thawed in freezing medium was attained 2.0M glycerol, 2.0M DMSO, 1M or 2.0M propanediol. 2. The high in vitro developmental rates of bovine frozen embryos after rapidly thawed in freezing medium was obtained single cryoprotectant(6.7~17.4%) than mixed cryoprotectants(6.7~16.7%). 3. In vitro developmental rate of bovine embryos after rapid frozen-thawing in the freezing medium added 0.25M and 0.50M sucrose were higher cleavage rate than those of sucrose concentration of 0.75M and 1.00M. 4. The freezing methods on in vitro developemental rates of bovine embryos was attained slow freezing method(9.70~15.6%) higher than rapid freezing method(9.4~13.3%).

• 한국식품과학회지
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• 제20권3호
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• pp.447-452
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• 1988

냉동육에 대한 동결속도의 영향을 살펴보고자 송풍동 결기를 이용하여 직경 10cm, 높이 10cm규격의 세절된 원통형 쇠고기를 0.97cm/hr, 2.05cm/hr, 3.71cm/hr의 속도로 동결시켰으며 $-20^{\circ}C$ 냉동고에서 16주간 저장하여 저장 중 쇠고기의 이화학적 성질을 조사하였다. 저장기간이 길어짐에 따라 pH는 다소 증가하여 16주 저장육에서 $0.1{\sim}0.2\;unit$의 pH상승을 보여주고 있으며, 동결속도가 빠를수록 pH 변화는 적었다. 유리지방산 함량 및 TBA가 역시 저장기간이 길어질수록 계속 증가하고 있으나 3. 71cm/hr의 동결속도에서 가장 낮은값을 유지 하고 있었으며, TBA 가와 유리지방산 함량과의 상관계수가 0.804로 나타나 지방산패 에 유리 지방산이 관여하는 것으로 나타났다. 0.97cm/hr, 2.05cm/hr, 3.71cm/hr의 동결속도에서 16주 저장육의 염용성단백질 추출성은 각각 17.7%, 6.1%, 1.6% 감소를 보여준 반면 수용성단백질 추출성은 26.0%, 21.2%, 18.5%의 감소를 나타내고 있어 동결속도의 영향이 염용성단백질추출성에 더욱 크게 나타났으나 동결변성은 오히려 수용성단백질이 더욱 빨리 진행되는 것으로 나타났다.

• 한국수정란이식학회지
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• 제29권1호
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• pp.13-19
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• 2014

This study was carried out to study the survival rate of thawed Hanwoo embryos frozen by the slow-rate freezing or the cryotop vitrification method. Hanwoo cumulus-oocyte complexes were recovered from ovaries at a slaughter house, matured for 20~22 hours, fertilized with Hanwoo semen for 5~6 hours, and cultured for 7~9 days in $38.5^{\circ}C$, 5% $CO_2$ incubator. For freezing, Day 7~9 blastocysts were collected. Embryos for the slow-rate freezing were equilibrated in 1.8 M ethylene glycol (EG) with Dulbecco's phosphate-buffered saline (D-PBS). Programmable cell freezer was precooled down to $-7^{\circ}C$, and the straw was seeded during 8 minutes-holding time, and was cooled to $-35^{\circ}C$ at the cooling rate of $0.3^{\circ}C/min$, and then was plunged and stored in liquid nitrogen. Embryos for the cryotop vitrification were treated in TCM199 with 0.5 M sucrose, 16% EG, 16% dimethylsulfoxide (DMSO). Embryos were then loaded individually onto cryotop and plunged directly into liquid nitrogen. The survival rates of embryos frozen by these two freezing methods were evaluated at 12 to 24h post-thawing. The survival rates of frozen/thawed Hanwoo embryos by the cryotop vitrification method ($56.86{\pm}26.53%$) were slightly higher than those by the slow-rate freezing method ($55.07{\pm}26.43%$) with no significant difference. Using the cryotop vitrification and the slow-rate freezing of Hanwoo blastocysts on Day 7 following in-vitro fertilization (IVF) treatment, the survival rates of frozen/thawed Hanwoo embryos were $72.65{\pm}18.3%$ and $79.06{\pm}17.8%$, respectively. The survival rates by the cryotop vitrification were higher than those by the slow-rate freezing on both Day 8 and 9 with significantly higher survival rate on Day 9 (p<0.05). Using the cryotop vitrification and the slow-rate freezing of Hanwoo embryos to compare between three different blastocyst stages, the survival rates of the blastocyst stage embryos were $66.22{\pm}18.8%$ and $45.76{\pm}12.8%$, respectively with higher survival rate by the vitrification method (p<0.05). And the survival rate of expanded blastocysts was higher than those of early blastocysts and blastocysts in two freezing methods with significantly higher survival rate by the slow-rate freezing method (p<0.05).

• 한국가금학회지
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• 제14권2호
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• pp.145-151
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• 1987

식육을 장기저장 할 때 발생되는 단백질의 변성과 해동시 육량감소를 줄이기 위한 방법의 하나로써 최적동결방법을 모색하고자 동결속도에 따른 저장중 닭고기의 물리화학적 변화를 조사한 결과 동결속도가 빠를수록 pH증가가 지연되어 장기보존의 가능성을 보였다. TBA가 및 유리지방산 생성율은 저장기간이 길어짐에 따라 증가하는 추세를 보이고 있으나 부위육의 종류에 따라 동결속도의 영향이 다르게 나타나고 있다. 그러나 단백질추출성을 비교할 때 염용성단백질과 수용성단백질의 추출성은 부위육의 종류에 관계없이 3cm/hr이상의 동결속도에서 저장기간 중 추출성이 가장 높게 나타났으며, 이러한 경향은 다리살 보다 가슴살의 추출성이 더욱 높게 유지되고 있다. 이상의 결과에서 송풍동결기를 이용하여 닭고기를 동결시킬 경우 3cm/hr이상의 동결속도로 동결시켜 냉동저장 할 때 좋은 품질의 냉동육을 얻을 수 있는 것으로 판단되었다.

• International Journal of Industrial Entomology and Biomaterials
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• 제47권2호
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• pp.126-133
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• 2023

Genetic resources of mulberry trees are commonly preserved as trophosomes, which are vulnerable to environmental factors, such as natural disasters, diseases, and pests. This study establishes a basic protocol for ultra-low temperature cryopreservation of mulberry trees using a two-step freezing process. The procedure was established using the "Daeshim" variety and then tested on genetic resources from 24 other mulberry varieties. Samples were first dried to a moisture content of 33-43% in a low-temperature forced-air chamber at -5 ℃, then slowly frozen from -5 ℃ to -20 ℃, and preserved in liquid nitrogen (-196 ℃). To determine the regeneration rate, isolated dormant buds were inoculated into MS basal medium, and grown shoots were grafted onto 1-year-old rootstock via chip budding and then cultured. After freezing in liquid nitrogen, the "Daeshim" variety exhibited a survival and regeneration rate of more than 70% and 50%, respectively. Applying the two-step freezing process to genetic resources from 24 mulberry species yielded average survival and regeneration rates of 85.3% and 75.5%, respectively. Morus alba showed survival and regeneration rates of 100%, confirming the efficacy of the two-step freezing method. These results indicate the high feasibility of ultra-low-temperature cryopreservation through two-step freezing of dormant buds from mulberry genetic resources. Additional research is required into the variations in regeneration rates with freezing period in liquid nitrogen.

• 한국가축번식학회지
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• 제16권2호
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• pp.125-131
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• 1992

This study was carried out to investigate the effects of concentration and equilibration time of cryoprotective agents on the survival rate of slowly and ultrarapidly frozen porcine embryos. The porcine embryos following dehydration by cryoprotective agents and a various concentration of sucrose were directly plunged into liquid nitrogen and thawed in 38$^{\circ}C$ water bath. Survival rate was defined as development rat to the morula and blastocyst stage after in vitro culture or by FDA test. The results are summarized as follows : 1. The survival rates of porcine embryos after ultrarapid frozen-thawing in the freezing medium of 0.25M sucrose added 2.0, 2.5, 3.0, 3.5, or 4.0M glycerol was 65.3, 61.8, 64.3, 59.4 or 39.4%, respectively. Addition of 0.25M sucrose into the freezing medium containing 2.0M glycerol showed higher survival rate than those of 2.5~4.0M glycerol. 2. The survival rates of porcine embryos after ultraradpid frozen-thawing in the freezing medium of 0.25M sucroese added 2.0, 2.5, 3.0, 3.5 or 4.0M DMSO was 65.6, 67.6, 68.6, 60.6 or 23.6%, respectively. However, addition of 0.25M sucrose into the freezing medium containing 3.0M DMSO showed higher survival rate than those of 2.0, 2.5, 3.5 or 4.0M DMSO. 3. The survival rates of porcine embryos after ultrapid frozen-thawing in the freezing medium of 0.25M sucrose added 2.0, 2.5, 3.0, 3.5 or 4.0M propanediol was 63.2, 60.3, 62.1, 52.3 or 24.3%, respectively. Addition of 0.25M sucroese into the freezing medium containing 2.0M propanediol showed higher survival rate than those of 2.5~4.0M glycerol. 4. The survival rates of porcine embryos after ultrarapid frozen-thawing the freezing medium of 2.0M glycerol added 0.10, 0.25, 0.50 or 0.75M sucrose was 61.8, 70.8, 67.6 or 52.2%, respectively. Addition of 2.0M glycerol into the freezing medium containing 0.25M sucreose showed higher survival rate than that those of 0.10, 0.50 or 0.75M sucrose. 5. The higher suvival rate of porcine embryos were attained at short period of equilibration time 92.5~5min.) in the freezing medium added 0.25M sucreose and 3.0M compared to those of 10 or 20min. equilibration time in the same condition.