Abstracts: CryoLetters 36 (1), 2015

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



Volume 36, No. 1 January/February 2015

ISSN 0143-2044



Lutein, Trolox, ascorbic acid and combination of Trolox with ascorbic acid can improve boar semen quality during cryopreservation
Florin Varo-Ghiuru, Ileana Miclea, Andrea Hettig, Ioan Ladoşi,
Vasile Miclea, Istvn Egerszegi and Marius Zăhan




Successful production of piglets derived from mature oocytes vitrified using OPS method
Barbara Gajda, Marzena Skrzypczak-Zielińska,
Barbara Gawrońska, Ryszard Słomski and Zdzisław Smorąg




A feasibility study towards cryopreservation of silkworm eggs: response of non-diapause silkworm eggs to low temperature
Anuradha H Jingade, Lekha G, Srinivasa Babu GK,
Manjula A and Sivaprasad V




Cryopreservation effect on proliferation and differentiation potential of cultured chorion cells
Volkova N. A. and Goltsev A. N.




Effects of mechanical delipation in porcine oocytes on mitochondrial distribution, ros activity and viability after vitrification
Liang Ren, Bo Fu, Hong Ma and Di Liu




Can additives ameliorate oxidative stress and improve development of greenshell™ mussel (Perna canaliculus) oocytes during cryopreservation?
Samantha L. Gale, David J. Buritt, H. Robin Tervit,
Lindsay T McGowan and Serean L. Adams




Hydroxyapatite nanoparticles improved survival rate of vitrified porcine oocytes and its mechanism
Xinli Zhou, Weijie Li, Lu Fang, Defu Zhang and Jianjun Dai




Cryopreservation of sugarcane using the V cryo-plate technique
Tariq Rafique, Shin-ichi Yamamoto, Kuniaki Fukui,
Zahid Mahmood and Takao Niino




Vapor pressures above the vitrified sucrose solution at low temperatures
Shaozhi Zhang, Yu Peng, Bo Wang and Guangming Chen






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CryoLetters 36 (1), 1-7 (2015)
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lutein, trolox, ascorbic acid and combination of trolox with ascorbic acid can improve boar semen quality during cryopreservation

Florin Varo-Ghiuru1, Ileana Miclea1, Andrea Hettig1, Ioan Ladoşi2,
Vasile Miclea1, Istvn Egerszegi3 and Marius Zăhan1*

1Department of Animal Reproduction, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, 3-5 Manastur Street, 400372 Cluj-Napoca, Romania
2PIC Romania SRL, 8 Caimatei Street, 021056 Bucharest, Romania
3Research Group of Reproductive Biology, Research Institute for Animal Breeding and Nutrition, 1 Gesztenyes Street, 2053 Herceghalom, Hungary
*Corresponding author: e-mail:


BACKGROUND: Due to pour quality of cryopreserved boar semen, artificial innsemination with frozen-thawed semen is quite limited. Developing protocols of boar semen cryopreservation represents a priority but also a challange. OBJECTIVE: The goal of the present study was to evaluate the antioxidant potential of lutein, Trolox, ascorbic acid, and certain combinations of Trolox with ascorbic acid on boar semen cryopreservation procedure.  MATERIALS AND METHODS: Antioxidants were added to lactose-egg yolk extender, containing a final concentration of 3% glycerol and 0.5% Equex-STM. Semen of six boars was cryopreserved using straw-freezing procedure. After cryopreservation semen was thawed and evaluated for motility, normal apical ridge (NAR), hypo-osmotic swelling test (HOST) and DNA fragmentation index (DFI). Data were analyzed by one-way ANOVA.  RESULTS: The results showed better motility after thawing at the concentration of 10 μM lutein, 200 μM Trolox, 200 μM ascorbic acid and 400-200 μM Trolox and ascorbic acid. The supplementation on boar freezing extender with 10 μM lutein increased post-thawed motility, NAR and HOST values (P < 0.01), and decrease DFI (P < 0.05) in comparison with control group. Similar results were obtained using 400-200 μM Trolox and ascorbic acid, with better results in the case of DFI (P < 0.01). In comparison with the control group, a concentration of 200 μM Trolox and 200 μM ascorbic acid provided significant differences (P < 0.01) of motility and NAR. CONCLUSION: The analysis of sperm characteristics showed that lutein and the mix between Trolox and ascorbic acid used in boar semen cryopreservation can improve the quality of spermatozoa.

Keywords: cryopreservation, boar spermatozoa, lutein, Trolox, ascorbic acid




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CryoLetters 36 (1), 8-18 (2015)
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Successful production of piglets derived from mature oocytes vitrified using OPS method

Barbara Gajda1*, Marzena Skrzypczak-Zielińska2,
Barbara Gawrońska3, Ryszard Słomski2,3 and Zdzisław Smorąg1

1Department of biotechnology of Animal Reproduction, National Research Institute of Animal Production, Krakowska 1, 32-083 Balice/Krakw, Poland.
2Institute of Human Genetics, Polish Academy of Sciences, Strzeszyńska 32, 60-479 Poznań, Poland
3Department of Biochemistry and Biotechnology, University of Life Sciences, Dojazd 11, 60-632 Poznań, Poland
*Corresponding author email:


OBJECTIVE: Examination of effect of vitrification solution with or without foetal calf serum (FCS) on the in vitro and in vivo survival of matured pig oocytes. MATERIALS AND METHODS: Exp. 1: oocytes were exposed to vitrification solutions: VSa (15% DMSO, 15% EG, 0.5 M sucrose dissolved in TCM-199 with FCS) or VSb (VSa without FCS). Exp. 2: oocytes were vitrified in VSa or VSb using OPS. A fraction of vitrified oocytes were transferred to 6 synchronised and inseminated recipients. RESULTS: Survival rate after exposure and vitrification was the same for VSa and VSb. Transfer of 48 oocytes vitrified in VSb resulted with two pregnancies and 12 live piglets. Molecular analysis results: eight piglets originated from the surrogate mothers oocytes, four piglets from vitrified oocytes.  CONCLUSION: The use of DMSO and EG as cryoprotectants without serum supplementation was advantageous for the in vivo development of vitrified mature porcine oocytes.

Keywords: pig, oocyte, OPS vitrification, serum, in vivo, viability




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CryoLetters 36 (1), 19-24 (2015)
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Anuradha H Jingade*, Lekha G, Srinivasa Babu GK, Manjula A
and Sivaprasad V

Central Sericultural Germplasm Resources Centre, Central Silk Board, Govt. of India, Thally Road, Hosur-635 109, Tamil Nadu, India
Corresponding author email:
Telephones: +91 4344 222013/221148; Fax: +91 4344 220520


Insect embryos are very sensitive to chilling temperatures and vary with species and their developmental stages. Insect eggs are small and can supercool to temperatures ranging from -5C to -40C. In general, insects rely on a variety of ecological and physiological adaptations to survive low temperatures, making cryopreservation technique significantly complex. Mulberry silkworm (Bombyx mori L., Lepidoptera: Bombycidae) eggs are cleidoic with chorion (approximately 20-25m thick). Preservation of non-diapause eggs to a limited period is practiced usually to delay hatching. The advantage of early embryonic periods having resistance to low temperature is utilized for chilling of eggs and preservation for long periods. However, technique for cryopreservation of silkworm eggs is not yet developed and the identification of precise embryonic stage and chill sensitivity is necessary for effective silkworm cryopreservation. The paper reports the chill-sensitivity and tolerance of non-diapause silkworm embryos of mulberry silkworm at various embryonic stages. Silkworm embryo of 48h-age are relatively chill-sensitive as compared to other embryonic ages. This is vital information for the development of cryopreservation protocol for silkworm eggs.

Keywords: Cryopreservation, silkworm, embryonic ages, chill sensitivity, non-diapause




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CryoLetters 36 (1), 25-29 (2015)
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Volkova N. A.* and Goltsev A. N.

Institute for Рroblems of Сryobiology and Сryomedicine, National Academy of Science of Ukraine, Kharkov.
Corresponding author:


BACKGROUND: Fetoplacental tissues including the early chorion contain stem cells with various morphological and functional characteristics. Cultured chorionic cells may be used in perspective therapies of different pathologies. OBJECTIVE: To investigate the effect of cryopreservation on proliferation and differentiation potential of chorion cell culture (ChCC). MATERIALS AND METHODS: Five freezing programs for ChCC were compared: Program 1, cooling from 25C down to -30C at 0.5C/min; Program 2, cooling from 25C down to -30C at 1C/min; Program 3, cooling from 25C down to -10C at 1C/min with further cooling down to -80C at 10C/min; Program 4, cooling from 25C down to -5C at 1C/min with further cooling down to -80C at 10C/min; Program 5, cooling from 25C down to -6C at 1C/min with further crystal seeding by adding the surplus nitrogen into the chamber, and cooling down to -80C at 10C/min. Viability, adhesion, proliferation and directed differentiation were examined. RESULTS: Freezing program 5 achieved the best result, with the highest viability, adhesion, proliferation and directed differentiation. CONCLUSION: The data may help establishing better cryopreservation protocols for perspective chorionic cell lines and their further application in biotechnology.

Keywords: cryopreservation, chorion cell culture, proliferation, differentiation.




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CryoLetters 36 (1), 30-36 (2015)
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Liang Ren1**, Bo Fu2,3,4**, Hong Ma4, and Di Liu1,4*

1College of Animal Science and Technology, Northeast Agriculture University, Harbin;
2Heilongjiang Academy of Agricultural Sciences Postdoctoral Station, Harbin;
3Northeast Forestry University Postdoctoral Station, Harbin;
4Institute of Animal Husbandry Research, Heilongjiang Academy of Agricultural Sciences, Harbin, China
**Two authors contributed equally.
*Corresponding author email:


BACKGROUND: Porcine oocytes were sensitive to cooling because of the presence of excessive lipids. OBJECTIVE: To evaluate the effect of lipid removal on the mitochondrial distribution, the formation of reactive oxygen species (ROS) and the viability of porcine oocytes after vitrification. MATERIALS AND METHODS: Porcine oocytes were cultured in vitro, and lipid droplets were removed at the MII stage by mechanical delipation via micromanipulation. Mitochondrial distribution, ROS activity and oocyte viability were assessed after delipation and vitrification. RESULTS: Vitrification disrupted mitochondrial distribution in oocytes. The vitrified groups had a significantly lower rate of oocytes with normal mitrochondrial distribution than the fresh control group (39.6% and 58.5% versus 88.9%). The percentage of oocytes with normal mitochondrial distribution was significantly lower in the delipated group than that in the undelipated group after vitrification (39.6% vs 58.5%, p < 0.05). Vitrification also increased the ROS activity (p < 0.05); but there was no significant difference between the delipated and the undelipated groups (p > 0.05). Delipated oocytes developed into blastocysts via parthenogenetic activation without vitrification. Delipation significantly decreased the rate of blastocyst formation. Vitrification also decreased the rates of cleavage and blastocyst formation (p < 0.05). The delipated group had a significantly higher cleavage rate than the undelipated group after vitrification (21.4% versus 10.4%). CONCLUSION: Lipid removal at the MII stage via micromanipulation impaired their subsequent development of porcine oocytes. Although vitrification causes an abnormal distribution of mitochondria and an increase in ROS production in porcine oocytes, the removal of lipid droplets improves subsequent development after vitrification.

Keywords: mitochondrial distribution, reactive oxygen species, porcine oocyte, vitrification




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CryoLetters 36 (1), 37-44 (2015)
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Samantha L. Gale1*, David J. Buritt2, H. Robin Tervit1,
Lindsay T McGowan3 and Serean L. Adams1

1Cawthron Institute, 98 Halifax Street East, Nelson, New Zealand;
2Department of Botany, 464 Great King Street, University of Otago, Dunedin, New Zealand;
3AgResearch, Private Bag 3123, Hamilton, New Zealand.
*Corresponding author email:


BACKGROUND: Cryopreservation of P. canaliculus oocytes has not yet been achieved. OBJECTIVE: The present study is to investigate whether the incorporation of: DMSO (0.09%), α-tocopherol (0.1mM) plus taurine (1mM) and ethylenediaminetetraacetic acid (EDTA; 0.1mM), is beneficial during cryopreservation. METHODS: These three additives were incorporated to both the cryoprotectant (CPA) and recovery media, and evaluated in terms of development and oxidative stress at three key stages of cryopreservation: 1) cryoprotectant addition [10% v/v ethylene glycol plus 0.2M trehalose; final concentration], 2) cooling to 6C, and 3) cooling to 35C and liquid nitrogen immersion. RESULTS: Over all treatments (including controls) progressive cryopreservation steps resulted in a decrease in fertilization and development to D-larvae, an increase in macromolecular oxidative damage markers (protein carbonyls, lipid hydroperoxides and oxidized DNA), and a decrease in enzymatic (superoxide dismutase, catalase, glutathione S-transferase and glutathione reductase) and non-enzymatic antioxidants. CONCLUSION: Whilst results varied, the major effects of the additives were the improved percentage fertilization and a decrease in macromolecular damage.

Keywords: DNA damage, shellfish, reactive oxygen species, embryo development.




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CryoLetters 36 (1), 45-50 (2015)
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Xinli Zhou1, Weijie Li1, Lu Fang2, Defu Zhang3 and Jianjun Dai3

1Institute of Biomedical Technology, University of Shanghai for Science and Technology, Shanghai, 200093, China.
2Centre for Reproductive Medicine of Shanghai Jiaotong University Affiliated Sixth Peoples Hospital, Shanghai, 200233, China.
3Animal and Veterinary Research Institute, SAAS, Shanghai Municipal Key Laboratory of Agri-Genetics and Breeding, Shanghai, 201106, China.
Corresponding author email:


BACKGROUND: Cryopreservation of oocyte has become most essential for the long-term conservation and widespread dispersion of animal genetic resources, but the efficiency of oocyte cryopreservation remains low. OBJECTIVE: The present study is to study the effect of hydroxyapatite (HA) nanoparticles on the survival rate of vitrified oocytes and the possible mechanism of cryopreserving oocytes with nanoparticles. MATERIALS AND METHODS: Porcine oocytes were vitrified in cryoprotectant (CPA) with biocompatible HA nanoparticles by Cryotop. The recrystallization of nano-CPA was observed with cryomicroscope. RESULTS: When 0.01%, 0.02%, 0.05% and 0.1% HA nanoparticles were added into vitrification solution, the survival rate of oocytes after in vitro maturation ranged from 25.9% to 35.4%, which is significantly higher than group without HA nanoparticles (14.7%). The microphotographs of oocytes in different solutions during freezing, thawing and melting showed that HA nanoparticles at a certain concentration can hinder the recrystallization of vitrification solution during rewarming. CONCLUSION: We speculate that preventing the recrystallization is the reason that nano-CPA can promote the survival rate of vitrified oocytes.

Keywords: oocytes, nanoparticles, vitrification, recrystallization.




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CryoLetters 36 (1), 51-59 (2015)
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Tariq Rafique1, 2*, Shin-ichi Yamamoto1, Kuniaki Fukui1,
Zahid Mahmood2 and Takao Niino1, 3

1Genebank, National Institute of Agrobiological Sciences (NIAS), 2-1-2 Kannondai, Tsukuba 305-8602, Japan.
2Plant Genetic Resource Institute, National Agricultural Research Center, Islamabad 45500, Pakistan.
3University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-0006, Japan
*Corresponding author enail:


BACKGROUND: Sugarcane is a tropical crop of major importance primarily for its high sucrose content. It is difficult to conserve it in the field or in vitro because of biotic and abiotic stresses. Cryopreservation of sugarcane germplasm is an appropriate approach for conserving its genetic diversity. OBJECTIVE: This study was carried out to develop an efficient and practical cryopreservation protocol for sugarcane with high post-cryopreservation recovery. MATERIALS AND METHODS: Factors affecting regrowth after cryopreservation using the V cryo-plate method including preculture medium, size of shoot tips, sucrose concentration in loading solution, exposure time to PVS2, light conditions after liquid nitrogen exposure, presence and absence of alginate gel and recovery medium composition were studied. RESULTS: Shoot tips with a length of 1.5 to 2.0 mm, precultured on semi-solid 1/2 MS medium for 1 day and semi-solid MS medium with 0.5 M sucrose for 1 day, treated with LS containing 2.0 M glycerol + 1.6 M sucrose for 30 min and exposed to PVS2 for 30 min showed maximum (100%) recovery after cryopreservation. It was also observed that removing the alginate gel and keeping the cultures in the dark for 7 days after cryopreservation significantly improved recovery. After optimizing the cryopreservation conditions using sugarcane variety Ni-1, 10 additional varieties were cryopreserved using the optimized protocol, with regrowth ranging from 56.7% to 100%. CONCLUSION: This study showed that V cryo-plate is an efficient and practical method for cryopreservation of sugarcane shoot tips in genebanks.

Keywords: cryopreservation, PVS2, sugarcane, V cryo-plate, vitrification.




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CryoLetters 36 (1), 60-67 (2015)
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Shaozhi Zhang1, Yu Peng1, Bo Wang2 and Guangming Chen1*

1Institute of Refrigeration and Cryogenics, Zhejiang University, Hangzhou, China.
2 No.117 Hospital, Hangzhou, China
*Corresponding author email:


BACKGROUND: Vitrified solutions are encountered in food and biomaterial preservation. Data of the equilibrium water vapor pressure above the vitrified solution are scarce at temperatures below 0C.  OBJECTIVE: The study measures the water vapor pressures above vitrified sucrose solutions. MATERIALS AND METHODS: Sucrose solutions with concentration ranging from 75% to 79% (wt) were vitrified by cooling in liquid nitrogen, and placed in a thermostat bath. Vapor pressure was measured with a high precision pressure gauge using static method. The vitrification of sucrose solution was verified with DSC.  RESULTS: Experimental data were compared with two models. The empirical Zobrist model fits the experimental data well. The free volume model was improved by fitting the interaction parameter to experimental data. The average discrepancy could be reduced from 5.57% to 2.86%. CONCLUSION: The data have significant implications in the driving force of water removal in secondary drying process.

Keywords: freeze drying, sucrose solution, vitrification, water vapor pressure.

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