Abstracts: CryoLetters 36 (2), 2015

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



Volume 36, No. 2 March/April 2015

ISSN 0143-2044



Evaluation of different methods of cryopreservation of Ehrlich tumor cells
Kamilla M. Santos, Fernanda P. S. Maciel,
Cristina M. Souza, Geovanne D. Cassali, Ralph G. Thomé,
Hélio B. Santos and Rosy. I. M. A Ribeiro




Differential proteomic expression of himalayan psychrotrophic diazotroph Pseudomonas palleroniana N26 under low temperature diazotrophic conditions
Ravindra Soni, Deep Chandra Suyal,Komal Agrawal,
Amit Yadav, Yogesh Shouche and Reeta Goel




Sage (Salvia officinalis) and fennel (Foeniculum vulgare) improve cryopreserved boar epididymal semen quality study
A.Montón, L. Gil, C. Malo, M. Olaciregui, N. González,
and I. de Blas




Cryopreservation of gemmae of Marchantia polymorpha L. (Marchantiophyta, Marchantiaceae) without prior pretreatment
S-P Wu, Z-Z Qin, T-Z Xiao, Q-P Li, B-B Lu, L-J Jiang,
J Wang and R-L Zhu




Effect of the pH pre-adjustment in the freezing extender on post-thaw boar sperm quality
Cristina Tomás, José Gómez-Fernández,
Emilio Gómez-Izquierdo, Ernesto Gómez-Fidalgo,
Raúl Sánchez-Sánchez, Antonio González-Bulnes and
Eduardo de Mercado




Evaluation of the new vacuum infiltration vitrification (VIV) cryopreservation technique for native Australian plant shoot tips
Bryn Funnekotter, Susan E. Whiteley, Shane R. Turner,
Eric Bunn and Ricardo L. Mancera




Strain preservation of rats: vitrification of two-cell stage embryos for multiple inbred strains
Tomoo Eto




Similar cold stress induces sex-specific neuroendocrine and working memory responses
Rima Solianik, Albertas Skurvydas, Daiva Urboniene,
Nerijus Eimantas, Laura Daniuseviciute and Marius Brazaitis




First steps of in vitro gastrulation in rabbit vitrified embryos
J.S. Vicente, A. Parrilla-Ocon, M.D. Saenz-de-Juano,
C. Naturil-Alfonso and F. Marco-Jiménez




Cryopreservation of rainbow trout spermatozoa (Onchorhynchus mykiss) using different cryodiluents
Makwana Nayan P, Sanjay K Gupta, Sathis K Srivastva
and Gopal Krishna






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CryoLetters 36 (2), 68-73 (2015)
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Kamilla M. Santos, Fernanda P. S. Maciel1, Cristina M. Souza2,
Geovanne D. Cassali2, Ralph G. Thomé1, Hélio B. Santos1
and Rosy. I. M. A Ribeiro1*

Federal University of São João del Rei - CCO. Laboratory of Experimental Pathology. Av Sebastião Gonçalves Coelho, 400, Chanadour, Divinópolis, Minas Gerais, Brazil. CEP: 35501-296.
2Federal University of Minas Gerais. Laboratory of Comparative Pathology. Av. Antônio Carlos, 6627,  Belo Horizonte – MG, Brazil. CEP: 31270-901
*Corresponding author email: /


BACKGROUND: The Ehrlich Ascitic Carcinoma (EAC) is an experimental transplantable neoplasm that develops in several species of mice. The maintenance of the tumor occurs in vivo. Thus, freezing the cells would reduce the number of passages between animals, ensuring genetic stability and storage for longs period of experimentation. OBJECTIVE: Search by EAC cryoprotectants. MATERIALS AND METHODS: The combinations of nutrient medium (Tris, hen egg yolk, and DEMEN) and cryoprotective agent (Glicerol, Trehalose and DMSO) on freezing EAC cells and the transplantability after defrosting were evaluated. The cooling was conducted at 2°C/min. until -180°C and the thawing by immersion in water at 37°C. The transplantability was evaluated from cell inoculation in mice for 14 days. RESULTS: The best results were the associations IA (Cryoprotective agent Glycerol 6% and medium containing 3.0% Tris w / v, 1.8% Citric acid w / v, 1.3% D-fructose w / v and 20% hen egg yolk v / v) and IIB (Cryoprotective agent Trehalose 100mM and medium containing 50% coconut water v / v, 25% sodium citrate 5% v / v and 20% hen egg yolk v / v) with 85.2% and 55.1% viable cells, respectively. CONCLUSION: These transplantable cells were efficient for tumor development, therefore demonstrating that this method of cryopreservation is simple and affordable.

Keywords: ascitic tumor, glycerol, trehalose, transplantability, hen egg yolk, cryoprotective agent




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CryoLetters 36 (2), 74-82 (2015)
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Ravindra Soni1,2ψ, Deep Chandra Suyal ,Komal Agrawal1,
Amit Yadav3, Yogesh Shouche3 and Reeta Goel1*

1Department of Microbiology, College of Basic Sciences and Humanities, G.B. Pant University of Agriculture and Technology, Pantnagar-263145, Uttarakhand, India
2Department of Agricultural Microbiology, College of Agriculture, Indira Gandhi Krishi Viswavidyalaya, Raipur (C.G.) India
3Microbial Culture Collection, National Centre for Cell Science, Pune University Campus, Ganeshkhind, Pune-411 007, India
*Corresponding author email:
ψ Authors contributed equally to this manuscript


BACKGROUND: In low temperature nitrogen-deficient ecosystems, native microorganisms must possess adaptive mechanisms to cope with environmental stress as well as nitrogen (N) starvation-like conditions. However, moderate information is available about the cold adapted diazotrophs and diazotrophy. OBJECTIVE: The aim of this study was to examine the proteomic response(s) of Himalayan psychrotrophic diazotroph under low temperature nitrogen fixing conditions.  MATERIALS AND METHODS: Proteomic analysis of Pseudomonas palleroniana N26 was carried out using two dimensional electrophoresis technique. RESULTS: Altogether, fifty three protein spots were found to be differentially expressed revealing several mechanisms thought to be involved in low temperature adaptation and nitrogen fixation, including general stress adaptation, protein synthesis and modifications, and energy metabolism. Expression profiling of the spots revealed the up-regulation of low molecular weight acidic proteins; a majority of which were stress proteins. The largest group of down-regulated proteins were related to biosynthetic processes; thereby, providing the evidence for stress-associated metabolic adaptations.  CONCLUSION: The present study, which provides an overview of the cold diazotrophy of a Himalayan psychrotrophic bacterium and its adaptive responses, can facilitate further studies of low temperature nitrogen fixing mechanisms, psychrophilic diazotrophic markers, and transgenic microorganism(s)/crop(s) development.

Keywords: psychrotrophs; diazotrophs, proteomics, Western Indian Himalaya




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CryoLetters 36 (2), 83-90 (2015)
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Sage (Salvia officinalis) and fennel (Foeniculum vulgare) improve cryopreserved boar epididymal semen quality study

A.Montón1, L. Gil1, C. Malo1*, M. Olaciregui1, N. González1, I. de Blas2

1Department of Animal Pathology, Obstetrics and Reproduction Area, Faculty of Veterinary, University of Zaragoza, Zaragoza, Spain.
2Department of Animal Pathology, Infectious Diseases Area, Faculty of Veterinary, University of Zaragoza, Zaragoza, Spain.
*Corresponding author email:
Tel: 976761544 ; Fax: 976761612


The aim of this study was to evaluate the effect of fennel and sage extracts and the influence of the egg yolk source (fresh or pasteurized) on the success of freezing boar epididymal spermatozoa. In experiment 1, epididymal sperm was recovered by flushing and cryopreserved in a lactose-egg yolk solution supplemented with various concentrations (10, 5 and 2.5 g/L) of sage or fennel. Sperm quality was evaluated (motility, viability, HOST and acrosome integrity) at 0h and 2 h after thawing. Fennel 10 g/L and sage 5 g/L and control (no extracts) were selected for experiment 2 which also compared fresh or pasteurized egg yolk in the freezing extender and measured DNA integrity of the frozen sperm. Results showed that the interaction between fennel and sage antioxidants with fresh egg yolk significantly improved post thaw sperm quality and protected boar epididymal spermatozoa from cryopreservation damage as a result of oxidative stress.

Keywords: Cryopreservation; natural antioxidant; boar; epididymis; spermatozoa




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CryoLetters 36 (2), 91-96 (2015)
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Cryopreservation of gemmae of Marchantia polymorpha L. (Marchantiophyta, Marchantiaceae) without prior pretreatment

S-P Wu, Z-Z Qin, T-Z Xiao, Q-P Li, B-B Lu, L-J Jiang, J Wang *
and R-L Zhu*

Bryology Laboratory, School of Life Science, East China Normal University, 500 Dongchuan Road, Shanghai 200241, China.
*Corresponding author email:


BACKGROUND: Successful cryopreservation of gametophytic material of bryophytes requires pretreatment with sucrose or abscisic acid. Compared to gametophyte materials, spore and gemmae cryopreservation may be more efficient, simple and stable systems for storing large amounts of genetic diversity of bryophytes within a small space. However, there has still been no attempt at cryopreserving bryophyte gemmae.  OBJECTIVE: The aim of this study is to determine whether bryophyte gemmae with differing levels of desiccation tolerance could survive and germinate after cryopreservation without prior encapsulation and pretreatment.  MATERIALS AND METHODS: Gemmae of Marchantia polymorpha L. were dried with silica gel for different times and then rapidly cooled in liquid nitrogen.  RESULTS: The germination level of fresh gemmae was 95%. After 3 h predrying and 1 d in LN, germination was 68%, and was still up to 59% after storage for 75 days.  CONCLUSION: We conclude that the natural desiccation tolerance of bryophyte gemmae permits cryopreservation without prior pretreatment other than drying.

Keywords: liverwort; gemmae; Marchantia polymorpha; cryopreservation




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CryoLetters 36 (2), 97-103 (2015)
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Cristina Tomás2, José Gómez-Fernández1, Emilio Gómez-Izquierdo1,
Ernesto Gómez-Fidalgo3, Raúl Sánchez-Sánchez3,
Antonio González-Bulnes3 and Eduardo de Mercado1*

1Centro de Pruebas de Porcino. Área de Investigación Ganadera, Subdirección de investigación y Técnología. Instituto Tecnológico Agrario. Consejería de Agricultura y Ganaderia. Junta de Castilla y León. Crta Riaza-Toro s/n 40353. Hontalbilla, Segovia, Spain.
2Centro de Investigación y Tecnología Animal – Instituto Valenciano de Investigaciones Agrarias (CITA-IVIA), 12400 Segorbe, Castellón, Spain. 3Dpto. De Reproducción Animal, INIA, Avda. Puerta de Hierro s/n, 28040 Madrid, Spain.
*Corresponding author email:


BACKGROUND: During freezing the selective precipitation of substances in the medium may provoke a pH shift and lead to sperm damage. OBJECTIVE: To study the effect of the pH pre-adjustment in the freezing extender on post-thaw boar sperm quality. METHODS: A total of 15 ejaculates from different boars were obtained and divided into six aliquots prior to a standard straw cryopreservation in freezing extender (lactose-egg yolk-glycerol-Orvus ES Paste) with different pH. After thawing, sperm quality (plasma membrane integrity, motility and acrosome status) were assessed at 30 and 90 minutes of post-thaw incubation at 37ºC. RESULTS: When the boar sperm were frozen in a freezing media with pH basic, and particularly at pH 8, it had higher post-thaw sperm quality. CONCLUSION: The pre-adjustment at pH 8 of the freezing extender (lactose-egg yolk-glycerol-Orvus ES Paste) is able to improve the post-thaw boar sperm quality.

Keywords: Boar sperm, cryopreservation, freezing extender, pH sperm quality




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CryoLetters 36 (2), 104-113 (2015)
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Bryn Funnekotter1, 2, Susan E. Whiteley2, 3, Shane R. Turner2, 3,
Eric Bunn2, 3 and Ricardo L. Mancera1*

1School of Biomedical Sciences, CHIRI Biosciences, Curtin University, GPO Box U1987, Perth, WA 6845, Australia.
2 Botanic Gardens and Parks Authority, Fraser Avenue, West Perth WA 6005, Australia.
3School of Plant Biology, Faculty of Natural and Agricultural Sciences, University of Western Australia, Crawley WA 6009, Australia.
*Correspondence author email:


BACKGROUND: The application of a vacuum during the incubation in cryoprotective agents such as PVS2 allows for increased penetration, reducing total incubation times required before vitrification and post-cryopreservation regeneration is achieved. OBJECTIVE: This study compared a conventional droplet-vitrification protocol to the new vacuum infiltration vitrification protocol in four Australian plant species. MATERIALS AND METHODS: The new vacuum infiltration vitrification applied an 80 kPa vacuum during incubations in loading solution and PVS2. Infiltration of the cryoprotective agents into shoot tips was determined by differential scanning calorimetry measuring ice formation in the thermographs comparing a range of loading solution and PVS2 incubation times. RESULTS AND CONCLUSION: The application of the vacuum infiltration vitrification technique resulted in a significantly reduced PVS2 incubation time for cryogenic survival and regeneration for all four species, reducing the time needed to adequately protect shoot tips by half to a quarter when compared to a conventional droplet-vitrification technique.

Keywords: conservation, PVS2, rare and endangered, biodiversity




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CryoLetters 36 (2), 114-119 (2015)
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Tomoo Eto*

Central Institute for Experimental Animals, 3-25-12 Tonomachi, Kawasaki-ku, Kawasaki, 210-0821 Japan.
*Corresponding author email:


OBJECTIVE: We examined whether the vitrification method using P10 and PEPeS is suitable for the cryopreservation of two-cell stage embryos collected from multiple rat strains with the objective of strain preservation of inbred rat strains.  RESULTS: The average numbers of two-cell stage embryos collected per female for F344/Jcl, ACI/N, BUF/N, and WKY/N strains were 7.0 to 12.0, and survival rates of the embryos after vitrification were 94.2 to 96.3%. The in vitro development rates of vitrified embryos transferred were 47.1 to 60.8%. CONCLUSION: At least two offspring produced from the embryos collected from one female are required for strain preservation of inbred strain. Taken together, the results of the experiment indicated expected numbers of surviving fetuses for embryos collected from one female were 3.2 to 6.7, and all were usable for strain preservation. The results suggest that this vitrification method is suitable for strain preservation of multiple inbred rat strains.

Keywords: Strain preservation, Rat Inbred, Cryopreservarion, Vitrification, P10 and PEPeS, Rat 2-cell stage embryos




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CryoLetters 36 (2), 120-127 (2015)
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Rima Solianik1*, Albertas Skurvydas1, Daiva Urboniene2,
Nerijus Eimantas1, Laura Daniuseviciute3 and Marius Brazaitis1

1Institute of Sports Science and Innovations, Lithuanian Sports University, Sporto str. 6, LT-44221 Kaunas, Lithuania
2Department of Laboratory Medicine, Medical Academy, Lithuanian University of Health Sciences, Eiveniu str. 2, LT-50028, Kaunas, Lithuania
3Department of Physical Education, Kaunas University of Technology, K. Donelaicio str. 73, LT-44029 Kaunas, Lithuania
* Corresponding author email:


BACKGROUND: Men have higher cold-induced neuroendocrine response than women; nevertheless, it is not known whether a different stress hormone rise elicits different effects on cognition during whole body cooling. OBJECTIVE: The objective was to compare the effect of cold-induced neuroendocrine responses on the performance of working memory sensitive tasks between men and women. MATERIALS AND METHODS: The cold stress continued until rectal temperature reached 35.5°C or for a maximum of 170 min. Working memory performance and stress hormone concentrations were monitored. RESULTS: During cold stress, body temperature variables dropped in all subjects (P<0.001) and did not differ between sexes. Cold stress raised plasma epinephrine and serum cortisol levels only in men (P<0.05). Cold stress adversely affected memory performance in men but not in women (P<0.05). CONCLUSION: The present study indicated that similar moderate cold stress in men and women induces sex-specific neuroendocrine and working memory responses.

Keywords: cognition, cold water immersion, cortisol, epinephrine, female




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CryoLetters 36 (2), 128-136 (2015)
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J.S. Vicente*, A. Parrilla-Ocon, M.D. Saenz-de-Juano,
C. Naturil-Alfonso and F. Marco-Jiménez

Instituto de Ciencia y Tecnología Animal, Universidad Politécnica de Valencia, 46022-Valencia, Spain.
*Corresponding author email:


BACKGROUND: The in vitro rabbit embryo production and their cryopreservation methodologies such as vitrification generate less viable embryos, and occasionally, with significant differences from those that are not subjected to any treatment. Besides, in vitrified rabbit embryos little information is available about exactly when and where begin to emerge the first differences that finally result in foetal losses comparing with non-vitrified embryos. OBJECTIVE: The aim of this study was to evaluate the vitrification effects on the early in vitro gastrulation events. MATERIALS AND METHODS: After oviductal transfers of vitrified and non-vitrified embryos (control) in rabbit recipients, blastocysts from 144h (6-day-old) were recovered and cultured into TCM199 supplemented with rabbit homologous serum media for 48 hours. Gastrula stage and measures of perimeter and area of blastocyst and gastrula were noted. Moreover, eight independent pools consisting of six embryos each one were generated for each experimental group (control and vitrified) and total RNA was isolated to study the OCT4 gene expression.  RESULTS: Of 151 control and 164 vitrified morulae transferred, 69.5% and 70.1% developed in vivo to 6-day-old blastocyst respectively. After 24 hour of in vitro culture, 41.8% of vitrified blastocyst had begun the neurulation (stage 5-) versus 22.8% of control group. Nevertheless, the vitrified group showed the highest percentage of collapsed blastocyst at 48 hours (26.8%). Non morphometric differences differences were observed in perimeter and area of blastocyst and gastrula between control and vitrified group at 0 and 24 hours. By contrast, perimeter and gastrula areas were slightly higher for the vitrified group than those for the control group at 48 hours of in vitro culture CONCLUSION: The study reveal the existence of the first morphological differences in vitrified blastocysts of 7 and 8-day-old, marked by a further development of gastrulation in the vitrified group.

Keywords: Blastocyst, gastrulation, vitrification, rabbit




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CryoLetters 36 (2), 137-147 (2015)
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Makwana Nayan P1, Sanjay K Gupta2*, Sathis K Srivastva2
and Gopal Krishna1

1Division of Fish Genetics and Biotechnology, Central Institute of Fisheries Education, Versova, Mumbai-61, India.
2Directorate of Coldwater Fisheries   Research, Chhirapani Fish Farm, Champawat, Uttrakhand-262523, India.
*Corresponding author email:
Tel.: +91 8859967653; Fax: +91 5965230052


An experiment was conducted to evaluate the potential of extenders on sperm motility and to delineate the effect of cyodiluent compostion on post thaw motility, fertilization and hatching percentage in rainbow trout (Onchorhynchus mykiss) sperm. The mature males of rainbow trout with an average  weight of (340 ± 15) g and average size (28.8 ± 0.6) cm were used. In the trials for testing the suitability of cryoprotectants, DMSO yielded higher motility percentage with extenders Zhang & Liu and 0.6 M Sucrose. Significantly (P<0.05) higher value of % post thaw motility and fertilization was recorded in cryodiluent composition of 8% DMSO and Zhang & Liu. Additionally, composition of 8% DMSO with Zhang & Liu led to significant (P<0.05) increase in percentage hatching.  Overall, results indicated that the combination of (8% DMSO + Zhang & Liu) produced best results in terms of percentage post thaw motility, fertilization and hatching.

Keywords: Extender, DMSO, Spermatozoa, Motility, Hatching


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