Volume 36, No. 5 September/October 2015 ISSN 0143-2044
	Long-term conservation of protocorms of Brassavola nodosa (L) LIND. (Orchidaceae): effect of aba and a range of cryoconservation techniques Martín Mata-Rosas and Elba Lastre-Puertos	289-298
	Zygotic embryo cryopreservation of Calamus vattayila renuka, an endangered Rattan Palm of India, as influenced by seed maturity Joemon Jacob and Sabarimuthan William Decruse	299-307
	Effect of semen extender supplementation with trehalose, vitamin C and E on post-thaw min pig sperm qualities Hong Ma, Di Liu, Wentao Wang, Liang Wang, Bo Fu, Zhongqiu Li and Xinmiao He	308-312
	Effect of solid medium during cooled storage on Stallion sperm parameters Fernanda Carlini Cunha dos Santos, Carine Dahl Corcini, Vitória Gasperin Guazzelli Costa, Stela Mari Meneghello Gheller, Carlos Eduardo Wayne Nogueira, Bruna da Rosa Curcio and Antonio Sergio Varela Junior	313-317
	Assessment of molecular genetic stability between long-term cryopreserved and tissue cultured wasabi (Wasabia japonica) plants Shinya Maki, Yuki Hirai, Takao Niino and Toshikazu Matsumoto	318-324
	Cryopreservation strategy for tissue engineering constructs consisting of human mesenhymal stem cells and hydrogel biomaterials Yingnan Wu, Feng Wen, Sok Siam Gouk, Eng Hin Lee and Lilia L. Kuleshova	325-335
	Effects of semen extender supplemented with L-methionine and packaging methods (straws and pellets) on post-thaw Goldfish (Carassius auratus) sperm quality and DNA damage Filiz Kutluyer, Fatih Öğretmen and Burak Evren İnanan	336-343
	Cryopreservation of Sweet Potato shoot tips using a droplet-vitrification procedure Sang-Un Park and Haeng-Hoon Kim	344-352

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CryoLetters 36 (5), 289-298 (2015)
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LONG-TERM CONSERVATION OF PROTOCORMS OF BRASSAVOLA NODOSA (L) LIND. (ORCHIDACEAE): EFFECT OF ABA AND A RANGE OF CRYOCONSERVATION TECHNIQUES

Martín Mata-Rosas^1* and Elba Lastre-Puertos¹

¹Red Manejo Biotecnológico de Recursos, Instituto de Ecología, A.C., Carretera Antigua a Coatepec 351, El Haya, Xalapa, Ver. 91070, Mexico.
^*Corresponding author e-mail: martin.mata@inecol.mx

Abstract

BACKGROUND: Populations of Brassavola nodosa have been severely affected by habitat destruction and illegal collecting, and as with the majority of orchid species, it is critical to take action to guarantee their continued survival. OBJECTIVE: The present study aimed to establish protocols for the long-term conservation of protocorms of species. MATERIALS AND METHODS: Four different cryogenic techniques were compared: encapsulation-dehydration (ED), encapsulation-vitrification (EV), encapsulation-dehydration-vitrification (EDV) and vitrification. RESULTS: Preculture of protocorms with ABA was a critical factor in obtaining high percentages of regrowth. With vitrification, 100% regrowth was achieved in five treatments, mainly when protocorms were dehydrated with PVS2 for 120 min. 100% regrowth was also obtained with EDV, where the protocorms were precultured with ABA 5 mg l^-1 for 3 days and incubated with PVS2 for 60 min. With the ED, regrowth of 72% was achieved with the preculture of protocorms with ABA 5 mg l^-1 for the three times of incubation used (3, 6 and 9 days). In the case of EV, 92% regrowth, was recorded when protocorms were precultured for 9 days with ABA 3 mg l^-1 and incubated with PVS2 for 90 min. CONCLUSION: Although regrowth of protocorms was obtained with all the techniques used, the vitrification technique is preferred since it requires less labour and is less costly.

Keywords: Orchids, vitrification, encapsulation-dehydration, encapsulation-vitrification, abscisic acid.

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CryoLetters 36 (5), 299-307 (2015)
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ZYGOTIC EMBRYO CRYOPRESERVATION OF CALAMUS VATTAYILA RENUKA, AN ENDANGERED RATTAN PALM OF INDIA, AS INFLUENCED BY SEED MATURITY

Joemon Jacob and Sabarimuthan William Decruse*

Jawaharlal Nehru Tropical Botanic Garden and Research Institute, Palode, Thiruvananthapuram-695562
*Corresponding author email: willdic@rediffmail.com

Abstract

BACKGROUND: Calamus vattayila Renuka is an endemic and endangered rattan palm of the Western Ghats, India where the development of a protocol for cryopreservation is important for their ex situ conservation in gene banks. OBJECTIVE: The objective of the study is to devise an efficient protocol for cryopreservation of the species, comparing the relative efficiency of seeds and zygotic embryos as storage material.  MATERIALS AND METHODS: Mature seeds extracted from ripened fruits were subjected to cryopreservation through a simple dehydration method and zygotic embryos isolated from seeds of three different maturity stages were cryopreserved through an encapsulation-dehydration method. RESULTS: The mature seeds did not tolerate desiccation and freezing but the isolated zygotic embryos tolerated both desiccation and freezing. Embryos isolated from immature, partially mature and mature seeds harvested respectively after 160-170, 210-220 and 250-260 days after flowering possessed 82 - 86% moisture content (m.c.) and tolerated desiccation down to 9% m.c. with 80% viability. Embryos from immature seeds gave a maximum 63±26% regeneration after LN exposure, which is nearly equal to the corresponding desiccated control (73%). LN tolerance declined with maturity of seeds with a maximum of 49% of embryos from partially mature seeds and 25% from mature seeds subjected to LN exposure showing post-freeze recovery and regeneration.  CONCLUSION: Although immature seeds collected during February-March were recalcitrant (desiccation sensitivity), their embryos tolerated cryopreservation through encapsulation-dehydration better than embryos isolated from more mature seeds.

Keywords: encapsulation-dehydration, seed maturity, recalcitrance

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CryoLetters 36 (5), 308-312 (2015)
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EFFECT OF SEMEN EXTENDER SUPPLEMENTATION WITH TREHALOSE, VITAMIN C AND E ON POST-THAW MIN PIG SPERM QUALITIES

Hong Ma, Di Liu*, Wentao Wang, Liang Wang, Bo Fu,
Zhongqiu Li and Xinmiao He

Heilongjiang Academy of Agricultural Sciences, Harbin, 150086, China.
*Corresponding author email: mahong_ok@163.com or m18104626703_1@163.com

Abstract

BACKGROUND: Cryopreservation of min pig sperm offers an effective method to protect this valuable genetic resource. OBJECTIVE: To determine whether the extender supplementation with some antioxidants is able to improve the quality of post-thawed sperm of min pig. MATERIALS AND METHODS: Ejaculated min pig sperm frozen in lactose-egg yolk (LEY) extender supplemented with trehalose, vitamin C and/or water-soluble vitamin E were thawed, and the quality of sperm samples were evaluated. RESULTS: The quality of post-thawed sperm samples was higher in the groups with supplementation than that of the control group (P < 0.05). Furthermore, the addition of 100 mM trehalose and 25mM water-soluble vitamin E to the freezing extender strongly preserved the quality of the sperm (P < 0.05). CONCLUSION: Trehalose and water-soluble vitamin E in the freezing extender effectively improved post-thaw qualities of min pig sperm.

Keywords: sperm quality, cryopreservation, min pig.

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CryoLetters 36 (5), 313-317 (2015)
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EFFECT OF SOLID MEDIUM DURING COOLED STORAGE ON STALLION SPERM PARAMETERS

Fernanda Carlini Cunha dos Santos^1,2,*, Carine Dahl Corcini²,
Vitória Gasperin Guazzelli Costa², Stela Mari Meneghello Gheller²,
Carlos Eduardo Wayne Nogueira¹, Bruna da Rosa Curcio¹
and Antonio Sergio Varela Junior^2,3*

¹Departamento de Clínicas Veterinárias, Faculdade de Veterinária (FV), Universidade Federal de Pelotas (UFPel), Campus Universitário, Pelotas, RS, 96010-900, Brasil.
² Departamento de Patologia Animal, Laboratório de Reprodução Animal, FV, UFPel;.
³Reprodução Animal Comparada, Instituto de Ciência Biológicas, Universidade Federal de Rio Grande (FURG), Rio Grande, Brasil.
*Corresponding author email: antoniovarela@furg.br and carlini@portoweb.com.br

Abstract

BACKGROUND: Solid storage medium prevents cellular sedimentation, reduces metabolic demand via limiting movement, and avoids the modification of an extender composition in the sedimentary microenvironment. It has been proven to prolong spermatozoa viability in mammalians. OBJECTIVE: This experiment aims to evaluate the effect of cool storage in solid phase extender on stallion sperms. MATERIALS AND METHODS: Semen was collected from 10 Crioulo stallions (n=30) and submitted to treatments: control group (semen extender) and groups with gelatin addition in different concentrations (semen extender + 1%; 2% and 3%). Seminal analyses included motility, mitochondrial functionality, plasma membrane integrity, DNA and acrosome at 0; 24; 48 and 72 hours during cooled storage at 5ºC. RESULTS: Motility, mitochondrial functionality, plasma membrane and acrosome integrity declined during storage time, with no statistical difference between treatments. DNA integrity did not significantly change during storage period.  CONCLUSION: Solid medium was not harmful and did not improved stallion sperm parameters during cooled storage.

Keywords: equine, gelatin, semen, spermatozoa, solid-phase extender

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CryoLetters 36 (5), 318-324 (2015)
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ASSESSMENT OF MOLECULAR GENETIC STABILITY BETWEEN LONG-TERM CRYOPRESERVED AND TISSUE CULTURED WASABI (WASABIA JAPONICA) PLANTS

Shinya Maki^1*, Yuki Hirai², Takao Niino³ and Toshikazu Matsumoto⁴

¹Department of Science of Technology Innovation, Nagaoka University of Technology, Nagaoka, Niigata 940-2188, Japan
²Department of Applied Chemistry and Biotechnology, Niihama National College of Technology, Niihama, Ehime 792-8580, Japan
³National Institute of Agrobiological Science, Tsukuba, Ibaraki 305-8518, Japan
⁴Faculty of Life and Environment Science, Shimane University, Matsue, Shimane 690-1060, Japan
*Corresponding author e-mail: maki@vos.nagaokaut.ac.jp

Abstract

BACKGROUND: Maintaining the genetic integrity in long-term tissue cultured and cryopreserved plants is important for the conservation of plant genetic resources. OBJECTIVE: In this study, the genetic stability of cryopreserved wasabi shoot tips stored for 10 years at -150°C was visualized using Amplified Fragment Length Polymorphism (AFLP) and Methylation Sensitive Amplified Polymorphism (MSAP). MATERIALS AND METHODS: The study included plants derived from cryopreserved shoot tips after 10.5 years storage at -150°C (LN10yr), after 2 h storage at -196°C (LN2hr), cryopreservation controls (No LN cooling (TC)) and non-treated controls without LN cooling (LC). The donor plants for LN2hr, TC and LC were also maintained in vitro at 20°C for the same period. RESULTS: Neither technique detected genetic variations in either control or cryopreserved plants. Some mutations were noted in plants maintained in tissue culture for 10 years. Comparison of genome stability for TC and LN2hr plants showed only a minor change in DNA. However, when comparing the LC and Ln10yr, many differences were found. CONCLUSION: We conclude that cryopreservation is a superior conservation method compared to tissue culture in maintaining genetic stability for a long-term storage of wasabi germplasm.

Keywords: Wasabi, long-term storage, cryopreservation, tissue culture, AFLP, MSAP

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CryoLetters 36 (5), 325-335 (2015)
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CRYOPRESERVATION STRATEGY FOR TISSUE ENGINEERING CONSTRUCTS CONSISTING OF HUMAN MESENHYMAL STEM CELLS AND HYDROGEL BIOMATERIALS

Yingnan Wu^1,2,#, Feng Wen^3,#, Sok Siam Gouk³, Eng Hin Lee^1,2
and Lilia L. Kuleshova^3,*

¹Tissue Engineering Program; ²Department of Orthopaedic Surgery, YLL School of Medicine; ³Low Temperature Preservation Unit, National University Medical Institutes, YLL School of Medicine, National University of Singapore, Singapore
^#These authors contributed equally.
*Corresponding author email: kuleshova@imp.uni-hannover.de

Abstract

BACKGROUND: The development of vitrification strategy for cell-biomaterial constructs, particularly biologically inspired nanoscale materials and hydrogels mimicking the in vivo environment is an active area. A cryopreservation strategy mimicking the in vivo environment for cell-hydrogel constructs may enhance cell proliferation and biological function. OBJECTIVE: To demonstrate the efficacy of vitrification as a platform technology involving tissue engineering and human mesenchymal stem cells (hMSCs). MATERIALS AND METHODS: Microcarriers made from alginate coated with chitosan and collagen are used. Conventional freezing and vitrification were compared. The vitrification strategy includes 10 min step-wise exposure to a vitrification solution (40% v/v EG, 0.6M sucrose) and immersion into liquid nitrogen. RESULTS: Confocal imaging of live/dead staining of hMSCs cultured on the surface of microcarriers demonstrated that vitrified cells had excellent appearance and prolonged spindle shape morphology. The proliferation ability of post-vitrified cells arbitrated to protein Ki-67 gene expression was not significantly different in comparison to untreated control, while that of post-freezing cells was almost lost. The ability of hMSCs cultured on the surface of microcarriers to proliferate has been not affected by vitrification and it was significantly better after vitrification than after conventional freezing during continuous culture. Collagen II related mRNA expression by 4 weeks post-vitrification and post-freezing showed that ability to differentiate into cartilage was sustained during vitrification and reduced during conventional freezing. No significant difference was found between control and vitrification groups only. CONCLUSION: Vitrification strategy coupled with advances in hMSC-expansion platform that completely preserves the ability of stem cells to proliferate and subsequently differentiate allows not only to reach a critical cell number, but also demonstrate prospects for effective utilization and transportation of cells with their support system, creating demand for novel biodegradable materials.

Keywords: vitrification, hydrogel, microcarrier, mesenchymal stem cell, chondrogenic differentiation

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CryoLetters 36 (5), 336-343 (2015)
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EFFECTS OF SEMEN EXTENDER SUPPLEMENTED WITH L-METHIONINE AND PACKAGING METHODS (STRAWS AND PELLETS) ON POST-THAW GOLDFISH (CARASSIUS AURATUS) SPERM QUALITY AND DNA DAMAGE

Filiz Kutluyer¹*, Fatih Öğretmen², Burak Evren İnanan²

¹Tunceli University, Fisheries Faculty, 62000, Tunceli, Turkey
²Muğla Sıtkı Koçman University, Faculty of Science, Department of Biology, 48000, Muğla, Turkey.

Abstract

BACKGROUND: Amino acids protect spermatozoa against cell damage during cryopreservation due to have antioxidant property and found in seminal plasma at high concentration. OBJECTIVE: The aim of the present work was to analyse the effect of extender supplementation with L-methionine on post-thawed sperm motility, duration and DNA damage and also it was tested the feasibility of using straws and pellets method for the cryopreservation of goldfish (Carassius auratus) sperm. MATERIALS AND METHODS: Extenders were supplemented with different L-methionine concentrations of 1 mM; 1.5 mM; 3 mM; 6 mM. Semen samples diluted at the ratio of 1:9 by the extenders were subjected to cryopreservation. After dilution the semen was aspirated into 0.25 ml straws and 0.1 ml pellets, the straws and pellets were placed on the tray, frozen in nitrogen vapor and plunged into liquid nitrogen. DNA damage was determined with comet assay after cryopreservation. RESULTS: Our results indicated that an increase in the concentration of L-methionine caused a significant increase in the motility rate and duration of sperm in goldfish (C. auratus) (p<0.05). In addition, duration and percentage of motility in pellets were higher than in straws. Comparing all concentrations of L-methionine, the best concentration of L-methionine was 1.5 mM. Highest post-thaw motility (45.00±7.07%) and duration of motility (17.00±0.71s) were obtained with the extender at concentration 1.5 mM in pellets. Addition of the extender with L-methionine was reduced DNA damage compared to control group.  CONCLUSION: Consequently, pellets could be use for goldfish sperm cryopreservation and the tested amino acid affected the motility parameters, and semen extenders could be supplement with L-methionine.

Keywords: Goldfish, Carassius auratus, L-methionine, amino acid.

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CryoLetters 36 (5), 344-352 (2015)
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CRYOPRESERVATION OF SWEET POTATO SHOOT TIPS USING A DROPLET-VITRIFICATION PROCEDURE

Sang-Un Park ¹ and Haeng-Hoon Kim^2*

Division of Plant Science and Resources, Chungnam National University, Daejeon 305-764, Korea.
Department of Well-being Resources, Sunchon National University, Suncheon, 540-742, Korea.
^*Corresponding author email: cryohkim@sunchon.ac.kr

Abstract

BACKGROUND: Sweet potato is a staple food worldwide, but a problematic species in terms of long term storage, as it is not suitable for germplasm conservation. OBJECTIVE: This study aimed to develop cryopreservation protocols for sweet potato shoot tips based on a droplet-vitrification procedure. METHODS: As a standard procedure, sweet potato shoot tips were precultured in a liquid MS medium supplemented with 10% sucrose (S-10%) and 17.5% sucrose (S-17.5%) for 31 and 17 h, respectively. They were then osmoprotected with C4-35% (17.5% glycerol + 17.5% sucrose) for 50 min and cryoprotected with PVS3 (50% glycerol + 50% sucrose) for 60 min. A set of experiments was designed to investigate critical factors, i.e. stepwise sucrose preculture, osmoprotection, cryoprotection with PVS2- and PVS3-based vitrification solutions, and their combinational effect, as well as temperature alteration through placement in a cooling/rewarming container. RESULTS: Sucrose preculture was determined to be necessary for the adaptation of sweet potato shoot tips to cryoprotection with PVS3, and the highest post-thaw (LN) regeneration rate was observed in a preculture with S-10% for 31 h → S-17.5% for 17 h (19.0%). The effect of one-step or two-step osmoprotection was not significant on survival or regeneration of either the cryoprotected-control (LNC) or LN shoot tips. Responses of sweet potato shoot tips to osmoprotection and cryoprotection were linked to the level of sucrose preculture. The use of alumimium foil strips (droplet-vitrification) resulted in significantly higher LN survival (89.8%) and regeneration (19.0%), compared to those using cryovials (vitrification, 67.2% and 0%, respectively). LN regeneration increased by 67.5% when cryopreserved shoot tips were transferred to a new postculture medium. CONCLUSIONS: This study demonstrates that the combination of stepwise sucrose preculture with a higher final concentration (up to 17.5%), cryoprotection with PVS3 and cooling with foil strip is crucial to the regeneration of LN sweet potato shoot tips.

Keywords: cooling and warming, cryoprotection, osmoprotection, preculture.

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