Abstracts: CryoLetters 35 (2), 2014

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



Volume 35, No. 2 March/April 2014

ISSN 0143-2044



Cryopreservation of native kazakhstan apricot (Prunus Armeniaca L) seeds and embryonic axes
Irina Kovalchuk, Timur Turdiev, Zinat Mukhitdinova,
Sergey Frolov and Barbara M. Reed




Vitrification of equine oocytes with a polyvinyl alcohol after in vitro maturation with equine growth hormone and insulin-like growth factor-i
Bruna da Rosa Curcio, Gabriel Ribas Pereira,
Lucas Insaurriaga Antunes, André Nunes Boff,
Fernanda Carlini Cunha dos Santos, Thomaz Lucia Jr,
Carlos Eduardo Wayne Nogueira, Carine Dahl Corcini,
Irwin Liu and João Carlos Deschamps




Effect of vascular network and nanoparticles on heat transfer and intracellular ice formation in tumor tissues during cryosurgery
Qianfeng Yu, Jingru Yi, Gang Zhao and Yuntian Zhang




Canine sperm cryopreservation using glucose in glycerol-free tris
Il-Jeoung Yu




Development of a method of vitrification, thawing, and transfer of mammalian blastocysts using a single closed cryo-straw
Young Tae Heo, Joon Kyo Lim, Yong Nan Xu ,
Woo in Jang, Soon Hong Jeon and Nam-Hyung Kim




Freeze-thawing induced structural and functional changes in glucose oxidase
Nardid O.A., Rozanova E.D., and Naumenko E.I.




Cryopreservation of thymus lotocephalus shoot tips and assessment of genetic stability
Natacha Coelho, María Elena González-Benito,
Carmen Martín and Anabela Romano




Cryopreservation of protocorm-like body-derived shoot tips of calanthe davidii by droplet-vitrification
Liang Lin, Qian Xu, Junchao Ma, Bin Yuan and Weiqi Li




Development of vitrification protocol in rubia akane (nakai) hairy roots using a systematic approach
Sang-Un Park, Hyunjung Kong, Dong-Jin Shin,
Chang-Hwan Bae, Sheong-Chun Lee,
Chang-Hyu Bae, Eui-Shik Rha and Haeng-Hoon Kim




IVF recovery of mutant mouse lines using sperm cryopreserved with mtg in cryovials
Ming-Wen Li, Jadine M Vallelunga, Kristy L Kinchen,
Karina L Rink, Jasmin Zarrabi, Armen O Shamamian
and K C Kent Lloyd




Pre and post lipocryolysis thermic conditioning enhances rat adipocyte destruction
Hernán Pinto, David Ricart-Jané and Eva Pardina






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CryoLetters 35 (2), 83-89 (2014)
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Irina Kovalchuk1, Timur Turdiev1, Zinat Mukhitdinova1,
Sergey Frolov1, and Barbara M. Reed2*

1Institute of Plant Biology and Biotechnology, Timiryazev Str. 45, 050040, Almaty, Republic of Kazakhstan.
2United States Department of Agriculture, Agricultural Research Service, National Clonal Germplasm Repository, Corvallis, OR 97333, USA
*Corresponding authors email: and


BACKGROUND: Preserving the genetic diversity of Central Asia includes conserving wild apricots found in the foothills of several mountain ranges. These include primitive and genetically diverse populations with important characteristics for crop improvement. Apricot seeds have a short storage life, so cryopreservation of the seeds of wild populations is important for conserving the genetic diversity. OBJECTIVE: This study was to determine a suitable protocol for long-term storage. METHODS: This study tested a range of protocols using embryos and embryonic axes for storage of an important population of wild apricots and to determine if seed size and the distribution of moisture in the seed play a role in successful cryopreservation. RESULTS: Germination of scarified whole seed from trees in the ‘Jungar’ population of Prunus armeniaca varied from 63% to 90% after 1 h in liquid nitrogen (LN) and was generally better at 7% moisture content (MC) than at the original 14% MC. Embryos (4% MC) from stratified seed had only 33% germination after LN exposure. Isolated embryonic axes from non-stratified seed germinated at 86% to 100% following drying to 4% or 7% MC. Examination of three seed sizes determined that the MC of whole seed, embryos and isolated axes varied with the seed size and shape. MC of whole seeds and embryos decreased as size decreased, however, the axis MC did not. MC of medium-size seed was more evenly distributed between the axis and endosperm than in the larger or smaller samples. Cryopreservation of axes from medium-sized seed was good at any moisture content and a 1-h drying time was significantly better than 90 min. for axes of all seed sizes. Cryopreservation of axes using vitrification protocols initially designed for shoot tips produced germination similar to or lower than seed and axis drying techniques. CONCLUSION: We recommend storing apricot germplasm as unstratified seed dried to 7% MC or as isolated embryonic axes.

Keywords: Embryonic axis, germplasm storage, seed, vitrification




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CryoLetters 35 (2), 90-94 (2014)
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Vitrification of equine oocytes with a polyvinyl alcohol after in vitro maturation with equine growth hormone and insulin-like growth factor-i

Bruna da Rosa Curcio1*, Gabriel Ribas Pereira2,
Lucas Insaurriaga Antunes1, André Nunes Boff1,
Fernanda Carlini Cunha dos Santos1; Thomaz Lucia Jr3,
Carlos Eduardo Wayne Nogueira1, Carine Dahl Corcini3;
Irwin Liu2 and João Carlos Deschamps4

1Veterinary Clinics Departament, Veterinary School, Federal University of Pelotas , Pelotas, Brazil.
2Department of Population Health & Reproduction, University of California, Davis, CA 95616, USA.
3Animal Reproduction Laboratory, Veterinary School, Federal University of Pelotas, Brazil.
4Animal Pathology Department, Veterinary  School, Federal University of Pelotas, Brazil.
*Corresponding author email:


BACKGROUND: In vitro fertilization (IVF) procedures are limited by the inability to mature equine oocytes on in vitro methods.  OBJECTIVE: The aim of this study was to evaluate structural integrity of equine oocytes subjected to vitrification with a synthetic polymer (PVA). METHODS: The effect of eGH and its relationship with IGF-I on in vitro maturation (IVM) were evaluated. Compact cumulus oocytes complexes (n=122) were cultured in TCM-199 with eGH, IGF-I or eGH+IGF-I for 30h at 38.5C in air with 5% CO2. Oocytes were fixed after IVM or subjected to the vitrification protocol. Cryopreserved oocytes were exposed to 1.4M DMSO+1.8M EG+1% PVA for 3min, and then transfer to 2.8M DMSO+3.6M EG+0,6M sucrose+1% PVA for 1min. After rewarming, oocytes were evaluated by confocal microscopy. RESULTS: Maturation rates of oocytes were not significant different among groups (P>0.05), however eGH+IGF-I group can develop the assessment of resumption of meiosis (MI+MII = 86.7%).  CONCLUSION: The oocyte did not show morphological alterations. The use of PVA-copolymer may represent a potential alternative for vitrification of equine oocytes after IVM.

Keywords: oocyte cryopreservation, PVA copolymer, in vitro maturation, horses




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CryoLetters 35 (2), 95-100 (2014)
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Qianfeng Yu1,2, Jingru Yi1, Gang Zhao1,* and Yuntian Zhang1

1Department of Electronic Science and Technology, University of Science and Technology of China, Hefei 230027, China;
2Institute of Nuclear Energy Safety Technology, Chinese Academy of Sciences, Hefei 230031, Anhui, China.
*Corresponding author email:


BACKGROUND: Cryosurgery is a physical therapy of tumor treatment which is welcome in clinics for its minimally invasive advantage. However, the high recurrence rate makes the conventional cryosurgery unsatisfactory, which needs adjuvant treatment such as introduction of nanoparticles. OBJECTIVE: This study is to examine the effects of vascular network and MgO nanoparticles on heat transfer and intracellular ice formation in tumor tissues during cryosurgery. METHOD: We developed a multi-scale model to study the efficiency of cryosurgery, including the macro-level (mass tumor tissue) heat transfer and the micro-level (tumor cells) probability of intracellular ice formation (PIF). The model is used to examine the effects of fractal vascular network (VN) and nanoparticles with different concentration on heat transfer and PIF during cryosurgery in the breast cancer tissue (MCF-7 cells). The nucleation rate kinetic parameter (Ω0) and the thermodynamic parameter (κ0) of MCF-7 cells are determined by nonlinear curve-fitting the published experimental data, and then the probability of intracellular ice formation of the picked points in the tumor tissue are determined using the classic model for intracellular ice nucleation with the simulated thermal profiles at those points during cryosurgery. RESULTS AND CONCLUSION: The introduction of nanoparticles have significantly enhanced the heat transfer in the mass tumor tissue and increased the PIF of tumor cells, indicating the nanocryosurgery is more efficient than conventional cryosurgery.

Keywords: Cryosurgery, Nanocryosurgery, IIF, PIF, Fractal vascular network




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CryoLetters 35 (2), 101-107 (2014)
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Il-Jeoung Yu

Department of Theriogenology and Reproductive Biotechnologies, College of Veterinary Medicine and Bio-safety Research Institute, Chonbuk National University, Jeonju, Republic of Korea.
Corresponding author email:


OBJECTIVE: The study is to develop a glycerol-free extender using glucose for canine sperm cryopreservation,. METHODS: Tris [hydroxymethyl] aminomethane (TRIS) extender. Canine sperm were cooled to 4°C in TRIS containing 44.4 mM glucose for 100 min, and then cooled at 4°C in TRIS with glucose concentrations of 0 mM, 44.4 mM, 100 mM, 200 mM, or 300 mM for 30 min followed by cryopreservation. After thawing at 37°C for 25 sec, sperm motility, viability, and morphological abnormalities were evaluated. In addition, 300 mM glucose-TRIS was compared to TRIS extenders with a final concentration of 5% glycerol. Sperm phosphatidylserine (PS) translocation after freezing and thawing was assayed by flow cytometry using Annexin V-FITC apoptosis detection kit.  RESULTS: Progressive motility and viability (42% and 41%, respectively) were significantly higher in the 300mM group than the other groups with lower concentrations of glucose (P<0.05). PS translocation index was significantly lower in 300mM glucose-TRIS than those in extenders with glycerol (85 vs 93, P<0.05). CONCLUSION: These results indicate that cryopreservation of canine sperm using glycerol-free 300 mM glucose-TRIS is feasible and yields more motile sperm with lower PS translocation compared with extenders containing glycerol.

Keywords: canine, spermatozoa, cryopreservation, glucose, glycerol-free TRIS, PS translocation




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CryoLetters 35 (2), 108-113 (2014)
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Young Tae Heo, Joon Kyo Lim, Yong Nan Xu1, Woo in Jang1,
Soon Hong Jeon1 and Nam-Hyung Kim1

1Department of Animal Sciences, Chungbuk National University, Cheongju, South Korea
2 Min Byung Yul Infertility Hospital, Cheongju, South Korea
§Co-first authors.  Corresponding author email: Nam-Hyung Kim (


BACKGROUND: There are different methods for cryopreservation of mammalian embryos with variable degrees of success. These methods require specific vessels for embryo vitrification, thawing, and transfer.  OBJECTIVE: Here, we report a simple and inexpensive way to vitrify, thaw and transfer mammalian blastocysts in one straw. METHODS: This in-straw vitrification solution with microdrop (ISVDM) was compared with EM grid and normal 0.25 mL straw methods. RESULTS: There were no differences in the rates of re-expanded and hatching-to-hatched murine and bovine blastocysts exposed to 1, 0.5, and 0.3 M of sucrose in the diluent that was loaded into the straw. Low re-expanded and hatching-to-hatched rates of murine and bovine blastocysts were observed with PBS only. The pregnancy rates of control murine blastocysts (57.1%) and blastocysts exposed to 0.3 M sucrose in diluent and ISVDM (71.4%) were significantly higher (p<0.05) than those exposed to 1, 0.5, and 0 M sucrose and those loaded into 0.25 mL straws. The rate of offspring delivery was highest in the control group. There was no significant difference (p<0.05) in the rate of offspring delivery among ISVDM, 0.25 mL straw, and EM grid groups. CONCLUSION: Our results indicate that vitrified embryos can be warmed and diluted in a single straw and that this one-step method enables farm animal embryo transfer without a microscope or other laboratory equipment.

Key words: vitrification, mouse blastocyst, 0.25 mL straw, embryo cryopreservation.




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CryoLetters 35 (2), 114-118 (2014)
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Nardid O.A.*, Rozanova E.D., and Naumenko E.I.

Institute for Problems of Cryobiology and Cryomedicine, National Academy of Sciences of Ukraine, 61015 Kharkov, Ukraine
Corresponding author email:


BACKGROUND: Glucose oxidase enzyme may be a suitable model for studying the effect of low temperatures on structural and functional properties of biomacromolecules. OBJECTIVE: The research aim was to study the freeze-thawing effect on glucose oxidase isolated and immobilized by cross-linking with glutaraldehyde METHODS: Effects of freezing rates on conformation and activity of glucose oxidase isolated and cross-linked in solution with gluteraldehyde was studied. RESULTS: Freezing with slow rate (2ºC/min) induces significant protein aggregation, activity reduction and conformational changes in polar and hydrophobic regions. Freezing at 100ºC/min, however, causes conformational changes primarily in polar regions, insignificant aggregation, depending on the number of freeze-thawing cycles and increases enzyme activity. CONCLUSION: With the rise in glucose oxidase concentration in solution the low temperature-induced destabilization effect is reduced both during low and rapid freezing. At the slow cooling rate, cross-linking with glutaraldehyde results in more conformation alterations in polar regions of proteins, accompanied with an increase in enzyme activity.

Keywords: freezing, glucose oxidase, cross-linking, conformation.




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CryoLetters 35 (2), 119-128 (2014)
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Natacha Coelho1, María Elena González-Benito2,
Carmen Martín2 and Anabela Romano1,*

1IBB/CGB, Faculty of Sciences and Technology, University of Algarve, Campus de Gambelas, Faro, Portugal.
2Departamento de Biología Vegetal, Universidad Politécnica de Madrid, Ciudad Universitaria, Madrid, Spain
*Corresponding author email:


BACKGROUND: Thymus lotocephalus is a rare endemic species from the Algarve, Portugal, and is legally protected by Portuguese and European legislation. OBJECTIVE: The aim is to develop a cryopreservation protocol for T. lotocephalus shoot tips, as an alternative approach for the long-term conservation of this species. METHODS: Several methods (droplet-vitrification, vitrification and encapsulation-dehydration) were tested. Conditions regarding the subculture period, cold-hardening and preculture were optimized. Cryopreserved shoot tips were also assessed for their genetic stability using RAPD markers. RESULTS: Droplet-vitrification presented the best results. The best regrowth of cryopreserved shoot tips obtained eight weeks after rewarming was 67%. This was accomplished with four weeks subculture period of in vitro-donor plants at 25ºC, preculture of excised shoot tips for one day on MS medium containing 0.3 M sucrose, treatment in PVS2 for 60 min, and MS supplemented with 0.2 mg l-1 zeatin as recovery medium. The assessment using RAPD markers observed variation at a low frequency and shoots regenerated from cryopreserved apices showed normal development compared to the regular in vitro-grown shoots.  CONCLUSION: Droplet-vitrification is thus a viable method for the cryopreservation of T. lotocephalus shoot tips.

Keywords: Droplet-vitrification, vitrification, encapsulation-dehydration, genetic integrity, RAPD




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CryoLetters 35 (2), 129-137 (2014)
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Liang Lin1, 2, Qian Xu1, Junchao Ma1, Bin Yuan1 and Weiqi Li1*

1 Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China.
2 University of Chinese Academy of Sciences, Beijing 100039, China.
*Corresponding author email:


BACKGROUND: Evaluation of the conservation status of Calanthe plants in China indicated that 18 species were endangered or critically endangered. Comprehensive conservation solutions including ex situ methods, are urgently required to protect Calanthe species in ChinaOBJECTIVE: The study aims to develop a simple and efficient cryopreservation protocol using droplet-vitrification for Calanthe davidii. METHODS: Protocorm-like bodies (PLBs) were induced from nodal sections of in vitro shoots, and their proliferation was promoted by a thin cell layer culture procedure. Shoot tips excised from three leaf-stage PLBs were used in cryopreservation experiments. Key factors of the droplet-vitrification procedure including sucrose preculture, treatment with PVS2 solution and post-rewarming culture conditions were optimized to achieve a high level of regeneration. RESULTS: When the optimized procedure was applied, 77.8 ± 3.9% of cryopreserved shoot tips withstood liquid nitrogen exposure and regenerated into new PLBs. CONCLUSION: These results highlighted the importance of post-rewarming osmo-conditioning for regeneration of cryopreserved shoot tips.

Keywords: Orchidaceae, Calanthe davidii, sucrose preculture, thin cell layer culture, post-thawing osmo-conditioning




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CryoLetters 35 (2), 138-144 (2014)
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Sang-Un Park1, Hyunjung Kong2, Dong-Jin Shin3,
Chang-Hwan Bae4, Sheong-Chun Lee2, Chang-Hyu Bae2,
Eui-Shik Rha2 and Haeng-Hoon Kim2*

1Division of Plant Science and Resources, Chungnam National University, Daejeon, Korea.
2Dept. of Well-being Resources, Sunchon National University, Suncheon, Korea.
3Species Restoration Center, Korea National Park Service, Gurye, Korea.
4National Institute of Biological Resources, 42 Nanji-ro, Seo-gu, Incheon, Korea.
*Corresponding author email:


BACKGROUND: A solution-based vitrification protocol is a process of sequentially changing-solutions from which both influx of cryoprotectants (loading) and efflux of water (dehydration) were accomplished before cryo-exposure. Hence, we need to properly control the concentration /composition of the cryoprotectant solutions. OBJECTIVE: The study was, using a systematic approach, to develop a protocol for Rubia akane hairy roots, a very sensitive material to cytotoxicity of vitrification solutions. METHODS: Due to the poor response of 10-year in vitro maintained R. akane hairy roots to already established cryopreservation protocols, the following sets of experiments were designed: 1) combinational effect of preculture, osmoprotection and cryoprotection with PVS2-based (A3-70%) and PVS3-based (B5-80%) vitrification solutions; 2) different  cooling/warming rates and warming temperature; 3) varying unloading solutions (25%, 35% and 45% sucrose) and durations (7 min and 30 min) with or without changing the unloading solutions. RESULTS: Preculture and osmoprotection treatments were necessary to acquire cytotoxicity tolerance in both vitrification solutions tested and osmoprotection treatment was more critical, especially in B5-80%. A sequential osmoprotection treatment (C10-50%) following conventional osmoprotection (C4-35%) was needed to increase the post-cryopreservation regrowth. Aluminum foil strips were superior to cryovials, but the warming temperature tested (20°C and 40°C) did not affect post-cryopreservation recovery. In the unloading procedure, a longer duration (30 min) with a higher sucrose solution (S-45%) was harmful, possibly due to osmotic stress. CONCLUSION: R. akane hairy roots are very sensitive to cytotoxicity (both osmotic stress and chemical toxicity) and thus a proper process (preculture, osmoprotection, cryoprotection and unloading) is necessary for higher post-cryopreservation recovery.

Keywords: cryopreservation, cryoprotection, osmoprotection, preculture, vitrification solution.




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CryoLetters 35 (2), 145-153 (2014)
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Ming-Wen Li, Jadine M Vallelunga, Kristy L Kinchen, Karina L Rink,
Jasmin Zarrabi, Armen O Shamamian and K C Kent Lloyd*

Mouse Biology Program, University of California, 2795 Second Street, Suite 400, Davis, California 95618, USA.
*Corresponding author email:



BACKGROUND: Modification of cryoprotective medium (CPM) R18S3 (18% raffinose and 3% skim milk) by addition of monothioglycerol (MTG) or L-glutamine (Glu) has been shown to improve in vitro fertilization (IVF) using mouse sperm cryopreserved in cryostraws. However, whether these CPMs can be applied effectively to sperm cryopreserved in cryovials is unknown. OBJECTIVE: The study was to determine the comparative effectiveness of using R18S3, R18S3+Glu (100mM and 87 mM), or R18S3+MTG (477 µM) to cryopreserve various sample volumes of mouse sperm in cryovials and cryostraws. METHODS: This study compared the effects of different CPMs on motility of fresh and frozen-thawed C57BL/6J sperm and on IVF rate of C57BL/6J sperm cryopreserved in different CPMs and containers with different volumes, and then used technologies developed to cryopreserve and recover sperm of knockout mouse lines on inbred C57BL/6 backgrounds. RESULTS: Glutamine at 100 mM inhibited, but MTG at 477 µM protected, fresh sperm motility significantly (P<0.05).  Sperm cryopreserved in R18S3+MTG had significantly better (P<0.05) post-thaw progressive motility and IVF rate than when cryopreserved in R18S3 alone, R18S3+Glu (100 mM), or RSGlu87 (15.7% raffinose, 2.6% skim milk, and 87 mM L-glutamine). There was no significant difference in IVF rates among sperm cryopreserved with R18S3+MTG in cryovials or in cryostraws (P>0.05). Sperm from 63 knockout mouse lines on C57BL/6 backgrounds cryopreserved using R18S3+MTG in cryovials were all recovered successfully to genotypically-confirmed offspring. CONCLUSION: Mouse sperm on C56BL/6 backgrounds can be successfully cryopreserved in cryovials using R18S3+MTG.

Keywords: Mouse, sperm, cryopreservation, cryovial, cryostraw, IVF




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CryoLetters 35 (2), 154-160 (2014)
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Hernán Pinto*1, David Ricart-Jané2 and Eva Pardina2

1Instituto de Investigaciones para las Especialidades Estéticas y del Envejecimiento, Barcelona (i2e3); 2 Centre de Recerca en Metabolisme, Barcelona (CEREMET).
*Corresponding author email: Dr. Hernán Pinto (


BACKGROUND: New knowledge about crystallization vs. lipid-to-gel transition has surfaced recently, since some of the latest publications on lipocryolysis have focused on its action mechanism. As a result, new opportunities for technical improvements and clinical outcome optimization have opened up. The food industry has been working with lipid crystal polymorphisms for decades, and tempering seems to be the easiest method of external conditioning, in addition to being harmless. OBJECTIVE: Evaluate if pre and post lipocryolyisis thermic conditioning enhances rat adipocyte destruction. METHODS: Several temperature treatment patterns (TTP) were applied to isolated rat adipocytes. The survival of the adipocytes exposed to the different TTPs and the formation of crystals in the surviving adipocytes were assessed and analyzed. RESULT: Pre and post lipocryolysis thermic conditioning changed lipocryolyisis crystallization process and showed an enhancement in adipocyte destruction that could represent an important step in improving clinical results. CONCLUSIONS: pre and post lipocryolyisis thermic conditioning enhances rat adipocyte destruction.

Keywords: Lipocryolysis; temperature treatment patterns; preconditioning; post-conditioning

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