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Abstracts: CryoLetters 23 (2), 2002

CryoLetters 23,  (2002)
 ©CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Andrew A. Gage1,2,* and John G. Baust2

1Roswell Park Memorial Institute, State University of New York at Buffalo, 666 Elm Street, Buffalo, NY 14263,USA. Email:
2Institute of Biomedical Technology, State University of New York at Binghamton University, Science III Suite 144, Binghamton, NY 13902-6000, USA. Email:


Advances in cryosurgery since 1990 were initiated by the development of improved cryosurgical equipment and by the availability of intraoperative ultrasound to monitor the tissue-freezing process. Interest in research on the effects of freezing on tissue and on new clinical applications was then stimulated. The research led to a better understanding of the mechanisms of cryogenic injury, including cell death by apoptosis, which has emerged as a potential key to the use of adjunctive chemotherapy in the treatment of cancer.  Optimization of cryosurgical technique will also improve clinical results.

Keywords: Cryosurgery.  Cryotherapy.  Apoptosis.  Necrosis.



CryoLetters 23,  (2002)
 ©CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Glass transition and enthalpy relaxation of polyphosphate compounds

Kiyoshi Kawai, Toru Suzuki* and Rikuo Takai

Department of Food Science and Technology, Tokyo University of Fisheries, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan. E-mail:


The glass transition behaviour of polyphosphate compounds such as di- and tri- polyphosphates used as food additives and ATP, ADP existing in biosystems, were investigated by using DSC. From the DSC heating curves of the frozen solutions, the glass transition temperatures of the maximum freeze concentrated solutions, Tg', were determined. It was found that Tg's for polyphosphates are relatively high. The lyophilized tripolyphosphates, ATP and ADP also showed the glass transition at a relatively high temperature, depending on the moisture content. In addition, the enthalpy relaxation behaviour of glassy ATP and ADP was examined and analyzed by using the Kohlrausch–Williams-Watts (KWW) and the Vogel-Fulcher-Tammann (VFT) equations. Judging from the parameters of the KWW and VFT equations, the amorphous states of ATP and ADP were suggested to be more fragile than trehalose and sucrose.

Keywords: polyphosphate, ATP, glass transition, state diagram, enthalpy relaxation, KWW



CryoLetters 23,  (2002)
 ©CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Chris J. Clarke, Sarah L. Buckley, Nigel Lindner*

Unilever, Colworth House, Sharnbrook, Beds, MK44 1LQ, UK


Antifreeze proteins (AFPs) have been reported in the academic literature for many years, and are increasingly arousing interest in the technical and popular media, particularly because of their potential applications. However, the term "antifreeze" does not always accurately describe their natural function, or their application in frozen systems, where they do not prevent freezing, but control the size, shape and aggregation of ice crystals. We survey the properties and applications of AFPs and propose a more generally applicable name based on the fact that all AFPs bind to ice and consequently influence crystal growth and interactions: "Ice Structuring Proteins".

Keywords: Antifreeze protein, antifreeze peptide, AFP, thermal hysteresis protein, THP



CryoLetters 23,  (2002)
 ©CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


F. Nawroth1, V. Isachenko1, S. Dessole2, G. Rahimi1, M. Farina2, N. Vargiu2, P. Mallmann1, M. Dattena2, G. Capobianco2, D. Peters1, I. Orth1 and E. Isachenko1*

1Department of Obstetrics and Gynecology, University of Cologne, Kerpener Str. 34, 50931 Cologne, Germany;
2Department of Obstetrics and Gynecology, University of Sassari, Italy


 The aim of our study was to investigate the effects of vitrification (cooling rate ~10000°C/min) without cryoprotectants on swim-up prepared human spermatozoa in comparison to standard conventional freezing with cryoprotectants. Motility, morphology, rate of viability and acrosome reaction of spermatozoa were evaluated. The described method of cryopreservation of human spermatozoa by direct plunging into liquid nitrogen slush without cryoprotectants was effective and could be recommended for routine IVF.

Keywords: conventional freezing, human spermatozoa, vitrification



CryoLetters 23,  (2002)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Y. Jitsuyama*, T. Suzuki, T. Harada and S. Fujikawa

Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan


The freezing tolerance of asparagus (Asparagus officinalis L.) embryogenic cells, as determined by electrolyte leakage, was increased by the incubation of samples in medium containing 0.8 M sucrose.  To elucidate the mechanism involved, we investigated the changes in soluble carbohydrates, cell ultrastructure and proteins accompanying the increase in freezing tolerance following incubation in sugar-rich medium.  During sugar incubation, the intracellular sucrose content increased from 67 µmol·g-1FW to 429 µmol·g-1FW; it was also metabolized into fructose and glucose, as determined by high-performance liquid chromatography.  Microscopy revealed that sugar incubation induced plasmolysis of embryogenic cells and drastic changes in cell ultrastructure with the appearance of rough endoplasmic reticulum (rER). Furthermore, immunoblotting analysis with anti-dehydrin antiserum revealed that a dehydrin-like protein appeared only when maximal freezing tolerance was induced by sugar incubation. These results suggest that freezing tolerance of asparagus embryogenic cells is increased by a complex mechanism involving notably changes in cell ultrastructure and accumulation of certain sugars and proteins during sugar incubation.

Keywords: Asparagus officinalis L.; cryopreservation; sugar incorporation; ultrastructural changes; plasmolysis; protein accumulation; late-embryogenesis abundant proteins; dehydrins.



CryoLetters 23,  (2002)
 ©CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

A simple GC method for determination of cryoprotector diols 1,4-butanediol or 2,3-butanediol in isolated rat hepatocytes

Luciana Almada1, Edgardo E. Guibert2 and Joaquin V. Rodriguez*1

1Farmacología, Depto Cs. Fisiológicas and 2Biología Molecular, Depto Cs. Biológicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, Rosario (S2002LRK), Argentina. * E-mail:


We devised a simple method for determining the cryoprotectant agents 1,4-butanediol or 2,3-butanediol in isolated rat hepatocytes. After extraction of of hepatocytes with water (containing internal standard - ethylene glycol 1.25 mg/mL) the diol content was analyzed by gas chromatography. The method shows a linear response in the range 0.125 to 2.50 mg/mL for 1,4-butanediol and 0.25 to 3.75 mg/mL for 2,3-butanediol. The accuracy and precision of the method were evaluated and the coefficients of variation were found to be within ≤ 6.0 %. The recoveries from hepatocyte samples containing 0.50, 1.00 and 2.00 mg/mL were 91.0 to 108 % for 1,4-butanediol and 80.6 to 100.3 % for 2,3-butanediol, respectively. This method allowed the determination of the intracellular concentration of diols in hepatocytes preserved for up to 120 hours at - 4 °C in UW solution + 8 % w/v 1,4-butanediol (or 2,3-butanediol).

Keywords: 1,4-butanediol, 2,3-butanediol, hepatocyte, GC, UW, cold preservation



CryoLetters 23,  (2002)
 ©CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


R. K. Browne*, J. Clulow and M. Mahony

School of Life and Environmental Sciences, University of Newcastle, University Drive, Callaghan 2308, NSW, Australia. *Email:


The effect of monosaccharides (glucose, fructose) and disaccharides (maltose, sucrose, trehalose) as diluents, in cryoprotective additives containing 15% (v/v) Me2SO or glycerol as cryoprotectants, were investigated on the recovery of sperm motility after cryopreservation of cane toad (Bufo marinus) spermatoazoa at low (5˚C min-1) and high cooling rates (35˚C min-1). The results show that: 1. recovery of percentage motility was higher with slow cooling than with high cooling rates (37.0  2.5%, 15.3  1.6%, P<0.001, respectively), 2. disaccharides were more effective than monosaccharides in protecting spermatozoa with slow cooling (43.9 1.2%, 26.8 2.5%, P<0.02, respectively), 3. glycerol was more effective than Me2SO with fast cooling (18.3 2.2%, 12.6 2.3%, P<0.02, respectively), 4. trehalose with glycerol was the most effective cryoprotective additive with fast cooling (31.0 3.2%, P<0.05), and 5. overall the recovery of degree (vigour) of motility (range, 1.9 - 3.2) was more resilient to cryopreservation than recovery of percentage motility (range, 8.9 - 51.5 %). Comparison of post-thaw percentage and vigour of sperm motility up to 24 minutes after activation showed disaccharides supported greater duration sperm motility than monosaccharides This result and the recovery of spermatozoa immediately after freeze-thaw, show the main effect of saccharides are as  cryoprotectants and not as exogenous energy substrates.

Keywords: Sperm, Cryopreservation, Saccharides, Frog, Amphibian, Bufo marinus



CryoLetters 23,  (2002)
 ©CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


R. K. Browne*, J. Clulow and M. Mahony

School of Life and Environmental Sciences, University of Newcastle, University Drive, Callaghan 2308, NSW, Australia.  Email: *


The short-term storage (at 0˚C) and cryopreservation of spermatozoa may be useful for providing gametes for fertilisations performed in programmes for the conservation and management of endangered amphibians. The current study was undertaken to examine the applicability of amphibian spermatozoa storage protocols developed with the cane toad (Bufo marinus) to a wider range of amphibian species, with a view to ultimately using these protocols for endangered species.  In Australia, at least 29 species of recently extinct or endangered frogs are from the families the Myobatrachidae and the Hylidae. This study investigated the applicability of short-term storage and cryopreservation protocols developed for cane toad (Bufo marinus) spermatozoa to those of hylid and myobatrachid species. 

Storage of spermatozoa in intact testes or in suspensions for six days at 0˚C showed spermatozoa maintained higher motility in suspensions than those in testes, and hylid spermatozoa maintained greater motility than myobatrachid spermatozoa. However, the protocols for optimal storage at 0C varied with testis size when spermatozoa were stored in whole testes. Spermatozoa from 13 frog species representing both families were cryopreserved using sucrose as diluent with Me2SO or glycerol as cryoprotectants. After cryopreservation hylid spermatozoa showed a greater recovery than myobatrachid spermatozoa and Me2SO provided higher recovery than glycerol. The freeze-thaw recovery of spermatozoa was independent of testes weight of the species studied. These results show spermatozoa from the Hylidae and Myobatrachidae may be stored both in the short-term (at 0˚C) and long-term by cryopreservation using protocols established for B. marinus.

Keywords: short-term storage; spermatozoa; frog; cryopreservation; testes


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