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ABSTRACT ARCHIVE |
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CryoLetters is a bimonthly, international journal for low temperature science and technology |
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CryoLetters 35 (6), 451-461 (2014) CRANIAL BONE REGENERATION AFTER CRANIOPLASTY USING CRYOPRESERVED AUTOGENOUS BONE BY A PROGRAMMED FREEZER WITH A MAGNETIC FIELD IN RATS *Masato Kaku, Hiroyuki Koseki, Shunich Kojima, Hiromi Sumi, Address correspondence to: Masato Kaku, Department of Orthodontics and Craniofacial Developmental Biology, Hiroshima University Graduate School of Biomedical Sciences, 1-2-3
Kasumi, Minami-ku, Hiroshima 734-8553, Japan Abstract BACKGROUND: The purpose of this study was to develop a bone tissue bank using a programmed freezer with a magnetic field. Parietal bones were removed from rats and used for organ culture examination (non-cryopreserved, cryopreserved with a magnetic field (CAS) and cryopreserved without a magnetic field group). Next, other parietal bones were used for histological examination. The cryopreserved bones by a CAS freezer and dried bones were transplanted respectively. Control bones were replanted without cryopreservation. Animals were sacrificed at 4, 8, 12 and 24 weeks after surgery. After organ culture, the isolated osteoblasts from parietal bones which were cryopreserved by a CAS freezer can survive and proliferate as much as non-cryopreserved group. Histological examinations showed new bone formation in control and CAS group. These results suggest that bone tissue cryopreservation by CAS freezer can be successfully used for bone grafting which may be a novel option for regeneration medicine. Keywords: cryopreservation, magnetic field, bone grafting
CryoLetters 35 (6), 462-472 (2014) EFFECT OF PRE-STORAGE ON LILIUM MARTAGON L. SEED LONGEVITY FOLLOWING CRYOPRESERVATION M. Urbaniec-Kiepura* and A. Bach Department of Ornamental Plants, University of Agriculture in Kraków, Al. 29 Listopada 54, 31-425 Kraków, Poland Abstract BACKGROUND: The aim of the study was to compare different strategies for the cryopreservation of Lilium martagon L. seeds. MATERIALS AND METHODS: The starting material was seeds of the martagon lily (without or with a special pretreatment involving sucrose pre-culture dehydratation and desiccation) subjected to cryopreservation after preliminary storage under conventional conditions (-5°C, 5°C, or 15°C). The effects of storage, pretreatment and cryopreservation on the seeds were assessed in terms of seed germination and phenotypic changes in the seedlings derived from those seeds. RESULTS: Subjecting the seeds to cold hardening or pretreatment did not affect significantly their germination capacity or the average germination time after cryopreservation. Combination of these strategies, however, significantly affected the capacity of the seeds to germinate. The highest germinability (100%) after cryopreservation was observed in the seeds stored at 15°C and subjected to sucrose pre-culture dehydration and air flow desiccation (seed moisture content [MC] 13.1%), and in those seeds stored at the same temperature but subjected subsequently to 5 h desiccation, not pretreated, with MC of 7.6%. Cryopreservation did not affect the germination capacity of the seeds but shortened the average germination time from 40.7 to 34.4 days. CONCLUSIONS: The obtained results demonstrate that a simple cryopreservation method without any special pretreatment of seeds can be used successfully for preserving L. martagon seeds. Neither the low temperature nor the sucrose concentration used affected the germination capacity or the average germination time of L. martagon seeds following cryopreservation. Keywords: biodiversity, cryopreservation, seeds, Lilium martagon
CryoLetters 35 (6), 473-481 (2014) CAN LINOLEIC ACID IMPROVE THE QUALITY OF FROZEN THAWED BULL SPERM? Serhat Büyükleblebici1, Umut Taşdemir2*, Pürhan Barbaros Tuncer2, 1Department of Reproduction and Artificial Insemination, Faculty of Veterinary, Aksaray University, Aksaray, Turkey Abstract BACKGROUND: Cryopreservation is known to have a detrimental effect on the motility, viability and membrane integrity of sperm cells. OBJECTIVE: The aim of this study was to investigate the effect of various amount of linoleic acid supplementation to the Tris extender, on bull sperm parameters, DNA integrity and oxidative stress after freeze-thawing. METHODS: Ejaculates were split into five aliquots and extended to a final concentration of 18x106 spermatozoa/ml with the base extender containing different doses of linoleic acid 0.125 ml, (L125); 0.250 ml (L250); 0.5 ml (L500), 1 ml (L1000) and no additive (control; L0). The extended samples were equilibrated slowly to 4°C for 4 h and then froze using a digital freezing machine. Frozen straws were thawed individually in water bath at 37°C for 30 s to analyse progressive motility and sperm motion characteristics as well as membrane integrity. Biochemical assays were performed in a spectrophotometer using commercial kits. DNA damage was evaluated by Comet Assay. RESULT: The addition of various linoleic acid did not improve the sperm subjective, CASA and progressive motilities, sperm motility characteristics and DNA integrity (P>0.05). L500 exhibited the greatest values for membrane integrity than that of the other groups (P<0.001). All supplementation groups led to lower percentages of tail abnormalities in comparison to the control (P<0.001). L500 and L1000 significantly decreased total abnormalities. In conclusion, our findings showed that L500 linoleic acid supplementation in semen extender was of great beneficial effect on frozen-thawed bull semen in terms of morphology and plasma membrane integrity. Keywords: Bull sperm, cryopreservation, DNA integrity, linoleic acid, oxidative stress
CryoLetters 35 (6), 482-494 (2014) SEA URCHIN (PARACENTROTUS LIVIDUS) CRYOPRESERVED EMBRYOS SURVIVAL AND GROWTH: EFFECTS OF CRYOPRESERVATION PARAMETERS AND REPRODUCTIVE SEASONALITY E. Paredes1* and J. Bellas2 1Departamento de Ecoloxía e Bioloxía Animal, Universidade de Vigo, Estrada Colexio Universitario s/n, 36310 Vigo, Galicia, Spain. Abstract The cryopreservation of embryos can be a powerful biotechnological tool to supply all year-round biological material for sea urchin aquaculture production. This study investigates different methodological and biological factors that may affect the result of the cryopreservation process of sea urchin (Paracentrotus lividus) embryos. Our data indicate that neither embryo density nor the use of different cryopreservation containers presented effect on the cryopreservation outcome. Contrary to other marine invertebrates, for sea urchin embryo cryopreservation ultrapure water cannot be used as CPA solvent, yielding zero survival. After studying the reproductive parameters along the reproductive season, we found a positive correlation between both male and female Condition Index (C.I.), and between the oocyte weight and C.I. Both the histology study of female gonads and the C.I. variation, suggest that the sea urchin natural spawning period in the Ría de Vigo occurs between June and July. We found no correlation between any of the reproductive parameters monitored and the cryopreservation outcome. Keywords: Cryopreservation, sea urchin, Paracentrotus lividus, Aquaculture
CryoLetters 35 (6), 495-500 (2014) INFLUENCES OF DEVELOPMENTAL STAGES, PROTECTIVE ADDITIVES AND CONCENTRATIONS OF CRYOPROTECTIVE AGENTS ON THE CRYOPRESERVATION OF PACIFIC OYSTER (Crassostrea gigas) LARVAE Youn Hee Choi1 and Young Jin Chang2* 1Institute of Fisheries Sciences, and 2Department of Marine Bio-materials and Aquaculture, Pukyong National University, Busan, Korea.
Abstract BACKGROUND: The selection of the optimal developmental stage, appropriate cryoprotectants (CPAs) and their concentrations is important for successful cryopreservation. OBJECTIVE: To investigate the effects of developmental stage and CPA concentrations on the cryopreservation of Pacific oyster (Crassostrea gigas) larvae. MATERIALS AND METHODS: Seven developmental stages, various CPAs and concentrations were investigated for optimizing cryopreservation of Pacific oyster larvae. The morphologies of pre-frozen and frozen-thawed larvae were evaluated using scanning electron microscopy and transmission electron microscopy. RESULTS: The survival rate of frozen-thawed larvae increased with developmental stage; late umbo veligers exhibited a survival as high as 98.6%. The addition of 0.2M or 0.5M sucrose improved the survival of larvae, and 2.0M ethylene glycol (EG) positively influenced the survival of frozen-thawed larvae. Moreover, the frozen-thawed larvae possessed irregularly arranged cilia and displayed a rough surface shell and a round-lumped cilium head. CONCLUSION: The findings indicate that the most desirable cryopreservation of Pacific oyster larvae may occur at any developmental stage except for the early trochophore stage. Sucrose at 0.2M or 0.5M and EG at 2.0M or 2.5M are appropriate cryoprotectant additives. Keywords: Pacific oyster, Crassostrea gigas, developmental stage, ethylene glycol.
CryoLetters 35 (6), 501-506 (2014) MALDI-ToF STUDY OF PEPTIDES FROM CRYOPRESERVED TISSUE FRAGMENTS Liliia Rohoza, Iryna Bespalova, Sergiy Galchenko* and Boris Sandomirsky Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine, Kharkiv, 61015, Ukraine. Abstract BACKGROUND: Previously it was shown that the extracts of cryopreserved organ fragments, containing tissue specific peptides, stimulates the reparative regeneration. In order to define the mechanism of action, it is necessary to determine the peptidic composition of extracts. OBJECTIVE: To examine molecular-mass distribution of peptides in the extracts of cryopreserved fragments of pigs’ heart and spleen as well as piglets’ heart and skin. MATERIALS AND METHODS: The extracts were obtained from cryopreserved fragments of porcine organs. MALDI-ToF method was used to study the peptidic composition of the extracts. RESULTS: The most intense mass spectra peaks, which may correspond to single peptidic molecules, were compared with Protein Knowledgebase (UniProtKB) data. A number of recorded peptides in the extracts expresses high biological activity. Keywords: extract, organ, cryopreservation, peptides, MALDI-ToF.
CryoLetters 35 (6), 507-515 (2014) CRYOPRESERVATION OF Populus Trichocarpa AND Salix DORMANT BUDS WITH RECOVERY BY GRAFTING OR DIRECT ROOTING Remi Bonnart1, John Waddell1,2, Kathy Haiby3, 1USDA-ARS National Center for Genetic Resources Preservation, 1111 S. Mason St. Abstract BACKGROUND: Methods are needed for the conservation of clonally maintained trees of Populus and Salix. In this work, Populus trichocarpa and Salix genetic resources were cryopreserved using dormant scions as the source explant. OBJECTIVE: We quantified the recovery of cryopreserved materials that originated from diverse field environments by using either direct sprouting or grafting. MATERIALS AND METHODS: Scions (either at their original moisture content of 48 to 60% or dried to 30%) were slowly cooled to -35°C, transferred to the vapor phase of liquid nitrogen (LNV, -160°C), and warmed before determining survival. RESULTS: Dormant buds from P. trichocarpa clones from Westport and Boardman, OR had regrowth levels between 42 and 100%. Direct rooting of cryopreserved P. trichocarpa was also possible. Ten of 11 cryopreserved Salix accessions, representing 10 different species, exhibited at least 40% bud growth and rooting after 6 weeks when a bottom-heated rooting system was implemented. CONCLUSION: We demonstrate that dormant buds of P. trichocarpa and Salix accessions can be cryopreserved and successfully regenerated without the use of tissue culture. Keywords: dormant buds, Salix, Populus, grafting, rooting, cryopreservation, liquid nitrogen
CryoLetters 35 (6), 516-520 (2014) COMPARATIVE ANALYSIS OF CRYOPROTECTIVE AGENTS INFLUENCE ON THERMODYNAMIC AND KINETIC PARAMETERS OF EQUINE AND HUMAN HEMOGLOBIN MOLECULES Aleksandra V. Zinchenko and Yuliana S. Govorova Department of Cryobiophysics, Institute for Problems of Cryobiology and Cryomedicine, National Academy of Sciences of Ukraine, Kharkov, Ukraine Abstract BACKGROUND: Critical to the understanding the mechanism of destruction and protection during cryopreservation of biological objects is the knowledge of the conformational transitions of biopolymers experiencing low temperatures in the presence of cryoprotective agents. This information may be derived from the kinetic and thermodynamic parameters of macromolecular thermal denaturation kinetics under different environmental conditions. OBJECTIVE: The study deals with the influence of cryoprotective agents (glycerol, 1.2-propanediol (1.2-PD), and dimethyl sulfoxide (DMSO)) on thermodynamic and kinetic parameters of equine and human hemoglobin. METHODS: Thermograms were recorded with differential scanning adiabatic microcalorimeter (DASM-4, Biopribor, Russia). RESULTS: Temperatures and enthalpy changes in the denaturation of hemoglobins (ΔHcal) in the presence of cryoprotective agents from 0–40% (w/w) were determined. Within the whole concentration range glycerol slightly alters the hemoglobin denaturation temperature while DMSO and 1.2-PD lead to a significant decrease. The addition of cryoprotectants to hemoglobin solutions induces a complex behavior of ΔHcal concentration dependences of denaturation caused by intra- and intermolecular processes such as hydration of the porphyrin cycle, heme cleavage from heme-containing proteins, disorder in hydrophobic contacts with globin etc. These factors may decrease thermal stability by loosening hemoglobin molecules and spatial disruption of fragments of the protein. Activation energy of irreversible unfolding of equine hemoglobin at heating was estimated using the approach of Sanchez-Ruiz et al. DMSO and 1.2-PD decreased activation energy values. CONCLUSIONS: The data indicate the reduction of protein thermal stability by DMSO and 1.2-PD. Glycerol slightly increases hemoglobin thermal stability within the studied range of concentrations. Keywords: thermal denaturation, glycerol, 1.2-propanediol, dimethyl sulfoxide
CryoLetters 35 (6), 521-529 (2014) Approaches for the cryopreservation of Plantago algarbiensis, a rare endemic species of the Algarve Natacha Coelho1, María Elena González-Benito2 and Anabela Romano1,* 1Faculty of Sciences and Technology, MeditBio, University of Algarve, Campus de Gambelas, Ed. 8, 8005-139 Faro, Portugal. Abstract BACKGROUND: Plantago algarbiensis is an endangered endemic species from the Algarve, Portugal. OBJECTIVE: The main goal of this study was to investigate the viability of cryopreservation procedures in the conservation of seeds and nodal explants from this species. MATERIALS AND METHODS: Seeds were directly immersed in liquid nitrogen (LN) for 30 days. Two methods were tested for the cryopreservation of nodal explants, namely droplet-vitrification and encapsulation-dehydration. For both methods, nodal segments were precultured on Murashige and Skoog (MS) medium and recovered on MS supplemented with 0.2 mg l1 6-benzyladenine (BA), after freezing. RESULTS: After 30 days in LN, the germination capacity of seeds was not affected. The regrowth percentages of cryopreserved nodal segments were approximately 60%. With the droplet-vitrification method, a regrowth percentage of 60.0 ± 15.2% was obtained after 120 min exposure to PVS2 (plant vitrification solution 2) and with encapsulation-dehydration method the highest percentage, 63.3 ± 9.6%, was achieved after 3 h desiccation. CONCLUSION: Seed cryopreservation and cryopreservation of nodal segments by droplet-vitrification and encapsulation-dehydration are therefore effective approaches for the conservation of P. algarbiensis. Keywords: conservation, droplet-vitrification, encapsulation-dehydration, recovery medium, seed cryopreservation
CryoLetters 35 (6), 530-536 (2014) EFFECT OF CAFFEINE TREATMENT BEFORE VITRIFICATION ON MPF AND MAPK ACTIVITY AND SPONTANEOUS PARTHENOGENETIC ACTIVATION OF IN VITRO MATURED OVINE OOCYTES F. Ariu1*, L. Bogliolo1, G. Leoni2, L. Falchi 1; D. Bebbere , 1Obstetric and Gynecological Section, Department of Veterinary Medicine, Sassari, Italy. Abstract BACKGROUND: Molecules that stabilize protein kinases may be useful in overcoming the deleterious effects of cryopreservation. OBJECTIVE: To evaluate the effect of caffeine treatment before vitrification of in vitro matured ovine oocytes on the activity of MPF and MAPK as well as the spontaneous parthenogenetic activation after 24 h culture. MATERIALS AND METHODS: Oocytes obtained from slaughterhouse sheep ovaries were in vitro matured for 21 h, incubated for 3 h with or without caffeine and then vitrified. After warming, oocytes were processed for the analysis of chromatin configuration and the evaluation of spontaneous parthenogenetic activation (24 h in vitro culture). Fresh in vitro matured oocytes were used as control. RESULTS: Caffeine treatment before vitrification maintained the MPF activity at a level similar to that of fresh oocytes, and reduced the spontaneous parthenogenetic activation in comparison with oocytes that were not-treated with caffeine. CONCLUSION: Caffeine treatment prolongs the meiotic arrest of vitrified MII oocytes, likely via its action of stabilizing the MPF level. Keywords: Vitrification, caffeine, cryotop, parthenogenetic activation, protein kinases.
CryoLetters 35 (6), 537-542 (2014) INFLUENCE OF THE TOXICITY OF CRYOPROTECTIVE AGENTS ON THE INVOLVEMENT OF INSULIN-LIKE GROWTH FACTOR-I RECEPTOR IN SURF CLAM (SPISULA SACHALINENSIS) LARVAE Youn Hee Choi and Taek Jeong Nam* Institute of Fisheries Sciences, Pukyong National University, Busan 619-911, South Korea Abstract BACKGROUND: The signaling of insulin-like growth factor-I (IGF-I) is involved in the development, growth, reproduction and aging of vertebrates. However, few studies have investigated the involvement of IGF-I during states of extreme shock, such as those induced by potently toxic cryoprotective agents (CPAs) or low temperature conditions, in bivalves. OBJECTIVE: We investigated the toxicity of CPAs and the potential relationship between larval viability and the IGF-I receptor (IGF-IR) after treatment with CPAs or freezing in surf clam (Spisula sachalinensis) larvae. MATERIALS AND METHODS: The umbo larvae and different concentrations of CPAs (dimethyl sulfoxide, DMSO; ethylene glycol, EG) were used to investigate the toxicity of CPAs and the vitrification of surf clam larvae. The relationship between larval viability and the IGF-I receptor (IGF-IR) after treatment with CPAs or freezing was investigated using immunoblot analysis. RESULTS: An increase in concentration greater than 4M DMSO was fatal in larvae; however, 5M EG combined with a mixture of CPAs had no harmful effects. Moreover, live larvae immersed in a 5M EG solution remained intact and maintained their normal shape and organs. However, even though the larvae survived the CPA toxicity test, none of the vitrified larvae survived. After immersion into CPAs and vitrification, 97-kDa IGF-IR ß-subunits could be detected in all larvae; but tyrosine phosphorylation of the intracellular β-subunits was detected only in the control and live groups. CONCLUSION: IGF-IR was activated in the umbo larvae but not in dead surf clam larvae treated with CPA and frozen. Activation of IGF-IR has relevance to the umbo larval stage in live surf clams treated with CPAs. Keywords: Surf clam, umbo larvae, cryoprotective agent, toxicity, vitrification, IGF-I
CryoLetters 35 (6), 544-548 (2014) CONFOCAL MICROSCOPIC ANALYSIS OF THE MICROFILAMENT CONFIGURATIONS FROM HUMAN VITRIFICATION-THAWED OOCYTES MATURED IN VITRO Qianqian Ci1 2 3, Mei Li1 3, Yingchun Zhang2 3, Shuiying Ma1, 1Center for Reproductive Medicine, Shandong Provincial Hospital, Shandong University, Jinan; Abstract BACKGROUND: The alteration in microfilaments of human oocytes by vitrification has not been understood well. OBJECTIVE: To evaluate the effect of vitrification on the microfilaments of human in vitro matured oocytes and the time needed for the repair. MATERIALS AND METHODS: Human in vitro matured oocytes were divided into the control group (Group 1) and the vitrified group. The vitrified oocytes were further divided into four sub-groups that were cultured after thawing for 1h, 2h, 3h and 4h, respectively (Groups 2, 3, 4 and 5). RESULTS: The survival rate of oocytes was 87.4% after vitrification and thawing. The percentage of oocytes with a normal configuration of microfilaments in Group 2 and Group 3 was significantly lower than that of the fresh control group (Group 1), whereas the percentage of oocytes with normal microfilament configuration in Group 4 and Group 5 was comparable to the control group. CONCLUSION: Vitrification alters microfilament structure of oocytes, which takes at least 3h after thawing for the repair and recovery. Keywords: oocyte, in vitro maturation, vitrification, microfilament, confocal microscopy |
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