• 제목/요약/키워드: thawing methods

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Viability of cells in aspirated fat tissue after 1 year cryopreservation (1년 이상 냉동 보관한 흡인 지방조직 내의 세포 생존)

  • Son, Daegu;Oh, Jaehoon;Choi, Taehyun;Kim, Junhyung;Han, Kihwan
    • Archives of Plastic Surgery
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    • v.36 no.2
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    • pp.135-139
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    • 2009
  • Purpose: The use of an autogenous fat graft has become a common procedure in plastic surgery. However, questions remain concerning on the viability of fat cells and preservation method of aspirated fat. The purpose of this study was to examine the viability of fat cells stored at $-20^{\circ}C$ in the freeze for 1 year after harvest from abdominal liposuction. Methods: Eighteen adults (aged 24 to 65 years old, 16 female and 2 male) were recruited for this study. Harvested aspirated fat tissues were obtained by suction - assisted lipectomy and frozen at $-20^{\circ}C$ commercial refrigerator for one year (average 12.5 months). The viability off at cells in specimens were measured after thawing. The numbers of viable cells were measured on a fluorescence microscope after staining with fluorescein diacetate and propidium iodide. GPDH (Glycerol - 3 - phosphate dehydrogenase) activity was measured. Cell culture was done for 3 weeks. Results: There were no viable cells under the fluorescence microscope, no detectable GPDH activity, and no cultured cells. Conclusion: These findings suggest that aspirated fat after frozen storage for one year at $-20^{\circ}C$ freezer is inadequate to reuse.

Assessment of Sperm Characteristics in Fresh and Frozen Semen of Miniature-Pig

  • Lee S. H.;Kim T. S.;Cheong H. T.;Yang B. K.;Kim C. I.;Park C. K.
    • Reproductive and Developmental Biology
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    • v.28 no.4
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    • pp.261-265
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    • 2004
  • The present study was conducted to assess sperm characteristics in miniature-pig. The semen samples were transported to the laboratory at 17℃ within 3 hours after collection. The extended semen was stored at 17℃, and sperm quality was evaluated at 0, 1, 3, 5 and 7 days after storage. The semen volume of miniature-pig (62±22㎖) was significantly (p<0.05) lower than that of Duroc (155±25㎖) and Yorkshire (154±23㎖). Significant differences were also observed in sperm concentrations. During 3 days of storage, sperm viability did not differ among miniature-pig, Duroc and Yorkshire. However, the viability was significantly (p<0.05) lower in miniature-pig than in Duroc and Yorkshire semen after Day 3 of storage. In abnormality, acrosome intactness and intensity, there were no differences among miniature-pig, Duroc and Yorkshire semen. On the other hand, the viability of frozen-thawed sperm in miniature-pig was significantly (p<0.05) lower than in that of Duroc and Yorkshire. This study also examined CTC patterns in frozen-thawed spermatozoa. The rates of AR pattern were higher in miniature-pig than in Duroc and Yorkshire. However, no difference was found in F, B and AR patterns. The results of present study suggest that further research is necessary to develop of semen extender and freezing methods to improve sperm quality in miniature-pig.

Current status of assisted reproductive technology in Korea, 2010

  • Lee, Gyoung Hoon;Song, Hyun Jin;Lee, Kyu Sup;Choi, Young Min
    • Clinical and Experimental Reproductive Medicine
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    • v.42 no.1
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    • pp.8-13
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    • 2015
  • Objective: Great advances have been made in the field of assisted reproductive technology (ART) since the first in vitro fertilization (IVF) baby was born in Korea. This study was designed to report on the current status of ART therapy in South Korea between January 1 and December 31 of 2010. Methods: A revised survey, originally developed by the International Committee Monitoring Assisted Reproductive Technologies, was sent to all available ART centers via email in 2013. Fresh embryo transfer (FET) cases were categorized into standard IVF or intracytoplasmic sperm injections. These cases, the thawing embryo transfer (TET) cases, and other related procedures were surveyed. Results: Data from 30,785 ART procedures were provided by 78 clinics. Of the 28,200 cycles in which oocytes were retrieved, 92.2% of these cycles were completely transferred. In addition, 8,075 cycles were confirmed to be clinical pregnancies in the FET cycles, which represent a pregnancy rate of 28.6% per oocyte pick-up and 31.1% per embryo transfer. The most common number of embryos transferred in the FET was three embryos (37.3%) followed by two embryos (36.3%) and one embryo (14.0%). Of the 6,648 TET cycles transferred, 2,356 clinical pregnancies were confirmed by ultrasonography. The most common number of embryos in the TET group was two embryos (43.4%) followed by three embryos (25.4%) and one embryo (18.9%). Conclusion: The clinical pregnancy rate per transfer in the FET cycles was similar in 2009 and 2010. Among the FET cycles where one or two embryos were transferred, the clinical pregnancy rate per transfer slightly increased from 2009 (28.7%) to 2010 (32.9%).

A Study to Evaluate Performance of Poly-Urethane Polymer Concrete for Long-Span Orthotropic Steel Bridge (장경간 강바닥판 케이블교량에 적용하기 위한 폴리우레탄 폴리머콘크리트의 공용특성 연구)

  • Park, Heeyoung;Lee, Junghun;Kwak, Byeongseok;Choi, Iehyun;Kim, Taewoo
    • International Journal of Highway Engineering
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    • v.15 no.1
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    • pp.1-9
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    • 2013
  • PURPOSES: The purpose of this study is to evaluate physical properties, durability, fatigue resistance, and long-term performance of poly-urethane concrete (PU) which can be possible application of thin layer on long-span orthotropic steel bridge and to check structural stability of bridge structure. METHODS : Various tests of physical properties, such as flexural strength, tensile strength, bond strength and coefficient of thermal expansion tests were conducted for physical property evaluation using two types of poly urethane concrete which have different curing time. Freezing and thawing test, accelerated weathering test and chloride ion penetration test were performed to evaluate the effect of exposed to marine environment. Beam fatigue test and small scale accelerated pavement test were performed to assess the resistance of PU against fatigue damage and long-term performance. Structural analysis were conducted to figure out structural stability of bridge structure and thin bridge deck pavement system. RESULTS: The property tests results showed that similar results were observed overall however the flexural strength of PUa was higher than those of PUb. It was also found that PU materials showed durability at marine environment. Beam fatigue test results showed that the resistances of the PUa against fatigue damage were two times higher than those of the PUb. It was found form small scale accelerated pavement test to evaluate long-term performance that there is no distress observed after 800,000 load applications. Structural analysis to figure out structural stability of bridge structure and thin bridge deck pavement system indicated that bridge structures were needed to increase thickness of steel deck plate or to improve longitudinal rib shape. CONCLUSIONS: It has been known that the use of PU can be positively considered to thin layer on long-span orthotropic steel bridge in terms of properties considered marine environment, resistance of fatigue damage and long-term performance.

In vitro Follicular Growth and Ovulation of Mouse Preantral Follicles Cryopreserved by Vitrification (초자화동결된 생쥐 Preantral Follicle의 체외성장과 배란)

  • Park, Ji-Kwon;Paik, Won Young
    • Clinical and Experimental Reproductive Medicine
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    • v.32 no.2
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    • pp.91-99
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    • 2005
  • Objective: To define an appropriate vitrification condition of preantral follicle that yields high survival and to evaluate growth and ovulation rate of mouse follicles during in vitro culture after vitrification. Methods: Preantral follicles were isolated mechanically from mouse ovaries that were surgically recovered from mice aged 14 days. Retrieved preantral follicles were placed in EG (Ethylene Glycol) for 2, 5, 10 minutes and transferred to EFS-40 (40% EG, 18% Ficoll-70, 0.5 M sucrose) for 0.5, 1, 2 minutes. And then, preantral follicles were placed onto an EM grid and submerged immediately in liquid nitrogen. Thawing was carried out at room temperature. After defining the most appropriate vitrification condition that yields high survival, in vitro growth and ovulation rate of follicles were evaluated. Results: Appropriate vitrification condition that yield high survival rate ($83.2{\pm}2.1%$) of preantral follicle was EG for 5 minutes and EFS-40 for 0.5 minutes. In vitro survival rate of the vitrified preantral follicles were $85.5{\pm}0.5%$, $67.9{\pm}0.8%$ and $40.2{\pm}0.5%$ on day 2, 6 and 10. And in vitro growth of the vitrified preantral follicles were $107.1{\pm}16.1{\mu}m$, $117.1{\pm}18.4{\mu}m$, $178.4{\pm}45.6{\mu}m$ and $325.4{\pm}54.4{\mu}m$ on day 0, 2, 6 and 10. Although in vitro survival rate and growth of vitrified preantral follicles were lower than that of non-vitrified preantral follicles, the patterns of survival and growth were similar in vitrified and non-vitrified preantral follicles. The ovulation rate of antral follicles that was grown from vitrified preantral follicles was $32.6{\pm}1.2%$. Conclusion: Vitrified preantral follicles could be grown to antral sizes, and mature oocytes that can be used for IVF-ET programs were produced successfully. These data suggest that cryopreservation of preantral follicle by vitrification can be used for the preservation of the fertility.

Effects of Mito-TEMPO on the survival of vitrified bovine blastocysts in vitro

  • Jeong, Jae-Hoon;Yang, Seul-Gi;Park, Hyo-Jin;Koo, Deog-Bon
    • Journal of Animal Reproduction and Biotechnology
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    • v.36 no.4
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    • pp.299-306
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    • 2021
  • Vitrification methods are commonly used for mammalian reproduction through the long-term storage of blastocyst produced in vitro. However, the survival and quality of embryos following vitrification are significantly low compared with blastocyst from in vitro production (IVP). This study evaluates that the survival of frozen-thawed bovine embryos was relevant to mitochondrial superoxide derived mitochondrial activity. Here we present supplementation of the cryopreservation medium with Mito-TEMPO (0.1 µM) induced a significant (p < 0.001; non-treated group: 56.8 ± 8.7%, reexpanded at 24 h vs Mito-TEMPO treated group: 77.5 ± 8.9%, re-expanded at 24 h) improvement in survival rate of cryopreserved-thawed bovine blastocyst. To confirm the quality of vitrified blastocyst after thawing, DNA fragmentation of survived embryos was examined by TUNEL assay. As a result, TUNEL positive cells rates of frozen-thawed embryos were lower in the Mito-TEMPO treated group (4.2 ± 1.4%) than the non-treated group (7.1 ± 3.5%). In addition, we investigated the intracellular ROS and mitochondrial specific superoxide production using DCF-DA and Mito-SOX staining in survived bovine embryos following vitrification depending on Mito-TEMPO treatment. As expected, intracellular ROS levels and superoxide production of vitrified blastocysts after cryopreservation were significantly reduced (p < 0.05) according to Mito-TEMPO supplement in freezing medium. Also, mitochondrial activity measured by MitoTracker Orange staining increased in the frozen-thawed embryos with Mito-TEMPO compared with non-treated group. These results indicate that the treatment of Mito-TEMPO during cryopreservation might induce reduction in DNA fragmentation and apoptosis-related ROS production, consequently increasing mitochondrial activation for developmental capacity of frozen-thawed embryos.

The relationship between reactive oxygen species, DNA fragmentation, and sperm parameters in human sperm using simplified sucrose vitrification with or without triple antioxidant supplementation

  • Juanpanich, Theesit;Suttirojpattana, Tayita;Parnpai, Rangsun;Vutyavanich, Teraporn
    • Clinical and Experimental Reproductive Medicine
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    • v.49 no.2
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    • pp.117-126
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    • 2022
  • Objective: This study examined whether the addition of triple antioxidants (3A)-10 µM acetyl-L-carnitine, 10 µM N-acetyl-L-cysteine, and 5 µM α-lipoic acid-in freezing-thawing medium during human sperm cryopreservation using the sucrose vitrification (SuV) and liquid nitrogen vapor (Vapor) techniques could improve post-thaw survival of spermatozoa. Methods: We analyzed 30 samples from healthy human sperm donors. Each sample was allocated into one of five groups: fresh control, SuV, SuV+3A, Vapor, and Vapor+3A. The sperm motility, morphology, viability, intracellular and extracellular reactive oxygen species (ROS) levels, and sperm DNA fragmentation (SDF) were evaluated. Results: The cryopreserved spermatozoa had significantly reduced percentages of motility (p<0.05) and viability (p<0.05). Antioxidant supplementation non-significantly improved these parameters (p>0.05). No significant differences were found in sperm morphology between the fresh and frozen-thawed groups (p>0.05). After freezing, the extracellular ROS levels in the frozen-thawed groups were significantly higher (p<0.05) than in the fresh group. However, we did not find any differences in intracellular ROS parameters among these groups (p>0.05). The SDF was higher in the SuV and Vapor groups than in the fresh group, but without statistical significance (p=0.075 and p=0.077, respectively). Conclusion: Cryopreservation had detrimental effects on sperm motility, viability, and extracellular ROS levels, without changing the morphology or intracellular ROS levels. Antioxidant supplementation was slightly effective in preventing SDF in frozen-thawed spermatozoa.

Quality Enhancement of Frozen Chicken Meat Marinated with Phosphate Alternatives

  • Mahabbat Ali;Shine Htet ,Aung;Edirisinghe Dewage Nalaka Sandun Abeyrathne;Ji-Young Park;Jong Hyun Jung;Aera Jang;Jong Youn Jeong;Ki-Chang Nam
    • Food Science of Animal Resources
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    • v.43 no.2
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    • pp.245-268
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    • 2023
  • The effects of phosphate alternatives on meat quality in marinated chicken were investigated with the application of chilling and freezing. Breast muscles were injected with solution of the green weight containing 1.5% NaCl and 2% sodium tripolyphosphate (STPP) or phosphate alternatives. Treatment variables consisted of no phosphate [control (-)], 0.3% sodium tripolyphosphate [control (+)], 0.3% prune juice (PJ), 0.3% oyster shell, 0.3% nano-oyster shell, and 0.3% yeast and lemon extract (YLE) powder. One-third of the meat samples were stored at 4℃ for 1 d, and the rest of the meats were kept at -18℃ for 7 d. In chilled meat, a lower drip loss was noted for control (+) and YLE, whereas higher cooking yield in YLE compared to all tested groups. Compared with control (+), the other treatments except PJ showed higher pH, water holding capacity, moisture content, lower thawing and cooking loss, and shear force. Natural phosphate alternatives except for PJ, improved the CIE L* compared to control (-), and upregulated total protein solubility. However, phosphate alternatives showed similar or higher oxidative stability and impedance measurement compared to control (+), and an extensive effect on myofibrillar fragmentation index. A limited effect was observed for C*, h°, and free amino acids in treated meat. Eventually, the texture profile attributes in cooked of phosphate alternatives improved except for PJ. The results indicate the high potential use of natural additives could be promising and effective methods for replacing synthetic phosphate in chilled and frozen chicken with quality enhancement.

Cryopreservation of in vitro Grown Shoot Tips of Korean Potato Varieties by Droplet-vitrification

  • Ji-Won Han;Jinjoo Bae;Jae-Young Song;Ho Chul Ko;Sung-Hee Nam;Jung-ro Lee
    • Proceedings of the Plant Resources Society of Korea Conference
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    • 2023.04a
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    • pp.33-33
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    • 2023
  • Potatoes are the world's 4th major food crop after maize, rice, and wheat and also are a staple food for 1.3 billion people. Due to their wide adaptability to various environmental conditions, their yeild capacity, and high commercial value, potatoes have contributed to global food security. Many potato germplasms are commonly preserved as whole plants in fields or in storage to maintain their particular genetic combinations. However, field maintenance is expensive and has the risk of potential losses from diseases, pests, plant ageing and climate change. Over the past four decades, meaningful efforts have been made toward the safe long-term conservation of potatoes through cryopreservation methods such as droplet-vitrification. In this study, we tested 4 Korean potato varieties('Golden Egg', 'Golden Ball', 'Ja-Young' and 'Ha-Ryeong') with the modified potato droplet -vitrification protocol. Potato shoot tips are precultured in a sucrose-enriched medium(0.3 and 0.7M for 7 and 17hrs, respectively) and submitted to a loading step with C4 solution for osmoprotection. The treated explants were dehydrated with Plant Vitrification Solution(PVS)2 which is 80% A3 solution in ice for 30 minutes. Thawing and unloading steps were performed with 0.8M sucrose solution for 30 sec(40℃) followed by 30min(25℃, room temperature). In a potato post-culture medium(MS+0.1 mg·L-1 GA3+0.1 mg·L-1 kinetin), we obtained a survival rates of post-thawed explants ranging 16.1-82.2%. The results suggest that modified and optimized protocols are required dependinig on every cultivar, genetic and ecological types. To achieve higher survival and regeneration rates, each step within the cryoprocedure must be carefully optimized.

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Enhancement of cryopreserved rooster semen and fertility potential after oral administration of Thai ginger (Kaempferia parviflora) extract in Thai native chickens

  • Vibuntita Chankitisakul;Supakorn Authaida;Wuttigrai Boonkum;Sarunya Tuntiyasawasdikul
    • 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.