Abstracts: CryoLetters 39 (3), 2018

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



Volume 39, No. 3 May/June 2018

ISSN 0143-2044



The influence of fatty acids on cold hardiness of Eogystia hippophaecolus larvae
Bin Tian, Yuqian Feng, Lili Ren, Tao Wang and Shixiang Zong




Micropropagation and cryoconservation of the endangered plant species Artemisia laciniata (Asteraceae)
Andrea Kodym, Angelika Senula, Eva M. Temsch,
Rebecca Hood-Nowotny, Frank Schumacher, 
Olga N. Sekurova, Sergey B. Zotchev and Michael Kiehn




The improvement of memory deficits after whole-body cryotherapy – The first report
Joanna Rymaszewska, Katarzyna M Urbańska,
Dorota Szcześniak, Bartłomiej Stańczykiewicz,
Elżbieta Trypka and Agnieszka Zabłocka




A dual phosphodiesterase inhibitor, zardaverine (type 3/4), enhances motility of frozen-thawed boar sperm
Jiyeon Jeong, Soo Jin Sa, Hak-Jae Chung, Sunyoung Baek
and Inchul Choi




Role of autophagy in ovarian cryopreservation by vitrification
Yi Xian, Bo Li, Pengge Pan, Yanrong Wang, Xiuying Pei
and Yanzhou Yang




Exposure to cryogenic temperature and exercises
And changes in blood pressure

Katarzyna Zarzycka, Ireneusz Pieszyński, Bartłomiej Szrajber,
Jolanta Kujawa, Maciej Zarzycki and Paweł Kolasa




Does the addition of salubrinal to in vitro maturation medium enhance bovine blastocyst yields and embryo cryotolerance?
Van Huong Do, Simon Walton, Sally Catt and
Andrew W. Taylor-Robinson






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CryoLetters 39 (3), 166-176 (2018)
© CryoLetters,


Bin Tian1, Yuqian Feng1, Lili Ren1, Tao Wang2 and Shixiang Zong1*

1Beijing Key Laboratory for Forest Pest Control, Beijing Forestry University, Beijing 100083, PR China
2Mentougou Forestry Station, Beijing, 102300, PR China
*Corresponding author email:


BACKGROUND: Larvae of the Cossidae family moth Eogystia hippophaecolus bore into and overwinter in the roots of sea buckthorn, which damages this plant in China. OBJECTIVE: The primary aims of the current study were to investigate the effects of fatty acids on cold hardness in overwintering larvae.  MATERIALS AND METHODS: The supercooling point (SCP), low temperature mortality and fatty acid composition of different overwintering larvae were assessed. RESULTS: E. hippophaecolus larvae could survive for a long time at temperatures far below the SCP. Saturated fatty acids became less abundant as overwintering proceeded, while unsaturated fatty acids did the opposite. C10:0, C16:1, C16:0, C18:0, C20:0, C20:5, C22:0 and C24:0 fatty acids showed significant seasonal variation during the overwintering period. CONCLUSION: E. hippophaecolus is “freezing-tolerant” and cold hardiness is enhanced by increasing fatty acid unsaturation and degrading medium- and long-chain fatty acids and eicosapentaenoic acid.

Keywords: Eogystia hippophaecolus, cold hardness, supercooling point, fatty acid




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CryoLetters 39 (3), 177-189 (2018)
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Andrea Kodym1,2, Angelika Senula3, Eva M. Temsch4,
Rebecca Hood-Nowotny5, Frank Schumacher2, Olga N. Sekurova1,
Sergey B. Zotchev1 &  Michael Kiehn2

1Department of Pharmacognosy, University of Vienna, 1090 Vienna, Austria.
2Core Facility Botanical Garden, University of Vienna, 1030 Vienna, Austria.
3Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), 06466 Seeland OT Gatersleben, Germany.
4Dept. of Botany and Biodiversity Research, University of Vienna, 1030 Vienna, Austria.
5Energy Department, AIT Austrian Institute of Technology GmbH, 3430 Tulln, Austria.
*Corresponding author email:


BACKGROUND: Artemisia laciniata, mainly distributed in Siberia and Central Asia, is classified as critically endangered in Europe. OBJECTIVES: This study developed a protocol for its micropropagation and cryopreservation. MATERIALS AND METHODS: In vitro cultures from fresh seed and in vivo shoots were initiated. Micropropagation and cryopreservation protocols were developed. Bacteria detected after cryopreservation were investigated using 16S rRNA analysis. Genome size measurements of regenerated plants after cryopreservation using flow cytometry and carbon isotope measurements to evaluate stress status were also carried out. RESULTS: A. laciniata from both starting materials could be successfully propagated on MS medium with 0.5 µM BAP. Material initiated from in vivo shoots yielded lower regeneration percentages (16%) after cryopreservation than material generated from seed (57 and 63%) using the droplet-vitrification method and PVS3. Bacteria occurring after cryopreservation belonged to the genera Sphingomonas, Staphylococcus, Curtobacterium and Gordonia. There was no significant difference in the genome size and stress status between non-cryopreserved and cryopreserved plants. CONCLUSION: A. laciniata could be readily micropropagated and cryopreserved. No negative effects of cryopreservation on plant water use efficiency or on genetic stability were found.

Keywords: droplet vitrification, endophytes, GSPC, latent infections




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CryoLetters 39 (3), 190-195 (2018)
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Joanna Rymaszewska1, Katarzyna M Urbańska1*, Dorota Szcześniak1,
Bartłomiej Stańczykiewicz2, Elżbieta Trypka1 and Agnieszka Zabłocka3

1Wroclaw Medical University, Department of Psychiatry, L. Pasteur 10, 50-367 Wroclaw, Poland
2Wroclaw Medical University, Department of Nervous System Diseases, Wroclaw, Poland
3Ludwik Hirszfeld Institute of Immunology and Experimental Therapy- Polish Academy of Sciences, Department of Immunochemistry, Wroclaw, Poland
*Corresponding author email:


BACKGROUND: Mild Cognitive Impairments (MCI) and dementia are still incurable. The Whole-Body Cryotherapy (WBC) - short, cyclic exposure to extremely low temperatures - has proven anti-inflammatory and anti-oxidative effects. It can also induce hormonal, lipid and neural changes. OBJECTIVE: To evaluate the WBC effect on cognitive functioning and blood parameters of MCI patients. MATERIALS AND METHODS: Participants with MCI undertook 10 WBC sessions. Cognitive functions and depressive symptoms were assessed before the first session, after the last session and 2 weeks later. Whole blood samples were collected. RESULTS: The cognitive functioning improved after 10 WBC sessions (p<0.05), especially memory processes. WBC caused a significant (p<0.05) increase of NO plasma level, BDNF concentration (p<0.05) and reduction of IL-6 (p<0.05). The moderate relationship between NO and cognitive functioning was noticed after WBC. CONCLUSION: The preliminary results of the first study evaluating WBC on memory deficits suggest that WBC may be useful as a supportive therapy of MCI.

Keywords: whole-body cryotherapy; mild cognitive impairment; memory improvement; supportive therapy




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CryoLetters 39 (3), 196-200 (2018)
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Jiyeon Jeong1,3, Soo Jin Sa2,3, Hak-Jae Chung2, Sunyoung Baek2
and Inchul Choi1*

1Division of Animal and Dairy Sciences, College of Agriculture and Life Sciences, Chungnam University, 99 Daehak-ro, Yuseong-gu, Daejeon, 34134, Republic of Korea.
2National Institute of Animal Science, Rural Development Administration, 1500, Kongjwipatjwi-ro, Iseo-myeon, Wanjugun, Jeollabuk-do, 55356, Republic of Korea.
3Both authors contributed equally to this work.
*Corresponding author email:;


BACKGROUND: Cryopreserved semen is useful for animal breeding via artificial insemination (AI); however, the use of frozen-thawed boar sperm is limited due to cryodamage. OBJECTIVE: The goal of this study was to improve post-thaw motility of boar semen by supplementing the thawing medium with a phosphodiesterase inhibitor, Zardaverine. MATERIALS AND METHODS: Thawed boar semen samples were treated with different concentrations of Zardaverine (0, 20, 50, 75, 100 µM) and the motility was evaluated using a computer-assisted sperm analyser. Toxic effects (sperm viability, DNA integrity, mitochondrial activity) were examined by eosin-nigrosin staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and MitoTracker. RESULTS: Sperm motility values included curvilinear velocity, rectilinear speed, average value, linearity index, straightness index, and progressive motility. The kinetic values were significantly higher with the 50 µM Zardaverine supplementation compared to non-treated control. Furthermore, there were no toxic effects of the Zardaverine treatment. CONCLUSION: The dual phosphodiesterase inhibitor (type 3/4) Zardaverine significantly enhanced the motility of thawed spermatozoa without adverse effects.

Keywords: Phosphodiester inhibitor (PDEI), Zardaverine, boar spermatozoa




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CryoLetters 39 (3), 201-2106 (2018)
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Yi Xian#, Bo Li#, Pengge Pan, Yanrong Wang, Xiuying Pei*
and Yanzhou Yang*

Key Laboratory of Fertility Preservation and Maintenance, Ministry of Education, Key Laboratory of Reproduction and Genetics in Ningxia, Department of Histology and Embryology, Department of Biochemistry, Ningxia Medical University, Yinchuan, Ningxia, 75004, P.R. China
#Yi Xian  and Bo Li are equally contributed to this work.
*Corresponding author emails:;


BACKGROUND: Ovarian cryopreservation by vitrification and transplantation are useful methods to recover female fertility after radiotherapy and chemotherapy. As type II programmed cell death, autophagy plays important roles in ovarian follicle development, ovarian follicle atresia and anti-stress injury. OBJECTIVE: The potential role of autophagy in ovarian vitrification was investigated.  MATERIALS AND METHODS: Mouse ovaries were cryopreserved by vitrification, and autophagy was treated, after which the ovarian histology was checked, and ovarian follicles were counted. The apoptotic rate was detected by TUNEL, and apoptotic molecular marker cleaved caspase-3 was checked by immunofluorescence and western blot analysis. RESULTS: Our results suggested that autophagy was increased in the process of vitrification compared with the fresh ovaries (p<0.05). The number of primordial follicles was decreased through inhibiting or over-activating the autophagy by autophagy inhibitor or activator (p<0.05). However, the number of primary follicles, antral follicles and atretic follicles was not significantly different compared with vitrified/warmed groups. The apoptotic rate was significantly increased in the vitrified/warmed, autophagy-inhibiting and over-activating groups compared with the fresh group (p<0.05), and this result was further confirmed by western blot analysis. CONCLUSIONS: Taken together, autophagy was activated in the ovarian cryopreservation by vitrification and plays a role in a natural adaptive response to cold stress in ovarian cryopreservation by vitrification.

Keywords: ovarian vitrification, autophagy, apoptosis




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CryoLetters 39 (3), 211-218 (2018)
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Katarzyna Zarzycka1*, Ireneusz Pieszyński2, Bartłomiej Szrajber3, Jolanta Kujawa2, Maciej Zarzycki4 and Paweł Kolasa3

1NZOZ Krio-Med Center, Lodz, Poland  
2Department of Medical Rehabilitation, Medical University of Lodz, Poland
3University of Social Sciences of Lodz, Poland 
4NZOZ Profimedic, Lodz, Poland
*Corresponding author email:


BACKGROUND: Cryotherapy is one of the methods used in physical medicine to achieve analgesic, anti-inflammatory, antiedematous effects and to reduce muscle tension. OBJECTIVE: The aim of the study was to assess the impact of systemic cryotherapy combined with kinesitherapy on systolic and diastolic blood pressure. MATERIALS AND METHODS: A group of 215 individuals of both sexes was selected and submitted to a two-week cycle of the systemic cryotherapy treatment combined with kinesiotherapy. RESULTS: The decrease of average values was observed in systolic and diastolic blood pressure. In the case of systolic blood pressure there was a significant decrease of average value from 125 mm Hg on the first day to the value of 121.7 mmHg on 10th treatment day. In the case of diastolic blood pressure a significant decrease in the average blood pressure value of 80 mmHg to a value of 76.8 mmHg was observed in the further course of treatment.  CONCLUSION: One cryostimulation treatment causes a decrease of the average values of the systolic blood pressure and diastolic blood pressure while the series of 10 treatments leads to stabilization of pressure. Stabilization of systolic blood pressure in men was significantly slower than in women, achieving statistically higher mean values. The women were more responsive to applied cryotherapy.

Keywords: Whole-body cryotherapy, systolic blood pressure, diastolic blood pressure, kinesiotherapy




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CryoLetters 39 (3), 219-226 (2018)
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Van Huong Do1,2, Simon Walton3, Sally Catt4
and Andrew W. Taylor-Robinson1*

1School of Health, Medical and Applied Sciences, Central Queensland University, Rockhampton, Australia. 
2National Key Laboratory of Animal Cell Technology, National Institute of Animal Sciences, Hanoi, Vietnam. 
3Australian Reproductive Technologies, Mt Chalmers, Australia. 
4Education Program in Reproduction and Development, Department of Obstetrics and Gynaecology, Monash University, Clayton, Australia.
*Corresponding author email:


BACKGROUND: Modifications to in vitro maturation (IVM) media are made to improve rates of blastocyst formation and quality of mammalian embryos. Embryo quality is an important factor in the viability of embryos following cryopreservation. Salubrinal is a specific inhibitor of endoplasmic reticulum stress-induced apoptosis in eukaryotic cells. Here, it was added to IVM medium in an attempt to increase blastocyst formation and to enhance embryo quality in cattle. OBJECTIVE: To assess the effect on blastocyst formation and cryotolerance of the supplementation of salubrinal to bovine IVM medium. MATERIALS AND METHODS: Cumulus-oocyte complexes (COCs) collected from slaughterhouse ovaries were assigned randomly to two groups, either cultured in IVM medium that was supplemented with 400 mM salubrinal (treated, 262 COCs) or that was not supplemented (control, 263 COCs). All oocytes of the matured COCs were fertilized in vitro with sperm from the same proven bull and cultured for 6-7 d. At the time of blastocyst collection, expanded blastocysts were chosen for cryopreservation, while early, hatching and hatched blastocysts and those of poor quality were not used. There were 83 expanded blastocysts classified to be of good quality in both the control and salubrinal-treated groups that were subjected to vitrification. After 5 to 10 months of cold storage, the embryos were warmed and cultured in vitro for 24 h to assess the survival rate and for 48 h to assess the hatching rate. RESULTS: The blastocyst developmental rate in the salubrinal-treated group was similar to that in the control group, 61.5% compared with 62.7% (P > 0.05). The survival rate of blastocysts after vitrification was also similar, at or very close to 100%. In addition, there was no statistically significant difference in the hatching rate of expanded blastocysts derived from the COCs cultured with (treated) and without (control) addition of salubrinal to the IVM medium (91.6% compared with 85.5%; P > 0.05). CONCLUSION: Supplementation of salubrinal to the IVM medium neither improved nor reduced rates of bovine blastocyst formation and of embryo cryotolerance as determined by post-warming viability.

Keywords: cattle; in vitro maturation; in vitro-derived embryo; blastocyst; cryotolerance; salubrinal.

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