Abstracts: CryoLetters 34 (6), 2013

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



Volume 34, No. 6 November/December 2013

ISSN 0143-2044



Dehydration improves cryopreservation of mat rush (Juncus decipiens Nakai) basal stem buds on cryo-plates
Takao Niino, Shin-ichi Yamamoto, Kuniaki Fukui,
Carlos Roman Castillo Martínez, Miriam Valle Arizaga,
Toshikazu Matsumoto and Florent Engelmann




Cryopreservation of Phaeocystis Antarctica
S Gäbler-Schwarz, C Rad-Menéndez, UEM Achilles-Day,
CN Campbell and JG Day




Cryopreservation of Arachis pintoi (leguminosae) somatic embryos
Hebe Y. Rey, Mirta Faloci, Ricardo Medina, Natalia Dolce,
Florent Engelmann and Luis Mroginski




Programmed cell death and necrosis during cryopreparative drying of in vitro Eucalyptus grandis axillary buds
Ida Risenga, Paula Watt and David Mycock




A new modified cut standard straw vitrification technique reduces the apoptosis of mouse blastocysts and generates more live mouse offspring
Joon Gyo Lim, Young Tae Heo, Seung Eun Lee, Woo In Jang
Seung Gi Min, Sang Jun Uhm and Nam Hyung Kim




Cryopreservation of Eucalyptus genetic resources
E. Kaya, A. Alves, L. Rodrigues, M. Jenderek,
M. Hernandez-Ellis, A. Ozudogru and D. Ellis




X-ray diffraction study confirms intra-adipocitary lipid crystallization after lipocryolysis stimulus
Hernán Pinto, David Ricart-Jané and Eva Pardina




Cryosurvival of in vitro produced embryos as affected by health status effect of oocyte donor cow
Sayyed Morteza Hosseini, Mehdi Hajian, Vajiheh Asgari,
Mohsen Forouzanfar, Somayyeh Ostadhosseini,
Fariba Moulavi, Parvaneh Abedi, Maryam Kiani,
Nima Tanhaei Vash N, Mehdi Safahani-Langroodi
and Mohammad Hossein Nasr-Esfahani




Effect of cytochalasin b pretreatment on developmental potential of ovine oocytes vitrified at the germinal vesicle stage
Adel R. Moawad, Jie Zhu, Inchul Choi, Dasari Amarnath
and Keith H.S. Campbell




Index for Volume 34
Author Index
Keyword Index






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CryoLetters 34 (6), 549-560 (2013)
© CryoLetters,


Takao Niino1*, **, Shin-ichi Yamamoto1, Kuniaki Fukui1,
Carlos Roman Castillo Martínez2, Miriam Valle Arizaga2,
Toshikazu Matsumoto3 and Florent Engelmann4

1National Institute of Agrobiological Sciences (NIAS), Tsukuba 305-8602, Japan.
2Centro Nacional de Recursos Genéticos, INIFAP, Tepatitlán de Morelos, Jalisco, México.
3Faculty of Life and Environmental Science, Shimane Univ., Matsue, 690-1102, Japan.
4Institut de recherche pour le développement (IRD), UMR DIADE, 34394 Montpellier cedex 5, France.
*Corresponding author  e-mail:
**Present address: Gene Research Cente, Univ. of Tsukuba, Tsukuba 305-8572, Japan.


Two cryopreservation procedures using aluminium cryo-plates, termed V-Cryo-plate and D-Cryo-plate, were successfully developed for in vitro mat rush (Juncus decipiens Nakai) basal stem buds. Multiple stems induced in liquid MS medium containing 8.9 μM BA by roller culture were cut into small clumps, plated on solid MS medium and cultured for 1 week at 25°C. Clumps that had produced many buds were cold-hardened at 5°C for 1-2 months. The buds with basal stems were dissected from small clumps and precultured overnight at 25°C on solid MS medium containing 0.3 M sucrose. Precultured buds were placed on aluminium cryo-plates and embedded in calcium alginate gel. Osmoprotection was performed by immersing the cryo-plates for 30 min at 25°C in loading solution (2 M glycerol + 1.0 M sucrose). In the D-Cryo-plate procedure, the buds were dehydrated to 27-25% moisture content (fresh weight) by placing the cryo-plates in the air current of a laminar flow cabinet for 2 to 3 h. In the V-Cryo-plate procedure, buds were dehydrated by immersing the cryo-plates in PVS2 vitrification solution for 40 min at 25°C. In both procedures, cooling was performed by placing the cryo-plates in uncapped cryotubes, which were immersed in liquid nitrogen. For rewarming, cryo-plates were immersed in medium with 1.0 M sucrose for 20 min at room temperature. Regrowth of cryopreserved buds of line ‘Kitakei 2’ using D-Cryo-plate and V-Cryo-plate procedures, was 90% and 80%, respectively. The two procedures were applied to 20 additional mat rush lines. Using the V-Cryo-plate procedure resulted in regrowth ranging between 13.3 and 86.7%, with an average of 52.5%. The D-Cryo-plate led to regrowth ranging between 73.3 and 96.7%, with an average of 86.3%. The D-Cryo-plate procedure will facilitate cryostorage of mat rush germplasm.

Keywords: aluminium plate, cryo-plate, D-Cryo-plate method, mat rush, V-Cryo-plate method




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CryoLetters 34 (6), 561-570 (2013)
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CRYOPRESERVATION OF Phaeocystis Antarctica

S Gäbler-Schwarz1*, C Rad-Menéndez2, UEM Achilles-Day2,
CN Campbell2, and JG Day2

1Alfred Wegener Institute for Polar and Marine Research, Bremerhaven, Germany.
2Culture Collection of Algae and Protozoa, Scottish Association for Marine Science, Oban, UK


A large number of clonal isolates of the prymnesiophyte Phaeocystis antarctica have been established at the Alfred Wegener Institute in Bremerhaven, Germany, to address questions on the genetic diversity and ecological response patterns to climate change. However, at present the wider scientific community cannot access these strains and their long-term conservation, (currently by serial transfer), cannot be assured. Cryopreservation could provide the solution to these issues, as it would guarantee the long-term security of this genetically and ecological invaluable collection. This study outlines the successful application of conventional approaches and the use of novel, combined non-penetrating and penetrating cryoprotective strategies that have been successfully applied to the different life-stages of this alga.

Keywords: Phaeocystis antarctica; cryopreservation; cryoprotectant




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CryoLetters 34 (6), 571-582 (2013)
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Hebe Y. Rey1*, Mirta Faloci1, Ricardo Medina1, Natalia Dolce1,
Florent Engelmann2 and Luis Mroginski1

1Instituto de Botánica del Nordeste (IBONE), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Facultad de Ciencias Agrarias, Universidad Nacional del Nordeste (UNNE), 3400 Corrientes, Argentina.
2Institut de Recherche pour le Développement (IRD), UMR DIADE, 911 avenue Agropolis, BP 64501, 34394 Montpellier cedex 5, France.
*Corresponding author email:


In this study, we successfully cryopreserved cotyledonary somatic embryos of diploid and triploid Arachis pintoi cytotypes using the encapsulation-dehydration technique. The highest survival rates were obtained when somatic embryos were encapsulated in calcium alginate beads and precultured in agitated (80 rpm) liquid establishment medium (EM) with daily increasing sucrose concentration (0.50, 0.75, and 1.0 M). The encapsulated somatic embryos were then dehydrated with silica gel for 5 h to 20% moisture content (fresh weight basis) and cooled either rapidly (direct immersion in liquid nitrogen, LN) or slowly (1ºC min-1 from 25ºC to -30ºC followed by immersion in LN). Beads were kept in LN for a minimum of 1 h and then were rapidly rewarmed in a 30ºC water-bath for 2 min. Finally, encapsulated somatic embryos were post-cultured in agitated (80 rpm) liquid EM with daily decreasing sucrose concentration (0.75 and 0.5 M) and transferred to solidified EM. Using this protocol, we obtained 26% and 30% plant regeneration from cryopreserved somatic embryos of diploid and triploid cytotypes. No morphological abnormalities were observed in any of the plants regenerated from cryopreserved embryos and their genetic stability was confirmed with 10 isozyme systems and nine RAPD profiles.

Key words: somatic embryo, cryopreservation, encapsulation-dehydration, genetic stability.




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CryoLetters 34 (6), 583-597 (2013)
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Ida Risenga*1, Paula Watt2 and David Mycock3

1School of Animal, Plant and Environmental Sciences, University of the Witwatersrand, Johannesburg, Private Bag 3, Wits, 2050, South Africa.
2School of Life Sciences, University of KwaZulu-Natal,  Private Bag X 54001, Durban, 4000, South Africa.
3School of Animal, Plant and Environmental Sciences, University of the Witwatersrand, Johannesburg, Private Bag 3, Wits, 2050, South Africa.
*Corresponding author email:


In preparation for cryopreservation, Eucalyptus grandis in vitro axillary buds were dried over silica gel. Pretreatment of the buds with 5 mg l-1 ABA resulted in partial resistance to water loss (0.76 to 0.45 g g-1 fresh mass basis) as compared with untreated buds (0.76 to 0.33 g g-1) and was associated with the retention of viability (70 vs. 55%). The loss of viability of the dried buds was protracted over several days. Ultrastructural examination and vital staining demonstrated cellular and tissue responses to drying. The meristem appeared to withstand drying and 72 h of rehydration whilst the leaf primordia were destroyed immediately after drying. High reactive oxygen species (ROS) activity was associated with bud excision and drying. Caspase-3-like protease activity was detected after rehydration, thereby providing evidence that the dried buds, that had ultimately died, had undergone programmed cell death. ROS production is considered to be the trigger for programmed cell death.

Keywords: abscisic acid, programmed cell death, axillary buds, caspase-3-like protease, cryo-preparative drying, Eucalyptus grandis, necrosis, reactive oxygen species.




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CryoLetters 34 (6), 598-607 (2013)
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Joon Gyo Lim1,2*, Young Tae Heo1*, Seung Eun Lee1, Woo In Jang1
Seung Gi Min3, Sang Jun Uhm4, and Nam Hyung Kim1

1Department of Animal Sciences, Chungbuk National University, Cheongju, South Korea.
2Min Byeong Yeol Obstetrics and Gynecology, Cheongju, South Korea.
3School of Medicine, Chungbuk National University, Cheongju, South Korea.
4Industrial Education Field, Sangji Youngseo College, Wonju, South Korea.
*Corresponding author email:


Effects of freezing on apoptosis and autophagy in embryos are poorly understood. This study introduces a simple and successful method (modified cut standard straw, M-CSS) for cryopreservation of mouse zygotes. Apoptosis and autophagy were investigated in cultured mouse blastocysts derived from vitrified zygotes using two vitrification containers (M-CSS vs 0.25-ml straw). The percentages of zygotes that survived and developed into blastocysts and the number of cells per blastocyst were higher in the M-CSS group than in the 0.25 ml straw group; whereas the rate of apoptosis in blastocysts was significantly lower in the M-CSS group than in the 0.25-ml straw group. The expression of the apoptosis-related gene Caspase 3 in blastocysts was higher in the 0.25-ml straw group than in the M-CSS group; however, there were no significant differences in autophagy between these two groups. Vitrified-thawed mouse zygotes were transferred into recipients. The percentage of recipients that became pregnant and the percentage of transferred zygotes that developed into live offspring were significantly lower in the 0.25-ml straw group than in the M-CSS (10.2% vs. 17.5%). In conclusion, the novel M-CSS procedure improves oocyte and embryo vitrification. The standard 0.25-ml straw vitrification procedure induces mitochondrial apoptosis in zygotes in an autophagy-independent manner.

Keywords: Vitrification, apoptosis, autophagy, modified cut standard straw




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CryoLetters 34 (6), 608-618 (2013)
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Cryopreservation of Eucalyptus Genetic Resources

E. Kaya1, 2, A. Alves1, 3, L. Rodrigues1, 4, M. Jenderek1,
M. Hernandez-Ellis1, A. Ozudogru5 and D. Ellis1

1USDA-ARS National Center for Genetic Resources Preservation, Fort Collins, CO  80526, USA.
2Gebze Institute of Technology, Molecular Biology and Genetics Department, 41400, Gebze, Kocaeli, Turkey.
3 mbrapa Labex-USA, USDA-ARS, Fort Collins, CO, USA.
4Universidade Federal de Lavras, Lavras, MG, Brazil.
5CNR-IVALSA Istituto per la Valorizzazione del Legno e Delle Specie Arboree, Florence, Italy.
*Corresponding author email:


The long-term preservation of forest genetic resources is a vital part of preserving our forest crops for future generations. Unfortunately, there are few genebanks dedicated to forest trees and very few methods for long-term preservation of forest genetic resources collections aside from field plantings of a limited number of seed-derived or elite clonal individuals. The use of cryopreservation for the long-term storage of elite germplasm is increasingly being used for the long-term preservation of clonal agronomic crops but for forest trees, such as Eucalyptus, the methodology for cryopreservation of diverse genetic resources collections has not been established. We report the successful cryopreservation of a germplasm collection of in vitro shoot cultures of thirteen Eucalyptus spp. lines consisting of two E. grandis x E. camaldulensis lines, seven E. urophylla x E. grandis lines, one E. grandis line, two E. grandis x E. urophylla lines, and one E. camaldulensis line. In a comparison of two cryopreservation methods, sucrose sensitivity limited the application of encapsulation-dehydration. However, with droplet-vitrification, all thirteen lines had good survival after cryopreservation in liquid nitrogen. A 30 min exposure to Plant Vitrification Solution 2 (PVS2) yielded post-liquid nitrogen survival between 38% and 85% depending on the line. One hundred shoot tips from all thirteen lines are currently in long-term storage as a germplasm collection.

Keywords: Conservation, forest genetic resources, germplasm, liquid nitrogen, plant genetic resources, PVS2 vitrification.




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CryoLetters 34 (6), 619-623 (2013)
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Hernán Pinto1 *, David Ricart-Jané2 and Eva Pardina2

1Instituto de Investigaciones para las Especialidades Estéticas y del Envejecimiento, Barcelona; 2 Centre de Recerca en Metabolisme, Barcelona (CEREMET).
*Corresponding author email:


Lipocryolysis always claimed localized-fat-reduction to be a consequence of local apoptotic adipocyte destruction triggered by intracellular triglyceride crystallization. The idea is now under debate, for it has been suggested that the physical changes occurring in adipocytes after lipocryolysis could be better explained by a lipid gel-like transition process rather than by lipid crystallization. Since a) lipocryolysis claims apoptosis to be the key to adipocyte destruction and clinical-result achievement and b) it considers crystallization to be a necessary step for the apoptotic stimulus unleashing, any effort to untangle, prove or discard this process is very important.

Keywords: Crystallization; gel-like transition; lipocryolisis; x-ray diffraction.




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CryoLetters 34 (6), 624-633 (2013)
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Sayyed Morteza Hosseini1, Mehdi Hajian1, Vajiheh Asgari1,
Mohsen Forouzanfar1, Somayyeh Ostadhosseini1, Fariba Moulavi1,
Parvaneh Abedi1, Maryam Kiani1, Nima Tanhaei Vash N1,
Mehdi Safahani-Langroodi 2 and Mohammad Hossein Nasr-Esfahani1*

1Department of Reproduction and Development, Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Iran.
2FKA dairy complex, Isfahan, Iran
*Corresponding author


In vitro embryo production and embryo vitrification of genetically superior cows that culled inevitably due to health problems can accelerate genetic progress. This study was carried out to investigate whether maternal age and health status effects of high genetic merit cows affect cryosurvival and developmental competence of IVP embryos. In this sense, the effects of ageing and four common culling causes of dairy cows [repeat breeding (RPB), udder problems (UPM), chronic endometritis (CRE), and lameness (LAM)] on in vitro embryo development, and in vivo developmental competence after embryo vitrification were evaluated. The mean number of oocytes obtained per cow did not vary significantly between donors indifferent groups. Cleavage rates in RPB (86.0±4.2%), SEN (81.3±2.5%) and CRE (77.6±6.3%) cows which were comparable to control (95.9±1.5%) but were significantly higher than the related rate of UPM donors (50.6±2.6%). Importantly, there was no significant difference between the blastocyst rates of different groups. Mean overall survival rate was not different between the groups and was not affected by the blastocyst production rate. There was no significant difference between pregnancy rates of different groups. The results of the present study indicated that in cattle, neither ageing, nor these four diseases affect ovarian potential in terms of the yield and quality of in vitro embryo development.

Keywords: vitrification, in vitro, embryo development, survival, health problem, ageing




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CryoLetters 34 (6), 633-643 (2013)
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Adel R. Moawad1, 2, 3, Jie Zhu1, Inchul Choi1, Dasari Amarnath1
and Keith H.S. Campbell1**

1Division of Animal Sciences, School of Biosciences, The University of Nottingham, Sutton Bonington Campus, Loughborough, LE12 5RD, UK.
2Department of Theriogenology, Faculty of Veterinary Medicine, Cairo University, Giza, P.O. Box 12211, Egypt.
3Present address: Department of Obstetrics and Gynecology, Urology Research Laboratory, H6-19, Royal Victoria Hospital, McGill University, 687 Pins Avenue West, Montreal, Quebec H3A 1A1, Canada.
*Corresponding author e-mail:
** Deceased


Oocyte cryopreservation remains a challenge in most mammalian species because of their sensitivities to chilling injuries. Relaxation of the cytoskeleton during vitrification may improve post-thaw viability and pre-implantation embryo development. The aim of this study was to investigate the effect of cytochalasin B (CB) pre-treatment before vitrification on viability, frequencies of in vitro fertilisation (IVF) and subsequent development of ovine cumulus-oocyte complexes (COCs) vitrified at the germinal vesicle (GV) stage using cryoloop. COCs obtained at slaughter were randomly divided into two groups and incubated with or without 7.5µg/mL CB for 60 min. Oocytes from each group were then vitrified using a cryoloop or used as toxicity and controls. Oocytes were then matured, fertilised, and cultured in vitro for 7 days. Viability following vitrifiaction and warming, fertilisation events following IVF and subsequent pre-implantation embryo development were evaluated. No significant differences were observed in survival rates between CB treated and non-treated oocytes in both vitrified and toxicity groups. Frequencies of fertilisation were increased in CB-vitrified group (oocytes pre-treated with CB before vitrification) than those vitrified without CB pre-treatment (57.0% vs 40.7%). Cleavage was significantly lower (P< 0.05) in vitrified and CB-vitrified oocytes at both 24 hpi (12.5% vs 9.1%) and 48 hpi (25.0% vs 16.2%) than in other groups. Based on the numbers of cleaved oocytes, (48 hpi), 16.1% and 18.8% of the cleaved embryos developed to blastocysts in both vitrified and CB-vitrified groups. These values did not differ significantly from those obtained in CB-control group (37.8%). No significant differences were observed in mean cell numbers per blastocyst between all groups. In conclusion, pre-treatment of ovine GV oocytes with cytochalasin B as cytoskeleton stabilizer before vitrification increased frequencies of in vitro fertilisation and subsequently resulted in production of good quality late stage pre-implantation embryos following IVF.

Keywords: oocyte, vitrification, ovine

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