Abstracts: CryoLetters 38 (6), 2017

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



Volume 38, No. 6 November/December 2017

ISSN 0143-2044



Cryopreservation of in vitro shoot tips of ulluco (Ullucus tuberosus Cal.) using D cryo-plate method
Miriam Valle Arizaga, Shin-ichi Yamamoto, Daisuke Tanaka,
Kuniaki Fukui,Naoko Nohara, Tomotaro Nishikawa,
Kazuo Watanabe and Takao Niino




Cryoelectrolysis for treatment of atrial fibrillation: A first order feasibility study
Mohammad Hjouj, Hanush Krishnan and Boris Rubinsky




Optimization of human semen extender components for cryopreservation using statistical tools
Ramesh Pathy M, Vickram A S, Sridharan T B, Parameswari R,
Archana K, Nithya S and Mishika A




Analysis of stress indicators during cryopreservation of seeds of landrace maize (Zea mays)
Jason Pérez, Emanuel Araya-Valverde, Giovanni Garro and
Ana Abdelnour-Esquivel




Vitrfication and slow freezing for cryopreservation of germinal vesicle-stage human oocytes: A bayesian

Hyun Sik Youm, Jong-Ryeol Choi, Daesik Oh and Yong Ho Rho




Cryopreservation of arecanut (Areca catechu L.) pollen
Anitha Karun, K.K. Sajini K.S. Muralikrishna, M.K. Rajesh and
Florent Engelmann




Effect of dilution on cryosurvival of low sperm doses:
A review

SA Lone, TK Mohanty, M Bhakat, RK Baithalu and R Kumar




The ability of pectins to modulate the action of glycerol in the freezing of nucleated cells
M.I. Sergushkina, А.N. Khudyakov, T.V. Polezhaeva
and O.M. Bezmeltseva




Authors Index

Keyword Index







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CryoLetters 38 (6), 419-427 (2017)
© CryoLetters,


Miriam Valle Arizaga1, Shin-ichi Yamamoto2*, Daisuke Tanaka2,
Kuniaki Fukui2, Naoko Nohara2, Tomotaro Nishikawa2,
Kazuo Watanabe3 and Takao Niino3

1Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba 305-8572, Japan.
2NARO Genetic Resources Center, Tsukuba 305-8602, Japan.
3Gene Research Center, University of Tsukuba, Tsukuba 305-8572, Japan,
Corresponding author e-mail:


BACKGROUND: Maintenance of in vitro collections of ulluco (Ullucus tuberosus Cal.) is cumbersome and costly in an ex-situ genebank. An alternative method for long term preservation which is safe and cost-effective is required. OBJECTIVE: To apply a novel cryopreservation procedure using the cryo-plate system to improve the long-term conservation of ulluco. MATERIALS AND METHODS: Initially V and D cryo-plate methods were tested, subsequently the D cryo-plate method was selected for ulluco cryopreservation. The D cryo-plate procedures were optimized for post-LN regrowth procedures including cold-hardening, sucrose addition in alginate gel, and duration of LS treatment. Optimized procedures were tested with 11 ulluco lines. RESULTS: Shoot tips were isolated from cold-hardened shoots for 3-4 weeks at 5°C were excised to 1.0–1.5 mm long and 0.5 mm wide and precultured for 16h at 25°C on MS with 0.3 M sucrose. The shoot tips were attached on the cryo-plates by alginate gel with 0.4M sucrose. The cryo-plates with attached shoot tips were treated with 2.0 M glycerol and 1.0 M sucrose solution for 90 min at 25°C and dehydrated on filter paper in a Petri dish by air current flow at 25°C for 45 min before direct immersion in LN. This optimized procedure was applied to shoot tips of 11 ulluco lines, resulting regrowth ranging from 73% to 97%, with an average of 90% post-LN regrowth. CONCLUSION: D cryo-plate is a practical and simple procedure for cryo-storage of in vitro grown ulluco shoot tips in an ex situ genebank.

Keywords: air dehydration, cryopreservation, D cryo-plate, glycerol, loading solution, ulluco




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CryoLetters 38 (6), 428-433 (2017)
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Mohammad Hjouj1*, Hanush Krishnan2 and Boris Rubinsky2

1The Medical Imaging Department / Faculty of Health Professions Al Quds University – Jerusalem, Palestinian Authority
2Department of Mechanical Engineering, University of California Berkeley, Berkeley CA 94720, USA
*Corresponding author email:


BACKGROUND: Catheter based treatment of atrial fibrillation (AFib) involves ablation of groups of cells near the pulmonary vein. OBJECTIVE: The goal of this study was to evaluate the technological feasability of a new technology for catheter-based ablation for treatment of AFib that combines freezing with electrolysis.  MATERIALS AND METHODS: The study was performed on a pH dye stained gel phantom of the pulmonary vein. Freezing was induced with a cryosurgical probe inserted in the vein and electrolysis was delivered through the probe with a DC power supply.  RESULTS: Visual recording of colorimetric changes in pH demonstrate that electrolytic products can propagate through the frozen tissue phantom. For example, a voltage of 9 V and a current of 40 mA delivered through a -15°C cryosurgical probe produced an electrolysis impacted rim of over 7 mm width in 2 min.  CONCLUSION: This early stage experimental work suggests that cryoelectrolysis may have potential for treatment of AFib.

Keywords: atrial fibrillation, cryoelectrolysis, heart, electrolytic ablation, cryosurgery




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CryoLetters 38 (6), 434-444 (2017)
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Ramesh Pathy M*, Vickram A S, Sridharan T B, Parameswari R,
Archana K, Nithya S and Mishika A

School of Biosciences and Technology, VIT University, Near Katpadi Road, Vellore, Tamil Nadu 632014, India.
*Corresponding author email:


BACKGROUND: Human sperm cell preservation is an important part of assisted reproductive technology (ART).  OBJECTIVE: This study aimed to find the essential and significant components in semen preservation extender required to prolong the shelf life of human spermatozoa. MATERIALS AND METHODS: By using the statistical tool ‘Plackett-Burman design’ the significant components present in E4 extender (formulated in our previous study) was determined by reducing the unacceptable large number of trial experiments from the full factorial method.  RESULTS: It was found that vitamin E, taurine and vitamin C were highly significant in maintaining the stability of sperm cells; and egg yolk, vitamin C and glucose were highly significant in sustaining the motility of the sperm cells. R2 values for the models were 0.9950 and 0.9960 respectively. In the optimized E4 extender 75% and 81% of the total motility was retained by the sperm cells from infertile and fertile samples respectively after cryopreservation. Also an increase in zeta potential was observed indicating a reduction in stability in both fertile and infertile sample (4% and 18% respectively) after cryopreservation in E4 medium, which was much less when compared with the sample preserved only with glycerol as cryoprotectant (11% and 69% for infertile and fertile samples respectively). CONCLUSION: The major components present in E4 semen extender was successfully optimized for further use.

Keywords: Human male infertility, response surface methodology, correlation, fertilizing capacity, zeta potential, sperm stability




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CryoLetters 38 (6), 445-454 (2017)
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Jason Pérez1, Emanuel Araya-Valverde2,3, Giovanni Garro1
and Ana Abdelnour-Esquivel1*

1Centro de Investigación en Biotecnología, Instituto Tecnológico de Costa Rica, Cartago, Costa Rica.
2Escuela de Biología, Instituto Tecnológico de Costa Rica, Cartago, Costa Rica.
3Centro Nacional de Innovaciones Biotecnológicas (CENIBiot-CENAT), San José, Costa Rica.
*Corresponding author email:


BACKGROUND: Maize breeding programs focus on the development of hybrid varieties and the cultivation of landrace materials is discouraged; however, they are a valuable source of genes and their conservation is advisable. OBJECTIVE: Analyzing some stress indicators during cryopreservation of maize landrace seeds. MATERIALS AND METHODS: Seeds of 35 accessions of landrace maize were collected in two regions of Costa Rica and cryopreserved by direct immersion in liquid nitrogen (LN). Membrane integrity, germination of seeds and DNA methylation in tissues were analyzed 5, 7 and 9 days after rewarming. RESULTS: Germination of landrace maize seeds was near 100% for most accessions. No statistically significant differences in germination were observed between non-cryopreserved controls and seeds stored in LN for 1 h or 1 year. Membrane integrity, number of leaves and root and shoot length of plantlets were similar after cryostorage of seeds for 1 h and 1 year.  A short delay in growth of cryostored seed compared to non-frozen controls was observed. Changes in the proportion of DNA methylation were noted from 0 to day 9 in the organs studied depending on the germination stage and cryopreservation treatment. CONCLUSION: It may be inferred that many of the methylated genes were related to growth and development. In addition, a cryobank of maize landraces from two regions of Costa Rica was established.

Keywords: cryopreservation, Costa Rica, cryobank, DNA methylation, membrane integrity, seed germination




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CryoLetters 38 (6), 455-462 (2017)
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Hyun Sik Youm*, Jong-Ryeol Choi, Daesik Oh and Yong Ho Rho

Center for Reproductive Medicine, Eroom Women’s Clinic, Busan, Republic of Korea *Corresponding author email:


BACKGROUND: The most commonly used methods for the cryopreservation of oocytes and embryos are vitrification and slow freezing. OBJECTIVE: The aim of this study was to to investigate whether there are differences in survival, in vitro maturation (IVM), and fertilization rates between cryopreserved immature oocytes, especially germinal vesicle (GV)-stage human oocytes, following vitrification and slow freezing. MATERIALS AND METHODS: A literature search was performed using the MEDLINE, Cochrane Central Register of Controlled Trials, and Embase databases. A total of three studies were included in the Bayesian meta-analysis. RESULTS: There was no difference in survival rates between vitrification and slow freezing. Additionally, there was no difference in IVM rates and fertilization rates between vitrification and slow freezing. CONCLUSION: The superiority of vitrification over slow freezing for cryopreservation of GV-stage human oocytes remains unclear. Additional studies on cytoarchitecture and modification of the cryopreservation protocol are essential to achieve strong conclusions.

Keywords: vitrification, slow freezing, cryopreservation, germinal vesicle stage, human oocytes, in vitro maturation




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CryoLetters 38 (6), 463-470 (2017)
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Anitha Karun1, K.K. Sajini1*, K.S. Muralikrishna1, M.K. Rajesh1
and Florent Engelmann2

1ICAR-Central Plantation Crops Research Institute, Kasaragod 671124, Kerala, India
2IRD, UMR DIADE, BP 64501, 34394 Montpellier, Cedex 05, France
*Corresponding author email:


BACKGROUND: Cryopreservation opens new avenues in the field of genetic resource conservation, especially in recalcitrant seeded palms such as arecanut for which field genebanks are exposed to pest and disease attacks and natural calamities. It is only through cryopreservation that the safety of the conserved germplasm can be assured at a relatively low cost for extended periods. OBJECTIVE: The objective of this work was to standardize various aspects of arecanut pollen cryopreservation, viz. collection and desiccation of pollen, in vitro germination, viability and fecundity studies. MATERIALS AND METHODS: Pollens of three arecanut genotypes (Sumangala, Hirehalli Dwarf and Hirehalli Dwarf x Sumangala) were collected in December 2013-February 2014. In vitro viability tests were conducted using fresh and desiccated pollen. Desiccated pollen was cryopreserved by direct immersion in liquid nitrogen and cryostored for different durations (24 hours to 2 years). Viability and fertility studies were conducted using cryopreserved pollen. RESULTS: Pollen extraction was achieved from fully opened male flowers by desiccation at room temperature (33-34ºC). A medium containing 2.5 g/L sucrose was found to be best for in vitro germination at room temperature. There was no significant difference in germination between desiccated and cryopreserved pollen whereas pollen tube length decreased significantly after cryopreservation. Fertility studies using HD x Sumangala pollen cryostored for various durations (1 month, 1 year and 2 years) showed the setting of 70, 43 and 62%, respectively. Normal nut set was observed using cryopreserved pollen. CONCLUSION: Pollen cryopreservation is a viable option for germplasm conservation and hybridization programmes in arecanut

Keywords: Areca catechu L., arecanut, pollen, cryopreservation, germination, vigor, fertility




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CryoLetters 38 (6), 471-476 (2017)
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SA Lone*, TK Mohanty1, M Bhakat1, RK Baithalu2 and R Kumar1

1Artificial Breeding Research Center (ABRC), ICAR- National Dairy Research Institute, Karnal, Haryana 132001 India
2Livestock Research Center (LRC), ICAR-National Dairy Research Institute, Karnal, 132001, Haryana, India.
*Corresponding author email:


BACKGROUND: Sperm concentration needed for artificial insemination (AI) to obtain reasonable fertility, taking into consideration genetic value of bull and numerous others components is one of the essential constituents for successful breeding program. AI-doses containing low sperm numbers are increasingly widespread to optimize the benefit of elite bulls, as well as to accommodate an eventual wider application of sex-sorted semen. Viability is reduced when higher dilutions of semen are carried out for production of low sperm doses. The reduction in viability may be due to dilution effect as well as due to dilution of numerous essential constituents in seminal plasma at higher dilutions. Fertility level of bull may play a pivotal role in determining the cryosurvival of low sperm doses and viability of low sperm doses may vary among bulls. This paper attempts to highlight studies dealing with the effect of dilution on cryosurvival of low sperm doses.

Keywords: Dilution; Cryosurvival; Low; Sperm Doses




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CryoLetters 38 (6), 477-481 (2017)
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M.I. Sergushkina*, А.N. Khudyakov, T.V. Polezhaeva
and O.M. Bezmeltseva

Physiology Institute at the Komi Scientific Center of the Russian Academy of Sciences, Komi Republic, Syktyvkar, Russian Federation
*Corresponding author email:


BACKGROUND: Pectins have a large number of active -OH and -COOH groups (3), suggesting the presence of this group of substances specific cryoprotective effect. OBJECTIVE: This research is aimed at studying the ability of pectins (zosteran, apple pectin АU-701, E-440) to change the freezing temperature of glycerol, which can increase the effect in the cryopreservation of nuclear cells. METHODS: Using the cryoscopic method we studied the ability of pectins (zosteran, apple pectin AU-701, E-440) to change the freezing temperature of glycerol. Heparinized venous blood was frozen under the protection of a complex cryoprotectant and stored in an electric freezer at -80 °C for 1 day. RESULT: The research shows that pectins in a concentration of 0.6 - 1.0% increase in varying degrees the osmolality of the glycerol solution, which helps to low its freezing temperature. According to the viability of leukocytes which had been frozen to -80°C in glycerol habitat and one of the pectins, we found out that all pectins in concentration (0.6%) are capable to increase the cryoprotective effect of glycerol. CONCLUSIONS: The obtained data on the preservation of leukocyte membranes and the phagocytic activity of neutrophils after freezing) in glycerol habitat and glycerol with pectin confirm the authors' supposition that decrease in freezing temperature of a solution of glycerol in the presence of pectin helps to reduce the risk of cell damage during freezing. Apple pectin AU-701 is able to strengthen the cryoprotective effect of glycerol in a greater grade. It is possible due to the large number of hydroxyl and carboxyl groups (the galacturonic acid content is 95%) and the presence of methyl groups in the pectin molecule (the degree of methyl etherification is 38-40%).  These groups are the most often found in the molecules of effective cryoprotectants.

Keywords: pectins, crystallization temperature, leukocytes, galacturonic acid, degree of etherification, cell viability.

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