Abstracts: CryoLetters 29 (4), 2008

CryoLetters is a bimonthly, international journal for low temperature science and technology

CryoLetters 29(4), 271-276 (2008)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Félix Pérez-García* and M. Elena González-Benito

Departamento de Biología Vegetal, Escuela Universitaria de Ingeniería Técnica Agrícola, Universidad Politécnica de Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain


Seed germination of four Halimium sapecies [H. atriplicifolium (Lam.) Spach, H. halimifolium (L.) Willk., H. ocymoides (Lam.) Willk., H. umbellatum (L.) Spach ssp. viscosum (Willk.) O. Bòlos & Viso] and eight Helianthemum species [H. almeriense Pau, H. appeninum (L.) Mill., H. cinereum (Cav.) Pers., H. hirtum (L.) Mill., H. marifolium (L.) Mill., H. nummularium (L.) Mill., H. syriacum (Jacq.) Dum. Cours., H. squamatum (L.) Dum. Cours.], all Cistaceae species from the Mediterranean region, was studied after seed storage in liquid nitrogen (LN,  -196ºC) for four months. In all samples assayed, mechanical scarification of the seed coat was carried out to enhance seed germination. For most samples studied, final germination percentages were unaffected by storage of seeds in LN, both for intact seeds and for scarified seeds. The germination rate of cryopreserved seeds, expressed as days to reach 50% of the final germination percentage (T50), was lower only for four samples of intact seeds and for three samples of scarified seeds. Therefore, this study shows that seed cryopreservation could be a suitable procedure for the long-term seed conservation of several Halimium and Helianthemum species.

Keywords: Cistaceae, cryopreservation, liquid nitrogen, seed germination, seed scarification



CryoLetters 29(4), 277-283 (2008)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Comparative Study of Different Mechanical Models for Identification of Viscoelastic Parameters of Cryopreserved Rabbit Carotid Arteries

Gang Zhao1*, Yu-xuan Zheng1, Fei Yu1, Zhi-feng Liu2, Dong Lei1, Da-yong Gao3

1Department of Modern Mechanics, University of Science and Technology of China, Hefei 230027, P.R. China.
2Department of Thermal Science and Energy Engineering, University of Science and Technology of China, Hefei 230027, P.R. China.
3Department of Mechanical Engineering, University of Washington, Seattle, WA 98195-2600, USA.
*To whom correspondence should be addressed; E-mail:


To characterize quantitatively the viscoelastic behaviors of cryopreserved rabbit carotid arteries, five mechanical models, i.e. standard linear model, and four kinds of generalized Maxwell models, were comparatively applied. Specific aim of this study was to test the validity of these models to simulate the stress relaxation behaviors of the cryopreserved arteries and to find a best-fit model for identification of viscoelastic parameters. Non-linear curve fittings were applied to the stress-relaxation data measured with dynamic mechanical thermal analyzer (DMTA IV; Rheometric. Scientific Inc.; Piscataway, NJ, USA), it was found that 5-element model (generalized Maxwell body composed of an elastic element and two Kelvin body) could provide satisfactory description of the cryopreserved rabbit carotid arteries.

Keywords: viscoelasticity, stress relaxation, cryopreserved, DMTA



CryoLetters 29(4), 285-292 (2008)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Xin-Li Zhou1, Hui He1, Bao-Lin Liu1, Tse-Chao Hua1* and Ying Chen2

1Institute of Biothermal Sciences, University of Shanghai for Science and Technology, Shanghai 200093, China.
2Institute of Blood test, Shanghai Antu Hospital, Shanghai 200093, China.


Successful storage of red blood cells (RBCs) by freeze-drying technique has important implications in blood transfusion and clinical medicine. We presented a method of preservation RBCs by pretreating them with glycerol solution and then freeze-drying. The effects of glycerol pretreatment on recovery and antioxidant enzyme activities of lyophilized RBCs were investigated. Rehydration of pretreated in 40% glycerol and freeze-dried RBCs resulted in 55.3±4.26% numerical recovery and 53.5±3.85% hemoglobin recovery, which were significantly higher than freeze-dried RBCs without glycerol pretreatment (P<0.01). Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-PX) activities of 40% glycerol pretreated and freeze-dried RBCs almost remained fully active after freeze-drying. After 180 days storage, the activities decreased by 25.6%, 21.5% and 18.4%, respectively, which are significantly less than those of freeze-dried RBCs without glycerol pretreatment (P<0.1). These data demonstrated that glycerol pretreatment had beneficial effects on the recovery and antioxidant enzyme activities of lyophilized RBCs.

Keywords: Glycerol; Freeze-drying; Red Blood Cells



CryoLetters 29(4), 293-300 (2008)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


C. Wilkens1 and H. Ramløv*

Department of Science, Systems and Models, Roskilde University, P.O. Box 260, DK-4000 Roskilde, Denmark.
*Corresponding author, e-mail:
1Current address: Department of Molecular Biology, University of Copenhagen, Ole Måløevsvej 5, DK-2200 Copenhagen N, Denmark


Larvae of Rhagium mordax empty their guts in preparation for the winter, which alone may enable the larvae to supercool down to -20°C or below. This should be sufficient for the larvae to over winter in Denmark if they can prevent inoculation. Antifreeze proteins (AFP) prevent inoculation in adult Rhagium inquisitor and this is also likely in the larvae of R. mordax, as they are in contact with ice in their hibernacula during the winter.

Larvae of R. mordax probably produce AFPs in the early autumn, however, in some individuals thermal hysteresis (TH) as high as 5.01 °C was observed in June. Whether or not these individuals have a constant level of TH in their haemolymph all year or if they produce further antifreeze proteins during the autumn is unknown. The lowest measured in January was 7.49°C (the highest during this month was 9.08ºC) so it is likely that the individuals with the highest TH in June also produce AFPs. Haemolymph osmolality in R. mordax is relatively low compared to other freeze avoiding insects, samples taken in January peak at 741 mOsm (±127 mOsm). The results of this study are compared with similar data for the closely related R. inquisitor.

Key words: Antifreeze proteins; Rhagium mordax; thermal hysteresis; Coleoptera; Cerambycidae.

Abbreviations: Tm – melting point; Th – hysteresis freezing point; AFP – antifreeze protein; Tf – freezing point; TH – thermal hysteresis



CryoLetters 29(4), 301-314 (2008)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Evgenia Isachenko1,2*, Vladimir Isachenko1,2, Frank Nawroth1,3, Gohar Rahimi1, Rolf Kreienberg2, Jochen Reinsberg4, and Juergen Weiss2

1Department of Obstetrics and Gynaecology, University of Cologne, Kerpener Str. 43, 50931 Cologne, Germany, E-mail:
2Department of Obstetrics and Gynaecology, University of Ulm, Prittwitzstrasse 43, 89075 Ulm, Germany
Endokrinologikum Hamburg, Centre of Hormone and Metabolic Diseases, Reproductive Medicine and Gynaecological Endocrinology, Lornsenstraße 22767 Hamburg, Germany
4Department of Gynecological Endocrinology and Reproductive Medicine, University of Bonn, Sigmund-Freud-Str. 25, 53127 Bonn, Germany


The aim of this study was to evaluate the most successful vitrification protocol.  The ovarian tissue pieces were randomly distributed into seven groups including fresh control. Each experimental group was divided into three subgroups according to the following cooling modes: a) in 1.8 ml cryo-vials with 1ml vitrification medium, b) in 1.8 ml cryo-vials with 100µl vitrification medium, or c) by direct dropping with 50 µl vitrification medium into liquid nitrogen. The best results were observed in the protocol using 2.62 M dimethylsulphoxide + 2.6 M acetamide + 1.31 M propylene glycol + 0.0075M polyethylene glycol in combination with direct dropping of ovarian tissue pieces into liquid nitrogen.  The vitrified and rewarmed samples after in vitro culture with this protocol showed 86% normally developed follicles, compared with 92 % in fresh non-treated control. The concentrations of hormones in spent medium from culture of the same samples were 319 pg/ml for 17βestradiol and 2.6 ng/ml for progesterone compared with fresh non-treated control (253 pg/ml and 6 ng/ml, respectively). The results obtained by vitrification of ovarian tissue with this protocol were compatible with those of the fresh ovarian tissue.

Keywords: human ovarian tissue, vitrification, hormone



CryoLetters 29(4), 315-320 (2008)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

comparison of efficiency of open pulled straw (OPS) and cryotop vitrification for cryopreservation of in vitro matured pig oocytes

Ying Liu1, Yutao Du1,2,3, Lin Lin1,2,3, Juan Li 1,2, Peter M. Kragh1,2, Masashige Kuwayama4, Lars Bolund2, Huanming Yang3 and Gábor Vajta5*

1Institute of Genetics and Biotechnology, Faculty of Agricultural Sciences, and
2Institute of Human Genetics, University of Aarhus, Denmark;
3Beijing Institute of Genomics at Shenzhen, Shenzhen, China;
4Kato Ladies' Clinic, Shinjuku, Tokyo, Japan;
Pivet Medical Centre, 166-168 Cambridge St, Leederville, Perth, WA-6007 Australia,
*corresponding author email address:


During the past few years vitrification has been acknowledged as a viable alternative to traditional slow-rate freezing in both animal and human embryology. However, few data are available regarding the comparative efficiency of published and commercially available vitrification methods. The purpose of our work was to compare the OPS and Cryotop technology for cryopreservation of porcine in vitro matured oocytes. In a 2 x 2 factorial experiment, OPS and Cryotop devices and solutions were used for vitrification and warming. Two hours after warming oocytes were parthenogenetically activated and cultured in vitro. In 6 replicates a total of 1153 oocytes were vitrified. The cleavage rate after vitrification with Cryotop device and Cryotop solution (34.7%) were higher than those after vitrification with Cryotop device and OPS solution, or OPS device with both OPS and Cryotop solution (11.5, 5.1 and 11.3%, respectively). Further embryo development has shown a similar difference: Cryotop device applied with Cryotop solution resulted in 11.6% blastocyst/oocyte rates, higher than those achieved with Cryotop device and OPS solutions, or OPS device with both Cryotop and OPS solution (1.6, 1,65 and 0.6%, respectively). Our results indicate that for cryopreservation of some highly sensitive biological specimen including porcine oocytes Cryotop vitrification is superior to the OPS technique.

Keywords: pig, cryopreservation, chilling, parthenogenetic activation.



CryoLetters 29(4), 321-328 (2008)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Avik Ray* and Sabita Bhattacharya

Department of Botany, Bose Institute, 93 / 1 A.P.C. Road, Kolkata – 700 009, West Bengal, India.
*Author for correspondence (e-mail:,; phone: 91-033-2303-1132; fax : 91-33-350-6790)


This paper describes the cryopreservation by PVS2 vitrification of Rauvolfia serpentina (L.) Benth ex kurz, an important tropical medicinal plant. The effects of type and size of explants, sucrose preculture (duration and concentration) and vitrification treatment were tested. Preliminary experiments with PVS1, 2 and 3 produced shoot growth only for PVS2. When optimizing the PVS2 vitrification of nodal segments, those of 0.31 – 0.39 cm in size were better than other nodal sizes and or apices. Sucrose preculture had a positive role in survival and subsequent regrowth of the cryopreserved explants. Seven days on 0.5 M sucrose solution significantly improved the viability of nodal segments. PVS2 incubation for 45 minutes combined with a 7-day preculture gave the optimum result of 66%. Plantlets derived after cryopreservation resumed growth and regenerated normally.

Keywords: PVS2; sucrose preculture; Rauvolfia serpentina; nodal segment; explant size; vitrification



CryoLetters 29(4), 329-338 (2008)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Cryopreservation of resting cysts of the freshwater ciliate Meseres corlissi BY conventional two-step methods and one-step vitrification ProtocolS

Helga Müller1*, Undine E.M. Achilles-Day2 and John G. Day2

1Private Laboratory, Jacob-Burckhardt-Str. 18, D-78464 Konstanz, Germany
2Culture Collection of Algae and Protozoa (CCAP), Scottish Association for Marine Science, Dunstaffnage Marine Laboratory, Dunbeg, Argyll, PA37 1QA, UK


Trophic cells of the freshwater ciliate Meseres corlissi CCAP 1647/1 proved to be recalcitrant to all the cryopreservation methods tested; however, resting cysts of this strain were amenable to both conventional two-step cryopreservation and ultra-rapid vitrification methods. Conventional controlled rate cooling and Mr. Frosty cooling methods, employing 5% dimethylsulphoxide (DMSO) as a cryoprotectant were effective in preserving cysts in either liquid medium or soil suspensions. Alternative cryopreservation methods involving dehydration of soil/cyst suspensions, in the absence of any colligitative cryoprotectant, and rapid cooling over liquid nitrogen or plunge freezing were effective. The level of residual moisture was the critical factor with no survival observed in any samples with >35% residual moisture, and high levels of survival (>50%) in samples with <14% residual moisture. Trophic cells obtained from cryopreserved cysts appeared healthy and did not differ obviously from the controls with respect to morphology, movement, division and encystment/excystment reactions. In a test culture derived from material which had been cryopreserved by the rapid cooling method a maximum growth rate of 1.32 d-1 (at 22°C) was determined, a value which agrees well with earlier observations on the original strain.

Keywords: Ciliate, culture collections, cryopreservation, Meseres corlissi, protozoa, vitrification



CryoLetters 29(4), 339-350 (2008)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Cryopreservation by encapsulation–dehydration of plumules of coconut (Cocos nucifera L.)

Oulo N’Nan1,4, Valérie Hocher1, Jean-Luc Verdeil2, Jean-Louis Konan3,
Koffi Ballo3, Fanja Mondeil4 and Bernard Malaurie1*

1IRD (Institut de Recherche pour le Développement), BP 64501, 911 Av Agropolis 34394 Montpellier Cedex 5 – France,
2CIRAD, TA40/02 Avenue Agropolis, 34398 Montpellier Cedex 5, France.
3Station Marc Delorme CNRA 07 BP 13 Abidjan 07 Côte d’Ivoire.
4Université d’Abidjan Cocody, laboratoire de génétique, UFR Biosciences 22 BP 582 Abidjan 22 Côte d’Ivoire.


This study describes the use of an encapsulation-dehydration cryopreservation technique on coconut plumules (apical dome with three or four leaf primordia) excised from embryos. In order to establish a reliable cryopreservation process for plumules, several different key factors were tested: pretreatment duration, sugar concentration, dehydration period and freezing. In parallel, histological studies were performed to describe the structural changes of tissues and plumule cells subjected to dehydration and freezing. A good survival level of around 60% was obtained. However, after 8 months culture regrowth, this level decreased to a maximum of 20% which was achieved using sucrose treatment. In this paper we report for the first time the regeneration of leafy shoots from coconut plumules after cryopreservation.

Keywords: Cocos nucifera L., cryopreservation, encapsulation-dehydration, histology, plumule, sucrose.



CryoLetters 29(4), 351-361 (2008)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Expression and antioxidant enzymes in Chaetoceros neogracile, An Antarctic Alga

Seul-Ki Park1, Eon Seon Jin2 and Mi-Young Lee1*

1Department of Medical Biotechnology, SoonChunHyang University, Asan PO Box 97, Chungnam, 336-600, Korea.
2Department of Life Science, Hanyang University, Seoul, Korea
*Corresponding author:


We examined low temperature-induced protein profile alterations in the Antarctic alga Chaetoceros neogracile using a proteomic approach. Chaetoceros neogracile was cultured at 4°C and then cooled to 0°C, and the resultant cold-induced alterations in protein expression patterns were analyzed by two-dimensional gel electrophoresis. Of the approximately 150 protein spots detected by Coomassie staining, we identified 15 with a greater than two-fold change in amount. Of these, ten proteins were up-regulated and five were down-regulated after cold exposure. Three cellular protein quality control proteins, such as chaperone protein DnaK, chaperone ClpB, and 26S protease regulatory subunit 6B homolog were prominently increased, whereas chaperone protein HtpG was decreased in response to cold stress. Moreover, changes in enzyme activity and isozyme profiles for superoxide dismutase, glutathione reductase, and glutathione S-transferase were also detected in the gel, using an enzyme activity staining method. These alterations in protein expression and antioxidant enzyme activity may be related to survival mechanisms of C. neogracile at low temperatures.

Keywords: proteomics, activity staining, low temperature, antarctic alga; Chaetoceros neogracile

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