Volume 32, No. 1 January/February 2011 ISSN 0143-2044
	Study on the mitochondrial activity and membrane potential after exposing later stage oocytes of two gorgonian corals (junceella juncea and junceella fragilis) to cryoprotectants S. Tsai, E. Spikings, I.C. Huang and C. Lin	1-12
	Histone deacetyltransferase1 expression in mouse oocyte and their in vitro-fertilized embryo: effect of oocyte vitrification Jun-Jie Li, Yan Pei , Guang-Bin Zhou, Lun Suo, Yan-Ping Wang, Guo-Quan Wu, Xiang-Wei Fu, Yun-Peng Hou and Shi-En Zhu	13-20
	Uniformity trials in plant freezing assays involving microtitre plates Dimitris Zaragotas and Elias Anastassopoulos	21-27
	The effects of desiccation and exposure to cryogenic temperatures on embryonic axes of Landolphia kirkii Provain Kistnasamy, Patricia Berjak and N.W. Pammenter	28-39
	Studies on cryoprotectant toxicity to zebrafish (danio rerio) ovarian tissue fragments S. Anil, F. Ghafari, T. Zampolla, D. M. Rawson and T. Zhang	40-50
	Sheep ovarian tissue vitrification by two different dehydration protocols and needle immersing methods Rouhollah Fathi, Mojtaba Rezazadeh Valojerdi Hussein Eimani, Fatemeh Hasani, Poopak Eftekhari Yazdi, Zahra Ajdari and Leila Sadat Tahaei	51-56
	Cryopreservation of goldfish caudal fin explants using glycerol as a cryoprotecant Casiano H. Choresca Jr., Dennis K. Gomez, Ji Hyung Kim, Jee Eun Han, Sang Phil Shin, Byeong Chun Lee and Se Chang Park	57-61
	Aseptic minimum volume vitrification technique for porcine parthenogenetically activated blastocySTS Lin Lin, Yutao Du, Xiuqing Zhang, Huanming Yang, Lars Bolund, Henrik Callesen and Gábor Vajta	62-68
	Structural, ultrastructural and functional studies of human cardiac valve allografts that suffered an increment of the cryostorage temperature Victoria Bessone, María D. Pizarro, María F. Izaguirre, María E. Biancardi, Graciela Furno, Norberto Baumgartner, Joaquín V. Rodríguez and Alejandra B. Quintana	69-80
	Cryopreservation of umbilical cord blood-derived mesenchymal stem cells without dimethyl sulfoxide Hai-Yan Wang, Zhao-Rong Lun and Shu-Shen Lu	81-88

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CryoLetters 32 (1), 1-12 (2011)
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STUDY ON THE MITOCHONDRIAL ACTIVITY AND MEMBRANE POTENTIAL AFTER EXPOSING LATER STAGE OOCYTES OF TWO GORGONIAN CORALS (JUNCEELLA JUNCEA AND JUNCEELLA FRAGILIS) TO CRYOPROTECTANTS

S. Tsai³, E. Spikings², I.C. Huang¹ and C. Lin^1*

¹National museum of Marine Biology & Aquarium, 2 Houwan Road, Checheng, Pingtung, 944, Taiwan.
²LIRANS institute of Research in the Applied Natural Sciences, University of Bedfordshire, 250 Butterfield, Great Marlings, Luton, Bedfordshire, LU2 8DL, UK
³Department of Biotechnology, Mingdao University, Peetow, Chang Hua, Taiwan
*Corresponding author e-mail: chiahsin@nmmba.gov.tw

Abstract

Coral reefs provide a valuable habitat for many economically valuable fish and invertebrates. However, they are in serious jeopardy, threatened by increasing over-exploitation, pollution, habitat destruction, disease and global climate change. Here, we examined the effect of cryoprotectant exposure on mitochondrial activity and membrane potential (ΔΨm) in coral oocytes in order to find suitable cryoprotectants towards their successful cryopreservation. According to the No Observed Effect Concentrations (NOECs), methanol was found to be the least toxic cryoprotectant whilst DMSO was the most toxic cryoprotectant. The results also demonstrated that there were no significant differences (p > 0.05) in ATP concentrations between Junceella juncea and Junceella fragilis after exposure to all concentrations of all cryoprotectants for 30 min. Using confocal microscopy, JC-1 (5,50,6,60-tetrachloro-1,10,3,30-tetraethyl-imidacarbocyanine iodide) staining indicated that the mitochondrial membrane potential of Junceella fragilis oocytes reduced after 1 M and 2 M methanol treatment and a loss of the mitochondrial distribution pattern and poor green fluorescence after 3M methanol treatment. Therefore, even oocytes that show no adverse effect of cryoprotectants on survival might suffer some more subtle impacts. The results obtained from this study will provide a basis for development of protocols to cryopreserve the oocytes of gorgonian corals.

Keywords: gorgonian coral, oocyte, mitochondrial membrane potential, cryoprotectants

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CryoLetters 32 (1), 13-20 (2011)
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HISTONE DEACETYLTRANSFERASE1 EXPRESSION IN MOUSE OOCYTE AND THEIR IN VITRO-FERTILIZED EMBRYO: EFFECT OF OOCYTE VITRIFICATION

Jun-Jie Li^1,2#, Yan Pei^1# , Guang-Bin Zhou¹, Lun Suo¹, Yan-Ping Wang¹, Guo-Quan Wu¹, Xiang-Wei Fu¹, Yun-Peng Hou¹ and Shi-En Zhu^1*

¹College of Animal Science and Technology, State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing, 100193, P.R.China.
²College of Animal Science and Technology, Agricultural University of Hebei, Baoding, 071000, P.R.China.
^#These authors contributed equally to this work.
^*Corresponding author e-mail: zhushien@cau.edu.cn

Abstract

This study was conducted to investigate the expression of Histone Deacetyltransferase1 (HDAC1) in mouse embryos derived from the vitrified-warmed oocytes. Firstly, the mouse oocytes at metaphaseII (MII) stage were randomly allocated into three groups: A untreated (control), B exposed to vitrfication solution (VS) without being plunged into liquid nitrogen (toxicity), or C vitrified by open-pulled straw(OPS) method (vitrification). After warming, they were fertilized in vitro. Fresh oocytes were used as control. Expression of HDAC1 was then examined in MII mouse oocytes and embryos by immunofluorescence with anti-HDAC1 polyclonal antibody and fluorescein isothiocyanate-conjugated goat anti-rabbit IgG. Results showed that after in vitro fertilization (IVF), developmental rates to two-cell embryos (38.52%), 4-cell embryos (35.25%), morula (31.97%) and blastocysts (26.23%) in cryopreserved oocytes were all significantly lower than those of fresh oocytes (P <0.01). In addition, HDAC1 expression in the vitrified group was significantly lower (P<0.05) than that in the control and toxicity groups at all developmental stages except for the blastocyst. Moreover, the vitrified-warmed oocytes showed significantly lower (P<0.05) HDAC1 expression compared with that of control and toxicity groups. In conclusion, HDAC1 was expressed both in oocytes and in their in vitro-fertilized embryos. This decreased expression of HDAC1 in mouse oocytes and the embryos due to the cryopreservation may have a negative impact on embryo development.

Keywords: OPS Vitrification, Oocyte, Early Embryo, Histone Deacetyltransferase 1, Mouse

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CryoLetters 32 (1), 21-27 (2011)
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UNIFORMITY TRIALS IN PLANT FREEZING ASSAYS INVOLVING MICROTITRE PLATES

Dimitris Zaragotas¹ and Elias Anastassopoulos^2*

¹Department of Forestry and Management of Natural Environment, Technological Educational Institute of Larissa (Karditsa), 43100 Karditsa, Greece.
^2*Department of Plant Production, Technological Educational Institute of Larissa, 41110 Larissa, Greece.
*Corresponding author email: anastaso@teilar.gr

Abstract

The primary aim of this study was to measure the inherent experimental variability in plant freezing assays involving microtitre plates. Laurus nobilis leaf strips were used as experimental material. Data analysis involved variability measurements among and within microtitre plates. Statistically significant variability (p < 0.05) was observed in both cases. The second aim was to test the effectiveness of five experimental designs, in controlling the experimental error. According to our results the variability in microtitre plate freezing assays can be controlled by the use of blocked experimental designs and single well plots.

Keywords: uniformity trial, experimental design, microtitre plate, freezing assay, plant, high-throughput screening, Laurus nobilis.

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CryoLetters 32 (1), 28-39 (2011)
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THE EFFECTS OF DESICCATION AND EXPOSURE TO CRYOGENIC TEMPERATURES ON EMBRYONIC AXES OF LANDOLPHIA KIRKII

Provain Kistnasamy, Patricia Berjak* and N.W. Pammenter

School of Biological and Conservation Sciences, University of KwaZulu-Natal (Westville Campus), Durban, South Africa
*Corresponding author email: berjak@ukzn.ac.za

Abstract

   The present study reports on the effects of rapid dehydration, chemical cryoprotectants and various cooling rates on survival, assessed by the ability for both root and shoot development, of embryonic axes excised with a small portion of each cotyledon, from mature, recalcitrant seeds of Landolphia kirkii. All axes withstood rapid (flash) drying to a water content of c. 0.28 g g^-1 (dry mass basis); however, the use of chemical cryoprotectants before flash drying was lethal. Rapid cooling rates were detrimental to axes flash-dried to 0.28 g g^-1, reducing survival to 10% and 0% after exposure to -196C and -210C, respectively. Ultrastructural examination revealed that decompartmentation and loss of cellular integrity were associated with viability loss after rapid cooling to cryogenic temperatures, although lipid bodies retained their morphology. Hence, lipid crystallisation was not implicated in cell death following dehydration, exposure to cryogenic temperatures and subsequent rewarming and rehydration. Cooling at 1C min^-1 facilitated survival of 70% of axes with attached cotyledonary segments at 0.28 g g^-1 after exposure to -70C, with 45% viability retention when further cooled at 15C min^-1 to -180C. However, no axes excised without attached cotyledonary segments produced shoots after cryogenic exposure. The use of slow cooling rates is promising for cryopreservation of mature axes of L. kirkii, but only when excised with a portion of each cotyledon left attached.

Keywords: cooling rate, cryopreservation, dehydration, excision damage, Landolphia kirkii, lipid body, recalcitrant

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CryoLetters 32 (1), 1-12 (2011)
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STUDIES ON CRYOPROTECTANT TOXICITY TO ZEBRAFISH (Danio rerio) OVARIAN TISSUE FRAGMENTS

S. Anil, F. Ghafari, T. Zampolla, D. M. Rawson and T. Zhang*

LIRANS Institute of Research in the Applied Natural Sciences, University of Bedfordshire, 250 Butterfield, Great Marlings, Luton, Bedfordshire, LU2 8DL, UK

Abstract

Cryopreservation of ovarian tissue is a viable alternative to cryopreservation of oocytes and embryos in many species but it has not been studied in fish. Selection of cryoprotectant is an important step in designing cryopreservation protocols. In order to identify the optimum cryoprotectant (CPA) in a suitable concentration for zebrafish ovarian tissue cryopreservation, studies on toxicities of five commonly used cryoprotectants methanol, ethanol, dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propylene glycol (PG) were carried out. Experiments were conducted on ovarian tissue fragments consisting of stage I and stage II ovarian follicles. Ovarian tissue fragments were incubated in 90% L-15 medium (pH 9) containing 1-4M cryoprotectants for 30min at 22°C. Three different tests were used to assess ovarian tissue fragment viability: trypan blue (TB) staining, fluorescein diacetate (FDA) combined with propidium iodide (PI) staining and adenosine 5´- triphosphate (ATP) assay. Results from these tests showed that ATP assay was more sensitive than FDA+PI or TB staining for assessing cryoprotectant toxicity to follicles in tissue fragments. Methanol and ethanol were the least toxic cryoprotectants tested. Cryoprotectant toxicity increased in the order of methanol/ethanol, DMSO, PG and EG. Ethanol was used for zebrafish ovarian tissue for the first time and the results showed that the effect of methanol and ethanol on ovarian tissue fragments were comparable. As methanol has been shown to be the most effective cryoprotectant for zebrafish ovarian follicles in our laboratory, the use of ethanol will also be considered in assisting future freezing protocol design. The present study also showed that stage II ovarian follicles are more sensitive to cryoprotectant treatment than stage I follicles in tissue fragments. The results obtained in this study provided useful information for ovarian tissue fragment cryopreservation protocol design in the future.

Keywords: zebrafish (Danio rerio), ovarian tissue fragments, cryopreservation, cryoprotectants, TB, FDA-PI, ATP assay

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CryoLetters 32 (1), 51-56 (2011)
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SHEEP OVARIAN TISSUE VITRIFICATION BY TWO DIFFERENT DEHYDRATION PROTOCOLS AND NEEDLE IMMERSING METHODS

Rouhollah Fathi¹, Mojtaba Rezazadeh Valojerdi^{1, 2}*, Hussein Eimani¹, Fatemeh Hasani¹, Poopak Eftekhari Yazdi¹, Zahra Ajdari¹ and Leila Sadat Tahaei¹

¹Department of Embryology, Infertility and IVF Unit, Royan Institute, East Hafez Ave., Bani Hashem Sq., Resalat St., P.O. Box 19395-4644, Tehran, Iran
²Department of Anatomy, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran
^*Corresponding author e-mail: mr_valojerdi@yahoo.com / mr_valojerdi@modares.ac.ir

Abstract

The present research investigated the effects of two vitrification methods on sheep ovarian tissue. The base medium (BM) of the vitrification solutions contains 60% HEPES tissue culture medium (HTCM), 15% ethylene glycol (EG) and 15% dimethyl sulfoxide (DMSO). Ovarian cortex pieces were dehydrated by two different regimens, the 2-step which consisted of 50% BM and a 90% solution of 0.25 mol/L sucrose in BM for 10 minutes each at 4˚C and the 4-step method which utilized: a) 25% BM, b) 50% BM, c) 75% BM and d) a 90% solution of 0.25 mol/L sucrose in BM for 5 minutes each at 4˚C. After warming, the proportion of intact antral follicles in the control (non-vitrified) and 2-step vitrification groups was significantly higher than in the 4-step vitrification group. The number of apoptotic follicles in the ovarian pieces was significantly different between the control and 4-step vitrification groups. These results indicated that sheep ovarian tissue vitrification by the 2-step method was simpler and more effective than the 4-step method.

Keywords: vitrification, dehydration, sheep ovary

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CryoLetters 32 (1), 57-61 (2011)
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CRYOPRESERVATION OF GOLDFISH CAUDAL FIN EXPLANTS USING GLYCEROL AS A CRYOPROTECANT

Casiano H. Choresca Jr.¹, Dennis K. Gomez³, Ji Hyung Kim¹, Jee Eun Han¹, Sang Phil Shin¹, Byeong Chun Lee²* and Se Chang Park¹*

¹Laboratory of Aquatic Animal Medicine, College of Veterinary Medicine, Seoul National University, Seoul 151-742, South Korea
²Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, South Korea
³Brain Korea 21 Program for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul 151-742, South Korea.
*Corresponding author email: parksec@snu.ac.kr, bclee@snu.ac.kr

Abstract

The objective of this study was to investigate the effect of glycerol on the cryopreservation fin explants of goldfish, Carassius auratus. Four different concentrations, 5, 10, 15, and 20% (v/v) of glycerol and a control were tested. These were prepared in Dulbecco's modified Eagle's medium with 20% (v/v) Fetal Bovine Serum. Attachment and outgrowing rates were monitored from day 3 to day 14. Results showed that fin explants cryopreserved in 20% concentration of glycerol was significantly higher (P<0.05) with a 100% attachment rate compared to 5, 10, and 15% concentrations with 36.67, 84.19 and 86.51% attachment rate, respectively. Fin explants cryopreserved in 20% glycerol concentration also had significantly higher (P<0.05) outgrowth of cells (72.50%) than the other three concentrations on day 3. Moreover, a 100% outgrowth of cells in all concentrations was achieved after 14 days of culture.  No attachment and out growth of cells were observed in control group. Goldfish caudal fin explants cryopreserved in glycerol can produce live cells efficiently, regardless of concentration.

Keywords: caudal fin, cryopreservation, fish explants, glycerol, goldfish

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CryoLetters 32 (1), 62-68 (2011)
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ASEPTIC MINIMUM VOLUME VITRIFICATION TECHNIQUE FOR PORCINE PARTHENOGENETICALLY ACTIVATED BLASTOCYST

Lin Lin^{1, 2, 3, *}, Yutao Du³, Xiuqing Zhang³, Huanming Yang³, Lars Bolund², Henrik Callesen¹ and Gábor Vajta¹

¹Institute of Genetics and Biotechnology, Faculty of Agricultural Sciences, Aarhus University, DK-8830 Tjele, Denmark
²Institute of Human Genetics, Aarhus University, DK-8000 Aarhus, Denmark
³BGI/HuaDa Shenzhen, 518000 Shenzhen, China
^*Corresponding author email: linlin@genomics.org.cn
Phone: 0086-755-25273786 Fax: 0086-755-25274307

Abstract

Minimum volume vitrification may provide extremely high cooling and warming rates if the sample and the surrounding medium contacts directly with the respective liquid nitrogen and warming medium. However, this direct contact may result in microbial contamination. In this work, an earlier aseptic technique was applied for minimum volume vitrification. After equilibration, samples were loaded on a plastic film, immersed rapidly into factory derived, filter-sterilized liquid nitrogen, and sealed into sterile, pre-cooled straws. At warming, the straw was cut, the filmstrip was immersed into a 39°C warming medium, and the sample was stepwise rehydrated. Cryosurvival rates of porcine blastocysts produced by parthenogenetical activation did not differ from control, vitrified blastocysts with Cryotop. This approach can be used for minimum volume vitrification methods and may be suitable to overcome the biological dangers and legal restrictions that hamper the application of open vitrification techniques.

Keywords: pig, sterile, embryo, oocyte, chilling, storage

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CryoLetters 32 (1), 69-80 (2011)
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STRUCTURAL, ULTRASTRUCTURAL AND FUNCTIONAL STUDIES OF HUMAN CARDIAC VALVE ALLOGRAFTS THAT SUFFERED AN INCREMENT OF THE CRYOSTORAGE TEMPERATURE

Victoria Bessone¹, María D. Pizarro², María F. Izaguirre³, María E. Biancardi¹, Graciela Furno⁴, Norberto Baumgartner⁵, Joaquín V. Rodríguez^2,6 and Alejandra B. Quintana¹*

¹Morfología, ⁶Farmacología, Departamento de Ciencias Fisiológicas, ⁴Estadística, Departamento de Matemáticas y Estadística; Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531- S2002LRK Rosario, Argentina.
²Centro Binacional (Argentina – Italia) de Investigaciones en Criobiología Clínica y Aplicada (CAIC). Avda. Arijón 28 bis, 2000 Rosario, Argentina.
³Laboratorio de Microscopia Aplicada a Estudios Moleculares y Celulares (LAMAE); Facultad de Ingeniería, Universidad Nacional de Entre Ríos, Ruta 11 Km 10^½, Oro Verde, Entre Ríos, Argentina
⁵Banco de Homoinjertos valvulares y vasculares, CRAISur, C.U.C.A.I.B.A. Ministerio de Salud de la Pcia. de Buenos Aires, Hospital Interzonal Especializado de Agudos y Crónicos San Juan de Dios. Calles 27 y 70, 1900 La Plata, Buenos Aires, Argentina.
*Address correspondence and reprints requests to Dra. Alejandra Beatriz Quintana, Área Morfología, Dto. de Ciencias Fisológicas, Universidad Nacional de Rosario, Suipacha 531- S2002LRK Rosario, Argentina.
*Correspondence author e-mail: aquintan@fbioyf.unr.edu.ar

Abstract

Human cardiac valve allografts (HVAs) suffer injuries during the cryopreservation period. Here, we described structural, ultrastructural and functional damages suffered by HVAs after an increment of their cryostorage temperature (100ºC). Two experimental groups of pulmonary and aortic HVAs were compared: cryopreserved (HVA_cryo) and cryopreserved with temperature changes (HVA_ΔT). Transmission electron microscopy (TEM) was used to analyze valve fibroblasts and extracellular matrix morphology. Total collagen amount was estimated using two different methods and fibroblast viability was assessed measuring their oxygen consumption rate. Porcine heart grafts valves were used to set the techniques.

Disorganized collagen network was seen in HVA_ΔT by TEM. Fibroblasts showed damages in the cellular membrane and many secretor vesicles. Mitochondria and chromatin were also altered. HVA_ΔT had less amount of collagen and fibroblasts showed an oxygen consumption rate markedly diminished compared to HVA_cryo.

The increment of 100ºC suffered by HVAs caused damages that made them unsuitable for clinical purposes.

Keywords: human heart valve allografts, cardiac valve fibroblasts, cryopreservation, collagen, fibroblast oxygen consumption rate.

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CryoLetters 32 (1), 81-88 (2011)
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CRYOPRESERVATION OF UMBILICAL CORD BLOOD-DERIVED MESENCHYMAL STEM CELLS WITHOUT DIMETHYL SULFOXIDE

Hai-Yan Wang¹, Zhao-Rong Lun² and Shu-Shen Lu^1,*

¹School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou 510275, China.
²School of life Sciences, Sun Yat-sen University, Guangzhou 510275, China.
*Corresponding author e-mail: lvshsh@mail.sysu.edu.cn

Abstract

Cryopreservation of umbilical cord blood-derived mesenchymal stem cells (UCB-derived MSCs) is crucial step for its clinical applications in cell transplantation therapy. In the cryopreservation of MSCs, dimethyl sulfoxide (Me₂SO) has been widely used as a cryoprotectant (CPA). However, it has been proved that Me₂SO has toxic side effects to human body.  In this study, Me₂SO-free CPA solutions which contained ethylene glycol (EG), 1, 2-propylene glycol (PG) and sucrose as basic CPAs, supplemented with polyvinyl alcohol (PVA) as an additive, were developed for the cryopreservation of UCB-derived MSCs.  The cryopreservation of UCB-derived MSCs was achieved by vitrification via plunging into liquid nitrogen and by programmed freezing via an optical-DSC system respectively. The viability of thawed UCB-derived MSCs was tested by trypan blue exclusion assay.  Results showed that the viability of thawed UCB-derived MSCs was enhanced from 71.2% to 95.4% in the presence of PVA for vitrification, but only < 10.0% to 44.8% of viability was found for programmed freezing.  These results indicate that PVA exerts a beneficial effect on the cryopreservation of UCB-derived MSCs and suggest the vitrification in combination with the Me₂SO-free CPA solutions supplemented with PVA would be an efficient protocol for the cryopreservation of UCB-derived MSCs.

Keywords: Cryopreservation, mesenchymal stem cell, vitrification, PVA

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