• 한국자원식물학회지
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• 제28권6호
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• pp.697-703
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• 2015

Twenty apple germplasm accessions from the Korean Genebank were successfully cryopreserved using two-step freezing to back up genetic resources maintained by field collections. This study examined the morphological and genetic stability of cryopreserved dormant apple buds that were stored in liquid nitrogen, and then rewarmed and regrown. Whole plants were regenerated directly from dormant buds through budding without an intermediary callus phase. The cryopreserved buds produced high levels of shoot formation (76.2-100%), similar to those of noncryopreserved buds (91.3-100%), with no observed differences between cryopreserved and noncryopreserved materials. Three of the twenty cryopreserved apple germplasm accessions were used to assess morphological and genetic stability. No differences in morphological characteristics including shoot length, leaf shape, leaf width/length ratio, and root length were observed between controls (fresh control and noncryopreserved) and cryopreserved plantlets. The genetic stability of regenerants (before and after cryopreservation) was investigated using inter simple sequence repeat (ISSR) markers. The ISSR markers produced 253 bands using four primers, ISSR 810, SSR 835, ISSR 864, and ISSR 899. These markers showed monomorphic banding patterns and revealed no polymorphism between the mother plant and regenerants before and after cryopreservation, suggesting that cryopreservation using two-step freezing does not affect the genetic stability of apple germplasm. These results show that two-step freezing cryopreservation is a practical method for long-term storage of apple germplasms.

• 한국양식학회지
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• 제17권1호
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• pp.46-50
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• 2004

In the present study, attempts were made to cryopreserve sea urchin, Anthocidaris crassispina sperm in liquid nitrogen, to evaluate the effects of various cryoprotectants and freezing rates on motility, survival rate and fertilization rate of the post-thawing sperm, and the ultrastructural changes of sperm after cryopreservation were observed. The highest values of sperm motility (motility index: 3.3$\pm$0.37) and survival rate (72$\pm$3.5%) were obtained with 15% dimethyl sulfoxide (DMSO), and these values were significantly higher than those of sperm preserved with glycerol. Comparisons of motilities and survival rates between treatments of difference freezing rates showed that there was no difference between procedures (a) 5$0^{\circ}C$/min to -8$0^{\circ}C$ (motility index: 3.3$\pm$0.31 ; survival late 70$\pm$2.7%) and (b) 3$0^{\circ}C$/min to -8$0^{\circ}C$ (motility index: 3.1$\pm$0.29; survival rate 69$\pm$3.7%), while the results of (c) 1$0^{\circ}C$/min to -8$0^{\circ}C$ were significantly lower than the others (motility index: 2.2$\pm$0.33 ; survival rate 42$\pm$4.6%). There was no significant difference in fertilization rate between fresh sperm and sperm preserved with 15% DMSO as cryoprotectant and freezing rate (3$0^{\circ}C$/min to -8$0^{\circ}C$). Some ultrastructural changes of sperm, such as the detachment of plasma membrane, the destruction of mitochondria, and the flagellum rolling up head, were observed after cryopreservation. Morphological normality of the sperm in 15% DMSO frozen at the ratio of 5$0^{\circ}C$/min to -8$0^{\circ}C$ was better than the others.

• 한국포장학회지
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• 제11권2호
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• pp.75-79
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• 2005

With three kinds of agricultural products such as perilla leaves, this research is focussed on how to keep a freshness of agricultural products during long time transport by sea. In case of export fresh agricultural products by sea, research on products loading method onto the pallet is very important factor for keeping effective cold air circulation inside the freezing marine container. Details are as follows: Clear examination of palletization for cold air ventilation inside the container. Optimization of package dimension for best loading efficiency onto the standard pallet. Best layout of palletized products inside the container. Research for the change of circumstance and product quality inside the pack.

• 한국식품과학회지
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• 제20권2호
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• pp.205-212
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• 1988

마늘의 영하온도에서 저장시 나타나는 내한성(耐寒性)의 원인규명을 위하여 인편의 성분조성이 빙점 강하에 미치는 영향에 대하여 시험하였다. 생체(生體)마늘의 성분조성의 특징은 가용성고형분이 $40^{\circ}$ Brix(건물기준액 90%)로 일반과채류의 $10^{\circ}$ Brix에 비(比)하여 월등히 높았으며 그 구성은 총 90%중 당류가 70% 단백질이 20%였다 그리고 불용성 고형분 10%의 조성은 회분 3% 조섬유 3%였고 그외 지방, 탄수화물 및 단백질을 합(合)하여 4%정도로 되어 있었다. 마늘 성분중 당류는 fructosan이 주종(主種)이었으며 이들은 $1{\sim}29$ 중합도(Polymerization degree)의 것이 혼재되어 있으며 특히 $4{\sim}5$중합도의 것이 전체 50%이상으로 가장 많았다. 마늘의 내한성 지표인 빙점은 수용성고형분 농도가 높을수록 낮았고 동일농도일 경우에는 저중합도(低重合度)의 fructosan일수록 낮았다.

• Fisheries and Aquatic Sciences
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• 제24권2호
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• pp.63-77
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• 2021

The aim of this research was to investigate different factors, including cryoprotective agents (CPAs), diluents, dilution ratios, equilibrium times, freezing rates, and thawing methods to optimize cryopreservation protocols for large yellow croaker (Larimichthys crocea). The parameters evaluated were sperm motility, sperm activity index (SAI), survival rate, and DNA damage. Different types of CPAs, such as dimethyl sulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), methanol, and glycerol, were tested for sperm preservation. The highest motility, SAI, and survival rate were observed when EG was used. Different diluents such as Stein's solution, Hank's balanced salt solution, marine fish Ringer's solution, artificial seminal plasma (ASP) of small yellow croaker, and Cortland solution were investigated. The highest post-thaw motility was observed upon using ASP as the diluent. Different concentrations of EG were then mixed with ASP to identify the optimal EG concentration. Experimental results showed that the motility (70.33 ± 1.20%), SAI (5), and survival rate (78.30 ± 0.42%) of post-thaw sperm were optimum when 10% EG and ASP were used as the CPA and diluent of cryopreservation, respectively. Post-thaw sperm motility was high at equilibration times below 150 s and at an optimum dilution ratio of 1:1 (sperm: CPA + diluent) and was not significantly different compared with fresh sperm motility. The freezing rate was found to be slow below -10℃/min. The thawing temperature of 45℃ was identified as ideal. The percentage of tail DNA in post-thaw sperm at 10% EG and ASP was also investigated and was found to have more significant DNA damage than that in fresh sperm but significantly lower damage than that in post-thaw sperm at EG concentrations of 5%, 15%, and 20% (p < 0.05). The cryopreservation protocols obtained in this study will be useful in large yellow croaker hatcheries.

• Clinical and Experimental Reproductive Medicine
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• 제20권2호
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• pp.165-176
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• 1993

These studies were carried out to investigate the optimal freezing protocol for 2 cell mouse embryos and to find the probability of quality control with 2-cell embryos frozen. The embryos showed the best survival by the protocol composed of a freezing solution with the cryoprotectants(1.5M propanediol + 0.1M sucrose), and a 2-steop thawing method(room temperature, 20 sec-37$^{\circ}C$, 20 sec). The developmental ability of frozen-thaw 2-cell embryos did not differ from that of fresh 2-cell embryos in m-KRB medium with 0.4% bovine serum albumin. But development of frozen-thaw embryos was depended on the supplements of the medium. In the albumin-free medium, the developmental rate(rate of blastocysts) was significantly reduced, compared with that in the medium with 0.4% BSA. Also, when frozen-thaw embryos were cultured in the meduim with human fetal cord serum(HCS), the developmental rate of frozen-thaw embryos was sligtly reduced, compared with that of fresh 2-cell embryos. Finally, frozen-thaw 2-cell mouse embryos were more sensitive to the toxic agent of disposable-plastic syringe. Therefore, toxicity of medium could be effectively detected by frozen-thaw 2-cell mouse embryos.

• Asian-Australasian Journal of Animal Sciences
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• 제22권4호
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• pp.507-515
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• 2009

A series of experiments were conducted to determine the suitable freezing and thawing temperatures for the freezing of boar semen in 5 ml maxi-straws. The ultrastructure, in vitro fertilization (IVF) and artificial insemination (AI) of frozen-thawed semen were also be evaluated. The 5 cm freezing height gave the best results not only in post-thaw motility rate (54.00%), but also in normal acrosome morphology rate (NAR) (80.23%). There was no significant difference in the post-thaw motility between different thawing temperatures and corresponding thawing times (p>0.05); the group of $52^{\circ}C$ and 25 s gave the highest motility rate (45.00%). As a whole, not only from the motility but also the NAR, thawing at $42^{\circ}C$ was better than the other two treatments. In the freezing packages, 5 ml maxi-straw gave a little lower mobility (40%), viability rate (49.58%), plasma membrane integrity rate (53.91%) and NAR (52.65%) than the 0.25 ml straw, but there was no significant difference between the two straw volumes (p>0.05). The IVF capacity of frozen-thawed semen in this experiment was similar to fresh semen. From ultrastructure observation, the main damage to boar spermatozoa after freezing was seen in the acrosome, such as swelling and formation of vesicles. After AI in recipient Shanghai White sows, frozen-thawed semen from 5 ml maxi-straws and pellets produced 72.2% and 80% conception rate and 7.8 and 8 litter sizes, respectively, and there was no significant difference between the 5 ml maxi-straw and the pellet (p>0.05).

• 한국식품영양과학회지
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• 제36권11호
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• pp.1444-1450
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• 2007

대추술의 가열살균으로 인한 품질저하를 개선하고자 초고압(500 MPa, 5분) 및 냉동처리($-20^{\circ}C$에서 3일간 냉동 후 $20^{\circ}C$에서 4시간 동안 해동)를 이용하여 대추술을 살균한 다음 가열처리구($63^{\circ}C$, 10분) 및 시판대추술과 비교하면서 품질특성을 조사하였다. 미생물은 초고압 및 시판대추술에서는 검출되지 않았고, 가열처리구에서는 세균류만이 10 CFU/mL 이하로 검출되었으며, 냉동처리구에서는 $10^2{\sim}10^3CFU/mL$ 수준으로 검출되어 $30{\sim}60%$의 살균효과를 보였다. 초고압, 냉동 및 가열처리는 대추술의 pH, 산도, 아미노산도, 환원당 및 에탄올함량 등의 성분에 유의적인 영향을 미치지 않았으나, 색도에는 유의적으로 영향을 미쳤으며 변화폭은 초고압 처리가 가장 작았고 다음으로 가열 및 시판, 냉동처리 순으로 크게 나타났다. 관능검사 결과에서는 가열처리 시 신맛 및 화한 맛이 감소하면서 전체적인 품질이 낮아진 데 비하여, 초고압 및 냉동처리 시에는 신맛과 화한 맛이 변하지 않으면서 품질이 유지되었다. 한편 시판 대추술에서는 탄내, 단내 및 단맛이 뚜렷이 증가하고 신내 및 신맛이 감소하면서 가장 낮은 품질을 보였다. 이상으로부터 초고압처리는 살균효과가 우수하고 제품의 성분 및 색도에 큰 영향을 미치지 않으면서 본래 대추술의 관능적인 특성을 최대 유지 할 수 있는 효과적인 비열살균법임을 알 수 있었고, 냉동처리 역시 관능적으로 받아들일 수 있는 수준으로 품질이 유지되었으나 불완전한 살균으로 인한 저장성 연구가 추가되어야 할 것이다.

• Clinical and Experimental Reproductive Medicine
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• 제49권2호
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• pp.87-92
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• 2022

Objective: The aim of this study was to determine the effects of melatonin and selenium in freezing extenders on frozen-thawed rat sperm. Methods: Semen samples were collected from 20 adult male Wistar albino rats. Following dilution, the samples were divided into six groups: four cryopreserved groups with 1 mM and 0.5 mM melatonin and selenium supplements, and two fresh and cryopreserved control groups. The rapid freezing technique was used to freeze the samples. Flow cytometry was used to assess plasma membrane integrity, mitochondrial membrane potential, and DNA damage, while computer-assisted sperm analysis was used to assess motility. Results: Total motility was higher in the 1 mM melatonin supplementation group than in the cryopreserved control group (mean±standard error of the mean, 69.89±3.05 vs. 59.21±1.31; p≤0.05). The group with 1 mM selenium had the highest plasma membrane integrity (42.35%±1.01%). The cryopreserved group with 0.5 mM selenium had the highest mitochondrial membrane potential, whereas the cryopreserved control group had the lowest (45.92%±4.53% and 39.45%±3.52%, respectively). Conclusion: Cryopreservation of rat semen supplemented with 1 mM melatonin increased sperm motility after freeze-thawing, while supplementation with 0.5 mM selenium increased mitochondrial activity.

• Molecules and Cells
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• 제26권6호
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• pp.558-565
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• 2008

Although the fertilizing ability of spermatozoa is greatly reduced after freezing, complete understanding of alterations induced by cryopreservation has not been elucidated. The present study evaluates the effects of cryopreservation on the $Ca^{2+}$ handling of boar spermatozoa using several sperm activators. Intracellular $Ca^{2+}$ signals from single spermatozoa were measured using confocal $Ca^{2+}$ imaging of unfrozen samples and of other spermatozoa after having been frozen. Elevation of the external $K^{2+}$ concentration elicited a three times larger $Ca^{2+}$ increase in fresh spermatozoa than in cryopreserved spermatozoa. Caffeine elicited $Ca^{2+}$ transients with some oscillations in the fresh spermatozoa, but not in the thawed spermatozoa. Depletion of the $Ca^{2+}$ store with thapsigargin induced a rapid rise in $Ca^{2+}$ in the control but generated a smaller increase of $Ca^{2+}$ after thawing. Exposure to progesterone induced a biphasic rise of the $Ca^{2+}$ level in the fresh spermatozoa only. Sperm viability was reduced by cryopreservation. Resting $Ca^{2+}$ levels in fresh and cryopreserved spermatozoa were similar. Longer incubation (2.5 h) of thawed spermatozoa partly recovered the $Ca^{2+}$ response to the interventions. These results suggest that cryopreservation reduces the responsiveness of spermatozoa to depolarization, modulators of the internal $Ca^{2+}$ store and progesterone in terms of the $Ca^{2+}$ signal, thus providing a possible mechanism for reduced fertility observed in cryopreserved boar spermatozoa.