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Abstracts: CryoLetters 38 (4), 2017

 

 

Volume 38, No. 4 July/August 2017

ISSN 0143-2044

 

 


Changes in the membrane carbohydrates from sperm cryopreservedwith dimethylsulfoxide or polyvinylpyrrolidone of red-tailed hawk (Buteo jamaicencis)
José A Herrera, Gustavo Calderón, Cuauhtemoc Cruz,
Marco A Ávila, Gustavo E Quintero and Reyna C Fierro

257-262

 

 


Canonical correlation analysis to identify the semen characteristics used to forecast the freeze survival of curimba (Prochilodus lineatus) spermatozoa
Aline F.S. Carvalho, Luis D.S. Murgas,
Monica R. Ferreira-Machado, Estefânia S. Andrade,
Viviane O. Felizardo, Ivan B. Allaman and Fernanda G. de Paula

263-268

 

 


Characterization of miRNAs modulated by torpor in the hibernating ground squirrel Ictidomys tridecemlineatus liver by next-generation sequencing
Mathieu D. Morin, Daneck Lang-Ouellette, Pierre J. Lyons,
Nicolas Crapoulet and Pier Jr Morin

269-277

 

 


Survival and death of seeds during liquid nitrogen storage: A case study on seeds with short lifespans
Daniel Ballesteros and Valerie C Pence

278-289

 

 


Development of a pvs2 droplet-vitrification cryopreservation technique for Aranda Broga Blue orchid protocorm-like bodies (PLBs)
Khor Soo Ping, Ranjetta Poobathy, Rahmad Zakaria and
Sreeramanan Subramaniam

290-298

 

 


Quercetin in equine frozen semen
Jorge Squeff Filho, Carine Dahl Corcini,
Fernanda Carlini Cunha dos Santos, Andréia Nobre Anciuti,
Norton Luis Souza Gatti, Edenara Anastácio, Rafael Mielke,
Carlos Eduardo Wayne Nogueira, Bruna da Rosa Curcio and
Antonio Sergio Varela Junior

299-304

 

 


A thermal protective urethral heater applied to modulate the prostate cryoablation area
Bin Qian, Hongli Zhao, Binkai Xu and Minbo Lan

305-314

 

 


Cryomicroscopic analysis of intracellular ice formation in porcine iliac endothelial cells upon cooling
Yufang Li, Fazil Panhwa, Zhongrong Chen, Fuquan Yuan, Xing Ji,
Peng Hu and Gang Zhao

315-320

 

 


Molecular and pathological evaluation of cryopreserved colorectal cancerous tissues: effects of freezing method and cryoprotection
Wei Liang, Fei Ding, Meixia Wang, Baolin Liu and Menghong Sun

321-329

 

 


Temporal plasticity in cold hardiness and cryoprotectant contents in northern versus temperate Colias butterflies (lepidoptera: pieridae)
Pavel Vrba, OldřichNedvěd, Helena Zahradníčková and
Martin Konvička

330-338

 

 


Cloning and expression analysis of glucose transporter 4 mRNA in the cold hardiness frog, Rana dybowskii
Baihong Guo, Shanshan Gong, Jingyu Zhang, Longhui Chai,
Yu Zhang, Boju Wang, Jie Shao and Xianghong Xiao

339-346

 

 

 

 

 

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CryoLetters 38 (4), 257-262 (2017)
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CHANGES IN THE MEMBRANE CARBOHYDRATES FROM SPERM CRYOPRESERVED WITH DIMETHYLSULFOXIDE OR POLYVINYLPYRROLIDONE OF RED-TAILED HAWK (Buteo jamaicencis)

José A Herrera1, Gustavo Calderón2, Cuauhtemoc Cruz3,
Marco A Ávila 4,5, Gustavo E Quintero5 and Reyna C Fierro*6.

1Department of Agricultural and Animal Production, and
2Mastery in Agricultural and livestock Sciences. UAM-X. Calzada del Hueso 1100, Villa Quietud, Coyoacán, CDMX. C.P. 04960.
3Mastery in Biology of the Animal Reproduction, and
6Department of Health Sciences. UAM-I. San Rafael Atlixco 186, C.P., 09340, CDMX.
4Ministry of Environment. Environmental Education Center "Rodolfo Landeros Gallegos" INE/CITES/DGVS-CR-IN-AV-0035-AGS./00; Ags, Ags, México. Convención Poniente 1626, Fraccionamiento La Concordia, Aguascalientes, Ags. México. CP 20040.
5Department of Biology, Center of Basic Sciences, UAA México

Abstract

BACKGROUND: That cryopreservation can induce alterations in sperm. OBJECTIVE: The goal of this study was evaluate sperm quality and distribution of N-acetylglucosamine, sialic acid and mannose residues in sperm cryopreserved of red-tailed hawk (Buteo jamaicensis). MATERIALS AND METHODS: We studied twenty samples of ejaculated semen for each cryoprotectant dimethylsulfoxide or polyvinylpyrrolidone. Carbohydrate identification was carried out with lectins Triticum vulgaris agglutinin to N-acetylglucosamine and sialic acid and Concanavalia ensiformis for mannose residues. Sperm viability was not altered but motility decreased significantly with both crioprotectants compared with fresh sperm. RESULTS: Neither the number of WGA positive sperm nor the distribution of N-acetylglucosamine and/or sialic acid residues were affected by the cryopreservation procedure. The sperm proportion with fluorescence associated with the presence of mannose residues was higher in thawed sperm.  CONCLUSION: Values obtained with the cryopreservation technique proposed in this study by freezing drops in liquid nitrogen, were within normal parameters established for good quality fresh semen. We can say that it can be used for assisted reproduction of Buteo jamaicenc.

Keywords: Bird prey, cryopreservation, mannose, sialic acid

 

 

 

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CryoLetters 38 (4), 263-268 (2017)
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CANONICAL CORRELATION ANALYSIS TO IDENTIFY THE SEMEN CHARACTERISTICS USED TO FORECAST THE FREEZE SURVIVAL OF CURIMBA (PROCHILODUS LINEATUS) SPERMATOZOA

    Aline F.S. Carvalho1, Luis D.S. Murgas 1*,
    Monica R. Ferreira-Machado2, Estefânia S. Andrade1,
    Viviane O. Felizardo1, Ivan B. Allaman3,  Fernanda G. de Paula4.

1Department of Veterinary Medicine, Federal University of Lavras, Lavras, Minas Gerais
2
Department of Biological Sciences, Federal University of Goiás, Campus Jataí, Jataí, Goiás;
3Departament of Exact and Technological Sciences, State University of Santa Cruz, Ilhéus, Bahia,
4Departament of Animal Production, School of Veterinary and Zootechny, Federal University of Goiás, Goiânia, Brazil.
*Corresponding email:
lsmurgas@dmv.ufla.br

Abstract

OBJECTIVE: To identify which sperm characteristics were able to predict more accurately the quality of curimba (Prochilodus lineatus) semen upon freezing using canonical correlation analysis.  METHODS: Eleven fish breeders with initial mean weight of 705.21±111 g were used. For cryopreservation, 200 µL of semen were taken from each animal and diluted in the cryoprotectant solution (10% dimethyl sulfoxide and 5% Beltsville Thawing Solution Minitüb®) in a 1:4 ratio and placed into 0.5-mL straws. Sperm characteristics (motility, sperm abnormalities, total antioxidant activity and lipid peroxidation) were evaluated. A randomized block design with duplicate samples per treatment (fresh and frozen semen) was used. The block factor was the animals, and the experimental unit the ejaculates. Canonical correlation was used to evaluate the association between sperm characteristics of fresh semen and thawed semen. RESULT: There was a significant association (P = 0.10) among the variables measured in fresh semen with the variables measured in thawed semen, and 78.6% of the difference observed in the thawed semen can be attributed to variation of variables measured in fresh semen. Sperm motility, motility duration and antioxidant activity of the thawed semen showed an inverse relationship with those of the fresh semen; whereas the minor sperm abnormalities, major sperm abnormalities and lipid peroxidation showed a direct relationship with those of the fresh semen. Only the rate and motility duration of the thawed semen presented high correlation (-0.63 and -0.73, respectively) with the canonical variable represented by the sperm characteristics of fresh semen. CONCLUSIONS: The rate and motility duration of fresh semen may be used to predict the quality of the thawed sperm in Prochilodus lineatus.

Keywords: Antioxidant, cryopreservation, fish. sperm morphology, motility, lipid peroxidation.

 

 

 

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CryoLetters 38 (4), 269-277 (2017)
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CHARACTERIZATION OF miRNAS MODULATED BY TORPOR IN THE HIBERNATING GROUND SQUIRREL Ictidomys tridecemlineatus LIVER BY NEXT-GENERATION SEQUENCING

Mathieu D. Morin1, Daneck Lang-Ouellette1, Pierre J. Lyons1,
Nicolas Crapoulet2 and Pier Jr Morin1*

1Department of Chemistry and Biochemistry, Université de Moncton, New Brunswick, Canada.
2Atlantic Cancer Research Institute, Pavillon Hôtel-Dieu, New Brunswick, Canada.
*Corresponding author email
pier.morin@umoncton.ca

Abstract

BACKGROUND: Mammalian hibernation is a fascinating phenomenon that involves multiple molecular and biochemical changes to proceed. While the molecular picture associated with torpor has become clearer in recent years, the function of non-coding RNAs, and especially of microRNAs, solicited during this process is not well understood.  OBJECTIVE: To better characterize a signature of cold torpor-associated miRNAs in the hibernating thirteen-lined ground squirrel Ictidomys tridecemlineatus.  MATERIALS AND METHODS: Next-generation sequencing and qRT-PCR approaches were conducted in euthermic and hibernating ground squirrel liver tissues. RESULTS: This high-throughput approach notably revealed modulation during hibernation of various miRNAs previously associated with lipid metabolism, glucose metabolism and antioxidant responses such as miR-145a-3p, miR-22-3p and miR-25-3p, respectively.  CONCLUSION: Overall, these results present a group of miRNAs differentially expressed in hibernating ground squirrel liver and provide additional knowledge on the underlying functions of these small non-coding molecules during cold torpor.

Keywords: hibernation, torpor, hypometabolism, microRNAs, next-generation sequencing

 

 

 

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CryoLetters 38 (4), 278-289 (2017)
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SURVIVAL AND DEATH OF SEEDS DURING LIQUID NITROGEN STORAGE: A CASE STUDY ON SEEDS WITH SHORT LIFESPANS

Daniel Ballesteros1,2,* and Valerie C Pence1

1Center for Research of Endangered Wildlife (CREW). Cincinnati Zoo and Botanical Garden. 3400 Vine Street. Cincinnati, OH-45220, USA.
2(present address) Department of Comparative Plant and Fungal Biology, Royal Botanic Gardens, Kew, Welcome Trust Millennium Building, Wakehurst Place, Ardingly, West Sussex, RH17 6TN, United Kingdom.
Corresponding author Email:
d.ballesteros@kew.org

Abstract

BACKGROUND: The low temperature of liquid nitrogen is assumed to stop ageing and preserve viability indefinitely, however there are few validating data sets. The use of seeds to test these assumptions is important because other cryopreserved systems lack quantitative measures of viability to allow comparisons among timed points. OBJECTIVE: To evaluate survival of a collection of seeds with short lifespans stored 12-20 years in liquid nitrogen.  MATERIALS AND METHODS: Seeds from 11 species (26 accessions) were removed from cryostorage and evaluated for germination and normal growth. RESULTS: Germination of Plantago cordata and Betula spp. seeds did not decrease significantly during cryostorage. However, Populus deltoides and most Salix spp. accessions showed a significant decrease in germination, with further loss observed when P. deltoides seedlings were followed to the young plant stage. Seeds of initial low quality showed greater deterioration during cryostorage.  CONCLUSION: Cryostorage maintained viability of Salix and Populus seeds longer than other temperatures. However, ageing was not completely stopped and seed longevity was shorter than that predicted for many other species. A high initial seed quality is important in order to obtain the maximum benefit of cryostorage.

Keywords: cryopreservation, seed longevity, germination, seedling development, ex situ conservation, wild species.

 

 

 

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CryoLetters 38 (4), 290-298 (2017)
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DEVELOPMENT OF A PVS2 DROPLET-VITRIFICATION CRYOPRESERVATION TECHNIQUE FOR Aranda Broga Blue ORCHID PROTOCORM-LIKE BODIES (PLBs)

Khor Soo Ping, Ranjetta Poobathy, Rahmad Zakaria
and Sreeramanan Subramaniam*

School of Biological Sciences, Universiti Sains Malaysia (USM), 11800 Gelugor, Penang, Malaysia.
*Corresponding author email:
sreeramanan@gmail.com / sreeramanan@usm.my

Abstract

BACKGROUND: Conservation of commercially important ornamental plants is important to maintain its unique beauty to cater the market demands. OBJECTIVE: The main objective is to develop an efficient cryopreservation technique for Aranda Broga Blue orchid PLBs using droplet-vitrification method. MATERIALS AND METHODS: Several critical factors in cryopreservation were accessed such as preculture concentrations and durations, choice of vitrification solutions, two-step or three-step vitrification, growth recovery medium and PVS2 exposure duration. RESULTS: The best growth regeneration percentage (5%) was obtained when 3-4mm PLBs were precultured in 0.2M sucrose for 3 days, followed by osmoprotection for 20 minutes, dehydration in PVS2 for 20 minutes at 0°C, LN storage, thawed and unloading for 20 minutes, and growth regeneration in VW10 medium. PLBs were found to be very sensitive to osmotic stress imposed by high molecular weight cryoprotectant such as sucrose and glycerol. Osmotic potential of growth recovery medium is one of the main factors that affect growth recovery in cryopreserved PLBs. CONCLUSION: Current report showed possibilities in cryopreserving Aranda Broga Blue PLBs using droplet-vitrification technique. However, further improvement of growth recovery can be done by focussing on approaches that facilitate sufficient water removal from PLBs without causing severe osmotic injuries to the plant cells.

Keywords: Aranda, protocorm-like bodies, organic additives, droplet-vitrification, cryopreservation.

 

 

 

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CryoLetters 38 (4), 299-304 (2017)
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QUERCETIN IN EQUINE FROZEN SEMEN

Jorge Squeff Filho1, Carine Dahl Corcini3,
Fernanda Carlini Cunha dos Santos1, Andréia Nobre Anciuti1
Norton Luis Souza Gatti1, Edenara Anastácio2,
Rafael Mielke2, Carlos Eduardo Wayne Nogueira3,
Bruna da Rosa Curcio3 and Antonio Sergio Varela Junior 4*

1Pós-Graduação em Veterinária, Faculdade de Veterinária, Universidade Federal de Pelotas, Campus Universitário, Pelotas, RS, 96010-900, Brasil.
2
Departamento de Patologia Animal, Laboratório de Reprodução Animal, Faculdade de Veterinária, Universidade Federal de Pelotas, Campus Universitário, Pelotas, RS, 96010-900, Brasil.
3
Departamento de Clínicas Veterinárias, Faculdade de Veterinária, Universidade Federal de Pelotas, Campus Universitário, Pelotas, RS, 96010-900, Brasil.
4Reprodução Animal Comparada, Instituto de Ciência Biológicas, Universidade Federal de Rio Grande, Rio Grande, Brasil.
*Corresponding author email
antoniovarela@furg.br

Abstract

BACKGROUND: Supplementation of sperm diluents to reduce the damage caused by the freeze-thaw cycle is broadly used in equine semen cryopreservation.  OBJECTIVE: The present study aimed at determining the most appropriate quercetin supplementation in equine freezing extender. MATERIALS AND METHODS: Quercetin at four different concentrations (0.25, 0.5, 0.75 or 1 mM) was added in the sperm freezing diluent before the freeze-thaw cycle. The spermatozoa population was analyzed by flow cytometry and a statistical analysis was conducted to detect significant differences between control and treated samples. RESULTS: The statistical analysis did not reveal any significant modification of seminal parameters.  CONCLUSION: Within the concentrations tested, quercetin supplementation in equine freezing extender did not affect progressive motility, mitochondrial functionality, acrosome reaction, membrane integrity or DNA fragmentation index in post-thaw equine semen.

Keywords: Antioxidant, ATP, spermatozoa, ROS, flavonoid.

 

 

 

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CryoLetters 38 (4), 305-314 (2017)
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A THERMAL PROTECTIVE URETHRAL HEATER APPLIED TO MODULATE THE PROSTATE CRYOABLATION AREA

Bin Qian1, Hongli Zhao1, Binkai Xu2 and Minbo Lan1*

1Shanghai Key Laboratory of Functional Materials Chemistry School of Chemistry and MolecularEngineering, East China University of Science and Technology Shanghai 200237, China
2AccuTarget MediPharma Co., LTD., Shanghai, 201318, China, Corresponding author at: Shanghai Key Laboratory of Functional Materials Chemistry and School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai 200237, China. Tel.: +86 21 64253574.
Corresponding author email:
minbolan@ecust.edu.cn (M. Lan)

Abstract

BACKGROUND: Urethral warmer and cryoheater are invented and applied in cryoablation to overcome urethral cryoinjury, but these devices cannot be fixed and precisely control the released heat which excessively reduces the effective ablation area. Current warmers enlarge the operation difficulty and decrease the precision in temperature control. OBJECTIVE: A reformed catheter termed urethral heater aims to protect the urethra and simultaneously control the released heat so as to meet the aid of doctors' convenient operation in effective therapy, device fixation and precise heat controllability. MATERIALS AND METHODS: In this paper, the temperature controller combined with temperature monitor was used to control the heating behavior of the urethral heater with the initial active temperature. The controllability and thermal protection of the urethral heater was simulated and tested, which compared with that of urethral warmer.  RESULTS: During the trials in vitro, the lowest temperature at the urethra surface is -3.7°C when one cryoprobe was introduced in the cryoablation for 15 min and -15.3°C with two cryoprobes. Above all, the effective cryoablation area increased with the decline of initial active temperatures. CONCLUSION: The urethral heater is able to prevent the urethra from irreversible damage and modulate the ablation area. The delay of heat is a new way to decline the recurrence rate and facilitate the desire of aconuresis during the cryoablation.

Keywords: urethral heater, cryoablation, temperature controllability, catheter, immune response

 

 

 

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CryoLetters 38 (4), 315-320 (2017)
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CRYOMICROSCOPIC ANALYSIS OF INTRACELLULAR ICE FORMATION IN PORCINE ILIAC ENDOTHELIAL CELLS UPON COOLING

Yufang Li1,2#, Fazil Panhwa1,2#, Zhongrong Chen1,2#, Fuquan Yuan1,2,
Xing Ji1,2, Peng Hu3* and Gang Zhao1,2*

1Centre for Biomedical Engineering, Department of Electronic Science & Technology, University of Science and Technology of China;
2Anhui Provincial Engineering Technology Research Center for Biopreservation and Artificial Organs;
3Department of Thermal Science and Energy Engineering, University of Science and Technology of China, Hefei, China.
#These authors contributed equally.
*Corresponding author email:
zhaog@ustc.edu.cn or hupeng@ustc.edu.cn

Abstract

BACKGROUND: Intracellular ice formation (IIF) plays an important role in cryopreservation and cryosurgery. IIF in porcine iliac endothelial cells (PIECs) has not been fully investigated. OBJECTIVE: To analyze the phenomenon of IIF in PIECs during freezing. MATERIALS AND METHODS: The cryomicroscopy system was used for observation of the cell morphology and for the count of IIF during freezing, while the theoretical model for probability IIF (PIF) was used to determine the nucleation kinetic and thermodynamic parameters. RESULTS: PIF was observed at 40, 80 and 100 ºC/min upon cooling and 100 ºC/min upon warming, in the presence and absence of 1 M dimethyl sulfoxide (Me2SO). The kinetic and thermodynamic parameters were obtained by fitting the PIF model to experimental data. CONCLUSION: PIF increases along with the increased cooling rate. The addition of 1 M dimethyl sulfoxide (Me2SO) decreases the onset IIF temperature, but increases PIF. Cooling rate and Me2SO concentration significantly affect IIF. The finding has significant implications in both cryopreservation and cryosurgery.

Keywords: cryomicroscope, porcine iliac endothelial cells, intracellular ice formation

 

 

 

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CryoLetters 38 (4), 321-329 (2017)
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MOLECULAR AND PATHOLOGICAL EVALUATION OF CRYOPRESERVED COLORECTAL CANCEROUS TISSUES: EFFECTS OF FREEZING METHOD AND CRYOPROTECTION

Wei Liang1a, Fei Ding2a, Meixia Wang1, Baolin Liu1*
and Menghong Sun2

1Institute of Biothermal Science, School of Medical Instruments and Food Engineering, University of Shanghai for Science and Technology;
2Department of Pathology, Fudan University Shanghai Cancer Center, Shanghai, China.
aThese authors contributed equally to the work.
*Corresponding author email: Dr. BL Liu (
blliuk@163.com)

Abstract

BACKGROUND: Snap freezing and RNAlater stabilization are methods that were wildly used in biospecimen depositories to preserve cancer tissues. Both methods have its own limitations. An ideal method for preservation of diseased tissues should permit the broader uses of stored tissue samples, including not just for molecular diagnostic analysis, histopathological evaluation, but also for the recovery of functional live cells and tissues as well as the regeneration of patient-derived xenograft (PDX) models for the drug screening study. MATERIALS AND METHODS: This study investigated molecular and pathological evaluation of cryopreserved colorectal cancer tissues, with an emphasis on effects of freezing method and cryoprotection. Global gene expression analysis with microarrays and histological examination of tissue samples were performed to compare tissue specimens after snap freezing, cryoprotectant permeation and subsequent cryopreservation. RESULTS: Compared with the fresh tissue samples (immediately stabilized in RNAlater after collection), samples preserved by snap freezing exhibited the largest number of differentially-expressed genes. Some genes relate to neuron, drug addiction and drug binding, but the rest of differentially-expressed genes were functionally dispersive. Cryoprotectant permeation into tissue samples and subsequent cryopreservation via a rate-controlled freezing resulted in much less differentially expressed genes. Histological structures of tissue specimens were both well preserved by snap freezing and cryoproservation. CONCLUSION: Snap freezing may not be as reliable as commonly considered. The pilot study demonstrates the feasibility of using cryopreservation to retain viable diseased tissues for multiple applications.

Keywords: depository, cryopreservation, histopathology, molecular diagnostics, snap freezing.

 

 

 

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CryoLetters 38 (4), 330-338 (2017)
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TEMPORAL PLASTICITY IN COLD HARDINESS AND CRYOPROTECTANT CONTENTS IN NORTHERN VERSUS TEMPERATE COLIAS BUTTERFLIES (LEPIDOPTERA: PIERIDAE)

Pavel Vrba1*, OldřichNedvěd1,2, Helena Zahradníčková1
and Martin Konvička1,2

1Institute of Entomology, Biology Centre CAS;
2Faculty of Sciences, University South Bohemia, Branišovská 31, 370 05 ČeskéBudějovice, Czech Republic
*Corresponding author email:
vrba_pavel@centrum.cz

Abstract

BACKGROUND: Butterflies Colias hyale and C. palaeno differ in distribution, habitat, voltinism and cold hardiness. OBJECTIVE: To compare changes in supercooling point (SCP) and cryoprotectant concentration(CPAc) of outdoor-reared caterpillars. MATERIALS AND METHODS: SCP was measured with a thermocouple and CPAc by gas chromatography monthly during winter. RESULTS: Seasonal changes in SCP and CPAc in overwintering larvae followed the pattern of change in ambient temperature. Only in warm November, SCP was low and CPAc high in boreal univoltine C. palaeno, whereas the opposite appeared in temperate multivoltine C. hyale. In mild December, SCP increased and CPAc decreased in C. palaeno, and acclimation reappeared in cold January. Both species differed in monthly cryoprotectant profiles, regarding both constitutive and inducible compounds. CONCLUSION: Seasonal pattern of SCP/CPAc enables C. palaenoto survive early frosts, but the costs of repeated acclimation during mild winters may set southern or low altitude limits of its distribution.

Keywords: butterfly ecology, cold hardiness, cryoprotectant compounds

 

 

 

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CryoLetters 38 (4), 339-346 (2017)
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CLONING AND EXPRESSION ANALYSIS OF GLUCOSE TRANSPORTER 4 mRNA IN THE COLD HARDINESS FROG, RANA DYBOWSKII

Baihong Guo, Shanshan Gong, Jingyu Zhang, Longhui Chai, Yu Zhang,
Boju Wang, Jie Shao and Xianghong Xiao*

Institute of Wildlife Resources, Northeast Forestry University, Harbin 150040, China.
*Corresponding author email:
xiaoxh2010@sina.com

Abstract

BACKGROUND: The Rana dybowskii distribute in northeast region of China which have seasonally cold climates. During winter they survival freezing by biosynthesizing carbohydrate cryoprotectants such as high concentrations glucose into blood and all tissues. The essential role of glucose transporter 4 is a high-affinity glucose transporter, which can increase glucose uptake in cells when it stimulated by insulin. OBJECTIVE: In this study, we analysis the full-length GLUT4 mRNA detect the gene levels of GLUT4 in R. dybowskii main tissues by qPCR during low temperature.  RESULTS: We found in heart, fat body, skeletal muscle and skin four tissues all express GLUT4, and the levels of GLUT4 decreased on initial cold exposure stage, 8~12 hours, followed 24 hours it recovered.  CONCLUSION: This study we firstly indentified and characterized GLUT4 in amphibious, and provide a novel insight into the role of GLUT4 in cryoprotectant synthesis and cell protection in cold hardiness amphibians.

Keywords: Glucose transporter 4, cold hardiness, glucose uptake, cryoprotectant.

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