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Abstracts: CryoLetters 29 (6), 2008

CryoLetters 29 (6), 447-461 (2008)
© CryoLetters, c/o University of Bedfordshire, Luton LU2 8DL

 COINCIDENCE ERROR DURING MEASUREMENT OF CELLULAR OSMOTIC PROPERTIES BY THE ELECTRICAL SENSING ZONE METHOD

Adam Z. Higgins1 and Jens O.M. Karlsson2*

1School of Chemical, Biological and Environmental Engineering, Oregon State University, Corvallis, Oregon 97331-2702, USA.
2Department of Mechanical Engineering, Villanova University, 800 Lancaster Avenue, Villanova, Pennsylvania 19085-1699, USA.
*Correspondence author email:
karlsson@alum.mit.edu

Abstract

Coincidence is a phenomenon that occurs when electrical sensing zone instruments fail to temporally resolve two or more particles passing through the sensing zone in close proximity. We have investigated the potential for coincidence errors to confound the estimation of cellular osmotic properties.  A mathematical model was developed to predict the magnitude of coincidence error as a function of the instrument sensing volume, the cell concentration, and the degree of cell aggregation.  The model was validated in a representative instrument (Coulter counter model Z2, with a 100-µm aperture tube), for which the sensing volume was estimated to be approximately 2 nL. Furthermore, we measured the degree of cell aggregation in trypsinized cultures of MIN6 cells, and used these data to estimate the effect of coincidence on MIN6 cell volume measurements. Finally, we simulated water transport experiments, and determined the sensitivity of estimates of the osmotically inactive volume and the membrane water permeability to coincidence error. Our results revealed that coincidence can result in significant overestimation of these two parameter values for high cell concentrations and for suspensions containing cell aggregates.

Keywords: Coulter counter, hydraulic conductivity, permeability, osmotically inactive volume, coincidence, cell aggregation

 

 

CryoLetters 29 (6), 463-475 (2008)
© CryoLetters, c/o University of Bedfordshire, Luton LU2 8DL

EVALUATION OF ZEBRAFISH (Danio rerio) OVARIAN FOLLICLE VIABILITY BY SIMULTANEOUS STAINING WITH FLUORESCEIN DIACETATE AND PROPIDIUM IODIDE

T. Zampolla, T. Zhang and D.M. Rawson*

LIRANS Institute of Research in the Applied Natural Sciences, University of Bedfordshire, 250 Butterfield, Great Marlings, Luton, Bedfordshire, LU2 8DL, United Kingdom
*Correspondence author e-mail:
david.rawson@beds.ac.uk

Abstract

Reliable fish oocyte quality assessment methods are essential in developing protocols for cryopreservation as well as their in vitro maturation and fertilisation. Current ovarian follicle viability assessment methods either lack sensitivity (e.g. Trypan Blue staining-TB) or are stage dependent (e.g. in vitro maturation and observation of germinal vesicle breakdown-GVBD). The aim of the present study was to develop a new viability assessment method for zebrafish ovarian follicles that is reliable, sensitive and not-stage specific. Fluorescein Diacetate (FDA) and Propidium Iodide (PI) were used for the first time to assess viability of zebrafish ovarian follicles. After preliminary studies to evaluate the efficacy of FDA and PI, a combination of these two fluorochromes was subsequently chosen and compared with TB staining and GVBD test in a series of cryoprotectant toxicity studies and following cryopreservation using stage III ovarian follicles. In all cases the FDA-PI test proved to be more sensitive than TB staining but less sensitive than the GVBD test. Ovarian follicle survivals after 4M Methanol treatment for 30 min at 22ºC were 67.4 ± 4.4%, 43.9 ± 3.8% and 19.6 ± 1.9% using TB, FDA-PI and GVBD test respectively. Survivals after cryopreservation procedure were 38.9 ± 4.0% and 28.9 ± 2.5% using TB and FDA-PI respectively when Hank's solution was used as medium and 45.2 ± 4.3% and 35.2 ± 3.5% when KCl buffer was used. The results showed the method to be promising, and it may offer a new approach for viability assessment of fish ovarian follicles.

Keywords: fluorescein diacetate, propidium iodide, ovarian follicles, zebrafish

 

 

CryoLetters 29 (6), 477- 483 (2008)
© CryoLetters, c/o University of Bedfordshire, Luton LU2 8DL

STUDIES ON CRYOPROTECTANT TOXICITY TO EARLY STAGE ZEBRAFISH (Danio rerio) OVARIAN FOLLICLES

S. Tsai, D. M. Rawson, and T. Zhang*

LIRANS Institute of Research in the Applied Natural Sciences, University of Bedfordshire, 250 Butterfield, Great Marlings, Luton, Bedfordshire, LU2 8DL, UK
*Correspondence author   e-mail:
tiantian.zhang@beds.ac.uk  

Abstract

Cryoprotectants are substances characterised by their ability to reduce cryoinjury of biological materials during the course of freezing. Unfortunately cryoprotectants can be toxic for cells. The aim of this study is to investigate the toxicity of cryoprotectants to early stage ovarian follicles of zebrafish (Danion rerio) before designing protocols for their cryopreservation. Commonly used cryoprotectants methanol, dimethyl sulfoxide (DMSO), propylene glycol (PG) and ethylene glycol (EG) were studied. Experiments were conducted with stage I and II zebrafish ovarian follicles, which were incubated in 50% L-15 medium containing different concentrations of cryoprotectants (0.25-5M) for 30 min at room temperature. Stage III zebrafish ovarian follicles were also used as comparisons. Two different tests were used to assess ovarian follicle viability: trypan blue (TB) and Fluorescein diacetate (FDA) + propidium iodide (PI) staining. Both TB and FDA+PI tests indicated that cryoprotectant toxicity to ovarian follicles increased in the order of methanol, DMSO, PG and EG. FDA+PI test was shown to be more sensitive than TB staining. No Observed Effect Concentrations (NOECs) for stage I and II follicles were 2M, 1M, 0.5M, and 0.25M for methanol, dimethyl sulfoxide (DMSO), propylene glycol (PG) and ethylene glycol (EG) respectively when assessed with FDA+PI. Stage III ovarian follicles appeared to be more sensitive than stage I and II ovarian follicles.

Keywords: cryopreservation, cryoprotectants, zebrafish (Danio rerio), ovarian follicles, toxicity, TB, FDA+PI

 

 

CryoLetters 29 (6), 485-491(20 08)
© CryoLetters, c/o University of Bedfordshire, Luton LU2 8DL

SEASONAL VARIATION OF TREHALOSE AND GLYCEROL CONCENTRATIONS IN WINTER SNOW-ACTIVE INSECTS

Stefano Vanin, Luigi Bubacco and Mariano Beltramini*

Department of Biology, University of Padua, via U. Bassi 58/b, 35121 Padova, Italy
*Correspondence author e-mail:
mariano.beltramini@unipd.it

Abstract

Different kinds of molecules were identified as antifreezing agents in the body fluids of cold tolerant invertebrates: sugars, polyols and proteins. While none of the active arthropods were so far reported to accumulate polyols, these compounds are present in the haemolymph of species that hibernate in a passive stage such as diapause. In this work we investigated insect species that are active during winter and we demonstrated the ability of the mecopteran Boreus hiemalis (Mecoptera, Boreidae), the wingless fly Chionea sp. (Diptera, Limoniidae) and cantharid larvae (Coleoptera, Cantharidae) to accumulate sugars in their haemolymph to survive during winter. We report, for the first time, that for snow-active insects, trehalose comprises an important haemolymph component, its concentration changing as a function of the season, suggesting that the same adaptive strategies against cold conditions have evolved both in winter active and winter diapausing insects.

Keywords: Supercooling point, trehalose, Boreus, Chionea, Cantharidae

 

 

CryoLetters 29 (6), 493-504 (2008)
© CryoLetters, c/o University of Bedfordshire, Luton LU2 8DL

PRESERVATION OF Quercus robur GERMPLASM BY CRYOSTORAGE OF EMBRYOGENIC CULTURES DERIVED FROM MATURE TREES AND RAPD ANALYSIS OF GENETIC STABILITY

C Sánchez*, M T. Martínez, N. Vidal, M. C. San-José, S. Valladares and A.M. Vieitez

Instituto de Investigaciones Agrobiológicas de Galicia, CSIC, Avda de Vigo s/n, Apartado 122, 15080 Santiago de Compostela, Spain.
*Correspondence author e-mail:
conchi@iiag.csic.es

Abstract

This study reports on the cryostorage of embryogenic lines derived from selected mature Quercus robur trees, following application of the PVS2-vitrification based procedure. In seven oak genotypes, embryo recovery levels ranging from 57-92% were obtained when 4-6 mg embryo clumps were precultured for 3 days on 0.3 M sucrose basal medium, treated with PVS2 solution for 60 min at 24ºC, and then immersed in liquid nitrogen (LN). Embryos of six out of seven lines were cryostored for one week and one year and used to evaluate cryopreservation tolerance, germination ability and to assess genetic fidelity by random amplified polymorphic DNA (RAPD) markers. There were no significant differences between the recovery frequencies of samples retrieved from LN after 1 week and 1 year of cryostorage. In five out of six lines, RAPD profiles of cryopreserved somatic embryos and regenerated plantlets were identical to those of the controls. Although polymorphisms were detected in only one cryostored embryo of one genotype, no genetic instability was found in the regenerated plantlets. This methodology appears to be suitable for long-term storage of this valuable germplasm, as the recovered plantlets were found to be genetically stable.

Keywords: cryopreservation, genetic fidelity, oak, plant regeneration, somatic embryogenesis, vitrification.

 

 

CryoLetters 29 (6), 505-515 (2008)
© CryoLetters, c/o University of Bedfordshire, Luton LU2 8DL

METABOLOMIC FINGERPRINT OF CRYO-STRESS IN A FREEZE TOLERANT INSECT

T.C. Hawes1,3, A. Hines1, M.R. Viant1, J.S. Bale1, M.R. Worland2, and P. Convey2

1School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K.
2British Antarctic Survey, Natural Environment Research Council, High Cross, Madingley Road, Cambridge CB3 OET, U.K.
3Current address: Department of Zoology, University of Otago, PO Box 56, Dunedin, New Zealand
*Correspondence author e-mail:
tinstone12@hotmail.com

Abstract

This study employed 1H NMR spectroscopy to assay the metabolome of the high Arctic freeze-tolerant dipteran larvae, Heleomyza borealis, after recovery from exposure to a range of sub-zero temperature treatments. Our data demonstrate the resilience of freeze tolerance in individuals of this permanently freeze-tolerant species that were acclimated to summer temperatures (5°C): recovery of homeostasis after 48h was not significantly disturbed by 2h exposures to -3, -12, or -20°C. Evidence of homeostatic perturbation to cryo-stress – both in terms of changes in specific metabolite concentrations as well as systemic changes in metabolism determined using multivariate pattern recognition techniques – was expressed almost entirely at a temperature coincident with the significant onset of mortality (-25°C) and considerably below the minimum winter temperatures of its over-wintering habitat (c.-12°C).

Keywords: freeze tolerance, NMR, metabolomics, Heleomyza borealis, homeostasis, proline

 

 

CryoLetters 29 (6), 517-526 (2008)
© CryoLetters, c/o University of Bedfordshire, Luton LU2 8DL

CRYOPRESERVATION OF Bletilla striata MATURE SEEDS, 3-DAY GERMINATING SEEDS AND PROTOCORMS BY DROPLET-VITRIFICATION

Nipawan Jitsopakul1, Kanchit Thammasiri2, 3* and Keiko Ishikawa4

1Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
2Department of Plant Science, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
3Institute of Science and Technology for Research and Development, Mahidol University, Nakhonpathom 73170, Thailand.
4Department of Research and Development, Japan Horticultural Production and Research Institute, Chiba 270-2221, Japan.
*Correspondence author email:
kanchitthammasiri@gmail.com

Abstract

Droplet-vitrification was studied for the cryopreservation of Bletilla striata mature seeds (0 day after sowing), 3-day germinating seeds and protocorms (6, 9 and 12 days after sowing). Mature seeds, 3-day germinating seeds and 6-day old protocorms were precultured in liquid medium supplemented with 0.3 M sucrose for 3 h on a shaker (110 rpm) and then dehydrated with 2 M glycerol and 0.4 M sucrose in liquid medium (loading solution) for 15 min and exposed to PVS2 solution for 60 min at 25°C. The plant materials were then immersed in liquid nitrogen, rewarmed rapidly and cultured on solidified ND medium supplemented with 3% sucrose for recovery. After cryopreservation, the highest germination percentage of mature seeds, 3-day germinating seeds and survival of cryopreserved 6-day old protocorms was 93%, 91% and 84%, respectively. For 9-day old protocorms, highest survival (66%) after cryopreservation was achieved after preculture with 0.5 M sucrose for 3 h on a shaker, dehydration with loading solution for 15 min, exposure to PVS2 solution for 40 min at 25°C, and culture on solidified ND medium supplemented with 480 mg l-1 ammonium nitrate and 3% sucrose. No survival was observed in cryopreserved 12-day old protocorms.

Keywords: Bletilla striata, cryopreservation, seeds, protocorms, droplet-vitrification

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