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Abstracts: CryoLetters 23 (6), 2002

CryoLetters 23, 345-352 (2002)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

POLYAMINES AND FATTY ACIDS IN SUCROSE PRECULTURED BANANA MERISTEMS AND CORRELATION WITH SURVIVAL RATE AFTER CRYOPRESERVATION

Matthew Ramon1, Jan M.C. Geuns1, Rony Swennen2, Bart Panis*2

1Laboratory of Plant Physiology, K.U.Leuven, Kasteelpark Arenberg 31, 3001 Leuven, Belgium,
2Laboratory Tropical Plant Improvement, K.U.Leuven, Kasteelpark Arenberg 13, 3001 Leuven, Belgium.

Abstract

Polyamines and fatty acids were studied in proliferating meristem cultures of 3 banana cultivars with high (Cachaco), medium (Williams Bronze free) and low (Mbwazirume) survival rates after cryopreservation. A 2-week preculture on medium containing 0.4 M sucrose which is essential to obtain survival after cryopreservation resulted in increased polyamine levels, especially putrescine. This increase in putrescine content was positively correlated with the survival rate after simple freezing or after vitrification. The total fatty acid content also increased after a 0.4 M sucrose pretreatment. However, only the ratio of unsaturated/saturated fatty acids correlated positively with the survival rate after cryopreservation. This is the first report showing a correlation of both putrescine increase and level of unsaturation of membrane lipids after sucrose treatment with survival rate after cryopreservation.

Keywords: Banana, Musa spp., cryopreservation, vitrification, fatty acids, polyamines, survival, sucrose.

 

 

CryoLetters 23, 353-360 (2002)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

RELATIONSHIP BETWEEN THE CHANGES IN CELLULAR VOLUME OF FISH SPERMATOZOA AND THEIR CRYORESISTANCE

B.B. Dzuba* and E.F.Kopeika

Institute for Problems of Cryobiology & Cryomedicine of the National Academy of Sciences of the Ukraine, 23, Pereyaslavskaya str., 61015, Kharkov, Ukraine
*Corresponding author; e-mail:
cryo@online.kharkov.ua

Abstract

We have investigated the hypothesis that the spermatozoa of marine fish are more resistant than freshwater species to the dynamic changes in osmotic pressure that occur during the process of cryopreservation. We show that while the spermatozoa of marine fish can be successfully activated across a wide range of osmotic pressures (0-2000 mOsmol/l), those of the freshwater species only survive activation within a more restricted range (0-300 mOsm/l). After freeze-thawing, up to 30% of motile cells were found in silver carp samples, while up to 90% of motile cells were observed in samples from the haarder (Mugil soiuy B). Haarder spermatozoa showed no change of cell volume after dilution in activating or cryoprotective media, while the silver carp spermatozoa responded by swelling and eventual cell disruption. We propose that the differences in cryoresistance of silver carp (Hypophthalmichthys molitrix V.) and haarder spermatozoa may be determined by the ability to preserve cellular volume under non-isotonic conditions.

Keywords: Sperm, freeze-thawing, cellular volume, haarder, silver carp

 

 

CryoLetters 23, 361-374 (2002)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

THE THERMAL EFFECT OF URETHRAL WARMING DURING CRYOSURGERY

Yoed Rabin* and Thomas F.Stahovich

Department of Mechanical Engineering, Carnegie Mellon University, Pittsburgh, PA 15213. Email: rabin@cmu.edu ; Tel: (412) 268 2204 ; Fax: (412) 268 3348

Abstract

The heating effect of urethral warming during cryosurgery has been investigated theoretically, via heat transfer simulations. Two warmer configurations have been considered: (i) the clinically available urethral warmer, which has a configuration of a counter flow fluid heat exchanger; (ii) a newly designed urethral warmer, based on a temperature controlled electrical heater, termed a "cryoheater". A dramatic effect of thermal resistance to heat transfer through the heat exchanger wall has been identified, which is absent in the cryoheater. It follows that the cryoheater is expected to be more efficient in generating an unfrozen region around the urethra. It is shown that the conventional heat exchanger may fail to prevent freezing around the urethra in a significant number of prostate cases, depending on the layout of cryoprobes around the urethra. On the other hand, clinical reports exist which suggest that the heat exchanger improves in many cases the outcome of cryosurgery, in terms of long term complications. It is speculated in the current report that the cryoheater can further improve the outcome of cryosurgery, by providing protection from freezing in a wider range of cases. It is suggested that a future study be conducted to examine the correlation between the layout of cryoprobes and surgical outcome.

Keywords: Cryosurgery, Cryoheater, Thermal Analysis, Prostate, Urethral Warmer

 

 

CryoLetters 23, 375-384 (2002)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

Sucrose preculture to simplify cryopreservation of banana meristem cultures

Bart Panis*, Hannelore Strosse, Sofie Van Den Hende and Rony Swennen

Laboratory of Tropical Crop Improvement, Catholic University of Leuven, Kasteelpark Arenberg 13, 3001 Leuven, Belgium. *Email: bart.panis@agr.kuleuven.ac.be

Abstract

A simple cryopreservation method is described for proliferating meristem cultures of banana (Musa spp.). It relies on a 2-week preculture on media containing 0.4 M sucrose followed by rapid cooling in liquid nitrogen. Different preculture media were screened for efficient protection of banana meristems against cryopreservation. Sucrose can be replaced by both fructose and glucose without significantly affecting post-thaw survival rates. A high BA concentration (100 µM) in the preculture medium results in less material available for cryopreservation, but does not affect cryoprotection. Culture in liquid media significantly improved post-thaw regeneration. The optimized cryopreservation protocol was applied on 36 banana accessions belonging to 8 different genomic groups. It is shown that post-thaw regeneration frequencies (ranging between 0 and 66 %) are highly dependent on the genomic constitution of the banana cultivar.

Keywords: banana, Musa spp., cryopreservation, cryoprotection, benzyladenine, fructose, glucose, meristem culture, sugar, sucrose

 

 

CryoLetters 23, 385-388 (2002)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

VITRIFICATION OF CULTURED AND NON-CULTURED EXPANDED AND HATCHED PORCINE BLASTOCYSTS

Barbara Gajda* and Zdzisław Smorąg

Department of Animal Reproduction, National Research Institute of Animal Production 32-083 Balice/Kraków, Poland
* Corresponding author, email:
bgajda@izoo.krakow.pl

Abstract

The aim of this experiment was to examine the survival of porcine embryos following exposure or vitrification in EFS solution. The work was carried out on cultured and non-cultured expanded and hatched blastocysts. The viability of treated embryos was assessed by their ability to develop in in vitro culture. The results showed a higher viability of cultured expanded blastocyst (35.1%) vitrified in EFS medium compared to those non-cultured (12.5%) (P<0.001). The proportion of cultured hatched blastocyst vitrified in this medium was 13.2%, while none of the non-cultured hatched blastocyst survived vitrification. .It is concluded that: 1.The EFS solution was more toxic to hatched blastocyst than expanded blastocyst 2.The expanded blastocyst was a more suitable developmental stage for vitrification than hatched blastocyst. Moreover, we demonstrated that the survival rate of vitrified cultured pig blastocyst was higher compared to non-cultured.

Keywords: vitrification, pig, blastocyst, culture, in vitro survival

 

 

CryoLetters 23, 389-396 (2002)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

AN EXPERIMENTAL STUDY OF THE MECHANICAL BEHAVIOR OF FROZEN ARTERIES AT LOW TEMPERATURES

Aili1 Zhang, Shuxia1* Cheng, Dong2 Lei, Liqun1 He, Dawei1 Luo, Dayong1 Gao

1 Department of Thermal Science and Energy Engineering, University of Science & Technology of China, Hefei, Anhui, 230027,P.R.China,
2 Department of Mechanics, USTC, Hefei, Anhui, P.R. China,
*
E-mail:
sxcheng@ustc.edu.cn, ailizh@mail.ustc.edu.cn.

Abstract

An experimental study of the mechanical response of frozen arteries to tensile stresses at low temperatures is presented. The Dynamic Mechanical Analyzer was used to perform the mechanical experiments. It was found that the frozen artery shows a kind of elastic-plasticity when the temperature is between -20oC and -40oC. And with the decrease of the temperature, the plasticity deformation decreases. Thus at the temperature of -120oC no plasticity deformation is observed before the artery's fracture and the tissue shows quite perfect elastic brittleness, both peripherally and axially. These kinds of mechanical characteristics help explain the fracture phenomena occurring during cryopreservation of the arteries. The mechanical properties, including elastic modulus and fracture strength, are also given.

It is known that Cryoprotectant (CPA) used in cryopreservation is necessary in maintaining the tissue's biological functions. Our investigation of its effect on the artery's mechanical properties found that the existence of CPA can soften the tissue at low temperatures, thus may decrease the possibility of fractures during the cryopreservation.

Keywords: Mechanical Behavior, Artery, Low temperatures, Cryoprotectant (CPA)

 

 

CryoLetters 23, 397-404 (2002)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

A CUSTOM-BUILT CONTROLLED-RATE FREEZER FOR SMALL SAMPLE CRYOPRESERVATION STUDIES

A. Medrano123*, W.J. Anderson1, J.D. Millar1, W.V. Holt2 and P.F. Watson1

1Department of Veterinary Basic Sciences, Royal Veterinary College. Royal College Street, London NW1 OTU UK.
2Institute of Zoology, The Zoological Society of London, Regents Park, London NW1 4RY UK. 
3*Current address: Department of Animal Sciences, Facultad de Estudios Superiores - Cuautitlan, National Autonomous University of Mexico. 54740. Cuautitlan Izcalli, Mexico.  email:
amedrano@servidor.unam.mx

Abstract

A custom-built controlled-rate freezing machine is described. It operates by automatically raising and lowering the sample carrier in a static column of liquid nitrogen vapour. It is controlled by a computer-assisted thermocouple feedback system that operates a stepper motor driving the sample carrier. The cooling protocol is divided into three phases: cooling from +5 to -5C, initiation of ice nucleation, and cooling from –5 to -80C. Experiments are described to validate the device over a range of different cooling rates. A freezing protocol is established to cool samples in plastic straws over a range of rates up to 80C min-1, with rapid and consistent absorption of the latent heat.

Keywords: Cooling rate, latent heat of fusion, boar spermatozoa, controlled-rate freezer.

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