Abstracts: CryoLetters 39 (4), 2018

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



Volume 39, No. 4 July/August 2018

ISSN 0143-2044



Effect of reduced glutathione, water soluble vitamin E analogue and butylated hydroxytoluene on the post thaw characteristics of boar spermatozoa
Santosh Kumar Baishya, Ranjan Kumar Biswas,
Kadirvel Govindasamy, Bharat Chandra Deka, Sudip Sinha
and Mahak Singh




Multivariate statistical analyses and predictive model of cold resistance associated with eleven crabapples and fuji apple
Ya HU, Yan-xiu WANG, Yan-fang ZHU and Bai-hong CHEN




Cryopreservation of winter-dormant apple buds IV: critical temperature variation that can compromise survival
C. Vogiatzi, B.W.W. Grout, A. Wetten, M. Ordidge
and S.K. Clausen




Comparison of residual dimethyl sulfoxide (DMSO) and ethylene glycol (EG) concentration in bovine ovarian tissue during warming steps between slow freezing and vitrification
Ryuichiro Obata, Yusuke Nakumura, Noriyuki Okuyama,
Chisa Sasaki, Yurina Ogura, Nobuya Aono, Seizo Hamano,
Tomoko Hashimoto and Koichi Kyono




Effects of embryonic development stage and cryoprotective agents on Galleria mellonella embryo cryopreservation
Muhamad Abidalla and Elena Cosi




Water-transport and intracellular ice formation of porcine adipose-derived stem cells during freezing
Kaixuan Zhu, SM. Chapal Hossain, Fazil Panhwar,
Zeeshan Haider, Peng Hu, Gang Zhao




Comparison of cryopreservation techniques for cells of the marine sponge Dysidea etheria
Stephanie Munroe, Dirk E. Martens, Detmer Sipkema
and Shirley A. Pomponi






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CryoLetters 39 (4), 227-234 (2018)
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Santosh Kumar Baishya1*, Ranjan Kumar Biswas2,
Kadirvel Govindasamy1, 3, Bharat Chandra Deka2,
Sudip Sinha2 and Mahak Singh1,3,4

1Krishi Vigyan Kendra, ICAR Research Complex for NEH Region Nagaland Centre, Longsachung, Nagaland;
2Department of Animal Reproduction, Gynaecology and Obstetrics, College of Veterinary Science, Assam Agricultural University, Khanapara, Assam;
3Division of Livestock Production, ICAR Research Complex for NEH Region, Umiam, Meghalaya;
4ICAR Research Complex for NEH Region Nagaland Centre, Jharnapani, Dimapur, Nagaland, India.
*Corresponding author email:


BACKGROUND: Antioxidant in freezing extender of boar semen improved post thaw sperm function. OBJECTIVE: The study compared the effects of reduced glutathione (GSH), water soluble vitamin E analogue Trolox and butylated hydroxytoluene (BHT) on quality of cryopreserved boar spermatozoa. MATERIALS AND METHODS: Using split sample technique three different antioxidants namely, GSH (1 mM), vitamin E (0.2 mM) and BHT (0.2 mM) were added to the freezing medium of lactose-egg yolk-glycerol extender, and samples were frozen using controlled freezing rate of 40C/min from -6 to -140C. Samples were evaluated for sperm motility, acrosomal status, plasma membrane integrity, mitochondrial membrane potential, lipid peroxidation and sperm DNA integrity after equilibration and after freezing. RESULTS: The supplementation of GSH, vitamin E and BHT resulted in significantly higher post thaw motility, live intact acrosome and plasma membrane intact sperm. The incidence of post thaw sperm lipid peroxidation was significantly reduced after addition of antioxidants. However, antioxidants treatment neither significantly improved mitochondrial membrane potential of live sperm sub-population nor sperm DNA integrity after freezing. There was no significant difference of the post thaw sperm characteristics among three antioxidants. Protective effect of GSH, vitamin E and BHT are comparable on cryopreserved boar spermatozoa.

Keywords: Antioxidants, boar spermatozoa, cryopreservation, semen quality




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CryoLetters 39 (4), 235-244 (2018)
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Ya HU, Yan-xiu WANG*, Yan-fang ZHU and Bai-hong CHEN

College of Horticulture, Gansu Agricultural University, Lanzhou 730070, China
*Corresponding author email:


BACKGROUND: Cold is one of the bottlenecks restricting the development of apple industry in the Northwest Loess Plateau of China. Crabapples play indispensable roles in the apple industry as excellent rootstocks and potential pollination trees. OBJECTIVE: The purpose of this experiment was to compare the cold resistance of crabapples and Fuji and provide reference for the simple evaluation of cold resistance. MATERIALS AND METHODS: 11 crabapples and ‘2001Fuji’ were employed as test materials. Nine physiological parameters were measured under low temperature (0, -15, -20, -30, -35 and -40ºC) treatments and the anatomical structures of the leaves were observed. Principal component analysis (PCA), regression analysis (RA) and system cluster analysis (SCA) were adopted to analyze the data of physiological parameters and leaf anatomical structure. RESULTS: According to PCA, the cold resistance of 12 genotypes was identified and the order of cold resistance is as follows: HS, HB, HF, HY, FS, HS, XQ, XK, XL, DG, Q, and HL. Moreover, the regression equation evaluating cold resistance was obtained by RA and 12 genotypes were divided into 4 categories by SCA. CONCLUSION: Multivariate statistical analysis methods can overcome the defects of single index evaluation. The lethal-temperature (LT50) can more accurately reflect the cold resistance of plants than other physiological parameters.

Keywords: Malus; Crabapple; cold resistance; pollination tree; multivariate statistical analyses; principal component analysis




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CryoLetters 39 (4), 245-250 (2018)
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C. Vogiatzi1, B.W.W. Grout1*, A. Wetten2, M. Ordidge2
and S.K. Clausen1

1Department of Plant & Environmental Sciences, University of Copenhagen, Højbakkegård Allé 13, DK-2630 Taastrup, Denmark.
2Crop Production; School of Agriculture Policy & Development; Faculty of Life Sciences, University of Reading, Harborne Building, RG6 6AS, UK
*Corresponding author email:


BACKGROUND: Precise temperature control in several key areas during cryopreservation of dormant, winter apple buds is critical for maximal survival. OBJECTIVE: To consider the effects of pre-harvest temperature, the duration of incubation at -30°C and variation in rewarming rate on survival.  MATERIALS AND METHODS: Dormant winter buds of Malus x domestica cultivars were harvested with two different acclimation histories and cryopreserved with variation in incubation time at -30°C. Recovery from LN using slow, intermediate and rapid rewarming was investigated as well as preservation after prolonged storage at -4°C.  RESULTS: The effects on survival of preharvest temperature regime and an altered -30°C incubation regime are cultivar dependent and an increase in rewarming rate has a strong negative effect on recovery.   CONCLUSION: Post-thaw survival of the winter-dormant buds can be compromised by increased temperature over a period as short as 5 days prior to bud harvest. Varying incubation times at -30°C produce variable, cultivar dependent, survival and moderate increases in rewarming rates can also radically reduce survival.

Keywords: Malus x domestica, cryopreservation, dormant bud, environmental temperature, cooling rate, warming




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CryoLetters 39 (4), 251-254 (2018)
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Comparison of residual dimethyl sulfoxide (DMSO) and ethylene glycol (EG) concentration in bovine ovarian tissue during warming steps between slow freezing and vitrification

Ryuichiro Obata1, Yusuke Nakumura2, Noriyuki Okuyama1,
Chisa Sasaki2, Yurina Ogura1, Nobuya Aono1,2, Seizo Hamano3,
Tomoko Hashimoto1, and Koichi Kyono1,2*

1Kyono ART Clinic Takanawa, Takanawa 3-13-1 Takanawa Court 5F, Minato-ku, Tokyo, 108-0074, Japan
2Kyono ART Clinic Mitsuiseimei, Sendai Honcho Bl, 3F 1-1-1 Honcho, Aoba-ku, Sendai, Miyagi, 980-0014, Japan
3Animal Bio-Technology Center, Livestock Improvement Association of Japan, Inc, 3-21-10 Higashi Shinagawa, Shinagawa-ku, Tokyo, 140-0002, Japan
*Information and permanent address: Koichi Kyono, Kyono ART Clinic Takanawa, Takanawa 3-13-1 Takanawa Court 5F, Minato-ku, Tokyo, 108-0074, Japan. Corresponding author email:


BACKGROUND: DMSO and EG have been used as cryoprotectants for human ovarian tissue cryopreservation, but residual cryoprotectants concentration and safety have rarely been reported. OBJECTIVE: We aimed to compare residual cryoprotectants (DMSO, EG) concentration in bovine ovarian tissue during warming steps between one kind of common slow freezing method and two kinds of vitrification methods, which are usually used for cryopreservation of human ovarian tissue in Japan. MATERIALS AND METHODS: In this study, we used five bovine ovaries with an average age of 24.2 months divided into three kinds of cryopreservation methods. All ovarian cortices cut to 1 mm thickness were cryopreserved in slow freezing and two kinds of vitrification methods. Residual cryoprotectants before, during and after warming of cryopreserved ovarian cortices were measured using GC-MS and compared. RESULTS: Concentrations of residual cryoprotectants in the ovarian tissue just before transplantation into the body after warming were high after both vitrification methods but almost zero with the slow freezing method. CONCLUSION: We are concerned about the residual cryoprotectants in ovarian tissue, and continue to study the safety of cryopreservation methods to the woman after reimplantation and her baby.

Keywords: ovarian tissue cryopreservation, residual cryoprotectant, slow freezing, vitrification




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CryoLetters 39 (4), 255-262 (2018)
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Muhamad Abidalla1* and Elena Cosi2

1Dipartimento di Scienze, Università degli Studi della Basilicata, Potenza;
2CREA – Agricultural Research Council, Centre for Agrobiological and Pedological Research, Florence, Italy
*Present address: Institute of Biology, Karl-Franzens-Universität Graz, Graz, Austria
*Corresponding author email:


BACKGROUND: The increased research use of the honeycomb pest Galleria mellonella has created the need for cryopreservation. The diverse characteristics of G. mellonella embryos at different stages may affect embryo survival upon cryopreservation due to differential dehydration, cold resistance, cryoprotectant toxicity and permeability. OBJECTIVE: The study aimed to determine the ability of G. mellonella to survive cryogenic condition in the early and late embryonic developmental stages (24h and 75h post-oviposition, PO). MATERIALS AND METHODS: A modified protocol was used to select the proper embryonic stage by comparing the early and late embryonic stages under two procedures of dechorionation–permeabilization (D/P) dilutions. The embryos at the early stage were used for testing the toxicity and viscosity effects of various cryoprotective agents. Various cryoprotectant treatments for improving the hatch rate were studied. RESULTS: The survival rates of embryos at the early stages (24h PO) were 51.5% and 69.5% respectively after cryoprotectant loading in 12% ethylene glycol (EG) for 30 min and dehydration in vitrification solution for for 10min. These survival rates of embryos at the late stages (75h PO), however, decreased to 22.9% and 34.0%, respectively. D/P treatment with Tween 20 and Tween 80 (2:6) slightly improved the survival of embryos at the 24h PO stage after immersion in liquid nitrogen (LN) from 0.1% to 0.6%. The increased exposure time in dehydration treatment from 14 to 30min in two vitrification solutions, EG and methanol (MeOH), slightly increased the survival rate of cryopreserved embryos from 0% and 0.3% to 1.1% and 1.4%, respectively, while the vitrification solution containing dimethyl sulfoxide (Me2SO or DMSO) slightly decreased the hatch rate from 1% to 0.6%. CONCLUSION: The survival rate of G. mellonella embryos after cryopreservation was affected by the oviposition period, eggshell permeability to cryoprotectant, cryoprotectant type, as well as their concentration and exposure times.

Keywords: Galleria mellonella, lepidoptera, embryogenesis, cryopreservation, cryoprotectant




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CryoLetters 39 (4), 263-268 (2018)
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Kaixuan Zhu1,#, SM. Chapal Hossain1,#, Fazil Panhwar1,#,
Zeeshan Haider1, Peng Hu2,*, Gang Zhao1,*

1Department of Electronic Science & Technology; 2 Department of Thermal Science & Energy Engineering, University of Science and Technology of China, Hefei, China, Hefei, China
# These authors contributed equally.
*Corresponding author email: or


BACKGROUND: Water transport and intracellular ice formation are important processes that relate to cryoinjury of cells upon freezing. To date, no study is reported on the characteristics of water transport and intracellular ice formation in porcine adipose-derived stem cells (pADSC). OBJECTIVE: To study water transport and intracellular ice formation upon freezing of pADSCs at different cooling rates. MATERIALS AND METHODS: The pADSCs were isolated using collagenase digestion from a subcutaneous adipose tissue of a 28-day-old Landrace pig. Freeze experiments were performed in a gas tight chamber of cryomicroscopy stage at different cooling rates between 40ºC and 150ºC. RESULTS: Water permeability coefficient Lpg and the activation energy ELP decrease with increasing cooling rates for pADSCs. The probability of intracellular ice formation increases with increasing cooling rates, being 0.35, 0.4 and 0.5 for cooling rates at 20, 30 and 60 ºC/min respectively. CONCLUSION: Based on the characteristics of water transport and intracellular ice formation in pADSCs, slow freezing is perhaps more suitable for pADSC cryopreservation.

Keywords: adipose-derived stem cells, ice nucleation, intracellular ice formation, water-transport.




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CryoLetters 39 (4), 269-278 (2018)
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Stephanie Munroe1,2,*, Dirk E. Martens1, Detmer Sipkema3,
and Shirley A. Pomponi1,2

1Wageningen University & Research, Bioprocess Engineering (Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands)
2Harbor Branch Oceanographic Institute, Florida Atlantic University (5600 US 1N, Fort Pierce, FL 34946, USA)
3Wageningen University & Research, Laboratory of Microbiology (Stippeneng 4, 6708 WE Wageningen, The Netherlands)
*Corresponding author email:


BACKGROUND: Cryopreservation is a commonly used method for the long-term storage of cell lines and provides a stable source of cells for experiments, allowing researchers to study species that are not geographically nearby, and useful to progress studies on sponge cell biotechnology. OBJECTIVE: The marine sponge Dysidea etheria was chosen as our model organism to evaluate the impact and effectiveness of two commonly used cryoprotectants, dimethyl sulfoxide (DMSO) and glycerol. MATERIALS AND METHODS: By testing a range of concentrations (3-10% DMSO, 10-50% glycerol), we determined the optimal cryoprotectant for D. etheria based on its ability to preserve viable cells and optimize recovery after cryopreservation. RESULTS: Cells cryopreserved in DMSO had significantly higher viability after cryopreservation than those cryopreserved in glycerol. Cells cryopreserved in glycerol had irregular morphology as well as lower recovery of viable cells than those from DMSO treatments. CONCLUSION: Our results demonstrate that the optimal cryoprotectant for sponge cells, without a significant loss of viability, is 5-8% DMSO. This approach can be used to optimize cryopreservation methods for cells of other marine invertebrate species.

Keywords: Cryopreservation, Porifera, glycerol, DMSO, cryoprotectants

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