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Abstracts: CryoLetters 35 (1), 2014

 

 

Volume 35, No. 1 January/February 2014

ISSN 0143-2044

 

 


Application of a functional marker for the effect of cryoprotectant agents on gorgonian coral (Junceella juncea and J. Fragilis) sperm sacs
S. Tsai, V. Kuit, Z.G. Lin and C. Lin

1-7

 

 


Milt cryopreservation for rheophilic fish threatened by extinction in the Rio Grande, Brazil
Estefânia de Souza Andrade, Daniella Aparecida
de Jesus Paula, Viviane de Oliveira Felizardo,
Luis David Solis Murgas, Galileu Crovatto Veras
and Priscila Vieira e Rosa

8-14

 

 


Evaluation of two distinct cryoprotectants for cryopreservation of human red blood cell concentrates
Jolanta Korsak, Agnieszka Goller,
Agnieszka Rzeszotarska and Katarzyna Pleskacz

15-21

 

 


Cryopreservation of adventitious shoot tips of Paraisometrum mileense by droplet vitrification
Liang Lin, Bin Yuan, Dandan Wang and Weiqi Lin

22-28

 

 


Effects of seed cryopreservation, stratification and scarification on germination for five rare species of pitcher plants
Sruti Khanna, Heather Jenkins, Kylie Bucalo,
Ron O. Determann, Jennifer M. Cruse-Sanders
and Gerald S. Pullman

29-39

 

 


Effect of shooting medium and source of material on grapevine (Vitis vinifera l.) Shoot tip recovery after cryopreservation
Zvjezdana Marković, Philippe Chatelet, Darko Preiner,
Isabelle Sylvestre, Jasminka Karoglan Kontić
and Florent Engelmann

40-47

 

 


Nuclear status and DNA fragmentation of oocytes from porcine, bovine and feline ovaries stored at 4°C for 5 days
Vien Viet Luu, Zhao Namula, Lanh Thi Kim Do,
Yoko Sato, Masayasu Taniguchi,
Ni Wayan Kurniani Karja, and Takeshige Otoi

48-53

 

 


Cold pretreatment amplifies the responses of in vitro Eucalyptus grandis shoots to cryopreparative drying
Chao-Hsuan Ting, David J. Mycock
and Kershree Padayachee

54-62

 

 


Controlled rate cooling of fungi using a stirling cycle freezer
Matthew J. Ryan, Daiva Kasulyte-Creasey,
Anthony Kermode, Shwe Phue San and Alan G. Buddie

63-69

 

 


Screening of transgenic frost-resistant cotton using a porous silicon biosensing platform
Liangliang Chen, Peng Li, Jie Zhao, Jieqiong Li,
Xiaoyi Lv, Zhenhong Jia and Ji Ma

70-76

 

 


Comparison of slow freezing and vitrification on ovine immature oocytes
M H Bhat, V Sharma , F A Khan, N A Naykoo,
S H Yaqoob , Ruby, H M Khan , M R Fazili ,
N A Ganai and R A Shah

77-82

 

 

 

 

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CryoLetters 35 (1), 1-7 (2014)
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APPLICATION OF A FUNCTIONAL MARKER FOR THE EFFECT OF CRYOPROTECTANT AGENTS ON GORGONIAN CORAL (JUNCEELLA JUNCEA AND J. FRAGILIS) SPERM SACS

S. Tsai1, V. Kuit2, Z.G. Lin3 and C. Lin2, 4*

1Department of Biotechnology, Mingdao University, Peetow, Chang Hua, Taiwan.
2Institute of Marine Biology, Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia.
3Institute of Marine Biotechnology, National Dong Hwa University, Checheng, Pingtung, Taiwan.
4National Museum of Marine Biology and Aquarium, Checheng, Pingtung, Taiwan.
*Corresponding author email: chiahsin@nmmba.gov.tw

Abstract

BACKGROUND: The establishment of coral sperm repositories which retain good post-rewarming viability and fertility play a vital role in species conservation. OBJECTIVE: This study aimed at obtaining baseline information regarding the effects of cryoprotectant agents (CPAs) on gorgonian coral (Junceella juncea and J. fragilis) sperm sacs. METHODS: The adenosine triphosphate assay was used to determine the energy level of the gorgonian sperm sacs as an indicator of sperm viability after exposure to cryoprotectants. RESULTS: The 'no observed effect concentrations' (NOECs) of methanol, dimethyl sulfoxide (DMSO), polypropylene glycol (PG), ethylene glycol (EG) and glycerol for J. juncea sperm sacs were 3 M, 3 M, 1 M, 2 M and 1 M respectively after 20 min exposure; whilst the NOECs for J. fragilis oocytes were 2 M, 3 M, 1 M, 2 M and 2 M, respectively. Methanol and DMSO had the least impact. PG was the most toxic CPA after 10 min exposure. ATP content of J. juncea and J. fragilis sperm sacs did not differ significantly from the control with incubation times of 10-20 min with 2 M EG. However, ATP content dropped significantly after exposing sperm sacs to 2 M EG for 40 min with average values of 2.34 ± 0.12 and 1.97 ± 0.48 μg/ml respectively. ATP content for J. juncea and J. fragilis sperm sacs was significantly decreased to 1.79 ± 0.31 and 2.40 ± 0.36 μg/ml after 20 min incubation in 2 M PG when compared to the control with 2.98 ± 0.16 and 4.14 ± 0.42 μg/ml respectively. Normalized ATP content for sperm sacs of two different gorgonian coral after incubation in methanol, DMSO, PG, EG and glycerol showed that J. juncea sperm sacs were slightly less tolerant to CPAs compared to J. fragilis sperm sacs. CONCLUSIONS: DMSO or methanol can be considered as efficient CPAs for gorgonian sperm sacs cryopreservation. The ATP luminescence assay provided sensitive and rapid quantification of mitochondrial activity in gorgonian coral sperm sacs. The study on the impact of CPA will contribute to the development of a cryopreservation protocol for coral sperm conservation.

Keywords: gorgonian coral; ATP; sperm; cryoprotectant; cryopreservation.

 

 

 

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CryoLetters 35 (1), 8-14 (2014)
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MILT CRYOPRESERVATION FOR RHEOPHILIC FISH THREATENED BY EXTINCTION IN THE RIO GRANDE, BRAZIL

Estefânia de Souza Andrade1, Daniella Aparecida de Jesus Paula2,
Viviane de Oliveira Felizardo1*, Luis David Solis Murgas1*,
Galileu Crovatto Veras1 and Priscila Vieira e Rosa2

1Department of Veterinary Medicine, and
2Department of Animal Production, Federal University of Lavras, Mailbox: 3037, Lavras, Minas Gerais 37200000, Brazil.
*Corresponding author email: viviofbio@yahoo.com.br

Abstract

BACKGROUND: Specific protocols for milt cryopreservation have been established for some freshwater fish species. However, cryopreservation reduces sperm quality, giving unsatisfactory results in reproduction. OBJECTIVE: The objective of this work was to evaluate the effect of different cryoprotectants on the quality of Prochilodus lineatus, Brycon orbignyanus and Piaractus mesopotamicus milt after cryopreservation. METHODS: The milt was diluted in different cryoprotectant solutions containing 10% methanol, dimethyl sulfoxide, glycerol, propylene glycol or ethylene glycol combined with the Beltsville Thawing Solution extender (5%), then placed in the vapour of a liquid nitrogen (LN) storage tank for 24 h, after which they were immersed in LN. After rewarming, the rate (%) and duration (s) of milt motility and abnormal morphology were evaluated. RESULTS: All of cryoprotectant solutions tested used maintained the viability of P. lineatus and P. mesopotamicus milt. However, in P. lineatus, glycerol ensured a lower percentage of abnormal morphology. In case of P. mesopotamicus, all of the cryoprotectant solutions tested may be used in the cryopreservation process, with the exception of those containing glycerol. CONCLUSION: For B. orbignyanus, cryoprotectant solutions containing methanol and ethylene glycol are recommended for use in the cryopreservation process, although they reduced the quality of sperm post-rewarming.

Keywords: Brycon orbignyanus, Piaractus mesopotamicus, Prochilodus lineatus, reproduction.

 

 

 

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CryoLetters 35 (1), 15-21 (2014)
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EVALUATION OF TWO DISTINCT CRYOPROTECTANTS FOR CRYOPRESERVATION OF HUMAN RED BLOOD CELL CONCENTRATES

Jolanta Korsak*, Agnieszka Goller, Agnieszka Rzeszotarska and Katarzyna Pleskacz

Department of Clinical Transfusiology, Military Institute of Medicine, Warsaw, Poland.
Corresponding author:  Prof. Jolanta Korsak, MD, PhD zt@wim.mil.pl

Abstract

BACKGROUND: Cryopreservaton of packed human red blood cells requires the use of cryoprotectants. OBJECTIVE: The study assessed physiological parameters of 40 RBC units frozen with either 40% glycerol or 6.7% HES. METHODS: After thawing, they were suspended in NaCl or in 6% HES. Tests of Hct, Hb, Na+ and K+ ions, ATP, 2,3-DPG, pH and erythrocyte stability were measured 30 minutes and 24 hours after thawing. RESULTS: Hct was lower after thawing but did not differ significantly between two groups. Hb was lower after thawing, but was statistically significant higher in the HES group (43.8 g/unit vs 35.4 g/unit). K+ concentration increased after thawing and was significantly higher after 24 hours in the glycerol group (29.0 mEq/l vs 8.7 mEq/l). ATP concentration in the HES group was significantly lower (2.15 µmol/g) in comparison with the glycerol group (6.30 µmol/g) 24 hours after thawing. 2,3-DPG levels did not differ significantly between the methods. Stability of RBCs frozen in glycerol were better (94.58%) than RBCs frozen in HES (80.75%) measured 24 hours after thawing. ATP is better protected in erythrocytes frozen in glycerol than in HES. CONCLUSION: Erythrocytes frozen with HES preserved more hemoglobin than with glycerol. Membrane permeability for Na+ and K+ ions was preserved better with HES. HES compared to glycerol offered better protection for erythrocytes.

Keywords: hydroxyethyl starch, glycerol, red blood cells, freezing.

 

 

 

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CryoLetters 35 (1), 22-28 (2014)
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CRYOPRESERVATION OF ADVENTITIOUS SHOOT TIPS OF PARAISOMETRUM MILEENSE BY DROPLET VITRIFICATION

Liang Lin1,2, Bin Yuan1, Dandan Wang1 and Weiqi Lin1*

1Key Laboratory of Biodiversity and Biogeography and Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China
2University of Chinese Academy of Sciences, Beijing 100039, China
*Corresponding author email:
weiqili@mail.kib.ac.cn

Abstract

BACKGROUND: Gesneriaceae family contains numerous species endemic to China, and many of them are listed as endangered species. There is a need for a simple and efficient method for long term conservation of these species.  OBJECTIVE: The study aimed to establish an efficient procedure for cryopreserving Paraisometrum mileense, a critically endangered species endemic to Yunnan, China. METHODS: Effects of sucrose concentration of preculture solution, duration of sucrose preculture, duration of plant vitrification solution 2 (PVS2) treatment, and cold acclimation on regeneration of cryopreserved adventitious shoot tips (ASTs) were assessed. RESULTS AND CONCLUSION: Among different sucrose preculture regimes tested, preculture with 0.3M sucrose for 24h resulted in best regeneration of cryopreserved ASTs. PVS2 treatment also affected regeneration considerably with the maximum survival of ASTs after incubation in PVS2 for 90 min at 0C. With the optimised parameters, the level of shoot regeneration from cryopreserved ASTs reached 86%. No morphological abnormalities were observed during one year's growth of the plantlets developing from cryopreserved ASTs.  Procedure established in this research is a promising technique for the cryopreservation of ASTs of this species.

Keywords: Gesneriaceae, endangered species, sucrose preculture, cold acclimation

 

 

 

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CryoLetters 35 (1), 29-39 (2014)
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EFFECTS OF SEED CRYOPRESERVATION, STRATIFICATION AND SCARIFICATION ON GERMINATION FOR FIVE RARE SPECIES OF PITCHER PLANTS

Sruti Khanna1, Heather Jenkins1, Kylie Bucalo2, Ron O. Determann3, Jennifer M. Cruse-Sanders3 and Gerald S. Pullman*1,2

1School of Biology, and
2Institute of Paper Science and Technology, Georgia Institute of Technology, Atlanta, GA 30332, USA;
3Department of Conservation Research, Atlanta Botanical Garden, Atlanta GA 30309, USA
*Corresponding author email: jerry.pullman@ipst.gatech.edu

Abstract

BACKGROUND: Habitat loss and over collection have caused North American pitcher plants to become rare, including U.S. federally endangered Sarracenia alabamensis and S. oreophila, and S. leucophylla, S. psittacina and S. purpurea spp. venosa, endangered in several states.  OBJECTIVE: To develop reliable seed cryopreservation protocols for endangered Sarracenia species enabling similar germination percentages before and after storage in liquid nitrogen (LN) either in vivo or using in vitro tools.  METHODS: Seed germination pre- and post-cryopreservation were compared following seed drying with germination in soil, aseptic environment with wet filter paper or enriched medium, and using scarification or stratification for dormancy removal. RESULTS: After cryostorage, germination in vitro (1/6- or 1/3-strength MS medium) increased compared to germination on peat moss. Germination pre- and post-cryopreservation was similar for S. alabamensis and S. oreophila when seeds were stratified and grown in vitro. S. leucophylla and S. psittacina also showed high germination after cryopreservation when germinated on medium following stratification. CONCLUSION: Rapid liquid nitrogen exposure and rewarming induced seed coat cracking that damaged seeds, likely allowing internal damage during acid scarification and microbial entry during germination in non-sterile environments.

Keywords: carnivorous plants, conservation, cryopreservation, endangered species, liquid nitrogen, scarification, stratification, seed coat cracks.

 

 

 

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CryoLetters 35 (1), 40-47 (2014)
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EFFECT OF SHOOTING MEDIUM AND SOURCE OF MATERIAL ON GRAPEVINE (VITIS VINIFERA L.) SHOOT TIP RECOVERY AFTER CRYOPRESERVATION

Zvjezdana Marković1, 2, 3, Philippe Chatelet2, 3, Darko Preiner1,
Isabelle Sylvestre3, Jasminka Karoglan Kontić1
and Florent Engelmann3*

1University of Zagreb, Department of Viticulture and Enology, Zagreb, Croatia.
2INRA, UMR AGAP, Equipe DAVEM, 34060 Montpellier, France.
3IRD, UMR DIADE, 911 Avenue Agropolis, 34394 Montpellier, France.
*Corresponding author e-mail: florent.engelmann@ird.fr

Abstract

BACKGROUND: Selecting experimental material at the optimal physiological stage is of paramount importance for successful cryopreservation. OBJECTIVE: The study was to investigate the effect of the physiological state of grapevine buds on their regrowth after liquid nitrogen exposure. METHODS: In a first set of experiments, we tested the regrowth of cryopreserved buds sampled from microcuttings cultured on shooting medium containing benzylaminopurine or zeatin riboside for various durations. In a second set of experiments, we studied the regrowth after liquid nitrogen exposure of buds sampled from different positions on the stem of in vitro plantlets. RESULTS: Regrowth of cryopreserved buds sampled from microcuttings was higher (30%), compared to buds sampled directly from in vitro plantlets (23%), for all culture durations of microcuttings on shooting medium tested (2-6 weeks). Addition of cytokinin in the shooting medium improved regrowth of cryopreserved buds compared to buds sampled from microcuttings cultured on medium devoid of growth regulators; however similar results were obtained with the two cytokinins tested. Buds sampled on nodes 3-4 and 6-7 (from the top of the stem) displayed higher regrowth compared to shoot tips. No significant differences were noted in regrowth after cryopreservation between buds sampled from microcuttings produced from the terminal node, or nodes 3-4 and 6-7. CONCLUSION: The physiological state of the plant material is important for cryopreservation success. Actively growing buds sampled from microcuttings displayed higher regrowth compared to buds sampled directly on in vitro plantlets.

Keywords: droplet-vitrification, shoot tips, growth regulators, position of buds.

 

 

 

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CryoLetters 35 (1), 48-53 (2014)
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NUCLEAR STATUS AND DNA FRAGMENTATION OF OOCYTES FROM PORCINE, BOVINE AND FELINE OVARIES STORED AT 4°C FOR 5 DAYS

Vien Viet Luu, Zhao Namula, Lanh Thi Kim Do, Yoko Sato,
Masayasu Taniguchi, Ni Wayan Kurniani Karja
and Takeshige Otoi*

Laboratory of Animal Reproduction, the United Graduate School of Veterinary Science,
Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan
*Corresponding author email:
otoi@yamaguchi-u.ac.jp

Abstract

BACKGROUND: The cooling of mammalian oocytes to sub-physiological temperatures is widely known to affect their viability through the induction of various abnormalities at all stages of meiosis. OBJECTIVE: This study was to compare the kinetics of nuclear status and oocyte damage in porcine, bovine and feline ovaries stored at 4°C for 5 days.  METHODS: The nuclear status and oocyte quality during storage were evaluated before and after maturation culture. RESULTS: The cold storage of ovaries decreased the proportions of porcine and bovine oocytes that remained at the germinal vesicle stage before maturation culture. The maturation rates of oocytes decreased with increasing storage time, independent of species. None of the porcine oocytes reached metaphase II (MII) after 1 day of storage. In contrast, bovine and feline oocytes from ovaries that were stored for 2 days and 3 days reached MII. DNA fragmentation in porcine oocytes from ovaries stored for 1 day was significantly higher than that in bovine and feline oocytes. CONCLUSION: The maturation competency of oocytes after the cold storage of ovaries could be related to the meiotic resumption of oocytes during storage and the occurrence of DNA fragmentation in oocytes during maturation culture.

Keywords: Chilling sensitivity, DNA damage, long-term storage, meiotic competency

 

 

 

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CryoLetters 35 (1), 54-62 (2014)
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COLD PRETREATMENT AMPLIFIES THE RESPONSES OF IN VITRO EUCALYPTUS GRANDIS SHOOTS TO CRYOPREPARATIVE DRYING

Chao-Hsuan Ting, David J. Mycock and Kershree Padayachee*

School of Animal, Plant and Environmental Sciences, University of the Witwatersrand,
Johannesburg, Private Bag 3, Wits, 2050, South Africa.
*Corresponding author email: Kershree.Padayachee@wits.ac.za

Abstract

BACKGROUND: Eucalyptus grandis is an important commercial forestry species in South Africa. Little is known about its response (which is both drought and cold sensitive) to cold exposure and subsequent drying. OBJECTIVE: The study was to investigate the responses of E. grandis in vitro shoots to cold pretreatment.  METHODS: E.grandis in vitro shoots were subjected to cold pretreatment (5, 10 and 15oC at different exposure periods of 1 or 3 days), different drying times (20, 40, 60 and 80 min over activated silica gel), and the combination of the selected cold pretreatment regime and subsequent drying. RESULTS: Cold pretreatment alone did not cause detrimental effects but elicited the accumulation of phenolic acids. Shoots exposed to 5oC for 3 days had significantly higher levels of reactive oxygen species as well. The accumulation of ROS and phenolic acids was also noted in unacclimated, dried shoots, especially after 80 min of drying. In addition, these shoots had significantly higher levels of total soluble sugars, lower levels of starch and elevated proline levels.  CONCLUSION: This osmotic adjustment strategy was amplified in cold acclimated, dried shoots (10oC, 3 days; 80 min drying), which also had significantly lower levels of ROS, increased levels of phenolic acid, and higher water content and viability.

Keywords: Cold pretreatment, drying, cross-tolerance, Eucalyptus grandis, osmotic adjustment

 

 

 

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CryoLetters 35 (1), 63-68 (2014)
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CONTROLLED RATE COOLING OF FUNGI USING A STIRLING CYCLE FREEZER

Matthew J. Ryan*, Daiva Kasulyte-Creasey, Anthony Kermode,
Shwe Phue San and Alan G. Buddie

CABI, Bakeham Lane, Egham Surrey TW20 9TY UK.
*Corresponding author email:
m.ryan@cabi.org

Abstract

BACKGROUND: The use of a Stirling cycle freezer for cryopreservation is considered to have significant advantages over traditional methodologies including 'N2-free' operation, application of low cooling rates, reduction of sample contamination risks and control of ice nucleation. OBJECTIVE: The study assesses the suitability of an 'N2-free' Stirling Cycle controlled rate freezer for fungi cryopreservation. METHODS: In total 77 fungi, representing a broad taxonomic coverage, were cooled using the 'N2-free' cooler following a cooling rate of -1°C min-1. Of these, 15 strains were also cryopreserved using a traditional 'N2 gas chamber' controlled rate cooler and a comparison of culture morphology and genomic stability against non-cryopreserved starter cultures was undertaken. RESULTS: In total of 75 fungi survived cryopreservation, only a recalcitrant Basidiomycete and filamentous Chromist failed to survive. No changes were detected in genomic profile after preservation, suggesting that genomic function is not adversely compromised as a result of using 'N2-free' cooling. CONCLUSION: The results demonstrate the potential of 'N2-free' cooling for the routine cryopreservation of fungi in Biological Resource Centres.

Keywords: cryopreservation, genomic stability, PCR fingerprinting

 

 

 

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CryoLetters 35 (1), 70-76 (2014)
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SCREENING OF TRANSGENIC FROST-RESISTANT COTTON USING A POROUS SILICON BIOSENSING PLATFORM

Liangliang Chen1, Peng Li2, Jie Zhao1, Jieqiong Li1, Xiaoyi Lv3, Zhenhong Jia3 and Ji Ma1,*

1Key Laboratory of Xinjiang Biological Resources and Gene Engineering, College of Life Sciences and Technology, and
2School of Physics, and
3College of Information Science and Engineering, Xinjiang University, Urumqi 830046, China
*Corresponding author email: majiuci@xju.edu.cn

Abstract

BACKGROUND: The genetic screening of exogenous gene expression is critical for the transgenic plant breeding. OBJECTIVE: Here, a novel label-free porous silicon (PSi)-based biosensor is reported for the identification of frost-resistant cotton. METHODS: Changes in optical response signal in the presence of antifreeze protein (AFP) were detected by Fourier transform infrared (FTIR) spectromicroscopy when binding of the target AFP protein with anti-AFP-antibodies was selectively captured on the PSi biosensor. RESULTS: Compared with non-transgenic plants, significant red shifts were observed for transgenic frost-resistant cotton lines except a transgenic line 2. CONCLUSION: The approach is highly efficient with lower cost and high sensitivity for detecting large number of transgenic samples in the trial field simultaneously. This PSi biosensing platform has potential commercial applications for the rapid assessment of transgenic plants in the field.

Keywords: transgenic cotton; antifreeze protein; antibody; porous silicon; protein biosensor

 

 

 

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CryoLetters 35 (1), 77-82 (2014)
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COMPARISON OF SLOW FREEZING AND VITRIFICATION ON OVINE IMMATURE OOCYTES

M H Bhat1*, V Sharma 2, F A Khan1, N A Naykoo1, S H Yaqoob 3, Ruby1, H M Khan 4, M R Fazili 5, N A Ganai1 and R A Shah1

1Division of Biotechnology, and
4Mountain research Centre for Sheep and Goat, and
5Teaching Veterinary Clinical Services Complex, Faculty of Veterinary Sciences, Sher-e-Kashmir University of Agricultural Sciences and Technology, Jammu and Kashmir, India
2Department of Bio-Science & Biotechnology, Banasthali University, Rajasthan, India.
3Department of Animal production, College of Food and Agricultural Sciences, King Saud University, Riyadh, KSA.
*Corresponding author email: maajidhassan@yahoo.com

Abstract

BACKGROUND: Immature oocytes are more sensitive to cold injury than mature oocytes. OBJECTIVE: The study was to evaluate the post thaw normal oocytes, cleavage and blastocyst rates of ovine cumulus oocyte complexes (COC's) using different cryoprotectants by slow freezing and Open pulled straw (OPS) vitrification. METHODS: In five replicates, abattoir derived COC's were collected and distributed into three groups. In Experiment 1, COC's were cryopreserved by a slow freezing protocol using 10% concentration of ethylene glycol (EG), 10% dimethyl sulphoxide (DMSO) or 5% EG and 5% DMSO mixture. In Experiment 2 and 3 embryos were cryopreserved by OPS vitrification using either 33% or 40% (EG, DMSO or an equal mixture of EG and DMSO mixture. Normal oocytes post thaw were in vitro matured and parthenogenetically activated. RESULTS: Although, there was no difference in the number of post thaw normal oocytes between the groups, cleavage and blastocyst rates were higher in 10% slow freezing group than any of the vitrified groups. CONCLUSION: The study demonstrates better cryopreservation of ovine COC's by controlled slow freezing than OPS vitrification.

Keywords: Slow Freezing, Vitrification, ethylene glycol, DMSO, immature oocytes

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