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Abstracts: CryoLetters 35 (4), 2014

 

 

Volume 35, No. 4 July/August 2014

ISSN 0143-2044

 

 


Effects of cryopreservation on motility characteristics and enzyme activity of sperm in a chinese fish, Nibea albiflora
Xiaorong Huang, Ping Zhuang, Longzhen Zhang,
Feng Zhao, Jianyi Liu, Guangpeng Feng and Tao Zhang

267-276

 

 


Effect of different disaccharides on the integrity and fertilising ability of freeze-dried boar spermatozoa: a preliminary study
Andrés García, Lydia Gil, Clara Malo, Felisa Martínez,Claire Kershaw-Young and Ignacio de Blas

277-285

 

 


Generation of live offspring from vitrified embryos with synthetic polymers supercool X-1000 and supercool Z-1000
Francisco Marco-Jiménez, Estrella Jiménez-Trigos,
Raquel Lavara and Jose S. Vicente

286-292

 

 


Cryoprotectant-free freezing of the goat epididymal sperm
Hoda Katanbafzadeh, Farid Barati and
Mohammadreza Tabandeh

293-298

 

 


Effects of some cryopreservation procedures on recalcitrant zygotic embryos of Ammocharis coranica
Nomali Z. Ngobese, Sershen, Patricia Berjak
and N.W. Pammenter

299-307

 

 


Effect of glutathione and y27632 on the viability of cryopreserved porcine adiposederived stem cells
Chang-Qing Qu, Dong-Wei Li, Nou-Shen,
Jing-Hong JiangYang and Yun-Tao Ji

308-311

 

 


Wide-scale pollen banking of ornamental plants through cryopreservation
Jin Xu, Bingling Li, Qian Liu, Yin Shi, Jingen Peng,
Mengxue Jia and Yan Liu

312-319

 

 


Use of cyclodextrins to increase cytoplasmic cholesterol in rabbit embryos and their impact on live kits derived from vitrified embryos
Francisco Marco-Jiménez*, Estrella Jiménez-Trigos,
Raquel Lavara and Jose S. Vicente

320-326

 

 


Effect of cryopreservation on in vitro seed germination and protocorm growth of mediterranean orchids
Andrea Pirondini and Elisabetta Sgarbi

327-335

 

 


Chelation of trace elements in preservation medium influences the quality of boar spermatozoa during liquid preservation at 5°c for 4 weeks
Masayasu Taniguchi, Manita Wittayarat, Kota Morinaga,
Yoko Sato, Lanh Thi Kim Do, Kaywalee Chatdarong,
Mongkol Techakumphu, Masahiro Nii and Takeshige Otoi

336-344

 

 


Liquidus tracking: controlled rate vitrification for the cryopreservation of larger volumes and tissues
E.Puschmann, C.Selden, S. Butler and B.Fuller

345-355

 

 

 

 

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CryoLetters 35 (4), 267-276 (2014)
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EFFECTS OF CRYOPRESERVATION ON MOTILITY CHARACTERISTICS AND ENZYME ACTIVITY OF SPERM IN A CHINESE FISH, Nibea albiflora

Xiaorong Huang, Ping Zhuang, Longzhen Zhang,
Feng Zhao, Jianyi Liu, Guangpeng Feng and Tao Zhang

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:
pzhuang@online.sh.cn

Abstract

BACKGROUND: Growing evidence suggests that among the causes which deteriorate qualitative and functional characteristics of sperm after freezing and thawing, there are those linked to decrease of sperm motility and release of various enzymes in the cells and seminal plasma. OBJECTIVE: In the present study, the motility, fertilization and enzyme activity of sperm were analyzed after cryopreservation. MATERIALS AND METHODS : Computer-assisted sperm motility analysis (CASA) was used to evaluate the effect of cryopreservation on sperm motility of Nibea albiflora. RESULTS: The activities of total adenosine triphosphatase (ATPase), creatine kinase (CK), succinate dehydrogenase (SDH), lactate dehydrogenase (LDH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and glutathione reductase (GR) in fresh and frozen seminal plasma and spermatozoa were measured respectively. Cryopreservation led to a decline in the percentage of motile sperm, moreover, other parameters of sperm motion, curvilinear and straight line velocities, linearity were changed observably (p<0.05), the fertilizing capacity of post-thaw sperm was lower than that of the fresh sperm significantly. After cryopreservation, the activities of total ATPase, CK, SDH, LDH, SOD, CAT and GSH-Px increased in seminal plasma and decreased in spermatozoa respectively, but GR activity varied contrarily, GR activity dropped in seminal plasma and increased in spermatozoa. CONCLUSION: Cryopreservation had significant effects on the motility characteristics, fertilization ability and enzyme activity of the sperm of Nibea albiflora.

Keywords: Cryopreservation; Nibea albiflora; sperm motility; enzyme activity; fertilizing capacity

 

 

 

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CryoLetters 35 (4), 277-285 (2014)
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EFFECT OF DIFFERENT DISACCHARIDES ON THE INTEGRITY AND FERTILISING ABILITY OF FREEZE-DRIED BOAR SPERMATOZOA: A PRELIMINARY STUDY

Andrés García1, Lydia Gil1, Clara Malo1*, Felisa Martínez1,
Claire Kershaw-Young 2 and Ignacio de Blas3

1Department of Animal Pathology, Obstetrics and Reproduction Area, Faculty of Veterinary Medicine, Universidad de Zaragoza, C/ Miguel Servet, 177 CP: 50013 Zaragoza (Spain)
2Department of Animal Production, Welfare and Veterinary Sciences, Harper Adams University, Newport, Shropshire, UK
3Department of Animal Pathology, Infectious Disease Area, Faculty of Veterinary Medicine, Universidad de Zaragoza, C/ Miguel Servet, 177 CP: 50013 Zaragoza (Spain)
*Corresponding author e-mail address:
claramalo@hotmail.com

Abstract

Freeze-drying spermatozoa is a developing technique that facilitates semen storage and transport. However, freeze-dried sperm exhibits impaired DNA integrity, which is associated with reduced fertilizing ability. Boar spermatozoa were freeze-dried in three different freeze-drying EDTA buffers with trehalose (75mM) and lactose (75mM) (EDTA-TL), (2) with sucrose (75mM) and lactose (75mM) (EDTA-SL) or just lactose (150mM) (EDTA-LL) using two freeze-drying protocols. In experiment 1 a one-step protocol was used and in experiment 2 a two-steps protocol was used. Spermatozoa were stored in1.5 mL cryo-tubes and 1.5 mL glass ampules at both 16ºC and 25ºC for 1 month. Successfully freeze-dried spermatozoa were stained with acridine-orange to assess chromatin stability. Freeze-drying was most successful when the 2-step protocol was used (experiment 2). Chromatin stability was greater in samples stored in glass ampules compared to cryo tubes. Chromatin stability was also greater in samples freeze-dried in EDTA-LL compared to EDTA-SL or EDTA-TL buffers. Spermatozoa freeze-dried in EDTA-LL and stored for 14 and 28 days at either 16C or 25C were utilized for ICSI. Two pronuclear formation wasgreatest using spermatozoa stored at 25ºC (69.23%) and for 28 days (50%). Although 16°C spermatozoa samples had better stable chromatin, 25ºC spermatozoa samples offered better two pronuclear formation results.

In conclusion, boar spermatozoa freeze-dried using media containing disaccharides exhibit high chromatin stability and are able to fertilise oocytes following ICSI. Disaccharides may therefore advance the development of freeze-drying techniques for spermatozoa enabling ease of sperm storage and transportation.

Keywords: freeze-drying; boar; spermatozoa; monosaccharides; disaccharides;

 

 

 

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CryoLetters 35 (4), 286-292 (2014)
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GENERATION OF LIVE OFFSPRING FROM VITRIFIED EMBRYOS WITH SYNTHETIC POLYMERS SUPERCOOL X-1000 AND SUPERCOOL Z-1000

Francisco Marco-Jiménez*, Estrella Jiménez-Trigos,
Raquel Lavara and Jose S. Vicente

Institute of Animal Science and Technology, Laboratorio de Biotecnología de la Reproducción, Universidad Politécnica de Valencia, Valencia, Spain. 46022.
*Corresponding author email:
fmarco@dca.upv.es

Abstract

BACKGROUND: Ice growth and recrystallisation are considered important factors in determining vitrification outcomes. Synthetic polymers inhibit ice formation during cooling or warming of the vitrification process. OBJECTIVE: The aim of this study was to assess the effect of adding commercially available synthetic polymers SuperCool X-1000 and SuperCool Z-1000 to vitrification media on in vivo development competence of rabbit embryos. METHODS: Four hundred and thirty morphologically normal embryos recovered at 72 h of gestation were used. The vitrification media contained 20% dimethyl sulphoxide and 20% ethylene glycol, either alone or in combination with 1% of SuperCool X-1000 and 1% SuperCool. RESULT: Our results show that embryos can be successfully vitrified using SuperCool X-1000 and SuperCool Z-1000 and when embryos are transferred, live offspring can be successfully produced. CONCLUSIONS: In conclusion, our results demonstrated that we succeeded for the first time in obtaining live offspring after vitrification of embryos using SuperCool X-1000 and SuperCool Z-1000 polymers.

Keywords: Rabbit; ice blockers; mucin coat; offspring; in vitro; in vivo

 

 

 

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CryoLetters 35 (4), 293-298 (2014)
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CRYOPROTECTANT-FREE FREEZING OF THE GOAT EPIDIDYMAL SPERM

Hoda Katanbafzadeh1, Farid Barati1*
and Mohammadreza Tabandeh2

1Department of Clinical Sciences, and
2Department of Basic Sciences, Faculty of Veterinary Medicine, Shahid Chamran University, Ahvaz, Iran, P.O.Box:61355-145,
Phone (work): +986113330073, Fax: +986113360807.
*Correspondence author email:
fabrtir@yahoo.com

Abstract

BACKGROUND: Cryoprotectant free approach successfully removed the impact of physical and chemical damages in preserving human sperm in a vitrification protocol. There is no any report on this technology in farm animal sperm freezing. OBJECTIVE: The aim of the present study was to find the efficacy of cholesterol-loaded cyclodextrin (CLC; 1 mg/60×106) and sucrose (0.1 and 0.2 M) on freezing of the goat epididymal sperm. METHODS: Caudal epididymides (n=5 pairs) were dissected, incised and incubated in the Tris-BSA solution for 15 min, followed by swim-up at room temperature. Sperm was loaded in 0.25 mL French straws and cooled on nitrogen vapor for 3 min then immersed in liquid nitrogen and remained for 48 h. Then the straws thawed by immersing in 37 C waterbath for 30 sec and analyzed. RESULT: The results showed the impact of freezing on the goat epididymal sperm motility, viability and DNA fragmentation that were improved by incorporation of CLC and sucrose (0.2 M). CONCLUSIONS: In conclusion, the goat epididymal sperm was frozen in a cryoprotectant-free freezing model. CLC and 0.4 M sucrose protected the goat epididymal sperm against freezing-induced damages.

Keywords: Cryoprotectant–free freezing; Goat; Epididymal sperm; Cholesterol-Loaded Cyclodextrin; Sucrose

 

 

 

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CryoLetters 35 (4), 299-307 (2014)
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EFFECTS OF SOME CRYOPRESERVATION PROCEDURES ON RECALCITRANT ZYGOTIC EMBRYOS OF AMMOCHARIS CORANICA

Nomali Z, Ngobese, Sershen, Patricia Berjak* and N.W. Pammenter

School of Life Sciences, University of KwaZulu-Natal (Westville Campus), Durban, 4001,South Africa.
*Corresponding author email:
berjak@ukzn.ac.za

Abstract

BACKGROUND: Cryopreservation, the most promising method for the long-term conservation of recalcitrant (desiccation-sensitive) seed germplasm, is often associated with high viability losses. Cryo-procedures involve a sequence of steps which must be optimised to reduce the impact of the stresses. OBJECTIVE: This study reports on the effects of some of the steps of cryopreservation on the recalcitrant zygotic embryos of the amaryllid, Ammocharis coranica. MATERIALS AND METHODS: Embryos were subjected to cryoprotection with glycerol and/or DMSO, rapid (flash) drying, and rapid (>100°C s-1) or slow (1°C s-1) cooling. RESULTS: Rapid dehydration (from c. 2.7 to 0.9 g g-1 over 60 min) and cooling had a detrimental effect on the viability of the embryos, which was exacerbated when these steps were applied sequentially. After cooling, seedling production (30%) was obtained only from embryos that had been cryoprotected with glycerol prior to drying and rapid cooling, while 30% of non-treated embryos and 70% of those that had undergone cathodic protection during flash drying produced callus. CONCLUSION: Noting that no post-cryo survival of A. coranica embryos had previously been obtained, this study identified cryoprotection with glycerol and the incorporation of cathodic protection during flash drying as promising intervention points for future studies.

Keywords: cathodic protection, cryopreservation, glycerol, recalcitrant seeds

 

 

 

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CryoLetters 35 (4), 308-311 (2014)
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EFFECT OF GLUTATHIONE AND Y27632 ON THE VIABILITY OF CRYOPRESERVED PORCINE ADIPOSE-DERIVED STEM CELLS

Chang-Qing Qu, Dong-Wei Li, Nou-Shen,
Jing-Hong JiangYang and Yun-Tao Ji*

School of Life Science, FuYang Teachers College, Fuyang, Anhui 236041, China.
*Corresponding author email:
qucq518@163.com

Abstract

BACKGROUND: Recently it has been reported that reduced glutathione (GSH) and/or Rho-associated kinase (ROCK) inhibitor supplemented in cryopreservation solution could improve the viability of cells. OBJECTIVE: To identify the cryopreservation efficiency of GSH and ROCK inhibitor on porcine ADSCs. MATERIALS AND METHODS: Porcine ADSCs were separated and cultured. Cells at the 4th passage were suspended in cryopreservation solution supplemented with Y-27632 and GSH or both, and then frozen and thawed. The viability of cryopreserved ADSCs was compared using a MTT assay. RESULTS: The a ddition of GSH and Y-27632 to cryopreservation solution (dimethyl sulfoxide) and post-thaw culture medium significantly improves the post-thaw viability of ADSCs. CONCLUSION: GSH and Y-27632 are able to increase the survival of ADSCs, and they have an additive effect, as compared to GSH or Y27632 alone.

Keywords: porcine adipose-derived stem cells; glutathione; Y-27632.

 

 

 

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CryoLetters 35 (4), 312-319 (2014)
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WIDE-SCALE POLLEN BANKING OF ORNAMENTAL PLANTS THROUGH CRYOPRESERVATION

Jin Xu, Bingling Li, Qian Liu, Yin Shi, Jingen Peng,
Mengxue Jia and Yan Liu*

Beijing Key Laboratory of Ornamental Plants Germplasm Innovation & Molecular Breeding, National Engineering Research Center for Floriculture, College of Landscape Architecture, Beijing Forestry University, Beijing, 100083, P.R. China
*Corresponding author e-mail:
chblyan@163.com

Abstract

BACKGROUND: Cryopreservation has been proved to be an efficient method for the long-term storage of pollen, based on our previous studies establishing cryo pollen banks for Camellia, Paeonia, and Prunus mume. OBJECTIVE: In the present study, we aimed to extend and verify the applicability of the cryopreservation protocol for ornamental plants. MATERIALS AND METHODS: 71 species/cultivars from 19 families were cryopreserved using the established protocol. RESULTS: Pollen from 60 species/cultivars (85% of those experimented on) retained viability after 1 year's cryopreservation; of these, 36 (51% of the total investigated) retained a sufficiently high germination capability compared with fresh pollen to enable longer term cryopreservation. CONCLUSION: This study confirms that wide-scale pollen banking of ornamental plants is feasible.

Keywords: ornamental plants, pollen, cryopreservation, pollen bank

 

 

 

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CryoLetters 35 (4), 320-326 (2014)
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USE OF CYCLODEXTRINS TO INCREASE CYTOPLASMIC CHOLESTEROL IN RABBIT EMBRYOS AND THEIR IMPACT ON LIVE KITS DERIVED FROM VITRIFIED EMBRYOS

Francisco Marco-Jiménez*, Estrella Jiménez-Trigos, Raquel Lavara and Jose S. Vicente

Institute of Animal Science and Technology, Laboratorio de Biotecnología de la Reproducción, Universidad Politécnica de Valencia, Valencia, Spain. 46022.
*Corresponding author email:
fmarco@dca.upv.es

Abstract

BACKGROUND: Low cryotolerance in oocytes and embryos is frequently associated with lipid accumulation in the cytoplasm. OBJECTIVE: This study aimed to evaluate the effect of cyclodextrin used as a cholesterol loader to change cytoplasmic cholesterol content of embryos and raise their tolerance to cryopreservation. METHODS: In the first experiment compact morulae-early blastocysts were exposed to CLC (0.11 mM and 0.23 mM cholesterol) for 1 hour. In the second experiment, embryos were exposed to CLC (0.11 mM and 0.23 mM cholesterol) and then vitrified. RESULT: Using both concentrations, cytoplasmic cholesterol content was increased. Vitrified groups demonstrated a lower capacity for embryonic development (in vitro and in vivo) compared to the control groups. Nevertheless, live young were obtained in all groups. CONCLUSIONS: In conclusion, we have demonstrated the feasibility of using cyclodextrin as a carrier for cholesterol into rabbit embryo cytoplasm, although further studies are required to clarify the usefulness of CLC use in embryo cryopreservation.

Keywords: rabbit; cholesterol; cyclodextrin; mucin coat; offspring

 

 

 

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CryoLetters 35 (4), 327-335 (2014)
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EFFECT OF CRYOPRESERVATION ON IN VITRO SEED GERMINATION AND PROTOCORM GROWTH OF MEDITERRANEAN ORCHIDS

Andrea Pirondini and Elisabetta Sgarbi*

Department of Life Sciences, University of Modena and Reggio Emilia, via Amendola, 2 - Padiglione Besta - 42122 Reggio Emilia (Italy).
*Corresponding author email:
elisabetta.sgarbi@unimore.it

Abstract

BACKGROUND: Cryopreservation is an effective method for the long-term conservation of plant germplasm. OBJECTIVE: In the present study the effect of liquid nitrogen on seed structure, germination and protocorm development of eight Mediterranean orchid species was evaluated. MATERIALS AND METHODS: A scanning electron microscopy analysis was conducted to investigate the effect of the immersion in liquid nitrogen on seed integuments. Germination and protocorms growth were obtained applying in vitro cultures techniques. RESULTS: No micro-morphological differences emerged between cryopreserved and untreated seeds. The effect of the treatment on the seed germination varied on the basis of the species. For Ophrys sphegodes ssp. passionis and Orchis mascula, freezing resulted in a significant increase of germination percentages. Protocorms of all species showed a positive growth trend although some significant differences of size occurred among the protocorms derived from treated and untreated seeds. CONCLUSION: Cryogenic techniques seem to have great potential in orchid germplasm conservation.

Keywords: Orchidaceae, germplasm, ex situ conservation, liquid nitrogen, in vitro cultures

 

 

 

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CryoLetters 35 (4), 336-344 (2014)
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CHELATION OF TRACE ELEMENTS IN PRESERVATION MEDIUM INFLUENCES THE QUALITY OF BOAR SPERMATOZOA DURING LIQUID PRESERVATION AT 5°C FOR 4 WEEKS

Masayasu Taniguchi1, Manita Wittayarat1,2, Kota Morinaga1,
Yoko Sato1, Lanh Thi Kim Do1, Kaywalee Chatdarong2,
Mongkol Techakumphu2, Masahiro Nii3, and Takeshige Otoi1*

1Laboratory of Animal Reproduction, The United Graduate School of Veterinary Science, Yamaguchi University, Yamaguchi 753-8515, Japan
2Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Sciences, Chulalongkorn University, Bangkok 10330, Thailand
3Tokushima Prefectural Livestock Research Institute, Tokushima 771-1310, Japan
*Corresponding author email:
otoi@yamaguchi-u.ac.jp

Abstract

BACKGROUND: The addition of a metal chelator, ethylenediaminetetraacetic acid (EDTA), to semen extender has the purpose of capturing trace element ions. OBJECTIVE: This study was conducted to evaluate the effects of EDTA on the quality and in vitro fertilisability of liquid-preserved boar spermatozoa. METHODS: In Experiment 1, semen samples were preserved in the semen extender supplemented with 0, 3, 6, or 12 mM of Na-EDTA at 5°C for 4 weeks. In Experiment 2, semen samples were preserved in the extender supplemented with 3 mM of Na-EDTA, Ca-EDTA, or Zn-EDTA and without chelator EDTA. RESULTS: When Na-EDTA was used as a chelating substance in the extender, 3 mM was a most suitable concentration for sperm motility and viability after cold preservation. The supplementation of 3 mM Ca-EDTA had advantages regarding sperm motility, viability and plasma membrane integrity. CONCLUSION: Our findings indicate that 3 mM Ca-EDTA is the most suitable metal-chelating substance for the liquid preservation of boar semen.

Keywords: CASA, cold storage, in vitro fertilisation, liquid preservation, spermatozoa

 

 

 

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CryoLetters 35 (4), 345-355 (2014)
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LIQUIDUS TRACKING: CONTROLLED RATE VITRIFICATION FOR THE CRYOPRESERVATION OF LARGER VOLUMES AND TISSUES

E.Puschmann1*, C.Selden1, S. Butler3 and B.Fuller2

1 UCL, Institute for Liver & Digestive Health, Royal Free Campus, London, UK
2 UCL, Department of Surgery, Royal Free Campus, London, U.K.
3 Planer PLC,110 Windmill Road, Sunbury-on-Thames, UK
*Corresponding author email:
eva.puschmann.10@ucl.ac.uk

Abstract

BACKGROUND: Vitrification of cells or tissue at controlled cooling rates suitable for larger volumes, and with reduced cryoprotectant toxicity. OBJECTIVE: To set out the current understanding of the LiquidusTracking (LT) vitrification technique, and to discuss the challenges and benefits of translating the method into laboratory protocols more generally applicable to meet requirements of large volume and 3-D cryo-banking in the era of regenerative medicine. METHODS: By adding small amounts of cryoprotectants at each step and subsequently cooling the sample just above its freezing point before further increasing CPA concentration, cryoprotectant toxicity is minimized. RESULT: CPA toxicity can be reduced by lowering the temperature. Different manual approaches to LT were evaluated and further improved. CONCLUSIONS: Manual liquidus tracking is complicated and exhibits potential high variability. Nevertheless, this approach offers the possibility of testing several conditions simultaneously and could be used to pre-test conditions prior to automatic LT development.

Keywords: Vitrification, high cryoprotectant concentrations, Liquidus Tracking, liquidus curve

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