• Title/Summary/Keyword: fresh and freezing

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Morphological and Genetic Stability of Dormant Apple Winter Buds After Cryopreservation

  • Yi, JungYoon;Lee, GiAn;Chung, JongWook;Lee, YoungYi;Kwak, JaeGyun;Lee, SeokYoung
    • Korean Journal of Plant Resources
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    • v.28 no.6
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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.

Effects of Cryoprotectants and Freezing Rates on Cryopreservation of Sea Urchin, Anthocidaris crassispina Sperm

  • Kang, Kyoung-Ho;Kho, Kang-Hee;Kim, YoungHun
    • Journal of Aquaculture
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    • v.17 no.1
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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.

Research on Packaging and Palletization for Improving Distribution Efficiency of Exported Fresh Agricultural Products (신선농산물의 수출 포장개발 및 파렛트화에 관한 연구)

  • Lee, Soo-Keun;Lee, Myung-Hoon
    • KOREAN JOURNAL OF PACKAGING SCIENCE & TECHNOLOGY
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    • v.11 no.2
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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.

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Physico-Chemical Characteristics of Components and Their Effects on Freezing Point Depression of Garlic Bulbs (마늘의 성분조성(成分組成)과 내한성(耐寒性) 연구(硏究))

  • Park, Moo-Hyun;Kim, Jun-Pyong;Kwon, Dong-Jin
    • Korean Journal of Food Science and Technology
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    • v.20 no.2
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    • pp.205-212
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    • 1988
  • The effect of physicochemical characteristics on the freezing point depression of garlic bulb was studied to examine the reasons of cryoprotectivity in garlic bulb stored at subzero temperature. The composition of fresh garlic was characterized by having high soluble solids($40^{\circ}$ Brix; 90% on dry basis), comparing with $10^{\circ}$ Brix in case of other fruits and vegetables. Soluble solids were composed of 70% sugars, 20% protein, and 10% insoluble solids(including 3% of ash, 3% of crude fiber, and 4% of fat). The main component of sugars in garlic was fructosan with 1-29 degree of polymerization(D.P) and the fructosan of 4-5 D.P was over 50% of total fructosan. Freezing point of garlic bulb, which is a parameter of cryoprotectivity, was depressed as the concentration of soluble solids increased, and as the D.P value decreased in the same concentration of soluble solids.

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Effect of diluent variation on cryopreservation of large yellow croaker Larimichthys crocea

  • Lim, Han Kyu;Irfan, Zidni;Lee, Hyo Bin;Song, Ji Hoon;Lee, Yun Ho
    • Fisheries and Aquatic Sciences
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    • v.24 no.2
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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.

Studies on Quality Control by Frozen-Thaw 2-Cell Mouse Embryos (냉동보존된 생쥐배아를 이용한 정도관리에 관한 연구)

  • Han, Sun-Nam;Kim, Hyang-Mee;Jung, Hae-Won;Oh, Seung-Eun;Son, Young-Soo;Yu, Han-Ki;Ahn, Jung-Ja;Woo, Bock-Hee
    • Clinical and Experimental Reproductive Medicine
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    • v.20 no.2
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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.

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Some Factors Affecting Freezing of Boar Semen in 5 ml Maxi-straws

  • Dai, J.J.;Wu, C.F.;Zhang, Defu;Yin, F.Z.;Zhang, T.Y.;Liu, D.;Wu, H.L.;Li, L.L.;Yang, S.T.;Wang, L.
    • Asian-Australasian Journal of Animal Sciences
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    • v.22 no.4
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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).

Quality of Jujube Wine with Hydrostatic Pressure and Freezing Treatment (초고압 및 냉동 처리한 대추술의 품질특성)

  • Park, Hee-Joeng;Kim, Kwang-Yup;Han, Gwi-Jung;Jeong, Heon-Sang
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.36 no.11
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    • pp.1444-1450
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    • 2007
  • To prevent the deterioration of jujube wine quality due to commercial heating process for sterilization, hydrostatic pressure (500 MPa, 5 min) or freezing (frozen at $-20^{\circ}C$ for 3 days, followed by thawing at room temperature for 4 hr) treatment was applied. Their microbial count, physicochemical property and sensory quality were investigated in comparison to heat-treated jujube wine ($63^{\circ}C$, 10 min) and commercial wine. Pressure-treated and commercial jujube wine were completely sterilized and heat-treated wine was decreased to <10 CFU/mL while freezing-treated jujube wine was partially sterilized $(30{\sim}60%)$. Hydrostatic pressure and freezing, and heat treatment had no influence on chemical compositions such as pH, acidity, amino acidity, reducing sugar and ethanol content, but significantly induced the changes of instrumental color. While sensory quality of heat-treated jujube wine was significantly deteriorated, reducing sour and burning taste, that of pressure and freezing-treated jujube wine was maintained fresh without decrease in sour and burning taste.

Melatonin and selenium supplementation in extenders improves the post-thaw quality parameters of rat sperm

  • Shahandeh, Erfan;Ghorbani, Mahboubeh;Mokhlesabadifarahani, Tahereh;Bardestani, Fateme
    • Clinical and Experimental Reproductive Medicine
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    • v.49 no.2
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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.

Effects of Cryopreservation on Ca2+ Signals Induced by Membrane Depolarization, Caffeine, Thapsigargin and Progesterone in Boar Spermatozoa

  • Kim, Joon-Chul;Li, Yuhua;Lee, Sunwoo;Yi, Young-Joo;Park, Chang-Sik;Woo, Sun-Hee
    • Molecules and Cells
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    • v.26 no.6
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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.