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Volume 40, No. 1 January/February 2019
ISSN 0143-2044
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Safer
vitrification of mouse and human embryos using the novel cryoroom vitrification system for assisted reproductive technology
Hiroaki Inui, Jinji Mizuno, Eiko Kikuchi, Kaori Noguchi,
Yuri Tanji, Miki Hamabata, Chiho Kotsuzumi,
Makiko Komiyama, Yukiko Noguchi and Midori Tamura
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1-10
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Effect of
different cryoprotectants (glycerol, methanol and dimethyl sulfoxide) on post-thaw quality, viability, fertilization ability and DNA damage of cryopreserved Nile tilapia (Oreochromis
niloticus) spermatozoa
Yusuf Bozkurt, İlker Yavaş, Mustafa Numan Bucak
and Deniz Yeni
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11-17
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Development
of cryopreservation protocol for Aquilaria malaccensis Lam., a recalcitrant seeded tropical tree species
Seram Devika Devi, Suman Kumaria and Meera C. Das
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18-27
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Cryopreservation
of encapsulated axillary buds of Clitoria ternatea (L.)
Deepa S. Nair, B. R. Reghunath, K. B. Soni and Swapna Alex
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28-35
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Seed
cryostorage enhances subsequent plant productivity in the forage species Teramnus labialis (L.f.) Spreng
Yanier Acosta, Lázaro Hernández, Carlos Mazorra,
Nicolás Quintana, Byron E. Zevallos, Inaudis Cejas, Sershen,
José Carlos Lorenzo, Marcos Edel Martínez-Montero
and Dayamí Fontes
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36-44
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Simulation
of hypothermic perfusion of rabbit livers for cryopreservation applications
Pable Barroso Rodríguez, Alberto Olmo Fernández,
Ariadna Corral, Marcin Balcerzyk and Ramón Risco
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45-50
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Isochoric
containers and its frontier between cryopreservation and sterilization
Abril Angulo-Sherman
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51-57
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Texture
feature differences between fresh and frozen-thawed ex-vivo porcine liver tissue in B-mode ultrasonic imaging
Gubing Wang, Jing Liu, Jianwen Luo, Lei Sheng
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58-63
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Time-dependent
effects of pressure during preservation of rat hearts in an isochoric system at subfreezing temperatures
Lili Wan, Matthew J. Powell-Palm, Mark G. Clemens
and Boris Rubinsky
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64-70
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CryoLetters 40 (1), 1-10 (2019)
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SAFER VITRIFICATION OF MOUSE AND HUMAN EMBRYOS USING THE NOVEL CRYOROOM VITRIFICATION SYSTEM FOR ASSISTED REPRODUCTIVE TECHNOLOGY
Hiroaki Inui1, Jinji Mizuno1*, Eiko Kikuchi1, Kaori Noguchi1, Yuri Tanji1,
Miki Hamabata1, Chiho Kotsuzumi1, Makiko Komiyama1,
Yukiko Noguchi2 and Midori Tamura3
1 Inui Institute for Frontier Reproductive Medicine and Infertility, Inui Maternity Clinic,
3-5-18 Namiki, Koriyama, Fukushima, 963-8021 Japan Tel: +81-24-925-0705 Fax: +81-24-925-0706
2 Department of Obstetrics and Gynecology, The Jikei University School of Medicine,
3-19-18 Nishishimbashi, Minato-ku, Tokyo, 105-8471 Japan
3 Department of Obstetrics and Gynecology, St. Marianna University School of
Medicine, 2-16-1, Sugao, Miyamae, Kawasaki 216-8511, Japan
* Corresponding author e-mail: jinmizuno@nifty.com
Abstract
BACKGROUND: Vitrification is widely used for assisted reproductive technology
(ART). Most vitrification devices require the skillful placement of embryos into the carrier and aspiration of excessive vitrification solution. OBJECTIVE: To evaluate the
efficacy and safety of the Cryoroom as a vitrification device. MATERIALS AND METHODS: Mouse and human embryos were vitrified with Cryoroom or Cryotop, and the developmental potency was assessed in vitro. Mouse monozygotic twin
blastocysts were vitrified with Cryoroom or Cryotop for microarray analysis. RESULTS AND DISCUSSION: In mouse and human embryos, there were no differences between
the survival and developmental progress in each device. In silico, the Cryoroom device showed no changes, particularly in DNA methylation after vitrification compared with
the Cryotop. These results showed that the form and function of the device may affect the gene expression levels in vitrified embryos. CONCLUSION: The Cryoroom
represents a safe and potentially revolutionary vitrification device for ART.
Keywords: safe vitrification device, automatic volume contro
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CryoLetters 40 (1), 11-17 (2019)
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EFFECT OF DIFFERENT CRYOPROTECTANTS (GLYCEROL, METHANOL AND DIMETHYL SULFOXIDE) ON POST-THAW QUALITY, VIABILITY, FERTILIZATION
ABILITY AND DNA DAMAGE OF CRYOPRESERVED NILE TILAPIA (Oreochromis niloticus) SPERMATOZOA
Yusuf Bozkurt1*, İlker Yavaş2, Mustafa Numan Bucak3 and Deniz Yeni4
1İskenderun Technical University, Faculty of Marine Sciences and Technology, Hatay, Turkey.
2Mustafa Kemal University, Faculty of Veterinary Medicine, Hatay, Turkey.
3Selçuk University, Faculty of Veterinary Medicine, Konya, Turkey.
4Afyon Kocatepe University, Faculty of Veterinary Medicine, Afyon, Turkey.
*Corresponding author email: yusuf.bozkurt@iste.edu.tr
Abstract
BACKGROUND: Cryopreservation of sperm from different fish species requires different
protocols. Therefore, it is necessary to perform studies to establish reliable procedures for each species. OBJECTIVE: Experiments were designed to analyse the effect of
different types of cryoprotectants on post-thaw motility, viability and fertility as well as cryoresistance of cryopreserved Nile tilapia (Oreochromis niloticus) sperm. MATERIALS AND METHODS:
Sperm samples were diluted with an ionic extender containing glycerol (Gly), methanol (MeOH) and dimethyl sulfoxide (DMSO) at ratios of
5, 10 and 15 % respectively. Diluted samples were aspirated into 0.25 ml French straws and frozen 3 cm above the surface of liquid nitrogen (LN) in a styrofoam box
and stored in a LN tank. DNA damage was evaluated with the comet assay technique following cryopreservation. RESULTS: Supplementation of extender with 10% glycerol
gave the highest motility rate compared with the other cryoprotectant groups (P<0.05). Differences in terms of post-thaw motility duration, cell viability and fertilization rates
were not significant among treatments (P>0.05). Although Gly gave the best score (5.0 ± 0.1%, P>0.05) at the concentration of 10%, 5% Me2SO caused significant DNA
damage (15.0 ± 1.0%, P<0.05) with the comet test. CONCLUSION: Gly or MeOH are more suitable cryoprotectants than DMSO for the cryopreservation of Nile tilapia sperm.
Keywords: cryodamage, fish sperm motility, cryoprotectant, DNA damage, Nile tilapia
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CryoLetters 40 (1), 18-27 (2019)
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DEVELOPMENT OF CRYOPRESERVATION PROTOCOL FOR Aquilaria malaccensis Lam., A RECALCITRANT SEEDED TROPICAL TREE SPECIES
Seram Devika Devi, Suman Kumaria and Meera C. Das*
Plant Biotechnology Laboratory, Botany Department, North-Eastern Hill University, Shillong-22, Meghalaya, India
*Corresponding author's email dasmeera73@gmail.com
Abstract
BACKGROUND: Cryopreservation is the only possible process for long-term germplasm storage of species with recalcitrant behavior. Aquilaria malaccensis is a
recalcitrant seeded tropical tree that produces a distinctive fragrance which has high value in the commercial market. OBJECTIVES: In the present study, we aimed to
develop possible long-term storage techniques for A. malaccensis germplasm. MATERIALS AND METHODS: We used zygotic embryos and in vitro derived nodal
buds as explants. The experiments were performed based on dehydration and encapsulation-dehydration methods. RESULTS: When the dehydration technique was
applied, survival (43%) and regeneration (23%) was found to be higher for zygotic embryos than for in vitro derived nodal buds (13% & 10% respectively). In addition to
moisture content within the tissue during cryogen exposure, dehydration duration has an important contributory role in post cryo-survival and regeneration. A slight increase
in survival (47%) and regeneration (30%) were observed in zygotic embryos with a modification to rehydration after re-warming, while such a change did not improve
success with in vitro derived nodal buds. In contrast, the encapsulation-dehydration technique was found to be more effective for in vitro derived nodal buds than zygotic
embryos. All the encapsulated nodal buds that survived (27%) regenerated into plantlets while encapsulated zygotic embryos failed to regenerate into plantlets. CONCLUSION:
This is the first report on the cryopreservation of A. malaccensis and the developed protocol conveys a comprehensive idea of its reliability for the long-term
storage of this desiccation sensitive (recalcitrant) seeded tree species.
Keywords: agarwood, desiccation, encapsulation-dehydration, germplasm, nodal buds, zygotic embryos
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CryoLetters 40 (1), 28-35 (2019)
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CRYOPRESERVATION OF ENCAPSULATED AXILLARY BUDS OF Clitoria ternatea (L.)
Deepa S. Nair*1, B. R. Reghunath1, K. B. Soni2 and Swapna Alex2
1*Department of Plantation Crops and Spices, Kerala Agricultural University, College
of Agriculture, Vellayani, Trivandrum, Kerala, India-695 522; *corresponding author email: deepaanil13@gmail.com; deepanair.s@kau.in
2 Department of Plant Biotechnology, Kerala Agricultural University, College of
Agriculture, Vellayani, Trivandrum, Kerala, India-695 522
Abstract
BACKGROUND: Clitoria ternatea is a brain revitalizing legume, with immense
pharmacological value including antimicrobial, antioxidant and anticancer. The lack of a commercial cultivation has led to its' over collection from the wild to meet the
demand of herbal pharmaceutical sector and the species is now rare in the wild. Hence, the plant needs to be conserved. Cryopreservation of the species would
supplement the conventional conservation strategies in the field or seed bank. OBJECTIVE: The objective of the study was to evolve an efficient and simple protocol for the cryopreservation of Clitoria ternatea using an encapsulation-dehydration
technique. MATERIALS AND METHODS: An in vitro culture system via axillary shoot proliferation was developed using nodal segments in MS medium and optimization of
the levels of cytokinins and auxins. Calcium alginate encapsulated axillary buds were subjected to 0-5 h of dehydration, to determine the optimum drying time and moisture
content for effective cryopreservation. RESULTS: The beads dehydrated for 4 h to 20.1 % moisture content had 60 % survival after freezing in LN, of which 65 % regrew.
Based on RAPD analysis, the plants regrowing after cryostorage were genetically stable. CONCLUSION: A simple and efficient cryopreservation protocol has been established for C. ternatea using an encapsulation-dehydration technique, and this
could be effectively utilized for germplasm conservation of this species.
Keywords: Clitoria ternatea, encapsulation-dehydration, cryopreservation, genetic
fidelity, random amplification of polymorphic DNA (RAPD)
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CryoLetters 40 (1), 36-44 (2019)
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SEED CRYOSTORAGE ENHANCES SUBSEQUENT PLANT PRODUCTIVITY IN THE FORAGE SPECIES Teramnus labialis (L.F.) SPRENG
Yanier Acosta1, Lázaro Hernández2, Carlos Mazorra1,
Nicolás Quintana3, Byron E. Zevallos4, Inaudis Cejas1, Sershen5,
José Carlos Lorenzo2*, Marcos Edel Martínez-Montero2
and Dayamí Fontes1
1 Faculty of Agricultural Sciences, 2 Laboratory for Plant Breeding & Conservation of Genetic Resources, Bioplant Center, and 3 Faculty of Informatics, University of Ciego
de Ávila, Ciego de Ávila, 69450, Cuba.
*Corresponding author email: jclorenzo@bioplantas.cu
4 Escuela Superior Politécnica Agropecuaria de Manabí Manuel Félix López
(ESPAMMFL), Campus Politécnico El Limón, Carrera de Ingeniería Agrícola, Calceta, Manabí, Ecuador.
5 School of Life Sciences, University of Kwazulu-Natal, Durban, 4001, South Africa.
Abstract
BACKGROUND: Teramnus labialis is an herbaceous legume that serves as a source
of carbohydrates and proteins for animals and humans, and is valued for its nitrogen contribution to soil. The benefits of this species are, however, limited by low seed
availability, small seed size and low in situ seed germination levels, due to physical dormancy. Cryostorage has been shown to be beneficial for both seed storage and
breaking physical dormancy in seeds of various species. However, its potential effects on subsequent seedling emergence, plant growth and seed production need to be
studied before large-scale implementation for T. labialis. OBJECTIVE: To record agricultural traits of T. labialis after seed exposure to liquid nitrogen. MATERIALS
AND METHODS: Seeds were maintained at 5°C (control) or stored in LN before sowing. Seedling emergence percentage and traits related to plant growth and seed production were evaluated for 6 months.
RESULTS: Except for seed weight, all traits differed significantly between seedlings generated from cryostored and control seeds.
Except for pod number, seedling emergence and plant growth traits were enhanced by cryostorage to a greater extent than seed production traits. Cryostorage resulted in
cracks and breaks in the seed coat which were absent in control seeds (scanning electron microscopy), and in breaking physical dormancy may have facilitated more
rapid seedling emergence than for control seeds. CONCLUSION: Seed cryostorage enhances subsequent plant productivity in terms of growth and to a lesser extent seed production in Teramnus labialis, validating its use for commercial growth of this
species.
Keywords: animal feed; crops; cryopreservation; legumes; nitrogen fixation; seed dormancy
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CryoLetters 40 (1), 45-50 (2019)
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SIMULATION OF HYPOTHERMIC PERFUSION OF RABBIT LIVERS FOR CRYOPRESERVATION APPLICATIONS
Pable Barroso Rodríguez1, Alberto Olmo Fernández2, Ariadna Corral3,
Marcin Balcerzyk3 and Ramón Risco1,3*
1 Departamento de Física Aplicada III. Escuela Superior de Ingenieros, Universidad de Sevilla;
2 Departamento de Tecnología Electrónica. Escuela Técnica Superior de Ingeniería
Informática. Universidad de Sevilla;
3 Centro Nacional de Aceleradores (Universidad de Sevilla-CSIC-Junta de Andalucía), Spain.
*Corresponding author email: ramon@us.es
Abstract
BACKGROUND: In cryopreservation of biological material, it is very important to
precise control the perfusion of cryoprotectants (CPA) inside the sample. CPA concentration inside tissues and organs during the process was measured in a few
studies. The simulation of the CPA perfusion inside the organ is still necessary to understand and optimize this complex process. OBJECTIVE: This study simulates the diffusion of Me2SO and PBS in a rabbit liver.
MATERIALS AND METHODS: The software COMSOL for computational fluid-dynamics has been used. A hypothermic perfusion is simulated where temperature and pressure at the entrance of the organ
are constant. RESULTS: The simulation shows that Me2SO concentration increases within the porous medium until saturation. The variation of perfusion speed and
pressure inside the organ is almost null with time. CONCLUSION: Finite elements modelling shows that under hypothermic conditions it is possible a full and even loading of this organ with Me2SO, keeping constant the perfusion parameters.
Keywords: dimethylsulfoxide, perfusion, rabbit liver, simulation
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CryoLetters 40 (1), 51-57 (2019)
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ISOCHORIC CONTAINERS AND ITS FRONTIER BETWEEN CRYOPRESERVATION AND STERILIZATION
Abril Angulo-Sherman*
Centro Universitario Tonalá, Universidad de Guadalajara, Tonalá Jalisco, México.
* Corresponding author email: abril.angulo@academicos.udg.mx
Abstract
BACKGROUND: The use of isochoric containers below the freezing point was proposed for the reduction of freezing damage.
OBJECTIVE: To determine mathematically the dielectric constant (k') of a sample inside an isochoric container depending on a suggested nucleated volume, and to compare the values with the
reported k' for an isochoric container. METHODS: Different nucleation arrangements inside a cylindrical capacitor filled with water was considered, and the way that ice Ih
changes the capacitance and the expected k' was examined. RESULTS: Dielectric constant for different nucleation arrangements decreases proportionally with the
nucleated volume, reaching smaller values when the nucleation is supposed only over the internal electrode. However, the nucleation proposed don't reproduce the experimental behavior. CONCLUSION:
When compared with experimental results, k' values suggest the water inside an isochoric container remains in liquid state (-4 ~
0°C), which may explain that there is no biological damage for this temperature range.
Keywords: isochoric, ice Ih, freezing damage, cryopreservation
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CryoLetters 40 (1), 58-63 (2019)
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TEXTURE FEATURE DIFFERENCES BETWEEN FRESH AND FROZEN-THAWED EX-VIVO PORCINE LIVER TISSUE IN B-MODE ULTRASONIC IMAGING
Gubing Wang1, Jing Liu2, Jianwen Luo2, Lei Sheng3*
1 Industrial Design Engineering, Delft University of Technology, Delft, Netherlands.
2 Department of Biomedical Engineering, Tsinghua University, Beijing, China.
3 Key Laboratory of Cryogenics, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing, China.
* Corresponding author email: shenglei@mail.ipc.ac.cn
Abstract
BACKGROUND: Ultrasound guidance of cryotherapy could be improved if changes
of tissue characterization parameters during freezing could be used to monitor freezing profiles and estimate frostbite distributions. OBJECTIVE: To investigate if B-mode
ultrasonic imaging can provide sufficient information to define the damaged zone during cryotherapy by quantifying the differences of texture features between fresh and
frozen-thawed porcine liver tissue. MATERIALS AND METHODS: The B-mode ultrasonic images were obtained from ten porcine liver tissue samples before and after
frozen-thawing treatment, then 20 texture parameters of the grey level histogram (GLH) and the grey level co-occurrence matrix (GLCM) from each image were extracted and analyzed statistically.
RESULTS: It was found that the differences in two texture parameters between the fresh and frozen-thawed tissue are significant enough for diagnostic purpose (variance: 317.0%, contrast: 144.4%).
CONCLUSION: Texture feature analysis of B-mode ultrasonic images can effectively differentiate the fresh and frozen-thawed porcine liver tissues, which offers a prospect for the future practice of
evaluating the effectiveness of cryotherapy using Ultrasound.
Keywords: B-mode ultrasonic images, cryotherapy, frozen-thawed liver tissue, image processing
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CryoLetters 40 (1), 64-70 (2019)
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TIME-DEPENDENT EFFECTS OF PRESSURE DURING PRESERVATION OF RAT HEARTS IN AN ISOCHORIC SYSTEM AT SUBFREEZING TEMPERATURES
Lili Wan1,2, Matthew J. Powell-Palm2*, Mark G. Clemens3
and Boris Rubinsky2
1 Department of Cardiovascular Surgery, Tongji Hospital, Tongji Medical College,
Huazhong University of Science and Technology, Wuhan, China.
2 Department of Mechanical Engineering & Department of Bioengineering, University of California, Berkeley, USA.
3 Department of Biological Sciences, University of North Carolina, Charlotte, USA.
* Corresponding author email: mpowellp@berkeley.edu
Abstract
BACKGROUND: Isochoric freezing systems enable ice-free preservation of biological
matter at subfreezing temperatures under the increased hydrostatic pressure. OBJECTIVE: To examine the effects of pressure and exposure period on rat hearts preserved in an isochoric chamber.
MATERIALS AND METHODS: Rat hearts were preserved in the UW solution in isochoric chambers at temperatures from -2ºC to -8ºC
and pressure from the atmospheric level to 78 MPa for up to eight hours, with and without the addition of glycerol. Hearts were evaluated via Langendorff perfusion and HE histology. RESULTS:
Hearts were compromised quickly as pressure increased, suggesting an acute time-pressure sensitivity. With the addition of 1 M glycerol, which
reduces the pressure experienced at a given temperature, the survival time at -4ºC was doubled. CONCLUSION: The enhanced hydrostatic pressure encountered during
isochoric preservation yields time-dependent negative effects on the heart, which can potentially be alleviated by the addition of a cryoprotectant.
Keywords: isochoric freezing, heart preservation, high hydrostatic pressure, cryoprotectants
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