Abstracts: CryoLetters 37 (3), 2016

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



Volume 37, No. 3 May/June 2016

ISSN 0143-2044



Effects of vitrification protocol on the lactate dehydrogenase and total ATPase activities of Chinese Mitten crab Eriocheir sinensis embryos
Xiaorong Huang , Guangpeng Feng , Feng Zhao,
Jianyi Liu , Tao Zhang, Yu Wang and Ping Zhuang




Vitrification-based cryopreservation of protocorm-like bodies of an endangered lady’s slipper orchid: Paphiopedilum niveum (Rchb.f.) Stein
Supornchai Chaireok, Kanchit Thammasiri
and Upatham Meesawat




Cryopreservation of in vitro shoot tips of strawberry by the vitrification method – establishment of a duplicate collection of Fragaria germplasm
Monika Höfer




Effect of cryoprotectant solution and of cooling rate on crystallization temperature in cryopreserved Hypericum perforatum cell suspension cultures
Anna Mišianiková, Daniela Zubrická, Linda Petijová,
Katarína Bruňáková and Eva Čellárová




Positive effect of apoptotic inhibitor Z-VAD-FMK on vitrified-thawed porcine mii stage oocytes
Yingfang Niu, Jianjun Dai, Yaning Chen, Caifeng Wu,
Shushan Zhang and Defu Zhang




Evolving trends in cryopreservation and parameters influencing semen extender preparation - A prospective review
Sridharan T B and Vickram A S




Supercooling capacity and cryoprotectants of overwintering larvae from different populations of Holcocerus hippophaecolus
Bin Tian, Lili Xu, Miao Zhang, Yuqian Feng and Shixiang Zong




Meetings Announcements & Corrections Volume 37(2)






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CryoLetters 37 (3), 142-153 (2016)
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Xiaorong Huang , Guangpeng Feng , Feng Zhao,
Jianyi Liu , Tao Zhang, Yu Wang and Ping Zhuang *

East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of East China Sea & Oceanic Fishery Resources Exploitation and Utilization, MOA, Shanghai 200090, China
*Corresponding author E-mail address:


BACKGROUND: Vitrification is the most promising option for the cryopreservation of fish embryos but requires high concentrations of potentially toxic cryoprotectants that can also cause cell injury, and affect cell division, enzymatic activities and cell metabolism. The effect of cryopreservation on the enzyme activity in crustacean embryos has not been reported. OBJECTIVES: The aim of this study was to investigate the effects of a vitrification protocol on the enzyme activity of different stages of Eriocheir sinensis embryos. The goal was to select an appropriate stage and vitrifying solution for the cryopreservation of embryos from this crustacean. MATERIALS AND METHODS: Embryos from E.sinensis at five development stages (cleavage, blastula, gastrula, eyed stage and heart beating stage) were submitted to six kinds of cryoprotectant incorporation protocol with a five stepwise method. Six vitrifying solutions were prepared by combining cryoprotectants PG, MeOH, Me2SO and DMF in different proportions (A: 20% PG + 20% DMF; B: 20%MeOH+20% DMF; C: 20%PG+20%MeOH; D:20% PG+10% MeOH+10% DMF; E:20% Me2SO+20% PG;F:20% Me2SO+20% MeOH). After incubation in the six kinds of vitrification solutions, embryos were loaded into cryo-tubes and plunged into liquid nitrogen. The activities of two cytoplasmic enzymes, LDH and total ATPase were analyzed in control embryos, those subjected to the cryoprotectant solutions and in frozen/thawed embryos. RESULTS: The cryoprotectant incorporation protocol had important effects on the enzymatic activities, and different vitrifying solutions had distinct effects on the enzymatic activities. The early stage embryo was sensitive to the toxic effect of the cryoprotectants, with a significant drop in total ATPase in comparison with fresh, control embryos. Enzymatic activities dropped significantly after vitrification, indicating cell damage and loss of cytoplasmic enzymes. CONCLUSION: The composition of vitrifying solutions had different effects on the loss of the enzyme activity, and the later stage embryo was more resistant to the effect of vitrification.

Keywords: Chinese mitten crab; vitrifying solution; embryos; enzyme activity




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CryoLetters 37 (3), 154-162 (2016)
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Supornchai Chaireok1, Kanchit Thammasiri2 and Upatham Meesawat1*

1Department of Biology, Faculty of Science, Prince of Songkla University, Hat-Yai 90110, Thailand
2Department of Plant Science, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
*Corresponding author e-mail:


BACKGROUND: Paphiopedilum niveum (Rchb.f.) Stein, an endangered lady’s slipper orchid, is listed in CITES Appendix I and thus requires conserving. OBJECTIVE: The study aimed to cryopreserve protocorm-like bodies (PLBs) by a vitrification technique. MATERIALS AND METHODS: Four-month-old PLBs were precultured in modified Vacin and Went liquid medium with various sucrose concentrations for 24 h and 5 d with daily increasing sucrose concentrations followed by different exposure times to plant vitrification solution 2 (PVS2). RESULTS: Precultured PLBs in 0.75 M sucrose for 5 d with stepwise increase of sucrose and PVS2 for 90 min provided the highest viability after drying to low moisture content. The cryopreserved PLBs exhibited fewer dark stained nuclei and accumulated starch grains than those of the control. Cryopreserved PLBs had some shrunken epidermal cells, no change in ploidy level, a survival level of 22% and the regenerated plantlets grew well. CONCLUSION: This study is the first report evaluating the success of cryopreserved PLBs of P. niveum via a vitrification protocol.

Keywords: cryopreservation, DNA content, Paphiopedilum, protocorm-like bodies, vitrification, histology




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CryoLetters 37 (3), 163-172 (2016)
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Cryopreservation of in vitro shoot tips of strawberry by THE vitrification method – Establishment of a duplicate collection of fragaria germplasm

Monika Höfer

Julius Kühn-Institute, Federal Research Centre for Cultivated Plants, Institute for Breeding Research on Fruit Crops, Pillnitzer Platz 3a, D-01326 Dresden, Germany
Corresponding author email:


BACKGROUND: The National German Strawberry Genebank, which includes 183 cultivars and 270 accessions of wild Fragaria species, is maintained in a field collection of the Fruit Genebank at Dresden-Pillnitz.  OBJECTIVE: A duplicate collection stored in liquid nitrogen would provide a higher level of security for these irreplaceable genetic resources. MATERIALS AND METHODS: Four distinct cryopreservation protocols were tested earlier and the best method, a PVS2 vitrification method with a 14-day alternating-temperature cold acclimation, was modified for further genotype screening. RESULTS: A comprehensive genotype spectrum was tested by screening 107 Fragaria ×ananassa cultivars and 20 Fragaria wild species (51 accessions). The average recovery for the cultivars using the optimized medium was 89.55% and for the wild species 85.50%. CONCLUSION: These results indicate that the method could be used to establish a backup collection in liquid nitrogen. Meristem cultures will be established and the collection will be cryopreserved using this vitrification technique to provide cost effective long-term storage.

Keywords: cold acclimation, cryopreservation, Fragaria, germplasm conservation, PVS2




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CryoLetters 37 (3), 173-187 (2016)
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Effect of cryoprotectant SOLUTION and OF cooling rate on crystalliZation temperature in Cryopreserved Hypericum perforatum cell suspension cultures

Anna Mišianiková, Daniela Zubrická, Linda Petijová,
Katarína Bruňáková* and Eva Čellárová

Institute of Biology and Ecology, Faculty of Science, P. J. Šafárik University in Košice, Mánesova 23, 041 54 Košice, Slovakia
*Corresponding author email:


BACKGROUND: The increasing demand for hypericins and hyperforins, the unique pharmaceuticals found in the Hypericum genus, requires the development of effective tools for long-term storage of cells and tissues with unique biochemical profiles. OBJECTIVE: To determine the temperature of crystallization (TC) and of ice formation of 14 cryoprotectant mixtures (CMs) for their use in cryoprotection of H. perforatum L. cell suspensions and to evaluate the impact of the lowest Tc on post-cryogenic recovery. MATERIALS AND METHODS: TC was determined by real-time microscopy of ice formation during slow cooling to -196°C and heating to 20°C. RESULTS: Exposure of cells to CMs CM2 (PVS3) containing sucrose and glycerol or CM12 and CM13 containing sucrose, glycerol, dimethylsulfoxide and ethylene glycol decreased TC below -60°C, prevented intracellular crystallization and considerably reduced both the size of crystals and the rate of extracellular ice propagation. CONCLUSION: The selected CMs proved suitable for cryopreservation of H. perforatum cell suspensions with the maximum of 58% post-thaw recovery.

Keywords: light microscopy, cooling stage, real-time observation, ice crystals, slow cooling, rapid cooling




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CryoLetters 37 (3), 188-195 (2016)
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Yingfang Niu, Jianjun Dai*, Yaning Chen, Caifeng Wu,
Shushan Zhang and Defu Zhang*

Institute of Animal Science and Veterinary Medicine, Shanghai Academy of Agricultural Sciences; Division of Animal Genetic Engineering, Shanghai Municipal Key Laboratory of Agri-Genetics and Breeding, Shanghai, 201106, China
*Corresponding author email: and


BACKGROUND: The developmental potential of vitrified porcine oocytes is very lower, and apoptosis is considered as one of the key factors involved. OBJECTIVE: To investigate the effects of apoptotic inhibitor Z-VAD-FMK addition into the incubation medium after warming on apoptosis and developmental ability of vitrified porcine MII-stage oocytes. MATERIALS AND METHODS: The activities of several caspases, mitochondrial membrane potential (ΔΨm) and early apoptotic levels were measured. Parthenogenetic developmental ability and relative expression levels of apoptosis related genes were also detected. RESULTS: Caspase activity and early apoptotic level of the Z-VAD-FMK group were significantly lower than those of the group without Z-VAD-FMK addition, but were much higher than those of fresh group (P < 0.05). The ΔΨm of Z-VAD-FMK group was 1.19, higher than the vitrified group (0.91) and lower than the fresh group (1.33). The cleavage rate and blastocyst rate after parthenogenetic activation in the Z-VAD-FMK group were much higher than those in the vitrified group, and much lower than those in the fresh group (P < 0.05). Vitrified porcine oocytes exhibited increased expression of pro-apoptotic genes (caspase 3, 8, 9, TNF-α) and decreased genes expression levels of anti-apoptotic genes (Bcl-2, CuZnSOD), and the Z-VAD-FMK addition in incubaiton medium significantly decreased the transcripts levels of caspase 3,8,9, Bax, TNF-α and increased Bcl-2 and CuZnSOD genes expression. CONCLUSION: The addition of apoptotic inhibitor Z-VAD-FMK into the incubation medium after warming improved the in vitro developmental ability of vitrified porcine oocytes by increasing mitochondrial function, reducing apoptotic level and changing apoptosis-elated gene expression.

Keywords: Z-VAD-FMK, apoptosis, development, vitrification, porcine oocytes.




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CryoLetters 37 (3), 196-205 (2016)
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Sridharan T B and Vickram A S*

Postdoctoral Fellow, SBST, VIT University, Vellore-14, India
*Corresponding author email:


BACKGROUND: Cryopreservation is a technique by which, semen can be preserved to sub zero temperature, usually at -196°C. The freezing of semen desires vitrification mediators that diminish wreck to the cells (spermatozoan) during the freeze and thaw process. Using cryopreservation, the quality of the semen has been increased in the latest years, by which the achievement rate for the insemination techniques has increased in an agreed way. The area need to be focused is to enhance the quality of the semen extender preparation before cryopreservation. Many researchers are working in the area of cryopreservation of human semen with different semen extenders. Several parameters influence the properties of semen extender essential for better post thaw results. This review is mainly focused on a range of parameters which influence the best semen extender for cryopreservation that includes glycerol and its importance, buffer and novel usage of antimicrobial peptides as antimicrobial agents.

Key Words: cryopreservation, semen extenders, glycerol, antimicrobial peptides




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CryoLetters 37 (3), 206-217 (2016)
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Bin Tian, Lili Xu, Miao Zhang, Yuqian Feng and Shixiang Zong*

Beijing Key Laboratory for Forest Pest Control, Beijing Forestry University, Beijing, 100083, P.R. China
*Corresponding author e-mail:


BACKGROUND: Holcocerus hippophaecolus is the most serious pest occurred in seabuckthorn forest of three north areas. OBJECTIVE: The primary aims of the current study were to explore the physiological mechanisms and adaptability of H. hippophaecolus to low temperatures. MATERIALS AND METHODS: Assessing supercooling point, freezing point, and cryoprotectants of different larval instars from three different populations. RESULTS: Supercooling capacity of larvae from the 8–13 instar groups was relatively independent of temperature and other indicators such as latitude. Larvae from the 14–16 instar groups were sensitive to temperature and latitude, with generally lower limits and a wider range of SCPs than those of the other instar groups. CONCLUSION: For each population, the differences in the supercooling capacity of different instar stages for the identical period were not significant. The metabolism of fat and glycogen might not be the primary factors affecting the supercooling capacity.

Keywords: Holcocerus hippophaecolus, overwintering larvae, different populations, different instars, supercooling point, cryoprotectant content

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