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Abstracts: CryoLetters 38 (2), 2017



Volume 38, No. 2 March/April 2017

ISSN 0143-2044



Cryopreservation of Lilium martagon L. meristems by droplet-vitrification and evaluation of their physiological stability
M. Urbaniec-Kiepura and A. Bach




Cooling strategies for Brazilian flounder Paralichthys orbignyanus embryos
Antonio Sergio Varela Junior, Tainã Figueiredo Cardoso,
Estela Fernandes e Silva, Karina Lemos Goularte,
Marcelo Hideo Okamoto, Luis André Sampaio,
Rodrigo Desseards Jardim and Carine Dahl Corcini




Tea polyphenol-T. arjuna bark as sperm antioxidant extender in
infertile smokers

Parameswari R, Kamini A Rao, Manigandan P, Vickram AS,
Archana K and Sridharan TB




Kidney preservation at subzero temperatures using a novel storagesolution and insect ice-binding proteins
Heather E. Tomalty, Erin F. Hamilton, Andrew Hamilton,
Olga Kukal, Thomas Allen, and Virginia K. Walker




The presence of zinc in the mouse ovary vitrification medium:
histological evaluation and follicle growth

Shirin Geravandi and Mehri Azadbakht




Experimental determination of surface heat transfer coefficient in a dry ice-ethanol cooling bath using a numerical approach
M.V. Santos, M. Sansinena, N. Zaritzky and J. Chirife




Carbon nanomaterials enhance survival of Agapanthus praecox callus after cryopreservation by vitrification
Shu-Min Chen, Li Ren, Di Zhang, Ya-Fei Zhang
and Xiao-Hui Shen




The effect of thawing protocols on follicle conservation in human ovarian tissue cryopreservation
T. Morewood, N. Getreu, B. Fuller, J. Morris and P. Hardiman




Freezability of water buffalo spermatozoa is improved with the addition of catalase in cryodiluent
Liaqat Ali, S. Murtaza Hassan Andrabi, Hussain Ahmed
and S. Aftab Hussain Shah




Cryo-mesh: A simple alternative cryopreservation protocol
Bryn Funnekotter, Eric Bunn and Ricardo L. Mancera






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CryoLetters 38 (2), 78-89 (2017)
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M. Urbaniec-Kiepura* and A. Bach

Department of Ornamental Plants, University of Agriculture in Krakow, Al. 29 Listopada 54, 31-425 Krakow, Poland
*Corresponding author email:


BACKGROUND: The aim of the present study was to compare different strategies for cryopreservation of martagon lily meristems and to evaluate the physiological status of the regenerants. MATERIALS AND METHODS: The bulblets were stored at 5oC or 20oC and pretreated with 3% or 6% sucrose prior to droplet-vitrification. The meristems were then assessed for their survival and regeneration. Their photochemical activity was investigated using a Photosynthesis Yield Analyzer MINI PAM 2000 Portable Chlorophyll Fluorometer and their photosynthesis oxygen production was evaluated with a Plant Vital 5030 device. RESULTS: The plant material stored at 5oC on medium containing 3% sucrose exhibited lower survival (40.8%) and regeneration (75%) of meristems following cryopreservation compared with material stored at 20oC on medium containing 3% sucrose, for which survival was 65% and regeneration 87%. Treatment of lily meristems for 30 min with PVS2 yielded high survival and regeneration. The implemented cryopreservation protocol did not induce any physiological changes in regenerants. Chlorophyll fluorescence (Fv/Fm) was 0.822 for cryopreserved samples (+LN) and 0.824 for non-cryopreserved ones (-LN). Photosynthetic oxygen production (KphA) was 1.531 (+LN) and 1.410 (-LN). CONCLUSION: Droplet-vitrification seems to be an effective method for cryopreservation of martagon lily meristems with the aim of its ex situ protection.

Keywords: Lilium martagon, cryopreservation, meristems, droplet-vitrification




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CryoLetters 38 (2), 90-94 (2017)
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Antonio Sergio Varela Junior1*, Tainã Figueiredo Cardoso2,
Estela Fernandes e Silva2, Karina Lemos Goularte1,
Marcelo Hideo Okamoto3, Luis André Sampaio4,
Rodrigo Desseards Jardim1 and Carine Dahl Corcini5

1Instituto de Ciências Biológicas, Universidade Federal de Rio Grande – FURG, Av.Italia s/n; 96201-900; Campus Carreiros – Rio Grande- RS - Brasil; 
2Programa em Fisiologia Animal Comparada; Universidade Federal de Rio Grande FURG, Rio Grande, RS
3Doutor em Aqüicultura; Universidade Federal de Rio Grande FURG, Rio Grande, RS
4Professor do Departamento de Oceanografia; Universidade Federal de Rio Grande - FURG
5Professora da Faculdade de Veterinária; Universidade Federal de Pelotas - UFPEL, Pelotas, RS
*Corresponding author email:


BACKGROUND: Paralichthys orbignyanus is the species of the greatest potential for marine and estuarine fish farming in southern Brazil. Consequently, embryo cryopreservation becomes an important tool for increasing their production. OBJECTIVE: To evaluate the effects of cooling protocols on the viability of embryos of P. orbignyanus at two stages of development (neurula and early differentiation of the tail). MATERIALS AND METHODS: Control embryos were maintained at 23°C and treated embryos were cooled to 15°C, 10°C and 5°C at rapid, moderate and slow cooling rates. Then embryos were maintained at these different temperatures for 30, 60 and 90 min and the loss of viability assessed as hatching rates (HR) and morphologically normal larvae (MNL). RESULTS: The average HR for embryos following cooling was higher for those at the tail stage compared to the neurula stage (P<0.05). In both stages there was no statistical difference between the HR of control embryos and those exposed to rapid cooling. Also for tail stage embryos, there was no difference between MNL of control and rapidly cooled embryos. CONCLUSION: As first steps in the development of cryopreservation methods for P. orbignyanus embryos, the use of a rapid cooling and holding at 5ºC for 30 min are recommended.

Keywords: cryopreservation, cooling protocol, viability, fish




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CryoLetters 38 (2), 95-99 (2017)
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Parameswari R, Kamini A Rao, Manigandan P, Vickram AS, Archana K
and Sridharan TB*

Department of Industrial Biotechnology, School of Biosciences and Technology, VIT University, Vellore, India.
Corresponding author email:


BACKGROUND: Antioxidants protect spermatozoa against lipid peroxidation during freezing. OBJECTIVE: The study is designed to elucidate the suitable extender to preserve infertile smoker's semen against ROS damage using natural Tea polyphenol (T. arjuna bark extract). MATERIALS AND METHODS: Forty-two infertile subjects with smoking habit and 28 fertile subjects without smoking habit were considered for the study. Four semen extenders including our naturally derived antioxidant component were prepared and used to preserve semen sample from the study subjects for a period of one month. Standard semen parameters, biochemical and sperm DNA damage marker with inhibition were measured before and after cryopreservation. RESULTS: The motility and morphology of sperm cells were maintained better in E4 extender, and DNA damage is reduced. CONCLUSION: Extender recipe with natural antioxidants (E3&E4) was found to be apt for infertile semen preservation.

Keywords: polyphenol, reactive oxygen species, Terminalia arjuna, smoking, sperm DNA damage




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CryoLetters 38 (2), 100-107 (2017)
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Heather E. Tomalty1, Erin F. Hamilton1, Andrew Hamilton2,
Olga Kukal1,3, Thomas Allen3, and Virginia K. Walker1,4*

1Department of Biology, Queen's University, Kingston, ON K7L 3N6, Canada
2Department of Surgery, Queen's University, Kingston, ON K7L 2R2, Canada
3CryoStasis Ltd., Westport, ON K0G 1X0, Canada
4Department of Biomedical and Molecular Sciences, Queen's University, Kingston, ON K7L 3N6, Canada
*Corresponding author email:


BACKGROUND: Contemporary kidney preservation methods involve storing at 4°C up to 24 h prior to transplantation. By decreasing the storage temperature to below 0°C, we hypothesized that the "safe" storage time could be significantly lengthened. OBJECTIVE: The efficacy of a proprietary CryoStasis (CrS) storage solution for the subzero preservation of kidneys was tested, with or without addition of a hyperactive insect antifreeze protein (TmAFP). MATERIALS AND METHODS: Rat kidneys were stored in either University of Wisconsin (UW) solution (4°C, 24 h), CrS (-2°C, 48 h), or CrS with 61.5 µM TmAFP (-4.4°C, 72 h).  Following storage, viability was assessed with MTT reduction assays and live vs. dead cell (FDA/PI) staining. Markers of ischemic damage were analyzed using fluormetric substrates for caspase-3 and calpain activity. RESULTS: Kidneys stored in CrS for 48 h and CrS with TmAFP for 72 h displayed similar levels of enzymatic activity compared to 24 h UW controls. CONCLUSION: This methodology shows promise to prolong the safe storage time of kidneys and offers the potential of increased organ availability for renal transplants.

Keywords: Organ preservation, hypothermic storage, ice-binding proteins




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CryoLetters 38 (2), 108-118 (2017)
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Shirin Geravandi and Mehri Azadbakht*

Department of Biology, Faculty of Basic Sciences, Razi University, Kermanshah, Iran
*Corresponding author email:


BACKGROUND: Cryopreservation of ovary is a relevant option for preserving fertility of cancer patients undergoing chemotherapy. OBJECTIVE: To evaluate the effect of zinc in the vitrification medium on histology and follicle growth. MATERIALS AND METHODS: Mouse ovaries were vitrified in vitrification medium supplemented with 0, 100, 150 or 200 μg/dl zinc, identified as V0, V1, V2 and V3 groups, respectively. Histological evaluation of ovaries was carried out. The isolated pre-antral follicles were cultured. The size and growth of follicles were assessed. RESULTS: The percentage of morphologically normal follicles increased with increasing zinc concentration in the vitrification medium (P < 0.05). Follicle viability was higher in the V3 than V0 group at the beginning and end of culture (P < 0.05). The highest follicle diameter was obtained in the V3 group after 6 days of culture (P < 0.05). CONCLUSION: Supplementation of the vitrification medium with zinc can improve follicle viability and growth.

Keywords: Ovary, vitrification, zinc, follicle growth, mouse




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CryoLetters 38 (2), 119-124 (2017)
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M.V. Santos1,3,*, M. Sansinena2,3*, N. Zaritzky1,3 and J. Chirife2

1Depto. de Ingeniería Química, Facultad de Ingeniería, Universidad Nacional de La Plata and Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CONICET-UNLP), La Plata;  Argentina.
2 Facultad de Ciencias Agrarias, Pontificia Universidad Católica Argentina, CABA;
3 Consejo Nacional de Investigaciones Científicas y Técnicas, CABA, Argentina.
*Corresponding author email:


BACKGROUND: Dry ice-ethanol bath (-78ºC) have been widely used in low temperature biological research to attain rapid cooling of samples below freezing temperature. The prediction of cooling rates of biological samples immersed in dry ice-ethanol bath is of practical interest in cryopreservation. The cooling rate can be obtained using mathematical models representing the heat conduction equation in transient state. Additionally, at the solid cryogenic-fluid interface, the knowledge of the surface heat transfer coefficient (h) is necessary for the convective boundary condition in order to correctly establish the mathematical problem.  OBJECTIVE: The study was to apply numerical modeling to obtain the surface heat transfer coefficient of a dry ice-ethanol bath.  MATERIALS AND METHODS: A numerical finite element solution of heat conduction equation was used to obtain surface heat transfer coefficients from measured temperatures at the center of polytetrafluoroethylene and polymethylmetacrylate cylinders immersed in a dry ice-ethanol cooling bath. The numerical model considered the temperature dependence of thermophysical properties of plastic materials used. RESULTS: A negative linear relationship is observed between cylinder diameter and heat transfer coefficient in the liquid bath, the calculated h values were 308, 135 and 62.5 W/(m2K) for PMMA 1.3, PTFE 2.59 and 3.14 cm in diameter, respectively. CONCLUSION: The calculated heat transfer coefficients were consistent among several replicates; h in dry ice-ethanol showed an inverse relationship with cylinder diameter.

Keywords: heat transfer coefficient, dry ice-ethanol cooling bath, unsteady state heat conduction




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CryoLetters 38 (2), 125-136 (2017)
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Shu-Min Chen1, Li Ren1, Di Zhang1, Ya-Fei Zhang2 and Xiao-Hui Shen1*

1Key Laboratory of Urban Agriculture (South) Ministry of Agriculture, School of Agriculture and Biology, Shanghai Jiao Tong University, No. 800, Rd. Dong Chuan, Shanghai 200240, P.R. China.
2Key Laboratory for Thin Film and Microfabrication of Ministry of Education, Research Institute of Micro/Nano Science and Technology, Shanghai Jiao Tong University, Shanghai 200240, P.R. China.
*Corresponding author E-mail:


BACKGROUND: Adding exogenous compounds is an effective way to improve cell survival after cryopreservation. Carbon nanomaterials (CNMs) are novel exogenous substances with small particle size and good biocompatibility. OBJECTIVE: In this work, four types of CNMs were used for the cryopreservation of Agapanthus praecox callus and their possible effects and mechanism of action were analyzed. MATERIALS AND METHODS: The thermal properties of the vitrification solutions tested were detected by differential scanning calorimetry. Raman spectroscopy and transmission electron microscopy analyses were used to study the distribution of CNMs inside cells. The MDA/H2O2 contents were measured to evaluate the toxicity of CNMs to cells. RESULTS AND CONCLUSION: Supplementation of PVS2 with various CNMs at different concentrations could enhance survival. The most effective concentration was 0.3 g/L C60, which increased survival by 159% compared to untreated controls and decreased the MDA and H2O2 contents. Single-walled carbon nanotubes (SWCNTs) and C60 entered callus cells. C60 was found only in mitochondria, whereas SWCNTs were located only around the cell walls.

Keywords: Carbon nanomaterials, cryopreservation, survival, differential scanning calorimetry, Raman spectroscopy, transmission electron microscopy, Agapanthus praecox callus




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CryoLetters 38 (2), 137-144 (2017)
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T. Morewood1, N. Getreu1*, B. Fuller2, J. Morris3 and P. Hardiman1

1Institute for Women's Health, University College London, UK.
2Division of Surgery and Interventional Science, University College London, UK.
3Asymptote, St. John's Innovation Centre, Cambridge CB4 0WS, UK.
*Corresponding author email:



BACKGROUND: Ovarian tissue cryopreservation has the potential to improve fertility preservation for a growing number of patients undergoing sterilising therapy, particularly where oocyte or embryo cryopreservation is not suitable. However, its success is limited by significant follicular apoptosis upon thawing, and there is wide variation in thawing protocols used with little evidence of efficacy. OBJECTIVE: To determine the best warming rates to maintain tissue viability. MATERIALS AND METHODS: Ovarian tissue biopsies from 11 patients were taken with informed consent and divided into four pieces, which were allocated to either fresh assessment or to one of several freeze-thaw protocols. Cryopreservation was undertaken using a Stirling cycle cryo-cooler and cryopreserved samples were exposed to different warming protocols. Tissue conservation was then assessed using a marker, neutral red, to identify viable follicles. RESULTS: The results showed greatest follicle conservation rates in fresh samples, followed by those thawed using a rapid thawing protocol (Protocol 1). Tissue thawed using an ultra fast protocol (Protocol 2) and slow warming (Protocol 3) resulted in greater follicle loss.  CONCLUSION: These preliminary results indicate thawing conditions significantly affect follicle conservation in cryopreserved human ovarian tissue.

Keywords: fertility preservation, cryopreservation, thawing protocols, human ovarian tissue, and follicle conservation




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CryoLetters 38 (2), 145-154 (2017)
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Liaqat Ali, S. Murtaza Hassan Andrabi*, Hussain Ahmed
and S. Aftab Hussain Shah

Animal Reproduction Laboratory, Animal Sciences Institute, National Agricultural Research Center, Islamabad 45500, Pakistan
*Corresponding author email:


BACKGROUND: Catalase enzyme is usually distributed in mammalian seminal plasma, where it decomposes hydrogen peroxide into water and oxygen and enhances sperm survivability. OBJECTIVE: To evaluate the effect of catalase (0, 100, 200 or 300 IU/ml) added in tris-citric acid (TCA) based extender on motion characteristics, viability and DNA integrity of bubaline spermatozoa at post dilution (PD) and post thawing (PT) stages of cryopreservation. MATERIALS AND METHODS: Collection of semen was done in four Nili-Ravi bulls with an artificial vagina (42°C). Qualified semen samples from each bull were further subdivided into four aliquots for dilution with the experimental TCA extender containing either 0.0 (T1), 100 IU (T2), 200 IU (T3) or 300 IU (T4) catalase (activity12660 U/mg). RESULTS: At PT, mean computer progressive motility, average path velocity, straight line velocity, curvilinear velocity, visual motility and DNA integrity were higher (P < 0.05) in catalase fortified treatment groups as compared with control. Regarding plasma membrane integrity and supra-vital plasma membrane integrity, at PT the mean values were higher (P < 0.05) in T4 as compared with control. At PD and PT, mean acrosomal integrity of buffalo bull spermatozoa was higher (P < 0.05) in T4 group as compared with control. CONCLUSION: Addition of catalase at a concentration of 300IU/ml in TCA cryodiluent improved the freezability of water buffalo spermatozoa.

Keywords: buffalo spermatozoa; egg yolk; cryopreservation; catalase; casa




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CryoLetters 38 (2), 155-159 (2017)
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Bryn Funnekotter1,2, Eric Bunn2,3 and Ricardo L. Mancera1*

1School of Biomedical Sciences, Curtin Health Innovation Research Institute, Curtin University, GPO Box U1987, Perth WA 6845, australia.
2Botanic Ggardens and Parks Authority, Fraser Avenue, West Perth WA 6005, australia.
3School of Plant Biology, Faculty of Science, The University of Western Australia, Crawley, Australia, 6009
*Corresponding author email:


BACKGROUND: The continued development of new cryopreservation protocols has improved post-cryogenic success rates for a wide variety of plant species. Methods like the cryo-plate have proven beneficial in simplifying the cryopreservation procedure.  OBJECTIVE: This study assessed the practicality of a stainless steel mesh strip (cryo-mesh) for cryopreserving shoot tips from Anigozanthos viridis.  MATERIALS AND METHODS: Shoot tips of A. viridis (Kangaroo Paw) were precultured on 0.4 M sucrose medium for 48 h. Precultured shoot tips were coated in a 2% alginate solution and placed onto the cryo-mesh (a 25 x 7 mm, 0.4 mm aperture, 0.224 mm diameter wire stainless steel mesh strip). The alginate was set for 20 min in a loading solution containing 100 mM CaCl2, anchoring the shoot tips to the cryo-mesh. The cryo-mesh was then transferred to PVS2 on ice for 20, 30 or 40 min prior to plunging the cryo-mesh into liquid nitrogen. The cryo-mesh protocol was compared to the droplet-vitrification protocol.  RESULTS: A maximum of 83% post-cryogenic regeneration was achieved with the cryo-mesh when exposed to PVS2 for 30 min. No significant difference in post-cryogenic regeneration was observed between the cryo-mesh and droplet-vitrification protocols. CONCLUSION: Anigozanthos viridis shoot tips were successfully cryopreserved utilising the new cryo-mesh. The cryo-mesh thus provides a simple and successful alternative for cryopreservation.

Keywords: Plant cryopreservation, cryo-mesh, vitrification, PVS2

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