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Abstracts: CryoLetters 27 (4), 2006

CryoLetters 27 (4), 203-209 (2006)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


K.M. van der Straten1*, L. K-P. Leung 1, R. Rossini2 and S.D. Johnston1.

1School of Animal Studies, The University of Queensland, Gatton, Australia.
School of Integrative Biology, The University of Queensland, St Lucia, Australia.
* Corresponding author, email address:


As a first step towards the development of a method for the cryopreservation of black marlin spermatozoa, this study investigated the effect of dimethylsulfoxide (DMSO) concentration and pellet size on post-thaw spermatozoal motility. Spermatozoa were recovered from the spermatic duct of testes retrieved post-mortem from four adult black marlin caught in the Coral Sea spawning grounds of Australia.  Undiluted spermatozoa were stored on ice for 4 to 10 hours during transport to shore, then evaluated for motility after activation in seawater (1:10 v:v).  Spermatozoa were prepared for cryopreservation in pellets by extension (1:3 v:v) in a defined fish Ringer's solution to give two final DMSO concentrations of 2.5% or 5.0%.  Diluted spermatozoa were frozen directly on a dry ice block in pellet sizes of either 0.25 ml or 0.50 ml. Frozen pellets were thawed in a water bath at 40C for 60 seconds and assessed for post-thaw motility following activation in seawater. Spermatozoa recovered within 50 minutes of death and chilled on ice for 4 to 10 hours showed a mean (± SEM) motility immediately following activation of 91.6  7.9%. 50% of the spermatozoa remained motile for approximately 4 to 5 minutes. Following cryopreservation, mean motility declined significantly across all cryoprotectant and pellet size combinations (P < 0.001) but spermatozoa frozen in 2.5% DMSO showed higher motility than those frozen in 5.0% DMSO (P = 0.014). Pellet size had no effect on post-thaw motility (P = 0.179).

Keywords: Teleostei, Istiophoridae, sperm cryopreservation, reproduction, billfish.



CryoLetters 27 (4), 211-222 (2006)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Ju-Won Yoon1, Haeng-Hoon Kim1*, Ho-Cheol Ko1, Hae-Sung Hwang1, Eun-Sun Hong1, Eun-Gi Cho1 and Florent Engelmann2, 3

1National Institute of Agricultural Biotechnology, RDA, Suwon 441-707, Korea. *Correspondence:
2Cirad, Station de Roujol, 97170 Petit-Bourg, Guadeloupe, French West Indies (present address).
3International Plant Genetic Resources Institute (IPGRI), Via dei Tre Denari 472/a, 00057 Maccarese (Fiumicino), Rome, Italy.


In this paper, we studied the effect of subculture of mother-plants and of preculture of shoot tips of two potato varieties (Dejima, cultivated and STN13, wild) cryopreserved using the droplet-vitrification technique. The subculture conditions, i.e. light intensity, aeration and planting density significantly affected survival of both non-cryopreserved and cryopreserved shoot-tips in both varieties. The subculture duration and the position of the shoot tips on the axis of the in vitro plantlets had a significant (P < 0.0001) effect on survival of cryopreserved shoot tips. The optimal subculture duration was 7 and 5 weeks and the optimal size of shoot tips was 1.5-2.0 and 1.0-1.5 mm for var. Dejima and STN13, respectively. Survival of cryopreserved shoot tips was influenced by the sucrose concentration in the preculture medium and the preculture duration. The highest survival of cryopreserved shoot tips was observed after preculture with 0.3 M sucrose for 8 h followed by 0.7 M sucrose for 18 h. These results indicate that the parameters of the subculture of mother-plants and of preculture of shoot tips should be carefully optimized, especially in the case of wild species.

Keywords: droplet-vitrification, Solanum tuberosum L, shoot tips, subculture, preculture.



CryoLetters 27 (4), 223-234 (2006)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Haeng-Hoon Kim1*, Ju-Won Yoon1, Young-Eun Park2, Eun-Gi Cho1, Jae-Keun Sohn3, Tae-San Kim1, and Florent Engelmann4, 5

1National Institute of Agricultural Biotechnology, RDA, Suwon 441-707, Korea.
2National Institute of Highland Agriculture, RDA, Pyungchang 232-955, Korea.
3Department of Agronomy, Kyungpook National University, Taegu, Korea.
4Cirad, Station de Roujol, 97170 Petit-Bourg, Guadeloupe, French West Indies (present address).
5International Plant Genetic Resources Institute (IPGRI), Via dei Tre Denari 472/a, 00057 Maccarese (Fiumicino), Rome, Italy.


The applicability of cryopreservation protocols to a broad range of genotypes is a key issue for genebanks. We tried to identify the critical factors causing differences in survival of cryopreserved shoot tips using potato varieties coming from cultivated and wild species. The droplet-vitrification method, a combination of droplet-freezing and solution-based vitrification, was selected from several protocols. High survival after freezing was observed after dehydration with PVS2 for 20 min, cooling shoot tips placed in a droplet of PVS2 solution on aluminum foil strips by immersing the foil strips in liquid nitrogen, warming them by plunging the foil strips into a 0.8 M sucrose solution (at 40°C) for 30 s and unloading in 0.8 M sucrose for 30 min. This optimized protocol was successfully applied to 12 accessions with survival ranging between 64.0 and 94.4%.

Keywords: droplet-vitrification, PVS2/PVS3 loading, shoot tips, Solanum tuberosum L.



CryoLetters 27 (4), 235-242 (2006)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


E. Flachsland, G. Terada, A. Scocchi, H. Rey, L. Mroginski1 and F. Engelmann2,3

1 Facultad de Ciencias Agrarias (UNNE), IBONE (Instituto de Botánica del Nordeste), CC 209, Corrientes (3400), Argentina. E-mail:
2 Cirad, Station de Roujol, 97170 Petit-Bourg, Guadeloupe, French West Indies (present address).
3 International Plant Genetic Resources Institute (IPGRI), Via dei Tre Denari 472/a, 00057 Maccarese (Fiumicino), Rome, Italy.


Encapsulation-dehydration was employed for cryopreserving seeds and in vitro cultured protocorms of Oncidium bifolium. Freshly harvested seeds, 120 days after pollination, were encapsulated in beads containing ½ MS medium with 3% sucrose and 3% calcium alginate and subsequently pretreated in agitated (80 rpm) liquid medium supplemented with 0.15 M sucrose (24 h) followed by 0.25 M sucrose (48 h), 0.5 M sucrose (24 h) and 0.75 M sucrose (24 h). The beads with seeds were dehydrated with silica gel for 5 h to 19.2% moisture content and immersed in liquid nitrogen for 1 h, thawed at 30 ºC for 2 min, post-treated using the same series of liquid media [0.5 M sucrose (24 h), 0.25 M sucrose (48 h), 0.15 M sucrose (24 h)], and recultured on ½ MS medium with 0.1M sucrose and 0.7% agar. As much as 4.8% of the cryopreserved seeds produced complete plants. In-vitro cultured protocorms were successfully cryopreserved following the same procedure, allowing 11.3% of them to produce plants.

Keywords: Oncidium bifolium, Orchidaceae, cryopreservation, encapsulation -dehydration, seeds, protocorms.



CryoLetters 27 (4), 243-252 (2006)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Numerical Simulation of tissue freezing by Liquid Nitrogen based Cryoprobe

Aili Zhang1, Xiaodong Luo2, Chao Chen1, Liqun He2 and Lisa X. Xu1*

1School of Life Sciences and Technology, Shanghai Jiao Tong University, 800 Dong Chuan Rd., Shanghai 200240, PR China
2Department of Energy Engineering and Thermal Science, University of Science and Technology of China


A model is presented to simulate the cooling processes during tumor cryosurgery with different kinds of flows through the cryoprobe. The heat flux between the cryoprobe wall and the tumor, the heat transfer coefficient under different inflow conditions are obtained numerically. The impact of the inlet mass flow rate, gas volume fraction on these parameters is investigated. It is found that the heat transfer coefficient decreased significantly when inflow changed from two-phase annular flow to droplet flow, and to gas flow. The inlet gas volume fraction and flow velocity only significantly affect the freezing ability of the probe when the inflow is gas or in droplet phase. Simulation of the tumor temperature profiles under different flow conditions show that the heat transfer coefficient is a crucial parameter in temperature prediction during cryosurgery. Results indicate that when the cryoprobe wall is assumed at a constant temperature conventionally, the cooling effect could be overestimated. It would be more reasonable to use the constant wall heat transfer coefficient to simulate the cooling progress under a specific flow.

Keywords: cryoprobe, two-phase flow, tumor, liquid-nitrogen



CryoLetters 27 (4), 253-260 (2006)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


Rhett McClean1*, Catriona MacCallum1, 2, David Blyde2, William Holt3 and Steve Johnston1

1 School of Animal Studies, University of Queensland, Gatton, 4343, Australia.
2 Western Plains Zoo, Obley Rd, Dubbo, 2830, Australia.
3 Institute of Zoology, Regent's Park, London, UK NW1 4RY, UK.


This study examined the hypothesis that filamentous actin associated with the complex cytoskeleton of the kangaroo sperm head and tail may be contributing to lack of plasma membrane plasticity and a consequent loss of membrane integrity during cryopreservation. In the first study, the distribution of G and F actin within Eastern Grey Kangaroo (EGK, Macropus giganteus) cauda epididymidal spermatozoa was successfully detected using DNAse-FITC and a monoclonal F-actin antibody (ab205, Abcam), respectively. G-actin staining was most intense in the acrosome but was also observed with less intensity over the nucleus and mid-piece. F-actin was located in the sperm nucleus but was not discernable in the acrosome or sperm tail. To investigate whether cytochalasin D (a known F-actin depolymerising agent) was capable of improving the osmotic tolerance of EGK cauda epididymal spermatozoa, sperm were incubated in hypo-osmotic media (61 and 104 mOsm) containing a range of cytochalasin D concentrations (0-200μM). Cytochalasin D had no beneficial effect on plasma membrane integrity of sperm incubated in hypo-osmotic media. However, when EGK cauda epididymidal sperm were incubated in isosmotic media, there was a progressive loss of sperm motility with increasing cytochalasin D concentration. The results of this study indicated that the F-actin distribution in cauda epididymidal spermatozoa of the EGK was surprisingly different from that of the Tammar Wallaby (M. eugenii) and that cytochalasin-D does not appear to improve the tolerance of EGK cauda epididymidal sperm to osmotically induced injury.

Keywords: kangaroo, spermatozoa, cryopreservation, osmotic tolerance, F-actin, cytochalasin D



CryoLetters 27 (4), 261-268 (2006)
© CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK


F. Marco-Jiménez1, R. Lavara2, J.S. Vicente2 and M.P. Viudes-de-Castro*1

1Animal Research and Technology Center. Valencian Institute of Agrarian Research.  Segorbe 12400, Spain.
2Laboratory of Biotechnological Reproduction Department of Animal Science. Polytechnic University of Valencia, Valencia 46071, Spain.


This study researches the effects of supplementation with reduced glutathione (GSH, 0.5 mM) and oxidised glutathione (GSSG, 0.5 mM) freezing extenders on different semen parameters after equilibration with DMSO preservation solution (45 min at 5º C) and post-thawing. The main findings that emerged from this study are that (i) addition of GSH and GSSG to the freezing media did not result in any improvement in functional sperm tests after equilibrium phase. (ii) No differences were observed after cryopreservation in functional sperm tests and embryo recovery rate.  In conclusion, the addition of 0.5 mM GSH or GSSG appears not to play an important role in sperm antioxidant defence during cooling and freezing in rabbit spermatozoa.

Keywords: Sperm, Rabbit, Glutathione, Cryopreservation, Flow cytometry.

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