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Abstracts: CryoLetters 37 (1), 2016



Volume 37, No. 1 January/February 2016

ISSN 0143-2044



Cryotherapy as a method for reducing the virus infection of apples (Malus sp.)
Natalya V. Romadanova, Svetlana A. Mishustina,
Dilyara A. Gritsenko, Madina Y. Omasheva,
Nurbol N. Galiakparov, Barbara M. Reed
and Svetlana V. Kushnarenko




Heat inducible expression of antifreeze protein genes from the beetles Tenebrio molitor and Microdera punctipennis
Jieqiong Li, Wenjing Ma and Ji Ma




Viscosities of concentrated NaCl-H2O AND Me2SO-NaCl-H2O solutions at subzero temperatures
Zhang Shaozhi, Jiang Qing, Fan Juli and Chen Guangming




High incidence of polyspermic fertilization in bovine oocytes matured in vitro after cryotop vitrification
In-Sul Hwang, Dae-Jin Kwon, Gi-Sun Im, Kazuya Tashima,
Shinichi Hochi and Seongsoo Hwang




Protective effects of glutathione supplementation against oxidative stress during cryopreservation of human spermatozoa
Marzieh Ghorbani, Akram Vatannejad, Iraj Khodadadi,
Iraj Amiri and Heidar Tavilani




Cryopreservation of goldfish (Carassius auratus) spermatozoa: effects of extender supplemented with taurine on sperm motility and dna damage
Filiz Kutluyer, Fatih Öğretmen and Burak Evren Inanan




First steps towards organ banks: vitrification of renal primordial
X. Garcia-Dominguez, C.D. Vera-Donoso, E. Jimenez-Trigos,
J.S. Vicente and F. Marco-Jiménez




Effects of lyophilization and rehydration on membrane surface antigens of human red blood cells
Xiaoming Zhou, Xueliang Zhang, Ying Zhang, Shuqing Hong,
Lin Li and Zhong Liu




Improved survival and developmental rates in vitrified-warmed pig oocytes after recovery culture with coenzyme Q10
In-Sul Hwang, Dae-Jin Kwon, Tae-Uk Kwak,
Jeong-Woong Lee, Gi-Sun Im, and Seongsoo Hwang






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CryoLetters 37 (1), 1-9 (2016)
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Natalya V. Romadanova1*, Svetlana A. Mishustina1,
Dilyara A. Gritsenko1, Madina Y. Omasheva1, Nurbol N. Galiakparov1,
Barbara M. Reed2 and Svetlana V. Kushnarenko1

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, 33447 Peoria Road, Corvallis, OR 97333.
*Corresponding author e-mail:


BACKGROUND: There is an urgent need in Kazakhstan for virus-free nursery stock to reinvigorate the industry and preserve historic cultivars. An in vitro collection of apples could be used for virus testing and elimination and to provide virus-free elite stock plants to nurseries. METHODS: Malus sieversii Ledeb. M. Roem. and Malus domestica Borkh. accessions were initiated in vitro for virus identification and elimination. Reverse transcription and multiplex PCR were used to test for five viruses. PVS2 vitrification was used as a tool for cryotherapy. RESULTS: Four viruses, Apple chlorotic leaf spot virus (ACLSV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV) and Apple mosaic virus (ApMV) were detected in 17 accessions. Tomato ringspot virus (ToRSV) was not detected. ACLSV affected 53.8% of the accessions, ASPV 30.8%, ASGV 5.1%, and ApMV was found only in 'Aport Alexander'. Cryotherapy produced virus-free shoot tips for seven of nine cultivars tested. Six cultivars had 60-100% elimination of ACLSV. CONCLUSIONS: An in vitro collection of 59 accessions was established. Virus elimination using cryotherapy produced virus-free shoots for seven of nine cultivars and is a promising technique for developing a virus-free apple collection.

Keywords: cryotherapy, Malus cryobank, multiplex PCR, viruses, vitrification, virus-free




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CryoLetters 37 (1), 10-18 (2016)
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Jieqiong Li, Wenjing Ma and Ji Ma*

Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, 14 Shengli Road, 830046 Urumqi, China
*Corresponding author e-mail:


BACKGROUND: Antifreeze proteins (AFPs ) play important roles in protecting poikilothermic organisms from cold damage. The expression of AFP genes (afps) is induced by low temperature. However, it is reported that heat can influence the expression of afps in the desert beetle Microdera punctipennis.  OBJECTIVE: To further detect whether heat also induce the expression of afps in other insects, and to determine the expression profiling of insect afps at different temperatures.  MATERIALS AND METHODS: The expression of antifreeze protein genes in the two beetles, Microdera punctipennis and Tenebrio molitor that have quite different living environment, under different temperatures were studied by using real–time quantitative PCR. RESULTS: Mild low temperatures (5°C~15°C), high temperature (38°C~47°C for M. punctipennis, or 37°C~42°C  for T. molitor) and temperature difference (10°C~30°C ) all stimulated strongly to the expression of AFP genes (Mpafps) in M. punctipennis which lives in the wild filed in desert. The mRNA level of Mpafps after M. punctipennis were exposed to these temperatures for 1h~5h was at least 30-fold of the control at 25°C. For T. molitor which is breeding in door with wheat bran all these temperatures stimulated significantly to the expression of Tmafps, while the extent and degree of the temperature stimulation on Tmafps expression were much lower than on Mpafps. After T. molitor were exposed to 5 °C.and 15°C.for 1h~5h, the mRNA level of Tmafps was over 6-fold and 45-fold of the control at 25°C. High temperature (37°C~42°C ) for 1h~3h treatments increased Tmafps mRNA level 4.8-fold of the control. Temperature difference of 10°C was effective in stimulating Tmafps expression. CONCLUSION: The expression of insect antifreeze protein genes both in M. punctipennis and T. molitor was induced by heat, suggesting that this phenomenon may be common in insects; the extent and degree of the influence differ in species that have different living conditions. The heat inducible expression of antifreeze protein genes hints that antifreeze proteins may involve in other functions except for antifreeze.

Keywords: desert beetle; Microdera punctipennis; Tenebrio molitor; antifreeze protein; heat




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CryoLetters 37 (1), 19-26 (2016)
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Zhang Shaozhi1, Jiang Qing1, Fan Juli2 and Chen Guangming1*

1Key Laboratory of Refrigeration and Cryogenic Technology of Zhejiang Province, Institute of Refrigeration and Cryogenics, Zhejiang University, Hangzhou;
2 College of Aerospace Engineering, Nanjing University of Aeronautics and Astronautics, Nanjing, China
*Corresponding author email:


BACKGROUND: Viscosity is an important property of cryoprotectant solutions because it directly influences diffusion and reaction at liquid state. OBJECTIVE: To measure the viscosities of concentrated NaCl-H2O and Me2SO-NaCl-H2O solutions at subzero temperatures down to -50°C.  MATERIALS AND METHODS: Solution viscosity was measured with a rotary viscometer coupled with a thermostat bath. Two viscosity models, Laliberté model and Williams–Landel–Ferry (WLF) model, were employed in the correlation and prediction. RESULTS AND CONCLUSION: The Laliberté model performed well to correlate the viscosities of NaCl-H2O solutions measured here and the viscosities of Me2SO-H2O solutions found in the literature. The parameters obtained were used to predict the viscosities of Me2SO-NaCl-H2O solutions. The average discrepancy between the calculation and the experimental data was 19.0%. With the WLF model, the average discrepancy between the calculation and the experimental data for ternary solution was 5.4%.

Keywords: viscosity, sodium chloride, dimethyl sulfoxide, solution, subzero temperature




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CryoLetters 37 (1), 27-33 (2016)
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In-Sul Hwang1, Dae-Jin Kwon1, Gi-Sun Im1, Kazuya Tashima2,
Shinichi Hochi2* and Seongsoo Hwang1*

1Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Jeollabuk-do 55365, Republic of Korea.
2Faculty of Textile Science and Technology, Shinshu University, Nagano 386-8567, Japan
*Corresponding author email:,


BACKGROUND: Vitrification with the Cryotop device is the most promising technique for oocyte cryopreservation, but the high post-warming morphological survival of bovine oocytes does not guarantee high developmental competence after in vitro fertilization (IVF). OBJECTIVE: This study was designed to examine achievement of normal fertilization in bovine oocytes vitrified-warmed with the Cryotop device. MATERIALS AND METHODS: Oocytes were matured in vitro and vitrified-warmed after complete removal of the cumulus layers. Distribution of cortical granules (CGs) was assessed by Lens culinaris agglutinin (LCA) lectin staining. Ten hours after IVF, presumptive zygotes were analyzed for pronuclear formation. Day-8 blastocysts were harvested and stained with Hoechst-33342 for total cell counting.  RESULTS: Both yield and mean cell number of the blastocysts were impaired by Cryotop vitrification. Incidence of polyspermic fertilization was three-times higher in vitrified oocytes compared to fresh oocytes. No difference in CG distribution was found between vitrified and fresh oocytes. CONCLUSION: Polyspermic fertilization induced in vitrified-warmed bovine oocytes may be one of the possible causes responsible for their low developmental potential.

Keywords: bovine oocyte, cortical granules, Cryotop vitrification, polyspermy




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CryoLetters 37 (1), 34-40 (2016)
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Marzieh Ghorbani1, Akram Vatannejad3, Iraj Khodadadi2,
Iraj Amiri4 and Heidar Tavilani5*

1Students Research Center;
2Department of Biochemistry;
3Department of Clinical Biochemistry;
4Research Center for Molecular Medicine;
5Urology and Nephrology Research Center, Hamadan University of Medical Sciences, Hamadan, Iran.
*Corresponding author email


BACKGROUND: Freeze damage is one of the most important factors which impair the membrane and DNA integrity of sperm cells. OBJECTIVE: The present study aims to investigate glutathione (GSH) supplementation on human spermatozoa cryopreservation. We determined sperm motility and viability, sperm lipid peroxidation, DNA damage, and the amount of hydrogen peroxide (H2O2) and superoxide (O2). MATERIALS AND METHODS: Twenty pooled semen samples were freeze with 5mM GSH for 10 minute (test) and without GSH as control and stored in liquid nitrogen. RESULTS: After thawing, cryovials supplemented with 5mM GSH led to higher sperm viability compared with control samples (p<0.05). Furthermore, the addition of 5mM GSH decreased sperm lipid peroxidation, DNA fragmentation, and H2O2 and O2 content compared with controls (p<0.05). CONCLUSION: GSH can be a good free radical scavenger in the freezing media and can support function of sperm cell after a cycle of freezing and thawing.

Keywords: cryopreservation, DNA fragmentation, glutathione, spermatozoa




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CryoLetters 37 (1), 41-46 (2016)
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Filiz Kutluyer1*, Fatih öğretmen2 and Burak Evren Inanan2

1Tunceli University, Fisheries Faculty, Tunceli;
2Muğla Sıtkı Koçman University, Faculty of Science, Department of Biology, Muğla, Turkey. 


BACKGROUND: Amino acids, present in seminal plasma at high concentration, protect spermatozoa against cell damage during cryopreservation. OBJECTIVE: Experiments were designed to analyze the effect of semen extender supplemented with taurine on post-thawed sperm motility and duration, as well as DNA damage. MATERIALS AND METHODS: Extenders were supplemented with 1, 2 or 4 mM taurine. Semen samples were diluted at the ratio of 1:9 with the extenders. Diluted samples were aspirated into 0.25 ml French straws and 0.1 ml pellets. DNA damage was assessed with the comet assay after cryopreservation. RESULTS: The percentage and duration of sperm motility were significantly increased by taurine. Additionally, sperm motility and the motility period in pellets were higher than in straws. The best concentration of taurine was 4 mM, and the highest post-thaw motility rate (72.50±3.54%) and duration (17.50±0.71 s) were obtained from the extender with 4 mM in pellets. DNA damage was decreased after taurine supplementation. CONCLUSION: Pellets could be used for goldfish sperm cryopreservation. T he addition of 4 mM taurine increases the post-thaw motility and decreases DNA damage on goldfish semen.

Keywords: Amino acid, Carassius auratus, goldfish, taurine, sperm quality.




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CryoLetters 37 (1), 47-52 (2016)
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X. Garcia-Dominguez1, C.D. Vera-Donoso2, E. Jimenez-Trigos3,
 J.S. Vicentea and F. Marco-Jiménez1,*

1Instituto de Ciencia y Tecnología Animal, Universidad Politécnica de Valencia, 46022- Valencia, Spain.
2Servicio de Urología, Hospital Universitari i Politècnic La Fe, 46026- Valencia, Spain.
3Instituto de Ciencias Biomédicas, Universidad CEU-Cardenal Herrera, 46115-Alfara del Patriarca, Valencia, Spain.
*Corresponding author email:


BACKGROUND: Kidney transplantation from deceased or living human donors has been limited by donor availability as opposed to the increasing demand, by the risks of allograft loss rejection and immunosuppressive therapy toxicity and by limitations of organ preservation protocols, which is essential to organise staff and facilities, transport organs, and perform necessary laboratory tests. However, the cryopreservation of composite tissues poses technical challenges beyond those seen in the preservation of single tissue types or organs.  OBJECTIVE: The purpose of our study was to establish a protocol for long-term storing of renal primordia, that generates new adult kidneys after transplant into a syngeneic non-immunosuppressed host. MATERIALS AND METHODS: Metanephroi from 16-days-old embryos were microdissected and vitrified following the minimum essential volume method and using Cryotop as a device and VM3 as vitrification solution. After 3 months of storage in liquid nitrogen (-196ºC), 20 metanephroi were warmed and transplanted using minimally invasive laparoscopic surgery into retroperitoneal fat of 5-month-old immune-competent New Zealand rabbits. In the same way, 22 fresh metanephroi were transplanted. Twenty-one days after transplantation, hosts were euthanized and developed kidneys were recovered and evaluated morphologically and histologically. RESULTS: Significant growth and fully differentiated mature glomeruli and tubule were observed in all kidney graft explants recovered. In total, 5 metanephroi (25.0%) were successfully grown after vitrification. In the same way, 12 metanephroi (54.5%) were successfully grown in the fresh group. CONCLUSION: These encouraging results reported that metanephroi not only survive vitrification, but they vascularized and developed morphologically normal glomeruli after their allotransplantation. These results suggest that it's possible to create a long-term biobank of kidney precursors as an unlimited source of organs for transplantation, and open new therapeutic possibilities for the patients with chronic renal failure.

Keywords: Biobank, metanephros, transplantation, vitrification, Cryotop®, Rabbit.




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CryoLetters 37 (1), 53-58 (2016)
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Xiaoming Zhou1, Xueliang Zhang1, Ying Zhang2,
Shuqing Hong2, Lin Li2 and Zhong Liu2,*

1School of Mechanical, Electronic, and Industrial Engineering, University of Electronic Science and Technology of China, Chengdu, 611731, China;
2Institute of Blood Transfusion, Chinese Academy of Medical Science, Chengdu, 610052, China.
*Corresponding author email:


BACKGROUND: Previous studies have demonstrated that human RBCs can survival from freeze-drying, but there is still some uncertainty on whether and how the cell properties change during the processes.  OBJECTIVE: The present study is to evaluate the effects of lyophilization and rehydration on the membrane surface antigens, such as CD antigens and blood group antigens, of human RBCs. MATERIALS AND METHODS: Human RBCs were lyophilized first through a simplified protocol, in which glycerol were used in the lyoprotectant solution instead of trehalose. Upon rehydration, a series of in-vitro experiments were performed to examine the variation of cell count, cell level of ATP and 2,3 DPG, and the surface antigens on cell membrane.  RESULTS: The cell count recovery was 58.87%±0.74%, and the cell levels of ATP and 2, 3 DPG maintained well upon rehydration. Those results are comparable to the literature, demonstrating the simplified protocol has similar effects on RBCs comparing with the reported approaches. Flow cytometry assays demonstrated that there were no significant difference in CD35, CD44, CD45, CD47 and CD71 expressions before and after freeze-drying. In blood group tests, most of the blood group antigens maintained after lyophilization and rehydration, except that Lea, Leb and JK1 showed high probabilities of falling off during the processes. 

Keywords: lyophilization; rehydration; CD antigens; blood group antigens.




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CryoLetters 37 (1), 59-67 (2016)
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In-Sul Hwang1, Dae-Jin Kwon1, Tae-Uk Kwak1, Jeong-Woong Lee2,
Gi-Sun Im1, and Seongsoo Hwang1*

1Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Jeollabuk-do 55365, Republic of Korea.
2Functional Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Republic of Korea
*Corresponding author email:


BACKGROUND: The primary problems with porcine oocyte vitrification are their low viability and development; both need improvement. OBJECTIVE: This study was designed to improve the survival and developmental rates in vitrified-warmed porcine oocytes. MATERIALS AND METHODS: Porcine oocytes matured in vitro were vitrified-warmed with Cryotop. Then the oocytes were supplemented with Q10 during recovery culture. RESULTS: The survival rates immediately after warming were 92.9% by morphological inspection and 39.3% by fluorescein diacetate (FDA) assay. The group of recovery culture with Q10 (VC+Q10) showed significantly higher viability compared to the group of recovery culture without Q10 (VC+) analyzed by morphology and the FDA. The VC+Q10 group showed a low Bax/Bcl-xl ratio and a high expression of MAP3K12 and TGFB3 compared to the VC+. The cleavage rate did not differ in both groups but, blastocyst yield was higher in VC+Q10 than the VC+ group. CONCLUSION: Supplementation of Q10 during recovery culture led to a higher blastocyst yield by increasing survival rates and regulating mRNA expressions.

Keywords: vitrification, coenzyme Q10, pig oocyte, cryopreservation, antioxidant

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